An antibody that binds to IL-11, an antigen-binding fragment thereof, and uses thereof

Abstract

The present invention relates to the field of biomedicine, and specifically provides an antibody or an antigen-binding fragment thereof that binds to IL-11, including three heavy-chain complementarity-determining regions represented by HCDR1, HCDR2, and HCDR3 respectively, and three light-chain complementarity-determining regions represented by LCDR1, LCDR2, and LCDR3 respectively. The antibody or its antigen-binding fragment is selected from A-I, A-II, A-III, or A-IV. The antibody or its antigen-binding fragment provided by the present invention has a high binding ability to the IL-11 antigen, can block the binding of the IL-11 antigen to its receptor, and further effectively inhibit the profibrotic effect of IL-11, inhibit or prevent the production or proliferation of fibroblasts, and can be effectively used for treating or preventing human fibrotic diseases, inflammation, cancer, or autoimmune diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to an antibody that binds to IL-11, an antigen-binding fragment thereof, and applications thereof. Background Art

[0002] Fibrosis is a repair response after tissue injury to protect the relative integrity of tissues and organs. A variety of harmful stimuli (including toxins, infectious pathogens, autoimmune responses, and mechanical stress) can induce fibrotic responses in cells. In response to tissue injury, fibroblasts from multiple sources (including resident fibroblasts, mesenchymal cells, circulating fibroblasts, and transdifferentiation of other cell types) can initiate wound healing responses by remodeling the extracellular environment to restore tissue integrity and promote the replacement of parenchymal cells. However, persistent injury and damage can lead to the dysregulation of this process, resulting in the pathological over-deposition of extracellular matrix (ECM) proteins (including collagen, laminin, and fibronectin), along with an upregulation of fibroblast activity, creating a chronic inflammatory environment with macrophage and immune cell infiltration. Cytokines and growth factors are released in large amounts, including members of the transforming growth factor-β (TGF-β) family and Wingless / Int-1 (Wnt1) protein, which are the main effectors in the fibrotic process. TGF-β and Wnt1 bind to their stem cell surface receptors and initiate downstream signal transduction, leading to an upregulation of target gene expression, and their functions further enhance fibroblast differentiation and the production and secretion of ECM proteins.

[0003] Worldwide, tissue fibrosis is the main cause of disability and death in many diseases. The main pathological change is the increase of fibrous connective tissue in organ tissues, the decrease of parenchymal cells, and continuous progression can lead to the destruction of organ structure and the decline of function, and even failure, seriously threatening human health and life. According to relevant statistical data in the United States, nearly 45% of the patients who died from various diseases in this country can be attributed to tissue fibroplasia diseases. Currently, about 25% of the people in the world suffer from non-alcoholic fatty liver disease. Although non-alcoholic fatty liver disease can improve, untimely treatment will lead to its deterioration into non-alcoholic hepatitis. Non-alcoholic hepatitis is characterized by liver inflammation, hepatocyte death, and liver fibrosis, and ultimately leads to the occurrence of cirrhosis and liver cancer. Pulmonary fibrosis is the end-stage change of a large category of lung diseases characterized by fibroblast proliferation, massive extracellular matrix aggregation, accompanied by inflammatory damage and tissue structure destruction, that is, the normal alveolar tissue is damaged and then undergoes abnormal repair, resulting in abnormal structure (scar formation). The incidence and mortality of pulmonary fibrosis increase year by year. Pulmonary fibrosis is mostly sporadic, with an incidence of about 3-5 / 100,000, accounting for about 65% of all interstitial lung diseases. The average survival period after diagnosis is only 2.8 years, and the mortality rate is higher than that of most tumors, so it is called a "tumor-like disease". Therefore, the research and development of drugs for treating anti-fibrosis diseases is particularly urgent.

[0004] Mature IL-11 (Interleukin-11) is a polypeptide consisting of 178 amino acids with a molecular weight of approximately 23KD. Studies on the three-dimensional structure of IL-11 have shown that it is a four-helix bundle structure composed of 4 α-helices and loop rings connecting the α-helices. IL-11 is a pleiotropic cytokine and a member of the IL-6 cytokine family, sharing the same signal transduction receptor subunit GP130. This family plays a crucial role in the occurrence, development, and metastasis of tumors. The production of IL-11 is induced by known major profibrotic factors, including TGFβ, FGF, PDGF, CTGF, or IL-13, and local overexpression of IL-11 leads to local tissue fibrosis. The activation of the IL-11 signaling pathway depends on the binding of IL-11 to cell surface receptors. The receptor of IL-11 consists of two glycoprotein chains, IL-11Ra and GPl30. IL-11Ra has the ability to bind ligands. IL-11 first binds to IL-11Ra with low affinity to form an IL-11 / IL-11Ra heterodimer, which then binds to GP130 with high affinity to form a heterotrimeric protein. The IL-11 / IL-llRa / GP130 heterotrimer homodimerizes to form a hexamer, which further phosphorylates and activates the downstream STAT signaling pathway or MAPK cascade. Finally, IL-11 transmits signals into the cell through the GP130 signaling chain, enabling the cell to obtain signals for proliferation and activation. It has been found through research that blocking this signaling pathway can be an effective treatment method for various tumors, chronic fibrosis, and inflammatory diseases. Therefore, the development of antibodies that bind to IL-11 and their antigen-binding fragments has important clinical significance. Summary of the Invention

[0005] In order to meet the needs of patients with fibrotic diseases at home and abroad, the present invention has screened antibodies and their antigen-binding fragments that can specifically bind to IL-11 and block the binding of IL-11 to its receptor.

[0006] The specific technical solution of the present invention is as follows:

[0007] The present invention provides an antibody that binds to IL-11 or its antigen-binding fragment, including 3 heavy-chain complementarity-determining regions represented by HCDR1, HCDR2, and HCDR3 respectively, and 3 light-chain complementarity-determining regions represented by LCDR1, LCDR2, and LCDR3 respectively. The antibody or its antigen-binding fragment is selected from any one of the following:

[0008] A-I: The amino acid sequence of the heavy chain complementarity determining region HCDR1 is as shown in SEQ ID No:1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is as shown in SEQ ID No:2, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is as shown in SEQ ID No:3, the amino acid sequence of the light chain complementarity determining region LCDR1 is as shown in SEQ ID No:4, the amino acid sequence of the light chain complementarity determining region LCDR2 is as shown in SEQ ID No:5, and the amino acid sequence of the light chain complementarity determining region LCDR3 is as shown in SEQ ID No:6;

[0009] A-II: The amino acid sequence of the heavy chain complementarity determining region HCDR1 is as shown in SEQ ID No:1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is as shown in SEQ ID No:2, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is as shown in SEQ ID No:3, the amino acid sequence of the light chain complementarity determining region LCDR1 is as shown in SEQ ID No:4, the amino acid sequence of the light chain complementarity determining region LCDR2 is as shown in SEQ ID No:5, and the amino acid sequence of the light chain complementarity determining region LCDR3 is as shown in SEQ ID No:7;

[0010] A-III: The amino acid sequence of the heavy chain complementarity determining region HCDR1 is as shown in SEQ ID No:8, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is as shown in SEQ ID No:9, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is as shown in SEQ ID No:10, the amino acid sequence of the light chain complementarity determining region LCDR1 is as shown in SEQ ID No:11, the amino acid sequence of the light chain complementarity determining region LCDR2 is as shown in SEQ ID No:12, and the amino acid sequence of the light chain complementarity determining region LCDR3 is as shown in SEQ IDNo:13;

[0011] A-IV: The amino acid sequence of the heavy chain complementarity determining region HCDR1 is as shown in SEQ ID No:1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is as shown in SEQ ID No:14, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is as shown in SEQ ID No:15, the amino acid sequence of the light chain complementarity determining region LCDR1 is as shown in SEQ ID No:4, the amino acid sequence of the light chain complementarity determining region LCDR2 is as shown in SEQ ID No:5, and the amino acid sequence of the light chain complementarity determining region LCDR3 is as shown in SEQ ID No:6.

[0012] The above-mentioned antibodies or antigen-binding fragments thereof provided by the present invention can all specifically bind to IL-11, block the binding of IL-11 antigen to its receptor, inhibit IL-11-mediated signal transduction, and the above-mentioned antibodies provided by the present invention have good biological activity.

[0013] The present invention further includes a heavy chain variable region and a light chain variable region, and the antibody or its antigen-binding fragment is selected from any one of the following:

[0014] MA-I: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 16, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 17;

[0015] MA-II: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 16, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 18;

[0016] MA-III: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 19, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 20;

[0017] MA-IV: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 21, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 17.

[0018] The present invention further includes a heavy chain constant region and a light chain constant region. The amino acid sequence of the heavy chain constant region is one of those shown in SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25 or SEQ ID No: 26; the amino acid sequence of the light chain constant region is as shown in SEQ ID No: 22.

