An antibody protein targeting degradation of calprotectin and application thereof

Abstract

This invention relates to HeartTAC, an antibody protein that targets and degrades calprotectin, and its applications, belonging to the field of biomedical technology. This invention provides HeartTAC, an antibody protein that targets and degrades calprotectin, the amino acid sequence of which is shown in SEQ ID NO. 6 or SEQ ID NO. 4. This invention employs an extracellular targeted protein degradation scheme to construct HeartTAC, an antibody protein that targets and degrades calprotectin. This protein exhibits good targeting specificity to the heart, can degrade calprotectin in the heart, can significantly improve cardiac function, alleviate cardiac fibrosis, and has good safety profile. It opens up an important new direction for the prevention and control of cardiovascular diseases and has promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to HeartTAC, an antibody protein that targets and degrades calprotectin, and its applications. Background Technology

[0002] Targeted protein degradation technology is a novel drug development approach that uses small or chimeric molecules to deliver target proteins to intracellular degradation systems for clearance. Currently, technologies like PROTACs and molecular gels are representative of this approach and have entered the clinical translation stage, demonstrating significant potential in fields such as oncology and autoimmune diseases. The main advantage of this technology lies in its ability to target "undruggable" proteins that are difficult for traditional drugs to act on, efficiently degrading them through catalysis, overcoming drug resistance, and exhibiting more sustained action and greater selectivity. However, it still has significant limitations: the degrading molecules are typically large in molecular weight, resulting in poor cell membrane permeability and oral absorption, leading to insufficient drug-likeness; off-target degradation, hook effects, and the widespread tissue distribution of E3 ligases also exist. Furthermore, limited ligand resources and the high difficulty of delivery and regulation restrict its clinical application and further development.

[0003] S100A8 / A9 (also known as calprotectin) is an important heterodimer in the S100 calcium-binding protein family, primarily expressed and secreted by immune cells, especially neutrophils and monocytes / macrophages. It plays a central role in inflammation and innate immune responses, serving as a key molecule connecting the immune system with various disease pathological processes. Plasma S100A8 / A9 levels are significantly elevated in patients with acute coronary syndromes, acting as an independent risk factor for predicting major adverse cardiovascular events, even superior to traditional hs-CRP. Following myocardial infarction, necrotic cells release S100A8 / A9, exacerbating local and systemic inflammatory storms, recruiting more neutrophils, expanding the area of ​​myocardial damage, and potentially inhibiting myocardial repair. In summary, S100A8 / A9 is a core bridging molecule connecting inflammation and cardiovascular disease. It is not only an "alarm" reflecting disease activity and prognosis but also an "accelerator" driving the progression of atherosclerosis, plaque rupture, myocardial damage, and heart failure. Research and targeted therapy for it have opened up an important new direction for the prevention and control of cardiovascular diseases. Currently, there are no reports on targeted degradation of calprotectin.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide an antibody protein HeartTAC that targets and degrades calprotectin and its application, so as to solve the problem of the lack of targeted degradation technology for calprotectin in the prior art.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides an antibody protein HeartTAC that targets and degrades calprotectin, the amino acid sequence of which is shown in SEQ ID NO.6 or SEQ ID NO.4.

[0007] The present invention provides a nucleotide sequence encoding the antibody protein HeartTAC, the nucleotide sequence being shown in SEQ ID NO.5 or SEQ ID NO.3.

[0008] This invention provides the use of the antibody protein HeartTAC or the nucleotide sequence described herein in the preparation of a medicament for treating cardiovascular diseases.

[0009] This invention provides an expression vector comprising a nucleotide sequence and an empty vector; The nucleotide sequence is the nucleotide sequence described above; The empty carrier includes the pSecTag2A carrier.

[0010] This invention provides the application of the expression vector in the preparation of drugs for treating cardiovascular diseases.

[0011] This invention provides a medicament for treating cardiovascular diseases, the medicament comprising a core ingredient and a pharmaceutically acceptable carrier; The core components include the expression vector or the antibody protein HeartTAC.

[0012] Preferably, the dosage form of the drug is a tablet, an injection, or a capsule.

