Application of LukS-PV and its combination with PD-1 inhibitors in the preparation of drugs for treating hepatocellular carcinoma

Abstract

This invention discloses the application of LukS-PV and its combination with a PD-1 inhibitor in the preparation of drugs for treating hepatocellular carcinoma, belonging to the field of biomedical technology. LukS-PV of this invention exerts its anti-tumor effect by promoting the polarization transformation of tumor-associated macrophages from M2 to M1 types, thereby improving the tumor immunosuppressive microenvironment. Furthermore, the combined use of LukS-PV and a PD-1 inhibitor can enhance CD8+ T cell infiltration and anti-tumor immune function, alleviate T cell exhaustion, and improve the therapeutic effect of liver cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of LukS-PV and its combination with PD-1 inhibitors in the preparation of drugs for treating hepatocellular carcinoma. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in clinical practice, characterized by insidious onset, rapid progression, high recurrence and metastasis rates, and poor prognosis. Most patients are diagnosed at an advanced stage, making radical surgical treatment difficult. In recent years, molecular targeted therapy and immune checkpoint inhibitors, especially programmed death-1 (PD-1) / programmed death-ligand 1 (PD-L1) inhibitors, have made some progress in the treatment of advanced HCC. However, the overall objective response rate remains limited, and some patients exhibit primary or secondary drug resistance, further restricting clinical benefit. Therefore, developing new therapeutic targets and combination therapy strategies is of great significance for improving the efficacy of immunotherapy for HCC.

[0003] The tumor immune microenvironment is a crucial factor influencing the development, progression, and treatment response of liver cancer. Tumor-associated macrophages (TAMs) are an important component of this microenvironment. Current research indicates that TAMs, particularly M2-like macrophages, can promote tumor cell proliferation, invasion, angiogenesis, and immune escape by secreting immunosuppressive factors such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), while inhibiting the infiltration and killing function of CD8+ T cells. Enrichment of M2-like macrophages typically indicates an immunosuppressive state in the tumor and is closely associated with poor response to immune checkpoint blockade therapy. Therefore, targeting and regulating the polarization state of TAMs to reverse the immunosuppressive microenvironment is a vital direction for improving the efficacy of immunotherapy for liver cancer.

[0004] Complement component 5a receptor 1 (C5aR1) is an important G protein-coupled receptor that plays a crucial role in inflammatory responses, immune cell recruitment, and tumor microenvironment regulation. Previous studies have suggested that aberrant activation of the C5a / C5aR1 signaling axis in various tumors can promote the aggregation of immunosuppressive myeloid cells, drive macrophage shift towards an M2-like phenotype, and inhibit the anti-tumor function of effector T cells. In liver cancer, C5aR1 is considered closely related to the formation of an immunosuppressive microenvironment and tumor progression. However, effective interventions targeting C5aR1 in current technologies remain limited, especially lacking combined application strategies that can both regulate myeloid immunosuppression and enhance the efficacy of PD-1 blockade.

[0005] LukS-PV is a component of Staphylococcus aureus-derived leukocyte toxin. Previous studies have suggested that LukS-PV can specifically bind to human C5aR1, thereby affecting C5aR1-mediated signal transduction. However, current research on the application of LukS-PV in tumor therapy is limited, especially regarding its systematic application in liver cancer treatment. Therefore, developing new uses for LukS-PV in the preparation of anti-hepatocellular carcinoma drugs has significant theoretical and practical value for improving the efficacy of liver cancer immunotherapy. Summary of the Invention

[0006] Based on the technical problems existing in the background art, the present invention proposes the application of LukS-PV and its combination with PD-1 inhibitors in the preparation of drugs for treating hepatocellular carcinoma, which can reshape the tumor immune microenvironment, improve the sensitivity of immune checkpoint blockade therapy and enhance the anti-tumor efficacy.

[0007] The application of LukS-PV proposed in this invention in the preparation of drugs for treating hepatocellular carcinoma is characterized in that LukS-PV improves the tumor immunosuppressive microenvironment by promoting the transformation of tumor-associated macrophages from an M2-like phenotype to an M1-like phenotype.

