Fusion protein pegtepskinase with targeted thrombolytic effect, preparation method therefor, and use thereof

Abstract

Provided is a fusion protein t-PA-SAK PegtepskinaseTM with a targeted thrombolytic effect. The fusion protein not only retains the high thrombolytic activity of staphylokinase itself, but also has the ability to target and bind to fibrin on the surface of a thrombus and has lower immunogenicity, thereby exhibiting superior safety relative to similar drugs. Further, modifying the fusion protein by means of the PEG modification technology further reduces the immunogenicity of SAK, which satisfies clinical application scenarios that require multiple drug administrations for thrombolysis; the introduction of PEG also prolongs the half-life of a drug in vivo, achieves drug administration by intravenous bolus injection just a few times or once alone while enhancing the efficacy, and simplifies the therapeutic process while ensuring the therapeutic effect, which better fits the application form of clinical thrombolysis rescue treatment, and is suitable for thrombolysis treatment and secondary prevention of thrombotic diseases such as stroke, myocardial infarction, pulmonary embolism, and lower extremity venous thrombosis.

Description

A fusion protein pectinase with targeted thrombolytic activity, its preparation method and application Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology and relates to a fusion protein pectinase with targeted thrombolytic function, its preparation method and application. Background Technology

[0002] With an aging population and changes in lifestyles and habits, thrombotic diseases are increasingly becoming a major global health problem. Thrombotic diseases involve two pathological processes: thrombosis and thromboembolism. Thrombosis refers to the pathological process in which, under certain conditions, blood components form an embolus within a blood vessel, causing partial or complete blockage and impaired blood supply to the affected area. Thromboembolism is the pathological process in which a thrombus breaks off from its formation site and, during its journey through the bloodstream, partially or completely blocks certain blood vessels, causing ischemia, hypoxia, and necrosis (arterial thrombosis) and congestion and edema (venous thrombosis) in the corresponding tissues and / or organs. Thrombotic diseases are mainly classified into arterial thrombotic diseases and venous thrombotic diseases based on their location. Common arterial thrombotic diseases include stroke and myocardial infarction; common venous thrombotic diseases include lower extremity venous thrombosis and deep vein thrombosis. Deep vein thrombosis, if detached, can easily cause pulmonary embolism. Treatment for thrombotic diseases includes anticoagulation therapy, thrombolytic therapy, surgical treatment, or interventional therapy. In addition, since blood vessel walls are damaged after thrombosis, thrombosis can easily recur if preventive measures are not taken, which can worsen the condition. Therefore, secondary prevention of thrombotic diseases is also very important. Patients can reduce the risk of recurrence by adjusting their lifestyle or using medication.

[0003] Currently, thrombolytic drugs used clinically can be divided into fibrin-specific and non-fibrin-specific thrombolytics based on their binding specificity. Representative fibrin-specific thrombolytics include tissue plasminogen activator (t-PA) and succinyl kinase (SAK), while representative non-fibrin-specific thrombolytics include streptokinase (SK) and urokinase (UK). Non-fibrin-specific thrombolytics act on fibrin in normal plasma, activating plasminogen in systemic circulation, leading to systemic fibrinolysis and thus posing a risk of systemic bleeding in clinical applications. Fibrin-specific thrombolytics primarily act on fibrin on the surface of the thrombus, activating plasminogen to form plasmin. Plasmin degrades the fibrin on the thrombus surface into soluble fragments, thereby dissolving the thrombus. Therefore, fibrin-specific thrombolytics have better clinical safety compared to non-fibrin-specific thrombolytics. t-PA, SAK and other drugs have proven efficacy and have significantly reduced thrombosis in patients with early acute myocardial infarction (AMI) in previous clinical practice, thus reducing the risk of death and disability. In addition, SAK also has corresponding clinical value in thrombotic diseases such as acute ischemic stroke, deep vein thrombosis (DVT), pulmonary embolism (PE), arteriovenous fistula occlusion, ductus venosus-related thrombosis, and peripheral arterial occlusive disease (PAOD).

[0004] t-PA is a typical fibrin-specific thrombolytic agent. It binds to thrombus fibrin, activating plasminogen to plasmin. While it has good fibrinogen targeting, its short in vivo half-life (approximately 5 minutes) leads to poor patient compliance with continuous infusion. Furthermore, because plasminogen activator inhibitor 1 (PAI-I) inhibits t-PA's activation of plasminogen, continuous high-dose administration is necessary, potentially causing systemic depletion of fibrinogen and plasminogen, leading to an increased risk of intrinsic bleeding. SAK is another fibrin-specific thrombolytic agent that inhibits fibrinogen less in vivo. The thrombolytic dose of SAK is one-third that of SK or t-PA, and it has less impact on platelet function. Studies suggest that t-PA has a significantly smaller and simpler structure, lower production cost, and higher thrombolytic activity compared to rt-PA. However, although SAK is called a fibrin-specific thrombolytic agent, or a fibrin-selective thrombolytic agent, it does not possess protease activity and cannot directly bind to fibrin. Its activation is achieved by forming a complex with plasminogen. Specifically, SAK first forms an SAK-Plg complex with plasminogen (Plg) in a 1:1 ratio. This complex then needs the action of trace amounts of plasmin on the thrombus surface to convert Plg into plasmin (Plm), forming the plasminogen activator SAK-Plm. This plasminogen activator efficiently activates plasminogen to plasmin, thereby completing the thrombolytic process. Because SAK cannot target and accumulate on fibrin, the rate at which SAK-Plg is activated on the thrombus surface to form SAK-Plm is very slow. Furthermore, after the SAK-Plm complex is formed, it cannot effectively accumulate around the thrombus, easily causing systemic plasminogen depletion and increasing the risk of bleeding. Additionally, SAK-Plg that does not accumulate on the fibrin surface is neutralized by α2-anti-plasmin in the body, leading to the consumption of thrombolytic active ingredients and affecting the thrombolytic effect. Meanwhile, SAK, being a foreign protein derived from microorganisms, can produce neutralizing antibodies and immunogenicity in clinical applications, occasionally causing allergic reactions. Furthermore, most patients develop high titers of neutralizing antibodies after administration. The product information for marketed glucoskinase drugs states that "after using r-Sak (recombinant glucoskinase), due to the potential production of neutralizing antibodies, r-Sak should not be used again. If infarction occurs again, other thrombolytic drugs should be used." This significantly limits the clinical application of glucoskinase. Therefore, in recent years, the modification of glucoskinase to produce low immunogenicity has become one of the research directions for glucoskinase. In addition, glucoskinase has a short half-life of only about 5 minutes, requiring intravenous infusion for up to 30 minutes to maintain an effective thrombolytic concentration, which is inconvenient for clinical procedures.

[0005] On the other hand, PAI-1 (Plasminogen Activator Inhibitor-1) is a fast-acting serine protease inhibitor produced by vascular endothelial cells, the liver, etc., and is a major negative regulator of the fibrinolytic system in the human body. It inhibits plasmin production and hinders thrombus dissolution by rapidly binding to and inactivating tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA). SAK itself is not a serine protease and does not possess the protease active site recognized by PAI-1. PAI-1 cannot directly bind to SAK, nor can it directly inhibit SAK activity. Therefore, SAK has a greater advantage in resisting PAI-1.

[0006] This invention selects to fuse SAK with a partial fragment of t-PA to form a fusion protein. The designed fusion protein not only retains the high thrombolytic activity of SAK itself, but also introduces the ability of t-PA to target and bind to thrombus fibrin, thus possessing a higher degree of targeted fibrin aggregation than similar drugs, ensuring the safety of treatment. Furthermore, the introduction of the humanized t-PA fragment is expected to reduce the immunogenicity of SAK, improving the safety and efficacy of SAK application. Further, modifying the fusion protein with PEG technology can further reduce the immunogenicity of SAK, improving safety while meeting the clinical application scenarios requiring multiple-dose thrombolysis; the introduction of PEG can also prolong the drug's half-life in vivo, enhancing efficacy while requiring only a few intravenous bolus injections or a single administration, simplifying the treatment process while ensuring therapeutic effect, and better aligning with the application of clinical thrombolytic rescue therapy. Summary of the Invention

[0007] To overcome the problems of existing thrombolytic drugs, this application first provides a fusion protein with targeted thrombolytic activity. This fusion protein is formed by fusing the non-protease region or part of the non-protease region of human tissue-type plasminogen activator (ht-PA) with mature Staphylococcus aureus styrax kinase (SAK), namely t-PA-SAK. The fusion protein of this application has both the thrombolytic activity of SAK and the targeted recognition ability of t-PA for fibrin; furthermore, the introduction of the humanized fragment of t-PA can reduce the immunogenicity of SAK, thereby improving the safety and efficacy of SAK application.

[0008] The t-PA-SAK fusion protein of the present invention contains a t-PA fragment and a SAK fragment, which are directly linked or linked via a linker peptide. The t-PA fragment is located at the N-terminus or C-terminus of the SAK fragment. The linker peptide can use conventional rigid linkers or flexible linkers. Common rigid linkers include (EAAAK)n, (EAAAAK)n, etc., where n represents the number of repeats of EAAAK or EAAAAK; common flexible linkers include (GGGGGS)n, etc., where n represents the number of repeats of GGGGGS.

[0009] One object of this invention is to provide a design of the t-PA moiety in a t-PA-SAK fusion protein. The selected t-PA moiety structure can endow the t-PA-SAK fibrin with affinity and targeting.

[0010] The non-protease domain of natural human t-PA includes a finger domain (F), an EGF-like domain (E), a Kringle 1 domain (K1), a Kringle 2 domain (K2), and connecting peptides between these domains. In the design of fusion proteins, the portion derived from t-PA is referred to as the targeting module (TM). The TM can be fused with two or more domains from the non-protease domain of natural human t-PA. Different TM designs, when fused with SAK, produce fusion proteins with varying affinities for fibrin, and the ease of expression and preparation of fusion proteins from different TM designs also varies considerably.

[0011] Preferably, the t-PA fragment consists of two or more domains selected from F, E, K1, and K2, with F and K2 being the most preferred. The exemplified F amino acid sequence is shown in SEQ ID NO: 1; the E amino acid sequence is shown in SEQ ID NO: 2; the K1 amino acid sequence is shown in SEQ ID NO: 3; and the K2 amino acid sequence is shown in SEQ ID NO: 4.

[0012] The domains in the t-PA fragment are linked by linker peptides. Preferably, the linker peptide connecting the K1 domain to its preceding domain in the t-PA fragment is a natural linker peptide with the amino acid sequence shown in SEQ ID NO: 5, or a conventional rigid linker or flexible linker can be used. The amino acid sequence of the natural linker peptide connecting the K2 domain to its preceding domain can be SEQ ID NO: 6, or a conventional rigid linker or flexible linker can be used. The F domain and the E domain can be directly linked without a linker peptide. Common rigid linkers include (EAAAK)n, (EAAAAK)n, etc., where n represents the number of repeats of EAAAK or EAAAAK; common flexible linkers include (GGGGGS)n, etc., where n represents the number of repeats of GGGGGS, and n is an integer from 1 to 4.

[0013] When the K2 domain is selected as the target module in t-PA-SAK, the P in the C-terminal CDVPSC sequence of the K2 domain can be mutated to K or R to form a K2 domain mutant (the K2 domain mutant formed by mutating the P amino acid to K is abbreviated as K2). m1 The K2 domain mutant, formed by the mutation of amino acid P to R, is abbreviated as K2. m2 The mutated K2, compared to the unmutated version, results in a fusion protein that is not only more conducive to the characterization and analysis of disulfide bonds, but also, as confirmed by research, does not alter the key properties of the K2 domain, nor does it change the fibroin affinity or SAK activity of the fusion protein. The main reason for this is that existing disulfide bond identification techniques require the use of proteases to cleave proteins into peptides, commonly including trypsin and chymotrypsin. However, there are no conventional protease cleavage sites between the two carbons in CDVPSC, making accurate identification impossible. During identification, the two carbons in this peptide segment are assumed to be paired, when in fact they are not, leading to disorder and misjudgment in the overall disulfide bond pairing identification. Introducing the mutated K2... m1 and K2 m2 This design incorporates a trypsin site, enabling cleavage with commonly used trypsin and avoiding interference from the sequence itself, thus preventing misinterpretations in disulfide bond analysis. When selecting the K2 domain as the targeting module in t-PA-SAK, other K2 domain mutants or the natural K2 domain reported in existing technologies can be chosen.

[0014] The SAK fragment in the fusion protein of the present invention endows the t-PA-SAK fusion protein with thrombolytic activity. The SAK can be a natural SAK protein or a mutant. The amino acid sequence of the mature peptide containing 136 amino acids exemplified in the examples is shown in SEQ ID NO: 9.

[0015] Mature SAK peptides contain 136 amino acids, and their N-terminus is prone to degradation, resulting in the removal of 6-10 amino acids. This removal typically does not affect SAK activity. However, when the SAK fragment in a fusion protein is formed by removing the first 10 amino acids from the N-terminus and is directly fused to the targeting module in the order of t-PA fragment followed by the SAK fragment from the N-terminus to the C-terminus, the thrombolytic activity of the fusion protein is lost or very low. However, when a linker peptide is introduced between the targeting module and the SAK fragment (with the first 10 amino acids removed from the natural mature peptide) during fusion protein design, the thrombolytic activity gradually recovers. This may be because the 11th amino acid of the natural mature SAK peptide is the key active site of SAK; if directly fused with the targeting module, the module may mask this active site, leading to a loss of SAK activity. When the N-terminal amino acid of the SAK fragment is not missing, or only a few are missing, the t-PA fragment and the SAK fragment can be directly fused in the order of t-PA fragment followed by the SAK fragment from the N-terminus to the C-terminus, or they can be linked by a linker peptide. The SAK fragment in the fusion protein of this application is not limited to this; other SAK or SAK mutants reported in the prior art may also be used in this application.

[0016] Another object of the present invention is to provide a recombinant expression host for the t-PA-SAK fusion protein. Escherichia coli is preferred. The Escherichia coli includes Top10, DH5α, JM109, BL21(DE3), Rosetta(DE3), and BL21AI(DE3).

[0017] Another object of the present invention is to provide a recombinant expression vector for the t-PA-SAK fusion protein. The recombinant expression vector includes: pET28a, pET26b, pBV220, and pDEST14.

[0018] Another object of the present invention is to provide a method for purifying and preparing t-PA-SAK fusion protein:

[0019] 1. After induction of expression, E. coli cells were resuspended in PBS (pH 7.4) buffer to 100-150 g / L to ensure the system was homogeneous and free of lumps.

[0020] 2. The resuspended bacterial solution from step 1 is broken up three times using a high-pressure homogenizer, with the pressure controlled at 900-1000 bar and the process temperature not exceeding 35℃.

