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

Abstract

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. The OX40 activating antibody is OX86, and the anti-angiogenic agent is verteporfin. OX40 causes 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 used in combination, and while activating the OX40 "anti-tumor" signal in T cells, the anti-angiogenic agent is used to block the "pro-tumor" effect of the OX40 activating antibody in endothelial cells. The two components act synergistically, showing a better tumor inhibition effect than the combined treatment of OX40 activating antibody and PD-1 monoclonal antibody (CD279).

Description

Technical Field

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

[0002] At present, there are two main ways of tumor immunotherapy: first, by designing immune checkpoint inhibitors, "blocking" immune checkpoints, and enhancing 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, CD28 activation monoclonal antibodies, etc. In recent years, immunotherapy has shown significant efficacy in many cancers, but the treatment effect is not ideal in some patients: about 4% to 29% of malignant tumor patients will experience enlargement of primary lesions or new lesions after receiving immunotherapy, and their condition will rapidly deteriorate, which greatly limits the clinical application of immunotherapy.

[0003] OX40 (TNFRSF4) is a member of the tumor necrosis factor (TNF) superfamily and 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 antigen presenting cell membrane, induces T cell activation and enhances effector function, and plays an important role in initial T cell response and secondary response. To date, many domestic and foreign pharmaceutical companies have invested billions of dollars in research and development funds, and a total of 11 OX40 activating antibodies have been used in clinical trials for tumor immunotherapy. In clinical trials, tissue staining analysis of patients' puncture samples showed that OX40 activating antibodies did play a role in enhancing anti-tumor immunity (such as activating CD4+, CD8+ effector T cells and NK cells, inhibiting CD4+ Treg cell function, etc.), but the partial remission rate of these agonist monotherapy did not exceed 10%, which may also be the main reason why OX40 activating antibodies have not been able to cross Phase II of clinical trials.

[0004] In addition, in clinical trials, OX40 activating antibodies are mainly used in combination with immune checkpoint inhibitors (ICB) PD-1, CTLA-4 monoclonal antibodies, etc. Their combined use can dual-activate T cells in a short period of time, but the objective remission rate is still less than 13%, and there is a risk of inducing cytokine release syndrome and leading to multiple organ failure.

[0005] In summary, the clinical transformation problems faced by OX40 activators are: the effect is poor when used alone, and there is a risk of over-activation of the immune system when used in combination with PD-1 and other drugs. Therefore, in order to solve the dilemma of clinical application of OX40 activators, it is necessary to study new combined treatment plans. Summary of the invention

[0006] 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 poorly effective in treating tumors when used alone or in combination with immunoblockers".

[0007] A second object of the present invention is to provide a pharmaceutical composition.

[0008] One of the purposes of the present invention is achieved by the following technical solution:

[0009] 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.

[0010] Furthermore, the OX40 activating antibody and the anti-angiogenic agent are administered simultaneously.

[0011] Furthermore, the OX40 activating antibody and anti-angiogenic agent are used in the preparation of drugs for treating colon cancer.

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

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

[0014] The second object of the present invention is achieved by adopting the following technical solution:

[0015] A pharmaceutical composition comprises the OX40 activating antibody and an anti-angiogenic agent, and pharmaceutically acceptable excipients.

[0016] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0017] The present invention provides an application of an OX40 activating antibody and an anti-angiogenic agent in the preparation of an anti-tumor drug, and the OX40 activating antibody and the anti-angiogenic agent are used in combination in the treatment of tumors. OX40 causes 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 used in combination, and while activating the OX40 "anti-tumor" signal in T cells, the anti-angiogenic agent is used to block the "pro-tumor" effect of the OX40 activating antibody in endothelial cells. The two components act synergistically, showing a better tumor inhibition effect than the combined treatment of the OX40 activating antibody and the PD-1 monoclonal antibody (CD279). BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The growth curves of tumors after injection of five different drugs into three subcutaneous tumor model mice.

[0019] Figure 2 Normal mice (A), T cell immunodeficient mice (B), and fully immunodeficient mice (C) were inoculated with colon cancer MC38 cells to construct subcutaneous tumors, and then treated with OX40L recombinant protein or OX86 antibody for 21 days. The tumor size and growth curve;

[0020] Figure 3 In the figure, A is the imaging and fluorescence value of the in vivo imaging system after 3 weeks of subcutaneous inoculation of MC38-LUC-GFP cells in the colon of fully immunodeficient mice and OX40 activation treatment; B is the liver metastasis nodules after 60 days of subcutaneous inoculation of MC38-LUC-GFP cells in the colon of fully immunodeficient mice and OX40 activation treatment; C is the survival time of mice treated with subcutaneous inoculation of MC38-LUC-GFP cells in the colon of fully immunodeficient mice and OX40 activation treatment;

