Membrane protein targeted degradation agent based on cell autophagy and application thereof in targeted protein degradation drug

Abstract

The application provides a membrane protein targeting degradation agent based on cell autophagy, which comprises a targeting membrane protein antibody, a nano-second antibody and a cationic polymer; the nano-second antibody is connected with the cationic polymer; and the nano-second antibody is combined with the targeting membrane protein antibody. The membrane protein targeting degradation agent based on cell autophagy connects the nano-second antibody capable of being combined with the antibody of the targeting membrane protein with the cationic polymer having the cell autophagy induction property to form a module unit of the degradation agent. Since the nano-second antibody can be combined with many membrane protein antibodies derived from specific species, different membrane protein antibodies can be matched according to requirements for the degradation of target proteins, and there is no need to couple each membrane protein antibody, the purpose of membrane protein degradation is achieved, and the convenience of use is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an autophagy-based membrane protein targeted degrader, and the application of the autophagy-based membrane protein targeted degrader in targeted protein degradation drugs. Background Technology

[0002] Targeted protein degradation technology is a novel technique that specifically identifies target proteins and directly degrades them using inherent intracellular protein degradation pathways. It holds significant promise for applications in drug development and biomedical research. Inherent intracellular protein degradation pathways can be divided into two types: the ubiquitin-proteasome pathway and the lysosomal pathway. In recent years, various targeted protein degradation technologies have been developed using both pathways. These technologies degrade proteins located within cells, on cell membranes, and extracellularly.

[0003] Current technologies for degrading cell membrane proteins include LYTAC, AbTAC, PROTAB, GlueTAC, and KineTAC. AbTAC, similar to PROTAB, is a bispecific antibody. One Fab fragment of the antibody recognizes and binds to membrane proteins, while the other Fab fragment recognizes and binds to E3 ligases on the cell membrane, such as RNF43. This spatially brings the membrane protein closer to the E3 ligase, promoting ubiquitination of the membrane protein by the E3 ligase, and subsequently inducing endocytosis of the membrane protein into lysosomes for degradation. LYTAC technology involves conjugating glycopeptides to a membrane protein antibody. These glycopeptides act as ligands and bind to the mannose-6-phosphate receptor M6PR on the cell membrane. When the antibody-glycopeptide conjugate simultaneously binds to both the membrane protein and M6PR, the conjugate utilizes the endocytotic transport properties of M6PR to deliver the membrane protein into the lysosome for degradation. KineTAC technology involves an antibody-cytokine complex. When this complex simultaneously binds to a membrane protein and the cytokine receptor CXCR7, it uses CXCR7 to transport the membrane protein to lysosomes for degradation. GlueTAC technology, on the other hand, involves developing and screening nanobodies that target membrane proteins. These nanobodies are simultaneously coupled with a membrane-penetrating peptide and a lysosomal sorting peptide. When the antibody binds to the membrane protein, the membrane-penetrating peptide enters the cell, and the lysosomal sorting peptide delivers the membrane protein to lysosomes for degradation using biological signals. As can be seen, current membrane protein degradation technologies, except for GlueTAC, require the degrading agent to simultaneously bind to the target protein and an E3 ligase or lysosomal delivery receptor on the cell membrane. Therefore, the preparation of the degrading agent is complex and requires consideration of spatial conformation. Furthermore, the significant differences in the expression of E3 ligases and lysosomal delivery receptors across various cell types greatly limit the versatility of these technologies across different cell types.

[0004] Reported cell membrane protein degradation technologies can be categorized into four technical routes based on their specific mechanisms of action. Their drawbacks and limitations are as follows: 1) LYTAC-related technologies rely on lysosomal transport proteins on the cell membrane, such as M6PR, ASGPR, IGFIIR, and Integrin. The expression of these proteins varies significantly across different cells, resulting in poor versatility and consistency. The constructed degradative agent needs to spatially bind both the target protein and these lysosomal transport receptors, making design and synthesis challenging. 2) PROTAB and AbTAC technologies rely on E3 ligases on the cell membrane. Their development is limited by the expression level of this enzyme and requires bispecific antibodies, leading to high development costs. 3) KineTAC technology relies on cytokine receptors on the cell membrane. Its versatility across different cells is limited by the expression of these receptors, and the degradative agent is a conjugate of antibodies and cytokines, making design complex. 4) GlueTAC technology uses nanobodies containing non-natural amino acids, raising concerns about safety. It also has a short in vivo half-life and high screening costs.

[0005] Autophagy is a fundamental cellular function. Through autophagy, cells can effectively remove damaged organelles and misfolded protein molecules that have occurred during the cell's life cycle, transporting them to lysosomes for degradation. Therefore, developing targeted protein degradation technologies using the autophagy-lysosome pathway is an important strategy in this field. Currently, there are four existing autophagy-based protein degradation technologies: ATTEC, AUTAC, AUTOTAC, and molecular chaperone-mediated autophagy. These technologies are all limited to degrading intracellular proteins and organelles, and cannot degrade cell membrane proteins.

[0006] Based on this, the present invention establishes an autophagy-based membrane protein degradation technology and provides a membrane protein targeted degrader based on cell autophagy. A nano-second antibody is linked with a cationic polymer that has the property of inducing cell autophagy to construct a membrane protein degrader module. By combining it with different membrane protein antibodies to synergistically degrade the targeted membrane protein, a single degrader can simultaneously degrade multiple membrane proteins, greatly improving ease of use and applicability. Summary of the Invention

[0007] The purpose of this invention is to provide a cell autophagy-based membrane protein targeted degrader, which overcomes the shortcomings of existing technologies such as poor versatility in cell membrane protein degradation and complex and costly design.

[0008] The present invention also aims to provide the application of an autophagy-based membrane protein targeting degrader in cell membrane protein degradation drugs.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] An autophagy-based membrane protein targeting degrader comprises a membrane protein targeting antibody, a nano-second antibody, and a cationic polymer; the nano-second antibody is linked to the cationic polymer; and the nano-second antibody binds to the membrane protein targeting antibody.

[0011] In this invention, the nano-second antibody is connected to the cationic polymer via coupling.

[0012] Furthermore, the nano-second antibody is covalently coupled to the cationic polymer.

[0013] In this invention, the cationic polymer is one that can induce autophagy in endosomes. The choice of a cationic polymer is based on its ability to induce autophagy in endosomes, promoting the degradation of endosomes by lysosomes. Since this autophagy can be universally induced in various cells and its mechanism does not depend on the binding of degrading agents to autophagy-related molecules, the technology of this invention has the advantages of being more versatile and convenient compared to previous technologies.

