Autophagy-based degrader targeting membrane protein and use thereof in drug for targeted protein degradation

Abstract

Provided is an autophagy-based degrader targeting a membrane protein, which degrader comprises a membrane protein-targeting antibody, a nano secondary antibody and a cationic polymer, wherein the nano secondary antibody is linked to the cationic polymer, and the nano secondary antibody binds to the membrane protein-targeting antibody. In the autophagy-based degrader targeting a membrane protein, the nano secondary antibody that can bind to the membrane protein-targeting antibody is linked to a cationic polymer with autophagy-inducing properties to form a modular unit of the degrader. Since the nano secondary antibody can bind to numerous membrane protein antibodies derived from specific species, different membrane protein antibodies can be selected for the degradation of target proteins according to requirements without the need to couple each membrane protein antibody, which achieves the purpose of membrane protein degradation while greatly improving the convenience of use.

Description

Membrane protein targeted degraders based on cell autophagy and their applications in targeted protein degradation drugs Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a membrane protein targeted degrader based on cell autophagy, and also to the application of the membrane protein targeted degrader based on cell autophagy in targeted protein degradation drugs. Background Art

[0002] Targeted protein degradation technology is a novel technique that specifically identifies target proteins and directly degrades them through intrinsic cellular protein degradation pathways. It holds significant promise for drug development and biomedical research. Intrinsic cellular protein degradation pathways can be divided into two types: the ubiquitin-proteasome pathway and the lysosomal pathway. In recent years, a variety of targeted protein degradation technologies have been developed utilizing both pathways. These technologies target proteins located within the cell, on the cell membrane, and outside the cell.

[0003] Current technologies targeting cell membrane protein degradation include LYTAC, AbTAC, PROTAB, GlueTAC, and KineTAC. Similar to PROTAB technologies, AbTAC is a bispecific antibody. One Fab fragment of the antibody is used to recognize and bind to the membrane protein, while the other Fab fragment is used to recognize and bind to an E3 ligase on the cell membrane, such as RNF43. This spatially brings the membrane protein and the E3 ligase closer, promoting ubiquitination of the membrane protein by the E3 ligase, thereby inducing endocytosis of the membrane protein into the lysosome for degradation. LYTAC technology involves coupling glycopeptide chains to membrane protein antibodies. These glycopeptide chains act as ligands that bind to the mannose-6-phosphate receptor (M6PR) on the cell membrane. When the antibody-glycopeptide conjugate binds to both the membrane protein and the M6PR, the conjugate leverages the M6PR's endocytic transport to the lysosome to deliver the membrane protein to the lysosome for degradation. KineTAC technology is a complex of an antibody and a cytokine. When the complex binds to a membrane protein and the cytokine receptor CXCR7 at the same time, the complex will use CXCR7 to transport the membrane protein to the lysosome for degradation. GlueTAC technology develops and screens a nano-antibody targeting a membrane protein, and simultaneously couples a membrane-penetrating peptide and a lysosomal sorting peptide to the antibody. When the antibody binds to the membrane protein, it uses the membrane-penetrating peptide to enter the cell and uses the biological signal of the lysosomal sorting peptide to deliver the membrane protein to the lysosome for degradation. As can be seen from the above, these current membrane protein degradation technologies, except for GlueTAC, require the degrader to simultaneously bind to the target protein and the E3 ligase or lysosomal delivery receptor on the cell membrane. Therefore, the preparation technology of the degrader is relatively complex and the spatial conformation needs to be considered. Moreover, since the expression of E3 ligases and lysosomal delivery receptors in various cells varies greatly, this will greatly limit the versatility of the technology in various cells.

[0004] Reported cell membrane protein degradation technologies can be categorized into four approaches based on their specific mechanisms of action. Their shortcomings 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 cell types, resulting in limited versatility and consistency. The degraders constructed must spatially bind to both the target protein and these lysosomal transport receptors, making their design and synthesis challenging. 2) PROTAB and AbTAC technologies rely on E3 ligases on the cell membrane, which are limited by their expression levels. Furthermore, these bispecific antibodies present high development costs. 3) KineTAC technology relies on cytokine receptors on the cell membrane, which are limited by their expression. This results in poor versatility across different cell types, and the degraders are conjugated antibodies to cytokines, making their design more complex. 4) GlueTAC technology uses nanobodies for screening, which contain unnatural amino acids, requiring further safety assessment. The in vivo half-life is short, and the screening cost is high.

[0005] Autophagy is a fundamental cellular function. Through the autophagy mechanism, cells can effectively remove damaged organelles and misfolded protein molecules during life, transporting them to lysosomes for degradation. Therefore, the development of 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 chaperone-mediated autophagy. These technologies are limited to degrading intracellular proteins and organelles, and cannot degrade cell membrane proteins.

[0006] Based on this, the present invention establishes a membrane protein degradation technology based on autophagy, provides a membrane protein targeted degrader based on cell autophagy, connects the nano second antibody with a cationic polymer with the property of inducing cell autophagy, constructs a module of membrane protein degrader, and synergistically degrades targeted membrane proteins by combining different membrane protein antibodies. In this way, one degrader can degrade multiple membrane proteins at the same time, greatly improving the convenience and applicability of use. Technical issues

[0007] The purpose of the present invention is to provide a membrane protein targeted degrader based on cell autophagy to solve the shortcomings of the prior art in cell membrane protein degradation, such as low versatility, complex design and high cost.

[0008] The present invention also aims to provide an application of a membrane protein targeted degrader based on cell autophagy in cell membrane protein degradation drugs. Technical Solutions

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

[0010] A membrane protein targeting degradation agent based on cell autophagy comprises a membrane protein targeting antibody, a nanometer second antibody and a cationic polymer; the nanometer second antibody is connected to the cationic polymer; and the nanometer second antibody is combined with the membrane protein targeting antibody.

