Nanomicellar fludox-MIC, and methods of making and using the same

Abstract

This invention belongs to the field of biomedical technology, specifically disclosing a nanomicelle FluDox-MIC, its preparation method, and its uses. This nanomicelle is formed by the self-assembly of flumatinib and doxorubicin co-loaded in mPEG-DSPE, exhibiting pH-responsive release properties. Upon entering the tumor microenvironment, it releases the drug. The released flumatinib induces the degradation of the poliovirus receptor (PVR / CD155), relieving its inhibitory signal on NK cells and reactivating innate immune surveillance. Simultaneously, low-dose doxorubicin induces significant immunogenic cell death (ICD), initiating and amplifying tumor-specific T cell-mediated adaptive immunity. Through the synergistic activation of innate and adaptive immunity, the nanomicelle of this invention can effectively inhibit melanoma growth and overcome immunotherapy resistance, showing promising clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a nanomicelle FluDox-MIC, its preparation method and uses. Background Technology

[0002] While immune checkpoint blockade (ICB) targeting the PD-1 / PD-L1 axis has demonstrated significant clinical efficacy in melanoma, its therapeutic potential is severely limited by the emergence of resistance mechanisms. Key factors contributing to poor treatment response include low T-cell sensitization efficiency, insufficient lymphocyte recruitment to the tumor bed, and compensatory upregulation of alternative immune checkpoints in the immunosuppressive tumor microenvironment. Strong evidence suggests that combination therapies, such as dual checkpoint inhibition or a rational combination with targeted drugs / chemotherapy, can synergistically remodel the immune microenvironment, thereby overcoming resistance to PD-1 / PD-L1 blockade and increasing therapeutic benefit. Therefore, developing multi-faceted immunotherapeutic strategies targeting complementary pathways is an urgent and unmet need in clinical practice and translational research.

[0003] NK cells are core effector components of the innate immune system, playing an indispensable role in anti-tumor immune surveillance. As the first line of defense in immune defense, NK cells can recognize and eliminate abnormal cells without prior antigen sensitization, and their activity is strictly regulated by a dynamic balance between activating and inhibitory receptors. When activating signals dominate, NK cells induce tumor cell apoptosis by releasing perforin and granzymes, or by expressing death receptor ligands such as FAS ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL). In addition, NK cells secrete cytokines, including interferon, to recruit and activate T cells, thereby coordinating innate and adaptive immune responses. In the highly immunosuppressive tumor microenvironment, such as melanoma, the poliovirus receptor PVR (CD155), as a key immune checkpoint ligand, is highly expressed in various solid tumors, including melanoma, and mediates tumor immune escape by binding to the inhibitory receptor TIGIT on the surface of NK cells and T cells. Although current research has attempted to block the PVR-TIGIT axis with antibody drugs, the complexity of the tumor microenvironment makes the development of small-molecule modulators that can effectively induce PVR degradation on the surface of tumor cells a significant challenge. Furthermore, while flumatinib, a clinically used multi-target tyrosine kinase inhibitor, has shown some potential in inhibiting tumor cell proliferation, its role in regulating the immune microenvironment, particularly in the degradation of specific immune checkpoint ligands, remains largely unexplored. Additionally, its low bioavailability and dose-related toxicity when administered systemically limit its clinical application in synergistic immunotherapy for melanoma.

[0004] Therefore, how to effectively induce the degradation of PVR on the surface of tumor cells to relieve their immunosuppression of NK cells, and how to construct a multi-level synergistic immunotherapy regimen to improve the immunotherapy resistance of melanoma, are technical problems that urgently need to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a FluDox-MIC nanomicelle, its preparation method, and its applications. This nanomicelle uses mPEG-DSPE as a carrier to co-encapsulate flumatinib and doxorubicin. The core of this invention lies in the first-time discovery that flumatinib can induce the degradation of PVR (CD155) via the ubiquitin-proteasome pathway. After entering the tumor microenvironment, the nanomicelles release the drug. The released flumatinib induces the degradation of PVR on the cell surface, relieving PVR-mediated NK cell inhibitory signals and thus reactivating innate immune surveillance. Simultaneously, low-dose doxorubicin induces immunogenic cell death (ICD) in tumor cells, initiating an adaptive immune response. Through this dual activation of innate and adaptive immunity, this invention achieves synergistic immunotherapeutic effects, effectively overcoming immune escape and treatment resistance in melanoma.

[0006] One objective of this invention is to provide a FluDox-MIC nanomicelle with the function of degrading the immune checkpoint ligand PVR. This invention unexpectedly discovers that flumatinib possesses a novel function of mediating the ubiquitination and degradation of the PVR protein. By co-loading it with drugs that induce ICD, it aims to address the challenges of the lack of effective small-molecule PVR modulation mechanisms in the prior art and the severe immune escape from melanoma.

[0007] The raw materials for preparing the FluDox-MIC nanomicelles include fluoromatinib, doxorubicin, and mPEG-DSPE.

[0008] In some embodiments of the present invention, the mass ratio of flumatinib, doxorubicin, and mPEG-DSPE is 1:0.5~4:20~60, preferably 1:0.5~3:30~50.

[0009] In some embodiments of the present invention, the number-average molecular weight of the mPEG-DSPE is 0.8~3 kDa, preferably 1.5~2.5 kDa.

[0010] In some embodiments of the present invention, the average diameter of the nanomicelles, as determined by transmission electron microscopy, is 60-120 nm.

[0011] In some embodiments of the present invention, the average hydration diameter of the nanomicelles, as determined by dynamic light scattering, is 110-180 nm.

[0012] The second objective of this invention is to provide a method for preparing the above-mentioned nanomicelles, which achieves efficient encapsulation and spatiotemporal synergy of two active ingredients through a self-assembly process.

[0013] The above-mentioned method for preparing nanomicelles includes the following steps: the flumatinib and the mPEG-DSPE form solution A; the doxorubicin forms solution B; under ultrasonic conditions, solution A is added to solution B, the ultrasonic reaction is continued, and after post-processing, the nanomicelles FluDox-MIC are obtained.

[0014] In some embodiments of the present invention, the concentration of flumatinib in solution A is 0.2~1 mg / mL.

[0015] In some embodiments of the present invention, the concentration of doxorubicin in solution B is 0.01~0.2 mg / mL.

[0016] In some embodiments of the present invention, the power of the ultrasound is 20~60 W.

[0017] In some embodiments of the present invention, the post-treatment includes at least one step of stirring under ventilated conditions, ultrafiltration, and rinsing with ultrapure water, preferably including all three steps.

[0018] A third objective of this invention is to provide the use of flumatinib or the aforementioned nanomicelles in the preparation of a poliovirus receptor (PVR) protein degrader.

[0019] The fourth objective of this invention is to provide the use of the above-mentioned nanomicelles FluDox-MIC in the preparation of a medicament for treating melanoma.