[0014] Further, the cationic polymer includes one or more of the following: polyethylenimine and its derivatives, polylysine, polyallylamine, poly[N-(2-hydroxypropyl)methacrylamide], poly[2-(dimethylamino)ethyl methacrylate], polyornithine, polyarginine, and chitosan.

[0015] Furthermore, the polyethyleneimine derivative includes one of acetylated PEI and PEGylated PEI.

[0016] In this invention, the membrane protein includes one or more of PD-L1, EGFR, CD73, C-Met, IGF1R, FGFR4, HER2, and PDGFR.

[0017] In this invention, the antibody targeting membrane proteins is an antibody that can recognize the extracellular domain of membrane proteins.

[0018] Furthermore, the antibodies targeting membrane proteins include antibodies that bind to PD-L1, antibodies that bind to EGFR, and antibodies that bind to CD73.

[0019] Furthermore, the antibody targeting the membrane protein includes one or more of Atezolizumab (Atz), Cetuximab (Ctx), Oleclumab (Ole), and Integrin antibody.

[0020] In this invention, the cells include one of the following: MDA-MB-231 cells, U87-MG cells, NCI-H292 cells, NCI-H1975 cells, A375 cells, HeLa cells, A549 cells, Huh7 cells, and PANC-1 cells. The autophagy-based membrane protein targeted degrader of this invention is applicable to all cells, but its targeting effect is more pronounced in cells that highly express specific membrane proteins.

[0021] A method for preparing a membrane protein-targeting degrader based on autophagy includes the following steps:

[0022] (1) The cationic polymer was linked to the nano-second antibody to obtain a linker of the nano-second antibody and the cationic polymer;

[0023] (2) The linker from step (1) is combined with the antibody targeting the membrane protein to obtain the degradation agent.

[0024] In this invention, the cationic polymer is linked to the nano-second antibody using one of the following methods:

[0025] a. Modify the cationic polymer and the nano-second antibody respectively, and then couple the modified products;

[0026] b. Modify only the cationic polymer, and then conjugate it with the nano-second antibody;

[0027] c. Modify only the nano-second antibody, and then couple it with the cationic polymer;

[0028] d. The nano-second antibody is directly coupled to the cationic polymer.

[0029] In this invention, the modifiers used to modify the cationic polymer include one or more of DBCO, maleimide, and avidin; the modifiers used to modify the nano-second antibody include one or more of azide C5 succinimide ester, non-natural amino acids, and biotin.

[0030] In some embodiments of the present invention, the cationic polymer is modified with DBCO, the second antibody is modified with azide C5 succinimide ester, and the two modified products are then coupled.

[0031] In some embodiments of the present invention, the cationic polymer is modified with maleimide, but the second antibody is not modified, and then the two are coupled.

[0032] In some embodiments of the present invention, the second antibody is modified with non-natural amino acids, the cationic polymer is not modified, and then the two are coupled.

[0033] In some embodiments of the present invention, the cationic polymer is modified with avidin, the second antibody is modified with biotin, and then the two are coupled together.

[0034] Application of the above-mentioned autophagy-based membrane protein targeted degrader in the preparation of targeted protein degradation drugs.

[0035] Furthermore, the membrane protein targets include one or more of PD-L1, EGFR, CD73, C-Met, IGF1R, FGFR4, HER2, and PDGFR.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The present invention is based on the membrane protein targeted degradation agent of cell autophagy. It connects a nano-second antibody that can bind to the antibody that targets the membrane protein with a cationic polymer that has the property of inducing cell autophagy to form a module unit of the degradation agent. Since the nano-second antibody can bind to many membrane protein antibodies from a specific species, different membrane protein antibodies can be matched as needed for the degradation of the target protein without the need to couple each membrane protein antibody. This achieves the purpose of membrane protein degradation and greatly improves the convenience of use.

[0038] (2) The degrading agent of this invention does not require the separate synthesis of a degrading agent for each membrane protein. It only requires the preparation of a Nano-AUTAB and its combination with a commercially available membrane protein antibody to efficiently degrade the target site, which has the advantage of convenient preparation. The process of coupling the nano-second antibody with the cationic polymer is simple and does not require consideration of steric hindrance or other factors in the preparation. Membrane protein antibodies are readily available in large quantities on the market, which greatly reduces the construction cost of this degrading agent. Attached Figure Description

[0039] The present invention will now be described in detail with reference to the embodiments and accompanying drawings, wherein:

[0040] Figure 1 This is a schematic diagram of membrane protein targeted degradation based on autophagy;

[0041] Figure 2 This is a diagram showing the synthesis and characterization of Atz-AUTAB;

[0042] Figure 3This is a diagram showing the autophagy induced by the cationic polymer PEI in endosomes;

[0043] Figure 4 This diagram illustrates how AUTAB binds to its target protein, induces endocytosis in the endosome, and triggers autophagy.

[0044] Figure 5 This is a diagram illustrating how AUTAB promotes the endocytosis and transport of target proteins to lysosomes;

[0045] Figure 6 This is a flow cytometry graph showing AUTAB clearing target proteins on the cell membrane;

[0046] Figure 7 This is a diagram showing the AUTAB-induced degradation of target proteins;

[0047] Figure 8 This is a graph of experimental data demonstrating that AUTAB promotes target protein degradation through the autophagy-lysosomal pathway;

[0048] Figure 9 This diagram illustrates how AUTAB coupled with various PEIs and poly-L-Lys promotes the degradation of target proteins.

[0049] Figure 10 This is a diagram illustrating the degradation of PD-L1 in tumor tissue by Atz-AUTAB in animals.

[0050] Figure 11 It is a membrane protein degradation strategy based on Nano-AUTAB;

[0051] Figure 12 This is the synthetic route for Nano-AUTAB;

[0052] Figure 13 Nano-AUTAB, combined with membrane protein antibodies, synergistically degrades various target proteins;

[0053] Figure 14 Nano-AUTAB-m synergistically promotes the degradation of membrane protein Notch1. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of this invention clearer, the embodiments of this invention will be described in further detail below with reference to specific examples. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0055] The process of membrane protein targeted degradation based on autophagy in this invention is as follows: Figure 1 , Figure 11 As shown, this invention provides a technology for developing targeted protein degradation using the autophagy-lysosome pathway. Figure 1 The method involves linking antibodies targeting membrane proteins with cationic polymers to construct a degradation agent. This polymer damages endosomes during endocytosis, inducing autophagy and thereby inducing membrane proteins to be endocytosed into lysosomes for degradation. Figure 11 Based on Figure 1 Further improvements to the pathway involve linking a nano-second antibody that can bind to an antibody targeting membrane proteins with a cationic polymer, and then pairing it with the antibody targeting membrane proteins to synergistically degrade the target protein.

