Methods of targeting protein degradation

Abstract

The invention relates to the technical field of biology, and provides a targeted protein degradation method. The method comprises the following steps: carrying out first incubation treatment on a biological sample and an enzyme-labeled antibody to obtain a first incubation product; and carrying out second incubation treatment on the first incubation product and a labeled substrate to realize degradation of the target protein in the biological sample. Therefore, compared with the traditional antibody-based degradation agent, the targeted protein degradation method disclosed by the invention can directly use the commercially available antibody, and does not need complex antibody engineering modification or chemical modification. By labeling adjacent to the target protein, efficient degradation can be realized, and the method is applicable to both intracellular protein and extracellular protein (including membrane protein and secretory protein). The method not only simplifies the experimental process, reduces the cost, but also improves the flexibility and universality of operation, and has application potential and value in the fields of biological coupling and biological medicine.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to a polymer proximity labeling method for targeted protein degradation. Background Technology

[0002] Targeted protein degradation (TPD) is an emerging strategy for treating diseases by inducing the degradation of disease-related proteins. Unlike traditional small molecule inhibitors (which only block protein function), TPD technology utilizes the cell's own degradation systems (such as the ubiquitin-proteasome system or lysosomal pathway) to directly remove target proteins, achieving therapeutic goals. Currently, the main TPD technologies (Nat. Rev. Drug Disc. 2022, 21, 181-200) include molecular glues and heterobifunctional degraders, represented by protein degradation-targeted chimeras (PROTACs).

[0003] Molecular gel design relies on serendipitous discovery, as it requires simultaneous binding of E3 ligases and target proteins and induction of interactions, making it more complex than traditional inhibitor design and lacking a systematic approach. PROTACs are complex to synthesize, have large molecular weights, poor pharmacokinetics, and both techniques mostly only degrade intracellular proteins, failing to cover extracellular proteins. To overcome these limitations, antibody-based degradative agents have attracted attention and become a research hotspot in the TPD field. Antibody-based degradative agents promote the internalization of antibody-target protein complexes through engineered modification or ligand conjugation, leading to lysosomal degradation of the target protein. However, bispecific antibodies require complex engineered design and optimization, increasing development time and cost. Even simple ligand conjugation involves multi-step synthesis, relies on specific reagents, and may lead to increased costs, time and effort, affect antibody activity, and potentially be occupied by natural ligands or trigger off-target effects. Furthermore, when antibody-based degradative agents are applied to new target proteins, they need to be individually designed for different diseases and cell types.

[0004] Therefore, developing a universal strategy to degrade extracellular proteins without engineering or modifying antibodies is a pressing technical problem that needs to be solved. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems existing in the prior art. To this end, the inventors have developed a novel method for targeted protein degradation through extensive experiments. This method can directly utilize commercially available antibodies, enzyme-labeled antibodies, aniline, and other labeled substrates, without the need to synthesize complex heterobifunctional degradative agents or antibody / aptamer-type degradative agents. Furthermore, it can label and degrade intracellular or extracellular proteins as needed, and has advantages such as simple operation, high flexibility, and strong versatility.

[0006] Therefore, in a first aspect, the present invention proposes a method for targeted protein degradation. According to an embodiment of the present invention, the method includes: incubating a biological sample with an enzyme-labeled antibody to obtain a first incubation product; and incubating the first incubation product with a labeled substrate to achieve degradation of the target protein in the biological sample. Thus, compared with traditional antibody-based degradative agents, the targeted protein degradation method of the present invention can directly use commercially available antibodies without complex antibody engineering or chemical modification. Highly efficient degradation can be achieved by labeling near the target protein, applicable to both intracellular and extracellular proteins (including membrane and secretory proteins). This not only simplifies the experimental procedure and reduces costs but also improves operational flexibility and versatility, possessing application potential and value in the fields of bioconjugation and biomedicine.

[0007] According to embodiments of the present invention, the enzyme-labeled antibody includes at least one of horseradish peroxidase-labeled antibody, ascorbate peroxidase-labeled antibody, tyrosinase-labeled antibody, and photosensitizing protein-labeled antibody.

[0008] According to embodiments of the present invention, the labeled substrate includes at least one of an easily oxidizable monomer, an acrylic monomer, and acetylacetone.

[0009] According to embodiments of the present invention, the easily oxidizable monomer includes at least one of aniline, naphthylamine, dopamine, pyrrole, and N-phenyl-p-phenylenediamine.

[0010] According to embodiments of the present invention, the acrylic monomer includes at least one of acrylamide and N-isopropylacrylamide.

[0011] According to an embodiment of the present invention, the biological sample comprises cells or tissues expressing the target protein.

[0012] According to embodiments of the present invention, the target protein is an intracellular protein or an extracellular protein.

[0013] According to an embodiment of the present invention, the ratio of the biological sample to the enzyme-labeled antibody is (10) 5 -10 6 ) cells: (0.01-5) μg / mL.

[0014] According to an embodiment of the present invention, the incubation time for the first incubation treatment is 0.5h-2h.

[0015] According to an embodiment of the present invention, in the second incubation treatment, the concentration of the labeled substrate is 0.01 μM-100 μM.

[0016] According to an embodiment of the present invention, the second incubation treatment time is 6h-48h.