[0019] Furthermore, the antibody or its antigen-binding fragment is a chimeric antibody molecule, and the chimeric antibody molecule further includes a human antibody constant region.

[0020] The present invention further includes a heavy chain variable region and a light chain variable region, and the antibody or its antigen-binding fragment is selected from any one of the following:

[0021] HA-I-A: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 32;

[0022] HA-I-B: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 33, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 34;

[0023] HA-I-C: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 35;

[0024] HA-I-D: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 36, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 37;

[0025] HA-II-A: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 38;

[0026] HA-II-B: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 39;

[0027] HA-II-C: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 33, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 39;

[0028] HA-II-D: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 36, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 40.

[0029] In the present invention, the antibody or its antigen-binding fragment further comprises a human antibody constant region.

[0030] Furthermore, the human antibody constant region comprises a human heavy chain antibody constant region and a human light chain antibody constant region. The amino acid sequence of the human heavy chain antibody constant region is one of those shown in SEQ ID No: 27, SEQ ID No: 28 or SEQ ID No: 29; the amino acid sequence of the human light chain antibody constant region is as shown in SEQ ID No: 30.

[0031] Furthermore, the antibody or its antigen-binding fragment is one or a combination of several of Fab, F(ab)2, Fv or ScFv.

[0032] The present invention also provides a nucleic acid molecule encoding the antibody or its antigen-binding fragment that binds to IL-11.

[0033] The present invention also provides a recombinant DNA expression vector comprising the nucleic acid molecule.

[0034] The present invention also provides a host cell transfected with the recombinant DNA expression vector described above. The host cell includes prokaryotic cells, yeast cells, insect cells or mammalian cells;

[0035] Preferably, the host cell is a mammalian cell, and the mammalian cell is a HEK293 cell, a CHO cell or an NS0 cell.

[0036] The present invention also provides a drug, which contains the antibody that binds to IL-11 or its antigen-binding fragment.

[0037] The present invention also provides a detection reagent, which is used to detect the antibody that binds to IL-11 or its antigen-binding fragment.

[0038] The present invention also provides a combined medication composition, which includes the antibody that binds to IL-11 or its antigen-binding fragment, and also includes a second composition, and the second composition is selected from the antibody that binds to PD-1, PD-L1, VEGF, VEGFR, TNF-α or TSLP or its antigen-binding fragment.

[0039] The present invention further provides the application of the antibody that binds to IL-11 or its antigen-binding fragment in the preparation of drugs for treating or preventing human fibrotic diseases, inflammation, cancer or autoimmune diseases.

[0040] Preferably, the fibrotic diseases include fibrosis of the heart, liver, kidney, lung, gallbladder, bladder, stomach, bone marrow, penis, breast, blood vessel, eye, pancreas, spleen, brain, intestine, muscle or skin.

[0041] Preferably, the inflammation includes hepatitis, myocarditis, nephritis, pneumonia, cholecystitis, cystitis, gastritis, osteomyelitis, prostatitis, mastitis, pancreatitis, enteritis, arthritis, polymyositis, dermatomyositis or dermatitis.

[0042] Preferably, the cancer includes leukemia, lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer or bladder cancer.

[0043] Preferably, the autoimmune diseases include psoriasis, Crohn's disease, primary biliary cirrhosis, systemic lupus erythematosus or multiple sclerosis.

[0044] The beneficial effects of the present invention are as follows: The antibody or its antigen-binding fragment provided by the present invention has a high binding ability to the IL-11 antigen, can block the binding of the IL-11 antigen to its receptor, and thus effectively inhibit the profibrotic effect of IL-11, inhibit or prevent the generation or proliferation of fibroblasts, and can be effectively used for the treatment or prevention of human fibrotic diseases, inflammation, cancer or autoimmune diseases. Among them, fibrotic diseases include but are not limited to fibrosis of the heart, liver, kidney, lung, gallbladder, bladder, stomach, bone marrow, penis, breast, blood vessels, eyes, pancreas, spleen, brain, intestine, muscle or skin; inflammation includes but is not limited to hepatitis, myocarditis, nephritis, pneumonia, cholecystitis, cystitis, gastritis, osteomyelitis, prostatitis, mastitis, pancreatitis, enteritis, arthritis, polymyositis, dermatomyositis or dermatitis; cancer includes but is not limited to leukemia, lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer or bladder cancer; autoimmune diseases include but are not limited to psoriasis, Crohn's disease, primary biliary cirrhosis, systemic lupus erythematosus or multiple sclerosis. Brief Description of the Drawings

[0045] Figure 1 It is the plasmid map of the pScFv-Disb-HS vector in Example 2 of the present invention;

[0046] Figure 2 It is the comparison chart of the affinity of the gradient-diluted ELISA anti-IL-11 phage monoclonal antibody in Example 3 of the present invention;

[0047] Figure 3 It is the map of the vector pTSE in Example 5 of the present invention;

[0048] Figure 4 It is the denaturing polyacrylamide gel electrophoresis map of the murine antibody molecule in Example 5 of the present invention;

[0049] Figure 5 It is the comparison chart of the binding ability of the murine antibody molecule to IL-11 in Example 6 of the present invention;

[0050] Figure 6 It is the comparison chart of the competitive inhibition experiment of the murine antibody with the IL-11 receptor protein IL-11RA in Example 7 of the present invention;

[0051] Figure 7 It is the comparison chart of the murine antibody inhibiting the binding of IL-11 to the IL-11RA receptor on the surface of BaF / 3-IL-11RA cells in Example 8 of the present invention;

[0052] Figure 8 It is the comparison chart of the murine antibody inhibiting the secretion of TIMP-1 by the embryonic lung fibroblast MRC-5 in Example 9 of the present invention;

[0053] Figure 9 This is the denaturing polyacrylamide gel electrophoresis pattern of the humanized antibody molecule in Example 14 of the present invention;

[0054] Figure 10 This is the comparison chart of the binding ability between the humanized antibody molecule and IL-11 in Example 18 of the present invention;

[0055] Figure 11 This is the comparison chart of the inhibition of the binding between the humanized antibody molecule and the IL-11RA receptor on the surface of BaF / 3-IL-11RA cells by the humanized antibody molecule in Example 19 of the present invention;

[0056] Figure 12 This is the comparison chart of the inhibition of the binding between the humanized antibody molecule and the GP130 receptor on the surface of BaF / 3-GP130 cells by the humanized antibody molecule in Example 20 of the present invention;

[0057] Figure 13 This is the comparison chart of the biological activity detection (reporter gene) of the humanized antibody molecule in Example 21 of the present invention;

[0058] Figure 14 This is the comparison chart of the inhibition of the secretion of TIMP-1 by the humanized antibody molecule in MRC-5, embryonic lung fibroblasts in Example 22 of the present invention;

[0059] Figure 15 This is the comparison chart of the cross-binding experiment between the humanized antibody molecule and IL-11 of different species in Example 23 of the present invention;

[0060] Figure 16 This is the bar chart of the change in the ratio of lung to body weight in the mouse pulmonary fibrosis model in Example 24 of the present invention;

[0061] Figure 17 This is the hematoxylin and eosin (HE) staining and Masson staining diagrams of the lung tissue sections in the mouse pulmonary fibrosis model in Example 24 of the present invention;

[0062] Figure 18 This is the bar chart of the change in the ratio of heart to body weight in the mouse cardiac fibrosis model in Example 25 of the present invention;

[0063] Figure 19 This is the hematoxylin and eosin (HE) staining and Masson staining diagrams of the heart tissue sections in the mouse cardiac fibrosis model in Example 25 of the present invention;

[0064] Figure 20 This is the bar chart of the urinary protein content in the kidney in the mouse renal fibrosis model in Example 26 of the present invention;

[0065] Figure 21It is the hematoxylin-eosin (HE) staining and Masson staining diagrams of kidney tissue sections in the mouse kidney fibrosis model of Example 26 of the present invention;

[0066] Figure 22 It is the bar graph of the liver weight change in the mouse liver fibrosis model of Example 27 of the present invention;

[0067] Figure 23 It is the bar graph of the changes in the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of mice in the mouse liver fibrosis model of Example 27 of the present invention;

[0068] Figure 24 It is the hematoxylin-eosin (HE) staining and Masson staining diagrams of liver tissue sections in the mouse liver fibrosis model of Example 27 of the present invention;

[0069] Figure 25 It is the evaluation diagram of the thermal stability of the anti-IL-11 monoclonal antibody HA-I-A in Example 28 of the present invention. Detailed implementation manners

[0070] To make the present invention easier to understand, before describing the embodiments, the following explanations are made for some technical and scientific terms of the present invention:

[0071] The term "antibody" used herein includes whole antibodies and any antigen-binding fragment thereof. Antibodies include murine antibodies, humanized antibodies, bispecific antibodies or chimeric antibodies. Antibodies can also be fragments such as Fab, F(ab)2, Fv or ScFv (single-chain antibody). Antibodies can be naturally occurring antibodies or antibodies modified (such as by mutation, deletion, substitution, etc.), as long as they show binding to the relevant target molecule. The "antibody" herein includes its fragments and derivatives, including synthetic antibodies and fragments. As used herein, an antibody is a polypeptide capable of specifically binding to a relevant target molecule (i.e., the antigen to which the antibody is specific).

