Antibodies that bind to IL-11, their antigen-binding fragments, and their use

Abstract

The present invention relates to the field of biopharmaceuticals, and more specifically, provides an antibody or antigen-binding fragment thereof that binds to IL-11. The antibody or antigen-binding fragment comprises 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, and the antibody or antigen-binding fragment is selected from AI, A-II, A-III, or A-IV. The antibody or antigen-binding fragment provided by the present invention has a high binding ability to the IL-11 antigen, inhibits the binding of the IL-11 antigen to its receptor, thereby effectively inhibiting the fibrotic effect of IL-11, inhibiting or preventing the production or proliferation of fibrous cells, and can effectively treat or prevent human fibrotic diseases, inflammation, cancer, or autoimmune diseases.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of Chinese Patent Application No. 202310652547.9, filed on June 5, 2023, the content of which is incorporated herein by reference.

[0002] Technical Field The present invention relates to the field of biopharmaceutical technology, and particularly relates to an antibody that binds to IL - 11, its antigen - binding fragment, and uses thereof.

Background Art

[0003] Fibrosis is a repair reaction after tissue injury and maintains the relative integrity of tissues and organs. Various harmful stimuli (including toxins, infectious pathogens, autoimmune reactions, and mechanical stress) can induce the fibrotic response of cells. In response to tissue damage, fibroblasts of diverse origins (including resident fibroblasts, mesenchymal cells, circulating fibroblasts, and trans - differentiation of other cell types) can initiate a wound - healing reaction by remodeling the extracellular environment, restore tissue integrity, and promote the replacement of parenchymal cells. However, persistent damage and disorders cause dysregulation of this process, leading to pathological over - deposition of extracellular matrix (ECM) proteins (including collagen, laminin, and fibronectin), enhanced fibroblastosis, and the formation of a chronic inflammatory environment due to infiltration of macrophages and immune cells. A large amount of cytokines and growth factors are released, including members of the transforming growth factor - β (TGF - β) family and Wingless / Int - 1 (Wnt1) protein, which are major effectors of the fibrosis process. TGF - β and Wnt1 bind to their stem - cell surface receptors, initiate downstream signal transduction, cause up - regulation of target gene expression, and their functions further promote the differentiation of fibroblasts and the production and secretion of ECM proteins.

[0004] Globally, tissue fibrosis is a leading cause of disability and death in many diseases. Its main pathological changes are an increase in fibrous connective tissue and a decrease in parenchymal cells within organ tissues. If it progresses persistently, it leads to the destruction and dysfunction of organ structures, and eventually failure, seriously threatening human health and life. According to relevant statistics from the United States, approximately 45% of deaths from various diseases in that country are due to histiofibrosis. Currently, about 25% of the world's population suffers from non-alcoholic fatty liver disease (NALF). While NALF is treatable, delayed treatment can worsen it into NALF steatohepatitis. NALF is characterized by liver inflammation, hepatocyte death, and liver fibrosis, eventually progressing to cirrhosis and liver cancer. Pulmonary fibrosis is the end-stage change of lung disease, characterized by fibroblast proliferation and the accumulation of large amounts of extracellular matrix, accompanied by inflammatory damage and destruction of tissue structure. In other words, it refers to the occurrence of structural abnormalities (scar formation) due to abnormal repair after damage to normal alveolar tissue. The incidence and mortality rates of pulmonary fibrosis are increasing year by year. Pulmonary fibrosis is mainly sporadic, with an incidence rate of approximately 3-5 per 100,000 people, accounting for about 65% of all interstitial lung diseases. The average survival time after diagnosis is only 2.8 years, and the mortality rate is higher than that of most tumors, leading to it being called a "tumor-like disease." For this reason, the development of treatments for fibrotic diseases is particularly urgent.