[0020] The fifth objective of this invention is to provide a pharmaceutical composition comprising the above-mentioned nanomicelles FluDox-MIC and a pharmaceutically acceptable carrier.

[0021] In some embodiments of the present invention, the concentration of the nanomicelles in the pharmaceutical composition is 20-1000 ng / mL.

[0022] The sixth objective of this invention is to provide the use of the above-described pharmaceutical composition in the preparation of a medicament for use in combination with anti-PD-1 therapy for the treatment of melanoma. This medicament achieves synergistic effects with immune checkpoint blockade therapies (such as anti-PD-1 therapy) through a dual activation pathway of "innate immune desuppression" and "adaptive immune initiation."

[0023] The mechanism of action of this invention is as follows:

[0024] The FluDox-MIC of this invention is constructed through a simple self-assembly process. Upon entering the tumor microenvironment, FluDox-MIC rapidly dissociates into its two active components—flumatinib and DOX. Flumatinib penetrates the cell membrane and selectively targets PVR. By promoting the ubiquitination of PVR, flumatinib labels the receptor for proteasome degradation, thereby depleting PVR from the plasma membrane. The subsequent reduction of surface PVR weakens its inhibitory signal on NK cells, leading to strong NK cell activation and enhanced cytotoxicity. Simultaneously, intracellular DOX embeds into nuclear DNA and inhibits topoisomerase II, thereby disrupting DNA synthesis and function. These damages trigger a cascade of reactions in the endoplasmic reticulum and oxidative stress pathways, ultimately leading to the release of damage-associated molecular patterns (DAMPs). The resulting ICD promotes dendritic cell maturation and cross-presentation, indirectly authorizing antigen-specific CD8 T cells. Activated CD8 T cells... + T cells therefore upregulate granzyme B (GZMB) and IFN, exerting effective tumor-killing activity. When combined with CD8 blockade... + When combined with anti-PD-1 antibodies that target the PD-1-mediated inhibitory axis on T cells, this approach synergistically amplifies the function of cytotoxic T lymphocytes and achieves superior inhibition of tumor development.

[0025] Compared with the prior art, the present invention has the following significant advantages:

[0026] 1. Achieved targeted degradation of PVR and reactivation of innate immunity: Overcame the limitation of existing technologies that rely on antibodies to block PVR, and for the first time used small molecule flumatinib to effectively deplete cell surface PVR through the ubiquitin-proteasome pathway, thereby relieving immunosuppression of NK cells.

[0027] 2. A spatiotemporally coupled dual immune activation mechanism was constructed: cleverly combining flumatinib-induced NK cell activation (innate immunity) with DOX-induced ICD effects and CD8. + T-cell activation (adaptive immunity) is significantly more effective than monotherapy.

[0028] 3. Significant advantages in reducing toxicity and enhancing efficacy: Compared with free drugs, the nanomicelles of the present invention utilize acid-responsive release characteristics to achieve significant tumor growth inhibition under extremely low drug burden, effectively reducing systemic toxicity.

[0029] 4. Overcoming immunotherapy resistance and possessing extremely high clinical translational potential: This regimen, when used in combination with anti-PD-1 antibodies, has demonstrated outstanding synergistic tumor suppression effects, providing a new strategy for addressing the resistance problem of immune checkpoint blockade therapy in melanoma. Attached Figure Description

[0030] Figure 1 The difference in PVR gene expression between tumor and normal tissues in the pan-cancer cohort;

[0031] Figure 2 A heatmap of PVR gene expression in microregions of different cell types in a pan-cancer spatial transcriptome section;

[0032] Figure 3 Spatial expression analysis of PVR in cutaneous melanoma (SKCM);

[0033] Figure 4 To analyze the correlation between PVR expression, tumor cell abundance, and NK cell infiltration at the cellular level, as well as the transcriptional analysis results of PVR expression and NK cell infiltration across multiple independent SKCM cohorts;

[0034] Figure 5 A violin plot showing the differential expression of PVR between the R (responders) and NR (non-responders) groups in multiple SKCM immunotherapy clinical cohorts;

[0035] Figure 6 A schematic diagram of high-throughput virtual drug screening and a Venn diagram of the intersection of the top 35 candidate compounds;

[0036] Figure 7 Western blot analysis of PVR protein expression in A375 cells after intervention with nilotinib, flumatinib, pralatinib, and panatinib;

[0037] Figure 8 A visual model of the binding of flumatinib to PVR protein, and the results of Western blot analysis, flow cytometry and quantitative analysis of PVR protein expression in A375 cells treated with different concentrations of flumatinib.

[0038] Figure 9 This diagram illustrates the protein degradation pathway and shows the PVR protein level and PVR ubiquitination assessment results after flumatinib treatment of A375 cells.

[0039] Figure 10 The results of Gene Set Enrichment Analysis (GSEA) and the relative cell viability of A375 cells after treatment with different concentrations of flumatinib are presented.

[0040] Figure 11 The results of the colony-forming cell count, EdU binding qualitative and quantitative analysis, EdU staining pattern qualitative and quantitative analysis, and DNA content quantitative analysis of A375 cells treated with different concentrations of flumatinib are presented.

[0041] Figure 12A schematic diagram illustrating the construction of a subcutaneous melanoma xenograft model;

[0042] Figure 13 The curves showing changes in tumor volume and mouse body weight at different time points, as well as representative tumor photographs and tumor weight statistics collected on day 14 after treatment;

[0043] Figure 14 Representative images and quantitative results of PVR detection in B16F10 tumor masses based on flow cytometry;

[0044] Figure 15 CD45 from B16F10 tumor masses based on flow cytometry + Detection of NK1.1 in TIL + Representative figures and quantitative results from B16F10 tumor masses, NK1.1 + Detection of CD107a in cells + Representative figures and quantitative results;

[0045] Figure 16 For flow cytometry sorting strategies and various T cells (CD45) in B16F10 tumor tissue + CD3 + CD8 + and GZMB + Quantitative analysis results of infiltration level;

[0046] Figure 17 TEM images and DLS analysis results of FluDox-MIC;

[0047] Figure 18 The UV-Vis absorption (Abs) and fluorescence (FL) spectra of free DOX and FluDox-MIC are shown.

[0048] Figure 19 A schematic diagram of the pH-responsive degradation of FluDox-MIC and the release behavior of flumatinib and DOX under different conditions;

[0049] Figure 20 The molecular cluster changes of flumatinib and DOX during a 100 ns simulation are shown (green ball: flumatinib; blue ball: DOX).

[0050] Figure 21 The results of RMSD, SASA, bond number, and binding energy analysis in FluDox-MIC are shown.

