Membrane protein targeted degradation agent based on cell autophagy and application of membrane protein targeted degradation agent in targeted protein degradation medicine

Abstract

The invention provides a membrane protein targeted degradation agent based on cell autophagy. The membrane protein targeted degradation agent comprises a targeted membrane protein antibody, a nano second antibody and a cationic polymer, the second nano antibody is connected with the cationic polymer; the second nano antibody is combined with the targeted membrane protein antibody. According to the membrane protein targeted degradation agent based on cell autophagy, the nano second antibody capable of being combined with the antibody of the targeted membrane protein is connected with the cationic polymer with the characteristic of inducing cell autophagy to form a module unit of the degradation agent, and the nano second antibody can be combined with a plurality of membrane protein antibodies derived from specific species; therefore, different membrane protein antibodies can be matched according to needs to be used for degrading the target protein, and each membrane protein antibody does not need to be coupled, so that the purpose of degrading the membrane protein is achieved, and the use convenience is greatly improved.

Description

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 new technology that specifically identifies target proteins and directly degrades target proteins using the intrinsic protein degradation pathways in cells. It has important application prospects in drug development and biomedical research. The intrinsic protein degradation pathways in cells can be divided into two types, namely the ubiquitin-proteasome pathway and the lysosomal pathway. In recent years, various targeted protein degradation technologies have been developed using these two pathways. The range of proteins degraded by these technologies includes proteins located in cells, proteins on cell membranes, and proteins outside cells.

[0003] At present, the degradation technologies for cell membrane proteins include LYTAC, AbTAC, PROTAB, GlueTAC, KineTAC, etc. AbTAC is similar to PROTAB technology, which is a bispecific antibody. One Fab fragment of the antibody is used to recognize and bind to membrane proteins, and the other Fab fragment is used to recognize and bind to E3 ligases on the cell membrane, such as RNF43, thereby spatially bringing the membrane protein and the E3 ligase closer, promoting the ubiquitination of the membrane protein by the E3 ligase, and then inducing the endocytosis of the membrane protein into the lysosome for degradation. LYTAC technology is to couple glycopeptide chains to membrane protein antibodies. These glycopeptide chains can bind to the mannose-6-phosphate receptor M6PR on the cell membrane as ligands; when the antibody-glycopeptide chain conjugate binds to the membrane protein and M6PR at the same time, the conjugate will use the characteristics of M6PR endocytosis and transport to lysosomes to deliver the membrane protein to the lysosome for degradation. KineTAC technology is a complex of antibodies and cytokines. When the complex binds to membrane proteins and cytokine receptor CXCR7 at the same time, the complex will use CXCR7 to transport membrane proteins to lysosomes for degradation. GlueTAC technology is to develop and screen a nano-antibody targeting membrane proteins, and simultaneously couple membrane-penetrating peptides and lysosomal sorting peptides to the antibody. When the antibody binds to membrane proteins, it will use the membrane-penetrating peptide to enter the cell, and use 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, the current membrane protein degradation technologies, except for GlueTAC, require the degrader to bind to the target protein and the E3 ligase or lysosomal delivery receptor on the cell membrane at the same time. Therefore, the preparation technology of the degrader is relatively complicated and the spatial conformation needs to be considered; and because the expression of E3 ligase and lysosomal delivery receptor in various cells is quite different, this will greatly limit the versatility of the technology in various cells.

[0004] The reported cell membrane protein degradation technology can be divided into four technical routes according to the specific working principle. Their shortcomings and deficiencies are as follows: 1) LYTAC-related technology relies on lysosomal transport proteins on the cell membrane, such as M6PR, ASGPR, IGFIIR, Integrin, etc. The expression of these proteins in different cells varies greatly, resulting in poor versatility and consistency of the technology. The constructed degrader needs to bind to the target protein and these lysosomal transport receptors at the same time in space, which makes the design and synthesis difficult. 2) PROTAB and AbTAC technologies rely on E3 ligases on the cell membrane, which are limited by the expression level of the enzyme, and are bispecific antibodies with high development costs. 3) KineTAC technology relies on cytokine receptors on the cell membrane, which are limited by the expression of cytokine receptors. It has poor versatility in different cells, and the degrader is a coupling of antibodies and cytokines, which is more complicated to design. 4) GlueTAC technology, the nanoantibodies used for screening contain non-natural amino acids, the safety needs to be evaluated, the half-life in vivo is short, and the screening cost is high.