[0072] The terms "variable region" and "constant region" used herein refer to the sequence regions near the N-terminus of the heavy and light chains of an antibody as the variable region (V region), and the remaining amino acid sequences near the C-terminus are relatively stable, which are the constant region (C region). The variable region includes 3 complementarity-determining regions (CDRs) and 4 framework regions (FRs). Each light chain variable region and heavy chain variable region are composed of 3 CDR regions and 4 FR regions. The 3 CDR regions of the heavy chain are represented by HCDR1, HCDR2 and HCDR3 respectively, and the 3 CDR regions of the light chain are represented by LCDR1, LCDR2 and LCDR3 respectively.

[0073] The term "murine antibody molecule" used herein refers to an antibody obtained by immunizing a mouse with a human IL-11 antigen.

[0074] As used herein, the term "chimeric antibody molecule" refers to an antibody formed by fusing the variable regions of a murine antibody with the constant regions of a human antibody, which can reduce the immune response induced by murine antibodies in the human body. Chimeric antibodies are produced using DNA recombination technology by inserting the variable region genes of the light and heavy chains of murine monoclonal antibodies into an expression vector containing human antibody constant regions. In the antibody molecules thus expressed, the variable regions of the light and heavy chains are murine, while the constant regions are human. Approximately two-thirds of the entire antibody molecule is human. Such antibodies produced have reduced immunogenicity of murine antibodies while retaining the ability of the parental antibody to specifically bind to antigens.

[0075] As used herein, the term "humanized antibody molecule" refers to a molecule in which the CDRs of a murine monoclonal antibody are transplanted into the variable regions of a human antibody, replacing the human antibody CDRs, enabling the human antibody to acquire the antigen-binding specificity of the murine monoclonal antibody while reducing its heterogenicity.

[0076] The term "CHO cell" refers to Chinese hamster ovary cell; the term "HEK293E cell" refers to human embryonic kidney 293E cell, and the term "NS0 cell" refers to murine NS0 thymoma cell.

[0077] In this specification, "IL-11" refers to IL-11 from any species and includes isotypes, fragments, variants, or homologs of IL-11 from any species. As used herein, a "fragment", "variant", or "homolog" of a protein can optionally be characterized as having a sequence identity of at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of a reference protein. In some embodiments, fragments, variants, isotypes, and homologs of the reference protein can be characterized as being capable of having the functions of the reference protein.

[0078] The present invention will be further described in detail below in conjunction with the following examples.

[0079] Example 1

[0080] Example 1 of the present invention provides an antibody or an antigen-binding fragment thereof that binds to IL-11, including three heavy-chain complementarity-determining regions represented by HCDR1, HCDR2, and HCDR3 respectively, and three light-chain complementarity-determining regions represented by LCDR1, LCDR2, and LCDR3 respectively. The antibody or its antigen-binding fragment is selected from any one of the following.

[0081]

[0082] Example 2 Screening of Murine Antibody Molecules

[0083] In the present invention, mice were immunized with IL-11 antigen (in subsequent experiments, IL-11 protein, IL-11-Fc antigen, and IL-11-mFc ligand protein were all human IL-11), the immunization method was optimized, and a phage display library was created. The construction, screening, and identification of the specific phage display library are as follows:

[0084] Step 1: Immunization of Mice with IL-11 Antigen

[0085] 1. Experimental Animals:

[0086] Species and Strain: BALB / c, female, mice;

[0087] Body Weight: 18 - 20 g;

[0088] Provider of Experimental Animals: Beijing Huafukang Biotechnology Co., Ltd.

[0089] 2. Immunization: The mice were immunized with human IL-11 (synthetic gene from Nanjing Genscript Biotechnology Co., Ltd., and the vector was constructed and expressed and purified by our company).

[0090] Step 2: Construction of Phage Antibody Library

[0091] Spleen cells of mice with higher titers were taken, and total RNA in the spleen cells of mice was extracted using Trizol reagent (purchased from Ambion, catalog number: 15596026). cDNA was obtained by RT-PCR. Using cDNA as a template, degenerate primers (the degenerate primers used refer to the reference: Journal of Immunological Methods 233 (2000) 167 - 177) were used for PCR amplification to obtain the immunized mouse heavy chain variable region gene library (VH) and light chain variable region gene library (VL). The pScFv-Disb-HS vector was constructed by modifying the vector pComb3 vector (purchased from the China Plasmid Vector Strain Cell Line Gene Preservation Center) using a series of gene cloning methods for the construction and expression of phage single-chain antibody libraries. The modified vector was named pScFv-Disb-HS vector, and its plasmid map is as Figure 1 shown, and based on this vector, a murine immunized phage antibody library was constructed. The light and heavy chain variable region gene libraries were double digested and ligated to the vector pScFv-Disb-HS that had been digested in the same step-by-step manner to construct the pScFv-Disb-HS-VH-VL gene library.

[0092] Step 3: Coat the immunization tube with IL-11 as the antigen. The antigen coating amount is 5 μg / 500 μL / tube, and coat overnight at 4°C. Then, block the immunization tube and the immunized phage antibody library with 4% skim milk / PBST respectively, and block at room temperature for 1 h. Add the blocked immunized phage antibody library into the immunization tube for antigen-antibody binding. The input amount of phage is about 10 9 ~10 12 individuals. After reacting at room temperature for 1 h, use PBST-PBS to wash away the unbound phages, elute with 0.1 M Glycine-HCl at pH 2.2, and finally neutralize the eluted phage antibody solution with 1.5 M Tris-HCl at pH 8.8 to about pH 7.0.

[0093] Step 4: Infect the neutralized phages above with 10 ml of TG1 bacterial liquid grown to the logarithmic phase, let it stand in a 37°C incubator for 30 min, take out part of the bacterial liquid for gradient dilution, and coat it on a 2YTAG plate for calculating the phage output. Centrifuge the remaining bacterial liquid to discard the supernatant, resuspend the bacterial cell precipitate in a small amount of medium, aspirate it and coat it on a large 2YTAG plate to prepare for the next round of screening.

[0094] Step 5: Scrape the bacteria coated on the plate after the above infection from the large plate, inoculate them into 2YTAG liquid medium, shake until the logarithmic phase, then add M13KO7 helper phage for superinfection, and culture overnight at 28°C and 220 rpm to prepare phages. Purify the phages by PEG / NaCl precipitation for the next round of screening. A total of one round of phage library enrichment screening is carried out.

[0095] Step 6: Screening of IL-11 phage single-chain antibody positive clones: After one round of screening, pick well-separated monoclonal colonies, inoculate them into a 96-well deep-well plate containing 2YTAG liquid medium, and culture at 37°C and 220 rpm until the logarithmic growth phase. Add about 10 10 helper phages M13KO7 to each well, and let it stand and infect at 37°C for 30 min. Centrifuge at 4000 rpm for 15 min, discard the supernatant, resuspend the bacterial cells with 2YTAK precipitate, and culture overnight at 28°C and 220 rpm. After centrifuging at 4000 rpm at 4°C for 15 min, aspirate the amplified phage supernatant for ELISA identification. Finally, four murine antibody molecules with higher affinity are screened and named MA-I, MA-II, MA-III, and MA-IV respectively. Determine the correct antibody sequences for the above-mentioned monoclonal antibodies by gene sequencing. After sequencing, the sequences of the 4 monoclonal antibodies screened above are as follows:

[0096]

[0097] Specifically, SEQ ID No: 16 (amino acid sequence of the heavy chain variable region of MA-I and MA-II):

[0098] EVKLEESGGGLVKPGGSLKLSCAASGFTFSDYYMFWVRQTPEKRLEWVATISDGGTYTYYPDSVKGRFTISRDNAKNNLYLQMTSLKSEDTAMYYCARDGGYVSSPEAMDYWGQGTSVTVSS;

[0099] SEQ ID No: 17 (amino acid sequence of the light chain variable region of MA-I and MA-IV):

[0100] DIVLTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPPTFGGGTKLEIK;

[0101] SEQ ID No: 18 (amino acid sequence of the light chain variable region of MA-II):

[0102] DIVLTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK;

[0103] SEQ ID No: 19 (amino acid sequence of the heavy chain variable region of MA-III):

[0104] EVKLEQSGAEVVKPGALVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGVIDPSDSYTTYNQKFKGKATLTVDTSSSTGYMQLSSLTSEDSAVYYCSQYGYDVNWYFDVWGAGTTVTVSS;

[0105] SEQ ID No: 20 (amino acid sequence of the light chain variable region of MA-III):

[0106] DIVMTQTTLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK;

[0107] SEQ ID No: 21 (Amino acid sequence of the heavy chain variable region of MA-IV):

[0108] EVQLEESGGGLVKPGGSLKLSCVASGFTFSDYYMFWVRQTPEKRLEWVATISDGGSYSYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTAMYYCARDGGYISSPEAMDYWGQGTSVTVSS.