[0011] In the present invention, the second nano antibody is connected to the cationic polymer by coupling.

[0012] Furthermore, the nano secondary antibody is connected to the cationic polymer by covalent coupling.

[0013] In the present invention, the cationic polymer is a cationic polymer that acts on endosomes to induce autophagy. This cationic polymer was selected based on its ability to induce autophagy within endosomes, promoting their degradation by lysosomes. Because this autophagy can be universally induced in various cells and its mechanism is independent of the binding of degradation agents to autophagy-related molecules, the technology of the present invention offers the advantages of greater versatility and convenience compared to previous technologies.

[0014] Furthermore, the cationic polymer includes one or more of 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 the present invention, the membrane protein includes one or more of PD-L1, EGFR, CD73, C-Met, IGF1R, FGFR4, HER2, and PDGFR.

[0017] In the present invention, the membrane protein-targeting antibody is an antibody that can recognize the extracellular domain of the membrane protein.

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

[0020] In the present invention, the cells include one of 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 degradation agent of the present invention is applicable to all cells, but the targeting effect of the degradation agent is more pronounced for cells that highly express the above-mentioned specific membrane proteins.

[0021] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, comprising the following steps:

[0022] (1) connecting the cationic polymer to the nano-second antibody to obtain a conjugate of the nano-second antibody and the cationic polymer;

[0023] (2) The linker from step (1) is combined with a membrane protein-targeting antibody to obtain a degrader.

[0024] In the present invention, the cationic polymer is linked to the nano secondary antibody by one of the following methods:

[0025] a. Modifying the cationic polymer and the nanosecond antibody respectively, and then coupling the two modified products;

[0026] b. Only the cationic polymer is modified and then coupled with the nano secondary antibody;

[0027] c. Only the second nanobody is modified and then coupled with the cationic polymer;

[0028] d. directly coupling the nano secondary antibody to the cationic polymer.

[0029] In the present invention, the modifiers for modifying the cationic polymer include one or more of DBCO, maleimide and avidin; the modifiers for modifying the nanosecond 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, and the second antibody is modified with azido C5 succinimidyl ester, and then the two modifications are coupled.

[0031] In some embodiments of the present invention, the cationic polymer is modified with maleimide, and 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, and 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, and the second antibody is modified with biotin, and then the two are coupled.

[0034] An application of the above-mentioned membrane protein targeted degrader based on cell autophagy 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. Beneficial effects

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

[0037] (1) The membrane protein targeted degrader based on cell autophagy of the present invention connects a nanometer second antibody that can bind to an antibody targeting a membrane protein with a cationic polymer that has the property of inducing cell autophagy to form a modular unit of the degrader. Since the nanometer second antibody can bind to many membrane protein antibodies derived from a specific species, different membrane protein antibodies can be used for target protein degradation as needed without the need to couple each membrane protein antibody. This achieves the purpose of membrane protein degradation while greatly improving the convenience of use.

[0038] (2) The degrader of the present invention does not require a separate synthesis of a degrader for each membrane protein. Instead, a single Nano-AUTAB can be prepared and combined with a commercial membrane protein antibody to efficiently degrade the target, which has the advantage of convenient preparation. The nano-second antibody is coupled to the cationic polymer in a simple step, and the preparation does not need to consider factors such as steric hindrance. Membrane protein antibodies are readily available on the market, which greatly reduces the construction cost of the degrader. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The present invention is described in detail below with reference to the embodiments and accompanying drawings, in which:

[0040] FIG1 is a schematic diagram of targeted degradation of membrane proteins based on autophagy;

[0041] FIG2 is a diagram showing the synthesis and characterization of Atz-AUTAB;

[0042] FIG3 is a diagram showing the autophagy associated with endosomes induced by cationic polymer PEI;

[0043] FIG4 is a diagram showing the situation in which AUTAB binds to the target protein for endocytosis and induces autophagy in the endosome;

[0044] FIG5 is a diagram showing how AUTAB promotes endocytosis and transport of target proteins to lysosomes;

[0045] FIG6 is a flow cytometry data diagram of AUTAB clearing target proteins from cell membranes;

[0046] FIG7 is a diagram showing the degradation of target proteins induced by AUTAB;

[0047] FIG8 is an experimental data diagram demonstrating that AUTAB promotes target protein degradation through the autophagy-lysosome pathway;

[0048] FIG9 is a diagram showing how AUTAB coupled with various PEIs and poly-L-Lys promotes target protein degradation;

[0049] FIG10 is a diagram showing the in vivo validation of Atz-AUTAB degradation of PD-L1 in tumor tissues;

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

[0051] FIG12 is a synthetic route of Nano-AUTAB;

[0052] Figure 13 shows the synergistic degradation of various target proteins by Nano-AUTAB combined with membrane protein antibodies;

[0053] FIG14 shows that Nano-AUTAB-m synergistically promotes the degradation of membrane protein Notch1. Modes for Carrying Out the Invention

[0054] To make the purpose, technical solutions and advantages of the present invention more clear, the following will further describe the embodiments of the present invention in detail with reference to specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0055] The process of targeted degradation of membrane proteins based on cellular autophagy of the present invention is shown in Figures 1 and 11, providing a technology for developing targeted protein degradation using the autophagy-lysosome pathway. Figure 1 shows how an antibody targeting a membrane protein is linked to a cationic polymer to construct a degrader. The polymer is internalized and simultaneously damages the endosome to induce autophagy, thereby inducing endocytosis of the membrane protein to lysosomes for degradation. Figure 11 is a further improvement on the pathway of Figure 1, where a nanosecond antibody capable of binding to the antibody targeting the membrane protein is linked to a cationic polymer, which is then used in conjunction with the antibody targeting the membrane protein to synergistically degrade the target protein.