[0017] According to an embodiment of the present invention, the first incubation treatment of the biological sample with the enzyme-labeled antibody further includes: a third incubation treatment of the biological sample with the first antibody to obtain a third incubation product; and a fourth incubation treatment of the third incubation product with a second antibody to obtain the first incubation product.

[0018] According to an embodiment of the present invention, the first antibody comprises an unlabeled specific antibody capable of binding to a target protein in the biological sample.

[0019] According to an embodiment of the present invention, the second antibody comprises at least one of horseradish peroxidase-labeled secondary antibody, ascorbate peroxidase-labeled secondary antibody, tyrosinase-labeled secondary antibody, and photosensitizing protein-labeled secondary antibody, and the second antibody is capable of binding to the first antibody.

[0020] According to an embodiment of the present invention, the ratio of the biological sample to the first antibody is (10) 5 -10 6 ) cells: (0.01-5) μg / mL.

[0021] According to an embodiment of the present invention, in the fourth incubation treatment, the concentration of the second antibody is 1-5 times that of the first antibody.

[0022] According to an embodiment of the present invention, the third incubation treatment time is 1h-2h.

[0023] According to an embodiment of the present invention, the fourth incubation treatment time is 1h-2h.

[0024] In a second aspect, the present invention provides a kit. According to embodiments of the present invention, the kit comprises at least one of a first antibody, a second antibody, an enzyme-labeled antibody, and a labeled substrate. Thus, the kit of the present invention allows for the degradation of target proteins through a simple experimental procedure, greatly simplifying experimental steps, reducing variability in experimental operations, and improving the repeatability and reliability of the experiment.

[0025] According to an embodiment of the present invention, the kit includes the first antibody, the second antibody, and the labeled substrate.

[0026] According to an embodiment of the present invention, the kit includes the enzyme-labeled antibody and the labeled substrate.

[0027] According to an embodiment of the present invention, the first antibody comprises an unlabeled specific antibody capable of binding to a target protein in a biological sample.

[0028] According to an embodiment of the present invention, the second antibody comprises at least one of horseradish peroxidase-labeled secondary antibody, ascorbate peroxidase-labeled secondary antibody, tyrosinase-labeled secondary antibody, and photosensitizing protein-labeled secondary antibody, and the second antibody is capable of binding to the first antibody.

[0029] According to embodiments of the present invention, the enzyme-labeled antibody includes at least one of horseradish peroxidase-labeled antibody, ascorbate peroxidase-labeled antibody, tyrosinase-labeled antibody, and photosensitized protein-labeled antibody, wherein the enzyme-labeled antibody is capable of binding to a target protein in a biological sample.

[0030] According to an embodiment of the present invention, the labeled substrate includes at least one of an easily oxidizable monomer and an acrylic monomer.

[0031] According to embodiments of the present invention, the easily oxidizable monomer includes at least one of aniline, naphthylamine, dopamine, pyrrole, and N-phenyl-p-phenylenediamine.

[0032] According to embodiments of the present invention, the acrylic monomer includes at least one of acrylamide and N-isopropylacrylamide.

[0033] In a third aspect, the present invention provides a combination drug. According to embodiments of the present invention, the combination drug comprises at least one of a first antibody, an enzyme-labeled antibody, and a labeled substrate. Thus, the combination drug of the present invention can achieve the degradation of targeted proteins, particularly beneficial for the degradation of cancer cells. By selecting different antibodies, the antibodies can bind to proteins of different cancer cells, thereby promoting the degradation of different cancer cells. According to embodiments of the present invention, the combination drug of the present invention can achieve the degradation of targeted proteins through injection or other methods, offering advantages such as ease of operation, high accuracy, and efficiency.

[0034] According to an embodiment of the present invention, the combination drug comprises the first antibody, the second antibody, and the labeled substrate.

[0035] According to an embodiment of the present invention, the combination drug includes the enzyme-labeled antibody and the labeled substrate.

[0036] According to an embodiment of the present invention, the first antibody comprises an unlabeled specific antibody capable of binding to a target protein in a biological sample.

[0037] According to an embodiment of the present invention, the second antibody comprises at least one of horseradish peroxidase-labeled secondary antibody, ascorbate peroxidase-labeled secondary antibody, tyrosinase-labeled secondary antibody, and photosensitizing protein-labeled secondary antibody, and the second antibody is capable of binding to the first antibody.

[0038] According to embodiments of the present invention, the enzyme-labeled antibody includes at least one of horseradish peroxidase-labeled antibody, ascorbate peroxidase-labeled antibody, tyrosinase-labeled antibody, and photosensitized protein-labeled antibody, wherein the enzyme-labeled antibody is capable of binding to a target protein in a biological sample.

[0039] According to an embodiment of the present invention, the labeled substrate includes at least one of an easily oxidizable monomer and an acrylic monomer.

[0040] According to embodiments of the present invention, the easily oxidizable monomer includes at least one of aniline, naphthylamine, dopamine, pyrrole, and N-phenyl-p-phenylenediamine.

[0041] According to embodiments of the present invention, the acrylic monomer includes at least one of acrylamide and N-isopropylacrylamide.

[0042] In a fourth aspect of the invention, the invention proposes the use of the kit described in the second aspect or the combination drug described in the third aspect in in vitro targeted protein degradation.

