An antibody degrader and its application

Abstract

The present invention discloses an antibody degrader and its application. The antibody degrader consists of three parts: a monoclonal antibody that recognizes the target protein or its antigen-binding fragment (collectively referred to as an antibody), a degrading ligand that can induce protein-targeted degradation, and a chemical linker that connects the two. The protein-targeted degrader provided by the present invention uses an antibody as the recognition group, and a specific antibody can be selected according to the actual degradation requirements. Considering the differences in the expression levels and activities of the endogenous degradation systems in different cells, in order to meet the protein-targeted degradation requirements of different types of cells, the antibody degrader of the present invention encompasses the degrading ligands involved in currently known classical degradation strategies (including proteolysis-targeting chimeras (PROTACs), autophagosome-anchoring compounds (ATTECs), chaperone-mediated autophagy chimeras (CMAs), hydrophobic tags (HyTs), etc.). The scope of application of this strategy is wide, and it can target the degradation of cell membrane proteins and intracellular proteins.

Description

Technical Field

[0001] The present invention relates to a preparation method of a class of antibody-based protein-targeting degrader (hereinafter referred to as antibody degrader) and its application in protein-targeting degradation. Background Art

[0002] Most existing drugs block or regulate the function of target proteins by binding to specific sites of the target proteins. However, the activities of many proteins cannot be effectively regulated in this way. Targeted protein degradation is another hot direction in the field of drug research and development after protein kinase inhibitors, monoclonal antibodies, and gene knockout, and has become an emerging strategy for regulating the concentration of undruggable proteins in cells (such as scaffold proteins, transcription factors, and other non-enzymatic proteins in cells). The research on targeted protein degradation at home and abroad mainly focuses on strategies such as proteolysis-targeting chimeras (PROTAC) based on the proteasome degradation system, hydrophobic tag (HyT) modification, and lysosome-targeting chimeras (such as LYTAC) based on the lysosome degradation system. Currently, the developed small molecule degraders (such as PROTAC molecules) provide an effective means for the specific degradation of intracellular proteins, but there are limitations such as a high threshold for protein-targeting design, limited applicable targets, and lack of tissue specificity. In contrast, antibodies are classical biomolecular recognition groups, and their preparation technology is mature, and theoretically can target any protein. However, there are only a few reports on applying antibodies to construct targeted degradation technologies for membrane proteins, such as the LYTAC technology. Summary of the Invention

[0003] The purpose of the present invention is to provide a preparation method and application of an antibody degrader in view of the deficiencies of the prior art; the present invention combines the specific recognition ability of antibody macromolecules and the characteristics of small molecule degradation ligands to induce protein degradation, and constructs a series of protein-targeting degraders based on antibodies to target and degrade cell membrane proteins and intracellular proteins respectively. The "general-purpose" protein-targeting degradation strategy based on antibodies constructed by the present invention uses monoclonal antibodies or their antigen-binding fragments as target protein recognition groups, and covers a variety of degradation ligands that can induce proteasome degradation pathways or lysosome degradation pathways (such as PROTAC, HyT, CMA, ATTEC, etc.), and assembles a series of antibody degraders.

[0004] The technical solution adopted by the present invention is as follows:

[0005] An antibody degrader, comprising an antibody and a degrading ligand coupled by a chemical linker chain, wherein the antibody is responsible for recognizing and binding to a target protein, and the degrading ligand induces the targeted degradation of the target protein (a small molecule compound or a polypeptide); the degrading ligand is a small molecule or a polypeptide containing an azide group that can induce proteasome- or lysosome-dependent protein degradation, and the chemical linker chain has a strained alkyne, and the azide group can undergo a metal-free catalytic bioorthogonal ligation reaction with the strained alkyne to achieve specific connection with the chemical linker chain.

[0006] Further, the general formula of the antibody degrader is (Ab-L-R), where Ab is a monoclonal antibody or an antigen-binding fragment of a monoclonal antibody, L is a chemical linker chain, and R is a degrading ligand.

