Application of ANGPT2 monoclonal antibody in preparation of medicine for treating non-small cell lung cancer of subjects subjected to heavy ion treatment

Abstract

The invention provides an application of an ANGPT2 monoclonal antibody in preparation of a medicine for treating non-small cell lung cancer of a subject subjected to heavy ion treatment. Research results show that the promotion effect of the combined treatment of the nevapsumab and the heavy ion irradiation on ANGPT2 overexpression A549 cell cycle arrest and cell apoptosis rate and the inhibition effect on cell invasion, migration and clone forming ability are obviously better than those of the single treatment of the nevapsumab and the heavy ion irradiation. In a nude mouse transplantation tumor model, the inhibition effect of the combined treatment on the growth of ANGPT2 overexpression A549 cell transplantation tumor is more obvious than that of the single treatment of nevapsumab and heavy ion irradiation, and the negative influence on the physiological state of a host is smaller. A new strategy is provided for clinical treatment of the non-small cell lung cancer, and it is indicated that combined application of heavy ion treatment and ANGPT2 monoclonal antibody is expected to improve the treatment effect, prolong the lifetime of a patient and reduce treatment-related side effects, so that the life quality of the patient is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the use of an anti-ANGPT2 monoclonal antibody in the preparation of a medicament for treating non-small cell lung cancer in subjects undergoing heavy ion therapy. Background Art

[0002] Non-small cell lung cancer (NSCLC) is the most common type of lung cancer globally, accounting for approximately 85% of all lung cancer cases. In recent years, the emergence of targeted therapy and immunotherapy has brought new treatment hopes to NSCLC patients. Although the latest technologies in targeted therapy and immunotherapy have improved the survival rate of some patients in recent years, the treatment effect of patients with advanced or recurrent NSCLC is still not ideal. Traditional treatment methods, such as surgery, chemotherapy, and radiotherapy, have limited efficacy for locally advanced or metastatic tumors and are often accompanied by severe side effects. In particular, radiotherapy and chemotherapy may cause normal tissue damage and drug resistance. Despite the continuous progress of the latest treatment methods, the treatment of NSCLC still faces many challenges.

[0003] As an advanced radiotherapy method, heavy ion therapy has unique physical and biological characteristics. Compared with traditional X-rays or γ-rays, heavy ions can more precisely concentrate high energy within tumor tissues, reducing damage to normal tissues. Heavy ions have a high linear energy transfer (LET), which can cause double-strand breaks in cellular DNA, resulting in irreversible DNA damage. Existing studies have shown that heavy ion therapy has achieved excellent treatment effects in various tumor types such as lung cancer, liver cancer, prostate cancer, and glioblastoma multiforme, showing broad application prospects.

[0004] Angiogenesis is a key process in tumor growth and metastasis. As one of the angiogenesis regulatory factors, angiopoietin-2 (ANGPT2) plays an important role in the process of tumor angiogenesis. The overexpression of ANGPT2 is related to tumor progression, metastasis, and drug resistance. By binding to its receptor Tie-2, ANGPT2 disrupts the stability of blood vessels and promotes the disordered growth of tumor-related blood vessels, thereby providing sufficient oxygen and nutritional support for tumors. Therefore, inhibiting the activity of ANGPT2 is considered one of the effective strategies for anti-angiogenesis. In recent years, anti-ANGPT2 monoclonal antibodies (such as REGN910, AMG386) have demonstrated potential efficacy in clinical trials. Especially when combined with chemotherapy and immunotherapy, they can significantly inhibit the formation of tumor blood vessels, improve the tumor microenvironment, and further enhance the anti-tumor effect. Therefore, the anti-angiogenesis targeting ANGPT2 combined with heavy ion irradiation has potential clinical practical value for the treatment of non-small cell lung cancer. Summary of the Invention

[0005] The object of the present invention is to provide the use of an anti-ANGPT2 monoclonal antibody in the preparation of a medicament for treating non-small cell lung cancer in a subject.

[0006] In the said use, the subject receives heavy ion therapy;

[0007] Furthermore, the subject has received heavy ion therapy before the administration of the monoclonal antibody, the subject receives heavy ion therapy simultaneously with the administration of the monoclonal antibody, or the subject receives heavy ion therapy after the administration of the monoclonal antibody.