[0043] In a fifth aspect of the invention, the invention provides for the use of the combined medicament described in the third aspect in the preparation of a medicament for treating diseases associated with overdose of a target protein.

[0044] According to an embodiment of the present invention, the disease includes cancer.

[0045] In a sixth aspect, the present invention provides a method for reducing the content of a target protein in cells. According to embodiments of the invention, the method includes targeting the cells with the protein according to the method described in the first aspect, thereby reducing the content of the target protein in the cells; and the method is a non-diagnostic and non-therapeutic in vitro method. As described above, the targeted protein degradation method of the present invention enables the degradation of the target protein in cells, thereby reducing the content of the target protein in the cells.

[0046] The beneficial effects of this invention are: (1) Compared with traditional methods of modifying antibodies through chemical conjugation or genetic engineering, this method uses commercially available antibodies, eliminating the need for complex and cumbersome design and reducing costs. Furthermore, this method can label antibody-target protein in situ immediately, without relying on cell receptors, making it more versatile.

[0047] (2) The method for targeted protein degradation of the present invention does not require pretreatment or premodification of the antibody, thereby maximizing the antibody’s ability to recognize the antigen.

[0048] (3) The targeted protein degradation method of the present invention is more comprehensive than PROTACs that can only degrade intracellular proteins and antibody / aptamer degradative agents that can degrade extracellular proteins. It can use commercially available antibodies to degrade extracellular proteins directly. At the same time, as needed, genetic engineering technology can be used to express relevant fusion proteins in cells to mark the target protein with adjacent degradation tags, thereby achieving the degradation of intracellular proteins, which is more universal.

[0049] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0050] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a schematic diagram illustrating the principle of using a proximity-labeled polymer for targeted protein degradation according to an embodiment of the present invention; Figure 2 This is a Western blot (WB) image of the degradation of the secretory protein Serpine1 using N-phenyl-p-phenylenediamine substrate according to Example 1 of the present invention. Figure 3 This is a Western blot (WB) diagram of the degradation of the membrane protein EGFR using N-phenyl-p-phenylenediamine substrate according to Example 2 of the present invention; Figure 4 This is a comparison of Western blot (WB) images of the degradation of the membrane protein EGFR using different substrates according to Example 3 of the present invention; Figure 5 This is a graph showing the in vivo antitumor results of the antibody and N-phenyl-p-phenylenediamine according to Example 4 of the present invention. Detailed Implementation

[0051] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0052] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0053] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0054] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0055] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0056] This invention proposes methods, kits, combination drugs, and applications for targeting protein degradation, which will be described in detail below.

[0057] Methods for targeting protein degradation In a first aspect, the present invention provides a method for targeted protein degradation. According to an embodiment of the present invention, the method includes: subjecting a biological sample to a first incubation treatment with an enzyme-labeled antibody to obtain a first incubation product; and subjecting the first incubation product to a second incubation treatment with a labeled substrate to achieve degradation of the target protein in the biological sample.

[0058] In this invention, the inventors developed a novel method for targeted protein degradation through extensive experimentation. Given the cytotoxicity of hydrogen peroxide, this invention attempts to achieve protein degradation solely through the cell's own hydrogen peroxide and / or reactive oxygen species, without the addition of additional hydrogen peroxide. Figure 1As shown, after incubating biological samples with enzyme-labeled antibodies, the antibodies bind to the target extracellular protein. Subsequent incubation with a labeled substrate allows for the absorption of endogenous hydrogen peroxide produced by the cells. This peroxidase-catalyzed polymerization and labeling of the labeled substrate, generated by the reduced coenzyme II (NADPH) in the extracellular space, through diffusion via water channels, drives the labeling process. This results in in-situ labeling of the protein near the enzyme without affecting cellular physiological activity. The labeled antibody-target protein complex is internalized and then transferred to lysosomes for degradation. Therefore, this method can directly use commercially available antibodies without complex antibody engineering or chemical modification. Highly efficient degradation can be achieved by labeling near the target protein, applicable to both intracellular and extracellular proteins (including membrane and secretory proteins). This simplifies the experimental procedure, reduces costs, and enhances operational flexibility and versatility, demonstrating significant application potential and value in bioconjugation and biomedicine.

[0059] The invention's requirement of not requiring engineered or chemically modified antibodies specifically refers to designing antibodies against the target substance as bispecific antibodies without using gene recombination and expression technologies, or using conjugation methods such as click chemistry or EDC-NHS to conjugate cell surface receptor-related ligands (such as folic acid, glycans) or polymerized macromolecules (such as polyethyleneimine) or nanoparticles (such as micelles, liposomes, gold nanoparticles, etc.) to antibodies, and using only commercially available enzyme-labeled antibodies for target protein degradation.

[0060] In this article, the term "enzyme-labeled antibody" refers to an enzyme-labeled antibody, which is formed by linking an enzyme with a specific antibody using an appropriate method.

[0061] The targeted protein degradation method disclosed in this invention has clear and important application value in the field of scientific research. In particular, it can be efficiently applied to in vitro research scenarios, such as in cell culture systems, cell-free biochemical reaction systems and other in vitro experimental models, to achieve precise degradation regulation of specific target proteins, providing key technical support for studying the physiological functions, molecular interaction mechanisms and related signaling pathways of target proteins.

