Fusion peptide with PD-L1 targeting and cell penetration, conjugate with paclitaxel, and preparation method and application thereof

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a fusion peptide with PD-L1 targeting and cell penetrating properties, a conjugate thereof with paclitaxel, and a preparation method and application thereof. Specifically, the present invention uses a MMP-2 enzyme sensitive peptide segment to target the PD-L1 peptide. D PPA-1 was connected with the selective penetrating peptide TH to construct a new fusion peptide PATH, which was further coupled with paclitaxel to obtain the prodrug-type nanomicelle PATH-VC-PTX with dual targeting and penetrating effects, providing a promising strategy for targeted therapy and combination therapy of tumors, and therefore has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a fusion peptide with PD-L1 targeting and cell penetrating properties, a conjugate thereof with paclitaxel, and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Cancer, also known as malignant tumor, is a disease caused by abnormal cell growth, characterized by high morbidity and mortality, and poses a serious threat to human life and health. In recent years, with the rapid development of tumor biology and immunology, immunotherapy has greatly changed the landscape of tumor treatment. Cancer immunotherapy has made breakthrough progress in the clinical treatment of various cancers by stimulating the host immune response to kill and eliminate local or metastatic tumor cells, and is a promising treatment method. At present, the most commonly used immune drugs in clinical cancer treatment are immune checkpoint inhibitors (ICIs). However, due to individual differences in patients and the heterogeneity of the tumor microenvironment, the response rate and efficacy of immunotherapy are limited. Combining it with other types of anti-tumor drugs may provide new opportunities to reduce side effects and improve treatment effects. In addition, in the context of the great era of immunity, chemotherapy is still the cornerstone of tumor treatment and plays an important role in tumor treatment. Based on the above statement, the combination of traditional chemotherapy and immunotherapy has spawned a new type of cancer treatment option, namely chemotherapy-immunotherapy, in order to enhance the ability of the immune system to fight tumors while chemotherapy drugs act on cancer cells, thereby exerting potential synergistic effects and improving tumor treatment effects.

[0004] Paclitaxel (PTX) is a classic anti-tumor drug widely used in the clinical treatment of various cancers. The mechanism of action of PTX is mainly to inhibit the disassembly of tubulin polymers, interfere with the dynamic balance of microtubules, thereby interfering with the normal mitotic process of cells and ultimately leading to cell apoptosis. In addition, under certain conditions, PTX may also have an immunomodulatory effect and enhance the body's immune response. Based on the diversity of its mechanism of action and the effectiveness of anti-tumor therapy, PTX has become a drug of choice in a variety of anti-tumor combination therapy regimens.

[0005] Peptide-drug conjugates (PDCs) are an emerging type of drug delivery method, mainly composed of three parts: drug molecules, peptides, and linkers. Using the action of active peptides, PDCs can deliver cytotoxic drugs to tumor sites in a targeted manner, providing an opportunity for combined therapy by delivering two different therapeutic entities (peptides and drugs) in an integrated mode. Among them, radionuclides and cytotoxic drugs can both be used as drug options in PDCs. According to the mechanism of action, cytotoxic drugs can be divided into two categories, namely DNA interactors and tubulin inhibitors.

[0006] For the selection of peptides, cell penetrating peptides (CPPs) and tumor targeting peptides (TTPs) are commonly used. CPPs can usually transport insoluble small molecule drugs, proteins and nucleic acids directly into cells in a non-invasive manner without destroying membrane integrity, thereby enhancing molecular permeability. However, the application of CPPs is limited due to low specificity and low circulation half-life. TTPs have high specificity and affinity for receptors overexpressed on tumor cells and can deliver drug molecules to tumor cells in a targeted manner. However, due to poor membrane permeability, TTPs-drug conjugates are difficult to enter cells effectively. In order to solve the shortcomings of drug conjugates based on CPPs or TTPs, the two are integrated into a new type of fusion peptide (TTPs-CPPs). In order to ensure the performance of their respective activities, the two polypeptides can be connected using a linker that can be recognized and cut by characteristic molecules of the tumor microenvironment. The resulting fusion peptide should have the ability to enhance tumor targeting and cell penetration.

[0007] As a bridge between peptides and drug molecules, linkers are crucial for the successful construction of PDCs. An ideal linker should have three key properties: first, it should be water-soluble to avoid affecting the specificity of the peptide; second, it should remain stable in the body's circulation to avoid premature release of the drug; and finally, it should have specific responsiveness and be able to effectively break to release toxic drugs after reaching the tumor site. According to the characteristics of the tumor microenvironment, commonly used linkers can be divided into acid-sensitive, redox-sensitive, hypoxia-sensitive, enzyme-sensitive and other linkers. Summary of the invention

[0008] The purpose of the present invention is to provide a fusion peptide with PD-L1 targeting and cell penetrating properties and a conjugate thereof with paclitaxel, as well as a preparation method and application. Specifically, the present invention uses a MMP-2 enzyme sensitive peptide segment to target the PD-L1 peptide. DPPA-1 is connected with the selective penetrating peptide TH to construct a new fusion peptide PATH, and further coupled with paclitaxel to obtain the prodrug nano-micelle PATH-VC-PTX with dual effects of PD-L1 targeting and penetration, providing a promising strategy for targeted therapy and combined drug use of tumors. Based on the above research results, the present invention is completed.

