A bispecific peptide nanomedicine targeting PD-L1 and its preparation method and application

Abstract

The present invention relates to a bispecific polypeptide nanodrug targeting PD-L1, a preparation method thereof, and an application thereof. The bispecific polypeptide nanodrug targeting PD-L1 comprises a tumor microenvironment targeting regulatory unit, a connecting unit, a self-assembly unit, and a PD-L1 blocking unit, which are sequentially connected by amide bonds. The bispecific polypeptide nanodrug involved in the present invention specifically binds to the CXCR4 protein overexpressed on the surface of tumor cells, blocks the CXCR4 / SDF-1 signaling pathway, improves the tumor microenvironment, and increases the infiltration of T cells; on the other hand, it specifically binds to the PD-L1 protein overexpressed on the surface of tumor cells, blocks the interaction between PD-1 and PD-L1, restores T cell killing of tumor cells, and enhances the immunotherapy of bladder cancer. After binding to the protein on the surface of tumor cells, the fragments with assembly ability are restricted due to the movement of the polypeptide molecules, which reduces the activation entropy required for their self-assembly, thereby triggering their self-assembly in situ to form nanofibers, thereby achieving long-term blocking of the receptor signaling pathway.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and relates to a tumor immunotherapy drug, a preparation method and applications thereof, and specifically to a bispecific polypeptide nanomedicine targeting PD-L1, a preparation method and applications thereof. Background Art

[0002] Bladder cancer is one of the three most common malignancies of the urinary system, ranking 12th in incidence among malignant tumors. Immunotherapy has brought significant breakthroughs in the treatment of bladder cancer, particularly immune checkpoint inhibitors such as PD-1 / PD-L1. Although PD-L1 immunosuppressants emerged later than PD-1, they are more diverse. Atezolizumab, avelumab (MSB0010718C), MP-DL3280A (RG7446), durvalumab (MEDI4736), AMP-514, and AMP-22 are all monoclonal antibodies that recognize and bind to PD-L1, achieving similar efficacy as PD-1 monoclonal antibodies. Drugs that inhibit the PD-1 checkpoint and its ligand PD-L1 have demonstrated promising therapeutic effects in a variety of cancers, particularly bladder cancer. While immune checkpoint blockade has yielded significant results in bladder cancer, it still faces the significant challenge of low response rates due to the high fibrosis and insufficient T cell infiltration within solid tumors. Therefore, exploring a new method to enhance bladder cancer immunotherapy has become a practical problem that urgently needs to be solved in clinical practice.

[0003] Small molecule peptides have become the preferred material system due to their biological activity, specificity, chemical modifiability, targeting, and biostability. Targeted peptides are peptides that can specifically bind to tumor cells or tissues and have advantages such as high affinity, high stability, and low toxicity. They can be coupled with anticancer drugs for diagnosis and specific delivery of early tumor lesions, and have good prospects in the treatment of cancer. With the deepening of oncology research, progress has been made in the screening, synthesis, discovery, and treatment of small molecule tumor-targeted peptide drugs. The peptide molecules targeting PD-L1 screened by Smriti Gurunga et al. using phage display technology have the advantages of strong targeting and high specificity.

[0004] Chemokine receptor 4 (CXCR4) belongs to the chemokine receptor subfamily and is a specific receptor for the chemokine stromal cell-derived factor-1 (SDF-1). It has a seven-transmembrane structure and is a G protein-coupled receptor containing seven transmembrane domains, an extracellular N-terminus, and an intracellular C-terminus. CXCR4 is highly expressed in bladder cancer cells, whereas it is almost absent in normal bladder mucosa. Studies have shown that high CXCR4 expression is associated with the infiltration of cells such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and tumor-associated neutrophils (TANs), exerting a pro-tumor effect. CXCR4 has been shown to enhance tumor immune evasion by recruiting Tregs and MDSCs to promote angiogenesis and promote tumor-associated fibroblast (CAF)-dependent immunosuppression. CXCR4 / SDF-1 signaling promotes CAF recruitment, activation, and matrix production. The above results indicate that CXCR4 plays an important role in the immunosuppression process of bladder cancer. Therefore, blocking the CXCR4 / SDF-1 signaling pathway is a potential method to assist in the immunotherapy of bladder cancer.

