A protein degrading agent based on endosome-phagosome junction assembly and its application

Abstract

This invention discloses a protein degrader based on an endosome-phagosome linker assembly and its applications, belonging to the fields of protein degradation and nanobiomedicine. The endosome-phagosome linker assembly can degrade proteins by inserting into the endosome membrane and labeling endosomes. Its unique degradation strategy expands the target spectrum of traditional intracellular autophagy degradation to include multiple target proteins on the cell membrane / extracellularly, potentially revolutionizing the current development of targeted protein degradation tools. The preparation method of this invention is simple, the raw materials are readily available, and it is suitable for research and application transformation. Utilizing the endosome-phagosome linker assembly breaks with traditional degradation approaches, achieving the degradation of most target proteins through an "endosome-autophagy-lysosome" pathway. The novel design has a wide range of applications, is low-cost, and highly efficient, demonstrating excellent degradation effects and potential for application in various complex disease areas, showing great development and translational prospects.

Description

Technical Field

[0001] This invention belongs to the field of protein degradation and nanobiomedicine technology, and particularly relates to a protein degrading agent based on endosome-phagosome linkage assembly and its application. Background Technology

[0002] In eukaryotic cells, damaged proteins or organelles can be cleared by proteasomes or lysosomes. Proteasomes eliminate short-lived proteins and soluble misfolded proteins via the ubiquitin-proteasome system (UPS). In contrast, lysosomes are responsible for degrading long-lived proteins, insoluble protein aggregates, and even entire organelles, macromolecules, and intracellular parasites (such as some bacteria) through endocytosis, phagocytosis, or autophagy. Targeted protein degradation (TPD) via proteasome and lysosome pathways is a novel tool for exploring cellular pathways and a promising therapeutic approach. UPS-dependent TPD strategies, such as protein degradation-targeting chimeras (PROTACs), molecular gels, and degradation tags (dTAGs), primarily degrade intracellular proteins. Lysosome-dependent TPD strategies, such as lysosome-targeting chimeras (LYTACs), utilize specific lysosomes to deliver proteins to degrade membrane proteins, extracellular proteins, and protein aggregates. Meanwhile, degradation technologies based on the traditional autophagy-lysosome pathway, such as autophagosome-binding complexes (ATTECs) and autophagy-targeting chimeras (AUTACs), anchor targets and recruit autophagic vesicles to deliver specific targets to autophagolysosomes for degradation, primarily targeting cytoplasmic components (proteins or non-proteins).

[0003] However, degradation strategies targeting specific membrane / extracellular proteins face a series of problems and challenges. First, constructing LYTAC molecules with lysosomal targeting function typically requires complex design and synthesis. Second, degradation efficiency is highly dependent on the expression levels and tissue distribution of lysosomal directed delivery receptors (such as the cation-independent mannose-6-phosphate receptor CI-M6PR and the insulin-like growth factor 2 receptor IGF2R). Third, degradation via bifunctional chimeras may be interfered with by potential "hook" effects. Therefore, there is an urgent need for a simple, universal new technological paradigm to upgrade the targeted protein degradation technology platform for specific membrane / extracellular proteins. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention develops a novel protein degradation tool, which is an endosome-phagosome linkage assembly. The degradation strategy involves degrading cell membrane and / or extracellular proteins through insertion into the endosome membrane, labeling endosomes, recruiting phagosomes, and an "endosome-phagosome-lysosome" pathway.

[0005] The first object of the present invention is to provide a protein degrading agent comprising an assembly, wherein the monomers forming the assembly contain the following connected portions:

[0006] Target protein (protein to be degraded) specific ligands;

[0007] Phagosome-targeting peptides;

[0008] The endosome intercalation polypeptide is located between the target protein-specific ligand and the phagosome-targeting polypeptide, and the endosome intercalation polypeptide is a C-terminal intercalation polypeptide (i.e., after insertion into the endosome membrane, the C-terminus is located on the cytoplasm side and the N-terminus is located on the endosome lumen side).

[0009] Furthermore, the target protein ligand is located at the C-terminus of the endosome intercalation polypeptide.

[0010] Furthermore, it must include at least one of the following:

[0011] (1) The target protein-specific ligand is chemically linked to the endosome intercalation polypeptide;

[0012] (2) The phagosome-targeting polypeptide and the endosome-intercalating polypeptide are chemically linked;

[0013] (3) The monomer contains hydrophilic modification;

[0014] (4) The target protein is an extracellular protein or a cell membrane protein;

[0015] (5) The target protein-specific ligands include target protein-specific polypeptide ligands.

[0016] Furthermore, it must include at least one of the following:

[0017] (1) The target protein-specific ligand is covalently linked to the endosome intercalation polypeptide;

[0018] The target protein-specific ligand and the endosome intercalation peptide can be linked in any way. For example, when the target protein-specific ligand is a peptide ligand, they can be directly linked. Preferably, the target protein-specific ligand and the endosome intercalation peptide are linked by a covalent bond formed by an amino group and a thiol group (that is, the target protein-specific ligand is modified with a thiol group or an amino group, and the endosome intercalation peptide is modified with a thiol group or an amino group, and the modifications on the two are different).

[0019] Most preferably, the endosome intercalation peptide is modified with maleimide, and the target protein-specific ligand is modified with a thiol-containing amino acid (such as cysteine), and the linkage is achieved through an addition reaction in the presence of an organic solvent; the organic solvent is a solvent capable of dissolving the peptide, including but not limited to... N , N -Dimethylformamide, dimethyl sulfoxide, etc.;

[0020] (2) The phagosome-targeting polypeptide and the endosome-intercalating polypeptide are covalently linked;

[0021] The phagosome-targeting polypeptide and the endosome-intercalating polypeptide can be linked in any way, such as by a reaction of modified groups. Preferably, the phagosome-targeting polypeptide and the endosome-intercalating polypeptide are linked by peptide bonds.

[0022] (3) The monomer contains hydrophilic groups for modification;

[0023] Preferably, the source of the hydrophilic group includes, but is not limited to, hydrophilic amino acids;

[0024] Preferably, the hydrophilic group includes, but is not limited to, hydroxyl, carboxyl, amino, and amide groups;

[0025] Preferably, the hydrophilic group is modified on the target protein-specific ligand; more preferably, the hydrophilic group is modified on the end of the target protein-specific ligand near the endosome intercalation polypeptide.

[0026] Furthermore, it must include at least one of the following:

[0027] (1) The amino acid sequence of the endosome intercalation polypeptide is shown in SEQ ID NO.5;

[0028] (2) The amino acid sequence of the phagosome-targeting polypeptide is shown in SEQ ID NO.4;

[0029] (3) The target proteins include, but are not limited to, one or more of the following: programmed death receptor-ligand 1, β-amyloid protein, advanced glycosylation ligand end products, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), prostate-specific membrane antigen (PAMA), and mitochondrial p32 molecular chaperone protein (p32);

[0030] Preferably, when the target protein includes programmed death receptor-ligand 1, the target protein-specific ligand includes a programmed death receptor-ligand 1-specific polypeptide ligand, the amino acid sequence of which is shown in SEQ ID NO.1;

[0031] Preferably, when the target protein includes β-amyloid protein, the target protein-specific ligand includes a β-amyloid protein-specific polypeptide ligand, the amino acid sequence of which is shown in SEQ ID NO.2;

[0032] Preferably, when the target protein includes late glycosylation ligand end products, the target protein-specific ligand includes a late glycosylation ligand end product-specific polypeptide ligand, the amino acid sequence of which is shown in SEQ ID NO.3.

[0033] This invention discloses an endosome-phagosome linker assembly that achieves targeted degradation of extracellular proteins via an "endosome-phagosome-lysosome" pathway by binding to target proteins, triggering endocytosis, inserting into the endosome membrane, labeling endosomes, and recruiting phagosomes. An endosome-anchored phagosome-targeting peptide is constructed by functionalizing one end of a polypeptide capable of inserting into the lipid membrane under acidic conditions with maleimide and the other end with a phagosome-targeting peptide. The specific ligand of the target protein is functionalized with histidine to improve hydrophilicity and with cysteine. The endosome-anchored phagosome-targeting peptide and the ligand of the target protein are linked via an addition reaction of maleimide and thiol groups and self-assemble to form the endosome-phagosome linker assembly. This endosome-phagosome linker assembly specifically recognizes and binds to target proteins, entering the cellular endosomes via constitutive endocytosis or receptor-mediated propagation. In the acidic environment of the endosome, the endosome-phagosome linker assembly inserts its intercalation peptide into the endosome lipid membrane, anchoring the terminal phagosome-targeting peptide to the endosome surface. It then recruits cytoplasmic phagosomes to phagocytose the endosome, degrading it via the autophagy-lysosome pathway. The degradation of PD-L1 and the late glycation end product receptor RAGE on the cell membrane, as well as extracellular amyloid Aβ, effectively demonstrates that the endosome-phagosome linker assembly proposed in this invention possesses the ability to efficiently target and degrade cell membrane proteins, especially extracellular proteins, via the endosome-autophagy-lysosome pathway.

[0034] A second object of the present invention is to provide a method for preparing the protein degrading agent, including the preparation of an assembly, the preparation of the assembly comprising the following steps: dispersing the monomer at a set concentration in an aqueous solution, and the monomer self-assembling to form the assembly.

[0035] Furthermore, the set concentration is not lower than the critical concentration for micelle formation. For example, when the protein degrading agent is a degrading agent for programmed death receptor-ligand 1, the critical concentration for micelle formation is 1.3-4.2 nM. Of course, other choices can be made based on existing technologies to determine the corresponding "critical concentration for micelle formation".

[0036] Furthermore, firstly, the phagosome-targeting peptide and the endosome-intercalating peptide (with modifications) are prepared to obtain the endosome-anchored phagosome-targeting peptide, and then the target protein-specific ligand (with modifications) is prepared. When preparing the monomer, the endosome-anchored phagosome-targeting peptide and the target protein-specific ligand are mixed and reacted in the presence of an organic solvent.