[0013] The present invention has the following technical effects and advantages: This invention employs an extracellular targeted protein degradation protocol to construct an antibody protein, HeartTAC, that targets and degrades calprotectin. HeartTAC comprises an Fc segment lacking ADCC (antibody-dependent cell-mediated cytotoxicity) activity, a conjugate specifically recognizing NPR1 and the disease-related targets S100A8 / A9, and a cathepsin-sensitive linker specifically activated in lysosomes. This protein exhibits excellent targeting specificity to the heart, degrading calprotectin within the heart, significantly improving cardiac function, alleviating cardiac fibrosis, and demonstrating good safety. It opens up an important new direction for the prevention and control of cardiovascular diseases and shows promising application prospects. Attached Figure Description

[0014] Figure 1 The structural diagram of an antibody protein that targets and degrades calprotectin; Figure 2 The spectrum of the pSecTag2A vector; Figure 3Results of S100A8 / A9 protein content determination in the purified supernatant of different recombinant cells; Figure 4 Results of S100A8 / A9 protein content determination in purified supernatant of different recombinant cells; Figure 5 A flowchart of the protocol for animal experiments; Figure 6 TTC staining results of heart tissue from mice in different treatment groups; Figure 7 The results of ultrasound examination of mice in different treatment groups; Figure 8 Results of myocardial fibrosis assessment in mice under different treatment groups; Figure 9 The results of plasma liver and kidney function tests in mice of different treatment groups; Figure 10 Immunohistochemical staining and analysis results for mice in different treatment groups. Detailed Implementation

[0015] The present invention provides an antibody protein HeartTAC that targets and degrades calprotectin, wherein the amino acid sequence of the antibody protein HeartTAC is shown in SEQ ID NO.6 or SEQ ID NO.4; The amino acid sequence shown in SEQ ID NO.6 is: QVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWNWVRQAPGKGLEWVSVIESKGNYIFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRYSMIYSYGAGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGFLGGVRGVDGEVQLQQSGPELVKSGASVKISCKASGYSFTDYNINWVKQSNGKSLEWIGLINPNYGTTFYNQKFKGKATLTVDQSSSTAYMQLNSLTSEDSAVYYCARETLLLRYFDYWGQGTTLTVSGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASESVDIYGISFMNWFQLKPGQPPKLLIYAASKQGSGVPARFSGSGSGTDFSLNIHSMEEDDTAMYFCQQSKEVPRTFGGGTKLEIK ; The SEQ ID The amino acid sequence shown in NO.4 is: QVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWNWVRQAPGKGLEWVSVIESKGNYIFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRYSMIYSYGAGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGFLGGVRGVDGQVQLQQSGAELVKPGASVKLSCTASGFNIKDTYLHWVKQRPEQGLEWVGRIDPANGDTKYD PKFQAKATITADTTSNTAYVHLNSLTSEDTAVYFCTGGWQMGGRYFDVWGAGTTVTVSGGGGSGGGGSGGGGSDVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK .

[0016] The present invention provides a nucleotide sequence encoding the antibody protein HeartTAC, the nucleotide sequence being shown in SEQ ID NO.5 or SEQ ID NO.3;

[0017] This invention provides the use of the antibody protein HeartTAC or the nucleotide sequence described herein in the preparation of a medicament for treating cardiovascular diseases.

[0018] This invention provides an expression vector comprising a nucleotide sequence and an empty vector; The nucleotide sequence is the nucleotide sequence described above; The empty carrier includes the pSecTag2A carrier.

[0019] This invention provides the application of the expression vector in the preparation of drugs for treating cardiovascular diseases.

[0020] This invention provides a medicament for treating cardiovascular diseases, the medicament comprising a core ingredient and a pharmaceutically acceptable carrier; The core components include the expression vector or the antibody protein HeartTAC.

[0021] In this invention, the dosage form of the drug is tablets, injections, or capsules.

[0022] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Example 1: Construction of an antibody protein targeting the degradation of calprotectin

[0024] To construct an antibody capable of targeting and degrading cardiac tissue, the inventors, following the method described in the paper "Transferrin receptor targeting chimeras for membrane protein degradation" (published September 25, 2024), designed a nanobody αNPR1 that specifically recognizes the cardiac tissue surface receptor NPR1. Then, based on the NPR1-specific nanobody αNPR1, the inventors linked the C-terminus of the anti-S100A8 / A9 protein to the NPR1-specific nanobody αNPR1, obtaining an antibody protein targeting the degradation of calprotectin, namely αNPR1-S100A8 / A9, named HeartTAC. This protein contains an Fc segment lacking ADCC (antibody-dependent cell-mediated cytotoxicity) activity, a conjugate specifically recognizing NPR1 and the disease-related target S100A8 / A9, and a cathepsin-sensitive linker specifically activated in lysosomes. Its structure is as follows: Figure 1 As shown.

