Application of CXCL16 neutralizing antibody as medicine for treating radioactive and idiopathic pulmonary fibrosis

Abstract

The invention relates to the technical field of medicines, in particular to application of a CXCL16 neutralizing antibody as a medicine for treating radioactive and idiopathic pulmonary fibrosis. Experiments find that the expression level of CXCL16 in serum of a patient receiving radiotherapy is remarkably increased, in an idiopathic pulmonary fibrosis model and a radiation-induced pulmonary fibrosis model, the CXCL16 mRNA level of lung tissue and the expression level of CXCL16 protein are remarkably increased, meanwhile, immunohistochemical staining shows that the expression of CXCL16 in the lung is increased, and the CXCL16 expression level of the lung tissue is increased. In addition, accumulation of expression of CXCL16 in macrophages can be observed from IHC images of fibrotic lungs, and subsequent experiments find that radiation-induced pulmonary fibrosis and idiopathic pulmonary fibrosis can be relieved by adopting the CXCL16 neutralizing antibody, so that the CXCL16 neutralizing antibody can be used as a wide therapeutic drug for radiation-induced pulmonary fibrosis and idiopathic pulmonary fibrosis.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202410219585X filed on February 28, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of medical technology, and in particular to the use of a CXCL16 neutralizing antibody as a therapeutic drug for radiation and idiopathic pulmonary fibrosis. Background Art

[0004] Radiotherapy is one of the main treatment methods for several common malignant tumors (such as lung cancer, breast cancer, and esophageal cancer). However, radiation-induced pulmonary fibrosis (RIPF) is a phenomenon that occurs when these malignant tumors receive chest radiotherapy, and it will affect the treatment effect. Therefore, it is one of the main limiting factors that need to be considered when formulating chest radiotherapy plans for these malignant tumors. Currently, the incidence of RIPF in patients receiving chest radiotherapy is about 9% to 30%. Once RIPF occurs, it will significantly reduce the patient's quality of life, and in severe cases it can cause death. At the same time, the mechanism of RIPF is still unclear, and there is a lack of effective therapeutic targets and drugs. Therefore, research on RIPF has always been a hot topic in radiobiology research. Recent studies have shown that the application of immunotherapy in lung cancer and esophageal cancer, especially the combination of PD-1 inhibitors and radiotherapy, can increase the incidence of radiation pneumonitis above grade 2. Radiation pneumonitis is closely related to the occurrence of RIPF, and late-stage RIPF is irreversible. Therefore, the prevention and treatment of RIPF is particularly urgent. The overall incidence of radiation-induced pulmonary fibrosis (RIPF) is 9% to 30%. Once it occurs, it is difficult to reverse, and the symptoms such as cough and dyspnea it causes will seriously affect the patient's quality of life. There is currently no effective drug for treatment.

[0005] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic interstitial lung disease with no clear cause. It is identified as one of the most common and severe idiopathic interstitial pneumonias, accompanied by dyspnea, cough, and worsening lung function. The global incidence and prevalence of IPF is 0.09 to 1.30 cases per 10,000 people, and it is increasing year by year. In addition, IPF has an extremely high mortality rate, with a median survival of 2 to 3 years after diagnosis. Currently, respiratory failure is the biggest cause of death in IPF patients, and other causes of death include coronary heart disease, pulmonary embolism, and lung cancer. However, only pirfenidone and nintedanib are currently approved for the treatment of IPF. The therapeutic principles of these two classes of drugs are to interfere with the proliferation and migration of fibroblasts and reduce the contraction of collagen gels embedded in fibroblasts and excessive extracellular matrix (ECM) secretion; however, gastrointestinal and skin-related adverse events (AEs) have been reported in pirfenidone treatment, and diarrhea and elevated liver enzymes have been reported in nintedanib treatment, which prevents the two classes of drugs from being widely used. Therefore, there is an urgent need to find other appropriate treatments for IPF. Summary of the invention

[0006] The present application provides a use of a CXCL16 neutralizing antibody as a therapeutic drug for radiation-induced pulmonary fibrosis and idiopathic pulmonary fibrosis, so as to solve the lack of a wide range of therapeutic drugs for radiation-induced pulmonary fibrosis and idiopathic pulmonary fibrosis in pulmonary fibrosis in the prior art.

[0007] In a first aspect, the present application provides a use of a CXCL16 neutralizing antibody as a therapeutic drug for radiation and idiopathic pulmonary fibrosis.

[0008] Optionally, the CXCL16 neutralizing antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0009] Optionally, the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO.3, and the nucleotide sequence of the light chain variable region is shown as SEQ ID NO.4.

[0010] In a second aspect, the present application provides a method for preparing a CXCL16 neutralizing antibody in the application of the first aspect, the method comprising:

[0011] The CXCL16 gene in the preset region is selected, and the whole gene is synthesized into an expression vector for protein expression to obtain the CXCL16 protein antigen;

[0012] Immunizing an animal model with the CXCL16 protein antigen, and then extracting and detecting antiserum to confirm that the animal immunization is complete, thereby obtaining an animal immune model;

[0013] Performing cell fusion on the animal immune model and performing subclone screening to obtain multiple groups of immune cell strains;

[0014] Conducting antibody neutralization effect screening and binding affinity screening on the multiple groups of immune cell lines, and performing protein immunoblotting analysis to obtain the best cell line;

[0015] The optimal cell line is cultured in an expanded manner to obtain ascites antibodies, which are then purified to obtain CXCL16 neutralizing antibodies.

