CN-121991174-A - EGFR-targeting double immune checkpoint co-blocker and application thereof

Abstract

The invention provides a dual immune checkpoint co-blocker targeting EGFR, which is prepared into target polypeptide molecules by adopting an Fmoc solid-phase polypeptide synthesis method, wherein the polypeptide molecules have self-assembly behavior in a solution containing EGFR protein, the critical assembly concentration of the polypeptide molecules under the triggering of the EGFR protein is 18.2 mu M, the self-assembly behavior forms nanofibers with the diameter of 8.5nm, and the nanofibers capture membrane cholesterol through high-density hydrophobic domains of the nanofibers and induce membrane vesicle shedding so as to simultaneously remove EGFR and cholesterol, so that the dual effects of blocking the biochemical immune checkpoints, reducing the STAT3 phosphorylation level caused by the reduction of the EGFR and further reducing PD-L1 expression are realized, and blocking the biomechanical immune checkpoints, namely, the removal of the cholesterol obviously improves the rigidity of tumor cell membranes and enhances the sensitivity of the nanofibers to T cell mechanical attack are realized. The blocking agent can obviously improve the killing effect of T cell immunotherapy such as CAR-T cells and the like on solid tumors.

Inventors

• ZHAO YONGDAN
• ZHANG MIN
• DU YAN
• JIN NING
• SUN YIDAN

Assignees

• 山西医科大学

Dates

Publication Date

20260508

Application Date

20260121

Claims (8)

1. 1. An EGFR-targeted dual immune checkpoint co-blocker, wherein the blocker is a polypeptide molecule having a structure according to formula I: Formula I.
2. 2. The EGFR targeted dual immune checkpoint co-blocker of claim 1, wherein the blocker is prepared by Fmoc solid phase polypeptide synthesis.
3. 3. The EGFR-targeting dual immune checkpoint co-blocker of claim 2, wherein the Fmoc solid phase polypeptide synthesis comprises the steps of: a) Providing a solid phase resin with N-terminal protected by Fmoc group and modified with cysteine; b) After swelling the resin overnight, deprotecting the resin with a DMF solution containing 20% by volume of piperidine, and washing to remove Fmoc protecting groups; c) Under the catalysis of a coupling agent, 10 times of molar equivalent of proline and benzotriazole-N, N, N ', N' -tetramethyl urea hexafluorophosphate are subjected to oscillation reaction with the deprotected resin, and the proline is coupled to the amino group of cysteine; d) Repeating steps b) and c) until the coupling of all amino acids in the sequence of formula I is completed; e) And (3) using a lysate to cleave the synthesized polypeptide from the resin, removing the side chain protecting group, and purifying to obtain the target polypeptide molecule.
4. 4. The dual immune checkpoint co-blocker targeting EGFR according to claim 3, wherein the solid phase resin in step a) is Wang resin and the cysteine has a modification density of 0.37 mM; swelling the resin in step b) overnight with anhydrous DMF, the deprotection time being 15min, washing with DMF and dichloromethane alternately 3 times; the coupling agent in the step c) is DMF solution of 5% of nitrogen methyl morpholine, and the oscillation reaction time is 60min; the pyrolysis liquid in the step e) is trifluoroacetic acid solution containing 2.5% of water, 2.5% of triisopropylsilane and 2.5% of 1, 2-ethanedithiol in volume ratio, and the purification process comprises the steps of drying trifluoroacetic acid by using low-flow nitrogen, precipitating the obtained crude polypeptide product by using anhydrous diethyl ether, washing and drying.
5. 5. The EGFR-targeted dual immune checkpoint co-blocker of claim 1, wherein the polypeptide molecule has self-assembly behavior in a solution containing EGFR protein.
6. 6. The EGFR-targeted dual immune checkpoint co-blocker of claim 5, wherein the critical assembly concentration of the polypeptide molecule upon triggering of EGFR protein is 18.2 μm.
7. 7. The EGFR-targeted dual immune checkpoint co-blocker of claim 6, wherein the self-assembly action forms nanofibers having a diameter of 8.5 nm.
8. 8. Use of an EGFR-targeted dual immune checkpoint co-blocker according to any of claims 1-7.

