A multi-effect synergistic dual-targeting peptide-based molecule and its application in the preparation of tumor therapeutic drugs

Abstract

The present invention provides a dual-targeted polypeptide-based molecule with multi-effect synergistic promotion and its use in the preparation of tumor therapeutic drugs, wherein the dual-targeted polypeptide-based molecule includes a nanofiber that can be self-assembled to form a β-sheet secondary structure and a tumor neovascularization targeting sequence and a PD-L1 targeting sequence connected to the nanofiber with a β-sheet secondary structure. The polypeptide-based molecule exerts the function of immune checkpoint inhibition while inducing vascular normalization through the long-term drug delivery window brought about by the self-assembly effect. Tumor vascular normalization can promote the infiltration of T cells, realize the transformation of the tumor immune microenvironment from "cold" to "hot", synergistically promote the immune checkpoint inhibition effect, and ultimately achieve the effect of inhibiting tumor proliferation and metastasis.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a multi-effect synergistically promoted dual-targeting polypeptide-based molecule and its application in the preparation of tumor therapeutic drugs. Background Art

[0002] Kidney cancer is one of the three major urinary tract cancers. According to statistics, in 2020, the death toll from kidney cancer worldwide exceeded 180,000. Kidney cancer is characterized by high vascular density and high vascular permeability, which contributes to its high metastatic potential. Over 30% of patients with kidney cancer have metastases at the time of initial diagnosis, and the five-year survival rate for metastatic kidney cancer is only 12%, with a median survival of only 6-12 months. Related research indicates that tumor growth and metastasis are primarily dependent on angiogenesis, leading to the development of a series of treatments targeting tumor angiogenesis. However, with the development of antivascular drugs, clinical trials have shown that the long-term benefits of antivascular therapy alone are limited, while combined antivascular therapy and chemotherapy have demonstrated significant efficacy. This combination of advantages contradicts common sense: antivascular therapy, while effectively disrupting the drug delivery route, exhibits a stronger tumor-killing effect. Professor Folkman's in-depth research has uncovered the underlying mechanism and proposed the "vascular normalization theory."

[0003] The theory of "vascular normalization" refers to restoring the balance between pro-angiogenic and anti-angiogenic factors, repairing structural and functional abnormalities in tumor vasculature before vascular regression, and normalizing tumor vasculature. Subsequent studies have shown that low-dose anti-angiogenic therapy can normalize immature, leaky tumor vessels. Unlike inhibiting angiogenesis, vascular normalization promotes the formation of functional vascular networks in tumors, reversing hypoxia and increasing blood perfusion, while also enhancing immune cell infiltration within the tumor and achieving tumor immune killing. Interferon produced by T cells in tumor tissue increases the expression of endothelial adhesion molecules, further promoting immune cell infiltration and, in turn, T cell-mediated immune killing. In various preclinical tumor models, anti-angiogenic therapy has enhanced the effectiveness of immunotherapy. However, the efficacy of this combination therapy requires that immune checkpoint inhibitors act within the window of vascular normalization. Therefore, due to the differences in the pharmacokinetic properties of the two different drugs, the time window for true synergistic effect is extremely uncontrollable despite the combination of drugs. At the same time, the existing clinical combination of anti-vascular and immune checkpoint inhibitory drugs is mainly based on antibody drugs, and the off-target toxicity of antibody drugs and the inherent penetrability problems of antibodies are inevitable.

[0004] Therefore, designing and constructing a new multifunctional drug platform based on the synergistic combination strategy of anti-vascular targeting and immune checkpoint inhibition to overcome the bottleneck problem of clinical combined drug administration and effectively inhibit the progression and metastasis of renal cancer has become a practical problem that needs to be urgently solved in clinical practice.

