Bioorthogonal immunomodulator prodrug delivery systems and their anticancer applications

Abstract

The application relates to a biological ortho immunomodulator prodrug delivery system and application thereof, and belongs to the technical field of biological medicine. The delivery system comprises two core components: one is a biological ortho prodrug TCO-IMQ which is composed of a TLR7 / 8 agonist imiquimod and a trans-cyclooctene (TCO) covalently connected; the other is a self-assembled polypeptide containing a tetrazine (Tz) group, an alkaline phosphatase (ALP) response unit and an epidermal growth factor receptor (EGFR) targeting unit. The self-assembled polypeptide can selectively form nanofibers in situ on the membrane of cancer cells with high expression of ALP and EGFR, and then realize extracellular specific release of IMQ through Tz and TCO reaction; the released IMQ can effectively activate antigen-presenting cells, promote the infiltration of cytotoxic T lymphocytes at the tumor site, and avoid the immune-related adverse reactions caused by systemic administration of TLR7 / 8 agonists.

Description

Technical Field

[0001] This invention relates to a prodrug delivery system for a biological orthogonal immunomodulator and its application, belonging to the field of biomedical technology. Background Technology

[0002] T-cell-centered cancer immunotherapy has limited efficacy in the treatment of solid tumors. Innate immune response is key to activating and maintaining T-cell immunity. Among them, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, as immunomodulators, can effectively activate antigen-presenting cells and promote the infiltration of cytotoxic T lymphocytes, thereby killing tumor cells. Currently, small molecule TLR7 / 8 agonists such as imiquimod and ralsimod have been approved by the FDA for anti-tumor therapy. However, when these small molecule TLR7 / 8 agonists are administered systemically, they can cause dose-dependent immune-related adverse reactions, such as fatal cytokine storms, which severely limit their clinical application.

[0003] Currently, although some TLR7 / 8 agonist delivery systems utilizing stimuli such as endogenous enzymes, glutathione, and acidic environments have been developed, they still face the risk of off-target activation. Bioorthogonal chemistry, especially the tetrazine-trans-cyclooctene pair, provides an ideal exogenous tool for the controlled release of drugs due to its high selectivity, efficiency, and biocompatibility. However, the key to the successful application of this technology lies in how to specifically enrich and retain the tetrazine group at the target site for a long time to maximize the therapeutic effect and avoid off-target side effects. Enzyme-guided peptide self-assembly technology can construct functional nanomaterials in situ at specific cellular or subcellular sites, and the resulting assemblies have enhanced target enrichment, retention, and anti-degradation capabilities. In summary, this provides new ideas for the precise delivery of bioorthogonally reactive tetrazine groups and the design of prodrug delivery systems for immunomodulators. Summary of the Invention

[0004] This invention aims to provide a bioorthogonal immunomodulator prodrug delivery system. This invention is a safe, efficient, and highly targeted TLR7 / 8 agonist delivery system that avoids immune-related adverse reactions caused by systemic use. This system comprehensively utilizes enzyme-guided peptide self-assembly and bioorthogonal reactions to achieve spatiotemporal specific activation of TLR7 / 8 agonist prodrugs on cancer cell membranes.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A bioorthogonal immunomodulator prodrug delivery system comprises: a self-assembled polypeptide consisting of a tetrazine group, an alkaline phosphatase response unit, and an epidermal growth factor receptor targeting unit; and a bioorthogonal prodrug TCO-IMQ formed by covalently linking imiquimod (IMQ) and 2E-cyclooctene (TCO); imiquimod (IMQ) is an immunomodulatory TLR7 / 8 agonist.

[0006] The self-assembling polypeptide is a compound of chemical formula I or a pharmaceutically acceptable salt thereof; (I); The alkaline phosphatase response unit is phosphorylated tyrosine.

[0007] The tetrazine group is selected from any one or more of 2-(4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)acetic acid, 4-(6-methyl-1,2,4,5-tetrazinecyclo-3-yl)phenyl)methylamine and its derivatives.

