Polypeptide degradation agent for targeted degradation of CD26 as well as preparation method and application of polypeptide degradation agent

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a polypeptide degradation agent for targeted degradation of CD26 as well as a preparation method and application thereof, and the polypeptide degradation agent is obtained by coupling tetrapeptide and lenalidomide; the amino acid sequence of the tetrapeptide is any one of SEQ ID NO. 1 and SEQ ID NO. 2. The polypeptide degradation agent disclosed by the invention can be used for specifically degrading DPP4, namely CD26 in a targeted manner; however, isoenzymes DPP7, DPP8, DPP9 and the like of DPP4 cannot be degraded, and high selectivity of the isoenzymes DPP7, DPP8, DPP9 and the like is embodied. Besides, the polypeptide degradation agent is small in molecular weight, one end of the polypeptide degradation agent is a natural short peptide, the polypeptide degradation agent has good histocompatibility, target protein can be rapidly degraded within 6 hours, and good tissue permeability is embodied.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a polypeptide degrading agent for targeted degradation of CD26 and a preparation method and application thereof. BACKGROUND

[0002] CD26, also known as dipeptidyl peptidase 4, is a II-type transmembrane glycoprotein dimer with a molecular weight of 110 kDa, and its extracellular region has serine protease activity, which can specifically recognize the X-proline or X-alanine sequence at the N-terminal of polypeptide and catalyze dipeptide cleavage. CD26 is highly expressed in immune cells such as kidney, small intestine, endothelial cells and T lymphocytes, and participates in immune regulation, tumor metastasis and metabolic disease progression through enzyme-dependent and non-enzyme-dependent dual mechanisms. Notably, the interaction of CD26 with extracellular matrix proteins such as collagen and fibronectin has been confirmed to activate the integrin signaling pathway and promote tumor cell migration.

[0003] At present, the clinical intervention strategy for CD26 mainly focuses on small molecule enzyme activity inhibitors such as alogliptin and sitagliptin, which inhibit dipeptide cleavage function by competitive binding to the catalytic pocket. However, such inhibitors have two major technical defects: (1) unable to block the non-enzyme-dependent function of CD26, such as the protein interaction network of CD26 with CD45 and caveolin-1; (2) long-term use is prone to cause compensatory up-regulation of the target and drug resistance. In addition, although monoclonal antibodies such as Begelomab can achieve the removal of membrane surface CD26, they have the risk of immunogenicity and are difficult to penetrate the solid tumor microenvironment.

[0004] In recent years, the protein degradation targeting chimera technology realizes the specific degradation of target proteins through the ubiquitin-proteasome system, providing a new idea to overcome the limitations of traditional inhibitors. However, the existing CD26 degrading agent development faces the following challenges: (1) small molecule PROTAC is limited by steric hindrance effect, and has insufficient binding efficiency for flat targets; (2) antibody-based protein degrading agent AbTAC has penetration limitation due to excessive molecular weight. Therefore, it is of important clinical significance to develop a new type of CD26 degrading agent with high selectivity and good tissue penetration. SUMMARY

[0005] To solve the limitations of traditional CD26 inhibitors, the application provides a polypeptide degrading agent for targeted degradation of CD26, which has high selectivity and good tissue penetration.

[0006] The technical scheme adopted by the application is as follows: The application provides a polypeptide degrading agent for targeted degradation of CD26, which is obtained by coupling a tetrapeptide and lenalidomide. The amino acid sequence of the tetrapeptide is any one of SEQ ID NO. 1 and SEQ ID NO. 2.

[0007] The second aspect of the present application provides a preparation method of the polypeptide degrading agent, comprising the following steps: According to the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, the tetrapeptide is synthesized by solid phase synthesis; The tetrapeptide is coupled with lenalidomide to obtain the polypeptide degrading agent.

