UNDERGLYCOSYLATED KALLIKREIN I, AND POLYETHYLENE GLYCOL MODIFIER THEREOF AND PHARMACEUTICAL USE THEREOF

Abstract

Provided are low glycosylated kallikrein I with no or a small amount of glycosylation at the NFS sequence and polyethylene glycol modified product and pharmaceutical applications. KLK1 with lower glycosylation at NFS is more active than KLK1 with higher glycosylation at NFS. A recombinant KLK1 mutant without N-glycosylation at the NFS sequence is also provided, containing only two N-glycosylation sites at NMS and NHT. Glycosylation of the recombinant KLK1 mutant is relatively more consistent, the molecular weight of the product is relatively more homogeneous, the yield is higher, the purification process is simpler, and the biological activity is higher, and the quality is more controllable.

Claims

1. Low glycosylated kallikrein I or its derivative, which is primate kallikrein I containing three N-glycosylation sites of native kallikrein I at NMS, NHT and NFS, of which asparagine at NFS has no glycosylation or a small amount of glycosylation, low glycosylated or small amount of glycosylation means that the proportion of glycosylated asparagine at NFS is 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%.

2. Recombinant kallikrein I mutant or its derivative, which is primate kallikrein I with only two N-glycosylation sites.

3. The recombinant kallikrein I mutant or its derivative of claim 2, which retains the N-glycosylation at NMS and NHT of the native kallikrein I and does not contain the N-glycosylation at NFS of the native kallikrein I

4. The recombinant kalinolysin I mutant or its derivative of claim 2, wherein the asparagine at NFS of the kalinolysin I is mutated to any other amino acid except asparagine, and 0, 1 or 2 amino acids of F and S at NFS are mutated to any other amino acid.

5. The recombinant kallikrein I mutant or its derivative of claim 2, wherein the phenylalanine at NFS of the kallikrein I is mutated to proline, and 0, 1, or 2 amino acids of N and S at NFS are mutated to any other amino acid.

6. The recombinant kalinolysin I mutant or its derivative of claim 2, wherein the serine at NFS of the kalinolysin I is mutated to any other amino acid except serine and threonine, and 0, 1 or 2 amino acids of N and F at NFS are mutated to any other amino acid.

7. The recombinant kallikrein I mutant or its derivative of claim 4, wherein the asparagine at the NFS of the kallikrein I is mutated to neutral polar amino acid, acidic amino acid, basic amino acid or aliphatic amino acid.

8. The recombinant kallikrein I mutant or its derivative of claim 7, wherein the asparagine at the NFS of the kallikrein I is mutated to glutamine (Gln), aspartic acid (Asp), arginine (Arg) or alanine (Ala).

9. The recombinant kallikrein I mutant or its derivative of any one of claims 2 to 8, wherein said kallikrein I is human kallikrein I, the asparagine, phenylalanine and serine at NFS are the 141st, 142nd, and 142rd amino acid of human kallikrein I respectively; the amino acid sequences of native human kallikrein I are shown in Genbank accession numbers AAA59455.1, NP002248.1, AAA36136.1, AAP35917, or AAU12569.

10. The recombinant kallikrein I mutant or its derivative of any one of claims 2 to 8, amino acid sequence of said mutant is shown as SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5 or SEQ ID No: 6.

11. A composition containing kallikrein I or its derivative of claim 1, or recombinant kallikrein I mutant or its derivative of any one of claims 2-10.

12. Application of Kallikrein I or its derivative of claim 1, or recombinant kallikrein I mutants or its derivative of any one of claims 2-10 in the preparation of drugs for the treatment, prevention, recovery, and prevention of recurrence of acute ischemic stroke, peripheral neuropathy, retinopathy, fundus disease, hypertension, diabetic nephropathy, IgA nephritis, and chronic kidney disease.

13. Pegylated kallikrein I, said kallikrein I is modified by polyethylene glycol modifier, kallikrein I is the kallikrein I of claim 1 or recombinant kallikrein I mutant of any one of claims 2-10.

14. The pegylated kallikrein I of claim 13, the PEG modifier is straight chain PEG succinimidyl propionate with molecular weight of 5 kDa-10 kDa, the general formula is as shown in (1), ##STR00007## where n is an integer from 105 to 225.

15. The pegylated kallikretin I of claim 13, the pegylated modifier is branched polyethylene glycol propionaldehyde with molecular weight of 30 kDa-40 kDa, the general formula is shown in (2), ##STR00008## where n is an integer from 335 to 455.

16. A composition containing pegylated kallikrein I of any one of claims 13 to 15.

17. Application of pegylated kallikrein I of any one of claims 13-15 in the preparation of drugs for the treatment, prevention, recovery, and prevention of recurrence of acute ischemic stroke, peripheral neuropathy, retinopathy, fundus disease, hypertension, diabetic nephropathy, IgA nephritis, and chronic kidney disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG. 1: Molecular design of recombinant hKLK1 and mutants

[0044] FIG. 2: Schematic diagram of the pZHK2.0 expression vector

[0045] FIG. 3: Hydrophobic chromatogram of the fermentation broth supernatant of CHO cells expressed wild-type recombinant hKLK1. The arrow indicates the isolated high glycosylated product (left) and low glycosylated product (right).

[0046] FIG. 4: SDS-PAGE electrophoretograms of high and low glycosylated proteins of CHO cells expressed wild-type recombinant hKLK1 after purification. Lane M represents the protein marker, lane 1 represents the high glycosylated hKLK1, and lane 2 represents the low glycosylated hKLK1.

[0047] FIG. 5: Hydrophobic chromatogram of the fermentation brothsupernatant of CHO cells expressed hKLK1X1. The arrow indicates isolated single low glycosylated product.

[0048] FIG. 6: SDS-PAGE electrophoretogram of low glycosylated recombinant hKLK1X1 expressed in CHO cells after purification. Lane M represents the protein marker and lane 1 represents the low glycosylated hKLK1 mutant.

[0049] FIG. 7: SDS-PAGE electrophoretogram of CHO cells expressed wild-type recombinant hKLK1 containing unseparated high and low glycosylated protein. Lane M represents the protein marker, and lane 1 represents the mixture of high and low glycosylated wild-type recombinant hKLK1.

[0050] FIG. 8: Sequence Coverage map of the high glycosylated hKLK1 sample after trypsin digestion

[0051] FIG. 9: Sequence Coverage map of the low glycosylated hKLK1 sample after trypsin digestion

[0052] FIG. 10: The TIC spectrum of three batches of high glycosylated hKLK1 after trypsin digestion

[0053] FIG. 11: The TIC spectrum of three batches of low glycosylated hKLK1 after trypsin digestion

[0054] FIG. 12: Sequence Coverage map of hKLK1X1 sample after trypsin digestion

[0055] FIG. 13: Anti-drug antibody detection in animals given PEG-hKLK1(low glycosylated)

[0056] FIG. 14: Anti-drug antibody detection in animals given PEG-hKLK1(highly glycosylated)

[0057] FIG. 15: Effect of the test article on neurological defect symptoms in example 10

[0058] FIG. 16: Effect of the test article on cerebral infarction area in example 10

[0059] FIG. 17: Brain slice map of the model group and the sham operation group in example 10

[0060] FIG. 18: Brain slice map of urinary kallikrein group and pancreatic kallikrein I group in example 10

[0061] FIG. 19: Effect of the test article on neurological defect symptoms in example 11

[0062] FIG. 20: Effect of the test article on cerebral infarction area in example 11

[0063] FIG. 21: Brain slice map of the model group and the sham operation group in example 11

[0064] FIG. 22: Brain slice map of the drug administration group in example 11

[0065] FIG. 23: Effect of the test article on neurological defect symptoms in example 12

[0066] FIG. 24: Effect of the test article on cerebral infarction area in example 12

[0067] FIG. 25: Brain slice map of model group, sham operation group and positive drug group in example 12

[0068] FIG. 26: Brain slice map of different PEG modified hKLK1 in example 12

[0069] FIG. 27: Effect of the test article on neurological defect symptoms in example 13

[0070] FIG. 28: Effect of the test article on cerebral infarction area in example 13

[0071] FIG. 29: Brain slice map of the model group and the sham operation group in example 13

[0072] FIG. 30: Brain slice map of different PEG modified hKLK1 mutants in Example 13

[0073] FIG. 31: Effect of the test article on neurological defect symptoms in example 14

[0074] FIG. 32: Effect of the test article on cerebral infarction area in example 14

[0075] FIG. 33: Brain slice map of the model group and the shame operation group in example 14

[0076] FIG. 34: Brain slice map of drug administration group in example 14

DETAILED DESCRIPTION OF THE INVENTION

[0077] Unless otherwise specified, the technical terms or abbreviations of this application have the following meanings:

[0078] Kallikrein I: Specifically tissue kallikrein I, KLK1.

[0079] hKLK1: unmutated human tissue kallikrein I with sequence homology to native human tissue kallikrein I; It covers various homologs of human KLK1, including but not limited to KLK1 as shown in Genbank accession numbers AAA59455.1, NP002248.1, AAA36136.1, AAP35917, AAU12569, etc.

[0080] Three glycosylation sites N78, N84 and N141 of KLK1: asparagine at position 78, 84 and 141 in KLK1 amino acid sequence, respectively. The corresponding N-glycosylation motifs are NMS, NHT, and NFS, with N for asparagine, M for methionine, S for serine, H for histidine, T for threonine, and F for phenylalanine.

[0081] High glycosylated hKLK1: unmutated hKLK1 with high glycosylation, that is, there are three glycosylation sites N78, N84 and N141 in high glycosylated hKLK1, each N-glycosylation are glycosylated.

[0082] Low glycosylated hKLK1: unmutated hKLK1 with low glycosylation, that is, there are three glycosylation sites N78, N84 and N141 in low glycosylated hKLK1, but no glycosylation, or only a small amount of glycosylation at N141.

[0083] PEG-hKLK1(high glycosylated): polyethylene glycol modified high glycosylated hKLK1, said high glycosylated hKLK1 refers to the above unmutated hKLK1 with high glycosylation.