[0016] Optionally, the detection includes detection using an enzyme-linked immunosorbent assay;

[0017] When the titer of the test is ≥1:64000, it means that the animal immunization process has been completed.

[0018] Optionally, the preset region is 27aa to 201aa of the CXCL16 gene sequence.

[0019] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0020] The present application provides an application of a CXCL16 neutralizing antibody as a therapeutic drug for radiation and idiopathic pulmonary fibrosis. Through a large number of creative experiments, it was found that the expression level of CXCL16 in the serum of patients receiving radiotherapy increased significantly, and in the BLM-induced mouse pulmonary fibrosis model (idiopathic pulmonary fibrosis model) and the RT-induced mouse pulmonary fibrosis model (radiation pulmonary fibrosis model), the CXCL16 mRNA level and the expression level of CXCL16 protein in the lung tissue increased significantly. At the same time, immunohistochemical staining of CXCL16 showed increased expression of CXCL16 in the lung. In addition, from the IHC images of fibrotic lungs, it can be observed that the expression of CXCL16 accumulates in macrophages. The above experimental results indicate that the upregulation of CXCL16 in fibrotic lungs increases the possibility that it may play a role in the progression of pulmonary fibrosis. At the same time, subsequent experiments found that the use of CXCL16 neutralizing antibodies can alleviate radiation pulmonary fibrosis and idiopathic pulmonary fibrosis. Therefore, CXCL16 neutralizing antibodies can be used as a wide range of therapeutic drugs for radiation pulmonary fibrosis and idiopathic pulmonary fibrosis in pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0022] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0023] Figure 1 The relationship diagram of CXCL16 in the process of pulmonary fibrosis provided in the embodiment of the present application, wherein: Figure 1 A is the single-cell sequencing result of mouse lung tissue in the control group and three months after radiotherapy. Figure 1 B is the result of the serum CXCL16 level of mice three months after lung radiotherapy. Figure 1 C is the immunofluorescence staining of CXCL16 in the lungs of mice 3 months after radiotherapy. Figure 1 D is a schematic diagram of the expression of CXCL16 in mouse lung homogenate 3 months after radiotherapy. Figure 1 E is the result of the expression level of CXCL16 mRNA in the lungs of mice 3 months after radiotherapy. Figure 1 F is the immunofluorescence co-localization result of the mouse lungs 3 months after radiotherapy. Figure 1 G is the result of single-cell sequencing analysis of the differential expression of CXCL16 in lung tissue of mice;

[0024] Figure 2 This is a schematic diagram of the radiological results of establishing a radiation lung injury mouse model provided in the examples of the present application, wherein: Figure 2 A is a schematic diagram of CT scanning and tissue staining of the radiation lung injury mouse model. Figure 2 B is a weekly record of body weight changes in the radiation lung injury mouse model. Figure 2 C is a curve of the survival percentage recorded weekly in the radiation lung injury mouse model. Figure 2 D is a schematic diagram of the fibrosis level in the radiation lung injury mouse model. Figure 2 E is a schematic diagram of hydroxyproline levels in the radiation-induced lung injury mouse model. Figure 2 F is the protein expression level result of fibrosis index in the radiation lung injury mouse model. Figure 2 G is a bar graph of mRNA expression levels in fibrosis indicators in the radiation lung injury mouse model;

[0025] Figure 3 This is a schematic diagram of the radiological results of the BLM mouse model of idiopathic pulmonary fibrosis established in the embodiments of the present application, wherein: Figure 3 A is a schematic diagram of CT scanning and tissue staining of the idiopathic pulmonary fibrosis mouse model. Figure 3 B is a weekly record of body weight changes in the idiopathic pulmonary fibrosis mouse model. Figure 3C is a curve of the survival percentage recorded weekly in the idiopathic pulmonary fibrosis mouse model. Figure 3 D is a schematic diagram of the fibrosis level in the idiopathic pulmonary fibrosis mouse model. Figure 3 E is a schematic diagram of hydroxyproline levels in the idiopathic pulmonary fibrosis mouse model. Figure 3 F is the protein expression level result diagram of fibrosis indexes in the idiopathic pulmonary fibrosis mouse model. Figure 3 G is a bar graph of mRNA expression levels in fibrosis indicators of the idiopathic pulmonary fibrosis mouse model;

[0026] Figure 4 This is a graph showing the results of the in vivo biosafety assessment of the CXCL16 neutralizing antibody in wild mice provided in the examples of the present application, wherein: Figure 4 A is the staining result of CXCL16 neutralizing antibody in the heart tissue of wild mice. Figure 4 B is the staining result of CXCL16 neutralizing antibody in liver tissue of wild mice. Figure 4 C is the staining result of CXCL16 neutralizing antibody in lung tissue of wild mice. Figure 4 D is the result of CXCL16 neutralizing antibody staining in spleen tissue of wild mice. Figure 4 E is the result of staining the kidney tissue of wild mice with CXCL16 neutralizing antibody. Figure 4 F is the data graph of serum glutathione aminotransferase, glutamate oxaloacetate aminotransferase and creatinine levels in wild mice;

