Low-glycosylation modified kallikrein 1 and its polyethylene glycol modified form and their applications in pharmaceuticals

Abstract

To provide a type of hypoglycosylated modified kallikrein 1 and its PEG-modified derivatives and their application in medicine. The present invention provides KLK1 with no or only a small amount of glycosylation modification in the NFS sequence, which realizes high activity. The present invention also provides a recombinant KLK1 mutant with N-glycosylation modification only in NMS and NHT, which has the advantages of consistent sugar modification, uniform product molecular weight, high yield, simple purification, high biological activity and quality control. The present invention also provides PEGylated KLK1 with long-acting action, quality control, low immunogenicity and high biological activity, which reduces the frequency of administration and improves patient compliance, and realizes the application of drugs in the entire disease process, such as prevention, treatment, prognosis recovery, and recurrence prevention of acute ischemic stroke, peripheral neuropathy, retinopathy, ocular fundus disease, hypertension, diabetic nephropathy, IgA nephritis, chronic kidney disease, etc.

Description

[Technical Field]

[0001] This invention relates to low-glycosylation-modified kallikrein 1 (KLK1), its polyethylene glycol modified form, and its application as a drug. In particular, it relates to KLK1 with no glycosylation modification or only a very small amount of glycosylation modification in the NFS sequence, KLK1 mutants in which the NFS sequence of recombinant KLK1 is mutated, polyethylene glycol modified forms of these KLK1s, and their application as drugs. [Background technology]

[0002] Kallikreins, also known as kininogenases or kallidinogenases, are a type of serine protease and are divided into two types: plasma kallikreins (PK) and tissue kallikreins (TK), both of which exert very important physiological functions. Currently, human tissue kallikreins are thought to consist of at least 15 members (KLK1-KLK15), and among them, tissue kallikrein 1 (KLK1) has been studied relatively extensively. KLK1 converts kininogen to kinin and acts on the corresponding receptor to exert a series of biological effects. Many studies have shown that KLK1 acts in the nervous system, circulatory system, respiratory system, diabetes, cancer, kidney disease, and other conditions.

[0003] Currently, there are two types of KLK1 products sold domestically and internationally. One type is pancreatic kallikrein 1 extracted from pig pancreas; for example, Yi Kai from Changzhou Qianhong Pharmaceutical belongs to this type. This type of KLK1 product is restricted by its animal-derived raw materials. The other type is KLK1 extracted from human urine; for example, Urinary Kallikrein Injection from Guangdong Tianpu is this type of KLK1. Human urine is difficult to collect, yields are low, there is a risk of viral contamination, and urinary KLK1 exhibits Glu / Lys polymorphism at the 162nd amino acid position, so this type of KLK1 product presents significant challenges in drug quality control. In addition to the aforementioned natural extract KLK1, there are numerous research reports on recombinant human kallikrein 1. The most advanced among these is DM199 from DiaMedica, which is currently undergoing a Phase 2 / 3 clinical trial for the treatment of stroke.

[0004] KLK1 is an enzyme with abundant glycosylation modifications, and the degree of glycosylation modification significantly affects its enzyme activity. N-glycosylation is the most notable type of glycosylation modification for glycoprotein drugs, where a sugar chain is bound via the free radical -NH2 of a specific asparagine molecule in the nascent peptide chain. The triad sequence motif of the N-glycosylation site must be either NXS or NXT, where N represents asparagine, S represents serine, T represents threonine, and X represents an amino acid other than proline. If these conditions are not met, N-glycosylation does not occur in asparagine. Different N-glycosylation motifs are located at different positions in the tertiary structure of glycoproteins, and the structure of the glycoprotein determines which glycosylation site binds to which glycosyltransferase, resulting in different glycosylation modifications. In conventional technology, there is no established theory as to how different degrees of glycosylation affect the efficacy and safety of KLK1. In Chinese patent CN107058269A (Patent Document 1), three types of protein products with varying degrees of glycosylation—highly glycosylated, moderately glycosylated, and lowly glycosylated—were prepared by purifying porcine pancreatic kallikrein 1. Among these, the lowly glycosylated porcine pancreatic kallikrein 1 had low enzyme activity stability. By removing this lowly glycosylated kallikrein 1 through the purification process, the biological activity of the product was increased and side effects were reduced. In Guangdong Tianpu's patent CN101134952A (Patent Document 2), high molecular weight kallikrein 1 and low molecular weight kallikrein 1 were separated from an intermediate of urine-derived kallikrein 1 to obtain two types of components. From these, a high molecular weight component was selected as the drug component. This single-component high molecular weight human urinary kallikrein 1 had significantly fewer side effects compared to a mixture of human urinary kallikrein 1 containing high and low molecular weight kallikrein 1. Guangdong Tianpu Co., Ltd. has also attempted to express recombinant human kallikrein 1 using CHO, and its patent CN101134953A (Patent Document 4) discloses a high molecular weight KLK1 containing three glycosylation modification sites. DiaMedica's patent US20130323222A (Patent Document 3) discloses that recombinant human kallikrein 1 was expressed and purified using CHO, and that high glycosylated KLK1 and low glycosylated KLK1 were obtained, respectively. While there was no significant difference in in vivo activity between high-glycosylated kallikrein 1 and low-glycosylated kallikrein 1, a mixture of high-glycosylated and low-glycosylated kallikrein 1 in a 1:1 ratio showed higher activity compared to single high-glycosylated KLK1 or single low-glycosylated KLK1.

[0005] Furthermore, even if the most promising glycosylated KLK1 is identified, controlling the level of glycosylation of the glycoprotein remains a significant technical challenge in modern pharmaceutical industrial production. Typically, extensive optimization of recombinant expression and purification conditions is required to obtain the desired glycosylated product to the maximum extent. For example, Chinese patent CN101134953A (Patent Document 4) expresses recombinant KLK1 using CHO, and paragraph

[0010] states that "the degree of glycosylation of the expression product was increased by optimizing the culture conditions." Chinese patent CN101092598A (Patent Document 5) expresses recombinant KLK1 using Pichia yeast. In the expression product, highly glycosylated KLK1 has a molecular weight of 32871.16D and accounts for a small proportion, while low glycosylated KLK1 has a molecular weight of 28975.79D and accounts for a relatively large proportion. Although the molecular weights of highly glycosylated KLK1 and low glycosylated KLK1 are similar, they were separated by a three-step purification process.

[0006] In addition to challenges related to KLK1 glycosylation, KLK1 products have other issues that need to be addressed, such as low biological stability, a short half-life, the need for repeated administration, and the fact that recombinant KLK1 protein has some degree of immunogenicity. For example, DM199 is administered as an intravenous infusion of KLK1 within 24 hours of stroke onset, followed by subcutaneous injections every three days for 22 days. Clinical trial results have shown that KLK1 can reduce the incidence of stroke in high-risk groups and prevent stroke recurrence. As a drug that improves the compensatory function of collateral circulation in the brain, KLK1 requires long-term and multiple administrations to achieve therapeutic effects. While long-term administration is an important method of applying KLK1, frequent administration may reduce patient compliance.

[0007] Polyethylene glycol (PEG) modification technology is a technique for chemically modifying proteins and polypeptides using polyethylene glycol modifiers. Currently, more than 10 types of PEG-modified protein pharmaceuticals are commercially available. PEG-modified proteins can offer benefits such as increased protein solubility, improved protein stability, extended in vivo half-life, and reduced immunogenicity. There are still few reports on PEG-modified recombinant kallikrein 1 both domestically and internationally. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Chinese Patent Application Publication No. 107058269 Specification [Patent Document 2] Chinese Patent Application Publication No. 101134952 Specification [Patent Document 3] U.S. Patent Application Publication No. 20130323222 Specification [Patent Document 4] Chinese Patent Application Publication No. 101134953 Specification [Patent Document 5] Chinese Patent Application Publication No. 101092598 Specification

Summary of the Invention

Problems to be Solved by the Invention

[0009] The first technical problem to be solved by the present invention is the selection of KLK1 glycosylation modification. The second technical problem to be solved by the present invention is to solve the problems existing in existing KLK1 products, such as low biological stability, short half-life, the need for repeated administration, and the fact that recombinant KLK1 protein has a certain degree of immunogenicity.

Means for Solving the Problems

[0010] The first technical problem to be solved by the present invention is the selection of KLK1 glycosylation modification. As described in the background art, there is no established theory in the prior art on how different degrees of glycosylation affect the efficacy and safety of KLK1. Also, even if the most promising glycosylation-modified KLK1 is determined, in order to obtain the target glycosylated product to the maximum extent, a large amount of optimization of the recombinant expression and purification conditions must be carried out to control the glycosylation level of the glycoprotein.

[0011] Through characterization analysis, the applicant of this application discovered that human kallikrein 1 (hKLK1) has three glycosylation modification sites: N78 (N in the NMS sequence), N84 (N in the NHT sequence), and N141 (N in the NFS sequence). Of these, highly glycosylated KLK1 has glycosylation modifications at all three sites, while low-glycosylated KLK1 has glycosylation modifications at two sites, N78 and N84, and either no glycosylation modification at N141 or only a very small amount. Furthermore, when the applicant compared the biological activity of highly glycosylated KLK1 and low-glycosylated KLK1, they unexpectedly discovered that the activity of low-glycosylated KLK1 was far higher than that of highly glycosylated KLK1. Furthermore, in order to obtain low-glycosylated KLK1, the applicant did not focus on optimizing recombinant expression conditions or purification conditions as in conventional methods, but rather, based on research on high- and low-glycosylated KLK1, mutated one or more amino acids in the NFS sequence motif in a way that would prevent the formation of an N-glycosylation motif, thereby creating mutants. This resulted in a more uniform product and a higher yield of low-glycosylated hKLK1. The applicant then made the surprising discovery that the mutant low-glycosylated KLK1 was superior to the unmutated low-glycosylated KLK1 in terms of enzymatic properties and activity.

[0012] Many natural mutants (homologs) exist for hKLK1. For example, there are natural mutants registered under Genbank registry numbers AAA59455.1, NP002248.1, AAA36136.1, AAP35917, and AAU12569. In terms of composition, the natural mutants of hKLK1 have the same number of amino acids, differing only in a few amino acids (as shown in the table below), showing very high homology, and their in vivo and in vitro activities are basically the same. The inventors also discovered that the glycosylation modification sites of these natural mutants are the same, located at N78, N84, and N141, and that the triad sequence motif of the N-glycosylation modification site is also the same, being NMS, NHT, and NFS.

[0013] [Table 1]

[0014] This invention is based on hKLK1, registered under Genbank registration number AAA59455.1, and involves mutating one or more amino acids in the NFS sequence motif to obtain KLK1 containing only two N-glycosylation modification sites (low-glycosylated KLK1). As described above, the amino acid composition and glycosylation modification sites of the hKLK1 native mutants are highly identical, and similar technical effects can be achieved by mutating the corresponding positions (i.e., one or more amino acids in the NFS sequence motif) of other hKLK1 native mutants. In other words, the product is more uniform, the yield of low-glycosylated hKLK1 is higher, and the target product has higher activity.