[0084] PEG-hKLK1(low glycosylated): polyethylene glycol modified low glycosylated hKLK1, said low glycosylated hKLK1 refers to the above unmutated hKLK1 with low glycosylation.

[0085] hKLK1X: hKLK1 mutant. hKLK1X1, hKLK1X2, hKLK1X3, and hKLK1X4 represent different mutants.

[0086] PEG-hKLK1X: Polyethylene glycol modified hKLK1 mutant.

[0087] KLK1 derivatives: It includes not only full-length protein of the KLK1 mutant described in this application, but also partial protein of the KLK1 mutant described in this application and the protein obtained by further mutation, fusion protein (including but not limited to albumin fusion, Fc fusion, etc.) and various forms of modification (except pegylated modification) on the basis of the KLK1 mutant described in this application.

[0088] Polyethylene glycol: PEG, usually formed by the polymerization of ethylene oxide, has branched, linear, and multi-arm forms. In general, those with molecular weight below 20,000 are referred to as PEG and those with larger molecular weight are referred to as PEO. Ordinary polyethylene glycol has one hydroxyl group at each end. If one end is blocked with methyl group, methoxy polyethylene glycol (mPEG) is obtained.

[0089] Polyethylene glycol modifiers: PEG modifiers refer to PEG derivatives with functional groups, which are activated polyethylene glycol and can be used for protein and peptide drug modification. The polyethylene glycol modifier used in this application was purchased from ZonHon Biopharma Institute, INC. or Beijing Jenkem Technology Co., LTD. The actual molecular weight of PEG modifier can be 90%-110% of the labeled value. For example, molecular weight of PEG5K may be 4.5-5.5 kDa.

[0090] PEG5K used specifically refers to M-SPA-5K, straight chain monomethoxy polyethylene glycol succinimidyl propionate with molecular weight of approximately 5 kDa, with structure as shown in formula (1), where n is an integer from 105 to 110,

##STR00005##

[0091] PEG10K used specifically refers to M-SPA-10K, straight chain monomethoxy polyethylene glycol succinimidyl propionate with molecular weight of about 10 kDa, with structure as shown in formula (1), where n is an integer from 220 to 225.

[0092] PEG30K used specifically refers to Y-PALD-30K, which is branched polyethylene glycol propionaldehyde with molecular weight of about 30 kD, with structure as shown in formula (2), where m is an integer from 335 to 340,

##STR00006##

[0093] PEG40K used specifically refers to Y-PALD-40K, branched polyethylene glycol propionaldehyde with molecular weight of about 40 kD, with structure as shown in formula (2), where m is an integer of 450 to 455.

Example 1 Gene Design, Expression Vector Construction, Expression, Purification of Recombinant Human Kallikrein I(hKLK1)

(1). Gene Design and Expression Vector Construction

[0094] According to the available hKLK1 sequence in GenBank (GenBank:AAA59455.1), the mature amino acid sequence were determined (SEQ ID No: 1), the hKLK1 cDNA sequence(SEQ ID No: 2) encoding signal peptide and propeptide was obtained after codon optimization based on CHO system. AvrII sequence was added before the recombinant hKLK1 gene (SEQ ID No: 2), BstZ17I sequence was added after the sequence. The artificial sequence was synthesized and constructed into pUC57 plasmid to obtain long-term preservation plasmid, which was denoted as puc57-hKLK1 plasmid. Primers were used to amplify the hKLK1 gene from the pUC57-hKLK1 plasmid. The PCR products were purified after 1% agarose electrophoresis, and

[0095] The target gene PCR product(FIG. 1) and pZHK2.0 vector (FIG. 2) were both digested with AvrII+BstZ17I and purified. The target gene was ligated into the pZHK2.0 vector by T4 ligase. They were transformed into Top10 competent cells and plated on LB plates containing kanamycin resistance for overnight culture at 37 C. The next day, positive clones were screened and sequenced, which were completely consistent with the expected sequence. The expression vector pZHK2.0-hKLK1 was formed.

(2) Stable Expression

[0096] The constructed plasmid was linearized by overnight digestion with NruI(R0192S, NEB) and electrotransfected into CHOS cells. Stable cell lines were obtained by pressure selection. The CHO cell line which can stablely and highly express recombinant hKLK1 was inoculated in Dynamis medium (A2617501, Thermo Fisher) at 37 C., 8% CO.sub.2, 130 rpm, fed-flow was added to the culture in batches and the culture medium was harvested after 2 weeks of culture.

(III) Purification

1. Pretreatment of Culture Medium

[0097] The above culture medium containing recombinant hKLK1 was collected, centrifuged at 6000 rpm for 15 min to remove cells, concentrated by ultrafiltration, and filtered through a 0.22 m membrane to remove cell debris. Then add 1.5M (NH.sub.4).sub.2SO.sub.4, and the mixture was activated by stirring at room temperature for 3 days. The activated culture medium was filterated through 0.45 m microfiltration membrane.

2. Hydrophobic Chromatography

[0098] The column was first equilibrated with buffer (20 mM Tris-HCl, 1.5M ammonium sulfate, pH=8.0) until baseline equilibrium. The pretreated supernatant was then run through POROS Benzyl medium (A32563, purchased from Thermobody) to capture recombinant hKLK1 from the medium. Then gradient elution was performed by the eluent(20 mM Tris-HCl, pH=8.0), and two obvious peaks through hydrophobic separation were observed. Each elution peak was collected. The recombinant hKLK1 proteins with inconsistent glycosylation were separated, and the chromatogram was shown in FIG. 3.

3. Anion Exchange

[0099] The elution peak containing recombinant hKLK1 protein collected in step 2 was ultrafiltration with a 10 kDa ultrafiltration membrane to a conductance of 10-15 ms/cm with 20 mM Tris-HCl, pH=8.0 phosphate buffer. The column was equilibrated with 20 mM Tris-HCl, 100 mM NaCl, pH=8.0 buffer until baseline equilibrium. The ultrafiltrated supernatant was then passed through Q FF medium (17515601, GE Company). Finally, the column was eluted equally with 20 mM Tris-HCl, 1M NaCl, pH=8.0 elution buffer, and the main elution peak was collected.

4. Cation Exchange

[0100] Sample of step 3 was ultrafiltrated and changed to 10 mM NaAcHAc, 50 mM NaCl, pH=3.5-3.7. Recombinant hKLK1 with different molecular weights collected in the previous step was purified with SP FF medium (17072904, purchased from GE). Equilibration buffer was 10 mM NaAcHAc, 50 mM NaCl, pH=3.7, and elution buffer was 50 mM Tris-HCl, 1M NaCl, pH 8.0.

[0101] The purity of the purified samples was analyzed by SDS-PAGE gel electrophoresis. The SDS-PAGE electrophoretogram showed that the molecular weight of the high glycosylated hKLK1 was slightly higher than that of the low glycosylated hKLK1, as shown in FIG. 4.

Example 2 Production and Quality Characterization of Three Continuous Batches of Recombinant Human Kallikrein I(hKLK1)

[0102] Wild-type recombinant hKLK1 was cultured in three consecutive batches at high density in a 7 L stirred reactor. The initial culture methods were as follows: 100 rpm, 40% dissolved oxygen, 37 C., pH7.0, Dynamis AGT Medium(purchased from Thermo Fisher Scientific) was used as the basal medium, and the seed suspension was inoculated into the bioreactor at a density of 510.sup.5 cells/mL. On the third day, the temperature was adjusted to 33 C., and the feed medium (EfficientFeed C+AGT supplement, purchased from Thermo Fisher Scientific) and glucose were respectively supplemented to maintain the sugar content in the culture medium not less than 2 g/L. When the cell viability was less than 90%, the culture medium was collected and purified using the purification process described in Example 1 to obtain three batches of recombinant hKLK1 with high and low glycosylation, respectively.

[0103] These samples were characterized, including yield, SEC-HPLC purity, amino acid sequence analysis of N/C terminus (Thermo Q Exactive), peptide mapping, glycosylation sites and glycosylation modification by LC/MS, and the results were summarized as follows.

TABLE-US-00002 TABLE 2 Yield SEC Amino acid sequence analysis of N/C Peptide Samples Batch (mg) purity (%) terminus mapping high glycosylated NO. 1 139.62 98.95 The N/C terminus were consistent with Peptide map hKLK1 the theoretical sequence (Table 3) characterization high glycosylated NO. 2 121.45 99.20 Parts of N/C terminus was consistent of three hKLK1 with the theoretical sequence; Parts of batches were C terminus was consistent with the inconsistent theoretical sequence, while parts of the (FIG. 10) IVG sequence of N terminus was missing (table 4) high glycosylated NO. 3 129.00 100.00 The N/C terminus were consistent with hKLK1 the theoretical sequence (Table 5) low glycosylated NO. 1 349.20 97.99 The N/C terminus were consistent with Peptide map hKLK1 the theoretical sequence (Table 6) characterization low glycosylated NO. 2 331.82 100.00 The N/C terminus were consistent with of three hKLK1 the theoretical sequence (Table 7) batches were low glycosylated NO. 3 290.10 100.00 The N/C terminus peptides were consistent consistent hKLK1 with the theoretical sequence (Table 8) (FIG. 11)

[0104] The analysis results in the above table showed that the purity of isolated high and low glycosylated recombinant hKLK1 is more than 9700. High and low glycosylated proteins had different molecular weights. Based on Trypsin digestion, the amino acid coverage of high and low glycosylated hKLK1 was detected, and the low glycosylated hKLK1 reached 100% coverage, and part of the high glycosylated hKLK1 reached 100% coverage, and N terminus of some high glycosylated hKLK1 was missing (FIG. 8, 9). N/C terminus of the low glycosylated hKLK1 was consistent with the theory (Tables 6 to 8), and peptide map characterization of the three batches was consistent (Figure. 11). However, the N-terminus of some high glycosylated protein in batch 2 of three consecutive batches was inconsistent with the theoretical sequence and some N terminus sequence was missing(Table 3-5). The peptide map of batch 2 was inconsistent with that of batch 1 and batch 3 (FIG. 10), indicating that the high glycosylated recombinant hKLK1 may have molecular stability defects, which may bring huge risks in the process of drug production. At the same time, the separation and purification process of high and low glycosylated hKLK1 was also complex, and the instability of the process also made it difficult to control the glycosylation level of purified products among different batches.