[0009] Specifically, the present invention relates to the following technical solutions:

[0010] In a first aspect of the present invention, a fusion peptide PATH having PD-L1 targeting and cell penetrating properties is provided, wherein the fusion peptide comprises a PD-L1 targeting peptide D PPA-1 and the selective penetrating peptide TH; and the PD-L1 targeting peptide D PPA-1 is linked to the selective penetrating peptide TH via an enzyme-sensitive linker peptide.

[0011] Wherein, the PD-L1 targeting peptide D The amino acid residue sequence of PPA-1 is nyskptdrqyhf (SEQ ID NO. 1);

[0012] The amino acid residue sequence of the selective penetrating peptide TH is AGYLLGHINLHHLAHL(Aib)HHIL-NH2 (SEQID NO.2);

[0013] Furthermore, the enzyme-sensitive connecting peptide is an MMP-2 enzyme-sensitive peptide, and its amino acid residue sequence is PVGLIG (SEQ ID NO. 3).

[0014] In a specific embodiment of the present invention, the amino residue sequence of the fusion peptide is AGYLLGHINLHHLAHL(Aib)HHILCPVGLIGnyskptdrqyhf (SEQ ID NO.4).

[0015] Among them, peptide D PPA-1 is a type D peptide screened using mirror phage technology. It can specifically bind to the recombinant PD-L1 protein to block the binding of PD-L1 and PD-1, and significantly inhibit tumor growth by activating the body's anti-tumor immune system. D PPA-1 also has the ability to target tumor tissue for drug delivery. D PPA-1 may be a promising drug delivery vehicle with tumor immunotherapy effects.

[0016] Peptide TH is a new type of activatable cell-penetrating peptide. Due to the change in the protonation state of the histidine residues in the sequence, peptide TH has a weak cell internalization ability under physiological pH conditions, but is protonated and positively ionized in the acidic environment of the tumor, thereby binding to the cell membrane and causing membrane perturbations and enhancing cell internalization. In addition, peptide TH has low toxicity under physiological conditions. Therefore, peptide TH is a potential drug delivery carrier with pH selectivity and low toxicity.

[0017] Peptides D PPA-1 exerts drug targeted delivery and anti-tumor effects by binding to the PD-L1 receptor on the cell membrane, while the polypeptide TH enhances the anti-tumor effect of the drug payload by delivering the payload into the cell under acidic conditions. There are differences in the specific locations where the two polypeptides play a role. In order to avoid affecting the activity of both, a MMP-2 enzyme-sensitive amino acid residue sequence (PVGLIG) was used to connect the two polypeptides, and finally the fusion peptide PATH was successfully prepared. It has been experimentally verified that the fusion peptide PATH provided by the present invention exhibits good MMP-2 enzyme sensitivity, PD-L1 targeting, pH response penetration and immune activation activity.

[0018] The second aspect of the present invention provides a conjugate PATH-VC-PTX, which comprises a fusion peptide PATH and PTX conjugated to the fusion peptide.

[0019] In view of the good targeting, activatable penetration and immune activity of the fusion peptide PATH, it can be used as a drug targeted delivery carrier and can be used in combination with other drugs to exert a synergistic anti-tumor effect. Based on the PDCs strategy, PATH was designed to be connected with PTX to synthesize a PTX prodrug, in order to improve the shortcomings of PTX, enhance the anti-tumor effect of PTX and reduce toxicity. Cathepsin B is a ubiquitous cysteine ​​protease that exists in cells, is highly expressed in a variety of tumor cells, and is also present in the damaged area of ​​tumor tissue. Therefore, the structure Val-Cit-PABC (VC) that can be recognized and broken by cathepsin B was selected as a bridge to connect the fusion peptide PATH and PTX to construct the conjugate PATH-VC-PTX. The fusion peptide PATH and the connecting arm Val-Cit-PABC are hydrophilic, while PTX is hydrophobic. Therefore, the conjugate PATH-VC-PTX of the two has amphiphilicity and a tendency to self-assemble in aqueous solution, thereby enhancing the water solubility of PTX.

[0020] Therefore, the conjugate PATH-VC-PTX has good water solubility and can self-assemble into uniform spherical nanomicelles in aqueous solution with a particle size of 87.85±7.08nm. The conjugate PATH-VC-PTX can also release free PTX under the action of cathepsin B. In addition, the conjugate PATH-VC-PTX inherits the good properties of the fusion peptide PATH, can specifically bind to the PD-L1 receptor on the cell, and is protonated under low pH conditions to enhance cellular internalization. In summary, the present invention provides a prodrug conjugate PATH-VC-PTX with dual targeting and penetration effects.