[0109] Example 3 Gradient Dilution ELISA to Compare the Affinity of Antibodies

[0110] The 4 murine antibody molecules (MA-I, MA-II, MA-III, and MA-IV) obtained in Example 2 were subjected to monoclonal phage display and purification, and then the phage gradient dilution ELISA experiment was carried out to identify the affinity. The specific method is as follows:

[0111] Coat the IL-11 antigen with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, and coat overnight at 4 °C. Wash three times with PBST. The 4 phage monoclonal antibodies screened in Example 2 were respectively diluted five-fold with PBST, and 100 μl of the diluted sample was added to each well and allowed to stand at room temperature for 1 hour. Wash the ELISA plate with PBST, add the HRP-anti-M13 monoclonal antibody (purchased from Bio-viewshine, product number: GE27-9421-01) diluted with 1% BSA-PBST to the ELISA plate, and place it at room temperature for 1 h. Color development was carried out using a TMB color development kit (purchased from ComWin Biotech, product number: CW0050S), color development was carried out at room temperature for 10 minutes, terminated with 2M H2SO4, and the absorbance was read by an enzyme-linked immunosorbent assay (ELISA) reader at 450 nm / 630 nm, and the corresponding EC50 value was calculated. The specific data is as follows:

[0112]

[0113] From the above data and as Figure 2 shown, the 4 different murine antibody molecules screened in Example 2 were all able to bind to IL-11, indicating that the monoclonal antibodies provided by the present invention all have a high affinity for IL-11.

[0114] Example 4

[0115] Example 4 of the present invention further limits on the basis of Example 2 that the antibody or antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region, and the amino acid sequence of the heavy chain constant region is one of those shown in SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25 or SEQ ID No: 26; the amino acid sequence of the light chain constant region is as shown in SEQ ID No: 22, and the specific sequence is as follows:

[0116] SEQ ID No: 22 (amino acid sequence of the light chain constant region of murine C k type):

[0117] ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC;

[0118] SEQ ID No: 23 (amino acid sequence of the heavy chain constant region of murine IgG1 type):

[0119] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG;

[0120] SEQ ID No: 24 (amino acid sequence of the heavy chain constant region of murine IgG2a type):

[0121] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK;

[0122] SEQ ID No:25 (Amino acid sequence of the heavy chain constant region of murine IgG2b):

[0123] AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK;

[0124] SEQ ID No:26 (Amino acid sequence of the heavy chain constant region of murine IgG3):

[0125] ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPELELNETCAEAQDGELDGLWTTITIFISLFLLSVCYSASVTLFKVKWIFSSVVQVKQTAIPDYRNMIGQGA。

[0126] Example 5 Preparation of Murine Antibody Molecule

[0127] In Example 5 of the present invention, on the basis of Example 4, it is preferably defined that the murine antibody molecule includes a heavy chain constant region of murine IgG1 type (the amino acid sequence of which is shown in SEQ ID No: 23) and a murine C k type light chain constant region (the amino acid sequence of which is shown in SEQ ID No: 22). The antibody preparation method is as follows:

[0128] 1. The coding genes of the heavy chain VH and light chain VL of the 4 antibody molecules screened out in Example 2 are respectively cloned into the vector pTSE equipped with the heavy chain and light chain constant region genes (as Figure 3 shown). Preferably, the heavy chain constant region is a murine IgG1 type constant region (the amino acid sequence is shown in SEQ ID No: 23), and the light chain constant region is a murine C k chain (the amino acid sequence is shown in SEQ IDNo: 22). The structure of the pTSE vector is as Figure 3 shown (for the preparation process of the pTSE vector, see paragraph

[0019] on page 3 of the specification of CN103525868A).

[0129] 2. Transiently transfected HEK293 cells (purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences, catalog number GNHu43) were used for antibody expression. Four monoclonal antibodies were purified by protein A affinity column using an AKTA instrument. At the same time, a BCA kit (purchased from Beijing Huitian Dongfang Technology Co., Ltd., catalog number BCA0020) was used to measure the protein concentration. Then, the protein size was identified by SDS-PAGE. The results are as Figure 4 shown. From left to right are non-reduced MA-I, MA-II, MA-III, and MA-IV, protein molecular weight Marker1, protein molecular weight Marker2, and reduced MA-I, MA-II, MA-III, and MA-IV murine anti-IL-11 monoclonal antibodies. The molecular weight of each band is consistent with the theory.

[0130] Example 6 Binding experiment of murine antibody molecule to IL-11

[0131] Coat the IL-11 antigen with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, and coat overnight at 4°C. Wash five times with 300 μL / well PBST, then add 1% BSA-PBST, 280 μL / well, and block for 1 h at 37°C. Add murine antibody molecules of MA-I, MA-II, MA-III, and MA-IV at different dilution concentrations. The initial highest concentration of the 4 antibody molecules is 5 μg / mL, and they are diluted 8 gradients by 5-fold dilution respectively. Incubate for 1 h at 37°C. Wash five times with 300 μL / well PBST, then add Goat Anti-MouseIgG-HRP diluted 1:2000 with 1% BSA-PBST (purchased from solarbio, catalog number SE131), and incubate for 1 h at 37°C. Color development was carried out using a TMB color development kit, 100 μL / well, color development at room temperature for 8 min, and then the color development was terminated with 2M H2SO4. Read the absorbance at 450 nm / 630 nm with an ELISA reader and calculate the corresponding EC50 value. The specific data are as follows:

[0132]

[0133] From the above data and as Figure 5 shown, the 4 different murine antibody molecules screened can all bind to IL-11 and have relatively high affinities.

[0134] Example 7 Competitive inhibition experiment of murine antibody with IL-11 receptor protein IL-11RA

[0135] Coat IL-11-Fc with carbonate buffer at pH 9.6, 200 ng / well / 100 μL, and coat overnight at 4°C. Wash five times with 300 μL / well PBST, then add 1% BSA-PBST, 280 μL / well, and block at 37°C for 1 h. First, add IL-11RA-Fc (IgG4 type) diluted to 0.5 μg / mL with 1% BSA-PBST, 50 μL / well, then add murine antibodies MA-Ⅰ, MA-Ⅱ, MA-Ⅲ, and MA-Ⅳ at different dilution concentrations, 50 μL / well. The initial highest concentration of the 5 antibodies is 100 μg / mL, and they are serially diluted 2-fold. Each antibody is diluted for 13 gradients in total, and incubated at 37°C for 3 h. Wash five times with 300 μL / well PBST, then add Anti-Human IgG4-HRP Mouse monoclonal antibody (purchased from Sigma, catalog number: SAB4200770) diluted 1:5000 with 2% BSA-PBST, and incubate at 37°C for 1 h. Color development is carried out using a TMB color development kit, 100 μl / well, at room temperature for 15 min, and then the color development is terminated with 2 M H2SO4. Read the absorbance at 450 nm / 630 nm using an ELISA reader and calculate the corresponding IC50 values. The specific data are as follows:

[0136]

[0137] Based on the above data and as Figure 6 shown, the 4 different murine antibodies screened out can all compete with the receptor protein IL-11RA, indicating that they can effectively inhibit the binding of IL-11 to the receptor protein IL-11RA.

[0138] Example 8 Inhibition of the Binding of Murine Antibodies to the IL-11RA Receptor on the Surface of BaF / 3-IL-11RA Cells

[0139] Count the BaF / 3-IL-11RA cell line, take a certain number of cells, resuspend them in PBS buffer after centrifugation, adjust the cell density to 1E+6 cells / mL, add 100 μL per well to a 96-well plate. Dilute the IL-11-mFc ligand protein with PBS to a concentration of 18 μg / mL, add 50 μL per well to the corresponding positions of the 96-well plate containing BaF / 3-IL-11RA cells. After gently mixing, place the 96-well plate at 4°C and incubate for 1 h. Gradient dilute 4 murine antibody molecules MA-I, MA-II, MA-III, and MA-IV with PBS. The initial concentration is 800 μg / mL, and dilute it in 3-fold gradients for a total of 10 gradients, add 50 μL per well to the corresponding positions of the 96-well plate containing the mixture of BaF / 3-IL-11RA cells and IL-11-mFc ligand protein. After mixing evenly, place it at 4°C and incubate for 2 h. After the incubation, centrifuge at 3000 rpm, wash the cells once with PBS buffer, and collect the cell pellet. Add the pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) to the cell pellet, incubate at 4°C for 30 min, then centrifuge at 3000 rpm, wash once with PBS buffer, resuspend in 100 μL of PBS buffer, and detect on a flow cytometer to collect the fluorescence signal in the FL1-A channel. Plot the dose-effect curve and calculate the corresponding IC50 value. The specific data is as follows:

[0140]

[0141] From the above data and Figure 7 it can be seen that the 4 different murine candidate molecules screened can effectively inhibit the binding of IL-11 ligand protein to the IL-11RA receptor on the cell surface.