[0056] Example 1

[0057] like Figure 2 The method for preparing a membrane protein-targeting degrader based on autophagy, as shown, includes the following steps:

[0058] (1) DBCO tag modification of linear polyethyleneimine (PEI)

[0059] Taking L25K-PEI as an example: Weigh 10 mg of linear PEI material (L25K-PEI, Mw = 25000) into a clean centrifuge tube, add 500 μL of ultrapure water and 250 μL of 1M hydrochloric acid solution in sequence, and use sonication to dissolve the PEI material to form a clear solution. Then, add 1M sodium carbonate solution to the above solution to adjust the pH to 9.0, and then add 20.1 μL of DBCO-NHS stock solution (1.0 equivalent, 8 mg / mL, DMSO as solvent). Finally, add PBS solution with pH = 9 until the final concentration of the reaction solution is 5 mg / mL (based on PEI material). Shake the reaction system at room temperature for 24 hours. The obtained L25K-DBCO solution can be used directly for subsequent reactions without further purification.

[0060] Various other linear PEIs of different molecular weights, including but not limited to Mw=2500, Mw=5000, and Mw=10000, were modified with DBCO according to the above synthesis method for subsequent antibody conjugation.

[0061] Various linear PEI derivatives, including but not limited to acetylated PEI and PEGylated PEI, are modified with DBCO according to the above synthesis method for subsequent antibody conjugation.

[0062] (2) Preparation of antibody-PEI material conjugate

[0063] Taking Atz-N3 as an example: Measure 29.2 μL of Atz antibody (Atezolizumab) stock solution (0.25 mg, 1.0 equivalent, 8.58 mL / mg, PBS as solvent, pH=7.3) and add it to a clean centrifuge tube. Then add 100 μL of PBS solution (pH=7.3) and 3 μL of 1M sodium carbonate solution. At this point, the pH of the mixture is slightly higher than 9.0. Finally, add 5.7 μL of N3-C5-NHS (Azide C5 Succinimide Ester) (22 μg, 50.0 equivalent, 3.82 mg / mL, DMSO as solvent). Shake the reaction solution at room temperature for 24 hours. After the reaction is complete, transfer the reaction solution to an ultrafiltration tube (4 mL, 30 kDa), concentrate and wash 3 times with 2% DMSO / PBS solution as washing buffer. Finally, obtain an Atz-N3 antibody stock solution of 1.0 μg / μL.

[0064] 250 μL of Atz-N3 stock solution (250 μg, PBS, pH 7.3) and 216 μL of L25K-DBCO stock solution (1.08 mg, 25.0 equivalent, 5.0 mg / mL) were mixed thoroughly and shaken at room temperature for 24 hours. After the reaction was complete, the reaction solution was transferred to an ultrafiltration tube (4 mL, 30 kDa), concentrated, and washed three times with 2% DMSO / PBS solution as the washing buffer, finally yielding an Atz-AUTAB stock solution of 1.0 μg / μL.

[0065] Other antibodies include, but are not limited to, Ctx antibody (Cetuximab) and Ole antibody (Oleclumab), whose modification and conjugation with PEI are exactly the same as the preparation process described above.

[0066] Example 2

[0067] A method for preparing a membrane protein-targeting degrader based on autophagy differs from Example 1 in that the tag modification process is different; specifically, linear PEI materials are modified with FITC fluorescent tags.

[0068] 20 mg of linear PEI material (L25K-PEI, Mw = 25000) was weighed into a clean centrifuge tube. 500 μL of ultrapure water and 500 μL of 1M hydrochloric acid solution were added sequentially, and the PEI material was dissolved by sonication to form a clear, transparent solution. Then, 1M sodium carbonate solution was added to adjust the pH to 9.0. Finally, 1.25 mg of FITC powder (4.0 equivalents) was added, and the mixture was thoroughly mixed. The reaction solution was protected from light and shaken at room temperature for 24 hours. After the reaction was complete, the reaction solution was dialyzed and freeze-dried to obtain 12.8 mg of red powder (L25K-FITC), with a yield of 60%.

[0069] Example 3

[0070] A method for preparing a membrane protein-targeting degrader based on autophagy differs from Example 1 in that the tag modification process is different; specifically, the branched PEI material is modified with a DBCO tag.

[0071] Taking B2K-PEI as an example: Weigh 4 mg of branched PEI material (B2K-PEI, Mw = 2000) into a clean centrifuge tube, add 500 μL of PBS solution (pH = 9.0) to dissolve it, then add 50.3 μL of DBCO-NHS stock solution (1.0 equivalent, 8 mg / mL, DMSO as solvent), and finally add PBS solution at pH = 9 until the final concentration of the reaction solution is 1 mg / mL (based on PEI material). Shake the reaction system at room temperature for 24 hours. The obtained B2K-DBCO solution can be used directly for subsequent reactions without further purification.

[0072] Various other branched PEIs of different molecular weights, including but not limited to Mw=800 and Mw=25000, were modified with DBCO according to the above synthesis method for subsequent antibody conjugation.

[0073] Various branched PEI derivatives, including but not limited to acetylated PEI and PEGylated PEI, are modified with DBCO according to the above synthetic method for subsequent antibody conjugation.

[0074] Example 4

[0075] A method for preparing a membrane protein-targeting degrader based on autophagy includes the following steps:

[0076] (1) DBCO tag modification of polylysine (PolyLys)

[0077] Taking PolyLys(15-30K)-DBCO as an example: Weigh 7.37 mg of polylysine hydrobromide (Poly-Lys, Mw = 15-30K) into a clean centrifuge tube, add 500 μL of PBS solution (pH = 9.0) and 16.5 μL of DBCO-NHS stock solution (1.0 equivalent, 8 mg / mL, DMSO as solvent) in sequence, and finally add PBS solution with pH = 9 until the final concentration of the reaction solution is 5 mg / mL (based on PEI material). Shake the reaction system at room temperature for 24 hours. The obtained PolyLys(15-30K)-DBCO solution can be used directly for subsequent reactions without further purification.

[0078] Various other polylysine molecules of different molecular weights, including but not limited to Mw=3000-7000 and Mw=4000-15000, were modified with DBCO according to the above synthesis method for subsequent antibody conjugation.