[0027] Figure 5 A schematic diagram of the process for preparing a CXCL16 neutralizing antibody provided in an embodiment of the present application;

[0028] Figure 6 This is a graph showing the ELISA test results of the antigen-immunized Balb / C mouse antiserum provided in the examples of the present application;

[0029] Figure 7 This is a diagram showing the exposure and development results of the CXCL16 neutralizing antibody provided in the examples of the present application on a chemiluminescent gel imager;

[0030] Figure 8 This is a diagram of Western blot analysis results of mouse primary lung fibroblasts under the influence of neutralizing antibodies provided in the examples of the present application, wherein: Figure 8 A is the result of fibroblasts crawling out of lung tissue. Figure 8 B is the result of Western Blot detection of protein levels after CXCL16 intervention in primary fibroblasts for 24 hours. Figure 8 C is a graph showing the protein level of primary fibroblasts cultured at a CXCL16 concentration of 400 ng / mL by Western Blot;

[0031] Fig. 9 This is a schematic diagram of the results of cellular fibrosis indicators of primary mouse fibroblasts stimulated by the conditioned medium rich in CXCL16 neutralizing antibodies provided in the examples of the present application, wherein: Fig. 9 A is the expression level of cell fibrosis protein detected by Western Blot. Fig. 9 B is a bar graph showing the expression levels of cell fibrosis proteins detected by Western Blot. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0033] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0034] The creative thinking of this application is:

[0035] Chemokines play an important role in the pathogenesis of pulmonary fibrosis. They are not only involved in the migration of immune cells to sites of inflammation and angiogenesis, but also in tumorigenesis and metastasis. Chemokines are involved in multiple pathways, such as angiogenesis, the movement and invasion of endothelial cells and immune cells at tumor sites, and the proliferation and survival of tumor cells. CXC motif chemokine ligand 16 (CXCL16) belongs to the α-chemokine subfamily and has two forms: membrane-bound (mCXCL16) and soluble (sCXCL16). The two forms of CXCL16 have different functions. mCXCL16 has a CXC chemokine domain and a transmembrane domain, while sCXCL16 only has a chemokine domain. Therefore, sCXCL16 is a type of chemokine that is mainly responsible for the chemotaxis of cells carrying CXCR6 receptors, while mCXCL16 is a type of transmembrane protein that is regulated by proinflammatory cytokines and plays an important role in the accumulation of immune cells at inflammatory sites. At the same time, mCXCL16 also mediates the adhesion of Gram-negative and Gram-positive bacteria and guides cells expressing mCXCL16 (such as macrophages and comb cells) to phagocytose bacteria. Therefore, mCXCL16 can also act as a receptor to transduce extracellular signals into cells.

[0036] Currently, macrophages play an important role in both RIPF and IPF, two fibrotic diseases. The preliminary study of this application found that in the early stage of RIPF formation, CXCL16 secreted by CXCL16+IMs activated the TGFβ signaling pathway dominated by BMPs, induced the transformation of fibroblasts to myofibroblasts, and promoted the proliferation of fibroblasts, thereby promoting the occurrence of RIPF.

[0037] Therefore, by inhibiting the expression of CXCL16, we can obtain better broad-based therapeutic drugs for radiation-induced pulmonary fibrosis and idiopathic pulmonary fibrosis in pulmonary fibrosis.

[0038] The present application provides an application of a CXCL16 neutralizing antibody as a therapeutic drug for radiation and idiopathic pulmonary fibrosis.

[0039] In some optional embodiments, the CXCL16 neutralizing antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0040] In some optional embodiments, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.3, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.4

[0041] In the examples of the present application, defining the specific composition of the CXCL16 neutralizing antibody and defining the amino acid sequence and nucleotide sequence of the heavy chain variable region and the light chain variable region of the CXCL16 neutralizing antibody respectively can make the CXCL16 neutralizing antibody better as a broad-based therapeutic drug for radiation and idiopathic pulmonary fibrosis.

[0042] like Figure 5 As shown, based on a general inventive concept, the present application embodiment provides a method for preparing a CXCL16 neutralizing antibody in the application, the method comprising:

[0043] S1. Select the CXCL16 gene in the preset region, synthesize the whole gene into the expression vector for protein expression, and obtain the CXCL16 protein antigen;

[0044] S2. Immunizing the animal model with the CXCL16 protein antigen, and then extracting and detecting antiserum to confirm that the animal immunization is complete, thereby obtaining an animal immune model;

[0045] S3. performing cell fusion on the animal immune model and performing subclone screening to obtain multiple groups of immune cell strains;

[0046] S4. performing antibody neutralization effect screening and binding affinity screening on the multiple groups of immune cell lines, and performing protein immunoblotting analysis to obtain the best cell line;

[0047] S5. The optimal cell line is cultured on an expanded scale to obtain ascites antibodies, which are then purified to obtain CXCL16 neutralizing antibodies.

