Targeted protein degradation and uses thereof

Abstract

The present application relates to the technical field of biological medicine. The present application provides a kind of targeted degradation protein and its application, the amino acid sequence of the targeted degradation protein is as shown in SEQ ID NO.3.The targeted degradation protein of the present application can actively mediate the endocytosis and degradation process of target protein based on extracellular protein targeted degradation technology, reduce the abundance of target protein from the source, so as to more fundamentally weaken its biological effect. Only need to screen or develop the binding molecule with high affinity to target protein, it can trigger its degradation. Significantly reduce the design difficulty, and greatly expand the range of target protein that can be intervened.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a targeted degradation protein and its applications. Background Technology

[0002] Extracellular soluble proteins play a central role in mediating various cellular functions and are frequently targets for drug action. However, strategies for specifically blocking the activity of these proteins are currently insufficient. Therefore, developing effective technologies for degrading extracellular proteins has significant basic research value and therapeutic intervention implications.

[0003] Compared to traditional small molecule inhibitor therapies, an ideal treatment strategy should combine low toxicity with high efficacy. Its advantages lie in: reduced dependence on sustained target binding; the ability to completely or significantly attenuate the functional activity of target proteins; and avoiding efficacy reduction caused by factors such as target protein overexpression, competitive inhibition by endogenous ligands, or blocked binding. Targeted protein degradation (TPD) has become a rapidly developing and important direction in drug development. As an innovative mechanism, it effectively complements traditional drug models, providing new ideas for intervention on challenging targets and enhancing the efficacy of existing drug therapies.

[0004] Current TPD research largely focuses on intracellular and membrane proteins, with relatively little attention paid to extracellular soluble proteins. Current mainstream extracellular targeted protein degradation (eTPD) protocols primarily employ bispecific molecular structures. These structures simultaneously bridge soluble target proteins with specific "effect molecules," promoting the co-internalization of target proteins along with effector molecules and their degradation via the intracellular lysosomal pathway. Classic effector molecules include membrane-bound E3 ubiquitin ligases (such as PROTAB), membrane cytokine receptors (such as KineTAC), membrane glycan receptors (such as LYTAC), and transferrin receptors (such as TransTAC). However, the effectiveness of such methods is often limited by the tissue specificity and expression abundance of the effector molecules. Given this limitation, existing eTPD technologies struggle to effectively address the challenges of cardiovascular disease. Developing effector molecules that are specifically or highly expressed in cardiovascular disease tissues or the heart holds promise for significantly expanding the indications for eTPD and improving its targeting specificity.

[0005] NPR1 (Natriuretic Peptide Receptor 1), also known as NPR-A, is a guanylate cyclase that functions in a dimer form and can be activated by the atrial natriuretic peptide (ANP) and the brain natriuretic peptide (BNP). NPR1 possesses an extracellular ligand-binding domain, a transmembrane domain, an intracellular granule (GC) domain, and an intracellular cGMP-dependent protein kinase (PKG) domain. Activated NPR1 catalyzes the conversion of GTP to cGMP. As an intracellular second messenger, cGMP can regulate multiple pathways, including ion channels, protein phosphorylation, nuclear translocation, and gene expression, by activating PKG and phosphodiesterase (PDE). The ANP / BNP-NPR1 system in the heart primarily functions in regulating cardiovascular homeostasis and influencing the physiological and pathological states of the heart. Therefore, NPR1 holds promise as an effector molecule for cardiovascular diseases, enabling the specific degradation of soluble target proteins in cardiac tissue.

[0006] ANGPTL4 (angiopoietin-like protein 4) is a secreted glycoprotein with an N-terminal coil-coil domain and a C-terminal fibroinogen-like domain. As a multifunctional secreted protein, ANGPTL4 inhibits the activity of lipoprotein lipases and plays an important physiological role in lipid metabolism. ANGPTL4 plays a regulatory role in angiogenesis, glucose and lipid energy metabolism, tumor development and progression, and aging, suggesting that ANGPTL4 may be a potential therapeutic target for related diseases. Summary of the Invention

[0007] The purpose of this invention is to provide a targeted degradation protein and its application, and to develop a drug that can directly degrade ANGPTL4 in cardiac tissue.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a targeted degradation protein, the amino acid sequence of which is shown in SEQ ID NO.3.