TABLE-US-00003 TABLE 3 N/C terminus analysis results of the first batch 20200701 of high glycosylated hKLK1 Mono Avg. Delta RT Charge Mass Mass Theoretical Peptide Sequence Modification Site (ppm) (min) M/Z State Exp. Exp. Mass MS Area N terminus: Amino (C7, C26) 3.88 51.82 780.972 5 3897.8408 3897.8257 2.48E+07 methylation C terminus: WIEDTIAENS None \ 2.26 26.06 1177.535 1 1176.5272 1177.11 1176.5299 3.74E+07 indicates data missing or illegible when filed

TABLE-US-00004 TABLE 4 N/C terminus analysis results of the second batch 20200703 of high glycosylated hKLK1 (IVG sequence of N-terminus missing) Mono Avg. Delta RT Charge Mass Mass Theoretical Peptide Sequence Modification Site (ppm) (min) M/Z State Exp. Exp. Mass MS Area N terminus: Amino (C7, C26) 3.51 51.7 780.972 5 3897.8394 3900.09 3897.8257 1.07E+08 methylation N terminus: Amino (C4, C23) 8.32 49.92 908.67 4 3628.682 3630.79 3628.652 9.17E+07 methylation C terminus: WIEDTIAENS None \ 2.88 26.02 1177.534 1 1176.5263 1177.11 1176.5299 7.33E+07 indicates data missing or illegible when filed

TABLE-US-00005 TABLE 5 N/C terminus analysis results of the third batch 20200705 of high glycosylated hKLK1 Mono Avg. Delta RT Charge Mass Mass Theoretical Peptide Sequence Modification Site (ppm) (min) M/Z State Exp. Exp. Mass MS Area N terminus: Amino (C7, C26) 3.44 51.66 780.972 5 3897.8391 3900.09 3897.8257 1.53E+08 methylation C terminus. WIEDTIAENS None \ 1.54 26.03 1177.533 1 1176.5281 1177.11 1176.5299 5.45E+07 indicates data missing or illegible when filed

TABLE-US-00006 TABLE 6 N/C terminus analysis results of the first batch 20200702 of low glycosylated hKLK1 Mono Avg. Delta RT Charge Mass Mass Theoretical Peptide Sequence Modification Site (ppm) (min) M/Z State Exp. Exp. Mass MS Area N terminus: Amino (C7, C26) 5.01 51.57 780.972 5 3897.8452 3900.09 3897.8257 2.47E+08 methylation C terminus: WIEDTIAENS None \ 0.81 25.98 1177.536 1 1176.5289 1177.11 1176.5299 6.71E+07 indicates data missing or illegible when filed

TABLE-US-00007 TABLE 7 N/C terminus analysis results of the second batch 20200704 of low glycosylated hKLK1 Mono Avg. Delta RT Charge Mass Mass Theoretical Peptide Sequence Modification Site (ppm) (min) M/Z State Exp. Exp. Mass MS Area N terminus: Amino (C7, C26) 4.45 51.6 780.973 5 3897.843 3900.1 3897.8257 2.34E+08 methylation C terminus: WIEDTIAENS None \ 26 1177.536 1 1176.5287 1177.11 1176.5299 7.34E+07 indicates data missing or illegible when filed

TABLE-US-00008 TABLE 8 N/C terminus analysis results of the third batch 20200706 of low glycosylated hKLK1 Mono Avg. Delta RT Charge Mass Mass Theoretical Peptide Sequence Modification Site (ppm) (min) M/Z State Exp. Exp. Mass MS Area N terminus: Amino (C7, C26) 4.26 51.7 780.972 5 3897.8423 3900.09 3897.8257 1.40E+08 methylation C terminus: WIEDTIANS None \ 0.71 26.04 1177.536 1 1176.5291 1177.11 1176.5299 5.65E+07 indicates data missing or illegible when filed

TABLE-US-00009 TABLE 9 Glycopeptide characterization analysis results of N78/N84/N141 sites in low glycosylated hKLK1(20200702) Modification N-Glycan Category Sequence Confidence Abundance N78 A3 A G N-Glycan HNLFDDENTAQFVHVSESFPHPGFNM ENHT QADEDYSHDLMLLR 100 43.57% N A A G N-Glycan HNLFDDENTAQFVHVSESFPHPGFNM ENHT QADEDYSHDLMLLR 100 .43% N A A G N-Glycan HNLFDDENTAQFVHVSESFPHPGFNM ENHT QADEDYSHDLMLLR 100 100% N141 A A G N-Glycan VVELPT EPEVGST LASGWGS DDLQCVDLK 100 1. % N141 A A G N-Glycan VVELPT EPEVGST LASGWGS DDLQCVDLK 100 0.29% N141 A A G N-Glycan VVELPT EPEVGST LASGWGS DDLQCVDLK 100 0.12% N141 A A N-Glycan VVELPT EPEVGST LASGWGS DDLQCVDLK 100 0.39% N141 A A G N-Glycan VVELPT EPEVGST LASGWGS DDLQCVDLK 100 0.46% N141 N-Glycan VVELPT EPEVGST LASGWGS DDLQCVDLK 100 0.59% N141 M M N-Glycan VVELPT EPEVGST LASGWGS DDLQCVDLK 100 0.29% N141 U ylated U lated N-Glycan VVELPT EPEVGST LASGWGS DDLQCVDLK 100 9 . %.sup. indicates data missing or illegible when filed

[0105] In addition, the analysis of the glycosylation sites of recombinant hKLK1 showed that N78 and N84 in recombinant low glycosylated hKLK1 were glycosylated, N141 was almost not glycosylated (96.39%), and only a small amount of N141 in hKLK1 (less than 4%) was glycosylated (Table 9). The high glycosylated hKLK1 contains three N-glycosylation sites (N78, N84 and N141), and contains multiple types of glycosylation, and the types of glycosylation are complex.

[0106] High and low glycosylated protein obtained from the hydrophobic chromatography elution of Example 1 was combined, and the same anion and cation purification was performed to obtain recombinant hKLK1 protein with high and low glycosylation. The purity of the purified sample was analyzed by SDS-PAGE gel electrophoresis, shown in FIG. 7. Bands of high and low glycosylated protein was diffused. A larger molecular weight component was present above the high glycosylated protein. As shown in FIG. 3, the separation of unmutated hKLK1 with high and low glycosylation by hydrophobic purification was poor, and it was difficult to completely separate high and low glycosylated hKLK1. In conclusion, the unmutated hKLK1 proprotein was composed of high and low glycosylated hKLK1 with different molecular weights, and N141 contains inhomogeneous and complex glycosylation, which brought a great challenge for the quality control of recombinant hKLK1.

[0107] Further comparison of the biological activity of hKLK1 with high and low glycosylation (Example 6) revealed unexpectedly that the activity of hKLK1 with low glycosylation was much higher than that of hKLK1 with high glycosylation.

Example 3 Design, Expression Vector Construction, Expression and Purification of Recombinant Human Kallikrein I Mutant (hKLK1X)

[0108] Further, in order to obtain low glycosylated hKLK1, the applicant did not strive to optimize the expression and purification methods of recombinant hKLK1 as the conventional method, but on the basis of the research on high and low glycosylated hKLK1, the applicant mutated one or more amino acids of the NFS motif, so that the corresponding position could not be glycosylated. Thus, more homogeneous and higher yield of low glycosylated hKLK1 mutant was obtained. In surprise, the low glycosylated hKLK1 mutant had further advantages in enzymatic properties and activity compared with the unmutated low glycosylated hKLK1.

[0109] The mutant exemplified in this example mutated N in the NFS motif (i.e., N141), and F and S in the NFS motif were not mutated. However, the embodiments were provided for illustrative purposes only and not to limit the scope of this application. All the following schemes can change the original NFS sequence so that it did not constitute N-glycosylation motif, thus there was no glycosylation at N141. For example N at NFS (i.e., N141) was mutated, 0, 1 or 2 amino acids of F and S at NFS were mutated to any other amino acid. Or F(F142) at NFS was mutated to proline, and 0, 1, or 2 amino acids of N and S at NFS were mutated to any other amino acid. Or S(i.e. S143) at NFS was mutated to any amino acid except threonine, and 0, 1, or 2 amino acids of N and F at NFS were mutated to any other amino acid. The above scheme not only obtained more homogeneous and higher yield of low glycosylated hKLK1, but also found that the obtained mutants had further advantages in enzymatic properties and activity compared with the unmutated product, which may be related to the lack of glycosylation at the original NFS sequence.

[0110] Further, this embodiment exemplized mutant based on the hKLK1 sequence shown in Genbank accession number AAA59455.1, but the embodiment was for illustrative purposes only and not to limit the scope of the application. The amino acids and glycosylation sites of other known natural variants of hKLK1 were highly consistent. Mutations in the corresponding position (i.e., one or more amino acids in the NFS motif) of other hKLK1 variants can achieve the same technical effect, that was, to obtain more homogeneous and low glycosylated hKLK1 with higher yield and better activity. Other primate KLK1 contained the same three N-glycosylation sites as hKLK1, and the motif is the same, which is NMS, NHT, and NFS. The same technical effect can be achieved by mutating the corresponding position of other primate KLK1 (that was, one or more amino acids of the NFS motif). That was, more homogeneous and low glycosylated KLK1 with higher yield and better activity was obtained.

[0111] Specific mutant schemes exemplified in this embodiment were as follows: Asparagine of the glycosylation site N141 was mutated to four different types of amino acids, namely neutral polar amino acid glutamine (Gln), acidic amino acid aspartic acid (Asp), basic amino acid arginine (Arg), and aliphatic amino acid alanine (Ala). The corresponding 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), respectively. Each mutant was prepared as described in Example 1.

[0112] The recombinant hKLK1 mutant described above was purified using the three-step purification of Example 1. In the hydrophobic purification profiles of the mutants, only one main elution peak was observed in all cases. For example, the hydrophobic chromatogram of hKLK1X1 was shown in FIG. 5. This indicated that the product was more homogeneous after mutation. For unmutated recombinant hKLK1, the separation of high and low glycosylated hKLK1 was poor (FIG. 3), and it was difficult to completely separate high and low glycosylated hKLK1. Hydrophobic chromatography of the mutant recombinant hKLK1 only need to collect one major peak and the purification process was simpler.