[0056] Example 1

[0057] As shown in FIG2 , the method for preparing a membrane protein targeted degradation agent based on cell autophagy comprises 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 dissolve the PEI material by ultrasonic treatment to form a transparent clear solution. Then, add 1M sodium carbonate solution to the above solution to adjust the pH value to 9.0, followed by adding 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 reaches 5 mg / mL (based on PEI material). The reaction system is shaken at room temperature for 24 hours. The resulting L25K-DBCO solution can be used directly in subsequent reactions without further purification.

[0060] Linear PEI with various other molecular weights, including but not limited to Mw=2500, Mw=5000, and Mw=10000, were DBCO-modified according to the above synthesis method for subsequent antibody coupling.

[0061] Various linear PEI derivatives, including but not limited to acetylated PEI and PEGylated PEI, were DBCO-modified according to the above-mentioned synthesis method for subsequent antibody coupling.

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

[0063] Take Atz-N3 as an example: Measure 29.2 μl of Atz antibody (Atezolizumab) stock solution (0.25 ml

[0064] 100 μL of PBS solution (pH = 7.3) and 3 μL of 1 M sodium carbonate solution were added to a clean centrifuge tube, followed by the addition of 100 μL of PBS solution (pH = 7.3) and 3 μL of 1 M sodium carbonate solution. The pH of the mixture was slightly higher than 9.0. Finally, 5.7 μL of N3-C5-NHS (azido C5 succinimidyl ester) (22 μg, 50.0 equivalents, 3.82 mg / mL, DMSO as solvent) was added. The reaction solution was 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 solution to obtain 1.0 ug / uL of Atz-N3 antibody stock solution.

[0065] Mix 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 equivalents, 5.0 mg / mL) and shake at room temperature for 24 hours. Once the reaction is complete, transfer the solution to an ultrafiltration tube (4 mL, 30 kDa), concentrate, and wash three times with 2% DMSO / PBS to obtain a 1.0 μg / μL Atz-AUTAB stock solution.

[0066] Other antibodies include but are not limited to Ctx antibody (Cetuximab) and Ole antibody (Oleclumab), and their modification and coupling with PEI are exactly the same as the above preparation process.

[0067] Example 2

[0068] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, which is different from Example 1

[0069] The difference lies in the label modification process. Specifically, the linear PEI material is modified with FITC fluorescent label:

[0070] Weigh 20 mg of linear PEI material (L25K-PEI, Mw=25000) in a clean centrifuge tube.

[0071] 500 μL of ultrapure water and 500 μL of 1 M hydrochloric acid were added sequentially, and the PEI material was dissolved by sonication to form a clear, transparent solution. 1 M sodium carbonate solution was then added to adjust the pH to 9.0. Finally, 1.25 mg of FITC powder (4.0 equivalents) was added and mixed thoroughly. The reaction mixture was protected from light and shaken at room temperature for 24 hours. Upon completion of the reaction, the solution was dialyzed and freeze-dried to yield 12.8 mg of a red powder (L25K-FITC) in a 60% yield.

[0072] Example 3

[0073] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, which is different from Example 1

[0074] The difference lies in the tag modification process. Specifically, the branched PEI material is modified with DBCO tag:

[0075] Taking B2K-PEI as an example: Weigh 4 mg of branched PEI material (B2K-PEI, Mw=2000) into a clean centrifuge tube and dissolve it in 500 μL of PBS solution (pH=9.0). Next, add 50.3 μL of DBCO-NHS stock solution (1.0 equivalent, 8 mg / mL, DMSO as solvent). Finally, add more PBS solution (pH=9) until the final concentration of the reaction solution reaches 1 mg / mL (based on PEI material). Shake the reaction system at room temperature for 24 hours. The resulting B2K-DBCO solution can be used directly in subsequent reactions without further purification.

[0076] Branched PEIs of various other molecular weights, including but not limited to Mw=800 and Mw=25000, were DBCO-modified according to the above synthesis method for subsequent antibody coupling.

[0077] Various branched PEI derivatives, including but not limited to acetylated PEI and PEGylated PEI, were DBCO-modified according to the above-mentioned synthesis method for subsequent antibody coupling.

[0078] Example 4

[0079] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, comprising the following steps:

[0080] (1) DBCO tag modification of polylysine (PolyLys)

[0081] 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. Finally, add pH=9 PBS solution until the final concentration of the reaction solution reaches 5 mg / mL (based on PEI material). Shake the reaction system at room temperature for 24 hours. The resulting PolyLys(15-30K)-DBCO solution can be used directly in subsequent reactions without further purification.

[0082] Polylysine of various other molecular weights, including but not limited to Mw=3000-7000 and Mw=4000-15000, is DBCO-modified according to the above synthesis method for subsequent antibody coupling.

[0083] (2) Preparation of antibody-Poly-Lys material conjugates

[0084] Take Atz-N3 as an example: Measure 29.2 μl of Atz antibody stock solution (0.25 mg, 1.0 equivalent, 8.58

[0085] mL / mg, PBS as solvent, pH=7.3) was added to a clean centrifuge tube, followed by the addition of 100 μL PBS solution (pH=7.3) and 3 μL 1 M sodium carbonate solution. At this time, the pH of the mixture was slightly higher than 9.0. Finally, 5.7 μL N3-C5-NHS (22 μg, 50.0 equivalents, 3.82 mg / mL, DMSO as solvent) was added. The reaction solution was 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 solution to obtain 1.0 ug / uL Atz-N3 antibody stock solution.

[0086] Mix 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) and shake at room temperature for 24 hours. Once the reaction is complete, transfer the solution to an ultrafiltration tube (4 mL, 30 kDa), concentrate, and wash three times with 2% DMSO / PBS to obtain a 1.0 μg / μL stock solution of PolyLys-Atz-AUTAB.