[0005] Mature IL-11 (interleukin-11) is a polypeptide consisting of 178 amino acids with a molecular weight of approximately 23 kD. Three-dimensional structural studies of IL-11 have shown that it has a tetrahedral bundle structure consisting of four α-helices and a loop connecting the α-helices. IL-11 is a cytokine with multifaceted effects, a member of the IL-6 cytokine family, and shares the same signaling receptor subunit GP130. This family plays a crucial role in tumor development, progression, and metastasis. IL-11 production is triggered by known major pro-fibrotic factors, such as TGFβ, FGF, PDGF, CTGF, or IL-13, and local overexpression of IL-11 leads to localized tissue fibrosis. Activation of the IL-11 signaling pathway depends on the binding of IL-11 to its cell surface receptor, which consists of two glycoprotein chains: IL-11Ra and GP130. IL-11Ra can bind to ligands. IL-11 first binds to IL-11Ra with low affinity, forming an IL-11 / IL-11Ra heterodimer. This heterodimer then binds to GP130 with high affinity, forming a heterotrimeric protein. The IL-11 / IL-11Ra / GP130 heterotrimer homodimerizes to form a hexamer, which phosphorylates and activates the downstream STAT signaling pathway or MAPK cascade. Ultimately, IL-11 transmits signals into the cell via the GP130 signaling pathway, signaling cell proliferation and activation. Research has revealed that blocking this signaling pathway could be an effective treatment for various tumors, chronic fibrosis, and inflammatory diseases. Therefore, research and development of antibodies that bind to IL-11 and their antigen-binding fragments have significant clinical importance. [Overview of the Initiative]

[0006] This invention was obtained by screening antibodies and their antigen-binding fragments that specifically bind to IL-11 and inhibit the binding of IL-11 to its receptor, in order to meet the needs of patients with fibrotic diseases both domestically and internationally.

[0007] The specific technical solutions of this invention are as follows:

[0008] The present invention relates to an antibody or antigen-binding fragment that binds to IL-11, comprising 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 The amino acid sequence of the heavy chain complementarity determination region HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain complementarity determination region HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the heavy chain complementarity determination region HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the light chain complementarity determination region LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of the light chain complementarity determination region LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain complementarity determination region LCDR3 is shown in SEQ ID NO: 6. The amino acid sequence of the heavy chain complementarity determination region HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain complementarity determination region HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the heavy chain complementarity determination region HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the light chain complementarity determination region LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of the light chain complementarity determination region LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain complementarity determination region LCDR3 is shown in SEQ ID NO: 7, A-II, The amino acid sequence of the heavy chain complementarity determination region HCDR1 is shown in SEQ ID NO: 8, the amino acid sequence of the heavy chain complementarity determination region HCDR2 is shown in SEQ ID NO: 9, the amino acid sequence of the heavy chain complementarity determination region HCDR3 is shown in SEQ ID NO: 10, the amino acid sequence of the light chain complementarity determination region LCDR1 is shown in SEQ ID NO: 11, the amino acid sequence of the light chain complementarity determination region LCDR2 is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain complementarity determination region LCDR3 is shown in SEQ ID NO: 13 for A-III, and The present invention provides an antibody or antigen-binding fragment that binds to IL-11, which is one of the antibodies selected from the group consisting of A-IV, where the amino acid sequence of the heavy chain complementarity determination region HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain complementarity determination region HCDR2 is shown in SEQ ID NO: 14, the amino acid sequence of the heavy chain complementarity determination region HCDR3 is shown in SEQ ID NO: 15, the amino acid sequence of the light chain complementarity determination region LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of the light chain complementarity determination region LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain complementarity determination region LCDR3 is shown in SEQ ID NO: 6.

[0009] The antibodies or antigen-binding fragments provided by the present invention can specifically bind to IL-11, inhibit the binding of the IL-11 antigen to its receptor, and inhibit IL-11-mediated signal transduction. The antibodies provided by the present invention have good biological activity.

[0010] The present invention further includes a heavy chain variable region and a light chain variable region. The antibody or its antigen-binding fragment is The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17, MA-I, MA-II, in which the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18. MA-III, in which the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 19 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 20, and MA-IV, in which the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 21 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17. It is one of the following selected from the group consisting of [the specified elements].

[0011] The present invention further comprises a heavy chain steady region and a light chain steady 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, and SEQ ID NO: 26. The amino acid sequence of the light chain constant region is shown in Sequence ID No. 22.