[0113] The purity of the purified samples was analyzed by SDS-PAGE gel electrophoresis, and it was found that the molecular weight of the purified mutant samples was very close to that of the unmutated low glycosylated hKLK1, and the high glycosylated hKLK1 containing more N141 glycosylation completely disappeared (FIG. 6). The proportion of low glycosylated hKLK1 was higher, the yield was higher, and the product was more homogeneous.

[0114] The amino acid coverage detection of recombinant hKLK1X1 was completed by LC-MS, and the coverage rate was 100% with the theoretical amino acid sequence (FIG. 12), indicating that the primary sequence of recombinant hKLK1X1 was correct. The glycosylation sites were identified (Table 10). Recombinant hKLK1X1 contained only two glycosylation sites, N78 and N84, which were consistent with the goal of molecular design. Similar results were obtained for the other mutants.

TABLE-US-00010 TABLE 10 Identification of glycosylation sites in Chymotrypsin digested hKLK1X1-20210811 samples Mono Avg Peptide Delta RT Charge Mass Mass Theoretical Sequence Modification Site (ppm) (min) M/Z State Exp. Exp. Mass MS Area VHVSESFP Nonspecific N78 1.09 31.66 529.242 3 1584.7048 1585.64 1584.7031 1.15E+08 HPGFNMS deamidation ENHTROAD Nonspecific N84 6.18 14.93 654.607 3 1960.7979 1961.82 1960.7857 4.76E+07 EDYSHDLM deamidation

Example 4 Deglycosylated Mass Determination of Mutated and Unmutated Protein

[0115] The deglycosylated mass of wild-type and mutated hKLK1 were detected by LC-MS after denaturation with guanidine hydrochloride and deglycosylation by PNGaseF. The molecular weight of high glycosylated hKLK1 after deglycosylation was 26380.338 Da, which was basically consistent with the theoretical molecular weight of 26377.493 Da (Table 11). The molecular weight of low glycosylated hKLK1 after deglycosylation was 26379.346 Da, which was basically consistent with the theoretical molecular weight of 26377.493 Da (Table 11). The molecular weight of hKLK1X1 after deglycosylation was 26418.82 Da, which was basically consistent with the theoretical mass of 26418.48 Da (Table 11). The molecular weights of hKLK1X2, hKLK1X3 and hKLK1X4 were also basically consistent with the theoretical mass. These results further confirmed the correctness of the primary structure of the recombinant protein.

TABLE-US-00011 TABLE 11 Summary of deglycosylated mass Theoretical monoisotopic Measured monoisotopic Possible Sample name molecular weight (Da) molecular weight (Da) Mass(Da) Modifications high glycosylated hKLK1 26377.493 26380.338 +2.845 3 deamidation low glycosylated hKLK1 26377.493 26379.346 +1.853 2 deamidation hKLK1X1 26418.480 26418.82 +0.34 /

Example 5 Preparation, Purification and Purity Analysis of PEG Modified hKLK1/hKLK1X Samples

[0116] Different PEG modified hKLK1/hKLK1X can be prepared and purified by conventional methods, as exemplified in the following example.

1. Preparation of PEG-Modified Samples

(1) Pretreatment

[0117] The unmutated low glycosylated recombinant hKLK1 was treated with 3 kDa ultrafiltration membrane package (or other equivalent buffer displacement method) with sodium dihydrogen phosphate/disodium hydrogen phosphate buffer(pH7.0) as the replacement buffer. And concentrate to 15 mg/mL.

(2) Modification

[0118] PEG5K-hKLK1 was prepared by random modification: PEG was added to the pretreated hKLK1 protein solution according to 1:25 mass ratio of hKLK1 protein to M-SAP-5K PEG. The mixture was stirred slowly until it was evenly mixed, and the reaction was carried out for 24 hours at 4 C.

[0119] PEG10K-hKLK1 was prepared by random modification: PEG was added to the pretreated hKLK1 protein solution according to 1:20 mass ratio of hKLK1 protein to M-SAP-10K PEG, and the mixture was evenly mixed after slow stirring. The reaction was carried out for 24 hours at 4 C.

[0120] Preparation of site-modified PEG30K-hKLK1: PEG was added to the pretreated hKLK1 protein solution at 1:6 molar ratio of hKLK1 protein to Y-PALD-PEG 30K. Reducing agent was added to the mixture solution at 1:50 molar ratio of PEG to reducing agent (sodium cyanoborohydride). The mixture was slowly stirred until it was evenly mixed. The reaction was carried out for 24 hours at 4 C.

[0121] Preparation of site-modified PEG40K-hKLK1: PEG was added to the pretreated hKLK1 protein solution at 1:6 molar ratio of hKLK1 protein to Y-PALD-PEG 40K. Reducing agent was added to the mixture solution at 1:50 molar ratio of PEG to reducing agent (sodium cyanoborohydride). The mixture was slowly stirred until it was evenly mixed. The reaction was carried out for 24 hours at 4 C.

2. Purification of the Reaction Mixture

[0122] The chromatographic conditions were as follows:

[0123] GE Q Sepharose High Performance medium was used as the purification filler, and the purification mobile phase was BufferA: 50 mM Tris-HCl 9.0. BufferB: 50 mM Tris-HCl+1M NaCl 9.0.

[0124] Loading: The above PEG-hKLK1 modification mixture after reaction was diluted about 10 times by double distilled water, and then diluted about 5 times by Buffer A solution before loading and purification. At the end of loading, the chromatographic column was washed with BufferA for more than 5 column volumes.

[0125] Elution: A gradient of 0-50% BufferB was set to elute 10 column volumes, elution samples were collected step by step according to UV280 trend.

[0126] PEG modified hKLK1 mutant (PEG-hKLK1X) was prepared and purified as described above.

3. Purity Analysis of PEG Modified Samples

(1) HPLC Purity Analysis

[0127] The HPLC detection was carried out according to the General rule 0512 of Chinese Pharmacopoeia, 2020 edition. The chromatographic type was SEC(Size exclusion chromatography), the mobile phase was 20 mM PB 7.0 containing 5% isopropanol, the chromatographic column was BEH450SEC 3.5 m, and the acquisition condition was 280 nm. The acquisition time ranged from 20 to 25 minutes.

[0128] The results showed that the purity of the prepared PEG-hKLK1/hKLK1X series proteins was 95%.

(2) Purity Analysis by SDS-PAGE

[0129] The purity of the samples was determined by SDS-polyacrylamide gel electrophoresis method, the fifth method of electrophoresis method in Chinese Pharmacopoeia (2020 edition), and 12.5% SDS-PAGE was used to detect the samples.

[0130] Electrophoresis results showed that the bands of PEG-hKLK1/hKLK1X series proteins were homogeneous, no impurity bands were observed, and the purity was good.

(3) PEG Binding Number Detection

[0131] a) Solution preparation: 1, 2, 4, 6, 8 mg/ml PEG standard solution, 1 mg/ml recombinant hKLK1 and corresponding PEG modified sample solution was prepared. [0132] b) Detection method: [0133] Column: XBridge BEH SEC 3.5 m 450A; Column temperature: 25 C. [0134] Mobile phase: 20 mM PB 7.0+10% IPA, at a flow rate of 0.4 ml/min. [0135] Detector: PDA detector, detection wavelength 280 nm; RI detector, detection wavelength 280 nm. [0136] c) Data analysis:

[0137] Measured concentration of test sample(PEG modified)=peak area detected by PDA detector of test sample(PEG modified)/peak area detected by PDA detector of unmodified protein1.0

[0138] PEG peak area in sample=(peak area of test sample(PEG modified) detected by RI detector/measured concentration of test sample (PEG modified))(peak area of unmodified proprotein detected by RI detector/concentration of unmodified proprotein)

[0139] PEG peak area in the sample was substituted into the PEG standard curve to calculate PEG concentration in the sample.

[0140] PEG binding number in sample=(PEG concentration in sample/molecular weight of PEG)/(protein concentration in sample/molecular weight of protein 26 kD)

TABLE-US-00012 TABLE 12 measured concentration PEG peak area in PEG concentration PEG binding Sample name of test sample (mg/ml) the sample (AU) in sample (mg/ml) number PEG10K-hKLK1 (low glycosylated) 0.96 319216 2.24 6.52 PEG10K-hKLK1X1 0.81 276707 1.93 6.24

Example 6 In Vitro Activity Detection of Recombinant Human Kallikrein I(hKLK1) and its Mutant (hKLK1X)

1. In Vitro Activity Evaluation Based on Artificial Substrates

[0141] KLK1 exerts its biological function in vivo through catalyzing the hydrolysis of LMWK to release lysyl bradykinin, which involves the cleavage of the carboxy-terminus peptide bond of arginine (Arg). P-nitroaniline (PNA) was generated by hydrolyzating the amide bond between Arg and p-nitroaniline in the synthetic chromogenic substrate S-2266(H-D-Val-Leu-Arg-PNA). Therefore, PNA was detected at 405 nm to evaluate the in vitro biological activity of recombinant hKLK1 and its mutants. One unit IU of activity was defined as the amount of enzyme that hydrolyzed 1 mol S-2266 to PNA per minute at 37 C. and pH8.0. The reaction system consisted of 200 l 20 mM trometamol buffer, 10 l test article, and 20 l 20 mM S-2266 substrate solution, which were placed in water bath at 37 C. for accurate reaction for 10 min. The reaction was terminated by adding 20 l 50% acetic acid solution. The amount of PNA produced in the reaction system was quantified based on a standard curve fitted with different concentrations of PNA standards. The in vitro biological activities of hKLK1, hKLK1 mutants and hKLK1-PEG modified samples were detected by the above method.