[0005] Due to their good biocompatibility and diverse bioactivities, peptides have become ideal building blocks for nanobiomedical materials. With the development of supramolecular chemistry, the self-assembly behavior of peptides and the structure-activity relationship have gradually become clear. In situ constructed self-assembling peptide fiber networks can be used for high signal-to-noise ratio imaging of tumors and long-term inhibition of tumor invasion and metastasis. Based on these findings, the introduction of self-assembling peptide fragments and the construction of peptide-based molecules through the design strategy of "single molecule, dual targeting, dual function, and synergistic promotion" are expected to break through the existing bottleneck of tumor treatment. However, the ideal peptide molecule that can target tumor blood vessels and immune checkpoints to achieve dual-effect synergy has not yet been found. Therefore, providing a dual-targeted peptide-based molecule that promotes multi-effect synergy has important application value in tumor treatment. Summary of the Invention

[0006] In response to the shortcomings of the prior art, the present invention aims to provide a dual-targeted peptide-based molecule with synergistic effects and its use in the preparation of tumor therapeutics. The peptide-based molecule, through its self-assembly effect, provides a long-lasting drug delivery window, inducing vascular normalization while also exerting immune checkpoint inhibition. Normalization of tumor vasculature promotes T cell infiltration, shifting the tumor immune microenvironment from a "cold" to a "hot" state, synergistically promoting immune checkpoint inhibition, and ultimately inhibiting tumor proliferation and metastasis.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a dual-targeting polypeptide-based molecule with multi-effect synergistic promotion, wherein the dual-targeting polypeptide-based molecule includes a tumor neovascularization targeting sequence and a PD-L1 targeting sequence that can self-assemble to form nanofibers with a β-sheet secondary structure and connected to the nanofibers with a β-sheet secondary structure.

[0009] Preferably, the amino acid sequence that can self-assemble to form nanofibers with a β-sheet secondary structure is GNNQQNYGGK.

[0010] Preferably, the tumor angiogenesis targeting sequence is an amino acid sequence that can target and recognize NRP-1 that is highly expressed in tumor angiogenesis endothelial cells, and the amino acid sequence of the tumor angiogenesis targeting sequence is RPPLWTA.

[0011] Preferably, the PD-L1 targeting sequence is an amino acid sequence that targets and recognizes PD-L1 that is highly expressed on the surface of a tumor, and the amino acid sequence of the PD-L1 targeting sequence is NYSKPTDRQYHF.

[0012] Preferably, the tumor angiogenesis targeting sequence is connected to the assembly sequence through (PEG)4 as a linker; and the PD-L1 targeting sequence is connected to the assembly sequence through octanoic acid as a linker.

[0013] In a second aspect, the present invention provides the use of the multi-effect synergistically promoted dual-targeting polypeptide-based molecule described in the first aspect in the preparation of anti-tumor drugs.

[0014] Preferably, the tumor is a tumor with high vascular density or high vascular permeability.

[0015] Preferably, the tumor comprises bladder cancer, kidney cancer, glioblastoma or liver cancer.

[0016] The polypeptide-based molecules described in this invention exert their anti-cancer effects by promoting normalization of tumor blood vessels and enhancing immune killing. Furthermore, they act on tumor vascular endothelial cells by targeting NRP-1, promoting normalization of blood vessels and T cell infiltration, shifting the tumor immune microenvironment from "cold" to "hot," synergistically promoting immune checkpoint inhibition, and ultimately inhibiting tumor proliferation and metastasis.

[0017] In a third aspect, the present invention provides the use of the multi-effect synergistically promoted dual-targeting polypeptide-based molecule described in the first aspect in the preparation of a drug that inhibits tumor angiogenesis and blocks immune checkpoints to enhance immune killing.

[0018] In a fourth aspect, the present invention provides the use of the multi-effect synergistic dual-targeting polypeptide-based molecule described in the first aspect in the preparation of drugs for inhibiting tumor cell proliferation and inhibiting tumor cell metastasis.

[0019] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the multi-effect synergistically promoted dual-targeting polypeptide-based molecule described in the first aspect.

[0020] Preferably, the composition further comprises a pharmaceutically acceptable excipient.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention discloses a dual-targeted peptide-based molecule (designated SA-AI) with synergistic effects. It is composed of an amino acid sequence that self-assembles into nanofibers with a β-sheet secondary structure, an amino acid sequence that targets NRP-1, which is highly expressed on tumor neovascular endothelial cells, and an amino acid sequence that targets PD-L1, which is highly expressed on tumor surfaces. One end of the SA-AI molecule targets the NRP-1 target, which is highly expressed on tumor vascular endothelial cells. Ligand-receptor binding triggers assembly onto the target surface to form a dense fibrous structure, thereby blocking the NRP-1 target and promoting normalization of vascular structure and function, thereby increasing T cell infiltration. Simultaneously, the other end of the dual-targeting sequence targets the PD-L1 immune checkpoint, which is highly expressed on the surface of tumor cells, thereby blocking the immune checkpoint and enhancing immune killing. This dual-targeted peptide-based molecule combines enhanced immune recruitment with immune killing, achieving synergistic effects that maximize the inhibition of tumor proliferation and metastasis.