[0087] Other antibodies include but are not limited to Ctx antibodies and Ole antibodies, and their modification and coupling with polylysine are exactly the same as the above preparation process.

[0088] Example 5

[0089] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, which is different from Example 1

[0090] The method is to only modify PEI with maleimide without modifying the antibody, and then couple PEI to the antibody using the disulfide bond on the antibody.

[0091] Example 6

[0092] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, which is different from Example 1

[0093] The method is to introduce only non-natural amino acid modifications to the antibody without modifying PEI, and then use the non-natural amino acid to undergo a condensation reaction with PEI to complete the coupling of PEI and the antibody.

[0094] Example 7

[0095] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, which is different from Example 1

[0096] The method comprises modifying the antibody with biotin and modifying PEI with avidin, and then utilizing the non-covalent binding between biotin and avidin to form a non-covalent coupling between PEI and the antibody.

[0097] Example 8

[0098] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, different from Examples 1 to 7

[0099] The difference is that the cationic polymer used is polyallylamine, and the other preparation processes are the same.

[0100] Embodiment 9

[0101] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, different from Examples 1 to 7

[0102] The difference is that the cationic polymer used is poly[N-(2-hydroxypropyl)methacrylamide], and the other preparation processes are the same.

[0103] Example 10

[0104] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, different from Examples 1 to 7

[0105] The difference is that the cationic polymer used is poly(2-(dimethylamino)ethyl methacrylate) and the other preparation processes are the same.

[0106] Example 11

[0107] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, different from Examples 1 to 7

[0108] The difference is that the cationic polymer used is polyornithine, and the other preparation processes are the same.

[0109] Example 12

[0110] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, different from Examples 1 to 7

[0111] The difference is that the cationic polymer used is polyarginine, and the other preparation processes are the same.

[0112] Example 13

[0113] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, different from Examples 1 to 7

[0114] The difference is that the cationic polymer used is chitosan, and the other preparation processes are the same.

[0115] In the above examples of the present invention, different antibodies were chemically coupled to different cationic polymers with autophagy-inducing properties to prepare different membrane protein-targeted degraders. By coupling different cationic polymers to the antibody Atz, the corresponding Atz-AUTABs were obtained. This method was also validated on different antibodies, ultimately resulting in the synthesis of a variety of membrane protein degraders, including Atz-AUTAB, Ctx-AUTAB, and Ole-AUTAB. The experimental conditions were reproducible and the yields were stable.

[0116] Example 14

[0117] As shown in FIG12 , the method for preparing a membrane protein targeted degradation agent based on cell autophagy comprises the following steps:

[0118] (1) MA modification of PEI material

[0119] For example, consider L2.5K-MA: 6 mg of linear polyethyleneimine (PEI) (L2.5K-PEI, Mw=2500) was weighed into a clean centrifuge tube. 200 μL of ultrapure water and 200 μL of 1 M hydrochloric acid were added, sequentially. The PEI was dissolved by sonication to form a clear solution. Tris-HCl buffer (pH=8.68) was added to the solution to adjust the pH to 8.0. Next, 148 μL of MA-C6-NHS solution (1.0 equivalent, 5 mg / mL, in DMSO) was added, resulting in a final concentration of 3.0 mg / mL (based on PEI). The reaction was shaken at room temperature for 3 hours. The resulting L2.5K-MA solution was used directly in subsequent reactions without further purification.

[0120] Linear PEI of various other molecular weights, including but not limited to Mw=5000 and Mw=10000, were MA-modified according to the above synthesis method for subsequent nanobody coupling.

[0121] Branched PEI of various molecular weights, including but not limited to Mw=800, Mw=2000, and Mw=25000, were MA-modified according to the above-mentioned synthesis method for subsequent nanobody coupling.

[0122] Various PEI derivatives, including but not limited to acetylated PEI and PEGylated PEI, were modified with MA according to the above-mentioned synthesis method for subsequent nanobody coupling.

[0123] In addition to PEI, the above MA modification method and preparation process are also applicable to polylysine (PolyLys), polyallylamine, poly[N-(2-hydroxypropyl)methacrylamide], poly[2-(dimethylamino)ethyl methacrylate], polyornithine, polyarginine, and chitosan.

[0124] (2) The nano-second antibody reacts with the L2.5K-MA solution to obtain a conjugate of the nano-second antibody and the cationic polymer, Nano-AUTAB.

[0125] Mix 125 μL of 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) and shake at room temperature for 3 hours. Once the reaction is complete, add 80.5 μL of N-acetylcysteine ​​solution (25.0 equivalents, 1 mg / mL) and continue shaking for 30 minutes to consume any unreacted starting material. The final mixture is transferred to an ultrafiltration tube (4 mL, 3 KDa), concentrated, and washed three times with 2% DMSO / PBS to obtain a 1.0 μg / μL stock solution of Nano-AUTAB.

[0126] (3) The Nano-AUTAB stock solution was combined with the Atz antibody targeting PD-L1 to obtain a membrane protein targeted degrader based on cell autophagy.

[0127] Other membrane protein antibodies include but are not limited to Ctx antibody (Cetuximab) and Ole antibody (Oleclumab), and their preparation process is exactly the same as above.

[0128] Example 15

[0129] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, comprising the following steps:

[0130] (1) DBCO tag modification of PEI material

[0131] 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 dissolve the PEI material by ultrasonic treatment to form a transparent clear solution. Then, add 1M sodium carbonate solution to the above solution to adjust the pH value to 9.0, followed by adding 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 reaches 5 mg / mL (based on PEI material). The reaction system is shaken at room temperature for 24 hours. The obtained L2.5K-DBCO solution can be used directly in subsequent reactions without further purification.

