Preparation method of KLK1 fusion protein

Abstract

A method of preparing a fusion protein comprising tissue kallikrein 1 (KLK1), a fusion protein comprising KLK1, a pharmaceutical composition comprising said fusion protein and use thereof in the treatment or prevention of disease.

Description

Preparation method of KLK1 fusion protein Technical Field

[0001] The present application relates to the field of biomedicine, and specifically, to a method for preparing a fusion protein comprising tissue kallikrein 1 (KLK1) (e.g., a KLK1-Fc fusion protein), a fusion protein comprising KLK1, a pharmaceutical composition comprising the fusion protein, and use thereof in treating or preventing diseases (e.g., acute ischemic stroke). Background Art

[0002] The kallikrein-kinin system (also known as "KKS") is a complex endogenous multi-enzyme system composed of kininogen, kallikrein (also known as "KLK" or "kininogenase"), and kinin. It is involved in regulating physiological functions such as blood pressure, kidneys, glucose metabolism, and the nervous system. KLK can catalyze the cleavage of kininogen to produce biologically active kinins. KLK is divided into two major categories: plasma KLK and tissue KLK. Plasma KLK and tissue KLK have significant differences in substrates, molecular weight, and functions. For example, plasma KLKs are encoded by the KLKB1 gene, located on chromosome 4q34-35, and have no other known paralogs. Tissue KLKs are encoded by the KLK1 to KLK15 genes, located on chromosome 19q13, and include 15 known paralogs (KLK1 to KLK15). KLK1 is the only member of the tissue KLK family that can produce biologically active kinins. KLK1 has a dilating effect on isolated arteries, resulting in vasodilation, anti-inflammatory effects, cell repair, and reduced apoptosis.

[0003] Currently, the KLK1 available on the market is mainly extracted from human urine or pig pancreas, which is an endogenous protein. Due to its complex production process and short half-life (e.g. Its half-life in humans is less than 4 hours), and the frequency of administration is high (e.g. Its clinical instructions require a once-daily intravenous injection for 21 consecutive days, which is costly and difficult to control, limiting its application. Consequently, there is a significant unmet clinical need. There is an urgent need to develop a KLK1 protein product with an extended half-life, less frequent dosing, and better production cost and quality control.

[0004] Summary of the Invention

[0005] In one aspect, the present application provides a method for preparing a fusion protein comprising tissue kallikrein 1 (KLK1), the method comprising step (1): placing a recombinant vector containing a nucleic acid molecule encoding the fusion protein in a suitable protein expression system for expression, wherein the fusion protein comprises a masking peptide, a first polypeptide and a second polypeptide, the masking peptide, the first polypeptide and the second polypeptide are directly linked or covalently linked via a linker, and,

[0006] a) the masking peptide has an amino acid sequence that can be specifically recognized and cleaved by a protease;

[0007] b) the first polypeptide comprises KLK1 or a variant thereof; and

[0008] c) the second polypeptide is used to extend the half-life of the first polypeptide.

[0009] In some embodiments, the method further comprises step (2): mixing the fusion protein obtained in step (1) with the protease to perform a specific enzymatic cleavage reaction to remove the masking peptide. In some embodiments, the fusion protein obtained in step (1) is purified before performing the specific enzymatic cleavage reaction in step (2).

[0010] In certain embodiments, the amino acid sequence of the masking peptide is shown in SEQ ID NO: 5. In certain embodiments, the amino acid sequence of the first polypeptide is shown in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122. In certain embodiments, the amino acid sequence of the second polypeptide is shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0011] In another aspect, the present application provides a fusion protein comprising a masking peptide, a first polypeptide and a second polypeptide, wherein the masking peptide, the first polypeptide and the second polypeptide are directly connected or covalently connected through a linker, and

[0012] a) the masking peptide has an amino acid sequence that can be specifically recognized and cleaved by a protease;

[0013] b) the first polypeptide comprises KLK1 or a variant thereof; and

[0014] c) the second polypeptide is used to extend the half-life of the first polypeptide.

[0015] In certain embodiments, the amino acid sequence of the masking peptide is shown in SEQ ID NO: 5. In certain embodiments, the amino acid sequence of the first polypeptide is shown in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122. In certain embodiments, the amino acid sequence of the second polypeptide is shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In certain embodiments, the amino acid sequence of the KLK1 fusion protein is shown in any one of SEQ ID NOs: 36-55, SEQ ID NOs: 100-109, SEQ ID NOs: 111-113, or SEQ ID NOs: 125-127.

[0016] In another aspect, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the KLK1 fusion protein described herein.

[0017] In another aspect, the present application provides a vector comprising the nucleic acid molecule described in the present application.

[0018] In another aspect, the present application provides a protein expression system comprising the nucleic acid molecule described herein or the vector described herein.

[0019] In another aspect, the present application provides a pharmaceutical composition comprising the KLK1 fusion protein described herein, the nucleic acid molecule described herein, the vector described herein, or the protein expression system described herein, and a pharmaceutically acceptable carrier.

[0020] In another aspect, the present application provides a method for constructing a protein expression system, comprising: introducing a nucleic acid molecule encoding the KLK1 fusion protein described herein into a vector to construct an expression vector; and introducing the expression vector into a protein expression system.

[0021] In another aspect, the present application provides a method for treating or preventing a disease, comprising administering an isolated KLK1 fusion protein prepared according to the methods described herein to a subject in need of treatment or prevention, wherein the disease is selected from the group consisting of an ischemic disease, a hemorrhagic disease, a kidney disease, a metabolic disease, and a neurodegenerative disease. In certain embodiments, the ischemic disease is acute ischemic stroke. In certain embodiments, the kidney disease is chronic kidney disease or acute kidney injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG1 shows an exemplary plasmid map, wherein “gene” indicated by an arrow represents a target gene.

[0023] Figure 2 shows the enzymatic activity of the positive control (Figure 2A), the KLK1 fusion protein prepared with an EK enzyme recognition sequence and cleaved by EK (Figure 2B), and the KLK1 fusion protein prepared without an EK enzyme recognition sequence (Figure 2C) or with an EK enzyme recognition sequence but not cleaved by EK (Figure 2D).

[0024] FIG3 shows the enzymatic catalytic activity of KLK1 fusion proteins with different sequences obtained after EK enzyme cleavage and carrying the EK enzyme recognition sequence during the preparation process.

[0025] Figure 4 shows the total ion current (TIC) spectrum (Figure 4A), mass spectrum (MS) spectrum (Figure 4B), deconvolution spectrum (Figure 4C), and a local magnified view of the deconvolution spectrum (Figure 4D) in the deglycosylation reduction molecular weight analysis experiment of fusion protein 2.4.

[0026] Figure 5 shows the total ion current (TIC) spectrum (Figure 5A), mass spectrometry (MS) spectrum (Figure 5B), deconvolution spectrum (Figure 5C), and a partial magnified view of the deconvolution spectrum (Figure 5D) in the desugared intact molecular weight analysis experiment of fusion protein 2.4.

[0027] Figure 6 shows the fusion protein 2.1 and PK curve in mice.

[0028] Figure 7 shows the behavioral scores of the positive control (edaravone), low-dose (25 μg / kg) fusion protein 2.1 and high-dose (150 μg / kg) fusion protein 2.1 on the transient cerebral ischemia-reperfusion (tMCAO) model in rats (#P<0.05, ##P<0.01, ###P<0.001 vs. sham-operated group; *P<0.05, **P<0.01, ***P<0.001 vs. model group).

[0029] Figure 8 shows the TTC staining results of the positive control (edaravone), low-dose (25 μg / kg) fusion protein 2.1 and high-dose (150 μg / kg) fusion protein 2.1 on the cerebral infarction area of ​​the rat tMCAO model (#P<0.05, ##P<0.01, ###P<0.001 vs. sham operation group; *P<0.05, **P<0.01, ***P<0.001 vs. model group, one-way ANOVA Dunnett test).

[0030] Figure 9 shows the effects of positive control (edaravone), low-dose (25 μg / kg) fusion protein 2.1, and high-dose (150 μg / kg) fusion protein 2.1 on brain tissue after ischemia-reperfusion in rat tMCAO model.

[0031] Detailed Description of the Invention

[0032] Although the present application will disclose various aspects and embodiments below, it is obvious that various equivalent changes and modifications can be made to it by those skilled in the art without violating the spirit and scope of the subject matter of the present application. The various aspects and embodiments disclosed in the present application are only for illustration and are not intended to limit the present application. The actual scope of protection of the present application is subject to the claims. Unless otherwise noted, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present application belongs. All references, patents, and patent applications cited in this application are incorporated herein by reference in their entirety.

[0033] definition

[0034] When referring to a polypeptide or protein, the term "wild-type" as used in this application refers to a naturally occurring polypeptide or protein that does not include an artificial mutation, insertion, deletion, or modification at one or more amino acid positions; when referring to a nucleic acid, nucleotide, or polynucleotide, the term "wild-type" as used in this application refers to a naturally occurring nucleic acid, nucleotide, or polynucleotide that does not include an artificial mutation, insertion, deletion, or modification at one or more nucleotide positions. However, polynucleotides encoding wild-type polypeptides are not limited to naturally occurring polynucleotides, but also include any polynucleotides encoding wild-type polypeptides (e.g., artificially synthesized polynucleotides).

[0035] The term "subject" as used in this application includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals. A "subject" may also be a livestock animal, such as cattle, pigs, sheep, poultry, and horses; or a rodent, such as a rat or mouse; or a primate, such as an ape, monkey, chimpanzee, gorilla, orangutan, or baboon; or a domesticated animal, such as a dog or cat. A "subject" may be male or female and may be elderly, adult, adolescent, child, or infant.

[0036] As used herein, the terms "protein," "polypeptide," and "peptide" are used interchangeably to refer to polymers of amino acids. The proteins, polypeptides, or peptides described herein may contain natural amino acids, non-natural amino acids, or amino acid analogs or mimetics. The proteins, polypeptides, or peptides described herein may be obtained by any method known in the art, such as, but not limited to, natural isolation, recombinant expression, or chemical synthesis.

[0037] As used herein, the term "amino acid" refers to an organic compound containing amino (-NH2) and carboxyl (-COOH) functional groups, as well as side chains unique to each amino acid. Amino acid names are also represented in this application using standard single-letter or three-letter codes, which are summarized below:

[0038] In the present application, when "conservative substitution" is used for amino acid sequences, it refers to replacing one amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between amino acid residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between amino acid residues with acidic side chains (e.g., Asp, Glu), between amino acid residues with basic side chains (e.g., His, Lys, and Arg), or between amino acid residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions generally do not cause significant changes in the conformational structure of the protein and therefore can retain the biological activity of the protein.

[0039] As used herein, the term "homologous" refers to a nucleic acid sequence (or its complement) or an amino acid sequence that has at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with another sequence when optimally aligned.

[0040] When "percent (%) sequence identity" is used for amino acid sequences (or nucleic acid sequences), it refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence, after alignment and, if necessary, introduction of gaps to maximize the number of identical amino acids (or nucleic acids). In other words, the percentage (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) identical to the reference sequence to be compared by the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence (whichever is shorter). Conservative substitutions of amino acid residues may or may not be considered identical residues. Sequences can be aligned to determine the percentage sequence identity of amino acid (or nucleic acid) sequences using tools disclosed in the art, such as BLASTN, BLASTp (National Center for Biotechnology Information (NCBI), see also Altschul SF et al., J. Mol. Biol., 215: 403-410 (1990); Stephen F et al., Nucleic Acids Res., 25: 3389-3402 (1997)), ClustalW2 (European Bioinformatics Institute website, see Higgins DG et al., Methods in Enzymology, 266: 383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters of the tool or appropriately adjust the parameters according to the needs of the comparison, for example, by selecting a suitable algorithm.

[0041] "Isolated" material has been artificially altered from its natural state. If an "isolated" composition or substance occurs in nature, it has been altered or removed from its original state, or both. For example, a polynucleotide or polypeptide naturally present in a living animal is not "isolated," but it can be considered "isolated" if it is sufficiently separated from the materials with which it coexists in nature and is present in a substantially pure state. An "isolated nucleic acid sequence" refers to the sequence of an isolated nucleic acid molecule. In some embodiments, "isolated KLK1" refers to a KLK1 protein or a nucleic acid molecule encoding a KLK1 protein that is at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure, wherein purity is determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatography (e.g., ion exchange chromatography or reverse phase HPLC).

[0042] The term "vector" in this application refers to a vehicle into which a genetic element can be operatively inserted and into which the genetic element is expressed, for example, to produce a protein, RNA or DNA encoded by the genetic element, or to replicate the genetic element. A vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries is expressed in the host cell. For example, vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector can also contain a replication initiation site. A vector can also include components that assist its entry into cells, including but not limited to viral particles, liposomes or protein coats. A vector can be an expression vector or a cloning vector. The vectors provided herein (e.g., expression vectors) contain the nucleic acid sequence encoding the KLK1 fusion protein described herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.

[0043] As used herein, "treatment" or "treatment" of a disease, disorder or condition includes preventing or alleviating a disease, disorder or condition, reducing the rate of onset or development of a disease, disorder or condition, reducing the risk of developing a disease, disorder or condition, preventing or delaying the development of symptoms associated with a disease, disorder or condition, reducing or stopping symptoms associated with a disease, disorder or condition, producing complete or partial reversal of a disease, disorder or condition, curing a disease, disorder or condition, or a combination of the above.

[0044] The term "pharmaceutically acceptable" means that the specified carrier, vehicle, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients that make up the formulation and physiologically compatible with the recipient thereof.

[0045] Method for preparing fusion protein

[0046] In one aspect, the present application provides a method for preparing a fusion protein comprising tissue kallikrein 1 (KLK1), the method comprising step (1): placing a recombinant vector containing a nucleic acid molecule encoding the fusion protein in a suitable protein expression system for expression, wherein the fusion protein comprises a masking peptide, a first polypeptide and a second polypeptide, the masking peptide, the first polypeptide and the second polypeptide are directly linked or covalently linked via a linker, and,

[0047] d) the masking peptide has an amino acid sequence that can be specifically recognized and cleaved by a protease;

[0048] e) the first polypeptide comprises KLK1 or a variant thereof; and

[0049] f) the second polypeptide is used to extend the half-life of the first polypeptide.

[0050] "Tissue kallikrein 1," "Kallikrein-1," "KLK1 protein," or "KLK1 enzyme" is a serine protease encoded by the KLK1 gene and a member of the tissue kallikrein family that releases active kinin from kininogen. In this application, "tissue kallikrein 1," "Kallikrein-1," "KLK1 protein," and "KLK1 enzyme" are used interchangeably. The KLK1 protein described herein may refer to a KLK1 protein derived from any vertebrate source, including mammals, such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats). An exemplary sequence of a human KLK1 protein is shown in UniProtKB Accession No. P06870 (its amino acid sequence is shown in SEQ ID NO: 1). An exemplary sequence of a mouse KLK1 protein is shown in UniProtKB Accession No. P15947 (its amino acid sequence is shown in SEQ ID NO: 3).