[0021] Specifically, the structural formula of the conjugate PATH-VC-PTX is as follows:

[0022]

[0023] The third aspect of the present invention provides a method for preparing the above-mentioned conjugate, wherein the preparation method comprises the following synthetic route:

[0024]

[0025] The fourth aspect of the present invention provides the use of the above-mentioned fusion peptide PATH or conjugate PATH-VC-PTX in the preparation of anti-tumor drugs.

[0026] It should be noted that tumors in the present invention include benign tumors and / or malignant tumors as known to those skilled in the art. Benign tumors are defined as excessive proliferation of cells that cannot form aggressive, metastatic tumors in the body. Conversely, malignant tumors are defined as cells with multiple cellular abnormalities and biochemical abnormalities that can form systemic diseases (e.g., tumor metastasis in distant organs).

[0027] In another specific embodiment of the present invention, the medicine of the present invention can be used to treat malignant tumors. Examples of malignant tumors that can be treated with the medicine of the present invention include solid tumors and hematologic tumors. Solid tumors can be tumors of the breast, bladder, bone, brain, central and peripheral nervous systems, colon, endocrine glands (such as thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, kidney, small intestine, soft tissue, testicle, stomach, skin (such as melanoma), ureter, vagina and vulva. Hematologic tumors are a type of malignant tumors originating from the hematopoietic system, including leukemia, lymphoma, multiple myeloma, etc., which are not specifically limited here. In addition, malignant tumors include primary tumors in the organs and corresponding secondary tumors (tumor metastasis) in distal organs.

[0028] Specifically, in the in vivo distribution experiment, compared with PTX, PATH-VC-PTX can actively target and accumulate at the tumor site, PATH enhances the intratumoral distribution of PTX and reduces the nonspecific accumulation of PTX in normal tissues. The conjugate PATH-VC-PTX can play an immune anti-tumor effect in vivo, which is specifically manifested in the effective activation and proliferation of T cells and the promotion of the release of cytokines INF-γ and TNF-α. In addition, the conjugate PATH-VC-PTX can also play a PTX-related cytotoxic effect, inhibiting microtubule depolymerization and ultimately leading to tumor cell apoptosis. Therefore, the conjugate PATH-VC-PTX provided by the present invention plays a dual role of immunity and chemotherapy in the anti-tumor process.

[0029] Therefore, the fifth aspect of the present invention provides an anti-tumor drug, wherein the active ingredient of the anti-tumor drug comprises the above-mentioned fusion peptide PATH or conjugate PATH-VC-PTX.

[0030] According to the present invention, the drug may further include at least one other inactive pharmaceutical ingredient.

[0031] The inactive ingredients of the drug can be carriers, excipients and diluents commonly used in pharmacy, etc. Moreover, according to the usual method, it can be made into oral preparations, external preparations, suppositories and sterile injection solutions in the form of powders, granules, suspensions, emulsions, syrups, sprays, etc.

[0032] The non-drug active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0033] A sixth aspect of the present invention provides a method for treating tumors, comprising administering a therapeutically effective amount of the above-mentioned drug to a subject.

[0034] In another specific embodiment of the present invention, the medicine of the present invention can be administered to the body by known means. For example, it is delivered to the tissue of interest by intravenous systemic delivery or local injection (such as intratumoral injection). Such administration can be carried out via a single dose or multiple doses. It is understood by those skilled in the art that the actual dose to be administered in the present invention can vary depending on a variety of factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0035] In another specific embodiment of the present invention, the subjects of drug administration can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc.

[0036] The subject refers to an animal that has been the object of treatment, observation or experiment, preferably a mammal, and most preferably a human. The "therapeutically effective amount" refers to the amount of active compounds or agents, including the compounds of the present invention, which can cause the biological or medical response of the tissue system, animal or human sought by the researcher, veterinarian, doctor or other medical personnel, including the alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated. It must be recognized that the optimal dosage and interval of the active ingredients of the present invention are determined by their properties and external conditions such as the form, route and site of administration and the specific mammal being treated, and this optimal dosage can be determined using conventional techniques. It must also be recognized that the optimal course of treatment, that is, the daily dosage of the compound over a specified period of time, can be determined by methods known in the art.

[0037] The tumor has been discussed in detail above and will not be repeated here.

[0038] Beneficial technical effects of the above technical solution:

[0039] Compared with the prior art, the above technical solution targets PD-L1 through a MMP-2 enzyme sensitive peptide segment. D PPA-1 was connected with the selective penetrating peptide TH to construct a new fusion peptide PATH. The fusion peptide PATH continues the D PPA-1-related PD-L1 targeting and immune activation and TH-related selective penetration, in addition, have MMP-2 enzyme sensitivity, which provide guarantees for the active effects related to the two polypeptide molecules.