[0008] The self-assembled peptide preparation method includes the following steps: Using a solid-phase peptide synthesis method, 2-chlorotriphenylmethyl chloride resin was used as the solid-phase carrier, and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) was used as the condensing agent. Under alkaline conditions, amino acids were condensed sequentially from the C-terminus to the N-terminus according to a predetermined amino acid sequence. The amino acid sequence included an epidermal growth factor receptor (EGFR) targeting sequence, an enzyme-responsive group, and a tetrazine group. Subsequently, the peptide was cleaved, deprotected, and purified to obtain the self-assembled polypeptide molecule.

[0009] The condensation reaction is carried out in an alkaline environment with a pH of 8-9.

[0010] The reaction reagent used for the cutting is a mixed solution of trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 95:2.5:2.5, and the cutting reaction time is 2-3 hours.

[0011] The deprotecting reagent is a 20% piperidine N,N-dimethylformamide solution, and the reaction time is 20-30 minutes.

[0012] The aforementioned bioorthogonal prodrug TCO-IMQ is a compound of chemical structural formula II or a pharmaceutically acceptable salt thereof; (II); The preparation method of the bioorthogonal prodrug TCO-IMQ, and the synthetic route of the compound with chemical structure II are as follows:

[0013]

[0014] DCM: dichloromethane; Et3N: triethylamine; EA: ethyl acetate; DIPEA: N,N-diisopropylethylamine; Microwave: microwave reaction.

[0015] The microwave reaction conditions are set as follows: temperature 65~100℃, power 95~125W, pressure 100~150PSI, pre-stirring for 120s, followed by microwave irradiation reaction for 30~90 minutes.

[0016] A pharmaceutical composition comprising a therapeutically effective amount of a bioorthogonal immunomodulator prodrug delivery system and a pharmaceutically acceptable carrier thereof (the compound).

[0017] The application of the aforementioned (compound) bioorthogonal immunomodulator prodrug delivery system or its solvates in the prevention or treatment of tumor diseases.

[0018] The pharmaceutical composition is used in the prevention or treatment of tumor diseases.

[0019] The beneficial effects of this invention are: The bioorthogonal immunomodulator prodrug delivery system of the present invention has a self-assembled peptide that can selectively form an in situ membrane anchored nanofiber on the membrane of cancer cells that highly express alkaline phosphatase and epidermal growth factor receptor through enzyme-guided self-assembly. This nanofiber then acts as a bioorthogonal platform to activate the bioorthogonal prodrug TCO-IMQ, achieving extracellular specific release of imiquimod. Delivery can significantly promote the maturation of dendritic cells and the infiltration of T lymphocytes in tumor tissues, and effectively avoid immune-related adverse reactions caused by systemic TLR7 / 8 activation.

[0020] The bio-orthogonal immunomodulator prodrug delivery system of this invention has demonstrated its anti-tumor effect using a 4T1-hEGFR tumor model mouse. Eight hours after tail vein injection, the self-assembled peptide can be specifically enriched at the tumor site, providing an in vivo activation site and dosing reference for TCO-IMQ. The combined use of the self-assembled peptide and the bio-orthogonal prodrug TCO-IMQ can significantly inhibit tumor growth, with a tumor inhibition rate of >80% (P<0.001), and no obvious immune-related adverse reactions, proving that it can improve the efficacy and safety of tumor treatment. Attached Figure Description

