A novel anti-pulmonary fibrosis neutralizing antibody and drug

Abstract

The present invention discloses a novel anti-pulmonary fibrosis neutralizing antibody and a drug, belonging to the field of antibody drug preparation. Based on cutting-edge artificial intelligence technologies such as Alphafold and de novo protein design, the present invention designs a neutralizing antibody targeting the binding site of Gremlin2 and TGF-β receptor 1, prepares the antibody using genetic engineering techniques, inserts the gene fragment of the target antibody into a plasmid through in vitro recombination, and then transfers the edited plasmid into a cell line and performs 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 artificial intelligence method adopted by the present invention for designing the neutralizing antibody is efficient and reliable, the process for preparing the neutralizing antibody is simple, the neutralizing antibody has high homogeneity, is safe and effective, has a good anti-pulmonary fibrosis function in animal models, and has the potential for clinical treatment of pulmonary fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of antibody drug preparation, and relates to a novel anti-pulmonary fibrosis neutralizing antibody and drug. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a typical fibrotic and fatal disease.

[0003] The mechanism of continuous activation of fibroblasts may be the key to the rapid and irreversible fibrosis process. Transforming growth factor-β (TGF-β) signaling has been proven to play an important role in the process of fibroblast-induced activation. Research shows that activated TGFβ1 (transforming growth factor β1) can bind to TGFBR2 (TGF-β Receptor 2) on the cell membrane, and then recruit TGFBR1 (TGF-β Receptor1) inside the cell to the membrane. After TGFBR1 is phosphorylated, signal transduction occurs. Subsequently, this signal is terminated through ubiquitination and degradation of TGFBR1 on the membrane. The present invention discovers that Gremlin2 binds to TGFBR1, stabilizes the signal transduction of TGFBR1 on the membrane, and thus leads to the continuous activation of TGFβ signaling. Through basic research, it is found that Gremlin2 is a ligand of TGFBR1, and through basic research and in-depth analysis of artificial intelligence (AI), the key binding epitope is identified, and monoclonal antibodies against the key antigenic epitope are designed through an optimized AI algorithm.

[0004] The TGFβ superfamily protein Gremlin2 is a potential anti-fibrosis treatment target. Different from the extensive regulatory role of TGFβ signaling in various cells, Gremlin2 is an important protein that specifically targets and regulates the activation of fibroblasts. The clinical transformation of Gremlin2 neutralizing antibody may bring new breakthroughs to the treatment of pulmonary fibrosis patients. Summary of the Invention

[0005] The purpose of the present invention is to provide a novel anti-pulmonary fibrosis neutralizing antibody and drug in view of the deficiencies of the prior art.

[0006] To achieve the above object, the present invention provides the following technical solutions: In the first aspect, a neutralizing antibody against pulmonary fibrosis is provided, which is composed of a heavy chain and a light chain. The heavy chain includes a heavy chain variable region, and the heavy chain variable region includes a heavy chain CDR1 shown in SEQ ID NO.1, a heavy chain CDR2 shown in SEQ ID NO.2, and a heavy chain CDR3 shown in SEQ ID NO.3; the light chain includes a light chain variable region, and the light chain variable region includes a light chain CDR1 shown in SEQ ID NO.4, a light chain CDR2 shown in SEQ ID NO.5, and a light chain CDR3 shown in SEQ ID NO.6.

[0007] Further, the amino acid sequence of the heavy chain is as shown in SEQ ID NO.8, and the amino acid sequence of the light chain is as shown in SEQ ID NO.9.

[0008] Further, the neutralizing antibody against pulmonary fibrosis targets the binding site of Gremlin2 and TGFBR1.

[0009] In the second aspect, a drug is provided, which includes the above-mentioned neutralizing antibody against pulmonary fibrosis and is used for treating pulmonary fibrosis diseases caused by Gremlin2.

[0010] Further, the drug further includes: a pharmaceutically acceptable carrier.