[0023] In addition, the present invention focuses on practical issues of clinical translational applications and uses polypeptides with high biosafety as materials for design and construction. Therefore, SA-AI has strong biosafety and great potential for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A This is a schematic diagram of the molecular structure of SA-AI.

[0025] Figure 1B This is a diagram of the molecular structure of AI.

[0026] Figure 2 Transmission electron microscopy images of SA-AI that can undergo conformational change and self-assemble into hydrophobic nanofibers in aqueous solutions of PD-L1 and NRP1 proteins, where a: transmission electron microscopy images of SA-AI at 100 μM and 400 μM solution concentrations; b: transmission electron microscopy images of SA-AI in solutions with PD-L1 protein alone, NRP-1 protein alone, and PD-L1 and NRP-1 protein added, respectively; scale bar: 200 nm.

[0027] Figure 3 The experimental molecule SA-AI and the control molecule AI and blank group PBS were used to incubate the 786-O cell line (PD-L1 + ) and HUVEC cell lines (NRP-1 + ) of the scanning electron microscope image.

[0028] Figure 4 These are the circular dichroism spectra of SA-AI after incubation for 1 hour with PD-L1 protein alone, NRP-1 protein alone, and PD-L1 and NRP-1 protein solutions.

[0029] Figure 5The target protein expression results of HUVEC cells, 786-O cells and HK-2 cells were verified.

[0030] Figure 6 : The KD value was measured by incubating SA-AI with NRP-1 protein solution, the KD value was measured by incubating SA-AI with PD-L1 protein solution, the KD value was measured by incubating AI with NRP-1 protein solution, and the KD value was measured by incubating AI with PD-L1 protein solution.

[0031] Figure 7 These are the results of verifying the long-term retention ability of materials at the cellular level, and are confocal images of the co-localization of SA-AI and AI with cells.

[0032] Figure 8A Microscopic images of scratch assays of HUVECs and 293T cells treated with SA-AI, AI, and PBS. The white areas in the images indicate cells that did not migrate.

[0033] Figure 8B The above figure is the statistical graph of the wound healing rate of the scratch experiment. The upper figure is the statistical graph of HUVECs cells, and the lower figure is the statistical graph of 293T cells.

[0034] Figure 9A Microscope images of HUVECs and 293T cells treated with SA-AI, AI, and PBS in a Transwell migration assay. The purple area indicates the migrated cells.

[0035] Figure 9B This is a statistical graph of the migration rate in the Transwell migration experiment.

[0036] Figure 10A These are confocal images of HUVECs cells after being treated with SA-AI, AI, and PBS, respectively.

[0037] Figure 10B These are the corresponding fluorescence statistical results after HUVECs cells reacted with SA-AI, AI, and PBS respectively.

[0038] Figure 11A Fluorescence images of SA-AI, SA-A (targeting tumor blood vessel NRP-1), SA-I (targeting tumor cell PD-L1), and AI injected into mice at different time periods.

[0039] Figure 11B The fluorescence statistics of SA-AI, SA-A (targeting tumor blood vessel NRP-1), SA-I (targeting tumor cell PD-L1), and AI injected into mice at different time periods.

[0040] Figure 12 This is the result of verifying the therapeutic ability of the material in a living subcutaneous tumor model. The figure shows the statistical curves of tumor growth after treatment in five groups: SA-AI, AI, SA-A mixed with SA-I, Bevacizumab and Atelizumab monoclonal antibody combination, and PBS group. DETAILED DESCRIPTION

[0041] 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.

