Novel Anti-pulmonary fibrosis neutralizing antibody and drug

Abstract

The present application to the field of antibody drug preparation, and provides a novel anti-pulmonary fibrosis neutralizing antibody and a drug. The present application, based on cutting-edge AI technology such as AlphaFold and de novo protein design, designs a neutralizing antibody targeting the binding site between Gremlin 2 and TGF-β receptor 1, and prepares the antibody using genetic engineering technology. By means of in vitro recombination, a gene fragment of a target antibody is inserted into a specific plasmid, and the edited plasmid is subsequently introduced into a specific cell line, followed by in vitro amplification and culture of the cell line to obtain transcription and translation of the gene fragment in vitro, thereby preparing the antibody. The AI algorithm design of the neutralizing antibody in the present application is efficient and reliable, the process for preparing the neutralizing antibody is simple, and the resulting neutralizing antibody exhibits high homogeneity, safety, and efficacy. The neutralizing antibody has demonstrated favorable anti-pulmonary fibrosis function in animal models and has the potential to be used for the clinical treatment of pulmonary fibrosis.

Description

A novel anti-pulmonary fibrosis neutralizing antibody and drug

[0001] This application claims priority to Chinese Patent Application No. 202510487050.5, filed on April 18, 2025, entitled "A Novel Anti-pulmonary Fibrosis Neutralizing Antibody and Drug", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application belongs to the field of antibody drug preparation and relates to a novel anti-pulmonary fibrosis neutralizing antibody and drug. Background Technology

[0003] Tissue fibrosis is the final outcome of almost all chronic diseases, and many patients die from organ failure caused by fibrosis. Idiopathic pulmonary fibrosis (IPF) is a typical example of a fatal fibrotic disease. Currently, medical treatments for IPF are very limited, resulting in poor quality of life and short survival times for patients. There is an urgent need to develop new anti-fibrotic drugs.

[0004] The mechanism of sustained fibroblast activation may be key to the rapid and irreversible fibrosis process, and transforming growth factor-β (TGF-β) signaling has been shown to play an important role in fibroblast activation. Studies have shown that activated TGF-β1 can bind to TGFBR2 on the cell membrane, recruiting intracellular TGFBR1 (TGF-β Receptor 1) onto the membrane, leading to phosphorylation of TGFBR1 and subsequent signal transduction. This signal is then terminated by ubiquitination and degradation of TGFBR1 on the membrane. Basic research has identified Gremlin2 as a ligand for TGFBR1, and through basic research and AI-based deep analysis, key binding epitopes have been identified. Optimized AI algorithms have been used to design monoclonal antibodies targeting these key antigenic epitopes.

[0005] Gremlin2, a TGF-β superfamily protein, is a potential therapeutic target for anti-fibrosis. Unlike the broad regulatory role of TGFβ signaling in various cells, Gremlin2 is a crucial protein that specifically targets and regulates the activation of fibroblasts. The clinical translation of Gremlin2 neutralizing antibodies may bring a new breakthrough in the treatment of pulmonary fibrosis patients. Summary of the Invention

[0006] The purpose of this application is to provide a novel anti-pulmonary fibrosis neutralizing antibody and drug that addresses the shortcomings of existing technologies.

[0007] To achieve the above objectives, this application provides the following technical solution: Firstly, it provides an anti-pulmonary fibrosis neutralizing antibody, composed of a heavy chain and a light chain. The heavy chain includes a heavy chain variable region, which includes heavy chain CDR1 as shown in SEQ ID NO: 1, heavy chain CDR2 as shown in SEQ ID NO: 2, and heavy chain CDR3 as shown in SEQ ID NO: 3. The light chain includes a light chain variable region, which includes light chain CDR1 as shown in SEQ ID NO: 4, light chain CDR2 as shown in SEQ ID NO: 5, and light chain CDR3 as shown in SEQ ID NO: 6.

[0008] Furthermore, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 8, and the amino acid sequence of the light chain is shown in SEQ ID NO: 9.

[0009] Furthermore, the anti-pulmonary fibrosis neutralizing antibody targets the binding sites of Gremlin2 and TGFBR1.

[0010] Secondly, the purpose of this application is also to provide a drug, including the aforementioned anti-pulmonary fibrosis neutralizing antibody, for the treatment of pulmonary fibrosis caused by Gremlin2.