[0062] In some embodiments of the present invention, the enzyme-labeled antibody includes at least one of horseradish peroxidase-labeled antibody, ascorbate peroxidase-labeled antibody, tyrosinase-labeled antibody, and photosensitizing protein-labeled antibody. Thus, the above-mentioned types of enzyme-labeled antibodies can not only specifically recognize and bind to the target protein, but also efficiently catalyze the polymerization reaction of the labeled substrate, achieving in-situ labeling and cell internalization, ultimately promoting the degradation of the target protein. In some embodiments of the present invention, the type of antibody in the enzyme-labeled antibody can be selected by those skilled in the art according to specific experimental needs. For example, the antibody in the enzyme-labeled antibody is a primary antibody, specifically it can be a plasminogen activator inhibitor-1 (Serpine1), an anti-epidermal growth factor receptor (EGFR) primary antibody, a programmed cell death ligand 1 (PD-L1) antibody, or a c-Met antibody. Thus, the enzyme-labeled antibody can specifically recognize and bind to the target protein. This specific binding is achieved through the antigen-binding site of the antibody, ensuring the precision of the labeling and degradation process.

[0063] It should be noted that any antibody used in this invention is a naturally occurring antibody, an antibody containing its antigen-binding fragment, or an extended genetically engineered antibody (i.e., an antigen-binding fragment largely identical to that of a naturally occurring antibody, thus maintaining the same antigen-binding specificity). In some embodiments of this invention, the antibodies are commercially available. Each antibody should meet the requirements of relevant industry standards.

[0064] In some embodiments of the present invention, the labeled substrate can be catalyzed and oxidized by an enzyme in the enzyme-labeled antibody, undergoing a polymerization reaction, and labeled onto a protein adjacent to the enzyme. Exemplarily, the labeled antibody includes, but is not limited to, at least one of easily oxidizable monomers and acrylic monomers. In some embodiments of the present invention, the easily oxidizable monomer includes at least one of aniline, naphthylamine, dopamine, pyrrole, and N-phenyl-p-phenylenediamine. In some embodiments of the present invention, the acrylic monomer includes at least one of acrylamide and N-isopropylacrylamide. Thus, the above-mentioned types of labeled substrates, after polymerization, are labeled onto the antibody-target protein complex, promoting internalization within cells.

[0065] In some embodiments of the present invention, the biological sample comprises cells or tissues expressing the target protein. In some embodiments of the present invention, the target protein is an intracellular protein or an extracellular protein.

[0066] In some embodiments of the present invention, the ratio of the biological sample to the enzyme-labeled antibody is (10) 5 -10 6 ) cells: (0.01-5) μg / mL. For example, it can be 105 Cells: 0.01 μg / mL, 10 5 Cells: 1 μg / mL, 10 5.2 Cells: 2 μg / mL, 10 5.5 Cells: 3 μg / mL, 10 5.8 Cells: 4 μg / mL, 10 6 The concentration per cell is 5 μg / mL, or any range of the above values. Therefore, the antibody can effectively bind to the target protein and incubate with an appropriate amount of enzyme to initiate the catalytic reaction, avoiding the waste caused by too few antibodies failing to target most sites and too many antibodies.

[0067] In some embodiments of the present invention, the first incubation treatment time is 0.5h-2h. For example, it can be 0.5h, 1h, 1.5h, 2h, etc., or a range of any of the above values. Thus, by keeping the first incubation treatment time within the above range, the effects of too short a time (until the antibody has not bound) and too long a time (causing increased background and cell damage) are avoided.

[0068] In some embodiments of the present invention, the concentration of the labeled substrate in the second incubation treatment is 0.01 μM-100 μM. For example, it can be 0.01 μM, 1 μM, 10 μM, 30 μM, 50 μM, 80 μM, 100 μM, etc., or any range of the above values. In some embodiments of the present invention, the time of the second incubation treatment is 6 h-48 h. For example, it can be 6 h, 10 h, 20 h, 30 h, 40 h, 48 h, etc., or any range of the above values. Therefore, the labeled substrate can sufficiently diffuse to the vicinity of the target protein, without producing significant cytotoxicity within the defined concentration and time range, and achieving a good labeling effect.

[0069] In some embodiments of the present invention, the incubation of the biological sample with the enzyme-labeled antibody can be performed using a one-step incubation method, where the biological sample is directly incubated with the enzyme-labeled antibody for a first incubation treatment, or a two-step incubation method, where the biological sample is first incubated with a first antibody for a third incubation treatment, and then incubated with a second antibody for a fourth incubation treatment. In the two-step incubation method, the first antibody specifically binds to the target protein in the biological sample, and the subsequent second antibody specifically binds to the first antibody, thereby introducing the enzyme label into the vicinity of the target protein. One first antibody molecule can bind to one target protein, but each first antibody molecule can bind multiple second antibody molecules; this binding method can significantly amplify the signal. Therefore, it can be ensured that sufficient second antibodies bind to the target protein, thereby improving the polymerization efficiency of the labeled substrate and achieving more efficient labeling and degradation.

