Application of neutralizing antibody of CXCL13 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 CXCL13 in preparation of a drug for improving radiation resistance. Experiments prove that the neutralizing antibody of CXCL13 improves radiation resistance of tumor patients by reducing immunosuppressive mononuclear cells induced by radiotherapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor therapeutic drug research, and specifically relates to the use of a CXCL13 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 monocytes with immunosuppressive functions in tumor tissues, which can inhibit CD8 + The function of T cells, thereby affecting the control of tumors by radiation.

[0003] Previous studies have reported that administering anti-Gr1 or anti-Ly6C to mice can eliminate immunosuppressive monocytes. However, this approach only nonspecifically eliminates all monocytes and neutrophils in the mouse body and fails to specifically eliminate immunosuppressive monocytes. Furthermore, human monocytes are characterized by high expression of CD14 on their cell membranes, but small amounts of CD14 are also present on the surfaces of monocytes, macrophages, dendritic cells, and activated neutrophils. Therefore, even with the current CD14-targeting drug atibolizumab (primarily used to treat ST-segment elevation myocardial infarction, acute decompensated heart failure, and adult respiratory distress syndrome), it is unable to specifically eliminate immunosuppressive monocytes in cancer patients and has yet to be used to enhance anti-tumor immune responses and improve the efficacy of radiotherapy. Summary of the Invention

[0004] Based on the above technical problems, the present invention provides a use of a CXCL13 neutralizing antibody in the preparation of a drug for improving radioresistance. The CXCL13 neutralizing antibody improves radioresistance in tumor patients by reducing immunosuppressive monocytes induced by radiotherapy.

[0005] The specific technical solutions provided by the present invention are as follows: In a first aspect, the present invention provides use of a CXCL13 neutralizing antibody in the preparation of a medicament for improving radioresistance, wherein the radioresistance is caused by immunosuppressive monocyte infiltration induced by radiotherapy.

[0006] As a preferred embodiment of the present invention, a neutralizing antibody against CXCL13 is used to prepare a drug for enhancing radiation-mediated anti-tumor immune response.

[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 CXCL13 neutralizing antibody is Mouse CXCL13 / BLC / BCA-1 Antibody. When the CXCL13 neutralizing antibody is used in human, a corresponding anti-human neutralizing antibody can be selected.

[0009] In a second aspect, the present invention provides a drug for improving radioresistance, which contains the CXCL13 neutralizing antibody as the sole active ingredient.

[0010] As a preferred embodiment of the present invention, the drug is prepared by compounding the CXCL13 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, 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, micropowder silica gel, polyethylene glycol, etc.; the regulator is selected from pigments, flavors, sweeteners, mucilages, 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.; the emulsifier Selected from Spans, Tweens, Melts, Benzyls, glycerol fatty acid esters, higher fatty acid salts, sulfates, sulfonates, gum arabic, tragacanth, gelatin, pectin, phospholipids, agar, sodium alginate, hydroxides, silicon dioxide, bentonite, etc.; antioxidants selected from sulfites, pyrosulfites, bisulfites, ascorbic acid, gallic acid and its esters, etc.; preservatives selected from parabens, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols and volatile oils, etc.; pH regulators 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 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 CXCL13 neutralizing antibodies in the preparation of drugs for improving radioresistance. Experiments have shown that the use of CXCL13 neutralizing antibodies can reduce CXCR5 + Monocytes infiltrate into irradiated tumor tissues, enhancing radiation-mediated anti-tumor immune responses and thus improving the efficacy of radiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 CXCL13 and CXCR5 in tumor tissue after irradiation + Increased monocytes; a, CXCL13 protein level, b, CXCR5 + Monocyte infiltration, c, CXCR5 - infiltration of mononuclear cells; Figure 2 CXCR5 + Monocytes inhibit the efficacy of radiation therapy; a, CD8 +T cell proliferation, b, CD8 + T cells TNF- Expression of c, CD8 + T cells IFN- Expression of d, CXCR5 + Monocytes suppress the efficacy of radiation therapy; Figure 3 CXCR5 + Deletion of monocytes or inhibition of their recruitment after radiation improves the efficacy of radiation; a, CXCR5 - / - The tumors of C57B6 mice were significantly controlled after irradiation compared with those of WT mice. b. Neutralization of CXCL13 in tumor tissues enhanced the radiation effect. 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, and approximately 50-60% of cancer patients require radiotherapy. However, some of these patients may develop radiation resistance, which affects the effectiveness of radiotherapy and even the patient's prognosis.