[0005] Currently, supramolecular assembly strategies have been widely used as promising approaches for the treatment of bladder cancer. Most of these supramolecular assembly-based nanoparticles or mucoadhesive biomaterials focus on prolonging drug retention and enhancing drug permeability to enhance the efficacy of intravesical chemotherapeutic drug delivery. Although improved efficacy has been observed compared to simple drug instillation, severe side effects such as urethral irritation, cystitis, and hematuria limit their application due to their lack of active targeting capabilities. Therefore, new therapeutic approaches are needed to reduce postoperative recurrence of bladder cancer. The primary driving forces of peptide self-assembly are weak interactions, such as hydrogen bonding, van der Waals forces, and electrostatic interactions. Furthermore, controllable supramolecular self-assembly with specific morphologies can be achieved through amino acid sequence design. Furthermore, peptide assembly can be achieved in complex physiological environments. The assembly-induced retention (AIR) effect can effectively optimize the biodistribution of bioactive molecules in vivo and increase drug tumor penetration, providing new insights into the development of novel, highly effective, and low-toxic biomaterials.

[0006] Therefore, how to use the AIR effect to construct peptide-based nanofibers in situ on the tumor cell membrane, thereby reducing the density of tumor-associated fibroblasts in the tumor microenvironment, reshaping the tumor immune microenvironment, and promoting T cell infiltration; at the same time, blocking the signal transmission of PD-1 / PD-L1, so that the functionally suppressed T cells can restore their recognition function of tumor cells, achieve anti-cancer effects through the body's own immune system, and ultimately enhance the immunotherapy of bladder cancer is an issue that needs to be solved urgently. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a tumor immunotherapy drug and its preparation method and application, specifically a bispecific polypeptide nanodrug targeting PD-L1 and its preparation method and application.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a bispecific polypeptide nanodrug targeting PD-L1, wherein the bispecific polypeptide nanodrug targeting PD-L1 comprises a tumor microenvironment targeting regulation unit, a connection unit, a self-assembly unit and a PD-L1 blocking unit sequentially connected by amide bonds.

[0010] The bispecific peptide nanomedicine targeting PD-L1 involved in the present invention contains a tumor microenvironment targeting regulation unit and a PD-L1 blocking unit, which has target recognition function, tumor microenvironment regulation function and immune checkpoint blocking function. It actively targets the tumor site through molecular specificity. On the one hand, it specifically binds to the CXCR4 protein overexpressed on the surface of tumor cells, blocks the CXCR4 / SDF-1 signaling pathway, improves the tumor microenvironment, and increases T cell infiltration; on the other hand, it specifically binds to the PD-L1 protein overexpressed on the surface of tumor cells, blocks the interaction between PD-1 and PD-L1, restores T cell killing of tumor cells, and enhances the immunotherapy of bladder cancer. After binding to the protein on the surface of tumor cells, the fragments with assembly ability are restricted due to the movement of the peptide molecules, which reduces the activation entropy required for their self-assembly and triggers their in situ self-assembly to form nanofibers, achieving long-term blockade of the receptor signaling pathway. This bispecific peptide nanomedicine provides a new method for tumor immunotherapy. At the same time, it does not produce obvious side effects in the body and has good biocompatibility.

[0011] The design principle of the bispecific peptide nanomedicine is as follows: Figure 1 As shown, the bispecific peptide nanomedicine targets and binds to the CXCR4 and PD-L1 proteins of tumor cells through the tumor microenvironment targeting regulatory unit and the PD-L1 blocking unit respectively. The fragments with assembly ability reduce the activation entropy required for their self-assembly due to the binding of the peptide molecules to the target proteins, thereby triggering their in situ self-assembly to form nanofibers, achieving long-term blocking of the PD-1 / PD-L1 and CXCR4 / SDF-1 signaling pathways, activating T cells, increasing T cell infiltration, promoting T cell killing of tumor cells, and enhancing the immunotherapy effect of bladder cancer.