[0042] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0043] Example 1 Preparation and molecular structure of multi-effect synergistic dual-targeting polypeptide-based molecules

[0044] 1. Preparation of the multi-effect synergistic dual-target peptide-based molecule SA-AI[GNNQQNYGGK (Peg4-RPPLWTA)-(Aoc)-NYSKPTDRQYHF]

[0045] The multi-effect synergistically promoted dual-targeting peptide-based molecule targets tumor vascular NRP-1 and tumor cell PD-L1 and forms water-insoluble nanofibers, which are composed of the following three parts:

[0046] 1) An amino acid sequence that can self-assemble into β-sheet secondary structure nanofibers, wherein the amino acid sequence is (SEQ ID NO: 1) GNNQQNYGGK: Due to hydrogen bond interactions, the nanofibers can self-assemble into β-sheet secondary structure water-insoluble nanofibers;

[0047] 2) Targeted recognition of the amino acid sequence of tumor vascular NRP-1, whose amino acid sequence is (SEQ ID NO: 2) RPPLWTA: A sequence that can target and recognize NRP-1, which acts as a target head in SA-AI and specifically binds to the target NRP-1.

[0048] 3) Targeted recognition of the amino acid sequence of PD-L1 in tumor cells, the amino acid sequence of which is (SEQ ID NO: 3) NYSKPTDRQYHF: a sequence that can target and recognize PD-L1, which acts as a target head in SA-AI and specifically binds to the target PD-L1.

[0049] 2. Preparation of the control peptide AI [GGGQGGYGGK(Peg4-RPPLWTA)-(Aoc)-NYSKPTDRQYHF]

[0050] SA-AI and AI were prepared by solid phase synthesis. Figure 1A and Figure 1B As shown, Figure 1A The molecular structure diagram of SA-AI; Figure 1B This is a diagram of the molecular structure of AI.

[0051] The specific steps of the solid-phase synthesis method are as follows: the resin loaded with the first amino acid is swollen in anhydrous N,N-dimethylformamide (DMF) in a solid-phase reactor for 4 hours. After swelling, the Fmoc protecting group at the amino acid's N-terminus is removed using a deprotection agent (hexahydropyridine:anhydrous DMF = 1:4, v / v) for 15 minutes. The deprotection process is verified using a ninhydrin reagent (ninhydrin:phenol:ascorbic acid = 1:1:1, v / v / v) colorimetric assay. The amino acids are sequentially dissolved in a coupling agent (N-methylmorpholine:anhydrous DMF = 5:95, v / v) along with HBTU (each in a 10-fold excess relative to the amino acid loading on the resin). This solution is then mixed with the resin in a solid-phase reactor and coupled on a shaker for 60 minutes. After verifying the coupling process using the ninhydrin colorimetric method, the Fmoc protecting group on the amino acid is removed again using a deprotecting agent, completing the synthesis of a single amino acid. Repeat these steps until the last amino acid in the amino acid sequence is synthesized. The completed resin is mixed with the cleavage solution and stirred in an ice-water bath for 3 hours. This is to cleave the synthesized peptide from the resin. After the amino acid is separated from the resin, the mixture is purged with dry nitrogen to remove trifluoroacetic acid. The resulting product is then reprecipitated with ice-cold anhydrous ether. The precipitated product is collected by centrifugation and vacuum-dried overnight to obtain a white powder, which is the synthesized peptide.

[0052] Example 2: SA-AI polypeptide undergoes conformational change after binding to NRP-1 protein or PD-L1 protein, and self-assembles into water-insoluble nanofibers

[0053] SA-AI aqueous solutions were prepared at 100 μM and 400 μM, and the morphology was observed under a biological electron microscope to analyze the effect of different concentrations on SA-AI assembly. NRP-1 or PD-L1 protein was then added to the SA-AI solution (100 μM) and incubated for 1 hour. The samples were observed under a biological electron microscope after 1 hour.

[0054] Transmission electron microscopy images of SA-AI that can transform conformations and self-assemble into hydrophobic nanofibers in aqueous solutions of PD-L1 and NRP1 proteins are shown in Figure 2. Figure 2 Figure 1: (a) Pure SA-AI peptide solutions at different concentrations. A 400 μM solution exhibited allosteric behavior and self-assembled into fibers. A 100 μM solution, however, did not undergo allosteric behavior and exhibited a nanoparticle morphology. (b) SA-AI solutions (100 μM) exhibited allosteric behavior and self-assembled into hydrophobic nanofibers when incubated with either NRP-1 or PD-L1, either individually or simultaneously. Simultaneous incubation with both proteins resulted in a denser fiber network.