[0048] In the examples of the present application, the specific preparation steps of the CXCL16 neutralizing antibody are defined. The CXCL16 gene can be designed first and then protein expression can be performed to obtain the CXCL16 protein antigen, and the CXCL16 protein antigen can be used as an antigen for animal immunization, and then antiserum extraction and detection can be performed to obtain an animal immune model, and finally cell fusion and subclone screening can be performed, so that multiple groups of immune cell lines can be obtained, and finally antibody neutralization effect screening and binding affinity screening are performed, combined with immunoblot analysis, so that the expected CXCL16 neutralizing antibody can be secreted, and finally expanded culture and purification are performed to finally obtain pure CXCL16 neutralizing antibody.

[0049] The method is implemented based on the application of the above-mentioned CXCL16 neutralizing antibody as a therapeutic drug for radiation and idiopathic pulmonary fibrosis. The specific principle of the application of the CXCL16 neutralizing antibody as a therapeutic drug for radiation and idiopathic pulmonary fibrosis can be referred to the above-mentioned embodiment. Since the method adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be repeated here one by one.

[0050] In some optional embodiments, the detection includes detection using an enzyme-linked immunosorbent assay;

[0051] When the titer of the test is ≥1:64000, it means that the animal immunization process has been completed.

[0052] In the examples of the present application, the specific method of identification and detection and the specific titer standard can clarify the progress of animal immunization and confirm the endpoint of animal immunization completion, so that sufficient CXCL16 neutralizing antibodies can be obtained subsequently.

[0053] In some optional embodiments, the preset region is 27aa to 201aa of the CXCL16 gene sequence.

[0054] In the embodiments of the present application, limiting the specific CXCL16 gene sequence length of the preset region can better enable the CXCL16 gene to express the antigen protein with the expected function, facilitate the subsequent animal immunization and obtain enough antibodies, so as to screen out suitable CXCL16 neutralizing antibodies.

[0055] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are usually measured according to industry standards. If there is no corresponding industry standard, then the conditions recommended by the manufacturer are followed.

[0056] Example 1

[0057] like Figure 5 As shown, the method for preparing the CXCL16 neutralizing antibody comprises:

[0058] Step 1: Preparation of CXCL16 protein antigen: The CXCL16 gene sequence was consulted on NCBI, and using it as a template, 27aa to 201aa were selected and synthesized into pGEX-4T-AB1 and pET-28a-SUMO expression vectors for protein expression. After identification, it was confirmed that the vector was expressed in inclusion bodies, and the antigen preparation was considered complete; wherein, the CXCL16 gene sequence involved in the template includes the nucleotide sequence of the heavy chain variable region of CXCL16: (SEQ ID NO.3) and the nucleotide sequence of the light chain variable region of CXCL16 is: atgatgagtcctgcccagttcctgtttctgttagtgctctggattcaggaaaccaacggtgatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgcaagtcaagtcagagcctcttatatagtaatggaaaaacctatttgaattggttattacagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacactgaaaatcagcagagtggaggctgaggatttgggagtttattactgcgtgcaaggtacacattttccttacacgttcggaggggggaccaagctggaaataaaacgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagcacc(SEQ ID NO.4); additionally, the amino acid sequence of the heavy chain variable region of CXCL16 in the amino acid sequence from 27aa to 201aa is selected as: MAWVWTLLFLMAAAQRIQAQIQLVQSGPELKKPGETVKISCKASGYTITDYSMHWVKQAPGKGLKWMGWINTETGVPTYADAFKGRFAFSLETSASTAYLQINNLINEDTATYFCTRGFFITTVIHWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIE(SEQ ID NO.1) and the amino acid sequence of the light chain variable region of CXCL16 is: MMSPAQFLFLLVLWIQETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSST (SEQID NO.2). .

[0059] Step 2: Animal immunization and polyantibody detection: 5Balb / C mice were immunized with the immunogen, and the mouse antiserum was tested by ELISA after 3 immunizations. The results are as follows: Figure 6 As shown in the figure, if the ELISA titer reaches 1:64000 or above, it means that the immunization is qualified.

[0060] Step 3. Cell fusion and subclone screening: Select one mouse for fusion, and plate 10 96-well cell plates for fusion. Use the screening source to perform ELISA test on the fusion supernatant. A positive result indicates that antibodies have been produced.

[0061] Step 4: Antibody neutralization effect screening and binding affinity determination:

[0062] 1. To determine whether the neutralizing antibody can bind to CXCL16, perform Western blot analysis. The specific steps are as follows:

[0063] Protein electrophoresis was performed using a precast 10% Bis-Tris gel. Primary mouse macrophages were extracted and cultured at 10% 6 100 μL of RIPA lysis buffer was added to each cell, incubated on ice for 30 minutes, centrifuged at 12000 rpm for 15 minutes at 4°C, and the supernatant was added with loading buffer, and heated at 95°C for 5 minutes. The sample loading volume was 10 μL per lane, and the loading volume for the marker was 3 μL per lane. The gel was run at 80V for 120 minutes at room temperature. Using the sandwich transfection system, 200 mA constant flow transfer membrane was performed for 120 minutes to transfer the protein to PVDF. The membrane was blocked in blocking buffer (3% skim milk in phosphate buffered saline) at room temperature for 1 hour. After the membrane was washed three times for 15 minutes in washing buffer (phosphate buffered saline + 0.05% Tween 20), the PVDF membrane was incubated with the screened neutralizing antibody (1 μg / mL in antibody diluent) at 4°C overnight.