[0025] (SEQ ID NO.2)。

[0026] (SEQ ID NO.6).

[0027] Example 2: Cell validation experiment

[0028] I. Experimental Principle

[0029] An expression vector was constructed using the pSecTag2A vector (purchased from Thermo Fisher Scientific (China) Co., Ltd., catalog number V90020), then transfected into human embryonic kidney cells, cultured in serum-free medium, and the supernatant was collected to obtain a crude extract containing the target protein, which was then purified.

[0030] II. Experimental Materials and Reagents

[0031] Expression vector: The spectrum of the pSecTag2A vector is as follows Figure 2 As shown.

[0032] Cells: HEK239T cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number CL-0005) and AC16 cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number CL-0790).

[0033] Culture medium: The complete culture medium was DMEM high glucose (purchased from Thermo Fisher Scientific (China) Co., Ltd., catalog number: 11965118) + 10% fetal bovine serum (FBS, purchased from Thermo Fisher Scientific (China) Co., Ltd., catalog number: A5256701) + 1% penicillin-streptomycin. The serum-free expression medium was DMEM high glucose (Opti-MEM) (purchased from Thermo Fisher Scientific (China) Co., Ltd., catalog number: 11965118).

[0034] Transfection reagent: Polyethyleneimine (PEI, 1 mg / mL, pH 7.0)

[0035] Purification materials: Ni-NTA agarose resin chromatography column (gravity column) (purchased from Sangon Biotech (Shanghai) Co., Ltd., item number C600791); Equilibration / binding buffer: 20mM Tris-HCl, 300mM NaCl solution, 10mM imidazole, pH 7.8 (filtered sterile before use). Washing buffer: 20 mM Tris-HCl, 300 mM NaCl solution, 40 mM imidazole, pH 7.8; Elution buffer: 20 mM Tris-HCl, 300 mM NaCl solution, 400 mM imidazole, pH 7.8; Detection reagents: SDS-PAGE gel, Western Blot related reagents, BCA protein quantification kit (Thermo Fisher Scientific (China) Co., Ltd., catalog number A55864).

[0036] III. Experimental Procedure

[0037] 1. Construction of expression carriers

[0038] The nucleotide sequences of αNPR1, αNPR1-S100A8 / A9-1, and αNPR1-S100A8 / A9-2 were ligated into the pSecTag2A vector using a cloning method. Sequencing confirmed the formation of the expression vectors (recombinant plasmids) pSecTag2A-αNPR1, pSecTag2A-αNPR1-S100A8 / A9-1, and pSecTag2A-αNPR1-S100A8 / A9-2, respectively. The specific method is as follows: First, the pSecTag2A empty vector and the target gene fragments (αNPR1, αNPR1-S100A8 / A9-1, and αNPR1-S100A8 / A9-2) were double-digested with restriction endonucleases (HindIII and XhoI) to obtain linearized vectors and insert fragments with compatible ends, respectively. Then, the purified linearized vectors and the three insert fragments were respectively incubated at T4... Recombinant plasmids were constructed by ligation at a 1:3 molar ratio using DNA ligase. The ligation products were then transformed into competent *E. coli*, plated on ampicillin-containing plates, and positive clones were screened and preliminarily identified by colony PCR and plasmid digestion. Finally, DNA sequencing was performed on the preliminarily correct clones to verify the accuracy of the inserted sequence and the correctness of the reading frame. This resulted in the successful construction of three validated expression vectors (recombinant plasmids): pSecTag2A-αNPR1, pSecTag2A-αNPR1-S100A8 / A9-1, and pSecTag2A-αNPR1-S100A8 / A9-2, for subsequent use.

[0039] 2. HEK239T cell experiment

[0040] (1) Cell culture: HEK239T cells were seeded into complete culture medium and cultured in a 37℃, 5% CO2 incubator until the logarithmic growth phase (confluence of 85%). (2) Cell plating: One day before transfection, cells were digested with trypsin at a concentration of 6 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / mL in 6-well cell culture plates; (3) Preparation of the transfection complex: a) Tube A: Dissolve 3 μg of recombinant plasmid in 150 μL of Opti-MEM and mix gently; b) Tube B: Dissolve 9 μg of PEI in 150 μL of Opti-MEM and let stand at room temperature for 5 min; c) Add the liquid from tube B dropwise to tube A, immediately vortex to mix, and let stand at room temperature for 20 minutes to obtain the transfection complex.