[0012] Furthermore, the antibody or its antigen-binding fragment is a chimeric antibody molecule. The aforementioned chimeric antibody molecule further comprises a constant region of a human-derived antibody.

[0013] The present invention further includes a heavy chain variable region and a light chain variable region. The antibody or its antigen-binding fragment is The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 32, HA-IA, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 34, HA-IB, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 35, HA-IC, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 37, HA-ID, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 38, HA-II-A, HA-II-B in which the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 39, HA-II-C in which the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 33 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 39, and HA-II-D in which the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 40, which is any one selected from the group consisting of.

[0014] In the present invention, the antibody or its antigen-binding fragment further comprises a human-derived antibody constant region.

[0015] Furthermore, the human-derived antibody constant region includes a human-derived antibody heavy chain constant region and a human-derived antibody light chain constant region, the amino acid sequence of the human-derived antibody heavy chain constant region is one of those shown in SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the amino acid sequence of the human-derived antibody light chain constant region is shown in SEQ ID NO: 30.

[0016] Furthermore, the antibody or its antigen-binding fragment is one or a combination of multiple ones of Fab, F(ab’)2, Fv and ScFv.

[0017] The present invention further provides a nucleic acid molecule encoding the antibody or its antigen-binding fragment that binds to the above IL-11.

[0018] The present invention further provides a recombinant DNA expression vector containing the above nucleic acid molecule.

[0019] The present invention is a host cell transfected with the above recombinant DNA expression vector, where the host cell includes a prokaryotic cell, a yeast cell, an insect cell or a mammalian cell, Preferably, the host cell is a mammalian cell, and the mammalian cell is a HEK293 cell, a CHO cell or an NS0 cell, and a host cell is further provided.

[0020] The present invention further provides a drug comprising an antibody that binds to the IL-11 or an antigen-binding fragment thereof.

[0021] The present invention further provides a detection reagent used for detecting an antibody that binds to the IL-11 or an antigen-binding fragment thereof.

[0022] The present invention further provides a combined drug composition comprising a first composition comprising an antibody that binds to the IL-11 or an antigen-binding fragment thereof, and a second composition selected from an antibody that binds to PD-1, PD-L1, VEGF, VEGFR, TNF-α or TSLP or an antigen-binding fragment thereof.

[0023] The present invention further provides the use of an antibody that binds to the IL-11 or an antigen-binding fragment thereof in the manufacture of a drug for treating or preventing human fibrotic diseases, inflammation, cancer or autoimmune diseases.

[0024] Preferably, the fibrotic disease includes fibrosis of the heart, liver, kidney, lung, gallbladder, bladder, stomach, bone marrow, penis, breast, blood vessel, eye, pancreas, spleen, brain, intestinal tract, muscle or skin.

[0025] Preferably, the inflammation includes hepatitis, myocarditis, nephritis, pneumonia, cholecystitis, cystitis, gastritis, osteomyelitis, prostatitis, mastitis, pancreatitis, enteritis, arthritis, polymyositis, dermatomyositis or dermatitis.

[0026] Preferably, the cancer includes leukemia, lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer or bladder cancer.

[0027] Preferably, the autoimmune disease includes psoriasis, Crohn's disease, primary biliary cirrhosis, systemic lupus erythematosus or multiple sclerosis.

[0028] The beneficial effects of the present invention are as follows: The antibodies or antigen-binding fragments provided by the present invention have a high binding ability to the IL-11 antigen, inhibit the binding of the IL-11 antigen to its receptor, thereby effectively inhibiting the fibrotic effect of IL-11, inhibiting or preventing the production or proliferation of fibrous cells, and thus effectively treating or preventing fibrotic diseases, inflammation, cancer, or autoimmune diseases in humans. Here, fibrotic diseases include, but are not limited to, fibrosis of the heart, liver, kidneys, lungs, gallbladder, bladder, stomach, bone marrow, penis, mammary glands, blood vessels, eyes, pancreas, spleen, brain, intestines, muscles, 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, stomach 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 explanation of the drawing]