[0015] Furthermore, the inventors discovered that KLK1 in other primates, such as NCBI Reference Sequence:XP_004061305.1 (gorilla), XP_003916022.1 (Anubis baboon), XP_003813685.1 (bonobo), XP_002829668.3 (Sumatran orangutan), and XP_032024960.1 (hoolock gibbon), also contains three N-glycosylation modification sites, similar to hKLK1, and that the triad sequence motif of the N-glycosylation modification site is the same, consisting of NMS, NHT, and NFS. Similar technical effects can be achieved by mutating the corresponding sites of the natural KLK1 amino acids in other primates (i.e., one or more amino acids in the NFS sequence motif). In other words, the product is more uniform, yields a higher amount of low-glycosylated hKLK1, and the target product has higher activity.

[0016] Based on the above findings, the present invention provides a type of low-glycosylation modified KLK1 or a derivative thereof. The KLK1 is a primate KLK1 and includes N-glycosylation modification in the NMS, NHT, and NFS sequences of natural KLK1, among which the asparagine in NFS has no glycosylation modification or only a very small amount of glycosylation modification. The small amount of glycosylation modification or low glycosylation modification is defined as the percentage of asparagine in NFS that has been glycosylated being ≤10%, ≤9%, ≤8%, ≤7%, ≤6%, ≤5%, ≤4%, ≤3%, ≤2%, ≤1%, ≤0.5%, or ≤0.1%. In specific examples, in the low-glycosylation KLK1 in question, 96.39% of the asparagine in NFS was not glycosylated, and less than 4% was glycosylated. This low-glycosylation KLK1 has higher activity compared to KLK1 with high glycosylation modification in NFS.

[0017] Furthermore, the present invention provides a recombinant KLK1 mutant or derivative thereof. The KLK1 is a primate KLK1, and the recombinant KLK1 or derivative thereof has N-glycosylation modification at only two sites, and does not contain KLK1 in which N-glycosylation modification occurs at three sites. The primate may be a human, or a non-human primate such as a gorilla, Anubis baboon, bonobo, Sumatran orangutan, or hoop's gibbon. That's fine.

[0018] Preferably, the recombinant KLK1 mutant or its derivative retains the N-glycosylation modification of natural KLK1 at NMS and NHT, but does not contain the N-glycosylation modification of natural KLK1 at NFS.

[0019] Preferably, the N (asparagine) in NFS of the KLK1 amino acid sequence is mutated to any amino acid other than asparagine. That is, the asparagine at position 141 is mutated to any other amino acid, and zero, one, or two F and S amino acids in NFS are mutated to any other amino acid. As a result, the mutant does not form an N-glycosylation motif at the relevant position, and therefore glycosylation modification does not occur at position 141. Compared to the unmutated low-glycosyl KLK1, all different mutants produce a more uniform product, yield a higher amount of low-glycosyl KLK1, and exhibit superior product activity. Preferably, the F (phenylalanine) in NFS of the KLK1 amino acid sequence is mutated to proline. That is, the phenylalanine at position 142 is mutated to proline, and zero, one, or two N and S amino acids of NFS are mutated to any other amino acid. As a result, the mutant does not form an N-glycosylation motif at the relevant position, and therefore glycosylation modification does not occur at position 141. Similarly, all mutants exhibit superior product activity, in addition to producing a more uniform product and a higher yield of low-glycosyl KLK1, compared to the unmutated low-glycosyl KLK1 product.

[0020] Preferably, the S (serine) of NFS in the KLK1 amino acid sequence is mutated to any amino acid other than serine or threonine. That is, the serine at position 143 is mutated to any amino acid other than serine or threonine, and zero, one, or two N and F amino acids of NFS are mutated to any other amino acid. As a result, the mutant does not form an N-glycosylation motif at the relevant position, and therefore glycosylation modification does not occur at position 141. Similarly, all mutants exhibit superior product activity compared to unmutated low-glycosyl KLK1, in addition to producing a more uniform product and higher yields of low-glycosyl KLK1.

[0021] In specific examples, asparagine was mutated into four different amino acids: a polar neutral amino acid (e.g., glutamine (Gln)), an acidic amino acid (e.g., aspartic acid (Asp)), a basic amino acid (e.g., arginine (Arg)), and an aliphatic amino acid (e.g., alanine (Ala)). As a result, the mutants do not form an N-glycosylation motif in NFS, and therefore glycosylation modification does not occur. Compared to the unmutated low-glycosyl KLK1, all of the different mutants produced more uniform products, yielded higher levels of low-glycosyl KLK1, and exhibited superior product activity.

[0022] Preferably, the KLK1 is hKLK1, and the amino acid sequence of the natural hKLK1 is a sequence registered in GenBank under registration numbers such as AAA59455.1, NP002248.1, AAA36136.1, AAP35917, and AAU12569.

[0023] Preferably, the amino acid sequence of the KLK1 mutant is SEQ ID No:3, SEQ This is the sequence indicated by ID No:4, SEQ ID No:5, or SEQ ID No:6.

[0024] The present invention also provides compositions comprising the low-glycosylated KLK1.

[0025] KLK1 converts kininogen to kinin and acts on corresponding receptors to exert a range of biological effects. KLK1 is involved in the nervous system, circulatory system, respiratory system, diabetes, cancer, kidney disease, and other conditions. Numerous studies have shown that it acts in these conditions. Currently available KLK1 has approved indications for microcirculatory disorders, such as diabetic nephropathy, peripheral neuropathy, retinopathy, fundus diseases, and adjunctive therapy for hypertension, and can also be used for mild to moderate acute thrombotic cerebral infarction. Indications currently under clinical trial include IgA nephritis and chronic kidney disease. The KLK1 mutants of the present invention have only a few amino acids mutated compared to natural KLK1. In specific examples, these mutants have been confirmed to exhibit activity when acting on the same substrate in vitro, and have also been demonstrated to exhibit activity in improving cerebral infarction symptoms in an MCAO cerebral ischemia-reperfusion model in vivo. Those skilled in the art can reasonably expect that the recombinant KLK1 mutants and their compositions of the present invention will be effective, like natural KLK1, in the treatment and prevention, prognosis recovery, and prevention of recurrence of acute ischemic stroke, peripheral neuropathy, retinopathy, fundus diseases, hypertension, diabetic nephropathy, IgA nephritis, and chronic kidney disease.

[0026] The second technical problem that this invention aims to solve is the problems of existing KLK1 products, such as low biological stability, short half-life, need for repeated administration, and the fact that recombinant KLK1 protein has a certain degree of immunogenicity. This invention provides KLK1 modified with a polyethylene glycol modifier that significantly enhances protein stability, reduces immunogenicity, improves the in vivo pharmacokinetic properties of the protein, and maintains protein activity to the maximum extent.

[0027] Preferably, the KLK1 modified by PEG is the low-glycosylated KLK1.

[0028] Preferably, the KLK1 is recombinant KLK1 or a derivative thereof in which N-glycosylation modification has occurred at two sites, and does not include KLK1 in which N-glycosylation modification has occurred at three sites.

[0029] Preferably, the polyethylene glycol modifier forms a covalent bond with the N-terminus of the KLK1 protein or the free amino group of lysine, and its general structural formula is as shown in formula (1-1) or formula (1-2), where R represents KLK1. In formula (1-1), n ​​is an integer between 335 and 455, and in formula (1-2), n is an integer between 105 and 225, and m is an integer between 1 and 8.

[0030] [ka] (1-1)

[0031] [ka] (1-2)

[0032] Preferably, the polyethylene glycol modifier has a molecular weight of 5 kDa to 10 kDa. It is a chain-type polyethylene glycol succinimide propionate, and its general structural formula is as shown in formula (2). In formula (2), n is an integer between 10⁵ and 22⁵.

[0033] [ka] (2)

[0034] Preferably, the polyethylene glycol modifier is a branched polyethylene glycol propionaldehyde with a molecular weight of 30 kDa to 40 kDa, and its general structural formula is as shown in formula (3). In formula (3), n is an integer between 335 and 455.

[0035] [ka] (3)

[0036] The present invention provides a method for producing polyethylene glycolated KLK1. The method comprises the following steps. Step 1: Buffer replacement The KLK1 to be modified is replaced with the modification buffer by methods such as desalting using a chromatography column, dialysis, concentration / dilution, or cross-flow ultrafiltration; Step 2: Modification reaction and purification of the modified product The KLK1 solution collected in Step 1 is reacted with a PEG modifier, and after the reaction is complete, it is purified by ion exchange chromatography.

[0037] The present invention also provides a composition comprising the polyethylene glycolated KLK1.

[0038] The present invention also provides applications of polyethylene glycolated KLK1 in the treatment, prevention, prognosis recovery, and relapse prevention of acute ischemic stroke, peripheral neuropathy, retinopathy, fundus diseases, hypertension, diabetic nephropathy, IgA nephritis, and chronic kidney disease. [Effects of the Invention]

[0039] Compared to conventional technology, the present invention has the following advantages. Firstly, the low-glycosylated KLK1 of the present invention has clear advantages. First, the low-glycosylated KLK1 of the present invention has no glycosylation modification or only a very small amount of glycosylation modification in NFS, and has higher activity compared to the highly glycosylated KLK1.

[0040] Next, the recombinant KLK1 mutant of the present invention has further advantages.

[0041] The recombinant KLK1 mutant of the present invention exhibits N-glycosylation modification compared to natural KLK1. One location is missing. In terms of the manufacturing process, the recombinant hKLK1 mutant of the present invention The purification process is simplified as it only requires the collection of the main peaks. In terms of quality, the present invention eliminates the need to separate highly glycosylated KLK1 from low glycosylated KLK1, resulting in a more uniform target product, easier quality control, and higher yields. In terms of activity and efficacy, the applicant made the surprising discovery that the mutant of the low glycosylated KLK1 of the present invention is superior to the unmutated low glycosylated KLK1 in terms of enzymatic properties and activity.