[0005] Autophagy is a basic function of cells. Through the autophagy mechanism, cells can effectively remove damaged organelles and misfolded protein molecules during the life process and transport them to lysosomes for degradation. Therefore, the development of targeted protein degradation technology using the autophagy-lysosome pathway is an important strategy in this field. There are currently four types of autophagy-based protein degradation technologies, namely ATTEC, AUTAC, AUTOTAC, and chaperone-mediated autophagy technology. These technologies are limited to degrading intracellular proteins and organelles, but 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. Summary of the invention

[0007] The purpose of the present invention is to provide a membrane protein targeted degradation agent based on cell autophagy, so as to solve the shortcomings of the prior art in that cell membrane protein degradation is not universal, has a relatively complex design and is costly.

[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.

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

[0010] A membrane protein targeted degradation agent based on cell autophagy comprises a membrane protein targeted 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 targeted 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 and can induce autophagy. The cationic polymer is selected based on its ability to induce autophagy in endosomes and promote the degradation of endosomes by lysosomes. Since this autophagy can be universally induced in various cells and does not rely on the combination of degradation agents with autophagy-related molecules in terms of mechanism, the technology of the present invention has the advantages of being more universal and more convenient than previous technologies.

[0014] Furthermore, the cationic polymer includes one or more of polyethyleneimine 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 antibody targeting membrane protein is an antibody that can recognize the extracellular domain of membrane protein.

[0018] Furthermore, the antibodies targeting membrane proteins include antibodies binding to PD-L1, antibodies binding to EGFR, and antibodies binding 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 for the above-mentioned cells that highly express specific membrane proteins, the targeting effect of the degradation agent is more obvious.

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

[0022] (1) connecting the cationic polymer to the nanosecond antibody to obtain a connection product of the nanosecond antibody and the cationic polymer;

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

[0024] In the present invention, the cationic polymer is connected 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 modified products of the two;

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

[0027] c. Only the nanosecond antibody 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 succinimidyl 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 azido C5 succinimidyl ester, and then the two modified products 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.

[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 nano-second antibody that can bind to an antibody targeting a membrane protein with a cationic polymer having the property of inducing cell autophagy to form a modular unit of the degrader. Since the nano-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 need to synthesize a degrader for each membrane protein separately. It only needs to prepare a Nano-AUTAB and match it with a commercial membrane protein antibody to efficiently degrade the target, which has the advantage of convenient preparation. The steps of coupling the nano second antibody to the cationic polymer are simple, and the preparation does not need to consider factors such as steric hindrance. Membrane protein antibodies can be obtained in large quantities 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, wherein:

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

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

[0042] Figure 3This is a diagram of cationic polymer PEI-induced endosome-related autophagy;

[0043] Figure 4 This is a diagram showing how AUTAB binds to target proteins for endocytosis and induces autophagy in endosomes;

[0044] Figure 5 This is a diagram showing how AUTAB promotes the endocytosis of target proteins into lysosomes;

[0045] Figure 6 This is the flow cytometry data diagram of AUTAB clearing the target protein on the cell membrane;

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

[0047] Figure 8 This is an experimental data diagram that proves that AUTAB promotes the degradation of target proteins through the autophagy-lysosome pathway;

[0048] Fig. 9 This is a diagram showing how AUTAB coupled with various PEIs and poly-L-Lys promotes the degradation of target proteins;

[0049] Fig.10 This is a diagram of the in vivo verification of Atz-AUTAB degrading PD-L1 in tumor tissues;

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

[0051] Fig.12 This is the synthetic route of Nano-AUTAB;

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

[0053] Fig.14 Nano-AUTAB-m synergistically promotes the degradation of membrane protein Notch1. DETAILED DESCRIPTION

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

[0055] The process of targeted degradation of membrane proteins based on cell autophagy of the present invention is as follows Figure 1 , Fig.11 As shown, a technology for developing targeted protein degradation using the autophagy-lysosome pathway is provided. Figure 1 The method is to connect antibodies targeting membrane proteins with cationic polymers to construct degraders. The polymers are internalized and at the same time damage endosomes to induce autophagy, thereby inducing the internalization of membrane proteins into lysosomes for degradation. Fig.11 is based on Figure 1 To further improve the pathway, the nano-secondary antibody that can bind to the antibody targeting membrane protein is connected to a cationic polymer, and then combined with the antibody targeting membrane protein to synergistically degrade the target protein.

[0056] Embodiment 1

[0057] like Figure 2 The method for preparing the 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: 10 mg of linear PEI material (L25K-PEI, Mw = 25000) was weighed into a clean centrifuge tube, 500 μl of ultrapure water and 250 μl of 1M hydrochloric acid solution were added in sequence, and the PEI material was dissolved by ultrasonic treatment to form a transparent clear solution, and then 1M sodium carbonate solution was added 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), and finally pH = 9 PBS solution was added until the final concentration of the reaction solution was 5 mg / ml (based on PEI material), and the reaction system was shaken at room temperature for 24 hours. The obtained L25K-DBCO solution can be directly used for 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, are DBCO-modified according to the above-mentioned synthesis method for subsequent antibody coupling.