[0132] (2) The nano-second antibody reacts with L2.5K-DBCO solution to obtain a conjugate of the nano-second antibody and cationic polymer, Nano-AUTAB

[0133] Taking the nano secondary antibody-N3 as an example: Measure 240 μL of nano antibody stock solution (0.24 mg, 1.0 equivalent, 1.0 mL / mg, PBS as solvent, pH=7.3) and add it to a clean centrifuge tube. Then add 6 μL of 1 M sodium carbonate solution. At this time, the pH of the mixture is slightly higher than 9.0. Finally, add 25.4 μL of N3-C5-NHS (0.203 mg, 50.0 equivalent, 8.0 mg / mL, DMSO as solvent). The reaction solution is shaken at room temperature for 24 hours. After the reaction is complete, the reaction solution is transferred to an ultrafiltration tube (4 mL, 3 KDa), concentrated and washed three times with 2% DMSO / PBS solution as the washing solution to finally obtain 1.0 ug / uL of nano secondary antibody-N3 stock solution.

[0134] Mix 240 μL of Nano-AUTAB secondary antibody 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) and shake at room temperature for 24 hours. Once the reaction is complete, transfer the solution to an ultrafiltration tube (4 mL, 3 kDa), concentrate, and wash three times with 2% DMSO / PBS solution to obtain a 1.0 μg / μL Nano-AUTAB stock solution.

[0135] (3) The Nano-AUTAB stock solution was combined with the Atz antibody to obtain a membrane protein targeted degrader based on cell autophagy.

[0136] Example 16

[0137] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, different from Example 15

[0138] The difference is that only the second antibody is modified with a non-natural amino acid, and the PEI is not modified. Subsequently, the non-natural amino acid is used to undergo a condensation reaction with PEI to complete the coupling of PEI and the second antibody.

[0139] Example 17

[0140] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, different from Example 15

[0141] The method is characterized in that the second antibody is modified with biotin, PEI is modified with avidin, and then the non-covalent binding between biotin and avidin is utilized to form a non-covalent coupling between PEI and the second antibody.

[0142] In the above embodiment of the present invention, the second antibody is coupled with different cationic polymers with the property of inducing cell autophagy by chemical methods to form a modular unit of the degrader, which is then matched with different membrane protein antibodies to obtain the corresponding Nano-AUTAB.

[0143] Example 18 Verification of AUTAB-induced endosome-related autophagy

[0144] (1) Verify that cationic polymers induce autophagy.

[0145] HeLa cells stably expressing mCherry-LC3C were cultured on slides. After reaching approximately 50% confluency, various types and sizes of PEI or FITC-labeled L25K-PEI were added to the culture medium and cultured for an additional hour. The cells were then fixed with 4% PFA and the formation of mCherry-LC3C-positive autophagosomes and the colocalization of endocytosed FITC-PEI with autophagosomes were observed under a laser confocal microscope.

[0146] HeLa cells stably expressing mCherry-LC3C were cultured on glass slides and transfected with the EGFP-Rab5 plasmid. 24 hours after transfection, PEI was added and incubated for 1 hour. The cells were then fixed and the colocalization of the autophagosome marker mCherry-LC3C and the early endosome marker EGFP-Rab5 was examined under a laser confocal microscope.

[0147] As shown in Figure 3a, punctate fluorescence signals of mCherry-LC3C appeared in cells after treatment with various sizes of linear (L2.5K, L25K) and branched (B800, B2K, B25K) PEI, demonstrating that PEI can induce LC3C-mediated autophagy. Furthermore, as shown in Figure 3b, the endocytosed FITC-PEI fluorescence signal colocalized with these mCherry-LC3C signals, indicating that autophagy occurs within PEI-containing endosomes. As shown in Figure 3c, these mCherry-LC3C signals colocalized with the early endosome marker EGFP-Rab5, indicating that autophagy occurs early during PEI internalization.

[0148] (2) Detection of AUTAB-induced autophagy.

[0149] In the present invention, cationic polymers are coupled to membrane proteins to construct AUTAB. Based on the autophagy-inducing properties of cationic polymers, a series of AUTAB-induced autophagy tests were conducted.

[0150] For Atz-AUTAB, HeLa cells stably expressing HA-PD-L1 and mCherry-LC3C were cultured on slides. After cells reached approximately 50% confluency, Atz-AUTAB or the control antibody Atz was added to the culture medium at a final concentration of 100 nM and incubated in an incubator for 1 hour. The cells were then fixed with 4% PFA and immunostained using a standard immunofluorescence protocol. Atz-AUTAB and the control Atz were labeled with anti-human IgG, anti-HA tag antibodies for PD-L1, anti-Rab5 antibodies for early endosomes, and anti-Rab7 antibodies for late endosomes. Following immunostaining, the slides were mounted with an anti-quencher and the colocalization of the autophagy marker mCherry-LC3C, Atz-AUTAB, HA-PD-L1, and early and late endosomes was observed and detected under a laser confocal microscope.

[0151] For PolyLys-Atz-AUTAB, the co-localization relationship among the autophagy markers mCherry-LC3C, PolyLys-Atz-AUTAB, and HA-PD-L1 was analyzed using the same method as described for Atz-AUTAB.

[0152] For Ctx-AUTAB (Cetuximab, abbreviated as Ctx), the colocalization of the autophagy marker mCherry-LC3C, Ctx-AUTAB, and EGFR was analyzed using a similar method. HeLa cells stably expressing mCherry-LC3C were cultured on slides and treated with Ctx-AUTAB or the control antibody Ctx at a final concentration of 100 nM for 1 hour. The cells were then fixed and labeled with anti-human IgG antibodies for Ctx-AUTAB and the control Ctx. Anti-EGFR antibodies were also used to label the membrane protein EGFR. After immunostaining, analysis and detection were performed using confocal laser scanning microscopy.