[0037] Furthermore, the molar ratio of the endosome-anchored phagosome-targeting peptide to the target protein-specific ligand is 1:(1-20).

[0038] Furthermore, after self-assembly, a purification step, such as dialysis, is included to obtain a purified assembly.

[0039] A third object of the present invention is to provide the use of the protein degrading agent or the protein degrading agent obtained by the preparation method in the preparation of preventive or therapeutic drugs.

[0040] Furthermore, the medical uses of the preventive or therapeutic drugs include at least one of the following: prevention or treatment of solid tumors (such as breast cancer, lung cancer, liver cancer, stomach cancer, kidney cancer, colon cancer, colorectal cancer, etc.), prevention or treatment of neurological diseases (such as Alzheimer's disease), and prevention or treatment of inflammatory diseases (such as diabetes and its vascular complications, cardiovascular diseases, etc.).

[0041] Furthermore, the prevention or treatment is achieved by degrading abnormal proteins, which is accomplished via the endosome-autophagy-lysosome pathway.

[0042] A fourth object of the present invention is to provide a pharmaceutical composition comprising the protein degrading agent or the protein degrading agent obtained by the preparation method.

[0043] Furthermore, the pharmaceutical composition is a pharmaceutical composition for antitumor purposes.

[0044] By means of the above-described solution, the present invention has at least the following advantages:

[0045] This invention discloses an endosome-phagosome linker assembly that, through binding to target proteins, triggering endocytosis, inserting into the endosome membrane, labeling endosomes, and recruiting phagosomes, utilizes the "endosome-phagosome-lysosome" pathway to achieve targeted degradation of cell membrane proteins, especially extracellular proteins. The degradation of PD-L1 and the receptor for advanced glycation end products (RAGE) on the cell membrane, as well as extracellular amyloid Aβ, effectively demonstrates that the endosome-phagosome linker assembly proposed in this invention possesses the ability to efficiently target and degrade extracellular proteins using the "endosome-autophagy-lysosome" pathway. This degradation strategy based on the endosome-phagosome linker assembly can expand the target spectrum of autophagy degradation to multiple target proteins on the cell membrane and extracellular surface, potentially revolutionizing the current development of targeted protein degradation tools. The assembly is simple to prepare and may have a profound impact on biomedical research and clinical treatment, possessing enormous potential for development and translation. Attached Figure Description

[0046] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0047] Figure 1 This is a schematic diagram illustrating the construction and intracellular behavior of the endosome-phagosome-based assembly and its technology tool for degrading cell membrane / extracellular proteins by inserting endosomes into the membrane and labeling endosomes.

[0048] Figure 2 This is a quantitative graph showing the hemolysis efficiency of different endosome-anchored phagosome-targeting peptides with maleimide at one end and phagosome-targeting peptide at the other end, as well as the effect of different ends of the acidic intercalation peptide on degradation.

[0049] Figure 3 This is a graph showing the particle size data of different assemblies characterized by dynamic light scattering.

[0050] Figure 4 This is a graph of polydispersity index data used to characterize different assemblies using dynamic light scattering.

[0051] Figure 5 This is a graph showing the electromotive force data of different assemblies characterized by dynamic light scattering.

[0052] Figure 6 This is a graph showing the change in electromotive potential of different assemblies within one week after preparation, characterized by dynamic light scattering.

[0053] Figure 7 This is a quantitative diagram of the critical micelle concentration of different assemblies determined by pyrene fluorescence method.

[0054] Figure 8 This is a full protein distribution map of the protein crown formed by different assemblies in mouse plasma, characterized by SDS-PAGE.

[0055] Figure 9 This is a quantitative graph showing the protein concentration of different assemblies forming a protein crown in mouse plasma, as determined by a full-wavelength microplate reader.

[0056] Figure 10 This is a quantitative graph of the electrokinetic potentials of different assemblies forming a protein crown in mouse plasma, determined by dynamic light scattering.

[0057] Figure 11 Confocal microscopy images and quantitative diagrams of the colocalization of PD-L1 and phagosomes in HEK293T cells after different assembly treatments.

[0058] Figure 12 Confocal microscopy images and quantitative diagrams of the co-localization of PD-L1 and lysosomes in HEK293T cells after different assembly treatments.

[0059] Figure 13Confocal microscopy images and quantitative diagrams of the colocalization of early endosomes and phagosomes in HEK293T cells after different assembly treatments.

[0060] Figure 14 Confocal microscopy images and quantitative diagrams of the colocalization of early endosomes and lysosomes in HEK293T cells after different assembly treatments.

[0061] Figure 15 Confocal microscopy images and quantitative diagrams of the localization of PD-L1 and phagosomes in HEK293T cells at different time points after treatment with PD-L1 and phagosome dual-target assembly.

[0062] Figure 16 Confocal microscopy images and quantitative diagrams of the localization of PD-L1 and lysosomes in HEK293T cells at different time points after treatment with PD-L1 and phagosome dual-targeting assemblies.

[0063] Figure 17 Confocal microscopy images and quantitative diagrams of the localization of endosomes and phagosomes in HEK293T cells at different time points after treatment with PD-L1 and phagosome dual-targeting assemblies.

[0064] Figure 18 Confocal microscopy images and quantitative diagrams of the early localization of endosomes and lysosomes in HEK293T cells at different time points after treatment with PD-L1 and phagosome dual-targeting assemblies.

[0065] Figure 19 This image shows the expression of PD-L1 in 4T1 cells after treatment with different concentrations of PD-L1 and phagosome dual-targeting assemblies using Western blotting.

[0066] Figure 20 This is a graph showing the expression of PD-L1 in 4T1 cells after treatment with different concentrations of PD-L1 single-target assemblies by Western blot.

[0067] Figure 21 This is a graph showing the expression of PD-L1 in 4T1 cells after treatment with PD-L1 and phagosome dual-targeting assemblies at different times, characterized by Western blot.

[0068] Figure 22 This is a graph showing the expression of PD-L1 in 4T1 cells after treatment with the PD-L1 single-target assembly at different times, characterized by Western blot.

[0069] Figure 23The image shows the expression of PD-L1 in 4T1 cells after treatment with different assemblies by Western blot at a concentration of 0.5 μM for 12 h.

[0070] Figure 24 This is a Western blot diagram to characterize the quantitative expression of PD-L1 in 4T1 cells after treatment with different inhibitors and a dual-targeting assembly of PD-L1 and phagosomes.

[0071] Figure 25 Confocal microscopy image of calcein leakage after cell treatment with PD-L1-related assemblages.

[0072] Figure 26 Characterization of mouse brain microvascular endothelial cells upregulated by RAGE.

[0073] Figure 27 This is a quantitative graph showing the expression of RAGE protein after treating mouse brain endothelial cells with RAGE-related assemblies.

[0074] Figure 28 Characterization of BV-2 cells upregulated by RAGE.

[0075] Figure 29 This is a quantitative graph showing the uptake of Aβ by microglia after treatment with different concentrations of Aβ single-target assemblies.

[0076] Figure 30 This is a quantitative graph showing the uptake of Aβ by microglia after cells have been treated with the Aβ single-target assembly for different time periods.

[0077] Figure 31 A graph quantifying the expression of Aβ and RAGE proteins in microglia treated with Aβ-associated assemblies.

[0078] Figure 32 Confocal microscopy image of the localization of FITC-labeled Aβ and phagosomes in microglia treated with Aβ and phagosome dual-targeting assemblies.

[0079] Figure 33 Confocal microscopy image showing the localization of FITC-labeled Aβ and lysosomes in microglia treated with Aβ and phagosome dual-targeting assemblies.

[0080] Figure 34 Confocal microscopy image of early endosome and phagosome localization in microglia treated with Aβ and phagosome dual-targeting assemblies.

[0081] Figure 35 Confocal microscopy image of microglia localizing to early endosomes and lysosomes when treated with Aβ and phagosome dual-targeting assemblies.

[0082] Figure 36 This is a schematic diagram of the treatment regimen for 4T1 breast cancer model mice using the PD-L1 and phagosome dual-targeting assembly designed in this invention.

[0083] Figure 37 Schematic diagrams illustrating changes in mouse tumor volume after intravenous injection of saline, PD-L1 single-target nanoparticles, PD-L1 and phagosome dual-target assembly, PD-L1 single-target assembly, phagosome-targeting peptide control group assembly, phagosome single-target assembly, and intraperitoneal injection of PD-L1 antibody, respectively.

[0084] Figure 38 Schematic diagrams illustrating the tumor weight in mice after intravenous injection of saline, PD-L1 single-target nanoparticles, PD-L1 and phagosome dual-target assembly, PD-L1 single-target assembly, phagosome-targeting peptide control group assembly, phagosome single-target assembly, and intraperitoneal injection of PD-L1 antibody, respectively.

[0085] Figure 39 This is a schematic diagram illustrating the quantitative characterization of the effects of intravenous injection of saline, PD-L1 single-target nanoparticles, PD-L1 and phagosome dual-target assembly, PD-L1 single-target assembly, phagosome-targeting peptide control group assembly, phagosome single-target assembly, and intraperitoneal injection of PD-L1 antibody on lung metastatic nodules in mice.

[0086] Figure 40 This is a quantitative schematic diagram illustrating the effects of intravenous injection of physiological saline, PD-L1 single-target nanoparticles, PD-L1 and phagosome dual-target assembly, PD-L1 single-target assembly, phagosome-targeting peptide control group assembly, phagosome single-target assembly, and intraperitoneal injection of PD-L1 antibody on the degradation of PD-L1 protein in mouse tumor tissue.