[0007] Further, considering the differences in the expression levels and activities of endogenous degradation systems in different cells, to meet the protein targeting degradation requirements of different types of cells, the antibody degrader of the present invention encompasses degrading ligands involved in currently known classical degradation strategies (including proteolysis-targeting chimeras (PROTACs), autophagosome-anchoring compounds (ATTECs), chaperone-mediated autophagy chimeras (CMAs), hydrophobic tags (HyTs), etc.). Specifically, the degrading ligand is one of the following structures:

[0008]

[0009] Further, the general formula of the chemical linker chain is as follows:

[0010]

[0011] Wherein, a is an integer from 1 to 4; b is an integer from 1 to 8.

[0012] Further, the antibody includes F(ab’) 2 (bivalent antibody fragment), Fab (monovalent antigen-binding fragment), scFv (single-chain variable fragment), Nanobody (nanobody), Affibody (affibody).

[0013] A pharmaceutical composition containing a therapeutically effective amount of the antibody degrader or a pharmaceutically acceptable salt thereof.

[0014] Use of the antibody degrader in the preparation of a protein drug for targeted degradation.

[0015] Further, the proteins that the drug can target for degradation include epidermal growth factor receptor EGFR and programmed cell death ligand 1 PD-L1.

[0016] The antibody degrader of the present invention can be prepared by a two-step method. First, one end of a chemical linker is coupled with an antibody, and then the other end is coupled with a degrading ligand to obtain the target molecule. The degrading ligand is a small molecule or polypeptide degrading ligand containing an azide group; one end of the chemical linker has a functional group that can be coupled with the free amino group of the antibody (preferably N-hydroxysuccinimide ester, abbreviated as NHS ester), and the other end has a functional group that can specifically connect with the azide group on the degrading ligand (preferably dibenzocyclooctyne, abbreviated as DBCO, which can undergo an efficient and specific bioorthogonal ligation reaction with the azide group), and the middle is a polyethylene glycol (PEG) chain.

[0017] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0018] (1) The recognition group used in the present invention is an antibody, which has a wide research foundation and a relatively mature preparation system. It not only has the ability to specifically bind to the antigen protein, but also theoretically can obtain antibodies targeting any protein; in addition, labeling some of the free amino groups on the surface of the antibody has little impact on its specific recognition with the antigen, and the feasibility of chemical modification is relatively high.

[0019] (2) By labeling different types of degrading ligands on the antibody to cover multiple protein degradation pathways, the present invention can expand the applicable scope of protein targeted degradation (including different degradation targets, different cell distribution situations, and different types of cell lines). Currently, the antibody-based protein targeted degradation strategy mainly targets membrane proteins. The present invention uses different types of degrading ligands, and according to the degradation requirements, antibody degraders can be assembled to induce specific endogenous degradation systems in cells to participate in the degradation of target proteins, and can target and degrade cell membrane and intracellular proteins respectively.

[0020] (3) The two-step labeling strategy provided by the present invention can systematically compare the protein degradation efficiencies induced by different degrading ligands, and can provide a reference for the research on the mechanism of protein targeted degradation and the optimization of methods. Description of the Drawings

[0021] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0022] Figure 1 It is a schematic diagram of using an antibody degrader for targeted degradation of a specific protein.

[0023] Figure 2 is Le-N 3 of 1 1H NMR spectrum.

[0024] Figure 3For Le-N 3 of 13 C NMR spectrum.

[0025] Figure 4 For VHL-N 3 of 1 H NMR spectrum.

[0026] Figure 5 For VHL-N 3 of 13 C NMR spectrum.

[0027] Figure 6 For RB-N 3 of 1 H NMR spectrum.

[0028] Figure 7 For RB-N 3 of 13 C NMR spectrum.

[0029] Figure 8 For Ada-N 3 of 1 H NMR spectrum.

[0030] Figure 9 For Ada-N 3 of 13 C NMR spectrum.

[0031] Figure 10 For CMA-N 3 ESI-HRMS spectrum.

[0032] Figure 11 For PTM-N 3 ESI-HRMS spectrum.

[0033] Figure 12 For ATc-N 3 of 1 H NMR spectrum.

[0034] Figure 13 For ATc-N 3 ESI-HRMS spectrum.

[0035] Figure 14 For ATI-N 3 of 1 H NMR spectrum.