[0021] Figure 1 This is a high-resolution mass spectrometry image of the self-assembled polypeptide Tz-pY from Example 1; Figure 2 The image shows the 1H NMR spectrum of the bioorthogonal prodrug TCO-IMQ from Example 1. Figure 3Example 1 (a) IMQ; (b) Molecular docking conformation of TCO-IMQ (green stick model) and TLR7 (PDB number: 5ZSF); Key amino acid residues are shown in pink stick model, hydrogen bonds are represented by green dashed lines, and salt bridges are represented by orange dashed lines; Figure 4 For Example 2, (a) optical images of the Tyndall effect before and after enzymatic self-assembly of Tz-pY peptide in PBS solution (200 μM, pH 7.4); (b) TEM image of Tz-pY peptide in PBS solution (200 μM, pH 7.4); (c) TEM image of Tz-pY peptide in PBS solution (200 μM, pH 7.4) after incubation at 37°C with ALP (10 U / mL) for 3 hours. Figure 5 Example 2: (a) Tz-pY (200 μM) incubation for 4 h; (b) Scanning electron microscopy image of untreated HeLa cell membrane; (c) Schematic diagram of the bioorthogonal reaction between tetrazine-containing polypeptide nanofibers and (4E)-TCO-Cy5.5 on the HeLa cell membrane; (d) Laser confocal microscopy image of HeLa cells treated with Tz-pY (200 μM) for 4 h, followed by co-incubation with (4E)-TCO-Cy5.5 (5 μM) for 45 min, with white dashed lines indicating the cell membrane outline; (e) Time-dependent microscopic image of Tz-pY on the HeLa cell membrane. Figure 6 This is a CLSM image of the concentration-dependent assembly behavior of Tz-pY on HeLa cell membranes after 4 hours of incubation in Example 2, with a scale bar of 20 μm. Figure 7 The image shown is a laser confocal image of HeLa treated with Tz-pY under alkaline phosphatase inhibitor (Na3VO4) or anti-EGFR antibody blocking conditions, with a scale value of 20 μm. Figure 8 The results of Example 2 show the effect of Tz-pY on the viability of HeLa cells and HUVECs. Figure 9 The image shown is a CLSM image obtained in Example 2 after HUVECs cells were treated with 200 μM Tz-pY for 4 h and then co-incubated with 5 μM (4E)-TCO-Cy5.5 for 45 min. The scale bar is 15 μm. Figure 10 This is a liquid chromatography image of the click reaction tracer between TCO-IMQ and the polypeptide assembly in Example 2, detected at a wavelength of 254 nm (peak 1: IMQ; peak 2: TCO-Tz adduct; peak 3: Tz-Y; peak 4: reaction intermediate; peak 5: TCO-IMQ). Figure 11The image shows a liquid chromatogram (detection wavelength 254 nm) of the serum-free culture medium supernatant from Example 2, characterizing the activation effect of HeLa cell membrane surface polypeptide nanofibers on TCO-IMQ. After HeLa cells were pretreated with 200 μM Tz-pY for 4 h, 16.5 μM TCO-IMQ was added and incubated at different time points. Figure 12 Example 3(a) shows the operation of peptide assembly activation of TCO-IMQ and in vitro stimulation of immature dendritic cells (iDCs); flow cytometry analysis: (b) CD86+ / CD80+ bone marrow dendritic cells (BMDCs); (c) CD40+ / MHC-II+ BMDCs; data are expressed as mean ± standard deviation (n=3); one-way ANOVA was used, ****P≤0.0001, ***P≤0.001, **P≤0.01, *P≤0.05, ns indicates no statistical difference; Figure 13 Near-infrared fluorescence imaging of the in vivo distribution of Cy5.5-labeled phosphorylated peptides in BALB / c Nude mice bearing HeLa tumors in Example 4; (b) Near-infrared fluorescence imaging of the in vivo distribution of Cy5.5-labeled non-phosphorylated peptides in BALB / c Nude mice bearing HeLa tumors in Example 4; (c) Near-infrared fluorescence imaging of the in vivo distribution of Cy5.5-labeled phosphorylated peptides in BALB / c mice bearing 4T1-hEGFR tumors in Example 4; (d) Near-infrared fluorescence imaging of the in vivo distribution of Cy5.5-labeled phosphorylated peptides in BALB / c mice bearing 4T1 tumors in Example 4. Figure 14 In Example 4, the hemolysis rate of different concentrations of TCO-IMQ was determined by ultraviolet-visible spectrophotometry; (a) optical image of the actual object; (b) hemolysis rate (detection wavelength 576nm). One-way ANOVA was used, ****P≤0.0001, **P≤0.01, ns indicates no statistical difference; Figure 15 The results of ELISA detection of (a) white blood cell (WBC) count in the serum of mice treated with PBS, IMQ and TCO-IMQ (at an IMQ equivalent of 6 mg / kg, administered via tail vein) in Example 4; and (b) IFN-γ, (c) IL-6 and (d) TNF-α in the serum of mice. Figure 16Example 5: (a) Schematic diagram of the construction and drug administration regimen of the 4T1-hEGFR tumor-bearing mouse model; (b) Tumor growth curves of each group; (c) Statistical analysis of tumor weight in different groups (n=4); (d) Optical image of the dissected tumor; (e) Typical staining images of tumor tissues in each group using H&E staining, TUNEL immunofluorescence, and Ki-67 immunohistochemistry; (f) Curves of weight change in mice in each treatment group; (g) Mature dendritic cells (CD11c) in the tumor draining lymph nodes. + CD80 + CD86 + DCs) proportion (n=3); (h) MHC-II in tumor draining lymph nodes + Dendritic cells (CD11c) + MHC-II + DCs) proportion (n=3); (i) tumor infiltration CD8a + T cell percentage (n=3); (j) Day 10 of drug administration, with CD3 + T-cell gated tumor infiltration CD4 + CD8a + Representative flow cytometry plots of T cells; data are expressed as mean ± standard deviation, and analyzed by one-way ANOVA; ****P≤0.0001, ***P≤0.001, **P≤0.01, *P≤0.05, ns indicates no statistical difference. Detailed Implementation