[0008] The heavy ion may specifically be a carbon ion, and the energy of the carbon ion is 80 - 300 MeV / u, and the LET is 31.3 - 50 keV / μm;

[0009] For the irradiation of the carbon ion peak region, the dose is 2 - 6 Gy, and specifically may be 4 Gy;

[0010] More specifically, the carbon ion is provided by the heavy ion beam research facility of the Institute of Modern Physics, Chinese Academy of Sciences - Lanzhou Heavy Ion Research Facility (HIRFL). The energy of the carbon ion therapy terminal of this facility is 80 MeV / u, and the LET is 50 keV / μm;

[0011] The anti-ANGPT2 monoclonal antibody may specifically be navasumab.

[0012] Furthermore, in the said use, the medicament has at least one of the following functions 1) - 5):

[0013] 1) Inducing apoptosis of tumor cells;

[0014] 2) Inducing cell cycle arrest of tumor cells at the G2 / M phase;

[0015] 3) Inhibiting the invasion and migration ability of tumor cells;

[0016] 4) Inhibiting the colony formation ability of tumor cells;

[0017] 5) Inhibiting tumor growth.

[0018] The tumor is cancer, and the cancer may specifically be non-small cell lung cancer.

[0019] The present invention also provides a new use of an anti-ANGPT2 monoclonal antibody medicament.

[0020] The new use of the anti-ANGPT2 monoclonal antibody medicament provided by the present invention is: the use of an anti-ANGPT2 monoclonal antibody in the preparation of a heavy ion radiation sensitizer for tumor cells.

[0021] The tumor cells are cancer cells, specifically non-small cell lung cancer cells, and more specifically A549 cells.

[0022] The heavy ions are specifically carbon ions, and the energy of the carbon ions is 80 - 300 MeV / u, and the LET is 31.3 - 50 keV / μm;

[0023] For the carbon ion peak region irradiation, the dose is 2 - 6 Gy, specifically 4 Gy;

[0024] More specifically, the carbon ions are provided by the Heavy Ion Beam Research Facility of the Institute of Modern Physics, Chinese Academy of Sciences - Lanzhou Heavy Ion Research Facility (HIRFL). The energy of the carbon ion treatment terminal of this device is 80 MeV / u, and the LET is 50 keV / μm;

[0025] The ANGPT2 monoclonal antibody is specifically nivolumab.

[0026] The present invention discovers that after the A549 cells stably overexpressing ANGPT2 are treated with the combination of nivolumab and heavy ion irradiation, the promotion effect on cell cycle arrest and apoptosis rate and the inhibition effect on cell invasion, migration and clone formation ability are significantly stronger than those of nivolumab and heavy ion irradiation alone. In vivo experiments in mice confirm that the combined treatment of nivolumab and heavy ion irradiation has a more significant inhibitory effect on tumor growth than nivolumab and heavy ion irradiation alone. The present invention proves that nivolumab can inhibit tumor growth by enhancing the radiation sensitivity of tumor cells. This exploration may become a new treatment strategy and is expected to provide theoretical basis and experimental support for future clinical treatment. Brief Description of the Drawings

[0027] Figure 1 It is a diagram of the ANGPT2 overexpression plasmid.

[0028] Figure 2 It is the detection of the expression of the FLAG-tagged protein in ANGPT2-overexpressing A549 cells by Western blotting.

[0029] Figure 3 It is the effect of nivolumab on the proliferation ability of ANGPT2-overexpressing A549 cells. A, The relationship between cell viability and nivolumab concentration, **p<0.01, ***p<0.001, n = 3; B, The IC 50 value of nivolumab acting on ANGPT2-overexpressing A549 cells.

[0030] Figure 4To investigate the effect of navasumab on enhancing the inhibitory effect of heavy ion radiation on the clonogenic survival of ANGPT2-overexpressing A549 cells. A, Effects of different doses of heavy ion radiation on the clonogenic survival of ANGPT2-overexpressing A549 cells; B, Effects of navasumab combined with heavy ion radiation on the clonogenic survival of ANGPT2-overexpressing A549 cells; C, D, Clonogenic survival counts. **p<0.01, ***p<0.001, n = 3.