[0040] The above technical solution constructs a prodrug nano-micelle PATH-VC-PTX. Compared with the prior art, by integrating chemotherapy and immunotherapy, an integrated drug nano-delivery system is constructed to reduce the metabolic burden. The conjugate PATH-VC-PTX wraps the PTX molecules in the nanocore by self-assembling into a nanostructure, thereby improving the water solubility of PTX. At the same time, the fusion peptide PATH gives the conjugate PATH-VC-PTX good tumor targeting, selective penetration and immune activation activity, which can enhance the specific aggregation and tissue penetration of PTX at the tumor site. The connecting arm Val-Cit-PABC (VC) gives the conjugate PATH-VC-PTX good enzyme sensitivity, which enhances the application safety of PTX. Combining the above advantages, the conjugate PATH-VC-PTX shows strong tumor targeting and penetration in in vivo experiments, and inhibits tumor growth through the dual effects of chemotherapy and immunity, reduces the amount of PTX, and enhances the safety of medication. Therefore, the above technical solution provides a promising strategy for targeted therapy and combined medication of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for use in the description of the embodiment will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0042] Figure 1 To determine the binding affinity between the fusion peptide PATH and the protein.

[0043] a. Determination of the binding affinity of the fusion peptide PATH to human PD-L1 (hPD-L1).

[0044] b. Determination of the binding affinity of the fusion peptide PATH and mouse PD-L1 (mPD-L1).

[0045] Figure 2 pH-dependent penetration of the fusion peptide PATH.

[0046] a. Fluorescence images of PATH uptake by EMT-6 cells captured by laser confocal microscopy under different pH conditions. Scale bar: 30 μm.

[0047] b. Fluorescence intensity of PATH uptake in EMT-6 cells measured by flow cytometry under different pH conditions. ****P<0.0001.

[0048] Figure 3 MMP-2 enzyme sensitivity of the fusion peptide PATH.

[0049] Figure 4 The T cell activation effect of fusion peptide PATH. Compared with the group with only PD-L1 protein, *P<0.05, **P<0.01, ***P<0.001.

[0050] a. Effect of fusion peptide PATH on IFN-γ secretion by human T cells.

[0051] b. Effect of fusion peptide PATH on IFN-γ secretion by mouse T cells.

[0052] Figure 5 This is the synthetic route of the conjugate PATH-VC-PTX.

[0053] Figure 6 For the structural characterization of compound PNO.

[0054] a. Compound PNO 1 H-NMR spectrum.

[0055] b. Q-TOF HRMS spectrum of compound PNO.

[0056] Figure 7 Structural characterization of compound VC-PAB-NBoc.

[0057] a. Compound VC-PAB-NBoc 1 H-NMR spectrum.

[0058] b. Q-TOF HRMS spectrum of compound VC-PAB-NBoc.

[0059] Figure 8 For the structural characterization of compound VC-PABC-PTX.

[0060] a. Compound VC-PABC-PTX 1 H-NMR spectrum.

[0061] b. Q-TOF HRMS spectrum of compound VC-PABC-PTX.

[0062] Fig. 9 Structural characterization of the conjugate PATH-VC-PTX.

[0063] a. Q-TOF HRMS spectrum of the conjugate PATH-VC-PTX.

[0064] b. HPLC spectrum of the conjugate PATH-VC-PTX.

[0065] Fig.10 Characterization of nanomicelles PATH-VC-PTX.

[0066] a. Particle size distribution and electron microscope image of nanomicelle PATH-VC-PTX.

[0067] b. Determination of the critical micelle concentration of nanomicelle PATH-VC-PTX.

[0068] c. In vitro release of PTX under the conditions of cathepsin B.

[0069] Fig.11 Cellular uptake of PATH-VC-PTX

[0070] a. Fluorescence image of EMT-6 cells taken by laser confocal microscopy. Scale bar is 30 μm.

[0071] b. Fluorescence intensity in EMT-6 cells measured by flow cytometry. ***P<0.001 compared with the Cy5-PTX group.

[0072] Fig.12The specific tumor sphere penetration of PATH-VC-PTX. The scale bar is 300 μm.

[0073] Fig.13 The in vivo distribution of PATH-VC-PTX.

[0074] a. Distribution of PATH-VC-PTX in tumor-bearing mice.

[0075] b. Statistics of fluorescence intensity of PATH-VC-PTX accumulation in tumor tissues in vivo. Compared with the Cy5-PTX group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0076] Fig.14 To evaluate the in vivo pharmacodynamics of PATH-VC-PTX.

[0077] a. Changes in tumor tissue volume during the experimental period.

[0078] b. The weight of tumor tissue after the experiment. *P<0.05, **P<0.01.

[0079] c. H&E, TUNEL, Tubulin and CD8 in tumor tissue + The staining results of T cells. The scale bar is 20 μm.

