Composition for subcutaneous administration comprising hyaluronidase polypeptide and drug

Abstract

The present application relates to a composition for subcutaneous administration comprising a hyaluronidase polypeptide and a drug.

Description

Composition for subcutaneous administration comprising hyaluronidase polypeptide and drug

[0001] The present application relates to a composition for subcutaneous administration comprising a hyaluronidase polypeptide and a drug.

[0002] Hyaluronidase, a general term for enzymes that degrade hyaluronic acid, was first known as a diffusion factor by Duran-Reynals, but was later observed to exhibit strong activity on hyaluronic acid, and was thus called hyaluronidase. Depending on its mechanism of action, this enzyme is classified into hyaluronate 4-glycanohydrolase (EC 3.2.1.35) distributed in testes, lysosomes, and bee venom, hyaluronate 3-glycanohydrolase (EC 3.2.1.36) present in leeches, and hyaluronate lyase (EC 4.2.2.1) present in bacteria.

[0003] In particular, hyaluronidase (PH-20) in the testis is an important enzyme that is attached to the glycosylphosphatidylinositol (GPI) anchor of the acrosome of the sperm, thereby degrading the thick outer wall layer of the egg and causing fertilization. In addition, PH-20 is known to hydrolyze the β(1-4) linkage of D-glucuronic acid and N-acetyl-D-glucosamine present in hyaluronic acid (HA), chondroitin, and chondroitin sulfate among glycosaminoglycans present in mammalian skin. The general molecular formula of these enzymes is C2455H3775N617O704S21, and the molecular weight is 53870.9 g / mol. In humans, six genes are associated with this enzyme, including HYAL1, HYAL2, HYAL3, and PH-20 / SPAM1.

[0004] The wide range of uses of hyaluronidase has been comprehensively reviewed since the 1950s, with the first use being subcutaneous injection of fluids. Other uses include infiltration and blocking anesthesia to increase the diffusion of local anesthetics and steroids in orthopedic, ophthalmic, plastic, dental, oral, gynecological, and otolaryngological surgeries, dispersing collections of fluid such as hematomas, preventing peritoneal adhesions, preventing stone formation, and treating infertility.

[0005] Currently, commercially available hyaluronidases are extracted from the testes of sheep (ovine) or cows (bovine). Examples include Vitrase (ISTA Pharmaceuticals, ovine source) and Amphadase (Amphastar Pharmaceuticals, bovine source). These raw hyaluronidases are dissolved to an appropriate concentration, filled into vials, and lyophilized to produce commercial products. However, commercialized animal-derived hyaluronidases contain foreign proteins, which can cause allergic reactions. In addition, their stability deteriorates over time, resulting in decreased physiological activity, leaving many problems for their diverse applications.

[0006] To address these issues, research on recombinant hyaluronidase has been conducted. Recombinant proteins can be expressed in various cell types, including Escherichia coli, yeast, insect cells, and animal cells. In particular, in the case of hyaluronidase, glycosylation, which occurs during the post-translational protein modification process, affects its activity. This is because glycans can affect the antigenicity, structural folding, solubility, and stability of glycoproteins. From this perspective, animal cells are suitable among various expression cell types because the post-translational protein modification process in yeast or insect cells differs from that in mammals. Among animal cells, CHO (Chinese Hamster Ovary) cells, which have been proven to be safe, are the most suitable.

[0007] The first recombinant hyaluronidase, PH-20, is marketed under the trade name Hylenex by Halozyme Therapeutics and is under development for various applications, including subcutaneous injection, vitrectomy, and ophthalmic disorders. However, hyaluronidase still has low yield and stability, and supply is low compared to demand, creating a need for hyaluronidase with improved yield and stability.

[0008] An example of the present application is to provide a composition for subcutaneous administration comprising a hyaluronidase polypeptide having excellent stability and increased activity; and a drug.

[0009] Another example of the present application is to provide a pharmaceutical composition for treating cancer, comprising a hyaluronidase polypeptide according to an example of the present application and an anticancer agent.

[0010] An example of the present application relates to a composition for subcutaneous administration comprising a polypeptide having a C-terminal truncated amino acid sequence of a wild-type hyaluronidase; and a drug.

[0011] Another example of the present application relates to a polypeptide having a sequence of amino acids of a wild-type hyaluronidase in which a selected number of amino acids from the C-terminus among 1 to 203 are consecutively deleted; and a composition for subcutaneous administration comprising a drug.

[0012] Another example of the present application relates to a pharmaceutical composition for treating cancer, comprising a polypeptide having a C-terminal truncated amino acid sequence of a wild-type hyaluronidase; and an anticancer agent.

[0013] Another example of the present application relates to a pharmaceutical composition for treating cancer, comprising a polypeptide having a sequence of amino acids of a wild-type hyaluronidase in which a selected number of amino acids from the C-terminus among 1 to 203 are consecutively deleted; and an anticancer agent.

[0014]

[0015] Hereinafter, the present application will be described in more detail.

[0016] An example of the present application relates to a polypeptide having at least 90% sequence homology with a C-terminally truncated polypeptide in the amino acid sequence of a wild-type hyaluronidase. The wild-type hyaluronidase may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

[0017] A polypeptide according to an example of the present application has hyaluronidase activity and has one or more of the following properties (1) to (6):

[0018] (1) pH 3 to 10, pH 3 to 9, pH 3 to 8, pH 3 to 7, pH 3 to 6.5, pH 3 to 6, pH 3 to 5.5, pH 3 to 5, pH 3 to 4.5, pH 4 to 10, pH 4 to 9, pH 4 to 8, pH 4 to 7, pH 4 to 6.5, pH 4 to 6, pH 4 to 5.5, pH 4 to 5, pH 4 to 4.5, pH 4.5 to 10, pH 4.5 to 9, pH 4.5 to 8, pH 4.5 to 7, pH 4.5 to 6.5, pH 4.5 to 6, pH 4.5 to 5.5, pH 4.5 to 5, pH 5 to 10, pH 5 to 9, pH 5 to 8, pH Stable properties in a pH range of 5 to 7, pH 5 to 6.5, pH 5 to 6, or pH 5 to 5.5, specifically, enzyme activity is at least 57% of the initial activity after 4 weeks of storage at a pH falling within a range of greater than pH 3 and less than or equal to 10 (e.g., greater than pH 3 and less than 7, greater than pH 3 and less than or equal to 6.5, greater than pH 3 and less than or equal to 6, greater than pH 3 and less than or equal to 5.5, greater than pH 3 and less than or equal to 5, for example, pH 5.0, pH 7.0, or pH 10), or enzyme activity is at least 32% of the initial activity after 4 weeks of storage at a pH falling within a range of greater than or equal to pH 3 and less than 5 (e.g., pH 3 to 4.5, or pH 3 to 4, for example, pH 3.0).

[0019] (2) Stable properties at temperatures ranging from -20 to 45°C, specifically, enzyme activity of at least 63% of the initial activity after 4 weeks of storage at a temperature below 0°C, enzyme activity of at least 83% of the initial activity after 4 weeks of storage at a temperature ranging from 0 to 40°C, or enzyme activity of at least 52% of the initial activity after 4 weeks of storage at a temperature ranging from 40°C or higher.

[0020] (3) High hyaluronidase activity, specifically, hyaluronidase activity that is 1 to 3 times higher than that of wild-type hyaluronidase,

[0021] (4) Having a potency that is 1 to 3 times greater than the potency of a polypeptide consisting of the amino acid sequence of sequence number 1;

[0022] (5) having an activity of 120,000 to 150,000 IU / mg, and

[0023] (6) One or more amino acid residues are glycated.

[0024] Specifically, in the present example, a polypeptide was prepared by truncating the C-terminus of the amino acid sequence of an ovine-derived wild-type hyaluronidase (CAS no. 488712-31-8), and the stability and activity of the prepared polypeptide were confirmed. As a result, it was found that the polypeptide exhibited significantly superior stability and activity compared to the wild-type hyaluronidase, and an equivalent drug absorption effect, drug diffusion promotion effect, and body fluid reabsorption promotion effect could be achieved using a small amount of protein. Accordingly, a polypeptide according to an example of the present application may be a hyaluronidase.

[0025] Specifically, a polypeptide according to an example of the present application may have a sequence identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more to a polypeptide having a consecutive deletion of an integer number of amino acids from 1 to 203 from the C-terminus in the amino acid sequence of a wild-type hyaluronidase. In this case, the polypeptide according to an example of the present application is not a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. In addition, the polypeptide according to an example of the present application may maintain the catalytic activity of a wild-type hyaluronidase. Specifically, the polypeptide according to an example of the present application may have an activity and / or stability equivalent to or greater than that of a wild-type hyaluronidase. The above wild-type hyaluronidase may be a polypeptide consisting of an amino acid sequence of sequence number 1.

[0026] Specifically, a polypeptide according to an example of the present application may be one in which n amino acids are consecutively deleted from the C-terminus in the amino acid sequence of a wild-type hyaluronidase (provided that n is a natural number from 1 to 203).

[0027] For example, a polypeptide according to an example of the present application comprises an amino acid sequence of a wild-type hyaluronidase, from the C-terminus, an integer selected from 1 to 203, an integer selected from 1 to 170, an integer selected from 1 to 136, an integer selected from 1 to 102, an integer selected from 1 to 68, an integer selected from 34 to 203, an integer selected from 34 to 170, an integer selected from 34 to 136, an integer selected from 34 to 102, an integer selected from 34 to 68, an integer selected from 68 to 203, an integer selected from 68 to 170, an integer selected from 68 to 136, an integer selected from 68 to 102, 34, 68, 102, 136, or 170 amino acids may be deleted consecutively. A polypeptide according to an example of the present application may be a polypeptide having an additional deletion of the first amino acid from the N-terminus in the amino acid sequence of a wild-type hyaluronidase. The amino acid sequence of the wild-type hyaluronidase may be the amino acid sequence of SEQ ID NO: 1.

[0028] For example, a polypeptide according to an example of the present application may be composed of 1 to m or 2 to m amino acids from the N-terminus in the amino acid sequence of a wild-type hyaluronidase (provided that m is a natural number from 315 to 517). The amino acid sequence of the wild-type hyaluronidase may be the amino acid sequence of SEQ ID NO: 1.

[0029] For example, a polypeptide according to an example of the present application may be composed of an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0030] For example, a polypeptide according to an example of the present application may be expressed using an animal cell as a host. Accordingly, a polypeptide according to an example of the present application may be expressed using an animal cell as a host, and may be glycosylated through a post-translational modification (PTM) process during protein expression.

[0031] A polypeptide according to an example of the present application may be stable at pH 3 to 10. Specifically, a polypeptide according to an example of the present application may exhibit less decrease in enzymatic activity compared to wild-type hyaluronidase when stored at pH 3 to 10.

