Application of neutralizing antibody of GM-CSF in preparation of medicine for improving radiation resistance

Abstract

The invention belongs to the technical field of tumor treatment drug research, and particularly relates to application of a neutralizing antibody of GM-CSF in preparation of a drug for improving radiation resistance. Tests prove that the neutralizing antibody of the GM-CSF improves radiation resistance of tumor patients by reducing M2 type macrophages induced by radiotherapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor therapeutic drug research, and particularly relates to the application of a GM-CSF neutralizing antibody in the preparation of a drug for improving radioresistance. Background Art

[0002] Radiotherapy is one of the main methods of local tumor treatment. About 50-60% of cancer patients need to receive radiotherapy. However, some of these patients may develop radioresistance, which affects the effectiveness of radiotherapy and even the patient's prognosis. Studies have shown that after tumors receive radiotherapy, they cause CD8 + The anti-tumor immune response centered on T cells is crucial to the efficacy of radiotherapy. At the same time, radiation also increases the number of M2 macrophages with immunosuppressive function in tumor tissues, which can inhibit CD8 + The function of T cells, thereby affecting the control of tumors by radiation.

[0003] Currently, some inhibitors and antibodies have been used to improve tumor resistance to drug therapy. However, since the immunological mechanism by which radiotherapy increases M2 macrophages is still unclear, there is a lack of specific drugs to improve the treatment resistance caused by the increase in M2 macrophages induced by radiotherapy. Summary of the Invention

[0004] Based on the above technical problems, the present invention provides a use of a GM-CSF neutralizing antibody in the preparation of a drug for improving radioresistance. The GM-CSF neutralizing antibody improves radioresistance in tumor patients by reducing M2 macrophages induced by radiotherapy.

[0005] The specific technical solutions provided by the present invention are as follows: In a first aspect, the present invention provides a use of a GM-CSF neutralizing antibody in the preparation of a drug for improving radioresistance, wherein the radioresistance is caused by an increase in M2 macrophages induced by radiotherapy.

[0006] As a preferred embodiment of the present invention, GM-CSF neutralizing antibodies are used to prepare drugs for enhancing radiation-mediated anti-tumor immune responses.

[0007] As a preferred embodiment of the present invention, the drug is prepared by intratumoral injection.

[0008] As a preferred embodiment of the present invention, the GM-CSF neutralizing antibody is Anti-mouse GM-CSF-InVivo or Punalimab. Punalimab is the corresponding anti-human neutralizing antibody selected when implementing in humans.

[0009] In a second aspect, the present invention provides a drug for improving radioresistance, which contains the GM-CSF neutralizing antibody as the sole active ingredient.

[0010] As a preferred embodiment of the present invention, the drug is prepared by compounding the GM-CSF neutralizing antibody with pharmaceutically acceptable excipients.

[0011] It is understood that the medicaments of the embodiments of the present invention may be prepared into suitable clinical dosage forms by adding various pharmaceutically acceptable excipients, including but not limited to the following dosage forms: tablets, capsules, granules, powders or oral liquid preparations, pills, and injections. These pharmaceutically acceptable excipients include but are not limited to diluents, wetting agents, adhesives, disintegrants, lubricants, regulators, solubilizers, cosolvents, emulsifiers, antioxidants, preservatives, pH regulators, isotonic or isotonic regulators, and the like.

[0012] Among them, the diluent is selected from starch, sucrose, cellulose, inorganic salts, etc.; the wetting agent is selected from water, ethanol, etc.; the binder is selected from starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, polyethylene glycol, etc.; the disintegrant is selected from starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, sodium cross-linked carboxymethyl cellulose, cross-linked polyvinylpyrrolidone, surfactant, effervescent disintegrant, etc.; the lubricant is selected from talc, calcium stearate, magnesium stearate, magnesium lauryl sulfate, micro powder silica gel, polyethylene glycol, etc.; the regulator is selected from pigments, flavors, sweeteners, mucilage agents, deodorants, etc.; the solubilizer is selected from Tweens, polyoxyethylene fatty alcohol ethers, soaps, sulfates, sulfonates, etc.; the cosolvent is selected from organic acids and their salts, amide and amine compounds, inorganic salts, polyethylene glycol, Glycerol, etc.; emulsifiers are selected from Spans, Tweens, glycerol fatty acid esters, higher fatty acid salts, sulfates, sulfonates, gum arabic, tragacanth, gelatin, pectin, phospholipids, agar, sodium alginate, hydroxide, silicon dioxide, etc.; antioxidants are selected from sulfites, pyrosulfites, bisulfites, ascorbic acid, gallic acid and its esters, etc.; preservatives are selected from parabens, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols and volatile oils, etc.; pH regulators are selected from hydrochloric acid, sulfuric acid, phosphoric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphates, acetates, citric acid, citrate, etc.; isotonic or isotonic regulators are selected from glucose, sodium chloride, sodium citrate, sorbitol and xylitol, etc.

[0013] It is understood that the drugs involved in the embodiments of the present invention can be prepared in different dosage forms based on different excipients, and accordingly, the administration methods can also be diverse.