[0008] Preferably, the coupling process is: After the tetrapeptide and lenalidomide are co-dissolved in N, N-dimethylformamide, a condensing agent is added, and the reaction is carried out in an alkaline environment under nitrogen protection at 24-26°C for 11-14 hours in the dark.

[0009] Preferably, the condensing agent is O-benzotriazol-N, N, N', N'-tetramethyluronium tetrafluoroborate.

[0010] Preferably, the molar ratio of the tetrapeptide to lenalidomide is 5:6-8.

[0011] Preferably, the reaction condition is 25°C in the dark for 12 hours.

[0012] The third aspect of the present application provides an application of the polypeptide degrading agent, and the polypeptide degrading agent is used for any one of the following: 1) preparing a drug for targeted degradation of CD26; 2) preparing a drug for improving the killing effect of NK cells; 3) preparing a drug for improving the content of cytokines in NK cells; 4) preparing a drug for treating tumors.

[0013] Preferably, the cytokines include at least one of interferon-γ and tumor necrosis factor-α.

[0014] Preferably, the tumor is esophageal cancer.

[0015] Compared with the prior art, the present application has the following beneficial effects: The application provides a polypeptide degrading agent for targeted degradation of CD26, which is obtained by coupling a tetrapeptide and lenalidomide; the amino acid sequence of the tetrapeptide is any one of SEQ ID NO. 1 and SEQ ID NO. 2. The polypeptide degrading agent can specifically target and degrade DPP4, i.e., CD26, but cannot degrade DPP4 isozymes such as DPP7, DPP8 and DPP9, thus showing high selectivity. In addition, the polypeptide degrading agent has a small molecular weight, one end of which is a natural short peptide, and the polypeptide skeleton has relatively strong modifiability compared with small molecules, and is more easily combined with a target with high affinity by flexible adjustment of the amino acid sequence; and has good tissue compatibility, can quickly degrade target proteins in 6 hours, and shows good tissue permeability.

[0016] The application finds and synthesizes two short peptides containing four amino acids, finds that the two short peptides have excellent abilities of targeted combination with CD26 and inhibition of CD26 enzyme activity. A PROTAC polypeptide degrading agent for targeting CD26 is synthesized based on the two short peptides, and it is confirmed that the polypeptide degrading agent has good ability of degrading CD26 in various cells. Meanwhile, in NK cells, it is confirmed that the polypeptide degrading agent has good abilities of promoting NK proliferation and purification. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 LWWY-Lenalidomide is a structural formula.

[0018] Figure 2 LWWH-Lenalidomide is a structural formula.

[0019] Figure 3 PLY and PLH are degradation effect verification.

[0020] Figure 4 PLY and PLH are effects on NK cell viability.

[0021] Figure 5 NK cell killing effect evaluation.

[0022] Figure 6 Cytokine INF-γ level statistics.

[0023] Figure 7 PLH is a degradation effect of DPP8 at different time points.

[0024] Figure 8 PLY is a degradation effect of DPP8 at different time points.

[0025] Figure 9The degradation effects of PLH and PLY on DPP7 and DPP9 at different time points, A: the degradation effects of PLH on DPP7 and DPP9 at different time points; B: the degradation effects of PLY on DPP7 and DPP9 at different time points. DETAILED DESCRIPTION

[0026] The application will be further described by specific examples, but the scope of the application is not limited. The details and forms of the technical solutions of the application can be modified or replaced without departing from the spirit and scope of the application, and these modifications or replacements all fall within the protection scope of the application.

[0027] The inventive concept of the application is as follows: Without re-encoding antigen recognition receptor genes, NK cells mainly recognize major histocompatibility complex through a series of specific cell surface receptors, activate NK cells to play corresponding biological functions through receptor and ligand interaction. According to domestic and foreign researches, NK cell immunotherapy is safe and effective in treating and preventing malignant tumors. Although NK cells show potential, their clinical application is still limited by multiple technical barriers, including: long culture period and insufficient expansion efficiency of conventional expansion scheme; up-regulation of NK cell inhibitory receptors such as NKG2A causes functional exhaustion; tumor microenvironment inhibits the killing effect of NK cells. These systemic defects seriously limit the production and clinical use of NK products, and technical innovation is urgently needed to achieve breakthroughs.