[0012] Preferably, the tumor microenvironment targeting regulatory unit is derived from a molecule that specifically binds to a tumor matrix-related protein, including any one of AMD070, AMD3100, BL-8040, LY2510924, POL5551, SP-13786, Talabostat or Linagliptin.

[0013] Preferably, the tumor microenvironment targeting regulatory unit is derived from AMD070 or SP-13786.

[0014] The target receptor corresponding to the molecule AMD070 is CXCR4; the target receptor corresponding to the molecule SP-13786 is FAP.

[0015] Preferably, the PD-L1 blocking unit is derived from a polypeptide that targets and binds to the PD-L1 protein, including any one of CVRARTR, CLQKTPKQC, WHRSYYTWNLNT or FSGTVTTAGLLF.

[0016] Preferably, the PD-L1 blocking unit is derived from the polypeptide sequence CVRARTR.

[0017] Preferably, the self-assembly unit is derived from a polypeptide with assembly ability, including any one of KLVFFG, KLVFF, FF, YFFGNNQQNY, GSNKGAIIGLM, ITSVV, SYSSYGQS, GNNQQNY, GNQQQQY or GNNNQNY.

[0018] Preferably, the self-assembly unit is derived from the polypeptide sequence KLVFFG or GNNQQNY.

[0019] Since the movement of the polypeptide molecules is restricted, the above-mentioned fragments with assembly ability reduce the activation entropy required for their self-assembly, thereby triggering their in situ self-assembly to form nanofibers, thereby achieving long-term blocking of the receptor signaling pathway.

[0020] Preferably, the linking unit is derived from a polypeptide that regulates the molecular length and hydrophilic-hydrophobic balance, including any one of DPGLGYL, D(OEG)4 or D(OEG)8.

[0021] Preferably, the linking unit is derived from the polypeptide sequence DPGLGYL or D(OEG)4.

[0022] As a preferred technical solution of the present invention, the bispecific polypeptide nanodrug targeting PD-L1 has a structure as shown in any one of Formula I, Formula II or Formula III.

[0023]

[0024] In the present invention, the molecule of the structure shown in Formula I is (AMD070)DPGLGYLKLVFFGCVRARTR, the molecule of the structure shown in Formula II is (AMD070)DPGLGYLGNNQQNYCVRARTR, and the molecule of the structure shown in Formula III is (AMD070)D-(OEG)4-KLVFFGCVRARTR, wherein KLVFFG and GNNQQNY are self-assembly sequences, which can realize in situ assembly on the tumor cell membrane, thereby achieving long-term blockade of tumor-related targets; wherein DPGLGYL and D(OEG)4 are connecting molecules, whose purpose is to connect different units of the molecule, adjust the length of the molecule and the hydrophilic-hydrophobic balance, thereby improving the properties of the molecule.

[0025] In a second aspect, the present invention provides a method for preparing the bispecific polypeptide nanodrug targeting PD-L1 according to the first aspect, the preparation method comprising:

[0026] Using amino acids with protected terminal amino groups and side chain amino groups and tumor microenvironment targeting regulatory molecules as raw materials, the PD-L1 blocking unit, self-assembly unit and linker unit are synthesized and connected by solid phase synthesis (SPPS), and then connected to the tumor microenvironment targeting regulatory molecules to obtain the bispecific polypeptide nanodrug targeting PD-L1.

[0027] For example, in the present invention, the steps for synthesizing the molecule of the structure shown in Formula I are as follows:

[0028] (1) Swelling the support resin; using amino acids with terminal amino groups protected by Fmoc and side chain amino groups protected by Boc as raw materials, first, the C-terminus of the first amino acid (arginine) is fixed on the resin, and the N-terminus is protected by Fmoc;

[0029] (2) removing the N-terminal protection of the first amino acid in step (1), and then connecting the next amino acid to react; finally, all amino acids are connected to form a polypeptide fixed on the resin;

[0030] (3) The polypeptide described in step (2) is coupled with the small molecule AMD070 through an amide condensation reaction, and the molecule having the structure shown in formula I is obtained by cleavage and purification.