[0014] Compared with the prior art, the beneficial effects of the present invention are: The present invention provides the use of GM-CSF neutralizing antibodies in the preparation of drugs for improving radioresistance. Experiments have shown that the use of GM-CSF neutralizing antibodies can reduce the expression of CXCR5 in M2 macrophages after radiotherapy. + Macrophages can enhance radiation-mediated anti-tumor immune responses and improve the efficacy of radiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Neutralize GM-CSF to improve the efficacy of radiotherapy; a. CXCR5 in tumor tissue after irradiation + The number of M2 macrophages increased, b, GM-CSF induced CXCR5 + CXCR5 in monocytes towards M2 phenotype + Macrophage differentiation, c. Neutralization of GM-CSF in tumor tissue after irradiation reduces the expression of M2 CXCR5 + Macrophages, d, CXCR5 + and CXCR5 - Macrophages on CD8 + Inhibition of T cells, e, neutralization of GM-CSF improves the efficacy of radiation. DETAILED DESCRIPTION

[0016] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0017] Radiotherapy is one of the main methods of local tumor treatment. About 50-60% of cancer patients need to receive radiotherapy. However, some patients may develop radioresistance, which affects the effectiveness of radiotherapy and even the patient's prognosis. Radiotherapy can increase the number of M2 macrophages with immunosuppressive function in tumor tissues, which can inhibit CD8 + The function of T cells, thereby affecting the control of tumors by radiation.

[0018] Currently, some inhibitors and antibodies have been used to improve tumor drug resistance. However, there is still a lack of specific drugs to improve the treatment resistance caused by radiation-induced increase in M2 macrophages.

[0019] Based on this, the present invention provides the use of a GM-CSF neutralizing antibody in the preparation of a drug for improving radioresistance. The GM-CSF neutralizing antibody improves radioresistance in tumor patients by reducing radiation-induced M2 macrophages.

[0020] The sources of materials involved in the embodiments of the present invention are as follows: GM-CSF neutralizing antibodies include Anti-mouse GM-CSF-InVivo, A2148, selleck; Gimsilumab, A2853, selleck; Otilimab, HY-P99795, MCE; and Plonmarlimab, HY-P99779, MCE. It should be noted that when GM-CSF neutralizing antibodies are administered in humans, corresponding commercially available anti-human neutralizing antibodies, such as pralizumab (I-Mab), should be used depending on the specific situation.

[0021] 0. Method 1.1. CXCR5 expression in tumor tissue after irradiation + Changes in macrophages and M2 macrophages On d0, mice received subcutaneous 10 6 MC38 tumor cells were inoculated. On d10, the tumors were grown to 100 mm. 3 At 12 Gy, the tumors of mice in the experimental group were irradiated. On day 17, the tumors of mice in the control and experimental groups were isolated, minced, and digested with 1 mg / ml collagenase IV (Sigma) and 0.2 mg / ml DNase I (Sigma) at 37°C for 1 hour. The cells were filtered to obtain a single-cell suspension. 100 μl of the cell suspension (approximately 2 × 10 6 Cells were then plated, 100µl of FACS buffer was added, and the cells were centrifuged at 1600rpm for 5 minutes. The supernatant was discarded. The membrane was first blocked with anti-FcR (BioXcell, 2.4G2) and washed with 2ml of FACS buffer (550g, 5 minutes, 4°C). Surface antibody staining was then performed by adding 10µl of an antibody cocktail to each sample (the cocktail included FITC-anti-F4 / 80, PE-anti-CD206, PE-Cy7-anti-CD45, PB-anti-MHC II, APC / CY7-anti-CD185 (CXCR5), and PerCP-Cy5.5-anti-CD11b, all Biolegend products).

[0022] GM-CSF induces CXCR5 + CXCR5 in monocytes towards M2 phenotype + Macrophage differentiation Obtain bone marrow-derived hematopoietic stem cells according to this procedure: https: / / www.thermofisher.cn / cn / zh / home / life-science / cell-analysis / cell-analysis-learning-center / immunology-at-work / immunology-protocols / mouse-bone-marrow-cell-isolation.html. The day of obtaining the bone marrow cell suspension is designated as D0. Culture the cells in induction medium (RPMI containing 20 ng / ml GM-CSF and 10% FBS) at 5% CO. 2, 37℃) to harvest mononuclear cells on D4. Take the tumor cell culture supernatant and use 0.44 Filter the cells through a microfilter. Add the filtered tumor supernatant to RPMI containing 10% FBS, based on the 50% tumor supernatant in the culture system. After 48 hours of culture, collect the supernatant and adherent monocytes. Aseptically stain the cells with APC / CY7-anti-CD185 or APC / CY7-anti-CXCR5. Resuspend the cells in induction medium and culture (5% CO2, 37°C) for 72 hours. Collect the supernatant and adherent macrophages. Stain with PE-anti-CD206 and PB-anti-MHC II for analysis.