[0021] Figure 4 is the expression level of OX40 in cells of different cancer types;

[0022] Figure 5 Tumor growth curves after OX40 activation therapy were performed in T cell immunodeficient mice subcutaneously inoculated with glioma and ovarian cancer cells;

[0023] Figure 6 In the figure, A is a scatter plot of subpopulation classification of single-cell sequencing of colon cancer tissue; B is the expression of OX40 in each cell population of colon cancer (CRC) and paracancerous (NT) tissue (n=5); C is a volcano plot of differentially expressed genes between colon cancer endothelial cells and paracancerous normal endothelial cells, where red dots represent upregulated genes and green dots represent downregulated genes;

[0024] Figure 7 is the expression of genes related to cell proliferation, migration and invasion, and apoptosis in TNFRSF4- and TNFRSF4+ endothelial cells;

[0025] Figure 8 In the figure, A is the co-localization of OX40 and CD31 detected in colon cancer and adjacent tissues; B is the expression level of OX40 in tumor endothelial cells (CRC CD31+cells) and normal endothelial cells (NT CD31+cells); C is the expression correlation of OX40 with endothelial marker genes CD31 and VWF;

[0026] Fig. 9 The effect of OX40L recombinant protein stimulation on the tubulogenesis of human umbilical vein endothelial cells (HUVEC);

[0027] Fig.10The differentially expressed genes and signaling pathways after HUVECs were stimulated with exogenous OX40L recombinant protein for 48 hours;

[0028] Fig.11 Cell phenotype after OX40L stimulation of endothelial cells for 5 days;

[0029] Fig.12 is the expression of EndMT-related genes in NFRSF4- and TNFRSF4+ endothelial cells;

[0030] Fig.13 The effect of OX40L stimulation on the expression of α-SMA and Vimentin in endothelial cells;

[0031] Fig.14 Analyze the correlation between OX40 and EndMT marker genes Vimentin and FSP1 for TCGA data;

[0032] Fig.15 In the figure, A is the protein spectrum identification result of cytoplasmic and nuclear proteins after exogenous OX40L stimulation of HUVEC for 48 hours; B is the subcellular localization of YAP protein observed by laser confocal microscopy after exogenous OX40L stimulation of HUVEC; C is the expression level 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;

[0033] Fig.16 Effects of Verteporfin on endothelial cell tubulogenesis regulated by activated OX40. DETAILED DESCRIPTION

[0034] The technical scheme of the present invention is 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 used to illustrate the present invention and should not be regarded as limiting the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0035] Table 1 Experimental reagents and sources

[0036]

[0037]

[0038] Example 1

[0039] Activating OX40 and combining it with YAP inhibitors to treat tumors:

[0040] The mice with normal immune function were divided into 18 groups, with 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).

[0041] Table 2 Mouse cancer models and drug treatment

[0042]

[0043]

[0044] Anesthetized mice were injected with 100 μL of 10 7 Subcutaneous tumors were constructed in nude mice using 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 similar size were selected and treated with intraperitoneal injections of OX86 activating antibody, Verteporfin, CD279, OX86, Verteporfin, OX86, and CD279, respectively. The treatment was performed twice a week for a total of 4 weeks. The tumor volume was measured every day from the first day of drug injection, and a tumor growth curve was drawn. The results are shown in the figure. Figure 1 As shown, although the single-drug treatment groups (OX86, CD279, Verteporfin) all showed a certain tumor inhibitory effect, the combination treatment groups (OX86 and Verteporfin, OX86 and CD279) showed a significant synergistic anti-tumor effect. In the later stage of the combined treatment of OX86 and Verteporfin, some subcutaneous tumors shrank or even disappeared, which was better than the combined treatment of OX86 and PD-1 monoclonal antibody (CD279).

[0045] Test Example 1

[0046] The role of activated OX40 signaling in the progression of colon cancer in normal immune mice and immunodeficient mice:

[0047] 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 The tumors were constructed by intraperitoneal injection of OX40L recombinant protein (10 mg / kg) or OX40 activating antibody OX86 (200 mg / kg) into the three tumor model mice to activate the OX40 signal. The treatment frequency was maintained 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 the figure. Figure 2 As shown, in the normal immune mouse subcutaneous tumor model, OX40L and OX86 antibody activation therapy both had tumor inhibition effects ( Figure 2 A); In T cell immunodeficient mice and fully immunodeficient mice models, OX40 activation therapy did not show anti-tumor effects, but promoted the growth of subcutaneous tumors to varying degrees ( Figure 2 B, C).