[0011] The beneficial effects of the present invention are as follows: The present invention designs a neutralizing antibody targeting the Gremlin2-TGFBR1 binding site through computer deep learning, overcoming a large number of experimental animals in the preparation of traditional neutralizing antibodies, and can use computer analysis to screen effective antibody sequences at an early stage, avoiding the problem of a large number of sequences with unknown sequences and unguaranteed effects prepared by traditional methods, reducing the workload of subsequent cell and animal model verification by researchers, and the process is stable, safe and efficient, which is a new method for clinical development of therapeutic antibodies; the Gremlin2 neutralizing antibody designed by the present invention is applicable to subsequent clinical studies for the treatment of pulmonary fibrosis, has a significant anti-fibrosis effect, and the process is systematic, low-cost and easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is an AI simulation diagram of the binding of Gremlin2 and TGFBR1;

[0013] Figure 2 It is a specific sequence diagram of the binding of Gremlin2 and TGFBR1; among them, Figure 2 in (A) is a ribbon diagram (Cartoons) of the antigen-antibody docking site, Figure 2 in (B) is an all-atom diagram of the antigen-antibody docking site, Figure 2In (C) is a schematic diagram of the antigen epitope, where green represents the epitope of the antigen and white represents the remaining part of the antigen except the epitope;

[0014] Figure 3 is a schematic diagram of Gremlin2 dimer and TGFBR1;

[0015] Figure 4 is a schematic diagram of the AI simulation of the binding site of Gremlin2-TGFBR1 and the neutralizing antibody;

[0016] Figure 5 is a schematic diagram of the production of Gremlin2 neutralizing antibody; among them, Figure 5 in (A) is the detection result diagram of SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis), Figure 5 in (B) is the detection result diagram of SEC-HPLC (Size Exclusion Chromatography-High Performance Liquid Chromatography);

[0017] Figure 6 is the detection result diagram of LC-MS (Liquid Chromatography-Mass Spectrometry Detection); among them, Figure 6 in (A) is the LC-MS detection result diagram of the intact antibody, Figure 6 in (B) is the LC-MS detection result diagram of the light chain (LC), Figure 6 in (C) is the LC-MS detection result diagram of the heavy chain (HC);

[0018] Figure 7 is the detection diagram of the inhibitory effect of Gremlin2 neutralizing antibody on fibroblast activation in vitro; among them, Figure 7 in (A) is the detection result diagram of the binding ability of TGFBR1 and Gremlin2, Figure 7 in (B) is a schematic diagram of labeling the activation index α-SMA (red) of fibroblasts and DAPI (blue) labeling the nucleus by immunofluorescence staining, and the scale bar is 50 microns, Figure 7 in (C) is the statistical histogram of α-SMA fluorescence signal, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and ANOVA variance analysis is used for multiple group comparisons;

[0019] Figure 8 It is a graph showing the anti-pulmonary fibrosis function of the neutralizing antibody against Gremlin2; among them, Figure 8 in (A) is the graph of the body weight change of mice in each group, Figure 8 in (B) is the graph of the lung tissue coefficient of mice in each group, Figure 8 in (C) is the schematic diagram of the hydroxyproline 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 variance analysis is used for multiple group comparisons; Figure 8 in (D) is the gross graph of the lung tissue of mice in each group;

[0020] Figure 9 It is the schematic diagram of Masson staining of the lung tissue of mice in each group; among them, Figure 9 in (A) is the schematic diagram of Masson staining of the PBS-IgG group, Figure 9 in (B) is the schematic diagram of Masson staining of the BLM-IgG group, Figure 9 in (C) is the schematic diagram of Masson staining of the BLM-AIAB group, and the scale bar is 200 microns;

[0021] Figure 10 It is the result graph of labeling the activation level of fibroblasts (α-SMA) and the extracellular matrix level (collagen 1 and fibronectin) in the lung tissue of mice by WB method (western blotting);

[0022] Figure 11 It is the statistical result graph of labeling the lung tissue of mice by RT-QPCR method (real-time fluorescence quantitative polymerase chain reaction); among them, Figure 11 in (A) is the statistical result graph of labeling Col1a1 (α1 chain of collagen 1) in the lung tissue of mice by RT-QPCR method, Figure 11 in (B) is the statistical result graph of labeling Col1a2 (α2 chain of collagen 1) in the lung tissue of mice by RT-QPCR method, Figure 11 in (C) is the statistical result graph of labeling Fibronectin in the lung tissue of mice by RT-QPCR method;

[0023] Figure 12 It is the graph of the toxicity detection of the neutralizing antibody against Gremlin2; among them, Figure 12 in (A) is the gross graph of the heart of mice in each group, Figure 12 in (B) is the gross graph of the liver of mice in each group, Figure 12 in (C) is the gross graph of the spleen of mice in each group, Figure 12 in (D) is the gross graph of the kidney of mice in each group. Specific implementation mode