[0022] A bioorthogonal immunomodulator prodrug delivery system comprises: a self-assembled polypeptide consisting of a tetrazine group, an alkaline phosphatase response unit, and an epidermal growth factor receptor targeting unit; and a bioorthogonal prodrug TCO-IMQ formed by covalently linking imiquimod (IMQ) and 2E-cyclooctene; imiquimod (IMQ) is an immunomodulatory TLR7 / 8 agonist.

[0023] The self-assembling polypeptide is a compound of chemical formula I or a pharmaceutically acceptable salt thereof; (I); The alkaline phosphatase response unit is phosphorylated tyrosine.

[0024] The tetrazine group is selected from any one or more of 2-(4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)acetic acid, 4-(6-methyl-1,2,4,5-tetrazinecyclo-3-yl)phenyl)methylamine and its derivatives.

[0025] The self-assembled peptide preparation method includes the following steps: Using a solid-phase peptide synthesis method, 2-chlorotriphenylmethyl chloride resin was used as the solid-phase carrier, and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) was used as the condensing agent. Under alkaline conditions, amino acids were condensed sequentially from the C-terminus to the N-terminus according to a predetermined amino acid sequence. The amino acid sequence included an epidermal growth factor receptor (EGFR) targeting sequence, an enzyme-responsive group, and a tetrazine group. Subsequently, the peptide was cleaved, deprotected, and purified to obtain the self-assembled polypeptide molecule.

[0026] The condensation reaction is carried out in an alkaline environment with a pH of 8-9.

[0027] The reaction reagent used for the cutting is a mixed solution of trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 95:2.5:2.5, and the cutting reaction time is 2-3 hours.

[0028] The deprotecting reagent is a 20% piperidine N,N-dimethylformamide solution, and the reaction time is 20-30 minutes.

[0029] The aforementioned bioorthogonal prodrug TCO-IMQ is a compound of chemical structural formula II or a pharmaceutically acceptable salt thereof; ; (II); The preparation method of the bioorthogonal prodrug TCO-IMQ, and the synthetic route of the compound with chemical structure II are as follows:

[0030]

[0031] DCM: dichloromethane; Et3N: triethylamine; EA: ethyl acetate; DIPEA: N,N-diisopropylethylamine; Microwave: microwave reaction.

[0032] The microwave reaction conditions are set as follows: temperature 65~100℃, power 95~125W, pressure 100~150PSI, pre-stirring for 120s, followed by microwave irradiation reaction for 30~90 minutes.

[0033] A pharmaceutical composition comprising a therapeutically effective amount of a bioorthogonal immunomodulator prodrug delivery system and a pharmaceutically acceptable carrier thereof (the compound).

[0034] The application of the aforementioned (compound) bioorthogonal immunomodulator prodrug delivery system or its solvates in the prevention or treatment of tumor diseases.

[0035] The pharmaceutical composition is used in the prevention or treatment of tumor diseases.

