Use of ox40 activating antibody and Anti-angiogenic agent in preparation of Anti-tumor drugs

Abstract

The present invention relates to the technical field of biomedicine, and particularly to a use of an OX40 activating antibody and an anti-angiogenic agent in the preparation of anti-tumor drugs. The OX40 activating antibody is OX86, and the anti-angiogenic agent is Verteporfin. OX40 induces an anti-tumor effect in T cells and induces a pro-tumor effect in endothelial cells. Therefore, the OX40 activating antibody and the anti-angiogenic agent are administered in combination, OX40 anti-tumor signaling is activated in the T cells, and the anti-angiogenic agent is used for blocking the pro-tumor effect of the OX40 activating antibody in the endothelial cells; and the two components act synergistically, showing a better anti-tumor effect compared to combination therapy of the OX40 activating antibody and a PD-1 monoclonal antibody CD279.

Description

Application of OX40 activating antibodies and anti-angiogenic agents in the preparation of anti-tumor drugs Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of OX40 activating antibodies and anti-angiogenic agents in the preparation of anti-tumor drugs. Background Art

[0002] Currently, there are two main approaches to tumor immunotherapy: first, by designing immune checkpoint inhibitors to "block" immune checkpoints and enhance the ability of immune cells to recognize and kill tumor cells, such as PD-1 and CTLA-4 monoclonal antibodies; second, by activating co-stimulatory molecules to enhance the anti-tumor activity of immune cells, such as OX40 and CD28 activating monoclonal antibodies. In recent years, immunotherapy has demonstrated significant efficacy in various cancers, but the treatment effect is not ideal in some patients: approximately 4% to 29% of malignant tumor patients experience enlargement of the primary lesion or new lesions after immunotherapy, leading to rapid deterioration of the disease, which greatly limits the clinical application of immunotherapy.

[0003] OX40 (TNFRSF4), a member of the tumor necrosis factor (TNF) superfamily, is a popular immunotherapy target after PD-1. As a key T cell co-stimulatory molecule, OX40 binds to the OX40 ligand (OX40L) on the membrane of antigen-presenting cells, inducing T cell activation and enhancing effector function, playing a vital role in both initial T cell responses and secondary responses. To date, numerous pharmaceutical companies, both domestic and international, have invested billions of dollars in research and development, and a total of 11 OX40-activating antibodies have been used in clinical trials for tumor immunotherapy. In clinical trials, tissue staining analysis of patient biopsy samples showed that OX40-activating antibodies did indeed enhance anti-tumor immunity (such as activating CD4+ and CD8+ effector T cells and NK cells, and inhibiting CD4+ Treg cell function). However, the partial remission rates of these agonist monotherapy treatments did not exceed 10%, which may be the main reason why OX40-activating antibodies have not been able to advance beyond Phase II clinical trials.

[0004] In addition, in clinical trials, OX40-activating antibodies are mainly used in combination with immune checkpoint inhibitors (ICBs) such as PD-1 and CTLA-4 monoclonal antibodies. Their combined use can dual-activate T cells in a short period of time, but the objective response rate still does not exceed 13%, and there is a risk of inducing cytokine release syndrome and leading to multiple organ failure.

[0005] In summary, the clinical translational challenges facing OX40 activators are: their efficacy is poor when used alone, while their combination with PD-1 and other agents carries the risk of overactivation of the immune system. Therefore, to address the challenges of OX40 activators' clinical application, new combination therapy approaches are needed.

[0006] Summary of the Invention

[0007] The first purpose of the present invention is to provide an application of an OX40 activating antibody and an anti-angiogenic agent in the preparation of an anti-tumor drug, which solves the clinical problem that "OX40 agonists are not effective in treating tumors when used alone or in combination with immunoblockers."

[0008] A second object of the present invention is to provide a pharmaceutical composition.