[0087] Figure 41 To quantitatively characterize the effects of intravenous injection of saline, PD-L1 single-target nanoparticles, PD-L1 and phagosome dual-target assemblies, PD-L1 single-target assemblies, phagosome-targeting peptide control group assemblies, phagosome single-target assemblies, and intraperitoneal injection of PD-L1 antibody on CD4+ in mouse tumor tissues, respectively. + T cells and CD8 + Schematic diagram illustrating the quantitative increase in T cell stimulation. Detailed Implementation

[0088] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0089] The solution involved in this invention is as follows:

[0090] This invention modifies a polypeptide that can be inserted into a lipid membrane under acidic conditions to construct an endosome-phagosome-based assembly and a technical tool for degrading cell membrane / extracellular proteins by inserting it into the endosome membrane and labeling the endosome. Specifically, this invention provides an endosome-phagosome linker assembly and its application in degrading cell membrane / extracellular proteins by inserting into the endosome membrane and labeling endosomes. This endosome-phagosome linker assembly is constructed by functionalizing peptides that can insert into lipid membranes under acidic conditions, creating peptides with endosome-anchored phagosome targeting functions. Subsequent reactions are performed by functionalizing cell membrane or extracellular protein-specific ligands. The endosome-anchored phagosome-targeting peptide and the target protein ligand chemically react and link together to form the endosome-phagosome linker assembly. This endosome-phagosome linker assembly specifically recognizes and binds to target proteins on the cell membrane or extracellularly, entering the endosome through constitutive endocytosis or receptor-mediated propagation. In the acidic environment of the endosome, the endosome-phagosome linker assembly inserts its intercalation peptide into the endosome lipid membrane, anchoring the terminal phagosome-targeting peptide to the endosome surface, recruiting phagosomes to phagocytose the endosome, and degrading it via the autophagy-lysosome pathway.

[0091] An endosome-phagosome connective assembly and its preparation strategy via endosome membrane insertion and labeling of endosome-degraded cell membrane / extracellular protein technology tools, comprising:

[0092] Step (1): Functional modification of each part of the endosome-anchored phagosome-targeting peptide: One end of the polypeptide that can insert into the lipid membrane under acidic conditions (i.e., "endosome membrane-intercalating polypeptide") is functionalized with maleimide, and the other end is functionalized with phagosome-targeting peptide to construct the endosome-anchored phagosome-targeting peptide. Histidine is functionalized on the specific ligands of cell membrane / extracellular proteins to improve hydrophilicity, and cysteine ​​is functionalized.

[0093] Step (2), Preparation of the dual-targeting assembly of target protein and phagosome: The endosome-phagosome linkage assembly is formed by linking the phagosome-targeting peptide with the target protein-specific ligand through the addition reaction of maleimide and cysteine ​​by the thiol group of the endosome-phagosome linkage peptide and the target protein-specific ligand, and then self-assembling.

[0094] Step (3), Endosome-phagosome junction assembly entry process: The constructed endosome-phagosome junction assembly can specifically recognize and bind to target proteins on the cell membrane or extracellular space, and enter the endosomes of the cell through constitutive endocytosis of the target protein or receptor-mediated propagation mechanism.

[0095] Please refer to the above-prepared endosome-phagosome connective assembly and the technical tools for inserting into the endosome membrane and labeling endosomes to degrade cell membrane / extracellular proteins. Figure 1 . Figure 1 This is a schematic diagram illustrating the construction of the endosome-phagosome linkage assembly-based technology tool for degrading cell membrane / extracellular proteins as described in this invention, and its degradation function after entering the cell: First, peptides that can insert into lipid membranes in an acidic environment are screened and modified to obtain peptides that can insert into the endosome membrane in an acidic endosome environment. The target protein ligand is functionalized with cysteine. An endosome-anchored phagosome-targeting peptide is constructed using the addition reaction of maleimide and cysteine. This peptide achieves targeted degradation of cell membrane / extracellular proteins by binding to the target protein, triggering endocytosis, inserting into the endosome membrane, labeling endosomes, and recruiting phagosomes through the "endosome-phagosome-lysosome" pathway.

[0096] In one embodiment of the present invention, the design method for maleimide functionalization modification of one end of the polypeptide that inserts into the lipid membrane in an acidic environment in step (1) is applicable to all polypeptides that insert into the lipid membrane in an acidic environment.

[0097] In one embodiment of the present invention, in step (1), the sequence used for the phagosome targeting peptide is: GIYVIWGPI.

[0098] In one embodiment of the present invention, in step (1), the endosome-anchored phagosome-targeting peptide is a combination of a maleimide-modified polypeptide that is inserted into the lipid membrane in an acidic environment and a phagosome-targeting peptide.

[0099] In one embodiment of the present invention, in step (1), the C-terminus of the polypeptide inserted into the lipid membrane under acidic conditions is functionalized with maleimide and the N-terminus is modified to connect to the phagosome-targeting peptide. The final sequence of the resulting polypeptide is preferably Mal-acid intercalation peptide 5-phagosome-targeting peptide, with the sequence: GIYVIWGPI-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT-maleimide modification.

[0100] In one embodiment of the present invention, in step (1), the design method for histidine functionalization and cysteine ​​functionalization of cell membrane / extracellular target protein ligands is applicable to all protein-targeting ligands.

[0101] In one embodiment of the present invention, in step (1), the PD-L1 protein targeting peptide: cysteine-CVRARTR (containing the hydrophilic modification sequence: cysteine-H) 15 CVRARTR); Late glycosylation ligand end product RAGE protein targeting peptide: cysteine-ELKVLMEKEL; Extracellular amyloid Aβ targeting peptide: cysteine-RGTFEGKF.

[0102] In one embodiment of the present invention, in step (2), the endosome-phagosome linker assembly is generated by adding maleimide of the endosome-anchored phagosome-targeting peptide to the thiol group on the cysteine ​​of the target protein-specific ligand and then self-assembling. This method is applicable to all chemical linking methods that can undergo this reaction.

[0103] In one embodiment of the present invention, in step (2), an appropriate amount of the endosome-anchored phagosome-targeting peptide is weighed and dissolved in... N , N -Dimethylformamide.

[0104] In one embodiment of the present invention, in step (2), the PD-L1 protein targeting peptide is weighed in an amount 5 times that of the endosome-anchored phagosome targeting peptide, the late glycosylation ligand end product RAGE protein targeting peptide is weighed in an amount 2 times that of the endosome-anchored phagosome targeting peptide, and the extracellular amyloid Aβ targeting peptide is weighed in an amount 2 times that of the endosome-anchored phagosome targeting peptide and dissolved in [the following solutions]. N , N -Dimethylformamide.

[0105] In one embodiment of the present invention, in step (2), the endosome-anchored phagosome targeting peptide and the PD-L1 protein targeting peptide are stirred at room temperature for 1-10 hours; the endosome-anchored phagosome targeting peptide and the late glycosylation ligand end product RAGE protein targeting peptide / extracellular amyloid Aβ targeting peptide are stirred at room temperature for 20-30 hours.

[0106] In one embodiment of the present invention, in step (2), the mixture formed by the endosome-anchored phagosome-targeting peptide and the cell membrane / extracellular protein-targeting peptide is injected into purified water and stirred at room temperature for 0.1-3 hours.

[0107] In one embodiment of the present invention, in step (2), the assembly formed by the endosome-anchored phagosome-targeting peptide and the cell membrane / extracellular protein-targeting peptide is dialyzed in water for 24-72 h using a dialysis bag.

[0108] In one embodiment of the present invention, in step (3), the endosome-phagosome connective assembly enters the endosome of the cell through constitutive endocytosis of the target protein or receptor-mediated propagation into the endosome by utilizing the endocytosis of the target protein itself on the cell membrane and, for extracellular proteins, indirectly through the propagation into the endosome mediated by the endocytic target protein receptor on the cell membrane.

[0109] Example 1

[0110] This embodiment demonstrates the preparation method of an endosome-phagosome-based assembly and its tool using endosome-mediated degradation of cell membrane / extracellular proteins via insertion into the endosome membrane and labeling:

[0111] (1) Preparation of PD-L1 (programmed death receptor-ligand 1) and phagosome dual-targeting assembly

[0112] Weigh 2.8 mg of the endosome-anchored phagosome-targeting peptide (composed of a maleimide-modified endosome insertion peptide and a phagosome-targeting peptide linked together, with the sequence: maleimide-modified-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT-GIYVIWGPI) and 8 mg of the PD-L1-targeting peptide (a cysteine ​​and histidine-modified peptide, with the sequence: cysteine-modified-HHHHHHHHHHHHHH-CVRARTR, hereinafter the same. Wherein CVRARTR is SEQ ID NO.1) (molar ratio 1:5) and dissolve them in... N , N The mixture was stirred in dimethylformamide for 4 h, then injected into 5 mL of purified water, and stirred for another 0.5 h at room temperature to prepare a preliminary PD-L1 and phagosome dual-targeting assembly. Subsequently, the assembly was dialyzed for 24 h using a 3500 Da dialysis bag, followed by dialysis for 24 h using a 7000 Da dialysis bag. Freshly prepared PD-L1 and phagosome dual-targeting assemblies were obtained.

[0113] (2) Preparation of PD-L1 single-target assembly

[0114] 2.8 mg of a maleimide-functionalized peptide capable of intercalating into a lipid membrane under acidic conditions and 9.9 mg of a PD-L1 targeting peptide (molar ratio 1:5) were weighed and dissolved in... N , N The mixture was stirred in dimethylformamide for 4 h, then injected into 5 mL of purified water, and stirred for another 0.5 h at room temperature to prepare the assembly. The assembly was then dialyzed for 24 h using a dialysis bag with a molecular weight of 3500 Da, followed by dialyzed for 24 h using a dialysis bag with a molecular weight of 7000 Da. Freshly prepared PD-L1 single-target assemblies were obtained.

[0115] (3) Preparation of Aβ (β-amyloid protein) and phagosome dual-target assembly

[0116] Weigh 2.8 mg of the endosome-anchored phagosome-targeting peptide and 1.14 mg of the Aβ-targeting peptide (Aβ-targeting peptide is a cysteine-modified polypeptide with the sequence: cysteine-modified-RGTFEGKF, the same below. RGTFEGKF is SEQ ID NO.2) (molar ratio 1:2) and dissolve them in... N , NThe mixture was stirred in dimethylformamide for 24 h, then injected into 5 mL of purified water and stirred for another 0.5 h at room temperature to form an assembly. The assembly was then dialyzed for 48 h using a dialysis bag with a molecular weight of 3500 Da to obtain a freshly prepared Aβ and phagosome dual-targeting assembly.