[0036] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0037] Synthesis of self-assembled peptides: Peptide chains were synthesized using the standard solid-phase peptide synthesis (SPPS) method: the reaction used 2-chlorotriphenylmethyl chloro resin as a solid support for the coupling of N-Fmoc-protected amino acids; the Fmoc protecting group was removed using 20% ​​piperidine / anhydrous N,N'-dimethylformamide (DMF), and then the Fmoc-protected amino acid (4 equivalents) was coupled to the free amino group under the condition of peptide coupling reagent (HBTU) (4 equivalents) as a condensing agent. The Fmoc-protected amino acids were added sequentially from the C-terminus to the N-terminus according to the GFFpYHWYGYTPQNVI sequence.

[0038] The peptides were lysed from the resin at room temperature using a lysis buffer (95% trifluoroacetic acid, 2.5% triisopropylsilane, 2.5% water) for 3 hours. After lysis, trifluoroacetic acid was removed using a rotary evaporator, and the peptides were precipitated with cold diethyl ether. The crude peptides were further purified by reversed-phase high-performance liquid chromatography (HPLC), and the products were characterized by mass spectrometry. Using 2-(4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl)acetic acid (Tz-COOH) as a capping group, it was chemically linked to the N-terminus of the peptide chain to obtain the target product Tz-GFFpYHWYGYTPQNVI (denoted as Tz-pY, molecular structure as shown in chemical structural formula I). ​​All synthesized self-assembled peptide compounds were purified by high-performance liquid chromatography (HPLC) and characterized by high-resolution mass spectrometry (HR-MS). See [link to relevant documentation]. Figure 1 .

[0039] Synthesis of TLR7 / 8 agonist prodrug TCO-IMQ: Synthesis route:

[0040]

[0041] (1) 1.0 equivalent (2E)-trans-cyclooctene and 3.0 equivalent triethylamine were dissolved in anhydrous dichloromethane; under an ice-water bath, 1.05 equivalent of anhydrous dichloromethane solution of chloro-substituted p-nitrobenzene was slowly added dropwise to the reaction system, and the reaction was stirred for 4 hours. After the reaction was completed, a white precipitate was precipitated by filtration, the solvent was removed under reduced pressure, and the crude product was separated by rapid silica gel column chromatography. The reactive intermediate TCO-pNb was obtained by using n-hexane-ethyl acetate (volume ratio 1:4) as the eluent.

[0042] (2) 1.0 equivalent of compound TCO-pNb, 2.0 equivalent of imiquimod, and 2.5 equivalent of diisopropylethylamine were dissolved in anhydrous ethyl acetate; the mixture was sealed in a microwave reactor, and the temperature was set to 95℃, the power to 125W, and the pressure to 150PSI. After pre-stirring for 120s, the mixture was microwave-irradiated for 45 minutes. The reaction solution was cooled to room temperature, and the insoluble matter was filtered off. The filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a white solid product. The product was then analyzed by nuclear magnetic resonance hydrogen spectroscopy (NMR 1H N ... 1 The structure of TCO-IMQ was characterized by H-NMR (see [reference]). Figure 2 .

[0043] Molecular docking simulations showed that, compared to free IMQ, TCO-IMQ lost its key interaction with the crucial amino acid Asp555 of the TLR7 protein due to the caged C4-amino group, resulting in a significant decrease in its binding affinity to TLR7. (See [link to relevant documentation]). Figure 3 This provides preliminary verification of its prodrug properties.

[0044] Enzymatic self-assembly behavior of Tz-pY: In phosphate-buffered saline (PBS) at pH 7.4, 200 μM Tz-pY pre-assembled into nanoparticles with a diameter of approximately 30 nm. After adding 10 U / mL alkaline phosphatase (ALP) and incubating at 37°C, Tz-pY underwent time-dependent dephosphorylation, reaching equilibrium after 3 hours with a dephosphorylation rate of approximately 90%. Transmission electron microscopy (TEM) characterization after enzymatic dephosphorylation showed that Tz-pY could further self-assemble into dense, long nanofibers with a diameter of approximately 15 nm. The system also exhibited a significant Tyndall effect. (See [link to TEM]). Figure 4 .