[0011] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0012] The beneficial effects of this application are as follows: This application discovers that the binding of Gremlin2 to TGFBR1 stabilizes the signal transduction of TGFBR1 on the membrane, thereby leading to the continuous activation of TGFβ signaling. This application designs neutralizing antibodies targeting the Gremlin2-TGFBR1 binding site using computer deep learning, overcoming the need for a large number of experimental animals in the preparation of traditional neutralizing antibodies. Effective antibody sequences can be screened early using computer analysis, avoiding the problems of preparing large quantities of unknown sequences and unreliable efficacy in traditional methods. This reduces the workload of researchers in subsequent cell and animal model validation. The process is stable, safe, and efficient, representing a new method for the clinical development of therapeutic antibodies. The Gremlin2 neutralizing antibody designed in this application is suitable for subsequent clinical research on the treatment of pulmonary fibrosis, exhibiting significant anti-fibrotic effects. Furthermore, the process is systematic, low-cost, and easy to promote. Attached Figure Description

[0013] Figure 1 is a schematic diagram of AI simulation combining Gremlin2 and TGFBR1;

[0014] Figure 2 shows the specific sequence diagram of Gremlin2 binding to TGFBR1; in Figure 2, (A) is a color map of the antigen-antibody docking site (Cartoons), (B) is a full atomic map of the antigen-antibody docking site, and (C) is a schematic diagram of the antigen epitope, with the green part being the epitope of the antigen and the white part being the remaining part of the antigen excluding the epitope.

[0015] Figure 3 shows a schematic diagram of Gremlin2 dimer and TGFBR1;

[0016] Figure 4 is a schematic diagram of AI simulation of Gremlin2-TGFBR1 binding site and neutralizing antibody;

[0017] Figure 5 shows a schematic diagram of the production of Gremlin2 neutralizing antibody; in Figure 5, (A) is the detection result of SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis), and (B) is the detection result of SEC-HPLC (Size Exclusion Chromatography-High Performance Liquid Chromatography).

[0018] Figure 6 shows the detection results of LC-MS (Liquid Chromatography-Mass Spectrometry Detection); in Figure 6, (A) is the LC-MS detection result of undegraded antibody (Intact), (B) is the LC-MS detection result of light chain (LC), and (C) is the LC-MS detection result of heavy chain (HC).

[0019] Figure 7 shows the detection effect of Gremlin2 neutralizing antibody on fibroblast activation inhibition in vitro; in Figure 7, (A) is the detection result of the binding ability of TGFBR1 to Gremlin2, (B) is a schematic diagram of the nucleus labeled with α-SMA (red) and DAPI (blue) activation indicators of fibroblasts by immunofluorescence staining, with a scale bar of 50 micrometers, and (C) is a statistical bar chart of α-SMA fluorescence signal. * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001. ANOVA analysis of variance was used for comparison of multiple groups.

[0020] Figure 8 shows the anti-pulmonary fibrosis function of Gremlin2 neutralizing antibody; in Figure 8, (A) is a graph of the change in body weight of mice in each group, (B) is a graph of the lung tissue coefficient of mice in each group, (C) is a graph of the hydroxyproline (HYP) content in the lung tissue of mice in each group, NS represents p>0.05, ** represents p<0.01, *** represents p<0.001, and ANOVA was used for comparison of multiple groups; Figure 8, (D) is a gross graph of the lung tissue of mice in each group.

[0021] Figure 9 shows schematic diagrams of masson staining in the lung tissue of mice in each group. In Figure 9, (A) is a schematic diagram of masson staining in the PBS-lgG group, (B) is a schematic diagram of masson staining in the BLM-lgG group, and (C) is a schematic diagram of masson staining in the BLM-AIAB group. The scale bar is 200 micrometers.

[0022] Figure 10 shows the results of labeling the activation level (α-SMA) and extracellular matrix level (collagen 1 and fibronectin) of fibroblasts in mouse lung tissue by Western blotting (WB).

