Use of gca-nab monoclonal antibody in the preparation of a medicament for preventing and / or treating pulmonary fibrosis

Abstract

The application relates to the field of biomedical technology, and discloses an application of a GCA-NAb monoclonal antibody in the preparation of a drug for preventing and / or treating pulmonary fibrosis. The application mainly relates to the application of an amino acid sequence of the GCA-NAb monoclonal antibody as shown in SEQ ID No. 2 in the preparation of the drug for preventing and / or treating pulmonary fibrosis. The GCA-NAb monoclonal antibody can significantly improve pulmonary fibrosis, and can prolong the survival cycle of a mouse model, reduce the proportion of TH17 cells in lung tissues, and slow down the progress of pulmonary fibrosis. In addition, the antibody can directly affect the transformation of fibroblasts, inhibit the transformation of the fibroblasts into myofibroblasts, and thus reduce the fibrosis. The GCA-NAb monoclonal antibody has the characteristics of high efficiency and safety.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and provides a GCA-NAb monoclonal antibody and its application in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis is a serious, progressive lung disease characterized by immune dysfunction, excessive proliferation of fibroblasts, and collagen deposition in lung tissue, leading to progressive impairment of lung function. Current clinical treatments mainly include anti-fibrotic drugs such as pirfenidone, oxygen therapy, and lung transplantation; however, these methods have limited efficacy and may be accompanied by serious side effects. Therefore, developing a novel, highly effective, and safe treatment method is an urgent problem to be solved.

[0003] Th17 cells, a subset of T cells in the immune system, are believed to play a crucial role in the pathogenesis of pulmonary fibrosis. Studies have shown that Th17 cells and their main secreted cytokine, IL-17, play a significant role in the development of pulmonary fibrosis. IL-17 can promote inflammatory responses, enhance the infiltration of inflammatory cells, and stimulate the fibrotic process, ultimately leading to structural and functional impairment of lung tissue. Furthermore, Th17 cells may also participate in the occurrence and development of pulmonary fibrosis through other pathways, such as regulating the activation state of fibroblasts and influencing cytokine production. Although further in-depth research is needed to elucidate the specific mechanisms of action of Th17 cells in pulmonary fibrosis, preliminary understanding of their role has already provided new insights for future treatment strategies.

[0004] Granular cadherin (GCA) is a member of the Penta-EF-hand (PEF) protein family of calcium-binding proteins with five EF hand motifs. GCA-NAb monoclonal antibodies, namely GCA neutralizing antibodies, can block the biological activity of GCA, but so far there have been no reports on the use of GCA neutralizing antibodies to prepare treatments for pulmonary fibrosis and related complications. Summary of the Invention

[0005] The technical problem solved by this invention is to overcome the lack of specific drugs in the prior art for the prevention and / or treatment of pulmonary fibrosis and its related complications. This application provides a GCA-NAb monoclonal antibody with high specificity that can inhibit the activity of GCA, thereby reducing its role in pulmonary fibrosis. The application of this antibody can significantly improve pulmonary fibrosis and prolong its survival period.

[0006] The technical problem solved by this invention is achieved by the following technical solution:

[0007] In a first aspect, this application provides the use of a GCA-NAb monoclonal antibody in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis, wherein the amino acid sequence of the GCA-NAb monoclonal antibody is shown in SEQ ID No. 2.

[0008] In some alternative embodiments, the GCA-NAb monoclonal antibody is secreted by a hybridoma cell line with accession number CCTCC NO: C2021182.

[0009] In some alternative implementations, the nucleic acid encoding the GCA-NAb monoclonal antibody is used.

[0010] In some alternative embodiments, the drug comprises a GCA-NAb monoclonal antibody.

[0011] In some optional embodiments, the drug comprises a mass concentration of 0.05% × 10 -2 Up to 0.15% × 10 -2 GCA-NAb monoclonal antibody.

[0012] In some alternative embodiments, the drug further includes pirfenidone.

[0013] In some alternative embodiments, the drug further includes pirfenidone at a mass concentration of 0.3%.

[0014] In some alternative implementations, the mass ratio of GCA-NAb monoclonal antibody to pirfenidone is 1:1400 to 1:466.