[0051] In the present application, KLK1 protein includes any variant, conformation, isoform or species homolog of KLK1 protein naturally expressed by cells or expressed by recombinant cells transfected with KLK1 gene. For example, "KLK1 protein" in the present application includes: 1) a naturally unprocessed form of KLK1 protein, a full-length KLK1 protein or a naturally occurring KLK1 variant (e.g., a splice variant or allelic variant); 2) any form of KLK1 protein produced by processing in cells; or 3) a full-length, fragment (e.g., truncated form, extracellular / transmembrane domain) or modified form (e.g., mutant form, glycosylated / pegylated, immunofluorescent fusion form) of KLK1 protein produced by recombinant methods.

[0052] In this application, a "fusion protein comprising KLK1" (also referred to herein as a "KLK1 fusion protein") refers to a protein in which a KLK1 protein is operably linked to at least one other heterologous peptide to form a single chain. The KLK1 fusion protein described herein can be produced by recombinant methods, chemical methods, or any other method.

[0053] In certain embodiments, the method for preparing a fusion protein comprising KLK1 provided herein comprises step (1). In certain embodiments, the method for preparing a fusion protein comprising KLK1 provided herein further comprises step (2). These two steps are described in detail below.

[0054] Step (1)

[0055] Step (1) is to place a recombinant vector containing a nucleic acid molecule encoding a KLK1 fusion protein in a suitable protein expression system for expression, wherein the KLK1 fusion protein comprises a masking peptide, a first polypeptide, and a second polypeptide, wherein the masking peptide, the first polypeptide, and the second polypeptide are directly connected or covalently connected via a linker, and the masking peptide has an amino acid sequence that can be specifically recognized and cleaved by a protease; the first polypeptide comprises KLK1 or a variant thereof; and the second polypeptide is used to extend the half-life of the first polypeptide.

[0056] a) Masking peptides

[0057] In certain embodiments, the masking peptides described herein have an amino acid sequence that can be specifically recognized and cleaved by a protease. In this application, a "masking peptide" refers to a peptide that is covalently linked to the first or second polypeptide described herein, either directly or through a linker, thereby blocking, shielding, inhibiting, or otherwise preventing the binding of the first or second polypeptide described herein to its receptor or substrate.

[0058] Because the KLK1 enzyme is a mammalian serine protease, it may interfere with the normal function of the protein expression system (e.g., host cells) expressing the KLK1 fusion protein, thereby causing the KLK1 fusion protein to lose activity or significantly reduce its activity after expression. The inventors of the present invention unexpectedly discovered that when, during the process of preparing a KLK1 fusion protein, the protein expression system expresses a KLK1 fusion protein containing a masking peptide, and then performs a specific enzymatic cleavage reaction, the KLK1 fusion protein obtained after removing the masking peptide has higher KLK1 enzyme activity. Without being bound by any theory, it is believed that the masking peptide helps to enhance the expression and / or activity of the KLK1 fusion protein, possibly because the presence of the masking peptide avoids or reduces the interference of the KLK1 fusion protein with the protein expression system.

[0059] In certain embodiments, the masking peptide has an amino acid sequence that can be specifically recognized and cleaved by a protease and can block, obstruct, or inhibit the binding of the KLK1 protein to its substrate. Those skilled in the art would reasonably anticipate that as long as the masking peptide possesses the aforementioned properties, it can be used in the methods for preparing the KLK1 fusion protein described herein.

[0060] In certain embodiments, the masking peptide is selected from the group consisting of an enterokinase (EK) recognition sequence, a thrombin recognition sequence, a coagulation factor Xa recognition sequence, a tobacco etch virus (TEV) protease recognition sequence, a SUMO protease recognition sequence, and a transpeptidase (sortase) recognition sequence. In certain embodiments, the masking peptide is an enterokinase (EK) recognition sequence. In certain embodiments, the enterokinase (EK) recognition sequence is SEQ ID NO:4 (DDDDK) or SEQ ID NO:5 (VDDDDK). In certain embodiments, the masking peptide comprises the amino acid sequence shown in SEQ ID NO:5. In certain embodiments, the amino acid sequence of the masking peptide is shown in SEQ ID NO:5.

[0061] In certain embodiments, the masking peptide is a thrombin recognition sequence. In certain embodiments, the amino acid sequence of the thrombin recognition sequence is shown in SEQ ID NO:6.

[0062] In certain embodiments, the masking peptide is a coagulation factor Xa recognition sequence. In certain embodiments, the amino acid sequence of the coagulation factor Xa recognition sequence is shown in SEQ ID NO:7.

[0063] In some embodiments, the masking peptide is a tobacco etch virus (TEV) protease recognition sequence. In some embodiments, the amino acid sequence of the tobacco etch virus (TEV) protease recognition sequence is shown in SEQ ID NO:8.

[0064] In certain embodiments, the masking peptide is a SUMO protease recognition sequence. In certain embodiments, the amino acid sequence of the SUMO protease recognition sequence is shown in SEQ ID NO:9.

[0065] In certain embodiments, the masking peptide is a sortase recognition sequence. In certain embodiments, the amino acid sequence of the sortase recognition sequence is shown in SEQ ID NO:10.

[0066] In certain embodiments, the half-life of the KLK1 fusion protein obtained after removal of the masking peptide by specific enzymatic cleavage is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

[0067] b) First polypeptide

[0068] In certain embodiments, the first polypeptide is a human KLK1 protein or a variant thereof. A KLK1 "variant" refers to a KLK1 polypeptide that has an amino acid mutation, deletion, insertion, or modification compared to the amino acid sequence of a wild-type KLK1 protein, but still retains the function or activity of KLK1. In this application, "human KLK1" is also referred to as "hKLK1."

[0069] In certain embodiments, the amino acid sequence of wild-type human KLK1 protein is as shown in SEQ ID NO: 1, wherein the polypeptide formed by amino acids 1-18 of SEQ ID NO: 1 is a signal peptide, the polypeptide formed by amino acids 19-24 is a propeptide (also referred to as "prepro" in this application), and the polypeptide formed by amino acids 25-262 is the mature protein of KLK1. In certain embodiments, the amino acid sequence of SEQ ID NO: 1, excluding the N-terminal signal peptide and propeptide, is as shown in SEQ ID NO: 2.

[0070] In certain embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2. In certain embodiments, the amino acid sequence of the first polypeptide is set forth in SEQ ID NO: 2. In certain embodiments, the amino acid sequence of the first polypeptide has at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, while still retaining the function or activity of KLK1.

[0071] The KLK1 protein or variant thereof of the present application may also contain non-natural amino acids, provided that the activity is not affected. Non-natural amino acids include, for example, β-fluoroalanine, 1-methylhistidine, γ-methyleneglutamate, α-methylleucine, 4,5-dehydrolysine, hydroxyproline, 3-fluorophenylalanine, 3-aminotyrosine, 4-methyltryptophan, and the like.

[0072] The KLK1 protein or variant thereof described herein can also be modified using methods known in the art, such as, but not limited to, PEGylation, glycosylation, amino-terminal modification, fatty acylation, carboxyl-terminal modification, phosphorylation, methylation, etc. It will be appreciated by those skilled in the art that the KLK1 protein or variant thereof described herein can retain substantially similar functions to the KLK1 protein after being modified using methods known in the art.

[0073] In certain embodiments, the KLK1 protein variants described herein have conservative amino acid substitutions at one or more positions based on the amino acid sequence as set forth in SEQ ID NO: 2, but still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants provided herein have at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to the amino acid sequence as set forth in SEQ ID NO: 2, and still retain the function or activity of KLK1.

[0074] In certain embodiments, the KLK1 protein variants described herein have a mutation at at least one amino acid position selected from the group consisting of positions 121, 162, and 164 of SEQ ID NO: 2. In certain embodiments, the KLK1 protein variants described herein have a mutation at at least one amino acid position selected from the group consisting of positions 121, 162, and 164 of SEQ ID NO: 2, and still retain the function or activity of KLK1.

[0075] In certain embodiments, the KLK1 protein variants described herein have a mutation at position 121 corresponding to SEQ ID NO: 2. In certain embodiments, the KLK1 protein variants described herein have a mutation at position 121 corresponding to SEQ ID NO: 2 and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein have a mutation at glutamic acid (E) corresponding to position 121 of SEQ ID NO: 2. In certain embodiments, the KLK1 protein variants described herein have a mutation at glutamic acid (E) corresponding to position 121 of SEQ ID NO: 2 and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein have a mutation at glutamic acid (E) corresponding to position 121 of SEQ ID NO: 2 to glutamine (Q), also referred to herein as "E121Q". In certain embodiments, the KLK1 protein variants described herein have the E121Q mutation and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variant described herein comprises the amino acid sequence shown in SEQ ID NO: 117. In certain embodiments, the amino acid sequence of the KLK1 protein variant described herein is shown in SEQ ID NO: 117.

[0076] In certain embodiments, the KLK1 protein variants described herein have a mutation at position 162 corresponding to SEQ ID NO: 2. In certain embodiments, the KLK1 protein variants described herein have a mutation at position 162 corresponding to SEQ ID NO: 2 and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein have a mutation at lysine (K) corresponding to position 162 of SEQ ID NO: 2. In certain embodiments, the KLK1 protein variants described herein have a mutation at lysine (K) corresponding to position 162 of SEQ ID NO: 2 and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein have a mutation at lysine (K) corresponding to position 162 of SEQ ID NO: 2 to glutamic acid (E), also referred to herein as "K162E." In certain embodiments, the KLK1 protein variants described herein have the K162E mutation and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variant described herein comprises the amino acid sequence shown in SEQ ID NO: 118. In certain embodiments, the amino acid sequence of the KLK1 protein variant described herein is shown in SEQ ID NO: 118.

[0077] In certain embodiments, the KLK1 protein variants described herein have a mutation at position 164 corresponding to SEQ ID NO: 2. In certain embodiments, the KLK1 protein variants described herein have a mutation at position 164 corresponding to SEQ ID NO: 2 and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein have a mutation at alanine (A) corresponding to position 164 of SEQ ID NO: 2. In certain embodiments, the KLK1 protein variants described herein have a mutation at alanine (A) corresponding to position 164 of SEQ ID NO: 2 and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein have a mutation at alanine (A) corresponding to position 164 of SEQ ID NO: 2 to valine (V), also referred to herein as "A164V". In certain embodiments, the KLK1 protein variants described herein have an A164V mutation and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variant described herein comprises the amino acid sequence shown in SEQ ID NO: 119. In certain embodiments, the amino acid sequence of the KLK1 protein variant described herein is shown in SEQ ID NO: 119.

[0078] In certain embodiments, the KLK1 protein variants described herein have mutations at positions 121 and 162 corresponding to SEQ ID NO: 2. In certain embodiments, the KLK1 protein variants described herein have mutations at positions 121 and 162 corresponding to SEQ ID NO: 2 and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein have mutations at glutamic acid (E) at position 121 and lysine (K) at position 162 corresponding to SEQ ID NO: 2. In certain embodiments, the KLK1 protein variants described herein have mutations at glutamic acid (E) at position 121 and lysine (K) at position 162 corresponding to SEQ ID NO: 2 and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein have a glutamic acid (E) mutation at position 121 corresponding to SEQ ID NO: 2 to a glutamine (Q), and a lysine (K) mutation at position 162 corresponding to SEQ ID NO: 2 to a glutamic acid (E), also referred to herein as "E121Q+K162E". In certain embodiments, the KLK1 protein variants described herein have the E121Q+K162E mutations and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein comprise the amino acid sequence set forth in SEQ ID NO: 120. In certain embodiments, the amino acid sequence of the KLK1 protein variants described herein is set forth in SEQ ID NO: 120.

[0079] In certain embodiments, the KLK1 protein variants described herein have mutations at positions 162 and 164 corresponding to SEQ ID NO: 2. In certain embodiments, the KLK1 protein variants described herein have mutations at positions 162 and 164 corresponding to SEQ ID NO: 2 and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein have mutations at lysine (K) at position 162 and alanine (A) at position 164 corresponding to SEQ ID NO: 2. In certain embodiments, the KLK1 protein variants described herein have mutations at lysine (K) at position 162 and alanine (A) at position 164 corresponding to SEQ ID NO: 2 and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein have a lysine (K) mutation at position 162 corresponding to SEQ ID NO: 2 to a glutamic acid (E), and an alanine (A) mutation at position 164 corresponding to SEQ ID NO: 2 to a valine (V), also referred to herein as "K162E+A164V". In certain embodiments, the KLK1 protein variants described herein have the K162E+A164V mutations and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein comprise the amino acid sequence set forth in SEQ ID NO: 121. In certain embodiments, the amino acid sequence of the KLK1 protein variants described herein is set forth in SEQ ID NO: 121.

[0080] In certain embodiments, the KLK1 protein variants described herein have mutations at positions 121 and 164 corresponding to SEQ ID NO: 2. In certain embodiments, the KLK1 protein variants described herein have mutations at positions 121 and 164 corresponding to SEQ ID NO: 2 and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein have mutations at positions 121 and 164 corresponding to SEQ ID NO: 2 to glutamic acid (E) and alanine (A) at position 164 corresponding to SEQ ID NO: 2. In certain embodiments, the KLK1 protein variants described herein have mutations at positions 121 and 164 corresponding to SEQ ID NO: 2 to glutamic acid (E) and alanine (A) at position 164 corresponding to SEQ ID NO: 2 and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein have a mutation of glutamic acid (E) at position 121 corresponding to SEQ ID NO: 2 to glutamine (Q), and alanine (A) at position 164 to valine (V), also referred to herein as "E121Q+A164V". In certain embodiments, the KLK1 protein variants described herein have the E121Q+A164V mutations and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein comprise the amino acid sequence set forth in SEQ ID NO: 115. In certain embodiments, the amino acid sequence of the KLK1 protein variants described herein is set forth in SEQ ID NO: 115.

[0081] In certain embodiments, the KLK1 protein variants described herein have mutations at positions 121, 162, and 164 corresponding to SEQ ID NO: 2. In certain embodiments, the KLK1 protein variants described herein have mutations at positions 121, 162, and 164 corresponding to SEQ ID NO: 2 and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein have mutations at glutamic acid (E) at position 121, lysine (K) at position 162, and alanine (A) at position 164 corresponding to SEQ ID NO: 2. In certain embodiments, the KLK1 protein variants described herein have mutations at glutamic acid (E) at position 121, lysine (K) at position 162, and alanine (A) at position 164 corresponding to SEQ ID NO: 2 and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein have a mutation of glutamic acid (E) at position 121 corresponding to SEQ ID NO: 2 to glutamine (Q), a mutation of lysine (K) at position 162 to glutamic acid (E), and a mutation of alanine (A) at position 164 corresponding to SEQ ID NO: 2 to valine (V), also referred to herein as "E121Q+K162E+A164V". In certain embodiments, the KLK1 protein variants described herein have the E121Q+K162E+A164V mutations and still retain the function or activity of KLK1. In certain embodiments, the KLK1 protein variants described herein comprise the amino acid sequence set forth in SEQ ID NO: 122. In certain embodiments, the amino acid sequence of the KLK1 protein variants described herein is set forth in SEQ ID NO: 122.

[0082] c) Second polypeptide

[0083] In certain embodiments, the second polypeptide is used to extend the half-life of the first polypeptide. In certain embodiments, the second polypeptide comprises an amino acid sequence that is used to extend the half-life of the first polypeptide. In certain embodiments, the second polypeptide comprises an amino acid sequence that is used to extend the half-life of the first polypeptide in a subject.