[0018] Previous studies have reported that in a mouse model, immunosuppressive monocytes were removed by administering anti-Gr1 or anti-Ly6C to the mice. However, this approach can only non-specifically remove all monocytes and neutrophils in the mouse body, and cannot specifically remove immunosuppressive monocytes. In addition, human monocytes are characterized by high expression of CD14 on the cell membrane surface, but a small amount of CD14 is also present on the surface of monocytes, macrophages, dendritic cells, and activated neutrophils. Therefore, even though atiniblimab, which targets CD14, is currently available, it cannot achieve specific removal of immunosuppressive monocytes in cancer patients, and has no application in enhancing anti-tumor immune responses and improving the efficacy of radiotherapy.

[0019] Based on this, the present invention provides the use of a neutralizing antibody against CXCL13 in the preparation of a drug for improving radioresistance. The neutralizing antibody against CXCL13 improves radioresistance in tumor patients by reducing radiation-induced immunosuppressive monocytes.

[0020] The sources of materials involved in the embodiments of the present invention are as follows: The CXCL13 neutralizing antibody is Mouse CXCL13 / BLC / BCA-1 Antibody, MAB470, from R&D Systems. It should be noted that when administering CXCL13 neutralizing antibodies to humans, commercially available anti-human neutralizing antibodies should be used as appropriate.

[0021] Example 1 CXCL13 increases in tumor tissue after radiation and promotes CXCR5 + Infiltration of mononuclear cells 4. Methods 1.1. Detection of CXCL13 protein levels in mouse tumor tissues On d0, mice received subcutaneous 10 6 MC38 tumor cells were inoculated. On d10, the tumors were grown to 100 mm. 3 On day 13, the tumors of mice in the experimental group received 12 Gy of irradiation. On day 13, tumors from mice in the control and experimental groups were isolated. Equal amounts of tumor tissue were cut into small pieces and homogenized in PBS containing protease inhibitors. Triton X-100 was then added to prepare the homogenate. CXCL13 expression in tumor tissue was detected using the LEGENDplex™ MU Proinflam Chemokine Panel (Biolegend), following the same procedures as the manufacturer's instructions.

[0022] 1.2. CXCR5 before and after radiation + Infiltration of mononuclear cells 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 in the IR group received 12 Gy irradiation, and the tumors of mice in the IR+anti-CXCL13 group received 12 Gy irradiation followed by 2 g anti-CXCL13 was injected intratumorally, and then injected intratumorally again one day later. On day 13, the tumors of the three groups of mice 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 h. The single-cell suspension was obtained after filtration. 100 μl of cell suspension (approximately 2×10 6Cells), then add 100µl FACS Buffer, centrifuge at 1600rpm for 5 minutes, and discard the supernatant. First, block the membrane surface with anti-FcR (BioXcell, 2.4G2), and wash with 2ml FACS Buffer (550G, 5min, 4℃). Then, add 10µl antibody mixture to each sample for surface antibody staining (the antibody mixture includes FITC-anti-Ly6C, PE-anti-Ly6G, PB-anti-CD45, APC / CY7-anti-CD185, PerCP-Cy5.5-anti-CD11b, where APC / CY7-anti-CD185 is APC / CY7-anti-CXCR5, and these antibodies are all Biolegend products). After staining for 45 minutes in the dark at 4℃, add 2ml FACS Buffer for washing (550G, 5min, 4℃). Discard the supernatant and resuspend with 100μl FACS Buffer for detection. Analysis of CXCR5 + Ly6C high Ly6G low and CXCR5 - Ly6C high Ly6G low The proportion of these two groups of cells in all cells in tumor tissue.