[0031] Preferably, the resin in step (1) is Wang resin with a modified density of 0.35 mM.

[0032] Preferably, the reagent for removing the N-terminal protection in step (2) is 20% by volume of hexahydropyridine in dimethylformamide (DMF).

[0033] Preferably, the deprotection detection reagent in step (2) is ninhydrin.

[0034] Preferably, the method used in step (2) to prepare the polypeptide by connecting amino acids is as follows: the amino acids to be connected are mixed with benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), dissolved with N-methylmorpholine (NMM) and DMF, added to the deprotected resin for reaction, and then the amino acids are connected in sequence to obtain the polypeptide.

[0035] Preferably, in step (3), the reaction conditions of the polypeptide and AMD070 are hydrazine hydrate:DMF (v / v=2 / 98) to remove the ODmab protecting group on the aspartic acid side chain of the polypeptide backbone, and the exposed carboxyl group of the deprotected polypeptide is connected to the amino group on the AMD070 molecule through an amide condensation reaction.

[0036] Preferably, the molar ratio of the polypeptide to AMD070 in step (3) is 1:5.

[0037] The synthesis steps of Formula II and Formula III in the present invention are carried out with reference to the synthesis method of Formula I.

[0038] In a third aspect, the present invention provides a use of the bispecific polypeptide nanodrug targeting PD-L1 according to the first aspect in the preparation of tumor immunotherapy drugs.

[0039] In a fourth aspect, the present invention provides a use of the bispecific polypeptide nanodrug targeting PD-L1 according to the first aspect in the preparation of an anti-tumor drug.

[0040] Preferably, the tumor comprises bladder cancer, pancreatic cancer or gastric cancer.

[0041] In a fifth aspect, the present invention provides a method for tumor immunotherapy, comprising administering to a patient a therapeutically effective dose of the bispecific polypeptide nanodrug targeting PD-L1 as described in the first aspect.

[0042] Preferably, the bispecific polypeptide nanomedicine is administered intravenously.

[0043] Preferably, the administration concentration is less than 800 μM, preferably 200-500 μM.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The bispecific peptide nanodrug targeting PD-L1 described in this invention precisely targets cancer cells, aligning with the current concept of "precision medicine." It contains a tumor microenvironment targeting and regulation unit and a PD-L1 blocking unit, each with target recognition, tumor microenvironment regulation, and immune checkpoint blockade capabilities. Through molecular specificity, it actively targets tumors. On the one hand, it specifically binds to the CXCR4 protein overexpressed on the surface of tumor cells, blocking the CXCR4 / SDF-1 signaling pathway, improving the tumor microenvironment and increasing T cell infiltration. On the other hand, it specifically binds to the PD-L1 protein overexpressed on the surface of tumor cells, blocking the interaction between PD-1 and PD-L1, restoring T cell killing of tumor cells, and enhancing bladder cancer immunotherapy. After binding to the protein on the tumor cell surface, the assembly-competent fragments are restricted due to the movement of the peptide molecules, reducing the activation entropy required for self-assembly, thereby triggering in situ self-assembly to form nanofibers, achieving long-term blockade of the receptor signaling pathway. This bispecific peptide nanodrug provides a new approach for tumor immunotherapy while also producing no significant side effects in vivo and exhibiting excellent biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the design principle of bispecific peptide nanomedicine.

[0047] Figure 2 This is the mass spectrometry characterization of the bispecific polypeptide nanomedicine in Example 1.

[0048] Figure 3 This is the enrichment fluorescence signal diagram of peptide 1 (Cy) on the surface of MB49 cells.

[0049] Figure 4 This is the result of the surface morphology of peptide 1 on MB49 cells.

[0050] Figure 5 This is a graph showing the results of peptide 1 inhibiting the invasion of bladder cancer cells.

[0051] Figure 6 This is the tumor-targeted enrichment fluorescence signal diagram of peptide 1 (Cy) in a mouse subcutaneous tumor model.

[0052] Figure 7 The graph shows the results of peptide 1 inhibiting tumor growth in a mouse subcutaneous tumor model.