[0055] Example 3 SA-AI undergoes conformational changes after binding to the target protein in the target-positive expression cell line and self-assembles to form nanofiber-like structures on the cell surface

[0056] 786O (PD-L1 + ), HUVEC (NRP-1 + ) cells were plated on six-well plates mounted on silicon wafers (200,000 cells / well) and cultured overnight in an incubator. 786O and HUVECs were incubated with peptides SA-AI and AI (20 μM), respectively, for 1 hour. The cells were then fixed with 4% paraformaldehyde for 30 minutes, dehydrated with a gradient of ethanol (10%-30%-50%-70%-90%-100%, 10 minutes each step), dried, and gold-sprayed for 100 seconds. Images were taken using a scanning electron microscope.

[0057] The experimental molecule SA-AI, the control molecule AI and the blank group PBS were detected in 786-O cell line (PD-L1 + ) and HUVEC cell lines (NRP-1 + ) as shown in the scanning electron microscope image of Figure 3 As shown, it can be found that the polypeptide SA-AI with assembly fragments can form a dense nanofiber network on the surface of both cell lines, while the polypeptide AI group without assembly did not form a dense fiber network on the surface of both cell lines, which is similar to the result of the control group PBS group.

[0058] Example 4: After the polypeptide SA-AI binds to the target protein NRP-1 or PD-L1, the secondary structure changes to form a β-sheet secondary structure.

[0059] After the peptide SA-AI was incubated with the target protein NRP-1 or PD-L1 for 1 h, the changes in the secondary structure in the circular dichroism spectrum were observed after 0 h and 1 h, respectively.

[0060] The circular dichroism spectra of SA-AI after incubation for 1 hour with PD-L1 protein alone, NRP-1 protein alone, and PD-L1 and NRP-1 protein solutions are shown in the following figure. Figure 4 As shown in the figure, after the peptide SA-AI was incubated with the target protein NRP-1 or PD-L1 for 1 h, a typical β-sheet characteristic signal was formed in the circular dichroism spectrum, and the β-sheet characteristic signal was stronger when incubated with the two proteins at the same time.

[0061] Example 5 Western-blot experiment verifies the expression level of target protein of peptide SA-AI targeting cell lines HUVECs and 786O

[0062] 786O (PD-L1 + ), HUVECs (NRP-1 + )、HK-2(PD-L1 - 、NRP-1 - ) cells, and the expression of target proteins in the three cell lines was verified by WB experimental method.

[0063] The target protein expression verification results of HUVEC cells, 786-O cells, and HK-2 cells are as follows Figure 5 As shown in the figure, higher levels of NRP-1 expression were observed in the HUVEC cell line, higher levels of PD-L1 expression were observed in the 786O cell line, while the expression levels of both targets were lower in the HK-2 cell line.

[0064] Example 6 Surface Plasmon Resonance Imaging (SPRI) Verification of the Affinity of Peptides SA-AI and AI to Target Proteins NRP-1 or PD-L1

[0065] Peptide SA-AI and AI solutions with different concentration gradients were prepared, and their affinity to the target protein NRP-1 or PD-L1 was verified by SPRI.

[0066] The results of the binding ability verification of SA-AI and AI with PD-L1 protein and NRP-1 protein are as follows: Figure 6 As shown, the KD values ​​were measured by incubating SA-AI with NRP-1 protein solution, SA-AI with PD-L1 protein solution, AI with NRP-1 protein solution, and AI with PD-L1 protein solution. Compared with peptide AI, peptide SA-AI exhibited higher affinity for both target proteins NRP-1 and PD-L1.

[0067] Example 7 Verification of the targeting and retention abilities of peptides SA-AI and AI on HUVECs cells

[0068] HUVECs cells were pre-plated in confocal culture dishes. After they adhered overnight, fluorescently labeled SA-AI and AI were added, respectively. The cells were cultured in an incubator, and cell fluorescence images were captured using a confocal microscope at 1 h, 4 h, 8 h, and 12 h.