[0070] In some embodiments of the present invention, the first antibody comprises an unlabeled specific antibody capable of binding to a target protein in the biological sample. It should be noted that those skilled in the art can specifically select the desired first antibody based on the target protein to be bound. For example, the first antibody may be a plasminogen activator inhibitor-1 (Serpine1), an anti-epidermal growth factor receptor (EGFR) primary antibody, a programmed cell death ligand 1 (PD-L1) antibody, a c-Met antibody, etc.

[0071] In some embodiments of the present invention, the second antibody comprises at least one of horseradish peroxidase-labeled secondary antibody, ascorbate peroxidase-labeled secondary antibody, tyrosinase-labeled secondary antibody, and photosensitizer-labeled secondary antibody, wherein the second antibody is capable of binding to the first antibody. It should be noted that those skilled in the art can select the desired secondary antibody based on the type of the selected first antibody.

[0072] In some embodiments of the present invention, the ratio of the biological sample to the first antibody is (10) 5 -10 6 ) cells: (0.01-5) μg / mL. For example, it can be 10 5 Cells: 0.01 μg / mL, 10 5 Cells: 1 μg / mL, 10 5.2 Cells: 2 μg / mL, 10 5.5 Cells: 3 μg / mL, 10 5.8 Cells: 4 μg / mL, 10 6 The concentration of the second antibody is 5 μg / mL per cell, or any range of the above values. In some embodiments of the present invention, in the fourth incubation treatment, the concentration of the second antibody is 1-5 times that of the first antibody. For example, it can be 1, 2, 3, 4, or 5 times that of the first antibody. Therefore, the antibody can effectively bind to the target protein and can incubate with an appropriate amount of enzyme to initiate the catalytic reaction, avoiding the waste caused by too little antibody failing to target most sites and too much antibody.

[0073] In some embodiments of the present invention, the third incubation treatment time is 1-2 hours. For example, it can be 1 hour, 1.2 hours, 1.5 hours, 1.8 hours, 2 hours, etc. According to embodiments of the present invention, the temperature of the third incubation treatment is 37°C. This allows the first antibody to sufficiently bind to the target protein in the biological sample.

[0074] In some embodiments of the present invention, the fourth incubation treatment time is 1-2 hours. For example, it can be 1 hour, 1.2 hours, 1.5 hours, 1.8 hours, 2 hours, etc. According to an embodiment of the present invention, the temperature of the fourth incubation treatment is 37°C. This allows the second antibody to fully bind to the first antibody, resulting in signal amplification and thus achieving the degradation of the target protein.

[0075] In some embodiments of the present invention, the targeted protein degradation method of the present invention is not for diagnostic or therapeutic purposes, but can be used for basic biological research, disease mechanism research, drug development, etc. Specifically, by selectively degrading specific proteins, the function of these proteins in cells can be studied, including their roles in the cell cycle, signal transduction, and metabolic processes; the degradation mechanisms of disease-related proteins (such as oncoproteins, mutant proteins, etc.) can be studied to explore their roles in disease occurrence and development. For example, specific oncoproteins in cancer cells can be degraded to study their effects on cell proliferation, apoptosis, and migration; this method can also be used to screen small molecule compounds that can induce the degradation of specific proteins, accelerating the drug discovery process. For example, high-throughput screening can be used to discover compounds that can enhance enzymatic reactions or promote protein degradation.

[0076] reagent kits and combination drugs In a second aspect, the present invention provides a kit. According to embodiments of the invention, the kit comprises at least one of a first antibody, a second antibody, an enzyme-labeled antibody, and a labeled substrate. Thus, the kit of the present invention allows for the degradation of target proteins through a simple experimental procedure, greatly simplifying experimental steps, reducing variability in experimental operations, and improving the repeatability and reliability of the experiment.

[0077] In some embodiments of the present invention, the kit includes the first antibody, the second antibody, and the labeled substrate. In some embodiments of the present invention, the kit includes the enzyme-labeled antibody and the labeled substrate. Therefore, the kit of the present invention allows for the degradation of target proteins through a simple experimental procedure, greatly simplifying experimental steps, reducing variability in experimental operations, and improving the repeatability and reliability of the experiment.

[0078] In some embodiments of the present invention, the first antibody comprises an unlabeled specific antibody capable of binding to a target protein in a biological sample.

[0079] In some embodiments of the present invention, the second antibody includes at least one of horseradish peroxidase-labeled secondary antibody, ascorbate peroxidase-labeled secondary antibody, tyrosinase-labeled secondary antibody, and photosensitizing protein-labeled secondary antibody, and the second antibody is capable of binding to the first antibody.

[0080] In some embodiments of the present invention, the enzyme-labeled antibody includes at least one of horseradish peroxidase-labeled antibody, ascorbate peroxidase-labeled antibody, tyrosinase-labeled antibody, and photosensitized protein-labeled antibody, wherein the enzyme-labeled antibody is capable of binding to a target protein in a biological sample.

[0081] In some embodiments of the present invention, the labeled substrate includes at least one of an easily oxidizable monomer and an acrylic monomer. In some embodiments of the present invention, the easily oxidizable monomer includes at least one of aniline, naphthylamine, dopamine, pyrrole, and N-phenyl-p-phenylenediamine. In some embodiments of the present invention, the acrylic monomer includes at least one of acrylamide and N-isopropylacrylamide.