[0009] One of the purposes of the present invention is achieved by the following technical solution:

[0010] Application of an OX40 activating antibody and an anti-angiogenic agent in the preparation of an anti-tumor drug, wherein the OX40 activating antibody is OX86 and the anti-angiogenic agent is verteporfin.

[0011] Furthermore, the OX40 activating antibody and the anti-angiogenesis agent are administered simultaneously.

[0012] Furthermore, the OX40 activating antibody and anti-angiogenic agent are used in the preparation of drugs for treating colon cancer.

[0013] Furthermore, the OX40 activating antibody and anti-angiogenic agent are used in the preparation of drugs for treating gliomas.

[0014] Furthermore, the OX40 activating antibody and anti-angiogenic agent are used in the preparation of drugs for treating ovarian cancer.

[0015] The second object of the present invention is achieved by adopting the following technical solution:

[0016] A pharmaceutical composition comprises the OX40 activating antibody and an anti-angiogenic agent, and pharmaceutically acceptable excipients.

[0017] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0018] The present invention provides the use of an OX40-activating antibody and an anti-angiogenic agent in the preparation of an anti-tumor drug. The OX40-activating antibody and the anti-angiogenic agent are used in combination in the treatment of tumors. OX40 induces an "anti-tumor" effect in T cells and a "pro-tumor" effect in endothelial cells. Therefore, the OX40-activating antibody and the anti-angiogenic agent are used in combination to activate the OX40 "anti-tumor" signal in T cells while simultaneously blocking the "pro-tumor" effect of the OX40-activating antibody in endothelial cells using the anti-angiogenic agent. The two components work synergistically, demonstrating superior tumor suppression efficacy compared to the combined treatment of the OX40-activating antibody and PD-1 monoclonal antibody (CD279). BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 shows the tumor growth curves of three subcutaneous tumor model mice after injection of five different drugs;

[0020] Figure 2 shows the tumor size and growth curves of normal mice (A), T cell immunodeficient mice (B), and fully immunodeficient mice (C) inoculated with colon cancer MC38 cells to construct subcutaneous tumors and treated with OX40L recombinant protein or OX86 antibody for 21 days;

[0021] In Figure 3, A shows the in vivo imaging system images and fluorescence values ​​of fully immunodeficient mice after subcutaneous inoculation of MC38-LUC-GFP cells in the colon and OX40 activation treatment for 3 weeks; B shows the liver metastasis nodules of fully immunodeficient mice after subcutaneous inoculation of MC38-LUC-GFP cells in the colon and OX40 activation treatment for 60 days; C shows the survival time of fully immunodeficient mice treated with subcutaneous inoculation of MC38-LUC-GFP cells in the colon and OX40 activation treatment;

[0022] Figure 4 shows the expression levels of OX40 in cells of different cancer types;

[0023] Figure 5 shows the tumor growth curves after OX40 activation therapy in T cell immunodeficient mice inoculated subcutaneously with glioma and ovarian cancer cells;

[0024] In Figure 6, A is a scatter plot of subpopulation classification of single-cell sequencing of colon cancer tissue; B is the expression of OX40 in various cell populations of colon cancer (CRC) and para-tumor (NT) tissue (n=5); C is a volcano plot of differentially expressed genes between colon cancer endothelial cells and para-tumor normal endothelial cells, with red dots representing upregulated genes and green dots representing downregulated genes;

[0025] Figure 7 shows the expression of genes related to cell proliferation, migration, invasion, and apoptosis in TNFRSF4- and TNFRSF4+ endothelial cells;

[0026] In Figure 8 , A shows the co-localization of OX40 and CD31 in colon cancer and adjacent adjacent tissues; B shows the expression level of OX40 in tumor endothelial cells (CRC CD31+ cells) and normal endothelial cells (NT CD31+ cells); C shows the correlation between the expression of OX40 and the endothelial marker genes CD31 and VWF;

[0027] FIG9 shows the effect of OX40L recombinant protein stimulation on the tubule formation ability of human umbilical vein endothelial cells HUVEC;