[0080] Fig.15 The secretion of cytokines in tumor tissue.

[0081] a. Secretion of cytokine IFN-γ in tumor tissues. Compared with the NS group, **P<0.01, ***P<0.001, ****P<0.0001.

[0082] b. Secretion of cytokine TNF-α in tumor tissue. Compared with NS group, *P<0.05. DETAILED DESCRIPTION

[0083] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0084] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0085] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0086] Example 1 Targeting of PATH Fusion Peptide

[0087] The fusion peptide PATH was commissioned to be synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. The surface plasmon resonance experiment was used to determine the binding ability of PATH to PD-L1 protein. First, the ligand proteins hPD-L1 and mPD-L1 were coupled to the CM5 chip through an amino reaction. Subsequently, a 1×PBS-P solution containing 5% DMSO was used as a running buffer to prepare peptide sample solutions with concentrations of 625, 156, 39, 9.7, and 2.4 nmol / L. Finally, the sample was injected and analyzed to calculate the equilibrium dissociation constant (K D ).

[0088] The binding response results of PATH and PD-L1 protein are as follows Figure 1 As shown, the K of its binding to human hPD-L1 and mouse mPD-L1 proteins D The values ​​are 7.436×10 -8 mol / L and 9.125×10 -8 mol / L, indicating that PATH has a strong binding and targeting effect on PD-L1 protein.

[0089] Example 2 pH-dependent penetration of the fusion peptide PATH

[0090] Laser confocal microscopy was used to capture the uptake images of EMT-6 cells, and the cellular uptake of PATH under different pH conditions was qualitatively studied. EMT-6 cells in the logarithmic growth phase were prepared to a density of 3×10 41 mL of the cell suspension was added to a confocal dish and placed in a cell culture incubator at 37°C and 5% CO2 for overnight culture. Two different culture media were prepared, one with pH 7.5 and one with pH 6.4 and MMP-2 enzyme. The fluorescent peptide solution was diluted with the above two culture media to a concentration of 1 μmol / L. After the cells adhered to the wall, the original culture medium was discarded and the fluorescent labeled peptide Cy5- D PPA-1, Cy5-TH and Cy5-PATH. After 1 hour of treatment, remove the supernatant, rinse twice with PBS, add 4% paraformaldehyde solution, and incubate in the dark for 30 minutes to fix the cells. Remove the fixative, rinse twice with PBS, add DAPI staining solution, and incubate in the dark for 5 minutes to stain the nuclei. Remove the nuclear staining solution, rinse twice with PBS, add an appropriate amount of PBS to keep it moist, and place it under a laser confocal microscope for observation and photography.

[0091] The fluorescence intensity in EMT-6 cells was measured by flow cytometry to quantitatively study the cellular uptake of PATH under different pH conditions. EMT-6 cells in the logarithmic growth phase were prepared at a density of 2×10 5 100 μmol / mL single cell suspension, take 2 mL of cell suspension and add it to a 6-well plate, and culture it in a cell culture incubator at 37°C and 5% CO2 overnight. Prepare two different culture media, one with pH 7.5 and one with pH 6.4 and MMP-2 enzyme, and dilute the fluorescent peptide solution with the above two culture media solutions to a concentration of 1 μmol / L. After the cells attach to the wall, discard the original culture medium and add the fluorescent labeled peptide Cy5- D PPA-1, Cy5-TH and Cy5-PATH. After 1 hour of treatment, the supernatant was removed, the cells were rinsed with PBS twice, and the cells were digested with trypsin for 1 minute. After adding culture medium to terminate cell digestion, the cells were transferred to a 15 mL centrifuge tube and centrifuged at 1000 r / min for 5 minutes. The supernatant was removed, and an appropriate amount of PBS was added for resuspending, and the cells were detected by flow cytometry.

[0092] The results are as follows Figure 2 As shown in Figure 2, when the pH value decreased, the uptake of Cy5-TH and Cy5-PATH by EMT-6 cells increased significantly, and the intracellular fluorescence was significantly enhanced. D The uptake of PPA-1 did not change much, indicating that PATH has a pH-dependent penetrating effect similar to TH.

[0093] Example 3 MMP-2 enzyme sensitivity of PATH fusion peptide

[0094] APMA was used to activate MMP-2 proenzyme, and a polypeptide solution with a concentration of 1 mg / mL was prepared. The activated MMP-2 enzyme solution was added to the polypeptide solution, and the cells were incubated in a 37°C water bath. After sampling at fixed points, HPLC was used for detection and analysis (0, 2, 4, 8, 12, 24, 36 and 48 h).

[0095] The results are as follows Figure 3 As shown, under the action of MMP-2 enzyme, PATH can undergo rapid and relatively complete cleavage, indicating that PATH is sensitive to MMP-2 enzyme.