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

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

[0063] Taking Atz-N3 as an example: 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) was added to a clean centrifuge tube, followed by adding 100 μL of PBS solution (PH=7.3) and 3 μL of 1M sodium carbonate solution in sequence. At this time, the pH of the mixed solution was slightly higher than 9.0. Finally, 5.7 μL of N3-C5-NHS (azide C5 succinimide ester) (22 μg, 50.0 equivalent, 3.82 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, 30 KDa), concentrated and washed 3 times, with 2% DMSO / PBS solution as washing solution, and finally 1.0 ug / uL of Atz-N3 antibody stock solution was obtained.

[0064] 250 μL Atz-N3 stock solution (250 μg, PBS, PH = 7.3) and 216 μL L25K-DBCO stock solution (1.08 mg, 25.0 equivalents, 5.0 mg / mL) were mixed evenly and shaken at room temperature for 24 hours. After the reaction was completed, the reaction solution was transferred to an ultrafiltration tube (4 mL, 30 KDa), concentrated and washed 3 times, with 2% DMSO / PBS solution as the washing solution, and finally 1.0ug / uL Atz-AUTAB stock solution was obtained.

[0065] 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.

[0066] Embodiment 2

[0067] A method for preparing a membrane protein targeted degradation agent based on cell autophagy is different from that of Example 1 in that the label modification process is different. Specifically, the linear PEI material is modified with a FITC fluorescent label:

[0068] Weigh 20 mg of linear PEI material (L25K-PEI, Mw = 25000) in a clean centrifuge tube, add 500 μl of ultrapure water and 500 μl of 1M hydrochloric acid solution in turn, and use ultrasonic treatment to dissolve the PEI material to form a clear and transparent solution. Then add 1M sodium carbonate solution to the above solution to adjust the pH value to 9.0, and finally add 1.25 mg of FITC powder (4.0 equivalents), mix well, protect the reaction solution from light, and shake at room temperature for 24 hours. After the reaction is complete, the reaction solution is dialyzed and freeze-dried to obtain 12.8 mg of red powder (L25K-FITC), with a yield of 60%.

[0069] Embodiment 3

[0070] A method for preparing a membrane protein targeted degradation agent based on cell autophagy is different from that of Example 1 in that the label modification process is different. Specifically, the branched PEI material is modified with a DBCO label:

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

[0072] Branched PEI with various other molecular weights, including but not limited to Mw=800 and Mw=25000, are DBCO-modified according to the above-mentioned synthesis method for subsequent antibody coupling.

[0073] Various branched PEI derivatives, including but not limited to acetylated PEI and PEGylated PEI, are DBCO-modified according to the above-mentioned synthesis method for subsequent antibody coupling.

[0074] Embodiment 4

[0075] A method for preparing a membrane protein targeted degradation agent based on cell autophagy comprises the following steps:

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

[0077] Taking PolyLys (15-30K) -DBCO as an example: 7.37 mg of polylysine hydrobromide (Poly-Lys, Mw = 15-30K) was weighed into a clean centrifuge tube, and 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) were added in sequence, and finally pH = 9 PBS solution was added until the final concentration of the reaction solution was 5 mg / ml (based on PEI material). The reaction system was shaken at room temperature for 24 hours. The obtained PolyLys (15-30K) -DBCO solution can be directly used in subsequent reactions without further purification.

[0078] Polylysine of various other molecular weights, including but not limited to Mw=3000-7000, Mw=4000-15000, is DBCO-modified according to the above-mentioned synthesis method for subsequent antibody coupling.

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

[0080] Taking Atz-N3 as an example: 29.2 μL of Atz antibody stock solution (0.25 mg, 1.0 equivalent, 8.58 ml / mg, PBS as solvent, PH=7.3) was added to a clean centrifuge tube, followed by adding 100 μL of PBS solution (PH=7.3) and 3 μL of 1M sodium carbonate solution in sequence. At this time, the pH of the mixed solution was slightly higher than 9.0. Finally, 5.7 μL of N3-C5-NHS (22 μg, 50.0 equivalent, 3.82 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, 30 KDa), concentrated and washed 3 times, with 2% DMSO / PBS solution as washing solution, and finally 1.0 ug / uL of Atz-N3 antibody stock solution was obtained.