[0009] The present invention also provides a nucleotide sequence encoding the targeted degradation protein, the nucleotide sequence being shown in SEQ ID. NO. 7.

[0010] The present invention also provides the application of the aforementioned targeted degradation protein in the preparation of drugs for treating heart failure.

[0011] The present invention also provides a medicament for treating heart failure, wherein the medicament contains the aforementioned targeted degradation protein.

[0012] Preferably, the drug is an injectable form.

[0013] Preferably, the drug also contains pharmaceutically acceptable excipients.

[0014] Compared with existing technologies, the present invention has the following significant advantages: Current mainstream strategies for intervening in extracellular protein function rely on neutralizing antibody blockade. However, compared to these neutralizing antibodies, the extracellular targeted protein degradation (eTPD) technology developed in this study exhibits several key differences: (1) Differences in mechanism of action: Neutralizing antibodies mainly block the activity of target proteins by binding to and blocking key functional domains (such as receptor binding sites). Some may also enter cells through Fc receptor-mediated endocytosis, but this process does not directly lead to a significant reduction in target proteins. In contrast, eTPD technology can actively mediate the endocytosis and degradation of target proteins, reducing the abundance of target proteins at the source, thereby more fundamentally weakening their biological effects.

[0015] (2) Differences in target binding requirements: The effectiveness of neutralizing antibodies typically depends on the precise recognition and binding of specific effector domains (i.e., functional epitopes) of the target protein. These epitopes are often difficult to design effective antibodies against, and are particularly challenging for target proteins with complex functions involving multiple receptor interactions. In contrast, eTPD technology only requires screening or developing binding molecules with high affinity for the target protein (without being limited to specific functional domains) to trigger its degradation. This significantly reduces the design difficulty and greatly expands the range of target proteins that can be intervened.

[0016] (3) Differences in tissue selectivity and side effect management: Many important target proteins are widely expressed in various tissues and perform complex physiological functions. Since neutralizing antibodies themselves lack tissue targeting, they mainly bind to and neutralize target proteins in the circulatory system. Their systemic effects often lead to unavoidable on-target, off-tissue effects. The innovation of this eTPD technology lies in its ingenious use of membrane proteins specifically highly expressed in myocardial tissue as effector molecules, which cleverly achieves the dual functions of targeting cardiac tissue and effectively mediating endocytosis, theoretically significantly reducing systemic side effects outside the tumor. Attached Figure Description

[0017] Figure 1 The affinity of different NPR1 antibodies for NPR1 was tested, and αnti-NPR1-1 had the smallest dissociation constant (human NPR1 Kd=0.259nM, mouse NPR1 Kd=0.346nM).

[0018] Figure 2To verify the specific binding of anti-NPR1-1 to NPR1 in AC16 and H9C2 cardiomyocytes endogenously expressing NPR1 and in 293T cells exogenously transfected with NPR1.

[0019] Figure 3 This study aimed to test the affinity of ANGPTL4 antibodies with different sequences for ANGPTL4 protein and to detect the degradation level of ANGPTL4 by CardioTAC with different sequences.

[0020] Figure 4 CardioTAC (anti-NPR1-1-ANGPTL4-2) effectively improves cardiopulmonary function in mice with preserved ejection fraction heart failure. In this diagram, A represents the mouse's gross and abdominal images, with "-" indicating 1 cm; B represents the animal's body weight; C represents the mouse's heart / tibia ratio (mg / mm); D represents the wet / dry weight ratio of lung tissue; E represents the distance traveled; F represents systolic blood pressure; BW represents body weight; HW represents heart weight; TL represents tibia length; LW represents lung weight; and HFpEF represents preserved ejection fraction heart failure. This means P < 0.05. This means P < 0.01.