[0045] Cellular experiments were conducted using HeLa cells that highly express ALP and EGFR. Bio-SEM observations showed that after treating HeLa cells with 200 μM Tz-pY for 4 hours, a dense nanofiber network with a fiber diameter of 10–20 nm was formed on the cell membrane surface; however, the untreated control group cells did not exhibit this characteristic structure (see product manual appendix). Figure 5 (a, 5b) To verify the reactive function of the Tz group, HeLa cells pretreated with Tz-pY were co-incubated with (4E)-TCO-Cy5.5 fluorescent dye. Laser confocal microscopy (CLSM) imaging showed that the red fluorescence signal was mainly enriched in the cell membrane region, confirming that the nanofibers constructed in situ by Tz-pY in the cell membrane can effectively mediate the inv-DA bioorthogonal reaction. (See [reference]) Figure 5d; Time- and concentration-dependent studies showed that when the peptide concentration was fixed at 200 μM, Tz-pY required at least 2 hours of incubation with HeLa cells to form stable nanofibers on the membrane and maintain them for 24 hours without endocytosis or clearance. See [link to relevant documentation]. Figure 5 e; When the incubation time is fixed at 4 hours, the minimum concentration of Tz-pY for effective assembly and inv-DA reaction is approximately 200 μM. See Figure 6 .

[0046] Treatment with ALP inhibitors (Na3VO4) or anti-EGFR antibodies significantly reduced or eliminated fluorescence signals and nanofiber formation on the membrane. See [link to relevant documentation]. Figure 7 This demonstrated that the process depends on ALP dephosphorylation and EGFR protein binding. Cytotoxicity experiments showed that at concentrations above 25 μM, Tz-pY was significantly less toxic to human umbilical vein endothelial cells (HUVECs, which express low levels of ALP and EGFR) than to HeLa cells. See [link to relevant documentation]. Figure 8 CLSM showed that the fluorescence signal produced by Tz-pY on the cell membrane of HUVECs was much weaker than that in HeLa cells. (See [link to CLSM]). Figure 9 This demonstrates that the self-assembly behavior of Tz-pY exhibits good cell selectivity.

[0047] Bioorthogonal activation of the prodrug TCO-IMQ: In a solution system, TCO-IMQ (187.5 μM) and the non-phosphorylated peptide Tz-Y (500 μM) were reacted in an acetonitrile / water (1:2) mixed solvent at 37 °C. The reaction process was monitored by liquid chromatography-mass spectrometry (LC-MS). The results showed that approximately 50% of TCO-IMQ was consumed after about 55 minutes of reaction, and the release of active IMQ, as well as the formation of TCO-Tz adduct and intermediate products, were detected. See [link to relevant documentation]. Figure 10 .

[0048] In a cell system, HeLa cells pretreated with Tz-pY (200 μM, 4 h) were co-incubated with TCO-IMQ (16 μM). LC monitoring showed that TCO-IMQ was completely consumed within 60 minutes, releasing approximately 10 μM of free IMQ into the extracellular culture medium. No other byproducts were detected, indicating that activation occurred at the cell membrane. (See [link to relevant documentation]). Figure 11 The above results demonstrate that self-assembled peptides can effectively trigger bioorthogonal reaction-mediated TCO-IMQ decage, thereby achieving bioorthogonal activation of TLR7 / 8 agonist prodrugs.

[0049] Bone marrow-derived dendritic cells (BMDCs) treated under different conditions were collected, and their maturation markers were analyzed by flow cytometry. Compared with the PBS group and the TCO-IMQ group, IMQ released after Tz-pY-mediated TCO-IMQ activation significantly increased the expression levels of co-stimulatory molecules CD80 and CD86, as well as maturation-related molecules CD40 and MHCII, on the surface of BMDCs. (See [link to relevant documentation]). Figure 12 The results showed that the TCO-IMQ prodrug itself had a weak immunostimulatory effect, but the IMQ released through Tz-mediated decageing could restore its biological activity and effectively promote DC maturation.