[0079] (2) Preparation of antibody-Poly-Lys conjugate

[0080] Taking Atz-N3 as an example: Measure 29.2 μL of Atz antibody stock solution (0.25 mg, 1.0 equivalent, 8.58 mL / mg, PBS as solvent, pH = 7.3) and add it to a clean centrifuge tube. Then add 100 μL of PBS solution (pH = 7.3) and 3 μL of 1M sodium carbonate solution. At this point, the pH of the mixture is slightly higher than 9.0. Finally, add 5.7 μL of N3-C5-NHS (22 μg, 50.0 equivalent, 3.82 mg / mL, DMSO as solvent). Shake the reaction solution at room temperature for 24 hours. After the reaction is complete, transfer the reaction solution to an ultrafiltration tube (4 mL, 30 kDa), concentrate and wash 3 times with 2% DMSO / PBS solution as washing buffer. Finally, obtain an Atz-N3 antibody stock solution of 1.0 μg / μL.

[0081] 250 μL of Atz-N3 stock solution (250 μg, 1.0 equivalent) and 195 μL of PolyLys(15-30K)-DBCO stock solution (973 μg, 25.0 equivalent, 5.0 mg / mL) were mixed thoroughly and shaken at room temperature for 24 hours. After the reaction was complete, the reaction solution was transferred to an ultrafiltration tube (4 mL, 30 kDa), concentrated, and washed three times with 2% DMSO / PBS solution as the washing buffer to obtain a PolyLys-Atz-AUTAB stock solution of 1.0 μg / μL.

[0082] Other antibodies, including but not limited to Ctx antibodies and Ole antibodies, are modified and conjugated with polylysine in exactly the same way as described above.

[0083] Example 5

[0084] A method for preparing a membrane protein-targeting degrader based on autophagy differs from Example 1 in that only PEI is modified with maleimide, without modifying the antibody. Subsequently, PEI is coupled to the antibody using disulfide bonds on the antibody.

[0085] Example 6

[0086] A method for preparing a membrane protein-targeting degrader based on autophagy differs from Example 1 in that only non-natural amino acid modifications are introduced into the antibody, without modifying PEI. Subsequently, the non-natural amino acids are used to perform a condensation reaction with PEI to complete the coupling of PEI and the antibody.

[0087] Example 7

[0088] A method for preparing a membrane protein-targeting degrader based on autophagy differs from Example 1 in that the antibody is modified with biotin and the PEI is modified with avidin. Subsequently, the non-covalent binding between biotin and avidin is used to form a non-covalent coupling between PEI and the antibody.

[0089] Example 8

[0090] A method for preparing a membrane protein-targeting degrader based on autophagy differs from Examples 1 to 7 in that the cationic polymer used is polyallylamine, while the other preparation processes are the same.

[0091] Example 9

[0092] A method for preparing a membrane protein-targeting degrader based on autophagy differs from Examples 1 to 7 in that the cationic polymer used is poly[N-(2-hydroxypropyl)methacrylamide], while the other preparation procedures are the same.

[0093] Example 10

[0094] A method for preparing a membrane protein-targeting degrader based on autophagy differs from Examples 1 to 7 in that the cationic polymer used is poly[2-(dimethylamino)ethylmethacrylate], while the other preparation processes are the same.

[0095] Example 11

[0096] A method for preparing a membrane protein-targeting degrader based on autophagy differs from Examples 1 to 7 in that the cationic polymer used is polyornithine, while the other preparation processes are the same.

[0097] Example 12

[0098] A method for preparing a membrane protein-targeting degrader based on autophagy differs from Examples 1 to 7 in that the cationic polymer used is polyarginine, while the other preparation processes are the same.

[0099] Example 13

[0100] A method for preparing a membrane protein-targeting degrader based on autophagy differs from Examples 1 to 7 in that the cationic polymer used is chitosan, while the other preparation processes are the same.

[0101] In the above embodiments of the present invention, different membrane protein targeted degraders were prepared by chemically coupling different antibodies with different cationic polymers possessing autophagy-inducing properties. Different cationic polymers were coupled to the antibody Atz to obtain corresponding Atz-AUTABs. This method was also validated on different antibodies, ultimately synthesizing a variety of membrane protein degraders, including Atz-AUTAB, Ctx-AUTAB, and Ole-AUTAB. The experimental conditions were reproducible, and the yields were stable.

[0102] Example 14

[0103] like Figure 12 The method for preparing a membrane protein-targeting degrader based on autophagy, as shown, includes the following steps:

[0104] (1) MA modification of PEI material

[0105] Taking L2.5K-MA as an example: Weigh 6 mg of linear polyethyleneimine (PEI) material (L2.5K-PEI, Mw = 2500) into a clean centrifuge tube, add 200 μL of ultrapure water and 200 μL of 1M hydrochloric acid solution sequentially, and dissolve the PEI material by sonication to form a clear solution. Add Tris-HCl buffer (pH = 8.68) to the above solution to adjust the pH to 8.0, then add 148 μL of MA-C6-NHS solution (1.0 equivalent, 5 mg / mL, DMSO as solvent). At this point, the final concentration of the reaction solution is 3.0 mg / mL (based on PEI material). Shake the reaction system at room temperature for 3 hours. The resulting L2.5K-MA solution can be used directly for subsequent reactions without further purification.

[0106] Various other linear PEIs of different molecular weights, including but not limited to Mw=5000 and Mw=10000, were modified with MA according to the above synthesis method for subsequent nanobody conjugation.

[0107] Branched PEIs of various molecular weights, including but not limited to Mw=800, Mw=2000, and Mw=25000, were modified with MA according to the above synthesis method and used for subsequent nanobody conjugation.

[0108] Various PEI derivatives, including but not limited to acetylated PEI and PEGylated PEI, are modified with MA according to the above synthesis method and used for subsequent nanobody conjugation.

[0109] In addition to PEI, the above-mentioned MA modification methods and preparation processes are also applicable to polylysine, polyallylamine, poly[N-(2-hydroxypropyl)methacrylamide], poly[2-(dimethylamino)ethylmethacrylate], polyornithine, polyarginine, and chitosan.

[0110] (2) The nano-second antibody was reacted with L2.5K-MA solution to obtain Nano-AUTAB, a conjugate of the nano-second antibody and a cationic polymer.