[0117] (4) Preparation of Aβ single-target assembly

[0118] 2.8 mg of a maleimide-functionalized peptide capable of intercalating into a lipid membrane under acidic conditions and 1.37 mg of an Aβ-targeting peptide (molar ratio 1:2) were weighed and dissolved in... N , N The mixture was stirred in dimethylformamide for 24 h, then injected into 5 mL of purified water and stirred for another 0.5 h at room temperature to form an assembly. The assembly was then dialyzed for 48 h using a dialysis bag with a molecular weight of 3500 Da to obtain a freshly prepared Aβ single-target assembly.

[0119] (5) Preparation of RAGE (late glycosylation ligand end product) and phagosome dual-target assembly

[0120] Weigh 2.8 mg of the endosome-anchored phagosome-targeting peptide and 1.4 mg of the RAGE-targeting peptide (the RAGE-targeting peptide is a cysteine-modified polypeptide with the sequence: cysteine-modified-ELKVLMEKEL, hereinafter the same. ELKVLMEKEL is SEQ ID NO.3) (molar ratio 1:2) and dissolve them in... N , N The mixture was stirred in dimethylformamide for 24 h, then injected into 5 mL of purified water and stirred for another 0.5 h at room temperature to prepare an assembly. The assembly was then dialyzed for 48 h using a dialysis bag with a molecular weight of 3500 Da to obtain a freshly prepared RAGE and phagosome dual-targeting assembly.

[0121] (6) Preparation of RAGE single-target assemblies

[0122] 2.8 mg of a maleimide-functionalized peptide capable of intercalating into a lipid membrane under acidic conditions and 1.7 mg of a RAGE-targeting peptide (molar ratio 1:2) were weighed and dissolved in... N , N The mixture was stirred in dimethylformamide for 24 h, then injected into 5 mL of purified water and stirred for another 0.5 h at room temperature to form an assembly. The assembly was then dialyzed for 48 h using a dialysis bag with a molecular weight of 3500 Da to obtain a freshly prepared RAGE single-target assembly.

[0123] Example 2

[0124] (1) To screen for peptides that can insert into lipid membranes under acidic conditions and conform to the assemblies constructed in this invention, the interaction between different peptides and the endosomal membrane after cell entry is indicated by the hemoglobin leakage rate. Please refer to [link to relevant documentation]. Figure 2 , Figure 2 This is a quantitative graph showing the damage to erythrocyte membranes by different peptides using a full-wavelength microplate reader. For example... Figure 2 As shown, the hemoglobin leakage results indicated that the Mal-acidic intercalation peptide 5-phagosome-targeting peptide had higher pH sensitivity, and that the Mal-acidic intercalation peptide 5-phagosome-targeting peptide and its parallel control group, Mal-acidic intercalation peptide 5, were more consistent with the design of this protocol. This result suggests that the Mal-acidic intercalation peptide 5-phagosome-targeting peptide was selected as the endosome-anchored phagosome-targeting peptide. Therefore, in subsequent experiments, the Mal-acidic intercalation peptide 5-phagosome-targeting peptide was used as the endosome-anchored phagosome-targeting peptide. The phagosome-targeting peptide sequence is shown in SEQ ID NO.4 (GIYVIWGPI), and the endosome intercalation peptide is shown in SEQ ID NO.5 (AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT).

[0125] (2) Effect of exposure at different ends (N-terminus or C-terminus) on acidic intercalation peptides: Acidic intercalation peptide 4 was designed, and an assembly targeting both PD-L1 and phagosomes was constructed according to the above method. The degradation effects were compared. The results are shown in […]. Figure 2 Acidic intercalation peptide 5 is C-terminal intercalated, while acidic intercalation peptides 3 and 4 are homologous sequences. The PD-L1 and phagosome dual-targeting assemblies formed by the two are analogs. However, acidic intercalation peptide 4 is C-terminal intercalated, while acidic intercalation peptide 3 is slightly N-terminal intercalated. Comparison of degradation effects revealed that although the PD-L1 and phagosome dual-targeting assembly prepared by acidic intercalation peptide 3 can achieve hemolysis, it has no significant effect on PD-L1 degradation. In contrast, acidic intercalation peptides 4 and 5 can achieve PD-L1 degradation. This proves that the assemblies formed by N-terminal intercalation cannot degrade PD-L1.

[0126] The peptides involved in the above experiments are as follows:

[0127] Mal-acidic intercalation peptide 1-phagosome targeting peptide: maleimide-IWLTALKFLGKHAAKHEAKQQLSKL-GIYVIWGPI;

[0128] Mal-acidic intercalation peptide 2-phagosome targeting peptide: maleimide-GALFLGFLGAAGSTMGAWSQPHHHHHV-GIYVIWGPI;

[0129] Mal-acidic intercalation peptide 3-phagosome targeting peptide: maleimide-FSGSGSGSGHQIHIWFQNHHMHWHHG-GIYVIWGPI;

[0130] Mal-acidic intercalation peptide 4-phagosome targeting peptide: maleimide-FSGSGSGSGRQIKIWFQAARMLWKK-GIYVIWGPI;

[0131] Mal-acidic intercalation peptide 5-phagosome targeting peptide: maleimide-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT-GIYVIWGPI.

[0132] Example 3

[0133] This embodiment examines the particle size of the assembly constructed according to the present invention. Please refer to [link / reference]. Figure 3 , Figure 3 To determine the particle size of all assemblies prepared in Example 1 using dynamic light scattering. Figure 3 As shown, the particle size of the phagosome-targeted peptide control group assembly was 89 nm; the particle size of the phagosome single-target assembly was 97 nm; the particle size of the PD-L1 single-target assembly was 176 nm; the particle size of the PD-L1 and phagosome dual-target assembly was 177 nm; the particle size of the Aβ single-target assembly was 128 nm; the particle size of the Aβ and phagosome dual-target assembly was 116 nm; the particle size of the RAGE single-target assembly was 134 nm; and the particle size of the RAGE and phagosome dual-target assembly was 157 nm. This indicates that the constructed nanocarrier may form an assembly-like structure and the particle size of the assembly system is relatively small.

[0134] The preparation methods for the phagosome-targeting peptide control group assembly and the phagosome-targeting assembly are the same as in Example 1, specifically:

[0135] Preparation of phagosome single-target assembly: 2.8 mg of endosome-anchored phagosome-targeting peptide and 5.3 mg of DSPE-PEG-NH2 (α-amino-ω-1,2-distearate-3-phosphatidylethanolamine polyethylene glycol) were weighed and dissolved in water. N , N The mixture was stirred in dimethylformamide for 4 h, then injected into 5 mL of purified water and stirred for another 0.5 h at room temperature to prepare the assembly. The assembly was then dialyzed for 24 h using a dialysis bag with a molecular weight of 3500D, followed by dialysis for 24 h using a dialysis bag with a molecular weight of 7000D. Freshly prepared phagosome-targeted assemblies were obtained.

[0136] Preparation of the phagosome-targeting peptide control group assembly: 2.8 mg of a maleimide-functionalized peptide capable of intercalating into the lipid membrane under acidic conditions and 6.6 mg of DSPE-PEG-NH2 (molar ratio 1:5) were weighed and dissolved in... N ,N The mixture was stirred in dimethylformamide for 4 h, then injected into 5 mL of purified water and stirred for another 0.5 h at room temperature to prepare the assembly. The assembly was then dialyzed for 24 h using a dialysis bag with a molecular weight of 3500D, followed by dialysis for 24 h using a dialysis bag with a molecular weight of 7000D. Freshly prepared phagosome-targeted assemblies were obtained.

[0137] Example 4

[0138] This embodiment examines the uniformity of the constructed nanoassemblies. Please refer to [link / reference]. Figure 4 , Figure 4 To determine the homogeneity of all assemblies using dynamic light scattering. For example... Figure 4 As shown, the polydispersity index (PDI) of the phagosome-targeted peptide control group assembly was 0.146, the PDI of the phagosome single-target assembly was 0.150, the PDI of the PD-L1 single-target assembly was 0.176, the PDI of the PD-L1 and phagosome dual-target assembly was 0.167, the PDI of the Aβ single-target assembly was 0.158, the PDI of the Aβ and phagosome dual-target assembly was 0.164, the PDI of the RAGE single-target assembly was 0.196, and the PDI of the RAGE and phagosome dual-target assembly was 0.196. This indicates that the constructed nanocarriers may form assemblies and the assembly system is homogeneous.

[0139] Example 5

[0140] This embodiment examines the electrokinetic potential of the nanoassemblies constructed above. Please refer to [link / reference]. Figure 5 , Figure 5 To determine the electrokinetic potential of all assemblies using dynamic light scattering. For example... Figure 5 As shown, the kinetic potential of the phagosome-targeted peptide control group assembly was -15 mV, and the kinetic potential of the phagosome-targeted assembly was -19 mV; the kinetic potential of the PD-L1-targeted assembly was +13 mV; the kinetic potential of the PD-L1 and phagosome dual-targeted assembly was +16 mV; the kinetic potential of the Aβ-targeted assembly was -14 mV; the kinetic potential of the Aβ and phagosome dual-targeted assembly was -15 mV; the kinetic potential of the RAGE-targeted assembly was -16 mV; and the kinetic potential of the RAGE and phagosome dual-targeted assembly was -14 mV, indicating that the constructed nanocarriers may form assemblies.

[0141] Example 6

[0142] This embodiment examines the stability of the constructed nanoassemblies by using dynamic light scattering to investigate the change in electrokinetic potential of the assemblies prepared under this method over one week. Please refer to [link to relevant documentation]. Figure 6 , Figure 6To determine the changes in electrokinetic potential of the prepared RAGE and phagosome dual-target assembly, Aβ single-target assembly, and Aβ and phagosome dual-target assembly within one week of preparation using dynamic light scattering. Figure 6 As shown, the three assemblies are structurally stable, and their electromotive potentials did not change significantly within one week of preparation.