[0021] 3. Collect the lysed system from step 2 by centrifugation. The collected precipitate is the inclusion body of the target protein.

[0022] 4. The inclusion bodies from step 3 were resuspended in PBS + 0.5-1.5% Triton X100 + 1-2M urea at pH 7.4, with 10g wet weight corresponding to 1L of buffer. The mixture was stirred and dispersed at room temperature for 2-5 hours, and then centrifuged again to collect the inclusion bodies. This process was repeated three times to obtain the crudely purified inclusion bodies.

[0023] 5. After crude purification, the inclusion bodies were resuspended at a ratio of 1-2 g / 100 mL in 20 mM Tris, 6 M guanidine hydrochloride, and 5 mM DTT at pH 8.0. The solution was stirred at room temperature until completely dissolved with no obvious solid particles. The supernatant of the denatured solution was collected by centrifugation. The supernatant was then diluted 1 / 10 and refolded. The refolding solution consisted of 20 mM Tris, 0.5 M guanidine hydrochloride, 3 mM reduced glutathione, and 1 mM oxidized glutathione at pH 8.0. The refolding temperature was 2-8℃, and the refolding time was 30-48 h.

[0024] 6. After refolding, the refolded solution is clarified by centrifugation or filtration, and then the clarified solution is treated by ultrafiltration, replacing it with a 10 mM PB solution at pH 7.4. This solution is then used for chromatographic purification.

[0025] 7. First step chromatography: The chromatography medium was Sepharose SP High Performance. The elution buffer ratio was linearly increased to 50% within 10 column volumes, and the sample was collected. Binding buffer: 10 mM PB, pH 7.4; Elution buffer: 10 mM PB + 0.5 M sodium chloride, pH 7.4.

[0026] 8. Second step chromatography: Add the chromatographic sample collected in the first step to a final concentration of 1M ammonium sulfate and adjust the pH to 7.4. The chromatography medium is Sepharose Phenyl High Performance. Within 10 column volumes, the elution buffer ratio passing through the column bed should reach 100% linearity. Collect the sample. Elution buffer: 10mM PB, pH 7.4; Binding buffer: 10mM PB + 1.0M ammonium sulfate, pH 7.4.

[0027] 9. Ultrafiltration buffer replacement: Replace the sample from the second step of chromatography with PBS (pH 7.4) for ultrafiltration.

[0028] This invention reveals that the t-PA-SAK fusion protein has good thrombolytic activity, and the fusion of t-PA does not affect the thrombolytic activity of SAK.

[0029] This invention further reveals that the design of the targeting module for the t-PA-SAK fusion protein affects the affinity, yield, and stability of the fusion protein for fibrin. When the targeting module TM is fused with a single finger domain to SAK, the resulting fusion protein has no affinity for fibrin. When two tandem finger domains are fused with SAK, the resulting fusion protein has very low affinity. When a fusion fragment of the FE-K1-K2 domain is fused with SAK, the resulting fusion protein has a strong affinity for fibrin, but the protein purification yield is significantly lower than other designs. This may be because the fused FE-K1-K2 module structure is too complex, making in vitro refolding difficult. When a fusion fragment of the F-K2 domain is fused with SAK, the resulting fusion protein has the strongest affinity for fibrin, and its protein purification yield is high, reaching 300 mg / L (pure product mass / fermentation volume). This is consistent with reports that F and K2 together form t-PA fibrin affinity.

[0030] This application also provides a polyethylene glycol-modified SAK fusion protein petimax enzyme. TM (Pegtepskinase TM It can significantly improve protein stability, reduce immunogenicity, improve the pharmacokinetic properties of the protein in vivo, and retain the original protein activity to the greatest extent, retain the original protein's ability to target fibrin to the greatest extent, and has a lower risk of bleeding.

[0031] Preferably, the polyethylene glycol-modified SAK fusion protein pectinase TM (Pegtepskinase TM The fusion protein in ) is the t-PA-SAK fusion protein mentioned above.

[0032] Preferably, the polyethylene glycol modifier is branched.

[0033] Preferably, the polyethylene glycol modifier is a branched polyethylene glycol modifier with a molecular weight of 5 kDa-30 kDa. More preferably, it is 10-20 kDa. The molecular weight refers to the molecular weight of the PEG chain.

[0034] Preferably, the general structural formula of the branched PEG is shown in formula (1).

[0035] Wherein, X1 and X2 are the same or different linking groups, and the linking groups are selected from -(CH2). i OCOO-、-(CH2) i OCONH-、-(CH2) i NHCOO-、-(CH2)i NHCONH-、-(CH2) i COO-, -(CH2) i The group consisting of CONH-, equation (2), and equation (3), where i is an integer from 0 to 10;

[0036] Y originates from the group consisting of Gly, Lys, Gly-Lys, and Glu-Lys.

[0037] m is an integer from 1 to 10; n is an integer from 50 to 375, preferably an integer from 102 to 250.

[0038] Preferably, each SAK fusion protein molecule is coupled with a polyethylene glycol modifier at its N-terminus.

[0039] The branched polyethylene glycol modifiers exemplified in the examples have the general structural formulas shown in formulas (4), (5-1), and (5-2).

[0040] The structural formulas exemplified in the embodiments are for illustrative purposes only and are not intended to limit the specific structure of the branched polyethylene glycol modifiers of this application. Those skilled in the art may also use other branched polyethylene glycol modifiers with structural formulas different from those exemplified.

[0041] The structural formulas of modified products obtained using PEG modifiers with specific structures are predictable to those skilled in the art. For example, if a branched polyethylene glycol modifier with a general structural formula as shown in formula (5-2) is used as the PEG modifier, the general structural formula of the resulting polyethylene glycol-modified SAK fusion protein is shown in formula (6), where R is the aforementioned t-PA-SAK fusion protein.

[0042] The present invention also provides a method for preparing the above-mentioned polyethylene glycol-modified t-PA-SAK, comprising the following steps:

[0043] Step 1, buffer replacement

[0044] The t-PA-SAK to be modified was replaced into the modification buffer by means of column desalting, dialysis, concentration and dilution, and tangential flow ultrafiltration.

[0045] The second step is the modification reaction and purification of the modification product.

[0046] After adding PEG modifier and reducing agent sodium cyanoborohydride to the t-PA-SAK solution collected in the first step, the reaction was carried out. After the reaction was completed, the solution was purified by ion exchange chromatography.

[0047] The ion-exchange chromatography eluted sample was concentrated by ultrafiltration and then purified using a molecular sieve chromatography column.

[0048] This invention also provides the application of the above-mentioned t-PA-SAK fusion protein and the above-mentioned polyethylene glycol-modified t-PA-SAK in thrombolytic therapy and secondary prevention of thrombotic diseases. Common thrombotic diseases include stroke, myocardial infarction, pulmonary embolism, and lower extremity deep vein thrombosis.

[0049] This application has the following advantages over the prior art:

[0050] This invention selects to fuse SAK with a partial fragment of t-PA to form a fusion protein. The designed fusion protein possesses both the thrombolytic activity of SAK and the fibrin-targeting recognition ability of t-PA. Furthermore, the introduction of the humanized t-PA fragment can reduce the immunogenicity of SAK, thereby improving the safety and efficacy of SAK application.

[0051] Furthermore, this application utilizes polyethylene glycolation technology to significantly improve protein stability, reduce immunogenicity, improve the pharmacokinetic properties of the protein in vivo, and maximize the preservation of the original protein's activity and its ability to target fibrin, while also reducing the risk of bleeding.

[0052] The drug molecules in this invention are expected to possess ideal thrombolytic drug characteristics, including: (1) better targeting. The polyethylene glycol-modified fusion protein of this invention introduces a t-PA targeting module on the basis of SAK. After PEG modification, the fusion protein still has the same affinity for fibrin as alteplase, allowing SAK to target and aggregate on fibrin, promoting the formation of plasminogen activator on the thrombus surface and its aggregation at the thrombus site, thereby more fully exerting the thrombolytic effect of SAK and reducing the risk of bleeding. (2) superior thrombolytic activity. In vitro and in vivo experiments have shown that the polyethylene glycol-modified fusion protein of this invention has high thrombolytic activity, good vascular recanalization rate, and thrombolytic effect superior to the original SAK protein, effectively reducing mortality. This indicates that the introduction of the t-PA targeting module and PEG does not affect the thrombolytic activity of SAK, and the fusion protein containing the targeting unit has better thrombolytic performance. Furthermore, the examples demonstrate that the polyethylene glycol fusion protein of the present invention can achieve a fibrinolytic effect of more than 50% with a low dose (2μg / 200μL), indicating that it has high thrombolytic performance and is suitable for different types of thrombi and different patient conditions. (3) It has higher safety. Bleeding is the most common adverse reaction of SAK thrombolytic agents. The incidence of bleeding in the clinical trials of marketed r-SAK preparations is 24%. The polyethylene glycol modified fusion protein of the present invention has a lower risk of causing bleeding complications and a significantly lower bleeding tendency than the original SAK protein, thus having better safety. On the other hand, the polyethylene glycol modified fusion protein of the present invention has extremely low immunogenicity compared to the original SAK protein, and the possibility of allergic reactions is extremely low, which can reduce immune-related side effects and complications and make up for the high immunogenicity of glucosamine. (4) It has the advantage of long-lasting effect. The half-life of SAK is only about 5 minutes, and thrombolytic administration requires an intravenous infusion of up to 30 minutes, so its clinical administration process is more complicated and inconvenient. The polyethylene glycol-modified fusion protein of the present invention significantly prolongs the drug half-life of SAK, making it feasible for intravenous bolus administration. This not only reduces the number of administrations and simplifies the treatment process, but also improves patient compliance and provides a more effective thrombolytic window for thrombosis patients. (5) It can be administered multiple times. Since SAK is a foreign protein of microbial origin, its immunogenicity will produce neutralizing antibodies after entering the human body. Therefore, the SAK preparations currently on the market can generally only be used once for thrombolysis. When thrombosis occurs again, other thrombolytic drugs need to be used. The polyethylene glycol-modified fusion protein of the present invention has extremely low immunogenicity and rarely produces anti-drug antibodies. Therefore, it can be administered multiple times, providing thrombosis patients with the opportunity for continuous or multiple thrombolysis. Compared with SAK thrombolytic agents that can only be used once, it is expected to achieve a more definite therapeutic effect. (6) It has broad application prospects.In vitro and in vivo studies show that the polyethylene glycol-modified fusion protein of the present invention has good safety and thrombolytic properties, and has advantages such as long-lasting effect, low immunogenicity, low-dose effectiveness, and intravenous injection. It meets the clinical expectation for an ideal thrombolytic drug and can be applied to the thrombolytic treatment of thrombotic diseases such as stroke, myocardial infarction, pulmonary embolism, and lower extremity venous thrombosis. It also has great potential for secondary prevention of thrombotic diseases. Attached Figure Description

[0053] Figure 1a Example 2F-K2 m2 -SAK fusion protein amino acid sequence coverage diagram using try+chy restriction enzyme digestion (non-reduction).

[0054] Figure 1b Example 2F-K2 m2 -LC-MS disulfide bond analysis results of SAK fusion protein

[0055] Figure 2 Example 2F-K2 m2 -LC-MS molecular weight analysis results of SAK fusion protein

[0056] Figure 3a Example 2F-K2 m2 -HPLC-SEC purity analysis results of SAK fusion protein

[0057] Figure 3b Example 2F-K2 m2 -HPLC-RP purity analysis results of SAK fusion protein

[0058] Figure 4. Fluorescence detection results of Example 6

[0059] Figure 5. Octet assay results showing the binding of each test sample to the fibrin-plasminogen complex.

[0060] Figure 6. Octet assay of fibrin binding to the test sample-plasminogen complex.

[0061] Figures 7a and 7b show the enzyme kinetics detection diagrams.

[0062] Figure 8. Octet detection of the binding of the test sample with PAI-1.

[0063] Figure 9. In vitro thrombolysis effect of the test sample

[0064] Figure 10. Results of rat blood drug concentration detection at different time points for each test sample.

[0065] Figure 11. Results of antibody titer detection at different dilutions of rat serum in Example 17

[0066] Figure 12. Pharmacokinetic curve fitting of ZHB130 and SAK after a single intravenous injection (n=3)

[0067] Figure 13. Effect of ZHB130 injection on serum creatinine levels in rats (n≥3, mean ± SEM)

[0068] Figure 14. Digital photographs of brain tissue in PT model mice after perfusion (n=8-9, Male)

[0069] Figure 15. Representative HE staining of the infarct area in PT model mice (n = 10⁻¹⁴, Male)

[0070] Figure 16. Semi-quantitative statistical plot of relative hemorrhage area in the infarct region of PT model mice stained with HE (n = 10⁻¹⁴, Male, Mean ± SEM). Note: ***: P < 0.001, vs ZHB130 group.

[0071] Figure 17. Representative TTC staining images of brain tissue from PT model mice (n=9)

[0072] Figure 18. Effect of ZHB130 injection on cerebral infarction volume in PT model mice (n=9, Male, Mean±SEM) Note: ***: P<0.001, **: P<0.01, *: P<0.05, vs PT model group

[0073] Figure 19. Inhibitory effect of ZHB130 injection on wet weight of arteriovenous bypass thrombus in rats (n=10, Male)

[0074] Figure 20. Inhibitory effect of ZHB130 injection on the dry weight of arteriovenous bypass thrombus in rats (n=10, Male)

[0075] Figure 21. Effect of ZHB130 injection on arterial thrombosis time in rats (n=10, Male)

[0076] Figure 22 Effect of ZHB130 injection on rat body weight

[0077] Figure 23 Effect of ZHB130 injection on the rate of change in rat body weight

[0078] Figure 24 Effects of ZHB130 injection on neurological deficit symptoms in rats

[0079] Figure 25 Effect of ZHB130 injection on cerebral infarction area in rats Detailed Implementation

[0080] Technical terms

[0081] Polyethylene glycol (PEG) is typically polymerized from ethylene oxide and comes in branched, linear, and multi-arm forms. Ordinary PEG has one hydroxyl group at each end; if one end is blocked with a methyl group, methoxylated polyethylene glycol (mPEG) is obtained.

[0082] Polyethylene glycol (PEG) modifiers: PEG modifiers refer to polyethylene glycol derivatives with functional groups. They are activated polyethylene glycols and can be used for the modification of proteins and peptide drugs. The actual molecular weight of a PEG modifier with a specific molecular weight can be 90% to 110% of its labeled value; for example, the molecular weight of PEG 5K can be 4.5 kDa to 5.5 kDa. The linear PEG modifiers used in this application were purchased from Beijing Keyk Technology Co., Ltd.; among the branched PEG modifiers, Y-type PEG 5K, 10K, and 20K were purchased from Beijing Keyk Technology Co., Ltd., and V-type PEG 10K, 20K, and 30K were purchased from Xiamen Sinobond Biotechnology Co., Ltd.