[0051] Figure 22 The results of flow cytometry and average quantitative fluorescence analysis of A375 cells at different drug concentrations or incubation times;

[0052] Figure 23 The relative cell viability of A375 cells treated with different concentrations of different drugs;

[0053] Figure 24 Western blot analysis of membrane PVR protein expression in A375 cells treated with different drugs, and crystal violet staining analysis of colony-forming cell number;

[0054] Figure 25 Qualitative and quantitative analysis results of EdU staining patterns in A375 cells treated with different drugs;

[0055] Figure 26 Western blot analysis, representative immunofluorescence images, and Pearson correlation coefficient (PCC) analysis results of HMGB1 protein expression in the membrane of A375 cells treated with different drugs;

[0056] Figure 27 To establish a subcutaneous melanoma tumor model in female C57BL / 6 mice, a schematic diagram of related treatment regimens, and photographs of tumor volume measured at different time points and representative tumors;

[0057] Figure 28 Representative flow cytometry maps and quantitative analysis results of PVR expression levels, NK cell and T cell subset infiltration and activation status in B16F10 tumor masses of each treatment group;

[0058] Figure 29 Representative sections and quantitative analysis results of immunohistochemical staining for DAPI, NK1.1 (yellow), CD8 (red) and GZMB (green) expression after various treatments for B16F10 tumors;

[0059] Figure 30 This is a schematic diagram illustrating the self-assembly process of FluDox-MIC and its mechanism of inhibiting tumor development. Detailed Implementation

[0060] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0061] The following content describes specific implementation methods of the cell experiments involved in this invention.

[0062] 1. Cell culture and treatment

[0063] Human A375 and mouse B16F10 melanoma cell lines were cultured in DMEM and RPMI-1640, respectively. Both media were supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Mycoplasma was not detected in any of the cell lines by routine testing. All cells were cultured in a 37°C incubator containing 5% CO2. Treatments were administered at specific concentrations and durations as required for each experiment. Cells were treated with 2.5 μg / mL flumatinib for 18 h, followed by co-treatment with 50 μM chloroquine (CQ) or 10 μM MG132 for 6 h. Total PVR protein expression was detected after incubation with 1 μg / mL or 2.5 μg / mL flumatinib for 24 h. To assess the expression of PVR and HMGB1, cells were treated for 24 hours with 300 ng / mL FluDox-MIC (Dox equivalent), 300 ng / mL free Dox, or 250 nM (140.7 ng / mL) free flumatinib. All compounds were diluted in complete culture medium and administered at the indicated concentrations and time points.

[0064] 2. Cell viability assessment

[0065] A375 cells were seeded into 96-well plates and allowed to adhere before being treated with free flumatinib, doxorubicin, or FluDox-MIC nanoparticles (0, 31.25, 62.5, 125, 250, 500, 1000 ng / ml) for 24 h. After drug exposure, 100 μL of a CCK-8 / DMEM mixture (CCK-8:DMEM = 1:9, v / v) was added to each well and incubated at 37°C for 2 h. The optical density (OD) at 450 nm was then measured using a microplate reader, and each condition was evaluated in 5 replicate wells. Cell viability was calculated using the following formula: Cell viability (%) = (OD_experimental - OD_control) / (OD_control) × 100%.

[0066] 3. Colony Formation Experiment

[0067] Collect A375 cells, resuspend them, and culture them at 3 × 10⁻⁶. 4 Cell suspensions were prepared at a density of cells / mL. Then, 100 μL of the cell suspension was seeded into each well of a 6-well cell culture plate. After cell attachment, the dose-dependent effects were assessed by treatment with 1 μg / mL and 2.5 μg / mL flumatinib, or by comparison with flumatinib, doxorubicin, and FluDox-MIC. After 7 days of culture, cells were stained with crystal violet and then analyzed.

[0068] 4. Cell proliferation experiment

[0069] Cell proliferation was assessed using the Alexa Fluor 488 (Beyotime, China) kit. Specifically, A375 cells were seeded into 12-well plates and treated with 1 μg / mL or 2.5 μg / mL flumatinib for dose-dependent assessment, or with flumatinib, doxorubicin, and FluDox-MIC for 24 h. Following treatment, cells were incubated with EdU for 2 h. Cells were then treated sequentially with the click reaction mixture and Hoechst 33342 at room temperature, following the manufacturer's instructions. Observation and imaging were performed using a fluorescence microscope. Additionally, samples were analyzed by flow cytometry based on fluorescence detection to quantify EdU incorporation.

[0070] 5. Cell cycle analysis

[0071] Cell cycle distribution was analyzed using a cell cycle and apoptosis analysis kit (Beyotime, China). Specifically, A375 cells were seeded in 6-well plates and treated with 1 μg / mL or 2.5 μg / mL flumatinib for 24 h to assess dose-dependent effects, or with flumatinib, doxorubicin, and FluDox-MIC for 24 h. After treatment, cells were collected and fixed in 70% ethanol for 2 h. Subsequently, the fixed cells were stained with propidium iodide (PI) and analyzed by flow cytometry according to the manufacturer's instructions.

[0072] The following content describes the preparation of FluDox-MIC nanomicelles according to this invention, as well as the performance characterization and analysis of FluDox-MIC.

[0073] 1. To confirm the correlation between pan-cancer PVR upregulation and immune rejection and impaired immunotherapy response.

[0074] A comprehensive analysis of transcriptome data from 29 cancer types in the TCGA database revealed the pan-cancer expression profile of PVR, such as... Figure 1 As shown, the results indicate that PVR is significantly upregulated in various solid tumors compared to corresponding normal tissues, with overexpression being particularly prominent in malignant tumors such as cutaneous melanoma (SKCM). This suggests that aberrant PVR expression may be a common molecular feature of various cancers.

[0075] To further characterize its spatial distribution within the tumor microenvironment, this invention integrates spatial transcriptome data from 13 cancer types, such as... Figure 2 As shown, in most cohorts, PVR expression is mainly enriched in malignant cells.

[0076] Further analysis revealed that PVR was significantly overexpressed in the tumor core region, while its level was extremely low in the surrounding stroma or normal tissue. Comparative analysis of malignant (Mal) and non-malignant (nMal) regions showed the following results: Figure 3 As shown in the figure, this unique spatial structure suggests that PVR may play a key role in crosstalk between tumor cells and the immune microenvironment, and its preferential enrichment in malignant regions may promote tumor immune escape. This suggests that PVR could serve as a potential therapeutic target or spatial biomarker for SKCM.

[0077] Further analysis of the correlation at the cellular level yielded the following results: Figure 4 As shown, PVR levels were significantly positively correlated with tumor cell abundance and negatively correlated with NK cell infiltration. The most significant “positive correlation with tumor cells / negative correlation with NK cells” phenomenon was observed in the SKCM cohort. Immune infiltration assessments across multiple independent transcriptome SKCM cohorts consistently showed a significant inverse relationship between PVR expression and NK cell infiltration. This means that elevated PVR expression is not only inherent to tumor cells but may also contribute to an immunosuppressive microenvironment characterized by NK cell reduction.