[0021] Figure 5 To demonstrate the effectiveness of CardioTAC in improving cardiac function in mice with preserved ejection fraction heart failure, the following data is presented: A represents representative ultrasound images in M-Mode, PW-Mode, and Tissue-Mode; B represents left ventricular ejection fraction; C represents the short-axis shortening rate of the left ventricle; D represents the ratio of early diastolic blood flow velocity at the mitral valve tip to early diastolic myocardial motion velocity at the mitral annulus, reflecting diastolic function; LVEF, left ventricular ejection fraction; LVFS, left ventricular short-axis shortening rate; E, early diastolic blood flow velocity at the mitral valve tip measured by pulsed Doppler; E', early diastolic myocardial motion velocity at the mitral annulus; Chow, control group; HFpEF, preserved ejection fraction heart failure model group. This means P < 0.05. P represents P < 0.01, and ns represents no difference. Detailed Implementation

[0022] The present invention provides a targeted degradation protein, the amino acid sequence of which is shown in SEQ ID NO.3.

[0023] SEQ ID NO.3: QVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWNWVRQAPGKGLEWVSVIESKGNYIFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRYSMIYSYGAGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGFLGGVRGVDGEVQLQQSGPELMKPGASVKMSCRTSGYTFTDYSIHWVKQSHGKRLEWIGYINPYNGDTYCQNFKGKATLTFNKASSTAYMEIPRLTSDDSAVYYCTRWKTIQAPFAYWGQGTLVTVSAGGGGSGGGGSGGGGSEIQMTQSPSSMSASLGDRITITCQATQDIVKNLNWYQQKPGKPPSFLIHYATELAEGVPSRFSGSGSGSDYSLTISNLESEDFADYYCLQSYDFPYTFGGGTKLEIN。

[0024] The present invention also provides a nucleotide sequence encoding the protein for targeted degradation, and the nucleotide sequence is as shown in SEQ ID.NO.7.

[0025] SEQ ID.NO.7:

[0026] The present invention also provides the application of the aforementioned targeted degradation protein in the preparation of drugs for treating heart failure.

[0027] The present invention also provides a medicament for treating heart failure, wherein the medicament contains the aforementioned targeted degradation protein.

[0028] In this invention, the drug is an injectable preparation.

[0029] In this invention, the drug also contains pharmaceutically acceptable excipients.

[0030] 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.

[0031] Example 1

[0032] The objective of this invention is to develop a drug capable of directly degrading ANGPTL4 within cardiac tissue. Given that ANGPTL4 is a key molecule in cardiovascular disease, a genetic fusion of ANGPTL4 and an antibody targeting the cardiac tissue surface receptor NPR1 was created. To avoid degradation of this antibody in FcR-expressing cells (such as dendritic cells and B cells), nanobodies were used instead of intact antibodies containing the Fc group. Therefore, this invention designs a conjugate by linking the C-terminus of ANGPTL4 to the anti-NPR1 nanobody αNPR1, namely αNPR1-ANGPTL4, named CardioTAC.

[0033] The pSecTag2A vector, containing an IL-2 secretion signal peptide, was used in the experiment to guide the target protein extracellularly; simultaneously, the vector encodes a 6×His tag for the C-terminal, facilitating the passage of nickel ions (Ni... 2+ Rapid one-step purification was performed using chelate affinity chromatography. The αANGPTL4 and αNPR1-ANGPTL4 constructs were cloned into the pSecTag2A vector, transiently transferred into human embryonic kidney 293 (HEK293) cells, and the medium was changed to serum-free medium (DMEM high-glucose Gibco™, catalog number: 11965118) the next day. The supernatant was continuously collected and purified using a Ni-NTA agarose Resin column. Multiple sequences of NPR1 and ANGPTL4 were used for experimental validation, and the most effective sequence was ultimately selected for animal experiments.