[0083] Y-type PEG 5K: A branched polyethylene glycol modifier with a molecular weight of approximately 5 kD, such as the PEG modifier shown in structural formula (1).

[0084] Y-type PEG 10K: A branched polyethylene glycol modifier with a molecular weight of approximately 10 kD, such as the PEG modifier shown in structural formula (1).

[0085] Y-type PEG 20K: A branched polyethylene glycol modifier with a molecular weight of approximately 20 kD, such as the PEG modifier shown in structural formula (1).

[0086] V-type PEG 10K: A branched polyethylene glycol modifier with a molecular weight of approximately 10 kD, such as the PEG modifier shown in structural formula (2-1) or (2-2). In specific embodiments, a modifier with general structural formula (2-2) is selected.

[0087] V-type PEG 20K: A branched polyethylene glycol modifier with a molecular weight of approximately 20 kD, such as the PEG modifier shown in structural formula (2-1) or (2-2). In specific embodiments, the modifier with general structural formula (2-2) is selected.

[0088] V-type PEG 30K: A branched polyethylene glycol modifier with a molecular weight of approximately 30 kD, such as the PEG modifier shown in structural formula (2-1) or (2-2). In specific embodiments, the modifier with general structural formula (2-2) is selected.

[0089] Linear PEG 5K: A linear polyethylene glycol modifier with a molecular weight of approximately 5 kD, such as the PEG modifier shown in structural formula (4).

[0090] Linear PEG 10K: A linear polyethylene glycol modifier with a molecular weight of approximately 10 kD, such as the PEG modifier shown in structural formula (4).

[0091] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that the embodiments are used only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0092] Example 1: Design of t-PA-SAK fusion protein

[0093] The t-PA-SAK fusion protein contains t-PA and SAK fragments, which are directly linked or linked via a linker peptide. The t-PA fragment is located at the N-terminus or C-terminus of the fusion protein. The fusion protein is composed of the t-PA fragment, linker peptide, and SAK fragment sequentially from the N-terminus to the C-terminus; or sequentially from the N-terminus to the C-terminus, it is composed of the SAK fragment, linker peptide, and t-PA fragment.

[0094] The t-PA fragment consists of one or more domains selected from F, E, K1, and K2, preferably domains that bind to fibroin, such as F and K2. Examples of the t-PA fragments include the amino acid sequence of F (SEQ ID NO: 1), the amino acid sequence of E (SEQ ID NO: 2), the amino acid sequence of K1 (SEQ ID NO: 3), and the amino acid sequence of K2 (SEQ ID NO: 4). The domains in the t-PA fragment are linked by a linker peptide, preferably a natural linker peptide. The amino acid sequence of the linker peptide connecting the K1 domain to its preceding domain, as illustrated in the examples, is shown in SEQ ID NO: 5. The amino acid sequence of the linker peptide connecting the K2 domain to its preceding domain can be SEQ ID NO: 6 (except for numbers 18-21 in the table below, the linker peptides connecting K2 to its preceding domain in the remaining fusion proteins), or SEQ ID NO: 7 (a single linker connecting the K2 domain to its preceding domain in the fusion proteins numbered 18 and 19 in the table below), or the sequence shown in SEQ ID NO: 8 (a single linker connecting the K2 domain to its preceding domain in the fusion proteins numbered 20 and 21 in the table below). The F domain and E domain are directly linked without a linker peptide. The domains or linkers that can be used in this application are not limited to the specific sequences illustrated in the examples. For example, individual amino acid deletions, substitutions, or insertions can be made based on the amino acid sequences of the natural domains and linkers. Mutants of the natural domains and linkers reported in the prior art can also be used in this application.

[0095] The linker peptide connecting the t-PA fragment and the SAK fragment can be a rigid linker or a flexible linker. The amino acid sequence of the rigid linker exemplified in the examples is shown in SEQ ID NO: 7; the amino acid sequence of the flexible linker is shown in SEQ ID NO: 8. The linker peptides used in the examples are for illustrative purposes only and are not intended to limit the specific form of the linker peptide. Those skilled in the art can select suitable linker peptides through conventional experiments, and are not limited to the specific linker peptides exemplified in the examples.

[0096] The SAK fragment is the natural SAK protein or a mutant. The amino acid sequence of the mature peptide containing 136 amino acids, exemplified in the examples, is shown in SEQ ID NO: 9. The SAK fragment in the fusion protein of this application is not limited to this; other SAK or SAK mutants reported in the prior art can also be used in this application.

[0097] Taking the fusion protein in Table 3 as an example, from the N-terminus to the C-terminus, it is composed of the F domain, E domain, the sequence shown in SEQ ID NO: 5, K1, the sequence shown in SEQ ID NO: 6, K2 domain, and SAK mature peptide, and its amino acid sequence is shown in SEQ ID NO: 10.

[0098] Table 1 Note: 1. The above K2 can be either wild-type K2 or K2 m1 (A K2 domain mutant is formed by mutating P in the CDVPSC sequence at the C-terminus of the K2 domain to K), or it can be K2 m2 (The K2 domain mutant is formed by mutating P in the CDVPSC sequence at the C-terminus of the K2 domain to R); 2. The F and E domains of the above-mentioned targeting module are directly connected.

[0099] Example 2: Construction, expression, and purification of t-PA-SAK fusion protein expression system in E. coli

[0100] With F-K2 m2 The construction, expression, and purification of SAK are used as an example for illustration. These examples are for illustrative purposes only and are not intended to limit the construction, expression, and purification methods of fusion protein expression systems. Those skilled in the art can use conventional construction and expression purification methods in the field. F-K2 m2 The -SAK amino acid sequence is shown in SEQ ID NO: 11. Its N-terminus to C-terminus consists of an F domain, two rigid linkers, and K2(K2) domain. m2 The fusion protein is composed of a 136-amino acid mature peptide (SAK). The first amino acid, M, is derived from the start codon of the *E. coli* expression system and is essential for expression, but not essential for the fusion protein's function; it may be cleaved after expression. It is well known to those skilled in the art that, in most cases, the start codon in prokaryotes is AUG, encoding formylmethionine; the start codon in eukaryotes is AUG, encoding methionine. Therefore, recombinant proteins expressed in prokaryotes such as *E. coli* usually contain formylmethionine at the N-terminus in addition to the natural amino acid sequence, and recombinant proteins expressed in eukaryotes such as yeast usually contain methionine at the N-terminus in addition to the natural amino acid sequence. After expression, this methionine may be completely retained, partially retained and partially cleaved, or completely cleaved.

[0101] 1. Obtaining the target genes: The recombinant human t-PA encoding DNA sequence and the recombinant Staphylococcus aureus stapkinase (SAK) encoding DNA sequence were designed and synthesized by Nanjing Genscript Genetics. Primers were designed, and F, rigid inniker, and K2 genes were obtained by PCR amplification. m2 The three-fragment fusion fragment, namely the coding fragment of the fusion protein targeting module, was obtained. Primers were designed, and the SAK fusion fragment was obtained by PCR amplification. The targeting module fragment and the SAK fragment were then fused by PCR to obtain F-K2. m2 -SAK complete DNA fragment, as shown in SEQ ID NO: 12.

[0102] 2. F-K2 m2 The -SAK PCR product was digested with XhoI and NdeI enzymes, and then cloned into the pET26b vector, ultimately forming pET26b-F-K2. m2 -SAK recombinant plasmid.

[0103] 3. pET26b-F-K2 m2 -SAK recombinant plasmid was transformed into BL21(DE3) competent cells by heat shock and cultured to form recombinants.

[0104] 4. Select typical single colonies of the recombinants from step 3 and inoculate them into LB kanamycin-resistant medium. Identify and sequence the recombinants by bacterial PCR to obtain the correctly sequenced ones.

[0105] 5. The correctly sequenced recombinants from step 4 are inoculated into LB liquid medium and cultured on a shaker (37℃, 220 rpm). When OD... 600 When the concentration reaches 1.0, add 0.2 mM IPTG to the culture medium to induce expression, and continue culturing for 4 hours to end the culture. Collect the bacterial cells by centrifugation.

[0106] 6. After induction of expression, E. coli cells were resuspended in PBS (pH 7.4) buffer to 120 g / L to ensure the system was homogeneous and free of lumps.

[0107] 7. The resuspended bacterial solution from step 6 is broken up three times using a high-pressure homogenizer, with the pressure controlled at 900-1000 bar and the process temperature not exceeding 35℃.

[0108] 8. Collect the lysed system from step 7 by centrifugation. The collected precipitate is the inclusion body of the target protein.

[0109] 9. The inclusion bodies from step 8 were resuspended in 1L of buffer at a ratio of 10g wet weight. The mixture was then dispersed in PBS + 1% Triton X100 + 2M urea (pH 7.4) at room temperature for 2 hours with stirring. The inclusion bodies were then collected by centrifugation. This process was repeated three times to obtain the crudely purified inclusion bodies.

[0110] 10. After crude purification, the inclusion bodies were resuspended at a ratio of 1 g / 100 mL in 20 mM Tris + 6 M guanidine hydrochloride + 5 mM DTT at pH 8.0. The solution was stirred at room temperature until completely dissolved with no obvious solid particles. The supernatant of the denaturing solution was collected by centrifugation. The supernatant was diluted 1 / 10 and refolded. The refolding solution consisted of 20 mM Tris + 0.5 M guanidine hydrochloride + 3 mM reduced glutathione + 1 mM oxidized glutathione at pH 8.0. The refolding temperature was 6-8℃, and the refolding time was 48 h.

[0111] 11. After refolding, centrifuge or filter to clarify the solution, then treat the clarified solution with ultrafiltration, replacing it with a 10 mM PB solution at pH 7.4. Then use this solution for chromatographic purification.

[0112] 12. First step chromatography: The chromatography medium was Sepharose SP High Performance. Within 10 column volumes, the elution buffer ratio was linearly achieved to 50%, and the sample was collected. Binding buffer: 10 mM PB, pH 7.4; Elution buffer: 10 mM PB + 0.5 M sodium chloride, pH 7.4;

[0113] 13. Second step chromatography: Add 1M ammonium sulfate to the sample from the first step chromatography and adjust the pH to 7.4. The chromatography medium is Sepharose Phenyl High Performance. The elution buffer ratio is linearly maintained at 100% within 10 column volumes. Collect the sample. Elution buffer: 10mM PB, pH 7.4; Binding buffer: 10mM PB + 1.0M ammonium sulfate, pH 7.4;

[0114] 14. Ultrafiltration buffer replacement: The ultrafiltration buffer for the second-step chromatography sample was replaced with PBS (pH 7.4). The purified sample after buffer replacement was analyzed for purity using HPLC-SEC and HPLC-RP, and disulfide bonds and molecular weight were analyzed using LC-MS. The results are shown in Figures 1a, 1b, 2, 3a, and 3b, respectively.

[0115] Figures 1a and 1b show that the target protein can be fully cleaved by trypsin, covering most of the sequence shown in SEQ ID NO: 11 (some sequences were not covered due to unsuitable cleavage sites). Combined with molecular weight analysis, the fusion protein sequence can be determined to be intact (the M of the N-terminal start codon was cleaved). Mass spectrometry analysis of the cleaved peptides showed that the detected disulfide bond pairings were consistent with the theory (theoretical disulfide bond pairings: C110 / C134, C35 / C44, C79 / C121, C58 / C139, C7 / C37).

[0116] As shown in Figure 2, the molecular weight of the recombinant fusion protein after purification is 131.19 Da smaller than expected. Mass spectrometry analysis indicates that this is due to the cleavage of the first amino acid, M, after expression. The first amino acid, M, originates from the start codon of the *E. coli* intracellular expression system and is not an amino acid of the target module itself. Therefore, it is unnecessary for the fusion protein to perform its physiological function, and its loss may further reduce immunogenicity. Therefore, it can be concluded that the molecular weight of the purified fusion protein is correct and highly homogeneous.

[0117] As shown in Figures 3a and 3b, the recombinant fusion protein, after purification, was analyzed by HPLC-SEC and HPLC-RP. The purity of both methods reached over 95%, which meets the general requirements for pharmaceuticals and shows potential for medicinal use.

[0118] Different design forms were expressed and purified using the same method, and the yields are shown in the table below:

[0119] Table 2 Notes: 1. The above yield calculations are based on a 100% purification yield of the F-K2-SAK (SAK contains 136 amino acids) fusion protein (number 19) per unit expression volume. 2. Except for number 3, which is wild-type, number 8 is K2. m1 The rest are K2 m2 When K2 is wild type or K2 m1 It also does not affect the yield of the target protein from fibroin. 1. The F and E domains of the above targeting module are directly connected.

[0120] Example 3: Affinity assessment of t-PA-SAK fusion proteins with different designs to fibrin

[0121] I. Experimental Methods

[0122] The affinity between the fusion protein and fibrin was detected using biomembrane layer interferometry (BLI) and compared with t-PA and SAK.

[0123] 1. Sample preparation

[0124] Receptor preparation: Since fibrin is poorly soluble, plasmin was used to dissociate fibrin during receptor preparation to effectively enable it to bind to the Fortebio probe, thereby obtaining characteristic fragments (i.e., the α, β, and γ chains of fibrin) for experimental use. Specifically, plasmin was added at a fibrin:platin mass ratio of 30:1, incubated at 37°C for 3 hours, and then the reaction was terminated by a 60°C water bath for 15 minutes. Unreacted enzyme was removed by desalting, and the characteristic fragments were coupled with biotin and then desalted for purification.

[0125] Protein preparation: Take the fusion protein samples separately, dilute them to 500nM with 1×Kinetics buffer, mix well, and set aside.

[0126] 2. Sample addition

[0127] Add 200 μL of sample to each well of a 96-well plate in the following order: Kinetics buffer, acceptor, Kinetics buffer, test sample / blank, Kinetics buffer.

[0128] II. Experimental Results

[0129] Run the program and perform data analysis using Fortebio Data Analysis 8.0 software. The calculated affinity binding values ​​are shown in the table below:

[0130] Table 3 Notes: 1. The above fibrin affinity is based on 100% of commercially available alteplase (rt-PA). 2. Except for number 3, which is wild-type, number 8 is K2. m1 The rest are K2 m2 When K2 is wild type or K2 m1 It also does not affect the target protein's affinity for fibroin. 3. Nd indicates undetectable. 4. The F and E domains of the above targeting module are directly linked.

[0131] In summary, it can be seen that

[0132] 1. Regardless of whether there is a linker connection between SAK and the target module, the presence of SAK will not interfere with the target module's affinity for fibrin.

[0133] 2. The F and K2 regions in the targeting module are key structural domains for exerting fibrin affinity. When both are present, the fusion protein molecule of the present invention has a fibrin binding strength comparable to that of alteplase molecules. When they exist alone or in combination with other structural domains, they do not have or have only very low fibrin affinity.