[0048] Test Example 2

[0049] Effects of OX40 activation on the growth and metastasis of colon cancer in immunodeficient mice:

[0050] The vector containing the Luc-GFP gene was transfected into the MC38 cell line using the LPP-HLUC-Lv201-100 virus, and the transgenic cells were screened for drugs using 2 μg / mL of puromycin, and finally the MC38-LUC-GFP cell line with stable high expression was 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 in situ 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 the live imaging system after 3 weeks. Starting from the first day of injection of OX40L or OX86, the survival rate of the mice was counted every day, 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 as follows Figure 3 As shown, in fully immunodeficient mice, activation of OX40 promoted tumor growth and metastasis and shortened the survival time of mice.

[0051] The above results indicate that in addition to playing an "anti-tumor" role in T cells, activated OX40 may also play a "pro-tumor" role through other microenvironment cells.

[0052] Test Example 3

[0053] Effects of activating OX40 signaling in other tumors with high OX40 expression:

[0054] Using the publicly available data from the TISCH Tumor Microenvironment Single Cell Transcriptome Database (http: / / tisch.comp-genomics.org / ), we analyzed the cancer types with high expression of OX40 in endothelial cells and screened out three cancer types with high expression of OX40 in endothelial cells: colon cancer, glioma, and ovarian cancer ( Figure 4 ).

[0055] 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 peritoneum to activate OX40 signal (8 mice in each tumor model mouse OX40L treatment group, OX86 treatment group, and Vehicle group). The treatment was continued 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. The results are shown in Figure 5 As shown, activation of OX40 signaling can significantly promote tumor growth, indicating that the "tumor-promoting" effect of activated OX40 is a universal phenomenon.

[0056] Test Example 4

[0057] Exploring the molecular mechanism of OX40's "tumor-promoting" function:

[0058] Human colon cancer tissues were sent to Guangzhou Kidio Biotechnology Co., Ltd. for single-cell sequencing to analyze the expression levels of OX40 in each cell subpopulation. Figure 6 As shown in the figure, 10 cell subsets were identified in colon cancer tissues and adjacent normal tissues: plasma cells, epithelial cells, B cells, T cells, endothelial cells, macrophages, fibroblasts, myeloid-derived suppressor cells (MDSC), innate lymphocytes (iLC), monocytes, and mast cells (Mast cells). Figure 6 A), and OX40 is expressed at the highest level in T cell subsets and also at a high level in tumor endothelial cells, second only to T cell subsets ( Figure 6 B). In addition, the expression level of OX40 in colon cancer endothelial cells was significantly higher than that in adjacent normal tissue endothelial cells ( Figure 6 B, C).

[0059] According to the results of single-cell sequencing, endothelial cells in human colon cancer tissues were divided into TNFRSF4 / OX40 negative (TNFRSF-) cells and TNFRSF4 / OX40 positive (TNFRSF+) cells based on whether TNFRSF4 expression could be detected, and the differentially expressed genes between the two were analyzed. Figure 7 As shown in the data, in colon cancer tissues, key oncogenes such as cell proliferation, migration and invasion (such as KMT2D, MYC, SOD2, SOX2, COX2, CCNB1, CDK1, AKT, etc.) were significantly overexpressed in TNFRSF+ endothelial cells, while apoptosis-related genes (such as RB, BAD, FAS, etc.) were significantly underexpressed in TNFRSF- endothelial cells, suggesting that OX40-highly expressed endothelial cells have a "tumor-promoting" effect.

[0060] The human colon cancer tissue samples were washed with PBS solution to remove blood, then fixed with 4% paraformaldehyde and sent to Heyuan Biotechnology (Shanghai) Co., Ltd. for multicolor immunofluorescence staining. The results showed that OX40 and CD31 were co-localized and expressed in vascular endothelial cells in colon cancer (CRC) tissues, and the expression level was higher than that in normal tissues adjacent to the cancer (NT) ( Figure 8 A).

[0061] Human colon cancer tissue samples were sorted using anti-CD31 magnetic beads to separate endothelial cells from colon cancer and corresponding adjacent tissues, and then qPCR detection 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 8 B).

[0062] The gene expression profile of colon cancer (COAD) was downloaded from the TCGA database (https: / / xenabrowser.net / datapages / ), and other genes related to the OX40 gene were found through correlation analysis. Figure 8 As shown in C, the expression level of OX40 was significantly positively correlated with the vascular endothelial cell marker gene CD31 or VWF.

[0063] The above results indicate that OX40 is very likely involved in the regulation of tumor blood vessels.

[0064] Test Example 5

[0065] Activation of OX40 signaling affects the tubulogenesis capacity of endothelial cells:

[0066] 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 to 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 The HUVEC cells were resuspended and incubated at 37°C and 5% CO2 for 6 hours. The culture medium was carefully removed, the cells were stained with Calcein AM, and the tubular network was imaged using a fluorescence microscope. Fig. 9 As shown, activation of OX40 significantly promoted the tubule formation ability of endothelial cells.