[0069] The results of the long-term retention capability verification of the cell-level materials are as follows: Figure 7 As shown in the figure, SA-AI still showed retention ability at 12 h, while AI molecules had no obvious fluorescence signal after 4 h.

[0070] Example 8 Cell scratch assay verifies the targeted killing ability of peptides SA-AI and AI on HUVECs and 293T cells

[0071] To verify the killing ability of peptides SA-AI and AI on NRP-1 target positive expression cells HUVECs, HUVECs (NRP-1 + ) and 293T (NRP-1 - ) were seeded in 6-well plates (500,000 cells / well). Once confluent, the culture dish surface was streaked with a pipette tip to simulate a wound model. SA-AI or AI materials were then added and incubated for 24 hours. Following incubation, the cells were fixed with 4% paraformaldehyde for 10 minutes and stained with crystal violet for 2 minutes. Finally, microphotographs were taken to observe the cell healing process in the wound model.

[0072] The results of the anti-vascular function verification of the cell-level material are as follows Figure 8A and Figure 8B As shown, Figure 8A Microscopic images of scratch assays of HUVECs and 293T cells treated with SA-AI, AI, and PBS. The white areas in the images represent cells that did not migrate. Figure 8B The wound healing rate of the scratch test is statistically shown in the upper figure for HUVECs cells and the lower figure for 293T cells. + ) can significantly inhibit cell migration, while AI molecules and PBS have no inhibitory effect. - ) have no inhibitory effect.

[0073] Example 9 Inhibitory Effects of Peptides SA-AI and AI on Migration of NRP-1 Target-Positively Expressing HUVECs

[0074] HUVECs and 293T cells in the logarithmic growth phase were taken and 1×10 5Cells were seeded at a density of 100 cells in the upper chamber of a Transwell containing SA-AI and AI serum-free culture medium, and complete culture medium was added to the lower chamber. After culturing in a cell culture incubator at 37°C for 48 h, the migrated cells in the lower layer were stained and counted.

[0075] The results of the anti-vascular function verification of the cell-level material are as follows Figure 9A and Figure 9B As shown, Figure 9A Microscope images of HUVECs and 293T cells treated with SA-AI, AI, and PBS in the Transwell migration assay, respectively. The purple area indicates the migrated cells. Figure 9B The figure shows the migration rate of HUVECs cells (NRP-1 + ) can significantly inhibit cell migration, while AI molecules and PBS have no inhibitory effect. - ) have no inhibitory effect.

[0076] Example 10 Inhibitory Effects of Peptides SA-AI and AI on Tube Formation Ability of NRP-1 Target-Positively Expressing HUVECs

[0077] 10 μL of Matrigel was pre-added to an Ibidi angiogenesis slide and placed in a cell culture incubator for 30 minutes to allow the Matrigel to solidify. A HUVEC cell suspension was added to the angiogenesis slide (10,000 cells / well) and cultured in an incubator for 8 hours. The peptides SA-AI and AI were then added and incubated in an incubator for 4 hours. Calcein AM (6.25 μg / mL) dye diluted in serum-free medium was then added and incubated at room temperature in the dark for 30 minutes. Images were taken using a confocal microscope.

[0078] The results of the tube formation experiment to verify the anti-angiogenic function of the cell-level material are as follows Figure 10A and Figure 10B As shown, Figure 10A These are confocal images of HUVECs cells after being treated with SA-AI, AI, and PBS respectively; Figure 10A As shown in the confocal images, for HUVEC cells that highly express the NRP-1 target, tube formation was obvious in the PBS control group, and the cells were connected into a network; however, in the experimental groups SA-AI and AI, the cells did not connect into a network due to the inhibition of NRP-1 expression, that is, their tube formation was inhibited. Among them, the SA-AI group with assembly function had the strongest inhibitory effect. Figure 10B The fluorescence statistics of HUVECs cells after being treated with SA-AI, AI and PBS are shown in the figure.+ ) can significantly inhibit cell migration.