[0023] Figure 11 shows the statistical results of RT-QPCR (real-time quantitative polymerase chain reaction) labeling of mouse lung tissue; where Figure 11(A) shows the statistical results of RT-QPCR labeling of Col1a1 (α1 chain of collagen 1) in mouse lung tissue, Figure 11(B) shows the statistical results of RT-QPCR labeling of Col1a2 (α2 chain of collagen 1) in mouse lung tissue, and Figure 11(C) shows the statistical results of RT-QPCR labeling of Fibronectin in mouse lung tissue; ns represents p > 0.05, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001;

[0024] Figure 12 shows the toxicity detection of Gremlin2 neutralizing antibodies; in Figure 12, (A) is a gross image of the heart of each group of mice, (B) is a gross image of the liver of each group of mice, (C) is a gross image of the spleen of each group of mice, and (D) is a gross image of the kidney of each group of mice. Detailed Implementation

[0025] This application utilizes optimized algorithms based on cutting-edge AI technologies such as Alphafold protein structure prediction and de novo protein design to design antibody sequences targeting the Gremlin2 and TGFBR1 binding sites (or Gremlin2 targets). Antibodies are prepared using genetic engineering techniques. Through in vitro recombination, the gene fragment of the target antibody is inserted into a specific plasmid. Subsequently, the edited plasmid is transformed into specific cell lines, and the cell lines are amplified and cultured in vitro to obtain the transcription and translation of the gene fragment, thereby preparing the antibody. Based on this, a neutralizing antibody targeting Gremlin2 (hereinafter referred to as Gremlin2 neutralizing antibody or AIAB) was developed. Treatment with Gremlin2 neutralizing antibody at both cellular and animal levels significantly reduced fibroblast activation and the degree of bleomycin-induced pulmonary fibrosis in mice. Further humanization of the Gremlin2 neutralizing antibody may be a novel treatment method for patients and potential patients with pulmonary fibrosis.

[0026] The embodiments, features, and aspects of this application will be described in detail below with reference to the accompanying drawings, but this is not intended to limit the application. Any embodiment extended from the embodiments of this application, and all other embodiments obtained by those skilled in the art without inventive effort, are within the scope of protection of this application.

[0027] The following detailed description of a novel anti-pulmonary fibrosis neutralizing antibody and drug provided in this application, with reference to specific embodiments, should not be construed as limiting the scope of protection of this application.

[0028] Example 1

[0029] Design, screening and preparation of Gremlin2 neutralizing antibodies.

[0030] The Gremlin2 neutralizing antibodies obtained in this application are mainly biologically active recombinant proteins with high purity, approximately 1–3 mg / L. They are dissolved in PBS concentrated via ultrafiltration and stored at -80 degrees Celsius.

[0031] (1) Sequence design: Antibody sequences targeting the binding sites of Gremlin2 and TGFBR1 (hereinafter referred to as Gremlin2 target) are designed using optimized algorithms based on cutting-edge AI algorithms such as Alphafold.

[0032] The modified alpha-fold2 algorithm was used to simulate the binding prediction of Gremlin2 dimer (blue and pink) and TGFBR1 extracellular segment, as shown in Figure 1. Since the Gremlin2 dimer structure is composed of two completely identical monomers, the extracellular segment of TGFBR1 binds to two sites of Gremlin2 dimer at the same time, and both are marked in the figure with gray outlines.

[0033] Based on the sequence characteristics of the two proteins, this application uses computer simulation technology to more clearly annotate the binding sequence of the two proteins. The main binding sequence on Gremlin2 is C-NSFYIPRHVKKEEDSFQS (SEQ ID NO: 7), as shown in Figure 2. The amino acids at positions 7, 12, 14, and 18 in the sequence are the specific binding sites that directly bind to the TGFBR1 receptor.

[0034] (2) Sequence Screening: The free energy of binding between Gremlin2 and the designed sequence was calculated using deep learning, and the stability of the Gremlin2-neutralizing antibody was measured through methods such as spatial allosteric transformation, thereby screening for antibody sequences with high affinity for Gremlin2. The amino acid sequence of the heavy chain of the antibody finally screened in this application is as follows:

[0035] The amino acid sequence of the heavy chain variable region CDR1 is: VSGDAPPAIGKY (SEQ ID NO: 1); the amino acid sequence of CDR2 is: AGGEDGI (SEQ ID NO: 2); and the amino acid sequence of CDR3 is: APFGGAAYIE (SEQ ID NO: 3).