[0064] After the initial incubation, the antibody solution was removed and the membrane was washed three times in a washing buffer (phosphate buffered saline + 0.05% Tween 20) for 15 min. The membrane was then incubated with a HRP-conjugated rabbit anti-mouse Fc-specific IgG secondary antibody (0.1 μg / mL in blocking solution) at room temperature for 1 h. After removing the secondary antibody, the membrane was washed four times with a washing buffer for 10 min. The membrane was then incubated with a working solution of a developer (SuperSignal West Pico Plus) for 5 min. After exposure and development in a chemiluminescent gel imager, the images were processed and analyzed using Image J software.

[0065] The results are as follows Figure 7 As shown, a clear band appeared at about 37 kDa, which was consistent with the molecular weight of CXCL16. The results proved that the neutralizing antibody could bind to mouse CXCL16, proving the binding ability of the neutralizing antibody.

[0066] 2. To determine whether the neutralizing antibody can neutralize CXCL16 and block the function of CXCL16, Western blot analysis was performed.

[0067] Primary mouse lung fibroblasts were extracted and fibroblasts were observed crawling out of the lung tissue on the third day after plating (e.g. Figure 8 A). Primary fibroblasts were treated with 0, 100 ng / mL, 200 ng / mL, and 400 ng / mL CXCL16 for 24 h, and the protein level was detected by Western Blot within 48 h. The results are shown in Figure 8 B and Figure 8 As shown in C, the expression of Collagen I, Collagen III, Fn and a-SMA in primary fibroblasts was positively correlated with the CXCL16 concentration in the range of 0-400 ng / mL, and TGF-β was also significantly activated. When primary fibroblasts were cultured at a CXCL16 concentration of 400 ng / mL, it was found that the activation of fibroblasts was most significant at 48 h. These results indicate that CXCL16 can significantly stimulate the activation of fibroblasts.

[0068] Since macrophages can stimulate fibroblast activation, the supernatant of mouse primary macrophage (BMDM) culture medium was centrifuged at 4°C, 1200rpm for 5 minutes, and the cell-free supernatant was taken to obtain the conditioned medium (CM) rich in activation factors. CM was used to stimulate mouse primary fibroblasts to activate the cells. At the same time, the CXCL16 neutralizing antibody produced above was added and cultured together. After 48 hours, Western Blot was used to detect the fibrosis index of each group of cells, and the cell line with the lowest fibrosis expression was selected. The results are shown in Fig. 9As shown in Figure 2, the group with the lowest expression of CXCL16 downstream molecules (α-SMA, Col-1) was selected, indicating that the neutralization effect was the best.

[0069] Step 5: Ascites preparation: The selected cell lines are expanded and cultured to prepare ascites antibodies, and CXCL16 neutralizing antibodies can be obtained after purification.

[0070] This application study established a mouse-derived neutralizing antibody for mouse CXCL16 and confirmed the neutralizing effect of the neutralizing antibody. At the same time, the CXCL16 neutralizing antibody established in this application has a strong neutralizing effect, is easy to produce, and has low cost, which is helpful for the treatment of CXCL16-related diseases, overcomes the current shortcomings of poor neutralizing ability and high price of CXCL16 antibodies, and provides an effective tool for subsequent in vitro and in vivo experiments on CXCL16.

[0071] Example 2

[0072] Based on the CXCL16 neutralizing antibody prepared in Example 1, further experiments were conducted:

[0073] 1. In vivo Idiopathic Pulmonary Fibrosis (IPF) Model in Mice

[0074] Bleomycin (BLM) is the most commonly used idiopathic pulmonary fibrosis model, in which BLM is a chemical irritant that can be injected into the lungs through tracheal administration to induce IPF-like acute phase IPF, which is characterized by the recruitment of inflammatory cells into the lungs and increased expression of fibrosis markers.

[0075] Obtain C57BL / 6 mice, 8-10 weeks old, weighing 18g-22g, and establish an IPF model by tracheal injection of BLM. On days 3, 10, and 17, mice receiving BLM were intraperitoneally injected with mouse IgG control antibody (5 mg / kg) or neutralizing antibody (5 mg / kg); healthy "no BLM" mice were intraperitoneally injected with IgG control antibody (5 mg / kg) or neutralizing antibody (5 mg / kg); and body weight was recorded every day; the number of mice used for each treatment was 10.

[0076] In vivo radiation-induced pulmonary fibrosis (RILF) model in mice

[0077] Adult female C57BL / 6 mice were anesthetized, fixed, covered with 1 cm thick compensation film, and received whole chest irradiation with a dose of 16 Gy / 1F. Different treatments were given regularly according to groups. The mice were randomly divided into 2 groups: Control IgG group and CXCL16mAb group.