[0015] Secondly, this application provides the use of a nucleic acid encoding a monoclonal antibody of the first aspect GCA-NAb in the preparation of a drug for the prevention and / or treatment of pulmonary fibrosis.

[0016] In some alternative implementations, the nucleotide sequence of the nucleic acid is shown in SEQ ID No. 1.

[0017] This application has at least the following beneficial effects:

[0018] The GCA-NAb monoclonal antibody of this application antagonizes the recruitment of TH17 cells by GCA, reducing the proportion of TH17 cells in the lungs. This mechanism of action is clearly defined by regulating the TH17 cell ratio and inhibiting the transformation of fibrotic cells into muscle fibrotic cells. The GCA-NAb monoclonal antibody also demonstrates significant efficacy in treating pulmonary fibrosis: it effectively improves the pathological manifestations of pulmonary fibrosis, increases patient survival rates, and, as verified in animal model experiments, exhibits good safety and tolerability during treatment.

[0019] Information on the preservation of biological materials:

[0020] The hybridoma cell line of this application was deposited on August 3, 2021, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Luojia Mountain, Wuchang District, Wuhan, Hubei Province, China, 430072, China, with accession number CCTCC NO: C2021182 and culture name GCA-NAB4 hybridoma cell line. Attached Figure Description

[0021] Figure 1 Survival curves of the control IgG antibody group and the GCA-NAb monoclonal antibody group in the embodiments of this application are shown.

[0022] Figure 2 The effect of the GCA-NAb monoclonal antibody of this application on the respiratory function of fibrotic mice is shown.

[0023] Figure 3 The images show HE and Masson staining patterns (n=5) of fibrotic mouse lung tissue obtained by the GCA-NAb monoclonal antibody according to the present application.

[0024] Figure 4 The figure shows a statistical analysis of the effect of flow cytometry analysis of the GCA-NAb monoclonal antibody on the number of CD4+IL17+Th17 cells in the bronchoalveolar lavage fluid of fibrotic mice, according to an embodiment of this application.

[0025] Figure 5 The figure shows a statistical analysis of the effect of the GCA-NAb monoclonal antibody of this application on hydroxyproline in the lung tissue of fibrotic mice.

[0026] Figure 6 The diagram shows a qPCR statistical graph of the GCA-NAb monoclonal antibody against fibrosis-related genes in the lung tissue of fibrotic mice, as described in this application.

[0027] Figure 7 The effect of the GCA-NAb monoclonal antibody from this application on fibrosis-related proteins in fibrotic mouse tissues is illustrated.

[0028] Figure 8 The effect of the GCA-NAb monoclonal antibody of this application on fibroblasts is shown.

[0029] Figure 9 The effect of the combination of GCA-NAb monoclonal antibody and pirfenidone on the life cycle of fibrotic mice is shown in the embodiments of this application. Detailed Implementation

[0030] To make the purpose, technical solution, and beneficial technical effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the embodiments described in this specification are merely for explaining this application and are not intended to limit it.

[0031] For simplicity, this application only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form a range not explicitly stated; and any lower limit can be combined with other lower limits to form a range not explicitly stated, just as any upper limit can be combined with any other upper limit to form a range not explicitly stated. Furthermore, although not explicitly stated, every point or individual value between the endpoints of the range is included within that range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value or with other lower or upper limits to form a range not explicitly stated.

[0032] In the description of this application, it should be noted that, unless otherwise stated, "above" and "below" include the stated number, and "multiple" in "one or more" means two or more.

[0033] The foregoing description of this application is not intended to describe every disclosed implementation or method. Instead, the following description provides more specific examples of exemplary embodiments. Throughout the application, guidance is provided through a series of embodiments that can be used in various combinations. The examples listed are representative only and should not be construed as exhaustive.

[0034] In a first aspect, this application provides the use of a GCA-NAb monoclonal antibody in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis, wherein the amino acid sequence of the GCA-NAb monoclonal antibody is shown in SEQ ID No. 2.

[0035] In related technologies, treatments such as oxygen therapy have long treatment cycles and limited efficacy, while adjuvant therapy with drugs such as pirfenidone and nintedani can cause adverse side effects such as gastrointestinal reactions and liver damage. Furthermore, although these drugs can alleviate disease progression to some extent, they cannot effectively improve patient survival rates. The GCA-NAb monoclonal antibody provided in this application can improve upon these issues.