[0134] Example 4: Evaluation of thrombolytic activity of t-PA-SAK fusion proteins with different designs

[0135] 1) Solution preparation

[0136] Working solution: Take 9g of NaCl, dilute to 1000mL with purified water, filter through a 0.45μm filter, and store at 4℃.

[0137] 1.5% agarose solution: Weigh 125 mg of agarose and dissolve it by heating in 23 mL of working solution.

[0138] Human plasminogen activator solution: Prepare the sample to 0.5 mg / mL using the working solution, and store the aliquoted sample at -20℃.

[0139] Human thrombin solution: Prepare the sample to 100 IU / mL using the working solution, and store the aliquoted sample at -20℃.

[0140] Human fibrinogen solution: Prepare before the experiment. Before preparation, let the human fibrinogen and working solution stand in a 37°C water bath for 15 minutes. Then, resuspend the human fibrinogen in an appropriate amount of working solution and keep it at 37°C for 30 minutes to completely dissolve it, and prepare a 6 mg / mL solution for later use.

[0141] Preparation of test solution: Replace the sample solution with the working solution, then use BCA for quantification. After quantification, dilute the sample with the working solution to 0.1 mg / mL.

[0142] 2) Measurement method

[0143] Redissolve the 1.5% agarose solution by heating, then incubate at 55-60℃. Add 14 μL of human thrombin solution and 280 μL of human plasminogen activator solution. While adding, shake well. Then add 2.2 mL of fibrinogen solution, shaking continuously until turbidity appears. Immediately pour the mixture into 8 cm diameter Petri dishes, allow to solidify horizontally, and incubate at 4℃ for at least 30 minutes before use (use within 2 days). Punch 2 mm wells in the Petri dishes, add 5 μL of the test sample to each well, and incubate horizontally at 37℃ for 16 hours. Measure the diameter of the clear zone and calculate the ratio of the clear zone of the test sample to that of the original protein (SAK). This ratio represents the relative activity of the candidate molecule and the original protein.

[0144] Table 4 Note: 1. The above in vitro thrombolytic activity is based on the recombinant SAK (containing 136 amino acids) with the amino acid sequence as shown in SEQ ID NO: 9 as 100%.

[0145] In summary, it can be seen that:

[0146] 1. When different design forms of the targeting module are fused with SAK (136 amino acid form) for expression, the targeting module will not interfere with the activity of SAK regardless of whether there is a linker connection between them;

[0147] 2. When the selected SAK is a 136-amino acid protein with the first 10 amino acids removed from the N-terminus, a linker must be introduced when fusing the SAK with the targeting module at the N-terminus; otherwise, the resulting fusion protein will have very low thrombolytic activity. This may be because the 11th amino acid is the key active site of the SAK, and if it is directly fused with the targeting module, the targeting module may mask this active site, leading to loss of activity.

[0148] SAK indirectly recognizes fibrin by interacting with plasmin on fibrin; it does not have the ability to directly bind to fibrin itself. Combining Examples 3 and 4, it can be seen that this invention, through the design of a fusion protein, retains the original biological effects of SAK while further increasing SAK's direct binding ability to fibrin, thus achieving a 1+1 specific recognition of fibrin and achieving the design objective of a thrombolytic molecule targeting thrombi.

[0149] Example 5: PEG-modified fusion protein pectinase TM (Pegtepskinase TM Sample preparation

[0150] I. Modification methods:

[0151] F-K2 with the amino acid sequence shown in SEQ ID NO: 11 m2 Taking -SAK (fusion protein number 19) and Y-type PEG 10K as examples, PEG-modified products were prepared. The purified fusion protein sample was concentrated and the modification buffer (50M disodium hydrogen phosphate-sodium dihydrogen phosphate, pH 6.0) was replaced to a concentration of approximately 5 mg / mL. Y-type PEG10K was added at a protein:PEG modifier molar ratio of 1:5, and a reducing agent (such as sodium cyanoborohydride) was added at a PEG modifier:reducing agent molar ratio of 1:50. The modification reaction was carried out at 4°C, and the reaction was terminated by diluting to 1 mg / mL after reacting overnight.

[0152] II. Purification Methods

[0153] Chromatographic conditions:

[0154] Step 1 chromatography: Mobile phase B was 50 mM NaAc + 1 M NaCl (pH 5.0), mobile phase A was 50 mM NaAc (pH 5.0), and the purification medium was SPHP (purchased from Cytiva).

[0155] Sample loading: After dilution, the modified sample was loaded at 10 mL / min onto a cation exchange chromatography column (Cytiva XK26).

[0156] Equilibration: After sample loading, rinse with solution A for approximately 5 column volumes.

[0157] Elution: Elute with 0-75% solution B, with an elution volume of 5 column volumes. Collect samples stepwise, perform electrophoresis and liquid chromatography analysis, and combine samples with a purity of ≥92% for the second purification step.

[0158] The second step of chromatography: the mobile phase was histidine buffer (pH 7.0) containing 5% trehalose and 4.5% sodium chloride, and the purification medium was Chromdex 200 pg (purchased from BorgL).

[0159] Separation: Ion exchange eluted samples were concentrated to approximately 8 mg / mL by ultrafiltration and then loaded for separation at a linear flow rate of 60 cm / h. Samples were collected stepwise, analyzed by electrophoresis and liquid chromatography, and samples with a purity of ≥95% were combined.

[0160] III. Results Analysis

[0161] The PEG-modified sample prepared in this embodiment was detected by SDS-PAGE, and the results showed that the sample bands were uniform, which met the research requirements.

[0162] Example 6: Detection of PEG-modified fusion protein targeting and binding to fibrin solids

[0163] I. Testing Steps:

[0164] (1) Coating fibrinogen: Dilute fibrinogen to 10 μg / mL with PB buffer at pH 7.0, then add 100 μL / well to a 96-well plate and incubate overnight at 4°C.

[0165] (2) Discard the coating solution in the wells, add 200 μL of PBS solution to each well for washing, wash 3 times, 1 min each time.

[0166] (3) Add 100 μL of 2% BSA solution to each well and incubate at 37°C for 1 h to seal.

[0167] (4) Discard the blocking solution in the wells, add 200 μL of PBS solution to each well for washing, wash 3 times, 1 min each time.

[0168] (5) Prepare a Tris-HCl buffer (pH 7.4) with a thrombin concentration of 1 U / mL and containing 20 mM CaCl2. Add 100 μL / well to a 96-well plate and incubate at 37°C for 2 h to generate fibrin.

[0169] (6) Discard the sample in the well and wash it with PBST solution. Add 200 μL of PBST to each well and wash it 3 times for 1 min each time. Note that the washing should be gentle to prevent the fibrin from being damaged.

[0170] (7) Prepare FITC-labeled protein samples. Weigh an appropriate amount of FITC, dissolve it in DMSO, and prepare a solution with a final concentration of 1 mg / mL. Take an appropriate amount of SAK and V-type PEG 20K modified fusion protein sample, add 1 / 10 volume of carbonate solution, mix well, add 30 μg FITC per milligram of protein, mix well, react at room temperature in the dark for 3.5 h, and remove unreacted fluorescein by ultrafiltration.

[0171] (8) Add the fluorescently labeled sample to a 96-well plate, 100 μL / well, and incubate at 37°C for 1 h.

[0172] (9) Discard the sample in the well, first wash with PBST solution, add 200 μL of PBST to each well, wash 3 times, 1 min each time; then wash with PBS, add 200 μL of PBS to each well, wash 3 times, 1 min each time.

[0173] (10) Add 100 μL of ultrapure water to each well, set the excitation light of the microplate reader to 490 nm and the emission light to 520 nm, and take the reading.

[0174] II. Experimental Results

[0175] Table 5

[0176] The experimental results are shown in the table above and Figure 4. Fibrinogen forms fibrin under the action of thrombin and calcium ions, which better simulates the composition and structure of thrombi in vivo. The results of this embodiment show that after washing, extremely strong fluorescence signals can still be detected in the wells of the PEG20K modified fusion protein sample. The fluorescence detection value is much higher than that of SAK and the blank group, indicating that the PEG20K modified fusion protein has bound to the fibrin in the 96-well plate. The polyethylene glycol modified fusion protein provided by this invention has the ability to target and bind fibrin thrombi.

[0177] Example 7: Detection of the activity and fibrin affinity of PEG-modified fusion protein samples

[0178] The fibrin affinity assay was performed using the same method as in Example 3, and the activity assay was performed using the same method as in Example 4. The results are as follows (the results are similar between different fusion protein modification products; the activity is presented as a percentage relative to the original SAK protein; there are certain fluctuations in the rt-PA affinity assay between different batches, and for the sake of accuracy, the rt-PA affinity is recorded as a range).

[0179] Table 6 Note: F-K2 in the table m2 The -SAK amino acid sequence is shown in SEQ ID NO: 11.

[0180] The activity results show that PEG modification has a slight masking effect on the activity of the sample, but the modified sample retains more SAK activity overall.

[0181] Examples 6 and 7 show that the polyethylene glycol-modified fusion protein has the ability to target and bind fibrin thrombi. Furthermore, based on biomembrane layer interference (BLI) technology, it is evident that despite the site-specific modification method where PEG is linked to the target fragment, surprisingly, the affinity of PEG-modified samples with molecular weights below 20K for fibrin did not decrease significantly. This maintained the same order of magnitude of fibrin recognition ability as rt-PA, demonstrating good drug-like properties.

[0182] Example 8: Interactions among fibrin, plasminogen (plg), and PEG-modified fusion protein

[0183] I. Experimental Methods

[0184] Using the Octet SA sensor array Fibrin-Biotin, a complex is first formed by binding plasminogen, followed by binding to a PEG-modified fusion protein (in this embodiment, F-K2 modified with V-type 20K). m2 -SAK as an example) or the control TNK (Tenecteplase).

[0185] Naming: Name the wells on the 96-well plate sequentially, according to the order in which they were added. Each column is named Buffer, Loading, Buffer, Reference-1 or Sample-1, Reference-2 or Sample-2, Buffer.

[0186] Parameters: Associate the first column Buffer with Sensor Check in sequence, running at 1000 rpm for 30 seconds; associate the second column Loading with Loading, running at 1000 rpm for 600 seconds; associate the third column Buffer with baseline, running at 1000 rpm for 120 seconds; associate the fourth column Reference-1 or Sample-1 with Association, running at 1000 rpm for 300 seconds; associate the fifth column Reference-2 or Sample-2 with Association, running at 1000 rpm for 300 seconds; and repeatedly associate the third column Buffer with Dissociation, running at 1000 rpm for 300 seconds.

[0187] Sensors: Select the sensor location, and select "remove" for the remaining areas.

[0188] Samples: Add samples sequentially. First column: Sensor Check: 1×Kinetics buffer; Second column: Loading: Receptor (Fibrin-Biotin) diluted to 20 μg / mL with 1×Kinetics buffer; Third column: Baseline: 1×Kinetics buffer; Fourth column: Association: Add 1×Kinetics buffer to the Reference well, and add 90 μg / mL (1000 nM) Glu-Pg to the Sample well; Fifth column: Association: Add 1×Kinetics buffer to the Reference well, and add 50 μg / mL (1000 nM) PEG-modified fusion protein and 60 μg / mL (1000 nM) TNK sequentially to the Sample well. The volume added to each well is 200 μL.

[0189] Run the program and perform data analysis using ForteBio Data Analysis 9.0 software.

[0190] II. Experimental Results

[0191] Table 7

[0192] The detection spectrum is shown in Figure 5. After fibrin binds to plasminogen to form a complex, it can further bind to PEG-modified F-K2. m2 The binding curves of the PEG fusion protein and TNK are consistent. Under these conditions, the affinity of the PEG fusion protein to the plasmin-plasminogen complex is 17.05 nM (the affinity between the protein and fibrin is approximately 80 nM in the absence of plasminogen), while the affinity of TNK to the plasmin-plasminogen complex is 26.85 nM (the affinity between TNK and fibrin is 51.7 nM in the absence of plasminogen). Therefore, under the plasmin-plasminogen complex condition, the binding affinity of the PEG fusion protein is stronger than that of TNK. These experimental results demonstrate that the PEG fusion protein possesses binding characteristics consistent with TNK, but with a higher bias towards the plasmin-plasminogen complex, effectively reducing activation of free plasminogen, decreasing non-specific systemic fibrinolysis, and exhibiting higher safety.

[0193] Example 9: Interaction between the PEG-modified fusion protein and plasminogen (Plg) after forming a complex and fibrin.

[0194] I. Experimental Methods

[0195] The detection was performed using the ForteBio Octet intermolecular interaction detection device, with fibrin-Biotin as the acceptor. The procedure was carried out according to Example 8.

[0196] Preparation of test sample: PEG-modified fusion protein (in this example, F-K2 modified with V-type 20K) m2 (Taking SAK as an example) Dilute to 100 μg / mL, SAK to 30 μg / mL, and plasminogen Lys-Pg to 160 μg / mL. Take plasminogen and mix it with PEG fusion protein and SAK solution at a volume ratio of 1:1. Incubate at 37°C with gentle shaking for 30 minutes.

[0197] Detection: After incubation, samples were added to the Association wells at a rate of 200 μL / well for detection.

[0198] II. Experimental Results

[0199] Table 8

[0200] As shown in Figure 6, the PEG-modified fusion protein, similar to SAK, can form a complex with plasminogen, further efficiently binding fibrin and activating plasminogen to exert a specific thrombolytic effect. Due to the presence of the target region, the complex formed by the PEG fusion protein and plasminogen exhibits higher fibrin affinity (23.26 nM vs 275.4 nM) and higher binding abundance compared to the complex formed by SAK and plasminogen, demonstrating a significant advantage.

[0201] Example 10: Evaluation of PEG fusion protease kinetics

[0202] I. Experimental Methods

[0203] Based on the principle of plasmin cleavage of chromogenic substrates to generate pNA, the PEG-modified fusion protein (F-K2 modified with V-type 20K) was measured. m2 (Taking SAK as an example) The generation of plasmin under different substrate concentrations was used to indirectly determine the enzymatic kinetic constant of the PEG-modified fusion protein.

[0204] Dilute the PEG-modified fusion protein to 1 mg / mL and the plasminogen to 1 mg / mL, and add them to the 96-well plates in the order shown in the table below (all units are μL). Incubate at 37℃ for 5 min. Add the protein to the S2288 microplate reader before reading the values. Detect the protein immediately after adding the protein.

[0205] Table 9

[0206] The microplate reader was set to 37℃, with a shaking time of 3 seconds, and kinetic detection for 15 minutes, taking one reading at 405 nm per minute. The slope of the curve at each substrate concentration was calculated as the conversion of enzyme activity units, and the Michaelis constant was calculated based on the substrate concentration.