[0008] Preferably, the polarization regulation of tumor-associated macrophages by the LukS-PV is C5aR1-dependent.

[0009] Preferably, the transformation of tumor-associated macrophages from M2-like phenotype to M1-like phenotype includes: a decrease in the expression of M2-like phenotype-related markers; and / or an increase in the expression of M1-like phenotype-related markers.

[0010] Preferably, the M2 sample phenotypic markers include one or more of CD206, CD163, Arg1, Tgfb1, and Il10; And / or, the M1 sample phenotypic markers include one or more of CD86, Cd80, Nos2, Ifng, and H2.

[0011] Preferably, the improvement of the tumor immunosuppressive microenvironment includes: Reduce the infiltration level of F4 / 80+CD11b+ macrophages in tumor tissue; And / or, increase the proportion of CD86-positive macrophages in tumor tissue; And / or, reduce the proportion of CD206-positive macrophages in tumor tissue.

[0012] The present invention proposes the application of the combination of LukS-PV and PD-1 inhibitor in the preparation of a drug for treating hepatocellular carcinoma, wherein the drug is used for: Enhanced CD8+ T cell infiltration in tumor tissue; And / or, increase the proportion of Granzyme B+IFN-γ+CD8+ T cells; And / or, reduce the proportion of TIM3+PD-1+CD8+ T cells.

[0013] Beneficial technical effects of the present invention: (1) This invention demonstrates through in vitro cell experiments and in vivo experiments in hC5aR1 humanized mice that LukS-PV can promote the transformation of macrophages from M2-like immunosuppressive phenotype to M1-like pro-inflammatory phenotype in a C5aR1-dependent manner, thereby reshaping the tumor immune microenvironment and exerting an anti-tumor effect.

[0014] (2) The present invention uses LukS-PV in combination with PD-1 inhibitor in the preparation of drugs for treating hepatocellular carcinoma, which can enhance CD8+ T cell infiltration and anti-tumor immune function, alleviate T cell depletion, and further improve the treatment effect of liver cancer. Attached Figure Description

[0015] Figure 1 This invention proposes LukS-PV to promote the polarization of THP-1-derived macrophages and BMDM towards the M1-like phenotype. Specifically, (A, B) show the changes in the expression of the M2 marker CD206 and the M1 marker CD86 on the surface of THP-1-derived macrophages after treatment with different concentrations of LukS-PV, detected by flow cytometry; (C) shows the mRNA expression levels of M2-related genes and M1-related genes in THP-1-derived macrophages detected by qRT-PCR. (D, E) show the changes in the expression of CD206 and CD86 on the surface of bone marrow-derived macrophages (BMDM) after treatment with different concentrations of LukS-PV, detected by flow cytometry; (F) shows the mRNA expression levels of M2-related genes and M1-related genes in BMDM, detected by qRT-PCR.

[0016] Figure 2This invention proposes a method to reduce the M1-like polarization effect of macrophages induced by LukS-PV knockdown using C5aR1. Specifically, (A) qRT-PCR was used to detect the knockdown efficiency of different interfering sequences of shC5aR1 on C5AR1 mRNA expression; (B) Western blot was used to detect the inhibitory effect of shC5aR1 on C5AR1 protein expression; (C) Flow cytometry was used to detect changes in CD86 expression in the control group (shCon), LukS-PV treatment group, and shC5aR1+LukS-PV group, and the mean fluorescence intensity (MFI) was quantitatively analyzed; (D) Flow cytometry was used to detect changes in CD206 expression in the above groups, and the MFI was quantitatively analyzed; and (E) qRT-PCR was used to detect the mRNA expression levels of M2-related genes and M1-related genes in different treatment groups.