[0053] Figure 8 This is a survival curve of mice treated with polypeptide 1 in a mouse subcutaneous tumor model. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0055] The experimental instruments, materials, and experimental solutions involved in the following examples are as follows:

[0056] Experimental instruments and materials:

[0057] Dimethylformamide (DMF), piperidine, Wang resin, dichloromethane (DCM), ninhydrin reagents (ninhydrin, vitamin C and phenol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), hexahydropyridine, triisopropylsilane (TIS), ethanedithiol (EDT), anhydrous ether, trifluoroacetic acid (TFA), N-methylmorpholine (NMM), N-fluorenylmethoxycarbonyl-6-aminohexanoic acid (Fmoc-e-Acp-OH), methanol, Fmoc-alanine (Fmoc-Ala-OH), Fmoc-cysteine (Fmoc-Cys(Trt)-OH), Fmoc-aspartic acid (Fmoc-Asp(OtBu)-OH), Fmoc-phenylalanine ( Fmoc-Phe-OH), Fmoc-glycine (Fmoc-Gly-OH), Fmoc-lysine (Fmoc-Lys(Boc)-OH), Fmoc-leucine (Fmoc-Leu-OH), Fmoc-aspartic acid (Fmoc-Asn(Trt)-OH), Fmoc-proline (Fmoc-Pro-OH), Fmoc-arginine (Fmoc-Arg(Pbf)-OH), Fmoc-threonine (Fmoc-Thr(tBu)-OH), Fmoc-valine (Fmoc-Val-OH), Fmoc-tyrosine (Fmoc-Tyr(Trt)-OH), AMD070, cyanine dye (Cy), peptide solid phase synthesis tube, etc.

[0058] Preparation of experimental solution:

[0059] Deprotection solution: Mix hexahydropyridine and DMF in a volume ratio of 1:4;

[0060] Reaction solution: Mix NMM and DMF in a volume ratio of 1:24;

[0061] Lysis solution: Combine TFA, TIS, and EDT to a solution volume fraction of 92.5% TFA, 2.5% TIS, and 2.5% EDT.

[0062] Ninhydrin test solution – one drop each of ninhydrin, vitamin C, and phenol;

[0063] Example 1

[0064] This example provides a bispecific peptide nanodrug (AMD070) DPGLGYLKLVFFGCVRARTR, whose structural formula can be represented by Formula I and is prepared by peptide solid-phase synthesis (hereinafter referred to as peptide 1).

[0065]

[0066] The steps for synthesizing peptides using solid phase synthesis are as follows:

[0067] (1) Fmoc (fluorenylmethoxycarbonyl) deprotection: Weigh 0.1g of Wang resin and place it in a peptide solid phase synthesis tube. Add DMF and swell for 30 minutes. Draw off the DMF and perform Fmoc deprotection reaction with deprotection solution. Place on a shaker for 10 minutes. Draw off the deprotection solution and wash with DMF and DCM three times. Take 10mg of Wang resin from the peptide solid phase synthesis tube and place it in a test tube. Wash it twice with ethanol. If the ninhydrin method shows a dark blue color, it is a positive result. Then prepare to connect the first amino acid (R) and enter the amino acid condensation reaction.

[0068] (2) Amino acid condensation: Take 10 equivalents of amino acids and HBTU according to the above amino acid sequence, dissolve them in 7 mL of reaction solution, and add them to the peptide solid phase synthesis tube. Stir and react. After 1 hour, take 10 mg of Wang resin from the peptide solid phase synthesis tube and place it in a test tube. Wash it twice with ethanol. If the ninhydrin method does not change color, it is a negative result, which proves that the condensation reaction is successful. Drain the liquid in the peptide solid phase synthesis tube and wash it twice with DMF and DCM to obtain the peptide resin after the first amino acid condensation.