[0036] Figure 15 For ATI-N 3 ESI-HRMS spectrum.

[0037] Figure 16Immunoblotting results of total EGFR protein content in cells after co-incubation of different concentrations of cetuximab (Ctx) or cetuximab-CMA degrader (Ctx-CMA) with HeLa cells for 24 h.

[0038] Figure 17 Results of tumor size after treatment of xenograft tumor models with cetuximab (Ctx) or cetuximab-CMA degrader (Ctx-CMA).

[0039] Figure 18 Immunoblotting results of total EGFR protein content in cells after co-incubation of cetuximab (Ctx) or cetuximab-Ada degrader (Ctx-Ada) with HeLa cells for 24 h. Detailed implementation manners

[0040] The antibody degrader provided by the present invention is prepared by first labeling the antibody with a chemical linker and then coupling the labeled antibody with a degrading ligand; the degrading ligand is a small molecule or polypeptide degrading ligand containing an azide group; the chemical linker is a bifunctional compound containing two reactive functional groups, one of which can be coupled to the amino group on the surface of the antibody, and the other can be specifically connected to the azide molecule, and these two functional groups do not react with each other.

[0041] The degrading ligands include:

[0042]

[0043] The degrading ligands shown in the present invention are prepared by the following method:

[0044] (1) Preparation of Le-N 3 Preparation

[0045]

[0046] At room temperature, phosphorus pentachloride (0.0206 g, 0.099 mmol) was added to a solution of azidoacetic acid (0.0100 g, 0.099 mmol) in tetrahydrofuran (THF; 1.0 mL), and then the reaction solution was heated to reflux for 1.5 h; after cooling to room temperature, potassium carbonate (0.0079 g, 0.057 mmol) and lenalidomide (0.0098 g, 0.038 mmol) were added to the reaction solution, and then heated to reflux for 1.5 h; finally, the reaction was terminated with saturated NaHCO 3 aqueous solution, extracted with ethyl acetate, the organic phase was dried over anhydrous MgSO 4 dried, the filtrate was collected and the organic solvent was removed by rotary evaporation to obtain a crude product, purified by silica gel column chromatography (using ethyl acetate as the eluent), monitored by thin layer chromatography (TLC; the developing agent was determined according to the product, preferably ethyl acetate as the developing agent), and the target product was collected (preferably Rf The value is 0.3 - 0.6), and the spectrogram is as Figure 2-3 shown.

[0047] (2) Preparation of VHL-N 3 Preparation

[0048]

[0049] To a solution of VHL032 (CAS: 1448188 - 62 - 2; 0.0255 g, 0.12 mmol) in N,N - dimethylformamide (DMF), azidoacetic acid (0.0060 g, 0.12 mmol) was added, and N,N - diisopropylethylamine (DIPEA; 0.0305 g, 0.47 mmol) was added dropwise. After the reaction solution was stirred at room temperature for 5 min, 2-(7 - azabenzotriazol - 1 - yl)-N,N,N',N' - tetramethyluronium hexafluorophosphate (HATU; 0.0245 g, 0.13 mmol) was added, and the reaction continued at room temperature for 1 h. After the reaction was completed, water was added, and the mixture was extracted with ethyl acetate; the organic phase was dried over anhydrous MgSO 4 dried, the filtrate was collected and the organic solvent was removed by rotary evaporation to obtain the crude product, which was purified by silica gel column chromatography (using n - hexane and acetone with a volume ratio of 1:2 as the eluent), monitored by TLC (the developing agent was determined according to the product, preferably using n - hexane and acetone with a volume ratio of 1:2 as the developing agent), and the target product was collected (preferably with an R f value of 0.2 - 0.5), and the spectrogram is as Figure 4-5 shown.

[0050] (3) Preparation of RB - N 3 Preparation

[0051]

[0052] Step 1:

[0053] Arginine (3.4800 g, 20 mmol) was added to a mixed solution of tert - butanol and water with a volume ratio of 1:1. The mixture was cooled to 0 °C in an ice bath, and sodium hydroxide (2.8000 g, 70 mmol) was added, and stirring was continued at 0 °C for 5 min; then di - tert - butyl dicarbonate (18.0 mL, 80 mmol) was added to the mixed solution, and the reaction solution was stirred at room temperature for 48 h. After the reaction was complete, the reaction solution was concentrated by rotary evaporation, and the residue was acidified to pH = 3 with citric acid. The above solution was extracted with ether, dried over anhydrous sodium sulfate and dried by rotary evaporation to obtain Hy - Boc.