[0142] Example 9 Inhibition of TIMP-1 Secretion by Murine Antibodies in Fetal Lung Fibroblasts MRC-5

[0143] After trypsin digestion of MRC-5 human embryonic lung fibroblasts, the cells were counted. A certain number of cells were taken, centrifuged, and resuspended in MEM complete medium (purchased from GIBCO, catalog number 10370-021), and the cell density was adjusted to 2E+5 cells / mL. 100 μL per well was added to a 96-well plate. The IL-11-mFc ligand protein was diluted with MEM complete medium to a concentration of 16 μg / mL, and 50 μL per well was added to the corresponding 96-well plate. Four murine antibody molecules, MA-I, MA-II, MA-III, and MA-IV, were serially diluted with MEM complete medium. The initial concentration was 40 μg / mL, and it was diluted in a 2-fold serial dilution for a total of 8 dilutions. 50 μL per well was added to the 96-well plate containing the cell suspension and the IL-11-mFc ligand protein suspension. After gently mixing, it was placed in a 37 °C CO2 incubator and incubated overnight for about 20 h. The cell culture supernatant was taken and detected using an ELISA kit for TIMP-1 (purchased from EKSEI Biotech Co., Ltd., catalog number EH021-96).

[0144] Human TIMP-1 detection kit: The cell supernatant and the standard were added to the sample wells, 100 μL per well. Immediately add the biotinylated antibody working solution (diluted 1:100), 50 μL per well, cover with a sealing film, and incubate with shaking at room temperature for 2 h. After incubation, wash the plate 4 times with the washing solution. Add the enzyme conjugate working solution (diluted 1:100) in the TIMP-1 detection kit, 100 μL per well. Cover with a sealing film and incubate with shaking at room temperature for 1 h. After incubation, wash the plate 4 times with the washing solution. Add the TMB chromogenic solution, 100 μL per well, incubate in the dark at room temperature for about 15 minutes, and terminate the reaction with 100 μL per well of Stop solution. Read the absorbance at 450 nm with an ELISA reader and calculate the corresponding IC50 value. The specific data is as follows:

[0145]

[0146] From the above data and Figure 8 it can be seen that the 4 different murine candidate molecules screened can effectively inhibit the release of TIMP-1 from human embryonic lung fibroblasts MRC-5 stimulated by the IL-11 ligand protein.

[0147] Example 10

[0148] In Example 10 of the present invention, the antibody or its antigen-binding fragment is further defined as a chimeric antibody molecule, and the chimeric antibody molecule further includes a human antibody constant region. The human antibody constant region includes a human heavy chain antibody constant region and a human light chain antibody constant region. The amino acid sequence of the human heavy chain antibody constant region is one of those shown in SEQ ID No: 27, SEQ ID No: 28, or SEQ ID No: 29; the amino acid sequence of the human light chain antibody constant region is as shown in SEQ ID No: 30.

[0149] SEQ ID No: 27 (Amino acid sequence of the heavy chain constant region of human IgG1):

[0150] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0151] SEQ ID No: 28 (Amino acid sequence of the heavy chain constant region of human IgG2):

[0152] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0153] SEQ ID No: 29 (Amino acid sequence of the heavy chain constant region of human IgG4):

[0154] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0155] SEQ ID No:30 (Human C k chain light chain constant region amino acid sequence):

[0156] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0157] Example 11 Preparation of Chimeric Antibody Molecule Antibodies

[0158] In Example 11 of the present invention, on the basis of Example 10, it is further defined that the human antibody constant region includes the heavy chain constant region of human IgG1 type (the amino acid sequence is as shown in SEQ ID No: 27) and human C k type light chain constant region (the amino acid sequence is as shown in SEQ ID No: 30).

[0159] Specific preparation method:

[0160] Keeping the murine sequences of the heavy chain variable regions VH (SEQ ID No: 16) of the murine antibody molecules MA-I and MA-II and the light chain variable region VL genes (SEQ ID No: 17) of MA-I and the light chain variable region VL gene (SEQ ID No: 18) of MA-II screened from the immunized phage antibody library in Example 2 unchanged, they were respectively cloned onto the vector pTSE containing the heavy chain constant region and light chain constant region genes (as Figure 3 shown), the heavy chain constant region is of human IgG1 type (the amino acid sequence is as shown in SEQ ID NO: 27), and the light chain constant region is human Ck Type (amino acid sequence as shown in SEQ ID NO: 30). Transiently transfect HEK293E cells (purchased from: Institute of Basic Medicine, Chinese Academy of Medical Sciences, catalog number: GNHu43) for antibody expression to obtain chimeric antibodies CA-I and CA-II.

[0161] Example 12 Humanization of Murine Antibody Molecules

[0162] First, select the sequences of murine antibody molecules MA-I and MA-II in Example 2 and compare them with the human antibody germline database (v-base) to find human antibody light and heavy chain germlines with relatively high homology as candidate sequences. Then, transplant the CDR sequences of murine antibody molecules MA-I and MA-II onto the human candidate sequences for homology modeling. Then, through three-dimensional structure simulation, calculate the key framework amino acid residues that may play an important role in maintaining the CDR loop structure, thereby designing the back mutations of the humanized antibody. The light and heavy chain variable region sequences of the designed humanized antibody containing back mutations were optimized and synthesized by Nanjing Genscript Biotech Co., Ltd. respectively, and then ligated to the transient expression vector. Analyze the light and heavy chain combinations obtained by humanization. For MA-I, the following humanized antibody molecules were obtained: HA-I-A, HA-I-B, HA-I-C, HA-I-D; for MA-II, the following humanized antibody molecules were obtained: HA-II-A, HA-II-B, HA-II-C, HA-II-D; the sequences of the 8 monoclonal antibodies screened above are as follows:

[0163]

[0164] Specifically, SEQ ID No: 31 (amino acid sequence of the heavy chain variable region of HA-I-A, HA-I-C, HA-II-A, and HA-II-B):

[0165] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATISDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVSSPEAMDYWGQGTLVTVSS;

[0166] SEQ ID No: 32 (amino acid sequence of the light chain variable region of HA-I-A):

[0167] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;

[0168] SEQ ID No:33 (Amino acid sequence of the heavy chain variable region of HA-I-B and HA-II-C):

[0169] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVSTISDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVSSPEAMDYWGQGTLVTVSS;

[0170] SEQ ID No:34 (Amino acid sequence of the light chain variable region of HA-I-B):

[0171] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;

[0172] SEQ ID No:35 (Amino acid sequence of the light chain variable region of HA-I-C):

[0173] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;

[0174] SEQ ID No:36 (Amino acid sequence of the heavy chain variable region of HA-I-D and HA-II-D):

[0175] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATISDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAMYYCARDGGYVSSPEAMDYWGQGTSVTVSS;

[0176] SEQ ID No:37 (Amino acid sequence of the light chain variable region of HA-I-D):

[0177] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;

[0178] SEQ ID No:38 (Amino acid sequence of the light chain variable region of HA-II-A):

[0179] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;

[0180] SEQ ID No:39 (Amino acid sequence of the light chain variable region of HA-II-B and HA-II-C):

[0181] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;

[0182] SEQ ID No:40 (Amino acid sequence of the light chain variable region of HA-II-D):

[0183] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPWTFGGGTKVEIK。

[0184] Example 13

[0185] In Example 13 of the present invention, on the basis of Example 12, the human antibody constant region is further defined to include a human antibody heavy chain constant region and a human antibody light chain constant region. The amino acid sequence of the human antibody heavy chain constant region is one of those shown in SEQ ID No:27, SEQ ID No:28 or SEQ ID No:29; the amino acid sequence of the human antibody light chain constant region is as shown in SEQ ID No:30.

[0186] The specific sequences of the above-mentioned human antibody constant regions are the same as those in Example 10.

[0187] Preparation of the humanized antibody molecule in Example 14

[0188] In Example 14 of the present invention, on the basis of Example 13, it is further defined that the human antibody constant region includes the heavy chain constant region of human IgG1 type (the amino acid sequence of which is shown in SEQ ID No: 27) and human C k type light chain constant region (the amino acid sequence of which is shown in SEQ ID No: 30).

[0189] The coding genes of the heavy chain VH and light chain VL of the 8 humanized antibody molecules HA-I-A, HA-I-B, HA-I-C, HA-I-D, HA-II-A, HA-II-B, HA-II-C, and HA-II-D humanized in Example 12 were respectively cloned into the vector pTSE containing the heavy chain constant region and light chain constant region genes (as Figure 3 shown), the heavy chain constant region is of human IgG1 type (the amino acid sequence is shown in SEQ ID NO: 27), and the light chain constant region is C k chain (the amino acid sequence is shown in SEQ ID No: 30).