[0143] Example 7

[0144] This embodiment examines the critical micelle concentration (CMC) of the assembly used in this scheme, and the CMC of the assembly is determined by the pyrene fluorescent probe method. Please refer to [link to relevant documentation]. Figure 7 ,like Figure 7 The critical micelle concentrations (CMCs) for the Aβ single-target assembly, RAGE single-target assembly, PD-L1 single-target assembly, and PD-L1 and phagosome dual-target assembly, as determined by the pyrene fluorescent probe method, are 1.25–5.8 nM, 3.5–6.8 nM, 1.2–4.7 nM, and 1.3–4.2 nM, respectively.

[0145] Example 8

[0146] A plasma adsorption control group assembly was prepared. An appropriate amount of polylactic acid-glycolic acid copolymer, activated with succinimide, was reacted with a certain amount of amino-modified polyethylene glycol carboxymethyl and supplemented with a small amount of N,N-diisopropylethylamine in dichloromethane for 2 h. The dichloromethane was then volatilized using argon gas, followed by the addition of an appropriate amount of... N , N After dimethylformamide, the successfully prepared control group assembly was injected into pure water. After reacting for 0.5 h, the assembly was dialyzed with pure water for 48 h and then freshly prepared was collected as a control for protein crown formation.

[0147] 100 μL of freshly prepared assembly (0.4 mg / mL in PBS 7.4) was incubated with 100 μL of PBS 7.4 or plasma at 37°C for 1 hour, followed by centrifugation at 70,000 g for 20 minutes at 4°C. The precipitate was then resuspended in PBS 7.4 and lysed with protein lysis buffer on ice for 30 minutes, followed by protein electrophoresis at 12,000 g for 15 minutes at 4°C to assess plasma protein adsorption. See also... Figure 8 , Figure 8 To characterize the protein corona formed by different assemblies adsorbed onto plasma using SDS-PAGE, a full protein map was generated, showing that all assemblies formed a protein corona after plasma incubation.

[0148] Example 9

[0149] Protein concentration was determined using the same sample preparation method as in Example 8. Please refer to [link to example]. Figure 9 , Figure 9 To measure the quantitative map of plasma protein concentration adsorbed by different assemblies using a full-wavelength microplate reader, it was found that all assemblies formed protein crowns after plasma incubation, and the protein concentrations were all higher than those in the phosphate buffer group.

[0150] Example 10

[0151] The plasma adsorption control group assembly was prepared using the same method as in Example 8.

[0152] 100 μL of freshly prepared assembly (0.4 mg / mL in PBS 7.4) was incubated with 100 μL of PBS 7.4 or plasma at 37°C for 1 hour, followed by centrifugation at 70,000 g for 20 minutes at 4°C. The precipitate was then resuspended in PBS 7.4 and analyzed by electrokinetic determination. See also: Figure 10 , Figure 10 To determine the quantitative electrokinetic potential of different assemblies after adsorbing plasma to form a protein crown using dynamic light scattering, it was found that all assemblies would form a protein crown after plasma incubation.

[0153] Example 11

[0154] To investigate whether the PD-L1 and phagosome dual-targeting assembly could transfer PD-L1 into the phagosome, HEK293T cells were transfected with pEGFP-N1 / PD-L1 and pmRFP-LC3 plasmids in serum-free medium for 48 h. The transfected cells were then treated with 0.5 μM of the different assemblies for 12 h, fixed with 4% PFA (paraformaldehyde) solution for 15 min, and finally imaged using laser confocal scanning microscopy. The conclusions of this example can be found in [link to relevant documentation]. Figure 11 , Figure 11 Confocal microscopy images showing the intracellular localization of PD-L1 and phagosomes after processing with different assemblages. Figure 11 As shown, in the blank control group, the phagosome-targeting peptide control group assembly, the phagosome single-targeting assembly, the PD-L1 single-targeting nanoparticle, and the PD-L1 single-targeting assembly, PD-L1 was located on the cell membrane and did not induce PD-L1 to reach the phagosome. However, in the PD-L1 and phagosome dual-targeting assembly group, PD-L1 was highly colocalized with the phagosome, indicating that the PD-L1 and phagosome dual-targeting assembly has the ability to bind PD-L1 and recruit phagosomes.

[0155] The preparation of PD-L1 single-target nanoparticles involved the following steps: 100 mg of polylactic acid-glycolic acid-polylysine was first dissolved in 2 mL of ethyl acetate as the base carrier. This mixture was then added dropwise to 4 mL of 2.5% polyvinyl alcohol under vortexing, and immediately sonicated at 300 W for 3 × 10⁻⁶ s to form an oil-in-water emulsion. This emulsion was immediately poured into 100 mL of 0.3% polyvinyl alcohol and stirred overnight to evaporate the ethyl acetate. The nanoparticle suspension was purified from polyvinyl alcohol by centrifugation at 70,000 g for 20 min, and the precipitate was resuspended in PBS 7.4 by sonication in a water bath. Subsequently, 11.2 mg of MAL-PEG-SCM (maleimide-polyethylene glycol-succinimide carboxymethyl ester, purchased from Beijing Jiankai Technology Co., Ltd., product number A5002) was added, and the reaction was carried out at room temperature for 1 hour. Subsequently, the excess MAL-PEG-SCM was removed by centrifugation at 70,000 g for 20 minutes. The nanosuspension was then resuspended and further modified for 1 hour at room temperature using an excess of cysteine-containing PD-L1-targeting ligand via maleimide-thiol reaction chemistry. Finally, excess unreacted peptides were removed by centrifugation at 70,000 g for 20 minutes, the nanosuspension was resuspended in water, and lyophilized to obtain PD-L1 single-target nanoparticles.

[0156] Example 12

[0157] To investigate whether the PD-L1 and phagosome dual-targeting assembly could transfer PD-L1 into lysosomes, HEK293T cells were transfected with two plasmids, pEGFP-N1 / PD-L1 and mTagBFP2-Lysosomes-20, in serum-free 48-well plates for 48 h. The transfected cells were then further treated with 0.5 μM of the different assemblies for 12 h, fixed with 4% PFA solution for 15 min, and finally imaged using laser confocal scanning microscopy. The conclusions of this example can be found in [link to relevant documentation]. Figure 12 , Figure 12 Confocal microscopy images showing the intracellular localization of PD-L1 and lysosomes after treatment with different assemblies. Figure 12 As shown, in the blank control group, the phagosome-targeting peptide control group assembly, the phagosome single-targeting assembly, the PD-L1 single-targeting nanoparticle, and the PD-L1 single-targeting assembly, PD-L1 was located on the cell membrane and did not induce PD-L1 to reach the lysosome. However, in the PD-L1 and phagosome dual-targeting assembly group, PD-L1 was highly co-localized with the lysosome, binding... Figure 11 This indicates that the PD-L1 and phagosome dual-target assembly has the ability to bind to PD-L1 and recruit phagosomes, as well as to degrade it via lysosomes.

[0158] Example 13

[0159] To investigate whether the PD-L1 and phagosome dual-targeting assembly could transfer PD-L1-containing endosomes into phagosomes, HEK293T cells were transfected with GFP-Rab5B and pmRFP-LC3 plasmids in serum-free medium in 48-well plates for 48 h. The transfected cells were then further treated with 0.5 μM of the different assemblies for 12 h, fixed with 4% PFA solution for 15 min, and finally imaged using laser confocal scanning microscopy. The conclusions of this example can be found in [link to example]. Figure 13 , Figure 13 This is a confocal microscopy image showing the intracellular localization of early endosomes and phagosomes after processing by different assemblages. (Example) Figure 13 As shown, in the blank control group, the phagosome-targeting peptide control group assembly, the phagosome single-targeting assembly, the PD-L1 single-targeting nanoparticle, and the PD-L1 single-targeting assembly, the early endosomes and phagosomes were clearly separated. However, in the PD-L1 and phagosome dual-targeting assembly group, the early endosomes and phagosomes were highly colocalized. This indicates that the PD-L1 and phagosome dual-targeting assembly can shorten the spatial distance between the early endosomes and phagosomes, which is beneficial for pulling the proteins contained in the endosomes into the phagosomes.

[0160] Example 14

[0161] To investigate whether the PD-L1 and phagosome dual-targeting assembly could ultimately transfer PD-L1-containing endosomes into lysosomes, HEK293T cells were transfected with two plasmids, GFP-Rab5B and mTagBFP2-Lysosomes-20, in serum-free 48-well plates for 48 h. The transfected cells were then further treated with 0.5 μM of the different assemblies for 12 h, fixed with 4% PFA solution for 15 min, and finally imaged using laser confocal scanning microscopy. The conclusions of this example can be found in [link to example]. Figure 14 , Figure 14 This is a confocal microscopy image showing the intracellular localization of early endosomes and lysosomes after processing with different assemblages. (Example) Figure 14 As shown, in the blank control group, the phagosome-targeting peptide control group assembly, the phagosome single-targeting assembly, the PD-L1 single-targeting nanoparticle, and the PD-L1 single-targeting assembly, early endosome and lysosome separation was obvious, while in the PD-L1 and phagosome dual-targeting assembly group, early endosomes and lysosomes were highly co-localized, binding... Figure 13 This demonstrates that the PD-L1 and phagosome dual-target assembly can shorten the spatial distance between the early endosome and the phagosome, pulling proteins contained in the early endosome into the phagosome and degrading them through lysosomes.

[0162] Example 15

[0163] To investigate the time required for the PD-L1 and phagosome dual-targeting assembly to transfer PD-L1 to the phagosome, HEK293T cells were transfected with pEGFP-N1 / PD-L1 and pmRFP-LC3 plasmids in serum-free medium for 48 h. Transfected cells were then treated with the PD-L1 and phagosome dual-targeting assembly for 0–12 h, fixed with 4% PFA solution for 15 min, and finally imaged using laser confocal scanning microscopy. The conclusions of this example can be found in [link to relevant documentation]. Figure 15 , Figure 15 Confocal microscopy images showing the intracellular localization of PD-L1 and phagosomes after 0-12 h of treatment with a dual-targeting PD-L1 and phagosome assembly. Figure 15 As shown, the PD-L1 and phagosome dual-target assembly can gradually bring PD-L1 and phagosome closer together in space over time.