[0081] 250 μL Atz-N3 stock solution (250 μg, 1.0 equivalent) and 195 μL PolyLys (15-30K) -DBCO stock solution (973 μg, 25.0 equivalent, 5.0 mg / mL) were mixed evenly and shaken at room temperature for 24 hours. After the reaction was completed, the reaction solution was transferred to an ultrafiltration tube (4 mL, 30 KDa), concentrated and washed 3 times, and 2% DMSO / PBS solution was used as the washing solution to finally obtain 1.0ug / uL PolyLys-Atz-AUTAB stock solution.

[0082] Other antibodies include but are not limited to Ctx antibody and Ole antibody, and their modification and coupling with polylysine are exactly the same as the above preparation process.

[0083] Embodiment 5

[0084] A method for preparing a membrane protein targeted degrader based on cell autophagy, which is different from Example 1 in that only PEI is modified with maleimide, and the antibody is not modified, and then PEI is coupled to the antibody using the disulfide bond on the antibody.

[0085] Embodiment 6

[0086] A method for preparing a membrane protein targeted degrader based on cell autophagy, which is different from Example 1 in that only non-natural amino acid modification is introduced into the antibody, and PEI is not modified, and then the non-natural amino acid is used to perform a condensation reaction with PEI to complete the coupling of PEI and the antibody.

[0087] Embodiment 7

[0088] A method for preparing a membrane protein targeted degrader based on cell autophagy, which is different from Example 1 in that the antibody is modified with biotin, the 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 antibody.

[0089] Embodiment 8

[0090] A method for preparing a membrane protein targeted degradation agent based on cell autophagy is different from that of Examples 1 to 7 in that the cationic polymer used is polyallylamine, and the other preparation processes are consistent.

[0091] Embodiment 9

[0092] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, which is different from Examples 1 to 7 in that the cationic polymer used is poly[N-(2-hydroxypropyl)methacrylamide], and the other preparation processes are consistent.

[0093] Embodiment 10

[0094] A method for preparing a membrane protein targeted degradation agent based on cell autophagy, which is different from Examples 1 to 7 in that the cationic polymer used is poly(2-(dimethylamino)ethylmethacrylate) and the other preparation processes are consistent.

[0095] Embodiment 11

[0096] A method for preparing a membrane protein targeted degradation agent based on cell autophagy is different from that of Examples 1 to 7 in that the cationic polymer used is polyornithine, and the other preparation processes are consistent.

[0097] Embodiment 12

[0098] A method for preparing a membrane protein targeted degradation agent based on cell autophagy is different from that of Examples 1 to 7 in that the cationic polymer used is polyarginine, and the other preparation processes are consistent.

[0099] Embodiment 13

[0100] A method for preparing a membrane protein targeted degradation agent based on cell autophagy is different from that of Examples 1 to 7 in that the cationic polymer used is chitosan, and the other preparation processes are consistent.

[0101] In the above embodiments of the present invention, different antibodies are coupled with different cationic polymers having the property of inducing cell autophagy by chemical methods to prepare different membrane protein targeted degraders. Different cationic polymers are coupled to the antibody Atz to obtain the corresponding Atz-AUTAB; this method has also been verified on different antibodies, and finally a variety of membrane protein degraders including Atz-AUTAB, Ctx-AUTAB and Ole-AUTAB are synthesized, and the experimental conditions are reproducible and the yield is stable.

[0102] Embodiment 14

[0103] like Fig.12 The method for preparing the membrane protein targeted degradation agent based on cell autophagy comprises the following steps:

[0104] (1) MA modification of PEI material

[0105] Take L2.5K-MA as an example: weigh 6 mg of linear polyethyleneimine (PEI) material (L2.5K-PEI, Mw = 2500) in a clean centrifuge tube, add 200 μl of ultrapure water and 200 μl of 1M hydrochloric acid solution in sequence, and use ultrasonic treatment to dissolve the PEI material to form a transparent clear solution. Add Tris-HCl buffer (pH = 8.68) to the above solution to adjust the pH value to 8.0, then add 148 μl of MA-C6-NHS solution (1.0 equivalent, 5 mg / ml, DMSO as solvent), at this time 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, and the obtained L2.5K-MA solution can be directly used for subsequent reactions without further purification.

[0106] Linear PEI of various other molecular weights, including but not limited to Mw=5000 and Mw=10000, were MA-modified according to the above-mentioned synthesis method for subsequent nanobody coupling.

[0107] Branched PEI of various molecular weights, including but not limited to Mw=800, Mw=2000, and Mw=25000, are MA-modified according to the above-mentioned synthesis method for subsequent nanobody coupling.