[0078] In the RILF trial, IgG control antibody (5 mg / kg) or neutralizing antibody (5 mg / kg) was intraperitoneally injected every two weeks from the 13th week to the 24th week after radiotherapy. After the administration, the body weight and survival rate of the mice (n=15) were recorded weekly, and the observation endpoint was set at the 24th week; the modeling and treatment procedures of each group were consistent.

[0079] 3. Single-cell sequencing experiment:

[0080] RILF model mice were killed with 1.5% sodium pentobarbital, and intact lung tissue was obtained and stored in a dry ice environment and then sent to Novogene for single-cell sequencing.

[0081] The steps for preparing the cell suspension during sequencing are:

[0082] 1. Put DMEM and complete DMEM on ice, and add PBS to a six-well plate for later use;

[0083] 2. Preparation of single cell suspension:

[0084] (1) Add some DMEM to the culture dish;

[0085] (2) Cut the lung tissue, put it into a six-well plate and soak it in PBS to clean the blood;

[0086] (3) Grinding using a blade and a 70 μm filter;

[0087] (4) Add the chopped tissue into the grinding tube and add 3 mL of digestive enzyme to each tube, including:

[0088] Digestive enzyme Mix: 10 mL of serum-free DMEM, containing;

[0089] IV glialase (100 mg / mL);

[0090] Hyaluronidase (100 mg / mL);

[0091] DNase (10 mg / mL);

[0092] (5) Digestion at 37°C for 45 min;

[0093] (8) Filter the tissue and collect the filtrate in a 50 mL centrifuge tube. Rinse the tube wall with complete DMEM to terminate digestion and fill the tube to 5 mL.

[0094] (10) Centrifuge at 1200 rpm to 1500 rpm for 5 min. Pay attention to the balance and placement during centrifugation.

[0095] 3. Red cracking:

[0096] (1) Discard the supernatant and add 3 mL of red blood cell lysis buffer to each tube;

[0097] (2) Leave it at room temperature for 5 minutes;

[0098] (3) Add complete DMEM to fill up to 5 mL to 15 mL;

[0099] (4) Centrifugation at 1200 rpm for 5 min;

[0100] 4. Micro-computed tomography

[0101] In RILF and IPF experiments, mice were anesthetized with 1.5% sodium pentobarbital and fixed in an appropriate posture. Chest computed tomography images were observed and reconstructed by a micro-CT scanner (SkyScan, 1176, Brooke, MA, USA) to evaluate lung inflammation and fibrosis.

[0102] 5. HE and Masson staining

[0103] HE (BH0001), Masson (BH0002), and Sirius red (B00039) staining kits were purchased from Boerfu Biotechnology (Hubei, China). Pathological staining was performed using the corresponding kits according to the manufacturer's instructions. Sirius red-stained sections were observed under a polarized light microscope. The Ashcroft scoring criteria were used to evaluate the HE staining of lung tissue in the RILF model to assess the severity of fibrotic lesions in different treatment groups. ImageJ software was used for visual quantitative analysis.

[0104] VI. Western blot detection

[0105] Protein extraction was performed on unfixed lung tissue from RILF model mice. Lung tissue was added to RIPA (G2002, Sevier Bio) and PMSF (G2007, Sevier Bio), crushed, ultrasonicated and centrifuged, and boiled at 100°C with 5× loading buffer (G2013, Sevier Bio). Electrophoresis was performed using a one-step PAGE gel rapid preparation kit (12%) (E304-01, Novozymes, Nanjing, China). Protein bands were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, Massachusetts, USA) and blocked with 5% BSA or fat-free milk for 1 h. The PVDF membrane was incubated with the following primary antibodies at 4°C overnight: anti-COL3A1 (1:1000, 66887S, CST, Massachusetts, USA), anti-COL1A2 (1:1000, A5786, Ibotek), anti-GAPDH (1:10000, A00227-1, Boster, Hubei, China), anti-Fn (1:1000, P02751, Ibotek), anti-α-SMA (also known as ACTA2, 1:1000, A17910, Ibotek), anti-TGF-β1 (1:1000, A2124, Ibotek). After incubation with the appropriate secondary antibodies at room temperature for 1 h, the membrane was developed using Super SignalWest Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Massachusetts, USA), and the protein bands were observed. Blot signals were detected using the G:BOX ChemiX system (Syngene, Cambridge, UK).

[0106] VII. Determination of Hydroxyproline (HYP) Content

[0107] The HYP content in lung tissue was detected to reflect the metabolism of collagen in fibrotic lesions. The detection process was strictly in accordance with the instructions of the kit purchased from Nanjing Jiancheng Bioengineering Institute (A030-2-1, Nanjing, China). Three mice were randomly selected from each group, and the intact right lung tissue was used for detection, and two secondary wells were made for the excised lung tissue.

[0108] 8. In vivo biosafety evaluation

[0109] ALT, AST and Cr in the serum of mice in all treatment groups were detected using Chemray 240 automatic biochemical analyzer (Rayto Science, Shenzhen, China) to evaluate the effect of CXCL16mAb on liver and kidney function. The heart, liver and kidney of mice treated with CXCL16mAb were fixed with 4% paraformaldehyde, sliced ​​and stained with HE to observe the effect of CXCL16 monoclonal antibody on important organs.