[0084] In certain embodiments, the second polypeptide is an Fc domain or albumin. In certain embodiments, the Fc domain comprises a hinge region. In certain embodiments, the Fc domain comprises a lower hinge region. In certain embodiments, the Fc domain comprises a core hinge region and a lower hinge region. In certain embodiments, the Fc domain comprises an upper hinge region, a core hinge region, and a lower hinge region. In certain embodiments, the Fc domain does not comprise a hinge region. In certain embodiments, the Fc domain is derived from a human IgG Fc domain. In certain embodiments, the Fc domain is derived from a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, or a human IgG4 Fc domain. In certain embodiments, the Fc domain is derived from a human IgG1 Fc domain or a human IgG4 Fc domain.

[0085] In certain embodiments, the Fc domain is derived from a human IgG1 Fc domain. In certain embodiments, the Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the Fc domain consists of the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the amino acid sequence of the Fc domain has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the amino acid sequence of the Fc domain has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 11, but still retains the function of the Fc domain shown in SEQ ID NO: 11 (e.g., being able to extend the half-life of the first polypeptide).

[0086] In certain embodiments, the Fc domain is derived from a human IgG4 Fc domain. In certain embodiments, the Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the Fc domain consists of the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the amino acid sequence of the Fc domain has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the amino acid sequence of the Fc domain has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 12, but still retains the function of the Fc domain shown in SEQ ID NO: 12 (e.g., being able to extend the half-life of the first polypeptide).

[0087] In certain embodiments, the Fc domain comprises a mutation at one or more (e.g., two, three, four, five or more) amino acid positions. In certain embodiments, the Fc domain has an amino acid mutation at position 252 (numbered according to the EU system) of the amino acid sequence corresponding to a natural human IgG Fc domain (such as shown in SEQ ID NO: 11 or SEQ ID NO: 12). In certain embodiments, the Fc domain has a methionine mutation at position 252 of the amino acid sequence corresponding to a natural human IgG Fc domain (such as shown in SEQ ID NO: 11 or SEQ ID NO: 12) to a tyrosine (also referred to herein as "M252Y").

[0088] In certain embodiments, the Fc domain has an amino acid mutation at position 254 (according to the EU numbering system) of the amino acid sequence corresponding to a native human IgG Fc domain (as shown in SEQ ID NO: 11 or SEQ ID NO: 12). In certain embodiments, the Fc domain has an amino acid mutation at position 254 (according to the EU numbering system) of the amino acid sequence corresponding to a native human IgG Fc domain (as shown in SEQ ID NO: 11 or SEQ ID NO: 12) of the serine at position 254 to a threonine (also referred to herein as "S254T").

[0089] In certain embodiments, the Fc domain has an amino acid mutation at position 256 (according to the EU numbering system) of the amino acid sequence corresponding to a native human IgG Fc domain (as shown in SEQ ID NO: 11 or SEQ ID NO: 12). In certain embodiments, the Fc domain has an amino acid mutation at position 256 (according to the EU numbering system) of the amino acid sequence corresponding to a native human IgG Fc domain (as shown in SEQ ID NO: 11 or SEQ ID NO: 12) of the threonine at position 256 to glutamic acid (also referred to herein as "T256E").

[0090] In certain embodiments, the Fc domain comprises one, two, or three mutations selected from the group consisting of: M252Y, S254T, and T256E. In certain embodiments, the Fc domain comprises M252Y, S254T, and T256E mutations.

[0091] In certain embodiments, the amino acid sequence of the Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the Fc domain consists of the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the amino acid sequence of the Fc domain has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the amino acid sequence of the Fc domain has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 13, but still retains the function of the Fc domain shown in SEQ ID NO: 13 (e.g., being able to extend the half-life of the first polypeptide).

[0092] In certain embodiments, the amino acid sequence of the Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the Fc domain consists of the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the amino acid sequence of the Fc domain has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the amino acid sequence of the Fc domain has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 14, but still retains the function of the Fc domain shown in SEQ ID NO: 14 (e.g., being able to extend the half-life of the first polypeptide).

[0093] In certain embodiments, the second polypeptide is albumin. In certain embodiments, the amino acid sequence of the albumin is as shown in SEQ ID NO: 15. In certain embodiments, the albumin comprises one or more domains of human serum albumin. In certain embodiments, the albumin comprises the D3 domain of human serum albumin.

[0094] In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:2, and a second polypeptide as set forth in SEQ ID NO:11. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:2, and a second polypeptide as set forth in SEQ ID NO:12. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:2, and a second polypeptide as set forth in SEQ ID NO:13. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:2, and a second polypeptide as set forth in SEQ ID NO:14. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:2, and a second polypeptide as set forth in SEQ ID NO:15.

[0095] In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 115, and a second polypeptide as set forth in SEQ ID NO: 11. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 115, and a second polypeptide as set forth in SEQ ID NO: 12. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 115, and a second polypeptide as set forth in SEQ ID NO: 13. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 115, and a second polypeptide as set forth in SEQ ID NO: 14. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 115, and a second polypeptide as set forth in SEQ ID NO: 15.

[0096] In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 117, and a second polypeptide as set forth in SEQ ID NO: 11. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 117, and a second polypeptide as set forth in SEQ ID NO: 12. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 117, and a second polypeptide as set forth in SEQ ID NO: 13. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 117, and a second polypeptide as set forth in SEQ ID NO: 14. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 117, and a second polypeptide as set forth in SEQ ID NO: 15.

[0097] In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:118, and a second polypeptide as set forth in SEQ ID NO:11. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:118, and a second polypeptide as set forth in SEQ ID NO:12. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:118, and a second polypeptide as set forth in SEQ ID NO:13. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:118, and a second polypeptide as set forth in SEQ ID NO:14. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:118, and a second polypeptide as set forth in SEQ ID NO:15.

[0098] In certain embodiments, the KLK1 fusion proteins described herein comprise a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:119, and a second polypeptide as set forth in SEQ ID NO:11. In certain embodiments, the KLK1 fusion proteins described herein comprise a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:119, and a second polypeptide as set forth in SEQ ID NO:12. In certain embodiments, the KLK1 fusion proteins described herein comprise a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:119, and a second polypeptide as set forth in SEQ ID NO:13. In certain embodiments, the KLK1 fusion proteins described herein comprise a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:119, and a second polypeptide as set forth in SEQ ID NO:14. In certain embodiments, the KLK1 fusion proteins described herein comprise a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:119, and a second polypeptide as set forth in SEQ ID NO:15.

[0099] In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 120, and a second polypeptide as set forth in SEQ ID NO: 11. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 120, and a second polypeptide as set forth in SEQ ID NO: 12. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 120, and a second polypeptide as set forth in SEQ ID NO: 13. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 120, and a second polypeptide as set forth in SEQ ID NO: 14. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 120, and a second polypeptide as set forth in SEQ ID NO: 15.

[0100] In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 121, and a second polypeptide as set forth in SEQ ID NO: 11. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 121, and a second polypeptide as set forth in SEQ ID NO: 12. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 121, and a second polypeptide as set forth in SEQ ID NO: 13. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 121, and a second polypeptide as set forth in SEQ ID NO: 14. In certain embodiments, the KLK1 fusion protein described herein comprises a masking peptide as set forth in SEQ ID NO: 5, a first polypeptide as set forth in SEQ ID NO: 121, and a second polypeptide as set forth in SEQ ID NO: 15.

[0101] In certain embodiments, the KLK1 fusion proteins described herein comprise a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:122, and a second polypeptide as set forth in SEQ ID NO:11. In certain embodiments, the KLK1 fusion proteins described herein comprise a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:122, and a second polypeptide as set forth in SEQ ID NO:12. In certain embodiments, the KLK1 fusion proteins described herein comprise a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:122, and a second polypeptide as set forth in SEQ ID NO:13. In certain embodiments, the KLK1 fusion proteins described herein comprise a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:122, and a second polypeptide as set forth in SEQ ID NO:14. In certain embodiments, the KLK1 fusion proteins described herein comprise a masking peptide as set forth in SEQ ID NO:5, a first polypeptide as set forth in SEQ ID NO:122, and a second polypeptide as set forth in SEQ ID NO:15.

[0102] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 11.

[0103] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 12.

[0104] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 13.

[0105] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 14.

[0106] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 15.

[0107] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 2, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 11.

[0108] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 2, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 12.

[0109] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 2, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 13.

[0110] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 2, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 14.

[0111] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 2, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 15.

[0112] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 115, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 11.

[0113] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 115, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 12.

[0114] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 115, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 13.

[0115] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 115, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 14.

[0116] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 115, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 15.

[0117] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 122, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 11.

[0118] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 122, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 12.

[0119] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 122, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 13.

[0120] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 122, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 14.

[0121] In certain embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In certain embodiments, the second polypeptide is located at the C-terminus of the first polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide. In certain embodiments, the KLK1 fusion protein comprises, from the N-terminus to the C-terminus, a masking peptide, a first polypeptide, and a second polypeptide, wherein the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 122, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 15.

[0122] In certain embodiments, the KLK1 fusion protein described herein comprises the following structure from N-terminus to C-terminus:

[0123] MP-L1-P1-L2-P2,

[0124] Wherein, MP represents a masking peptide, L1 represents a bond or a first linker, P1 represents a first polypeptide, L2 represents a bond or a second linker, and P2 represents a second polypeptide.

[0125] In certain embodiments, MP represents the masking peptide, L1 represents a bond, P1 represents the first polypeptide, L2 represents a second linker, and P2 represents the second polypeptide.

[0126] In certain embodiments, MP represents the enterokinase recognition sequence, L1 represents a bond, P1 represents human KLK1 protein, L2 represents a second linker, and P2 represents an Fc domain.

[0127] In certain embodiments, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:11.

[0128] In certain embodiments, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:12.

[0129] In certain embodiments, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:13.

[0130] In certain embodiments, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:14.

[0131] In certain embodiments, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:15.

[0132] In certain embodiments, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:11.

[0133] In certain embodiments, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:12.

[0134] In certain embodiments, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:13.

[0135] In certain embodiments, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:14.

[0136] In certain embodiments, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:15.

[0137] In certain embodiments, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:11.

[0138] In certain embodiments, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:12.

[0139] In certain embodiments, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:13.

[0140] In certain embodiments, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:14.

[0141] In certain embodiments, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:15.

[0142] In certain embodiments, the KLK1 fusion protein comprises the amino acid sequence of SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 113, or SEQ ID NO: 127. In certain embodiments, the KLK1 fusion protein consists of the amino acid sequence of SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 113, or SEQ ID NO: 127. In certain embodiments, the amino acid sequence of the KLK1 fusion protein is at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 113, or SEQ ID NO: 127.In certain embodiments, the amino acid sequence of the KLK1 fusion protein is the same as SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:113 or SEQ ID NO:114. The amino acid sequence of NO: 127 has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but still retains the function or activity of KLK1 after EK cleavage (e.g., capable of catalyzing the release of active kinin from kininogen).

[0143] d) Labels

[0144] In certain embodiments, the KLK1 fusion protein described herein further comprises a tag. In certain embodiments, the tag is selected from the group consisting of a fluorescent tag, a luminescent tag, a purification tag, and a chromogenic tag. In certain embodiments, the tag is a purification tag. In certain embodiments, the tag is selected from the group consisting of a HIS tag, a GST tag, an MBP tag, a Myc tag, a NusA tag, a SUMO tag, a FLAG tag, an Avi tag, a Halo tag, a SNAP tag, a Strep-II tag, and a TrX tag. In certain embodiments, the tag is a HIS tag. In certain embodiments, the tag is a HIS tag comprising 5, 6, 7, 8, 9, 10, or more histidines. In certain embodiments, the amino acid sequence of the tag is as shown in SEQ ID NO: 16.

[0145] In certain embodiments, the KLK1 fusion protein described herein comprises the following structure from N-terminus to C-terminus:

[0146] LB-MP-L1-P1-L2-P2,

[0147] Wherein, LB represents absence or a tag, MP represents a masking peptide, L1 represents a bond or a first linker, P1 represents a first polypeptide, L2 represents a bond or a second linker, and P2 represents a second polypeptide.

[0148] In certain embodiments, LB represents a tag, MP represents the masking peptide, L1 represents a bond, P1 represents the first polypeptide, L2 represents a second linker, and P2 represents the second polypeptide.

[0149] In certain embodiments, LB represents a HIS tag, MP represents the enterokinase recognition sequence, L1 represents a bond, P1 represents a human KLK1 protein, L2 represents a second linker, and P2 represents an Fc domain.

[0150] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121 or SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:11.

[0151] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121 or SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:12.

[0152] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121 or SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:13.

[0153] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121 or SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:14.

[0154] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121 or SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:15.

[0155] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 11.

[0156] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 12.

[0157] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 13.

[0158] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 14.

[0159] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:15.

[0160] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:11.

[0161] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 12.

[0162] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 13.

[0163] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 14.

[0164] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 15.

[0165] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:11.

[0166] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 12.

[0167] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:13.

[0168] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 14.

[0169] In certain embodiments, LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:15.

[0170] e) Linker

[0171] In certain embodiments, the first linker and the second linker are independently selected from the group consisting of a cleavable linker, a non-cleavable linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

[0172] In this application, the term "linker" refers to any chemical moiety that can covalently link one part to another. For example, a "linker" can be an artificial amino acid sequence having 1, 2, 3, 4, or 5 amino acid residues, or a length between 5 and 15, 20, 30, 50, or more amino acid residues, connected by peptide bonds, and used to connect one or more polypeptides. A linker may or may not have a secondary structure. Linker sequences are known in the art, for example, see Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); Poljak et al., Structure 2: 1121-1123 (1994).

[0173] In the present application, the term "cleavable linker" refers to a linker that is sensitive to factors such as proteases, pH or chemicals in the body and is easily cleaved in the presence of the above factors.

[0174] In this application, the term "non-cleavable linker" refers to a linker that is stable to factors such as proteases, pH or chemicals in the body and is not easily cleaved.

[0175] In the present application, the term "flexible linker" refers to a linker that can increase spatial extensibility when connecting different protein components, so that the spatial folding and conformation of the protein components are not affected by each other as much as possible.

[0176] In the present application, the term "rigid linker" refers to a linker that can maintain a fixed distance between protein components when connecting different protein components.

[0177] In the present application, the term "helical linker" refers to a linker in which the rigid unit can form a helix (such as an α-helix) within itself or with the same adjacent sequence, so that the formed fusion protein has a relatively stable three-dimensional conformation.

[0178] In this application, the term "non-helical linker" refers to a linker that is unable to form a helical structure.

[0179] In certain embodiments, the flexible linker comprises a linker comprising glycine and serine. In certain embodiments, the linker comprising glycine and serine comprises one, two, three, four or more repeats of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22. In certain embodiments, the first linker or the second linker comprises an amino acid sequence as shown in SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22. In certain embodiments, the first linker or the second linker consists of an amino acid sequence as shown in SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22.

[0180] In certain embodiments, the rigid linker comprises one, two, three, four or more repeats as shown in SEQ ID NO: 23. In certain embodiments, the first linker or the second linker comprises the amino acid sequence as shown in SEQ ID NO: 23. In certain embodiments, the first linker or the second linker consists of the amino acid sequence as shown in SEQ ID NO: 23.