[0164] Example 16

[0165] To investigate the time required for the PD-L1 and phagosome dual-targeting assembly to transfer PD-L1 to lysosomes, HEK293T cells were transfected with pEGFP-N1 / PD-L1 and mTagBFP2-Lysosomes-20 plasmids in serum-free medium for 48 h. Transfected cells were then treated with the PD-L1 and phagosome dual-targeting assembly for 0–12 h, fixed with 4% PFA solution for 15 min, and finally imaged using laser confocal scanning microscopy. The conclusions of this example can be found in [link to relevant documentation]. Figure 16 , Figure 16 Confocal microscopy images showing the intracellular localization of PD-L1 and lysosomes after 0-12 h of treatment with a dual-targeting PD-L1 and phagosome assembly. Figure 16 As shown, the PD-L1 and phagosome dual-target assembly can gradually bring PD-L1 and lysosomes closer together in space over time.

[0166] Example 17

[0167] To investigate the time it takes for the PD-L1 and phagosome dual-targeting assembly to transfer early endosomes containing PD-L1 to the phagosome, HEK293T cells were transfected with GFP-Rab5B and pmRFP-LC3 plasmids in serum-free medium for 48 h. Transfected cells were then treated with the PD-L1 and phagosome dual-targeting assembly for 0–12 h, fixed with 4% PFA solution for 15 min, and finally imaged using laser confocal scanning microscopy. The conclusions of this example can be found in [link to relevant documentation]. Figure 17 , Figure 17 Confocal microscopy images showing the intracellular localization of early endosomes and phagosomes containing PD-L1 after treatment with a dual-targeting PD-L1 and phagosome assembly for 0–12 h. Figure 17 As shown, with the extension of time, the colocalization of early endosomes and phagosomes in the PD-L1 and phagosome dual-target assembly group increases. This proves that the dual-target assembly group has the ability to recruit phagosomes, spatially reduce the distance between phagosomes and early endosomes anchored with PD-L1, and use phagosomes to engulf early endosomes anchored with PD-L1.

[0168] Example 18

[0169] To investigate the efficiency of the PD-L1 and phagosome dual-targeting assembly in transferring early endosomes containing PD-L1 to lysosomes, HEK293T cells were transfected with GFP-Rab5B and mTagBFP2-Lysosomes-20 plasmids in serum-free medium for 48 h. Transfected cells were then treated with the PD-L1 and phagosome dual-targeting assembly for 0–12 h, fixed with 4% PFA solution for 15 min, and finally imaged using laser confocal scanning microscopy. The conclusions of this example can be found in [link to relevant documentation]. Figure 18 , Figure 18 Confocal microscopy images showing the intracellular localization of early endosomes and lysosomes containing PD-L1 after treatment with a dual-targeting PD-L1 and phagosome assembly for 0–12 h. Figure 18 As shown, with the extension of time, the colocalization of early endosomes and lysosomes in the PD-L1 and phagosome dual-target assembly increases, which proves that the dual-target assembly can eventually bring early endosomes anchored with PD-L1 into lysosomes for degradation.

[0170] Example 19

[0171] To investigate the effect of PD-L1 and phagosome dual-targeting assembly concentrations on PD-L1 degradation, 4T1 mouse breast cancer cells in 6-well plates were treated with different concentrations of PD-L1 and phagosome dual-targeting assembly for 12 h. Western blot was used to examine the degradation of PD-L1 in 4T1 mouse breast cancer cells by different concentrations of PD-L1 and phagosome dual-targeting assembly. The conclusions of this example can be found in [link to relevant documentation]. Figure 19 , Figure 19 To quantitatively assess the effects of different concentrations of PD-L1 and phagosome dual-targeting assemblies on PD-L1 expression in 4T1 mouse breast cancer cells using Western blot, as shown in the figure... Figure 19 As shown, with the gradual increase in the concentration of the PD-L1 and phagosome dual-target assembly, the efficiency of the PD-L1 and phagosome dual-target assembly in degrading PD-L1 becomes higher and higher.

[0172] Example 20

[0173] To investigate the necessity of the phagosome-targeting ligand in the PD-L1 and phagosome dual-targeting assembly for PD-L1 degradation, 4T1 mouse breast cancer cells in 6-well plates were treated with different concentrations of the PD-L1 single-targeting assembly for 12 h. Western blot was used to examine the degradation of PD-L1 in 4T1 mouse breast cancer cells by different concentrations of the PD-L1 single-targeting assembly. The conclusions of this example can be found in [link to example]. Figure 20 , Figure 20 To quantitatively assess the effect of different concentrations of PD-L1 single-target assemblies on PD-L1 expression in 4T1 mouse breast cancer cells using Western blot, as shown in... Figure 20 As shown, although the concentration of PD-L1 single-target assembly gradually increases, PD-L1 single-target assembly does not have a degradation effect on cellular PD-L1.

[0174] Example 21

[0175] To investigate the effect of PD-L1 and phagosome dual-targeting assembly treatment time on PD-L1 degradation, PD-L1 and the phagosome dual-targeting assembly were incubated with 4T1 mouse breast cancer cells for 0, 2, 4, 8, 12, and 24 h, respectively. Western blot analysis was used to examine the effect of different incubation times on PD-L1 degradation. The conclusions of this example can be found in [link to example]. Figure 21 , Figure 21 To investigate the effects of the dual-targeting assembly of PD-L1 and phagosomes on cellular PD-L1 protein at different time points using Western blot, such as... Figure 21 As shown, as the treatment time of cells with the PD-L1 and phagosome dual-target assembly gradually increases, the efficiency of the PD-L1 and phagosome dual-target assembly in degrading PD-L1 becomes higher and higher.

[0176] Example 22

[0177] To investigate the necessity of the phagosome-targeted degradation ligand in the PD-L1 and phagosome dual-target assembly for PD-L1 degradation, 4T1 mouse breast cancer cells in 6-well plates were treated with the PD-L1 single-target assembly for 0-24 h. Western blot was used to examine the degradation of PD-L1 in 4T1 mouse breast cancer cells by the PD-L1 single-target assembly at different time points. The conclusions of this example can be found in [link to relevant documentation]. Figure 22 , Figure 22 To quantitatively assess the effect of PD-L1 single-target assembly on PD-L1 expression in 4T1 mouse breast cancer cells at different time points using Western blot, as shown in the figure... Figure 22As shown, although the treatment time of the PD-L1 single-target assembly on cells gradually increased, the PD-L1 single-target assembly did not have a degradation effect on PD-L1 in cells.

[0178] Example 23

[0179] To investigate the necessity of inserting various parts of the lipid membrane peptide and phagosome-targeting peptide into the PD-L1 targeting ligand under acidic conditions for PD-L1 degradation, please refer to the conclusions of this example. Figure 23 , Figure 23 To investigate the effect of different assemblies on PD-L1 expression in 4T1 cells, phagosome-targeting peptide control group assembly, phagosome single-targeting assembly, PD-L1 single-targeting nanoparticles, PD-L1 single-targeting assembly, and PD-L1 and phagosome dual-targeting assembly were treated with 0.5 μM for 12 h. Western blot was used to quantitatively examine the effect of different assemblies on PD-L1 expression in 4T1 cells. Figure 23 As shown, PD-L1 degradation in 4T1 cells can only occur when PD-L1 targeting ligands, pH-sensitive intercalation peptides, and phagosome-targeting peptides are all present.

[0180] Example 24

[0181] To investigate the potential mechanism of PD-L1 degradation mediated by the dual-targeting assembly of PD-L1 and phagosomes, please refer to the conclusions of this example. Figure 24 , Figure 24 4T1 cells were pretreated for 24 hours with bafloxacin A1 (lysosomal acidification inhibitor, 100 nM), chloroquine (autophagy flux inhibitor, 20 μM), NH4Cl (autophagy inhibitor, 10 mM), MG132 (proteasome inhibitor, 5 μM), or rapamycin (phagocytosis enhancer, 10 μM), followed by further treatment for 12 hours with a mixture of 0.5 μM PD-L1 and phagosome dual-targeting assemblies and the aforementioned functional molecules. Western blot was then used to quantify the PD-L1 protein level in the treated cells. Figure 24 As shown, bafimycin A1, chloroquine, and NH4Cl inhibited PD-L1 degradation, rapamycin enhanced PD-L1 degradation, while MG132 showed no effect, demonstrating that the PD-L1 and phagosome dual-target assembly degrades PD-L1 through an autophagy degradation mechanism.

[0182] Example 25

[0183] To investigate whether the degradation of the PD-L1 and phagosome dual-target assembly would damage the endosome, please refer to the conclusions of this embodiment. Figure 25 , Figure 25To simultaneously treat HEK293T cells with different assemblages and 0.1 mg / mL calcein, since calcein, which is membrane-impermeable, is typically isolated in the endosome after cellular uptake. For example... Figure 25 As shown, co-incubation with PD-L1 single-target nanoparticles revealed distinct calcein spots. However, cells treated with other assemblies containing pH-sensitive peptides showed calcein release from endosomes into the cytoplasm and uniform distribution within the cell, indicating that the PD-L1 and phagosome dual-target assembly disrupts endosomes during PD-L1 degradation.

[0184] Example 26

[0185] Preparation of Aβ oligomers. Human amyloid-β monomer 1-42 (Aβ1-42) powder was dissolved in cold hexafluoroisopropanol to a final concentration of 1 mM. The solution was incubated at room temperature for 60 min to ensure complete dissolution. The solution was then aliquoted into centrifuge tubes on ice, and the hexafluoroisopropanol was evaporated overnight at room temperature. The resulting dry, clear membrane was obtained as the Aβ monomer. 44 μL of dimethyl sulfoxide was added to each 1 mg of Aβ monomer to dissolve the peptide membrane and form a 5 mM solution. This solution was then diluted to a concentration of 100 μM with serum-depleted medium. The solution was further incubated at 4°C for 24 h and filtered through a 0.22 μm membrane to obtain homogeneous Aβ oligomers. RAGE-upregulated mouse brain microvascular endothelial cells were established by incubating with 10 μM Aβ oligomers for 24 h. Figure 26 The results showed that RAGE was upregulated in mouse brain microvascular endothelial cells after 24 hours of Aβ incubation.