[0029] [Figure 1] Figure 1 shows the plasmid profile of the pScFv-Disb-HS vector in Example 2 of the present invention. [Figure 2] Figure 2 shows a comparison of the affinity of anti-IL-11 phage monoclonal antibodies by serial dilution ELISA in Example 3 of the present invention. [Figure 3] Figure 3 shows the profile of the vector pTSE in Example 5 of the present invention. [Figure 4] Figure 4 is an electrophoresis diagram of a denatured polyacrylamide gel of a mouse-derived antibody molecule in Example 5 of the present invention. [Figure 5] Figure 5 is a comparative diagram of the binding ability of mouse-derived antibody molecules to IL-11 in Example 6 of the present invention. [Figure 6]Figure 6 is a comparative diagram of competitive inhibition experiments between mouse-derived antibodies and the IL-11 receptor protein IL-11RA in Example 7 of the present invention. [Figure 7] Figure 7 is a comparative diagram of the inhibition of IL-11 binding to the BaF / 3-IL-11RA cell surface IL-11RA receptor by mouse-derived antibodies in Example 8 of the present invention. [Figure 8] Figure 8 is a comparative diagram of the inhibition of TIMP-1 secretion from fetal lung fibroblasts (MRC-5) by mouse-derived antibodies in Example 9 of the present invention. [Figure 9] Figure 9 is an electrophoresis diagram of a denatured polyacrylamide gel of a humanized antibody molecule in Example 14 of the present invention. [Figure 10] Figure 10 is a comparative diagram of the binding ability of humanized antibody molecules to IL-11 in Example 18 of the present invention. [Figure 11] Figure 11 is a comparative diagram of the inhibition of IL-11 binding to the BaF / 3-IL-11RA cell surface IL-11RA receptor by humanized antibody molecules in Example 19 of the present invention. [Figure 12] Figure 12 is a comparative diagram of the inhibition of IL-11 binding to the BaF / 3-GP130 cell surface GP130 receptor by humanized antibody molecules in Example 20 of the present invention. [Figure 13] Figure 13 is a comparative diagram of the biological activity assay (reporter gene) of the humanized antibody molecule in Example 21 of the present invention. [Figure 14] Figure 14 is a comparative diagram of the inhibition of TIMP-1 secretion from fetal lung fibroblasts (MRC-5) by a humanized antibody molecule in Example 22 of the present invention. [Figure 15] Figure 15 is a comparative diagram of cross-binding experiments between humanized antibody molecules and IL-11 derived from different species in Example 23 of the present invention. [Figure 16] Figure 16 is a bar graph showing the change in the ratio of lung weight to body weight in a mouse lung fibrosis model in Example 24 of the present invention. [Figure 17]Figure 17 shows hematoxylin-eosin (HE) staining and Masson staining of lung tissue sections in a mouse lung fibrosis model in Example 24 of the present invention. [Figure 18] Figure 18 is a bar graph showing the change in the ratio of heart weight to body weight in a mouse cardiac fibrosis model in Example 25 of the present invention. [Figure 19] Figure 19 shows hematoxylin-eosin (HE) staining and Masson staining of cardiac tissue sections in a mouse cardiac fibrosis model in Example 25 of the present invention. [Figure 20] Figure 20 is a bar graph showing the urinary protein content in the kidney of a mouse renal fibrosis model in Example 26 of the present invention. [Figure 21] Figure 21 shows hematoxylin-eosin (HE) staining and Masson staining of kidney tissue sections in a mouse renal fibrosis model in Example 26 of the present invention. [Figure 22] Figure 22 is a bar graph showing the change in liver weight in the mouse liver fibrosis model in Example 27 of the present invention. [Figure 23] Figure 23 is a bar graph showing the changes in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mouse serum in a mouse liver fibrosis model in Example 27 of the present invention. [Figure 24] Figure 24 shows hematoxylin-eosin (HE) staining and Masson staining of liver tissue sections in a mouse liver fibrosis model in Example 27 of the present invention. [Figure 25] Figure 25 shows the thermal stability of the anti-IL-11 monoclonal antibody HA-IA in Example 28 of the present invention. [Modes for carrying out the invention]

[0030] To facilitate understanding of the present invention, some technical and scientific terms of the present invention will be explained below before describing the examples.