[0017] Figure 3 The present invention proposes that LukS-PV combined with anti-PD-1 significantly inhibits the in vivo growth of liver cancer; wherein, (A) is a schematic diagram of the drug administration and material collection process of Hepa1-6 tumor-bearing hC5aR1 mice; (B) is a representative image of the ex vivo tumor of each treatment group; (C) is the tumor weight statistics of each group at the endpoint; (D) is the tumor volume growth curve of each group; (E) is the individual tumor growth curve of each mouse group.

[0018] Figure 4 The curves show the weight changes of mice in different treatment groups during the treatment period as proposed in this invention.

[0019] Figure 5 This invention presents the LukS-PV combined with anti-PD-1 to reshape the polarization state of tumor-associated macrophages; wherein, (A) is a representative flow cytometry plot of F4 / 80+CD11b+ macrophages in each group of tumor tissues; (B) is a statistical analysis of the proportion of F4 / 80+CD11b+ cells in CD45+ cells; (C) is a representative flow cytometry plot of CD86 and CD206 expression in tumor-associated macrophages; (D) is a statistical analysis of the proportion of CD86-positive cells in F4 / 80+CD11b+ cells; and (E) is a statistical analysis of the proportion of CD206-positive cells in F4 / 80+CD11b+ cells.

[0020] Figure 6 This invention proposes LukS-PV combined with anti-PD-1 to promote the transformation of macrophages to the M1-like phenotype at the tissue level; wherein, (A) is a representative image of CD163 / F4 / 80 / DAPI and CD86 / F4 / 80 / DAPI immunofluorescence staining in tumor tissues of each group; (B) is a statistical representation of the proportion of CD163+ / F4 / 80+ cells; (C) is a statistical representation of the proportion of CD86+ / F4 / 80+ cells.

[0021] Figure 7The combined therapy proposed in this invention promotes the expression of M1-related genes and inhibits the expression of M2-related genes; wherein, (A) is the relative mRNA expression level of M1-related genes Cd80, Cd86, Nos2, Ifng and H2 in each group of tumor tissues; (B) is the relative mRNA expression level of M2-related genes Arg1, Cd163, Il4, Tgfb1 and Il10 in each group of tumor tissues.

[0022] Figure 8 The present invention proposes LukS-PV combined with anti-PD-1 to enhance CD8+ T cell infiltration and effector function and alleviate exhaustion; wherein, (A) is a representative flow cytometry diagram of CD8+ T cells in each group of tumor tissues; (B) is a statistical diagram of the proportion of CD8+ T cells in CD45+CD3+ cells; (C) is a representative flow cytometry diagram of Granzyme B and IFN-γ expression in CD8+ T cells; (D) is a statistical diagram of the proportion of Granzyme B+IFN-γ+ CD8+ T cells; (E) is a representative flow cytometry diagram of TIM3 and PD-1 expression in CD8+ T cells; (F) is a statistical diagram of the proportion of TIM3+PD-1+ CD8+ T cells. Detailed Implementation

[0023] The present invention will be further explained below with reference to specific embodiments.

[0024] Example 1 Application of LukS-PV in inducing macrophage polarization and inhibiting tumor growth To evaluate the regulatory effect of LukS-PV on macrophage polarization and its potential impact on tumor growth, in vitro experiments were conducted using THP-1-derived macrophages and mouse bone marrow-derived macrophages (BMDM). Patients were treated with 0 μM, 1 μM, 2 μM, and 4 μM LukS-PV, respectively. Flow cytometry was used to detect changes in the expression of the M2 marker CD206 and the M1 marker CD86. Real-time quantitative PCR was used to detect the expression levels of M2-related genes CD163, CD206, ARG1, TGFB1, IL10, and M1-related genes CD86 and NOS2.

[0025] Figure 1The results showed that in THP-1-derived macrophages, CD206 expression gradually decreased while CD86 expression gradually increased with increasing LukS-PV concentration. qPCR results further indicated that the expression of M2-related genes such as CD163, CD206, ARG1, TGFFB1, and IL10 was significantly downregulated, while the expression of M1-related genes such as CD86 and NOS2 was significantly upregulated. The same trend was observed in BMDM, where LukS-PV treatment decreased the expression of Cd163, Cd206, Arg1, TGFFB1, and IL10, while increasing the expression of Cd86 and Nos2.