[0036] GCA-NAb monoclonal antibody can reduce collagen deposition and fibrosis in lung tissue, improve respiratory function in mice with pulmonary fibrosis, and improve the survival rate of mice with pulmonary fibrosis.

[0037] In some alternative embodiments, the GCA-NAb monoclonal antibody is secreted by a hybridoma cell line with accession number CCTCC NO: C2021182.

[0038] In some alternative embodiments, the nucleic acid encoding the GCA-NAb monoclonal antibody is used. In some alternative embodiments, the nucleotide sequence of the nucleic acid is as shown in SEQ ID No. 1.

[0039] In some alternative embodiments, the drug comprises a GCA-NAb monoclonal antibody.

[0040] This drug reduces the proportion of TH17 cells in lung tissue. It can directly affect the transformation of fibroblasts, inhibiting their ability to transform into muscle fibroblasts, thereby alleviating fibrotic lesions and improving pulmonary fibrosis and survival.

[0041] In some optional embodiments, the drug comprises a mass concentration of 0.05% × 10⁻⁶. -2 Up to 0.15% × 10 -2 GCA-NAb monoclonal antibody.

[0042] In some alternative embodiments, the drug further includes pirfenidone.

[0043] Pirfenidone is a drug used for mild to moderate idiopathic pulmonary fibrosis (IPF).

[0044] In some alternative embodiments, the drug further includes pirfenidone at a mass concentration of 0.3%.

[0045] In the embodiments of this application, the combination of the drug and pirfenidone significantly improves the survival rate compared to pirfenidone monotherapy.

[0046] Treatment with GCA-NAb monoclonal antibody combined with pirfenidone significantly prolonged the survival time of mice compared with monotherapy.

[0047] In some alternative implementations, the mass ratio of GCA-NAb monoclonal antibody to pirfenidone is 1:1400 to 1:466.

[0048] In the embodiments of this application, the GCA-NAb monoclonal antibody and pirfenidone in the above mass ratio can work synergistically to improve pulmonary fibrosis and more significantly prolong the survival period of mice.

[0049] Secondly, this application provides the use of a nucleic acid encoding a monoclonal antibody of the first aspect GCA-NAb in the preparation of a drug for the prevention and / or treatment of pulmonary fibrosis.

[0050] In some alternative implementations, the nucleotide sequence of the nucleic acid is shown in SEQ ID No. 1.

[0051] To make the technical means, creative features, objectives, and effects of this invention easier to understand, the invention is further described below with reference to specific embodiments. All reagents and raw materials used in this invention are commercially available.

[0052] Example 1: Preparation of GCA-NAb monoclonal antibody

[0053] The hybridoma cell line GCA-NAB4, with accession number CCTCC NO: C2021182, was transferred into a 100mL culture flask. A certain amount of culture medium (purchased from GIBCO) was added periodically to expand the cells, and the culture was carried out for 10-12 days.

[0054] The culture was washed with ultrapure water using a Protein A affinity chromatography column, and then equilibrated with equilibration buffer. The treated hybridoma cell supernatant was loaded onto the affinity chromatography column, and after loading, it was washed with equilibration buffer. Elution was performed with elution buffer, the elution peak was collected, neutralized with Tris buffer, and desalted to PBS 7.4 to obtain the GCA-NAb monoclonal antibody. The amino acid sequence of the GCA-NAb monoclonal antibody includes the amino acid sequence shown in SEQ ID No. 2.

[0055] Example 2: GCA-NAb monoclonal antibody improves pulmonary fibrosis and survival time in mice by antagonizing GCA.

[0056] Eight-week-old mice were induced into a mouse model of pulmonary fibrosis by intratracheal injection of bleomycin (2 mg / kg). They were randomly divided into a control group (control IgG antibody) and an experimental group (GCA-NAb monoclonal antibody), with 15 mice in each group and an average weight of 24 g. The control group was subsequently injected with the control IgG antibody via tail vein, while the experimental group received GCA-NAb monoclonal antibody (1 mg / kg) via tail vein injection three times a week for three weeks. Body weight and survival rate were recorded. Respiratory function tests were performed after three weeks, and mice from each group were sacrificed. Serum and lung tissue or bronchoalveolar lavage fluid were collected.