[0041] (4) Transfection: Add 300 μL of the transfection complex dropwise evenly to the cell culture plate containing HEK239T cells. Add 300 mL to each well, gently shake to mix, and return to the incubator to continue culturing. (5) Change medium: 7 h after transfection, recombinant cells containing expression vectors pSecTag2A-αNPR1, pSecTag2A-αNPR1-S100A8 / A9-1 and pSecTag2A-αNPR1-S100A8 / A9-2 were obtained respectively, and the culture medium containing the transfection complex was discarded. (6) Serum-free induction: Slowly add preheated serum-free expression medium along the well wall (2 mL per well in a 6-well plate); (7) Culture and collection: Continue to culture the cells for 72 hours. During this period, the target protein will continue to be secreted into the supernatant; (8) Supernatant treatment: Carefully collect the cell culture supernatant with a pipette, transfer it to a centrifuge tube, centrifuge at 4℃ and 3500g for 20min, and store the supernatant at 4℃ for later use; (9) Column preparation: Take an appropriate amount of Ni-NTA agarose resin and pack it into the chromatography column. Rinse the column with equilibration buffer and equilibrate the resin. (10) Sample loading: Load the processed cell supernatant into the equilibrated Ni-NTA column at a slow flow rate (1 mL / min) and collect the flow-through liquid; (11) Washing: Rinse with equilibration buffer to remove unbound contaminating proteins. Then rinse with washing buffer to remove weakly nonspecifically bound contaminating proteins and collect the washing solution; (12) Elution: Elution was performed with elution buffer, and the eluted components were collected to obtain the purified supernatant of recombinant cells HEK239T containing expression vectors pSecTag2A-αNPR1, pSecTag2A-αNPR1-S100A8 / A9-1 and pSecTag2A-αNPR1-S100A8 / A9-2.

[0042] Using cells without the transfected vector as a control, the content of S100A8 / A9 protein in the purified supernatant of control cells and recombinant cells containing different expression vectors was determined by enzyme-linked immunosorbent assay (ELISA). The results of the determination of S100A8 / A9 protein content in the purified supernatant of different recombinant cells are as follows: Figure 3 As shown in the figure, " "represent P <0.001.

[0043] according to Figure 3 It was found that the content of S100A8 / A9 protein in the supernatant of recombinant HEK239T cells containing expression vectors pSecTag2A-αNPR1-S100A8 / A9-1 and pSecTag2A-αNPR1-S100A8 / A9-2 was significantly reduced, and the content of S100A8 / A9 protein in the supernatant of recombinant HEK239T cells containing expression vector pSecTag2A-αNPR1-S100A8 / A9-2 was lower than that in recombinant HEK239T cells containing expression vector pSecTag2A-αNPR1-S100A8 / A9-1.

[0044] 3. AC16 cell experiments

[0045] Recombinant AC16 cells containing the expression vectors pSecTag2A-αNPR1, pSecTag2A-αNPR1-S100A8 / A9-1, and pSecTag2A-αNPR1-S100A8 / A9-2 were constructed using the "2.HEK239T cell experiment" method. Cells without transfection were used as controls. The content of S100A8 / A9 protein in the purified supernatant of both control cells and recombinant cells containing different expression vectors was measured. The results of the S100A8 / A9 protein content measurement in the purified supernatant of different recombinant cells are shown below. Figure 4 As shown in the figure, " "represent P <0.001.

[0046] according to Figure 4 It can be seen that the content of S100A8 / A9 protein in the supernatant of recombinant AC16 cells containing expression vectors pSecTag2A-αNPR1-S100A8 / A9-1 and pSecTag2A-αNPR1-S100A8 / A9-2 was significantly reduced, and the content of S100A8 / A9 protein in the supernatant of recombinant AC16 cells containing expression vector pSecTag2A-αNPR1-S100A8 / A9-2 was lower than that in recombinant AC16 cells containing expression vector pSecTag2A-αNPR1-S100A8 / A9-1.

[0047] In summary, both the nucleotide sequences of αNPR1-S100A8 / A9-1 and αNPR1-S100A8 / A9-2 can reduce the content of S100A8 / A9 protein in cells, with the nucleotide sequence of αNPR1-S100A8 / A9-2 showing the best effect. Therefore, the nucleotide sequence of αNPR1-S100A8 / A9-2 will be used for subsequent experiments.