[0207] II. Experimental Results

[0208] Table 10

[0209] The experimental results, as shown in the table and Figures 7a and 7b above, indicate that plasmin was detected in the system only when the PEG-modified fusion protein and plasminogen were present simultaneously. This suggests that the fusion protein is identical to SAK and generates plasmin by activating plasminogen after forming a complex with it. The Michaelis constant Km of the 20K PEG-modified fusion protein was found to be 27.39 μM by software fitting.

[0210] Example 11: Anti-PAI-1 Inhibition

[0211] I. Experimental Methods

[0212] The assay was performed using the ForteBio Octet intermolecular interaction detection system, with His-PAI-1 (20 μg / mL) as the acceptor and Biosensor NTA as the sensor. The procedure was as described in Example 8. The test sample was diluted with 1×Kinetics buffer at a concentration of 1000 nM, i.e., PEG-modified F-K2. m2 -SAK fusion protein (in this example, F-K2 modified with type V 20K) m2 (Taking SAK as an example) Dilute to 50 μg / mL, F-K2 m2 - SAK was diluted to 30 μg / mL and TNK was diluted to 60 μg / mL. 200 μL / well was added to the Association well for detection.

[0213] II. Experimental Results

[0214] Table 11

[0215] As shown in Figure 8, the fusion protein F-K2 m2 -SAK and its PEG-modified products do not bind to PAI-1, meaning their thrombolytic activity is not regulated or inhibited by PAI-1; while TNK, despite undergoing PAI-1 resistance enhancement modifications, is still significantly affected by PAI-1.

[0216] Example 12: In vitro fibrinolytic effect of PEG-modified fusion protein samples

[0217] I. Experimental Methods

[0218] Blood was collected from the abdominal aorta of SD rats and placed in a heparin-anticoagulated blood collection tube. The blood collection tube was repeatedly inverted and centrifuged at 2000-3000 rpm for 15 min to collect plasma samples.

[0219] In a 24-well plate, 200 μL of plasma was added to each well, along with 2 mg of fibrin clots. The V-type PEG20K-modified fusion protein (F-K2) prepared according to the method described in Example 6 was then added in the dosages listed in the table below. m2 -SAK amino acid sequence as shown in SEQ ID NO: 11), incubate at 37℃ for 2h. After incubation, add 200μL of physiological saline to each well for dilution, take the sample from the well, centrifuge at 15000rpm, discard the supernatant, wash the precipitate once with physiological saline, centrifuge again and discard the supernatant, dry at 60℃, and weigh the total weight of solids.

[0220] II. Experimental Results

[0221] Table 12

[0222] This embodiment demonstrates that the PEG-modified sample exhibits superior in vitro thrombolytic performance. After incubation at 37°C for 2 hours, a minimum addition dose of 2 μg / 200 μL is sufficient to achieve a fibrinolytic effect of over 50%.

[0223] Example 13: Effect of PEG-modified fusion protein samples on in vitro dissolution of blood clots

[0224] I. Experimental Methods

[0225] Rat thrombus preparation: 2 mL of blood was collected from the orbital cavity of a healthy rat and injected into a catheter. The blood was allowed to coagulate at room temperature for 4 hours. After coagulation, the blood was cut into 1 cm lengths, and the blood clots in the catheter were blown out with physiological saline. Rat plasma preparation: Anticoagulant was added to a 1.5 mL EP tube. Blood was collected from the orbital cavity of a healthy rat, centrifuged at 3500 rpm for 10 min, and the supernatant was collected.

[0226] Sample loading for testing: Take a 24-well plate and add 200 μL of plasma + 100 μL of the test sample to each well (set three gradients: 0.5 mg / mL, 0.25 mg / mL, and 0.1 mg / mL; the PEG-modified fusion protein is F-K2 modified with V-type 20K). m2 -SAK (code ZHB130) as an example), add blood clot; place in a 37℃ constant temperature shaker and incubate at 400rpm for 35min, take 100μL of supernatant and detect the absorbance value at 540nm.

[0227] II. Experimental Results

[0228] Table 13

[0229] As shown in Figure 9 and the table above (left side is the photo before incubation, right side is the photo after incubation), in blank plasma containing thrombus clots, there was no sign of thrombus dissolution, and the A540 value was low; in the drug-treated group, as the thrombus dissolved and red blood cells were released, the A540 reading increased. The experimental results indicate that the PEG-modified fusion protein exhibits superior thrombolytic efficacy compared to TNK in vitro, with a stronger overall thrombolytic efficiency; and the in vitro thrombolytic effect is concentration-dependent.

[0230] Example 14: Pharmacokinetic assay of PEG-modified fusion protein samples

[0231] I. Experimental Methods

[0232] 1. Pharmacokinetic comparison of candidate molecules with different molecular weights

[0233] Male SD rats weighing 300-400g were anesthetized with 10% chloral hydrate via tail vein administration. The administration groups included the SAK group, the V-type 10K PEG-modified fusion protein group, the V-type 20K PEG-modified fusion protein group, and the V-type 30K PEG-modified fusion protein group. The fusion protein sequence was F-K2. m2 -SAK, the amino acid sequence is shown in SEQ ID NO: 11.

[0234] The drug was administered at a dose of 2 mg / kg (based on PEG modification content). Blood samples of 0.2 mL were collected from the orbital cavity at 5, 10, 30, 60, 120, 240, and 360 min after administration. The samples were placed in anticoagulant tubes containing 0.02 mL of 4% sodium citrate, mixed, and centrifuged at 3000 rpm to obtain plasma samples for testing.

[0235] Prepare thrombolytic ring detection petri dishes according to Example 4 and punch holes. Prepare standard solutions of 0.5, 1.5, 10, 50, and 100 μg / mL (based on protein concentration) using blank rat plasma from the corresponding groups. Add 10 μL / well of the standard solution and the post-administration plasma sample to the wells of the petri dish, respectively. Incubate at 37°C for 6 hours. Measure the diameter of the thrombolytic ring in the standard solution using calipers, plot a standard curve, and calculate the blood drug concentration at each administration time point.

[0236] II. Experimental Results

[0237] Table 14: Blood drug concentrations in rats at different time points for each test sample (unit: μg / mL)

[0238] The results, as shown in the table above and Figure 10, indicate that the pharmacokinetic process of the PEG-modified sample was significantly prolonged compared to the original SAK protein. As is generally true for PEG modification, the higher the molecular weight of the PEG-modified sample, the longer its drug half-life. At the same dosage, the PEG-modified fusion protein group maintained a plasma concentration close to or exceeding that of the SAK group at 5 minutes after 30 minutes in animals, demonstrating a significant pharmacokinetic advantage. Considering the literature reports that the half-life of SAK is around 5 minutes, and the clinical application background requiring continuous intravenous infusion for 30 minutes, the polyethylene glycol-modified fusion protein provided by this invention is feasible for clinical intravenous bolus administration.

[0239] Example 15: Evaluation of bleeding tendency in animals after administration of PEG-modified fusion protein samples

[0240] I. Experimental Methods

[0241] Animal administration was performed on SD rats. The test products were SAK, SAK modified with V-type PEG 10K, fusion protein modified with V-type PEG 20K, and fusion protein modified with V-type PEG 10K, wherein the fusion protein sequence was F-K2. m2 -SAK, amino acid sequence as shown in SEQ ID NO: 11. The drug was administered via tail vein injection at the designed dose (5 mg / kg for the first experiment, 10 mg / kg for the repeat experiment). Blood samples were collected before administration and at 0.5 h, 1 h, and 2 h after administration.

[0242] 1. Thrombin Time (TT) Detection:

[0243] (1) Preparation of the plasma to be tested: Take 1.8 mL of fresh venous blood to be tested and mix it with 0.2 mL of sodium citrate anticoagulant (109 mM) at a ratio of 9:1, and gently invert to mix. (Alternatively, use a plastic tube or siliconized glass tube containing 1 / 10 volume of sodium citrate anticoagulant to collect blood.)

[0244] (2) Centrifuge at 3000 rpm (or 2500 g) for 10-15 min, collect the supernatant (platelet-deficient plasma), and transfer it to a plastic test tube or centrifuge tube to prevent platelet activation; normal control plasma should also be provided.

[0245] (3) Take 0.1 mL of the anticoagulated plasma to be tested and place it in a 37℃ water bath for 5 min.

[0246] (4) Add 0.1 mL of thrombin solution and record the coagulation time. Repeat 2-3 times and take the average value.

[0247] 2. Prothrombin time (PT) test:

[0248] (1) Preparation of the plasma to be tested: Take 1.8 mL of fresh venous blood to be tested and mix it with 0.2 mL of sodium citrate anticoagulant (109 mM) at a ratio of 9:1, and gently invert to mix. (Alternatively, use a plastic tube or siliconized glass tube containing 1 / 10 volume of sodium citrate anticoagulant to collect blood.)

[0249] (2) Centrifuge at 3000 rpm (or 2500 g) for 10-15 min, collect the supernatant (platelet-deficient plasma), and transfer it to a plastic test tube or centrifuge tube to prevent platelet activation; normal control plasma should also be provided.

[0250] (3) Take 0.07 mL of the anticoagulated plasma to be tested, add 0.07 mL of the prothrombin detection working solution, and record the coagulation time. Repeat 2-3 times and take the average value.

[0251] 3. Fibrinogen content detection

[0252] (1) Take out the kit 30 minutes before the experiment and let it return to room temperature.

[0253] (2) Add 100 μL of standard working solution and test sample to each reaction well (standard curve range: 16.625~1000 ng / mL. If the sample concentration is higher than the detection range, it needs to be diluted with standard & sample diluent before sampling). The standard needs to be replicated. After sealing the plate, incubate it in a 37℃ incubator for 90 min.

[0254] (3) Discard the liquid, spin dry, add 100 μL of biotin-labeled fibrinogen antibody working solution to each reaction well, seal the plate and incubate at 37°C for 60 min.

[0255] (4) Washing: Discard the liquid, spin dry, add 350 μL of washing solution to each reaction well, soak for 1-2 min, and spin dry. Repeat 4 times.

[0256] (5) Add 100 μL of HRP-labeled streptavidin working solution to each reaction well, seal the plate and incubate at 37°C for 30 min.

[0257] (6) Washing: Add 300 μL of washing solution to each reaction well, and shake off the washing solution after 30 s intervals. Repeat 4 times.

[0258] (7) Add 90 μL of colorimetric reagent (protected from light) to each reaction well, seal the plate, and develop the color at 37°C in the dark for about 15 minutes.

[0259] (8) Add 50 μL of stop solution to each reaction well and immediately measure the OD value at 450 nm wavelength using an ELISA reader (within 5 min).

[0260] (9) OD value was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.

[0261] (10) Calculate the average OD value of standards and samples: the OD value of each standard and sample should be subtracted from the OD value of the zero well.

[0262] (11) Plot a standard curve using a four-parameter logistic model, with the standard concentration as the x-axis and the absorbance (OD) value as the y-axis. (Remove the values ​​of the blank group when plotting the curve.)

[0263] (12) If the OD value of the sample is higher than the upper limit of the standard curve, it should be appropriately diluted and retested, and the concentration should be multiplied by the dilution factor when calculating the concentration.

[0264] II. Experimental Results

[0265] The changes in bleeding tendency in animals were observed at doses of 5 mg / kg and 10 mg / kg, respectively.

[0266] Table 15: Prothrombin Time (s) of Rats at Different Time Points for Each Test Sample at a Dosage of 5 mg / kg

[0267] Table 16: Thrombin Time (in seconds) of Rats at Different Time Points for Each Test Sample at a Dosage of 5 mg / kg

[0268] Table 17: Fibrinogen levels in rat blood at different time points for each test sample at a dose of 5 mg / kg (unit: g / L)

[0269] Table 18: Prothrombin Time (s) of Rats at Different Time Points for Each Test Sample at a Dosage of 10 mg / kg

[0270] Table 19: Thrombin Time (s) of Rats at Different Time Points for Each Test Sample at a Dosage of 10 mg / kg

[0271] Table 20: Fibrinogen content in rat blood at different time points for each test sample at a dose of 10 mg / kg (unit: g / L)

[0272] This embodiment illustrates that, compared to the SAK protein group, the 10K-PEG modified fusion protein group and the 20K-PEG modified fusion protein group showed decreased thrombin time and prothrombin time after low-dose administration for 0.5h, 1h, and 2h, and significantly higher fibrinogen levels in the blood. Compared to the SAK protein group, the 20K-PEG modified fusion protein sample showed further decreased thrombin time and prothrombin time after high-dose administration for 0.5h, 1h, and 2h, and a more significant increase in fibrinogen levels in the blood. Its bleeding tendency in rats was significantly lower than that of the SAK protein, indicating that the PEG-modified fusion protein of this application has a lower bleeding tendency when administered in vivo.

[0273] As can be seen from Examples 14 and 15, the PEG-modified fusion protein sample of this application has significantly reduced bleeding side effects compared to SAK protein, while significantly improving pharmacokinetic properties. It can maintain blood drug concentration at the same dose with a low risk of bleeding, and has the feasibility to support intravenous bolus administration in clinical practice. While ensuring therapeutic effect, it greatly simplifies the treatment process and expands the application scenarios. Compared with commercially available intravenous drip products, it has advantages and potential.

[0274] Example 16: Effect of PEG-modified fusion protein sample on fibrinogen content in animals after administration

[0275] I. Experimental Methods

[0276] 1. Administration to rats: Healthy adult rats (weighing 200-500g) were administered TNK and PEG-modified fusion protein (F-K2 modified with V-type 20K) via tail vein injection at a dose of 5mg / kg. m2 (Taking SAK as an example), blood was collected at 0:00 before administration; blood was collected again at 0.5, 1, 2, 6, and 24 hours after administration. Plasma preparation: Anticoagulant was added to 1.5 mL EP tubes, blood was collected from the rat's orbital cavity, centrifuged at 3500 rpm for 10 min, and the supernatant was collected.

[0277] Fibrinogen content was detected using a purchased ELISA kit, following the same method as described in the previous examples.

[0278] 2. Cynomolgus monkeys were administered the drugs at doses of 0.05 mg / kg and 0.15 mg / kg, and blood samples were collected for testing.

[0279] II. Experimental Results

[0280] 1. Detection of fibrinogen content in rats after high-dose administration

[0281] Table 21

[0282] Table 22

[0283] The experimental results showed that the high dose (5 mg / mL) of PEG-modified fusion protein had a similar effect on the consumption of fibrinogen in rats as TNK. 30 min after administration of both drugs, fibrinogen levels decreased to about 80% of the pre-administration level, and recovered to about 95% after 2 h.