[0067] Test Example 6

[0068] Study on the mechanism of activating OX40 signaling to promote endothelial cell mesenchymal transition:

[0069] The HUVEC cells cultured in Experimental Example 5 were subjected to transcriptomic sequencing (RNA-Seq), and the results were as follows: Fig.10 As shown in the results, after activating the OX40 signal, epithelial-mesenchymal transition (EMT), endothelial cell proliferation, migration, angiogenesis and other signaling pathways were significantly activated, and the expression of genes related to the EMT signaling pathway was also significantly upregulated, suggesting that OX40 activation 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.

[0070] An Olympus inverted microscope ( CKX31 ) The HUVEC cells cultured in Experimental Example 5 were photographed and imaged. The results showed that after OX40L stimulation, the endothelial cells lost their cobblestone-like characteristics and gradually transformed into spindle-like shapes, with pseudopodia extending, showing a mesenchymal cell phenotype ( Fig.11 ).

[0071] Expression analysis of EndMT-related genes was performed on TNFRSF- and TNFRSF+ endothelial cells in colon cancer tissue (obtained from Experimental Example 4). The results showed that EndMT markers such as SNAIL, SLUG, TWIST, TGFβ and other genes were upregulated in TNFRSF4+ tumor endothelial cells, while adhesion molecules such as CDH1 and CDH5 were downregulated ( Fig.12 ).

[0072] WB semi-quantitative detection of EndMT markers α-SMA and Vimentin in HUVEC endothelial cells cultured in Experimental Example 5 showed that OX40L stimulated the expression of α-SMA and Vimentin ( Fig.13 ).

[0073] The gene expression profile of colon cancer was downloaded from the TCGA database (https: / / xenabrowser.net / datapages / ), and the endothelial-mesenchymal marker genes related to the OX40 gene were found through correlation analysis. It was found that OX40 was highly positively correlated with Vimentin and FSP1 ( Fig.14 ).

[0074] 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.

[0075] Test Example 7

[0076] Searching for key transcription factors that activate OX40 signaling to regulate endothelial cell function:

[0077] The cytoplasm and nuclear proteins of HUVEC cells cultured in Experimental Example 5 were extracted and protein spectrum was identified. The results showed that activation of OX40 significantly promoted YAP nuclear translocation. Compared with the control group, YAP protein nuclear translocation increased by 16.52 times ( Fig.15 A), and immunofluorescence also confirmed this result ( Fig.15 B).

[0078] Anti-CD31 magnetic beads were used to sort endothelial cells in colon cancer and corresponding adjacent tissues. qPCR detection 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 ( Fig.15 C).

[0079] Analysis of single-cell transcriptome data (experimental example 4) showed that YAP downstream target genes were also upregulated in TNFRSF4+ tumor endothelial cells ( Fig.15 D).

[0080] The above results suggest that activation of endothelial cell OX40 significantly promotes YAP nuclear translocation and activates its transcriptional regulatory activity.

[0081] Test Example 8

[0082] 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, OX40L protein (100ng / mL) and OX40L protein (100ng / mL) + Verteporfin (5μM) were added for 48 hours. 250μl BME was added to a 24-well plate to cover the bottom of the well and incubated for 30 minutes at 37°C and 5% CO2 to solidify. 300μL of culture medium and 10 5 The HUVEC cells were resuspended and incubated at 37°C and 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. Fig.16 As shown, the YAP-TEAD4 inhibitor Verteporfin blocked the biological functions of endothelial cells regulated by activated OX40, indicating that activated OX40 may regulate the biological functions of endothelial cells by promoting YAP nuclear translocation.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. On the basis of the present invention, some modifications or replacements may be made thereto, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of protection claimed by the present invention.

Claims

1. The use of OX40 activating antibodies and anti-angiogenic agents in the preparation of anti-tumor drugs, characterized in that: The OX40 activating antibody is OX86, the anti-angiogenic agent is verteporfin, the anti-tumor drug is a drug for treating colon cancer, and the OX40 activating antibody and the anti-angiogenic agent are administered simultaneously.

2. The use of OX40 activating antibodies and anti-angiogenic agents in the preparation of anti-tumor drugs, characterized in that: The OX40 activating antibody is OX86, the anti-angiogenic agent is verteporfin, the anti-tumor drug is a drug for treating glioma, and the OX40 activating antibody and the anti-angiogenic agent are administered simultaneously.

3. The use of OX40 activating antibodies and anti-angiogenic agents in the preparation of anti-tumor drugs, characterized in that: The OX40 activating antibody is OX86, the anti-angiogenic agent is verteporfin, the anti-tumor drug is a drug for treating ovarian cancer, and the OX40 activating antibody and the anti-angiogenic agent are administered simultaneously.

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