[0023] 5. Results The results showed that CXCL13 increased in irradiated tumor tissues and specifically promoted the expression of CXCR5 + Monocyte infiltration. The protein level of CXCL13 in mouse tumor tissue increased after radiation ( Figure 1 Middle (a), CXCR5 + Increased monocyte infiltration ( Figure 1 Middle b), CXCR5 - There was no significant change in the infiltration of mononuclear cells ( Figure 1 (C) Neutralization of CXCL13 in tumor tissue after radiation can reduce CXCR5 + Infiltration of mononuclear cells ( Figure 1 b) CXCR5 - There was no effect on the infiltration of mononuclear cells ( Figure 1 Middle c).

[0024] Example 2 CXCR5 + Monocyte suppression of radiotherapy efficacy 6. Methods 1.1、CXCR5 + Monocytes and CXCR5 - Monocytes on CD8+ The impact of T cells 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 - Monocytes were mixed with CD8 + T cells were co-cultured and cells were collected after 72 h. The staining steps were the same as in Example 1. CD8 + T cell CTV intensity and effector molecules (TNF- and IFN- ) expression. + or CXCR5 - Monocytes were obtained by sterile flow cytometry, stained under sterile conditions according to the steps in Example 1, and loaded onto the instrument.

[0025] 1.2、CXCR5 + Effects of monocytes on tumors On d0, mice received subcutaneous 10 6 MC38 tumor cells were inoculated. On d10, the tumors were grown to 100 mm. 3 When the tumors of mice treated with IR were irradiated with 12 Gy; IR+CXCR5 - The mice in the Mon group received 5×10 6 CXCR5 - Monocytes were adopted every 3 days for 4 times; IR+CXCR5 + The mice in the Mon group received 5×10 6 CXCR5+ Monocytes were adopted every 3 days for 4 times. 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] 7. Results The results showed that CXCR5 + Monocytes compared with CXCR5 - Monocytes on CD8 + T cells have a stronger inhibitory effect, which can inhibit the efficacy of radiation. + Monocytes significantly suppress CD8 + T cell proliferation ( Figure 2 a), TNF- ( Figure 2 b) and IFN- The generation of Figure 2 Middle (c). CXCR5 + Monocyte suppression of radiotherapy efficacy ( Figure 2 (d)

[0027] Example 3 Neutralizing CXCL13 improves tumor radiotherapy efficacy 8. Methods 1.1、CXCR5 - / - Changes of tumors in C57B6 mice after radiation WT mice and CXCR5 - / - 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 length, width and height of the tumors of mice were measured and the tumor volume was calculated as follows: Width high 0.5 to calculate tumor size.

[0028] 1.2 Neutralizing the Effects of CXCL13 on Tumors 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 in the IR group received 12 Gy irradiation, and the tumors of mice in the IR+anti-CXCL13 group received 12 Gy irradiation followed by 2 g Intratumoral injection of anti-CXCL13 was performed every other day for a total of 5 times.

[0029] 9. Results The results showed that CXCR5 - / - The tumors in C57B6 mice were significantly controlled after radiation ( Figure 3 Neutralizing CXCL13 in tumor tissue can enhance the effect of radiation ( Figure 3 Middle b).

[0030] The present invention reduces the production of CXCL13 in tumors after radiation by using CXCL13 neutralizing antibodies, thereby reducing the expression of CXCR5 + The infiltration of monocytes after radiation enhances the anti-tumor immunity induced by radiation, thereby improving the therapeutic effect of radiation.

[0031] 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 CXCL13 neutralizing antibody in the preparation of a drug for improving radioresistance, characterized in that: The radioresistance is caused by the infiltration of immunosuppressive monocytes induced by radiotherapy.

2. The use according to claim 1, characterized in that Neutralizing antibodies against CXCL13 are used to prepare drugs for enhancing 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 CXCL13 neutralizing antibody is Mouse CXCL13 / BLC / BCA-1 Antibody.

5. A drug for improving radioresistance, characterized in that: The neutralizing antibody against CXCL13 as claimed in claim 1 is used as the sole active ingredient.

6. The drug for improving radioresistance according to claim 5, characterized in that The drug is prepared by compounding the CXCL13 neutralizing antibody with pharmaceutically acceptable excipients.

7. The drug according to claim 6, characterized in that The medicine is an oral preparation or an injection preparation.

8. The drug according to claim 7, characterized in that The oral preparation is in the form of granules, capsules or tablets.

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