[0082] In a third aspect, the present invention provides a combination drug. According to embodiments of the present invention, the combination drug includes at least one of a first antibody, a second antibody, an enzyme-labeled antibody, and a labeled substrate. Thus, the combination drug of the present invention can achieve the degradation of targeted proteins, particularly beneficial for the degradation of cancer cells. By selecting different antibodies, the antibodies can bind to proteins of different cancer cells, thereby promoting the degradation of different cancer cells. According to embodiments of the present invention, the combination drug of the present invention can achieve the degradation of targeted proteins through injection or other methods, offering advantages such as ease of operation, high accuracy, and efficiency.

[0083] In some embodiments of the present invention, the combined drug comprises the first antibody, the second antibody, and the labeled substrate. In some embodiments of the present invention, the combined drug comprises the enzyme-labeled antibody and the labeled substrate. Therefore, the combined drug of the present invention can achieve the degradation of targeted proteins, especially beneficial for the degradation of cancer cells. By selecting different antibodies, the antibodies can bind to proteins of different cancer cells, thereby promoting the degradation of different cancer cells. According to embodiments of the present invention, the combined drug of the present invention can achieve the degradation of targeted proteins through injection or other methods, and has the advantages of simple operation, high accuracy, and high efficiency.

[0084] In some embodiments of the present invention, the first antibody comprises an unlabeled specific antibody capable of binding to a target protein in a biological sample.

[0085] In some embodiments of the present invention, the second antibody includes at least one of horseradish peroxidase-labeled secondary antibody, ascorbate peroxidase-labeled secondary antibody, tyrosinase-labeled secondary antibody, and photosensitizing protein-labeled secondary antibody, and the second antibody is capable of binding to the first antibody.

[0086] In some embodiments of the present invention, the enzyme-labeled antibody includes at least one of horseradish peroxidase-labeled antibody, ascorbate peroxidase-labeled antibody, tyrosinase-labeled antibody, and photosensitive protein-labeled antibody, wherein the enzyme-labeled antibody is capable of binding to the first antibody.

[0087] In some embodiments of the present invention, the labeled substrate includes at least one of an easily oxidizable monomer and an acrylic monomer. In some embodiments of the present invention, the easily oxidizable monomer includes at least one of aniline, naphthylamine, dopamine, pyrrole, and N-phenyl-p-phenylenediamine. In some embodiments of the present invention, the acrylic monomer includes at least one of acrylamide and N-isopropylacrylamide.

[0088] Uses and methods In a fourth aspect of the invention, the invention proposes the use of the kit described in the second aspect or the combination drug described in the third aspect in in vitro targeted protein degradation.

[0089] In a fifth aspect of the invention, the invention provides for the use of the combined medicament described in the third aspect in the preparation of a medicament for treating diseases associated with overdose of a target protein.

[0090] In some embodiments of the present invention, the disease includes cancer. The disease or cancer associated with the overdose of the target protein is related to the targeted protein. For example, when the target protein is plasminogen activator inhibitor-1 (Serpine1), the associated disease or cancer includes at least one of gastric cancer and colon cancer. When the target protein is human epidermal growth factor receptor (EGFR), the associated disease or cancer includes at least one of pancreatic cancer, nasopharyngeal carcinoma, and head and neck tumors. When the target protein is programmed cell death-ligand 1 (PD-L1), the associated disease or cancer includes at least one of non-small cell lung cancer, melanoma, gastric cancer, colon cancer, breast cancer, pancreatic cancer, and kidney cancer. When the target protein is cell-metabolite transition factor (c-Met), the associated disease or cancer includes at least one of colorectal cancer, non-small cell lung cancer, gastric cancer, and breast cancer.

[0091] In a fifth aspect, the present invention provides a method for reducing the content of a target protein in cells. According to embodiments of the invention, the method includes targeting the cells with the protein according to the method described in the first aspect, thereby reducing the content of the target protein in the cells; and the method is a non-diagnostic and non-therapeutic in vitro method. As described above, the targeted protein degradation method of the present invention enables the degradation of the target protein in cells, thereby reducing the content of the target protein in the cells.

[0092] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0093] Example 1: Degradation of secretory protein Serpine1 using N-phenyl-p-phenylenediamine substrate In this embodiment, colorectal cancer RKO cells were divided into a control group and an experimental group, as follows: Control group: Cells were not treated with any antibodies or substrates and served as a blank control for the experiment; Experimental group: First, incubate with anti-Serpine1 primary antibody (Ab1), then incubate with horseradish peroxidase-labeled secondary antibody (HRP-Ab2), and finally incubate with N-phenyl-p-phenylenediamine substrate.

[0094] The specific process is as follows: 1) There are 2×10 seeds. 5 Add 0.1 μg / mL of anti-Serpine1 primary antibody to a six-well plate of colorectal cancer RKO cells, incubate at room temperature for 1 h, and then wash three times with 1×PBS.

[0095] 2) Add 0.4 μg / mL of HRP-labeled secondary antibody, incubate at room temperature for one hour, and then wash three times with 1×PBS.

[0096] 3) Add 2 μM N-phenyl-p-phenylenediamine and incubate for 6 h.

[0097] 4) After the reaction, collect the supernatant directly and determine the protein concentration using a BCA protein analysis kit. Determine the loading amount based on the protein concentration, then separate the protein using gel electrophoresis (10% separating gel), transfer it to a PVDF membrane, and stain with the corresponding antibody.

