Double immune checkpoint co-blocking agent for targeting EGFR and application of double immune checkpoint co-blocking agent

Abstract

The invention provides a dual immune checkpoint co-blocking agent for targeting EGFR (epidermal growth factor receptor), the blocking agent is prepared into a target polypeptide molecule by an Fmoc solid phase polypeptide synthesis method, and the polypeptide molecule has a self-assembly behavior in a solution containing EGFR protein; the critical assembly concentration of polypeptide molecules triggered by EGFR protein is 18.2 [mu] M, and nanofibers with the diameter of 8.5 nm are formed through the self-assembly behavior; the nanofiber captures membrane cholesterol and induces membrane vesicles to fall off through a high-density hydrophobic area of the nanofiber, so that EGFR and cholesterol are removed at the same time, and the following dual effects are achieved: 1, biochemical immune checkpoint blocking: reduction of EGFR causes reduction of STAT3 phosphorylation level, and further down-regulation of PD-L1 expression; 2, biomechanical immune checkpoints are blocked, wherein cholesterol is removed, so that the rigidity of tumor cell membranes is remarkably improved, and the sensitivity of the tumor cell membranes to mechanical attack of T cells is enhanced; the blocking agent can significantly improve the killing effect of T cell immunotherapy such as CAR-T cells on solid tumors.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy and nanobiotechnology, specifically relating to a dual immune checkpoint co-blocker targeting EGFR and its application. Background Technology

[0002] Tumor immune escape is one of the core challenges in cancer treatment. Tumor cells evade the body's immune system through multi-dimensional and synergistic mechanisms, severely limiting clinical treatment efficacy. At the biochemical immunomodulation level, tumor cells often abnormally upregulate immune checkpoint molecules such as programmed death-ligand 1 (PD-L1). After this molecule binds to the PD-1 receptor on the surface of T cells, it initiates immunosuppressive signals in T cells, directly weakening the proliferative activity and cytotoxicity of effector T cells, allowing tumor cells to survive and proliferate under immune surveillance. Although single immune checkpoint blockade therapies such as anti-PD-1 / PD-L1 antibodies have achieved breakthroughs in the treatment of some tumors, they still face limited efficacy in solid tumors. The response rate for most patients is less than 30%, and drug resistance easily develops after long-term treatment. The core reason is that tumor cells can bypass single blockade targets by switching to other immune checkpoint pathways and reshaping the tumor microenvironment, leading to treatment failure.

[0003] Meanwhile, the remodeling of the mechanical properties of tumor cells has become another key mechanism for immune escape. Normal cells maintain a certain degree of cell membrane stiffness to ensure cell morphology and functional stability, while tumor cells reduce cell membrane stiffness by downregulating cytoskeletal protein expression and increasing cell membrane cholesterol content, making the cell membrane highly fluid. This change in mechanical properties directly affects the interaction between T cells and tumor cells: the soft tumor cell membrane is difficult for T cell surface TCR receptors to recognize effectively and cannot form stable immune synapses, leading to the obstruction of T cell killing signal transmission and further weakening the immune system's ability to clear tumor cells.

[0004] Currently, interventions targeting cell mechanical properties have been proven to have potential therapeutic value. For example, the cholesterol scavenger MeβCD can enhance the rigidity of tumor cells by removing cell membrane cholesterol, thereby improving the recognition efficiency of T cells. However, these methods have significant drawbacks: they lack tumor tissue targeting and can easily damage normal cells while acting on tumor cells, leading to off-target effects. More importantly, they can only regulate cell mechanical properties and cannot synergistically intervene in biochemical and immune checkpoint pathways such as PD-L1, making it difficult to break the pattern of multi-mechanism synergistic escape of tumor cells.

[0005] In summary, both single biochemical and immune checkpoint blockade therapies and biomechanical interventions have limitations and cannot achieve comprehensive regulation of tumor immune escape mechanisms. Therefore, developing a dual-function treatment strategy that can precisely target tumor tissue while synergistically intervening in biochemical and immune checkpoints and cellular biomechanical properties has become an urgent need to overcome existing treatment bottlenecks and improve the treatment efficacy of solid tumors, and is of great significance to promoting the development of tumor immunotherapy. Summary of the Invention

[0006] The purpose of this invention is to provide a dual immune checkpoint co-blocker targeting EGFR, which can specifically recognize EGFR on the surface of tumor cells and self-assemble in situ to form nanofibers, thereby achieving synergistic blocking of biochemical and biomechanical dual immune checkpoints to overcome the above-mentioned technical problems.

[0007] To achieve the above objectives, the technical solution provided by the present invention is as follows: A dual immune checkpoint co-blocker targeting EGFR, wherein the blocker is a polypeptide molecule having the structure shown in Formula I: Formula I.