[0032] In the present example, a polypeptide according to an example of the present application was stored in a pH range of 3 to 10 and enzyme activity was measured. As a result, enzyme activity was maintained even after 4 weeks of storage.

[0033] For example, a polypeptide according to an example of the present application may have an enzyme activity of at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, or at least 55% of the initial activity after storage for 4 weeks at a pH of less than pH 3 to 5, or a pH in the range of pH 3 to 4, for example, pH 3. At this time, the storage temperature of the polypeptide may be 5°C or 37°C.

[0034] For example, a polypeptide according to an example of the present application may have an enzyme activity of at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, or at least 65% of the initial activity after storage for 4 weeks at a pH ranging from pH 3 to 10, pH 4 to 10, or pH 5 to 10, for example, pH 5, pH 7, or pH 10. In this case, the storage temperature of the polypeptide may be 5°C or 37°C.

[0035] For example, a polypeptide according to an example of the present application has an enzyme activity after 4 weeks storage at a pH in the range of pH 3 to less than 10, pH 3 to 9, pH 3 to 8, pH 3 to 7, pH 4 to less than 10, pH 4 to 9, pH 4 to 8, pH 4 to 7, pH 5 to less than 10, pH 5 to 9, pH 5 to 8, or pH 5 to 7, for example, pH 5 or pH 7, of which the initial activity is at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74% The content may be 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, or 82% or more. At this time, the storage temperature of the polypeptide may be 5°C or 37°C.

[0036] Specifically, a polypeptide according to an example of the present application may be stable under slightly acidic conditions, for example, pH 3 to 6.5, pH 3 to 6, pH 3 to 5.5, pH 3 to 5, pH 3 to 4.5, pH 4 to 4.5, pH 4 to 5, pH 4 to 5.5, pH 4 to 6, pH 4 to 6.5, pH 4.5 to 5, pH 4.5 to 5.5, pH 4.5 to 6, pH 4.5 to 6.5, pH 5 to 5.5, pH 5 to 6, or pH 5 to 6.5. pH is an important factor in the stability of drugs, and particularly, antibody drugs have a range of pH 5 to 6.5 or pH 5 to 6. Conventional hyaluronidases are stable at neutral pH, and thus, when conventional hyaluronidases are mixed with antibody drugs and stored for long periods, stability due to pH may be an issue. A polypeptide according to an example of the present application is stable under slightly acidic conditions similar to the pH of the antibody drug (e.g., pH 5.0), and is thus advantageous for preparation as a formulation mixed with the antibody drug, thereby being advantageous for formulation and long-term storage.

[0037] A polypeptide according to an example of the present application may be stable under freezing, refrigeration, and high temperature conditions. Specifically, a polypeptide according to an example of the present application may exhibit a smaller decrease in enzymatic activity compared to wild-type hyaluronidase when stored under freezing, refrigeration, and high temperature conditions.

[0038] In the present example, a polypeptide according to an example of the present application was stored at frozen (e.g., -18°C to -20°C), refrigerated (e.g., 2°C to 8°C) and high temperature (e.g., 40°C to 45°C) temperatures, and enzyme activity was measured. As a result, enzyme activity was maintained even after 4 weeks of storage.

[0039] For example, a polypeptide according to an example of the present application has an enzyme activity of at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% of the initial activity after storage for 4 weeks at a temperature of -20°C. The polypeptide may have a purity of 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. At this time, the storage pH of the polypeptide may be 5 or 7.

[0040] For example, a polypeptide according to an example of the present application may have an enzyme activity of at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the initial activity after storage for 4 weeks at a refrigerated temperature, for example, a temperature in the range of 0 to 40°C, 0 to 10°C, or 2 to 8°C, for example, a temperature of 5°C. At this time, the storage pH of the polypeptide may be 5 or 7.

[0041] For example, a polypeptide according to an example of the present application may have an enzyme activity of at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, or at least 79% of the initial activity after storage for 4 weeks at a high temperature, for example, at a temperature of 40°C. At this time, the storage pH of the polypeptide may be 5 or 7.

[0042] A polypeptide according to an example of the present application may have a higher titer compared to wild-type hyaluronidase. For example, a polypeptide according to an example of the present application may have an activity of greater than 1 to 3 times, greater than 1 to 2.5 times, greater than 1 to 2 times, greater than 1 to 1.9 times, greater than 1 to 1.8 times, greater than 1 to 1.7 times, 1.1 to 3 times, 1.1 to 2.5 times, 1.1 to 2 times, 1.1 to 1.9 times, 1.1 to 1.8 times, 1.1 to 1.7 times, 1.2 to 3 times, 1.2 to 2.5 times, 1.2 to 2 times, 1.2 to 1.9 times, 1.2 to 1.8 times, 1.2 to 1.7 times, 1.3 to 3 times, 1.3 to It may have a potency of 2.5 times, 1.3 times to 2 times, 1.3 times to 1.9 times, 1.3 times to 1.8 times, 1.3 times to 1.7 times, 1.4 times to 3 times, 1.4 times to 2.5 times, 1.4 times to 2 times, 1.4 times to 1.9 times, 1.4 times to 1.8 times, 1.4 times to 1.7 times, 1.5 times to 3 times, 1.5 times to 2.5 times, 1.5 times to 2 times, 1.5 times to 1.9 times, 1.5 times to 1.8 times, or 1.5 times to 1.7 times.

[0043] For example, a polypeptide according to an example of the present application may have an IU / mg of 120,000 to 150,000 IU / mg, 120,000 to 145,000 IU / mg, 120,000 to 140,000 IU / mg, 120,000 to 135,000 IU / mg, 121,000 to 150,000 IU / mg, 121,000 to 145,000 IU / mg, 121,000 to 140,000 IU / mg, 121,000 to 135,000 IU / mg, 122,000 to 150,000 IU / mg, 122,000 to 145,000 IU / mg, 122,000 to It may have an activity of 140,000 IU / mg, or 122,000 to 135,000 IU / mg.

[0044] In one embodiment of the present invention, topical administration of a polypeptide according to an example of the present invention resulted in significantly superior drug absorption and diffusion promotion compared to wild-type hyaluronidase. Furthermore, in one embodiment of the present invention, topical administration of a polypeptide according to an example of the present invention resulted in significantly superior drug reabsorption promotion compared to wild-type hyaluronidase. Therefore, a polypeptide according to an example of the present invention can be used in combination with a drug for subcutaneous administration of the drug.

[0045] The drug included in the composition according to an example of the present application may be a protein drug, an antibody, an aptamer, mRNA, siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (micro RNA), aiRNA (asymmetric interfering RNA), RNAi (RNA interference), an antisense oligonucleotide, or a small molecule.

[0046] The drug may be a soluble receptor, or a soluble receptor and Fc fusion protein.

[0047] The above drug may be an anticancer agent, antidiabetic agent, anti-inflammatory agent, antiviral agent, or immunomodulator. The immunomodulator may be, for example, an autoimmune disease treatment agent.

[0048] The above protein pharmaceutical is a drug composed of amino acids and exhibits a disease treatment or prevention effect through the activity of the protein, and refers to a drug composed of proteins other than antibody pharmaceuticals, and may include, for example, one or more selected from the group consisting of cytokines, therapeutic enzymes, hormones, soluble receptors and fusion proteins thereof, insulin or analogues thereof, BMP (Bone Morphogenetic Protein), EPO (erythropoietin), and serum derived proteins.

[0049] The above cytokines may include one or more selected from the group consisting of interferon, interleukin, colony stimulating factor (CSF), tumor necrosis factor (TNF), and tissue growth factor (TGF).

[0050] The cytokines and their analogues include, but are not limited to, Oprelvekin, Peginterferon Beta-1a, Pegilodecakin, Rezpegaldesleukin, SAR 444245, GO-203-2c, Dekavil, Nemvaleukin alfa, Efavaleukin alfa, Efineptakin alfa, Onfekafusp alfa, Bifikafusp alfa, or Lerodalcibep.

[0051] The above therapeutic enzyme may include at least one selected from the group consisting of β-glucocerebrosidase and agalsidase β.

[0052] The above soluble receptor refers to the extracellular domain of the receptor, and its fusion protein refers to a protein in which the Fc region of an antibody, etc. is fused to the soluble receptor. The above soluble receptor is a soluble form of a receptor to which a disease-related ligand binds, such as a form in which an Fc region is fused to a TNF-α soluble receptor (e.g., a product with the generic name Etanercept and similar forms), a form in which an Fc region is fused to a VEGF soluble receptor (e.g., a product with the generic name Aflibercept, Conbercept or OPT-302 and similar forms), a form in which an Fc region is fused to a CTLA-4 (e.g., a product with the generic name Abatacept or Beladacept and similar forms), a form in which an Fc region is fused to an interleukin soluble receptor (e.g., a product with the generic name Rilonacept or Inbakicept and similar forms), a form in which an Fc region is fused to a LFA3 soluble receptor (e.g., Alefacept) Examples thereof include, but are not limited to, products with the active ingredient name of and similar forms), products in which the Fc region is fused to signal regulatory protein alpha (SIRPalpha) that interacts with CD47 (e.g., products with the active ingredient name of Evorpacept, Ontorpacept, or TTI-622 and similar forms), products with the active ingredient name of Sotatercept, Dalantercept, Acazicolcept, or Dazodalibep and similar forms, etc.

[0053] The above hormone refers to a hormone or analogue thereof that is injected externally to treat or prevent diseases caused by hormone deficiency, etc., and examples thereof include, but are not limited to, human growth hormone, estrogen, and progesterone.

[0054] The above plasma-derived proteins are proteins present in plasma, and refer to both those extracted from plasma and those produced recombinantly, and examples thereof include, but are not limited to, fibrinogen, von Willebrand Factor, albumin, thrombin, FII (Factor II), FV (Factor V), FVII (Factor VII), FVIII (Factor VIII), FIX (Factor IX), FX (Factor X), and FXI (Factor XI).

[0055] The above antibody drug product may be a monoclonal antibody drug or a polyclonal antibody drug. The antibody may be a complete antibody or a binding fragment thereof, and the "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure, which is a part of a polypeptide that includes a portion capable of binding an antigen. For example, it may be scFv, (scFv)2, scFvFc, Fab, Fab', or F(ab')2, but is not limited thereto. The antigen-binding fragment of the antibody in the present application may be selected from the group consisting of antibody fragments including one or more complementarity determining regions, for example, scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2. The above antigen-binding fragment can be obtained using a proteolytic enzyme (for example, Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab')2 fragment can be obtained by digestion with pepsin), and can be produced through genetic recombination technology.

[0056] The antibody or antigen-binding fragment may be isolated from a living organism or non-naturally occurring. The antibody or antigen-binding fragment may be synthetically or recombinantly produced.