[0111] 125 μL of the nano-AUTAB stock solution (250 μg, 2.0 mg / mL, pH 7.0) and 208 μL of L2.5K-MA stock solution (15.0 equivalents, 3 mg / mL, pH 8.0) were mixed thoroughly and shaken at room temperature for 3 hours. After the reaction was complete, 80.5 μL of N-acetylcysteine ​​solution (25.0 equivalents, 1 mg / mL) was added to the reaction system and the mixture was shaken for another 30 minutes to consume unreacted raw materials. The final mixture was transferred to an ultrafiltration tube (4 mL, 3 kDa), concentrated, and washed three times with 2% DMSO / PBS solution as the washing buffer to obtain a 1.0 μg / μL Nano-AUTAB stock solution.

[0112] (3) Nano-AUTAB stock solution was combined with Atz antibody targeting PD-L1 to obtain a membrane protein targeting degradation agent based on autophagy.

[0113] Other membrane protein antibodies, including but not limited to Ctx antibody (Cetuximab) and Ole antibody (Oleclumab), are prepared using the same process as described above.

[0114] Example 15

[0115] A method for preparing a membrane protein-targeting degrader based on autophagy includes the following steps:

[0116] (1) DBCO label modification of PEI material

[0117] Taking L2.5K-PEI as an example: Weigh 10 mg of linear PEI material (L2.5K-PEI, Mw = 2500) into a clean centrifuge tube, add 500 μL of ultrapure water and 250 μL of 1M hydrochloric acid solution in sequence, and use sonication to dissolve the PEI material to form a clear solution. Then, add 1M sodium carbonate solution to the above solution to adjust the pH to 9.0, followed by adding 20.1 μL of DBCO-NHS stock solution (1.0 equivalent, 8 mg / mL, DMSO as solvent), and finally add PBS solution with pH = 9 until the final concentration of the reaction solution is 5 mg / mL (based on PEI material). Shake the reaction system at room temperature for 24 hours. The obtained L2.5K-DBCO solution can be used directly for subsequent reactions without further purification.

[0118] (2) The nano-second antibody was reacted with L2.5K-DBCO solution to obtain Nano-AUTAB, a conjugate of the nano-second antibody and a cationic polymer.

[0119] Taking the nano-second antibody-N3 as an example: 240 μL of nano-antibody stock solution (0.24 mg, 1.0 equivalent, 1.0 mL / mg, PBS as solvent, pH = 7.3) was added to a clean centrifuge tube, followed by 6 μL of 1M sodium carbonate solution. At this point, the pH of the mixture was slightly higher than 9.0. Finally, 25.4 μL of N3-C5-NHS (0.203 mg, 50.0 equivalent, 8.0 mg / mL, DMSO as solvent) was added. The reaction solution was shaken at room temperature for 24 hours. After the reaction was completed, the reaction solution was transferred to an ultrafiltration tube (4 mL, 3 kDa), concentrated, and washed three times with 2% DMSO / PBS solution as the washing buffer. Finally, a 1.0 μg / μL nano-second antibody-N3 stock solution was obtained.

[0120] 240 μL of nano-AUTAB-N3 stock solution (240 μg, 1.0 equivalent) and 125 μL of L2.5K-DBCO stock solution (1.0 mg, 25.0 equivalent, 8.0 mg / mL) were mixed thoroughly and shaken at room temperature for 24 hours. After the reaction was complete, the reaction solution was transferred to an ultrafiltration tube (4 mL, 3 kDa), concentrated, and washed three times with 2% DMSO / PBS solution as the washing buffer to obtain a final Nano-AUTAB stock solution of 1.0 μg / μL.

[0121] (3) Nano-AUTAB stock solution was combined with Atz antibody to obtain a membrane protein targeted degradation agent based on cell autophagy.

[0122] Example 16

[0123] A method for preparing a membrane protein-targeting degrader based on autophagy differs from Example 15 in that only the second antibody is modified with non-natural amino acids, without modifying PEI. Subsequently, the non-natural amino acids are used to perform a condensation reaction with PEI to complete the conjugation of PEI and the second antibody.

[0124] Example 17

[0125] A method for preparing a membrane protein-targeting degrader based on autophagy differs from Example 15 in that the second antibody is modified with biotin and the PEI is modified with avidin. Subsequently, the non-covalent binding between biotin and avidin is used to form a non-covalent coupling between PEI and the second antibody.

[0126] In the above embodiments of the present invention, a second antibody is coupled with different cationic polymers with autophagy-inducing properties through a chemical method to form a modular unit of a degradation agent, and then combined with different membrane protein antibodies to obtain the corresponding Nano-AUTAB.

[0127] Example 18 verifies AUTAB-induced autophagy associated with endosomes.

[0128] (1) Verify that cationic polymers induce autophagy.

[0129] HeLa cells stably expressing mCherry-LC3C were cultured on glass slides. After the cells reached approximately 50% confluence, various types and sizes of PEI or FITC-labeled L25K-PEI were added to the culture medium, and the cells were cultured for another hour. Subsequently, the cells were fixed with 4% PFA, and the formation of mCherry-LC3C-positive autophagosomes and the co-localization of endocytosed FITC-PEI with autophagosomes were observed under a laser confocal microscope.

[0130] HeLa cells stably expressing mCherry-LC3C were cultured on glass slides and transfected with the EGFP-Rab5 plasmid. Twenty-four hours after transfection, the cells were incubated with PEI for one hour, then fixed, and the co-localization of the autophagosome marker mCherry-LC3C and the early endosome marker EGFP-Rab5 was detected under a laser confocal microscope.

[0131] Depend on Figure 3 As shown in Figure a, after treatment of cells with various sizes of linear (L2.5K, L25K) and branched (B800, B2K, B25K) PEI, punctate fluorescent signals of mCherry-LC3C were observed in all cells, indicating that PEI can induce LC3C-mediated autophagy. Further analysis... Figure 3As shown in b, the endocytic FITC-PEI fluorescence signal co-localizes with these mCherry-LC3C signals, indicating that autophagy occurs on PEI-containing endosomes. Figure 3 As shown in c, these mCherry-LC3C signals colocalize with the early endosome marker EGFP-Rab5, indicating that autophagy occurs early in PEI endocytosis.

[0132] (2) Detection of AUTAB-induced autophagy.

[0133] In this invention, a cationic polymer is coupled with a membrane protein to construct AUTAB. Based on the autophagy-inducing properties of cationic polymers, a series of AUTAB-induced autophagy experiments were conducted to verify these results.