[0034] The experimental materials and reagents used are as follows: Expression vectors: pSecTag2A-αANGPTL4 and pSecTag2A-αNPR1-ANGPTL4 Cell line: HEK293T cells (easy to transfect and culture at high density) Cell culture medium: Complete culture medium: DMEM high glucose (Gibco™, catalog number: 11965118) + 10% fetal bovine serum (FBS, Gibco™, catalog number: A5256701) + 1% penicillin-streptomycin antibiotics. Serum-free expression medium: DMEM high glucose (Gibco™, catalog number: 11965118) Transfection reagent: Polyethyleneimine (PEI, 1 mg / mL, pH 7.0) Purification material: Ni-NTA Agarose Resin Chromatography column (gravity column or syringe column) Equilibration / binding buffer: 20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 7.8-8.0 (filter sterilize before use) Washing buffer: 20 mM Tris-HCl, 300 mM NaCl, 30-50 mM imidazole, pH 7.8-8.0 Elution buffer: 20 mM Tris-HCl, 300 mM NaCl, 250-500 mM imidazole, pH 7.8-8.0 Detection reagents: SDS-PAGE gel, Western Blot related reagents, BCA protein quantification kit.

[0035] The specific experimental steps are as follows:

[0036] 1. Cell preparation and plasmid transfection

[0037] (1) Cell culture: HEK293 cells were cultured in a 37℃, 5% CO2 incubator until the logarithmic growth phase (confluence 80-90%).

[0038] (2) Cell plating: One day before transfection, cells were digested with trypsin at a concentration of 8 × 10⁻⁶ mcg. 5 Cells are seeded at a density of 1 / mL in cell culture plates / flasks. Cell confluence should reach 90-95% at transfection.

[0039] (3) Preparation of transfection complex (taking PEI transfection as an example, calculated per well of a 6-well plate): a) Tube A: Dissolve 3 μg of pSecTag2A-αANGPTL4 plasmid in 150 μL of Opti-MEM and mix gently.

[0040] b) Tube B: Dissolve PEI (plasmid mass: PEI mass = 1:3) in 150 μL of Opti-MEM and let stand at room temperature for 5 minutes.

[0041] c) Add the liquid from tube B dropwise to tube A, vortex immediately to mix, and let stand at room temperature for 20 minutes to form a DNA-PEI complex.

[0042] (4) Transfection: Add 300 μL of the transfection complex dropwise evenly to one well of a 6-well plate and gently shake to mix. Return to the incubator and continue culturing.

[0043] 2. Protein Expression and Supernatant Collection

[0044] (1) Change the medium: 7 hours after transfection, aspirate the medium containing the transfection complex.

[0045] (2) Serum-free induction: Slowly add preheated serum-free expression medium along the well wall (2 mL per well of a 6-well plate).

[0046] (3) Culture and collection: Continue to culture the cells for 48 hours. During this period, the target protein will continue to be secreted into the supernatant.

[0047] (4) Supernatant treatment: Carefully collect the cell culture supernatant with a pipette and transfer it to a centrifuge tube. Centrifuge at 4000×g for 20 minutes at 4°C to remove dead cells and debris. Transfer the supernatant after centrifugation to a new tube and store it on ice or at 4°C for short-term use or at -80°C for long-term storage.

[0048] 3. Ni-NTA affinity chromatography purification (all steps are performed in a 4°C cold room or on ice)

[0049] (1) Column preparation: Take an appropriate amount of Ni-NTA agarose resin (each milliliter of resin can bind 10 mg of His-tagged protein) and pack it into the chromatography column. Wash the column with 10 column volumes (CV) of equilibration buffer to equilibrate the resin.

[0050] (2) Sample loading and binding: Load the processed cell supernatant into the equilibrated Ni-NTA column at a rate of 0.5 mL / min. Collect the flow-through (FT) for analysis. The sample can be loaded twice to improve binding efficiency.