[0031] As used herein, the term “antibody” includes complete antibodies and any antigen-binding fragments thereof, and antibodies include mouse-derived antibodies, humanized antibodies, bispecific antibodies, or chimeric antibodies. Antibodies may also be fragments such as Fab, F(ab')2, Fv, or ScFv (single-chain antibodies), and antibodies may be naturally occurring antibodies or modified antibodies (e.g., mutations, deletions, substitutions, etc.), as long as they exhibit binding to the relevant target molecule. As used herein, “antibody” includes its fragments and derivatives, synthetic antibodies and fragments. As used herein, an antibody is a polypeptide that can specifically bind to the relevant target molecule (i.e., the antigen to which the antibody is specific).

[0032] As used herein, the terms "variable region" and "constant region" refer to the sequence region near the N-terminus of the antibody's heavy and light chains as the variable region (V region), and the remaining amino acid sequence near the C-terminus, which is relatively stable, as the constant region (C region). The variable region contains three complementarity-determining regions (CDRs) and four framework regions (FRs), and each light chain variable region and heavy chain variable region consists of three CDR regions and four FR regions. The three CDR regions of the heavy chain are represented as HCDR1, HCDR2, and HCDR3, respectively, and the three CDR regions of the light chain are represented as LCDR1, LCDR2, and LCDR3, respectively.

[0033] As used herein, the term "mouse-derived antibody molecule" refers to antibodies obtained from mice immunized with human IL-11 antigen.

[0034] As used herein, the term "chimeric antibody molecule" refers to an antibody formed by fusing the variable region of a mouse-derived antibody with the constant region of a human-derived antibody, which can mitigate the immune response induced in the human body by mouse-derived antibodies. Chimeric antibodies are produced by using DNA recombination technology to insert the light chain and heavy chain variable region genes of a mouse-derived monoclonal antibody into an expression vector containing the constant region of a human-derived antibody. In the antibody molecule thus expressed, the light chain and heavy chain variable regions are mouse-derived, while the constant region is human-derived, resulting in approximately two-thirds of the entire antibody molecule being human-derived. The antibody produced in this way reduces the immunogenicity of the mouse-derived antibody while maintaining its ability to specifically bind to the antigen of the parent antibody.

[0035] As used herein, the term "humanized antibody molecule" refers to a human-derived antibody in which the CDR of a mouse-derived monoclonal antibody has been transplanted into the variable region of a human-derived antibody, in order to acquire the antigen-binding specificity of a mouse-derived monoclonal antibody and to reduce its heterogeneity.

[0036] The term "CHO ​​cells" refers to Chinese hamster ovary cells. The term "HEK293E cells" refers to human embryonic kidney 293E cells. The term "NS0 cells" refers to mouse NS0 thymoma cells.

[0037] In this specification, “IL-11” refers to IL-11 from any species and includes isoforms, fragments, variants, or homologs of IL-11 from any species. As used herein, a “fragment,” “variant,” or “homolog” of a protein may be optionally characterized as having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of a reference protein. In some embodiments, the fragments, variants, isoforms, and homologs of the reference protein may be characterized as having the function of the reference protein.

[0038] The present invention will be described in more detail below with reference to examples. Example 1

[0039] Example 1 of the present invention provides an antibody or antigen-binding fragment that binds to IL-11, comprising 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 antigen-binding fragment is selected from the following, where the following CDRs are determined based on the Kabat numbering system.

[0040] [Table 1] Example 2: Screening of mouse-derived antibody molecules

[0041] This invention involves immunizing mice with the IL-11 antigen (in subsequent experiments, the IL-11 protein, IL-11-Fc antigen, and IL-11-mFc ligand protein are all human IL-11), optimizing the immunization method, and creating a phage display library. The specific construction and screening identification of the phage display library were as follows. Step 1: Immunizing mice with IL-11 antigen

[0042] 1. Experimental animals: Species: BALB / c, female, mouse, weight: 18~20g, Laboratory animal supplier: Beijing Huafukang Biotechnology Co., Ltd.