[0026] The above results indicate that LukS-PV can promote the transformation of macrophages from M2 to M1 types, reduce immunosuppressive phenotypes, and enhance pro-inflammatory and anti-tumor phenotypes. Since M2 macrophages typically promote tumor immune escape, tumor cell proliferation, and tumor progression, while M1 macrophages are beneficial for activating anti-tumor immune responses, LukS-PV-induced macrophage reprogramming can improve the tumor immune microenvironment, thereby inhibiting tumor growth. This example illustrates that LukS-PV can serve as an active molecule targeting macrophage functional states and thus intervening in tumor growth, for use in the preparation of anti-tumor drugs.

[0027] Example 2 Mechanism analysis of LukS-PV regulating macrophage polarization via C5aR1 To clarify whether LukS-PV-induced macrophage polarization transition depends on C5aR1, a C5aR1 knockdown macrophage model was first constructed. Three shRNA interference sequences targeting C5aR1 were designed and transfected into macrophages. The knockdown efficiency of C5aR1 was detected by real-time quantitative PCR and Western blot, and shC5aR1 with better knockdown effect was selected for subsequent experiments. Subsequently, shCon, LukS-PV treatment, and shC5aR1+LukS-PV groups were set up. The expression changes of the M1 marker CD86 and the M2 marker CD206 were detected by flow cytometry. Furthermore, the expression levels of polarization-related genes such as CD163, CD206, ARG1, TGFB1, IL10, CD86, and NOS2 were detected by real-time quantitative PCR.

[0028] Figure 2The results showed that all three shC5aR1 interference sequences could reduce the mRNA and protein expression of C5aR1, with some interference sequences exhibiting high knockdown efficiency, indicating that the C5aR1 knockdown model was successfully constructed. Flow cytometry results showed that, compared with the shCon group, LukS-PV treatment significantly increased CD86 expression and significantly decreased CD206 expression on the surface of macrophages; after C5aR1 knockdown, the LukS-PV-induced increase in CD86 was significantly weakened, while the decrease in CD206 was partially restored. qPCR results further showed that LukS-PV treatment downregulated the expression of M2-related genes CD163, CD206, ARG1, TGFB1, and IL10, and upregulated the expression of M1-related genes CD86 and NOS2; after C5aR1 knockdown, the above-mentioned LukS-PV-induced gene expression changes were significantly weakened.

[0029] The effect of LukS-PV in promoting the transformation of macrophages from M2 to M1 phenotypes depends on C5aR1. Knockdown of C5aR1 significantly inhibited the induction of pro-inflammatory phenotypes in macrophages by LukS-PV, suggesting that C5aR1 is an important target for LukS-PV to exert its immunomodulatory effects. Since M2 macrophages facilitate tumor immune escape and tumor growth, while M1 macrophages enhance anti-tumor immune responses, LukS-PV, through C5aR1-mediated macrophage reprogramming, helps improve the tumor immunosuppressive microenvironment, thereby inhibiting tumor growth. This example provides experimental evidence for the application of LukS-PV in the preparation of drugs that target C5aR1, regulate tumor-associated macrophage polarization, and inhibit tumor growth.

[0030] Example 3 Antitumor effect of LukS-PV combined with anti-PD-1 in Hepa1-6 tumor-bearing hC5aR1 mouse model A Hepa1-6 mouse model bearing hC5aR1 tumors was established. After tumor formation, the experimental animals were randomly divided into four groups: an IgG control group, a LukS-PV group, an anti-PD-1 group, and a LukS-PV combined with anti-PD-1 group. Treatment was administered periodically according to a pre-defined protocol. During treatment, the long and short diameters of the tumor were measured periodically, and the tumor volume was calculated. At the experimental endpoint, tumor tissue was dissected and its weight was measured. Simultaneously, changes in body weight were monitored in each group to evaluate treatment safety.