[0098] Experimental results are as follows Figure 2 As shown, compared with the blank control group (100% of the total Serpine1 protein), the experimental group achieved effective Serpine1 degradation with a degradation efficiency of 69%.

[0099] Example 2: Degradation of membrane protein EGFR using N-phenyl-p-phenylenediamine substrate In this embodiment, colorectal cancer RKO cells were divided into a control group and an experimental group. The experimental group was further subdivided into three subgroups, each undergoing different treatment steps. The specific groupings are as follows: Control group: Cells were not treated with any antibodies or substrates and served as a blank control for the experiment; Group Ab: Incubation was performed using only anti-EGFR primary antibody + horseradish peroxidase-labeled secondary antibody (Ab1 + HRP-Ab2); PD group: Incubation was performed using only N-phenyl-p-phenylenediamine substrate; Ab + PD group: First, incubate with anti-EGFR primary antibody (Ab1), then incubate with horseradish peroxidase-labeled secondary antibody (HRP-Ab2), and finally incubate with N-phenyl-p-phenylenediamine substrate; The specific process is as follows: 1) There are 2×10 seeds. 5 Add 0.1 μg / mL of anti-EGFR primary antibody to a six-well plate of colorectal cancer RKO cells, incubate at room temperature for 1 h, and then wash three times with 1×PBS.

[0100] 2) Add 0.4 μg / mL of HRP-labeled secondary antibody, incubate at room temperature for one hour, and then wash three times with 1×PBS.

[0101] 3) Add 2 μM aniline and incubate for 6 h.

[0102] 4) After the reaction, the cells were washed three times with cold 1×PBS, then lysed with cell lysis buffer. After centrifugation at 16000g for 5 min, the lysate in the supernatant was collected, and the protein concentration was determined using a BCA protein assay kit. The loading amount was determined based on the protein concentration, and the cells were then separated by gel electrophoresis (10% separating gel). The cells were then transferred to a PVDF membrane and stained with the corresponding antibody immunoblotting.

[0103] Experimental results are as follows Figure 3 As shown, compared with the blank control group (100% of the total EGFR protein), only the complete experimental group (Ab+ PD group) achieved effective EGFR degradation, with a degradation efficiency of 39%.

[0104] Example 3: Comparison of the degradation of the membrane protein EGFR by different substrates 1) There are 2×10 seeds. 5 Add 0.1 μg / mL of anti-EGFR primary antibody to a six-well plate of colorectal cancer RKO cells, incubate at room temperature for 1 h, and then wash three times with 1×PBS.

[0105] 2) Add 0.4 μg / mL of HRP-labeled secondary antibody, incubate at room temperature for one hour, and then wash three times with 1×PBS.

[0106] 3) Add 50 μM naphthylamine substrate, 50 μM pyrrole substrate, or 1, 10, 50 μM aniline substrate, or 1, 10, 50 μM N-phenyl-p-phenylenediamine (PD) substrate respectively, and incubate for 6 h.

[0107] 4) After the reaction, the cells were washed three times with cold 1×PBS, then lysed with cell lysis buffer. After centrifugation at 16000g for 5 min, the lysate in the supernatant was collected, and the protein concentration was determined using a BCA protein assay kit. The loading amount was determined based on the protein concentration, and the cells were then separated by gel electrophoresis (10% separating gel). The cells were transferred to a PVDF membrane and stained with the appropriate antibody for immunoblotting.

[0108] Experimental results are as follows Figure 4 As shown, all substrates can significantly degrade the target protein. Among them, aniline (lanes 4-6) and N-phenyl-p-phenylenediamine (PD, lanes 7-9) can be degraded in a dose-dependent manner. Furthermore, N-phenyl-p-phenylenediamine, as a dimer of aniline, is more easily catalyzed and polymerized, thus achieving better degradation results.

[0109] Example 4: In vivo anti-tumor experiment The antitumor effect of this invention was evaluated in NOG mice carrying xenograft tumors. All animal experiments were approved by the laboratory animal welfare and ethics review team of Zhongnan Hospital, Wuhan University. 1×10 6 RKO cells were subcutaneously injected into the right axillary region of 4-6 week old male BALB / c nude mice to establish a solid tumor model. The tumor volume was increased to approximately 50 mm. 3 Subsequently, every other day, 0.05 mg / kg of antibody and N-phenyl-p-phenylenediamine (Ab+PD) were injected into the tumor. Mice injected with an equal volume of PBS as controls followed the same procedure. Two dimensions of the tumor were measured using calipers and calculated according to the formula (tumor volume = length × width). 2 / 2) Calculate tumor volume. Mice were sacrificed on day 33 of the experiment and tumors were removed for analysis.

[0110] Experimental results are as follows Figure 5 As shown, the tumor volume and weight of mice in the experimental group with added antibodies and substrates were significantly lower than those in the control group, demonstrating that the present invention also has good anti-tumor application potential in live animals.

[0111] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0112] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for targeted protein degradation, characterized in that, include: The biological sample was subjected to a first incubation treatment with an enzyme-labeled antibody to obtain the first incubation product. The first incubation product is then subjected to a second incubation treatment with the labeled substrate to achieve the degradation of the target protein in the biological sample.