[0069] (3) Repeat the above "Fmoc deprotection-amino acid condensation" reaction steps for the obtained peptide resin until the reaction of the last amino acid (aspartic acid) is completed, add 2% hydrazine hydrate and shake on a shaker for 15 minutes to remove the ODmab protecting group, then take 5 times the equivalent of AMD070 and HBTU, dissolve them in 7mL of reaction solution, put them into the peptide solid phase synthesis tube, shake on a shaker for 45 minutes, connect AMD070 through amide condensation, and obtain a peptide with the target sequence. After the reaction is completed, wash the resin with DMF and DCM 3 times each, wash it with methanol 2 times, and continue to drain for 20 minutes. Take out the synthesized peptide resin from the peptide solid phase synthesis tube, cleave it in the cleavage solution at room temperature for 2 hours, and the cleavage solution is first ice-bathed for 20 minutes. After filtering the resin, evaporate it to dryness in a rotary evaporator and wash it with anhydrous ether 3 times under ice-bath conditions. The crude peptide is purified by preparative reverse phase HPLC, and the purity is >94.7% detected by HPLC. The obtained pure peptide is identified by mass spectrometry (MS, electrospray), as shown Figure 2As shown, the measured molecular weight is consistent with the target molecular weight. This indicates that the target molecule has been synthesized, indicating that the peptide nanoparticle drug with the above structure has been successfully synthesized. After lyophilization, store at -20°C until use.

[0070] Example 2

[0071] This embodiment provides a bispecific polypeptide nanodrug (AMD070) DPGLGYLGNNQQNYCVRARTR, whose structural formula can be represented by Formula II and is prepared by polypeptide solid-phase synthesis.

[0072]

[0073] The preparation method is as described in Example 1.

[0074] Example 3

[0075] This embodiment provides a bispecific polypeptide nanodrug (AMD070) D-(OEG)4-KLVFFGCVRARTR, whose structural formula can be represented by Formula III and is prepared by polypeptide solid-phase synthesis.

[0076]

[0077] The preparation method is as described in Example 1.

[0078] Example 4

[0079] Cell-level specific identification experiment:

[0080] First, a Cy-modified polypeptide nanodrug (hereinafter referred to as polypeptide 1 (Cy)) was prepared. The specific operation was as follows: polypeptide 1 and fluorescent molecule Cy were dissolved in Tris HCl solution and stirred at room temperature overnight, and then polypeptide 1 (Cy) was purified by extraction, dialysis and other operations.

[0081] The cells selected for the experiment were mouse bladder cancer cells MB49, which highly expressed both CXCR4 and PD-L1.

[0082] The cells were seeded into confocal culture dishes and incubated in an incubator for 24 h. Then, the Cy-modified peptide nanomedicine was co-incubated with the MB49 cells for 30 min. The cells were observed using a multi-beam laser confocal imaging system (U-Vox). Figure 3 As shown (the left picture is the fluorescence field picture, and the right picture is the superposition picture of the fluorescence field and the bright field), it can be seen from the figure that obvious fluorescence was observed on the surface of MB49 cells, indicating that the polypeptide nanomedicine can specifically bind to the cell membrane of MB49 cells, thereby preliminarily verifying its targeting.

[0083] Example 5

[0084] Cell surface morphology observation experiment:

[0085] The cells selected for the experiment were mouse bladder cancer cells MB49, which highly expressed both CXCR4 and PD-L1.

[0086] MB49 cells were seeded into a 24-well plate with a silicon wafer and incubated in an incubator for 24 h. The polypeptide nanoparticles prepared in Example 1 were then co-incubated with the cells for 6 h. The cells were fixed with 4% paraformaldehyde for 30 min, washed twice with PBS, and then dehydrated with 10%, 30%, 50%, 70%, 90%, and 100% ethanol solutions for 10 min, respectively. The cells were dried, sprayed with gold, and observed using a traceable metrology scanning electron microscope (JC-Zeiss). The results are shown in FIG. Figure 4 As shown, it was found that polypeptide nanomedicines formed short rod-shaped fiber structures on the cell membrane surface.

[0087] Example 6

[0088] Invasiveness inhibition test:

[0089] The cells selected for the experiment were mouse bladder cancer cells MB49, which highly expressed both CXCR4 and PD-L1.