[0050] In vivo distribution of self-assembled peptides: A Cy5.5-labeled phosphorylated peptide (Cy5.5-pY) and a non-phosphorylated peptide (Cy5.5-Y) were synthesized and injected intravenously into mice bearing HeLa tumors or 4T1-hEGFR tumors. In vivo imaging (IVIS) showed that Cy5.5-pY was significantly enriched at the tumor site, reaching a peak concentration 4-8 hours post-injection, and remained within the tumor for a relatively long time. Cy5.5-Y showed weaker tumor enrichment. In a stable 4T1 cell (4T1-hEGFR) tumor model expressing human EGFR, Cy5.5-pY accumulation in 4T1-hEGFR tumors was significantly higher than in parental 4T1 tumors, confirming EGFR targeting. (See [link to relevant documentation]). Figure 13 .

[0051] 2. Biosafety of TCO-IMQ prodrugs: Hemolysis experiments showed that the hemolysis rate of TCO-IMQ at concentrations of 25–200 μM was <5%. (See [link to relevant documentation]). Figure 14 Following systemic administration, compared to the PBS group, the peripheral blood leukocyte (WBC) count in the TCO-IMQ group showed no significant change, while the WBC count in the free IMQ group decreased significantly. (See [link to relevant documentation]). Figure 15 a; Enzyme-linked immunosorbent assay (ELISA) showed that serum levels of IL-6, TNF-α, and IFN-γ were significantly elevated in the free IMQ group, while there was no significant difference between the TCO-IMQ group and the PBS group, indicating that TCO-IMQ did not induce an acute cytokine storm. See [link to ELISA]. Figure 15 b~15d.

[0052] 1. Anti-tumor effects: A 4T1-hEGFR orthotopic tumor-bearing BALB / c mouse model was established. When the tumor volume reached approximately 80 mm³, mice were randomly divided into groups and administered PBS, Tz-pY, TCO-IMQ, IMQ, or Tz-pY+TCO-IMQ (8-hour interval between injections) via tail vein injection on days 1, 3, 5, 7, and 9. Tumor growth curves showed that the Tz-pY+TCO-IMQ combination group significantly inhibited tumor proliferation, while the PBS control group experienced rapid tumor growth. The tumor growth rates of the IMQ, Tz-pY, and TCO-IMQ single-drug groups showed no significant difference from the blank control group. Mice were sacrificed after 15 days of treatment, and tumors and major organs were dissected for analysis. Tumor weight results were consistent with volume changes, with the combination group showing significantly lower tumor weight than the other groups. Hematoxylin-eosin (H&E) staining, TUNEL apoptosis staining, and Ki67 proliferation staining confirmed that, compared to other groups, the combination group exhibited the deepest tumor tissue necrosis, the highest apoptosis rate, and the lowest proliferation level. (See [link to relevant documentation]). Figure 16 a~16e.

[0053] Monitoring of mouse body weight changes throughout the treatment revealed that, starting from the third administration (day 7), the body weight of mice in the IMQ monotherapy group was significantly lower than that in the PBS group. (See attached image.) Figure 16 f; Combined with relevant inflammatory factor data, it was shown that free IMQ caused a decrease in body weight and an increase in inflammatory factors in mice, resulting in immune-related adverse reactions. However, the body weight of mice in the Tz-pY+TCO-IMQ combination group was not significantly different from that in the blank group, and no obvious abnormal lesions were found in the pathological sections of heart, liver, spleen, lung, and kidney tissues. In summary, this biological orthogonal immunomodulator prodrug delivery system can effectively inhibit tumors in vivo without significant systemic toxic side effects.

[0054] Anti-tumor immune response: Following the established experimental procedure, tumor drainage lymph nodes and tumor tissues from mice were collected on day 10 of drug administration for analysis. Migrating dendritic cells, after entering the lymph nodes via the tumor drainage lymphatic vessels, could interact with T cells and jointly regulate CD4. + CD8 + T-cell-mediated immune responses were analyzed using flow cytometry to detect the maturation level of dendritic cells in lymph nodes. Results showed that the CD80 levels in the Tz-pY+TCO-IMQ combination group and the IMQ monotherapy group were significantly increased. + / CD86 + The proportions of mature dendritic cells were approximately 38.5% and 32.5%, respectively, both significantly higher than in the other experimental groups; the detection results of the key antigen-presenting molecule MHC-II showed that CD11c in the combination group and the IMQ monotherapy group were significantly higher. +The expression rates of MHC-II on the surface of dendritic cells were approximately 50.5% and 40.3%, respectively, demonstrating that in situ release of IMQ can effectively promote dendritic cell maturation; compared with the PBS control group, the Tz-pY+TCO-IMQ group showed a higher proportion of cytotoxic T cells (CD3+) infiltrating the tumor. + / CD8 + ), helper T cells (CD3) + / CD4 + The proportions increased by approximately 3.25 times and 2.64 times respectively. (See [reference]) Figure 16 In summary, this bioorthogonal immunomodulator prodrug delivery system can elicit a potent antitumor immune response in mice bearing 4T1-hEGFR tumors.