[0024] The antibody sequence targeting the binding site of Gremlin2 and TGFBR1 (or called the Gremlin2 target) designed by an optimized algorithm based on cutting-edge AI technologies such as AlphaFold protein structure prediction and de novo protein design. Antibodies are prepared using genetic engineering techniques. Through in vitro recombination, the gene fragment of the target antibody is inserted into a plasmid, and then the edited plasmid is transferred into a cell line and amplified and cultured in vitro to obtain transcription and translation of the gene fragment in vitro, thereby preparing the antibody. Based on this, a neutralizing antibody targeting Gremlin2 (hereinafter referred to as Gremlin2 neutralizing antibody or AIAB) is developed. After treatment with the Gremlin2 neutralizing antibody at the cellular and animal levels, the activation of fibroblasts and the degree of bleomycin-induced pulmonary fibrosis in mice are significantly reduced. The subsequent humanization modification of the Gremlin2 neutralizing antibody may be a new treatment method for patients with pulmonary fibrosis and potential patients.

[0025] Embodiments, features, and aspects of the present invention will be described in detail below with reference to the accompanying drawings, but this does not limit the present invention. Any embodiment extended based on the embodiments of the present invention, without creative efforts, belongs to the scope of protection of the present invention for all other embodiments obtained by those of ordinary skill in the art.

[0026] Example 1: Design, screening, and preparation of Gremlin2 neutralizing antibody.

[0027] The Gremlin2 neutralizing antibody screened in the present invention is mainly a class of bioactive recombinant proteins with high purity, about 1 - 3 mg / L, and is stored in PBS dissolved by ultrafiltration membrane concentration at -80 °C.

[0028] (1) Sequence design: The antibody sequence targeting the binding site of Gremlin2 and TGFBR1 (hereinafter referred to as the Gremlin2 target) designed by an optimized algorithm based on cutting-edge AI algorithms such as AlphaFold.

[0029] Using the improved alpha-fold2 algorithm to simulate the binding prediction of Gremlin2 dimer (blue and pink) and the extracellular segment of TGFBR1, as Figure 1 shown. Since the Gremlin2 dimer structure is composed of two identical monomers, the extracellular segment of TGFBR1 binds to two positions of the Gremlin2 dimer simultaneously, and both are highlighted in green boxes in the figure.

[0030] According to the sequence characteristics of the two proteins themselves, the present invention uses computer simulation technology to more clearly label the sequences of their binding, as Figure 2As shown, its main binding sequence on Gremlin2 is as shown in SEQ ID NO.7, and 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.

[0031] (2) Screening sequence: Calculate the binding free energy between Gremlin2 and the designed sequence through computer deep learning, and calculate the stability of the neutralizing antibody in Gremlin2 through spatial allostery and other methods, so as to screen out the antibody sequence with high affinity for Gremlin2. The amino acid sequence of the heavy chain of the antibody finally screened in the present invention is as follows:

[0032] EVQLVESGGGLVQPGGSLRLSCAASVSGDAPPAIGKYYMHWVRQAPGKGLEWVGLIAGGEDGITIYDPKFQDRATISADNSKNTAYLQMNSLRAEDTAVYYCARAPFGGAAYIEWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO.8).

[0033] The amino acid sequence of CDR1 in the heavy chain variable region is: VSGDAPPAIGKY (SEQ ID NO.1); The amino acid sequence of CDR2 is: AGGEDGI (SEQ ID NO.2); The amino acid sequence of CDR3 is: APFGGAAYIE (SEQ ID NO.3).

[0034] The amino acid sequence of the light chain is as follows:

[0035] DIQMTQSPSSLSASVGDRVTITCSGGSGADIAWYQQKPGKAPKVLIYNGSLPPGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCAACTRKLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO.9).

[0036] The amino acid sequence of the CDR1 of the light chain variable region is: SGGSGADIA (SEQ ID NO.4); the amino acid sequence of CDR2 is: NGSLPP (SEQ ID NO.5); the amino acid sequence of CDR3 is: AACTRKLT (SEQ ID NO.6).

[0037] As Figure 3 shown is a schematic diagram of Gremlin2 dimer and TGFBR1 (the pink and blue respectively represent the simulation diagrams of TGFBR1 receptors binding to the two monomers of Gremlin2 dimer), Figure 4 is an AI simulation schematic diagram of the binding site of Gremlin2-TGFBR1 and the neutralizing antibody. Calculate the binding free energy of the above sequences binding to the Gremlin2-TGFBR1 model. The smaller the value, the more stable the predicted sequence binds. The binding free energy in the original state is -7.13 kCal / mol, and the binding sites on both sides with the same sequence have the same binding probability.