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

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

[0110] (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

[0111] 125 μL of nano secondary antibody 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 evenly and shaken at room temperature for 3 hours. After the reaction was completed, 80.5 μL of N-acetylcysteine ​​solution (25.0 equivalents, 1 mg / mL) was added to the reaction system and continued to shake for 30 minutes to consume the unreacted raw materials; the final mixture was transferred to an ultrafiltration tube (4mL, 3KDa), concentrated and washed 3 times, 2% DMSO / PBS solution was used as the washing solution, and finally 1.0ug / uL of Nano-AUTAB stock solution was obtained.

[0112] (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.

[0113] Other membrane protein antibodies include but are not limited to Ctx antibody (Cetuximab) and Ole antibody (Oleclumab), and the preparation process is exactly the same as the above.

[0114] Embodiment 15

[0115] A method for preparing a membrane protein targeted degradation agent based on cell autophagy comprises the following steps:

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

[0117] Taking L2.5K-PEI as an example: 10 mg of linear PEI material (L2.5K-PEI, Mw = 2500) was weighed into a clean centrifuge tube, 500 μl of ultrapure water and 250 μl of 1M hydrochloric acid solution were added in sequence, and the PEI material was dissolved by ultrasonic treatment to form a transparent clear solution, and then 1M sodium carbonate solution was added 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), and finally pH = 9 PBS solution was added until the final concentration of the reaction solution was 5 mg / ml (based on PEI material), and the reaction system was shaken at room temperature for 24 hours. The obtained L2.5K-DBCO solution can be directly used for subsequent reactions without further purification.

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

[0119] Taking the nano secondary 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 adding 6 μL of 1M sodium carbonate solution. At this time, the pH of the mixed solution 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, 3KDa), concentrated and washed 3 times, with 2% DMSO / PBS solution as washing solution, and finally 1.0 ug / uL of nano secondary antibody-N3 stock solution was obtained.

[0120] Mix 240 μL of Nano-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. After the reaction is completed, transfer the reaction solution to an ultrafiltration tube (4 mL, 3 KDa), concentrate and wash 3 times, using 2% DMSO / PBS solution as the washing solution, and finally obtain 1.0ug / uL of Nano-AUTAB stock solution.

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

[0122] Embodiment 16

[0123] A method for preparing a membrane protein targeted degrader based on cell autophagy, which is different from Example 15 in that only a non-natural amino acid modification is introduced into the second antibody, and PEI is not modified, and then a condensation reaction is carried out between the non-natural amino acid and PEI to complete the coupling of PEI and the second antibody.

[0124] Embodiment 17

[0125] A method for preparing a membrane protein targeted degrader based on cell autophagy, which is different from Example 15 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.

[0126] In the above embodiment of the present invention, the second antibody is coupled with different cationic polymers having the property of inducing cell autophagy by a chemical method to form a modular unit of the degrader, and then combined with different membrane protein antibodies to obtain the corresponding Nano-AUTAB.

[0127] Example 18 Verification of AUTAB-induced endosome-related autophagy

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

[0129] HeLa cells stably expressing mCherry-LC3C were cultured on glass slides. After the cells reached about 50% confluence, various types and sizes of PEI or FITC-labeled L25K-PEI were added to the culture medium and cultured for 1 hour. The cells were then fixed with 4% PFA, and the formation of mCherry-LC3C-positive autophagosomes and the co-localization of endocytosed FITC-PEI and autophagosomes were observed under a laser confocal microscope.

[0130] HeLa cells stably expressing mCherry-LC3C were cultured on glass slides and transfected with EGFP-Rab5 plasmid. PEI was added 24 hours after transfection and incubated for 1 hour, then the cells were fixed and the co-localization of autophagosome marker mCherry-LC3C and early endosome marker EGFP-Rab5 was detected under a laser confocal microscope.

[0131] Depend on Figure 3 As shown in a, after the cells were treated with linear (L2.5K, L25K) and branched (B800, B2K, B25K) PEI of various sizes, mCherry-LC3C punctate fluorescence signals appeared in the cells, indicating that PEI can induce LC3C-mediated autophagy. Figure 3As shown in b, the internalized FITC-PEI fluorescence signal co-localizes with these mCherry-LC3C signals, indicating that autophagy occurs on the PEI-containing endosomes. Figure 3 As shown in c, these mCherry-LC3C signals colocalized with EGFP-Rab5, a marker of early endosomes, indicating that autophagy occurred at the early stage of PEI endocytosis.

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

[0133] In the present invention, cationic polymers are coupled to membrane proteins to construct AUTAB. Based on the property of cationic polymers to induce autophagy, a series of AUTAB-induced autophagy were verified below.