[0142] As shown in the table below, the activity of low glycosylated hKLK1(unmutated) samples was significantly higher than that of high glycosylated hKLK1(unmutated) samples. Among the pegylated unmutated proteins, random modification had higher in vitro activity than site-directed modification, and PEG10K-hKLK1(low glycosylated) was better. After mutation, the activity of mutant samples (hKLK1X1, hKLK1X2, hKLK1X3, hKLK1X4) was higher than that of unmutated low glycosylated hKLK1 samples. At the same time, the activity of PEG modified samples was slightly lower than that of the unmodified hKLK1X1 protein. The activity of PEG10K-hKLK1X1 was higher than that of PEG5K-hKLK1X1, and the original protein activity was basically preserved after PEG10K modification.

TABLE-US-00013 TABLE 13 Specific Relative Sample activity (IU/mg) activity low glycosylated hKLK1 5.1 100% high glycosylated hKLK1 3.0 58.8% hKLK1X1 7.0 137.3% hKLK1X2 7.1 139.2% hKLK1X3 5.7 111.8% hKLK1X4 5.7 111.8% urinary kallikrein 4.5 88.2%

2. In Vitro Activity Evaluation Based on Natural Substrates

Enzymatic Reaction and Liquid Phase Detection

[0143] KLK1 exerts its biological function in vivo through catalyzing the hydrolysis of LMWK to release lysyl bradykinin. This example compared the enzymatic reaction to produce the effector molecule bradykinin at different ratios of substrate (LMWK) to enzyme (KLK1 or its PEG modified KLK1). The produced effector molecules were separated by reverse phase chromatography, the peak area of the product is calculated, and the curve of bradykinin production at different ratios of substrate to enzyme was drawn. Compare the amount of effector molecules produced by different test samples under the same reaction methods. Through this in vitro method simulating the in vivo effect, the in vivo effects of different tested samples were indirectly compared.

[0144] hKLK1X1 was prepared according to Example 3, PEG10K-hKLK1X1 was prepared according to Example 5; Kb was KLK1 extracted from porcine pancreas. PEG-Kb was pancreatic kallikretin I for injection and was PEG10K-modified Kb, prepared as described in Example 5.

[0145] The samples were mixed according to the following table (in PEG modified samples, the molar mass of the modified active article was converted). The mixed samples were placed in a 37 C. thermostat, incubated for 15 min, accurately timed, and the reaction was terminated by adding 50% acetic acid solution according to the volume ratio of 10:1.

TABLE-US-00014 TABLE 14 System (substrate:enzyme Substrate Enzyme 50% acetic Total volume mol/mol) L l acid l of system l 1:1 10 10 2 22 5:1 50 10 6 66 10:1 50 5 5.5 60.5 15:1 60 4 6.4 70.4 20:1 60 3 6.3 69.3

[0146] After the termination of the reaction, the sample was placed in a desktop centrifuge, centrifuged at 12000 rpm for 5 min. Took the supernatant And detected with Conduct Waters ACQUITY UPLC H-Class, the mobile phase A was 0.1% TFA-H.sub.2O, the mobile phase B was 0.1% TFA-ACN, the detection wavelength was 214 nm, the column temperature was 30 C., the sample loading volume was 10 l, the flow rate was 0.2 ml/min, the running time was 35 min, and the running gradient was as follows:

TABLE-US-00015 TABLE 15 Run time min Flow ml/min A % B % initial 0.200 90.0 10.0 5.00 0.200 90.0 10.0 30.00 0.200 0.0 100.0 32.00 0.200 0.0 100.0 32.01 0.200 90.0 10.0 35.00 0.200 90.0 10.0

[0147] According to the UPLC chromatograph results of the enzymatic reaction mixture, the chromatographic peaks with retention time of 130.5 min(the peak position of bradykinin) were integrated and summed, and compared with the peak area of bradykinin (1 mg/ml, loading volume 10 l) to calculate the concentration of bradykinin generated by the enzymatic reaction. The amount of bradykinin produced by the total reaction system (Table 14) was calculated, and finally converted to the amount of bradykinin(g/mg) produced per milligram of enzyme at each molar ratio. The results were shown in Table 16.

TABLE-US-00016 TABLE 16 Molar ratio Bradykinin production g/mg(enzyme) of substrate PEG10K- to enzyme hKLK1X1 hKLK1X1 Kb PEG-Kb 1:1 17.22 14.52 20.92 11.28 5:1 122.71 73.01 90.81 44.63 10:1 249.44 142.69 166.01 79.99 15:1 339.03 192.45 217.86 103.85 20:1 451.52 248.25 287.65 119.89

[0148] The detection results based on natural substrates showed that the bradykinin produced by the modified protein was lower than that of the unmodified sample on the whole trend, which was consistent with the characteristics of PEG modified protein. Combined with the data from Examples 9-13, we believed that the PEG-modified protein can more modestly maintain the enzymatic process than the original protein, achieving sustained release of effector molecules. At the same time, the effect of KLK1 is based on the regulation of the KKS system in vivo, which includes the release and clearance of effector molecules. Obviously, a mild and continuous enzymatic process can effectively reduce the clearance mechanism of the KKS system, so that the drugs of the invention can play their biological role more stably and effectively.

[0149] In addition, comparing Kb and hKLK1X1 and their modified products, the sample of the present invention released more effector molecules, which was consistent with the in vivo efficacy results shown in Examples 10-13, further demonstrating that the hKLK1X1 protein of the present invention had a significant advantage over KLK1 of animal origin.

Example 7 In Vitro Enzymatic Kinetic Assay of PEG-Modified hKLK1/hKLK1X, Etc

1. Detection Method

[0150] (1) Standard dilution: The standard pancreatic kallikrein I(from Changzhou Qianhong Biochemical Pharmaceutical) was diluted to 101 U/mL using S2266 substrate buffer (20 mM Tris-HCl 8.5), and the diluted standard was diluted to 1, 2, 3, 4, 5, and 6IU/mL according to the table below to make standard curve samples:

TABLE-US-00017 substrate buffer 90 L 80 L 70 L 60 L 50 L 40 L 10 IU/mL standard 10 L 20 L 30 L 40 L 50 L 60 L

[0151] (2) Substrate with different concentrations: the S2266 substrate was diluted into 400, 200, 100, 50, 25 and 10 M using S2266 substrate buffer.

[0152] (3) Sample dilution: Samples were diluted to 1-6 IU/mL using S2266 substrate buffer.

[0153] (4) Sample addition: Add the standard in step (1) to the ELISA plate, 80 L/well, and add each sample to one well. The diluted samples in (3) above were added to the ELISA plate, 80 L/well of each sample, and 6 wells were added in parallel.

[0154] (5) Reading: The microplate reader was set at 37 C. and 405 nm for kinetic detection, the reading interval was 1 min, and the detection time was 15 min. When the temperature of the sample cell raised to 37 C., the substrate was added. 80 L/well of 200 M substrate was added to each standard well. Different concentrations of substrate as described in (2) were added to each of the six parallel wells, 80 L/well. Samples needed to be quickly added and blown and mixed. After the sample loading, the readings should be taken immediately and readings were taken at 1-min intervals.

[0155] (7) Results processing: GraphPad prism software was used for results processing, data fitting was performed according to Mie equation, and the enzyme kinetic parameters were calculated. Where Km is Mie constant, which means the concentration of substrate at half of the maximum speed (Vmax) during the enzymatic reaction. Generally, 1/Km is used to approximate the affinity of the enzyme to the substrate. The larger 1/Km means the higher affinity of the enzyme to the substrate, and the enzymatic reaction is easy to proceed.

2. Experimental Results

[0156] The enzyme kinetics results of each sample were shown in the table below:

TABLE-US-00018 TABLE 17 Sample K.sub.m (mol/L) low glycosylated hKLK1 43.76 PEG10K-hKLK1 (low glycosylated) 79.39 hKLK1X1 27.56 PEG5K-hKLK1X1 47.23 PEG10K-hKLK1X1 46.65

[0157] According to the results of enzymatic kinetics, the Km value of hKLK1X1 was lower than that of unmutated low glycosylated hKLK1, indicating that the affinity to the substrate increased after the mutation. The Km values of two pegylated mutants were lower than those of pegylated unmutated protein, indicating that two pegylated mutant had better enzymatic properties than pegylated unmutated protein.

Example 8 Comparison of Pharmacokinetics of PEG Modified hKLK1 in Rats

1. Grouping

[0158] According to the weight of SD rats measured on the first day of the experiment, they were randomly divided into 6 groups: PEG5K-hKLK1(high glycosylated) intravenous injection (0.5 mg/kg), PEG5K-hKLK1(high glycosylated) subcutaneous injection (0.5 mg/kg), PEG10K-hKLK1(low glycosylated) intravenous injection group (0.5 mg/kg), PEG10K-hKLK1(low glycosylated) subcutaneous injection group (0.5 mg/kg), PEG10K-hKLK1X1 intramuscular injection (0.1 mg/kg), PEG10K-hKLK1X1 intramuscular injection (0.02 mg/kg), 6 rats in each group, half male and half female, staining number. A single dose was administered on the first day of the experiment.

2. Blood Sample Collection and Test Sample Preparation

[0159] Detection time: blood samples were collected at 0 min(before drug administration), 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 2d, 3d, 5d, 7d, a total of 11 times, blood collection volume: 50-100 l serum, for the determination of pharmacokinetic parameters.

Sample Preparation:

[0160] 1) Pharmacokinetics samples: The samples collected were diluted with 10% SD rat mixed blank serum (SD rat mixed blank serum: blocking solution (2% BSA in PBST)=1:9) to the limit of quantitation (LOQ) concentration range of 2560 ng/ml to 80 ng/ml.

[0161] 2) Standard: The PEG modified hKLK1 with high and low glycosylation were diluted with mixed blank serum of SD rats to prepare a series of two-fold gradient concentration standards, 5120 ng/ml to 40 ng/ml.

[0162] 3) Quality control: quality control materials with high (1920 ng/ml), medium (480 ng/ml) and low concentration (240 ng/ml) were prepared by diluting PEG modified high and low glycosylated hKLK1 with mixed blank serum of SD rats.

3. pharmacokinetics Detection

[0163] Drug concentrations in serum were measured by ELISA. The analysis process was as follows:

[0164] 1) Coating: anti-hKLK1 antibody was diluted to 400 ng/well with 20 mM phosphate buffer (PB, pH7.4), and the diluted antibody solution was added into the well of ELISA plate (100 uL/well), the plate was sealed, and coated at 4 C. overnight. The coated plate was Thermo ELISA plate.