[0038] (3) Synthesize the antibody: Use genetic engineering technology to insert the gene fragment of the target antibody into a plasmid, and then transfer the edited plasmid into a cell line; amplify and culture the edited cell line in vitro; transcribe and translate the target gene in vitro to prepare the recombinant protein.

[0039] (4) Crude separation: Collect the cells, lyse the cells with a lysis buffer containing protease inhibitors at a low temperature environment (4°C), and break the cells by repeated freezing and thawing to release the recombinant protein.

[0040] (5) Concentration and purification: Adopt the ultrafiltration membrane concentration method, filter out liquids such as water through a microporous cellulose membrane under high pressure, and the recombinant protein remains on the membrane. After eluting with sterile 1xPBS (phosphate buffer), measure the concentration of the recombinant protein, and after aliquoting, maintain the stock solution concentration at 1-3 mg / L and store it at -80°C.

[0041] Use SDS-PAGE to detect the protein size of the antibody. The concentration range of the polyacrylamide gel is 4-12% (w / v), as Figure 5 shown in (A) below. The M lane (Marker) represents the standard example of the molecular weight size, the NR (Nonreduced) lane represents the size of the undegraded (heavy and light chains not separated) antibody (143.3 kDa), and the R lane (Reduced) represents the size of the antibody with separated heavy and light chains (the light chain is 22-30 kDa, and the heavy chain is about 53 kDa). The electrophoresis diagram indicates that the antibody is a dimer.

[0042] The purity of the protein was analyzed by SEC-HPLC (SEC-300 chromatographic column). SEC-HPLC is mainly used for analyzing and separating substances with different molecular weights. As shown in Figure 5 item (B) in Figure 5 and Table 1, it shows that this antibody is a single peak, indicating that the antibody exists in a homogeneous form. Among them, the injection volume is 20 μg, the flow rate is 0.35 mL / min, and ND means not detected.

[0043] Table 1: SEC-HPLC test result table

[0044]

[0045] The LC-MS mass spectrum is as shown in Figure 6 and Table 2, showing that the intact antibody (Intact) without degradation is mainly distributed at a size of 143 kDa, the heavy chain (HC) is mainly distributed at a size of 50 kDa, and the light chain (LC) is at a size of 22 kDa. Consistent with the above results, it again indicates that this antibody exists in the form of a dimer and has good homogeneity.

[0046] Table 2: LC-MS test result table

[0047]

[0048] Example 2: Verification of the inhibitory effect of Gremlin2 neutralizing antibody on the activation of fibroblasts in vitro.

[0049] TGFBR1 (TβRⅠ) and Gremlin2 proteins were overexpressed in 293T tool cells, each with tags Flag and His-Myc respectively. After 72 hours of successful overexpression, the 293T cells were lysed to obtain all the proteins in the cells. Subsequently, the binding level between Gremlin2 and TGFBR1 was detected by CO-IP experiment (Co-Immunoprecipitation). The magnetic beads were combined with Myc antibody, and according to the principle of antigen-antibody binding, the magnetic beads could successfully bind to the Gremlin2 protein with Myc tag. After adding different concentrations of Gremlin2 neutralizing antibody AIAB (concentrations were 0, 0.1, 0.2, 0.3 μg / mL respectively) to the protein lysate, the binding ability between TGFBR1 and Gremlin2 was detected. As shown in Figure 7 item (A) in Figure 7 , Input represents that both TGFBR1 and Gremlin2 proteins are present in the original cell lysate. Detection with Flag and His tags respectively represents TGFBR1 and Gremlin2 proteins, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is the internal reference protein. The IP group represents that with the increase of antibody addition, the binding between TGFBR1 and Gremlin2 is gradually inhibited.

[0050] 5 ng / mL of TGF-β1 stimulator was added to lung fibroblasts, and different concentrations of AI neutralizing antibody AIAB (concentrations were 0, 0.1, 0.2 μg / mL) were added. After that, the activation index α-SMA (red) of fibroblasts was labeled by immunofluorescence staining, and the nucleus was labeled by DAPI (blue). As Figure 7 shown in (B) and (C) of

[0051] Example 3: Verification of the anti-fibrotic effect of Gremlin2 neutralizing antibody in an animal model of pulmonary fibrosis.