[0048] Example 3: Animal Experiment

[0049] Construction of a model of ischemia-reperfusion heart failure (I / R)

[0050] Animals: 6-week-old male C57BL / 6N mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.); The protocol and procedure for animal experiments are as follows: Figure 5 As shown, mice were specifically divided into an experimental group (Ig group) and an intervention group (HeartTAC group). A mouse model of ischemia-reperfusion heart failure (I / R) was surgically established: mice were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (40 mg / kg). After confirming the absence of corneal and foot reflexes, the chest hair was shaved and disinfected with 75% ethanol. The mice were placed in a supine position and intubated and mechanically ventilated. After a left thoracotomy was performed in the fourth intercostal space, the left anterior descending coronary artery was exposed, and a PE-50 polyethylene tube was placed below the left anterior descending artery. The artery and the catheter were ligated together to induce myocardial ischemia for 45 minutes. Ischemia was confirmed by myocardial whitening. After ischemia, the ligature and PE-50 catheter were gently removed to restore blood flow and close the thoracic cavity, allowing the mice to recover and thus obtaining a mouse ischemia-reperfusion heart failure (I / R) model. During the construction of the mouse ischemia-reperfusion heart failure (I / R) model, both the control group and the model group received intraperitoneal injections of drugs 1 hour before surgery and on days 0 and 1 after surgery. The drug dosage was 10 mg / kg (concentration 2.5 mg / mL) for both groups. The experimental group was injected with IgG, and the intervention group was injected with the antibody protein HeartTAC (αNPR1-S100A8 / A9-2). The mice were cultured for 14 days.

[0051] 1. TTC staining

[0052] Three days later, some mice were euthanized, and their hearts were rapidly frozen at -80°C for 5 minutes. Sections were then prepared from the apex to the base of the heart at 1 mm intervals. The sections were incubated with 2% 2,3,5-triphenyltetrazolium chloride and photographed for infarct area analysis. TTC staining results of heart tissue from different treatment groups are shown below. Figure 6 As shown.

[0053] The red area represents surviving cardiomyocytes, and the white area represents dead cardiomyocytes. Figure 6It can be seen that the HeartTAC treatment group had a larger red area than the IgG group, indicating that more surviving cardiomyocytes were present, thus improving myocardial infarction.

[0054] 2. Ultrasonic testing

[0055] Three and fourteen days later, some mice were euthanized. Mice were anesthetized with isoflurane (2% v / v) and their heart rate was maintained at 450–550 beats / min during image acquisition. Cardiac function was assessed using a high-resolution imaging system equipped with a 30 MHz linear array transducer. The ultrasound results of mice in different treatment groups are shown below. Figure 7 As shown, Figure A shows representative ultrasound images of mice under different treatments after 3 days, Figure B shows a quantitative statistical chart of cardiac ejection fraction, Figure C shows representative ultrasound images of mice under different treatments after 14 days, and Figure D shows a quantitative statistical chart of cardiac ejection fraction.

[0056] according to Figure 7 The results showed that echocardiography revealed a significant increase in cardiac ejection fraction in mice in the HeartTAC intervention group 3 days post-surgery, with even more significant improvement observed 14 days post-surgery. This suggests that HeartTAC significantly improves ejection fraction in mice and has a long-term protective effect. In conclusion, HeartTAC significantly improves cardiac function in mice.

[0057] 3. Fibrosis assessment

[0058] Fourteen days later, some mice were euthanized, and their hearts were removed and fixed in 4% paraformaldehyde (pH=7.4) at room temperature for 24 hours. Subsequently, they were paraffin-embedded and stained with Masson's stain to assess myocardial fibrosis. The results of myocardial fibrosis assessment in different treatment groups are as follows: Figure 8 As shown, Figure A represents a representative image of Masson staining bands in cardiac tissue, Figure B represents a quantitative statistical graph of cardiac fibrosis, Figure C represents the RNA expression level of the fibrosis marker col1a1, Figure D represents the RNA expression level of the fibrosis marker col3a1, and Figure E represents the RNA expression level of the fibrosis marker ACTA2.

[0059] according to Figure 8 As can be seen, Masson staining showed that HeartTAC treatment significantly improved cardiac fibrosis in mice. Quantitative statistics revealed a significant difference, with a decrease in the level of RNA, a representative fibrosis marker, suggesting that HeartTAC can improve cardiac fibrosis. This indicates that HeartTAC can significantly improve cardiac fibrosis and related indicators.