[0028] FIG10 shows the differentially expressed genes and signaling pathways after HUVEC stimulation with exogenous OX40L recombinant protein for 48 hours;

[0029] Figure 11 shows the cell phenotype of endothelial cells after OX40L stimulation for 5 days;

[0030] Figure 12 shows the expression of EndMT-related genes in NFRSF4- and TNFRSF4+ endothelial cells;

[0031] Figure 13 shows the effect of OX40L stimulation on the expression of α-SMA and Vimentin in endothelial cells;

[0032] Figure 14 shows the correlation between OX40 and EndMT marker genes Vimentin and FSP1 according to TCGA data analysis;

[0033] In Figure 15, A is the protein spectrum identification results of cytoplasmic and nuclear proteins after HUVEC stimulation with exogenous OX40L for 48 hours; B is the subcellular localization of YAP protein observed by laser confocal microscopy after HUVEC stimulation with exogenous OX40L; C is the expression levels of CYR61, CTGF and ANKRD1 genes in tumor endothelial cells (CRC CD31+ cells) and normal endothelial cells (NT CD31+ cells); D is the expression of YAP downstream target genes in TNFRSF4- and TNFRSF4+ endothelial cells;

[0034] FIG16 shows the effect of Verteporfin on endothelial cell tubulogenesis regulated by activated OX40. DETAILED DESCRIPTION

[0035] The technical solution of the present invention will be further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only intended to illustrate the present invention and should not be construed as limiting the present invention. Specific conditions not specified in the examples are to be followed according to conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments used, unless otherwise specified, are conventional products obtained from commercial channels.

[0036] Table 1 Experimental reagents and sources

[0037] Example 1

[0038] Activating OX40 and combining it with YAP inhibitors to treat tumors:

[0039] Normal immune function mice were divided into 18 groups, 8 mice in each group, each corresponding to a tumor model and a treatment regimen. The vehicle group of the three cancer models was injected with DMSO (100 mg / kg) and IgG antibody (1 mg / mouse).

[0040] Table 2 Mouse cancer models and drug treatments

[0041] Anesthetize the mice and inject 100 μL of 10 7 Subcutaneous tumors were established in nude mice with mouse colon cancer cells MC38, mouse glioma cells GL261, and mouse ovarian cancer cells ID8 at a concentration of 100 μg / mL. Mice with tumor masses of approximately identical size were treated with intraperitoneal injections of the OX86 activating antibody, verteporfin, CD279, OX86 and verteporfin, and OX86 and CD279, twice weekly for four weeks. Tumor volume was measured daily starting on the first day of drug injection, and tumor growth curves were plotted. As shown in Figure 1, while the monotherapy groups (OX86, CD279, and verteporfin) all showed some tumor suppression, the combination therapy groups (OX86 and verteporfin, and OX86 and CD279) exhibited a significant synergistic antitumor effect. In the later stages of OX86 and verteporfin treatment, some subcutaneous tumors shrank or even disappeared, demonstrating superior efficacy compared to the combination of OX86 and PD-1 monoclonal antibody (CD279).

[0042] Test Example 1

[0043] The role of activated OX40 signaling in the progression of colon cancer in normal immune mice and immunodeficient mice:

[0044] Twenty-four 5-week-old normal immune mice (C57BL / 6J), T cell immunodeficient mice (BALB / c-nu), and fully immunodeficient mice (NVSG) were subcutaneously injected with 100 μL of colon cancer MC38 cells (10 7 / mL) to construct tumors. OX40L recombinant protein (10 mg / kg) or OX40 activating antibody OX86 (200 mg / kg) were injected intraperitoneally into the three tumor model mice to activate the OX40 signal, and the treatment frequency was maintained at 3 times a week for 3 weeks. The tumor volume was measured every day from the first day of injection of OX40L or OX86, and the tumor growth curve was drawn. After 8 treatments, the mice were killed, and the subcutaneous tumors were removed and photographed. The results are shown in Figure 2. In the subcutaneous tumor model of normal immune mice, both OX40L and OX86 antibody activation therapy had an inhibitory effect on tumors (Figure 2A); in the T cell immunodeficient mouse and full immunodeficient mouse models, OX40 activation therapy did not show an anti-tumor effect, but instead promoted the growth of subcutaneous tumors to varying degrees (Figures 2B, C).