[0028] The application provides a polypeptide degradation agent targeting CD26 to solve the above problems. The polypeptide degradation agent can target and degrade CD26, and can improve the proliferation activity of NK cells, and can enhance the killing function of NK cells by promoting the secretion of cytokines such as interferon-γ and tumor necrosis factor-α during the killing process.

[0029] Based on the technical bottleneck of existing CD26 targeted therapy, the application provides a polypeptide degradation agent for targeting and degrading CD26, wherein the polypeptide degradation agent is obtained by coupling a tetrapeptide and lenalidomide; the amino acid sequence of the tetrapeptide is any one of SEQ ID NO. 1 and SEQ ID NO. 2. SEQ ID NO. 1: LWWY; SEQ ID NO. 2: LWWH. The tetrapeptide provided by the application targets and recognizes CD26 protein and simultaneously inhibits enzyme activity, and lenalidomide recruits ubiquitination to cause protein degradation. The two are synthesized together to form a PROTAC drug, so as to achieve the purpose of targeting and degrading CD26.

[0030] The application aims to develop a new type of targeted degradation agent, which specifically solves the following core problems: 1. Limitations of traditional small molecule inhibitors.

[0031] Inadequate functional inhibition: current DPP4 enzyme activity inhibitors such as alogliptin can only block the catalytic function of CD26, and cannot intervene in the non-enzymatic activity signal pathway mediated by protein interaction, such as the integrin / FAK pathway driven tumor metastasis.

[0032] Drug resistance risk: long-term use leads to up-regulation of CD26 compensatory expression, reducing treatment effect.

[0033] 2. Technical defects of antibody drugs.

[0034] Permeability limitation: anti-CD26 mAb, with a molecular weight of about 150 kDa, is difficult to penetrate solid tumor tissue.

[0035] 3. Adaptability of PROTAC technology Target binding problem: CD26 lacks a flat surface structure of a deep binding pocket, resulting in insufficient driving effective degradation of traditional small molecule PROTAC Warhead modules, such as based on sitagliptin derivatives.

[0036] 4. Application scenario expansion requirements.

[0037] Current CD26 targeting agents mainly focus on type 2 diabetes treatment, and their application in tumor immune microenvironment regulation and drug-resistant tumors lacks systematic research.

[0038] The present application realizes the following breakthrough objectives by designing a polypeptide degrading agent targeting CD26: Dual mechanism blocking: simultaneous elimination of the enzyme activity function of CD26 and its protein interaction-mediated non-enzymatic activity signal transduction.

[0039] High permeability: using a polypeptide backbone of 4 amino acids, the molecular weight is less than 3 kDa.

[0040] Anti-drug resistance advantage: complete degradation of CD26 protein through the proteasome pathway, avoiding the risk of target compensatory up-regulation.

[0041] Multifunctional application: expanded to the field of tumor immunotherapy, enhancing NK cell proliferation and killing In order for those skilled in the art to better understand the technical solutions of the present application and to implement them, the present application will be further described below in conjunction with specific examples. In the description of the present application, if not specifically stated, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0042] The abbreviation table of the present application is shown in Table 1.

[0043] Table 1 Abbreviation table of the present application Example 1 A method for preparing a polypeptide degrader targeting CD26, specifically as follows: 1. Solid phase synthesis of tetrapeptide chain H-Leu-Trp-Trp-Tyr-OH.

[0044] 1.1. Loading of the starting amino acid Tyr.

[0045] Take Fmoc-Tyr(tBu)-Wang resin with a loading of 0.3 mmol / g, 0.5 g, and place it in a 30 mL polypeptide synthesis reactor. Add dichloromethane 15 mL, and swell at room temperature for 30 min, and remove the solvent by suction filtration. Wash the resin with N,N-dimethylformamide, 10 mL each time, and suction filter to dryness in 3 times.