[0031] Figure 3The results showed that, compared with the IgG control group, both the LukS-PV group and the anti-PD-1 group could inhibit tumor growth to some extent, while the LukS-PV combined with anti-PD-1 group showed the most significant tumor-suppressing effect. At the endpoint, the combined treatment group exhibited a significantly smaller tumor volume and a significantly lower tumor weight. Tumor growth curves and individual tumor growth trajectories both indicated that the combined treatment group showed a slower tumor growth trend throughout the observation period. Meanwhile, Figure 4 The results showed that the overall weight of mice in each group remained stable, and no significant weight loss was observed in the combined treatment group.

[0032] The above results indicate that the combined use of LukS-PV and anti-PD-1 significantly enhances the inhibitory effect on liver cancer, demonstrating a synergistic anti-tumor effect that is significantly superior to monotherapy. Weight monitoring results suggest that this combined treatment regimen exerts its anti-tumor effect without causing significant systemic toxicity, indicating good in vivo safety and promising application prospects.

[0033] Example 4 Effects of LukS-PV combined with anti-PD-1 on tumor-associated macrophage infiltration and polarization To evaluate the regulatory effect of LukS-PV combined with anti-PD-1 on macrophages in the tumor immune microenvironment, single-cell suspensions were prepared from tumor tissues of mice in each group. Flow cytometry was used to analyze the proportion of F4 / 80+CD11b+ macrophages in CD45+ cells, and the expression of CD86 and CD206 on the macrophage surface was further detected to assess their M1-like and M2-like polarization status. Simultaneously, immunofluorescence staining was performed on tumor tissues to detect the co-expression of CD163 / F4 / 80 and CD86 / F4 / 80, verifying macrophage polarization changes at the tissue level.

[0034] Figure 5 The results showed that, compared with the IgG control group, both the LukS-PV group and the anti-PD-1 group reduced the proportion of F4 / 80+CD11b+ macrophages among CD45+ cells in tumor tissue, with the most significant decrease observed in the combined treatment group. Further analysis of macrophage polarization phenotypes revealed that the proportion of CD86-positive cells among F4 / 80+CD11b+ cells was significantly increased, while the proportion of CD206-positive cells was significantly decreased in the combined treatment group. Figure 6 The immunofluorescence results were consistent with the flow cytometry results. In the combined treatment group, the number of CD163+ / F4 / 80+ cells in the tumor tissue was significantly reduced, while the number of CD86+ / F4 / 80+ cells was significantly increased.

[0035] The above results indicate that LukS-PV combined with anti-PD-1 can significantly reduce the infiltration of immunosuppressive macrophages in tumor tissues and promote the transformation of tumor-associated macrophages from an M2-like phenotype to an M1-like phenotype, thereby reshaping the tumor immune microenvironment. These results suggest that LukS-PV may provide key support for improving the efficacy of immune checkpoint blockade by targeting C5aR1 to intervene in myeloid cell immunosuppressive programs.

[0036] Example 5 Regulatory effect of LukS-PV combined with anti-PD-1 on macrophage polarization-related gene expression Total RNA was extracted from tumor tissues of mice in each group, and the expression changes of macrophage polarization-related genes were detected by real-time quantitative PCR. M1-type related genes included Cd80, Cd86, Nos2, Ifng, and H2; M2-type related genes included Arg1, Cd163, Il4, Tgfb1, and Il10. After standardization with internal reference genes, the relative expression levels of different treatment groups were compared.

[0037] Figure 7 The results showed that, compared with the IgG control group, both the LukS-PV group and the anti-PD-1 group upregulated the expression of M1-related genes Cd80, Cd86, Nos2, Ifng, and H2 to varying degrees, with the most significant upregulation in the combination therapy group. Conversely, the expression of M2-related genes Arg1, Cd163, Il4, Tgfb1, and Il10 was significantly downregulated in the combination therapy group, and the inhibitory effect was better than that in the single-drug group.