[0079] Example 11 Retention of polypeptide SA-AI in mice

[0080] In this experiment, Balb / C mice were used. Cy7-labeled peptides SA-AI, AI, SA-A, and SA-I (200 μM) were injected into the mice via the tail vein. Fluorescence imaging of the anesthetized mice was performed using IVIS at 0 h, 12 h, 24 h, 36 h, 48 h, and 60 h.

[0081] The results of the material targeting and retention ability verification at the in vivo level are as follows Figure 11A and Figure 11B As shown; Figure 11A Fluorescence images of SA-AI, SA-A (targeting tumor blood vessel NRP-1), SA-I (targeting tumor cell PD-L1), and AI injected into mice at different time periods; Figure 11B The fluorescence statistics for SA-AI, SA-A (targeting tumor vascular NRP-1), SA-I (targeting tumor cell PD-L1), and AI at different time intervals after injection into mice. The results show that the peptides SA-AI, AI, SA-A, and SA-I all exhibited excellent targeting capabilities for subcutaneous tumors in mice, but the peptide SA-AI exhibited the best long-term retention in mice.

[0082] Example 12 Verification of the Tumor Inhibitory Ability of Peptide SA-AI in a Mouse Subcutaneous Tumor Model

[0083] This experiment used Balb / C mice. When the subcutaneous tumor model of Renca cell line xenograft mice reached 100 mm³, the peptides SA-AI, AI, SA-A+SA-I, Bevacizumab, and Atelizumab combination (10 mg / kg) were injected into the mice via the tail vein (peptide group, once every two days for five consecutive doses; antibody group, once a week for two doses). The tumor volume of the mice was recorded every other day during the two weeks of treatment.

[0084] The results of the material's ability to treat subcutaneous tumors in vivo were as follows: Figure 12 As shown, the results show that the peptide SA-AI exhibits better tumor inhibition ability in the mouse subcutaneous tumor model.

[0085] In summary, the present invention provides a dual-targeting peptide-based molecule with synergistic effects. This peptide-based molecule, through its self-assembly effect, creates a long-lasting dosing window, simultaneously inducing vascular normalization and exerting immune checkpoint inhibition. This dual-targeting peptide-based molecule combines enhanced immune recruitment with immune killing, synergistically promoting maximal inhibition of tumor proliferation and metastasis.

[0086] The applicant declares that the embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A dual-targeting polypeptide-based molecule with multiple effects and synergistic promotion, characterized in that: The dual-targeting polypeptide-based molecule includes a tumor neovascularization targeting sequence and a PD-L1 targeting sequence that can self-assemble to form a nanofiber with a β-sheet secondary structure and is connected to the nanofiber with a β-sheet secondary structure; The PD-L1 targeting sequence is an amino acid sequence that targets and recognizes PD-L1 that is highly expressed on the surface of tumors. The amino acid sequence of the PD-L1 targeting sequence is NYSKPTDRQYHF; The amino acid sequence that can self-assemble to form nanofibers with a β-sheet secondary structure is GNNQQNYGGK; The tumor angiogenesis targeting sequence is an amino acid sequence that can target and recognize NRP-1 that is highly expressed in tumor angiogenesis endothelial cells. The amino acid sequence of the tumor angiogenesis targeting sequence is RPPLWTA.

2. The multi-effect synergistic dual-targeting polypeptide-based molecule according to claim 1, characterized in that: The tumor angiogenesis targeting sequence was connected to the assembly sequence using (PEG)4 as a linker; the PD-L1 targeting sequence was connected to the assembly sequence using octanoic acid as a linker.

3. Use of the multi-effect synergistically promoted dual-targeting polypeptide-based molecule according to claim 1 or 2 in the preparation of an anti-tumor drug, wherein the tumor is bladder cancer, kidney cancer, glioblastoma or liver cancer.

4. The use according to claim 3, characterized in that The multi-effect synergistically promoted dual-targeting polypeptide-based molecule can inhibit tumor angiogenesis and block immune checkpoints to enhance immune killing ability.

5. The use according to claim 3, characterized in that The multi-effect synergistically promoted dual-targeting polypeptide-based molecule can inhibit tumor cell proliferation and tumor cell metastasis.

6. A pharmaceutical composition, characterized in that The composition includes the multi-effect synergistically promoted dual-targeting polypeptide-based molecule according to claim 1 or 2.

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