[0165] 2) Sealing: Discard the liquid in the hole, wash the plate once with a washing machine, 300 uL/hole, and dry. After adding blocking solution (20 mM PBS containing 2% BSA and 0.05% Tween20), 200 ul/well, plates were sealed and incubated at 37 C. for about 2 h.

[0166] 3) Sample addition: the liquid in the well was removed, and the pharmacokinetics samples, standards and quality control solutions were diluted 10 times with blocking solution, then added to the ELISA plate, 100 uL/well, the plate was sealed, and incubated at 37 C. for 1.5 h;

[0167] 4) Adding detection antibody: Discard the liquid in the well, wash the plate with a washing machine for 3 times, 300 uL/well, dry, add the detection antibody working solution (dilute the enzyme-linked anti hKLK1 antibody for detection to 1 ug/ml with blocking solution), 100 uL/well, seal the plate, and incubate at 37 C. for 45 min;

[0168] 5) Color development: Discard the liquid in the well, wash the plate 5 times with a washing machine, 300 uL/well, dry, add TMB No. 1 color development solution, and incubate at 37 C. for 15 min, depending on the color development situation;

[0169] 6) Plate reading: 50 uL/well of terminating solution (2M H.sub.2SO.sub.4) was added to terminate the reaction and the OD value at 450 nm was measured immediately. Draw a standard curve with the standard concentration as the X-coordinate and OD value as the Y-coordinate. The sample concentration was calculated.

[0170] 7) Using Origin 8 software to draw the standard curve and calculate the sample concentration. Microsoft EXCEL was used to calculate the mean, standard deviation and coefficient of variation. GraphPad Prism 7.00 was used to calculate the area under the curve (AUC).

4. Test Results

[0171] At the same dose, the body exposure (AUC) of the drug was as follows:

TABLE-US-00019 TABLE 18 Administration AUC (h .Math. ng/ml) Sample route Gender value PEG5K-hKLK1 (high Intravenous Male 40256 glycosylated) (0.5 mg/kg) injection Female 47715 Subcutaneous Male 15575 injection Female 15891 PEG10K-hKLK1 (low Intravenous Male 383645 glycosylated) (0.5 mg/kg) injection Female 147389 Subcutaneous Male 105376 injection Female 67261 PEG10K-hKLK1X1 (0.1 mg/kg) Intramuscular Male 9501 injection PEG10K-hKLK1X1 (0.02 mg/kg) Intramuscular Male 2266 injection

[0172] According to the above pharmacokinetic results, the pharmacokinetic curve of PEG10K-hKLK1(low glycosylated) was significantly better than that of PEG5K-hKLK1(high glycosylated) when injected intravenously and subcutaneously, showing a longer drug half-life. PEG10K-hKLK1X1 still showed better absorption when injected intramusculally at lower doses.

Example 9 Comparison of Immunogenicity of PEG Modified hKLK1 with High and Low Glycosylation in Rats

1. Grouping

[0173] According to the weight of SD rats measured on the first day of the experiment, they were randomly divided into 2 groups: PEG5K-hKLK1(high glycosylated) group and PEG10K-hKLK1(low glycosylated) group, 12 rats in each group, half male and half female, and made a stain number.

2. Administration of Drugs

[0174] Administration route: intravenous injection.

[0175] Administration frequency: once a week for 8 doses.

[0176] Administration dose: the concentration of administration was 0.2 mg/mL, and the dose was 0.5 mg/kg.

3. Blood Sample Collection

[0177] Detection time: blood samples were collected at 0 min(before administration), 3 days and 7 days after administration. Blood samples were collected once a week for 2 weeks during the recovery period. About 500 L of whole blood was collected each time, stranded for 2 hours, and centrifuged at 3000 rpm for 10 min. The serum was separated and used for the determination of immunogenicity.

4. Test of Immunogenicity

[0178] 1) Coating: The coating antigen (PEG-modified hKLK1) working solution was added to the ELISA plate, 100 uL/well, and incubated at 2-8 C. overnight;

[0179] 2) Blocking: the liquid in the well was discarded, the plate was washed 3 times, dried, and the blocking solution (20 mM PBS containing 2% BSA and 0.05% Tween20) was added to the plate, 200 uL/well, incubated at 37 C. for about 2 hours.

[0180] 3) Sample processing: all animal serum samples were diluted 10-fold with blocking solution;

[0181] 4) Sample addition: Discard the liquid in the ELISA plate wells, dry, add the samples to each well, 100 uL/well, and then incubate at 37 C. and 200 rpm shaking for about 2 hours;

[0182] 5) Adding the detection antigen working solution: Discard the liquid in the well, wash the plate three times, dry. Add 100 uL/well of detection antigen working solution (dilute biotin-labeled PEG modified hKLK1 to 2.5 g/ml with blocking solution as the detection antigen working solution) to each well for screening test. In the confirmatory test, 100 ul/well of the confirmatory assay antigen working solution was added to each well (PEG modified hKLK1 was diluted to 200 g/ml with the above-mentioned detection antigen working solution as the confirmatory assay antigen working solution), and incubate at 37 C. with 200 rpm shaking for about 1 hour.

[0183] 6) Adding signal amplification detection solution (streptavidin-horseradish peroxidase was diluted to 0.05 g/ml with blocking solution): Discard the liquid in the well, wash the plate 3 times, then dry, add signal amplification detection solution, 100 uL/well, and incubate at 37 C. with 200 rpm shaking for about 1 h;

[0184] 7) Color development: Discard the liquid in the well, wash the plate 3 times, dry, add color development solution, 100 uL/well, and place in the dark at 37 C. for 15 min;

[0185] 8) Termination: The reaction was terminated by adding termination solution (2M H.sub.2SO.sub.4), 100 uL/well, and the OD value at 450 nm was immediately measured on a microplate reader.

5. Test Results

[0186] The immunogenicity evaluation results of FIGS. 13, 14 showed that anti-drug antibody (ADA) was not detected in the serum of all 12 animals in the PEG-hKLK1(low glycosylated) group, while high ADA values were detected in 2 out of 12 animals in the PEG-hKLK1(high glycosylated) group. This indicated that PEG modified low glycosylated protein was less immunogenic.

Example 10 In Vivo Activity Comparison of Urinary Kallikrein for Injection and Pancreatic Kallikrein I for Injection

1. Model Preparation

[0187] SD rats, male, SPF grade, weighing 270-300 g. The cerebral ischemia-reperfusion model of middle cerebral artery occlusion (MCAO) was prepared by using the intraluminal suture method in rats. Animals were anesthetized with gas (isoflurane), and then fixed them in a supine position. The skin was disinfected, and the right common carotid artery, external carotid artery, and internal carotid artery were separated from the midline incision in the neck. The vagus nerve was gently peeled off, and the external carotid artery was ligated and cut. The common carotid artery was clamped near the proximal end, and an incision was made from the distal end of the ligature line of the external carotid artery. The suture(model 2438-A5, purchased from Beijing Xinong Technology Co., LTD.) was inserted and passed through the bifurcation of the common carotid artery into the internal carotid artery. The suture was then slowly inserted until slight resistance was encountered (about 20 mm from the bifurcation), blocking the blood supply to the middle cerebral artery. The neck skin was sutured, disinfected, and the rat was returned to the cage. After 90 minutes of ischemia, the rat was anesthetized again, fixed on the rat board, and the neck skin was cut open to find the suture, which was gently removed. Reperfusion was performed, and the neck skin was sutured, disinfected, and the rat was returned to the cage for feeding.

[0188] Grouping: There were four groups, namely sham operation group, model group, and pancreatic kallikrein I group for injection (0.41 U/kg, intramuscular injection, pancreatic kallikrein I for injection was PEG10K-modified Kb, the preparation method was as described in Example 5, in which Kb was KLK1 extracted from pig pancreas). Urinary kallikrein group (0.1IU/kg, intravenous injection). In the administration group, the drug was administered 2 h after reperfusion. The specific activity of the samples in all animal experiments was calculated according to method 1 of Example 6.

2. Evaluation of Neurological Defect

[0189] The modified Bederson method was used to evaluate neurological defect symptoms.

TABLE-US-00020 0: When the tail is suspended, both forelimbs of the animal extend towards the floor without other behavioral deficits. 1: When the tail is suspended, the forelimb of the animal contralateral to the surgery (left) shows wrist and elbow flexion, shoulder internal rotation, elbow abduction, and tight adhesion to the chest wall. 2: When the animal is placed on a smooth plate, the resistance is reduced when the operated side shoulder is moved to the contralateral side. 3: When the animal walks freely, it circles or turns to the opposite side of the surgery. 4: Limb paralysis, no spontaneous movement.

Measurement of Cerebral Infarction Area

[0190] Animals were anesthetized with 10% chloral hydrate, the brain tissue was removed, the olfactory bulbs, cerebellum and lower brainstem were removed, and the bloodstain on brain surface was washed with SPSS. After removing surface residual water, the brain tissue was placed at 80 C. for 7 minutes, took out and cut into coronal sections perpendicular to the visual cross plane, and sliced at intervals of 2 mm to the posterior direction. The brain slices were placed in fresh TTC (20 g/L) dye solution prepared with SPSS at 37 C. for 90 minutes. Normal brain tissue was stained deep red, while ischemic brain tissue appeared pale. After washing with SPSS, the brain slices were quickly arranged in sequence from front to back, residual water on the surface was absorbed, and photographed. The image analysis software (Image Tool) was used to delineate the ischemic area (white area) and the right side area on the photos for statistical analysis. The percentage of cerebral infarction area was calculated using the following formula:

Cerebral infarction area (%)=100total ischemic area/total right hemisphere area.

4. Results

[0191] 1). The effect of the test article on the neurological defects symptoms As shown in FIG. 15 and Table 19, the urinary kallikrein group had a significant improvement in neurological defect symptoms compared with the model group(F.sub.7,87=20.80, P=0.018); Pancreatic kallikrein I for injection significantly improved the neurological defect symptoms compared with the model group(F.sub.7, 87=20.80, P=0.021).