[0008] The above-mentioned blocking agent was prepared using the Fmoc solid-phase peptide synthesis method.

[0009] The Fmoc solid-phase peptide synthesis method includes the following steps: a) Provide a solid resin modified with cysteine ​​and protected at the N-terminus by an Fmoc group; b) After the resin swells overnight, the resin is deprotected using a DMF solution containing 20% ​​hexahydropyridine by volume, and then washed to remove the Fmoc protecting groups. c) Under the catalysis of the coupling agent N-methylmorpholine, 10 molar equivalents of proline and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate are reacted with the deprotected resin by oscillation to couple proline to the amino group of cysteine. d) Repeat steps b) and c) until the coupling of all amino acids in the sequence shown in Formula I is complete; e) The synthesized peptide is lysed from the resin using a lysis buffer to remove the side-chain protecting groups, and the target peptide molecule is obtained after purification.

[0010] Preferably, in step a), the solid-phase resin is Wang resin, and the cysteine ​​modification density is 0.37 mM; in step b), the resin is swollen overnight with anhydrous DMF, the deprotection time is 15 min, and the washing is performed three times alternately with DMF and dichloromethane; in step c), the coupling agent is a DMF solution with a volume ratio of 5% N-methylmorpholine; the shaking reaction time is 60 min; in step e), the lysis buffer is a trifluoroacetic acid solution containing a volume ratio of 2.5% water, 2.5% triisopropylsilane, and 2.5% 1,2-ethylenedithiol, and the purification process is as follows: the trifluoroacetic acid is dried with low-flow-rate nitrogen gas, and the crude polypeptide product is precipitated with anhydrous diethyl ether, washed, and dried.

[0011] Furthermore, the polypeptide molecules exhibit self-assembly behavior in solutions containing EGFR protein.

[0012] Furthermore, the critical assembly concentration of the polypeptide molecule triggered by the EGFR protein was 18.2 μM.

[0013] Furthermore, the self-assembly behavior forms nanofibers with a diameter of 8.5 nm.

[0014] Application of a dual immune checkpoint co-blocker targeting EGFR: Nanofibers formed by the self-assembly behavior of the co-blocker can simultaneously clear cholesterol and downregulate PD-L1 expression on the surface of tumor cells, enhance T-cell immunotherapy, and be used in combination with CAR-T cells to treat solid tumors.

[0015] The beneficial effects of this invention are as follows: The EGFR-targeting dual immune checkpoint co-blocker provided by this invention can specifically recognize EGFR on the surface of tumor cells and self-assemble in situ into nanofibers. These nanofibers capture membrane cholesterol through their high-density hydrophobic water regions, inducing vesicle shedding and thus simultaneously removing EGFR and cholesterol, achieving the following dual effects: first, biochemical immune checkpoint blockade: the reduction of EGFR leads to a decrease in STAT3 phosphorylation levels, thereby downregulating PD-L1 expression; second, biomechanical immune checkpoint blockade: cholesterol clearance significantly increases tumor cell membrane stiffness, enhancing its sensitivity to T cell mechanical attack. This blocker can significantly improve the killing effect of CAR-T cell and other T-cell immunotherapies on solid tumors, such as a breast cancer lung metastasis model, exhibiting good targeting, synergistic effects, and therapeutic safety. Attached Figure Description

[0016] Figure 1 The figure shows the results of determining the critical assembly concentration of the peptide using the pyrene probe method.

[0017] Figure 2 This is a transmission electron microscope (TEM) image of the morphology of a polypeptide assembly.

[0018] Figure 3This represents the cholesterol content in the cell membrane after peptide treatment.

[0019] Figure 4 Cell stiffness as measured by atomic force microscopy.

[0020] Figure 5 The values ​​represent the levels of EGFR, PD-L1, and Vinculin in the cell membrane after peptide treatment.

[0021] Figure 6 This image shows the anti-tumor effect of T cells after peptide treatment.

[0022] Figure 7 This diagram illustrates how peptides can enhance the therapeutic effects of CAR-T cell therapy. Detailed Implementation

[0023] To make the technical objectives, solutions, and effects of this invention easier to understand, the invention will now be further described in conjunction with the embodiments and accompanying drawings.