[0186] Preparation of RAGE single-target nanoparticles. First, 100 mg of polylactic acid-glycolic acid-polylysine was dissolved in 2 mL of ethyl acetate as the base carrier. This mixture was added dropwise to 4 mL of 2.5% polyvinyl alcohol under vortexing, and immediately sonicated at 300 W for 3 × 10⁻⁶ s to form an oil-in-water emulsion. This emulsion was immediately poured into 100 mL of 0.3% polyvinyl alcohol and stirred overnight to evaporate the ethyl acetate. The nanoparticle suspension was purified from polyvinyl alcohol by centrifugation at 70,000 g for 20 min, and the precipitate was resuspended in PBS 7.4 by sonication in a water bath. Subsequently, 11.2 mg of maleimide-containing polyethylene glycol was added, and the reaction was carried out at room temperature for 1 h. Excess maleimide-containing polyethylene glycol was then removed by centrifugation at 70,000 g for 20 min, the nanoparticle suspension was resuspended, and further modified with excess cysteine-containing targeted RAGE ligands via maleimide-thiol reaction chemistry at room temperature for 1 h. Finally, excess unreacted peptides were removed by centrifugation at 70,000 g for 20 minutes, the nanosuspension was resuspended in water and freeze-dried to obtain RAGE single-target nanoparticles.

[0187] To investigate the degradation of RAGE upregulated in mouse brain endothelial cells by the RAGE and phagosome dual-targeting assembly, please refer to the conclusions of this example. Figure 27 , Figure 27 This study aimed to quantify RAGE expression in mouse brain endothelial cells after treating them with blank culture medium, phagosome-targeting peptide control group assemblies, phagosome single-targeting assemblies, RAGE single-targeting nanoparticles, RAGE single-targeting assemblies, and RAGE and phagosome dual-targeting assemblies at a concentration of 0.5 μM for 12 h using Western blot. Figure 27 As shown, the dual-targeting assembly of RAGE and phagosomes has a significant effect on the degradation of RAGE.

[0188] Example 27

[0189] Preparation of Aβ-targeted nanoparticles. First, 100 mg of polylactic acid-glycolic acid-polylysine was dissolved in 2 mL of ethyl acetate as the base carrier. This mixture was added dropwise to 4 mL of 2.5% polyvinyl alcohol under vortexing, and immediately sonicated at 300 W for 3 × 10⁻⁶ s to form an oil-in-water emulsion. This emulsion was immediately poured into 100 mL of 0.3% polyvinyl alcohol and stirred overnight to evaporate the ethyl acetate. The nanoparticle suspension was purified from polyvinyl alcohol by centrifugation at 70,000 g for 20 min, and the precipitate was resuspended in PBS 7.4 by sonication in a water bath. Subsequently, 11.2 mg of maleimide-linked polyethylene glycol was added, and the reaction was carried out at room temperature for 1 h. Excess maleimide-linked polyethylene glycol was then removed by centrifugation at 70,000 g for 20 min, the nanoparticle suspension was resuspended, and further modified with excess cysteine-containing targeted Aβ ligands via a maleimide-thiol reaction chemistry at room temperature for 1 h. Finally, by centrifuging at 70,000 g for 20 minutes to remove excess unreacted peptides, the nanosuspension was resuspended in water and freeze-dried to obtain Aβ single-target nanoparticles.

[0190] Given that Aβ oligomers can be internalized through RAGE-mediated endocytosis and spread among brain parenchymal cells, promoting the progression of Alzheimer's disease, a dual-targeting assembly of Aβ and phagosomes is expected to form a ternary complex with Aβ oligomers and RAGE for cellular uptake of Aβ and RAGE.

[0191] To investigate the effect of assemblies with Aβ-targeting activity on Aβ uptake concentration in RAGE-upregulated microglia, please refer to the conclusions of this example. Figure 29 , Figure 29 To establish RAGE-upregulated BV-2 microglia in 6-well plates (treatment method as described above, incubation with 10 μM Aβ oligomers for 24 hours to establish RAGE-upregulated BV-2 cells, results are shown in [link to results]). Figure 28A mixture of 10 μM Aβ oligomers and different concentrations of Aβ single-target assemblies was incubated for 12 hours. The protein expression of microglia upregulated by RAGE was quantified by western blot. Figure 29 As shown, with the increase of the concentration of Aβ single-target assembly in cells treated with RAGE, the uptake of Aβ by microglia upregulated by RAGE gradually increased.

[0192] Example 28

[0193] To investigate the effect of assemblies with Aβ-targeting activity on Aβ uptake time in RAGE-upregulated microglia, please refer to the conclusions of this example. Figure 30 , Figure 30 To set different incubation times (0, 2, 4, 6, 8, and 12 h), RAGE-upregulated BV-2 microglia were incubated with a mixture of 10 μM Aβ oligomers and Aβ single-target assemblies. Western blot analysis was used to determine the cellular uptake of Aβ protein. (See figure). Figure 30 As shown, with increasing treatment time of cells with the Aβ single-target assembly, the uptake of Aβ by RAGE-upregulated microglia gradually increased.

[0194] Example 29

[0195] To investigate the effect of the Aβ and phagosome dual-targeting assembly on the simultaneous degradation of both Aβ and RAGE proteins in RAGE-upregulated BV-2 cells, please refer to the conclusions of this example. Figure 31 , Figure 31 To investigate the protein expression quantification of Aβ and RAGE in RAGE-upregulated BV-2 microglia, we incubated them for 12 h with a mixture of 10 μM Aβ oligomers and different assemblies. Western blot analysis was then performed on the RAGE-upregulated microglia. Figure 31 As shown, both nanoparticles and assemblies with Aβ single-targeting activity enhanced cellular uptake of Aβ, while the assembly with dual-targeting activity of Aβ and phagosomes simultaneously degraded both Aβ and RAGE proteins in BV-2 cells with RAGE upregulation. This indicates that the dual-targeting assembly of Aβ and phagosomes can simultaneously bind to the endocytic receptor RAGE on the cell membrane and extracellular Aβ protein, and degrade them through endosome-phagosome-lysosome pathway.

[0196] Example 30

[0197] To examine the co-localization of Aβ and phagosomes after cell treatment with the dual-targeting assemblies of Aβ and phagosomes, please refer to the conclusions of this example. Figure 32 , Figure 32Confocal microscopy images of BV-2 cells transfected with pmRFP-LC3, followed by treatment with blank culture medium, phagosome-targeting peptide control group assemblies, phagosome single-target assemblies, Aβ single-target assemblies, and Aβ and phagosome dual-target assemblies, co-treated with FITC-labeled Aβ oligomers and RAGE-upregulated BV-2 cells for 12 h. The drug concentration of the assemblies was 10 μM. Figure 32 As shown, only cells treated with the Aβ and phagosome dual-targeting assembly exhibited high colocalization of internalized Aβ and phagosomes, indicating that the Aβ and phagosome dual-targeting assembly has the ability to recruit phagosomes.

[0198] Example 31

[0199] To investigate the co-localization of Aβ with lysosomes after treatment with the dual-targeting assemblies of Aβ and phagosomes, please refer to the conclusions of this example. Figure 33 , Figure 33 Confocal microscopy images of BV-2 cells transfected with mTagBFP2-Lysosomes-20, followed by treatment with blank culture medium, phagosome-targeting peptide control group assemblies, phagosome single-targeting assemblies, Aβ single-targeting assemblies, and Aβ and phagosome dual-targeting assemblies, co-treated with FITC-labeled Aβ oligomers and RAGE-upregulated BV-2 cells for 12 h. The drug concentration of the assemblies was 10 μM. Figure 33 As shown, only cells treated with the dual-targeting assembly of Aβ and phagosomes exhibited high colocalization of internalized Aβ and lysosomes, indicating that the dual-targeting assembly of Aβ and phagosomes enables internalized Aβ to be phagocytosed by phagosomes and then carried into lysosomes for degradation.

[0200] Example 32

[0201] To examine the co-localization of Aβ-containing endosomes and phagosomes after cell treatment with the Aβ and phagosome dual-targeting assemblies, please refer to the conclusions of this example. Figure 34 , Figure 34 Confocal microscopy images of BV-2 cells transfected with pmRFP-LC3, followed by treatment with blank culture medium, phagosome-targeting peptide control group assemblies, phagosome single-target assemblies, Aβ single-target assemblies, and Aβ and phagosome dual-target assemblies, co-treated with 10 μM Aβ oligomers and RAGE-upregulated BV-2 cells for 12 h. The drug concentration for the assemblies was 10 μM. Figure 34 As shown, only cells treated with the Aβ and phagosome dual-targeting assembly exhibited high colocalization of Aβ-containing endosomes and phagosomes, indicating that the Aβ and phagosome dual-targeting assembly facilitates the transfer of Aβ-containing endosomes to phagosomes.

[0202] Example 33

[0203] To investigate the co-localization of Aβ-containing endosomes and lysosomes after cell treatment with the Aβ and phagosome dual-targeting assemblies, please refer to the conclusions of this example. Figure 35 , Figure 35 Confocal microscopy images of BV-2 cells transfected with mTagBFP2-Lysosomes-20, followed by treatment with blank culture medium, phagosome-targeting peptide control group assemblies, phagosome single-targeting assemblies, Aβ single-targeting assemblies, and Aβ and phagosome dual-targeting assemblies, co-treated with 10 μM Aβ oligomers with RAGE-upregulated BV-2 cells for 12 h. The drug concentration of the assemblies was 10 μM. Figure 35 As shown, only cells treated with the Aβ and phagosome dual-targeting assembly exhibited high colocalization of Aβ-containing endosomes and lysosomes, indicating that the Aβ and phagosome dual-targeting assembly facilitates the transfer of Aβ-containing endosomes to phagosomes, which are then degraded by lysosomes.