[0046] 1.2. Assembly of the second position tryptophan Trp.

[0047] Remove the Fmoc protecting group: add 20% v / v piperidine / DMF solution 15 mL, protect with nitrogen, and oscillate the reaction at room temperature for 15 min, and suction filter; repeat the deprotection once at room temperature for 10 min.

[0048] Washing test: wash the resin with DMF 6 times, 10 mL each time; take 5 resin particles for ninhydrin test, and confirm that the free amino group is dark blue.

[0049] Coupling reaction: weigh Fmoc-Trp(Boc)-OH 0.6 mmol, condensing agent TBTU 0.6 mmol, and place them in a beaker, add DCM / DMF mixed solvent prepared according to the volume ratio of 1:1 8 mL, and then inject N,N-diisopropyl ethylamine 1.2 mmol. After pre-activation for 5 min, transfer the solution to the reactor, and oscillate the reaction at room temperature for 2 h under nitrogen protection.

[0050] Post-processing: remove the reaction liquid by suction filtration, and wash the resin with DMF 3 times, 10 mL each time.

[0051] 1.3. Assembly of the third position tryptophan Trp.

[0052] The same as step 1.2, and the same amount of reagent is used.

[0053] 1.4. Assembly of the fourth position leucine Leu.

[0054] Remove the Fmoc protecting group: remove the Fmoc group as in 1.2.

[0055] Coupling optimization: 0.6 mmol Fmoc-Leu-OH was dissolved in 8 mL pure DMF, and 0.6 mmol of condensing agent TBTU was added, and 1.2 mmol of DIEA was pre-activated for 5 min. The activated solution was transferred to the resin of the previous step, and the reaction was shaken for 3 h under nitrogen protection.

[0056] N-terminal deprotection and verification: the resin was treated with 15 mL of 20% piperidine / DMF (v / v) for 15 min twice. The 8 mL pure DMF washes were neutral, and the presence of free amino groups was confirmed by ninhydrin detection.

[0057] The final resin peptide was obtained.

[0058] 1.5, Cleavage and purification of the crude peptide.

[0059] The cleavage solution was prepared with the following formula: Trifluoroacetic acid: triisopropylsilane: water = 95:2.5:2.5, v / v / v, and 10 mL of cleavage solution was prepared for cleavage in this example.

[0060] The cleavage solution was added to the reactor, and the cleavage was carried out at room temperature for 2.5 h under light protection. The filtrate was collected by suction filtration, and the resin was washed with fresh TFA for 3 times, 2 mL each time, and the filtrates were combined.

[0061] The filtrate was slowly dropped into 50 mL of pre-cooled ether at -20°C, and the tetrapeptide was precipitated by ice bath standing for 30 min.

[0062] The precipitate was collected by centrifugation at 3000 rpm for 5 min, washed with cold ether for 3 times, and vacuum dried for 4 h to obtain the crude peptide LWWY. About 120 mg of crude peptide LWWY was obtained in this example.

[0063] 2, Coupling of LWWY with lenalidomide.

[0064] 2.1, Activation and condensation reaction.

[0065] 0.15 mmol of crude peptide LWWY and 0.18 mmol of lenalidomide were dissolved in 10 mL of anhydrous DMF. The ice bath was cooled to 4°C, and 0.18 mmol of condensing agent TBTU and 0.54 mmol of base DIEA were added in sequence; the ice bath was removed, and the reaction was carried out at 25°C under nitrogen protection for 12 h in the dark.

[0066] Process monitoring: samples were taken every 2 h for HPLC analysis. HPLC analysis: C18 column, gradient elution with acetonitrile / 0.1% v / v TFA aqueous solution, detection at 280 nm, and the target product had a retention time of about 18.5 min.

[0067] 2.2, Post-treatment and purification.