[0042] Secondly, the present invention has developed polyethylene glycolated recombinant hKLK1, which has long-acting properties, allows for quality control, exhibits low immunogenicity, and high biological activity, through polyethylene glycolation technology. Furthermore, through pharmacokinetic studies, the advantages of safety and long-acting properties of polyethylene glycolated drugs have been fully explored, enabling a reduction in administration frequency, improved patient compliance, and the application of the present invention's drugs throughout the entire disease process, covering stages such as prevention, treatment, prognosis recovery, and relapse prevention for diseases such as acute ischemic stroke, peripheral neuropathy, retinopathy, fundus diseases, hypertension, diabetic nephropathy, IgA nephritis, and chronic kidney disease. [Brief explanation of the drawing]

[0043] [Figure 1] This figure shows the molecular designs of recombinant hKLK1 and mutants. [Figure 2] This diagram shows the pZHK2.0 expression vector. [Figure 3] This figure shows a chromatogram of hydrophobic chromatography elution peaks from the fermentation supernatant of wild-type recombinant hKLK1 expressed in CHO. The arrows indicate the separated high-glycosylation product (left) and low-glycosylation product (right). [Figure 4] This figure shows the SDS-PAGE electrophoresis of highly and lowly glycosylated proteins obtained by purifying and isolating wild-type hKLK1 expressed in CHO. Lane M is the protein marker, lane 1 is highly glycosylated hKLK1, and lane 2 is lowly glycosylated hKLK1. [Figure 5]This figure shows a chromatogram of hydrophobic chromatography elution peaks from the fermentation supernatant of hKLK1X1 (recombinant hKLK1 mutant) expressed in CHO. The arrows indicate the isolated single low-glycosylated product. [Figure 6] This figure shows the SDS-PAGE electrophoresis of the low-glycosylated protein obtained by purifying and isolating hKLK1X1 (recombinant hKLK1 mutant) expressed in CHO. Lane M is the protein marker, and lane 1 is the low-glycosylated hKLK1 mutant. [Figure 7] This figure shows an SDS-PAGE electrophoresis map of wild-type recombinant hKLK1 expressed in CHO when high and low glycosylated proteins have not been separated. Lane M is the protein marker, and lane 1 is wild-type recombinant hKLK1 with a mixture of high and low glycosylated proteins. [Figure 8] This figure shows the sequence coverage of a highly glycosylated hKLK1 sample after trypsin enzyme digestion. [Figure 9] This figure shows the sequence coverage of a low-glycosylated hKLK1 sample after trypsin enzyme digestion. [Figure 10] This figure shows the TIC chromatograms after trypsin enzyme digestion of three batches of highly glycosylated hKLK1 samples. [Figure 11] This figure shows the TIC chromatograms after trypsin enzyme digestion of three batches of low-glycosylated hKLK1 samples. [Figure 12] This figure shows the sequence coverage of the hKLK1X1 sample after trypsin enzyme digestion. [Figure 13] This figure shows the results of measuring anti-drug antibodies in animals administered PEG-hKLK1 (low-glycosylated). [Figure 14] This figure shows the results of measuring anti-drug antibodies in animals administered PEG-hKLK1 (highly glycosylated). [Figure 15] This graph shows the effect of the test substance in Example 10 on nerve deficiency symptoms. [Figure 16]This graph shows the effect of the test substance in Example 10 on the area of ​​cerebral infarction. [Figure 17] This figure shows brain section images of the model group and the sham surgery group in the animal experiment of Example 10. [Figure 18] This figure shows brain section images of the Urinary Kallikrein group and the Injectable Pancreatic Kallikrein 1 group in the animal experiment of Example 10. [Figure 19] This graph shows the effect of the test substance in Example 11 on nerve deficiency symptoms. [Figure 20] This graph shows the effect of the test substance in Example 11 on the area of ​​cerebral infarction. [Figure 21] This figure shows brain section images of the model group and the sham surgery group in the animal experiment of Example 11. [Figure 22] This figure shows brain section photographs of the treatment group in the animal experiment of Example 11. [Figure 23] This graph shows the effect of the test substance in Example 12 on nerve deficiency symptoms. [Figure 24] This graph shows the effect of the test substance in Example 12 on the area of ​​cerebral infarction. [Figure 25] This figure shows brain section images of the model group, sham surgery group, and positive drug group in the animal experiment of Example 12. [Figure 26] This figure shows brain section images of the groups administered with different PEG-modified hKLK1 variants in the animal experiment of Example 12. [Figure 27] This graph shows the effect of the test substance in Example 13 on nerve deficiency symptoms. [Figure 28] This graph shows the effect of the test substance in Example 13 on the area of ​​cerebral infarction. [Figure 29] This figure shows brain section images of the model group and the sham surgery group in the animal experiment of Example 13. [Figure 30] This figure shows brain section images of the group administered with different PEG-modified hKLK1 mutants in the animal experiment of Example 13. [Figure 31] This graph shows the effect of the test substance in Example 14 on nerve deficiency symptoms. [Figure 32] This graph shows the effect of the test substance in Example 14 on the area of ​​cerebral infarction. [Figure 33] This figure shows brain section images of the model group and the sham surgery group in the animal experiment of Example 14. [Figure 34] This figure shows brain section photographs of the treatment group in the animal experiment of Example 14. [Modes for carrying out the invention]

[0044] Unless otherwise specified, technical terms or abbreviations in this invention have the following meanings: Kallikrein 1: Specifically refers to tissue kallikrein 1 (KLK1).

[0045] hKLK1: Refers to unmutated human tissue kallikrein 1 whose sequence is identical to that of natural human tissue kallikrein 1, and includes, but is not limited to, various homologs of human KLK1, including KLK1 registered under Genbank registry numbers AAA59455.1, NP002248.1, AAA36136.1, AAP35917, AAU12569, etc.

[0046] The three glycosylation modification sites of KLK1, N78, N84, and N141, refer to the asparagine at positions 78, 84, and 141 of the KLK1 amino acid sequence, respectively. The corresponding N-glycosylation triad sequence motifs are NMS, NHT, and NFS, respectively. N represents asparagine, M represents methionine, S represents serine, H represents histidine, T represents threonine, and F represents phenylalanine.

[0047] Highly glycosylated hKLK1: This is an unmutated hKLK1 that is highly glycosylated. Specifically, glycosylation sites N78, N84, and N141 all have many glycosylation modifications.

[0048] Low glycosylation hKLK1: This is an unmutated hKLK1 that has undergone low glycosylation modification. That is, both glycosylation modification sites N78 and N84 have many glycosylation modifications. Although ornamentation is present, the glycosylation modification site N141 either lacks glycosylation modification or has undergone only very small amounts of glycosylation modification.

[0049] PEG-hKLK1 (highly glycosylated): This is a highly glycosylated hKLK1 modified with polyethylene glycol. The aforementioned highly glycosylated hKLK1 refers to the unmutated hKLK1 having the high glycosylation modification.

[0050] PEG-hKLK1 (low glycosylation): This is a low glycosylation hKLK1 modified with polyethylene glycol. The low glycosylation hKLK1 refers to the unmutated hKLK1 having the low glycosylation modification.

[0051] hKLK1X is a mutant of hKLK1. hKLK1X1, hKLK1X2, hKLK1X3, and hKLK1X4 each represent different mutants.

[0052] PEG-hKLK1X: This is an hKLK1 mutant modified with polyethylene glycol.

[0053] KLK1 derivatives: If the KLK1 mutant full-length protein of the present invention is included, then the derivatives also include a portion of the KLK1 mutant protein of the present invention, or proteins obtained by further mutations based on the KLK1 mutant of the present invention, fusion proteins (including, but not limited to, albumin fusion and Fc fusion), and modified products modified in various ways (excluding polyethylene glycolated modified products).

[0054] Polyethylene glycol, abbreviated as PEG, is typically formed by the polymerization of ethylene oxide and comes in branched, linear, and multi-armed forms. Generally, those with a molecular weight of 20,000 or less are called PEG, while those with a larger molecular weight are called PEO. Ordinary polyethylene glycol has hydroxyl groups at both ends, and blocking one end with a methyl group yields methoxy-polyethylene glycol (mPEG).

[0055] Polyethylene glycol modifiers, abbreviated as PEG modifiers, refer to polyethylene glycol derivatives with functional groups and are activated polyethylene glycols used for the modification of proteins and polypeptides into drugs. The polyethylene glycol modifiers used in this invention are purchased from Jiangsu Zhonghong Bioengineering Drug Discovery Research Institute Co., Ltd. or Beijing Jiankai Technology Co., Ltd. The actual molecular weight of a PEG modifier of a specific molecular weight may be 90% to 110% of the indicated value. For example, the molecular weight of PEG5K may be 4.5 kDa to 5.5 kDa.

[0056] The PEG5K used in the examples specifically refers to M-SPA-5K, which is a linear methoxypolyethylene glycol succinimidylpropionate with a molecular weight of approximately 5 kDa, and its structural formula is as shown in formula (1). In the formula, n is an integer between 10⁵ and 110.

[0057] [ka] (1)

[0058] The PEG10K used in the examples specifically refers to M-SPA-10K, which is a linear methoxypolyethylene glycol succinimidylpropionate with a molecular weight of approximately 10 kDa, and its structural formula is as shown in formula (1). In the formula, n is an integer between 220 and 225.

[0059] The PEG30K used in the examples specifically refers to Y-PALD-30K, which is a branched polyethylene glycol propionaldehyde with a molecular weight of approximately 30 kDa, and its structural formula is as shown in formula (2). In the formula, m is an integer between 335 and 340.

[0060] [ka] (2)

[0061] The PEG40K used in the examples specifically refers to Y-PALD-40K, which is a branched polyethylene glycol propionaldehyde with a molecular weight of approximately 40 kDa, and its structural formula is as shown in formula (2). In the formula, m is an integer between 450 and 455. [Examples]

[0062] Example 1: Gene design, expression vector construction, expression, and purification of recombinant human kallikrein 1 (hKLK1). (1) Gene design and expression vector construction Based on the information of the hKLK1 sequence (GenBank:AAA59455.1) published in GenBank, codon optimization is performed to obtain the amino acid sequence (SEQ) of the mature product. The hKLK1 cDNA sequence (SEQ ID No: 2), including ID No: 1 and the signal peptide and propeptide, was determined. The recombinant hKLK1 gene (SEQ ID No: 2) was synthesized by adding an AvrII restriction enzyme site sequence before it and a BstZ17I restriction enzyme site sequence to the end of the sequence. The synthesized gene was constructed on the pUC57 plasmid, and the resulting long-term storage plasmid was named the pUC57-hKLK1 plasmid. The hKLK1 gene was amplified from the pUC57-hKLK1 plasmid using primers. PCR products were recovered by 1% agarose electrophoresis, and the target gene PCR product (Figure 1) and the pZHK2.0 vector (Figure 2) were double digested using AvrII and BstZ17I. The digested target gene was ligated to the pZHK2.0 vector using T4 ligase, and then converted to Top10 competent cells. These cells were spread on kanamycin-resistant LB plates and incubated overnight at 37°C. The following day, positive clones were screened, sequenced, and compared, and the sequences were found to be in perfect agreement with the expected sequences. This completed the preparation of the pZHK2.0-hKLK1 expression vector.

[0063] (ii) Stable expression The plasmid constructed above was linearized by overnight enzymatic digestion in NruI (R0192S, purchased from NEB), introduced into CHO-S cells by electroporation, and then screened under selective pressure to obtain stable cell lines. CHO stable expression cell lines that highly express recombinant hKLK1 were inoculated into Dynamis medium (A2617501, purchased from Thermo Fisher), cultured in fed-batch flow-batch at 37°C, 8% CO2, and 130 rpm, and the culture medium was collected after 2 weeks of culture.

[0064] (3) Purification 1. Pre-treatment of the culture medium The culture medium containing recombinant hKLK1 was collected, centrifuged at 6000 rpm for 15 minutes to remove cells, concentrated by ultrafiltration, and filtered through a 0.22 μm membrane to remove cell debris. 1.5 M (NH4)2SO4 was added, and the mixture was stirred at room temperature for 3 days to activate it. The activated culture medium was clarified by filtering through a 0.45 μm microfiltration membrane.

[0065] 2. Hydrophobic chromatography First, the column was equilibrated using a buffer solution (20 mM Tris-HCl, 1.5 M ammonium sulfate, pH=8.0) until the baseline was at equilibrium. Then, the pre-treated supernatant was passed through a medium POROS Benzyl (A32563, purchased from Thermo Fisher) to capture recombinant hKLK1 from the culture medium. Gradient elution was then performed with 20 mM Tris-HCl, pH=8.0, clearly revealing two peaks separated by hydrophobicity. Each elution peak was collected to separate recombinant hKLK1 proteins with different glycosylation modifications. The chromatograms of the elution peaks are shown in Figure 3.