[0036] The amino acid sequence of the light chain is as follows:

[0037] The amino acid sequence of the light chain variable region CDR1 is: SGGSGADIA (SEQ ID NO: 4); the amino acid sequence of CDR2 is: NGSLPP (SEQ ID NO: 5); and the amino acid sequence of CDR3 is: AACTRKLT (SEQ ID NO: 6).

[0038] Figure 3 shows a schematic diagram of Gremlin2 dimer and TGFBR1 (light gray and dark gray represent simulations of the TGFBR1 receptor binding to the two monomers of Gremlin2 dimer, respectively). The binding free energy of the above sequence with the Gremlin2-TGFBR1 model was calculated. The smaller the value, the more stable the predicted sequence binding. The original binding free energy is -7.13 kcal / mol. Identical binding sites on both sides have the same binding probability. Figure 4 shows an AI simulation diagram of the Gremlin2-TGFBR1 binding site and the neutralizing antibody. 。

[0039] (3) Antibody synthesis: Genetic engineering technology is used to insert the gene fragment of the target antibody into the plasmid, and then the edited plasmid is transferred into the cell line; the edited cell line is expanded and cultured in vitro; the target gene is transcribed and translated in vitro to prepare recombinant protein.

[0040] (4) Crude separation: Collect cells and lyse them in a low-temperature environment (4°C) using a lysis buffer containing protease inhibitors. Repeated freeze-thaw cycles are used to break the cells and release recombinant proteins.

[0041] (5) Concentration and purification: Ultrafiltration membrane concentration was used. Liquids such as water were filtered out through a microporous cellulose membrane under high pressure, and the recombinant protein remained on the membrane. After elution with sterile 1×PBS (phosphate buffer), the concentration of recombinant protein was measured. After aliquoting, the stock solution concentration was maintained at 1-3 mg / L and stored at -80℃.

[0042] The protein size of the antibody was detected using SDS-PAGE, with the concentration of polyacrylamide gel ranging from 4% to 12%, as shown in Figure 5(A). Lane M (Marker) represents a standard example of molecular weight, NR (Non-reduced) represents the size of the antibody that has not been degraded (light and heavy chains not separated) (143.3 kDa), and lane R (Reduced) represents the size of the antibody with separated light and heavy chains (light chain 22-30 kDa, heavy chain approximately 53 kDa). The electrophoresis pattern indicates that the antibody is a dimer.

[0043] SEC-HPLC (SEC-300 column) was used to analyze and detect protein purity. SEC-HPLC is mainly used to analyze and separate substances of different molecular weights. As shown in Figure 5 (B) and Table 1, the antibody is a single peak, indicating that the antibody exists in a homogeneous form. The injection volume was 20 μg and the flow rate was 0.35 mL / min. ND indicates that it was not detected.

[0044] Table 1: SEC-HPLC Detection Results

[0045] The LC-MS mass spectra are shown in Figure 6 and Table 2. They show that the undegraded antibody (Intact) is mainly distributed at 143 kDa, the heavy chain (HC) is mainly distributed at 50 kDa, and the light chain (LC) is at 22 kDa. This is consistent with the above results, which again suggests that the antibody exists in a dimer form and has good homogeneity.

[0046] Table 2: LC-MS Detection Results

[0047] Example 2

[0048] Verification of the inhibitory effect of Gremlin2 neutralizing antibody on in vitro fibroblast activation.

[0049] TGFBR1 (TβRI) and Gremlin2 proteins were overexpressed in 293T cells, tagged with Flag and His-Myc, respectively. After 72 hours of successful overexpression, the 293T cells were lysed to obtain all intracellular proteins. The binding levels of Gremlin2 and TGFBR1 were then detected using a CO-IP (Co-Immunoprecipitation) assay. Magnetic beads were used to bind to Myc antibodies, and based on the principle of antigen-antibody binding, the magnetic beads successfully bound to the Myc-tagged Gremlin2 protein. Different concentrations of the Gremlin2 neutralizing antibody AIAB (0, 0.1 μg / mL, 0.2 μg / mL, and 0.3 μg / mL) were added to the protein lysis buffer, and the binding ability of TGFBR1 to Gremlin2 was then detected.

[0050] The results are shown in Figure 7(A). Input represents the presence of both TGFBR1 and Gremlin2 proteins in the original cell lysate. Flag and His tags represent TGFBR1 and Gremlin2 proteins, respectively. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is an internal control protein. The IP group represents the gradual inhibition of TGFBR1 and Gremlin2 binding as the antibody is added.