[0284] 2. Detection of fibrinogen content in cynomolgus monkeys after drug administration

[0285] Table 23

[0286] The results of testing on cynomolgus monkeys showed that after administration of a low dose of 0.05 mg / kg, fibrinogen levels fluctuated by no more than 8%; after administration of a high dose of 0.15 mg / kg, fibrinogen levels fluctuated by no more than 10%, and basically recovered within 4 hours after administration. This demonstrates the good safety of the samples.

[0287] Example 17: Immunogenicity evaluation of PEG-modified fusion protein samples in animals after administration

[0288] I. Experimental Methods

[0289] Determination of serum IgG titers against SAK / t-PA-SAK fusion protein. Dosage regimen: BALB / c mice were used as experimental animals. The fusion protein F-K2 was modified with SAK and PEG-20K (type V). m2 -SAK (the amino acid sequence of the fusion protein is shown in SEQ ID NO: 11) was administered at 100 μg / animal (based on PEG modification content) via subcutaneous injection at multiple sites on days 0, 4, 11, and 18, and serum was collected for testing on day 21.

[0290] ELISA testing steps:

[0291] (1) Coating antigen: The concentration of SAK / fusion protein was diluted to 3 μg / mL with coating solution and then added to a 96-well plate at 100 μL / well and incubated overnight at 4°C.

[0292] (2) Discard the coating solution in the wells, add 200 μL of PBS solution to each well for washing, wash 3 times, 1 min each time.

[0293] (3) Add 100 μL PBSM (4 g skim milk powder dissolved in 100 mL PBS buffer) to each well and incubate at 37 °C for 1 h to block.

[0294] (4) Discard the blocking solution in the wells, add 200 μL of PBS solution to each well for washing, wash 3 times, 1 min each time.

[0295] (5) Use PBSM to perform serial dilutions of serum samples by 2-fold, starting from 1:50 and continuing until 1:400. Then add 100 μL of the diluted sample to each well and incubate at 37°C for 2 h.

[0296] (6) Discard the sample in the well and wash it. First, wash with PBST solution, add 200 μL of PBST to each well and wash 3 times for 1 min each time. Then wash with PBS, add 200 μL of PBS to each well and wash 3 times for 1 min each time.

[0297] (7) Add 100 μL of goat anti-mouse IgG-HRP diluted 5000 times with PBSM to each well and incubate at 37°C for 2 h.

[0298] (8) Discard the sample in the well and wash it. First, wash with PBST solution. Add 200 μL of PBST to each well and wash 3 times for 1 min each time. Then wash with PBS solution. Similarly, add 200 μL of PBS to each well and wash 3 times for 1 min each time.

[0299] (9) Add 100 μL of substrate color development solution to each well and incubate at 37°C for 30 min.

[0300] (10) Add 25 μL of stop solution to each well and measure the absorbance at 450 nm.

[0301] II. Experimental Results

[0302] Table 24: Antibody titers (A450) at different dilutions of rat serum

[0303] The experimental results are shown in the table above and Figure 11. At different dilutions, the absorbance values ​​of anti-SAK IgG induced by SAK and 20K PEG-modified fusion protein (containing F+K2 targeting units) samples in the ELISA reaction were compared. The results showed that compared with the original SAK protein, the PEG-modified fusion protein had significantly reduced immunogenicity, with almost no antibody production detected. The results of this embodiment demonstrate that the polyethylene glycol-modified fusion protein provided by this invention overcomes the limitation of SAK protein in repeated administration due to immunogenicity issues, meeting the clinical application scenarios requiring multiple-dose thrombolysis, and also possessing great potential for secondary prevention of recurrent thrombosis in patients.

[0304] Example 18: Pharmacological and toxicological study of ZHB130 for injection in a rat pulmonary embolism model

[0305] A pulmonary embolism thrombosis model was established using male SD rats. A PE tube was connected to a 1mL syringe. 150U of thrombin and 20μL of saturated calcium chloride solution were added to a 1.5 / 2mL centrifuge tube. Blood from a normal rat was collected and added to the centrifuge tube. The mixture was quickly and gently shaken, and the blood was aspirated into the PE tube within 10 seconds, ensuring the blood was homogeneous and free of air bubbles. After 2 minutes, the PE tube was divided into two equal segments and allowed to stand at room temperature for 1 hour. Once the blood had completely coagulated, the PE tube was cut into 4-6mm segments. The thrombus was blown into physiological saline using a pipette or syringe. Rats were anesthetized with gas (isoflurane) and fixed in a supine position on a rat board connected to a breathing mask. The skin was disinfected, the neck was incised, and the right jugular vein was isolated. The thrombus was aspirated into a 1mL syringe and injected into the jugular vein through a 10mL syringe needle. After successful thrombus injection, the number of successfully injected thrombi was recorded, and the model was considered complete. The wound was sutured for further testing. Based on this model, the pharmacokinetic / toxicokinetic and ADA bioanalyses of injectable ZHB130 in rats were evaluated. Specifically, injectable ZHB130 is the aforementioned V-type PEG 20K modified fusion protein F-K2. m2 -SAK, this applies to all the following embodiments.

[0306] I. Grouping and Experimental Design

[0307] Male SD rats weighing 300-360g were selected to establish a pulmonary embolism thrombosis model. After modeling, the rats were immediately divided into four groups: a ZHB130 injection model group (ZHB130 was injected intravenously after modeling), a ZHB130 injection control group (ZHB130 was injected directly intravenously into healthy rats), a SAK model group (SAK was injected intravenously after modeling), and a SAK control group (SAK was injected directly intravenously into healthy rats). There were three rats in each group. Serum samples were collected from the rats at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h after a single intravenous injection of 0.2 mg / kg of the corresponding drug.

[0308] Two healthy male SD rats weighing 300-360g were selected and administered ZHB130 intravenously multiple times (13 days, 1mg / kg, once a day). Serum samples were collected from the rats at time points of 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h after the multiple intravenous injections.

[0309] Serum samples were used for ELISA analysis to detect and evaluate the pharmacokinetics / toxicokinetics and ADA of injectable ZHB130 and SAK in normal rats and rats with pulmonary embolism.

[0310] II. Results

[0311] As shown in Figure 12 and Table 25, in rat serum, compared with SAK, ZHB130 had significantly prolonged pharmacokinetic distribution and elimination half-life. After pulmonary embolism modeling, administration of C...max and AUC last The decrease in area under the curve (AUC) suggests that ZHB130 has certain thrombosis targeting and significant long-term efficacy, and its drug release is milder compared to SAK.

[0312] As shown in Table 26, the pharmacokinetic results for Day 1 and Day 13 indicate that, after 13 consecutive days of injection, the levels of ZHB130 in the serum of healthy rats were significantly higher than those after the initial administration of THB130. 1 / 2 C max and AUC last A certain decrease was observed, but no significant difference was observed. Furthermore, ADA titers in rat serum were all 0 13 days after injection. Based on the above results and the test results of the kit in Figure 13, it can be concluded that ZHB130 did not produce immunogenicity in rats after 13 consecutive intravenous injections, and did not significantly affect the serum creatinine levels at each pharmacokinetic sampling time point (N56 and N58 in Figure 13 correspond to SD rats that were repeatedly administered the drug 13 times). In conclusion, ZHB130 did not produce significant toxicity in rats after 13 consecutive intravenous injections, and its immunogenicity was low.

[0313] Table 25 Pharmacokinetic parameters of ZHB130 and SAK after a single intravenous injection (n=3)

[0314] Table 26 Pharmacokinetic parameters of ZHB130 for injection after 13 consecutive intravenous injections (n=2)

[0315] III. Results Analysis

[0316] ZHB130 for injection, as a long-half-life thrombolytic drug, exerts its thrombolytic effect over a long period and can reduce immunogenicity. It has not produced toxicity even with long-term injection, and has advantages as a novel targeted long-acting thrombolytic drug.

[0317] Example 19: Pharmacodynamic and pharmacological study of ZHB130 for injection in a mouse model of photoinduced forelimb motor cortex infarction.

[0318] In this invention, a photoinduced forelimb motor cortex infarction model was established using male C57BL / 6J mice, and a focal ischemic stroke model of the forelimb motor cortex was established using the mouse photoirradiation method. Animals were anesthetized with isoflurane. First, mice were placed in the induction box of an R550IP small animal anesthesia machine for anesthesia. Then, the mice were fixed to a stereotaxic table, and pre-prepared rose red B (100 mg / kg) was injected intraperitoneally, while thrombin was injected via the tail vein. Five minutes later, the skin was disinfected, the hair on the brain was shaved, and the skull was exposed through a midline incision. Connective tissue was removed and dried. An optical fiber was positioned 1.5 mm to the right of the anterior fontanelle, irradiating the skull as close as possible to the bone for 15 minutes, during which time the mice's eyes were covered with light-shielding paper. After irradiation, iodine was applied locally to prevent infection, the brain skin was sutured, and the corresponding drug group was immediately injected via the tail vein. After observing vital signs, the mice were returned to their cages. Based on this model, the effect of injected ZHB130 on the infarct volume and hemorrhage risk in mice was evaluated.

[0319] I. Grouping and Experimental Design

[0320] C57BL / 6J mice weighing 20-25g were selected and a photoinduced forelimb motor cortex infarction model (PT model) was established. Mice were randomly divided into five groups: the PT model group, the positive control drug SAK group (1.5 mg / kg), the positive control drug TNK group (2.4 mg / kg), and the ZHB130 injection group (0.5 mg / kg). A blank control group was also included, for a total of 5 groups, with 27-32 animals in each group. Animals received a single intravenous injection 1 hour after model establishment.

[0321] Following drug administration, mice in each experimental group were divided into three batches and treated using different methods. Specifically: two batches were used to evaluate the effects of injectable ZHB130 and TNK on hemorrhage risk; 72 hours after administration, the mice were perfused and their brains were harvested. One batch was used for digital photography, with 8-9 animals per group. The other batch underwent fixation and HE staining, with 10-14 animals per group. The final batch was used to evaluate the effect of injectable ZHB130 on cerebral infarction volume in mice; 48 hours after administration, the mice's brains were harvested for TTC staining, with 9 animals per group.

[0322] II. Results

[0323] The effect of ZHB130 injection on the volume of cerebral infarction in mice is shown in Figure 14. The incidence of hemorrhage in the PT model group was about 4 / 9. ZHB130 injection can effectively reduce the incidence of hemorrhage (1 / 9). The incidence of hemorrhage in the TNK group was not significantly improved (3 / 8).

[0324] The effects of injectable ZHB130 and TNK on hemorrhage risk are shown in Figures 15 and 16. HE staining results indicate that the pathological photographs of mice in the PT model group showed significant hemorrhage points, accounting for approximately 0.64% of the infarct area, with obvious cellular infarction. Injectable ZHB130 effectively reduced the percentage of hemorrhage area (0.44%), while the percentage of hemorrhage area in the TNK group was significantly higher than in other groups (2.5%, P<0.001). SAK was only used for reference due to its inability to meet clinical requirements for intravenous administration. These results demonstrate that ZHB130 effectively reduces cerebral infarction volume and damage in mice in the in vivo PT model, and compared to TNK, reduces the risk and incidence of hemorrhage, further exhibiting thrombosis targeting and safety.

[0325] As shown in Figures 17 and 18, the TTC test results indicate that, compared with the model group, the injectable ZHB130, the positive control drugs SAK and TNK all significantly reduced the infarct volume (P<0.001, P<0.05, P<0.01). Among them, the infarct volume reduction was most significant in the ZHB130 group, but there was no significant difference compared with the SAK and TNK groups.

[0326] III. Results Analysis

[0327] Injectable ZHB130 effectively reduced cerebral infarction volume in mice and decreased the risk and incidence of bleeding compared to TNK, demonstrating preliminary thrombus targeting and safety. As a long-acting thrombolytic drug, injectable ZHB130 can activate plasminogen at the thrombus site, dissolve the thrombus, exert a prolonged thrombolytic effect, and reduce immunogenicity and bleeding side effects. It holds promise as a novel targeted long-acting thrombolytic drug.

[0328] Example 20: Acute toxicity test of maximum tolerated dose (MTD) in cynomolgus monkeys

[0329] In this invention, the maximum tolerated dose (MTD) of ZHB130 for injection was determined by administering a single rapid intravenous bolus injection to cynomolgus monkeys. A 7-day observation period was then conducted to assess the reversibility, persistence, or delayed effects of the compound's toxicity. Furthermore, the toxicokinetics (TK) characteristics of ZHB130 for injection in animals were evaluated.

[0330] I. Grouping and Experimental Design

[0331] Four cynomolgus macaques (2 per sex) were administered ZHB130 injection via intravenous injection every other day in escalating doses, using a single rapid intravenous bolus at doses of 0.15, 0.5, 1.5, and 4 mg / kg / dose. The administration volume was 4 mL / kg. At the start of administration, the animals were approximately 2.5 to 2.8 years old, with females weighing 2.40 to 2.65 kg and males weighing 2.36 to 3.07 kg. Day 1 of the experiment was the first day of administration. The animals were dissected on day 8 after a 7-day observation period. The toxicity of the test product was evaluated based on animal survival rate, clinical signs, body weight, clinicopathological findings, gross anatomical observations, and toxicokinetics.

[0332] II. Results

[0333] The toxicokinetic analysis of ZHB130 for injection is summarized as follows: the mean half-life ranged between 4.3 and 6.5 hours. There were no significant sex differences in systemic exposure across all dose groups. As the dose increased from 0.15 mg / kg / dose to 4 mg / kg / dose, the increase in systemic exposure in male and female monkeys was less than the dose-related proportion. No changes in body weight, food intake, or serum biochemistry related to the test product were observed. All animals survived to planned necropsy. The main clinical symptom related to the test product was swelling in both male and female animals, including mild or moderate swelling of the left and right forelimbs, hands, hind limbs, and hind paws, or tail swelling, or mild swelling of the dorsal abdomen. Except for one male animal where swelling was observed from Day 1, swelling was observed in all other animals from Day 5. By the end of the observation period, swelling had disappeared in all animals except for one male animal that still showed mild swelling of the left and right forelimbs, hind paws, hands, and hind limbs, and moderate tail swelling, and one female animal that showed mild swelling of the dorsal abdomen.

[0334] Notable hematological changes were primarily observed in erythroid-related parameters, including decreased red blood cell count (RBC), hemoglobin (HGB), and hematocrit (HCT), and increased red blood cell distribution width (RDW), absolute reticulocyte count, and percentage (#RET / %RET). Most of these parameters exceeded the laboratory background range after administration on day 4 or 6. By the end of the observation period, all parameters except RDW and #RET / %RET had returned to normal. Compared to pre-experiment levels, a notable coagulation change was observed from Day 5 onwards, with a prolonged thrombin time (TT), which completely recovered by the end of the observation period. Gross lesions were observed only in one male animal, presenting with diffuse or localized swelling of the bilateral hands, feet, and forelimbs. No gross lesions were observed in other animals.