[0096] Example 4 T cell activation effect of fusion peptide PATH

[0097] The night before, CD3 antibody was coated in a 96-well plate at a concentration of 0.5 μg / mL and incubated overnight at 4°C. The supernatant was discarded and the plate was rinsed twice with PBS for later use. CD8 + T cells were prepared at a concentration of 10 6 / mL of cell suspension was added to a 96-well plate at a volume of 200μL per well. Subsequently, CD28 antibody with a final concentration of 2μg / mL was added to the activation stimulation well, PD-L1 solution with a final concentration of 10μg / mL was added to the immunosuppression well, and polypeptide solution with a final concentration of 1μmol / L was added to the polypeptide action well. Incubate in a cell culture incubator at 37°C and 5% CO2 for 48h, take the supernatant, and determine the content of cytokine INF-γ by ELISA experiment.

[0098] The results are as follows Figure 4 As shown in the figure, compared with the PD-L1 protein-treated group, after PATH was co-incubated with PD-L1 protein, CD8 + The secretion of INF-γ by T cells increased significantly, indicating that PATH can block the immunosuppressive effect of PD-L1 protein and thus restore T cell activation.

[0099] Example 5 Synthesis and Characterization of Conjugate PATH-VC-PTX

[0100] (1) Synthesis and characterization of compound PNO

[0101] Add 1g of dry molecular sieves, 256mg of PTX, 50mg of 4-nitrophenyl chloroformate, 60mg of pyridine and 5mL of anhydrous dichloromethane into a flask and mix. The mixed solution was stirred and reacted in a -35°C cold trap under the protection of inert gas. The reaction progress was monitored by thin layer chromatography and the reaction was completed after 4h. Subsequently, ethyl acetate and petroleum ether were used as eluents and separated and purified by silica gel column chromatography. Finally, the solvent was rotary evaporated to obtain the product PNO.1 The compound PNO was characterized by H-NMR and Q-TOF HRMS methods.

[0102] The results are as follows Figure 6 As shown, 1 H-NMR and Q-TOF HRMS data showed that PNO had been successfully synthesized.

[0103] (2) Synthesis and characterization of compound VC-PAB-NBoc

[0104] Add 200 mg of Mc-Val-Cit-PABC-PNP, 96 μL of tert-butyl methyl (2-(methylamino)ethyl) carbamate, 112 μL of DIPEA and 20 mL of anhydrous tetrahydrofuran into a flask and mix. The mixture is stirred and reacted at room temperature under the protection of an inert gas. The reaction progress is monitored by thin layer chromatography and the reaction is completed after 12 hours. Subsequently, ethyl acetate and methanol are used as eluents and separated and purified by silica gel column chromatography. Finally, the solvent is rotary evaporated to obtain the product VC-PABC-NBoc. 1 The compound VC-PABC-NBoc was characterized by H-NMR and Q-TOF HRMS methods.

[0105] The results are as follows Figure 7 As shown, 1 H-NMR and Q-TOF HRMS data showed that VC-PAB-NBoc had been successfully synthesized.

[0106] (3) Synthesis and characterization of compound VC-PABC-PTX

[0107] Add 200 mg of VC-PABC-NBoc, 2 mL of trifluoroacetic acid and 4 mL of dichloromethane to a flask, mix and react for 15 minutes, and remove the trifluoroacetic acid by rotary evaporation. Add 300 mg of PNO, 244 μL of DIPEA and 10 mL of anhydrous N, N-dimethylformamide to a flask and mix. The mixed solution is stirred and reacted at room temperature under the protection of an inert gas, and the progress of the reaction is monitored by HPLC. The reaction is completed after 3 hours. Add saturated NaCl and ethyl acetate solutions for extraction, repeat 3 times, and rotary evaporate the organic phase solvent. Subsequently, semi-preparative HPLC is used for separation and purification, and the product VC-PABC-PTX is obtained after freeze-drying the sample solution. Adopt 1 The compound VC-PABC-PTX was characterized by H-NMR and Q-TOF HRMS methods.

[0108] The results are as follows Figure 8 As shown, 1 H-NMR and Q-TOF HRMS data showed that VC-PAB-PTX had been successfully synthesized.

[0109] (4) Synthesis and characterization of compound PATH-VC-PTX

[0110] Weigh 180 mg of PATH and add it to a flask, and dissolve it in 2 mL of PBS buffer with a pH of 7.4. Weigh 60 mg of VC-PABC-PTX and add it to a centrifuge tube, and dissolve it in 4 mL of methanol. Subsequently, the organic phase solution was slowly added dropwise to the aqueous phase polypeptide solution for a mixed reaction, and the reaction progress was monitored by HPLC. The reaction was completed after 2 hours. Semi-preparative HPLC was used for separation and purification, and the sample solution was freeze-dried to obtain the product PATH-VC-PTX. The purity and molecular weight of the compound PATH-VC-PTX were characterized by HPLC and Q-TOF HRMS methods.