[0057] The above monoclonal antibody drug refers to a protein containing a monoclonal antibody or monoclonal antibody fragment capable of specifically binding to an antigen associated with a specific disease. Monoclonal antibodies also include bispecific and multispecific antibodies, and proteins containing monoclonal antibodies or fragments thereof are used in the sense of including antibody-drug conjugates (ADCs).

[0058] For example, antigens associated with specific diseases include 4-1BB, 5T4, integrin, Activin, amyloid beta, angiopoietin (angiopoietin 1 or 2), angiopoietin-like substance 3, B cell maturation antigen (BCMA), B-cell activating factor (BAFF), B7-H3, complement 5, CCR4, CCR5, CCL11, CD2, CD3, CD4, CD6, CD11a, CD16A, CD19, CD20, CD22, CD25, CD27, CD28, CD30, CD32B, CD33, CD38, CD40, CD45, CD46, CD47, CD52, CD56, CD62, CD70, CD73, CD74, CD79b, CD80, CD105, CD123, CD154, CD166, CD262, CD278, CD319, CD326, Carcinoembryonic antigen (CEA), CGRP, Claudin-18, c-Met, CSF-1, CSF-1 receptor, CTLA4, DLL3, EGF receptor, hemophilia factor, Fc receptor, FGF23, folate receptor, GD2, Glucocorticoid-induced TNF receptor (GITR), Glypican 3, GM-CSF, HER2, HER3, Hepatocyte Growth Factor (HGF), Interferon receptor, Interferon gamma, IgE, IGF-1 receptor, Interleukin 1, Interleukin 2 receptor, Interleukin 4, Interleukin 4 receptor, Interleukin 5, interleukin 5 receptor, interleukin 6, interleukin 6 receptor, interleukin 8, interleukin 12 / 23, interleukin 13, interleukin 17A, interleukin 17 receptor A, interleukin 23, interleukin 31 receptor, interleukin 36 receptor,Lymphocyte-activation gene 3 (LAG3), lysyl oxidase homolog 2 (LOXL2), mesothelin, mucin-1, mucin-16, netin-4, nerve growth factor (NGF), OX40, proprotein convertase subtilisin / kexin type 9 (PCSK9), PD-1, PD-L1, phospholipase C, receptor activator of nuclear factors kappa Bligand (RANKL), tyrosine-protein kinase transmembrane receptor (ROR1), sialic acid binding Ig-like lectin 15 ig-like lectin 15 (Siglec-15), transforming growth factor beta (TGFβ), TIGIT (T-cell innunoreceptor with immunoglobulin and ITIM domain), T cell immunoglobulin and mucin-domain containing-3 (Tim-3), tissue factor, tissue factor pathway inhibitor (TFPI), TORP-2, tumor necrosis factor (TNF), thymic stromal lymphopoietin (TSLB), colony stimulating factor 1 receptor (CSF1R),Examples include, but are not limited to, vascular endothelial growth factor (VEGF), VEGF receptors, and von Willebrand Factor (vWF).

[0059] Proteins comprising the above monoclonal antibodies and monoclonal antibody fragments include ADG-106, EU-101, LVGN6051, Urelumab, Utomilumab, Bebtelovimab, Aducanumab, Bapinezumab, Crenezumab, Donanemab, Gantenerumab, Lecanemab, Solanezumab, Nesvacumab, Evinacumab, Enoblituzumab, Omburtamab, Belimumab, Ianalumab, Tabalumab, Bertilimumab, Mogamulizumab, Leronlimab, Siplizumab, Foralumab, Muromonab-CD3, Otelixizumab, Teplizumab, Ibalizumab, Tregalizumab, Zanolimumab, Itolizumab, Efalizumab, Inebilizumab, Tafasitamab, Tositumomab, Ocrelizumab, Ofatumumab, Rituximab, Ublituximab (Ublituximab), Veltuzumab, Epratuzumab, Basiliximab, Daclizumab, Varlilumab, Lulizumab pegol, Iratumumab, BI-1206, Lintuzumab,Daratumumab, Felzartamab, GEN3014, Isatuximab, Mezagitamab, CDX-1140, Bleselumab, Dacetuzumab, Iscalimab, Lucatumumab, Mitazalimab, SEA-CD40, Sotigalimab, SAR441344, Tegoprubart, Dapirolizumab pegol, I-131-Apamistamab, AO-176, Ligufalimab, Magrolimab Alemtuzumab, Crizanlizumab, Inclacumab, Cusatuzumab, Oleclumab, Milatuzumab, Galiximab, Carotuximab, Adecatumumab, Eptinezumab, Erenumab, Fremanezumab, Galcanezumab, TST001, ZL-1211, Zolbetuximab, Onartuzumab, Eculizumab, Pozelimab, Ravulizumab, Lacnotuzumab (Lacnotuzumab), PD-0360324, AMB-051, Axatilimab, Cabiralizumab, Emactuzumab, BA3071, Ipilimumab, Quavonlimab, Tremelimumab, Zalifrelimab, Cetuximab,Depatuxizumab, Futuximab, Imgatuzumab, Matuzumab, Modotuximab, Necitumumab, Nimotuzumab, Panitumumab, Tomuzotuximab, Zalutumumab, MK-2060, Batoclimab, Nipocalimab, Rozanolixizumab, Burosumab, Farletuzumab, Dinutuximab, Dinutuximab beta, Naxitamab, BMS-986156, Ragifilimab, Gimsilumab, Lenzilumab, Mavrilimumab, Namilumab, Otilimab, Plonmarlimab, Codrituzumab, Margetuximab, Pertuzumab, Trastuzumab, HMBD-001, Patritumab, Seribantumab, Duligotuzumab, Ficlatuzumab, Rilotumumab, Alomfilimab, Anifrolumab, Emapalumab (Emapalumab), FB825, Ligelizumab, Omalizumab, Cixutumumab, Dalotuzumab, Figitumumab, Ganitumab, Teprotumumab, Bermekimab,Canakinumab, Gevokizumab, Briakinumab, Ustekinumab, Anrukinzumab, Cendakimab, Lebrikizumab, Tralokinumab, Brodalumab, Bimekizumab, Ixekizumab, Secukinumab, Brazikumab, Guselkumab, Mirikizumab, Risankizumab, Tildrakizumab, Nemolizumab, Imsidolimab, Spesolimab (Spesolimab), Pascolizumab, CBP201, Dupilumab, Defemokimab, Mepolizumab, Reslizumab, Benralizumab, Clazakizumab, Olokizumab, Siltuximab, Sirukumab, Ziltivekimab, Levilimab, Sarilumab, Satralizumab, Tocilizumab, HuMax-IL8, Abituzumab, Fabezelimab, Fianlimab, GSK2831781, Ieramilimab (Ieramilimab), INCAGN02385, Relatlimab, Simtuzumab, Abagovomab, Oregovomab, Tanezumab, BMS-986178, GSK3174998, INCAGN01949,Ivuxolimab, Rocatinlimab, Tavolimab, Telazorlimab, Vonlerolizumab, Alirocumab, Bococizumab, Ebronucimab, Evolocumab, Frovocimab, Ongericimab, Tafolecimab, Dostarlimab, Balstilimab, Camrelizumab, Cemiplimab, Geptanolimab, MEDI0680, Nivolumab, Pembrolizumab Pembrolizumab, Penpulimab, Pidilizumab, Prolgolimab, Retifanlimab, Sasanlimab, Serplulimab, Sintilimab, Spartalizumab, Tislelizumab, Toripalimab, Ezabenlimab, Zimberelimab, Atezolizumab, Avelumab, Cosibelimab, Sugemalimab, Durvalumab, IMC-001, Envafolimab, Subratoxumab (Suvratoxumab), Denosumab, Zilovertamab, NC318, Elotuzumab, NIS793, BMS-986207, Domvanalimab, EOS-448, Etigilimab, Ociferlimab,Tiragolumab, Vibostolimab, Surzebiclimab, Cobolimab, Sabatolimab, TQB2618, Concizumab, Marstacimab, Adalimumab, Golimumab, Infliximab, Certolizumab pegol, Conatumumab, Tigatuzumab, Tezepelumab, Gatipotuzumab, Cabiralizumab, Bevacizumab, Brolucizumab, Ranibizumab (Ranibizumab), Olinvacimab, Icrucumab, Ramucirumab, Caplacizumab, Abrilumab, Etrolizumab, Vedolizumab, Intetumumab, Natalizumab, etc., but are not limited thereto.

[0060] The above bi / multispecific antibodies and antibody-like proteins include DSP107, RO7122290, Cinrebafusp alfa, GEN1042, Rozibafusp alfa, GEN1044, Obrindatamab, GEN1047, Elranatamab, Linvoseltamab, Teclistamab, Epcoritamab, Glofitamab, Mosunetuzumab, Odronextamab, Flotetuzumab, Vibecotamab, Catumaxomab, Cibisatamab, TAK-186, Talquetamab, Ubamatamab, NVG-111, Emfizatamab, HPN536, AFM13, Blinatumomab, ISB1442, CPO107, AFM24, Amivantamab, MCLA-129, SI-B001, Emicizumab, Mim8, Zenocutuzumab, Zanidatamab, Tibulizumab, GI-101, Vobarilizumab, REGN5668, HX009, Cadonilimab, Vudalimab, EMB-02, RO7247669, Tevotelimab (Tebotelimab), IBI318, AZD2936, AZD7789, RO7121661, Ivonescimab, IBI322, ES101, GEN1046, PRS-344, Erfonrilimab, FS118, Bintrafusp alfa, HB0036, HLX301, NM21-1480, Ozoralizumab,BI836880, Faricimab, Vanucizumab, Navicixizumab, IBI302, etc., but are not limited thereto.

[0061] The antibody-drug conjugates include Naptumomab estafenatox, Belantamab mafodotin, DS-7300, MGC018, Pivekimab sunirine, Praluzatamab ravtansine, Coltuximab ravtansine, Denintuzumab mafodotin, Loncastuximab tesirine, Ibritumomab tiuxetan, Inotuzumab ozogamicin, Epratuzumab-cys-tesirine, and Moxetumomab pasudotox. (Moxetumomab pasudotox), Brentuximab vedotin, Gemtuzumab ozogamicin, Vadastuximab talirine, STI-6129, FOR46, Lorvotuzumab mertansine, Polatuzumab vedotin, Tusamitamab ravtansine, Telisotuzumab vedotin, Rovalpituzumab tesirine, Depatuxizumab mafodotin, Farletuzumab ecteribulin, Mirvetuximab soravtansine, ARX788, Trastuzumab deruxtecan, Trastuzumab duocarmazine, A166, Trastuzumab emtansine,DP303c, MRG002, BDC-1001, SHR-A1811, Disitamab vedotin, Pertuzumab zuvotolimod, Patritumab Deruxtecan, Anetumab ravtansine, BMS-986148, Enfortumab vedotin, NBE-002, MRG004, ABBV-3373, Sacituzumab Govitecan, etc., but are not limited thereto.