[0024] All reagents used in this invention are commercially available and commonly used. Example

[0025] This embodiment provides a dual immune checkpoint co-blocker targeting EGFR, the structural formula of which is shown below: The blocking agent was specifically synthesized using the 9-fluorenylmethoxycarbonyl Fmoc solid-phase polypeptide synthesis method: Wang resin with N-terminus protected by Fmoc groups modified with cysteine ​​and a modification density of 0.37 mM was selected. The resin was swollen overnight with anhydrous N,N-dimethylformamide (DMF), and then deprotected for 15 min with a DMF solution containing 20% ​​hexahydropyridine by volume to remove the Fmoc protecting group. The resin was then washed three times alternately with DMF and dichloromethane (DCM). The success of deprotection was then detected by the ninhydrin test method, wherein the ninhydrin test solution was prepared by ninhydrin, vitamin C, and phenol in a volume ratio of 1:1:1. After confirming the removal of the protecting group: the resin color turns dark blue. Weigh out 10 molar equivalents of proline and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), dissolve them in a 5% (v / v) DMF solution of N-methylmorpholine, and add them to the deprotected resin. React on a shaker for 60 min. Under the catalysis of the coupling agent N-methylmorpholine, proline is covalently coupled to cysteine ​​through the condensation reaction between the amino group of cysteine ​​and the carboxyl group of proline. After the reaction was completed, the resin was washed three times with DMF and DCM alternately. Then, the ninhydrin test was used to check whether proline was coupled to cysteine. If the resin did not change color, it indicated that the amino acid coupling was successful. Repeat the above operation to complete the coupling of all amino acids. After removing the last amino acid protecting group, the synthesized peptide was lysed from the resin using a lysis buffer containing 2.5% water, 2.5% triisopropylsilane, and 2.5% 1,2-ethylenedithiol in a volume ratio of trifluoroacetic acid, while simultaneously removing the side chain protection of the amino acid. The trifluoroacetic acid was dried with nitrogen gas at a low flow rate, and the crude peptide product was precipitated with anhydrous diethyl ether, washed, and dried to obtain the target peptide fragment. The molecular weight was characterized by mass spectrometry, and the target molecule was finally confirmed.

[0026] The molecular assembly behavior of the peptides prepared above under EGFR triggering is as follows: The critical assembly concentration (CAC) of the peptide was determined using the pyrene probe method. Specifically, peptide molecular solutions containing EGFR protein at concentrations of 2 μM, 4 μM, 10 μM, 20 μM, 30 μM, 35 μM, 45 μM, 50 μM, and 60 μM were prepared. The same amount of pyrene was added to each solution, and after mixing, the fluorescence values ​​at 373 nm and 414 nm were measured at an excitation wavelength of 393 nm, and their ratio was calculated. The resulting fluorescence ratio-concentration curve is shown below. Figure 1 As shown, the results indicate that when the peptide molecule solution concentration is 18.2 μM, the ratio suddenly increases, indicating that the nonpolarity of the microenvironment in which pyrene is located suddenly increases. This suggests that pyrene is solubilized in the hydrophobic core formed by the assembly of peptide molecules, which means that the critical assembly concentration of peptide molecules is 18.2 μM.

[0027] The morphology of the assembly was characterized by transmission electron microscopy (TEM): lanthanum hexaboride TEM, Tecnai G2 20 S-TWIN (T-20), FEI Corporation, USA. The resulting assembly morphology image is shown below. Figure 2 As shown in the figure, the peptides assemble into nanofibers with a diameter of approximately 8.5 nm under EGFR triggering.

[0028] Cholesterol clearance, stiffness enhancement and PD-L1 downregulation effects of peptide nanofibers at the cellular level Cholesterol Filipin staining flow cytometry analysis: A549 cells pretreated with peptides or methyl-β-cyclodextrin (MβCD) were stained with 10 µg mL⁻¹ Filipin at 4°C in the dark for 30 minutes. After washing, the cells were resuspended in 10 µg mL⁻¹ propidium iodide solution for flow cytometry analysis. Results are as follows: Figure 3 As shown, the cholesterol content of cell membranes was significantly reduced after peptide treatment, and was lower than that of the positive control group MβCD.

[0029] Cell stiffness was measured using atomic force microscopy (AFM): Single cells were measured at 37°C, and cell stiffness was determined before and after treatment with either peptides or MβCD. An AFM cantilever probe contacted the cell surface, and deflection data resulting from the interaction were monitored and recorded in real time. The calculated Young's modulus reflects the change in cell stiffness. Results are as follows: Figure 4 As shown, the cell stiffness increased significantly after peptide treatment, exceeding that of the positive control group MβCD.

[0030] Western blot analysis of proteins: Pretreated cells were resuspended in a lysis buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton-X 100 (v / v), and protease inhibitors. Protein concentration was determined using a BCA kit. 60 µg of protein sample was mixed with loading buffer and heated at 95°C for 10 min to denature the protein. Electrophoresis was performed on a 4-20% Bis-Tris protein gel at 100 V for 1.5 h, followed by transfer to a PVDF membrane. The transferred membrane was blocked with TBST solution containing 5% skim milk, incubated overnight with primary antibody on a shaker at 4°C, and then incubated with the corresponding secondary antibody at room temperature for 1 h. Finally, the membrane was scanned using a multimodal imaging system. Band gray values ​​were analyzed using NIH ImageJ software. The primary antibodies used and their dilution ratios were as follows: EGFR (1:1000), PD-L1 (1:1000), and Vinculin (1:1000). Results are as follows: Figure 5 As shown, the levels of EGFR and PD-L1 in the cell membrane decreased significantly after peptide treatment.