[0066] 3. Anion exchange The elution peaks containing recombinant hKLK1 protein collected in Step 2 were ultrafiltered using a 20 mM Tris-HCl, pH=8.0 buffer until the electrical conductivity reached 10–15 ms / cm using a 10 kDa ultrafiltration membrane. The column was equilibrated using a buffer (20 mM Tris-HCl, 100 mM NaCl, pH=8.0) until the baseline was equilibrium. The supernatant pretreated by ultrafiltration was then passed through a medium-Q FF (17515601, purchased from GE), and finally, the column was isocratic eluted using an elution buffer (20 mM Tris-HCl, 1 M NaCl, pH=8.0) to collect the major elution peaks.

[0067] 4. Cation exchange Ultrafiltration was performed on the sample from Step 3, and the sample was replaced with 10 mM NaAc-HAc, 50 mM NaCl, pH=3.5–3.7. High-level purification was performed on recombinant hKLK1 of different molecular weights collected in the previous step using a medium SP FF (17072904, purchased from GE). The equilibrium buffer was 10 mM NaAc-HAc, 50 mM NaCl, pH=3.7, and the elution buffer was 50 mM Tris-HCl, 1 M NaCl, pH=8.0.

[0068] The purity of the purified sample was analyzed by SDS-PAGE gel electrophoresis. The results, shown in the SDS-PAGE gel electrophoresis graph, indicated that the molecular weight of the highly glycosylated hKLK1 protein was slightly higher than that of the lowly glycosylated hKLK1 protein. The results are shown in Figure 4.

[0069] Example 2: Three-batch continuous production and quality characterization analysis of recombinant human kallikrein 1 (hKLK1). Three batches of high-density continuous culture of wild-type recombinant hKLK1 were performed in a 7L agitated reactor. The initial culture conditions were as follows: rotation speed 100 rpm, dissolved oxygen 40%, temperature 37°C, pH 7.0, and basal medium Dynamis AGT Medium (purchased from Thermo Fisher Scientific). Seed suspension was collected and inoculated into the bioreactor at a density of 5 × 10 e5 cells / mL. On day 3, the temperature was adjusted to 33°C, and feed medium (EfficientFeed C+AGT supplement purchased from Thermo Fisher Scientific) and glucose were added separately to maintain the sugar content of the culture medium above 2 g / L. When the cell viability fell below 90%, the culture medium was collected and purified using the purification process described in Example 1. This process yielded three batches each of highly glycosylated recombinant hKLK1 and low glycosylated recombinant hKLK1.

[0070] Protein quality characteristics analysis was performed on these samples. The analysis included yield, SEC-HPLC purity, and analysis of N / C terminal amino acid sequences, peptide mapping, glycosylation modification sites, and glycosylation modifications using LC / MS (Thermo Q Exactive). The results are summarized below.

[0071] [Table 2]

[0072] The analysis results shown in the table above indicate the following: The purity of all isolated high-glycosylation recombinant hKLK1 and low-glycosylation recombinant hKLK1 reached over 97%, and there was a difference in molecular weight between the high-glycosylation and low-glycosylation proteins. Based on the measurement of amino acid sequence coverage of high-glycosylation hKLK1 and low-glycosylation hKLK1 based on trypsin enzyme digestion, low-glycosylation hKLK1 achieved 100% coverage, while high-glycosylation hKLK1 achieved 100% coverage in some areas, but some products had sequence deletions at the N-terminus (Figures 8 and 9). The N / C-terminal sequences of low-glycosylation hKLK1 were consistent with the theoretical sequence (Tables 6-8), and the characteristics of the peptide maps of the three batches were consistent (Figure 11). However, in batch 2 of the three-batch continuous production process, the N-terminal sequences of some protein components of the highly glycosylated protein did not match the theoretical sequences, resulting in sequence deletions (Tables 3-5). Furthermore, the peptide map of batch 2 did not match the peptide maps of batches 1 and 3 (Figure 10). This suggests a possible stability defect in the highly glycosylated recombinant hKLK1 molecule. This is considered to introduce a significant risk into the drug production process. In addition, the separation and purification processes for highly glycosylated and low-glycosylated proteins are relatively complex, and it is thought that process instability makes it difficult to control the glycosylation levels of purified products in different batches.

[0073] [Table 3]

[0074] [Table 4]

[0075] [Table 5]

[0076] [Table 6]

[0077] [Table 7]

[0078] [Table 8]

[0079] [Table 9]

[0080] Furthermore, the analysis of glycosylation modification sites in recombinant hKLK1 revealed the following: Recombinant low-glycosyl hKLK1 molecules all had glycosylation modifications at N78 and N84, with almost no glycosylation modification at N141 (96.39%). Only a very small amount of hKLK1 molecules (less than 4%) had glycosylation modification at N141 (Table 9). On the other hand, high-glycosyl hKLK1 molecules contained three N-glycosylation modification sites (N78, N84, and N141), exhibiting a wide variety of glycosylation modifications and complex glycoform types.

[0081] The elution peaks of the highly glycosylated and low glycosylated proteins obtained by hydrophobic chromatography in Example 1 were combined, and subsequent anion exchange purification and cation exchange purification were performed in the same manner to obtain recombinant hKLK1 protein containing both highly glycosylated and low glycosylated proteins. The purity of the purified sample was analyzed by SDS-PAGE gel electrophoresis, and the results are shown in Figure 7. The results show that the highly glycosylated and low glycosylated proteins formed a diffusive band, indicating the presence of a component with an even larger molecular weight on top of the highly glycosylated protein. As shown in Figure 3, the degree of separation when separating highly glycosylated hKLK1 and low glycosylated hKLK1 from unmutated recombinant hKLK1 by hydrophobic purification was not good, and it is difficult to completely separate highly glycosylated hKLK1 and low glycosylated hKLK1. In short, the proto-protein of unmutated hKLK1 contains a mixture of highly glycosylated and lowly glycosylated hKLK1 with different molecular weights, resulting in heterogeneous and complex glycosylation modifications at N141. This presents a significant challenge in quality control of recombinant hKLK1.

[0082] Furthermore, when the biological activity of highly glycosylated hKLK1 and low glycosylated hKLK1 was compared (Example 6), it was surprisingly found that the activity of low glycosylated hKLK1 was far higher than that of highly glycosylated hKLK1.

[0083] Example 3: Design, expression vector construction, expression, and purification of recombinant human kallikrein 1 mutant (hKLK1X). Furthermore, in order to obtain low-glycosylated hKLK1, the applicant of this invention did not focus on optimizing the expression and purification conditions of recombinant hKLK1 as in conventional methods, but rather, based on the aforementioned research on high- and low-glycosylated hKLK1, mutated one or more amino acids in the NFS sequence motif to prevent glycosylation modification at the relevant position. As a result, a mutant was obtained in which the product was more uniform and yielded a higher amount of low-glycosylated hKLK1. Surprisingly, subsequent experiments revealed that the low-glycosylated mutant had further advantages in terms of enzymatic properties and activity compared to the unmutated low-glycosylated hKLK1.

[0084] In this embodiment, the mutants exhibited mutations in N (i.e., N141) within the NFS sequence motif, while F and S within the NFS sequence motif remained unmutated. However, these examples are illustrative and do not limit the scope of application of the present invention. The following methods can all be used to prevent glycosylation modification at N141 by altering the original NFS sequence to prevent the formation of an N-glycosylation motif. For example, this could involve mutating the amino acid N (i.e., N141) in the NFS and mutating zero, one, or two amino acids in F and S to any other amino acid; mutating F (i.e., F142) in the NFS to proline and mutating zero, one, or two amino acids in N and S to any other amino acid; or mutating S (i.e., S143) in the NFS to any amino acid other than threonine and mutating zero, one, or two amino acids in N and F to any other amino acid. The aforementioned method results in a more uniform product and a higher yield of low-glycosylated hKLK1, while the resulting mutant exhibits further advantages in terms of enzymatic properties and activity compared to the pre-mutation mutant. This is thought to be related to the fact that glycosylation modification does not occur in the original NFS sequence.

[0085] Furthermore, this embodiment uses a mutant based on the hKLK1 sequence registered in Genbank under registration number AAA59455.1, but this is illustrative and does not limit the scope of application of the present invention. Although the amino acid composition and glycosylation modification sites are highly similar to other known hKLK1 natural mutants, similar technical effects can be achieved by mutating at the corresponding positions (i.e., one or more amino acids in the NFS sequence motif) of other hKLK1 natural mutants. That is, a target product with a more uniform product, a higher yield of low-glycosylated hKLK1, and better activity can be obtained. All KLK1s of other primates contain three types of N-glycosylation modification sites, just like hKLK1, and the triad sequence motif is also the same, all being NMS, NHT, and NFS. Similar technical effects can be achieved by mutating at the corresponding positions (i.e., one or more amino acids in the NFS sequence motif) of the natural amino acid sequence of other primate KLK1s. In other words, the product is more uniform, yields a higher amount of low-glycosylated hKLK1, and the target product has better activity.

[0086] The specific mutation patterns illustrated in this embodiment are as follows: Site-directed mutations were performed on the glycosylation modification site N141, mutating asparagine into four different amino acids: the polar neutral amino acid glutamine (Gln), the acidic amino acid aspartic acid (Asp), the basic amino acid arginine (Arg), and the aliphatic amino acid alanine (Ala). The resulting hKLK1 mutants were named hKLK1X1 (SEQ ID No:3), hKLK1X2 (SEQ ID No:4), hKLK1X3 (SEQ ID No:5), and hKLK1X4 (SEQ ID No:6). Each mutant was prepared using the method described in Example 1.

[0087] The recombinant hKLK1 mutant described above was purified by the three-step purification method of Example 1. In the hydrophobic purification chromatogram of the mutant, only one major elution peak appeared due to hydrophobic elution. Taking hKLK1X1 as an example, its hydrophobic chromatography elution chromatogram, shown in Figure 5, revealed that the product after mutation was more homogeneous. Unmutated recombinant hKLK1 does not have good separation capabilities for highly glycosylated hKLK1 and low glycosylated hKLK1 during the purification process (Figure 3), making it difficult to completely separate them. Since only one major peak can be collected during the hydrophobic chromatography purification of recombinant hKLK1 after mutation, the purification process is simpler.

[0088] The purity of the samples purified by SDS-PAGE gel electrophoresis was analyzed. It was confirmed that the molecular weight of the purified samples of each mutant was very close to that of the unmutated low-glycosylated hKLK1. As a result, it was found that highly glycosylated hKLK1, which contains a large amount of sugar modification at N141, was completely eliminated (Figure 6), the proportion of low glycosylated hKLK1 was higher, the yield was higher, and the product was more uniform.

[0089] The amino acid coverage of recombinant hKLK1 was measured and analyzed based on LC-MS. The coverage rate against the theoretical amino acid sequence was 100% (Figure 12), confirming the accuracy of the primary sequence of recombinant hKLK1. Furthermore, the glycosylation modification sites were identified, and recombinant hKLK1 contained only two glycosylation modification sites (N78 and N84), consistent with the molecular design objective. Other mutants showed similar results.