[0023] 1.3 Neutralizing GM-CSF reduces M2 CXCR5 expression in tumor tissues + macrophages On d0, mice received subcutaneous 10 6 MC38 tumor cells were inoculated. On d10, the tumors were grown to 100 mm. 3 The tumors of mice treated with IR were irradiated with 12 Gy; the tumors of mice in the IR+anti-GM-CSF group were irradiated with 12 Gy and then Intratumoral injections of anti-GM-CSF were performed every other day. Tumor tissue was isolated on day 17 and processed and stained as described in 1.1 (FITC-anti-F4 / 80, PE-anti-CD206, PE-Cy7-anti-CD45, PB-anti-MHC II, APC / CY7-anti-CD185 (CXCR5), and PerCP-Cy5.5-anti-CD11b, all Biolegend products).

[0024] 1.4. CXCR5 + Macrophages on CD8 + T cell suppression Mouse lymph nodes were isolated and single cell suspensions were prepared by collagenase digestion or grinding. + T cell magnetic bead separation kit (StemCell) to separate CD8 + T cells. Resuspend the cells in 500µl sorting buffer and add 50µl rat serum to incubate for 5 minutes to block. Add 50µl cocktail antibody and incubate for 15 minutes. Mix well and add 125µl magnetic beads (antibody: magnetic beads = 1:2.5), incubate for 5 minutes, add sorting buffer to 3mL, place on the sorting magnet, let it stand for 2.5 minutes, pour the supernatant into a centrifuge tube, centrifuge at 1600rpm for 5 minutes, and obtain CD8 + The T cells were discarded and resuspended in an appropriate volume of PBS. They were labeled with CellTraceViolet™ (Thermo). CD3 / CD28 antibodies were used to activate TCR signaling. CXCR5 was expressed in the presence of 5µg / ml anti-CD3 (BioLegend) and 2µg / ml anti-CD28 (BioLegend). + or CXCR5 - Macrophages were mixed with CD8 + T cells were co-cultured and cells were collected after 72 hours and stained. CD8 + T cell CTV intensity and effector molecules (TNF- and IFN- ) expression. + or CXCR5 - Macrophages were obtained by sterile flow cytometry sorting.

[0025] 1.5 Neutralizing GM-CSF to improve radiotherapy efficacy On d0, mice received subcutaneous 10 6 MC38 tumor cells were inoculated. On d10, the tumors were grown to 100 mm. 3 The mice in the IR+anti-GM-CSF group received 12 Gy irradiation and then 2 g anti-GM-CSF was injected intratumorally, and the injection was repeated 6 times every other day. The length, width and height of the mouse tumor were measured, and the tumor volume was calculated as follows: Width high 0.5 to calculate tumor size.

[0026] 1. Results The results showed that CXCR5 +Macrophages increased significantly, and this group of cells mainly originated from CXCR5 + Monocytes ( Figure 1 (a) Radiation can induce CXCR5 by increasing GM-CSF in tumor tissues. + CXCR5 in monocytes towards M2 phenotype + Macrophage differentiation ( Figure 1 (b) Neutralization of GM-CSF produced in tumor tissue after radiation can reduce the expression of M2 CXCR5 in tumor tissue. + Macrophages ( Figure 1 Middle c). Compared with CXCR5 - Macrophages, CXCR5 + Shows CD8 + Stronger inhibitory effect of T cells ( Figure 1 Neutralizing GM-CSF produced in tumor tissue after radiation can improve the efficacy of radiation ( Figure 1 Middle e).

[0027] Currently, several monoclonal antibodies targeting GM-CSF have entered commercial applications and are used in autoimmune diseases and infections. Therefore, the present invention proposes to use GM-CSF neutralizing antibodies to neutralize GM-CSF produced in tumors after radiation and inhibit M2 CXCR5 + Macrophage accumulation: a therapeutic strategy to improve radiation efficacy in cancer patients.

[0028] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. Use of a GM-CSF neutralizing antibody in the preparation of a drug for improving radioresistance, characterized in that: The radioresistance is caused by the increase of M2 macrophages induced by radiotherapy.

2. The use according to claim 1, characterized in that Neutralizing antibodies against GM-CSF are used to prepare drugs that enhance radiation-mediated anti-tumor immune responses.

3. The use according to claim 1, characterized in that The drug is prepared in a manner of intratumoral injection.

4. The use according to claim 1, characterized in that The GM-CSF neutralizing antibody is Anti-mouse GM-CSF-InVivo or pralizumab.

5. A drug for improving radioresistance, characterized in that: The GM-CSF neutralizing antibody according to claim 1 is used as the sole active ingredient.

6. The drug for improving radioresistance according to claim 5, characterized in that The GM-CSF neutralizing antibody is Anti-mouse GM-CSF-InVivo or pralizumab.

7. The drug for improving radioresistance according to claim 5, characterized in that The drug is prepared by compounding the GM-CSF neutralizing antibody with pharmaceutically acceptable excipients.

8. The drug according to claim 7, characterized in that The medicine is an oral preparation or an injection preparation.

9. The drug according to claim 8, characterized in that The oral preparation is a capsule, granule or tablet.

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