[0153] Metrics: The number of LC3 punctate signals shown in the image, and the colocalization of LC3 and AUTAB signals.

[0154] Taking Atz-AUTAB targeting PD-L1 as an example, as shown in Figure 4a, endocytosed Atz-AUTAB significantly colocalizes with HA-PD-L1, indicating that it can recognize and bind to PD-L1 and be internalized along with PD-L1. Furthermore, compared to Atz itself, Atz-AUTAB induces more mCherry-LC3C punctate signals, which colocalize with these LC3C autophagy signals, indicating that Atz-AUTAB can induce cellular autophagy after internalization. Furthermore, as shown in Figures 4b and 4c, these Atz-AUTAB colocalize with LC3C and the endosome markers Rab5 or Rab7, indicating that after Atz-AUTAB internalization, autophagy occurs in endosomes at both early and late stages of the endocytic pathway. Similar autophagy induction has also been demonstrated for Ctx-AUTAB targeting EGFR (see Figure 4d). More importantly, AUTAB constructed using other cationic polymers, such as poly-L-Lys, also induced a large number of mCherry-LC3C punctate signals in cells, which colocalized with these LC3C signals, indicating that they can also induce autophagy (see Figure 4e). These results demonstrate that AUTAB with autophagy effects can be constructed based on a variety of cationic polymers.

[0155] Example 19 Verification of the Degradation Effects of Various AUTABs on Various Membrane Proteins

[0156] (1) Detection of AUTAB promoting the endocytosis and transport of membrane proteins to lysosomes

[0157] For Atz-AUTAB, HeLa cells stably expressing HA-PD-L1 were cultured on slides and transfected with the RFP-Lamp1 plasmid. Twenty-four hours after transfection, cells were treated with Atz-AUTAB or the control antibody Atz at a final concentration of 5 nM for one hour. The cells were then fixed and immunofluorescently stained with an anti-HA tag antibody to label PD-L1. Following immunostaining, colocalization of the lysosomal marker RFP-Lamp1 with the target protein PD-L1 was analyzed using confocal laser scanning microscopy.

[0158] For Ctx-AUTAB, the method was similar to that described above. HeLa cells were cultured on glass slides and transfected with the RFP-Lamp1 plasmid. Twenty-four hours after transfection, cells were treated with Ctx-AUTAB or the control antibody Ctx at a final concentration of 5 nM for one hour. Immunofluorescence staining with an anti-EGFR antibody was then performed, and colocalization was observed under a microscope.

[0159] For Ole-AUTAB, HeLa cells stably expressing mCherry-CD73 were cultured on glass slides. When cells reached 80% confluency, they were treated with Ole-AUTAB or the control antibody Ole at a final concentration of 100 nM for 12 hours. Lysosomes were then labeled with the LysoTracker fluorescent probe in living cells, and the colocalization of lysosomes and mCherry-CD73 was observed and detected in real time under a laser confocal microscope.

[0160] As shown in Figure 5a, compared to the Atz-treated group, Atz-AUTAB treatment resulted in a significant amount of PD-L1 endocytosis and co-localization 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 both endocytosed into lysosomes in large quantities, as shown in Figures 5b and 5c.

[0161] (2) Verification that AUTAB weakens the localization of target proteins on the cell membrane

[0162] Live cell flow cytometry was used to analyze the changes in the localization of the target protein on the cell membrane under the action of AUTAB.

[0163] For Atz-AUTAB, MDA-MB-231 cells or HeLa cells stably expressing HA-PD-L1 were seeded into 12-well plates at 80% density and cultured overnight. The next day, cells were treated with 5 nM Atz-AUTAB or the control antibody Atz for a specified period of time (0.5, 2, 6, and 24 hours for 231 cells and 4 hours for HeLa cells). PD-L1 on the cell membrane was then labeled with an antibody that recognizes the extracellular domain of PD-L1 while living at 4°C. The signal was further amplified using a corresponding fluorescent secondary antibody. After immunofluorescence labeling, labeled PD-L1 was detected by flow cytometry.

[0164] For Ctx-AUTAB, the method was similar to that described above. HeLa cells were treated with 5 nM Ctx-AUTAB or the control antibody Ctx for 4 hours, and then EGFR on the cell membrane was labeled with an antibody that recognizes the EGFR extracellular domain. Fluorescent secondary antibody staining and flow cytometric analysis were performed.

[0165] For Ole-AUTAB, HeLa cells stably expressing mCherry-CD73 were treated with 100 nM Ole-AUTAB or control antibody Ole for 12 hours, and then the target protein on the membrane was labeled with anti-mCherry antibody, and further fluorescent secondary antibody staining and flow cytometry analysis were performed.

[0166] As shown in the flow cytometry data in Figure 6, treatment of cells with Atz-AUTAB, Ctx-AUTAB, and Ole-AUTAB all resulted in a sharp decrease in the corresponding target proteins from the membrane. Combined with the fluorophore localization shown in Figure 5, it was observed that treatment of cells with Atz-AUTAB, Ctx-AUTAB, and Ole-AUTAB resulted in a large amount of target proteins being internalized into lysosomes, indicating that under the action of AUTAB, the target proteins on the membrane can be rapidly internalized from the membrane and transported to the lysosome.

[0167] (3) Detection of AUTAB degradation of various membrane proteins

[0168] Western blotting (WB) was used to evaluate the degradation effects of a series of AUTABs on the corresponding target proteins in various cell lines.

[0169] 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 (using a concentration gradient) or the control Atz solution for 24 hours or the duration of the gradient. Total protein samples were then obtained using SDS lysis buffer and analyzed for changes in PD-L1 protein levels using standard Western blotting methods.

[0170] 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 that for Atz-AUTAB.