[0051] (3) Washing: Rinse with 10 CV equilibration buffer to remove unbound contaminating proteins. Then rinse with 10 CV washing buffer to remove weakly nonspecifically bound contaminating proteins. Collect the wash fraction.

[0052] (4) Elution: Elute with 3 CV of elution buffer. Gradient elution (e.g., using buffers containing 50 mM, 100 mM, 250 mM, and 500 mM imidazole) or stepwise elution can be performed. Collect the elution fraction (usually the 250 mM imidazole elution fraction contains the target protein), collecting 0.5 CV per tube.

[0053] 4. Protein Analysis and Preservation

[0054] (1) SDS-PAGE analysis: Take a small amount of flow-through buffer, washing buffer and elution buffer from each tube, perform SDS-PAGE electrophoresis (usually using 12% or 15% separating gel), stain with Coomassie brilliant blue, and analyze the purification effect.

[0055] (2) Western Blot verification: Western Blot was performed using anti-His tag or anti-ANGPTL4 antibody to confirm the specificity of the target protein.

[0056] (3) Desalting and concentration: Combine the eluent containing the target protein, use an ultrafiltration centrifuge tube (select an appropriate molecular weight cutoff according to the protein molecular weight) or a desalting column, replace it with a storage buffer (such as PBS or Tris-HCl buffer without imidazole), and concentrate it to the required concentration.

[0057] (4) Dispensing and storage: Determine the protein concentration, dispense in small quantities, and store at -80℃ to avoid repeated freeze-thaw cycles.

[0058] The complete amino acid and protein sequences of the construct are given below.

[0059] >NPR1-1 amino acid sequence (SEQ ID NO. 1)

[0060] QVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWNWVRQAPGKGLEWVSVIESKGNYIFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRYSMIYSYGAGAFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK .

[0061] Note: An asterisk at the end of a sequence indicates that it is a complete, predicted, or verified encoded sequence, signifying termination. The same applies below.

[0062] >NPR1-1-ANGPTL4-1 amino acid sequence (SEQ ID NO. 2)

[0063] QVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWNWVRQAPGKGLEWVSVIESKGNYIFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRYSMIYSYGAGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGFLGGVRGVDGEVKLVESGGGLVKPGGSLKLSCAASGFAFSRYDMSWVRQTPEKRLEWVATISTGGSYTYYPDSVKGRFTISRDNARNTLYLQMGSLRSEDTALYFCVRHEQSTVVPHYPLDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVSLSCRASQSIGDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSIDSVEPEDVGVYYCQNGHSFPFTFGSGTKLEIK 。

[0064] >Amino acid sequence of NPR1-1-ANGPTL4-2 (SEQ ID NO. 3)

[0065] QVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWNWVRQAPGKGLEWVSVIESKGNYIFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRYSMIYSYGAGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGFLGGVRGVDGEVQLQQSGPELMKPGASVKMSCRTSGYTFTDYSIHWVKQSHGKRLEWIGYINPYNGDTYCQNFKGKATLTFNKASSTAYMEIPRLTSDDSAVYYCTRWKTIQAPFAYWGQGTLVTVSAGGGGSGGGGSGGGGSEIQMTQSPSSMSASLGDRITITCQATQDIVKNLNWYQQKPGKPPSFLIHYATELAEGVPSRFSGSGSGSDYSLTISNLESEDFADYYCLQSYDFPYTFGGGTKLEIN 。

[0066] >Amino acid sequence of NPR1-1-ANGPTL4-3 (SEQ ID NO. 4)

[0067] QVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWNWVRQAPGKGLEWVSVIESKGNYIFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRYSMIYSYGAGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGFLGGVRGVDGQVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMDWVRQAPGKGLEWVSKISYDGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPSMSYYIIPAGFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIELTQPPSVSVSPGQTASITCSGDALGVKRASWYQQKPGQAPVLVIYKDNNRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCSVRTWSMNLYVFGGGTKLTVL 。

[0068] > Nucleotide sequence of NPR1-1 (SEQ ID NO. 5)