[0062] The above polyclonal antibody may be a serum antibody extracted from plasma, such as immune globulin, but is not limited thereto.

[0063] A composition for subcutaneous administration according to an example of the present application may further comprise a stabilizer. The stabilizer may comprise at least one selected from the group consisting of a buffer, a nonionic surfactant, a chelating agent, an alkali metal salt, and an alkaline earth metal salt.

[0064] The above buffer may be at least one selected from the group consisting of succinate buffer, acetate buffer, phosphate buffer, citrate buffer, malonate buffer, MES (2-(N-Morpholino)ethanesulphonic acid) buffer, Tris buffer, and glycine buffer.

[0065] The above nonionic surfactant may be a polyoxyethylene-sorbitan fatty acid ester.

[0066] The above nonionic surfactant may be at least one selected from the group consisting of polysorbate 20, polysorbate 80, and Triton X-100.

[0067] The above chelating agent may be EDTA.

[0068] The above alkaline earth metal salt may be MgCl2 and / or MgSO4.

[0069] Another example of the present application relates to a pharmaceutical composition for treating cancer, comprising a hyaluronidase polypeptide according to an example of the present application and an anticancer agent.

[0070] The above cancer is not particularly limited and includes both solid cancer and blood cancer. For example, it may be one or more selected from the group consisting of skin cancer such as melanoma, liver cancer, hepatocellular carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical spine cancer, ureteral cancer, osteosarcoma, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma, but is not limited thereto.

[0071] A composition according to an example of the present application may additionally include one or more active ingredients exhibiting the same or similar function in addition to the above-mentioned active ingredient.

[0072] In addition, a composition according to an example of the present application, for example, a composition for subcutaneous administration or a pharmaceutical composition, can be manufactured in a unit dosage form or manufactured by introducing it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier according to a method that can be clearly performed by a person having ordinary skill in the art to which the present invention pertains. The term “carrier” in the present application means a compound that facilitates the addition of a compound into a cell or tissue, and the term “pharmaceutically acceptable” means a composition that is physiologically acceptable and does not typically cause an allergic reaction such as gastrointestinal disorder or dizziness or a similar reaction when administered to a human.

[0073] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0074] In addition, the composition according to the present application, for example, a composition for subcutaneous administration or a pharmaceutical composition, may further include additives such as fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives in addition to the above components. In the present application, the content of the additives included in the composition is not particularly limited and may be appropriately adjusted within the content range used in conventional formulations.

[0075] As used herein, the term "excipient" refers to any substance, other than a therapeutic agent, that serves as a carrier or medium for the delivery of the therapeutic agent or is added to a pharmaceutical composition, thereby improving handling and storage characteristics or facilitating or facilitating the formation of a unit dosage of the composition.

[0076] The composition according to the present application, for example, a composition for subcutaneous administration or a pharmaceutical composition, can be formulated and used in various forms, such as an injection of a sterile injection solution, according to a conventional method according to each intended use, and can be administered through various routes, including local administration, for example, subcutaneous administration or intramuscular injection.

[0077] The preferred dosage of the composition according to the present application, for example, a composition for subcutaneous administration or a pharmaceutical composition, may vary in its range depending on the patient's condition and weight, age, sex, health status, dietary constitution, nature of the preparation, degree of disease, administration time of the composition, administration method, administration period or interval, excretion rate, and drug form, and may be appropriately selected by a person skilled in the art.

[0078] The term “effective dosage of a pharmaceutical composition” as used herein means the amount of an active ingredient in a composition sufficient to treat a specific symptom. This may vary depending on the formulation method, administration method, administration time, and / or administration route of the pharmaceutical composition, and may vary depending on various factors including the type and degree of response to be achieved by administration of the pharmaceutical composition, the type, age, weight, general health condition, symptoms or degree of disease, sex, diet, excretion, drugs used simultaneously or at the same time in the subject, other components of the composition, and similar factors well known in the medical field. A person of ordinary skill in the art can easily determine and prescribe an effective dosage for the desired treatment.

[0079] The pharmaceutical composition according to the present application may be administered once daily or divided into several doses. The composition may be administered as an individual treatment or in combination with another treatment, and may be administered sequentially or simultaneously with conventional treatments. Taking all of the above factors into consideration, the composition may be administered in an amount that achieves maximum efficacy with minimal side effects.

[0080] A polypeptide or composition according to an example of the present application may have a higher potency compared to wild-type hyaluronidase, thereby achieving an equivalent effect at a lower dosage. For example, a polypeptide or composition according to an example of the present application may have a daily dosage of 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, or 65% or less of the wild-type hyaluronidase. The dosage may be based on weight or weight percent. Additionally, the total daily dosage may be divided and administered continuously or discontinuously as needed.

[0081] A hyaluronidase polypeptide according to an example of the present application has an increased expression amount and stability in animal cells compared to mature wild-type PH-20, and has an enzymatic activity equivalent to or greater than that of mature wild-type PH-20. Therefore, a hyaluronidase polypeptide according to an example of the present application has an increased protein expression amount when expressed in CHO cells compared to mature wild-type PH-20, and has an increased protein stability, thereby having the effect of increasing industrial applicability for various purposes.

[0082] In addition, hyaluronidase has the property of decomposing hyaluronic acid, a component of the intercellular space, and thus regulates the binding force of tissues, facilitating drug penetration and diffusion, and promoting the reabsorption of body fluids that are excessively present in tissues. Therefore, its scope and application fields are gradually expanding. However, in the case of wild-type PH-20 of animal origin, there is a high possibility of infection from animal-derived materials. The hyaluronidase polypeptide according to an example of the present application is safe because the possibility of infection is low, and compared to wild-type PH-20 of animal origin, it has a higher activity per unit amount of protein, which can further increase the possibility of industrial use for various purposes.

[0083] FIGS. 1A to 1C are drawings showing the results of comparing the pH stability of a polypeptide according to an example of the present application with wild-type hyaluronidase through SDS-PAGE (FIG. 1A: storage week 0, FIG. 1B: storage week 2, FIG. 1C: storage week 4).

[0084] FIGS. 2A to 2C are drawings showing the results of comparing the temperature stability of a polypeptide according to an example of the present application with that of a wild-type hyaluronidase through SDS-PAGE (FIG. 2A: storage week 0, FIG. 2B: storage week 2, FIG. 2C: storage week 4; frozen: frozen, refrig.: refrigerated, high tem.: high temperature).

[0085] Figure 3 is a diagram showing the results of a plate titer test under various pH and temperature conditions to confirm whether the hyaluronic acid solution exhibits its own activity.

[0086] Figure 4a is a diagram showing the results of a titer test of wild-type hyaluronidase at temperature conditions of 20 to 60°C.

[0087] FIG. 4b is a diagram showing the results of a titer test at a temperature of 20 to 60°C for a polypeptide according to an example of the present application.

[0088] Figure 5a is a diagram showing the results of a titer test of wild-type hyaluronidase at temperature conditions of 35 to 40°C.

[0089] FIG. 5b is a diagram showing the results of a titer test at a temperature of 35 to 40°C for a polypeptide according to an example of the present application.

[0090] Figure 6a is a diagram showing the results of a titer test of wild-type hyaluronidase under pH 5 to 7 conditions.

[0091] Figure 6b is a drawing showing the results of a titer test under pH 5 to 7 conditions of a polypeptide according to an example of the present application.

[0092] FIG. 7 is a diagram showing the correlation between time and blood concentration of a polypeptide according to an example of the present application and Hirax in SD rats.

[0093] FIG. 8 is a diagram showing the bioavailability of a composition for subcutaneous administration comprising a polypeptide and a drug according to an example of the present application.

[0094] Hereinafter, the present application will be described in more detail with reference to the following examples. However, these examples are merely intended to illustrate the present application, and the scope of the present application is not limited by these examples.

[0095]

[0096] Example 1. Preparation of hyaluronidase

[0097] Hyaluronidase was prepared by truncating 34 (Example 1-2), 68 (Example 1-3), 102 (Example 1-4), 136 (Example 1-5), 170 (Example 1-6), or 204 (Example 1-7) amino acids from the C-terminus of the amino acid sequence of Ovine-derived wild-type hyaluronidase (CAS no. 488712-31-8) of SEQ ID NO: 1.

[0098] First, cDNA was synthesized based on the amino acid sequence of wild-type hyaluronidase (CAS no. 488712-31-8). The wild-type hyaluronidase gene was amplified using polymerase chain reaction (PCR), and expression and activity were confirmed in CHO-DG44 cells using the pcDNA 3.1 vector. Then, the gene was inserted into CHO-DG44 cells using the pOptiVEC vector. The number of CHO-DG44 cells was 4 to 6 x 10 6When the concentration reached 100 cells / mL, CHO-DG44 cells were transformed with a plasmid containing hyaluronidase cDNA inserted into the pOptiVEC vector using a gene injection method (electroporation). After transfection, CHO-DG44 cells were cultured in Power CHO 2CD (with L-Glutamine 4 mM) medium, and then centrifuged at 12,000 rpm for 10 minutes to collect the cell supernatant. The recovered culture medium was purified using various methods such as affinity chromatography, hydrophobic chromatography, and ion exchange chromatography, if necessary, and the purified hyaluronidase was replaced with water using Ultafiltration and Microfiltration. The sequence of the manufactured hyaluronidase is shown in Table 1.