[0051] 5 ng / mL of TGF-β1 stimulating factor was added to lung fibroblasts, and different concentrations of AI neutralizing antibody AIAB (0, 0.1 μg / mL, and 0.2 μg / mL, respectively) were added. The activation marker α-SMA (red) of fibroblasts was labeled by immunofluorescence staining, and the cell nucleus was labeled with DAPI (blue).

[0052] The results are shown in Figures 7(B) and (C). As the concentration of Gremlin2 neutralizing antibody increases, the average fluorescence intensity decreases, indicating that Gremlin2 neutralizing antibody can inhibit fibroblast activation in vitro.

[0053] Example 3

[0054] Validation of the anti-fibrotic effect of Gremlin2 neutralizing antibody in an animal model of pulmonary fibrosis.

[0055] Eight-week-old male C57BL / 6J rats were used in the study. A mouse pulmonary fibrosis model was established by instilling PBS or bleomycin (BLM) into the airway. On days 8, 11, 14 and 17, 50 μg of Gremlin2 neutralizing antibody AIAB or an equal volume of IgG were injected intraperitoneally. The rats were divided into three groups: PBS-IgG group, BLM-IgG group and BLM-AIAB group.

[0056] (1) Record the changes in mouse body weight and plot the changes in body weight, as shown in Figure 8(A). There was no significant difference between the PBS-IgG group and the BLM-AIAB group, but the mice in the BLM-IgG group had significantly lower body weight. The mouse lung tissue coefficient plot is shown in Figure 8(B), where the mouse lung coefficient is the proportion of mouse lung tissue weight to mouse body weight. Compared with the BLM-IgG group, the mouse lung coefficient of the BLM-AIAB group was significantly lower.

[0057] (2) Hydroxyproline (HYP) represents the severity of pulmonary fibrosis. The hydroxyproline content per milligram of wet weight lung tissue in mice was determined using a hydroxyproline assay kit. The results are shown in Figure 8(C). Compared with the BLM-IgG group, the hydroxyproline content in the lung tissue of mice in the BLM-AIAB group was significantly lower.

[0058] (3) The lung tissue of mice is shown in Figure 8(D). It can be observed that the lung tissue damage area of ​​mice increased after bleomycin modeling, while the lung tissue damage area decreased significantly after AIAB treatment. Masson staining was performed on the mouse lung tissue. The results are shown in Figure 9. Blue represents collagen deposition. It can be observed that the collagen deposition area of ​​mouse lung tissue significantly increased after bleomycin modeling, while the collagen deposition area of ​​lung tissue significantly decreased after AIAB treatment.

[0059] (4) The activation level of fibroblasts (α-SMA) and the level of extracellular matrix (collagen 1 and fibronectin) in mouse lung tissue were labeled by Western blotting, with β-tubulin as an internal reference protein. The results are shown in Figure 10. After bleomycin modeling, the activation level of fibroblasts in mouse lung tissue was significantly increased, as shown by a significant increase in α-SMA expression. The expression of extracellular matrix was also significantly increased, as shown by a significant increase in collagen 1 and fibronectin expression. However, these fibrosis indicators were significantly inhibited after AIAB treatment.

[0060] (5) The extracellular matrix level in mouse lung tissue was labeled by RT-QPCR. The results are shown in Figure 11. After bleomycin modeling, the expression of extracellular matrix in mouse lung tissue was significantly increased, with a significant increase in the expression of collagen 1 (Col1a1, Col1a2) and fibronectin. However, these fibrosis indicators were significantly inhibited after AIAB treatment.

[0061] The above indicates that the Gremlin2 neutralizing antibody AIAB has a good inhibitory effect on pulmonary fibrosis in mice.

[0062] Example 4

[0063] Toxicity detection of Gremlin2 neutralizing antibodies.

[0064] As shown in Figure 12, the gross images of the heart, liver, spleen, and kidney of mice in the PBS-IgG group, BLM-IgG group, and BLM-AIAB group in Example 3 were observed. From the overall characterization, bleomycin administered via the airway did not cause damage to other internal organs except for the lung tissue, and the exogenous application of Gremlin2 neutralizing antibody did not cause toxic damage to these organs.