[0190] The 2 chimeric antibodies CA-I and CA-II obtained in Example 11 and the 8 humanized antibody molecules HA-I-A, HA-I-B, HA-I-C, HA-I-D, HA-II-A, HA-II-B, HA-II-C, and HA-II-D obtained in Example 12 were respectively transiently transfected into HEK293 cells (purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences, catalog number: GNHu43) for antibody expression. Monoclonal antibodies were purified using an AKTA instrument through a protein A affinity column. At the same time, a BCA kit (purchased from: Beijing Huitian Dongfang Technology Co., Ltd., catalog number: BCA0020) was used for protein concentration determination, and then the protein size was identified by SDS-PAGE. The results are as Figure 9 shown. From left to right are the non-reduced protein molecular weights HA-I-A, HA-I-B, HA-I-C, HA-I-D, the chimeric antibody CA-I prepared in Example 11, the reduced protein molecular weight Marker, HA-II-A, HA-II-B, HA-II-C, HA-II-D, and the chimeric antibody CA-II. The molecular weight of each band is consistent with the theory.

[0191] Example 15

[0192] Example 15 defines that the antibody or its antigen-binding fragment disclosed in the present invention is one or several combinations of Fab, F(ab)2, Fv, or ScFv, not limited to the monoclonal antibodies disclosed in the present invention.

[0193] Example 16

[0194] Example 16 of the present invention also provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof that binds to IL-11.

[0195] The present invention also provides a recombinant DNA expression vector comprising the nucleic acid molecule described above.

[0196] The present invention also provides a host cell transfected with the recombinant DNA expression vector described above, and the host cell includes a prokaryotic cell, a yeast cell, an insect cell or a mammalian cell;

[0197] Further preferably, the host cell is a mammalian cell, and the mammalian cell is a HEK293 cell, a CHO cell or an NS0 cell.

[0198] The present invention also provides a drug comprising the antibody or antigen-binding fragment thereof that binds to IL-11.

[0199] The present invention also provides a detection reagent for detecting the antibody or antigen-binding fragment thereof that binds to IL-11.

[0200] The present invention also provides a combination drug composition, which includes the antibody or antigen-binding fragment thereof that binds to IL-11, and further includes a second composition, and the second composition is selected from the antibody or antigen-binding fragment thereof that binds to PD-1, PD-L1, VEGF, VEGFR, TNF-α or TSLP.

[0201] Preferably, the antibody or antigen-binding fragment thereof that binds to PD-1 is selected from DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12, DFPD1-13, Nivolumab, Pembrolizumab, Toripalimab, Sintilimab, Tirzepatide, Camrelizumab, Pamiparib or Sepalimab. Among them, the heavy chain constant region and light chain constant region sequences of DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12, DFPD1-13 are the same as those of the anti-PD-1 monoclonal antibody provided in the patent application number CN201510312910.8.

[0202] Preferably, the antibody or antigen-binding fragment thereof that binds to VEGF is selected from Aflibercept, Bevacizumab, or Ranibizumab.

[0203] Example 17

[0204] Example 17 of the present invention provides the use of the antibody or antigen-binding fragment thereof that binds to IL-11 in the preparation of drugs for treating or preventing human fibrotic diseases, inflammation, cancer or autoimmune diseases.

[0205] Preferably, the fibrotic diseases include, but are not limited to, fibrosis of the heart, liver, kidney, lung, gallbladder, bladder, stomach, bone marrow, penis, breast, blood vessel, eye, pancreas, spleen, brain, intestine, muscle or skin.

[0206] Preferably, the inflammation includes, but is not limited to, hepatitis, myocarditis, nephritis, pneumonia, cholecystitis, cystitis, gastritis, osteomyelitis, prostatitis, mastitis, pancreatitis, enteritis, arthritis, polymyositis, dermatomyositis or dermatitis.

[0207] Preferably, the cancers include, but are not limited to, leukemia, lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer or bladder cancer.

[0208] Preferably, the autoimmune diseases include, but are not limited to, psoriasis, Crohn's disease, primary biliary cirrhosis, systemic lupus erythematosus or multiple sclerosis.

[0209] The present invention also provides a method for treating or preventing a subject with fibrotic diseases, inflammation, cancer or autoimmune diseases in humans, the method comprising administering to the subject a therapeutically or prophylactically effective amount of an antibody that binds to IL-11 or an antigen-binding fragment thereof.

[0210] Example 18 Experiment on the Binding of Humanized Antibody Molecules to IL-11

[0211] Coat the IL-11 antigen with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, and incubate overnight at 4°C. Wash five times with 300 μL / well of PBST, and then add 280 μL / well of 1% BSA-PBST to block for 1 h at 37°C. Dilute the humanized antibodies HA-I-A, HA-I-B, HA-I-C, HA-I-D, HA-II-A, HA-II-B, HA-II-C, HA-II-D and the chimeric antibody CA-I, CA-II prepared in Example 11 with 1% BSA-PBST. The initial concentration of the humanized antibodies is 10 μg / mL, and they are diluted 5-fold in a total of 8 gradients and incubated for 1 h at 37°C. Wash five times with 300 μL / well of PBST, and then add goat anti-Human IgG Fab HRP diluted 1:5000 with 1% BSA-PBST (purchased from Invitrogen, catalog number: 31482) and incubate for 1 h at 37°C. Develop color with a TMB color development kit, 100 μL / well, develop color at room temperature for 5 min, and then terminate the color development with 2M H2SO4. Read the absorbance at 450 nm / 630 nm with an enzyme-linked immunosorbent assay reader and calculate the corresponding EC50 value. The specific data are as follows:

[0212]

[0213]

[0214] As shown by the above data and experimental results, Figure 10 all 8 different humanized antibody molecules can bind to IL-11, and the EC50 values of the humanized antibody molecules HA-I-A, HA-I-B, HA-I-C, and HA-I-D are all close to those of the chimeric antibody CA-I, and the EC50 values of the humanized antibody molecules HA-II-A, HA-II-B, HA-II-C, and HA-II-D are all close to those of the chimeric antibody CA-II, indicating that the humanized antibody molecules retain the high binding ability of the murine parental antibodies MA-I and MA-II to IL-11.

[0215] Example 19 Inhibition of the binding of humanized antibody molecules to the IL-11RA receptor on the surface of BaF / 3-IL-11RA cells

[0216] Four humanized antibody molecules HA-I-A, HA-I-B, HA-I-C, and HA-I-D with relatively excellent binding activity at the protein level were selected for cell activity evaluation experiments. The BaF / 3-IL-11RA cell line was counted, a certain number of cells were taken, resuspended in PBS buffer after centrifugation, and the cell density was adjusted to 1E+6 cells / mL, 100 μL / well, and added to a 96-well plate. Dilute the IL-11-mFc ligand protein with PBS to a concentration of 18 μg / mL, 50 μL / well, and add it to the corresponding positions of the 96-well plate containing BaF / 3-IL-11RA cells. After gently mixing, the 96-well plate was placed at 4°C and incubated for 1 h. Four humanized antibody molecules HA-I-A, HA-I-B, HA-I-C, and HA-I-D were serially diluted with PBS buffer. The initial concentration was 800 μg / mL, and 3-fold serial dilutions were made for a total of 10 gradients, 50 μL / well, and added to the corresponding positions of the 96-well plate containing the mixture of BaF / 3-IL-11RA cells and IL-11-mFc ligand protein. After mixing evenly, it was incubated at 4°C for 2 h. After the incubation, the cells were washed once with PBS buffer by centrifugation at 3000 rpm, and the cell pellet was collected. The pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) was added to the cell pellet, incubated at 4°C for 30 min, then centrifuged and washed once at 3000 rpm, resuspended in 100 μL PBS, and detected by flow cytometry. The fluorescence signal in the FL1-A channel was collected. A dose-effect curve was plotted, and the corresponding IC50 values were calculated. The specific data are as follows:

[0217]

[0218] From the above data and Figure 11 it can be seen that all 4 humanized candidate molecules screened can inhibit the binding of IL-11 ligand protein to IL-11RA receptor on the surface of BaF / 3-IL-11RA cells.