[0068] The reaction solution was concentrated to 3 mL at 30°C under reduced pressure, 10 mL of a 1:1 v / v TFA / DCM mixture was added, and the byproduct was quenched by stirring at room temperature for 30 min. The mixture was dropped into 50 mL of methyl tert-butyl ether pre-cooled at 0°C, and after ultrasonic dispersion for 1 min, it was placed in an ice bath for 1 h. The precipitate was collected by centrifugation at 4000 rpm for 10 min, and vacuum dried to obtain the crude conjugate.

[0069] 2.3, High-performance liquid purification.

[0070] The crude conjugate was dissolved in 500 μL of DMSO and 1.5 mL of a v / v 0.1% TFA aqueous solution, and was loaded into a preparative HPLC system, which had the following parameters: Chromatographic column: Kromasil C18 20 mm x 250 mm; mobile phase: A phase - v / v 0.1% TFA aqueous solution, B phase - v / v 0.1% TFA acetonitrile solution; gradient program: 0 min ~ 5 min 20% B phase → 30 min ~ 40 min 60% B phase; flow rate: 8 mL / min, detection wavelength: 280 nm.

[0071] The fractions with retention times of 17.5 min ~ 19.5 min were collected, and the operation was performed in the dark in an ice bath. The combined fractions were freeze-dried to obtain LWWY-Lenalidomide white powder, about 10 mg of LWWY-Lenalidomide was prepared in this example. The specific structure is shown in Figure 1 .

[0072] Example 2 A method for preparing a polypeptide degrading agent targeting CD26 degradation, specifically as follows: 1. Solid-phase synthesis of tetrapeptide chain H-Leu-Trp-Trp-His-OH.

[0073] 1.1, Loading of starting amino acid.

[0074] Take Fmoc-His(Trt)-Wang Resin resin with a loading of 0.3 mmol / g, 0.5 g, i.e. 0.15 mmol; the imidazole nitrogen of His is protected by triphenylmethyl, and after swelling in DCM for 30 min, wash with DMF for 3 times, 10 mL each time.

[0075] 1.2, Assembly of the second tryptophan Trp.

[0076] Remove the Fmoc protecting group: v / v 20% piperidine / DMF 15 mL, shake 2 times, 15 min each time.

[0077] Washing test: DMF washing 6 times, ninhydrin test is dark blue.

[0078] Coupling reaction: coupling Trp to the product of 1.1, reagent system as follows: Fmoc-Trp(Boc)-OH: 0.6 mmol, TBTU: 0.6 mmol, DIEA: 1.2 mmol, solvent: DCM / DMF 8 mL prepared with volume ratio of 1:1.

[0079] The coupling process is exactly the same as 1.2 of the example 1.

[0080] Removing Fmoc protecting group: same as 1.1 of the example.

[0081] 1.3, assembling the third position tryptophan Trp.

[0082] Same as 1.2 of the example, same equivalent reagents are used.

[0083] 1.4, assembling the fourth position leucine Leu.

[0084] Same as 1.4 of the example 1, finally the resin peptide is obtained.

[0085] 1.5, cleavage and purification of crude peptide.

[0086] Stepwise cleavage strategy is adopted to avoid the side reaction of His: First, 10 mL of v / v 1% TFA / DCM is used to oscillate for 10 min, suction filtration, and the Trt protecting group of His is removed; then the standard cleavage solution is replaced → oscillation for 2 h, cleavage of the peptide chain and removal of Boc → the filtrate is dropped into 50 mL of cold ether at -20℃ → precipitation → centrifugation → cold ether washing for 3 times → vacuum drying → crude peptide LWWH. The crude peptide LWWH obtained in this example is about 115 mg, which is a light yellow solid.

[0087] The formula of the standard cleavage solution is as follows: TFA: TIS: H2O: benzyl thioether = 90: 2.5: 2.5: 5, 10 mL is prepared for cleavage in this example.

[0088] 2, coupling LWWH with lenalidomide.