[0054] Step 2:

[0055] The product Hy-Boc (3.890 g, 8.2 mmol) obtained in Step 1 was cooled to 0 °C in an ice bath, then N-hydroxysuccinimide (0.9440 g, 8.2 mmol) was added, and then N,N'-dicyclohexylcarbodiimide (DCC; 2.530 g, 12 mmol, dissolved in 2.0 mL of THF) was added. The reaction was stirred at 25 °C for 12 h to obtain Hy-NHS, which was directly used for the next reaction.

[0056] Step 3:

[0057] In a 50 mL round-bottom flask, Hy-NHS (0.1500 g, 0.27 mmol) was dissolved in acetonitrile (ACN; 10.0 mL), then 3-azido-1-propylamine (0.0333 g, 0.32 mmol) was added, and the reaction was stirred at 25 °C for 12 h. After the reaction was completed, the organic phase was evaporated to remove the organic solvent to obtain a crude product, which was purified by silica gel column chromatography (using a petroleum ether and ethyl acetate mixture with a volume ratio of 2:1 as the eluent), monitored by TLC (the developing agent was determined according to the product, and preferably a petroleum ether and ethyl acetate mixture with a volume ratio of 2:1 was used as the developing agent), and the target product was collected (preferably with an Rf value of 0.2 - 0.4). The spectrum is as Figure 6-7 shown.

[0058] (4) Preparation of Ada-N 3 Preparation

[0059]

[0060] In a dry round-bottom flask, adamantane carboxylic acid (0.0250 g, 0.14 mmol), 4-dimethylaminopyridine (DMAP; 0.0169 g, 0.14 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl; 0.03170 g, 0.17 mmol) and dichloromethane DCM (10.0 mL) were added, and the mixture was stirred at room temperature until all solids were dissolved. 3-azido-1-propylamine (0.0167 g, 0.17 mmol) was added dropwise, and the mixture was stirred at room temperature for 24 h. The reaction was quenched with 1M aqueous HCl solution (3.0 mL), diluted with DCM (10.0 mL) and water (25.0 mL) and extracted. The organic phases were combined, washed with saturated brine, and then dried over Na 2 SO 4 After drying, it was concentrated by rotary evaporation. It was purified by silica gel column chromatography (using a petroleum ether and ethyl acetate mixed solution with a volume ratio of 1:2 as the eluent), monitored by TLC (the developing agent was determined according to the product, and preferably a petroleum ether and ethyl acetate mixed solution with a volume ratio of 1:2 was used as the developing agent), and the target product was collected (preferably with an R f value of 0.3 - 0.6). The spectrum is as Figure 8-9 shown.

[0061] (5) Preparation of CMA-N 3 / PTM-N 3 Preparation

[0062] The sequence of CMA-N is N 3 -CH 3 CO-KFERQKILDQRFFE, and the sequence of PTM-N is N 2 -CH 3 CO-KFERQ. Both can introduce N groups by modifying the amino group of the lysine side chain in the polypeptide, and their spectra are as shown in 3 -CH 2 Figure 3 and Figure 10 respectively. Figure 11 shown.

[0063] (6) Preparation of ATc-N 3 Preparation

[0064]

[0065] To a DMF solution of 5,7-dihydroxy-4-phenylcoumarin (0.0508 g, 0.20 mmol), potassium carbonate (0.0276 g, 0.20 mmol) and 1-azido-4-iodobutane (0.0449 mg, 0.20 mmol) were added. The reaction solution was heated under reflux for 16 h under argon protection. After the reaction was completed, the reaction was quenched with water, extracted with DCM, the combined organic layers were washed with saturated brine, and then dried over MgSO 4 and concentrated by rotary evaporation. The concentrate was purified by silica gel column chromatography (using petroleum ether and ethyl acetate with a volume ratio of 4:1 as the eluent), monitored by TLC (the developing agent was determined according to the product, and preferably a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1 was used as the developing agent), and the target product was collected (preferably with an R f value of 0.3 - 0.6), and the spectrum is as shown in Figure 12-13 Figure