[0171] 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.

[0172] AUTAB coupled with different types and sizes of PEI (L25K, L2.5K, B25K, B2K, B800) was tested for degradation of target proteins PD-L1, EGFR, or CD73 in MDA-MB-231 cells, HeLa cells, and U87-MG cells, respectively, using the same method as described above for Atz-AUTAB.

[0173] For Atz-AUTAB conjugated with poly-L-Lys, its degradation of PD-L1 was detected in MDA-MB-231 cells using the same method as described above for Atz-AUTAB.

[0174] Western blotting experiments demonstrated that the constructed series of AUTABs can all degrade target proteins. As shown in Figures 7a-c, Atz-AUTAB, at an extremely low concentration (0.09 nM), significantly degraded PD-L1 within 24 hours. At 6.25 nM, target protein degradation was observed in as little as 12 hours. As shown in Figure 7d, Atz-AUTAB was validated for target protein degradation in various cell lines (U87-MG, NCI-H292, NCI-H1975, and A375). Furthermore, using HeLa cells overexpressing PD-L1, Figures 7e-g demonstrate that Atz-AUTAB can degrade even highly expressed PD-L1. Similarly, as shown in Figures 7h-j, Ctx-AUTAB demonstrated its ability to degrade EGFR at lower concentrations and in a shorter time, with consistent efficacy across multiple cell lines. In addition to targeting transmembrane proteins, Ole-AUTAB was also developed for the membrane-anchored protein CD73. Its concentration- and time-dependent effects were also verified, and its applicability to multiple cells was also demonstrated (see Figures 7k-m). These results demonstrate the universal applicability of the AUTAB technology of the present invention.

[0175] (4) Verification that AUTAB degrades target proteins through the autophagy-lysosome pathway

[0176] AUTAB was shown to degrade target proteins through the autophagy pathway. Using Atz-AUTAB as an example, RNAi was used to knock down the expression of the autophagy core genes ATG5 or LC3C in MDA-MB-231 cells. The cells were then treated with Atz-AUTAB (6.25 nM) for 12 hours. Total protein was then extracted from the cells and the changes in PD-L1 expression were detected using Western blotting.

[0177] To demonstrate that AUTAB degrades target proteins through the lysosome, MDA-MB-231, HeLa, and U87-MG cells were treated with AUTAB and the lysosomal inhibitors bafilomycin A1 (100 nM) or chloroquine (200 μM) were added to the culture medium and cultured for 24 hours. Total protein was then extracted from the cells and the target protein levels were determined by Western blotting.

[0178] As shown in Figure 8a, under conditions of autophagy inhibition, Atz-AUTAB was unable to degrade PD-L1, indicating that it requires autophagy to function. Subsequently, cells were treated with lysosomal inhibitors (bafilomycin A1 and chloroquine) to test whether Atz-AUTAB could still function as a degradation agent. The results showed that Atz-AUTAB was completely unable to promote target degradation, indicating that its function depends on lysosomal activity (see Figure 8b). Furthermore, the same method was used to demonstrate that both Ctx-AUTAB and Ole-AUTAB degrade target proteins through the lysosomal pathway (see Figures 8c and 8d). These results demonstrate that AUTAB degrades target proteins through the autophagy-lysosomal pathway.

[0179] In addition to the aforementioned demonstration of degradation efficacy using AUTAB conjugated with L25K-PEI, the targeted degradation efficacy of AUTAB conjugated with other PEIs and other cationic polymers, poly-L-Lys, was further demonstrated. As shown in Figures 9a-d, both linear (L2.5K, L25K) and branched (B800, B2K, B25K) PEIs significantly promoted target protein degradation after conjugation to the corresponding antibodies. Atz-AUTAB constructed using two poly-L-Lys size ranges (4-15K and 15-30K) also significantly degraded PD-L1 within a 1 nM concentration range (see Figure 9e). These results demonstrate that a variety of cationic polymers can be used in the membrane protein targeted degradation strategy proposed in this invention.

[0180] (5) Verification of AUTAB degradation of membrane proteins in animals

[0181] Using Atz-AUTAB as an example, a subcutaneous tumor model was first established in nude mice (8 weeks old, BALB / c strain) bearing MDA-MB-231 cells. When the tumor volume reached approximately 50 mm³, Atz-AUTAB or the control antibody Atz was injected peritumorally (at a dose of 0.01 nmol / 100 μL, once daily for two consecutive days). The day after injection, tumor tissue was harvested and lysed for total protein extraction. Changes in PD-L1 expression in the tumor tissue were then detected using Western blotting.

[0182] As shown in Figure 10, a nude mouse tumor model bearing MDA-MB-231 cells was established. Atz-AUTAB (0.01 nmol / injection, once daily for two times) was injected peritumorally. Changes in the amount of PD-L1 in tumor tissue were measured. The results showed that Atz-AUTAB could rapidly degrade PD-L1 in vivo. This demonstrates that it can also degrade target proteins in animals.

[0183] Example 20 Verification of the effect of Nano-AUTAB and membrane protein antibody on synergistic degradation of target protein

[0184] (1) Verification of Nano-AUTAB combined with membrane protein antibodies to weaken the localization of target proteins on the cell membrane

[0185] For example, targeting PD-L1, HeLa cells stably expressing HA-PD-L1 were seeded at 80% density onto glass slides and cultured overnight. A premix of Atz and Nano-AUTAB or a control nanobody (final concentration: 6.25 nM Atz and 25 nM Nano-AUTAB or 25 nM nanobody) was then added to the culture medium, and the cells were cultured for another 24 hours. The cells were fixed with 4% PFA, and PD-L1 on the cell membrane was labeled under non-permeabilized conditions using an anti-HA antibody. Following immunofluorescence staining, changes in PD-L1 expression on the cell membrane were analyzed under a laser confocal microscope.