[0335] In summary, after a 7-day observation period, animals tolerated ZHB130 injection at doses of 0.15, 0.5, 1.5, and 4 mg / kg / dose via a single rapid intravenous bolus injection well, with no deaths or near-death experiences. Under the conditions of this experiment, the maximum tolerated dose (MTD) is considered to be 4 mg / kg / dose, corresponding to CO and AUC0 24h of 58,800 ng / mL and 265,000 h*ng / mL for male animals, and 57,000 ng / mL and 319,000 h*ng / mL for female animals, respectively.

[0336] The mean values ​​of C0, T1 / 2, Vss, CL, and AUC0-24h, expressed as mean values, were obtained after a single intravenous bolus administration of 0.15, 0.5, 1.5, or 4 mg / kg / dose of ZHB130 to male and female monkeys.

[0337] Table 27. C0, T1 / 2, Vss, CL, and AUC0-24h values ​​after a single intravenous bolus injection in male and female monkeys.

[0338] Example 21 ZHB130: Toxicity and toxicokinetics study of cynomolgus monkeys with repeated intravenous administration for 14 days and a recovery period of 28 days.

[0339] In this invention, the test substance ZHB130 was administered intravenously to cynomolgus monkeys once a day for 14 consecutive days to detect its potential toxicity and evaluate the toxicokinetics (TK) characteristics of ZHB130 in animals.

[0340] I. Grouping and Experimental Design

[0341] Forty cynomolgus macaques (20 per sex) were randomly divided into four groups and administered ZHB130 via intravenous injection once daily at doses of 0 (0.9% sodium chloride injection), 0.15, 0.5, and 1.5 mg / kg / day for 14 days. At the start of administration, the age of males and females was approximately 3–3.5 years; females weighed between 2.19 and 2.66 kg, and males between 2.43 and 3.52 kg. Females were nulliparous and non-pregnant. Evaluation criteria included survival rate (dying / death), clinical observation (including injection site irritation), body weight, food intake, ophthalmological examination, electrocardiogram, body temperature, clinicopathology (hematology, serum biochemistry, hemagglutination, and urinalysis), gross (necropsy) observation, organ weight, histopathology, immunogenicity (antibody detection), and toxicokinetics.

[0342] II. Results

[0343] All animals survived until their planned euthanasia.

[0344] The toxicokinetic analysis of ZHB130 is summarized as follows: the median half-life ranged from 0.5 hours to 6.7 hours. Except for the 0.15 mg / kg / day dose groups, where female monkeys had higher systemic exposures on day 14 than males (possibly due to ADA influence), there were no significant sex differences in systemic exposures among female monkeys in the other dose groups. As the dose increased from 0.15 to 1.5 mg / kg / day, systemic exposures on days 1 and 7 increased proportionally with the dose in both male and female monkeys, but the increase in systemic exposure on day 14 was generally higher than the dose-related proportion. When comparing day 7 to day 1, no significant drug accumulation was observed in systemic exposures in either male or female monkeys across all dose groups; when comparing day 14 to day 1, systemic exposures in either male or female monkeys across all dose groups were generally lower (possibly due to ADA influence).

[0345] The immunogenicity assay analyzed 136 samples, with 50 showing a positive signal in the screening test. Of these, 46 samples were positive in the confirmatory test and underwent titration. In group 2 (0.15 mg / kg / day), all animals were confirmed as ADA-positive on day 14 (before administration), with no negative animals serving as a control. In group 3 (0.5 mg / kg / day), one female animal was confirmed as ADA-positive on day 7 (before administration), and her serum drug concentration was not significantly different from that of animals confirmed as ADA-negative. All animals were confirmed as ADA-positive on day 14 (before administration), with no negative animals serving as a control. In group 4 (1.5 mg / kg / day), two male animals were confirmed as ADA-positive on day 7 (before administration), and their serum drug concentration was not significantly different from that of animals confirmed as ADA-negative. All animals were confirmed as ADA-positive on day 14 (before administration), with no negative animals serving as a control. No animal concentrations were excluded from the calculation of mean serum drug concentration, mean TK parameter, and mean drug-time curve. It is evident that ADA was detectable in only a few animals (1 at the medium dose and 2 at the high dose) after 7 days of continuous administration, demonstrating an advantage in immunogenicity compared to commercially available thrombolytic drugs.

[0346] No changes in body weight, food intake, ophthalmological findings, electrocardiogram, or urinalysis related to the test product were observed. Clinical symptoms related to the test product included multiple bruises, swelling, edema, ulcers, and abrasions in all treatment groups. These symptoms did not show a clear dose-dependent relationship. Edema and swelling primarily occurred during the first week of administration and did not completely resolve by the end of the treatment period, correlated with edema / hardening observed at the injection site. The bruises were likely caused by blood collection and administration procedures; given their high incidence, they may also be related to the pharmacological effects of the test product itself (acute thrombolysis, affecting blood clotting). Although some symptoms (bruises) were also observed in the control group, the test product-treated animals exhibited more bruises, greater severity, and longer duration, therefore these are considered test product-related. These symptoms did not affect animal activity and recovered during the recovery period, and are considered non-harmful changes related to the test product. A transient decrease in body temperature was observed only in two animals in the 0.15 mg / kg / day dose groups, 1-2 hours after administration on day 7. The body temperatures of these two animals were 34.5℃ and 36.6℃, respectively, and returned to normal on day 8. The two animals exhibited the largest decreases in hematological parameters (RBC, HGB, HCT) within their groups on day 7, possibly due to anemia indirectly causing the temperature drop. Given the short duration and low incidence of these temperature changes, coupled with good animal activity, they are considered non-harmful changes.

[0347] Compared with the control group, the clinical pathological changes related to the test product were mainly observed in erythroid-related parameters in male and / or female animals across all dose groups. These included decreased red blood cell (RBC), hemoglobin (HGB), and hematocrit (HCT), and increased absolute and percentage reticulocyte counts (#RET / %RET), platelet count (PLT), and total bilirubin (TBIL). These parameters were observed in all dosing groups, with significant changes on day 7, and most exceeded the laboratory background range with statistical significance. With repeated dosing, these parameters partially or completely recovered by the end of the dosing period, possibly due to gradual adaptation. Although exposure decreased in all groups at the last dose due to the influence of ADA, the exposure in the high-dose group remained relatively high at the last dose. No clinical signs of anemia were observed in the animals, and no histopathologically associated changes were observed at the end of the dosing / recovery period; therefore, these changes were considered non-harmful.

[0348] Compared with the control group, two hours after administration on the first day, test-sample-related hemocoagulation changes were observed in both male and female animals in the 1.5 mg / kg / day dose group, with prolonged activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT). On day 7, decreased fibrinogen (FIB) was observed in both male and female animals in all dose groups, and prolonged thrombin time (TT) was observed in both male and female animals in the 0.5 and 1.5 mg / kg / day dose groups. Most of these indicators were outside the laboratory background range, and most were statistically significant, related to the pharmacological effects of the drug itself (acute thrombolysis, affecting hemocoagulation). At the end of the administration and recovery periods, no test-sample-related hemocoagulation changes or histopathological changes were observed, and therefore these were considered non-harmful changes.

[0349] At the end of the dosing period, the organ-related weights of the test product were as follows: decreased thymus weight in male animals of the 0.15 and 0.5 mg / kg / day dose groups and decreased thymus weight in female animals of the 1.5 mg / kg / day dose group. At the end of the dosing period, the test product-related gross pathology was as follows: small thymus in male animals of the 0.15 and 0.5 mg / kg / day dose groups and decreased thymus size in female animals of the 1.5 mg / kg / day dose group. At the end of the dosing period, the test product-related histopathological changes included mild to moderate lymphopenia in the thymus of male and female animals of the ≥0.15 mg / kg / day dose group and mild mononuclear cell infiltration in the portal area of ​​the liver, mild increase in cell count in the hepatic sinusoids, and mild increase in cell count in the red pulp of the spleen in female animals of the 1.5 mg / kg / day dose group. Of these changes, the thymic changes did not lead to immunosuppression or related infections, and the degree of change was low in most animals; therefore, they were considered non-harmful changes. The changes in the liver and spleen were also considered non-harmful changes due to their low degree and lack of other alterations.

[0350] At the end of the administration period, the gross changes that could not be ruled out as related to the test product were the femoral prominences and substances in the muscles surrounding the femur in both male and female animals in the 0.5 mg / kg / day dose group and male animals in the 1.5 mg / kg / day dose group. The histopathological changes that could not be ruled out as related to the test product were mild to moderate bone increase and / or slight to mild fibrosis on the periosteum of the femur in both male and female animals in the 0.5 mg / kg / day dose group and male animals in the 1.5 mg / kg / day dose group. These changes were not observed during the recovery period and had no effect on animal activity; therefore, they were considered non-harmful changes.

[0351] In summary, cynomolgus monkeys were well-tolerated after 14 consecutive days of intravenous administration of ZHB130 at doses of 0.15, 0.5, and 1.5 mg / kg / day. No adverse changes related to the test product were observed in any dose group; therefore, under the conditions of this study, the no-adverse-effect level (NOAEL) was considered to be 1.5 mg / kg / day. At the NOAEL, after administration on day 7, the exposures (C0 and AUC0-24h) in males were 23300±2820 ng / mL and 125000±18400 ng*h / mL, respectively, while those in females were 28900±5080 ng / mL and 137000±27600 ng*h / mL, respectively. After the last administration on day 14, the exposure levels (C0 and AUC0-24h) in male animals were 46800±7250 ng / mL and 103000±37800 ng*h / mL, respectively, while those in female animals were 40300±18000 ng / mL and 78100±56000 ng*h / mL, respectively.

[0352] Male and female monkeys were administered ZHB130 intravenously once daily for 14 consecutive days at doses of 0.15, 0.5, or 1.5 mg / kg / day. The values ​​of C0, Vss, CL, and AUC0-24h, and the median (range) of T1 / 2, expressed as mean ± standard deviation, are shown in the table below. (All animals in the 0.15, 0.5, and 1.5 mg / kg / day dose groups were confirmed to be ADA-positive on day 14 (before administration), therefore caution should be exercised when assessing TK parameters on day 14.)

[0353] Table 28. 24-hour values ​​of C0, T1 / 2, Vss, CL, and AUC0 after 14 consecutive days of intravenous injection in male and female monkeys. C0, Vss, CL, and AUC are expressed as mean ± standard deviation. 0-24h The value of T is expressed as the median (range). 1 / 2

[0354] Example 22: Pharmacodynamic study of ZHB130 for injection in a rat model of arteriovenous bypass thrombosis.

[0355] A thrombotic model of arteriovenous bypass was established using male SD rats. After anesthetizing the rats, the left external jugular vein and right common carotid artery were isolated. A three-segment cannula was constructed using PE or PVC tubing of different diameters. A 7cm length of 3-0# medical-grade silk braided suture was pre-inserted into the middle PVC tubing (1.35mm inner diameter), with the silk braided suture extending 5cm in the middle section. The outer end of the PE tubing connecting the artery and vein had an outer diameter of 0.99mm. The cannula was filled with saline before insertion. One end of the cannula was inserted into the left external jugular vein and secured with suture, while the other end was inserted into the right common carotid artery and secured with suture. The arterial clamp was released, and blood flow was allowed to resume for 30 minutes. Based on this model, the effects of injectable ZHB130 on thrombus wet weight, dry weight, and thrombus inhibition rate were evaluated.

[0356] I. Grouping and Experimental Design

[0357] Male SD rats weighing 300–360 g were selected to establish an arteriovenous bypass model. After modeling, the rats were immediately divided into 7 groups: a model group, a TNK group (1.6 mg / kg), a SAK group (50,000 U / kg), and four ZHB130 dosage groups (0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, and 1.0 mg / kg), with 10 animals in each group. The animals were administered the drug intravenously 5 minutes before blood flow was established. The administration was stopped 30 minutes after blood flow was established, the suture was removed, and the wet weight of the thrombus was measured. After drying, the dry weight of the thrombus was measured.

[0358] II. Results

[0359] The effects of ZHB130 injection on arteriovenous bypass thrombosis in rats are shown in Tables 29-30 and Figures 19-20. Thrombus wet weight (mg, Mean±SEM) is detailed in Table 20 and Figure 19. Compared with the model group, at doses of 0.2 mg / kg and above, the thrombus wet weight in all ZHB130 injection groups was significantly reduced (P<0.01); compared with the SAK group, at doses of 0.5 mg / kg and above, the thrombus wet weight in all ZHB130 injection groups was significantly reduced (P<0.01). The thrombus inhibition rate of each group relative to the model group is detailed in the table below.

[0360] Table 29 Inhibitory effect of ZHB130 for injection on wet weight of arteriovenous bypass thrombus in rats (n=10, Male) Note: Compared with the model group, ** indicates P < 0.01. Compared with the SAK group, # indicates P < 0.05; ## indicates P < 0.01.

[0361] The effect of ZHB130 injection on arteriovenous bypass thrombosis in rats was investigated. Thrombus dry weight (mg, Mean±SEM) is detailed in Table 21 and Figure 20. Compared with the model group, at doses of 0.1 mg / kg and above, the thrombus dry weight in all ZHB130 injection groups was significantly reduced (P<0.05). Compared with the SAK group, at doses of 0.2 mg / kg and above, the thrombus wet weight in all ZHB130 injection groups was significantly reduced (P<0.05). The thrombus inhibition rate of each group relative to the model group is detailed in the table below.

[0362] Table 30 Inhibitory effect of ZHB130 injection on the dry weight of arteriovenous bypass thrombus in rats (n=10, Male) Note: Compared with the model group, * indicates P < 0.05; ** indicates P < 0.01. Compared with the SAK group, # indicates P < 0.05; ## indicates P < 0.01.

[0363] In a rat model of arteriovenous bypass thrombosis, ZHB130 injection significantly inhibited thrombosis. Compared with the model group, the wet weight of thrombi in the 0.2 mg / kg, 0.5 mg / kg, and 1.0 mg / kg ZHB130 injection groups was significantly reduced, and the thrombosis inhibition rate was significantly higher (P<0.01); the dry weight of thrombi in the 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, and 1.0 mg / kg ZHB130 injection groups was significantly reduced, and the thrombosis inhibition rate was significantly higher (P<0.05). The four dose groups of ZHB130 (0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, and 1.0 mg / kg) showed a certain dose-dependent relationship.

[0364] Compared with the SAK group, the wet weight of thrombi in all groups of ZHB130 for injection (0.5 mg / kg, 1.0 mg / kg) was reduced and the thrombus inhibition rate was higher, with significant differences (P<0.01); the dry weight of thrombi in all groups of ZHB130 for injection (0.2 mg / kg, 0.5 mg / kg, 1.0 mg / kg) was reduced and the thrombus inhibition rate was higher, with significant differences (P<0.05).