[0181] In certain embodiments, the KLK1 fusion protein comprises the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 112, or SEQ ID NO: 126. In certain embodiments, the KLK1 fusion protein consists of the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 112, or SEQ ID NO: 126. In certain embodiments, the amino acid sequence of the KLK1 fusion protein is at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 112, or SEQ ID NO: 126.In certain embodiments, the amino acid sequence of the KLK1 fusion protein is the same as SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 112 or SEQ ID NO: 113. The amino acid sequence shown in NO: 126 has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but still retains the function or activity of KLK1 after EK cleavage (e.g., capable of catalyzing the release of active kinin from kininogen).

[0182] f) Signal peptide

[0183] In certain embodiments, the KLK1 fusion protein further comprises a signal peptide.

[0184] The term "signal peptide" as used in this application refers to a sequence of amino acid residues that can participate in the secretion or directed transport of the mature or precursor form of a protein. The signal peptide is usually located at the N-terminus of the precursor or mature protein sequence. The signal peptide can be endogenous or exogenous. Signal peptides are generally not present in mature proteins. Typically, after protein transport, the signal peptide is cleaved from the protein by a signal peptidase.

[0185] In certain embodiments, the signal peptide is exogenous. In certain embodiments, the signal peptide is located at the N-terminus of the KLK1 fusion protein. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO:24 or SEQ ID NO:25. In certain embodiments, the signal peptide has an amino acid sequence set forth in SEQ ID NO:24 or SEQ ID NO:25, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:24 or SEQ ID NO:25. In certain embodiments, the signal peptide consists of the amino acid sequence set forth in SEQ ID NO:24. In certain embodiments, the signal peptide consists of the amino acid sequence set forth in SEQ ID NO:25.

[0186] In certain embodiments, the KLK1 fusion protein described herein comprises the following structure from N-terminus to C-terminus:

[0187] SP-L3-LB-MP-L1-P1-L2-P2,

[0188] Wherein, SP represents absence or signal peptide, L3 represents bond or third linker, LB represents absence or tag, MP represents masking peptide, L1 represents bond or first linker, P1 represents first polypeptide, L2 represents bond or second linker, and P2 represents second polypeptide.

[0189] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO: 24 or SEQ ID NO: 25, L3 represents a linker formed by two glycines (GG), LB represents a tag, MP represents a masking peptide, L1 represents a bond, P1 represents the first polypeptide, L2 represents a second linker, and P2 represents the second polypeptide.

[0190] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO: 24 or SEQ ID NO: 25, L3 represents a linker formed by two glycine (GG), LB represents a HIS tag, MP represents an enterokinase recognition sequence, L1 represents a bond, P1 represents human KLK1 protein, L2 represents a second linker, and P2 represents an Fc domain.

[0191] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine residues (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121 or SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:11.

[0192] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine residues (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121 or SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:12.

[0193] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine residues (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121 or SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:13.

[0194] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine residues (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121 or SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:14.

[0195] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine residues (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121 or SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:15.

[0196] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:11.

[0197] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:12.

[0198] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine residues (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:13.

[0199] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine residues (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:14.

[0200] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:15.

[0201] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine residues (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:11.

[0202] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine residues (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:12.

[0203] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine residues (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:13.

[0204] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine residues (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:14.

[0205] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:15.

[0206] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine residues (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:11.

[0207] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:12.

[0208] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:13.

[0209] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:14.

[0210] In certain embodiments, SP represents a signal peptide as shown in SEQ ID NO:24 or SEQ ID NO:25, L3 represents a linker formed by two glycine (GG), LB represents a HIS tag as shown in SEQ ID NO:16, MP represents a masking peptide as shown in SEQ ID NO:5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO:122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO:15.

[0211] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121 or SEQ ID NO: 122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 11.

[0212] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121 or SEQ ID NO: 122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 12.

[0213] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121 or SEQ ID NO: 122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 13.

[0214] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121 or SEQ ID NO: 122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 14.

[0215] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121 or SEQ ID NO: 122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 15.

[0216] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 11.

[0217] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 12.

[0218] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 13.

[0219] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 14.

[0220] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 2, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 15.

[0221] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 11.

[0222] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 12.

[0223] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 13.

[0224] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycine residues (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 14.

[0225] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 115, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 15.

[0226] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 11.

[0227] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 12.

[0228] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 13.

[0229] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 14.

[0230] In certain embodiments, SP represents absence, L3 represents a linker formed by two glycines (GG), LB represents a HIS tag as shown in SEQ ID NO: 16, MP represents a masking peptide as shown in SEQ ID NO: 5, L1 represents a bond, P1 represents a first polypeptide as shown in SEQ ID NO: 122, L2 represents a second linker, and P2 represents a second polypeptide as shown in SEQ ID NO: 15.

[0231] In certain embodiments, the KLK1 fusion protein comprises the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 110, or SEQ ID NO: 124. In certain embodiments, the KLK1 fusion protein consists of the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 110, or SEQ ID NO: 124. In certain embodiments, the amino acid sequence of the KLK1 fusion protein has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 110, or SEQ ID NO: 124.In certain embodiments, the amino acid sequence of the KLK1 fusion protein has at least 70%, at least 75%, or at least 80% similarity to the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 110, or SEQ ID NO: 124. At least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but still retains the function or activity of KLK1 after cleavage by EK (e.g., capable of catalyzing the release of active kinin from kininogen).

[0232] In certain embodiments, the KLK1 fusion protein comprises the amino acid sequence of SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 111, or SEQ ID NO: 125. In certain embodiments, the KLK1 fusion protein consists of the amino acid sequence of SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 111, or SEQ ID NO: 125. In certain embodiments, the amino acid sequence of the KLK1 fusion protein is at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 111, or SEQ ID NO: 125.In certain embodiments, the amino acid sequence of the KLK1 fusion protein is the same as SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 111 or SEQ ID NO: 112. The amino acid sequence of NO: 125 has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but still retains the function or activity of KLK1 after cleavage by EK (e.g., capable of catalyzing the release of active kinin from kininogen).

[0233] g) Nucleic acids

[0234] In certain embodiments, in the method for preparing the KLK1 fusion protein provided herein, the nucleic acid molecule encoding the KLK1 fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 77, and the nucleotide sequence shown in SEQ ID NO: 78.

[0235] In certain embodiments, in the method for preparing the KLK1 fusion protein provided herein, the nucleic acid molecule encoding the KLK1 fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 77, and the nucleotide sequence shown in SEQ ID NO: 79.

[0236] In certain embodiments, in the method for preparing the KLK1 fusion protein provided herein, the nucleic acid molecule encoding the KLK1 fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 77, and the nucleotide sequence shown in SEQ ID NO: 80.

[0237] In certain embodiments, in the method for preparing a KLK1 fusion protein provided herein, the nucleic acid molecule encoding the KLK1 fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 77, and the nucleotide sequence shown in SEQ ID NO: 81.

[0238] In certain embodiments, in the method for preparing a KLK1 fusion protein provided herein, the nucleic acid molecule encoding the KLK1 fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 123, and the nucleotide sequence shown in SEQ ID NO: 78.

[0239] In certain embodiments, in the method for preparing a KLK1 fusion protein provided herein, the nucleic acid molecule encoding the KLK1 fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 123, and the nucleotide sequence shown in SEQ ID NO: 79.

[0240] In certain embodiments, in the method for preparing the KLK1 fusion protein provided herein, the nucleic acid molecule encoding the KLK1 fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 123, and the nucleotide sequence shown in SEQ ID NO: 80.

[0241] In certain embodiments, in the method for preparing the KLK1 fusion protein provided herein, the nucleic acid molecule encoding the KLK1 fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 123, and the nucleotide sequence shown in SEQ ID NO: 81.

[0242] In certain embodiments, in the method for preparing a KLK1 fusion protein provided herein, the nucleic acid molecule encoding the KLK1 fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 130, and the nucleotide sequence shown in SEQ ID NO: 78.

[0243] In certain embodiments, in the method for preparing a KLK1 fusion protein provided herein, the nucleic acid molecule encoding the KLK1 fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 130, and the nucleotide sequence shown in SEQ ID NO: 79.

[0244] In certain embodiments, in the method for preparing a KLK1 fusion protein provided herein, the nucleic acid molecule encoding the KLK1 fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 130, and the nucleotide sequence shown in SEQ ID NO: 80.

[0245] In certain embodiments, in the method for preparing the KLK1 fusion protein provided herein, the nucleic acid molecule encoding the KLK1 fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 130, and the nucleotide sequence shown in SEQ ID NO: 81.

[0246] In certain embodiments, in the method for preparing the KLK1 fusion protein provided herein, the nucleic acid molecule encoding the KLK1 fusion protein further comprises the nucleotide sequence shown in SEQ ID NO: 82.

[0247] In certain embodiments, in the method for preparing the KLK1 fusion protein provided herein, the nucleic acid molecule encoding the KLK1 fusion protein further comprises a nucleotide sequence as shown in SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0248] In certain embodiments, in the method for preparing the KLK1 fusion protein provided herein, the nucleic acid molecule encoding the KLK1 fusion protein further comprises a nucleotide sequence as shown in SEQ ID NO: 87 or SEQ ID NO: 88.

[0249] In certain embodiments, in the methods for preparing KLK1 fusion proteins provided herein, the nucleic acid molecule encoding the KLK1 fusion protein comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence shown in SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 116, or SEQ ID NO: 129. In certain embodiments, in the methods for preparing KLK1 fusion proteins provided herein, the nucleic acid molecule encoding the KLK1 fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 116, or SEQ ID NO: 129. In the methods for preparing KLK1 fusion proteins provided herein, the nucleic acid molecule encoding the KLK1 fusion protein consists of the nucleotide sequence shown in SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 116, or SEQ ID NO: 129.

[0250] The term "nucleic acid" or "nucleotide" as used herein refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise indicated, a particular nucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985) and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)).

[0251] The nucleic acid molecules encoding the KLK1 fusion protein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the gene encoding the fusion protein). Encoding DNA can also be obtained synthetically.

[0252] h) Vectors and protein expression systems

[0253] In certain embodiments, in the method for preparing the KLK1 fusion protein provided herein, the recombinant vector containing the nucleic acid molecule encoding the KLK1 fusion protein is selected from the group consisting of a plasmid, an artificial chromosome, and a viral vector.

[0254] Using recombinant techniques known in the art, nucleic acid molecules encoding KLK1 fusion proteins can be inserted into vectors for further cloning (amplification of the DNA) or for expression. A variety of vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0255] Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phage and M13 phage, pcDNA plasmids (e.g., pcDNA3.4 plasmids), pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0256] In certain embodiments, the protein expression system used in the methods for preparing KLK1 fusion proteins provided herein is a cell expression system. In certain embodiments, the cell expression system is selected from the group consisting of bacteria, fungi, plant cells, or animal cells.

[0257] In certain embodiments, the bacterium is selected from the group consisting of Bacilli, Clostridia, Corynebacterium, Cupriavidus, Delftia, Escherichia, Enterobacter, Erwinia, Klebsiella, Lactobacillus, Lactococcus, Proteus, Pseudomonas, Rhodococcus, Salmonella, Serratia, Shigella, and Streptomyces. In certain embodiments, the bacteria is selected from the group consisting of Escherichia coli, Salmonella typhimurium, Serratia marcescens, Bacillus subtilis, Bacillus licheniformis, and Pseudomonas aeruginosa. In certain embodiments, the cell is an Escherichia coli cell.

[0258] In certain embodiments, the fungus is selected from the group consisting of Candida, Saccharomyces cerevisiae, Kluyveromyces, Pichia pastoris, and Schizosaccharomyces pombe.

[0259] In certain embodiments, the animal cell is a mammalian cell. In certain embodiments, the mammalian cell is a human cell or a Chinese hamster ovary (CHO) cell. In certain embodiments, the mammalian cell is a CHO-K1 cell or a CHO-S cell. In certain embodiments, the mammalian cell is a human embryonic kidney cell 293 (HEK293 cell).

[0260] Step (2)

[0261] Step (2) is to mix the KLK1 fusion protein obtained in step (1) with the protease to perform a specific enzymatic cleavage reaction to remove the masking peptide. In certain embodiments, the KLK1 fusion protein obtained in step (1) is purified before performing the specific enzymatic cleavage reaction in step (2).

[0262] Without being bound by any theory, it is believed that the KLK1 fusion protein containing the masking peptide is an intermediate produced during the preparation of an active KLK1 fusion protein and does not itself have KLK1 enzymatic activity, for example, it does not have the activity to cleave kallikrein. However, when the KLK1 fusion protein containing the masking peptide is mixed with a protease that can specifically recognize and cleave the masking peptide and subjected to a specific enzymatic cleavage reaction, after removing the masking peptide, the resulting KLK1 fusion protein (e.g., KLK1-Fc fusion protein) has KLK1 enzymatic activity, for example, it can cleave kallikrein. In certain embodiments, the KLK1 fusion protein formed after removing the masking peptide still retains the function or activity of wild-type KLK1 (e.g., it still retains the enzymatic activity to cleave kallikrein). Those skilled in the art can determine the enzymatic activity of the KLK1 fusion protein using methods known in the art, for example, the method described in Example 4 of this application. In certain embodiments, the activity of the KLK1 fusion protein prepared by the method described in the present application in cleaving kininogen-releasing kinin is 0.3-3 PNAU / mg (e.g., 0.4 PNAU / mg, 0.5 PNAU / mg, 0.6 PNAU / mg, 0.7 PNAU / mg, 0.8 PNAU / mg, 0.9 PNAU / mg, 1 PNAU / mg, 1.5 PNAU / mg, 2 PNAU / mg, 2.5 PNAU / mg, etc.).

[0263] In certain embodiments, the KLK1 fusion protein obtained in step (1) is mixed with the protease for specific enzymatic cleavage reaction, and the KLK1 fusion protein obtained after removing the masking peptide comprises an amino acid sequence as shown in SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 114 or SEQ ID NO: 128. In certain embodiments, the KLK1 fusion protein consists of an amino acid sequence as shown in SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 114 or SEQ ID NO: 128. In certain embodiments, the amino acid sequence of the KLK1 fusion protein is at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 114, or SEQ ID NO: 128.In certain embodiments, the amino acid sequence of the KLK1 fusion protein has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 114, or SEQ ID NO: 128, but still retains the function or activity of KLK1 (e.g., capable of catalyzing the release of active kinin from kininogen).

[0264] Fusion protein

[0265] In one aspect, the present application provides a fusion protein comprising a masking peptide, a first polypeptide and a second polypeptide, wherein the masking peptide, the first polypeptide and the second polypeptide are directly connected or covalently connected through a linker, and

[0266] a) the masking peptide has an amino acid sequence that can be specifically recognized and cleaved by a protease;

[0267] b) the first polypeptide comprises KLK1 or a variant thereof; and

[0268] c) the second polypeptide is used to extend the half-life of the first polypeptide.

[0269] The masking peptide, first polypeptide and second polypeptide described in the "Method for Preparing Fusion Protein" section of this application are also applicable to the masking peptide, first polypeptide and second polypeptide in the "Fusion Protein" section of this application, and therefore will not be repeated here.

[0270] In certain embodiments, the masking peptide is selected from the group consisting of an enterokinase (EK) recognition sequence, a thrombin recognition sequence, a factor Xa recognition sequence, a tobacco etch virus (TEV) protease recognition sequence, a SUMO protease recognition sequence, and a transpeptidase (sortase) recognition sequence. In certain embodiments, the masking peptide is an enterokinase (EK) recognition sequence. In certain embodiments, the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5 (VDDDDK).