[0066] (7) Preparation of ATI-N 3 Preparation

[0067]

[0068] Step 1:

[0069] To a solution of 3,5-dibromo-4-hydroxybenzaldehyde (0.0559 g, 0.20 mmol) was added potassium carbonate (0.0553 g, 0.40 mmol) and 1-azido-4-iodobutane (0.0675 mg, 0.30 mmol). The reaction mixture was heated under reflux for 16 h under argon protection. The reaction was monitored by TLC. After completion of the reaction, it was quenched with water and extracted three times with EA (15.0 mL each time). The combined organic layers were washed with saturated brine and dried over MgSO 4 and concentrated by rotary evaporation. Purification was carried out by silica gel column chromatography (using petroleum ether and ethyl acetate in a volume ratio of 9:1 as the eluent), monitored by thin-layer chromatography (the developing solvent was determined according to the product, preferably a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 9:1), and the target product was collected (preferably with an R f value of 0.4 - 0.6).

[0070] Step 2:

[0071] Piperidine (0.0053 g, 0.063 mmol) and 4-(4-azidobutoxy)-3,5-dibromobenzaldehyde (0.0200 g, 0.050 mmol) were added to a solution of 5-iodoindolin-2-one (0.0106 g, 0.041 mmol) in methanol (4.0 mL). The reaction mixture was heated to 65 °C and stirred for 16 h, then cooled to room temperature. Water (20.0 mL) was added to the mixture and it was extracted three times with DCM (20.0 mL each time). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate, and concentrated by rotary evaporation. The concentrate was purified by silica gel column chromatography (preferably using petroleum ether / ethyl acetate in a volume ratio of 2:1 as the eluent), monitored by TLC (the developing solvent was determined according to the product, preferably a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 2:1), and the target product was collected (preferably with an R f value of 0.4 - 0.6), and the spectrum is as shown in Figure 14-15 .

[0072] The structural general formula of the chemical linking chain (L) is (I).

[0073]

[0074] Wherein, a is an integer from 1 to 4; non-limiting examples are that a is selected from 1, 2, 3, and 4; b is an integer from 1 to 8. Preferably, in the examples, a = 2 and b = 4.

[0075] The chemical linking chain described in the present invention can be obtained through commercial customization (Dangang Biotech Co., Ltd.).

[0076] The monoclonal antibodies and their antigen-binding fragments are all antibodies that have an "antigen-antibody" response to a specific protein, including but not limited to cetuximab, anti-PD-L1 monoclonal antibody, bevacizumab, etc.

[0077] The antibody degrader of the present invention is prepared according to the following steps:

[0078] (1) Antibody labeling: Dissolve an antibody such as a monoclonal antibody in 50 mM phosphate buffer at pH = 7.4 to prepare an antibody solution (5 - 100 μM, preferably 30 μM). Add a chemical linker (preferably DBCO-PEG 4 -NHS ester) thereto, and react at 25 °C for 24 h. Then dialyze using a 100 kDa MWCO dialysis bag, and the retentate is the labeled antibody; the molar ratio of the antibody to the chemical linker in the antibody solution is 1:25;

[0079] (2) Antibody degrader: Configure the obtained antibody label to a preferred concentration of 10 μM, add 25-fold equivalents of a degrading ligand, and oscillate at 25 °C for 48 h. Dialyze the reaction solution using a 100 kDa MWCO dialysis bag, take the retentate to obtain the antibody degrader. The protein labeling situation can be detected by gel imaging and the sample concentration can be detected by the BCA method.

[0080] The present invention provides the application of the antibody degrader in protein targeted degradation. A series of protein targeted degraders based on antibodies are constructed to specifically degrade cell membrane and intracellular proteins. The applications are as follows: Co-incubate cells with the antibody degrader targeting membrane proteins, which can be directly used to degrade membrane proteins; Transfect or deliver the antibody degrader targeting intracellular proteins into cells using an electroporation or protein delivery system, which can be used to degrade intracellular proteins; This strategy has an ideal targeted degradation effect in both live cell and in vivo animal experiments.