[0057] 1) A portion of serum and lung tissue or bronchoalveolar lavage fluid was fixed with formaldehyde and dehydrated using a gradient of alcohol. After paraffin embedding and sectioning, immunohistochemical staining was performed, including HE staining and MASSON staining. Figure 3 As shown, from Figure 3 It can be seen that the GCA-NAb monoclonal antibody improved fibrosis in the lungs of mice with pulmonary fibrosis.

[0058] 2) Proteins and RNA were directly extracted from another portion of serum and lung tissue or bronchoalveolar lavage fluid to prepare protein and cDNA samples for Western blotting and qPCR analysis.

[0059] The results are shown below.

[0060] Figure 1 The study showed that the GCA-NAb monoclonal antibody significantly improved the survival rate of mice. Results analysis: All values ​​are expressed as the mean ± SD of three assays. * indicates a comparison with the control group treated with the control IgG antibody (P < 0.05), ** indicates a comparison with the control group treated with the control IgG antibody (P < 0.01), *** indicates a comparison with the control group treated with the control IgG antibody (P < 0.001). Figure 1 This demonstrates that the GCA-NAb monoclonal antibody significantly improved the survival rate of mice.

[0061] Table 1. Experimental results showing the effect of GCA-NAb monoclonal antibody on the expression of fibrosis genes in rat lung.

[0062]

[0063] As shown in Table 1 and Figure 6 As shown, compared with the control group treated with IgG antibody, the expression of fibrosis genes in the lungs of mice injected with GCA-NAb monoclonal antibody via the tail vein was significantly reduced, indicating that GCA-NAb monoclonal antibody treatment can reduce pulmonary fibrosis.

[0064] Table 2. Effects of GCA-NAb monoclonal antibody on CD4 in bronchoalveolar lavage fluid. + IL17 + Experimental results on the effect of Th17 cell number on the total number of alveolar cells.

[0065] Group <![CDATA[CD4 + IL17 + Th17 cell percentage / % Control IgG antibody 3.351±1.012 GCA-NAb monoclonal antibody 1.130±0.643**

[0066] As shown in Table 2 and Figure 4 It can be seen that, compared with the control group treated with IgG antibody, the experimental group had lower levels of CD4+ in the bronchoalveolar lavage fluid after intravenous injection of GCA-NAb monoclonal antibody in rats. + IL17 + The decrease in the number of Th17 cells indicates that treatment with the GCA-NAb monoclonal antibody can reduce the recruitment of Th17 cells that promote fibrosis. Figure 4 The horizontal axis represents APC-IL17A, and the vertical axis represents FSC-H. Figure 4 It is known that GCA-NAb monoclonal antibody can significantly improve the recruitment of Th17 cells in the lungs of mice.

[0067] Table 3. Experimental results showing the effect of GCA-NAb monoclonal antibody on hydroxyproline in lung tissue.

[0068] Group Hydroxyproline (μg / g) Control IgG antibody 270.320±73.982 GCA-NAb monoclonal antibody 178.505±43.785**

[0069] As shown in Table 3 and Figure 5As shown, compared with the control group treated with IgG antibody, the experimental group showed a decrease in fibrosis-related proteins in lung tissue after intravenous injection of GCA-NAb monoclonal antibody in the tail vein. Figure 5 The vertical axis HYP represents the hydroxyproline content. The hydroxyproline content in the GCA-NAb monoclonal antibody group was lower than that in the control IgG antibody group, indicating that the GCA-NAb monoclonal antibody improved early pulmonary fibrosis in mice.

[0070] The lung function of mice was tested using the buxco MAX II invasive pulmonary function test method. After the mice's breathing stabilized, the lung resistance was obtained by calculating the resistance encountered by the airflow through the airway.

[0071] Lung function in mice was assessed using a buxco MAX II invasive pulmonary function testing method. After the mice's breathing stabilized, lung compliance was determined by calculating the volume change per unit pressure change during dynamic respiration.