[0090] [Table 10]

[0091] Example 4: Measurement of molecular weight by MS after deglycosylation of protein samples before and after mutation. The molecular weights of recombinant proteins before and after mutation were measured by LC-MS after deglycosylation. Recombinant proteins were denatured with guanidine hydrochloride, enzymatically digested with the sugar-cleaving enzyme PNGaseF, and then measured for molecular weight. The deglycosylation molecular weight of highly glycosylated hKLK1 was 26380.338 Da, which is in close agreement with the theoretical molecular weight of 26377.493 Da (Table 11). The deglycosylation molecular weight of low-glycosylated hKLK1 was 26379.346 Da, which is in close agreement with the theoretical molecular weight of 26418.48 Da (Table 11). The deglycosylation molecular weight of hKLK1X1 was 26418.82 Da, which is in close agreement with the theoretical molecular weight of 26418.48 Da (Table 11). The deglycosylated molecular weights of hKLK1X2, hKLK1X3, and hKLK1X4 are also in close agreement with their theoretical molecular weights. These results further confirm the accuracy of the primary structures of these recombinant proteins.

[0092] [Table 11]

[0093] Example 5: Preparation, purification, and purity analysis of PEG-modified hKLK1 / hKLK1X samples hKLK1 / hKLK1X samples modified with different PEGs can be prepared and purified using conventional methods. An example of this is illustrated below.

[0094] 1. Preparation of PEG-modified samples (1) Pretreatment The protoprotein of unmutated low-glycosylated recombinant hKLK1 was taken and processed using a 3 kDa ultrafiltration membrane (or another buffer substitution method with equivalent effect). The buffer was then replaced with sodium dihydrogen phosphate / disodium hydrogen phosphate buffer, and the pH was adjusted to 7.0, while simultaneously concentrating the protein to 15 mg / mL.

[0095] (2) Feed and modification Preparation of randomly modified PEG5K-hKLK1: M-SAP-PEG5K was added to a pre-treated hKLK1 protein solution so that the mass ratio of hKLK1 protein to M-SAP-PEG5K was 1:25. The mixture was then slowly stirred to ensure homogeneity, and the mixture was reacted at 4°C for 24 hours.

[0096] Preparation of randomly modified PEG10K-hKLK1: M-SAP-PEG10K was added to a pre-treated hKLK1 protein solution so that the mass ratio of hKLK1 protein to M-SAP-PEG10K was 1:20. The mixture was then slowly stirred to ensure homogeneity, and the reaction was carried out at 4°C for 24 hours.

[0097] Preparation of site-directed modified PEG30K-hKLK1: Y-PALD-PEG30K was added to a pre-treated hKLK1 protein solution so that the molar ratio of hKLK1 protein to Y-PALD-PEG30K was 1:6. The reducing agent (sodium cyanoborohydride) was added to a mixed solution of PEG30K and protein so that the molar ratio of PEG to reducing agent (sodium cyanoborohydride) was 1:50. The mixture was slowly stirred to ensure homogeneity, and then the reaction was carried out at 4°C for 24 hours.

[0098] Preparation of site-directed modified PEG40K-hKLK1: Y-PALD-PEG40K was added to a pre-treated hKLK1 protein solution so that the molar ratio of hKLK1 protein to Y-PALD-PEG40K was 1:6. The reducing agent (sodium cyanoborohydride) was added to a mixed solution of PEG40K and protein so that the molar ratio of PEG to reducing agent (sodium cyanoborohydride) was 1:50. The mixture was slowly stirred to ensure homogeneity, and then the reaction was carried out at 4°C for 24 hours.

[0099] 2. Purification of the reaction mixture The chromatography conditions are as follows: The packing material for purification is GE's Q Sepharose. TM High Performance Medium was used, and the purification mobile phase Buffer A was 50 mM Tris-HCl9. The solution is 0, and Buffer B is 50 mM Tris-HCl + 1 M NaCl 9.0. . Sample Loading: The PEG-hKLK1 modified reaction mixture described above was taken after the reaction was complete, diluted approximately 10-fold with redistilled water, and then further diluted approximately 5-fold with buffer A. The sample was then loaded and purified. After sample loading was complete, the chromatographic column was washed with buffer A to a volume of at least 5 column beds.

[0100] Elution: Elution was performed using a 0-50% Buffer B gradient across 10 column bed volumes, and eluted samples were collected in stages based on the UV280 trend.

[0101] The PEG-modified hKLK1 mutant (PEG-hKLK1X) was prepared and purified using the method described above.

[0102] III. Purity analysis of PEG-modified samples (1) Purity analysis by HPLC The measurements were performed referencing General Rule 0512, High-Performance Liquid Chromatography, of the 2020 edition of the Chinese Pharmacopoeia. The type of chromatography used was SEC (Size Exclusion Chromatography), with a liquid phase of 20 mM PB pH 7.0 buffer containing 5% isopropanol, a BEH450 SEC 3.5 μm chromatography column, and a collection condition of 280 nm for a collection time of 20-25 minutes.

[0103] According to the results of the liquid-phase measurement, the series of PEG-hKLK1 / hKLK1X proteins that were prepared The purity of the quality was ≥95% for all samples.

[0104] (2) Purity analysis by SDS-PAGE Sample purity was measured based on the SDS-polyacrylamide gel electrophoresis method, method 5 of electrophoresis (0541) in the 2020 edition of the Chinese Pharmacopoeia. 12.5% ​​SDS-PAGE The solution was prepared and the sample was measured.

[0105] Electrophoresis results showed that the molecular bands of the prepared series of PEG-hKLK1 / hKLK1X proteins were uniform, no impurity bands were observed, and all had good purity.

[0106] (3) Measurement of PEG bond count a) Solution preparation: PEG standard solutions at concentrations of 1, 2, 4, 6, and 8 mg / mL were prepared, and recombinant hKLK1 and its PEG-modified solution at 1 mg / mL were prepared. b)Measurement method: Chromatography column: XBridge BEH SEC 3.5μm 450A Column temperature: 25℃. Mobile phase: 20mM PB pH7.0+10%IPA, flow rate 0.4mL / min. Detectors: PDA detector, detection wavelength 280 nm; RI detector, detection wavelength 280 nm.

[0107] c) Data analysis: Measured concentration of the test sample (PEG modified) = Peak area of ​​the test sample (PEG modified) measured by the PDA detector / Peak area of ​​the unmodified protoprotein measured by the PDA detector × 1.0 PEG peak area in the sample = (Peak area of ​​the test sample (PEG-modified) as measured by the RI detector / Measured concentration of the test sample (PEG-modified)) - (Peak area of ​​the unmodified protoprotein as measured by the RI detector / Concentration of the unmodified protoprotein)

[0108] The PEG concentration in a sample is calculated by substituting the PEG peak area in the sample into the PEG standard curve.

[0109] Number of PEG bonds in the sample = (PEG concentration in the sample / PEG molecular weight) / (Sample Protein concentration / Protein molecular weight (26kD)

[0110] [Table 12]

[0111] Example 6: In vitro activity measurement of recombinant human kallikrein 1 (hKLK1) and its mutant (hKLK1X) 1. Evaluation of in vitro activity using artificial substrates KLK1 exerts its biological function in vivo by catalyzing the hydrolysis of low molecular weight kininogen LMWK, thereby releasing lysylbradykinin. This hydrolysis reaction involves the cleavage of the peptide bond at the carboxyl end of arginine (Arg). Therefore, the in vitro biological activity of recombinant hKLK1 and its mutants can be evaluated by detecting the production of PNA at 405 nm, based on the cleavage of the amide bond between Arg and p-nitroaniline in the chromogenic artificially synthesized substrate S-2266 (H-D-Val-Leu-Arg-PNA) to produce p-nitroaniline (PNA). An active unit (IU) is defined as the amount of enzyme required to hydrolyze 1 μmol of S-2266 to produce PNA in one minute under conditions of 37°C and pH 8.0. The reaction system consists of 200 μl of 20 mM trishydroxymethylaminomethane buffer, 10 μl of the test sample, and 20 μl of 20 mM S-2266 substrate solution. The reaction system was precisely controlled in a 37°C water bath for 10 minutes, and the reaction was stopped by adding 20 μl of 50% acetic acid solution. The amount of PNA produced in the reaction system was quantified using a standard curve fitted based on PNA standards of different concentrations. Using the above method, the in vitro biological activity of highly glycosylated hKLK1, low-glycosylated hKLK1, hKLK1 mutants, and hKLK1-PEG modified samples was measured.

[0112] The results are shown in the table below. The activity of the low-glycosylation hKLK1 (unmutated) sample was significantly higher than that of the high-glycosylation hKLK1 (unmutated) sample. Among the series of PEG-modified products of the unmutated protein, randomly modified products showed higher in vitro activity compared to site-directed modified products, with PEG10K-hKLK1 (low-glycosylation) showing particularly superior activity. The activity of site-mutated mutant samples (hKLK1X1, hKLK1X2, hKLK1X3, hKLK1X4) was higher in all cases than that of the unmutated low-glycosylation hKLK1 sample. Furthermore, the activity of the PEG-modified samples was slightly lower than that of the unmodified hKLK1X1 protein, while the activity of PEG10K-hKLK1X1 was higher than that of PEG5K-hKLK1X1, indicating that the PEG10K-modified protein largely retained the activity of the original protein.

[0113] [Table 13]

[0114] 2. Measurement of in vitro activity using natural substrates 1. Enzyme reactions and liquid-phase measurements KLK1 exerts its biological function in vivo by catalyzing the hydrolysis of low molecular weight kininogen LMWK, thereby releasing lysyl bradykinin. In this example, the production of the effector molecule bradykinin is compared by enzymatically reacting a substrate (low molecular weight kininogen LMWK) with an enzyme (KLK1 or its PEG-modified form) in different ratios. The generated effector molecules were separated by reverse-phase chromatography, the peak area of ​​the product was calculated, and curves representing the bradykinin production when the substrate and enzyme reacted in different ratios were plotted. The amount of effector molecules produced by different test samples under the same reaction conditions was then compared. In this way, the mode of action in vivo was simulated in vitro, and the in vivo effects of different test samples were indirectly compared.

[0115] The hKLK1X1 in the test sample was prepared by the method described in Example 3, the PEG10K-hKLK1X1 was prepared by the method described in Example 5, Kb was KLK1 extracted from porcine pancreas, and PEG-Kb was PEG10K-modified Kb, which is injectable pancreatic kallikrein 1, prepared with reference to Example 5.

[0116] The samples were mixed and reacted according to the table below (for PEG-modified samples, the molar mass of the modified active substance was used). The mixed samples were placed in a 37°C incubator, timed precisely, and incubated for 15 minutes. The reaction was then stopped by adding a 50% acetic acid solution in a volume ratio of 10:1.

[0117] [Table 14]

[0118] After stopping the reaction, the sample was placed in a benchtop centrifuge and centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected. Waters ACQUITY UPLC H-Class measurement was performed. Mobile phase A was 0.1% TFA-H2O, mobile phase B was 0.1% TFA-ACN, the measurement wavelength was 214 nm, the column temperature was 30°C, the sample loading volume was 10 μl for all measurements, the flow rate was 0.2 mL / min, the run time was 35 minutes, and the run gradient was as follows.