[0081] The working principle of the antibody degrader of the present invention is as Figure 1 shown. Based on the high affinity and high selectivity binding between the antibody and the antigen in the degrader, specific recognition of the target protein (regarded as an antigen) is achieved. On this basis, the degrading ligand linked to the antibody has the property of inducing the antibody-antigen complex to enter the proteasome or lysosome degradation pathway, thereby targeting and degrading the antigen protein.

[0082] The following further describes the present invention with specific examples, but the protection scope of the present invention is not limited thereto:

[0083] Example 1: Preparation and application of the antibody degrader Ctx-CMA

[0084]

[0085] (1) Preparation of antibody degrader

[0086] 1) Antibody labeling: Cetuximab (abbreviated as Ctx) was dissolved in 50 mM phosphate buffer at pH = 7.4 to prepare a 30 μM antibody solution. DBCO-PEG 4 -NHS ester was added thereto, and the reaction was carried out at 25 °C for 24 h. Dialysis was performed using a 100 kDa MWCO dialysis bag, and the retentate was the labeled antibody (Ctx-DBCO); the molar ratio of the antibody to the chemical linker in the antibody solution was 1:25.

[0087] 2) Antibody degrader: The obtained antibody label was diluted with 50 mM phosphate buffer at pH = 7.4 to a final concentration of 10 μM, and CMA-N 3 (25-fold equivalent) was added. The reaction was shaken at 25 °C for 48 h. The reaction solution was dialyzed using a 100 kDa MWCO dialysis bag, and the retentate was taken to obtain the antibody degrader (Ctx-CMA). The protein labeling situation can be detected by gel imaging method and the sample concentration can be detected by BCA method.

[0088] (2) Application

[0089] 1. Targeted degradation of membrane proteins in living cells

[0090] The Ctx-CMA obtained in step (1) was dissolved in DMEM medium containing serum to a concentration of 0.1 - 50 nM to obtain a DMEM medium containing 0.1 - 50 nM antibody degrader.

[0091] Human cervical cancer cells HeLa (purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were inoculated in a 10 cm cell culture dish, and the cell count was 10 6 , and 10 mL of DMEM medium (Gibico) containing 10 vol% FBS and 1 wt% double antibody (penicillin with a final concentration of 100 units / ml and streptomycin with a final concentration of 100 μg / ml) was added. The cells were cultured at 37 °C in a 5 vol% CO 2 environment for 24 h. Then the cells were inoculated in a 12-well plate (corning), and 1000 μL of the above DMEM medium containing 10 vol% FBS and 1 wt% double antibody (penicillin with a final concentration of 100 units / ml and streptomycin with a final concentration of 100 μg / ml) was added. The cells were cultured at 37 °C until the cell density reached 70 - 85%. The medium was removed, and the cells were washed once with DMEM medium. 500 μL of DMEM medium containing Ctx-CMA or Ctx (0.1 - 50 nM) was added, and the cells were incubated at 37 °C for 24 h. The cells were collected for immunoblotting assay (WB) to detect the change in the content of EGFR. The results are shown in Figure 16From the results of immunoblotting, compared with the control group Ctx, Ctx-CMA could significantly reduce the content of EGFR protein in HeLa cells, and the degradation effect was positively correlated with the concentration.

[0092] 2. Targeted degradation of membrane proteins in mouse tumor models

[0093] A subcutaneous xenograft tumor model was established in nude mice using A549 cells. When the tumors grew to an appropriate size, 4 mg / kg of PBS, Ctx, or Ctx-CMA was injected into the tail vein every 3 days. The tumors were measured daily and the tumor volumes were calculated. After 16 days of treatment, the subcutaneous tumors were isolated, and the results are shown in Figure 17 . The results showed that treatment with 4 mg / kg Ctx-CMA or Ctx could significantly inhibit tumor growth, and the inhibitory effect of Ctx-CMA was more obvious.