[0219] Example 20 Binding inhibition of humanized antibody molecule to GP130 receptor on the surface of BaF / 3-GP130 cells by IL-11

[0220] Count the BaF / 3-GP130 cell line, take a certain number of cells, resuspend with PBS buffer after centrifugation, adjust the cell density to 1E+6 cells / mL, 100 μL / well, and add to a 96-well plate. Dilute the IL-11-mFc ligand protein with PBS to a concentration of 12 μg / mL, 50 μL / well, and add to the corresponding positions of the 96-well plate containing BaF / 3-GP130 cells. After gently mixing, place the 96-well plate at 4°C and incubate for 1 h. Gradient dilute 4 human antibodies HA-I-A, HA-I-B, HA-I-C, and HA-I-D with PBS buffer, prepare an initial concentration of 2000 μg / mL, and perform 2-fold serial dilution for a total of 10 gradients, 50 μL / well, and add to the corresponding positions of the 96-well plate containing BaF / 3-GP130 cells and IL-11-mFc ligand protein. After mixing evenly, place the 96-well plate at 4°C and incubate for 2 h. After incubation, centrifuge at 3000 rpm and wash the cells once with PBS buffer, and collect the cell pellet. Add the pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) to the cell pellet, 100 μL / well, and incubate at 4°C for 30 min. After centrifuging at 3000 rpm and washing once with PBS buffer, resuspend the cells with 100 μL / well of PBS buffer, detect on a flow cytometer, and collect the fluorescence signal in the FL1-A channel. Plot the dose-effect curve and calculate the corresponding IC50 value. The specific data is as follows:

[0221]

[0222] From the above data and Figure 12 it can be seen that all 4 humanized candidate molecules screened can block the binding of IL-11 ligand protein to GP130 receptor on the surface of BaF / 3-GP130 cells.

[0223] Example 21 Biological activity detection of humanized antibody molecule (reporter gene)

[0224] Count the BaF / 3-IL-11RA-GP130-STAT3-Luc engineered cell line. Use the sample diluent (its components include 90% IMDM, 10% FBS, 10 ng / mL mouse IL-3) to adjust the cell density to 2E+6 cells / mL. After gently mixing, add the cell suspension to a 96-well plate, 50 μL / well. Dilute the 4 humanized antibody molecules HA-I-A, HA-I-B, HA-I-C, and HA-I-D to an initial concentration of 200 μg / ml with the sample diluent, and perform 5-fold serial dilution, for a total of 10 gradients, 100 μL / well, and add them to the corresponding positions of the 96-well plate containing the engineered cell line. Set two replicates for each sample concentration. Prepare IL-11 protein with the sample diluent at a concentration of 10 μg / mL, 50 μL / well, and add it to the 96-well plate containing the engineered cell line and the humanized antibody molecules. Gently mix the cell culture plate and incubate it in a 37°C CO2 incubator for 6 h. Centrifuge to discard the supernatant, add lysis buffer, 10 μL / well to a 384-well plate, add an equal amount of luciferase reaction substrate (purchased from Promega Biotechnology Co., Ltd., catalog number E2610), react at room temperature for 5 min, read the fluorescence value under a microplate reader, and calculate the corresponding IC50 value. The specific data is as follows:

[0225]

[0226] As shown by the above data and Figure 13 All 4 humanized antibody molecules screened can block the binding of IL-11 to the IL-11RA and GP130 receptors and inhibit the signal pathway transduction.

[0227] Example 2 Inhibition of TIMP-1 secretion by humanized antibody molecules in embryonic lung fibroblasts MRC-5

[0228] After trypsin digestion of MRC-5 human embryonic lung fibroblasts, the cells were counted. A certain number of cells were taken, centrifuged, and resuspended in MEM complete medium. The cell density was adjusted to 2E+5 cells / mL, and 100 μL / well was added to a 96-well plate. The IL-11-mFc ligand protein was diluted with MEM complete medium to a concentration of 16 μg / mL, and 50 μL / well was added to the corresponding 96-well plate. Four humanized antibody molecules, HA-I-A, HA-I-B, HA-I-C, and HA-I-D, were serially diluted with MEM complete medium. The initial concentration was 40 μg / mL, and the dilution factor was 3-fold, with a total of 8 gradients. 50 μL / well was added to the 96-well plate containing the cell suspension and the IL-11-mFc ligand protein suspension. The mixture was gently mixed and incubated overnight at 37°C in a CO2 incubator for about 20 h. The cell culture supernatant was taken and detected using the TIMP-1 ELISA kit (the method was the same as in Example 9). The microplate reader was used to read the absorbance at 450 nm, and the corresponding IC50 values were calculated. The specific data are as follows:

[0229]

[0230] From the above data and Figure 14 it can be seen that all 4 screened humanized antibody molecules can effectively inhibit the release of TIMP-1 from human embryonic lung fibroblasts MRC-5 stimulated by the IL-11 ligand protein.

[0231] Example 23 Cross-binding experiment of humanized antibody molecules with IL-11 of different species

[0232] The humanized antibody molecule HA-I-A with better protein level and functional detection activity was selected for cross-binding detection with IL-11 of different species. Human IL-11, mouse IL-11 (purchased from Beijing Sino Biological Inc., product number: 50117-MNCE), rat IL-11 (purchased from Kanglang Biotechnology, product number: KL40001Ra), and cynomolgus monkey IL-11 (purchased from Sino Biological, product number: 90925-CNCE) were coated at 100 ng / well / 100 μL with carbonate buffer at pH 9.6 and incubated overnight at 4°C. Washed five times with 300 μL / well PBST, and then 1% BSA-PBST, 280 μL / well, was added and blocked at 37°C for 1 h. The humanized antibody HA-I-A was diluted with 1% BSA-PBST, with an initial concentration of 50 μg / mL, diluted in a 5-fold gradient, a total of 9 gradients, two replicates for each gradient, 100 μL / well was added to the 96-well plate, and incubated at 37°C for 1 h. Washed five times with 300 μL / well PBST, goat anti-Human IgG Fab HRP (purchased from invitrogen, product number: 31482) was diluted with 1% BSA-PBST, the working solution concentration was 1:5000, 100 μL / well was added to the 96-well plate, and incubated at 37°C for 1 h. Washed five times with 300 μL / well PBST, developed color with the TMB color development kit, 100 μL / well, developed color in the dark at room temperature for 5 min, and then terminated the color development with 2 M H2SO4. Read with an enzyme-linked immunosorbent assay (ELISA) reader at 450 nm / 630 nm, and calculate the corresponding EC50 value. The specific data is as follows:

[0233]

[0234] Based on the above data and as Figure 15 shown, the humanized antibody molecule HA-I-A can bind to human IL-11, mouse IL-11, rat IL-11, and cynomolgus monkey IL-11, and has a relatively high affinity.

[0235] Example 24 Therapeutic Efficacy Experiment of Anti-IL-11 Monoclonal Antibody on Pulmonary Fibrosis

[0236] Bleomycin (bLF) was used to establish a model to study the therapeutic effect of anti-IL-11 monoclonal antibody HA-I-A on pulmonary fibrosis.

[0237] Animal species: C57BL / 6J mice (purchased from Jiangsu GICC Bio-Tech Co., Ltd.)

[0238] Quantity, gender, and mouse age: 6 mice / group, male, 6 - 8 weeks old;

[0239] The control group was only injected with normal saline;

[0240] The administration group was given HA-I-A antibody molecules by injection twice a week for 4 consecutive weeks.

[0241] The body weight of the animals was measured once a week, and the animals were observed for any abnormalities; organ weight detection: the lung organs were collected, the weight of the lung organs was measured, and the ratio of lung to body weight was calculated; lung pathology detection: lung sections were made and hematoxylin-eosin (HE) and Masson staining were used to observe the degree of lung fibrosis.

[0242] The results were as Figure 16 shown. After administration of HA-I-A antibody molecules, the ratio of lung to body weight in the mice of the administration group was significantly lower than that of the control group; the results were as Figure 17 shown. Compared with the control group, the lung sections of the administration group showed significantly less lung fibrosis. Therefore, it can be shown that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the generation of lung fibrosis.

[0243] Example 25 Therapeutic Efficacy Experiment of Anti-IL-11 Monoclonal Antibody on Cardiac Fibrosis

[0244] Isoproterenol was used to establish a model to study the therapeutic effect of anti-IL-11 monoclonal antibody HA-I-A on cardiac fibrosis.

[0245] Animal species: C57BL / 6J mice (purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.)

[0246] Quantity, gender and mouse age: 6 per group, male, 6 - 8 weeks old;

[0247] The control group was only injected with normal saline;

[0248] The administration group was given HA-I-A antibody molecules by injection twice a week for 4 consecutive weeks.

[0249] The body weight of the animals was measured once a week, and the animals were observed for any abnormalities; organ weight detection: the heart was collected, the weight of the heart was measured, and the ratio of heart to body weight was calculated; heart pathology detection: heart sections were made and hematoxylin-eosin (HE) and Masson staining were used to observe the degree of heart fibrosis.

[0250] The results were as Figure 18 shown. The ratio of heart to body weight in the mice of the administration group was significantly lower than that of the control group; the results were as Figure 19 shown. Compared with the control group, the heart sections of the administration group showed significantly less heart fibrosis. Therefore, it can be shown that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the generation of heart fibrosis.

[0251] Example 26 Therapeutic Efficacy Experiment of Anti-IL-11 Monoclonal Antibody on Renal Fibrosis

[0252] Modeling with doxorubicin (dKF) to study the therapeutic effect of anti-IL-11 monoclonal antibody HA-I-A on renal fibrosis.