[0119] [Table 15]

[0120] 2, results Based on the UPLC chromatogram results of the enzyme reaction mixture, the sum was calculated by integrating the peaks with retention times of 13 ± 0.5 min (bradykinin peak position) on the chromatogram of the reaction mixture. The concentration of bradykinin produced by the enzyme reaction was calculated by comparing this with the peak area of ​​bradykinin per unit mass (1 mg / mL, 10 μL sample loading). The amount of bradykinin produced was calculated based on the total reaction system in Table 14, and finally converted to the amount of bradykinin produced per 1 mg of enzyme (μg / mg) under each molar ratio condition. The results are shown in Table 16.

[0121] [Table 16]

[0122] Measurement results using natural substrates showed that, as a general trend, the bradykinin produced by the enzymatic reaction in the modified proteins was lower than in the unmodified samples. This is consistent with the characteristics of PEG-modified proteins. When considered together with the data from Examples 9-13, it is thought that PEG-modified proteins can maintain a milder enzymatic reaction compared to the original proteins, enabling the sustained generation of effector molecules. Also, KLK1 The efficacy of these drugs is based on the regulatory action of the kallikrein-kinin system (KKS) in the body, which includes the generation and removal of effector molecules. Therefore, a gentle and sustained enzymatic reaction process effectively reduces the removal mechanism in the KKS, enabling the drugs provided by the present invention to exert their biological effects more stably and effectively.

[0123] Furthermore, when comparing Kb, hKLK1X1, and their modified products, the samples provided by the present invention produced higher amounts of effector molecules. This is consistent with the results of in vivo pharmacokinetic experiments using animals shown in Examples 10-13. These experiments demonstrated that the hKLK1X1 protein provided by the present invention has a significant advantage over animal-derived KLK1.

[0124] Example 7: In vitro enzyme kinetic measurement of PEG-modified hKLK1 / hKLK1X, etc. 1.Measurement method (1) Dilution of standard: Pancreatic kallikrein 1 (provided by Changzhou Qianhong Biochemical Pharmaceutical Co., Ltd.), a standard, was diluted to 10 IU / mL using S2266 substrate buffer (20 mM Tris-HCl 8.5). The diluted standard was then further diluted to 1, 2, 3, 4, 5, and 6 IU / mL according to the table below, and standard curve samples were prepared.

[0125] [Table 17]

[0126] (2) Preparation of substrates at different concentrations: Using S2266 substrate buffer, the already dispensed substrate S2266 was diluted to substrate solutions of 400, 200, 100, 50, 25, and 10 μM, respectively.

[0127] (3) Sample dilution: Samples were diluted to 1–6 IU / mL using S2266 substrate buffer.

[0128] (4) Adding the samples: The standard curve samples prepared in (1) above were added to the microplate at 80 μL / well, one well for each sample. The diluted samples from (3) above were added to the microplate at 80 μL / well, six wells in parallel for each sample.

[0129] (5) Reading: The microplate reader was set up and dynamic measurements were performed at 37°C and 405 nm, with a reading interval of 1 min and a measurement time of 15 min. When the temperature of the instrument's sample pool rose to 37°C, the substrate was added. At this time, 80 μL / well of 200 μm substrate was added to the standard curve sample, and 80 μL / well of substrate of different concentrations prepared in (2) was added to each of the six parallel wells of the sample to be measured. Care must be taken to add the samples quickly and blow them to mix them uniformly. After the addition of the samples was complete, readings were started immediately and then taken every minute thereafter.

[0130] (6) Processing of results: The results were processed using the software GraphPad Prism, the data were fitted based on the Michaelis-Menten equation, and the enzyme kinetic parameters were calculated. Here, K m This is the Michaelis-Menten constant, and it represents the maximum rate of the enzyme reaction (V). max This represents the substrate concentration that gives half the rate. Generally, 1 / K m This approximates the magnitude of the enzyme's affinity for its substrate, and 1 / K m The larger the value, the more the enzyme interacts with its substrate. Therefore, it has a strong affinity, and the enzymatic reaction proceeds easily.

[0131] 2. Experimental Results The results of the enzyme kinetic measurements for each sample are shown in the table below.

[0132] [Table 18]

[0133] Enzyme kinetic measurements showed that hKLK1X1 has a higher K content compared to unmutated low-glycosylated hKLK1. m The values ​​decreased, indicating improved affinity with the substrate after the mutation. Furthermore, the two PEG modifiers of the mutant showed a higher K value compared to the unmutated PEG modifier. m The values ​​were lower, indicating that the two modified mutant proteins exhibited superior enzymatic properties compared to the modified unmutated protein.

[0134] Example 8: In vivo pharmacokinetic comparison of PEG-modified hKLK1 in rats 1. Grouping Based on the body weight of SD rats measured on the first day of the experiment, the rats were randomly divided into six groups: PEG5K-hKLK1 (highly glycosylated) intravenous injection group (0.5 mg / kg), PEG5K-hKLK1 (highly glycosylated) subcutaneous injection group (0.5 mg / kg), PEG10K-hKLK1 (lowly glycosylated) intravenous injection group (0.5 mg / kg), PEG10K-hKLK1 (lowly glycosylated) subcutaneous injection group (0.5 mg / kg), PEG10K-hKLK1X1 intramuscular injection group (0.1 mg / kg), and PEG10K-hKLK1X1 intramuscular injection group (0.02 mg / kg). Each group consisted of six rats, half female and half male. The animals were stained and numbered. A single dose was administered on the first day of the experiment.

[0135] 2. Collection of blood samples and preparation of samples for measurement. Measurement times: Blood samples were taken once at 0 min (before administration), 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 2 day, 3 day, 5 day, and 7 day, for a total of 11 blood samplings.

[0136] Blood sample volume: 50-100 μL of serum was collected and used to measure pharmacokinetic parameters.

[0137] Sample preparation 1) Pharmacokinetic samples: Diluted using 10% SD rat mixed blank serum (SD rat mixed blank serum: blocking solution (2% BSA in PBST) = 1:9) to a limit of quantification concentration in the range of 2560 ng / mL to 80 ng / mL.

[0138] 2) Standard: Using SD rat mixed blank serum, PEG-modified products of high-glycosylated hKLK1 and low-glycosylated hKLK1 were diluted to obtain a concentration gradient range of 5120 ng / mL~ A standard was prepared in a 2-fold dilution series with a concentration of 40 ng / mL.

[0139] 3) Controls: Using SD rat mixed blank serum, PEG-modified products of hyperglycosylated hKLK1 and hypoglycosylated hKLK1 were diluted to prepare controls at high (1920 ng / mL), medium (480 ng / mL), and low (240 ng / mL) concentrations.

[0140] III. Pharmacokinetic measurements The drug concentration in the serum is measured by the ELISA method. The analytical process is as follows: 1) Coating: Anti-hKLK1 antibody was diluted to 400 ng / well with 20 mM phosphate buffer (PB, pH 7.4) and coated. The diluted and coated antibody was aspirated and added to the wells of a microplate (100 uL / well), the plate was sealed, and coated overnight at 4°C. The coated plate was a Thermo microplate.

[0141] 2) Blocking: Discard the liquid in the wells, wash the plate once with a microplate washer, shake, and dry. Add 200 μL / well of blocking solution (20 mM PBS containing 2% BSA and 0.05% Tween 20), seal the plate, and incubate at 37°C for approximately 2 hours.

[0142] 3) Add the sample: Remove the liquid from the wells, dilute the pharmacokinetic sample to be measured, the standard, and the control solution 10-fold with blocking solution, add them to the microplate at a rate of 100 μL / well, seal the plate, and incubate at 37°C for approximately 1.5 hours. did.

[0143] 4) Add detection antibody: Discard the liquid in the wells, wash the plate three times with a microplate washer, and shake to dry with 300 uL / well. Add 100 uL / well of detection antibody activator (enzyme-labeled anti-hKLK1 antibody for detection diluted to 1 ug / mL with blocking solution), seal the plate, and incubate at 37°C for approximately 45 minutes.

[0144] 5) Color development: Discard the liquid in the wells, wash the plate 5 times with a microplate washer, and shake to dry with 300uL / well. Add TMB color developer No. 1 and incubate at 37°C for 15 minutes (determined according to the color development).

[0145] 6) Plate reading: The reaction was stopped by adding 50 μL / well of stop solution (2 M H2SO4), and the OD value at 450 nm was immediately measured. A standard curve was drawn with the sample concentration on the X axis and the OD value on the Y axis, and the sample concentration was calculated.

[0146] 7) A standard curve was plotted and sample concentrations were calculated using Origin8 software. The mean, standard deviation, coefficient of variation, etc., were calculated using Microsoft Excel. The AUC (Area Under the Curve) was calculated using GraphPad Prism 7.00.

[0147] 4. Measurement results At equivalent doses, the in vivo area of ​​exposure (AUC) of the drug is as follows:

[0148] [Table 19]

[0149] The pharmacokinetic results described above show that PEG10K-hKLK1 (low glycosylation) exhibits significantly superior pharmacokinetic curves and a longer drug half-life compared to PEG5K-hKLK1 (high glycosylation) in both intravenous and subcutaneous injections. PEG10K-hKLK1X1 shows good absorption even at low doses when administered intramuscularly.

[0150] Example 9: Comparison of in vivo immunogenicity of PEG-modified products of highly glycosylated and low-glycosylated hKLK1 in rats. 1. Grouping Based on the body weight of the SD rats measured on the first day of the experiment, the rats were randomly divided into two groups: the PEG5K-hKLK1 (highly glycosylated) group and the PEG10K-hKLK1 (lowly glycosylated) group. Each group consisted of 12 rats, half female and half male. The animals were stained and numbered.

[0151] 2. Administration Route of administration: Intravenous injection. Dosage frequency: Administer once a week for 8 weeks. Dosage: Each group will be administered at a concentration of 0.2 mg / mL and a dose of 0.5 mg / kg.

[0152] 3. Collection of blood samples Measurement time: Blood samples were taken once at 0 min (before administration), and once each on day 3 and day 7 after administration. During the recovery period, blood samples were taken once a week. The recovery period is 2 weeks. Approximately 500 μL of whole blood was collected each time, allowed to stand for 2 hours, and then centrifuged at 3000 rpm for 10 minutes. The separated serum was used for immunogenicity measurement.

[0153] IV. Measurement of immunogenicity 1) Coating: The working solution of the coating antigen (PEG-modified hKLK1) was added to microwells at a rate of 100 μL / well and incubated overnight at 2-8°C. 2) Blocking: Discard the liquid in the wells, wash the plate three times, shake, and dry. Blocking solution (20 mM PBS containing 2% BSA and 0.05% Tween20) The solution was added at 200 μL / well and incubated at 37°C for approximately 2 hours. 3) Sample preparation: All animal serum samples were diluted 10-fold with blocking solution. 4) Adding the sample: Discard the liquid in the microplate wells and shake to dry. Add 100 μL / well of the treated sample to each well and incubate with shaking at 37°C and 200 rpm for approximately 2 hours.