[0072] The lung function of mice was tested using the buxco MAX II invasive pulmonary function test method. After the mice's breathing stabilized, the minute ventilation was obtained by calculating the total amount of gas inhaled or exhaled per minute.

[0073] Table 4. Effects of GCA-NAb monoclonal antibody on respiratory function in mice.

[0074]

[0075]

[0076] From Table 4 and Figure 2 It can be seen that, compared with the control group treated with IgG antibody, the respiratory function of mice injected with GCA-NAb monoclonal antibody via the tail vein was significantly improved.

[0077] Detection of lung tissue using immunoblotting yielded results such as... Figure 7 The expression results of α-SMA and COL1A1 proteins were obtained. Fibroblasts were stained with immunofluorescence to obtain the following results: Figure 8 The expression of α-SMA in cells.

[0078] Figure 7 The effects of the GCA-NAb monoclonal antibody from this application on fibrosis-related proteins in fibrotic mouse tissues, particularly COL1A1, α-SMA, and GAPDH, are shown. It can be seen that treatment with the GCA-NAb monoclonal antibody significantly reduces the expression level of α-SMA protein. Figure 8 The image shows the expression of α-SMA protein, by... Figure 7 and Figure 8It is known that GCA can directly promote the transformation of mouse fibroblasts into myofibroblasts, thereby aggravating fibrosis.

[0079] In summary, GCA-NAb monoclonal antibody can effectively block the promoting effect of GCA on pulmonary fibrosis, thereby significantly improving pulmonary fibrosis in mice and prolonging their survival and development.

[0080] Example 3

[0081] To compare the efficacy of bleomycin-induced pulmonary fibrosis treatment with existing drugs, bleomycin-induced pulmonary fibrosis mice were divided into a control group (0.005 g / mL carboxymethyl cellulose, 100 μL gavage, once daily) and a control group (3 mg / kg control IgG antibody, purchased from Cell Signaling). The study included several treatment groups: a GCA-NAb monoclonal antibody group (3 mg / kg control IgG antibody, 100 μL tail vein injection, 3 times a week), a pirfenidone monotherapy group (pirfenidone, 300 mg / kg, dissolved in 100 μL of 0.005 g / mL carboxymethyl cellulose, gavage, once daily), and a GCA-NAb monoclonal antibody + pirfenidone combination therapy group (3 mg / kg control IgG antibody, 100 μL tail vein injection, 3 times a week; pirfenidone, 300 mg / kg, dissolved in 100 μL of 0.005 g / mL carboxymethyl cellulose, gavage, once daily). Each group consisted of 15 mice, with an average weight of 24 g. The treatment lasted for 3 weeks, and mouse survival rates were recorded.

[0082] The 3 mg / kg control IgG antibody dosage is based on the mouse's body weight; each mouse weighing 1 kg corresponds to 3 mg of IgG antibody. Each control and experimental group contains 15 mice, with an average weight of 24 g, corresponding to a dosage of 3 mg / kg * 24 g of IgG antibody.

[0083] Depend on Figure 9 It can be seen that, compared with GCA-NAb monoclonal antibody or pirfenidone monotherapy, GCA-NAb monoclonal antibody combined with pirfenidone treatment can significantly improve the survival rate of mice with pulmonary fibrosis.

[0084] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. Use of GCA-NAb monoclonal antibody in the preparation of a drug for preventing and / or treating pulmonary fibrosis, wherein the GCA-NAb monoclonal antibody is secreted by the hybridoma cell strain with the preservation number CCTCC NO: C2021182.

2. Use according to claim 1, characterized in that, The drug comprises the GCA-NAb monoclonal antibody.

3. Use according to claim 1, characterized in that, The medicament includes GCA-NAb monoclonal antibody at a mass concentration of 0.05% x 10 -2 to 0.15% x 10 -2 .

4. Use according to claim 1, characterized in that, The drug further comprises pirfenidone.

5. The use according to claim 1, characterized in that, The drug further comprises pirfenidone with a mass concentration of 0.3%.

6. Use according to claim 1, characterized in that, The mass ratio of the GCA-NAb monoclonal antibody to pirfenidone is 1:1400 to 1:466.

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