[0031] Figure 5 To investigate the effect of navasumab on enhancing heavy ion radiation-induced G2 / M phase arrest in ANGPT2-overexpressing A549 cells. A, Results of flow cytometry analysis of the cell cycle; B, Analysis of cell cycle ratios; *p<0.05, **p<0.01, ***p<0.001, n = 3.

[0032] Figure 6 To investigate the effect of navasumab on enhancing heavy ion radiation-induced apoptosis in ANGPT2-overexpressing A549 cells. A, Results of flow cytometry analysis of cell apoptosis; B, Analysis of apoptosis rates; *p<0.05, ***p<0.001, n = 3.

[0033] Figure 7 To investigate the effect of navasumab on enhancing the inhibitory effect of heavy ion radiation on the invasion and migration abilities of ANGPT2-overexpressing A549 cells. A, Results of microscopic observation of invasion and migration; B, Statistical analysis of invasion counts; C, Statistical analysis of migration counts. *p<0.05, ***p<0.001, n = 3.

[0034] Figure 8 To investigate the effect of navasumab on enhancing the inhibitory effect of heavy ion radiation on the xenografts of ANGPT2-overexpressing A549 cells. A, Comparison of the volumes of xenografts at the end of treatment; B, Monitoring of the volumes of xenografts, *p<0.05, **p<0.01; C, Monitoring of the body weights of nude mice bearing tumors. There were 5 tumor-bearing mice in each group, n = 5.

[0035] Figure 9 For H&E staining analysis of ANGPT2-overexpressing A549 cell xenografts at the end of combination treatment. Detailed implementation manners

[0036] The present invention will be further described in detail below in combination with the specific implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0037] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0038] Example 1: Combined treatment of ANGPT2 monoclonal antibody and heavy ion radiation for non-small cell lung cancer

[0039] 1.1 Cell lines and culture conditions

[0040] Human non-small cell lung cancer A549 cells were purchased from Suzhou Haixing Biotechnology Co., Ltd. and cultured in F-12K containing 10% fetal bovine serum (FBS: ExCell, Suzhou, China) at 37 °C in an environment of 5% CO 2 . The cells used in the experiment were all in the logarithmic growth phase.

[0041] 1.2 Carbon ion irradiation

[0042] Carbon ions were provided by the Heavy Ion Beam Research Facility of the Institute of Modern Physics, Chinese Academy of Sciences - Lanzhou Heavy Ion Research Facility (HIRFL). The carbon ion treatment terminal energy of this facility is 80 MeV / u and the LET is 50 keV / μm.

[0043] 1.3 Construction of A549 cell line with overexpressed ANGPT2

[0044] The pcSLenti-CMV-MCS-3xFLAG-PGK-Puro-WPRE3 overexpression plasmid (Shanghai Heyuan Biotechnology) was used to construct ANGPT2-overexpressing cells. The accession number of the ANGPT2 gene is NM_001147.3, the gene size is 1491 bp, and the upstream and downstream cloning restriction enzyme sites are EcoRI and BamHI. The ANGPT2 overexpression plasmid is pcSLenti-CMV-ANGPT2-3xFLAG-PGK-Puro-WPRE3. A549 cells were seeded into 6-well plates (about 2×10 5 cells per well) at a confluence of 30%. After 20 h, A549 cells were infected with lentivirus expressing ANGPT2. Polybrene with a final concentration of 5 μg / mL was added to each well. After 20 h of infection, the medium was discarded, and 2 mL of fresh medium was added to each well. After 72 h, puromycin with a final concentration of 2 μg / mL was added. Fresh medium containing puromycin with a final concentration of 2 μg / mL was changed every 2 days. After two weeks of drug screening, the cell status was observed.

[0045] 1.4 Cell grouping and treatment

[0046] Nivastumab (product number HY-P99036) and IgG (product number HY-P99001) were purchased from MCE. The experiments were divided into a control group (IgG), a nivastumab group, a heavy ion irradiation group (IgG + 4 Gy), and a nivastumab combined with heavy ion irradiation group (nivastumab + 4 Gy). The cells in the control group were treated with 5 μg / mL IgG for 48 h; the cells in the nivastumab group were treated with 5 μg / mL nivastumab for 48 h; the cells in the heavy ion irradiation group were treated with 5 μg / mL IgG for 24 h, without changing the culture medium, then irradiated with 4 Gy carbon ions and continued to be cultured for 24 h (the IgG treatment time was 48 h); the cells in the nivastumab combined with heavy ion irradiation group were treated with 5 μg / mL nivastumab for 24 h, without changing the culture medium, then irradiated with 4 Gy carbon ions and continued to be cultured for 24 h (the nivastumab treatment time was 48 h).