[0099] <h2 style=";text-align:left;direction:ltr">명명서열 (N → C)서열번호실시예 1-1(CAS no.488712-31-8)LDFRAPPLIS NTSFLWAWNA PAERCVKIFK LPPDLRLFSV KGSPQKSATG QFITLFYADR LGYYPHIDEK TGNTVYGGIP QLGNLKNHLE KAKKDIAYYI PNDSVGLAVI DWENWRPTWA RNWKPKDVYR DESVELVLQK NPQLSFPEAS KIAKVDFETA GKSFMQETLK LGKLLRPNHL WGYYLFPDCY NHNYNQPTYN GNCSDLEKRR NDDLDWLWKE STALFPSVYL NIKLKSTPKA AFYVRNRVQE AIRLSKIASV ESPLPVFVYH RPVFTDGSST YLSQGDLVNS VGEIVALGAS GIIMWGSLNL SLTMQSCMNL GNYLNTTLNP YIINVTLAAK MCSQVLCHDE GVCTRKQWNS SDYLHLNPMN FAIQTGKGGK YTVPGKVTLE DLQTFSDKFY CSCYANINCK KRVDIKNVHS VNVCMAEDIC IEGPVKLQPS DHSSSQNEAS TTTVSSISPS TTATTVSPCT PEKQSPECLK VRCLEAIANV TQTGCQGVKW KNTSSQSSIQ NIKNQTTY1실시예 1-2DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLS LTMQSCMNLG NYLNTTLNPY IINVTLAAKMCSQVLCHDEG VCTRKQWNSS DYLHLNPMNF AIQTGKGGKY TVPGKVTLED LQTFSDKFYC SCYANINCKK RVDIKNVHSV NVCMAEDICI EGPVKLQPSD HSSSQNEAST TTVSSISPST TATTVSPCTP EKQSPECLKV RCL2실시예 1-3(BMI2004)DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLS LTMQSCMNLG NYLNTTLNPY IINVTLAAKM CSQVLCHDEG VCTRKQWNSS DYLHLNPMNF AIQTGKGGKY TVPGKVTLED LQTFSDKFYC SCYANINCKK RVDIKNVHSV NVCMAEDICI EGPVKLQPSD HSSSQNEAS3실시예 1-4DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSVGEIVALGASG IIMWGSLNLS LTMQSCMNLG NYLNTTLNPY IINVTLAAKM CSQVLCHDEG VCTRKQWNSS DYLHLNPMNF AIQTGKGGKY TVPGKVTLED LQTFSDKFYC SCYANINCKK RVDIK4실시예 1-5DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLS LTMQSCMNLG NYLNTTLNPY IINVTLAAKM CSQVLCHDEG VCTRKQWNSS DYLHLNPMNF AIQTGKGGKY T5실시예 1-6DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLS LTMQSCMNLG NYLNTTLNPY IINVTLAAKM CSQVLCH6실시예 1-7DFRAPPLISN TSFLWAWNAP AERCVKIFKLPPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLS LTM7

[0100] Example 2. Confirmation of stability according to hyaluronidase cleavage site

[0101] (1) Comparison of enzyme activity or content by storage pH according to cut area

[0102] The pH stability of six hyaluronidases, excluding Examples 1-7, which had no activity among the hyaluronidases manufactured in Example 1, was confirmed according to the cleavage site. After expressing each amino acid sequence in transient cells, the culture solution was concentrated in the same manner and the activity was measured. The experimental concentration was 1,500 IU / mL (=100%), and solutions were prepared according to the conditions described in Table 2 by substituting with water, and these were stored at 37°C at pH 3.0, pH 5.0, pH 7.0, and pH 10.0 for 4 weeks, and a titer test was performed at each time point every 2 weeks.

[0103] At each time point, the enzyme activity or content of the sample according to the cut site was tested according to the following test method:

[0104] 1) The activity of hyaluronidase according to the cut site is determined by ratio comparison with EP STD.

[0105] 1-1) Preparation of pH 6.4 phosphate buffer: Dissolve 2.5 g of sodium dihydrogen phosphate dodecahydrate, 2.5 g of sodium dihydrogen phosphate, and 8.2 g of sodium chloride in 950 mL of water, adjust to pH 6.4 with 1 M sodium hydroxide solution or 1 M hydrochloric acid solution, and add water to make 1,000 mL.

[0106] 1-2) Preparation of dilution: Mix 100 mL of pH 6.4 phosphate buffer and 100 mL of water, then add 0.140 g of gelatin reagent and dissolve at 37°C. Use the dilution within 2 hours.

[0107] 1-3) Preparation of substrate solution: Add 0.5 g of sodium hyaluronate to 100 mL of water little by little while stirring. Slowly add water until the sodium hyaluronate swells. Stir at 4°C for more than 12 hours. Store the substrate solution at 4°C and use within 4 days.

[0108] 1-4) Preparation of standard solution: Dissolve the EP STD (EDQM) standard in diluent to make approximately 50 IU / mL. Take exactly 3 mL of this solution and place it in a 250 mL volumetric flask, add diluent to make exactly 250 mL, and use it as a standard solution.

[0109] 1-5) Preparation of test solution: Dilute the hyaluronidase test solution according to the cut site to approximately 0.6 IU / mL, adjust the pH according to the conditions, and use.

[0110] 2) Operating method: Test using the standard solution and test solution according to the method below.

[0111] 2-1) After setting the constant temperature water bath to 37℃, add 7.5 mL of pH 6.4 phosphate buffer solution and 5.0 mL of substrate solution to a 50 mL conical tube, mix, place in the constant temperature water bath, and leave to stand until the temperature reaches 37℃.

[0112] 2-2) Add 2.5 mL of the test solution to a conical tube containing pH 6.4 phosphate buffer and substrate solution and mix for 1 minute.

[0113] 2-3) Uberde microviscometer (DIN 51 562, Part 2, Calillary type MIII, constant: approx. 0.1 mm 2 / s 2 Put the entire amount of mixed liquid in the conical tube into a viscometer (or equivalent viscometer).

[0114] 2-4) Using a stopwatch, measure the time it takes for the liquid to drop from the upper line to the lower line on the Uberde microviscometer.

[0115] 2-5) Repeat several times for about 20 minutes and measure the time.

[0116] 2-6) Repeat the above process 3 times and test.

[0117] 3) Calculation: Calculate the potency (IU / mg) using the following formula.

[0118] 3-1) Reaction time: T1+ T2 / 2

[0119] 3-2) ηr -1 : {(k X T2) / 0.6915} -1

[0120] T1: Time (in seconds) to reach the upper mark on the Uberde microviscometer

[0121] T2: T - T1

[0122] T: Time (in seconds) until the liquid reaches the lower mark on the Uberde microviscometer.

[0123] k: Uberde microviscometer constant (mm) 2 / s 2 ), see Uberde Microviscometer Certificate

[0124] 0.6915: Kinematic viscosity of the substrate solution at 37 ℃ (mm) 2 / s 2 )

[0125] 3-3) Activity calculation: (B T / B R )*(E R / E T )*A

[0126] B T : x-axis is reaction time, y-axis is ηr -1 The slope of the regression equation of the test solution obtained by taking the natural logarithm of

[0127] B R : x-axis is reaction time, y-axis is ηr -1 The slope of the regression equation of the standard solution obtained by taking the natural logarithm of

[0128] E T : Concentration of test solution (mg / mL)

[0129] E R : Concentration of standard solution (mg / mL)

[0130] A: Titer of standard solution (IU / mg)

[0131] Sample activityCleavage siteAmino acidEnzyme cleavage rate(%)Storage pHStorage temperatureExample 1-1hyaluronidase (Transient Cell)L - YApproximately 0%pH 7.037 ℃Example 1-2D - LApproximately 6%Example 1-3D - SApproximately 12%Example 1-4D - KApproximately 18%Example 1-5D - TApproximately 24%Example 1-6D - HApproximately 30%Example 1-7D - MApproximately 36%

[0132] The results of the pH stability experiment are shown in Table 3 below. The enzyme activity of each test is expressed as a percentage value compared to the activity (IU / mL) measured at each time point based on the initial enzyme activity of 1,500 IU / mL (=100%) at week 0, and the enzyme activity ratio was calculated according to the following mathematical formula:

[0133] Enzyme activity ratio (%) = (enzyme activity at the time of measurement) / (initial enzyme activity) * 100

[0134] Enzyme activity ratio (%) pH 3.0 pH 5.0 pH 7.0 pH 10.0 Sample 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week Example 1-1 100.476 1.313 1.62 100.43 72.84 55.30 100.36 73.1756.03 100.88 63.314 5.92 Example 1-2 102.00 84.77 55.05 101.1794.52 80.57 101.4795.88 1.24 101.25 86.29 64.98 Example 1-3102.6685.4855.26101.8095.0281.19101.4996.2282.55102.6889.9565.11Example 1-4101.2384.9154.76101.1994.9680.65101.3895.5081.73101.4988.2764.19Example 1-5100.4683.5952.94100.1594.8877.45100.6093.8778.81100.6786.7462.64Example 1-6100.8282.3250.58100.0892.6174.8899.3291.3677.6399.7585.7661.17

[0135] As shown in Table 3, the pH stability of hyaluronidase according to the cleavage site for about 4 weeks was confirmed, and a higher enzyme activity ratio (%) was shown in the range of pH 5.0 to 7.0. The hyaluronidases of Examples 1-2 to 1-6 had significantly higher pH stability compared to the wild-type hyaluronidase of Example 1-1, and in particular, the pH stability of the hyaluronidases of Examples 1-2 to 1-4 was excellent. A value exceeding 100% appeared to be a measurement error that generally occurs due to a large range of variation in potency tests of biological products such as vaccines and recombinant proteins (cytokines, monoclonal antibodies, etc.).

[0136]

[0137] (2) Comparison of enzyme activity or content by storage temperature according to cut area

[0138] The temperature stability of the hyaluronidase of Examples 1-1 to 1-6 was confirmed by the same method as (1) of Example 2. Solutions were prepared according to the conditions described in Table 2, stored in a freezer (-20°C), refrigerator (5°C), and at high temperature (40°C) for 4 weeks, and titer tests were performed at each time point every 2 weeks. The temperature stability of hyaluronidase according to the cut site for approximately 4 weeks is shown in Table 4.

[0139] Enzyme activity ratio (%) Freezing Refrigeration High temperature Sample 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week Example 1-1 100.228 1.476 2.51 100.549 0.898 2.559 8.136 5.095 1.55 Example 1-2 101.98 100.749 8.85 101.52 100.8199 459 8.96 88.92 78.54 Example 1-3 102.68 100.889 9.22 102.77 101.91 100.239 9.4789 9.90 79.63 Example 1-4101.07100.1798.03100.32100.7299.1898.1688.1177.95Example 1-5100.1298.8295.52100.5398.7097.5199.3985.3175.78Example 1-699.4996.9293.1299.6496.3295.6399.8782.6973.12

[0140] As shown in Table 4, the hyaluronidases of Examples 1-2 to 1-6 had higher temperature stability than those of Example 1-1, and in particular, the enzyme activity ratio (%) of the hyaluronidases of Examples 1-2 to 1-6 was maintained significantly higher under frozen and refrigerated storage conditions. In the following examples, the amino acid DS hyaluronidase corresponding to Example 1-3, which was truncated by about 12% and showed the highest activity among the recombinant hyaluronidases, was designated BMI2004 and used in the experiments.

[0141]

[0142] Example 3. Stability verification of wild-type hyaluronidase and BMI2004.

[0143] (1) Comparison of enzyme activity or content according to pH

[0144] The pH stability of BMI2004 manufactured in Example 1 was compared with that of wild-type hyaluronidase. BMI2004 and wild-type hyaluronidase (manufacturer; BMI Korea, trade name; Hirax, hereinafter referred to as Hirax) used in the pH stability test were purified by 95% or more, and the experimental concentration was 1,500 IU / mL (=100%). Solutions were prepared according to the conditions described in Table 5 by substitution with water, and these were stored at 5℃ at pH 3.0, pH 5.0, pH 7.0, and pH 10.0 for 4 weeks, and a titer test was performed at each time point every 2 weeks. The enzyme activity or content of the sample at each time point was confirmed by the same method as (1) of Example 2.