Claims

1. A prodrug delivery system for a biological orthogonal immunomodulator, characterized in that: The invention comprises a self-assembled polypeptide consisting of a tetrazine group, an alkaline phosphatase response unit, and an epidermal growth factor receptor targeting unit; and a bioorthogonal prodrug TCO-IMQ formed by covalently linking imiquimod and 2E-cyclooctene; wherein imiquimod is an immunomodulatory TLR7 / 8 agonist.

2. The prodrug delivery system for a biological orthogonal immunomodulator according to claim 1, characterized in that: The self-assembling polypeptide is a compound of chemical formula I or a pharmaceutically acceptable salt thereof; （I）。 3. The prodrug delivery system for a biological orthogonal immunomodulator according to claim 1, characterized in that: The aforementioned bioorthogonal prodrug TCO-IMQ is a compound of chemical structural formula II or a pharmaceutically acceptable salt thereof; (II)。 4. The prodrug delivery system for a biological orthogonal immunomodulator according to claim 1, characterized in that: The alkaline phosphatase response unit is phosphorylated tyrosine; The tetrazine group is selected from any one or more of 2-(4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)acetic acid, 4-(6-methyl-1,2,4,5-tetrazinecyclo-3-yl)phenyl)methylamine and its derivatives.

5. The prodrug delivery system for a biological orthogonal immunomodulator according to any one of claims 1 to 4, characterized in that: The self-assembled peptide preparation method includes the following steps: Using a solid-phase peptide synthesis method, 2-chlorotriphenylmethyl chloride resin was used as the solid-phase carrier, and O-benzotriazole-tetramethylurea hexafluorophosphate was used as the condensing agent. Under alkaline conditions, amino acids were condensed sequentially from the C-terminus to the N-terminus according to a predetermined amino acid sequence, which includes an epidermal growth factor receptor targeting sequence, an enzyme-responsive group, and a tetrazine group. Subsequently, the peptide was cleaved, deprotected, and purified to obtain the self-assembled peptide molecule.

6. The method for preparing self-assembled peptides according to claim 5, characterized in that: The condensation reaction is carried out in an alkaline environment with a pH of 8-9; The reaction reagent used for the cutting is a mixed solution of trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 95:2.5:2.5, and the cutting reaction time is 2-3 hours. The deprotecting reagent is a 20% piperidine N,N-dimethylformamide solution, and the reaction time is 20-30 minutes.

7. The prodrug delivery system for a biological orthogonal immunomodulator according to any one of claims 1 to 4, characterized in that: The preparation method of the bioorthogonal prodrug TCO-IMQ, and the synthetic route of the compound with chemical structure II are as follows: ； ； DCM: Dichloromethane; Et3N: Triethylamine; EA: Ethyl acetate; DIPEA: N,N-Diisopropylethylamine; Microwave: Microwave reaction; The microwave reaction conditions are set as follows: temperature 65~100℃, power 95~125W, pressure 100~150PSI, pre-stirring for 120s, followed by microwave irradiation reaction for 30~90 minutes.

8. A pharmaceutical composition, characterized in that: It contains a prodrug delivery system for a therapeutically effective amount of a biological orthogonal immunomodulator and a pharmaceutically acceptable carrier.

9. The use of the bioorthogonal immunomodulator prodrug delivery system of claim 1 or its solvates in the prevention or treatment of tumor diseases.

10. The use of the pharmaceutical composition of claim 8 in the prevention or treatment of tumor diseases.

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