[0047] 1.5 CCK-8 assay

[0048] ANGPT2-overexpressing cells in the logarithmic growth phase with good growth status were selected, and 3000 cells were inoculated into each well. There were 5 replicate wells in each group, and a blank group was set up simultaneously. The 96-well plate inoculated with cells was placed in an incubator at 37 °C and 5% CO 2 for 24 h, then drugs were added and incubation continued for 24 h. After incubation, 10 μL of CCK-8 reagent was added to each well. The mixture was shaken well and incubated at 37 °C for another 3 h, and then the absorbance value at 450 nm was measured on an enzyme-linked immunosorbent assay (ELISA) reader.

[0049] 1.6 Cell apoptosis

[0050] ANGPT2-overexpressing cells were inoculated at 2.5×10 5 / well into φ35 culture dishes. After the cells were treated with nivastumab and carbon ions for 48 h, they were digested with trypsin without EDTA, centrifuged at 800 rpm for 4 min, and washed twice with PBS. The cells were stained according to the instructions of the AnnexinV-7AAD apoptosis detection reagent. The apoptosis rate of the cells was detected and analyzed by a CUBE flow cytometer (FlowJo-V10).

[0051] 1.7 Cell cycle

[0052] Inoculate ANGPT2 overexpressing cells in a φ35 culture dish with the above cell number. After treating the cells by the same method as above, collect the cells in a centrifuge tube, centrifuge at 800 rpm for 4 min, discard the supernatant, and wash the cells twice with PBS. Add an appropriate amount of pre-cooled 70% ethanol, gently pipette to mix well, and suspend the cells in ethanol. Place the cell suspension in a -20°C refrigerator for overnight fixation. Wash the cells three times with PBS to remove the residual ethanol, centrifuge at 800 rpm for 4 min to collect the cell pellet, and discard the supernatant. Add an appropriate amount of RNase solution and incubate at room temperature for about 30 min, then add propidium iodide (PI) staining solution, gently pipette to mix well, incubate in the dark for more than 30 min, and detect by loading on a flow cytometer.

[0053] 1.8 Transwell Migration and Invasion Assays

[0054] Inoculate ANGPT2 overexpressing cells in the logarithmic growth phase at 3×10 4 / well in the upper chamber of Transwell for culture (use medium without FBS in the upper chamber), add medium containing FBS to the lower chamber, place the chamber in an incubator and incubate for 48 h to promote cell migration to the lower chamber. Gently rinse the upper chamber of Transwell with PBS to remove the non-migrated cells. After rinsing three times with PBS, fix the cells with 4% paraformaldehyde for 15 min. After fixation, rinse the cells twice with PBS to remove the excess fixative. Add 0.1% crystal violet solution to the upper chamber of Transwell and stain for 30 min. After staining, gently rinse three times with PBS to remove the excess dye. Observe the Transwell membrane using an inverted microscope, take images, and select random areas to count the cells that have migrated to the bottom of the membrane. Compare according to the number of migrated cells in each group, and statistically calculate the migration rate.

[0055] Similar to the Transwell migration assay, Matrigel needs to be added to the upper chamber for the invasion assay. First, place the thawed Matrigel on ice. Add an appropriate volume concentration of Matrigel to the bottom of the upper chamber of Transwell and incubate at 4°C for 3 h. Then add medium containing FBS to the lower chamber of Transwell. Inoculate the cells in the gelled upper chamber with the same cell number as in the migration assay, and collect the samples after treating in the incubator for 48 h. The specific steps for sample collection are the same as those for the migration assay.

[0056] 1.9 Colony Formation

[0057] Inoculate ANGPT2-overexpressing cells into φ60 culture dishes: control group (1000 cells), nivolumab group (1000 cells), 4 Gy heavy ion irradiation group (2000 cells), and nivolumab + 4 Gy heavy ion irradiation group (2000 cells). After culturing for 14 days, discard the culture medium, wash twice with PBS, fix with 4% paraformaldehyde for 30 min, stain with 1% crystal violet for 1 h, and count cell clusters containing 50 or more cells under a microscope.