[0090] 600 μL of culture medium containing 20% serum was added to the lower chamber of the Transwell invasion and migration culture dish, and 200 μL of cell suspension containing peptide 1, AMD070, and PBS was added to the upper chamber. The dish was cultured in an incubator for 24 hours, and then the lower surface was immersed in 4% paraformaldehyde solution for 30 minutes. The cells were stained with crystal violet and observed under a microscope. Figure 5 As shown in the figure, the number of cells penetrated by the MB49 cell group treated with peptide nanomedicine was significantly lower than that of the PBS group and AMD070 group, indicating that the peptide nanomedicine has good invasive inhibition ability.

[0091] Example 7

[0092] Specific identification and long-term retention experiments at the animal level:

[0093] The animals selected for the experiment were C57BL / 6 female mice, 6 weeks old.

[0094] Construction of mouse subcutaneous tumor model: bladder cancer cells were used to establish subcutaneous transplanted tumors in mice. 5×10 6 MB49 cells were injected subcutaneously into the right leg of mice. Two weeks later, tumors were formed, and a mouse subcutaneous tumor model was obtained.

[0095] The polypeptide 1 (Cy) prepared in Example 4 was injected into the mouse via the tail vein and imaged using a small animal in vivo imaging device (IVIS Spectrum). The imaging results are shown in FIG. Figure 6As shown, peptide 1 (Cy) has obvious signal accumulation in tumor tissue and can be retained for up to 120 hours.

[0096] Example 8

[0097] Subcutaneous tumor implantation inhibition experiment at the animal level:

[0098] The animals selected for the experiment were C57BL / 6 female mice, 6 weeks old.

[0099] Construction of mouse subcutaneous tumor model: bladder cancer cells were used to establish subcutaneous transplanted tumors in mice. 5×10 6 MB49 cells were injected subcutaneously into the right leg of mice. Two weeks later, tumors were formed, and a mouse subcutaneous tumor model was obtained.

[0100] The polypeptide nanomedicine prepared in Example 1 was injected into the body through the tail vein of mice, with 6 mice per group. The growth status of the subcutaneous tumor of the mice and the survival status of the mice were then statistically recorded. The results are as follows: Figure 7 and Figure 8 As shown, Figure 7 The results showed that the growth of tumors pretreated with peptide nanomedicine was significantly inhibited. Figure 8 The results showed that the peptide nanomedicine could significantly prolong the survival of mice.

[0101] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate a bispecific peptide nanodrug targeting PD-L1, its preparation method, and application, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacement of various raw materials in the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

[0102] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0103] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A bispecific peptide nanodrug targeting PD-L1, characterized in that: The bispecific polypeptide nanomedicine targeting PD-L1 comprises a tumor microenvironment targeting regulation unit, a connection unit, a self-assembly unit and a PD-L1 blocking unit connected in sequence by amide bonds; The tumor microenvironment targeting regulatory unit is derived from AMD070; The PD-L1 blocking unit is derived from the polypeptide sequence CVRARTR; The self-assembly unit is derived from the polypeptide sequence KLVFFG; The linking unit is derived from the polypeptide sequence DPGLGYL; The bispecific polypeptide nanodrug targeting PD-L1 has a structure as shown in Formula I; Formula I.

2. The method for preparing a bispecific polypeptide nanodrug targeting PD-L1 according to claim 1, characterized in that: The preparation method comprises: Using amino acids with protected terminal amino groups and side chain amino groups and tumor microenvironment targeting regulatory molecules as raw materials, the PD-L1 blocking unit, self-assembly unit and connection unit are synthesized and connected by solid-phase synthesis, and then connected to the tumor microenvironment targeting regulatory molecules to obtain the bispecific polypeptide nanodrug targeting PD-L1.

3. Use of the bispecific polypeptide nanodrug targeting PD-L1 according to claim 1 in the preparation of tumor immunotherapy drugs.

4. Use of the bispecific polypeptide nanodrug targeting PD-L1 according to claim 1 in the preparation of anti-tumor drugs.

5. The use according to claim 3 or 4, characterized in that The tumor includes bladder cancer, pancreatic cancer or gastric cancer.

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