[0134] For Atz-AUTAB, HeLa cells stably expressing HA-PD-L1 and mCherry-LC3C were cultured on glass slides. After the cells reached approximately 50% confluence, Atz-AUTAB or the control antibody Atz was added to the culture medium at a final concentration of 100 nM and incubated for 1 hour. Cells were then fixed with 4% PFA and subjected to standard immunofluorescence staining. Anti-Human IgG antibodies were used to label Atz-AUTAB and the control group's Atz, anti-HA-tagged PD-L1, anti-Rab5 antibodies to label early endosomes, and anti-Rab7 antibodies to label late endosomes. After immunostaining, slides were mounted with an antifluorescence quencher, and the co-localization of autophagy markers mCherry-LC3C, Atz-AUTAB, HA-PD-L1, and early / late endosomes was observed and detected under a laser confocal microscope.

[0135] For PolyLys-Atz-AUTAB, the colocalization relationship among autophagy markers mCherry-LC3C, PolyLys-Atz-AUTAB, and HA-PD-L1 was mainly analyzed, using the same method as Atz-AUTAB described above.

[0136] For Ctx-AUTAB (Cetuximab, abbreviated as Ctx), the co-localization relationship among the autophagy marker mCherry-LC3C, Ctx-AUTAB, and EGFR was analyzed, using a method similar to that described above. HeLa cells stably expressing mCherry-LC3C were cultured on slides and treated for 1 hour with either Ctx-AUTAB at a final concentration of 100 nM or the control antibody Ctx. Cells were then fixed, and Ctx-AUTAB and the control Ctx were labeled with anti-Human IgG antibodies, while the membrane protein EGFR was labeled with an anti-EGFR antibody. After immunostaining, analysis and detection were performed using laser confocal microscopy.

[0137] Measurement metrics: The amount of point signals in LC3 shown in the figure, and the co-location status of LC3 signals and AUTAB signals.

[0138] Taking Atz-AUTAB, which targets PD-L1, as an example, Figure 4 As shown in figure a, the endocytosed Atz-AUTAB significantly co-localizes with HA-PD-L1, indicating that it can recognize and bind to PD-L1 and be endocytosed into the cell along with PD-L1. Furthermore, compared to Atz itself, Atz-AUTAB induces more punctate signals of mCherry-LC3Cs, and co-localizes with the autophagy signals of these LC3Cs, indicating that Atz-AUTAB can induce autophagy after endocytosis. Further, from... Figure 4 As shown in b and 4c, these Atz-AUTABs co-localize with LC3C and the endosome markers Rab5 or Rab7, indicating that autophagy occurs on endosomes in both the early and late stages of the endocytosis pathway after Atz-AUTAB endocytosis. Ctx-AUTAB targeting EGFR has also been shown to induce similar autophagy, see [link to relevant documentation]. Figure 4 d. More importantly, AUTAB, constructed using other types of cationic polymers such as poly-L-Lys, also induced a large number of punctate signals of mCherry-LC3C after treatment of cells, and these signals co-localized with the signals of these LC3Cs, indicating that they can also induce autophagy. See [link to article]. Figure 4 e. These results demonstrate that AUTAB with autophagy effects can be constructed using a variety of cationic polymers.

[0139] Example 19 verifies the degradation effects of various AUTABs on multiple membrane proteins.

[0140] (1) Detection of AUTAB promoting membrane protein endocytosis and transport to lysosomes

[0141] For Atz-AUTAB, HeLa cells stably expressing HA-PD-L1 were cultured on glass slides and transfected with the RFP-Lamp1 plasmid. Twenty-four hours after transfection, cells were treated with Atz-AUTAB at a final concentration of 5 nM or the control antibody Atz for 1 hour. Cells were then fixed and immunofluorescence staining was performed using an anti-HA-tagged antibody to label PD-L1. After immunostaining, the co-localization of the lysosomal marker RFP-Lamp1 and the target protein PD-L1 was analyzed using laser confocal microscopy.

[0142] For Ctx-AUTAB, the method is similar to that described above: HeLa cells are cultured on a glass slide and transfected with the RFP-Lamp1 plasmid. Twenty-four hours after transfection, cells are treated with either Ctx-AUTAB at a final concentration of 5 nM or the control antibody Ctx for one hour. Immunofluorescence staining is then performed using an anti-EGFR antibody, and colocalization is observed under a microscope.

[0143] For Ole-AUTAB, HeLa cells stably expressing mCherry-CD73 were cultured on a glass slide. When the cells reached 80% confluence, they were treated with Ole-AUTAB or the control antibody Ole at a final concentration of 100 nM for 12 hours. Subsequently, in a live-cell state, lysosomes were labeled with LysoTracker fluorescent probes, and the colocalization of lysosomes and mCherry-CD73 was observed and detected in real time under a laser confocal microscope.

[0144] like Figure 5 As shown in Figure a, compared to the Atz treatment group, Atz-AUTAB treatment led to the endocytosis of a large amount of the target protein PD-L1 into the cells, and it co-localized with the lysosomal marker Lamp1, indicating that PD-L1 was transported into lysosomes. Similarly, under the action of Ctx-AUTAB and Ole-AUTAB, the corresponding target proteins EGFR and CD73 were also massively endocytosed into lysosomes, as shown in Figure a. Figure 5 b and 5c.

[0145] (2) Verify that AUTAB weakens the localization of target proteins on the cell membrane.

[0146] We used live-cell flow cytometry to analyze the changes in the localization of target proteins on the cell membrane under the action of AUTAB.

[0147] For Atz-AUTAB, MDA-MB-231 cells or HeLa cells stably expressing HA-PD-L1 were seeded at 80% confluence in 12-well plates and cultured overnight. The next day, cells were treated with 5 nM Atz-AUTAB or the control antibody Atz for a specified time (0.5, 2, 6, and 24 hours for 231 cells, and 4 hours for HeLa cells). Subsequently, in live cells, PD-L1 on the cell membrane was labeled at 4°C using an antibody recognizing the extracellular domain of PD-L1, and the labeling signal was further amplified using the corresponding fluorescent secondary antibody. After immunofluorescence labeling, the labeled PD-L1 was detected by flow cytometry.

[0148] For Ctx-AUTAB, the method is similar to that described above. HeLa cells were treated with 5 nM Ctx-AUTAB or the control antibody Ctx for 4 hours, followed by labeling of EGFR on the cell membrane with an antibody that recognizes the extracellular domain of EGFR, and further analysis with fluorescent secondary antibody staining and flow cytometry.