[0078] In addition, analyses of clinical immunotherapy cohorts, such as Figure 5 As shown, in multiple independent human SKCM datasets, PVR expression was significantly higher in non-responders (NR) than in responders (R), and the upregulation of PVR may confer resistance to immune checkpoint blockade (ICB) therapy.

[0079] Therefore, based on the above pan-cancer PVR and bioinformatics analysis focusing on SKCM, the key role of PVR in tumor-mediated immunosuppression and immune escape was established, and its overexpression is associated with an altered immune environment characterized by impaired NK cell activity and dysfunction and impaired response to immunotherapy.

[0080] 2. The degradation pathway of PVR mediated by flumatinib was confirmed.

[0081] To identify high-affinity ligands targeting the immune checkpoint protein PVR, this invention employed a structure-based virtual screening using a key compound library containing tyrosine kinase inhibitors and FDA-approved anticancer drugs. After standard ligand and receptor preparation (including structure optimization, protonation state assignment, and binding site definition), the compound libraries were docked to the PVR domains. Docking postures were evaluated based on binding affinity and interaction patterns, and the top 35 compounds from each library were selected for intersection analysis based on docking scores. The results are as follows: Figure 6As shown, this process identified four high-potential kinase inhibitors: nilotinib, flumatinib, pralsetinib, and panatinib.

[0082] To verify the functional effects of the candidate compounds, this invention treated A375 human melanoma cells with these four drugs for 24 hours, extracted cellular proteins, and performed Western blot (WB) analysis. The results are as follows: Figure 7 As shown, compared with the other three drugs, flumatinib treatment significantly reduced PVR protein levels, suggesting that the binding of flumatinib to PVR may trigger an atypical protein homeostasis regulation mechanism.

[0083] To elucidate the unique degradation effect of flumatinib, this invention conducted a detailed comparative analysis of the ligand binding mechanisms, such as... Figure 8 As shown, flumatinib forms a specific hydrogen bond with Asn147 of PVR, a residue located within a key flexible loop involved in protein-protein interactions. Furthermore, flumatinib participates in an extended hydrophobic network mediated by residues such as Lys144, Phe40, and Ala143. These results indicate that this unique binding mode induces local conformational rearrangement, potentially exposing hidden degraders or impairing global protein stability, thereby guiding PVR towards degradation. Moreover, flow cytometry and Western blotting consistently showed that flumatinib treatment significantly reduced membrane localization and total PVR protein levels, with the inhibitory effect being dose-dependent. Figure 8 As shown in the figure. These data indicate that flumatinib not only reduces intracellular PVR levels but also decreases its exposure as an immune checkpoint ligand on the cell surface.

[0084] Since intracellular proteins are mainly degraded through the autophagy-lysosomal pathway and the ubiquitin-proteasome pathway, such as Figure 9 As shown, this invention further investigated the molecular mechanism of flumatinib degradation of PVR. In the presence of the autophagy / lysosome inhibitor chloroquine (CQ) or the proteasome inhibitor MG132, PVR protein levels were detected after treating A375 cells with flumatinib. Specifically, A375 cells were pretreated with flumatinib (2.5 μg / ml), then treated with chloroquine (CQ) (50 μM) and MG132 (10 μM) for 6 hours, and PVR protein levels were detected by Western blotting. The results are as follows. Figure 9As shown, MG132 significantly rescued the reduction in PVR protein induced by flumatinib, while CQ had no significant effect, indicating that flumatinib mainly reduces PVR levels through the ubiquitin-proteasome pathway rather than the autophagy-lysosome pathway. To further confirm that flumatinib induces PVR degradation through the UPS pathway, this invention performed immunoprecipitation on PVR after flumatinib treatment and assessed its ubiquitination level. Specifically, A375 cells were incubated with flumatinib (2.5 μg / mL) for 18 hours, followed by co-incubation with chloroquine (CQ, 50 μM) or MG132 (10 μM) for another 6 hours. Cellular proteins were then extracted and PVR protein levels were detected by Western blotting. PVR was immunoprecipitated with an anti-PVR antibody, and the immunoprecipitate was detected with an anti-ubiquitin (UB) antibody. The results showed that flumatinib significantly enhanced PVR ubiquitination compared to the control group. Combined with early molecular docking data, the results suggest that flumatinib may bind to PVR or its regulatory complex, altering the balance between PVR and specific E3 ligases or deubiquitinating enzymes, thereby promoting its ubiquitination and proteasome degradation.

[0085] In summary, these results demonstrate that flumatinib reduces PVR levels on the surface of melanoma cells by promoting its ubiquitination and inducing proteasome-dependent degradation, providing structural and functional evidence for the pharmacological targeting of PVR by flumatinib. These experimental results confirm that flumatinib can specifically degrade PVR proteins on the surface of melanoma cells via the ubiquitination pathway. The discovery of this novel mechanism provides a solid pharmacological and molecular biological basis for the subsequent construction of nanomedicines (FluDox-MIC) that target PVR and reverse the tumor immunosuppressive microenvironment.

[0086] 3. To confirm the inhibitory effect of flumatinib on melanoma cell proliferation and its ability to remodel the PVR-mediated innate immune microenvironment.

[0087] Melanoma, a highly malignant solid tumor, is characterized by the persistent overactivation of proliferation signaling pathways. Numerous studies have shown that aberrant activation of multiple PTKs plays a crucial role in driving melanoma development and progression by mediating key proliferation cascades, including the MAPK and PI3K / AKT pathways. Given that flumatinib is a potent TKI, it is hypothesized that it may possess intrinsic antiproliferative activity against melanoma cells. Combined with proteomics analysis, this invention focuses on cellular processes related to cell cycle regulation and DNA metabolism. Results are as follows... Figure 10As shown, genes significantly downregulated after flumatinib treatment were highly enriched in these functional modules, indicating broad inhibition of cell cycle-related processes. Notably, all three core modules (cell cycle progression, mitosis, and DNA replication) were collectively inhibited, suggesting that flumatinib exerts a systemic remodeling effect on proliferation-related networks, rather than acting on a single molecular target. To validate this hypothesis, this invention evaluated the cytotoxicity of flumatinib in A375 human melanoma cells. CCK-8 assay results are shown below. Figure 10 As shown, it exhibited significant dose-dependent inhibition of cell proliferation, with an IC50 value of 9.13 ug / mL.