[0110] IX. Statistical Analysis

[0111] Data were analyzed using GraphPad Prism version 8.0. Results are expressed as mean ± standard deviation (SD) or mean ± standard error (SEM). Statistical significance was defined as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, respectively.

[0112] 10. Experimental Results

[0113] 1. CXCL16 is upregulated in fibrotic lungs:

[0114] Preliminary single-cell sequencing results showed that 3 months after radiotherapy, a group of CXCL16-positive interstitial macrophages accumulated in the lungs (e.g. Figure 1 A), which indicates that the proportion of interstitial macrophages was significantly different between the experimental group and the control group. In the two mouse lung fibrosis models induced by BLM and RT, the serum of mice treated with radiotherapy and BLM (as shown in Figure 1 As shown in B, three months after lung radiotherapy, the serum CXCL16 level of mice increased significantly) The CXCL16 mRNA level in lung tissue (as shown in Figure 1 E shows the expression level of CXCL16 mRNA in the lungs of mice 3 months after radiotherapy, which indicates that the expression of CXCL16 in the lungs of mice is increased) and the protein level (as shown in Figure 1 As shown in D, the expression of CXCL16 protein in mouse lung homogenate 3 months after radiotherapy was significantly upregulated. Immunohistochemical staining of CXCL16 showed that the expression of CXCL16 in the lung was increased in the fibrosis model induced by RT and BLM (as shown in Figure 1 As shown in C, 3 months after radiotherapy, CXCL16 immunofluorescence staining of mouse lungs showed an increase in CXCL16. In addition, from the IHC images of fibrotic lungs, it can be seen that CXCL16 expression accumulates in macrophages, which is consistent with the previous single-cell sequencing results (such as Figure 1 As shown in F, immunofluorescence co-localization of the lungs revealed that CXCL16 was mainly co-localized with the macrophage marker F4 / 80, indicating that macrophages are the main expressers of CXCL16. Figure 1 (G) 3 months after radiotherapy, single-cell sequencing was used to analyze the differential expression of CXCL16 in lung tissue of mice, and it was found that macrophages were the main expressers of CXCL16. These data indicate that CXCL16 is upregulated in fibrotic lungs, raising the possibility that it may play a role in the progression of pulmonary fibrosis.

[0115] 2. CXCL16 neutralizing antibodies alleviate radiation-induced pulmonary fibrosis (RIPF) and prolong survival:

[0116] A single dose of 16 Gy whole-thoracic irradiation was used to establish a radiation-induced lung injury mouse model (15 mice in each group). CT scanning and tissue staining were performed 6 months later (see Table 1). Figure 2 A). Radiological results showed obvious fused patchy shadows, solid lesions and a large amount of exudates in the Control IgG group, while only slightly increased bronchovascular shadows, a small amount of exudates and scattered streaky lesions were observed in the CXCL16mAb group. Left lung tissues of mice (n=3) were isolated for HE and Masson staining. The staining results showed that the lung tissues of the Control IgG group showed obvious inflammatory infiltration 6 months after radiotherapy, and the overall lung tissue structure was abnormal, accompanied by a large area of ​​solid tissue, alveolar collapse and atrophy and poor structure.

[0117] The CXCL16mAb group showed less inflammatory infiltration and tissue damage at the observation site than the Control IgG group (e.g. Figure 2 Western Blot and PCR were used to detect protein and gene expression in lung tissues, and it was found that the fibrosis indexes in the CXCL16mAb group were significantly reduced (as shown in Figure 2A). Figure 2 F and Figure 2 G). Ashcroft score can be used to evaluate HE results, and the score shows that the fibrosis level in the Control IgG group was significantly reduced (as shown in Figure 2A). Figure 2 D, p = 0.0004). The effect of RIPF on mice can be reflected by the weight change curve and survival percentage curve recorded weekly. Statistical analysis showed that the weight loss of mice in the CXCL16mAb group was slightly lower than that of mice in the Control IgG group (e.g. Figure 2 Compared with the ControlIgG group, although there was no significant difference, the weight curve showed a trend of continued increase 8 weeks after radiotherapy, which was opposite to the continuous decrease in the ControlIgG group (as shown in Figure 2B). Figure 2 In addition, compared with Control IgG mice, CXCL16 mAb significantly prolonged the survival of RIPF mice (as shown in Figure 2A). Figure 2 C, p = 0.0479). The lungs of mice were ground and the lung homogenates were obtained for determination of hydroxyproline content. The hydroxyproline level in the CXCL16mAb group was significantly lower than that in the ControlIgG group (as shown in Figure 5). Figure 2 E, P = 0.0014).