[0365] Example 23: Pharmacodynamic study of ZHB130 for injection in a rat arterial electrocoagulation thrombosis model.

[0366] An arterial electrocoagulation thrombosis model was established using male SD rats. After anesthesia, the right common carotid artery was isolated and clamped with an electrode. When current was applied to the stimulation electrode, thrombus formation was induced by the thermal effect of the current, causing vascular damage. The stimulation current intensity was 1 mA. The thrombus occlusion rate was observed in real time. Stimulation was stopped when the thrombus occlusion rate reached 95%, and the time of thrombus formation was recorded. Based on this model, the effect of injectable ZHB130 on the time of arterial thrombus formation was evaluated.

[0367] I. Grouping and Experimental Design

[0368] Male SD rats weighing 280–350 g were selected to establish an arterial electrocoagulation thrombosis model. They were divided into six groups: a model group, a TNK group (1.6 mg / kg), a SAK group (50,000 U / kg), and three ZHB130 dose groups (0.2 mg / kg, 0.5 mg / kg, and 1.0 mg / kg), with ten animals in each group. Five minutes before electrocoagulation began, the animals in each group received the intravenous medication. The right common carotid artery was stimulated with a 1 mA current, and the time from the start of electrical stimulation to the thrombus occlusion rate reaching 95% was recorded.

[0369] II. Results

[0370] The effects of ZHB130 injection on arterial thrombosis time in rats are shown in Table 31 and Figure 21. Compared with the model group, the arterial thrombosis time was significantly prolonged in the medium- and high-dose ZHB130 injection groups (P<0.01). Compared with the SAK group, the arterial thrombosis time was significantly prolonged in the high-dose ZHB130 injection group (P<0.01).

[0371] Table 31 Effect of ZHB130 injection on arterial thrombosis time in rats (n=10, Male) Note: Compared with the model group, ** indicates P < 0.01. Compared with the SAK group, ## indicates P < 0.01.

[0372] In a rat arterial electrocoagulation thrombosis model, injected ZHB130 significantly inhibited thrombus formation. Compared with the model group, the arterial thrombus formation time was significantly prolonged in the medium- and high-dose ZHB130 groups (0.5 mg / kg and 1.0 mg / kg), with statistically significant differences (P<0.01). Compared with the SAK group, the arterial thrombus formation time was significantly prolonged in the high-dose ZHB130 group (1.0 mg / kg), with statistically significant differences (P<0.01). The three dose groups of ZHB130 (0.2 mg / kg, 0.5 mg / kg, and 1.0 mg / kg) showed a certain dose-dependent relationship.

[0373] Example 24: Pharmacodynamic study of ZHB130 for injection in a rat autologous thrombotic stroke model

[0374] A rat autologous thrombotic stroke model was established using male SD rats. After anesthesia, the midline of the neck was incised, and the right common carotid artery, external carotid artery, and internal carotid artery were isolated. The vagus nerve was gently dissected, and the external carotid artery was ligated and cut. The proximal end of the common carotid artery was clamped, and prepared thrombus columns were sequentially aspirated into PE50 tubes (0.58 mm inner diameter) and slowly injected into the external carotid artery stump using 0.2 mL of phosphate buffer. 10-15 thrombus columns were injected into each animal. Animals in the sham-operated group received an equal volume of 0.9% sodium chloride injection. After thrombus column injection, the external carotid artery stump was ligated, the neck skin was sutured, and the area was disinfected. Based on this model, the effects of ZHB130 injection on mortality, body weight, neurological deficits, and infarct area in the model rats were evaluated.

[0375] I. Grouping and Experimental Design

[0376] Male SD rats weighing 280–350 g were selected to establish an autologous thrombotic stroke model. One hour after model establishment, animals with neurological deficit symptom scores of 2–3 were enrolled. The model group, TNK group (1.6 mg / kg), SAK group (50000 U / kg), and three ZHB130 dose groups (0.2 mg / kg, 0.5 mg / kg, and 1.0 mg / kg), with 12 animals in each group; a sham surgery group of 8 animals was also included, for a total of 7 groups. All animals received drug treatment after enrollment. The effects of the test substance on mortality, body weight, neurological deficit symptoms, and cerebral infarction area were observed the day after drug administration.

[0377] II. Results

[0378] The effect of injectable ZHB130 on rat mortality is shown in Table 32. During the experiment, 3 animals died in the model group, 1 animal died in the ZHB130 (0.2 mg / kg) group, and 1 animal each in the ZHB130 (0.5 mg / kg) and ZHB130 (1.0 mg / kg) groups showed epileptic symptoms but were not included in the statistics. No other abnormalities were observed in any group. Compared with the model group, the mortality rates of animals in the TNK group, SAK group, and the low, medium, and high dose groups of injectable ZHB130 showed an improving trend, but the changes were not statistically significant.

[0379] Table 32 Effect of ZHB130 injection on rat mortality

[0380] The effects of injectable ZHB130 on body weight are shown in Table 33, Figure 22, and Figure 23. There were no statistically significant differences in body weight among the groups during the experiment. Compared with the model group, the TNK group showed a significantly lower rate of body weight change (P<0.05); compared with the model group, the ZHB130 (0.5 mg / kg) group showed a highly significantly lower rate of body weight change (P<0.01).

[0381] Table 33 Effect of ZHB130 injection on rat body weight Note: Compared with the model group, * indicates P<0.05; ** indicates P<0.01.

[0382] The effects of ZHB130 injection on neurological deficit symptoms in rats are shown in Table 34 and Figure 24. Compared with the model group, the TNK group, SAK group, and low, medium, and high dose groups of ZHB130 injection showed a trend of improvement in neurological deficit symptoms, but no significant changes were observed.

[0383] Table 34 Effects of ZHB130 injection on neurological deficit symptoms in rats

[0384] The effects of ZHB130 injection on the cerebral infarction area in rats are shown in Table 35 and Figure 25. Compared with the model group, the cerebral infarction area in the ZHB130 (0.2 mg / kg) group showed a decreasing trend, but the change was not significant. Compared with the model group, the cerebral infarction area in the TNK group, SAK group, and ZHB130 (0.5 mg / kg) group was significantly reduced (P<0.05). Compared with the model group, the cerebral infarction area in the ZHB130 (1.0 mg / kg) group was significantly reduced (P<0.01). The three dose groups of ZHB130 (0.2 mg / kg, 0.5 mg / kg, and 1.0 mg / kg) showed a certain dose-dependent relationship.

[0385] Table 35 Effect of ZHB130 injection on cerebral infarction area in rats Note: Compared with the model group, * indicates P<0.05; ** indicates P<0.01.

[0386] In a rat autologous thrombotic stroke model, injectable ZHB130 exhibited significant thrombolytic activity and therapeutic effects on thrombosis-induced stroke. Compared with the model group, administration of injectable ZHB130 showed a trend towards improvement in mortality and neurological deficit symptoms, but no significant changes were observed. Compared with the model group, the ZHB130 (0.5 mg / kg) group showed significant improvements in body weight change rate, and the ZHB130 (0.5 mg / kg) and ZHB130 (1.0 mg / kg) groups showed significant improvements in cerebral infarction area (P<0.05). A dose-dependent relationship was observed among the three ZHB130 dosage groups (0.2 mg / kg, 0.5 mg / kg, and 1.0 mg / kg).

[0387] In summary, this application has the following advantages over the prior art:

[0388] This application proposes to fuse SAK with a partial fragment of t-PA to form a fusion protein. The designed fusion protein possesses both the thrombolytic activity of SAK and the fibrin-targeting recognition ability of t-PA. Furthermore, the introduction of the humanized t-PA fragment can reduce the immunogenicity of SAK, thereby improving the safety and efficacy of SAK application.

[0389] Furthermore, this application utilizes polyethylene glycolation technology to significantly improve protein stability, reduce immunogenicity, improve the pharmacokinetic properties of the protein in vivo, and maximize the preservation of the original protein's activity and its ability to target fibrin, while also reducing the risk of bleeding.

[0390] The drug molecules in this invention are expected to possess ideal thrombolytic drug characteristics, including: (1) better targeting. The polyethylene glycol-modified fusion protein of this invention introduces a t-PA targeting module on the basis of SAK. After PEG modification, the fusion protein still has the same affinity for fibrin as alteplase, allowing SAK to target and aggregate on fibrin, promoting the formation of plasminogen activator on the thrombus surface and its aggregation at the thrombus site, thereby more fully exerting the thrombolytic effect of SAK and reducing the risk of bleeding. (2) superior thrombolytic activity. In vitro and in vivo experiments have shown that the polyethylene glycol-modified fusion protein of this invention has high thrombolytic activity, good vascular recanalization rate, and thrombolytic effect superior to the original SAK protein, effectively reducing mortality. This indicates that the introduction of the t-PA targeting module and PEG does not affect the thrombolytic activity of SAK, and the fusion protein containing the targeting unit has better thrombolytic performance. Furthermore, the examples demonstrate that the polyethylene glycol fusion protein of the present invention can achieve a fibrinolytic effect of more than 50% with a low dose (2μg / 200μL), indicating that it has high thrombolytic performance and is suitable for different types of thrombi and different patient conditions. (3) It has higher safety. Bleeding is the most common adverse reaction of SAK thrombolytic agents. The incidence of bleeding in the clinical trials of marketed r-SAK preparations is 24%. The polyethylene glycol modified fusion protein of the present invention has a lower risk of causing bleeding complications and a significantly lower bleeding tendency than the original SAK protein, thus having better safety. On the other hand, the polyethylene glycol modified fusion protein of the present invention has extremely low immunogenicity compared to the original SAK protein, and the possibility of allergic reactions is extremely low, which can reduce immune-related side effects and complications and make up for the high immunogenicity of glucosamine. (4) It has the advantage of long-lasting effect. The half-life of SAK is only about 5 minutes, and thrombolytic administration requires an intravenous infusion of up to 30 minutes, so its clinical administration process is more complicated and inconvenient. The polyethylene glycol-modified fusion protein of the present invention significantly prolongs the drug half-life of SAK, making it feasible for intravenous bolus administration. This not only reduces the number of administrations and simplifies the treatment process, but also improves patient compliance and provides a more effective thrombolytic window for thrombosis patients. (5) It can be administered multiple times. Since SAK is a foreign protein of microbial origin, its immunogenicity will produce neutralizing antibodies after entering the human body. Therefore, the SAK preparations currently on the market can generally only be used once for thrombolysis. When thrombosis occurs again, other thrombolytic drugs need to be used. The polyethylene glycol-modified fusion protein of the present invention has extremely low immunogenicity and rarely produces anti-drug antibodies. Therefore, it can be administered multiple times, providing thrombosis patients with the opportunity for continuous or multiple thrombolysis. Compared with SAK thrombolytic agents that can only be used once, it is expected to achieve a more definite therapeutic effect. (6) It has broad application prospects.In vitro and in vivo studies show that the polyethylene glycol-modified fusion protein of the present invention has good safety and thrombolytic properties, and has advantages such as long-lasting effect, low immunogenicity, low-dose effectiveness, and intravenous injection. It meets the clinical expectation for an ideal thrombolytic drug and can be applied to the thrombolytic treatment of thrombotic diseases such as stroke, myocardial infarction, pulmonary embolism, and lower extremity venous thrombosis. It also has great potential for secondary prevention of thrombotic diseases.

Claims

1. A fusion protein comprising a t-PA fragment and a SAK fragment, wherein the t-PA fragment and the SAK fragment are directly linked or linked via a linker peptide; the t-PA fragment is located at the N-terminus or C-terminus of the SAK fragment; The t-PA fragment contains two or more of the F, E, K1, and K2 domains of the non-protease region of human tissue plasminogen activator (ht-PA), wherein the F, E, K1, and K2 domains are natural domains or mutants thereof; the F, E, K1, and K2 domains are directly linked or linked by linker peptides. The SAK fragment is the natural protein of glucosamine (SAK) or a mutant thereof.

2. The fusion protein of claim 1, wherein the t-PA fragment contains F and K2 domains.

3. The fusion protein as described in claim 1 or 2, wherein when the t-PA fragment is at the N-terminus of the SAK fragment, and the SAK fragment is in a form where the N-terminus of the native SAK protein is missing an amino acid, the t-PA fragment and the SAK fragment are linked by a linker peptide.

4. The fusion protein according to any one of claims 1-3, wherein the linker peptide connecting the t-PA fragment and the SAK fragment is a rigid linker (EAAAK)n or (EAAAAK)n; or a flexible linker (GGGGGS)n; where n is an integer from 1 to 4.

5. The fusion protein according to any one of claims 1 to 4, wherein the linker peptide connecting the K1 domain to its preceding domain is a natural linker peptide with an amino acid sequence as shown in SEQ ID NO: 5, or a conventional rigid linker or flexible linker. The linker peptide connecting the K2 domain to its preceding domain is a natural linker peptide with an amino acid sequence as shown in SEQ ID NO: 6, or a conventional rigid linker peptide or a flexible linker peptide. The F domain and the E domain can also be directly linked, resulting in a non-linked peptide. Rigid linkers include (EAAAK)n, (EAAAAK)n, etc., where n represents the number of repetitions of EAAAK or EAAAAK; common flexible linkers include (GGGGGS)n, where n represents the number of repetitions of GGGGGS, and n is an integer from 1 to 4.

6. The fusion protein according to any one of claims 1 to 5, wherein the amino acid sequence of the F domain of the t-PA fragment is as shown in SEQ ID NO: 1; the amino acid sequence of the E domain is as shown in SEQ ID NO: 2; the amino acid sequence of the K1 domain is as shown in SEQ ID NO: 3; the amino acid sequence of the K2 domain is as shown in SEQ ID NO: 4, or the P in the terminal CDVPSC sequence of the sequence shown in SEQ ID NO: 4 is mutated to K or R.

7. The fusion protein according to any one of claims 1 to 6, wherein the amino acid sequence is as shown in SEQ ID NO: 10 or 11.

8. A polyethylene glycol-modified t-PA-SAK fusion protein, wherein the t-PA-SAK fusion protein is the fusion protein according to any one of claims 1-7.

9. The polyethylene glycol-modified t-PA-SAK fusion protein as described in claim 8, wherein the polyethylene glycol modifier is a branched polyethylene glycol modifier with a molecular weight of 5kDa-30kDa.

10. The use of the fusion protein according to any one of claims 1-7 and the polyethylene glycol-modified t-PA-SAK fusion protein according to claim 8 or 9 in thrombolytic therapy and secondary prevention of thrombotic diseases.

11. The application according to claim 10, wherein the thrombotic disease includes stroke, myocardial infarction, pulmonary embolism, and lower extremity deep vein thrombosis.

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