[0058] 1.10 H&E staining

[0059] Place the tumor tissue in 4% paraformaldehyde solution for fixation for 24 h. After fixation, dehydrate the tissue through gradient ethanol (70%, 80%, 90%, 100%), clear with xylene, and finally embed in paraffin. The section thickness of the paraffin-embedded tissue sample is 4 μm. Spread the tissue sections on glass slides and dry them in an oven at 60 °C for later use. The sections are successively dewaxed with xylene (twice, 5 min each time) and hydrated through gradient ethanol (100%, 90%, 80%, 70%). Subsequently, stain the nuclei with hematoxylin stain for 10 min and rinse thoroughly with running water to remove background staining. Immerse the sections in 1% hydrochloric acid ethanol for rapid differentiation, and then rinse repeatedly with tap water until the tissue turns blue-violet. Next, place the sections in 1% eosin stain for 3 min and rinse with running water to remove excess stain. After staining, dehydrate the sections through gradient ethanol, clear with xylene, then add neutral gum to seal the slides and cover with cover slips. After the sealed slides are completely cured, observe and record the tissue morphological characteristics using an optical microscope, including the staining conditions of the cell nuclei and cytoplasm and the structural changes of the tumor tissue.

[0060] 1.11 Data statistics and analysis

[0061] Use GraphPad Prism 8 software to perform statistical analysis on the data. The experimental results are expressed in the form of "mean ± error". The comparison between two groups of data uses the t-test, and the comparison of multiple groups of data is performed by one-way ANOVA. The significance level is set at *p < 0.05, which is considered the standard for statistical significance.

[0062] 2. Animal experiments

[0063] 2.1 Establishment and grouping of animal models

[0064] In this study, immunodeficient female nude mice aged 4 - 6 weeks were used as experimental animals. Each nude mouse was inoculated with approximately 5 million ANGPT2 - overexpressing A549 cells via subcutaneous injection. Tumor growth was monitored by measuring tumor volume, and the nude mice were randomly divided into 4 groups for treatment: IgG control group, nivolumab treatment group, heavy ion irradiation treatment group, and combination treatment group of nivolumab and heavy ion irradiation. There were 5 nude mice in each group.

[0065] 2.2 Administration schedule for combination treatment:

[0066] (1) Control group (IgG): The administration dose of IgG was 25 μg / mouse, administered once every 3 days for a total of 5 times.

[0067] (2) Nivolumab treatment group: Received nivolumab treatment, with an administration dose of 25 μg / mouse, administered once every 3 days for a total of 5 times.

[0068] (3) Heavy ion irradiation treatment group (IgG + 4 Gy): The nude mice were given IgG 1 h in advance, and then received local irradiation in the peak region of heavy ions at 4 Gy. The administration method and frequency of IgG were the same as those in the IgG group.

[0069] (4) Combination treatment group of nivolumab and carbon ion irradiation (nivolumab + 4 Gy): The nude mice received nivolumab treatment 1 h in advance, and then received local irradiation in the peak region of heavy ions at 4 Gy. The administration method and frequency of nivolumab were the same as those in the nivolumab treatment group.

[0070] 2.3 Monitoring and evaluation of tumor growth

[0071] Tumor volume was measured on the 3rd day after the start of treatment, and tumor volume was monitored every 3 days until the end of the experiment. The measurement of tumor volume used the following formula: Tumor volume = 1 / 2 × long diameter × short diameter 2 . By measuring tumor volume, the inhibitory effect of different treatment methods on tumor growth was evaluated.

[0072] 3. Results and discussion

[0073] 3.1 Detection of ANGPT2 over - expressed cell expression level

[0074] The ANGPT2 - overexpressing plasmid is as Figure 1 shown. After the cells were completed with drug screening, cell pellets were collected, the cells were lysed with IP lysis buffer to separate the supernatant, and Western Blotting was used to analyze the protein expression status. The primary antibodies used were FLAG - tagged antibody and GAPDH antibody. The Western Blotting results are as Figure 2As shown, FLAG-tagged protein expression was detected in the ANGPT2 vector group A549 cells (the third lane), and the molecular weight was 70 KDa, which was consistent with the molecular weight of ANGPT2 protein. However, no tagged protein expression was detected in A549 and A549 empty vector cells, indicating that the stable overexpressing cells of ANGPT2 were successfully constructed and the tagged protein was successfully expressed.