[0038] LukS-PV combined with anti-PD-1 not only promotes the transformation of tumor-associated macrophages to M1-like structures at the cellular phenotypic level, but also significantly enhances the pro-inflammatory M1 program and inhibits the immunosuppressive M2 program at the transcriptional level. These results further demonstrate that LukS-PV can effectively reverse tumor immunosuppression and enhance the body's anti-tumor immune response by regulating C5aR1-related signaling.

[0039] Example 6 LukS-PV combined with anti-PD-1 enhances CD8+ T cell infiltration and effector function and alleviates exhaustion. To evaluate the effect of combination therapy on adaptive antitumor immunity, single-cell suspensions were prepared from tumor tissues in each group, and the proportion of CD8+ T cells in CD45+CD3+ cells was analyzed by flow cytometry. Granzyme B and IFN-γ expression were further detected to assess CD8+ T cell cytotoxicity and effector function; TIM3 and PD-1 expression were also detected to evaluate CD8+ T cell exhaustion status.

[0040] Figure 8The results showed that, compared with the IgG control group, both the LukS-PV group and the anti-PD-1 group increased the proportion of CD8+ T cells in tumor tissue, with the most significant increase observed in the combination therapy group. Functional analysis revealed a significant increase in the proportion of Granzyme B+IFN-γ+CD8+ T cells in the combination therapy group, indicating a marked enhancement in the cytotoxicity and effector function of CD8+ T cells. Simultaneously, the proportion of TIM3+PD-1+CD8+ T cells significantly decreased in the combination therapy group, suggesting an improvement in T cell exhaustion.

[0041] LukS-PV combined with anti-PD-1 inhibitors not only improves the tumor myeloid immune microenvironment but also enhances CD8+ T cell infiltration and killing function, alleviating their exhaustion state, thereby forming a stronger synergistic anti-tumor effect of innate and adaptive immunity against liver cancer. This further demonstrates the significant therapeutic potential of combining LukS-PV with PD-1 inhibitors.

[0042] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. The scope of this application is defined by the appended claims and their equivalents, all of which should be included within the protection scope of this application.

Claims

1. The application of LukS-PV in the preparation of drugs for treating hepatocellular carcinoma, characterized in that, The LukS-PV improves the tumor immunosuppressive microenvironment by promoting the transformation of tumor-associated macrophages from an M2-like phenotype to an M1-like phenotype.

2. The application of LukS-PV according to claim 1 in the preparation of drugs for treating hepatocellular carcinoma, characterized in that, The polarization regulation of tumor-associated macrophages by LukS-PV is C5aR1-dependent.

3. The application of LukS-PV according to claim 1 in the preparation of drugs for treating hepatocellular carcinoma, characterized in that, The transformation of tumor-associated macrophages from M2-like to M1-like phenotype includes: decreased expression of M2-like phenotype-related markers; and / or increased expression of M1-like phenotype-related markers.

4. The application of LukS-PV according to claim 3 in the preparation of drugs for treating hepatocellular carcinoma, characterized in that, The M2 sample phenotypic markers include one or more of CD206, CD163, Arg1, Tgfb1, and Il10; And / or, the M1 sample phenotypic markers include one or more of CD86, Cd80, Nos2, Ifng, and H2.

5. The application of LukS-PV according to claim 1 in the preparation of a drug for treating hepatocellular carcinoma, characterized in that, The improvement of the tumor immunosuppressive microenvironment includes: Reduce the infiltration level of F4 / 80+CD11b+ macrophages in tumor tissue; And / or, increase the proportion of CD86-positive macrophages in tumor tissue; And / or, reduce the proportion of CD206-positive macrophages in tumor tissue.

6. The application of LukS-PV and PD-1 inhibitors in the preparation of drugs for treating hepatocellular carcinoma, characterized in that, The drug is used for: Enhanced CD8+ T cell infiltration in tumor tissue; And / or, increase the proportion of Granzyme B+IFN-γ+CD8+ T cells; And / or, reduce the proportion of TIM3+PD-1+CD8+ T cells.

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