[0134] For Atz-AUTAB, HeLa cells stably expressing HA-PD-L1 and mCherry-LC3C were cultured on slides. After the cells reached about 50% confluence, Atz-AUTAB or control antibody Atz was added to the culture medium at a final concentration of 100nM and incubated in an incubator for 1 hour. The cells were then fixed with 4% PFA, and the Atz-AUTAB and control group Atz were labeled with anti-Human IgG antibodies, anti-HA tag antibodies were used to label PD-L1, anti-Rab5 antibodies were used to label early endosomes, and anti-Rab7 antibodies were used to label late endosomes according to the standard immunofluorescence staining process. After immunostaining, the slides were sealed with an anti-fluorescence 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 co-localization relationship among the autophagy markers mCherry-LC3C, PolyLys-Atz-AUTAB and HA-PD-L1 was mainly analyzed using the same method as the above-mentioned Atz-AUTAB.

[0136] For Ctx-AUTAB (Cetuximab, Ctx for short), the co-localization relationship between the autophagy markers mCherry-LC3C, Ctx-AUTAB and EGFR was mainly analyzed, and the method was similar to that described above. HeLa cells stably expressing mCherry-LC3C were cultured on slides, and the cells were treated with Ctx-AUTAB or control antibody Ctx at a final concentration of 100nM for 1 hour. The cells were then fixed, and anti-Human IgG antibodies were used to label Ctx-AUTAB and Ctx in the control group, and anti-EGFR antibodies were used to label the membrane protein EGFR. After immunostaining, laser confocal microscopy was used for analysis and detection.

[0137] Metrics: The number of LC3 punctate signals shown in the figure, and the co-localization of LC3 and AUTAB signals.

[0138] Take Atz-AUTAB targeting PD-L1 as an example. Figure 4 As shown in a, the internalized Atz-AUTAB co-localizes significantly with HA-PD-L1, indicating that it can recognize and bind to PD-L1 and be internalized together with PD-L1; and Atz-AUTAB can induce more mCherry-LC3C punctate signals compared to Atz itself, and co-localizes with these LC3C autophagy signals, indicating that Atz-AUTAB can induce cell autophagy after internalization. Figure 4 As shown in Figures 4b and 4c, these Atz-AUTABs can co-localize with LC3C and the endosomal markers Rab5 or Rab7, indicating that after Atz-AUTAB is internalized, autophagy occurs on endosomes in both the early and late stages of the endocytic pathway. For Ctx-AUTAB targeting EGFR, it has also been shown that it can induce similar autophagy, see Figure 4 d. More importantly, AUTAB constructed with other cationic polymers, such as poly-L-Lys, also induced a large number of mCherry-LC3C punctate signals after acting on cells, and co-localized with these LC3C signals, indicating that they can also induce the occurrence of autophagy, see Figure 4 e. These results indicate that AUTAB with autophagic effects can be constructed based on a variety of cationic polymers.

[0139] Example 19 Verification of the Degradation Effects of Various AUTABs on Various Membrane Proteins

[0140] (1) Detection of AUTAB promoting endocytosis of membrane proteins to lysosomes

[0141] For Atz-AUTAB, HeLa cells stably expressing HA-PD-L1 were cultured on slides and transfected with RFP-Lamp1 plasmid. 24 hours after transfection, cells were treated with Atz-AUTAB or control antibody Atz at a final concentration of 5nM for 1 hour. The cells were then fixed and immunofluorescence stained with an anti-HA tag antibody to label PD-L1. After immunostaining, the colocalization of the lysosomal marker RFP-Lamp1 and the target protein PD-L1 was analyzed using laser confocal microscopy.

[0142] For Ctx-AUTAB, the method was similar to that described above. HeLa cells were cultured on glass slides and transfected with RFP-Lamp1 plasmid. 24 hours after transfection, cells were treated with Ctx-AUTAB or control antibody Ctx at a final concentration of 5 nM for 1 hour. Immunofluorescence staining was then performed using anti-EGFR antibodies, and relevant co-localization was observed under a microscope.

[0143] For Ole-AUTAB, HeLa cells stably expressing mCherry-CD73 were cultured on glass slides, and when the cells grew to 80% confluence, the cells were treated with Ole-AUTAB or control antibody Ole at a final concentration of 100 nM for 12 hours. Lysosomes were then labeled with LysoTracker fluorescent probes in living cells, and the co-localization of lysosomes and mCherry-CD73 was observed and detected in real time under a laser confocal microscope.

[0144] like Figure 5 As shown in a, compared with the Atz-treated group, Atz-AUTAB-treated cells resulted in a large amount of target protein PD-L1 internalized into the cells and co-localized with the lysosomal marker Lamp1, indicating that PD-L1 was transported into the lysosome. Similarly, under the action of Ctx-AUTAB and Ole-AUTAB, the corresponding target proteins EGFR and CD73 were internalized into the lysosome in large quantities, as shown in Figure 5 b and 5c.