[0043] 2. Immunization: Mice were immunized, and the immune antigen used was human IL-11 (synthesized gene from Nanjing Jinsirui Biotechnology Co., Ltd., which our company constructed, expressed, and purified as a vector).

[0044] Step 2: Construction of the Phage Antibody Library: Splenocytes from mice with high titers were collected, and total RNA was extracted from the mouse splenocytes using Trizol reagent (purchased from Ambion, catalog number: 15596026). cDNA was obtained by RT-PCR. Using the cDNA as a template, PCR amplification was performed using degenerate primers (see reference: Journal of Immunological Methods 233(2000) 167-177 for the degenerate primers used), thereby obtaining the antibody heavy chain variable region gene library (VH) and light chain variable region gene library (VL) from immunized mice. The pScFv-Disb-HS vector was created by modifying the pComb3 vector (purchased from the China Plasmid Vector Cell Gene Preservation Center) using a series of gene cloning methods so that it could be used for the construction and expression of a phage single-chain antibody library. The modified vector was named the pScFv-Disb-HS vector, and its plasmid profile is shown in Figure 1. A mouse immunophage antibody library was constructed based on this vector. The light chain and heavy chain variable region gene libraries were each treated with double restriction enzymes and then ligated to the pScFv-Disb-HS vector, which had been similarly treated stepwise with restriction enzymes, to construct the pScFv-Disb-HS-VH-VL gene library.

[0045] Step 3: Immunotubules were coated with IL-11 as the antigen, with an antigen coating amount of 5 μg / 500 μL / tubule. After coating overnight at 4°C, the immunotubules and immunophage antibody library were blocked with 4% skim milk powder / PBST, respectively, and blocked at room temperature for 1 hour. The blocked immunophage antibody library was added to the immunotubules to perform antigen-antibody binding, and the phage input amount was approximately 10 9 ~10 12 After reacting the phages at room temperature for 1 hour, any phages that did not bind were washed with PBST-PBS, eluted with 0.1 M Glycine-HCl at pH 2.2, and finally neutralized the eluted phage antibody solution with 1.5 M Tris-HCl at pH 8.8 to approximately pH 7.0.

[0046] Step 4: The neutralized phages were infected with 10 ml of TG1 bacterial suspension grown in the logarithmic growth phase. After standing in a 37°C incubator for 30 minutes, a portion of the suspension was collected, serially diluted, and spread onto a 2YTAG plate to calculate the phage production. The remaining suspension was centrifuged, the supernatant was discarded, the cell precipitate was resuspended in a small amount of culture medium, aspirated, and spread onto a larger 2YTAG plate in preparation for the next screening.

[0047] Step 5: After infection, the bacterial cells spread on the plate were scraped from the larger plate, inoculated into 2YTAG liquid medium, and cultured with shaking until the logarithmic growth phase. Then, M13KO7 helper phage was added to co-infect the cells, and the cells were cultured overnight at 28°C at 220 rpm to prepare the phages. The purified phages were precipitated with PEG / NaCl and used for the next screening, performing a total of one phage library-rich screening.

[0048] Step 6: Screening of IL-11 phage single-chain antibody-positive clones: After one screening, well-isolated monoclonal colonies were selected and inoculated into 96-well deep-well plates containing 2YTAG liquid medium. They were cultured at 37°C and 220 rpm until the logarithmic growth phase. Approximately 10 per well 10Helper phage M13KO7 was added, and the cells were allowed to infect statically for 30 minutes at 37°C. The cells were centrifuged at 4000 rpm for 15 minutes, the supernatant was discarded, and the cells were resuspended in 2YTAK and precipitated. The cells were then cultured overnight at 28°C and 220 rpm. After centrifuging at 4000 rpm and 4°C for 15 minutes, the amplified phage supernatant was aspirated, and ELISA identification was performed. Finally, four highly affinity mouse-derived antibody molecules were obtained through screening and named MA-I, MA-II, MA-III, and MA-IV, respectively. Gene sequencing was performed on the obtained monoclonal antibodies to determine the correct antibody sequences. The sequences of the four monoclonal antibodies obtained in the above screening were as follows.