[0089] 2.1, activation condensation reaction.

[0090] The crude peptide LWWH and lenalidomide are dissolved in anhydrous DMF. Condensing agent TBTU and base DIEA are added successively under 4℃ ice bath; remove the ice bath, and react for 12 h under 25℃ in the dark under nitrogen protection. The material ratio is shown in Table 2.

[0091] Table 2 material ratio Process monitoring: sample every 2h for HPLC analysis. HPLC analysis: C18 column, acetonitrile / 0.1% v / v TFA aqueous solution gradient elution, 280nm detection, target product retention time about 17.8min.

[0092] 2.2, Post-treatment and purification.

[0093] Same as 2.2 of Example 1.

[0094] 2.3, High performance liquid purification.

[0095] The crude conjugate was dissolved with 500μL DMSO and 1.5mL aqueous solution containing 0.1% v / v TFA, and then loaded into a preparative HPLC system, and the parameters of the preparative HPLC system were as follows: Chromatographic column: Kromasil C18 20mm×250mm; mobile phase: A phase-0.1% v / v TFA aqueous solution, B phase-0.1% v / v TFA acetonitrile solution; gradient program: 0min~5min 15% B phase→30min 50% B phase; flow rate: 8mL / min, detection wavelength 280nm.

[0096] The fractions with retention time of 17.5min~18.2min were collected, and the operation was carried out in dark ice bath. The fractions were combined and freeze-dried to obtain LWWH-Lenalidomide, and the LWWH-Lenalidomide prepared in the present example was about 10mg. The specific structure was as follows: Figure 2 .

[0097] Example 3 Application of a polypeptide degrading agent targeting degradation of CD26, specifically as follows: LWWY-Lenalidomide prepared in Example 1 and LWWH-Lenalidomide prepared in Example 2 were subjected to effect verification, and for convenience of expression, LWWY-Lenalidomide was referred to as PLY, and LWWH-Lenalidomide was referred to as PLH hereinafter.

[0098] 1, Degradation effect evaluation.

[0099] PLH, PLY prepared by the present application and the 1st generation degrading agent P4-3 of CD26 were simultaneously used on KYSE-140 cells for 6h to explore the degradation effect on CD26. The concentration of PLH, PLY and P4-3 was 10μM.

[0100] The Western blot results are as follows: Figure 3, wherein the cell group is a blank control group, PLH, PLY and P4-3 can all degrade the CD26 of the cells, but the degradation effect of PLH and PLY is better than that of P4-3.

[0101] 2, Effect on cell viability.

[0102] PLH and PLY prepared by the present application were simultaneously used on NK cells for 24 hours with the 1st generation of CD26 degrading agent P4-3 to explore their effect on cell viability. The concentration of PLH, PLY and P4-3 was 10 μM.

[0103] The results are shown in Figure 4 , wherein the cell group is a blank control group, after adding PLH, PLY and P4-3 to NK cells for 24 hours, if the cell viability of the cell group is taken as 100%, the cell viability of P4-3 is increased by 37%, and the cell viability of PLH and PLY is increased by 129.9% and 126.5% respectively, P<0.05.

[0104] 3, Evaluation of promoting NK killing effect.

[0105] Esophageal cancer cells KYSE-140 were inoculated in a 96-well plate and cultured to a cell density of 80%, and then divided into 5 groups, NK cells, NK cells+PLH, NK cells+PLY and NK cells+P4-3 were added according to the effector-target ratio of 1:1, wherein the concentration of PLH, PLY and P4-3 was 10 μM, and the CCK8 of KYSE-140 was measured after 24 hours. The cell group is a blank control group, the simple NK cell group is recorded as cell+NK, the NK cell+PLH is recorded as cell+PLH, the NK cell+PLY is recorded as cell+PLY, and the NK cell+P4-3 is recorded as cell+P4-3.