[0111] The results are as follows Fig. 9 As shown, Q-TOF HRMS data showed that PATH-VC-PTX was successfully synthesized, and HPLC data showed that the purity of PATH-VC-PTX was 98.503%.

[0112] Example 5 Preparation and characterization of nanomicelles PATH-VC-PTX

[0113] A PATH-VC-PTX solution with a concentration of 4 mg / mL was prepared using deionized water. At 25°C, it was first stirred at a speed of 200 r / min for 30 min, and then ultrasonicated at a power of 40 W for 5 min, and finally a PATH-VC-PTX nanomicelle solution was successfully prepared. The morphology was observed and photographed using a transmission electron microscope, and the particle size was measured using a particle size potential analyzer.

[0114] The results are as follows Fig.10 As shown in ab, PATH-VC-PTX can form spherical nanomicelles with a particle size of 87.85±7.08 nm in aqueous solution, and its critical micelle concentration is 6.19×10 -6 mol / L.

[0115] Example 6 In vitro release of PTX

[0116] The in vitro release of PTX was studied by dialysis. Two conditions were set with and without MMP-2 enzyme and cathepsin B, where the MMP-2 enzyme content was 5μg / mL and the cathepsin B content was 10μg / mL. PATH-VC-PTX was dissolved in PBS buffer containing 1% Tween 80 at a concentration of 1.2mg / mL. 1mL of PATH-VC-PTX solution was added to the dialysis bag (1000Da) and placed in containers containing 30mL of different release media. Incubate at 37℃ and 100r / min, and at 0, 2, 4, 8, 12, 24, 36 and 48h, 1mL of solution was drawn from the container for PTX content detection, and 1mL of fresh solution was added to the container. The PTX content was determined by HPLC, and the total release of PTX was calculated based on the PTX content at different time points.

[0117] The results are as follows Fig.10 As shown in Figure c, under the action of MMP-2 and cathepsin B, PTX can be slowly and continuously released from PATH-VC-PTX, and the release rate can reach 71.72±0.95% at 48 h.

[0118] Example 8 Cellular uptake of PATH-VC-PTX

[0119] The peptide was fluorescently labeled with Cy5, and the fluorescence image of EMT-6 cells taking up PATH-VC-PTX was taken by laser confocal microscopy. EMT-6 cells in the logarithmic growth phase were prepared to a density of 3×10 4 Take 1 mL of the cell suspension and add it to the confocal dish, and culture it in a cell culture incubator at 37°C and 5% CO2 overnight. After the cells adhere to the wall, discard the original culture medium and add 1 mL of Cy5-PTX, Cy5-PATH and Cy5-PATH-VC-PTX solution at a concentration of 1 μmol / L respectively. After 4 hours of treatment, remove the supernatant, rinse twice with PBS, add 4% paraformaldehyde solution, and incubate in the dark for 30 minutes to fix the cells. Remove the fixative, rinse twice with PBS, add DAPI staining solution, and incubate in the dark for 5 minutes to stain the nuclei. Remove the nuclear staining solution, rinse twice with PBS, add an appropriate amount of PBS to keep it moist, and place it under a laser confocal microscope for observation and photography.

[0120] The fluorescence intensity of PATH-VC-PTX uptake in EMT-6 cells was measured by flow cytometry. EMT-6 cells in the logarithmic growth phase were prepared to a density of 2×10 5Take 2 mL of the cell suspension and add it to a 6-well plate, and culture it in a cell culture incubator at 37°C and 5% CO2 overnight. After the cells adhere to the wall, discard the original culture medium and add 1 mL of Cy5-PTX, Cy5-PATH and Cy5-PATH-VC-PTX solution at a concentration of 1 μmol / L respectively. After 4 hours of treatment, remove the supernatant, rinse twice with PBS, digest with trypsin for 1 minute, add culture medium to terminate cell digestion, transfer the cells to a 15 mL centrifuge tube, and centrifuge at 1000 r / min for 5 minutes. Remove the supernatant, add an appropriate amount of PBS to resuspend, and detect using a flow cytometer.

[0121] The results are as follows Fig.11 As shown, Cy5-PATH and Cy5-PATH-VC-PTX were taken up more in cells compared with Cy5-PTX, indicating that in the PATH-VC-PTX structure, PATH could deliver more PTX into PD-L1-overexpressing cells.

[0122] Example 9 Specific tumor sphere penetration of PATH-VC-PTX

[0123] 200 μL of 2% agarose solution was added to a 48-well plate and placed in a clean bench to solidify. EMT-6 cells were prepared at a density of 4×10 4 1 mL of single cell suspension was added to a 48-well plate and cultured at 37 °C and 5% CO2 for 7 days. After the cells were spherical and stable, the tumor spheres were transferred to the confocal dish. Cy5-PATH, Cy5-PTX and Cy5-PATH-VC-PTX were dissolved and diluted in pH 7.5 and pH 6.4 culture media, respectively, with a concentration of 1 μmol / L. 1 mL was added to the confocal dish, incubated for 2 hours, washed twice with PBS, fixed with 4% paraformaldehyde for 30 minutes, and then observed and photographed using LSCM.