[0075] 3.2 Effect of Nevasumab on the Proliferation of ANGPT2 Overexpressing Cells

[0076] After the cells were cultured in a 96-well plate for 24 h, different concentrations of nevasumab were added. After culturing the cells for another 24 h, the cell viability was detected using a CCK-8 kit. As Figure 3 shown in A below, compared with the control group, 5 μg / mL of nevasumab already showed a significant inhibitory effect on the cells, indicating that nevasumab has a strong inhibitory effect on the proliferation of ANGPT2 overexpressing cells. The IC 50 was 13.36 μg / mL ( Figure 3 shown in B below). Therefore, nevasumab at a concentration of 5 μg / mL was selected for subsequent experiments.

[0077] 3.3 Effect of Combination Therapy on the Clonal Survival of ANGPT2 Overexpressing Cells

[0078] The results were as Figure 4 shown in A below. The inhibitory effect of heavy ion radiation on the clonal survival of ANGPT2 overexpressing cells was dose-dependent. As Figure 4 shown in B, C, and D below, compared with the control group, the combination therapy had a stronger inhibitory effect on the clonal survival of ANGPT2 overexpressing cells than nevasumab and heavy ion irradiation, indicating that nevasumab enhanced the inhibitory effect of heavy ions on the clonal survival of tumor cells by increasing the radiation sensitivity of tumor cells.

[0079] 3.4 Effect of Combination Therapy on the Cell Cycle of ANGPT2 Overexpressing Cells

[0080] Flow cytometry of the cell cycle was as Figure 5 shown in A below, and the cell cycle ratio results were as Figure 5 shown in B below. The cell cycle of ANGPT2 overexpressing cells in the nevasumab, heavy ion irradiation, and combination therapy groups was blocked at the G2 / M phase, and the combination therapy had a stronger cell cycle blocking effect than nevasumab and heavy ion irradiation. Therefore, nevasumab may increase the inhibitory effect of heavy ions on tumor cells by increasing the G2 / M phase block of tumor cells.

[0081] 3.5 Effect of Combination Therapy on the Apoptosis of ANGPT2 Overexpressing Cells

[0082] Flow cytometry of cell apoptosis was as Figure 6As shown in A, the results of apoptosis rate analysis are as follows Figure 6 As shown in B, compared with the control group, there was no significant change in the apoptosis rate of the navasumab group, indicating that the use of navasumab alone had little effect on the apoptosis of ANGPT2 overexpressing cells. The G2 / M phase arrest caused by navasumab might not induce apoptosis in cells. The apoptosis rate of the heavy ion irradiation group increased significantly, indicating that heavy ions could effectively induce apoptosis. The apoptosis rate of the combination treatment group was higher than that of the navasumab and heavy ion irradiation groups, indicating that the combined use of navasumab and heavy ion irradiation could further enhance the apoptosis induced by heavy ions.

[0083] 3.6 Effects of combination treatment on the invasion and migration abilities of ANGPT2 overexpressing cells

[0084] The microscopic observation results of the Transwell chambers are as follows Figure 7 As shown in A, the cell counting results are as follows Figure 7 As shown in B, the results indicated that the combination treatment had a more significant inhibitory effect on the invasion and migration abilities of ANGPT2 overexpressing cells than navasumab and heavy ion irradiation treatments. This result suggested that navasumab might further inhibit the invasion and migration abilities of tumor cells by enhancing the sensitivity of tumor cells to heavy ion radiation.