[0253] Animal species: BALB / c mice (purchased from Jiangsu Genscript Biotech Co., Ltd.)

[0254] Number, sex and age: 6 mice / group, male, 6 - 8 weeks old;

[0255] The control group was only injected with normal saline.

[0256] The administration group was injected with HA-I-A antibody molecules twice a week for 4 consecutive weeks.

[0257] The animal body weight was measured once a week, and the animals were observed for any abnormalities; Organ weight detection: The heart was collected, the weight of the kidneys was measured, and the urine protein content was detected; Renal pathology detection: Kidney sections were made and stained with hematoxylin and eosin (HE) and Masson staining to observe the degree of renal fibrosis.

[0258] The results were as Figure 20 shown. Compared with the control group, the urine protein content in the kidneys of the mice in the administration group was significantly lower than that in the control group; The results were as Figure 21 shown. Compared with the control group, the kidney sections of the administration group showed significantly less renal fibrosis. Therefore, it can be shown that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the occurrence of renal fibrosis.

[0259] Example 27 Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody on liver fibrosis

[0260] Modeling with CCl4 to study the therapeutic effect of anti-IL-11 monoclonal antibody HA-I-A on liver fibrosis.

[0261] Animal species: C57BL / 6J mice (purchased from Jiangsu Genscript Biotech Co., Ltd.)

[0262] Number, sex and age: 6 mice / group, male, 6 - 8 weeks old;

[0263] The control group was only injected with normal saline.

[0264] The administration group was injected with HA-I-A antibody molecules twice a week for 4 consecutive weeks.

[0265] Body weight monitoring: The animal body weight was measured once a week, and the animals were observed for any abnormalities; Liver pathology detection: Liver sections were made and stained with hematoxylin and eosin (HE) and Masson staining to observe the degree of liver fibrosis; Organ weight detection: The kidneys were collected, the weight of the liver was measured, and HE staining was performed; Serum detection: Serum was collected, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the mouse serum were detected.

[0266] The results are as Figure 22 shown. Compared with the control group, the liver weight of the mice in the administration group was significantly lower than that of the control group; the results are as Figure 23 shown. The levels of ALT and AST in the serum of the mice in the administration group were significantly lower than those of the control group; the results are as Figure 24 shown. Compared with the control group, the liver sections of the administration group showed significantly less liver fibrosis. Therefore, it can be shown that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the generation of liver fibrosis.

[0267] Example 28 Evaluation of the thermal stability of anti-IL-11 monoclonal antibody HA-I-A

[0268] The thermal stability of anti-IL-11 monoclonal antibody HA-I-A was evaluated using a multi-functional protein thermal stability analysis system (purchased from Unchained Labs). By monitoring the change in the endogenous fluorescence of the protein with temperature (starting from 25°C and heating at a rate of 0.3°C / min to 95°C), the change in the protein conformation was detected, thereby determining the protein melting temperature Tm and evaluating the conformational stability of the protein. When the sample aggregates, it will cause interference in the scattered light wave and an increase in the scattered light signal. The colloidal stability of the protein was determined by static light scattering (characterized by Tagg), and the results are shown in the following table and appendix Figure 25 as follows.

[0269]

[0270] The melting temperature Tm of anti-IL-11 monoclonal antibody HA-I-A was 79.5°C, and the average Tagg was 75.9°C, showing good conformational stability and colloidal stability.

[0271] The present invention is not limited to the above best implementation manner. Anyone can obtain other various forms of products under the inspiration of the present invention. However, no matter what changes are made in its shape or structure, as long as it has the same or similar technical solutions as the present application, it falls within the protection scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that binds to IL-11, characterized in that, It includes three heavy-chain complementarity-determining regions represented by HCDR1, HCDR2, and HCDR3 respectively, and three light-chain complementarity-determining regions represented by LCDR1, LCDR2, and LCDR3 respectively. The antibody or its antigen-binding fragment is selected from any one of the following: A-I: The amino acid sequence of the heavy-chain complementarity-determining region HCDR1 is as shown in SEQ ID No:1, the amino acid sequence of the heavy-chain complementarity-determining region HCDR2 is as shown in SEQ ID No:2, the amino acid sequence of the heavy-chain complementarity-determining region HCDR3 is as shown in SEQ ID No:3, the amino acid sequence of the light-chain complementarity-determining region LCDR1 is as shown in SEQ ID No:4, the amino acid sequence of the light-chain complementarity-determining region LCDR2 is as shown in SEQ ID No:5, and the amino acid sequence of the light-chain complementarity-determining region LCDR3 is as shown in SEQ ID No:6; A-II: The amino acid sequence of the heavy-chain complementarity-determining region HCDR1 is as shown in SEQ ID No:1, the amino acid sequence of the heavy-chain complementarity-determining region HCDR2 is as shown in SEQ ID No:2, the amino acid sequence of the heavy-chain complementarity-determining region HCDR3 is as shown in SEQ ID No:3, the amino acid sequence of the light-chain complementarity-determining region LCDR1 is as shown in SEQ ID No:4, the amino acid sequence of the light-chain complementarity-determining region LCDR2 is as shown in SEQ ID No:5, and the amino acid sequence of the light-chain complementarity-determining region LCDR3 is as shown in SEQ ID No:

7.

2. The antibody or antigen-binding fragment thereof that binds to IL-11 according to claim 1, wherein It includes a heavy-chain variable region and a light-chain variable region. The antibody or its antigen-binding fragment is selected from any one of the following: MA-I: The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID No:16, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID No:17; MA-II: The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID No:16, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID No:

18.

3. The antibody or antigen-binding fragment thereof that binds to IL-11 according to claim 2, wherein It further includes a heavy-chain constant region and a light-chain constant region. The amino acid sequence of the heavy-chain constant region is one of those shown in SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, or SEQ ID No:26; the amino acid sequence of the light-chain constant region is as shown in SEQ ID No:

22.

4. The antibody or antigen-binding fragment thereof that binds to IL-11 according to claim 2, wherein The antibody or its antigen-binding fragment is a chimeric antibody molecule, and the chimeric antibody molecule further includes a human antibody constant region.

5. The antibody or antigen-binding fragment thereof that binds to IL-11 according to claim 1, wherein It includes a heavy-chain variable region and a light-chain variable region. The antibody or its antigen-binding fragment is selected from any one of the following: HA-I-A: The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID No:31, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID No:32; HA-I-B: The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID No:33, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID No:34; HA-I-C: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 35; HA-I-D: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 36, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 37; HA-II-A: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 38; HA-II-B: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 39; HA-II-C: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 33, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 39; HA-II-D: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 36, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No:

40.

6. The antibody or antigen-binding fragment thereof that binds to IL-11 according to claim 5, wherein The antibody or its antigen-binding fragment further comprises a human antibody constant region.

7. The antibody or antigen-binding fragment thereof that binds to IL-11 according to claim 4 or 6, wherein The human antibody constant region comprises a human heavy chain constant region and a human light chain constant region. The amino acid sequence of the human heavy chain constant region is one of those shown in SEQ ID No: 27, SEQ ID No: 28 or SEQ ID No: 29; the amino acid sequence of the human light chain constant region is as shown in SEQ ID No:

30.

8. The antibody or antigen-binding fragment thereof that binds to IL-11 according to claim 1, wherein The antibody or its antigen-binding fragment is one or a combination of Fab, F(ab)2, Fv or ScFv.

9. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or its antigen-binding fragment that binds to IL-11 as claimed in claim 1.

10. A recombinant DNA expression vector, characterized in that, The recombinant DNA expression vector comprises the nucleic acid molecule as claimed in claim 9.

11. A host cell transfected with the recombinant DNA expression vector as claimed in claim 10, characterized in that, The host cell includes a prokaryotic cell, a yeast cell, an insect cell or a mammalian cell.

12. The host cell according to claim 11, wherein The host cell is a mammalian cell, and the mammalian cell is a HEK293 cell, a CHO cell or an NS0 cell.

13. A drug, characterized in that, The drug comprises the antibody or its antigen-binding fragment that binds to IL-11 as claimed in claim 1.

14. A combined medicament composition, characterized in that, The combined drug composition comprises the antibody or its antigen-binding fragment that binds to IL-11 as claimed in claim 1, and further comprises a second composition, and the second composition is selected from antibodies or their antigen-binding fragments that bind to PD-1, PD-L1, VEGF, VEGFR, TNF-α or TSLP.

15. Use of the antibody or antigen-binding fragment thereof that binds to IL-11 in the preparation of a medicament for treating human fibrotic diseases, inflammation or cancer; wherein, The fibrotic disease is selected from fibrosis of the heart, liver, kidney, lung, bone marrow, eye, pancreas, spleen, brain, intestine or skin; The inflammation is selected from pancreatitis, enteritis, arthritis or dermatitis; The cancer is selected from lung cancer, gastric cancer, breast cancer or colorectal cancer.

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