[0045] Test Example 2

[0046] Effects of OX40 activation on the growth and metastasis of colon cancer in immunodeficient mice:

[0047] The vector containing the Luc-GFP gene was transfected into the MC38 cell line using the LPP-HLUC-Lv201-100 virus. The transgenic cells were screened with puromycin at a concentration of 2 μg / mL, and the MC38-LUC-GFP cell line with stable high expression was finally obtained. 100 μL MC38-LUC-GFP cell suspension (5×10 6 / mL) was inoculated into the subcutaneous part of the colon of NVSG mice (about 2-3 cm from the cecum to the anus) to construct a colon orthotopic tumor-liver metastasis model, and 8 replicates were performed. The growth of mouse colon cancer cells was detected and photographed using a live imaging system every week. The mice were then intraperitoneally injected with OX40L recombinant protein (10 mg / kg) or OX40 activating antibody OX86 (200 mg / kg) to activate the OX40 signal. The treatment was performed 3 times a week, and the fluorescence values ​​were statistically analyzed again using a live imaging system after 3 weeks. The survival rate of the mice was counted every day from the first day of injection of OX40L or OX86, and a survival curve was drawn. After 60 days of treatment, all mice were killed, the livers were dissected, and the metastatic nodules (GFP) were observed under a fluorescent stereomicroscope. The results are shown in Figure 3. In fully immunodeficient mice, activating OX40 promoted tumor growth and metastasis, and shortened the survival time of the mice.

[0048] The above results indicate that in addition to exerting an "anti-tumor" effect in T cells, activated OX40 may also exert a "pro-tumor" effect through other microenvironmental cells.

[0049] Test Example 3

[0050] Effects of activating OX40 signaling in other tumors with high OX40 expression:

[0051] Using publicly available data from the TISCH Tumor Microenvironment Single-Cell Transcriptome Database (http: / / tisch.comp-genomics.org / ), we analyzed cancer types with high OX40 expression in endothelial cells and identified three cancer types with high OX40 expression in endothelial cells: colon cancer, glioma, and ovarian cancer (Figure 4).

[0052] The hypothesis that activation of OX40 promotes tumor growth was further verified in glioma and ovarian cancer models. 100 μL of mouse glioma GL261 and ovarian cancer cell ID8 (10 7 / mL) were inoculated subcutaneously into T cell immunodeficient mice. After tumor formation, OX40L recombinant protein (10 mg / kg) or OX40 activating antibody OX86 (200 mg / kg) was injected into the peritoneal cavity to activate OX40 signaling (8 mice in each tumor model group, OX40L treatment group, OX86 treatment group, and Vehicle group). Treatment was done three times a week for 3 weeks. Tumor volume was measured every day from the first day of injection of OX40L or OX86, and a tumor growth curve was drawn. As shown in Figure 5, activation of the OX40 signaling significantly promoted tumor growth, indicating that the "tumor-promoting" effect of activating OX40 is a common phenomenon.

[0053] Test Example 4

[0054] Exploring the molecular mechanism of OX40's tumor-promoting function:

[0055] Human colon cancer tissues were sent to Guangzhou Kidio Biotechnology Co., Ltd. for single-cell sequencing, analyzing the expression levels of OX40 in various cell subsets. As shown in Figure 6, 10 cell subsets were identified in both colon cancer tissues and adjacent normal tissues: plasma cells, epithelial cells, B cells, T cells, endothelial cells, macrophages, fibroblasts, myeloid-derived suppressor cells (MDSCs), innate lymphocytes (iLCs), monocytes, and mast cells (Figure 6A). OX40 expression was highest in T cell subsets, and was also high in tumor endothelial cells, second only to T cell subsets (Figure 6B). Furthermore, OX40 expression was significantly higher in colon cancer endothelial cells than in adjacent normal tissue endothelial cells (Figures 6B, C).