[0271] In certain embodiments, the first polypeptide is a human KLK1 protein or a variant thereof. In certain embodiments, the first polypeptide is a human KLK1 protein. In certain embodiments, the amino acid sequence of the first polypeptide has at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122, while still retaining the function or activity of KLK1. In certain embodiments, the amino acid sequence of the first polypeptide is set forth in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122. In certain embodiments, the amino acid sequence of the first polypeptide has at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, while still retaining the function or activity of KLK1. In certain embodiments, the amino acid sequence of the first polypeptide has at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 115, while still retaining the function or activity of KLK1. In certain embodiments, the amino acid sequence of the first polypeptide has at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 115. In certain embodiments, the amino acid sequence of the first polypeptide has at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 122, while still retaining the function or activity of KLK1. In certain embodiments, the amino acid sequence of the first polypeptide is set forth in SEQ ID NO: 122.

[0272] In certain embodiments, the second polypeptide is an Fc domain or albumin. In certain embodiments, the amino acid sequence of the second polypeptide is shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0273] In certain embodiments, the KLK1 fusion protein further comprises a tag. In certain embodiments, the KLK1 fusion protein further comprises a HIS tag. In certain embodiments, the KLK1 fusion protein further comprises a HIS tag consisting of 5, 6, 7, 8, 9, 10 or more histidines.

[0274] In certain embodiments, the KLK1 fusion protein comprises the following structure from N-terminus to C-terminus:

[0275] LB-MP-L1-P1-L2-P2,

[0276] Wherein, LB represents absence or a tag, MP represents the masking peptide, L1 represents a bond or a first linker, P1 represents the first polypeptide, L2 represents a bond or a second linker, and P2 represents the second polypeptide.

[0277] In certain embodiments, the KLK1 fusion protein comprises the following structure from N-terminus to C-terminus:

[0278] LB-MP-L1-P1-L2-P2,

[0279] Wherein, LB represents a tag, MP represents the masking peptide, L1 represents a bond, P1 represents the first polypeptide, L2 represents a second linker, and P2 represents the second polypeptide.

[0280] In certain embodiments, the KLK1 fusion protein comprises the following structure from N-terminus to C-terminus:

[0281] LB-MP-L1-P1-L2-P2,

[0282] Wherein, LB represents a HIS tag, MP represents the enterokinase recognition sequence, L1 represents a bond, P1 represents human KLK1 protein, L2 represents a second linker, and P2 represents an Fc domain.

[0283] In certain embodiments, the KLK1 fusion protein comprises the following structure from N-terminus to C-terminus:

[0284] LB-MP-L1-P1-L2-P2,

[0285] Wherein, LB represents a HIS tag, MP represents the amino acid sequence shown in SEQ ID NO: 5, L1 represents a bond, P1 represents the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121 or SEQ ID NO: 122, L2 represents a second linker, and P2 represents the amino acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0286] In certain embodiments, the KLK1 fusion protein comprises the following structure from N-terminus to C-terminus:

[0287] LB-MP-L1-P1-L2-P2,

[0288] Wherein, LB represents a HIS tag, MP represents the amino acid sequence shown in SEQ ID NO: 5, L1 represents a bond, P1 represents the amino acid sequence shown in SEQ ID NO: 2, L2 represents a second linker, and P2 represents the amino acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0289] In certain embodiments, the KLK1 fusion protein comprises the following structure from N-terminus to C-terminus:

[0290] LB-MP-L1-P1-L2-P2,

[0291] Wherein, LB represents a HIS tag, MP represents the amino acid sequence shown in SEQ ID NO: 5, L1 represents a bond, P1 represents the amino acid sequence shown in SEQ ID NO: 115, L2 represents a second linker, and P2 represents the amino acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0292] In certain embodiments, the KLK1 fusion protein comprises the following structure from N-terminus to C-terminus:

[0293] LB-MP-L1-P1-L2-P2,

[0294] Wherein, LB represents a HIS tag, MP represents the amino acid sequence shown in SEQ ID NO: 5, L1 represents a bond, P1 represents the amino acid sequence shown in SEQ ID NO: 122, L2 represents a second linker, and P2 represents the amino acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0295] In certain embodiments, the KLK1 fusion protein comprises the following structure from N-terminus to C-terminus:

[0296] LB-MP-L1-P1-L2-P2,

[0297] Wherein, LB represents a HIS tag, MP represents the amino acid sequence shown in SEQ ID NO: 5, L1 represents a bond, P1 represents the amino acid sequence shown in SEQ ID NO: 2, L2 represents the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23, and P2 represents the amino acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0298] In certain embodiments, the KLK1 fusion protein comprises the following structure from N-terminus to C-terminus:

[0299] LB-MP-L1-P1-L2-P2,

[0300] Wherein, LB represents a HIS tag, MP represents the amino acid sequence shown in SEQ ID NO: 5, L1 represents a bond, P1 represents the amino acid sequence shown in SEQ ID NO: 115, L2 represents the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23, and P2 represents the amino acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0301] In certain embodiments, the KLK1 fusion protein comprises the following structure from N-terminus to C-terminus:

[0302] LB-MP-L1-P1-L2-P2,

[0303] Wherein, LB represents a HIS tag, MP represents the amino acid sequence shown in SEQ ID NO: 5, L1 represents a bond, P1 represents the amino acid sequence shown in SEQ ID NO: 122, L2 represents the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23, and P2 represents the amino acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0304] In certain embodiments, the KLK1 fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 26 to 65, 100 to 114, and 124 to 128. In certain embodiments, the KLK1 fusion protein consists of an amino acid sequence as shown in any one of SEQ ID NOs: 26 to 65, 110 to 114, and 124 to 128.

[0305] Nucleic Acids

[0306] In another aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the KLK1 fusion protein described herein.

[0307] In certain embodiments, the nucleic acid molecules provided herein comprise:

[0308] a) the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 77, and the nucleotide sequence shown in SEQ ID NO: 78;

[0309] b) the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 77, and the nucleotide sequence shown in SEQ ID NO: 79;

[0310] c) the nucleotide sequence shown in SEQ ID NO:76, the nucleotide sequence shown in SEQ ID NO:77, and the nucleotide sequence shown in SEQ ID NO:80;

[0311] d) the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 77, and the nucleotide sequence shown in SEQ ID NO: 81;

[0312] e) the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 123, and the nucleotide sequence shown in SEQ ID NO: 78;

[0313] f) the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 123, and the nucleotide sequence shown in SEQ ID NO: 79;

[0314] g) the nucleotide sequence shown in SEQ ID NO:76, the nucleotide sequence shown in SEQ ID NO:123, and the nucleotide sequence shown in SEQ ID NO:80;

[0315] h) the nucleotide sequence shown in SEQ ID NO:76, the nucleotide sequence shown in SEQ ID NO:123, and the nucleotide sequence shown in SEQ ID NO:81;

[0316] i) the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 130, and the nucleotide sequence shown in SEQ ID NO: 78;

[0317] j) the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 130, and the nucleotide sequence shown in SEQ ID NO: 79;

[0318] k) the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 130, and the nucleotide sequence shown in SEQ ID NO: 80; or

[0319] l) the nucleotide sequence shown in SEQ ID NO:76, the nucleotide sequence shown in SEQ ID NO:130, and the nucleotide sequence shown in SEQ ID NO:81.

[0320] In certain embodiments, the nucleic acid molecules provided herein further comprise the nucleotide sequence shown in SEQ ID NO: 82. In certain embodiments, the nucleic acid molecules provided herein further comprise the nucleotide sequence shown in SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86. In certain embodiments, the nucleic acid molecules provided herein further comprise the nucleotide sequence shown in SEQ ID NO: 87 or SEQ ID NO: 88.

[0321] In certain embodiments, the nucleic acid molecules provided herein comprise a nucleotide sequence as set forth in SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:116, or SEQ ID NO:129, or have at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the nucleic acid molecule provided herein consists of a nucleotide sequence as shown in SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO: 116 or SEQ ID NO:129.

[0322] Vectors and cells

[0323] In another aspect, the present application provides a vector comprising a nucleic acid molecule encoding the KLK1 fusion protein described herein.

[0324] In another aspect, the present application also provides a protein expression system, which comprises the nucleic acid molecule encoding the KLK1 fusion protein according to the present application or the vector according to the present application.

[0325] On the other hand, the present application also provides a method for constructing a protein expression system, comprising introducing a nucleic acid molecule encoding the KLK1 fusion protein described in the present application into a vector to construct an expression vector; and introducing the expression vector into the protein expression system.

[0326] Using recombinant techniques known in the art, the isolated polynucleotide encoding the KLK1 fusion protein can be inserted into a vector for further cloning (amplification of the DNA) or for expression. A variety of vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0327] In certain embodiments, the nucleic acid provided herein encodes a KLK1 fusion protein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection tag. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), phage lambda and M13, plasmids pcDNA3.4, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX , pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0328] The vector containing the nucleic acid sequence encoding the KLK1 fusion protein can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vector described in the present application are the prokaryotes, yeasts or higher eukaryotic cells described above. Prokaryotic cells suitable for use in the present application include eubacteria, such as Gram-negative bacteria or Gram-positive bacteria, for example, Enterobacteriaceae, for example, Escherichia (e.g., Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens). marcescans), Shigella, Bacilli (e.g., B. subtilis and B. licheniformis), Pseudomonas (e.g., P. aeruginosa), and Streptomyces. In certain embodiments, the cell is an Escherichia coli cell.

[0329] In addition to prokaryotes, eukaryotic cells, for example eukaryotic microorganisms such as filamentous fungi or yeast can also be used as suitable cloning or expression hosts for vectors encoding fusion proteins. Saccharomyces cerevisiae, or baker's yeast, is the most commonly used lower eukaryotic host microorganism. However, many other genera, species, and strains are commonly used and suitable for use in this application, for example, Schizosaccharomyces pombe; Kluyveromyces hosts, for example, K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia lipolytica (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, for example, Schwanniomyces occidentalis; and filamentous fungi, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus (for example, A. nidulans and A. niger). In certain embodiments, the eukaryotic cell is a mammalian cell. In certain embodiments, the mammalian cell is a human cell or a Chinese hamster ovary (CHO) cell. In certain embodiments, the mammalian cell is a CHO-K1 cell or a CHO-S cell. In certain embodiments, the mammalian cells are human embryonic kidney 293 cells (HEK293 cells).

[0330] Pharmaceutical composition

[0331] In another aspect, the present application provides a pharmaceutical composition comprising the KLK1 fusion protein described herein, the nucleic acid described herein, the vector described herein, or the cell described herein, and a pharmaceutically acceptable carrier.

[0332] The pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed in the present application may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous solvents, non-aqueous solvents, antimicrobial substances, isotonic substances, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components well known in the art, or various combinations thereof.

[0333] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylated methyl anisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, including one or more antioxidants such as methionine in a composition comprising a KLK1 fusion protein disclosed herein can reduce oxidation of the KLK1 fusion protein. The present application further provides various methods for preventing oxidation of the KLK1 fusion protein, extending its shelf life, and / or increasing its activity, for example, by mixing the KLK1 fusion protein provided herein with one or more antioxidants (e.g., methionine).

[0334] Further, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's solution injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, non-aqueous media such as fixed oils of plant origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial substances at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (Tween-80), chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Antimicrobial agents serving as carriers may be added to pharmaceutical compositions in multidose containers and include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, glucose, glycerol, or ethanol. Suitable nontoxic auxiliary substances may include, for example, wetting agents, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, sorbitan laurate, triethanolamine oleate, or cyclodextrins.

[0335] The pharmaceutical composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, saccharin sodium, cellulose, magnesium carbonate, etc.

[0336] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, for example, a liquid solvent, a suspending agent, an emulsifier, or a solid form suitable for producing a liquid solvent, a suspending agent, or an emulsifier. Injectable formulations can include ready-to-use sterile and / or pyrogen-free solutions, sterile dried solubles that are combined with a solvent before use, such as lyophilized powders, including subcutaneous tablets, sterile suspensions ready for injection, sterile dried insoluble products that are combined with a medium before use, and sterile and / or pyrogen-free emulsions. The solvent can be an aqueous phase or a non-aqueous phase.

[0337] In certain embodiments, the unit dose of the injection preparation is packaged in an ampoule, a tube or a syringe with a needle. As is known in the art, all preparations for injection should be sterile and pyrogen-free.

[0338] In certain embodiments, a sterile lyophilized powder can be prepared by dissolving a KLK1 fusion protein disclosed herein in a suitable solvent. The solvent may contain a component that enhances the stability of the powder or a reconstituted solution prepared from the powder, or other pharmacologically improves the powder or reconstituted solution. Suitable excipients include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable substances. The solvent may contain a buffer, such as a citrate buffer, sodium phosphate or potassium phosphate buffer, or other buffer known to those skilled in the art. In one embodiment, the buffer has a neutral pH. The dissolution is then sterilized by filtration under standard conditions known in the art, and then lyophilized to produce the desired formulation. In one embodiment, the resulting solution is dispensed into vials and lyophilized. Each vial can accommodate a single dose or multiple doses of the KLK1 fusion protein or composition thereof. The amount of each vial can be slightly more than that required for each dose or multiple doses (e.g., a 10% excess) to ensure accurate sampling and dosing. The lyophilized powder can be stored under appropriate conditions, such as at a temperature ranging from about 4°C to room temperature.

[0339] The lyophilized powder is reconstituted with water for injection to obtain a formulation for injection. In one embodiment, the lyophilized powder can be reconstituted in sterile pyrogen-free water or other suitable liquid carrier. The exact amount is determined by the selected therapy and can be determined based on empirical values.

[0340] Methods of treating or preventing disease

[0341] In another aspect, the present application provides a method for treating or preventing a disease, comprising administering the KLK1 fusion protein described herein (e.g., an isolated KLK1 fusion protein prepared according to the method described herein) to a subject in need of treatment or prevention, wherein the disease is selected from the group consisting of ischemic diseases, hemorrhagic diseases, renal diseases, metabolic diseases, and neurodegenerative diseases.

[0342] In another aspect, the present application provides the use of the KLK1 fusion protein described herein (e.g., an isolated KLK1 fusion protein prepared according to the methods described herein) in the preparation of a medicament for treating or preventing ischemic diseases, hemorrhagic diseases, kidney diseases, metabolic diseases, and neurodegenerative diseases. In certain embodiments, the present application provides the use of the KLK1 fusion protein described herein (e.g., an isolated KLK1 fusion protein prepared according to the methods described herein) in the preparation of a medicament for treating or preventing ischemic diseases, hemorrhagic diseases, kidney diseases, metabolic diseases, and neurodegenerative diseases, wherein the treatment or prevention comprises administering the medicament to a subject in need thereof.

[0343] In another aspect, the present application provides use of the KLK1 fusion protein described herein (e.g., an isolated KLK1 fusion protein prepared according to the method described herein) for treating or preventing a disease, wherein the disease is selected from the group consisting of ischemic diseases, hemorrhagic diseases, renal diseases, metabolic diseases, and neurodegenerative diseases.