[0145] (2) Verification of AUTAB's ability to weaken the localization of target proteins on the cell membrane

[0146] 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.

[0147] 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 5nM Atz-AUTAB or control antibody Atz for a certain period of time (231 cells were treated for 0.5, 2, 6, and 24 hours, and HeLa cells were treated for 4 hours). Subsequently, in the living cell state, PD-L1 on the cell membrane was labeled with an antibody that recognizes the extracellular domain of PD-L1 at 4 degrees Celsius, and the corresponding fluorescent secondary antibody was further used to amplify the labeled signal. After immunofluorescence labeling, the labeled PD-L1 was detected using a flow cytometer.

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

[0149] For Ole-AUTAB, HeLa cells stably expressing mCherry-CD73 were treated with 100 nM of Ole-AUTAB or control antibody Ole for 12 h, and then anti-mCherry antibody was used to label the target protein on the membrane, and further fluorescent secondary antibody staining and flow cytometry analysis were performed.

[0150] like Figure 6 As shown in the flow cytometry data, Atz-AUTAB, Ctx-AUTAB and Ole-AUTAB treatment of cells all led to a sharp reduction of the corresponding target proteins from the membrane. Figure 5 As shown in the localization of the fluorophore, it was observed that after Atz-AUTAB, Ctx-AUTAB and Ole-AUTAB were treated with cells, a large amount of target proteins were internalized into lysosomes, indicating that under the action of AUTAB, the target proteins on the membrane can be quickly internalized from the membrane and transported to the lysosomes.

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

[0152] Western blotting (WB) was used to evaluate the degradation effects of a series of AUTABs on the corresponding target proteins in various cell lines.

[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 (set concentration gradient) or control group Atz and continued to be cultured for 24 hours or gradient time. The cells were then treated with SDS lysis buffer to obtain total cell protein samples, and the changes in the amount of PD-L1 protein in the protein samples were analyzed according to the standard WB method.

[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 that of Atz-AUTAB.

[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 that of Atz-AUTAB.

[0156] For AUTAB coupled 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 the above-mentioned Atz-AUTAB.

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

[0158] The present invention proves through protein immunoblotting experiments that the constructed series of AUTAB can lead to the degradation of target protein. Figure 7 As shown in Figures ac, for Atz-AUTAB, it was detected that at an extremely low concentration (0.09 nM), it could cause significant degradation of PD-L1 within 24 hours; and at 6.25 nM Atz-AUTAB, the degradation of the target protein could be observed in as short as 12 hours. Figure 7 As shown in Figure d, in a variety of cells (U87-MG, NCI-H292, NCI-H1975, A375), it was verified that Atz-AUTAB can degrade the target protein. In addition, using HeLa cells overexpressing PD-L1, Figure 7 As shown in the figure, even highly expressed PD-L1 can be cleared by Atz-AUTAB. Figure 7As shown in hj, for Ctx-AUTAB, it was demonstrated that it can degrade EGFR at lower concentrations and in a shorter time, and that it can be effective in a variety of cell lines. In addition to targeting transmembrane proteins, Ole-AUTAB was also developed for the membrane-anchored protein CD73, and its concentration- and time-dependent effects were also verified, and its applicability to multiple cells was also demonstrated, see Figure 7 These results indicate that the AUTAB technology of the present invention is universal.

[0159] (4) Verification that AUTAB degrades target proteins through the autophagy-lysosome pathway

[0160] It was proved that AUTAB degraded the target protein through the autophagy pathway. Taking Atz-AUTAB as an example, RNAi technology was used to knock down the expression of autophagy core genes ATG5 or LC3C in MDA-MB-231 cells, and then the cells were treated with Atz-AUTAB (6.25nM) for 12 hours, and then the cells were lysed to extract total protein and the changes in PD-L1 were detected by WB technology.

[0161] Prove that AUTAB degrades target proteins through lysosomes. MDA-MB-231 cells, HeLa cells, and U87-MG cells were treated with AUTAB, and lysosomal inhibitors bafilomycin A1 (100nM) or chloroquine (200μM) were added to the culture medium and cultured for 24 hours. The cells were then lysed to extract total protein and the changes in the amount of target protein were detected using WB technology.

[0162] like Figure 8 As shown in a, under the condition of autophagy inhibition, Atz-AUTAB cannot degrade PD-L1, which indicates that it needs to work through autophagy. Then, cells were treated with lysosomal inhibitors (bafilomycin A1 and chloroquine) to detect whether Atz-AUTAB can still play a degradation role. The results showed that it was completely unable to promote the degradation of the target, which indicates that its function depends on the activity of lysosomes. Figure 8 b. In addition, the same method was used to prove that both Ctx-AUTAB and Ole-AUTAB degraded the target protein through the lysosomal pathway, see Figure 8 c, 8d. These demonstrate that AUTAB degrades target proteins through the autophagy-lysosome pathway.