[0106] The results are shown in Figure 5 , the killing effect of the simple NK cell group is 33%, and the killing effect is increased to 51.67% on the basis of NK by adding P4-3; the killing effect is increased to 81.09% by adding LWH, and the killing effect is increased to 77.59% by adding LWY, P<0.05.

[0107] Esophageal cancer cells KYSE-140 were inoculated in 96-well plates and cultured to a cell density of 80%, and then divided into 5 groups, and NK cells, NK cells+PLH, NK cells+PLY and NK cells+P4-3 were added according to an effector-to-target ratio of 1:1, wherein the concentrations of PLH, PLY and P4-3 were all 10 μM, and the cytokine levels in the culture medium were determined after 24 h. The cell group was NK cells without KYSE cell stimulation, the simple NK cell group was recorded as cell+NK, the NK cell+PLH group was recorded as cell+PLH, the NK cell+PLY group was recorded as cell+PLY, and the NK cell+P4-3 group was recorded as cell+P4-3.

[0108] The cytokine INF-γ level in the killing environment was determined, as shown in Table 2. Figure 6 The INF-γ levels of the cell group, the cell+NK group, the cell+p4-3 group, the cell+LWH group and the cell+LWY group were 21.83 ng / ml, 37.33 ng / ml, 68.83 ng / ml, 83.83 ng / ml and 84.17 ng / ml, respectively, and P<0.05.

[0109] 4. Selectivity and tissue penetration verification.

[0110] PLH, PLY and the degrading agent P4-3 prepared in the application were respectively used on KYSE-140 cells for 3 h, 6 h and 9 h, and the concentrations of PLH, PLY and P4-3 were all 10 μM. The results showed that the isozymes DPP7, DPP8 and DPP9 were not degraded, and the results are shown in Table 3, which embodies the high selectivity. Figures 7-9

[0111] In addition, the polypeptide degrading agent described in the application has a small molecular weight and a natural short peptide at one end, has good tissue compatibility, and can quickly degrade the target protein in 6 hours.

[0112] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, but as long as the combinations of the technical features do not contradict, they should be considered as within the scope of the present disclosure.

[0113] The above-described embodiments only express several implementation manners of the application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that, for those skilled in the art, without departing from the concept of the application, a number of modifications and improvements can be made, which are all within the protection scope of the application.​

Claims

1. A polypeptide degrader that targets degradation of CD26, characterized in that, The polypeptide degradation agent is obtained by coupling a tetrapeptide and lenalidomide; The amino acid sequence of the tetrapeptide is any one of SEQ ID NO. 1 and SEQ ID NO.

2.

2. The method for preparing the polypeptide degrading agent as described in claim 1, characterized in that, The method comprises the following steps: The tetrapeptide is synthesized by solid phase according to the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; The tetrapeptide is coupled with lenalidomide to obtain the polypeptide degradation agent; The coupling process is as follows: The tetrapeptide and lenalidomide are co-dissolved in N, N-dimethylformamide, and then a condensing agent is added, and the reaction is carried out in an alkaline environment under nitrogen protection at 24-26°C for 11-14 hours in the dark.

3. The production method according to claim 2, wherein The condensing agent is O-benzotriazole-N, N, N', N'-tetramethyluronium tetrafluoroborate.

4. The production method according to claim 2, wherein The molar ratio of the tetrapeptide to lenalidomide is 5:6-8.

5. The production method according to claim 2, wherein The reaction condition is 25°C in the dark for 12 hours.

6. The use of a polypeptide degrading agent according to claim 1, wherein The polypeptide degradation agent is used in any one of the following: 1) preparation of a drug for targeted degradation of CD26; 2) preparation of a drug for improving the killing effect of NK cells; 3) preparation of a drug for improving the content of cytokines in NK cells; 4) preparation of a drug for treating tumors.

7. Use according to claim 6, wherein The cytokines include at least one of interferon-γ and tumor necrosis factor-α.

8. Use according to claim 7, wherein the compound is ###0002### The tumor is esophageal cancer.

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