[0149] For Ole-AUTAB, HeLa cells stably expressing mCherry-CD73 were treated with 100 nM Ole-AUTAB or the control antibody Ole for 12 hours, followed by labeling of the target protein on the membrane with anti-mCherry antibody, and further fluorescent secondary antibody staining and flow cytometry analysis.

[0150] like Figure 6 Flow cytometry data showed that treatment with Atz-AUTAB, Ctx-AUTAB, and Ole-AUTAB all resulted in a sharp reduction in the corresponding target proteins from the membrane. Figure 5 The localization of the fluorophores shown indicates that treatment with Atz-AUTAB, Ctx-AUTAB, and Ole-AUTAB resulted in the endocytosis of a large number of target proteins into lysosomes, suggesting that under the action of AUTAB, target proteins on the membrane can be rapidly endocytosed from the membrane and transported into lysosomes.

[0151] (3) Detection of AUTAB degradation of various membrane proteins

[0152] We used Western blotting (WB) to evaluate the degradation effects of a range of AUTABs on their respective target proteins in various cell types.

[0153] For Atz-AUTAB, MDA-MB-231 cells, U87-MG cells, NCI-H292 cells, NCI-H1975 cells, A375 cells, and HeLa cells stably expressing HA-PD-L1 were incubated with Atz-AUTAB (with concentration gradients) or the control group Atz for 24 hours or the gradient time, respectively. Subsequently, the cells were treated with SDS lysis buffer to obtain total cellular protein samples, and the changes in PD-L1 protein levels in the protein samples were analyzed according to standard Western blotting.

[0154] For Ctx-AUTAB, its degradation of EGFR was detected in HeLa cells, A549 cells, Huh7 cells, and PANC-1 cells using the same method as Atz-AUTAB described above.

[0155] For Ole-AUTAB, its degradation of CD73 was detected in MDA-MB-231 cells, U87-MG cells, NCI-H292 cells, and PANC-1 cells, using the same method as Atz-AUTAB described above.

[0156] For AUTAB conjugated with different types and sizes of PEI (L25K, L2.5K, B25K, B2K, B800), the degradation of target proteins PD-L1, EGFR, or CD73 was detected in MDA-MB-231 cells, HeLa cells, and U87-MG cells, respectively, using the same method as Atz-AUTAB described above.

[0157] For Atz-AUTAB conjugated with poly-L-Lys, its degradation of PD-L1 in MDA-MB-231 cells was detected using the same method as described above for Atz-AUTAB.

[0158] This invention demonstrates through Western blotting experiments that the constructed series of AUTABs can all lead to the degradation of target proteins. For example... Figure 7 As shown in the diagram, Atz-AUTAB, even at a very low concentration (0.09 nM), caused significant degradation of PD-L1 within 24 hours; while at 6.25 nM Atz-AUTAB, degradation of the target protein was observed in as little as 12 hours. Figure 7 As shown in Figure d, Atz-AUTAB was verified to degrade target proteins in various cell types (U87-MG, NCI-H292, NCI-H1975, A375). Furthermore, HeLa cells overexpressing PD-L1, such as... Figure 7 The example demonstrates that even highly expressed PD-L1 can be cleared by Atz-AUTAB. Similarly, as shown... Figure 7As shown in hj, Ctx-AUTAB has been demonstrated to degrade EGFR at low concentrations and for short durations, and it is effective in a variety of cell lines. In addition to targeting transmembrane proteins, Ole-AUTAB targeting the membrane anchoring protein CD73 has also been developed, and its concentration- and time-dependent effects have been verified, demonstrating its applicability in multiple cell lines. (See [link to Hj]). Figure 7 km. These results demonstrate that the AUTAB technology of the present invention has universality.

[0159] (4) Verify that AUTAB degrades target proteins via the autophagy-lysosome pathway.

[0160] This study demonstrates that AUTAB degrades target proteins via autophagy. Using Atz-AUTAB as an example, RNAi technology was used to knock down the expression of the core autophagy genes ATG5 or LC3C in MDA-MB-231 cells. Cells were then treated with Atz-AUTAB (6.25 nM) for 12 hours, followed by cell lysis to extract total protein, and Western blotting to detect changes in PD-L1 levels.

[0161] This study demonstrated that AUTAB degrades target proteins via lysosomes. MDA-MB-231 cells, HeLa cells, and U87-MG cells were treated with AUTAB, with the lysosomal inhibitors bafilomycin A1 (100 nM) or chloroquine (200 μM) added to the culture medium, and cultured for 24 hours. Cells were then lysed to extract total protein, and changes in target protein levels were detected using Western blotting.

[0162] like Figure 8 As shown in Figure a, Atz-AUTAB failed to degrade PD-L1 under autophagy inhibition, indicating that it requires autophagy to function. Next, cells were treated with lysosomal inhibitors (bafilomycin A1 and chloroquine) to test whether Atz-AUTAB could still exert a degradative effect. The results showed that it completely failed to promote the degradation of the target site, indicating that its function depends on lysosomal activity. (See Figure a). Figure 8 b. Furthermore, using the same method, it was demonstrated that both Ctx-AUTAB and Ole-AUTAB degrade target proteins via the lysosomal pathway, see [link to documentation]. Figure 8 c, 8d. These demonstrate that AUTAB degrades target proteins via the autophagy-lysosomal pathway.

[0163] In addition to verifying the degradation effect using AUTAB coupled with L25K-PEI as described above, the targeted degradation effect of AUTAB coupled with other different PEIs and other cationic polymers poly-L-Lys was further demonstrated. Figure 9As shown in the ad, both linear (L2.5K, L25K) and branched (B800, B2K, B25K) PEIs, when conjugated to their respective antibodies, significantly promote the degradation of target proteins. Atz-AUTAB, constructed using two different size ranges of poly-L-Lys (4-15K, 15-30K), also significantly degrades PD-L1 at concentrations up to 1 nM. Figure 9 e. These results demonstrate that a variety of cationic polymers can be used in the membrane protein targeted degradation strategy proposed in this invention.

[0164] (5) Validation of AUTAB degradation of membrane proteins in animals

[0165] Taking Atz-AUTAB as an example, a subcutaneous tumor-bearing model of MDA-MB-231 cells was first established in nude mice (8 weeks old, BALB / c strain). The tumor volume was increased to approximately 50 mm. 3 At the time of administration, Atz-AUTAB or the control antibody Atz (0.01 nmol / 100 μL, once a day for two consecutive days) was injected around the tumor. The day after the injection, mouse tumor tissue was collected, and the tissue was lysed to extract total protein. Subsequently, Western blotting was used to detect changes in the amount of PD-L1 in the tumor tissue.