[0204] Example 34

[0205] To investigate the therapeutic efficacy of the dual-targeting PD-L1 and phagosome assembly, treatment was administered via intravenous injection of different assemblies and intraperitoneal injection of PD-L1 antibody (PD-L1 antibody dosage: 100 μg intraperitoneally every two days; 1.8 μmol / kg intravenously every other day for the other groups, the same below). Please refer to [link to relevant documentation]. Figure 36 , Figure 36 This is a schematic diagram of the PD-L1 and phagosome dual-targeting assembly treatment protocol designed in this invention. Specifically, a breast cancer model is constructed by subcutaneous injection of 4T1 breast cancer cells. Treatment is administered in groups starting 7 days after tumor implantation, and the therapeutic effect is subsequently evaluated by tumor volume, tumor weight, PD-L1 degradation efficiency, and T-cell enhancement.

[0206] The treatment results are as follows:

[0207] Treatment options included intravenous injection of saline (blank control group), PD-L1 single-target nanoparticles, PD-L1 and phagosome dual-target assembly, PD-L1 single-target assembly, phagosome-targeting peptide control group assembly, phagosome single-target assembly, and intraperitoneal injection of PD-L1 antibody. Please refer to [link to relevant documentation]. Figure 37 , Figure 37 Statistical analysis was conducted to quantitatively characterize changes in tumor volume. It was observed that the tumor volume growth in the group receiving intravenous injection of the PD-L1 and phagosome dual-targeting assembly was significantly inhibited compared to other groups, demonstrating a significant difference and highlighting the therapeutic potential of the PD-L1 and phagosome dual-targeting assembly. Figure 38 To quantitatively characterize tumor weight statistics, it was observed that the group receiving intravenous injection of PD-L1 and phagosome dual-targeting assembly significantly inhibited tumor weight compared to other groups, demonstrating a significant difference and highlighting the therapeutic value of PD-L1 and phagosome dual-targeting assembly.

[0208] Example 35

[0209] To investigate the therapeutic efficacy of the PD-L1 and phagosome dual-targeting assembly, treatments were administered via intravenous saline, PD-L1 single-targeting nanoparticles, PD-L1 and phagosome dual-targeting assemblies, PD-L1 single-targeting assemblies, phagosome-targeting peptide control group assemblies, phagosome single-targeting assemblies, and intraperitoneal injection of PD-L1 antibodies. Please refer to [link to relevant documentation]. Figure 39 , Figure 39 To quantitatively characterize the effects of different groups on PD-L1 degradation in mouse tumor tissue, the following groups were selected: intravenous injection of saline, PD-L1 single-target nanoparticles, PD-L1 and phagosome dual-target assembly, PD-L1 single-target assembly, phagosome-targeting peptide control group assembly, phagosome single-target assembly, and intraperitoneal injection of PD-L1 antibody. Figure 39 As shown, the PD-L1 protein in the group receiving intravenous injection of the PD-L1 and phagosome dual-target assembly was significantly degraded compared to other groups, demonstrating the high degradation efficiency of the PD-L1 and phagosome dual-target assembly.

[0210] Example 36

[0211] To investigate the therapeutic side effects of the PD-L1 and phagosome dual-targeting assembly, treatments were administered via intravenous saline, PD-L1 single-targeting nanoparticles, PD-L1 and phagosome dual-targeting assemblies, PD-L1 single-targeting assemblies, phagosome-targeting peptide control group assemblies, phagosome single-targeting assemblies, and intraperitoneal injection of PD-L1 antibodies. Please refer to [link to relevant documentation]. Figure 40 , Figure 40 To quantitatively characterize the effect of different groups on the number of lung nodules in mice, the results showed that the group receiving intravenous injection of PD-L1 and phagosome dual-targeting assembly significantly reduced the number of lung nodules compared to other groups, demonstrating the low toxicity and high safety of the PD-L1 and phagosome dual-targeting assembly.

[0212] Example 37

[0213] To investigate the therapeutic efficacy of the PD-L1 and phagosome dual-targeting assembly, treatments were administered via intravenous saline, PD-L1 single-targeting nanoparticles, PD-L1 and phagosome dual-targeting assemblies, PD-L1 single-targeting assemblies, phagosome-targeting peptide control group assemblies, phagosome single-targeting assemblies, and intraperitoneal injection of PD-L1 antibodies. Please refer to [link to relevant documentation]. Figure 41 , Figure 41 To quantitatively characterize the effects of different groups on CD4 in mouse tumor tissue + T cells and CD8 +Schematic diagram of increased T cell stimulation. Results show that the group receiving intravenous PD-L1 and phagosome dual-targeting assemblies showed higher CD4 levels in tumor tissue compared to other groups. + T cells and CD8 + The significant increase in T cells indicates that after the PD-L1 and phagosome dual-targeting assembly degrades PD-L1, tumor cells lose key immunosuppressive molecules, thereby relieving the suppression of T cells and restoring the anti-tumor immune response.

[0214] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A protein degradation agent, characterized in that, The protein degradation agent comprises an assembly, and the monomer forming the assembly comprises the following parts arranged in connection: a target protein specific ligand; a phagosome targeting polypeptide; an endosome membrane-inserting polypeptide, which is located between the target protein specific ligand and the phagosome targeting polypeptide, and the endosome membrane-inserting polypeptide is a C-terminal membrane-inserting polypeptide; wherein, the amino acid sequence of the C-terminal membrane-inserting polypeptide is FSGSGSGSGRQIKIWFQAARMLWKK or AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT, the amino acid sequence of the phagosome targeting polypeptide is GIYVIWGPI.

2. The protein degrader of claim 1, wherein, At least one of the following characteristics is contained: (1) the target protein specific ligand is chemically connected with the endosome membrane-inserting polypeptide; (2) the phagosome targeting polypeptide is chemically connected with the endosome membrane-inserting polypeptide; (3) the monomer contains a hydrophilic group modification; (4) the target protein is an extracellular protein or a cell membrane protein; (5) the target protein specific ligand comprises a target protein specific polypeptide ligand.

3. The protein degrader of claim 1, wherein, At least one of the following characteristics is contained: (1) the target protein specific ligand is covalently connected with the endosome membrane-inserting polypeptide; (2) the connection mode of the target protein specific ligand and the endosome membrane-inserting polypeptide comprises a covalent bond formed by amino and sulfydryl or a peptide bond; (3) the phagosome targeting polypeptide is covalently connected with the endosome membrane-inserting polypeptide; (4) the connection mode of the phagosome targeting polypeptide and the endosome membrane-inserting polypeptide comprises a peptide bond; (5) the monomer contains a hydrophilic group modification.

4. The protein degrader of claim 3, wherein, At least one of the following characteristics is contained: (1) the hydrophilic group comprises at least one of a hydroxyl group, a carboxyl group, an amino group and an amide group; (2) the source of the hydrophilic group comprises a hydrophilic amino acid; (3) the hydrophilic group modification is on the target protein specific ligand, and the modification comprises modifying the hydrophilic group on the end of the target protein specific ligand close to the endosome membrane-inserting polypeptide.

5. The protein degrader of claim 1, wherein: The target protein comprises one or more of programmed death receptor-ligand 1, beta amyloid, advanced glycosylation end product, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, prostate specific membrane antigen, and mitochondrial p32 molecular chaperone protein; wherein, when the target protein comprises programmed death receptor-ligand 1, the target protein specific ligand comprises a programmed death receptor-ligand 1 specific polypeptide ligand, and the amino acid sequence of the programmed death receptor-ligand 1 specific polypeptide ligand is shown in SEQ ID NO. 1; and / or, when the target protein comprises beta amyloid, the target protein specific ligand comprises a beta amyloid specific polypeptide ligand, and the amino acid sequence of the beta amyloid specific polypeptide ligand is shown in SEQ ID NO. 2; and / or, when the target protein comprises advanced glycosylation end product, the target protein specific ligand comprises an advanced glycosylation end product specific polypeptide ligand, and the amino acid sequence of the advanced glycosylation end product specific polypeptide ligand is shown in SEQ ID NO.

3.

6. A method of preparing a protein degradation agent according to any one of claims 1 to 5, characterized in that, The preparation of the assembly comprises the following steps: The monomer is dispersed in an aqueous solution at a set concentration, and the monomer self-assembles to form the assembly.

7. The production method according to claim 6, wherein At least one of the following features is included: (1) the set concentration is not lower than the critical concentration for forming micelles; (2) the specific steps include: S1, preparing a phagosome-targeting polypeptide with a connection setting and an endosome membrane-inserting polypeptide with a modification, to obtain an endosome-anchored phagosome-targeting peptide; preparing a target protein-specific ligand with a modification; S2, mixing and reacting the endosome-anchored phagosome-targeting peptide and the target protein-specific ligand in the presence of a solvent, to connect the modified parts on the endosome membrane-inserting polypeptide and the target protein-specific ligand, to obtain the monomer; (3) after the self-assembly is completed, a purification step is further included.

8. The preparation method according to claim 7, characterized in that, At least one of the following features is included: (1) the molar ratio of the endosome-anchored phagosome-targeting peptide to the target protein-specific ligand is 1:(1-20); (2) the solvent is an organic solvent that can dissolve the polypeptide, and the organic solvent includes N , N dimethylformamide or dimethyl sulfoxide; (3) the purification step includes dialysis.

9. Use of a protein degradation agent according to any one of claims 1 to 5 or a protein degradation agent obtainable by the process according to any one of claims 6 to 8 for the manufacture of a medicament for the prevention or treatment of a solid tumor, characterized in that, The target protein is programmed death receptor-ligand 1.

10. A pharmaceutical composition, characterized by, The protein degradation agent obtained by the preparation method of any one of claims 6-8. The protein degradation agent of any one of claims 1-5 or obtained by the preparation method of any one of claims 6-8.

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