[0088] In addition, such as Figure 11 As shown, crystal violet staining analysis of A375 cells treated with different concentrations of flumatinib revealed that flumatinib significantly reduced the long-term colony-forming potential of A375 cells in a dose-dependent manner, indicating a sustained inhibition of self-renewal and population expansion capabilities. Simultaneously, flow cytometry analysis of EdU binding and mean fluorescence quantification in A375 cells treated with different concentrations of flumatinib, as well as qualitative and quantitative analysis of EdU staining patterns, showed that EdU / Hoechst33258 double staining provided direct evidence of reduced DNA replication, indicating that flumatinib treatment significantly reduced EdU binding. The proportion of U-positive cells decreased significantly, which is consistent with the downregulation of DNA replication-related pathways revealed by proteomics data. Furthermore, quantitative analysis of cell cycle DNA content in A375 cells treated with different concentrations of flumatinib using flow cytometry showed that flumatinib increased the G1 phase cell population in a concentration-dependent manner while reducing the proportion of S phase cells. This indicates that it primarily inhibits DNA synthesis by blocking the G1 / S phase transition, thereby limiting mitosis and cell expansion. This aligns with previous findings that TKIs typically induce G1 phase arrest by inhibiting the Cyclin-CDK axis or upstream growth factor signaling. These results suggest that flumatinib may exert its anti-proliferative effect in melanoma cells through a similar mechanism.

[0089] To further investigate the clinical potential of flumatinib, this invention established a subcutaneous melanoma xenograft model in immunocompetent C57BL / 6 mice, such as... Figure 12 As shown, with n=5 mice in each group, the in vivo antitumor efficacy and potential immunological effects were evaluated. Considering the poor water solubility of flumatinib and the fact that its clinical formulation is an oral drug, a gavage administration route was adopted to simulate the clinical administration route as closely as possible (dose of 75 mg / kg, once daily for 8 consecutive days). Figure 13As shown, the results indicated that the tumor volume in the treatment group was moderately reduced compared to the control group, and no significant difference in mouse body weight was observed throughout the experiment. Flumatinib exhibited certain anti-tumor activity in vivo and was well-tolerated systemically. The experimental results suggest that although flumatinib monotherapy demonstrates clear dual potential for anti-proliferation and immunomodulation, its clinical benefit in the highly immunosuppressive melanoma microenvironment is limited by the delivery efficiency of single drugs and complex compensatory immune escape mechanisms, leaving room for further improvement. This provides important experimental evidence and technological entry points for developing nanosystems capable of achieving synergistic multi-drug delivery and improving pharmacokinetic characteristics.

[0090] To determine whether flumatinib targets PVR in vivo, this invention prepared single-cell suspensions from excised tumor tissue for flow cytometry analysis. The representative profile and quantification of PVR in B16F10 tumor masses based on flow cytometry (n=4 mice per group) are as follows: Figure 14 As shown, flumatinib treatment significantly downregulated PVR expression on the surface of tumor cells, consistent with in vitro characterization and proteomics data. These results demonstrate that flumatinib can effectively reach tumor sites and inhibit PVR expression, thereby remodeling PVR-related immune checkpoint pathways in the native tumor microenvironment. This observation links experimental evidence from molecular docking and cell studies with animal models, supporting the feasibility of flumatinib mediating intracellular immunomodulation through PVR degradation. In tumors, PVR overexpression preferentially activates the TIGIT-mediated inhibitory pathway, thereby contributing to an immunosuppressive microenvironment and impairing the effector functions of NK cells and T cells. Based on this, this invention experimentally validates that flumatinib may restore anti-tumor immunity by downregulating tumor-associated PVR and relieving immunosuppression. Based on flow cytometry, CD45 from B16F10 tumor masses... + Detection of NK1.1 in TIL + Representative profiles and quantifications (n=4 mice per group), and NK1.1 from B16F10 tumor masses based on flow cytometry. + Detection of CD107a in cells + Representative profiles and quantitative results (n=4 mice per group), such as Figure 15As shown, there was no significant difference in NK cell infiltration between the two groups, but NK cell activation was significantly enhanced, manifested by increased CD107a expression. Given that CD107a is a classic marker of NK cell degranulation, these results indicate that flumatinib primarily restored the cytotoxic function of NK cells, rather than increasing their number. This is consistent with the mechanism proposed in this invention, namely, that flumatinib relieves TIGIT-mediated inhibition by inhibiting PVR expression, thereby enhancing innate immune activation in vivo. In contrast, T cell-related changes were relatively limited, based on flow cytometry detection of CD45 in B16F10 tumor tissue. + TILs, CD45 + CD3 in TILs + Cells, CD3 + CD8 in TILs + T cells and CD3 + GZMB in TILs + CD8 + Quantitative results of T cells (4 mice per group) showed that, whether CD8 + No significant differences were observed between groups in T cell infiltration, activation levels, or effector phenotypes. Figure 16 These results indicate that, under current experimental models and administration conditions, flumatinib-induced immune remodeling primarily involves innate immune components (such as NK cells) and fails to adequately trigger a broad adaptive immune response. These results confirm that although flumatinib targets the PVR-TIGIT axis, T cell function is simultaneously regulated by multiple inhibitory pathways—including PD-1 / PD-L1, LAG-3, TIM-3, Treg cells, and MDSCs—therefore, partial blockade of PVR alone is insufficient to broadly remodel the T cell landscape.

[0091] In summary, flumatinib not only directly inhibits melanoma cell proliferation by inducing G1 / S phase arrest and suppressing DNA replication, but also effectively targets PVR to relieve its inhibitory effect on NK cells, thereby enhancing the cytotoxic activity of innate immune cells and promoting immune activation. However, in vivo experiments show that flumatinib monotherapy is insufficient to induce strong T cell infiltration and activation, resulting in a relatively limited adaptive immune response during anti-tumor development. In vivo experiments further reveal the complexity of the melanoma microenvironment: while flumatinib monotherapy can effectively activate innate immune components (such as NK cells), it faces challenges in inducing a broad and sustained adaptive immune response. This imbalance between "innate immune activation and weak adaptive immunity" points to the inherent bottleneck of single-target therapy. Therefore, this invention aims to integrate innate immune desuppression and adaptive immune initiation (such as DOX-induced ICD effects) by constructing FluDox-MIC nanomicelles, thereby coordinating and completing the immune activation loop in spatiotemporal space to achieve superior inhibition of tumor development.

[0092] 4. Synthesis of FluDox-MIC

[0093] To counteract the inhibitory effect of high tumor PVR expression on NK cell activity and thus synergistically enhance the efficacy of immunotherapy, this invention synthesizes flumatinib and doxorubicin (DOX) into FluDox-MIC via a one-pot self-assembly method. Furthermore, unless otherwise specified, all raw materials used in this invention are commercially available.

[0094] The general preparation steps of the FluDox-MIC nanomicelles of the present invention are as follows: Flumatinib and mPEG-DSPE are dissolved in an appropriate amount of organic solvent (such as tetrahydrofuran) to form solution A (the concentration of flumatinib is preferably 0.2~1 mg / mL); doxorubicin is dissolved in ultrapure water to form solution B (the concentration of doxorubicin is preferably 0.01~0.2 mg / mL). Under ultrasonic conditions of 20~60 W, solution A is added to solution B. After the ultrasonic reaction is completed, the organic solvent is removed by stirring under ventilation. Finally, the nanomicelles are obtained by ultrafiltration and washing with ultrapure water.