[0094] Example 2. Preparation and application of antibody degrader Ctx-Ada

[0095]

[0096] (I) Preparation of antibody degrader

[0097] 1) Antibody labeling: Cetuximab (abbreviated as Ctx) was dissolved in 50 mM phosphate buffer at pH = 7.4 to prepare a 30 μM antibody solution. DBCO-PEG 4 -NHS ester was added thereto, and the reaction was carried out at 25 °C for 24 h. Dialysis was performed using a 100 kDa MWCO dialysis bag, and the retentate was the labeled antibody (Ctx-DBCO); the molar ratio of the antibody to the chemical linker in the antibody solution was 1:25.

[0098] 2) Antibody degrader: The obtained antibody label was diluted with 50 mM phosphate buffer at pH = 7.4 to a final concentration of 10 μM, and Ada-N 3 (25-fold equivalent) was added, and the mixture was shaken at 25 °C for 48 h. The reaction solution was dialyzed using a 100 kDa MWCO dialysis bag, and the retentate was taken to obtain the antibody degrader (Ctx-Ada). The protein labeling situation could be detected by gel imaging method and the sample concentration could be detected by BCA method.

[0099] (II) Application

[0100] 1. Targeted degradation of membrane proteins in living cells

[0101] The Ctx-Ada obtained in step (I) was dissolved in DMEM medium containing serum to a concentration of 10 nM to obtain a DMEM medium containing 10 nM antibody degrader.

[0102] The human cervical cancer cell line HeLa (purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences) was seeded in a 10-cm cell culture dish, and the cell count was 10 6 , and 10 mL of DMEM medium (Gibico) containing 10 vol% FBS and 1 wt% double antibiotics (penicillin at a final concentration of 100 units / ml and streptomycin at 100 μg / ml) was added. The cells were cultured at 37 °C in a 5 vol% CO 2 environment for 24 h. Then, the cells were seeded in a 12-well plate (Corning), and 1000 μL of the above DMEM medium containing 10 vol% FBS and 1 wt% double antibiotics (penicillin at a final concentration of 100 units / ml and streptomycin at 100 μg / ml) was added. The cells were cultured at 37 °C until the cell density reached 70 - 85%. The medium was removed, and the cells were washed once with DMEM medium. Then, 500 μL of DMEM medium containing Ctx-Ada or Ctx (10 nM) was added, and the cells were incubated at 37 °C for 3 - 12 h. The cells were collected for immunoblotting (WB) to detect the change in the content of EGFR. The results are shown in Figure 18 . From the results of immunoblotting, compared with the control group Ctx, Ctx-Ada could significantly reduce the content of EGFR protein in HeLa cells, and the degradation effect was positively correlated with the incubation time.

Claims

1. An antibody degrader, characterized in that, it comprises an antibody and a degrading ligand coupled by a chemical linker, wherein the antibody is responsible for recognizing and binding to the target protein, and the degrading ligand induces the targeted degradation of the target protein; the degrading ligand is a small molecule or polypeptide containing an azide group that can induce proteasome- or lysosome-dependent protein degradation, one end of the chemical linker has a functional group that can be coupled to the free amino group of the antibody, and the other end has a strained alkyne, and the azide group undergoes a metal-free catalytic bioorthogonal ligation reaction with the strained alkyne to achieve specific connection with the chemical linker; the degrading ligand is one of the following structures: wherein, n is an integer from 1 to 8; The chemical linker uses DBCO-PEG 4 -NHS ester; the antibody is cetuximab.

2. A pharmaceutical composition containing a therapeutically effective amount of the antibody degrader according to claim 1 or a pharmaceutically acceptable salt thereof.

3. Use of the antibody degrader according to any one of claims 1-2 in the preparation of a drug for targeted degradation of proteins, characterized in that, From CMA-N 3 The protein that can be targeted for degradation by the antibody degrader Ctx-CMA obtained by conjugating CMA-N with cetuximab through a chemical linker is the epidermal growth factor receptor EGFR on the membrane of cervical cancer cells or lung adenocarcinoma cells; from Ada-N 3 The protein that can be targeted for degradation by the antibody degrader Ctx-Ada obtained by conjugating Ada-N with cetuximab through a chemical linker is the epidermal growth factor receptor EGFR on the membrane of cervical cancer cells.

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