[0186] (2) Detection of Nano-AUTAB synergistic degradation of various membrane proteins

[0187] Western blotting (WB) was used to evaluate the effect of Nano-AUTAB combined with corresponding membrane protein antibodies on the degradation of multiple membrane proteins.

[0188] For PD-L1, MDA-MB-231 cells were treated with a premix of Atz and Nano-AUTAB or a control nanobody for 24 hours. The cells were then lysed with SDS lysis buffer, and total protein was extracted. Protein samples were analyzed for changes in PD-L1 protein levels using standard Western blotting. The same method was used to examine the degradation effects of Nano-AUTAB in combination with other PD-L1 antibodies (Abcam, Cat. No. ab205921; Cell Signaling, Cat. No. 13684).

[0189] For CD73, Nano-AUTAB was used in combination with Ole to detect their synergistic degradation of the target protein using the same method as described above.

[0190] For Integrin α5, Nano-AUTAB and Integrin α5 antibody were used to detect their synergistic degradation of target protein using the same method as above.

[0191] For both PD-L1 and EGFR, cells were treated with a premix of Nano-AUTAB, Atz, and Ctx for 24 hours, and their synergistic degradation of the two target proteins was subsequently detected using the same method as described above.

[0192] As shown in Figure 13a, Nano-AUTAB combined with Atz can synergistically induce a dramatic reduction of the target protein PD-L1 from the cell membrane. Using Western blotting, Nano-AUTAB combined with Atz was found to be highly effective in degrading PD-L1 (see Figure 13b). Furthermore, Nano-AUTAB combined with other commercially available PD-L1 antibodies also induced PD-L1 degradation (see Figures 13c and 13d).

[0193] In addition to PD-L1, Nano-AUTAB was also shown to synergistically degrade membrane proteins such as CD73 and Integrin α5 (see Figures 13e and 13f). Furthermore, using Nano-AUTAB in combination with Atz and Ctx demonstrated its ability to synergistically degrade both PD-L1 and EGFR (see Figure 13g).

[0194] (3) Expanding the nano secondary antibody and verifying the synergistic degradation of membrane proteins by Nano-AUTAB-m

[0195] The aforementioned Nano-AUTAB recognizes both human and rabbit antibodies. To validate the versatility of this strategy, we prepared Nano-AUTAB-m, which recognizes mouse antibodies. For the membrane protein Notch1, a premix of anti-Flag antibody (mouse) and Nano-AUTAB-m was added to HUVEC cells stably expressing Flag-Notch1 and incubated for 24 hours. Notch1 degradation was then detected using Western blotting.

[0196] As shown in Figure 14, Nano-AUTAB-m was constructed using another nanobody that recognizes a mouse antibody. The results demonstrated that this Nano-AUTAB-m, when combined with a mouse anti-Flag antibody, can promote the degradation of Flag-Notch1. This result demonstrates the versatility of the present invention's strategy of constructing degraders using a conjugate of a nanosecondary antibody and a cationic polymer in combination with a membrane protein antibody.

[0197] The above disclosure is merely a preferred embodiment of the present invention and certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A membrane protein targeted degradation agent based on cell autophagy, characterized in that: The invention comprises a membrane protein targeting antibody, a nanometer second antibody and a cationic polymer; the nanometer second antibody is connected to the cationic polymer, and the nanometer second antibody is combined with the membrane protein targeting antibody.

2. The membrane protein targeted degradation agent based on cell autophagy according to claim 1, characterized in that: The nanometer second antibody is connected to the cationic polymer by coupling.

3. The membrane protein targeted degradation agent based on cell autophagy according to claim 2, characterized in that: The cationic polymer is a cationic polymer that acts on endosomes and can induce autophagy.

4. The membrane protein targeted degradation agent based on cell autophagy according to claim 3, characterized in that: The cationic polymer includes one or more of polyethyleneimine and its derivatives, polylysine, polyacrylamine, poly[N-(2-hydroxypropyl)methacrylamide], polydimethylaminoethyl methacrylate, polyornithine, polyarginine, and chitosan.

5. The membrane protein targeted degradation agent based on cell autophagy according to claim 2, characterized in that: The membrane protein includes one or more of PD-L1, EGFR, CD73, C-Met, IGF1R, FGFR4, HER2, and PDGFR.

6. The membrane protein targeted degradation agent based on cell autophagy according to claim 5, characterized in that: The antibody targeting membrane protein is an antibody that can recognize the extracellular domain of membrane protein.

7. A method for preparing a membrane protein targeted degradation agent based on cell autophagy, characterized in that: The following steps are involved: (1) connecting the cationic polymer to the nano-second antibody to obtain a connection product of the nano-second antibody and the cationic polymer; (2) The linker of step (1) is combined with a membrane protein targeting antibody to obtain a degrader.

8. The method for preparing a membrane protein targeted degradation agent based on cell autophagy according to claim 7, characterized in that: The cationic polymer is connected to the nano secondary antibody by one of the following methods: a. modifying the cationic polymer and the nanosecond antibody respectively, and then coupling the modified products of the two; b. Only the cationic polymer is modified and then coupled with the nano secondary antibody; c. Only the nanosecond antibody is modified and then coupled with the cationic polymer; d. directly coupling the nano secondary antibody to the cationic polymer.

9. The method for preparing a membrane protein targeted degradation agent based on cell autophagy according to claim 8, characterized in that: The modifiers for modifying the cationic polymer include one or more of DBCO, maleimide and avidin; the modifiers for modifying the nano secondary antibody include one or more of azide C5 succinimide ester, non-natural amino acids and biotin.

10. Use of the membrane protein targeted degrader based on cell autophagy according to any one of claims 1 to 6 in the preparation of targeted protein degradation drugs.

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