[0154] 5) Add the detection antigen agonist: Discard the liquid in the microplate wells, wash the plate three times, shake, and dry. Add 100 uL / well of the detection antigen agonist (prepared by diluting biotin-labeled PEG-modified hKLK1 to 2.5 μg / mL with blocking solution) to each well of the microplate for the screening test, and add 100 uL / well of the confirmation assay detection antigen agonist (prepared by diluting PEG-modified hKLK1 to 200 μg / mL using the detection antigen agonist as a diluent) to each well of the microplate for the confirmation test, and incubate with shaking at 37°C and 200 rpm for approximately 1 hour.

[0155] 6) Add signal amplification detection solution (streptavidin-wasabi peroxidase diluted to 0.05 μg / mL with blocking solution): Discard the liquid in the microplate wells, wash the plate three times, shake to dry, add 100 μl / well of signal amplification detection solution, and incubate with shaking at 37°C and 200 rpm for approximately 1 hour.

[0156] 7) Color development: Discard the liquid in the wells, wash the plate three times, shake, and dry. Add 100uL of color developing solution to each well and leave in the dark at 37°C for 15 minutes.

[0157] 8) Termination: The reaction was stopped by adding 100 μl / well of stop solution (2M H2SO4), and the OD value at 450 nm was immediately measured using a microplate reader.

[0158] 5. Measurement results The immunogenicity evaluation results in Figures 13 and 14 show that no serum anti-drug antibodies (ADAs) were detected in any of the 12 animals in the PEG-hKLK1 (low glycosylation) group, but relatively high ADA levels were detected in two of the 12 animals in the PEG-hKLK1 (high glycosylation) group. This indicates that the immunogenicity of the low glycosylation protein modification is lower.

[0159] Example 10: Comparison of in vivo activity of Urinary Kallikrein for injection and Pancreatic Kallikrein 1 for injection. 1. Model creation We used male, SPD-class SD rats weighing 270-300g. We created a middle cerebral artery occlusion (MCAO) cerebral ischemia-reperfusion model using middle cerebral artery embolization. The animals were anesthetized with isoflurane, fixed supine, their skin was disinfected, an incision was made in the midline of the neck, and the right common carotid artery, external carotid artery, and internal carotid artery were separated. The vagus nerve was gently dissected, and the external carotid artery was ligated and transected. The mesial end of the carotid artery was clamped, an incision was made distal to the ligation site of the external carotid artery, and an MCAO stent (model 2438-A5, purchased from Beijing Xinong Technology Co., Ltd.) was inserted. It passed through the common carotid artery bifurcation and entered the internal carotid artery, and was gradually inserted until slight resistance was felt (approximately 20mM from the bifurcation), thereby blocking blood flow in the middle cerebral artery. The skin of the neck was sutured, disinfected, and the rat was returned to its cage. 90 minutes after ischemia, the rat was anesthetized again, fixed to a board, the skin of the neck was incised, the duct plug was found and gently removed, blood flow was restored, and reperfusion was performed. The skin of the neck was sutured, disinfected, and the rat was returned to its cage for rearing.

[0160] Grouping: A total of 4 groups, namely a sham surgery group, a model group, and one group of pancreatic kallikrein for injection (0.4 IU). Two groups were established: a urinary kallikrein group (0.1 IU / kg, intramuscular injection) and a urinary kallikrein group (0.1 IU / kg, intravenous injection). The administration groups were given 2 hours after reperfusion. The specific activity of all samples used in the animal experiment was calculated using the method shown in Example 6. Furthermore, the above-mentioned injectable pancreatic kallikrein 1 is PEG10K-modified Kb, which was prepared by the manufacturing method described in Example 5. Kb is KLK1 extracted from the pancreas of a pig.

[0161] 2. Evaluation of neurological deficit symptoms The modified Bederson 5-point scale is used to evaluate neurological deficit symptoms.

[0162] [Table 20]

[0163] III. Measurement of Infarct Volume After the animals were anesthetized with 10% chloral hydrate, their heads were severed and the brains were removed. The olfactory bulbs, cerebellum, and lower brainstem were removed, and after washing away the bloodstains on the cerebral surface with physiological saline, the residual moisture on the surface was blotted, and the samples were left at -80°C for 7 minutes. Then, immediately after removal, downward coronal sections perpendicular to the plane of intersection of the visual lines were cut, and further sections were cut every 2 mm backward. The brain sections were placed in freshly prepared TTC (20 g / L) staining solution with physiological saline, warmed at 37°C for 90 minutes, and normal brain tissue was stained dark red, while ischemic brain tissue turned pale. After washing away with physiological saline, the brain sections were immediately arranged in order from front to back, the residual moisture on the surface was blotted, and photographs were taken. Based on the photographs, statistics were taken using image analysis software (Image Tool), and the ratio of the infarct area was calculated according to the following formula by surrounding the ischemic area (white area) on the right side and the right side area. Calculation formula: Infarct area % = total ischemic area ÷ total right side area × 100.

[0164] IV. Results 1. Influence of the test substance on neurological symptoms As shown in Figure 15 and Table 19, the Urinary Kallikrein group (F 7,87 = 20.80, P = 0.018) had a significant improvement effect on neurological symptoms compared with the model group. Also, the Pancreatic Kallikrein for Injection 1 group (F = 20.80, P = 0.021) had a significant improvement effect on neurological symptoms compared with the model group. 7,87 = 20.80, P = 0.021) had a significant improvement effect on neurological symptoms compared with the model group.

[0165] [Table 21]

[0166] 2. Influence of the test substance on the infarct area (%) of the brain As shown in Figures 16, 17, 18 and Table 20, the Urinary Kallikrein group (F 7,87 = 12.72, P = 0.042), the Pancreatic Kallikrein for Injection 1 group (F7,87 The group (=12.72, P=0.019) showed a significant improvement in cerebral infarction area compared to the model group.

[0167] [Table 22]

[0168] 3. Impact on the cumulative mortality rate (%) of the test subject. As shown in Table 21, all groups except the sham surgery group experienced animal deaths, but there was no difference in mortality rates between the groups. Urinary Kallikrein group (F 1,28 =0.18, P=0.6746), Kallikrein for injection group 1 (F 1,28 The difference between the model group and the model group (=0.18, P=0.6753) was not statistically significant.

[0169] [Table 23]

[0170] 4, Conclusion When the test substance was administered as treatment after ischemia-reperfusion, compared to the model group, the test substance Urina Both ry Kallikrein and pancreatic kallikrein 1 for injection had significant neuroprotective effects.

[0171] Example 11: In vivo activity evaluation of unmutated, highly glycosylated hKLK1 modified with PEG10K. The methods for creating the model, evaluating nerve deficiency symptoms, and measuring cerebral infarction volume are the same as in Example 10. Grouping and Medication: A total of four groups were established: a sham surgery group, a model group, a positive drug group (pancreatic kallikrein 1 for injection, 0.4 IU / kg, intramuscular injection), and a PEG10K-hKLK1 (hyperglycosylated) group (0.1 IU / kg, intravenous injection). The treatment groups received their medication 2 hours after reperfusion.

[0172] 2. Results 1. Effects on nerve deficiency symptoms in the test subjects As shown in Figure 19 and Table 22, the test substance PEG10K-hKLK1 (high glycosylation) group (F 3,43 = 63.32, P = 0.352) had a certain improvement effect on nerve deficit symptoms compared with the model group, but there was no statistically significant difference. The positive drug group (F 3,44 = 56.34, P = 0.006) had a significant improvement effect on nerve deficit symptoms compared with the model group.

[0173]

Table 24

[0174] 2. Influence of the test substance on the cerebral infarction area (%) As shown in Figures 20, 21, 22 and Table 23, the PEG10K-hKLK1 (high glycosylation) group (F 3,43 = 37.72, P = 0.197) had a certain improvement effect on the cerebral infarction area compared with the model group, but there was no significant difference. The positive drug group (F 3,44 = 34.58, P = 0.007) had a significant improvement effect on the cerebral infarction area compared with the model group.

[0175]

Table 25

[0176] 3. Influence of the test substance on the cumulative mortality rate (%) As shown in Table 24, all groups except the sham operation group had animal deaths, but there was no difference between groups in the mortality rate. The positive drug group (F 1,6 = 0.00, P = 1.0000), the PEG10K-hKLK1 (high glycosylation) group (F 1,6 = 0.00, P = 1.0000) had no statistically significant difference compared with the model group.

[0177]

Table 26

[0178] 4, Conclusion When the test substance was administered as treatment after ischemia-reperfusion, it tended to improve symptoms compared to the model group, but the difference was not statistically significant.

[0179] Example 12: In vivo activity evaluation of unmutated, low-glycosylated hKLK1 modified with different PEGs. 1. The methods for model preparation, evaluation of nerve deficiency symptoms, and measurement of cerebral infarction volume are the same as in Example 10. Grouping and Medication: A total of seven groups were established: a sham surgery group, a model group, a positive drug group (pancreatic kallikrein 1 for injection, 0.4 IU / kg, intramuscular injection), a PEG40K-hKLK1 (hypoglycosylated) group (0.1 IU / kg), a PEG30K-hKLK1 (hypoglycosylated) group (0.1 IU / kg), a PEG10K-hKLK1 (hypoglycosylated) group (0.1 IU / kg), and a PEG5K-hKLK1 (hypoglycosylated) group (0.1 IU / kg). The treatment groups received their medications 2 hours after reperfusion. The dosage for each treatment group was calculated based on the specific activity of the sample, and the same amount of PEG-hKLK1 modified compound with the same active units was administered per kilogram of body weight.

[0180] 2. Results 1. Effects on nerve deficiency symptoms in the test subjects As shown in Figure 23 and Table 25, the PEG40K-hKLK1 (low glycosylation) group (F 3,44 =46.28, P=0.209), PEG30K-hKLK1 (low glycosylation) group (F 3,43 =53.33, P=0.242), PEG5K-hKLK1 (low glycosylation) group (F 3,44 The PEG10K-hKLK1 (low glycosylation) group (F) showed some improvement in neuronal deficiency symptoms compared to the model group, but the difference was not statistically significant. 3,45 =46.43, P=0.008), positive drug group (F 3,45 The group (=46.43, P=0.003) showed a significant improvement in neuronal deficiency symptoms compared to the model group.

[0181] [Table 27]

[0182] 2. Effect on the cerebral infarction area (%) of the test subject. As shown in Figures 24, 25, 26 and Table 26, the PEG40K-hKLK1 (low glycosylation) group (F 3,44 =37.73, P=0.446), PEG30K-hKLK1 (low glycosylation) group (F 3,43 =36.56, P=0.502), PEG5K-hKLK1 (low glycosylation) group (F 3,44 The PEG10K-hKLK1 (low glycosylation) group (F) showed some improvement in stroke area compared to the model group, but the difference was not statistically significant. 3,45 =39.30, P=0.001), positive drug group (F 3,45 The group with a score of 39.30 (P=0.001) showed a significant improvement in cerebral infarction area compared to the model group.

[0183] [Table 28]

[0184] 3. Impact on the cumulative mortality rate (%) of the test subject. As shown in Table 27, all groups except the sham surgery group experienced animal deaths, but there was no significant difference in mortality rates between the groups. Positive drug group (F 1,3 =0.15, P=0.7239), PEG40K-hKLK1 (low glycosylation) group (F 1,3 =0.17, P=0.7100), PEG30K-hKLK1 (low glycosylation) group (F 1,3 =0.17, P=0.7106), PEG10K-hKLK1 (low glycosylation) group (F 1,3 =0.15, P=0.7239), PEG5K-hKLK1 (low glycosylation) group (F 1,3 The result (=0.00, P=0.9994) showed no statistically significant difference compared to the model group.