[0095] Example 1

[0096] This embodiment provides a FluDox-MIC nanomicelle, the preparation steps of which include:

[0097] 0.5 mg of flumatinib and 20 mg of mPEG-DSPE (molecular weight 2 kDa) were added to 1 mL of tetrahydrofuran to form solution A; 1 mg of doxorubicin was dissolved in 9 mL of ultrapure water to form solution B. Under ultrasonic conditions (35 w, 5 min), solution A was added to solution B in one step. After the ultrasonic reaction was completed, the reaction solution was placed in a fume hood and stirred for 12 h (300 rpm) to remove tetrahydrofuran. Finally, the reaction solution was ultrafiltered (ultrafiltration tube molecular weight: 30 kDa, 3500 rpm, 15 min), and washed three times with ultrapure water to obtain the FluDox-MIC nanomicelles.

[0098] Example 2

[0099] This embodiment provides a FluDox-MIC nanomicelle, the preparation steps of which include:

[0100] 0.5 mg of flumatinib and 20 mg of mPEG-DSPE (molecular weight 2 kDa) were added to 1 mL of tetrahydrofuran to form solution A; 0.5 mg of doxorubicin was dissolved in 9 mL of ultrapure water to form solution B. Under ultrasonic conditions (35 w, 5 min), solution A was added to solution B in one step. After the ultrasonic reaction was completed, the reaction solution was placed in a fume hood and stirred for 12 h (300 rpm) to remove tetrahydrofuran. Finally, the reaction solution was ultrafiltered (ultrafiltration tube molecular weight: 30 kDa, 3500 rpm, 15 min), and washed three times with ultrapure water to obtain the FluDox-MIC nanomicelles.

[0101] Example 3

[0102] This embodiment provides a FluDox-MIC nanomicelle, the preparation steps of which include:

[0103] 0.5 mg of flumatinib and 15 mg of mPEG-DSPE (molecular weight 2 kDa) were added to 1 mL of tetrahydrofuran to form solution A; 1 mg of doxorubicin was dissolved in 9 mL of ultrapure water to form solution B. Under ultrasonic conditions (35 w, 5 min), solution A was added to solution B in one step. After the ultrasonic reaction was completed, the reaction solution was placed in a fume hood and stirred for 12 h (300 rpm) to remove tetrahydrofuran. Finally, the reaction solution was ultrafiltered (ultrafiltration tube molecular weight: 30 kDa, 3500 rpm, 15 min), and washed three times with ultrapure water to obtain the FluDox-MIC nanomicelles.

[0104] 5. Characterization of the physical and chemical properties of FluDox-MIC

[0105] The morphology of FluDox-MIC obtained in Example 1 was observed by transmission electron microscopy (TEM), showing that FluDox-MIC has a bowl-shaped structure with an average diameter of 80-100 nm. The average hydrated diameter of the prepared FluDox-MIC was measured to be approximately 142 nm by dynamic light scattering (DLS). Due to the presence of the PEG hydration layer, this value is slightly higher than the value measured in the TEM image. Figure 17 ).

[0106] To further confirm the presence of different components, the present invention investigated the photophysical characteristics of the FluDox-MIC in Example 1, such as... Figure 18 As shown. UV-Vis spectrophotometric analysis revealed similar spectral characteristics among flumatinib, DOX, and FluDox-MIC, exhibiting characteristic absorption bands at 276 nm (flumatinib) and 488 nm (DOX), thus confirming the structural integrity of the payload. Furthermore, based on the calibration curve, the ratio of flumatinib to DOX in FluDox-MIC was determined to be 2:1. Free DOX molecules showed strong fluorescence intensity at approximately 600 nm, while this fluorescence band was significantly weakened in FluDox-MIC, further confirming their precise structure.

[0107] The potent synergistic antitumor efficiency of FluDox-MIC depends on the release behavior of flumatinib and DOX from the self-assembled nanocarrier. Here, the invention simultaneously investigated the release of flumatinib and DOX from the FluDox-MIC of Example 1 under different conditions, and the results are as follows... Figure 19 As shown, compared to only 9.17% of flumatinib release within 48 hours under neutral conditions, flumatinib release (assessed by UV-Vis spectroscopy) increased significantly to 77.42% during the same period at pH 5.2. Similarly, DOX release was also assessed by UV-Vis spectroscopy, and the trend of DOX release behavior was consistent with that of flumatinib. The results indicate that decreasing pH accelerates the drug release behavior of FluDox-MIC. This acid-accelerated drug release mechanism may be due to the protonation of the carboxyl group of DOX under acidic conditions. Therefore, the protonated carboxyl group of DOX under acidic conditions weakens the interaction between flumatinib and DOX, leading to drug release. This will minimize systemic toxicity caused by premature leakage and simultaneously promote precise spatiotemporal therapy at tumor sites.

[0108] To verify the self-assembly structural characteristics and driving force of FluDox-MIC, this invention used molecular dynamics (MD) simulations (Gromacs program) to analyze the co-assembly dynamics of the flumatinib / DOX system within 0-100 ns. Cluster analysis of the simulated trajectories (RMSD cutoff value set at 1.2 nm) showed that flumatinib and DOX molecules spontaneously and continuously aggregated under intermolecular interactions, eventually forming a stable spherical nanocluster structure. Figure 20 Its internal interaction network is mainly composed of weak hydrogen bonds between flumatinib and DOX, as well as hydrogen bonds and π-π stacking between flumatinib molecules.

[0109] Analysis of kinetic and thermodynamic parameters of the simulation process ( Figure 21 The high stability of the assembly was further confirmed: (1) The root mean square deviation (RMSD) of the system increased briefly in the initial stage and then quickly stabilized, with an average RMSD value of 7.569 ± 1.834 nm, indicating that a structurally stable composite aggregate system was formed; (2) The solvent accessible surface area (SASA) of the system decreased significantly with the simulation and eventually stabilized (average value of 137.182 ± 47.350 nm). 2 (2) The system formed a highly dense aggregate structure; (3) The average number of hydrogen bonds in the system reached 34.408, which greatly enhanced the intermolecular bonding strength; (4) The total interaction energy showed a significant decreasing trend with simulation time and approached the thermodynamic steady state, with the average interaction energy stabilizing at approximately -6625.74 kcal / mol. The above high negative energy characteristics are highly consistent with the conformational changes, objectively proving from both kinetic and thermodynamic perspectives that the fluoromatinib and DOX binary co-assembled system of this invention has excellent structural stability.

[0110] 6. Confirm the anti-tumor efficacy of FluDox-MIC in melanoma cells.

[0111] Thanks to the superior physicochemical properties of FluDox-MIC, its potent synergistic antitumor efficacy largely depends on the efficiency of its internalization into cells. Therefore, this invention systematically investigated the endocytosis kinetics of FluDox-MIC in melanoma cells as described in Example 1. Figure 22 As shown, FluDox-MIC exhibits significantly enhanced cellular uptake capacity compared to free DOX. Further studies confirmed that this internalization process is distinctly time- and dose-dependent, indicating that the self-assembled nanostructures effectively facilitate the transmembrane transport of therapeutic payloads via endocytosis, thus laying a solid biological foundation for subsequent synergistic therapy.