[0163] In addition to the above-mentioned use of AUTAB coupled with L25K-PEI to verify the degradation effect, the targeted degradation effect of AUTAB coupled with other different PEIs and other cationic polymers poly-L-Lys was further demonstrated. Fig. 9As shown in ad, both linear (L2.5K, L25K) and branched (B800, B2K, B25K) PEI, after coupling to the corresponding antibody, can significantly promote the degradation of the target protein. Atz-AUTAB constructed using two different size ranges of poly-L-Lys (4-15K, 15-30K) can also significantly degrade PD-L1 within a concentration range of 1nM, see Fig. 9 These results indicate that a variety of cationic polymers can be used in the membrane protein targeted degradation strategy proposed in the present invention.

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

[0165] Taking Atz-AUTAB as an example, we first established a subcutaneous tumor-bearing model of nude mice (8 weeks old, BALB / c strain) with MDA-MB-231 cells. When the tumor volume reached about 50 mm 3 Atz-AUTAB or control antibody Atz was injected peritumorally (injection dose was 0.01 nmol / 100 μL, once a day, for two consecutive days). The day after the injection, the mouse tumor tissue was taken, and the tissue was lysed to extract total protein, and then the change of PD-L1 amount in the tumor tissue was detected by WB technology.

[0166] like Fig.10 As shown, a nude mouse tumor model of MDA-MB-231 cells was established, Atz-AUTAB (0.01 nmol / time, once a day, twice in total) was injected around the tumor, and the changes in the amount of PD-L1 in the tumor tissue were detected. The results showed that Atz-AUTAB can rapidly degrade PD-L1 in vivo. This proves that it can also play the role of degrading target proteins in animals.

[0167] Example 20 Verification of the effect of Nano-AUTAB and membrane protein antibody on synergistic degradation of target protein

[0168] (1) Verification of Nano-AUTAB combined with membrane protein antibodies to weaken the localization of target proteins on the cell membrane

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

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

[0171] 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.

[0172] For PD-L1, MDA-MB-231 cells were treated with a premix of Atz and Nano-AUTAB or a control nanoantibody for 24 hours, and then the cells were lysed with SDS lysis buffer and total protein was extracted. The changes in the amount of PD-L1 protein in the protein samples were analyzed according to the standard WB method. The same method was used to detect the degradation effect of Nano-AUTAB combined with other PD-L1 antibodies (Abcam, catalog number ab205921; Cell Signaling, catalog number 13684) on the target.

[0173] 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.

[0174] For Integrinα5, Nano-AUTAB and Integrinα5 antibody were used to detect their synergistic degradation of target protein, using the same method as above.

[0175] For PD-L1 and EGFR, the 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 above.

[0176] like Fig.13 a, proved that Nano-AUTAB combined with Atz can synergistically induce a sharp reduction of the target protein PD-L1 from the cell membrane. Using the WB method, it was detected that Nano-AUTAB combined with Atz can efficiently degrade PD-L1, see Fig.13 b. In addition, Nano-AUTAB combined with other commercial PD-L1 antibodies can also induce PD-L1 degradation, see Fig.13 c, 13d.

[0177] In addition to PD-L1, Nano-AUTAB was also verified to be able to synergistically degrade membrane proteins such as CD73 and Integrinα5, see Fig.13 e, 13f. In addition, using Nano-AUTAB with Atz and Ctx simultaneously proved that it can synergistically degrade PD-L1 and EGFR at the same time, see Fig.13 g.

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

[0179] The above Nano-AUTAB recognizes human and rabbit antibodies. In order to verify the universality of the strategy, we prepared Nano-AUTAB-m that 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 the cells were incubated for 24 hours. The degradation of Notch1 was then detected using the WB method.

[0180] like Fig.14 , Nano-AUTAB-m was constructed using another nano antibody that can recognize mouse antibodies, and the results showed that this Nano-AUTAB-m combined with mouse anti-Flag antibodies can promote the degradation of Flag-Notch1. This result proves the versatility of the strategy of using the conjugate of the nano second antibody and cationic polymer combined with membrane protein antibodies to construct a degrader.

[0181] The above disclosure is only the preferred embodiment of the present invention, which 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 nanosecond antibody to obtain a connection product of the nanosecond antibody and the cationic polymer; (2) The linker of step (1) is combined with a membrane protein targeting antibody to obtain a degradation agent.

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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