[0145] pH sample storage temperature 3.0 BMI 2004 / Hirax (1,500 IU / mL) 2-8 ℃ 5.02-8 ℃ 7.02-8 ℃ 10.02-8 ℃

[0146] The results of the pH stability experiment are shown in Table 6. The enzyme activity of each test is expressed as a percentage value compared to the activity (IU / mL) measured at each time point based on the initial enzyme activity of 1,500 IU / mL (=100%) in week 0, and the enzyme activity ratio was calculated according to the following mathematical formula:

[0147] Enzyme activity ratio (%) = (enzyme activity at the time of measurement) / (initial enzyme activity) * 100

[0148] Enzyme activity ratio (%) pH 3.0 pH 5.0 pH 7.0 pH 10.0 Sample 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week BMI 2004 102.66 85.48 55.26 101.80 95.0 281.19 101.49 96.22 82.55 102.68 89.95 65.11 Hirax 102.04 51.11 31.03 103.39 71.20 50.40 102.33 70.76 50.95 101.88 62.07 41.03

[0149] As shown in Table 6, the pH-dependent stability of BMI2004 and Hirax was confirmed for approximately 4 weeks. The enzyme activity ratio (%) was higher at pH 5.0 and pH 7.0 than at pH 3.0 and pH 10.0, and the content (%) of Hirax tended to decrease rapidly compared to BMI2004 after 2 weeks. The values ​​exceeding 100% appeared to be a measurement error that generally occurs due to the large range of variation in potency tests of biological products such as vaccines and recombinant proteins (cytokines, monoclonal antibodies, etc.).

[0150] To further test the change in enzyme activity (or content) for the stability of Hirax and BMI2004 according to pH, SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) was performed under the conditions of Table 5 above, and the test was conducted according to the following test method:

[0151] 1) Preparation of Sample Buffer (5X): Use Pierce™ Lane Marker Reducing Sample Buffer (Thermo Scientific™, Cat No. 39000).

[0152] 2) Preparation of Running Buffer (1X): Use Novex™ Tris-Glycine SDS Running Buffer (10X) (Invitrogen, Cat No. LC2675). Take exactly 100 mL, place in a 1,000 mL volumetric flask, add water, mix, and make exactly 1,000 mL.

[0153] 3) Preparation of test solution: Replace Hirax and BMI2004 standard forms with water and adjust to suit the conditions. Take approximately 20 μL of this and 5 μL of Sample Buffer (5X), place in an EP tube, and mix.

[0154] 4) Operating method: Operate the test solution and PageRuler Prestained Protein Ladder standard (Thermo, Cat No. 26616) under the operating conditions below.

[0155] 4-1) Novex TM Wedgewell TM Remove the comb from one sheet of 8 to 16% Tris-Glycine Gel (Invitrogen, Cat No. XP08160BOX) or equivalent gel, and wash the preservative from the gel with water.

[0156] 4-2) Attach the washed gel to the Mini Gel Tank and fill the cathode area of ​​the Mini Gel Tank with Running Buffer (1X) until it is completely filled and the anode area is filled about 2 / 3.

[0157] 4-3) Inject 7 μL of PageRuler Prestained Protein Ladder standard and 25 μL of test solution into the gel.

[0158] 4-4) Connect the power supply to the Mini Gel Tank and operate as shown below to expand the gel to 90%.

[0159] Volt: 140 V

[0160] Ampere: 400 mA

[0161] Time: 60 minutes (Time can be changed depending on gel development.)

[0162] 4-5) Once the deployment is complete, separate the gel from the caster and wash it with water.

[0163] 4-6) Place the washed gel into a container containing staining solution (Coomassie Brilliant Blue R-250 Staining Solution, BIO-RAD, Cat No. 1610436).

[0164] 4-7) Place the container on the rocker and dye at 30 rpm for 30 minutes.

[0165] 4-8) Once staining is complete, place the gel in a container containing a bleaching solution (Coomassie Brilliant Blue R-250 Destaining Solution, BIO-RAD, Cat No. 1610438) and destain by replacing the bleaching solution on a rocker at 30 rpm until the gel is destained. When destaining is partially complete, wash the bleaching solution with water.

[0166] 4-9) When the bleaching solution is washed away, observe the gel on a white light.

[0167]

[0168] The experimental results are shown in Figures 1a to 1c. As shown in Figures 1a to 1c, the SDS-PAGE results indicate that BMI2004 has higher pH stability than Hirax, based on the generation of other bands over time in a water-substituted state.

[0169]

[0170] (2) Comparison of enzyme activity or content according to storage temperature

[0171] The temperature stability of BMI2004 manufactured in Example 1 was compared with that of the wild-type hyaluronidase Hirax. BMI2004 and Hirax used in the temperature stability test were purified by more than 95%, and the experimental concentration was 1,500 IU / mL (=100%). Solutions were prepared by substituting with water according to the conditions described in Table 7, and these were stored for 4 weeks in freezing (-20°C), refrigeration (5°C), and high temperature (40°C), and a titer test was performed at each time point every 2 weeks.

[0172] Storage temperature Sample storage pH ≤-20 ℃ Hirax / BMI2004 (1,500 IU / mL) pH 5.0 2-8 ℃ pH 5.0 ≥40 ℃ pH 5.0

[0173] The temperature stability of Hirax and BMI2004 for approximately 4 weeks was confirmed and is shown in Table 8 and Fig. 2 below. The highest content (%) was observed at a freezing temperature of -20℃ or lower, and the content (%) of BMI2004 remained significantly higher than that of Hirax under all temperature conditions after the 2nd week.

[0174] Enzyme activity ratio (%) Freezer Refrigeration High temperature Sample 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week BMI 2004102.68100.8899.22102.77101.91100.2399.4789.9079.63Hirax 102.7680.2459.75101.4291.5182.83102.1966.4040.15

[0175] Additional tests on the change in content for the stability test according to temperature of Hirax and BMI2004 were performed in the same manner as in Example 3, and SDS-PAGE was performed under the conditions of Table 7.

[0176] The results of the SDS-PAGE experiment are shown in Figures 2a to 2c. As shown in Figures 2a to 2c, the formation of bands other than the initial main band position over time under frozen, refrigerated, and high-temperature conditions in a water-displaced state suggests that BMI2004 has higher stability under storage conditions than Hirax.

[0177]

[0178] Example 4. Optimal enzyme activity according to temperature

[0179] In this example, the plate titer test was designed based on the cylindrical plate method, a microbiological titer test for antibiotics. Specifically, when a penicillin dispensed with a certain amount of hyaluronidase is placed on a solid agarose plate containing hyaluronidase, the hyaluronidase diffuses. Through this diffusion, the hyaluronidase degrades the hyaluronidase, and the undegraded hyaluronic acid is precipitated by cetylpyridinium chloride, forming a transparent ring. Therefore, in this example, the enzymatic activity of hyaluronidase can be confirmed by the size of the ring formed through a simple plate-based method. The plate titer test was conducted under each condition according to the following test method:

[0180] 1) Preparation of hyaluronic acid solution: Dissolve 0.2 g of hyaluronic acid in water until completely dissolved, then make 100 mL and add hydrochloric acid and sodium hydroxide to adjust the pH to 7.0 ± 0.1.

[0181] 2) Preparation of 1.5% agarose: Dissolve 1.5 g of agarose (SIGMA, Cat No. A9539) in 100 mL of water using a microwave.

[0182] 3) Preparation of 10% cetylpyridinium chloride: Prepare by adding 10 g of cetylpyridinium chloride (SIGMA, Cat No. C0732) to 100 mL of water.

[0183] 4) Test solution: Replace Hirax and BMI2004 standard forms with water and adjust to suit the conditions before use.

[0184] 5) Operating method: Test the test solution under the operating conditions below.

[0185] 5-1) Warm the hyaluronic acid solution at 37°C for approximately 20 minutes before mixing it with 1.5% agarose.

[0186] 5-2) When 100 mL of 1.5% agarose cools to about 60℃, add 100 mL of hyaluronic acid solution and stir to mix.

[0187] 5-3) Pour into a Petri Dish (SPL, Cat No. 10050) to a thickness of about 3 mm.

[0188] 5-4) When the agarose gel is completely solidified, place a penicillin (KisanBio, Cat No. KS-P0161) on a concentric circle of a Petri dish at a 90° angle, dispense 20 μL of the test solution into the penicillin, and react for 18 to 20 hours in an incubator adjusted to the temperature conditions listed in each table.

[0189] 5-5) After the reaction is complete, remove the penicillin, add 3 mL of 10% cetylpyridinium chloride, and check for a transparent ring after about 20 minutes.

[0190] 5-6) The diameter of the ring (mm) is measured accurately to 0.5 mm or less.

[0191]

[0192] (1) Confirmation of activity of hyaluronic acid solution

[0193] Before conducting the optimal enzyme activity test, plate titer tests were conducted under various pH and temperature conditions to confirm whether the hyaluronic acid solution exhibited its own activity. The experiments were conducted at pH 4.0, pH 7.0, or pH 10.0, with temperatures adjusted to 20°C, 25°C, 30°C, 35°C, and 40°C, and the experimental results are shown in Fig. 3. As shown in Fig. 3, it was confirmed that the hyaluronic acid solution did not exhibit its own activity.

[0194]

[0195] (2) Comparison of enzyme activity under temperature conditions of 20-40 ℃

[0196] Hirax and BMI2004 used in the enzyme activity test were purified by more than 95%, and were substituted with water to a concentration of 1,500 IU / mL (=100%). Solutions were prepared according to the conditions described in Table 9 below, and plate titer tests were performed at each temperature condition of 20°C, 25°C, 30°C, 35°C, and 40°C.

[0197] Storage pH Storage temperature Specimen condition 1 pH 4.0 20-40 ℃ Hirax / BMI2004 (1,500 IU / mL) Condition 2 pH 7.0 20-40 ℃ Condition 3 pH 10.0 20-40 ℃

[0198] The experimental results are shown in Fig. 4a and Fig. 4b. The enzyme activities of Hirax and BMI2004 were confirmed by plate titer test at temperature conditions of 20 to 40°C, and the ring sizes were large at 35°C and 40°C.

[0199] To quantify enzyme activity, the diameters of the rings in Figs. 4a and 4b were measured and shown in Table 10 below. The error range of the diameter (mm) in Table 10 is ±0.5 mm.

[0200] Temperature Sample pH 4.0pH 7.0pH 10.020℃Hirax8 mm10 mm11 mmBMI20048 mm12 mm13 mm25℃Hirax9 mm10 mm11 mmBMI20049 mm13 mm13 mm30℃Hirax9 mm13 mm14 mmBMI200410 mm13 mm15 mm35℃Hirax10 mm15 mm18 mmBMI200411 mm18 mm21 mm40℃Hirax10 mm15 mm18 mmBMI200411 mm18 mm21 mm

[0201] As shown in Table 10, when enzyme activity was compared under temperature conditions of 20 to 40℃, the activity of BMI2004 was higher than that of Hirax.