[0112] Furthermore, this invention compared the cytotoxicity profiles of flumatinib, free Dox, and FluDox-MIC from Example 1 in A375 cells, such as... Figure 23 As shown, flumatinib monotherapy did not exhibit significant cytotoxicity at low concentrations (0–1 μg / mL), consistent with its previously obtained IC50 values ​​from CCK-8 assays. This indicates that at lower exposure doses, flumatinib exerts its effects primarily through signal modulation rather than direct cytotoxicity. In contrast, both free Dox and FluDox-MIC exhibited typical dose-dependent cell proliferation inhibition. However, at equivalent Dox concentrations, FluDox-MIC resulted in significantly stronger cell viability inhibition: the IC50 of FluDox-MIC was 387.6 ng / mL, only about half that of free Dox (IC50 = 785.9 ng / mL). This suggests that FluDox-MIC possesses higher functional potency under matched drug loading conditions. Mechanistically, this enhancement may stem from the altered uptake and distribution patterns of the conjugated form, allowing for more efficient spatial and temporal co-delivery and accumulation of Dox and flumatinib within the cell, thereby increasing the effective drug exposure.

[0113] Based on the aforementioned verification that flumatinib promotes PVR degradation via UPS, this invention further investigates whether FluDoxMIC from Example 1 can amplify this immune checkpoint regulatory effect at the same low dose concentration. Western blot analysis results are as follows: Figure 24 As shown, while low-concentration flumatinib monotherapy only marginally inhibited PVR expression, FluDox-MIC, administered at the same flumatinib equivalent, significantly downregulated PVR levels. In contrast, Dox monotherapy had almost no effect on PVR expression. These results indicate that FluDox-MIC demonstrates a significant efficacy advantage in modulating PVR at low doses, achieving a greater downregulation with a lower drug burden. This characteristic is particularly promising for in vivo applications: it may enable stronger immune checkpoint modulation without significantly increasing systemic toxicity and creates opportunities to exert both cytotoxic and immunomodulatory effects within a lower therapeutic dose window. Colony formation assays further confirmed this advantage: at low concentrations, flumatinib alone had limited effects on long-term colony-forming ability, while FluDox-MIC significantly reduced colony number and size, indicating a deeper and more durable inhibition of proliferative potential.

[0114] Furthermore, DAPI staining indicates live cells, while EdU staining highlights DNA molecules undergoing replication; the dual staining results obtained via immunofluorescence microscopy are as follows: Figure 25As shown in the figure, the FluDox-MIC treatment group in Example 1 exhibited the most significant reduction in EdU-positive cells, reflecting a stronger inhibition of short-term DNA synthesis and proliferation activity. In summary, these data indicate that, in vitro, compared to monotherapy, FluDox-MIC not only enhances cytotoxicity and inhibition of tumor cell proliferation but also strengthens the negative regulation of PVR at low doses, highlighting its multi-layered synergistic advantages.

[0115] Dox, a classic anthracycline chemotherapy drug, can trigger ICD in tumor cells. This ICD process promotes the release of DAMPs (such as HMGB1). These signals are recognized by the immune system, thereby triggering a potent anti-tumor immune response. To assess whether FluDox-MIC in Example 1 affected the ICD-inducing ability of Dox, A375 cells were treated with flumatinib, free Dox, or FluDox-MIC for 24 hours. Western blot analysis and confocal immunoassay results are shown below. Figure 26 As shown: Western blot analysis revealed that flumatinib did not alter HMGB1 expression, while both Dox and FluDox-MIC significantly reduced intracellular HMGB1 levels, with FluDox-MIC showing a more pronounced decrease, suggesting enhanced nuclear-to-cytoplasmic translocation and robust ICD activation. Consistent with these results, confocal immunoassay demonstrated a significant reduction in HMGB1 fluorescence after Dox or FluDox-MIC treatment, which was more pronounced in the FluDox-MIC group, accompanied by weakened nuclear signaling, supporting HMGB1 translocation and extracellular release, and further confirming the superior ICD activity of FluDox-MIC.

[0116] In summary, these results indicate that FluDox-MIC demonstrates a significant advantage over monotherapy in melanoma cell models in the following three aspects: (1) at the same Dox dose, it enhances cytotoxicity and exhibits stronger inhibition of DNA replication and colony formation, indicating higher pharmacodynamic efficiency; (2) by promoting the translocation and release of HMGB1, it preserves and enhances ICD induction, thereby supporting subsequent immune activation; (3) at low doses, it further downregulates PVR, compensating for the limited efficacy of flumatinib alone. These results collectively demonstrate that FluDoxMIC can achieve spatiotemporally coordinated drug delivery and a synergistic triple-action antitumor mechanism within target cells, outperforming traditional free drug combinations in both efficiency and functional efficacy.

[0117] 7. Confirm the synergistic anti-tumor effect of FluDox-MIC combined with anti-PD-1 therapy.

[0118] Based on previous characterization results, flumatinib primarily activates innate immunity through the PVR-NK cell axis, but in improving CD8... + Limited efficacy in T-cell function treatment; therefore, this invention further proposes a combination therapy strategy utilizing FluDox-MIC. Given that FluDox-MIC exhibits enhanced PVR downregulation and induces ICD in vitro, this invention experimentally verifies that FluDox-MIC not only amplifies innate immunity but also effectively activates adaptive immunity. Based on this, this invention combines the FluDox-MIC from Example 1 with an anti-PD-1 antibody, a standard therapy for melanoma, anticipating a synergistic anti-tumor effect from simultaneously blocking different immunosuppressive pathways. To test this hypothesis, subcutaneous melanoma tumors were established in immunocompetent C57BL / 6 mice, with tumor volumes reaching 50-100 mm². 3 They were randomly assigned to six treatment groups. The specific dosing regimen was as follows: free flumatinib (1.6 mg / kg), free doxorubicin (3 mg / kg), or FluDox-MIC nanomicelles (3 mg / kg, based on Dox equivalents) were administered intraperitoneally (ip) every two days; the combination therapy group additionally received an anti-PD-1 monoclonal antibody (100 μg / animal, dissolved in 100 μL PBS) intraperitoneally every three days. Treatment regimens and tumor growth results are as follows. Figure 27 As shown, there was no significant difference in tumor growth between the flumatinib monotherapy group and the control group. This indicates that at the current extremely low doses (only about 1 / 40th of the conventional effective dose), free flumatinib cannot exert an effective anti-tumor effect. In contrast, at equivalent extremely low doses of flumatinib, FluDox-MIC showed significantly superior tumor-suppressive activity compared to all monotherapy groups, strongly demonstrating the strong targeting enrichment capacity and low-dose synergistic potential of the nanomicelles of this invention. More importantly, compared to FluDox-MIC alone, the combination of FluDox-MIC with anti-PD-1 therapy further enhanced the tumor-suppressive effect, strongly supporting the hypothesis of complementary mechanisms in this invention.