Description

EGFR-targeting double immune checkpoint co-blocker and application thereof Technical Field The invention belongs to the technical fields of tumor immunotherapy and nanobiology, and particularly relates to a dual immune checkpoint co-blocker targeting EGFR and application thereof. Background Tumor immune escape is one of the core challenges faced by cancer treatment, and tumor cells avoid the recognition and killing of the immune system of the organism through a multidimensional and synergetic mechanism, so that the clinical treatment effect is severely restricted. In the biochemical immune regulation level, tumor cells often abnormally up-regulate immune checkpoint molecules such as programmed death ligand 1 (PD-L1), and the like, and after the molecules are combined with PD-1 receptors on the surfaces of the T cells, immune inhibition signals of the T cells can be started, so that proliferation activity and cytotoxicity of effector T cells are directly weakened, and the tumor cells can survive and proliferate under immune monitoring. Although single immune checkpoint blocking therapies such as anti-PD-1/PD-L1 antibodies have made breakthroughs in the treatment of partial tumors, there is still a dilemma of limited efficacy in solid tumors, most patients have a response rate of less than 30%, and drug resistance is likely to occur after long-term treatment—the core reason is that tumor cells can bypass a single blocking target by switching other immune checkpoint pathways, remodelling tumor microenvironment, etc., leading to treatment failure. At the same time, remodeling of the mechanical properties of tumor cells has become another key mechanism of immune escape. Normal cells ensure stable cell morphology and function by maintaining certain cell membrane rigidity, while tumor cells reduce cell membrane rigidity by downregulating cytoskeletal protein expression, increasing cell membrane cholesterol content and the like, so that the cell membrane presents high fluidity. The interaction between T cells and tumor cells is directly affected by the change of the mechanical property, wherein soft tumor cell membranes are difficult to effectively identify by TCR receptors on the surfaces of the T cells, stable immune synapses cannot be formed, the T cells are prevented from killing signal transmission, and the clearance capacity of an immune system to the tumor cells is further weakened. At present, an intervention means aiming at the mechanical properties of cells has proved to have potential therapeutic value, for example, a cholesterol scavenger MeβCD can enhance the rigidity of tumor cells by removing cell membrane cholesterol, so that the recognition efficiency of T cells is improved, but the method has the obvious defects that the targeting of tumor tissues is lacking, normal cells are easily damaged while the targeting of the tumor cells is performed, the off-target effect is initiated, and more importantly, the method can only regulate the mechanical properties of the cells, can not cooperatively intervene in biochemical immune check point channels such as PD-L1 and the like, and is difficult to break the pattern of multi-mechanical cooperative escape of the tumor cells. In summary, single biochemical immune checkpoint blocking therapies or mechanical intervention approaches have limitations, and comprehensive regulation of tumor immune escape mechanisms cannot be realized. Therefore, developing a dual-function treatment strategy capable of precisely targeting tumor tissues and cooperatively intervening biochemical immune check points and cell mechanical characteristics becomes an urgent need for breaking through the existing treatment bottleneck and improving the treatment effect of solid tumors, and has important significance for promoting the development of tumor immunotherapy. Disclosure of Invention The invention aims to provide an EGFR-targeting double immune checkpoint co-blocker which can specifically identify EGFR on the surface of tumor cells and form nanofibers by in-situ self-assembly, so as to realize the synergistic blocking of biochemical and biomechanical double immune checkpoints and overcome the technical problems. In order to achieve the above purpose, the technical scheme provided by the invention is as follows: an EGFR-targeting dual immune checkpoint co-blocker, wherein the blocker is a polypeptide molecule with a structure shown in formula I: Formula I. The blocking agent is prepared by adopting Fmoc solid-phase polypeptide synthesis method. The Fmoc solid-phase polypeptide synthesis method comprises the following steps: a) Providing a solid phase resin with N-terminal protected by Fmoc group and modified with cysteine; b) After swelling the resin overnight, deprotecting the resin with a DMF solution containing 20% by volume of piperidine, and washing to remove Fmoc protecting groups; c) Under the catalysis of coupling agent nitrogen methyl morpholine, 10 times of molar equivalent o