[0202]

[0203] (3) Comparison of enzyme activity under temperature conditions of 35-40 ℃

[0204] Based on the activity comparison of Hirax and BMI2004 confirmed in (2) of the above Example 4, in order to conduct additional tests under detailed temperature conditions, a solution was prepared according to the conditions described in Table 11 below, and a plate titer test was performed at each temperature condition of 35°C, 37°C, and 40°C.

[0205] Storage pH Storage temperature Specimen condition 1 pH 4.035 ℃ Hirax / BMI2004 (1,500 IU / mL) 37 ℃ 40 ℃ Condition 2 pH 7.035 ℃ 37 ℃ 40 ℃ Condition 3 pH 10.035 ℃ 37 ℃ 40 ℃

[0206] The experimental results are shown in Fig. 5a and Fig. 5b. The enzyme activities of Hirax and BMI2004 at 35°C, 37°C, and 40°C were confirmed by a plate titer test. The ring size was largest at 37°C, and the ring diameters are shown in Table 12 below. The error range of the diameter (mm) is ±0.5 mm.

[0207] Category 35 ℃37 ℃40 ℃ SampleHiraxBMI2004HiraxBMI2004HiraxBMI2004pH 4.010 mm12 mm10 mm13 mm10 mm12 mmpH 7.016 mm19 mm17 mm20 mm16 mm19 mmpH 10.018 mm21 mm19 mm22 mm18 mm21 mm

[0208] As shown in Table 12, when enzyme activity was compared under temperature conditions of 35℃, 37℃, and 40℃, the activity of BMI2004 was higher than that of Hirax.

[0209]

[0210] Example 5. Optimal enzyme activity according to pH

[0211] Hirax and BMI2004 used in the optimal enzyme activity test were purified by more than 95%, and were substituted with water to a concentration of 1,500 IU / mL (=100%), and solutions were prepared according to the conditions described in Table 13 below. Enzyme activity was performed according to the test method of Example 4, and based on the activity results according to temperature confirmed in (3) of Example 4, a plate titer test according to each pH was performed under the condition of 37℃, where the ring size was the largest.

[0212] Storage pH Storage temperature Specimen condition 3 pH 5.0 37 ℃ Hirax / BMI2004 (1,500 IU / mL) Condition 4 pH 6.0 Condition 5 pH 7.0 Condition 6 pH 8.0 Condition 7 pH 9.0 Condition 8 pH 10.0

[0213] The experimental results are shown in Fig. 6a and Fig. 6b. The enzyme activities of Hirax and BMI2004 at 37°C and pH 5.0-10.0 were confirmed by plate titer test. As a result, the ring size tended to be particularly large at pH 7.0-10.0, and a comparison of the ring diameters is shown in Table 14 below. The error range of the diameter (mm) is ±0.5 mm.

[0214] Category 37 ℃ Sample HiraxBMI2004pH 5.014 mm16 mmpH 6.017 mm18 mmpH 7.019 mm20 mmpH 8.019 mm20 mmpH 9.019 mm20 mmpH 10.020 mm21 mm

[0215] As shown in Table 14 above, when comparing enzyme activities for pH 5.0-10.0 at 37°C, BMI2004 substituted with water showed superior enzyme activity compared to Hirax at pH 5.0 or higher, and particularly excellent activity at pH 7.0 or higher.

[0216]

[0217] Example 6. Confirmation of hyaluronidase activity

[0218] The titers of hyaluronidase according to an example of the present application and conventional Hirax were measured in the same manner as in (1) of Example 2, and are shown in Table 15.

[0219] sample no.BMI2004 (IU / mg)Hirax (IU / mg)1124,49781,3962133,87081,5283134,67880,6534122,43380,538Average128,87081,029

[0220] As shown in Table 15, BMI2004 exhibited an activity of approximately 122,433 to 134,678 IU per mg of protein, while Hirax exhibited an activity of approximately 80,538 to 81,528 IU per mg of protein. Therefore, the hyaluronidase according to one example of the present application can achieve equivalent activity with approximately 63% of the protein amount compared to conventional Hirax.

[0221]

[0222] Example 7. Effect of hyaluronidase on promoting drug absorption and diffusion (1)

[0223] In order to confirm the effect of hyaluronidase according to an example of the present application on promoting drug absorption and diffusion, a drug absorption and diffusion promotion test using Trypan Blue was conducted.

[0224] Specifically, Hirax and BMI2004 were mixed in 0.2% Trypan blue solution and administered subcutaneously once to balb / c nude mice, and the extent of Trypan blue diffusion was compared. Based on the conditions described in Table 16 and the potencies described in Table 15, all drugs were prepared at 10 IU / mL or 100 IU / mL and administered 0.05 mL each, and the diffusion area (πmm) at each time point was measured. 2 ) was confirmed.

[0225] Sample (IU / mL) Peptide Dose (ug / mL) 2.5 min 5 min 10 min 15 min 20 min Saline 0 7 7.38 ± 15.86 8 4.61 ± 13.9 2 9 8.11 ± 7.83 1 17.67 ± 10.35 1 2 1.84 ± 11.48 Hirax (10 IU / mL) Approximately 0.12 3 7 4.45 ± 22.40 1 2.84 ± 15.01* 146.69 ± 29.36** 165.98 ± 18.49* 189.90 ± 41.85** Hirax (100 IU / mL) Approximately 1.23 4 8 4.25 ± 15.8 1 133.90 ± 14.52*** 149.27 ± 27.41**184.80 ± 37.10**215.12 ± 20.60***BMI2004(10 IU / mL)about 0.07882.70 ± 22.95109.65 ± 12.70*153.17 ± 15.30***176.85 ± 14.08**187.63 ± 36.40**BMI2004(100 IU / mL)about 0.77687.02 ± 17.51126.70 ± 16.00***140.23 ± 16.95***180.98 ± 29.93***218.14 ± 26.85***

[0226] 1) Mean ± SD

[0227] 2) *p < 0.05, **p < 0.01, ***p < 0.001 compared to saline treated group by SPSS (one-way ANOVA, LSD test)

[0228]

[0229] As shown in Table 16 above, the diffusion area was determined over a period of 2.5 to 20 minutes. From 5 minutes after administration, the diffusion area significantly increased in Hirax and BMI2004 compared to the negative control group, Saline.

[0230]

[0231] Example 8. Effect of hyaluronidase on promoting drug absorption and diffusion (2)

[0232] In order to confirm the effect of hyaluronidase according to an example of the present application on promoting drug absorption and diffusion, a drug absorption and diffusion promotion test was conducted using an akinesia model.

[0233] Specifically, cynomolgus monkeys were used to confirm the increased drug penetration using the Akinesia Model. BMI2004 and Hirax, listed in Table 15, were used as test substances, and saline was used as a negative control. Lidocaine and bupivacaine were used as anesthetics mixed with the samples. 2 mL was administered to each of the right and left peribulbar regions of six monkeys, and eye movements were observed.

[0234] After administering anesthetics together with test substances, the time until pupil movement disappeared (Time to Akinesia) was measured, and the time from when the pupils were anesthetized until the anesthesia wore off and pupil movement was observed (Duration of Akinesia) was measured, and these are shown in Table 17.

[0235] Sample peptide dosage (ug / mL)Model(Monkey)EyeTime to Akinesia (min)Duration of Akinesia (min)Saline002LeftNo AkinesiaNo Akinesia03RightNo AkinesiaNo Akinesia05LeftNo AkinesiaNo Akinesia06RightNo AkinesiaNo AkinesiaHirax (500 IU / 0.2 mL) About 6.17㎍ / 0.2mL01Left42702Right42704Left21705Right232Mean3.025.8SD1.26.3BMI2004 (500 IU / 0.2 mL approx. 3.88㎍ / 0.2mL01Right13203Left22404Right21706Left141Mean1.528.5SD0.610.3

[0236] 1) Drug; Lidocaine 2% (0.9 mL), Bupivacaine 0.5% (0.9 mL) and the dosing article (0.2 mL)

[0237] As shown in Table 17 above, the akinesia effect was not observed in the negative control group, Saline, but when a polypeptide according to an example of the present application was administered together with an anesthetic, the penetration power of the anesthetic was enhanced, resulting in a paralyzing effect on the pupil, and exhibited an akinesia effect equivalent to that of the conventional Hirax, thereby inducing drug absorption and diffusion. In addition, the polypeptide according to an example of the present application exhibited equivalent activity at a protein dosage of about 63% of that of the conventional Hirax.

[0238]

[0239] Example 9. Confirmation that hyaluronidase promotes reabsorption of excess fluid.

[0240] To confirm the effect of hyaluronidase according to an example of the present application on promoting the reabsorption of excessive body fluid, a test was conducted using an edema model. In this example, C57BL / 6 mice were used to artificially induce lymphatic edema and confirm the effect using a lymphedema model. Hirax and BMI2004, as described in Table 15, were used as test substances, and saline was used as a negative control. The samples and dosages were as described in Table 18 below.

[0241] Specimen Lymphedema-induced or not Dosage ( / site) Route Number of animals Saline + -SC6 Hirax + 100 IU (approximately 1.234 ug) SC6 BMI 2004 + 100 IU (approximately 0.776 ug) SC6

[0242] Lymphedema induction was performed on the tail, which is easy and accurate to measure. A 2 mm wide ring-shaped skin was cut 1 cm from the base of the mouse's tail, and 4 mm of the ventral side was cut. 2 The degree was left uncut. To confirm the edema reduction effect, on the 15th day of induction, the diameter (mm) of the tail was measured at each time point from before administration (0 hours) to after the first administration, as described in Table 19 below. The second administration was administered 24 hours later, and the third administration was administered 48 hours later. The diameter (mm) of the tail was measured using a caliper at a point 10 mm from the defect site.

[0243] SpecimenSalineHiraxBMI20040 hours4.55 ± 0.434.53 ± 0.494.53 ± 0.381 hours4.57 ± 0.434.39 ± 0.494.57 ± 0.652 hours4.48 ± 0.364.45 ± 0.404.50 ± 0.374 hours4.52 ± 0.384.41 ± 0.384.48 ± 0.5124 hours4.56 ± 0.344.57 ± 0.394.55 ± 0.4525 hours4.68 ± 0.324.48 ± 0.354.53 ± 0.4426 hours4.56 ± 0.324.43 ± 0.384.45 ± 0.4728 Time 4.58 ± 0.37 4.42 ± 0.44 4.46 ± 0.48 48 Time 4.65 ± 0.39 4.49 ± 0.41 4.47 ± 0.38 49 Time 4.56 ± 0.27 4.22 ± 0.37 4.26 ± 0.37 50 Time 4.58 ± 0.25 4.13 ± 0.38 4.09 ± 0.36 52 Time 4.59 ± 0.25 4.16 ± 0.47 4.18 ± 0.36

[0244] As shown in Table 19 above, BMI2004 administration was shown to reduce lymphedema, promoting the reabsorption of excess fluid. Furthermore, the polypeptide according to one example of the present application exhibited equivalent activity at approximately 63% of the protein dosage compared to conventional Hirax.