[0119] To assess the safety profile of this treatment strategy, the present invention evaluated systemic toxicity in mice. Results showed no significant differences in body weight fluctuations among the treatment groups, no abnormalities in serum parameters of cardiac, hepatic, or renal function, and no significant histopathological damage in major organs was detected by H&E staining, indicating that the combined treatment regimen was generally well-tolerated at the administered doses.

[0120] To confirm the immunological mechanism behind this superior anti-tumor effect, this invention performed flow cytometry analysis on tumor tissue. Figure 28) and multiplex immunofluorescence staining analysis ( Figure 29 Consistent with in vitro studies, FluDox-MIC in Example 1 more effectively inhibited PVR expression compared to flumatinib monotherapy. Although the differences in NK cell infiltration rates among the control, flumatinib, Dox, and FluDox-MIC groups were small, FluDox-MIC treatment significantly enhanced NK cell degranulation activity, manifested by a significant upregulation of CD107a expression. The addition of anti-PD-1 to FluDox-MIC further increased intratumoral NK cell infiltration, compensating for FluDox-MIC's deficiency in increasing NK cell numbers, indicating that alleviating PD-1 / PD-L1-mediated immunosuppression helps mobilize innate immunity. At the adaptive immunity level, compared to monotherapy, FluDox-MIC significantly increased intratumoral CD8+. + T cell infiltration and significantly increased GZMB + CD8 + and IFN + CD8 + The proportion of T cells demonstrates its unique advantage in remodeling T cell effector function. The addition of PD-1 blockers further amplifies CD8... + The cytotoxic efficacy of T cells clearly demonstrated the synergistic effect between ICD-induced immune activation and PD-1 pathway inhibition relief. Significantly enhanced CD8... + T cell infiltration and effector activity directly confirmed that FluDox-MIC can effectively amplify adaptive anti-tumor immunity. Furthermore, multiplex immunofluorescence staining visually revealed NK cells and GZMB levels in each treatment group. + CD8 + The different spatial distribution of T cells is consistent with flow cytometry data and provides morphological support for the effects of immune remodeling.

[0121] Overall, in vivo data systematically demonstrated that FluDox-MIC maintains and enhances "innate immune components" by downregulating PVR and activating NK cell function, while significantly enhancing CD8 activation. + T cell tumor infiltration and effector function are enhanced, thereby achieving dual activation of innate and adaptive immunity. Combining with anti-PD-1 blockade agents further alleviates effector T cell depletion during chronic antigen exposure, thus promoting a sustained immune response. This establishes a therapeutic cascade: PVR downregulation, NK cell enhancement, ICD induction, and CD8 activation. +T-cell expansion and desuppression of the PD-1 pathway. This process coordinates the spatiotemporal correlation between innate immune reactivation and adaptive immune expansion. This multi-level strategy shows great promise for treating immunotherapy-resistant melanoma and provides a strong theoretical basis for developing precision combination therapies targeting the PD-1 pathway.

[0122] In summary, as Figure 30 As shown, FluDox-MIC in this invention is constructed through a simple self-assembly process. Upon entering the tumor microenvironment, FluDox-MIC rapidly dissociates into its two active components—flumatinib and DOX. Flumatinib penetrates the cell membrane and selectively targets PVR. By promoting the ubiquitination of PVR, flumatinib labels the receptor for proteasome degradation, thereby depleting PVR from the plasma membrane. The subsequent reduction of surface PVR weakens its inhibitory signal on NK cells, leading to strong NK cell activation and enhanced cytotoxicity. Simultaneously, intracellular DOX embeds into nuclear DNA and inhibits topoisomerase II, thereby disrupting DNA synthesis and function. These damages trigger a cascade of reactions in the endoplasmic reticulum and oxidative stress pathways, ultimately leading to the release of damage-associated molecular patterns (DAMPs). The resulting ICD promotes the maturation and cross-presentation of dendritic cells, indirectly authorizing antigen-specific CD8 T cells. Activated CD8 T cells... + T cells therefore upregulate granzyme B (GZMB) and IFN, exerting effective tumor-killing activity. When combined with CD8 blockade... + When combined with anti-PD-1 antibodies that target the PD-1-mediated inhibitory axis on T cells, this approach synergistically amplifies the function of cytotoxic T lymphocytes and achieves superior inhibition of tumor development.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that after reading this application specification, they can still modify or make equivalent substitutions to the specific implementation of the present invention, but these modifications or changes do not depart from the protection scope of the pending claims of the present invention.

Claims

1. A nanomicelle FluDox-MIC, characterized in that, The raw materials used in the preparation include flumatinib, doxorubicin, and mPEG-DSPE.

2. The FluDox-MIC nanomicelles according to claim 1, characterized in that, The mass ratio of flumatinib, doxorubicin, and mPEG-DSPE is 1:0.5~4:20~60; And / or, the number-average molecular weight of the mPEG-DSPE is 0.8~3 kDa.

3. The FluDox-MIC nanomicelles according to claim 1, characterized in that, The average diameter of the nanomicelles, as determined by transmission electron microscopy, is 60–120 nm; and / or, the average hydrated diameter of the nanomicelles, as determined by dynamic light scattering, is 110–180 nm.

4. The method for preparing FluDox-MIC nanomicelles according to any one of claims 1 to 3, characterized in that, The process includes the following steps: the flumatinib and the mPEG-DSPE form solution A; the doxorubicin forms solution B; under ultrasonic conditions, solution A is added to solution B, the ultrasonic reaction continues, and post-processing is performed to obtain the nanomicelles FluDox-MIC.

5. The preparation method of FluDox-MIC nanomicelles according to claim 4, characterized in that, The concentration of flumatinib in solution A is 0.2~1 mg / mL; And / or, the concentration of doxorubicin in solution B is 0.01~0.2 mg / mL; And / or, the power of the ultrasound is 20~60 W; And / or, the post-treatment includes at least one step of stirring under ventilated conditions, ultrafiltration, and rinsing with ultrapure water.

6. Use of flumatinib or the FluDox-MIC nanomicelles according to any one of claims 1 to 3 in the preparation of a poliovirus receptor PVR protein degrading agent.

7. Use of the FluDox-MIC nanomicelles according to any one of claims 1 to 3 in the preparation of a medicament for treating melanoma.

8. A pharmaceutical composition, characterized in that, Includes the FluDox-MIC nanomicelles as described in any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that, The concentration of the FluDox-MIC nanomicelles in the pharmaceutical composition is 20~1000 ng / mL.

10. Use of the pharmaceutical composition of claim 8 or 9 in the preparation of a medicament for use in combination with anti-PD-1 therapy for the treatment of melanoma.

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