[0085] 3.7 Effects of combination treatment on the growth of transplanted tumors

[0086] The anatomical diagrams of the tumor masses at the end of treatment are as follows Figure 8 As shown in A, compared with the navasumab group and the heavy ion irradiation group, the tumor volume of the combination treatment group was the smallest, and the inhibitory effect on the tumor mass was stronger, indicating that the combination treatment had a better therapeutic effect than navasumab and heavy ion irradiation, suggesting that navasumab could increase the radiation sensitivity of ANGPT2 overexpressing cells to heavy ions and enhance the therapeutic effect of heavy ions on tumors. The monitoring results of the transplanted tumor volume are as follows Figure 8 As shown in B, the tumor volume of the control group showed a continuous growth trend, indicating that the growth ability of untreated tumors was strong. Compared with the control group, navasumab and heavy ion irradiation showed an inhibitory effect on tumors, and the growth rate of the tumor mass slowed down significantly, and the inhibitory effect of heavy ion irradiation on tumors was better than that of navasumab. Compared with the navasumab group and the heavy ion irradiation group, the tumor volume of the combination treatment group was the smallest, and the inhibitory effect on the tumor mass was more significant, indicating that the combination treatment had a better therapeutic effect than navasumab and heavy ion irradiation, suggesting that navasumab could increase the radiation sensitivity of ANGPT2 overexpressing cells to heavy ions and enhance the therapeutic effect of heavy ions on tumors. As follows Figure 8 As shown in C, the body weight of the mice in the combination treatment group did not decrease significantly, and the body weight recovery trend of the combination treatment group was better, approaching the control group level, showing the advantages of the combination treatment in maintaining the health of mice and demonstrating good safety and application prospects.

[0087] The results of H&E staining were as Figure 9 shown. In the control group, the tumor tissue structure was compact, and the cell arrangement was relatively regular, showing a high proliferation activity. In contrast, the tumor tissues in the heavy ion irradiation treatment group and the nivolumab treatment group showed varying degrees of tissue necrosis and tumor cell apoptosis. Especially in the heavy ion irradiation treatment group, the phenomenon of cell necrosis was more obvious. The tumor tissue structure in the combination treatment group was more severely damaged, with large vacuolated areas and a large number of cell necroses. It is indicated that nivolumab enhanced the sensitivity of tumor cells to heavy ion radiation, resulting in a larger range of necrosis and disintegration of the tumor tissue.

[0088] Based on the above research results, nivolumab enhanced the sensitivity of tumor cells to heavy ion radiation through multiple mechanisms such as promoting apoptosis, inhibiting invasion and migration, and cell survival. The combined treatment of nivolumab and heavy ion radiation had a more significant inhibitory effect on the survival, invasion, and migration abilities of ANGPT2-overexpressing cells than the single treatment of nivolumab and heavy ion irradiation, and also had an advantage in inhibiting transplanted tumors. In addition, the combined treatment could reduce its potential toxicity compared with the single use of nivolumab, showing better safety. The above mechanisms indicate that the combined treatment of nivolumab and heavy ion irradiation has important clinical application potential in the treatment of non-small cell lung cancer.

[0089] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In general, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. Use of ANGPT2 monoclonal antibody in the preparation of a drug for treating non-small cell lung cancer in a subject.

2. The use according to claim 1, characterized in that: In the application, the subject receives heavy ion therapy.

3. The use according to claim 2, characterized in that: The subject has received heavy ion therapy before the administration of the monoclonal antibody, the subject has received heavy ion therapy simultaneously with the administration of the monoclonal antibody, or the subject has received heavy ion therapy after the administration of the monoclonal antibody.

4. The use according to claim 1, characterized in that: The heavy ions are carbon ions.

5. The use according to claim 4, characterized in that: The energy of the carbon ions is 80-300 MeV / u, and the linear energy transfer density LET is 31.3-50 keV / μm.

6. The use according to claim 4, characterized in that: The carbon ion peak region irradiation has a dose of 2-6 Gy.

7. The use according to claim 1, characterized in that: The ANGPT2 monoclonal antibody is nevasumab.

8. The use according to any one of claims 1 to 7, characterized in that: In the application, the drug has at least one of the following functions 1)-5): 1) Induce apoptosis of tumor cells; 2) Induce tumor cell cycle arrest at the G2 / M phase; 3) Inhibit the invasion and migration ability of tumor cells; 4) Inhibit the cloning ability of tumor cells; 5) Inhibit tumor growth; The tumor is cancer, and the cancer may specifically be non-small cell lung cancer.

9. Use of ANGPT2 monoclonal antibody in the preparation of a heavy ion radiation sensitizer for tumor cells, wherein the ANGPT2 monoclonal antibody may specifically be nevacuzumab.

10. The use according to claim 9, characterized in that: The tumor cells are cancer cells, and the cancer cells are non-small cell lung cancer cells; The heavy ions are carbon ions.

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