[0185] [Table 29]

[0186] 4, Conclusion According to the results of this embodiment, among the unmutated low-glycosylated hKLK1 strains, PEG10K-hKLK1 (low-glycosylated) showed a significant neuroprotective effect in an in vivo animal model, and PEG5K-hKLK1 (low-glycosylated), PEG40K-hKLK1 (low-glycosylated), and PEG30K-hKLK1 (low-glycosylated) also showed a tendency toward symptom improvement. Furthermore, the results of drug efficacy experiments conducted in vivo and in vitro showed a certain correlation, and in vitro activity of PEG10K-modified low-glycosylated hKLK1 was higher than that of PEG5K-modified low-glycosylated hKLK1 and higher than that of PEG30K / 40K-modified low-glycosylated hKLK1.

[0187] In summary, the results of Examples 11 and 12 show that, when evaluated at the same dosage, the efficacy of the PEG10K-hKLK1 (highly glycosylated) sample is inferior to that of the PEG10K-hKLK1 (lowly glycosylated) sample. Therefore, it is considered that, when the same PEG modification is applied, the unmutated low-glycosylated sample exhibits superior efficacy compared to the highly glycosylated sample.

[0188] Example 13: In vivo activity evaluation of hKLK1 mutants modified with different PEGs. 1. The methods for model preparation, evaluation of nerve deficiency symptoms, and measurement of cerebral infarction volume are the same as in Example 10. Grouping: A total of four groups were established: a sham surgery group, a model group, a PEG5K-hKLK1X1 group (0.1 IU / kg), and a PEG10K-hKLK1X1 group (0.1 IU / kg). The treatment groups received intravenous injection 2 hours after reperfusion.

[0189] 2. Results 1. Effects on nerve deficiency symptoms in the test subjects As shown in Figure 27 and Table 28, the PEG5K-hKLK1X1 group (F 2,38= 67.13, P = 0.017) showed a significant improvement effect on neurological deficit symptoms compared with the model group. The PEG10K-hKLK1X1 group (F 2,37 = 86.65, P = 0.003) showed a very significant improvement effect on neurological deficit symptoms compared with the model group.

[0190]

Table 30

[0191] 2. Influence of the test substance on the infarct area (%) of the brain As shown in Figures 28, 29, 30 and Table 29, the PEG5K-hKLK1X1 group (F 2,38 = 39.50, P = 0.001), the PEG10K-hKLK1 group (F 2,37 = 90.5, P = 0.000) showed a significant improvement effect on the infarct area compared with the model group.

[0192]

Table 31

[0193] 3. Influence of the test substance on the cumulative mortality rate (%) As shown in Table 30, all groups except the sham operation group had animal deaths, but there was no difference between groups in the mortality rate. The PEG5K-hKLK1X1 group (F 1,5 = 0.71, P = 0.4369), the PEG10K-hKLK1X1 group (F1,5 = 0.00, P = 1.0000) showed no statistical difference compared with the model group.

[0194]

Table 32

[0195] 4. Conclusion When the test substances were administered as treatment after ischemia-reperfusion, both PEG5K-hKLK1X1 and PEG10K-hKLK1X1 showed significant neuroprotective effects compared to the model group. The in vivo efficacy of PEG-hKLK1 (unmutated) / hKLK1X (mutant) from Examples 12 and 13 is summarized in Table 31.

[0196] [Table 33]

[0197] In other words, when modified with the same PEG modifier, the hKLK1 protein with a mutation at the N141 site exhibits significantly superior pharmacological efficacy compared to the pre-mutation protein. The mutated protein shows improved performance, resulting in better in vivo therapeutic effects.

[0198] Table 32 summarizes the drugs that showed in vivo efficacy in Examples 10-13.

[0199] [Table 34]

[0200] In other words, when modified with the same modifier, unmutated low-glycosylated hKLK1 exhibits superior efficacy compared to unmutated high-glycosylated hKLK1. Furthermore, hKLK1 proteins mutated by NFS exhibit superior efficacy compared to unmutated low-glycosylated hKLK1, demonstrating significantly superior efficacy even at low doses.

[0201] Example 14: Efficacy study of PEG-hKLK1 under different administration routes 1. The methods for model preparation, evaluation of nerve deficiency symptoms, and measurement of cerebral infarction volume are the same as in Example 10. Grouping: A total of three groups were established: a sham surgery group, a model group, and a PEG10K-hKLK1X1 intramuscular injection group (0.4 IU / kg). The treatment group received the drug 2 hours after reperfusion.

[0202] 2. Results 1. Effects on nerve deficiency symptoms in the test subjects As shown in Figure 31 and Table 33, the PEG10K-hKLK1X1 intramuscular injection group (F 2,36 The group (=122.84, P=0.000) showed a very significant improvement in neuronal deficiency symptoms compared to the model group.

[0203] [Table 35]

[0204] 2. Effects of the test substance on the area of ​​cerebral infarction (%) As shown in Figure 32 and Table 34, the PEG10K-hKLK1X1 intramuscular injection group (F 2,36 The group (=55.02, P=0.001) showed a very significant improvement in stroke area compared to the model group.

[0205] [Table 36]

[0206] 4, Conclusion Overall, Examples 13 and 14 show that PEG10K-hKLK1X1 exhibits very significant neuroprotective effects when administered via both intravenous injection (0.1 IU / kg) and intramuscular injection (0.4 IU / kg). Furthermore, it was confirmed that it effectively treats post-stroke neurological deficits in animals and significantly improves the area of ​​cerebral infarction.

[0207] In summary, the low-glycosylated KLK1 of the present invention exhibits higher activity compared to the high-glycosylated KLK1. Compared to unmutated KLK1, the recombinant KLK1 mutant has one fewer N-glycosylation modification site, simplifying the purification process, improving product uniformity, facilitating quality control, and increasing production volume. Furthermore, the KLK1 mutant of the present invention has further advantages in terms of enzymatic properties and activity compared to unmutated low-glycosylated KLK1. Moreover, the polyethylene glycolated recombinant hKLK1 of the present invention showed remarkable efficacy when administered by various methods. It also possesses the advantages of safety and long-acting properties of polyethylene glycol drugs, enabling reduced administration frequency and improved patient compliance. This allows for the application of the drug of the present invention throughout the entire disease process, covering stages such as prevention, treatment, recovery of prognosis, and prevention of recurrence of diseases including acute ischemic stroke, peripheral neuropathy, retinal diseases, fundus diseases, hypertension, diabetic nephropathy, IgA nephritis, and chronic kidney disease.

Claims

1. A recombinant kallikrein 1 mutant or derivative thereof, wherein the kallikrein 1 is primate animal kallikrein 1, has N-glycosylation modifications at only two sites, retains the N-glycosylation modifications at NMS and NHT in the natural kallikrein 1 amino acid sequence, and, as a result of the mutation, does not contain the N-glycosylation modification at NFS of natural kallikrein 1, wherein the derivative is an albumin fusion protein or an Fc fusion protein.

2. A recombinant kallikrein 1 mutant or derivative according to claim 1, characterized in that the asparagine in the NFS of the amino acid sequence of kallikrein 1 is mutated to any amino acid other than asparagine, and zero, one, or two F, S amino acids in the NFS are mutated to any other amino acid.

3. A recombinant kallikrein 1 mutant or derivative according to claim 1, characterized in that phenylalanine in the NFS of the amino acid sequence of kallikrein 1 is mutated to proline, and zero, one, or two N and S amino acids in the NFS are mutated to any other amino acid.

4. A recombinant kallikrein 1 mutant or derivative according to claim 1, characterized in that the serine of the NFS in the amino acid sequence of kallikrein 1 is mutated to any amino acid other than serine or threonine, and zero, one, or two N and F amino acids of the NFS are mutated to any other amino acid.

5. A recombinant kallikrein 1 mutant or derivative according to claim 2, characterized in that the mutant has been mutated in which the asparagine of NFS in the amino acid sequence of kallikrein 1 is changed to a polar neutral amino acid, an acidic amino acid, a basic amino acid, or an aliphatic amino acid.

6. The recombinant kallikrein 1 mutant or derivative thereof according to claim 5, wherein the protrusion The natural mutant is a recombinant kallikrein 1 mutant or a derivative thereof, characterized in that the asparagine in the NFS of the kallikrein 1 amino acid sequence is mutated to glutamine (Gln), aspartic acid (Asp), arginine (Arg), or alanine (Ala).

7. A recombinant kallikrein 1 mutant or derivative according to claim 1, wherein kallikrein 1 is human kallikrein 1, asparagine, phenylalanine, and serine of NFS are the amino acids at positions 141, 142, and 143 of human kallikrein 1, respectively, and the amino acid sequence of natural human kallikrein 1 is the sequence of SEQ ID No. 1, the sequence in which Q at position 121 is replaced with E, the sequence in which A at position 164 is replaced with V, the sequence in which K at position 162 is replaced with E, or the sequence in which D at position 90 is replaced with N, V ​​at position 115 is replaced with F, and K at position 162 is replaced with E.

8. A recombinant kallikrein 1 mutant or derivative thereof according to claim 1, wherein the amino acid sequence of the mutant is SEQ ID No: 3, SEQ ID No: 4, SEQ A recombinant kallikrein 1 mutant or its derivative, characterized by having the sequence shown in ID No: 5 or SEQ ID No:

6.

9. A composition comprising a recombinant kallikrein 1 mutant or a derivative thereof according to any one of claims 1 to 8.

10. The composition according to claim 9 for the treatment, prevention, improvement of prognosis, or prevention of recurrence of a disease selected from the group consisting of acute ischemic stroke, peripheral neuropathy, retinopathy, fundus disease, hypertension, diabetic nephropathy, IgA nephritis, and chronic kidney disease.

11. Polyethylene glycolated kallikrein 1, characterized in that the kallikrein 1 is a recombinant kallikrein 1 mutant according to any one of claims 1 to 8, wherein the kallikrein 1 is modified with a polyethylene glycol modifier.

12. Polyethylene glycolated kallikrein 1 according to claim 11, wherein the polyethylene glycol modifier is a linear polyethylene glycol succinimide propionate having a molecular weight of 5 kDa to 10 kDa, and its general structural formula is as shown in formula (1), wherein n is an integer from 105 to 225. 【Chemistry 1】 （１）

13. Polyethylene glycolated kallikrein 1 according to claim 11, wherein the polyethylene glycol modifier is a branched polyethylene glycol propionaldehyde having a molecular weight of 30 kDa to 40 kDa, the general structural formula is as shown in formula (2), and in formula (2), n is an integer from 335 to 455. 【Chemistry 2】 （２）

14. A composition comprising polyethylene glycolated kallikrein 1 as described in claim 11.

15. The composition according to claim 14 for the treatment, prevention, improvement of prognosis, or prevention of recurrence of a disease selected from the group consisting of acute ischemic stroke, peripheral neuropathy, retinopathy, fundus disease, hypertension, diabetic nephropathy, IgA nephritis, and chronic kidney disease.

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