TABLE-US-00021 TABLE 19 Effect of test article on neurological defect symptoms Total Neurological animals Dead Discarded Samples Defect Group (n) (n) (n) (n) Symptoms Score Sham operation 8 0 0 8 0.00 0.00 group Model group 21 9 0 12 2.92 0.15 Urinary 21 8 0 13 1.92 0.21* kallikrein group Pancreatic 21 8 1 12 1.92 0.15** kallikrein I group

2). The Effect of the Test Article on the Cerebral Infarction Area (%)

[0192] As shown in FIGS. 16, 17, 18 and Table 20, urinary kallikrein (F.sub.7,87=12.72, P=0.042) and pancreatic kallikrein I for injection (F.sub.7,87=12.72, P=0.019) significantly improved the infarct size compared with the model group.

TABLE-US-00022 TABLE 20 Effect of test article on cerebral infarction area (%) Total Dis- Cerebral animals Dead carded Samples infarction Group (n) (n) (n) (n) area (%) Sham operation 8 0 0 8 0.00 0.00 group Model group 21 9 0 12 39.71 2.30 Urinary kallikrein 21 8 0 13 22.74 3.71* group Pancreatic 21 8 1 12 21.05 3.01* kallikrein I group Mean SE. *P < 0.05, compared with model group.

3). The Effect of Test Article on Cumulative Mortality Rate (%)

[0193] As shown in Table 21, animals died in all groups except the sham operation group, and there was no difference between groups in mortality. The urinary kallikrein group (F.sub.1,28=0.18, P=0.6746), pancreatic kallikrein I for injection group (F.sub.1,28=0.18, P=0.6753) had no significant difference compared with the model group.

TABLE-US-00023 TABLE 21 Effect of test article on cumulative mortality rate(%) Group D0 D1 D5 Sham operation group 0.00 0.00 0.00 Model group 0.00 23.81 42.86 Urinary kallikrein group 0.00 23.81 38.10 pancreatic kallikrein I group 0.00 23.81 38.10

4). Conclusion

[0194] Compared with the model group, urinary kallikrein and pancreatic kallikrein I for injection had significant protective effect on the brain after ischemia-reperfusion.

Example 11 In Vivo Activity Evaluation of PEG10K Modified Unmutated Hyperglycosylated hKLK1

1. Model Preparation, Evaluation of Neurological Defect Symptoms, and Measurement of Cerebral Infarction Area were the Same as in Example 10.

[0195] Grouping and administration: There were 4 groups, namely sham operation group, model group, positive drug group (pancreatic kallikretin I for injection, 0.41 U/kg, intramuscular injection), PEG10K-hKLK1(high glycosylated) group (0.1IU/kg, intravenous injection), drug was given 2 hours after reperfusion.

2. Results

1) Effect of the Test Article on the Neurological Defect Symptoms

[0196] As shown in FIG. 19 and Table 22, PEG10K-hKLK1(high glycosylated) group (F.sub.3,43=63.32,P=0.352) had a certain improvement in neurological defect symptoms compared with the model group, but the difference was not statistically significant. Compared with the model group, the positive drug group (F.sub.3,44=56.34, P=0.006) had a significant improvement in neurological defect symptoms.

TABLE-US-00024 TABLE 22 Effect of test article on neurological defect symptoms Total Neurological animals Dead Discarded Samples Defect Symptoms Group (n) (n) (n) (n) Score Sham operation group 8 0 0 8 0.00 0.00 Model group 21 8 0 13 3.15 0.15 Positive drug group 21 7 1 13 2.23 0.17* PEG10K-hKLK1 (high 21 7 1 13 2.77 0.17 glycosylated) group Mean SE. *P < 0.05, compared with model group.

2) The Effect of the Test Article on the Cerebral Infarction Area (%)

[0197] As shown in FIGS. 20, 21, 22 and Table 23, PEG10K-hKLK1(high glycosylated) group (F.sub.3,43=37.72, P=0.197) had a certain improvement in cerebral infarction area compared with the model group, but the difference was not significant; Compared with the model group, the positive drug group (F.sub.3,44=34.58, P=0.007) had a significant improvement in cerebral infarction area.

TABLE-US-00025 TABLE 23 Effect of test article on cerebral infarction area (%) Total animals Dead Discarded Samples Cerebral infarction Group (n) (n) (n) (n) area (%) Sham operation group 8 0 0 8 0.00 0.00 Model group 21 8 0 13 44.76 2.46 Positive drug group 21 7 1 13 29.42 2.91* PEG10K-hKLK1 (high 21 7 1 13 36.40 3.35 glycosylated) group Mean SE. *P < 0.05, compared with model group.

3) The Effect of Test Article on Cumulative Mortality Rate (%)

[0198] As shown in Table 24 below, animals died in all groups except the sham operation group, and there was no difference between groups in mortality. There was no significant difference between positive drug group (F.sub.1,6=0.00,P=1.0000), PEG10K-hKLK1(high glycosylated) group (F.sub.1,6=0.00,P=1.0000) and model group.

TABLE-US-00026 TABLE 24 Effect of test article on cumulative mortality rate (%) Group D1 D2 Sham operation group 0.0 0.0 Model group 17.39 34.78 Positive drug group 21.74 30.43 PEG10K-hKLK1 (high glycosylated) group 21.74 30.43

4) Conclusion

[0199] When animals were treated with the test article after ischemia-reperfusion, PEG10K-hKLK1(high glycosylated) only tended to improve symptoms compared with the model group, but the difference was not statistically significant.

Example 12 In Vivo Activity Evaluation of Different PEG Modification Products of Unmutated Low Glycosylated hKLK1

1. Model Preparation, Evaluation of Neurological Defect Symptoms, and Measurement of Cerebral Infarction Area were the Same as in Example 10.

[0200] Grouping and administration: There were 7 groups, sham operation group, model group and positive drug group (pancreatic kallikrein I for injection, 0.41 U/kg, intramuscular injection), PEG40K-hKLK1(low glycosylated) group (0.1IU/kg), PEG30K-hKLK1(low glycosylated) group (0.1IU/kg), PEG10K-hKLK1(low glycosylated) group (0.1IU/kg), PEG5K-hKLK1(low glycosylated) group (0.1IU/kg). The drug was injected intravenously 2 h after reperfusion in the administration group. The dose of PEG-hKLK1 was converted to the specific activity, and the animals were administrated with the same activity unit of PEG-hKLK1 per kilogram.

2. Results

1) The Effect of the Test Article on the Neurological Defect Symptoms

[0201] As shown in FIG. 23 and Table 25 below, PEG40K-hKLK1(low glycosylated) group (F.sub.3,44=46.28,P=0.209), PEG30K-hKLK1(low glycosylated) group (F.sub.3,43=53.33,P=0.209), PEG5K-hKLK1(low glycosylated) group (F.sub.3,44=46.28, P=0.209) had a certain improvement in neurological defect compared with the model group, but the difference was not statistically significant. Compared with the model group, PEG10K-hKLK1(low glycosylated) group (F.sub.3,45=46.43, P=0.008) and positive drug group (F.sub.3,45=46.43, P=0.003) had a significant improvement in neurological defect.

TABLE-US-00027 TABLE 25 Effect of test article on neurological defect symptoms Total Neurological animals Dead Discarded Samples Defect Symptoms Group (n) (n) (n) (n) Score Sham operation group 8 0 0 8 0.00 0.00 Model group 21 8 0 13 3.08 0.14 Positive drug group 21 7 1 13 2.15 0.22* PEG40K-hKLK1 (low glycosylated) group 21 7 0 14 2.57 0.17 PEG30K-hKLK1 (low glycosylated) group 21 8 0 13 2.62 0.14 PEG10K-hKLK1 (low glycosylated) group 21 6 0 15 2.27 0.15* PEG5K-hKLK1 (low glycosylated) group 21 7 0 14 2.57 0.17 Mean SE. *P < 0.05, compared with model group.

2) The Effect of the Test Article on the Cerebral Infarction Area (%)

[0202] As shown in FIGS. 24, 25, 26 and Table 26, PEG40K-hKLK1(low glycosylated) group (F.sub.3,44=37.73, P=0.446), PEG30K-hKLK1(low glycosylated) group (F.sub.3,43=36.56, P=0.502), PEG5K-hKLK1(low glycosylated) group (F.sub.3,44=39.05,P=0.218) had a certain improvement in cerebral infarction area compared with the model group, but there was no significant difference. Compared with the model group, the PEG10K-hKLK1(low glycosylated) group (F.sub.3,45=39.30,P=0.001) and the positive drug group (F.sub.3,45=39.30,P=0.001) had a significant improvement in cerebral infarction area.

TABLE-US-00028 TABLE 26 Effect of test article on cerebral infarction area (%) Total Cerebral animals Dead Discarded Samples infarction Group (n) (n) (n) (n) area (%) Sham operation group 8 0 0 8 0.00 0.00 Model group 21 8 0 13 42.41 2.26 Positive drug group 21 7 1 13 27.36 3.29* PEG40K-hKLK1 (low glycosylated) group 21 7 0 14 36.48 2.72 PEG30K-hKLK1 (low glycosylated) group 21 8 0 13 36.66 2.97 PEG10K-hKLK1 (low glycosylated) group 21 6 0 15 27.72 2.09* PEG5K-hKLK1 (low glycosylated) group 21 7 0 14 34.92 2.47 Mean SE. *P < 0.05, compared with model group.

3) Effect of Test Article on Cumulative Mortality Rate (%)

[0203] As shown in Table 27, animals died in all groups except the sham operation group, and there was no difference between groups in mortality. Compared with the model group, there was no statistical differences between positive drug group (F.sub.1,3=0.15, P=0.7239), PEG40K-hKLK1(low glycosylated) group (F.sub.1,3=0.17, P=0.7100), PEG30K-hKLK1(low glycosylated) group (F.sub.1,3=0.17, P=0.7106), PEG10K-hKLK1(low glycosylated) group (F.sub.1,3=0.15, P=0.7239), PEG5K-hKLK1(low glycosylated) group (F.sub.1,3=0.00, P=0.9994).