[0052] Eight-week-old C57BL / 6J male mice were selected for the study. A mouse model of pulmonary fibrosis was constructed by intratracheal instillation of PBS or bleomycin (BLM). On the 8th, 11th, 14th, and 17th days, 50 μg of Gremlin2 neutralizing antibody AIAB or an equal volume of IgG (immunoglobulin G) was intraperitoneally injected. The groups were: PBS-IgG group, BLM-IgG group, BLM-AIAB group.

[0053] (1) The body weight changes of the mice were recorded, and a body weight change graph was plotted. As Figure 8 shown in (A) of Figure 8 there was no significant difference between the PBS-IgG group and the BLM-AIAB group, and the body weight of the mice in the BLM-IgG group was significantly lower. The mouse lung tissue coefficient graph is as

[0054] shown in (B) of Figure 8 where the mouse lung coefficient is the ratio of the weight of the mouse lung tissue to the body weight of the mouse. Compared with the BLM-IgG group, the mouse lung coefficient in the BLM-AIAB group was significantly lower.

[0055] (2) Hydroxyproline (HYP) represents the severity of pulmonary fibrosis. The content of hydroxyproline in each milligram of wet lung tissue of the mice was measured by a hydroxyproline detection kit. As Figure 8 shown in (C) of

[0055] compared with the BLM-IgG group, the content of hydroxyproline in the mouse lung tissue in the BLM-AIAB group was significantly lower. Figure 8 (3) The lung tissue map of the mice is as Figure 8 shown in (D) of Figure 9 It can be observed that the damaged area of the mouse lung tissue increased after bleomycin modeling, while the damaged area of the lung tissue decreased significantly after AIAB treatment. Masson staining was performed on the mouse lung tissue. As Figure 9 shown, blue represents the collagen deposition situation. It can be observed that the collagen deposition area in the mouse lung tissue increased significantly after bleomycin modeling, while the collagen deposition area in the lung tissue decreased significantly after AIAB treatment.

[0056] (4)The activation level of fibroblasts (α-SMA) and the level of extracellular matrix (collagen 1 and fibronectin) in mouse lung tissues were labeled by Western blotting (WB), with β-tubulin as the internal reference protein. As Figure 10 shown, after bleomycin-induced modeling, the activation level of fibroblasts in mouse lung tissues increased significantly, manifested as a significant increase in α-SMA expression, and the expression of extracellular matrix also increased significantly, manifested as a significant increase in the expression of collagen 1 (Collagen1) and fibronectin (Fibronectin). After treatment with AIAB, these fibrosis indicators were significantly inhibited.

[0057] (5)The level of extracellular matrix in mouse lung tissues was labeled by RT-QPCR. As Figure 11 shown, after bleomycin-induced modeling, the expression of extracellular matrix in mouse lung tissues increased significantly, manifested as a significant increase in the expression of collagen 1 (Col1a1, Col1a2) and fibronectin (Fibronectin). After treatment with AIAB, these fibrosis indicators were significantly inhibited.

[0058] The above results indicate that the Gremlin2 neutralizing antibody AIAB has a good inhibitory effect on mouse pulmonary fibrosis.

[0059] Example 4: Toxicity detection of the Gremlin2 neutralizing antibody.

[0060] As Figure 12 shown, the gross images of the hearts, livers, spleens, and kidneys of the mice in the PBS-IgG group, BLM-IgG group, and BLM-AIAB group in Example 3 were observed. From the overall characterization, intratracheal administration of bleomycin did not cause damage to other visceral organs except the lung tissues, and exogenous application of the Gremlin2 neutralizing antibody did not cause toxic damage to these organs.

[0061] It should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made without creative efforts shall be included within the protection scope of the present invention.

Claims

1. An anti-pulmonary fibrosis neutralizing antibody, 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, the heavy chain variable region includes a heavy chain CDR1 as shown in SEQ ID NO.1, a heavy chain CDR2 as shown in SEQ ID NO.2, and a heavy chain CDR3 as shown in SEQ ID NO.3; the light chain includes a light chain variable region, the light chain variable region includes a light chain CDR1 as shown in SEQ ID NO.4, a light chain CDR2 as shown in SEQ ID NO.5, and a 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. A drug, characterized in that The anti-pulmonary fibrosis neutralizing antibody according to claim 1 is used for treating pulmonary fibrosis caused by Gremlin2.

4. The drug according to claim 3, characterized in that The medicament further comprises a pharmaceutically acceptable carrier.

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