[0065] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A neutralizing antibody against pulmonary fibrosis, characterized in that, The anti-pulmonary fibrosis neutralizing antibody is composed of a heavy chain and a light chain. The heavy chain includes a heavy chain variable region, which includes heavy chain CDR1 as shown in SEQ ID NO: 1, heavy chain CDR2 as shown in SEQ ID NO: 2, and heavy chain CDR3 as shown in SEQ ID NO:

3. The light chain includes a light chain variable region, which includes light chain CDR1 as shown in SEQ ID NO: 4, light chain CDR2 as shown in SEQ ID NO: 5, and light chain CDR3 as shown in SEQ ID NO:

6.

2. The anti-pulmonary fibrosis neutralizing antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID NO: 8, and the amino acid sequence of the light chain is shown in SEQ ID NO:

9.

3. The anti-pulmonary fibrosis neutralizing antibody according to claim 1, characterized in that, The anti-pulmonary fibrosis neutralizing antibody targets the binding sites of Gremlin2 and TGFBR1.

4. The anti-pulmonary fibrosis neutralizing antibody according to claim 3, characterized in that, The Gremlin2 is a Gremlin2 dimer.

5. A nucleic acid molecule encoding the anti-pulmonary fibrosis neutralizing antibody as described in any one of claims 1 to 4.

6. A gene-derived product comprising the nucleic acid molecule of claim 5, characterized in that, This includes recombinant plasmids and / or recombinant cell lines.

7. A drug, characterized in that, The invention includes the anti-pulmonary fibrosis neutralizing antibody according to any one of claims 1 to 4, or the anti-pulmonary fibrosis neutralizing antibody prepared from the nucleic acid molecule according to claim 5 or the gene-derived product according to claim 6, for the treatment of pulmonary fibrosis caused by Gremlin2.

8. The medicament according to claim 7, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

9. The use of the anti-pulmonary fibrosis neutralizing antibody according to any one of claims 1 to 4, or the anti-pulmonary fibrosis neutralizing antibody prepared from the nucleic acid molecule according to claim 5 or the gene-derived product according to claim 6, in the preparation of a medicament for treating pulmonary fibrosis.

10. The use of the anti-pulmonary fibrosis neutralizing antibody according to any one of claims 1 to 4, or the anti-pulmonary fibrosis neutralizing antibody prepared from the nucleic acid molecule according to claim 5 or the gene-derived product according to claim 6, in the prevention and / or treatment of pulmonary fibrosis.

11. The application according to claim 9 or 10, characterized in that, The pulmonary fibrosis includes diseases caused by Gremlin2.

12. The application according to claim 9 or 10, characterized in that, The pulmonary fibrosis diseases mentioned include idiopathic pulmonary fibrosis.

13. The application according to claim 9, characterized in that, The anti-pulmonary fibrosis includes at least one of the following: reducing the hydroxyproline content in lung tissue, reducing the area of ​​lung tissue damage, reducing the area of ​​collagen deposition in lung tissue, reducing the activation level of fibroblasts in lung tissue, and inhibiting the expression level of extracellular matrix.

14. The application according to claim 13, characterized in that, The reduction in fibroblast activation levels in lung tissue includes a decrease in α-SMA expression levels.

15. The application according to claim 13, characterized in that, The inhibition of extracellular matrix expression levels includes inhibiting the expression levels of collagen 1 and / or fibronectin.

16. A method for treating pulmonary fibrosis, characterized in that, Includes the following steps: Inject the anti-pulmonary fibrosis neutralizing antibody according to any one of claims 1 to 4 or the drug according to claim 7 or 8.

17. A method for inhibiting fibroblast activation in vitro, characterized in that, Includes the following steps: Add the anti-pulmonary fibrosis neutralizing antibody of any one of claims 1 to 4 or the drug of claim 7 or 8 to a co-culture system of fibroblasts and stimulating factors for cell activation.

18. The method according to claim 17, characterized in that, The working concentration of the anti-pulmonary fibrosis neutralizing antibody is not less than 0.1 μg / mL.

19. The method according to claim 17, characterized in that, The working concentration of the anti-pulmonary fibrosis neutralizing antibody is 0.2 μg / mL.

20. The method according to any one of claims 13 to 15, characterized in that, The stimulating factor for cell activation includes TGF-β1, and the working concentration of TGF-β1 includes 5 ng / mL.

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