[0118] 3. CXCL16 neutralizing antibodies alleviate BLM-induced pulmonary fibrosis and prolong survival:

[0119] After anesthesia, mice were injected intratracheally with 5 mg kg-1 The idiopathic pulmonary fibrosis (IPF) mouse model was established with BLM (15 mice in each group), and CT scanning and tissue staining were performed 21 days later (as shown in Figure 2A). Figure 3 (As shown in A). Radiological results showed that the Control IgG group had obvious fused patchy shadows, solid lesions and a large amount of exudates, while only a slight increase in bronchial vascular shadows, a small amount of exudates and scattered streaky lesions were observed in the CXCL16mAb group. The left lung tissue of the mice (n=3) was isolated for HE and Masson staining. The staining results showed that the lung tissue of the Control IgG group showed obvious inflammatory infiltration 6 months after radiotherapy, and the overall lung tissue structure was abnormal, accompanied by a large area of ​​solid tissue, alveolar collapse and atrophy and poor structure. The CXCL16mAb group showed less inflammatory infiltration and tissue damage than the Control IgG group at the observation site (such as Figure 3 Western Blot and PCR were used to detect protein and gene expression in lung tissues, and it was found that the fibrosis indexes in the CXCL16mAb group were significantly reduced (as shown in Figure 2A). Figure 3 F and as shown in 3G). Ashcroft score can be used to evaluate HE results, and the score shows that the fibrosis level in the Control IgG group was significantly reduced (as shown in Figure 3 D, p = 0.0007). The effect of RIPF on mice can be reflected by the weight change curve and survival percentage curve recorded weekly. Statistical analysis showed that the weight loss of mice in the CXCL16mAb group was less than that of mice in the Control IgG group (e.g. Figure 3 B), compared with the NS group, although there was no significant difference, the weight curve showed a continued increase trend 8 weeks after radiotherapy, which was opposite to the continuous decrease trend of the ControlIgG group (as shown in Figure 3 In addition, compared with Control IgG mice, CXCL16 mAb significantly prolonged the survival of RIPF mice (as shown in Figure 2A). Figure 3 C, p = 0.0481). The lungs of mice were ground and the lung homogenates were obtained for determination of hydroxyproline content. The hydroxyproline level in the CXCL16mAb group was significantly lower than that in the ControlIgG group (as shown in Figure 5). Figure 3 E, P = 0.0007).

[0120] 4. In vivo biosafety evaluation of CXCL16 neutralizing antibodies:

[0121] Considering the potential adverse effects of CXCL16 neutralizing antibodies, wild mice were selected for in vivo biosafety assessment of CXCL16 neutralizing antibodies (n=5). The hearts, livers, lungs, spleens, and kidneys of mice were completely separated and HE stained. The myocardial fibers of mice were neatly arranged, the structures of liver lobules and kidney capsules were clear, and there was no acute damage such as exudation, congestion, and inflammatory cell infiltration in the tissues, indicating that the CXCL16 neutralizing antibodies administered in vivo did not cause significant damage to the major organs (such as Figure 4 A to Figure 4 E).

[0122] Serum glutathione aminotransferase (ALT), glutamate oxaloacetate aminotransferase (AST), and creatinine (Cr) levels were measured in the CXCL16mAb group and the control group mice 15 days after the start of treatment. The results showed that there were no significant differences in serum ALT, AST, and Cr among the three groups, indicating that the in vivo administration of CXCL16mAb did not affect liver and kidney function (such as Figure 4 F).

[0123] At the same time, the above results show that CXCL16mAb targets CXCL16 while reducing macrophage recruitment and acute and chronic lung toxicity induced by RT and BLM. Therefore, CXCL16 antibody can be used to safely and effectively treat radiation-induced pulmonary fibrosis and idiopathic pulmonary fibrosis in mice.

[0124] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0125] In the present application, in the absence of any contrary description, the directional words used, such as "upper" and "lower", are specifically the directions of the drawings in the accompanying drawings. In addition, in the description of the present specification, the terms "including", "comprising", etc. refer to "including but not limited to". In this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. In this article, "and / or" describes the association relationship of the associated objects, indicating that there may be three relationships, for example, A and / or B, which can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A, B can be singular or plural. In this article, "at least one" refers to one or more, and "plural" refers to two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of singular or plural items. For example, "at least one of a, b, or c", or "at least one of a, b and c", can both mean: a, b, c, ab (i.e. a and b), ac, bc or abc, where a, b, c can be single or plural, respectively.

[0126] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. Use of a CXCL16 neutralizing antibody as a therapeutic drug for radiation-induced and idiopathic pulmonary fibrosis.

2. The use according to claim 1, characterized in that: The CXCL16 neutralizing antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.3, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.

4.

4. A method for preparing a CXCL16 neutralizing antibody for use according to any one of claims 1 to 3, the method comprising: The CXCL16 gene in the preset region is selected, and the whole gene is synthesized into an expression vector for protein expression to obtain the CXCL16 protein antigen; Immunizing an animal model with the CXCL16 protein antigen, and then extracting and detecting antiserum to confirm that the animal immunization is complete, thereby obtaining an animal immune model; Performing cell fusion on the animal immune model and performing subclone screening to obtain multiple groups of immune cell strains; Conducting antibody neutralization effect screening and binding affinity screening on the multiple groups of immune cell lines, and performing protein immunoblotting analysis to obtain the best cell line; The optimal cell line is cultured in an expanded manner to obtain ascites antibodies, which are then purified to obtain CXCL16 neutralizing antibodies.

5. The method according to claim 4, characterized in that The detection includes detection by enzyme-linked immunosorbent assay; When the titer of the test is ≥1:64000, it means that the animal immunization process has been completed.

6. The method according to claim 4, characterized in that The preset region is 27aa to 201aa of the CXCL16 gene sequence.

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