[0031] The effect of peptide nanofiber-induced biological effects on T cell killing efficacy CD8⁺T cell cytotoxicity assay: To evaluate the antitumor effect of effector T cells, A549 tumor cells were seeded in 12-well plates at a density of 2.0 × 10⁶ cells per well. 5 Cells were cultured overnight, then the medium was replaced with low-glucose RPMI-1640 medium and cultured for another 24 hours, followed by peptide treatment for 24 hours. After washing three times with PBS, effector T cells were added at a 1:1 ratio to target cells and co-cultured for 24 hours. Cytotoxicity was assessed by detecting the release of lactate dehydrogenase (LDH) in the culture medium. Results are as follows: Figure 6 As shown, the killing ability of T cells treated with peptides was 2.4 times that of the untreated group.

[0032] Investigating the potential applications of peptide-enhanced CAR-T therapy in mouse tumor models In vivo CAR-T cell therapy: 4T1-EGFR-Luc cells were injected intravenously into the tail vein of female BALB / c mice. On day 5, tumor-bearing mice were randomly divided into four groups: saline group, P1 peptide group, CAR-T cell group, and combination therapy group. From day 6 to day 16, mice were administered 5 mg·kg⁻¹ peptide molecules or PBS intravenously, and EGFR-CAR T cells were adoptively infused intravenously at 3 × 10⁶ cells on days 6, 10, and 14. 6 One mouse was administered, or no infusion was given. The survival status of tumor-bearing mice was monitored for at least 60 days. Mice were euthanized when their body weight decreased by more than 15% of their baseline weight. Results were as follows: Figure 7 As shown, the peptides can significantly enhance the therapeutic effect of CAR-T cell therapy and significantly prolong the survival of tumor-bearing mice.

Claims

1. A dual immune checkpoint co-blocker targeting EGFR, characterized in that, The blocking agent is a polypeptide molecule having the structure shown in Formula I: Equation I.

2. The dual immune checkpoint co-blocker targeting EGFR as described in claim 1, characterized in that, The blocking agent was prepared using the Fmoc solid-phase peptide synthesis method.

3. A dual immune checkpoint co-blocker targeting EGFR as described in claim 2, characterized in that, The Fmoc solid-phase polypeptide synthesis method includes the following steps: a) Provide a solid resin modified with cysteine ​​and protected at the N-terminus by an Fmoc group; b) After the resin swells overnight, the resin is deprotected using a DMF solution containing 20% ​​hexahydropyridine by volume, and then washed to remove the Fmoc protecting groups. c) Under the catalysis of a coupling agent, 10 molar equivalents of proline and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate are reacted with the deprotected resin by oscillation to couple proline to the amino group of cysteine. d) Repeat steps b) and c) until the coupling of all amino acids in the sequence shown in Formula I is complete; e) The synthesized peptide is lysed from the resin using a lysis buffer to remove the side-chain protecting groups, and the target peptide molecule is obtained after purification.

4. A dual immune checkpoint co-blocker targeting EGFR as described in claim 3, characterized in that, The solid resin mentioned in step a) is Wang resin, and the cysteine ​​modification density is 0.37 mM; The resin described in step b) is swollen overnight with anhydrous DMF, the deprotection time is 15 min, and the washing is performed 3 times alternately with DMF and dichloromethane; The coupling agent in step c) is a DMF solution of 5% N-methylmorpholine by volume; the shaking reaction time is 60 min; The lysis buffer in step e) is a trifluoroacetic acid solution containing 2.5% water, 2.5% triisopropylsilane and 2.5% 1,2-ethylenedithiol by volume. The purification process is as follows: the trifluoroacetic acid is dried by blowing it with nitrogen gas at a low flow rate, and then the crude polypeptide product is precipitated with anhydrous diethyl ether, washed and dried.

5. The dual immune checkpoint co-blocker targeting EGFR according to claim 1, characterized in that, The polypeptide molecules exhibit self-assembly behavior in solutions containing EGFR protein.

6. The dual immune checkpoint co-blocker targeting EGFR according to claim 5, characterized in that, The critical assembly concentration of the polypeptide molecule triggered by EGFR protein is 18.2 μM.

7. A dual immune checkpoint co-blocker targeting EGFR according to claim 6, characterized in that, The self-assembly behavior forms nanofibers with a diameter of 8.5 nm.

8. The application of a dual immune checkpoint co-blocker targeting EGFR according to any one of claims 1-7.

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