[0056] Human colon cancer tissue samples were used to separate endothelial cells from colon cancer and corresponding adjacent tissues using anti-CD31 magnetic beads, and then qPCR was performed. The results showed that the expression level of OX40 in colon cancer endothelial cells was significantly higher than that in normal endothelial cells (Figure 7A).

[0057] Colon cancer (COAD) gene expression profiles were downloaded from the TCGA database (https: / / xenabrowser.net / datapages / ), and correlation analysis was performed to identify other genes associated with the OX40 gene. The results, shown in Figure 8C, showed that OX40 expression levels were significantly positively correlated with the vascular endothelial cell marker genes CD31 or VWF (Figure 7B).

[0058] The above results indicate that OX40 is very likely involved in the regulation of tumor blood vessels.

[0059] Test Example 5

[0060] Activation of OX40 signaling affects the tubule formation capacity of endothelial cells:

[0061] After culturing HUVEC cells in DMEM medium containing 0.2% fetal bovine serum, 2mM L-glutamine, 1mM sodium pyruvate, 100U / ml penicillin and 100μg / ml streptomycin for 24 hours, endothelial cells HUVEC were stimulated with 50ng / mL and 100ng / mL OX40L for 24 hours to activate OX40 signaling. 250μl of low growth factor basement membrane extract (BME) was added to the 24-well plate. Avoid bubbles during pipetting and ensure that the glue completely covers the bottom of the well. The 24-well plate was incubated at 37°C and 5% CO2 for 30 minutes to solidify the BME. Subsequently, 300μL of culture medium and 10 5 Resuspended HUVEC cells were incubated at 37°C, 5% CO₂ for 6 hours. The culture medium was carefully removed, and the cells were stained with Calcein AM. The tubular network was imaged using a fluorescence microscope. As shown in Figure 8, activation of OX40 significantly promoted the tubule formation ability of endothelial cells.

[0062] Test Example 6

[0063] Study on the mechanism of activating OX40 signaling to promote endothelial cell mesenchymal transition:

[0064] The HUVEC cells cultured in Experimental Example 5 were subjected to transcriptomic sequencing (RNA-Seq). The results are shown in Figure 9. After activating the OX40 signal, signaling pathways such as epithelial-mesenchymal transition (EMT), endothelial cell proliferation, migration, and angiogenesis were significantly activated, and the expression of genes related to the EMT signaling pathway was also significantly upregulated, suggesting that activation of OX40 may be involved in regulating endothelial mesenchymal transition (EndMT). EndMT is a necessary process for tumor hematogenous metastasis and is closely related to endothelial cell angiogenesis.

[0065] An Olympus inverted microscope ( CKX31 ) The HUVEC cells cultured in Experimental Example 5 were photographed and imaged, and the results showed that after OX40L stimulation, the endothelial cells lost their cobblestone-like characteristics and gradually transformed into spindle-shaped cells, extending pseudopodia and presenting a mesenchymal cell phenotype ( FIG. 10 ).

[0066] Expression analysis of EndMT-related genes was performed on TNFRSF- and TNFRSF+ endothelial cells in colon cancer tissue (obtained in Experimental Example 4). The results showed that EndMT markers such as SNAIL, SLUG, TWIST, and TGFβ were upregulated in TNFRSF4+ tumor endothelial cells, while adhesion molecules such as CDH1 and CDH5 were downregulated ( Figure 11 ).

[0067] Semi-quantitative Western blotting analysis of α-SMA and Vimentin, EndMT-promoting markers in cultured HUVEC endothelial cells in Experimental Example 5, showed that OX40L stimulated the expression of α-SMA and Vimentin ( FIG. 12 ).