[0344] In certain embodiments, the ischemic disease is selected from the group consisting of cerebral ischemia, ischemic stroke, myocardial ischemia, ischemic colitis, limb ischemia, skin ischemia, ischemic stroke, transient ischemic attack (TIA), and traumatic brain injury (TBI). In certain embodiments, the ischemic disease is acute ischemic stroke. In certain embodiments, the ischemic disease is mild to moderate acute thrombotic cerebral infarction.

[0345] In certain embodiments, the bleeding disorder is hemorrhagic stroke. In certain embodiments, the hemorrhagic stroke is intracerebral hemorrhagic stroke or subarachnoid hemorrhagic stroke.

[0346] In certain embodiments, the kidney disease is chronic kidney disease, diabetic nephropathy, or acute kidney injury.

[0347] In certain embodiments, the metabolic disease is selected from the group consisting of diabetes (eg, type 1 diabetes, type 2 diabetes), obesity, insulin resistance, hyperglycemia, and hyperinsulinemia.

[0348] In certain embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, motor neuron disease, Huntington's disease, and Parkinson's disease.

[0349] The SEQ ID NOs and their descriptions, and the corresponding amino acid sequences or nucleotide sequences mentioned in this application are shown in Table 1.

[0350] Table 1. SEQ ID NOs and their descriptions and represented amino acid or nucleotide sequences DETAILED DESCRIPTION

[0351] The biological materials involved in all the embodiments, such as bacterial strains, various cloning and expression plasmids, culture media, tool enzymes, buffers, as well as various culture methods, protein extraction and purification methods, and other molecular biology operation methods, are all familiar to technicians in this field. Reference can be made to "Molecular Cloning" (A Laboratory Manual, Cold Spring Harbor, 1989) compiled by Sambrook et al. and "The Concise Guide to Molecular Biology Experiments" (written by F. Osber et al., translated by Yan Ziying et al., Beijing, Science Press, 1998).

[0352] Example 1: Plasmid construction and expression of KLK1 fusion protein

[0353] The target gene encoding KLK1 fusion protein was synthesized, and various target genes were inserted into the AgeI and EcoRV sites of the pcDNA3.4 vector (as shown in FIG1 ) to construct the pcDNA3.4-KLK1-Fc plasmid.

[0354] The constructed pcDNA3.4-KLK1-Fc plasmid was transformed into Escherichia coli DH5α competent cells, and selected for resistance on LB agar plates containing ampicillin (Amp+). Then, single clones were picked and cultured in LB culture medium containing the corresponding antibiotics, and the plasmid was extracted. The extracted plasmid was then transfected into HEK293 cells and the cells were cloned using Expi293. TM The expression system expresses the target protein.

[0355] The constructed plasmids, inserted target genes, and expressed fusion proteins are shown below, where SP in the name of each plasmid indicates a signal peptide; GG indicates two glycines; His indicates a histidine tag; VDDDDK indicates an enterokinase recognition sequence; hKLK1 indicates human KLK1; GGGSSSS, GSSGSSG, GGGGSGGGGS, and EAAAK indicate linkers; IgG1 Fc indicates an Fc domain derived from human IgG1; YTE IgG1 Fc indicates an Fc domain derived from human IgG1 with M252Y, S254T, and T256E mutations; IgG4 Fc indicates an Fc domain derived from human IgG4; and YTE IgG4 Fc indicates an Fc domain derived from human IgG4 with M252Y, S254T, and T256E mutations.

[0356] The target gene inserted into the pcDNA3.4-SP-GG-His-VDDDDK-hKLK1-IgG1 Fc plasmid is shown in SEQ ID NO:66, and the expressed fusion protein (hereinafter referred to as "fusion protein 1 intermediate") comprises the amino acid sequence shown in SEQ ID NO:100;

[0357] The target gene inserted into the pcDNA3.4-SP-GG-His-VDDDDK-hKLK1-GGGSSSS-IgG1 Fc plasmid is shown in SEQ ID NO:67, and the expressed fusion protein (hereinafter referred to as "fusion protein 2.1 intermediate") comprises the amino acid sequence shown in SEQ ID NO:101;

[0358] The target gene inserted into the pcDNA3.4-SP-GG-His-VDDDDK-hKLK1-EAAAK-IgG1 Fc plasmid is shown in SEQ ID NO:68, and the expressed fusion protein (hereinafter referred to as "fusion protein 2.2 intermediate") comprises the amino acid sequence shown in SEQ ID NO:102;

[0359] The target gene inserted into the pcDNA3.4-IL2-GG-His-VDDDDK-hKLK1-GGGSSSS-IgG1 Fc plasmid is shown in SEQ ID NO:69, and the expressed fusion protein (hereinafter referred to as "fusion protein 2.3 intermediate") comprises the amino acid sequence shown in SEQ ID NO:103;

[0360] The target gene inserted into the pcDNA3.4-IL2-GG-His-VDDDDK-hKLK1-EAAAK-IgG1 Fc plasmid is shown in SEQ ID NO:70, and the expressed fusion protein (hereinafter referred to as "fusion protein 2.4 intermediate") comprises the amino acid sequence shown in SEQ ID NO:104;

[0361] The target gene inserted into the pcDNA3.4-SP-GG-His-VDDDDK-hKLK1-GSSGSSG-IgG1 Fc plasmid is shown in SEQ ID NO:71, and the expressed fusion protein (hereinafter referred to as "fusion protein 3.1 intermediate") comprises the amino acid sequence shown in SEQ ID NO:105;

[0362] The target gene inserted into the pcDNA3.4-SP-GG-His-VDDDDK-hKLK1-GSSGSSG-YTE IgG1 Fc plasmid is shown in SEQ ID NO:72, and the expressed fusion protein (hereinafter referred to as "fusion protein 3.2 intermediate") comprises the amino acid sequence shown in SEQ ID NO:106;

[0363] The target gene inserted into the pcDNA3.4-SP-GG-His-VDDDDK-hKLK1-GSSGSSG-IgG4 Fc plasmid is shown in SEQ ID NO: 73, and the expressed fusion protein (hereinafter referred to as "fusion protein 3.3 intermediate") comprises the amino acid sequence shown in SEQ ID NO: 107;

[0364] The target gene inserted into the pcDNA3.4-SP-GG-His-VDDDDK-hKLK1-GSSGSSG-YTE IgG4 Fc plasmid is shown in SEQ ID NO:74, and the expressed fusion protein (hereinafter referred to as "fusion protein 3.4 intermediate") comprises the amino acid sequence shown in SEQ ID NO:108;

[0365] The target gene inserted into the pcDNA3.4-SP-GG-His-VDDDDK-hKLK1-GGGGSGGGGS-IgG1 Fc plasmid is shown in SEQ ID NO:75, and the expressed fusion protein (hereinafter referred to as "fusion protein 3.5 intermediate") comprises the amino acid sequence shown in SEQ ID NO:109;

[0366] The target gene inserted into the pcDNA3.4-SP-prepro-hKLK1-EAAAK-IgG1 Fc plasmid is shown in SEQ ID NO: 97, and the expressed fusion protein (hereinafter referred to as "fusion protein 3.6") comprises the amino acid sequence shown in SEQ ID NO: 93;

[0367] The target gene inserted into the pcDNA3.4-SP-prepro-hKLK1 plasmid is shown in SEQ ID NO: 98, and the expressed protein (hereinafter referred to as "protein 4.1") comprises the amino acid sequence shown in SEQ ID NO: 94;

[0368] The target gene inserted into the pcDNA3.4-SP-prepro-VDDDDK-hKLK1 plasmid is shown in SEQ ID NO:99, and the expressed protein (hereinafter referred to as "Protein 4.2 Intermediate") comprises the amino acid sequence shown in SEQ ID NO:95;

[0369] The target gene inserted into the pcDNA3.4-SP-prepro-VDDDDK-hKLK1-EAAAK-IgG1 Fc plasmid is shown in SEQ ID NO:90, and the expressed fusion protein (hereinafter referred to as "fusion protein 4.3 intermediate") comprises the amino acid sequence shown in SEQ ID NO:96;

[0370] The target gene inserted into the pcDNA3.4-SP-GG-His-VDDDDK-hKLK1(E121Q+A164V)-GSSGSSG-IgG1 Fc plasmid is shown in SEQ ID NO: 116, and the expressed fusion protein (hereinafter referred to as "fusion protein 5.1 intermediate") comprises the amino acid sequence shown in SEQ ID NO: 111;

[0371] ●The target gene inserted into the pcDNA3.4-SP-GG-His-VDDDDK-hKLK1(E121Q+K162E+A164V)-GSSGSSG-IgG1 Fc plasmid is shown in SEQ ID NO:129, and the expressed fusion protein (hereinafter referred to as "fusion protein 6.1 intermediate") comprises the amino acid sequence shown in SEQ ID NO:125.

[0372] Example 2: Purification of KLK1 fusion protein

[0373] 2.1. Initial purification of KLK1 fusion protein

[0374] Several proteins prepared in Example 1 (e.g., fusion protein 1 intermediate, fusion protein 2.1 intermediate, fusion protein 2.2 intermediate, fusion protein 2.3 intermediate, fusion protein 2.4 intermediate, fusion protein 3.1 intermediate, fusion protein 3.2 intermediate, fusion protein 3.3 intermediate, fusion protein 3.4 intermediate, fusion protein 3.5 intermediate, fusion protein 3.6, protein 4.1, protein 4.2 intermediate, and fusion protein 4.3 intermediate) were passed through a Ni-NTA nickel column: the conditioned medium was harvested by centrifugation (4°C, 4000 rpm, 40 minutes), filtered to remove cell debris, and exchanged into PBS supplemented with 0.8 mM EDTA. 300 ml of the clarified medium was loaded onto a 5 ml Ni-NTA column (purchased from GE) pre-equilibrated with buffer A (25 mM Tris, 150 mM NaCl, 10 mM imidazole, pH 8.0) at a flow rate of 3 ml / min. The column was washed with 20 column volumes (CV) of equilibration buffer (25 mM Tris, 150 mM NaCl, 10 mM imidazole, pH 8.0). Elution was then performed with elution buffer (25 mM Tris, 150 mM NaCl, 500 mM imidazole, pH 8.0). The eluate was dialyzed into PBS (pH 7.4) and finally filtered through a MILLEX-MP 0.22 μm filter (available from MILLIPORE).

[0375] Then, the various protein intermediates purified by the Ni-NTA nickel column were purified by Protein A column. Specifically, the conditioned medium was harvested by centrifugation (4°C, 4000 rpm, 40 min) and then filtered to remove cell debris. 50 ml of clarified culture medium was loaded into a 1 ml MabSelect SuRe Protein A column (purchased from GE), which had been pre-equilibrated with equilibration buffer (25 mM Tris, 150 mM NaCl, pH 8.0) at a flow rate of 1 ml / min. The column was washed with 20 CV of equilibration buffer (25 mM Tris, 150 mM NaCl, pH 8.0). The protein-loaded column was eluted with elution buffer (50 mM sodium citrate, 150 mM NaCl, pH 3.0). The eluate (1 / 10 volume of the eluate) was then neutralized with neutralization buffer (1 M arginine, 400 mM succinic acid, pH 9.0). The eluate was directly dialyzed into PBS, pH 7.4, and finally filtered through MILLEX-MP 0.22 μm (purchased from MILLIPORE).

[0376] 2.2. EK digestion

[0377] The KLK1 fusion protein containing the EK enzyme recognition sequence (VDDDDK) obtained by preliminary purification in Example 2.1 was digested with EK enzyme with a His tag, wherein the KLK1 fusion protein: EK enzyme = 50:1 (mg:mg), and incubated at 20-25°C overnight. The amino acid sequences of the human KLK1 protein or KLK1 fusion protein obtained after EK digestion are shown in SEQ ID NO: 56 (also known as "fusion protein 1"), SEQ ID NO: 57 (also known as "fusion protein 2.1"), SEQ ID NO: 58 (also known as "fusion protein 2.2" or "fusion protein 4.3"), SEQ ID NO: 59 (also known as "fusion protein 2.3"), SEQ ID NO: 60 (also known as "fusion protein 2.4"), SEQ ID NO: 61 (also known as "fusion protein 3.1"), SEQ ID NO: 62 (also known as "fusion protein 3.2"), SEQ ID NO: 63 (also known as "fusion protein 3.3"), SEQ ID NO: 64 (also known as "fusion protein 3.4"), SEQ ID NO: 65 (also known as "fusion protein 3.5"), and SEQ ID NO: 2 (also known as "protein 4.2"), respectively.

[0378] 2.3. Repurification after enzyme digestion

[0379] The human KLK1 protein or KLK1 fusion protein obtained after enzymatic digestion in Example 2.2 was purified again using a nickel column. The mixed product after enzymatic digestion was once again passed through the nickel column. The final product was collected, the concentration was determined, and the product was filtered through a MILLEX-MP 0.22 μm filter membrane and stored at -80°C.

[0380] Example 3: Characterization of KLK1 fusion protein

[0381] 3.1. Disulfide bond analysis method

[0382] Prepare samples under reduced and non-reducing conditions using the protein prepared in Example 2.3. For reduced samples, place an appropriate amount in a centrifuge tube, add denaturing buffer, and mix thoroughly. Add DTT solution, mix thoroughly, and incubate to reduce the sample. Add iodoacetamide to the reduced sample, mix thoroughly, and incubate to alkylate the sample. For non-reducing samples, place an appropriate amount in a centrifuge tube, add buffer solution, and dilute the sample to the desired concentration. Label.

[0383] Transfer the prepared reduced and non-reduced samples to ultrafiltration tubes, add buffer solution, and centrifuge for ultrafiltration. Repeat the addition of buffer solution and centrifuge for ultrafiltration until the target concentration is reached and mix thoroughly. Add PNGase F enzyme to the desalted sample and incubate for enzymatic deglycolysis. Add an appropriate amount of trypsin / chymotrypsin / Glu-C to the deglycosylated sample and incubate for enzymatic digestion. Transfer the completed sample to an injection vial, separate the enzymatic fragments by UPLC, and then confirm the position and structure of the cysteine-SH residues of the disulfide bonds in the protein by mass spectrometry.

[0384] Molecular weight analysis method

[0385] 3.2.1. Desugared reduced molecular weight analysis

[0386] (a) Preparation of deglycosylated reductive protein samples

[0387] Label the sample name on the centrifuge tube, concentrate the sample using an ultrafiltration tube, add 50 μg of protein sample solution to the centrifuge tube, add an appropriate amount of 5xQuick reaction buffer (reduced), mix well, incubate at 75°C, cool to room temperature, add an appropriate amount of Quick PNGase F enzyme, incubate at 75°C, replace the sample buffer with 100 mM ammonium bicarbonate solution, centrifuge at 13000 rpm for 10 minutes, transfer the supernatant to a sample vial, and inject 2 μL for analysis.

[0388] After the reaction is completed, the sample is transferred to an injection bottle, the prepared sample is separated by HPLC, and the desugared molecular weight of the sample is determined by mass spectrometry.

[0389] (b) Instrument parameters

[0390] HPLC (purchased from Agilent, model 1290 Infinity II) parameters are as follows:

[0391] Column: Waters, BioResolve, RP mAb, Polyphenyl, 450A, 2.1 x 50 mm, 2.7 μm

[0392] Column temperature: 75°C

[0393] ●Autosampler temperature: 8℃

[0394] ●Mobile phase: Mobile phase A (deionized water, containing 0.1% formic acid); Mobile phase B (acetonitrile, containing 0.1% formic acid).