[0069]

[0070] >NPR1-1-ANGPTL4-1 nucleotide sequence (SEQ ID NO. 6)

[0071]

[0072] >NPR1-1-ANGPTL4-2 nucleotide sequence (SEQ ID NO. 7)

[0073]

[0074] >NPR1-1-ANGPTL4-3 nucleotide sequence (SEQ ID NO. 8)

[0075]

[0076] We designed three sequences targeting NPR1 and performed affinity tests on human and mouse NPR1. We found that αnti-NPR1-1 had the smallest dissociation constant (human NPR1 Kd = 0.259 nM, mouse NPR1 Kd = 0.346 nM), indicating that αnti-NPR1-1 has the strongest affinity for the NPR1 protein. Figure 1 In AC16 and H9C2 cardiomyocytes endogenously expressing NPR1, and in 293T cells exogenously transfected with NPR1, αnti-NPR1-1 was found to specifically bind to NPR1, further demonstrating the effectiveness of this sequence. Figure 2 Based on the above results, we designed a bispecific antibody for cardiac targeting (named CardioTAC), comprising an Fc segment lacking ADCC (antibody-dependent cell-mediated cytotoxicity) activity, a conjugate specifically recognizing NPR1 and disease-related targets, and a cathepsin-sensitive linker 1 specifically activated in lysosomes. The disease-related target selected in this study was ANGPTL4. We designed a sequence targeting ANGPTL4 for affinity and cellular-level ANGPTL4 degradation assays. We found that anti-ANGPTL4-2 had the strongest affinity, and the corresponding sequence of CardioTAC (anti-NPR1-1-ANGPTL4-2) showed good degradation efficiency. Figure 3 ).

[0077] To evaluate the targeting efficacy of CardioTAC in vivo, the inventors constructed a mouse model of heart failure with preserved ejection fraction. Six-week-old male C57BL / 6N mice were acclimatized to the animal facility environment for two weeks and then randomly assigned to three groups (control group, model group, and treatment group). They were fed a high-fat diet (HFD, high-fat diet D12492, fat calories 60 kcal%) and nitro-L-arginine methyl ester hydrochloride (L-NAME, added to drinking water at a ratio of 0.5 g / L) for seven weeks. The treatment group received drug intervention at four weeks of acclimatization, with intraperitoneal injections (10 mg / kg) every three days for three weeks. We found that three weeks of CardioTAC injection significantly improved mouse body weight, heart-to-tibia ratio, lung tissue wet-to-dry weight ratio, and movement distance, without affecting blood pressure. Figure 4 In mouse hyperotropic assays, CardioTAC significantly improved diastolic function (E / E') without affecting diastolic function (LVEF and LVFS). Figure 5 In conclusion, CardioTAC has promising prospects for clinical application.

[0078] As can be seen from the above embodiments, the present invention provides a targeted degradation protein and its application, the amino acid sequence of which is shown in SEQ ID NO.3. The targeted degradation protein of the present invention is based on extracellular protein targeted degradation technology, and can actively mediate the endocytosis and degradation process of target proteins, reducing the abundance of target proteins at the source, thereby more fundamentally weakening their biological effects. Degradation can be triggered simply by screening or developing binding molecules with high affinity for the target protein. This significantly reduces the design difficulty and greatly expands the range of target proteins that can be intervened.

[0079] 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 targeted degradation protein, characterized in that, The amino acid sequence of the targeted degradation protein is shown in SEQ ID NO.

3.

2. A nucleotide sequence encoding the targeted degradation protein of claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID.NO.

7.

3. The use of the targeted degradation protein according to claim 1 in the preparation of a drug for treating heart failure.

4. A method for preparing a drug for treating heart failure, characterized in that, The drug contains the targeted degradation protein as described in claim 1.

5. The drug according to claim 4, characterized in that, The drug is an injectable form.

6. The drug according to claim 4, characterized in that, The drug also contains pharmaceutically acceptable excipients.

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