[0068] The gene expression profile of colon cancer was downloaded from the TCGA database (https: / / xenabrowser.net / datapages / ), and correlation analysis was performed to search for endothelial-mesenchymal transition marker genes related to the OX40 gene. It was found that OX40 was highly positively correlated with Vimentin and FSP1 (Figure 13).

[0069] These results indicate that activation of OX40 can cause endothelial cell mesenchymal transformation, thereby inducing changes in the phenotype and biological function of endothelial cells.

[0070] Test Example 7

[0071] Searching for key transcription factors that activate OX40 signaling to regulate endothelial cell function:

[0072] The cytoplasmic and nuclear proteins of HUVEC cells cultured in Experimental Example 5 were extracted and protein spectrum identification was performed. The results showed that activation of OX40 significantly promoted YAP nuclear translocation. Compared with the control group, the nuclear translocation of YAP protein increased by 16.52 times (Figure 14A), and immunofluorescence also confirmed this result (Figure 14B).

[0073] Anti-CD31 magnetic beads were used to sort endothelial cells from colon cancer and corresponding adjacent tissues. qPCR analysis revealed that the expression levels of YAP downstream target genes CYR61, CTGF, and ANKRD1 in colon cancer endothelial cells were significantly higher than those in normal endothelial cells ( Figure 14C ).

[0074] The above results suggest that activation of OX40 in endothelial cells significantly promotes YAP nuclear translocation and activates its transcriptional regulatory activity.

[0075] Test Example 8

[0076] After culturing HUVEC cells for 24 hours in DMEM medium containing 0.2% fetal bovine serum, 2mM L-glutamine, 1mM sodium pyruvate, 100U / ml penicillin, and 100μg / ml streptomycin, OX40L protein (100ng / mL) and OX40L protein (100ng / mL) + Verteporfin (5μM) were added and treated for 48 hours. 250μl of BME was added to a 24-well plate to cover the bottom of the wells and incubated at 37°C, 5% CO2 for 30 minutes to solidify. 300μL of culture medium and 10 5 Resuspended HUVEC cells were incubated at 37°C, 5% CO₂ for 6 hours. The culture medium was removed, the cells were stained with Calcein AM, and the tubular network was imaged using a fluorescence microscope. As shown in Figure 15, the YAP-TEAD4 inhibitor Verteporfin blocked the endothelial cell biological functions regulated by activated OX40, suggesting that activated OX40 may regulate the biological functions of endothelial cells by promoting YAP nuclear translocation.

[0077] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. The basic principles and main features of the present invention have been described above using specific implementation schemes. Modifications or substitutions may be made based on the present invention, but such modifications or substitutions do not detract from the essence of the corresponding technical solutions from the scope of protection claimed by the present invention.

Claims

1. Use of an OX40 activating antibody and an anti-angiogenic agent in the preparation of an anti-tumor drug, characterized in that: The OX40 activating antibody is OX86, and the anti-angiogenic agent is verteporfin.

2. The use of the OX40 activating antibody and the anti-angiogenic agent in the preparation of an anti-tumor drug according to claim 1, wherein: The OX40 activating antibody and the anti-angiogenic agent are administered simultaneously.

3. The use of the OX40 activating antibody and the anti-angiogenic agent in the preparation of an anti-tumor drug according to claim 1, wherein: Application in the preparation of drugs for treating colon cancer.

4. The use of the OX40 activating antibody and the anti-angiogenic agent in the preparation of an anti-tumor drug according to claim 1, wherein: Application in the preparation of drugs for treating gliomas.

5. The use of the OX40 activating antibody and the anti-angiogenic agent in the preparation of an anti-tumor drug according to claim 1, wherein: Application in the preparation of drugs for treating ovarian cancer.

6. A pharmaceutical composition, characterized in that The invention comprises the OX40 activating antibody and anti-angiogenic agent according to claim 1, and pharmaceutically acceptable excipients.

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