[0060] 4. Plasma liver and kidney function tests

[0061] Blood plasma from mice was obtained for liver and kidney function testing. The results of blood plasma liver and kidney function testing in mice of different treatment groups are as follows: Figure 9As shown in the figure, ns represents no difference between the two groups. Figure A shows the detection of plasma alanine aminotransferase (ALT) concentration in mice in different treatment groups; Figure B shows the detection of plasma aspartate aminotransferase (AST) concentration in mice in different treatment groups; Figure C shows the detection of plasma uric acid (Uric Acid) concentration in mice in different treatment groups; and Figure D shows the detection of plasma urea nitrogen (BUN) concentration in mice in different treatment groups.

[0062] according to Figure 9 It can be seen that there was no difference between the two groups in the plasma test for representative indicators of liver and kidney function, indicating that HeartTAC does not affect the liver and kidney function indicators of mice, proving its good safety.

[0063] 5. Immunohistochemical staining and analysis

[0064] After euthanizing the mice, heart, liver, lung, spleen, and kidney tissues were obtained for staining. The specific methods are as follows: After dewaxing at 65°C for 30 min, paraffin sections were rehydrated using a gradient of ethanol / xylene and then subjected to antigen retrieval in boiling citrate buffer (pH 6.0) for 20 min. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide (30 min, protected from light), and nonspecific binding was inhibited using 10% normal goat serum (1 h, room temperature). Primary antibody against S100A8 / A9 (Sanying Biotechnology Co., Ltd.; catalog number 27455-1-AP) was incubated overnight at 4°C. Sections were incubated with species-matched secondary antibody (1 h, room temperature), followed by DAB staining and counterstaining with hematoxylin. After dehydration and mounting, sections were photographed and analyzed under a microscope. Immunohistochemical staining and analysis results of mice in different treatment groups are shown below. Figure 10 As shown, Figure A shows the staining results of heart, liver, lung, spleen and kidney tissues of mice in different treatment groups, and Figure B shows the content of S100A8 / A9 protein in heart, liver, lung, spleen and kidney tissues of mice in different treatment groups.

[0065] according to Figure 10 It can be seen that the HeartTAC intervention group significantly reduced the expression of S100A8 / A9 in cardiac tissue, without affecting the expression in the liver, lungs, spleen and kidneys, suggesting that HeartTAC specifically degrades S100A8 / A9 in cardiac tissue and has good cardiac targeting.

[0066] In conclusion, HeartTAC has promising prospects for clinical application.

[0067] As shown in the above embodiments, this invention provides an antibody protein, HeartTAC, that targets and degrades calprotectin, and its applications. This invention employs an extracellular targeted protein degradation scheme to construct an antibody protein, HeartTAC, that targets and degrades calprotectin. HeartTAC comprises an Fc segment lacking ADCC (antibody-dependent cell-mediated cytotoxicity) activity, a conjugate that specifically recognizes NPR1 and the disease-related targets S100A8 / A9, and a cathepsin-sensitive linker that is specifically activated in lysosomes. This protein exhibits good targeting specificity to the heart, can degrade calprotectin in the heart, can significantly improve cardiac function, alleviate cardiac fibrosis, and has good safety profile. It opens up an important new direction for the prevention and control of cardiovascular diseases and has promising application prospects.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A target antibody protein HeartTAC that degrades calprotectin, characterized in that, The amino acid sequence of the antibody protein HeartTAC is shown in SEQ ID NO.6 or SEQ ID NO.

4.

2. A nucleotide sequence encoding the antibody protein HeartTAC of claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.5 or SEQ ID NO.

3.

3. The use of the antibody protein HeartTAC of claim 1 or the nucleotide sequence of claim 2 in the preparation of a medicament for treating cardiovascular diseases.

4. An expression carrier, characterized in that, The expression vector includes a nucleotide sequence and an empty vector; The nucleotide sequence is the nucleotide sequence according to claim 2; The empty carrier includes the pSecTag2A carrier.

5. The use of the expression vector according to claim 4 in the preparation of a medicament for treating cardiovascular diseases.

6. A drug for treating cardiovascular diseases, characterized in that, The drug comprises a core ingredient and a pharmaceutically acceptable carrier; The core components include the expression vector of claim 4 or the antibody protein HeartTAC of claim 1.

7. The drug according to claim 6, characterized in that, The dosage form of the drug is tablets, injections, or capsules.

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