TABLE-US-00029 TABLE 27 Effect of test article cumulative mortality rate (%) Group D1 D2 Sham operation group 0.00 0.00 Model group 19.05 38.10 Positive drug group 19.05 33.33 PEG40K-hKLK1 (low glycosylated) group 19.05 33.33 PEG30K-hKLK1 (low glycosylated) group 23.81 38.10 PEG10K-hKLK1 (low glycosylated) group 23.81 28.57 PEG5K-hKLK1 (low glycosylated) group 23.81 33.33

4) Conclusion

[0204] The results showed that: for unmutated hKLK1 with low glycosylation, PEG10K-hKLK1(low glycosylated) showed significant brain protective efficacy in animal models, and PEG5K-hKLK1(low glycosylated), PEG40K-hKLK1(low glycosylated), and PEG30K-hKLK1(low glycosylated) all showed a trend of improving symptoms. At the same time, the in vivo efficacy results were also consistent with the in vitro efficacy. The in vitro activity of PEG10K modified low glycosylated hKLK1 was higher than that of PEG-5K modified low glycosylated hKLK1, and higher than PEG30K/40K modified low glycosylated hKLK1.

[0205] Combined with the results of examples 11 and 12, PEG10K-hKLK1(high glycosylated) is less effective than PEG10K-hKLK1(low glycosylated) with the same dosage. The results showed that the unmutated low glycosylated samples were better than the high glycosylated samples with the same PEG modification.

Example 13 In Vivo Activity Evaluation of Different PEG Modified hKLK1 Mutants

1. Model Preparation, Evaluation of Neurological Defect Symptoms, and Measurement of Cerebral Infarction Area were the Same as in Example 10.

[0206] Grouping: there were 4 groups, namely sham operation group, model group, PEG5K-hKLK1X1 group (0.1IU/kg), PEG10K-hKLK1X1 group (0.1IU/kg). The animals were injected intravenously 2 h after reperfusion.

2. The Results

1) The Effect of the Test Article on Neurological Defect Symptoms

[0207] As shown in FIG. 27 and Table 28, the PEG5K-hKLK1X1 group (F.sub.2,38=67.13, P=0.017) had a significant improvement in neurological defect compared with the model group; The PEG10K-hKLK1X1 group (F.sub.2,37=86.65, P=0.003) had a extremely significant improvement in neurological defect compared with the model group.

TABLE-US-00030 TABLE 28 Effect of test article on neurological defect symptoms Neurological Total Dis- Defect animals Dead carded Samples Symptoms Group (n) (n) (n) (n) Score Sham operation 8 0 0 8 0.00 0.00 group Model group 18 2 0 16 2.69 0.12 PEG5K- 18 1 0 17 2.12 0.17* hKLK1X1 group PEG10K- 18 2 0 16 2.06 0.14** hKLK1X1 group Mean SE. *P < 0.05, **P < 0.01, compared with model group.

2) The Effect of the Test Article on the Cerebral Infarction Area (%)

[0208] As shown in FIGS. 28, 29, 30 and Table 29, the PEG5K-hKLK1X1 group (F.sub.2,38=39.50, P=0.001) and PEG10K-hKLK1X1 group (F.sub.2,37=90.50, P=0.000) had a significant improvement in cerebral infarction area compared with the model group.

TABLE-US-00031 TABLE 29 Effect of test article on cerebral infarction area (%) Total animals Dead Discarded Samples Cerebral infarction Group (n) (n) (n) (n) area (%) Sham operation group 8 0 0 8 0.00 0.00 Model group 18 2 0 16 38.73 1.99 PEG5K-hKLK1X1 group 18 1 0 17 23.60 3.28** PEG10K-hKLK1X1 group 18 2 0 16 22.92 1.71*** Mean SE. *P < 0.05, **P < 0.01, ***P < 0.001 compared with model group.

3) Effect of Test Article on Cumulative Mortality Rate (0%)

[0209] As shown in Table 30, animals died in all groups except the sham operation group, and there was no difference between groups in mortality. There was no significant difference between the PEG5K-hKLK1X1 group (F.sub.1,5=0.71, P=0.4369) and the PEG10K-hKLK1X1 group (F.sub.1,5=0.00, P=1.0000) with the model group.

TABLE-US-00032 TABLE 30 Effect of test article on cumulative mortality rate (%) Group D0 D1 Sham operation group 0.0 0.0 Model group 0.00 11.11 PEG5K-hKLK1X1 group 0.00 5.56 PEG10K-hKLK1X1 group 0.00 11.11

4) Conclusion

[0210] Compared with the model group, PEG5K-hKLK1X1 and PEG10K-hKLK1X1 showed significant brain protection after ischemia-reperfusion.

[0211] In combination with examples 12 and 13, the in vivo efficacy results of PEG-hKLK1(unmutated)/hKLK1X(mutant) indicated that:

TABLE-US-00033 TABLE 31 Protein In vivo efficacy after PEG-5K modification In vivo efficacy after PEG-10K modification low trend of improvement in neurological defect, with significant therapeutic effect on neurological defect glycosylated no statistical difference; (*P < 0.05); hKLK1 improvement in the cerebral infarction area, with significant improvement in the cerebral infarction (unmutated) no statistical difference area (*P < 0.05) hKLK1X significant therapeutic effect on neurological extremely significant therapeutic effect on (mutant) defect (*P < 0.05); neurological defect (**P < 0.01); extremely significant improvement in the cerebral very significant improvement in the cerebral infarction area (*P < 0.01) infarction area (*P < 0.001)

[0212] The proteins with N141 mutation were modified with the same PEG. The PEG-modified protein with mutation had better efficacy, better performance, and better therapeutic effect in vivo that the PEG-modified protein without mutation.

[0213] Results of in vivo effects of drugs in examples 10-13:

TABLE-US-00034 TABLE 32 Dose Sample IU/kg In vivo effect urinary kallikrein 0.1 significant therapeutic effect on neurological defect (*P < 0.05) significant improvement in the cerebral infarction area (*P < 0.05) pancreatic kallikrein I 0.4 significant therapeutic effect on neurological defect (*P < 0.05); significant improvement in the cerebral infarction area (*P < 0.05) PEG10K-hKLK1 0.1 significant therapeutic effect on neurological defect (*P < 0.05); (low glycosylated) significant improvement in the cerebral infarction area (*P < 0.05) PEG10K-hKLK1 0.1 trend of improvement in neurological defect, with no statistical difference; (high glycosylated) trend of improvement in the cerebral infarction area, with no statistical difference PEG5K-hKLK1X1 0.1 significant therapeutic effect on neurological defect (*P < 0.05); extremely significant improvement in the cerebral infarction area (*P < 0.01) PEG10K-hKLK1X1 0.1 extremely significant therapeutic effect on neurological defect (**P < 0.01); very significant improvement in the cerebral infarction area (*P < 0.001) *Pancreatic kallikrein I for injection was administered intramuscularly, and the other samples were administered intravenously.

[0214] With the same polyethylene glycol modification, the efficacy of unmutated hKLK1 with low glycosylation was better than that of unmutated hKLK1 with high glycosylation; the hKLK1 protein with mutation at NFS had better efficacy than the unmutated low glycosylated hKLK1, and had obvious advantages at low doses.

Example 14 Pharmacodynamic Study of PEG-hKLK1X1 with Different Administration Methods

1. Model Preparation, Evaluation of Neurological Defect Symptoms, and Measurement of Cerebral Infarction Area were the Same as in Example 10.

[0215] Grouping: There were three groups, sham operation group, model group and PEG10K-hKLK1X1 (intramuscular injection) group(0.4IU/kg). The animals were administered 2 h after reperfusion.

2. Results

1) the Effect of the Test Article on the Neurological Defects Symptoms

[0216] As shown in FIG. 31 and Table 33, the PEG10K-hKLK1X1 intramuscular injection group (F.sub.2,36=122.84, P=0.000) had a very significant improvement in neurological defect symptoms compared with the model group.

TABLE-US-00035 TABLE 33 Effect of test article on neurological defect symptoms Total Neurological animals Dead Discarded Samples Defect Symptoms Group (n) (n) (n) (n) Score Sham operation group 8 0 0 8 0.00 0.00 Model group 20 5 0 15 3.00 0.10 PEG10K-hKLK1X1 group 20 4 0 16 2.25 0.14*** Mean SE. *P < 0.05, **P < 0.01, ***P < 0.001, compared with model group.

2) The Effect of the Test Article on the Area of Cerebral Infarction (%)

[0217] As shown in FIG. 32 and Table 34 below, PEG10K-hKLK1X1 intramuscular injection (F.sub.2,36=55.02, P=0.001) had extremely significant improvement in cerebral infarct size compared with the model group.

TABLE-US-00036 TABLE 34 Effect of test article on cerebral infarction area (%) Total Cerebral animals Dead Discarded Samples infarction Group (n) (n) (n) (n) area (%) Sham operation group 8 0 0 8 0.00 0.00 Model group 20 5 0 15 38.79 2.01 PEG10K-hKLK1X1 group 20 4 0 16 25.93 2.68** Mean SE. *P < 0.05, **P < 0.01, compared with model group.

3) Conclusion

[0218] In combination with examples 13 and 14, PEG10K-hKLK1X1 by intravenous injection (0.1IU/kg) and intramuscular injection (0.41 U/kg) all had a very significant brain protection effect, and can effectively treat the neurological defect symptoms and improve the cerebral infarction area in animals after stroke.

[0219] In summary, the low glycosylated KLK1 had higher activity than the high glycosylated KLK1; Compared with the unmutated KLK1, the recombinant KLK1 mutant lacked N-glycosylation, and the purification process was simpler, the product was more homogeneous, the quality was more controllable, and the yield was higher. Moreover, KLK1 mutant had further advantages in enzymatic properties and activity compared with the unmutated low glycosylated KLK1. In addition, the pegylated recombinant hKLK1 in the present application had significant efficacy in a variety of administration methods, and had the advantages of safety and long-term effect of pegylated drug, which can reduce the administration frequency and improve patient compliance. This application covered the prevention, treatment, prognosis recovery, and prevention of recurrence of acute ischemic stroke, peripheral neuropathy, retinopathy, ocular fundus disease, hypertension, diabetic nephropathy, IgA nephritis, chronic kidney disease and other diseases.