2. The method according to claim 1, characterized in that, The enzyme-labeled antibody includes at least one of horseradish peroxidase-labeled antibody, ascorbate peroxidase-labeled antibody, tyrosinase-labeled antibody, and photosensitizing protein-labeled antibody. Optionally, the labeled substrate includes at least one of an easily oxidizable monomer and an acrylic monomer; Optionally, the easily oxidizable monomer includes at least one of aniline, naphthylamine, dopamine, pyrrole, and N-phenyl-p-phenylenediamine; Optionally, the acrylic monomer includes at least one of acrylamide and N-isopropylacrylamide.

3. The method according to claim 1, characterized in that, The biological sample includes cells or tissues expressing the target protein; Optionally, the target protein is an intracellular protein or an extracellular protein; Optionally, the ratio of the biological sample to the enzyme-labeled antibody is (102)2. 5 -10 6 ) cells: (0.01-5) μg / mL; Optionally, the incubation time for the first incubation treatment is 0.5h-2h; Optionally, in the second incubation treatment, the concentration of the labeled substrate is 0.01 μM-100 μM; Optionally, the second incubation treatment lasts for 6-48 hours.

4. The method according to claim 3, characterized in that, The first incubation treatment of the biological sample with the enzyme-labeled antibody further includes: The biological sample was subjected to a third incubation treatment with the first antibody to obtain a third incubation product. The third incubation product is subjected to a fourth incubation treatment with the second antibody to obtain the first incubation product.

5. The method according to claim 4, characterized in that, The first antibody includes an unlabeled specific antibody that is capable of binding to the target protein in the biological sample; Optionally, the second antibody comprises at least one of horseradish peroxidase-labeled secondary antibody, ascorbate peroxidase-labeled secondary antibody, tyrosinase-labeled secondary antibody, and photosensitizing protein-labeled secondary antibody, wherein the second antibody is capable of binding to the first antibody; Optionally, the ratio of the biological sample to the first antibody is (102)2 5 -10 6 ) cells: (0.01-5) μg / mL; Optionally, in the fourth incubation treatment, the concentration of the second antibody is 1-5 times that of the first antibody; Optionally, the third incubation treatment lasts for 1-2 hours; Optionally, the fourth incubation treatment lasts for 1-2 hours.

6. A reagent kit, characterized in that, It includes at least one of enzyme-labeled antibody, primary antibody, secondary antibody, and labeled substrate.

7. The reagent kit according to claim 6, characterized in that, The kit includes the first antibody, the second antibody, and the labeled substrate; Optionally, the kit includes the first antibody, the enzyme-labeled antibody, and the labeled substrate; Optionally, the enzyme-labeled antibody includes at least one of horseradish peroxidase-labeled antibody, ascorbate peroxidase-labeled antibody, tyrosinase-labeled antibody, and photosensitized protein-labeled antibody, wherein the enzyme-labeled antibody is capable of binding to the target protein in the biological sample; Optionally, the first antibody includes an unlabeled specific antibody capable of binding to a target protein in a biological sample; Optionally, the second antibody comprises at least one of horseradish peroxidase-labeled secondary antibody, ascorbate peroxidase-labeled secondary antibody, tyrosinase-labeled secondary antibody, and photosensitizing protein-labeled secondary antibody, wherein the second antibody is capable of binding to the first antibody; Optionally, the labeled substrate includes at least one of an easily oxidizable monomer and an acrylic monomer; Optionally, the easily oxidizable monomer includes at least one of aniline, naphthylamine, dopamine, pyrrole, and N-phenyl-p-phenylenediamine; Optionally, the acrylic monomer includes at least one of acrylamide and N-isopropylacrylamide.

8. A combination drug, characterized in that, It includes at least one of enzyme-labeled antibody, primary antibody, secondary antibody, and labeled substrate.

9. The combination drug according to claim 8, characterized in that, The combination drug comprises the first antibody, the second antibody, and the labeled substrate; Optionally, the combination drug includes the enzyme-labeled antibody and the labeled substrate; Optionally, the first antibody comprises an unlabeled specific antibody capable of binding to a target protein in a biological sample; Optionally, the second antibody comprises at least one of horseradish peroxidase-labeled secondary antibody, ascorbate peroxidase-labeled secondary antibody, tyrosinase-labeled secondary antibody, and photosensitizing protein-labeled secondary antibody, wherein the second antibody is capable of binding to the first antibody; Optionally, the enzyme-labeled antibody includes at least one of horseradish peroxidase-labeled antibody, ascorbate peroxidase-labeled antibody, tyrosinase-labeled antibody, and photosensitized protein-labeled antibody, wherein the enzyme-labeled antibody is capable of binding to the target protein in the biological sample; Optionally, the labeled substrate includes at least one of an easily oxidizable monomer and an acrylic monomer; Optionally, the easily oxidizable monomer includes at least one of aniline, naphthylamine, dopamine, pyrrole, and N-phenyl-p-phenylenediamine; Optionally, the acrylic monomer includes at least one of acrylamide and N-isopropylacrylamide.

10. The kit according to claims 6-7 or the combination drug according to claims 8-9 has at least one of the following uses: Applications in in vitro targeted protein degradation; Use in the preparation of a medicament for treating diseases associated with an overdose of a target protein; Optionally, the disease includes cancer.

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