[0124] The results are as follows Fig.12 As shown, compared with the fluorescence distribution in tumor spheres at pH 7.5, when the pH value decreased, the fluorescence in the tumor spheres in the Cy5-PATH and Cy5-PATH-VC-PTX treatment groups increased significantly and moved inward, while the fluorescence distribution in the tumor spheres treated with Cy5-PTX did not change much, indicating that PATH-VC-PTX has a specific penetration effect.

[0125] Example 10 Distribution of PATH-VC-PTX in vivo

[0126] A mouse subcutaneous tumor model was established using EMT-6 cells, and the in vivo tumor targeting of fluorescently labeled Cy5-PATH, Cy5-PATH-VC-PTX, and Cy5-PTX was explored by in vivo distribution experiments through tail vein injection. At 2, 4, 8, 12, 24, and 48 hours after administration, the in vivo fluorescence distribution of each group of mice was observed and photographed using a mouse in vivo imaging system, and the fluorescence values ​​were statistically analyzed.

[0127] The results are as follows Fig.13 As shown, compared with Cy5-PTX, the contents of Cy5-PATH and Cy5-PATH-VC-PTX in mouse tumor tissues were significantly increased, indicating that modification of PATH enhanced the distribution and accumulation of PTX in tumor tissues.

[0128] Embodiment 11 In vivo tumor inhibition effect of PATH-VC-PTX

[0129] When the mouse subcutaneous tumor grows to about 100mm 3 At the same time, the tumor-bearing mice were randomly divided into groups, with 6 mice in each group. PTX (4 mg / kg), PATH and PATH-VC-PTX solutions of equimolar concentrations were prepared using physiological saline and injected into the tail vein once every 3 days. The mixed group was given sequentially, with PTX injected on the first day and PATH injected on the next day. The treatment cycle was 16 days, during which the tumor volume was measured and recorded every 2 days. After the treatment, the tumor tissue was dissected, weighed and recorded, and the tumor inhibition rate of each group was calculated. Subsequently, the tumor tissue was subjected to H&E, TUNEL, microtubule and CD8 + T cell staining was used to explore and analyze the anti-tumor response associated with PTX and PATH. Finally, the content of cytokines IFN-γ and TNF-α in tumor tissue grinding fluid was detected using ELISA experiments.

[0130] The results are as follows Fig.14 and 15 As shown, PATH-VC-PTX simultaneously exerts PTX-related antiproliferative effects and PATH-related immunomodulatory effects, thereby inhibiting tumor growth and having the strongest antitumor effect.

[0131] In summary, the conjugate PATH-VC-PTX provided by the present invention improves the shortcomings of poor water solubility, nonspecificity and weak penetration of PTX on the one hand, and enhances the intratumoral distribution of PTX. On the other hand, it integrates chemotherapy and immunotherapy for combined application, showing synergistic anti-tumor activity, and provides a promising therapeutic strategy for the treatment of tumors.

[0132] It should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given embodiments, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A conjugate, characterized in that: The conjugate comprises a fusion peptide PATH and PTX coupled to the fusion peptide; The structural formula of the conjugate is: The fusion peptide comprises a PD-L1 targeting peptide D PPA-1 and the selective penetrating peptide TH; and the PD-L1 targeting peptide D PPA-1 is linked to the selective penetrating peptide TH via an enzyme-sensitive linker peptide; The PD-L1 targeting peptide D The amino acid residue sequence of PPA-1 is nyskptdrqyhf (SEQ ID NO. 1); The amino acid residue sequence of the selective penetrating peptide TH is AGYLLGHINLHHLAHL(Aib)HHIL-NH2 (SEQ ID NO.2); The enzyme-sensitive connecting peptide is an MMP-2 enzyme-sensitive peptide, and its amino acid residue sequence is PVGLIG (SEQ ID NO.3); The amino acid residue sequence of the fusion peptide is AGYLLGHINLHHLAHL(Aib)HHILCPVGLIGnyskptdrqyhf (SEQ ID NO.4).

2. The method for preparing the conjugate according to claim 1, characterized in that: The synthetic route is as follows:

3. Use of the conjugate according to claim 1 in the preparation of anti-tumor drugs.

4. An anti-tumor drug, characterized in that: The active ingredient of the anti-tumor drug comprises the conjugate according to claim 1.

5. The anti-tumor drug according to claim 4, characterized in that: The medicament further comprises at least one other pharmaceutically inactive ingredient.

6. The antitumor drug according to claim 5, characterized in that: The inactive ingredients of the drug are carriers, excipients and diluents commonly used in pharmacy.

7. The anti-tumor drug according to claim 5, characterized in that: The subjects of drug administration are humans and non-human mammals.

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