[0166] like Figure 10 As shown, a nude mouse tumor-bearing model of MDA-MB-231 cells was established. Atz-AUTAB (0.01 nmol / time, once daily for two days) was injected around the tumor, and the changes in PD-L1 levels in the tumor tissue were detected. The results showed that Atz-AUTAB could rapidly degrade PD-L1 in vivo. This demonstrates that it can also exert its effect of degrading target proteins in animals.

[0167] Example 20 verifies the synergistic effect of Nano-AUTAB and membrane protein antibodies in degrading target proteins.

[0168] (1) Verify that Nano-AUTAB combined with membrane protein antibody weakens the localization of target protein on the cell membrane.

[0169] Taking PD-L1 targeting as an example, HeLa cells stably expressing HA-PD-L1 were seeded at 80% density onto glass slides and cultured overnight. Subsequently, a premixed solution of Atz and Nano-AUTAB or control nanobody (final concentration: 6.25 nM Atz and 25 nM Nano-AUTAB or 25 nM nanobody) was added to the culture medium, and the cells were cultured for another 24 hours. Cells were fixed with 4% PFA, and PD-L1 on the cell membrane was labeled with anti-HA antibody under non-permeable conditions. After immunofluorescence staining, changes in PD-L1 on the cell membrane were analyzed under a laser confocal microscope.

[0170] (2) Detection of Nano-AUTAB co-degradation of various membrane proteins

[0171] Using Western blotting (WB), the efficacy of Nano-AUTAB combined with corresponding membrane protein antibodies for target degradation was evaluated for multiple membrane proteins.

[0172] To target PD-L1, MDA-MB-231 cells were treated for 24 hours with a premix of Atz and Nano-AUTAB or a control nanobody. Cells were then lysed using SDS lysis buffer, and total protein was extracted. Changes in PD-L1 protein levels in the protein samples were analyzed using standard Western blotting. The degradation effects of Nano-AUTAB combined with other PD-L1 antibodies (Abcam, catalog number ab205921; Cell Signaling, catalog number 13684) on the target were also assessed using the same method.

[0173] For CD73, Nano-AUTAB and Ole were used in combination to detect its co-degradation of target proteins, using the same method as described above.

[0174] For Integrinα5, Nano-AUTAB and Integrinα5 antibody were used to detect its co-degradation of target proteins, using the same method as described above.

[0175] Simultaneously, for PD-L1 and EGFR, cells were treated with a premix of Nano-AUTAB, Atz, and Ctx for 24 hours, and then their synergistic degradation of the two target proteins was detected, using the same method as described above.

[0176] like Figure 13 a) This study demonstrated that Nano-AUTAB combined with Atz synergistically induces a sharp reduction in the target protein PD-L1 from the cell membrane. Western blotting showed that Nano-AUTAB combined with Atz efficiently degrades PD-L1. (See [reference needed]) Figure 13 b. In addition, Nano-AUTAB, when combined with other commercially available PD-L1 antibodies, can also induce PD-L1 degradation, see [link to relevant documentation]. Figure 13 c, 13d.

[0177] In addition to PD-L1, Nano-AUTAB was also verified to synergistically degrade membrane proteins such as CD73 and Integrinα5. (See [link to documentation]). Figure 13 e, 13f. Additionally, using Nano-AUTAB in combination with Atz and Ctx demonstrated its ability to synergistically degrade PD-L1 and EGFR simultaneously, see [reference needed]. Figure 13 g.

[0178] (3) Expanding the nano-second antibody and verifying the synergistic degradation of membrane proteins by Nano-AUTAB-m

[0179] The aforementioned Nano-AUTAB recognizes both human and rabbit antibodies. To verify the universality of the strategy, we prepared Nano-AUTAB-m, which recognizes mouse antibodies. For the membrane protein Notch1, a premixed solution of anti-Flag antibody (mouse-derived) and Nano-AUTAB-m was added to HUVEC cells stably expressing Flag-Notch1, and the cells were incubated for 24 hours. Subsequently, Western blotting was used to detect Notch1 degradation.

[0180] like Figure 14 Nano-AUTAB-m was constructed using another nanobody capable of recognizing murine antibodies. Results demonstrated that Nano-AUTAB-m, when combined with a murine anti-Flag antibody, promoted the degradation of Flag-Notch1. This result proves the versatility of the present invention's strategy of constructing degradative agents using conjugates of nano-second antibodies and cationic polymers combined with membrane protein antibodies.

[0181] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A membrane protein-targeting degrader based on autophagy, characterized in that, It is composed of a membrane protein-targeting antibody, a nano-second antibody, and a cationic polymer; the nano-second antibody is linked to the cationic polymer, and the nano-second antibody binds to the membrane protein-targeting antibody; the nano-second antibody and the cationic polymer are linked by coupling. The cationic polymer is a cationic polymer that can induce autophagy by acting on endosomes; the cationic polymer includes one or more of polyethyleneimine and its derivatives, and polylysine; the cationic polymer is modified, and the modifiers include one or more of DBCO and maleimide; the nano-second antibody is modified, and the modifiers include azide C5 succinimide ester. The antibody targeting the membrane protein is an antibody that can recognize the extracellular domain of the membrane protein; the antibody targeting the membrane protein includes one or more of Atezolizumab, Cetuximab, and Oleclumab antibodies.

2. The membrane protein targeted degrader based on autophagy according to claim 1, characterized in that, The membrane proteins include one or more of PD-L1, EGFR, CD73, C-Met, IGF1R, FGFR4, HER2, and PDGFR.

3. A method for preparing the membrane protein-targeting degrader based on autophagy as described in any one of claims 1-2, characterized in that, Includes the following steps: (1) The cationic polymer was linked to the nano-second antibody to obtain a linker of the nano-second antibody and the cationic polymer; (2) The linker from step (1) is combined with the antibody targeting the membrane protein to obtain the degradation agent.

4. The method for preparing the membrane protein-targeting degrader based on autophagy according to claim 3, characterized in that, The cationic polymer is linked to the nano-second antibody using one of the following methods: a. Modify the cationic polymer and the nano-second antibody respectively, and then couple the modified products; b. Modify only the cationic polymer, and then conjugate it with the nano-second antibody; c. Modify only the nano-second antibody, and then couple it with the cationic polymer; d. The nano-second antibody is directly coupled to the cationic polymer.

5. The use of any one of the membrane protein-targeting degraders based on autophagy in the preparation of targeted protein degradation drugs.

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