[0395] The mass spectrometer (purchased from Agilent, model 6545XT) parameters are as follows:

[0396] Scan range:50-3200m / z

[0397] Ion polarity: positive

[0398] ESI GAS Temp:325℃

[0399] Fragmentor Voltage:225V

[0400] (c) Test results

[0401] Taking fusion protein 2.4 as an example (characterization results of other proteins prepared in Example 2.3 are not shown), the total ion current (TIC) spectrum, mass spectrometry (MS) spectrum, and deconvolution spectrum of fusion protein 2.4 are shown in Figure 4. The test results show that the deglycosylated reducing molecular weight of fusion protein 2.4 is 51801 Da, which is consistent with the molecular weight of the target protein.

[0402] 3.2.2. Analysis of intact molecular weight of desugared

[0403] (a) Preparation of deglycosylated intact protein samples

[0404] Label the sample name on the centrifuge tube, add 50 μg of protein sample solution to the centrifuge tube, add an appropriate amount of 5xQuick reaction buffer (non-reducing), mix well, incubate at 75°C, cool to room temperature, add an appropriate amount of Quick PNGase F enzyme, incubate at 50°C, replace the sample buffer with 100 mM ammonium bicarbonate solution, centrifuge at 13000 rpm for 10 minutes, transfer the supernatant to a sample vial, and inject 2 μl for analysis.

[0405] (b) Instrument parameters

[0406] HPLC (purchased from Agilent, model 1290 Infinity II) parameters are as follows:

[0407] Column: Waters, BioResolve, RP mAb, Polyphenyl, 450A, 2.1 x 50 mm, 2.7 μm

[0408] Column temperature: 75°C

[0409] ●Autosampler temperature: 8℃

[0410] ●Mobile phase: Mobile phase A (deionized water, containing 0.1% formic acid); Mobile phase B (acetonitrile, containing 0.1% formic acid).

[0411] The mass spectrometer (purchased from Agilent, model 6545XT) parameters are as follows:

[0412] Scan range:500-5000m / z

[0413] Ion polarity: positive

[0414] ESI GAS Temp:325℃

[0415] Fragmentor Voltage:380V

[0416] (c) Test results

[0417] Taking fusion protein 2.4 as an example (characterization results of other proteins prepared in Example 2.3 are not shown), the total ion current (TIC) spectrum, mass spectrometry (MS) spectrum, and deconvolution spectrum of fusion protein 2.4 are shown in Figure 5. The test results show that the deglycosylated intact molecular weight of fusion protein 2.4 is 103,568 Da.

[0418] Sequence coverage analysis methods

[0419] 3.3.1. Sample denaturation, reduction, and alkylation

[0420] Calculate the required sample volume using the following formula:

[0421] Required sample volume (μL) = 300 (μg) / sample protein concentration (μg / μL).

[0422] Pipette 300 μg of the sample solution to be tested into an ultrafiltration tube and centrifuge at 12,000 rpm for 10 minutes. Add denaturing solution to the 100 μL mark on the ultrafiltration tube, mix thoroughly, and transfer to a 1.5 mL centrifuge tube. Add 200 μL of denaturing solution to a final volume of 300 μL and mix thoroughly. Add 5 μL of 1 M DTT solution and mix thoroughly. Incubate the mixture at 37°C for 60 minutes. Add 10 μL of 1 M iodoacetamide solution and mix thoroughly. Incubate the mixture at 10-30°C in the dark for 15 minutes (prepare three replicates: Group 1, Group 2, and Group 3).

[0423] 3.3.2. Sample desalting

[0424] Group 1 and Group 2: Label the sample names on the ultrafiltration tubes, add 300 μL of 100 mM ammonium bicarbonate solution to each ultrafiltration tube, and centrifuge at 12,000 rpm for 5 minutes. Transfer the samples to ultrafiltration tubes, centrifuge at 12,000 rpm for 10 minutes, and wash the samples twice with 300 μL of 100 mM ammonium bicarbonate solution, centrifuging at 12,000 rpm for 10 minutes each time. Add 300 μL of 100 mM ammonium bicarbonate solution to each ultrafiltration tube and centrifuge at 12,000 rpm for 15 minutes. Add 100 mM ammonium bicarbonate solution to the 100 μL mark on the ultrafiltration tube, mix thoroughly, transfer the sample solution to a centrifuge tube, add 100 μL of 100 mM ammonium bicarbonate solution (final sample concentration is approximately 1.5 mg / mL), and mix thoroughly.

[0425] Group 3: Replace the 100 mM ammonium bicarbonate solution in the above-mentioned Group 1 and Group 2 operation steps with 100 mM phosphate buffer solution. The rest of the experimental procedures are the same.

[0426] 3.3.3 Acid hydrolysis / desugaring

[0427] Enzymatic hydrolysis: 15 μL of trypsin solution was added to the first group of samples, 15 μL of chymotrypsin was added to the second group of samples, and 15 μL of Glu-C protease was added to the third group of samples. The samples were incubated at 37°C for 120 min.

[0428] Desugaring and deacidification: Pipette 2 μL of Quick PNGase F enzyme solution and 2 μL of α2-3,6,8,9Neuraminidase A into the samples of Group 1, Group 2, and Group 3, respectively. Incubate at 37°C for 60 minutes. Add 5 μL of 10% formic acid solution to each centrifuge tube and mix well. The samples are then ready.

[0429] The prepared samples are transferred to injection bottles, and the peptide amino acid sequences of the samples digested by different enzymes are analyzed by MS and MS / MS, and matched with the theoretical sequences of the samples to determine the sequence coverage of the samples to be tested.

[0430] Example 4: KLK enzyme activity assay

[0431] Methods

[0432] BCA protein assay kit (Bradford method) or Pierce TM The protein content in the purified sample of Example 2.3 was determined using a 660 nm protein detection reagent (Biuret method).

[0433] First, prepare the following reagents:

[0434] Incubation buffer: 200 mM Tris-HCl, pH 8.0

[0435] ●Sample dilution buffer: 20 mM Tris-HCl, pH 8.0

[0436] • Reagents: Prepare 100 mM DMSO stock solutions of 4-nitroaniline and HD-Val-Leu-Arg-pNA, respectively.

[0437] ● Sample: The purified human KLK1 protein or KLK1 fusion protein sample from Example 2.3 was stored at -80°C and thawed at room temperature. The undiluted stock solution was used for protein concentration determination, and the 2-fold, 5-fold, and 10-fold dilutions were used for KLK1 enzyme activity determination.

[0438] Positive control sample Dissolve the powder in the vial with 100 μl of sample dilution buffer. Take 50 μl of the sample solution for protein concentration determination, and dilute the remaining sample solution 2-, 5-, or 10-fold for KLK1 enzyme activity determination.

[0439] Then, the enzyme activity of each sample was determined as follows:

[0440] 1) Determine the appropriate range for the PNA standard curve. Read the spectrophotometer at 405 nm using concentrations of 0, 0.0625, 0.125, 0.25, 0.5, and 1 mM PNA in incubation buffer without sample or substrate.

[0441] 2) Add the diluted sample and incubation buffer to the assay plate, incubate for 5 minutes, and read the background value of the plate at a wavelength of 405 nm on a spectrophotometer;

[0442] 3) Add 20 ml of HD-Val-Leu-Arg-pNA to start the reaction and read the kinetics of the plate at 405 nm on a spectrophotometer at 37°C for 15 minutes;

[0443] 4) A standard curve of PNA (p-nitroaniline) concentrations ranging from 0 to 100 nmol is generated. The exact amount of PNA released from the sample is calculated based on the PNA standard curve, thereby calculating the KLK1 enzyme activity in the sample. KLK1 enzyme activity in the test sample is expressed as the amount of PNA produced per minute (μmole / min / mg) per mg of KLK1 fusion protein per minute (PNAU / mg). Higher values ​​indicate greater enzyme activity.

[0444] Results

[0445] As shown in Table 2 and Figure 2, the KLK1 fusion proteins obtained during the preparation process with the EK enzyme recognition sequence and after EK enzyme cleavage all have KLK1 enzyme catalytic activity, while the KLK1 fusion proteins obtained during the preparation process without the EK enzyme recognition sequence, or with the EK enzyme recognition sequence but without EK enzyme cleavage, all have no KLK1 enzyme catalytic activity.

[0446] Table 2. Comparison of enzyme catalytic activities of various KLK1 fusion proteins

[0447] As shown in Table 3 and Figure 3, human KLK1 proteins or KLK1 fusion proteins with different sequences that carry the EK enzyme recognition sequence during the preparation process and are obtained after EK enzyme digestion all have KLK1 enzyme catalytic activity.

[0448] Table 3. Comparison of enzyme catalytic activities of various KLK1 fusion proteins

[0449] Example 5: CD-1 mice were intravenously injected with fusion protein 2.1 and After serum fusion protein 2.1 and Pharmacokinetic study of protein concentration

[0450] CD-1 mice were intravenously injected with the test fusion protein 2.1 and Afterwards, the serum of mice was collected at each time point. The fusion protein 2.1 and Protein concentrations were measured and pharmacokinetic analysis was performed. Twenty-four female CD-1 mice with an average body weight of 25-30 g were divided into two groups and dosed according to the dosing schedule shown in Table 4. Each mouse was identified by a tail tag number and labeled on the experimental cage. All animals in each group were not fasted and had free access to water during the experiment.

[0451] Table 4. Dosage regimen

[0452] Table 5. Serum sample collection plan for PK testing

[0453] Serum samples for PK testing were collected and pretreated as follows: Whole blood was collected from the tip of the blood sample at the time points listed in Table 5. Approximately 85 μL was collected at each time point for Group 1 and approximately 130 μL was collected at each time point for Group 2. 260 μL of whole blood was collected at the pre-dose blood collection point and placed in a centrifuge tube without anticoagulant. After standing on ice for 30 minutes, the samples were centrifuged (3,000 g, 2-8°C, 10 minutes), and the supernatant was collected. Serum was stored in a centrifuge tube, snap-frozen on dry ice, and then stored in an ultra-low temperature freezer at -60°C or below.

[0454] All animals tolerated the drug well. No abnormalities were observed during and after the administration. CD-1 mice were intravenously injected with 1 mg / kg of fusion protein 2.1 or 0.25 mg / kg of Then, the commercial kit was used to detect the fusion protein 2.1 and The protein concentration was calculated and the fusion protein 2.1 and The average PK parameters of

[0455] Using a non-compartmental model, (Version 8.2, Pharsight, Mountain View, NCA) was used to analyze the plasma concentration-time data. PK parameters included peak concentration (C max ), peak time (T max ), elimination half-life (T 1 / 2 ), area under the serum concentration-time curve (AUC), clearance (CL), mean residence time (MRT), etc. The calculation formulas and results of PK parameters are shown in Table 6.

[0456] Table 6. Calculation and results of PK parameters

[0457] After a single intravenous injection of 1 mg / kg of fusion protein 2.1, the clearance (CL) of fusion protein 2.1 in CD-1 mice was 0.239 mL / min / kg, the apparent volume of distribution (Vz) was 0.832 L / kg, and the half-life (t 1 / 2 ) was 40.2h, the mean residence time (MRT) was 27.2h, and the area under the plasma concentration-time curve (AUC 0-t The last measurable blood draw was at 120 hours, with an average concentration of 108 ng / mL.

[0458] A single intravenous injection of 0.25 mg / kg After that, CD-1 mice The clearance (CL) is 3.01 mL / min / kg, the apparent volume of distribution (Vz) is 0.192 L / kg, and the half-life (t 1 / 2 ) was 0.737h, the mean residence time (MRT) was 0.391h, and the area under the plasma concentration-time curve (AUC 0-t The last measurable blood draw was at 4 hours, with an average concentration of 28.9 ng / mL.

[0459] CD-1 mice were given a single intravenous injection of 1 mg / kg of fusion protein 2.1 and 0.25 mg / kg of After that, the blood drug concentration-time curve in the animal is shown in Figure 6. In this experiment, the fusion protein 2.1 mice were only administered once, and the blood drug concentration of the fusion protein 2.1 was still detectable on Day 3 after administration. The drug was also administered once, but its blood concentration could not be detected after 24 hours. An increase of 54 times.

[0460] Example 6: Study on the efficacy of hKLK1 fusion protein in rat transient cerebral ischemia-reperfusion (tMCAO) model

[0461] 6.1. Experimental Drugs

[0462] The experimental drug information used in this example is as follows:

[0463] ● Edaravone injection: positive control, purchased from Nanjing Xiansheng Dongyuan Pharmaceutical Co., Ltd. (production batch number: 80-190516)

[0464] ● The fusion protein 2.1 prepared in Example 2

[0465] The reagent information used in the experiment is shown in Table 7:

[0466] Table 7. Reagent information

[0467] 6.2. Construction of tMCAO Animal Model

[0468] Fifty SPF-grade healthy 6-7 week old male SD rats (200 g to 250 g, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were enrolled in the experiment to study the efficacy of the test samples on the rat transient cerebral ischemia reperfusion (tMCAO) model.

[0469] On the day of the experiment, rats in each group were anesthetized with a combination of Zolpidem 50 (50 mg / kg, im) and Xylazine (8 mg / kg, ip). The rats were then fixed in the supine position. A midline incision was made in the neck, and the tissue layers were bluntly dissected to expose the left common carotid artery. The external and internal carotid arteries were then isolated. A slipknot was tied at the proximal end of the common carotid artery. Two lines were threaded through the external carotid artery, with the distal line tied with a knot and the proximal line tied with a loose knot for securing the line plug. The internal carotid artery was clamped with an artery clamp. A small incision was made between the two lines in the external carotid artery. A line plug was inserted from the incision toward the internal carotid artery. The fixed line was gently tied around the line plug. The artery clamp was released, and the line plug was gently pushed into the internal carotid artery until it reached the line plug mark (approximately 18 mm ± 0.5 mm). The fixed line was tightened at the external carotid artery to secure the line plug. The rat's neck was sutured. After 1.5 hours of ischemia, the line plug was removed to the external carotid artery to restore blood supply from the common carotid to the internal carotid artery. Successful modeling was determined when cerebral blood flow in the ischemic region decreased by 70% (rCBF ≥ 70%). Rats were maintained at 37°C postoperatively until awake. Laser Doppler blood flow was recorded twice before and after ischemia to verify the model. Meloxicam was administered intraoperatively for analgesia, and gentamicin was administered postoperatively to prevent infection.

[0470] The efficacy of experimental drugs is assessed using the following endpoints:

[0471] ● Cerebral blood flow detection in the ischemic area: once at baseline and once after MCAO modeling, for a total of 2 times;

[0472] ●Behavioral assessment: The rats were assessed using the Longa scoring method 24, 48, and 72 hours after ischemia, for a total of three times;

[0473] ●Cerebral infarction area: determined by TTC staining 72 hours after cerebral ischemia.

[0474] Experimental methods

[0475] The experimental rats were grouped and dosed according to Table 8. Blood was collected at the end of the experiment and serum was prepared for subsequent serum marker detection. After the experiment, all rats in the groups were euthanized by carbon dioxide inhalation.