[0245]

[0246] Example 10. Confirmation of in vivo stability of hyaluronidase

[0247] In order to confirm the stability of hyaluronidase according to an example of the present application in the body, pharmacokinetics were confirmed by intravenous administration. In this example, SD rats were used as test animals, and Hirax and BMI2004 were each administered by intravenous infusion for 30 minutes to a dose of approximately 180,000 IU. Blood samples were collected before administration, 15 minutes after the start of administration (mid-infusion), 30 minutes after the start of administration (end of infusion), and at 31, 33, 36, 40, 45, 60, 75, and 90 minutes, and at 2.5 hours, 4.5 hours, 24.5 hours, and 48.5 hours.

[0248] Pharmacokinetic parameters of a polypeptide according to an example of the present application and Hirax are shown in Table 20, and the blood concentration of the polypeptide over time is shown in FIG. 7. As can be seen in FIG. 7 and Table 20, in the case of Hirax, it is degraded so rapidly that the half-life in the body cannot be measured, whereas the polypeptide according to an example of the present application has a half-life of about 0.272 hours (about 16.3 minutes) that is longer than Hirax, showing the possibility of showing a greater effect than Hirax by remaining in the body for a longer time. In addition, there is a difference in the blood concentration in the body; in the case of Hirax, it reaches the peak within 0.25 hours (15 minutes), whereas BMI2004 shows a more gradual increase in blood concentration than Hirax at 0.5 hours (30 minutes), showing the possibility of showing a greater effect than Hirax by remaining in the body for a longer time.

[0249] Test substance administration dose (IU / kg)t max (h)t 1 / 2 (h)CL (mL / h / kg)Hirax1800000.250NCNCBMI20041800000.5000.272418

[0250] Example 11. Preparation of a drug for subcutaneous administration

[0251] A composition for subcutaneous administration containing hyaluronidase and a drug according to an example of the present application was prepared. BMI2004 of Example 1-3 was used as the hyaluronidase and Herceptin, which is commercially available, was used as the drug. Specifically, BMI2004 hyaluronidase of Example 1-3 was added to a formulation buffer (5 mM sodium acetate hydrate, 1 mM magnesium chloride hydrate, 0.9% sodium chloride, 1.33% lactose hydrate, 0.01% polysorbate 80, pH 5.0) to a concentration of 2,000 IU / mL, and 120 mg of Herceptin injection (trastuzumab, Roche Korea Co., Ltd.) powder was mixed to prepare a subcutaneous drug at a ratio of 120 mg Herceptin / 2,000 IU / mL BMI2004.

[0252]

[0253] Example 12. Effect of improving the bioavailability of subcutaneously administered drugs

[0254] To confirm the drug absorption-promoting effect of the subcutaneous administration preparation manufactured in Example 11, pharmacokinetics after subcutaneous administration were examined. In this example, mice (ICR, Female) were used as test animals, and in the single-dose study, Herceptin was administered to a total of 90 mice, and blood was collected from 2 mice per time point.

[0255] As described in Table 21, the administered dose was 6 mg for Herceptin IV in Group 1, the control group, 6 mg for Herceptin Hylecta in Group 2, the control group, and 6 mg for Herceptin / 100 IU rHuPH20 in Group 3, the test substance, and lastly, 6 mg for Herceptin / 100 IU BMI2004 in Group 3, which were administered subcutaneously. Specifically, in the negative control group that received only the drug (Group 1), 150 mg / vial powder of Herceptin Injection (trastuzumab, Roche Korea Co., Ltd.) was dissolved in 1.25 mL of formulation buffer (5 mM sodium acetate monohydrate, 1 mM magnesium chloride monohydrate, 0.9% sodium chloride, 1.33% lactose monohydrate, 0.01% polysorbate 80, pH 5.0) to prepare a concentration of 120 mg / mL and administered 0.05 mL per mouse. The positive control group, Herceptin Hylecta (Group 2), received Herceptin Hylecta, a Herceptin drug formulated with commercially available rHuPH20 hyaluronidase. TM 600 mg / 10,000 IU / 5 mL (120 mg / 2,000 IU / mL, liquid) was administered 0.05 mL per mouse. Group 3, the experimental group, was administered 0.05 mL per mouse of the subcutaneous drug Herceptin 120 mg / BMI2004 2,000 IU / mL prepared in Example 11. Blood samples were collected at each time point from 0 hour (before administration) to 0.17, 0.5, 0.83, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 240, 336, 504, 672, and 840 hours (total 21 time points) after administration, and Herceptin concentrations were measured using ELISA (Enzyme linked immunosorbent assay).

[0256] As shown in Table 22 and Figure 8, Herceptin with added hyaluronidase was absorbed into the body faster than Herceptin without added hyaluronidase. Furthermore, Herceptin drug formulated with hyaluronidase according to an example of the present application showed pharmacokinetic results similar to Herceptin hylecta with added commercially available rHuPH20 hyaluronidase. Therefore, hyaluronidase according to an example of the present application can be used in the subcutaneous administration formulation of drugs and has the effect of promoting drug absorption into the body.

[0257] Group Sample Name Animal Administration Method Administration Dose (Herceptin / Hyaluronidase) Blood Collection Time (hours) 1 Herceptin ICR, Female Subcutaneous Administration 6mg / 0IU 0 (before administration), 0.17, 0.5, 0.83, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 240, 336, 504, 672, 840 2 Herceptin Hylecta ICR, Female Subcutaneous Administration 6mg / 100IU rHuPH 20 3 Herceptin / BMI 2004 ICR, Female Subcutaneous Administration 6mg / 100IU BMI 2004

[0258] Group Sample Name Addition Dosage Method AUC (last) AUC (inf) T max C max t 1 / 2 (hr)1Herceptin-subcutaneous administration55545.4759593.3272.00198.08220.932Herceptin Hylecta-subcutaneous administration54907.1255127.1124.00282.99127.073Herceptin BMI2004subcutaneous administration61894.8562453.6724.00294.69142.41

Claims

1. A composition for subcutaneous administration, comprising a hyaluronidase polypeptide having a sequence of amino acids of sequence number 1, wherein a selected number of amino acids from 1 to 203 are consecutively deleted from the C-terminus of the polypeptide; and a drug.

2. A composition for subcutaneous administration in claim 1, wherein the polypeptide has a selected number of amino acids consecutively deleted from an integer of 34 to 170 from the C-terminus of the amino acid sequence of sequence number 1.

3. A composition for subcutaneous administration in the first paragraph, wherein the polypeptide has a selected number of amino acids consecutively deleted from an integer of 34 to 68 from the C-terminus of the amino acid sequence of sequence number 1.

4. A composition for subcutaneous administration in the first paragraph, wherein the polypeptide has 34, 68, 102, 136, or 170 amino acids consecutively deleted from the C-terminus of the amino acid sequence of sequence number 1.

5. A composition for subcutaneous administration in the first paragraph, wherein the polypeptide has the first amino acid additionally deleted from the N-terminus of sequence number 1.

6. A composition for subcutaneous administration in claim 1, wherein the polypeptide comprises an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO:

6.

7. A composition for subcutaneous administration according to claim 1, wherein the polypeptide is glycosylated.

8. A composition for subcutaneous administration according to claim 1, wherein the polypeptide is stable at pH 3 to 10.

9. A composition for subcutaneous administration according to claim 1, wherein the polypeptide is stable at a temperature of -20 to 45°C.

10. A composition for subcutaneous administration in claim 1, wherein the polypeptide has an enzyme activity of at least 57% of the initial activity after storage for 4 weeks at a pH ranging from pH 3 to 10.

11. A composition for subcutaneous administration in claim 1, wherein the polypeptide has an enzyme activity of at least 32% of the initial activity after storage for 4 weeks at a pH of 3 to 5.

12. A composition for subcutaneous administration, wherein the polypeptide has an enzyme activity of at least 63% of the initial activity after storage for 4 weeks at a temperature below 0°C.

13. A composition for subcutaneous administration, wherein the polypeptide has an enzyme activity of at least 83% of the initial activity after storage for 4 weeks at a temperature ranging from 0 to 40°C.

14. A composition for subcutaneous administration, wherein the polypeptide has an enzyme activity of at least 52% of the initial activity after storage for 4 weeks at a temperature of 40°C or higher.

15. A composition for subcutaneous administration, wherein the polypeptide in claim 1 has a potency that is 1 to 3 times greater than the potency of a polypeptide consisting of the amino acid sequence of sequence number 1.

16. A composition for subcutaneous administration according to claim 1, wherein the polypeptide has an activity of 120,000 to 150,000 IU / mg.

17. A composition for subcutaneous administration in claim 1, wherein the drug is a protein drug, an antibody, an aptamer, mRNA, siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (micro RNA), aiRNA (asymmetric interfering RNA), RNAi (RNA interference), an antisense oligonucleotide, or a small molecule.

18. A composition for subcutaneous administration in claim 17, wherein the drug is a soluble receptor or a soluble receptor and Fc fusion protein.

19. A composition for subcutaneous administration in paragraph 1, wherein the drug is an anticancer agent, an antidiabetic agent, an anti-inflammatory agent, an antiviral agent, or an immunomodulatory agent.

20. A composition for subcutaneous administration, further comprising a stabilizer according to claim 1.

21. A composition for subcutaneous administration in claim 20, wherein the stabilizer comprises at least one selected from the group consisting of a buffer, a nonionic surfactant, a chelating agent, an alkali metal salt, and an alkaline earth metal salt.

22. A composition for subcutaneous administration in claim 21, wherein the buffer is at least one selected from the group consisting of a succinate buffer, an acetate buffer, a phosphate buffer, a citrate buffer, a malonate buffer, a MES (2-(N-Morpholino)ethanesulphonic acid) buffer, a Tris buffer, and a glycine buffer.

23. A composition for subcutaneous administration in claim 21, wherein the nonionic surfactant is a polyoxyethylene-sorbitan fatty acid ester.

24. A composition for subcutaneous administration in claim 23, wherein the nonionic surfactant is at least one selected from the group consisting of polysorbate 20, polysorbate 80, and Triton X-100.

25. A composition for subcutaneous administration in claim 21, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

26. In paragraph 21, the alkaline earth metal salt is MgCl 2 and / or MgSO 4 A composition for subcutaneous administration.

27. A pharmaceutical composition for treating cancer, comprising a hyaluronidase polypeptide having a sequence of amino acids of the sequence number 1, wherein a selected number of amino acids from 1 to 203 are deleted sequentially from the C-terminus; and an anticancer agent.

Images