[0476] Table 8. Grouping and dosing regimen of rats

[0477] The behavioral scoring results of the animals in each group are shown in Figure 7. As shown in Figure 7, both low-dose (25 μg / kg) and high-dose (150 μg / kg) fusion protein 2.1 improved the behavior of rats 72 hours after ischemia-reperfusion, with the high-dose group (150 μg / kg) achieving significant improvement.

[0478] The TTC staining results of the cerebral infarct area of ​​each group of animals are shown in Figure 8, and the brain tissue images of each group of animals after ischemia-reperfusion are shown in Figure 9. As shown in Figures 8 and 9, compared with the model group, both the low dose (25 μg / kg) and the high dose (150 μg / kg) of fusion protein 2.1 showed a dose-dependent protective trend in the brain tissue of rats after ischemia-reperfusion.

[0479] Although the present application specifically presents and describes the invention by citing specific embodiments, those skilled in the art should understand that the above content may be changed in various forms and details without departing from the spirit and scope of protection disclosed in the present invention.

Claims

1. A method for preparing a fusion protein comprising tissue kallikrein 1 (KLK1), the method comprising the step (1): placing a recombinant vector containing a nucleic acid molecule encoding the fusion protein in a suitable protein expression system for expression, wherein: The fusion protein comprises a masking peptide, a first polypeptide and a second polypeptide, wherein the masking peptide, the first polypeptide and the second polypeptide are directly connected or covalently connected via a linker, and a) the masking peptide has an amino acid sequence that can be specifically recognized and cleaved by a protease; b) the first polypeptide comprises a KLK1 protein or a variant thereof; and c) the second polypeptide is used to extend the half-life of the first polypeptide.

2. The method according to claim 1 further comprises step (2): mixing the fusion protein obtained in step (1) with the protease to perform a specific enzymatic cleavage reaction to remove the masking peptide.

3. The method according to claim 2, wherein the fusion protein obtained in step (1) is purified before the specific enzyme cleavage reaction in step (2).

4. The method according to any one of the preceding claims, wherein the masking peptide is selected from the group consisting of an enterokinase (EK) recognition sequence, a thrombin recognition sequence, a coagulation factor Xa recognition sequence, a tobacco etch virus (TEV) protease recognition sequence, a SUMO protease recognition sequence, and a transpeptidase (Sortase) recognition sequence. The method according to claim 4 , wherein the masking peptide is an enterokinase (EK) recognition sequence.

6. The method according to claim 5, wherein the enterokinase (EK) recognition sequence consists of the amino acid sequence shown in SEQ ID NO: 4 (DDDDK) or SEQ ID NO: 5 (VDDDDK).

7. The method according to any one of the preceding claims, wherein the first polypeptide is human KLK1 protein or a variant thereof.

8. The method of claim 7, wherein the amino acid sequence of the first polypeptide has at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, but still retains the function or activity of KLK1.

9. The method according to claim 8, wherein the amino acid sequence of the first polypeptide is shown in SEQ ID NO:2, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121 or SEQ ID NO:

122.

10. The method according to any one of the preceding claims, wherein the second polypeptide is an Fc domain or albumin.

11. The method of claim 10, wherein the Fc domain is derived from a human IgG Fc domain.

12. The method of claim 11, wherein the Fc domain is derived from a human IgG1 Fc domain.

13. The method of claim 11, wherein the Fc domain is derived from a human IgG4 Fc domain.

14. The method of claim 12, wherein the Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO:

11.

15. The method according to claim 14, wherein the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 11; or the Fc domain consists of the amino acid sequence shown in SEQ ID NO:

11.

16. The method of claim 13, wherein the Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO:

12.

17. The method according to claim 16, wherein the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 12; or the Fc domain consists of the amino acid sequence shown in SEQ ID NO:

12.

18. The method according to any one of claims 10 to 17, wherein the Fc domain comprises mutations in one, two, three, four, five or more amino acid positions.

19. The method of claim 18, wherein the mutations are M252Y, S254T and T256E.

20. The method according to claim 19, wherein the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 13, or the Fc domain consists of the amino acid sequence shown in SEQ ID NO:

13.

21. The method according to claim 19, wherein the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 14, or the Fc domain consists of the amino acid sequence shown in SEQ ID NO:

14.

22. The method of claim 10, wherein the albumin comprises one or more domains of human serum albumin.

23. The method of claim 22, wherein the albumin comprises the D3 domain of human serum albumin.

24. The method according to any one of the preceding claims, wherein the second polypeptide is located at the N-terminus or C-terminus of the first polypeptide.

25. The method according to any one of the preceding claims, wherein the fusion protein comprises, from N-terminus to C-terminus, the masking peptide, the first polypeptide and the second polypeptide.

26. A method according to any one of the preceding claims, wherein the amino acid sequence of the masking peptide is shown as SEQ ID NO:5, the amino acid sequence of the first polypeptide is shown as SEQ ID NO:2, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121 or SEQ ID NO:122, and the amino acid sequence of the second polypeptide is shown as SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:

15.

27. The method according to any one of claims 2 to 26, wherein the amino acid sequence of the fusion protein obtained after removing the masking peptide in step (2) is as shown in SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:114 or SEQ ID NO:

128.

28. The method of any one of the preceding claims, wherein the fusion protein further comprises a tag.

29. The method of claim 28, wherein the tag is selected from the group consisting of a fluorescent tag, a luminescent tag, a purification tag, and a chromogenic tag.

30. The method of claim 29, wherein the tag is a purification tag.

31. The method of claim 29, wherein the tag is selected from the group consisting of a HIS tag, a GST tag, an MBP tag, a Myc tag, a NusA tag, a SUMO tag, a FLAG tag, an Avi tag, a Halo tag, a SNAP tag, a Strep-II tag, and a TrX tag.

32. The method of claim 31 , wherein the tag is a HIS tag.

33. The method of claim 32, wherein the tag is a HIS tag comprising 5, 6, 7, 8, 9 or 10 histidines.

34. The method according to any one of the preceding claims, wherein the fusion protein comprises the following structure from N-terminus to C-terminus: LB-MP-L1-P1-L2-P2, in, LB represents absence or tag, MP represents the masking peptide, L1 represents a bond or a first linker, P1 represents the first polypeptide, L2 represents a bond or a second linker, and P2 represents the second polypeptide.

35. The method of claim 34, wherein the first linker and the second linker are independently selected from the group consisting of a cleavable linker, a non-cleavable linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

36. The method of claim 35, wherein the rigid linker comprises one, two, three, four or more repeats as shown in SEQ ID NO: 23 (EAAAK).

37. The method of claim 35, wherein the flexible linker comprises a linker comprising glycine and serine.

38. The method of claim 37, wherein the glycine and serine-containing linker comprises one, two, three, four or more repeats as shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO:

22.

39. The method according to any one of claims 34 to 38, wherein the first linker or the second linker comprises an amino acid sequence as shown in SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23, or consists of an amino acid sequence as shown in SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:

23.

40. The method according to any one of the preceding claims, wherein the amino acid sequence of the fusion protein is shown in any one of SEQ ID NOs: 36-55, SEQ ID NOs: 100-109, SEQ ID NOs: 111-113, and SEQ ID NOs: 125-127.

41. The method of any one of the preceding claims, wherein the fusion protein further comprises a signal peptide.

42. The method of claim 41, wherein the amino acid sequence of the signal peptide is as shown in SEQ ID NO:24 or SEQ ID NO:25, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity with the amino acid sequence as shown in SEQ ID NO:24 or SEQ ID NO:

25.

43. The method according to any of the preceding claims, wherein the half-life of the fusion protein obtained after removing the masking peptide by specific enzymatic cleavage reaction in the blood circulation of a subject is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

44. The method according to any one of the preceding claims, wherein the nucleic acid molecule encoding the fusion protein comprises: (a) the nucleotide sequence shown in SEQ ID NO:76, the nucleotide sequence shown in SEQ ID NO:77, and the nucleotide sequence shown in SEQ ID NO:78; (b) the nucleotide sequence shown in SEQ ID NO:76, the nucleotide sequence shown in SEQ ID NO:77, and the nucleotide sequence shown in SEQ ID NO:79; (c) the nucleotide sequence shown in SEQ ID NO:76, the nucleotide sequence shown in SEQ ID NO:77, and the nucleotide sequence shown in SEQ ID NO:80; (d) the nucleotide sequence shown in SEQ ID NO:76, the nucleotide sequence shown in SEQ ID NO:77, and the nucleotide sequence shown in SEQ ID NO:81; (e) the nucleotide sequence shown in SEQ ID NO:76, the nucleotide sequence shown in SEQ ID NO:123, and the nucleotide sequence shown in SEQ ID NO:78; (f) the nucleotide sequence shown in SEQ ID NO:76, the nucleotide sequence shown in SEQ ID NO:123, and the nucleotide sequence shown in SEQ ID NO:79; (g) the nucleotide sequence shown in SEQ ID NO:76, the nucleotide sequence shown in SEQ ID NO:123, and the nucleotide sequence shown in SEQ ID NO:80; (h) the nucleotide sequence shown in SEQ ID NO:76, the nucleotide sequence shown in SEQ ID NO:123, and the nucleotide sequence shown in SEQ ID NO:81; (i) the nucleotide sequence shown in SEQ ID NO:76, the nucleotide sequence shown in SEQ ID NO:130, and the nucleotide sequence shown in SEQ ID NO:78; (j) the nucleotide sequence shown in SEQ ID NO:76, the nucleotide sequence shown in SEQ ID NO:130, and the nucleotide sequence shown in SEQ ID NO:79; (k) the nucleotide sequence shown in SEQ ID NO:76, the nucleotide sequence shown in SEQ ID NO:130, and the nucleotide sequence shown in SEQ ID NO:80; or (l) the nucleotide sequence shown in SEQ ID NO:76, the nucleotide sequence shown in SEQ ID NO:130 and the nucleotide sequence shown in SEQ ID NO:

81.

45. A method according to any of the preceding claims, wherein the nucleic acid molecule encoding the fusion protein comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the nucleotide sequence shown in SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:116 or SEQ ID NO:

129.

46. ​​The method of claim 44 or 45, wherein the nucleic acid molecule encoding the fusion protein comprises a nucleotide sequence as shown in SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:116 or SEQ ID NO:129; or the nucleic acid molecule encoding the fusion protein consists of a nucleotide sequence as shown in SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:116 or SEQ ID NO:

129.

47. The method according to any one of the preceding claims, wherein the recombinant vector is selected from the group consisting of a plasmid, an artificial chromosome and a viral vector.

48. The method of claim 47, wherein the recombinant vector is a pcDNA plasmid (eg, pcDNA3.4 plasmid).

49. The method according to any one of the preceding claims, wherein the protein expression system is a cell expression system selected from the group consisting of bacteria, fungi, plant cells or animal cells.

50. The method of claim 49, wherein the bacteria is selected from the group consisting of Bacilli, Clostridia, Corynebacterium, Cupriavidus, Delftia, Escherichia, Enterobacter, Erwinia, Klebsiella, Lactobacillus, Lactococcus, Proteus, Pseudomonas, Rhodococcus, Salmonella, Serratia, Shigella, and Streptomyces.

51. The method of claim 49, wherein the bacteria is selected from the group consisting of Escherichia coli, Salmonella typhimurium, Serratia marcescens, Bacillus subtilis, B. licheniformis, and P. aeruginosa.

52. The method of claim 49, wherein the fungus is selected from the group consisting of Candida, Saccharomyces cerevisiae, Kluyveromyces, Pichia pastoris, and Schizosaccharomyces pombe.

53. The method of claim 49, wherein the animal cell is a mammalian cell.

54. The method of claim 53, wherein the mammalian cell is a human cell or a Chinese Hamster Ovary (CHO) cell.

55. The method of claim 53, wherein the mammalian cells are human embryonic kidney 293 cells (HEK293 cells).

56. The method of claim 53, wherein the mammalian cell is a CHO cell (eg, a CHO-K1 cell, a CHO-S cell).

57. The fusion protein prepared by the method according to the preceding claim, which has an activity of cleaving kallikrein of 0.3-3 PNAU / mg.

58. A fusion protein comprising a masking peptide, a first polypeptide and a second polypeptide, wherein: The masking peptide, the first polypeptide and the second polypeptide are directly connected or covalently connected through a linker, and a) the masking peptide has an amino acid sequence that can be specifically recognized and cleaved by a protease; b) the first polypeptide comprises KLK1 or a variant thereof; and c) the second polypeptide is used to extend the half-life of the first polypeptide.

59. The fusion protein of claim 58, wherein the masking peptide is selected from the group consisting of an enterokinase (EK) recognition sequence, a thrombin recognition sequence, a coagulation factor Xa recognition sequence, a tobacco etch virus (TEV) protease recognition sequence, a SUMO protease recognition sequence, and a sortase recognition sequence.

60. The fusion protein of claim 59, wherein the enterokinase (EK) recognition sequence is SEQ ID NO: 5 (VDDDDK).

61. The fusion protein according to any one of claims 57 to 60, wherein the first polypeptide is human KLK1 protein or a variant thereof.

62. The fusion protein of claim 61, wherein the amino acid sequence of the first polypeptide has at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, but still retains the function or activity of KLK1.

63. The fusion protein of any one of claims 57 to 62, wherein the second polypeptide is an Fc domain or albumin.

64. The fusion protein of any one of claims 57 to 63, further comprising a tag.

65. The fusion protein of claim 64, wherein the tag is a HIS tag.

66. The fusion protein according to any one of claims 57 to 65, wherein the fusion protein comprises the following structure from N-terminus to C-terminus: LB-MP-L1-P1-L2-P2, in, LB represents absence or tag, MP represents the masking peptide, L1 represents a bond or a first linker, P1 represents the first polypeptide, L2 represents a bond or a second linker, and P2 represents the second polypeptide.

67. The fusion protein according to claim 66, wherein the amino acid sequence of the fusion protein is shown in any one of SEQ ID NOs: 36 to 55, SEQ ID NOs: 100 to 109, SEQ ID NOs: 111 to 113, and SEQ ID NOs: 125 to 127.

68. A nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of any one of claims 57 to 67.

69. A vector comprising the nucleic acid molecule according to claim 68.

70. A protein expression system comprising the nucleic acid molecule according to claim 68 or the vector according to claim 69.

71. A pharmaceutical composition comprising the fusion protein according to any one of claims 57 to 67, the nucleic acid molecule according to claim 68, the vector according to claim 69 or the protein expression system according to claim 70, and a pharmaceutically acceptable carrier.

72. A method for constructing a protein expression system, comprising: Introducing a nucleic acid molecule encoding the fusion protein according to any one of claims 57 to 67 into a vector to construct an expression vector; The expression vector is introduced into a protein expression system.

73. A method for treating or preventing a disease, comprising administering to a subject in need of treatment or prevention the isolated fusion protein prepared by the method of any one of claims 1 to 56, wherein: The disease is selected from the group consisting of ischemic diseases, hemorrhagic diseases, renal diseases, metabolic diseases and neurodegenerative diseases.

74. The method of claim 73, wherein the ischemic disease is selected from the group consisting of cerebral ischemia (ischemic stroke), myocardial ischemia, ischemic colitis, limb ischemia, skin ischemia, ischemic stroke (e.g., acute ischemic stroke), transient ischemic attack (TIA), and traumatic brain injury (TBI).

75. The method of claim 73, wherein the ischemic disease is acute ischemic stroke.

76. The method of claim 73, wherein the renal disease is chronic kidney disease or acute kidney injury.

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