[0049] [Table 2]

[0050] Specifically, Sequence ID: 16 (amino acid sequence of the heavy chain variable region of MA-I and MA-II): EVKLEESGGGLVKPGGSLKLSCAASGFTFSDYYMFWVRQTPEKRLEWVATISDGGTYTYYPDSVKGRFTISRDNAKNNLYLQMTSLKSEDTAMYYCARDGGYVSSPEAMDYWGQGTSVTVSS, Sequence ID: 17 (amino acid sequence of the light chain variable region of MA-I and MA-IV): DIVLTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPPTFGGGTKLEIK, Sequence ID: 18 (Amino acid sequence of the light chain variable region of MA-II): DIVLTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK, Sequence ID: 19 (Amino acid sequence of the heavy chain variable region of MA-III): EVKLEQSGAEVVKPGALVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGVIDPSDSYTTYNQKFKGKATLTVDTSSSTGYMQLSSLTSEDSAVYYCSQYGYDVNWYFDVWGAGTTVTVSS, Sequence ID: 20 (Amino acid sequence of the light chain variable region of MA-III): DIVMTQTTLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK, Sequence ID: 21 (Amino acid sequence of the heavy chain variable region of MA-IV): EVQLEESGGGLVKPGGSLKLSCVASGFTFSDYYMFWVRQTPEKRLEWVATISDGGSYSYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTAMYYCARDGGYISSPEAMDYWGQGTSVTVSS.

[0051] From another perspective, the present invention also relates to an antibody or antigen-binding fragment that binds to IL-11, specifically comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementarity-determining regions represented by HCDR1, HCDR2, and HCDR3, respectively, and the light chain variable region comprises three light chain complementarity-determining regions represented by LCDR1, LCDR2, and LCDR3, respectively. Here, HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NOs: 16, 19, or 21, respectively, and LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NOs: 17, 18, or 20, respectively. Those skilled in the art can determine the CDR sequence of a heavy chain variable region or light chain variable region whose amino acid sequence is known based on a common numbering system (e.g., Kabat, AbM, Chothia, Contact, or IMGT). When determining the CDR using the Kabat numbering system, the CDR sequences for the heavy chain variable region and the light chain variable region are as shown in Example 1. Example 3: Comparison of antibody affinity by serial dilution ELISA

[0052] The four mouse-derived 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 their affinity was identified by phage serial dilution ELISA experiments. The specific method was as follows.

[0053] IL-11 antigen was coated with 100 ng / well / 100 μl of carbonate buffer at pH 9.6 and coated overnight at 4°C. After washing three times with PBST, the four phage monoclonal antibodies obtained from the screening in Example 2 were each serially diluted 5-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. The ELISA plate was washed with PBST, and the HRP-anti-M13 monoclonal antibody (purchased from Bio-viewshine, catalog number: GE27-9421-01), diluted with 1% BSA-PBST, was added to the ELISA plate and allowed to stand at room temperature for 1 hour. The samples were developed using a TMB chromogenic reagent kit (purchased from Kangwei Century, catalog number: CW0050S) for 10 minutes at room temperature, then terminated with 2M H2SO4. The values ​​were then read at 450 nm / 630 nm using a microplate reader, and the corresponding EC50 values ​​were calculated. The specific data was as follows:

[0054] [Table 3]

[0055] As shown in the data above and Figure 2, all four different mouse-derived antibody molecules obtained in the screening of Example 2 were able to bind to IL-11. From this, it was clear that all monoclonal antibodies provided by the present invention have high affinity for IL-11. Example 4

[0056] Example 4 of the present invention is based on Example 2 and further comprises an antibody or antigen-binding fragment further comprising a heavy chain constant region and a light chain constant region, wherein 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 and SEQ ID NO: 26, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 22. The specific sequence is as follows.

[0057] Sequence ID: 22 (Amino acid sequence of the constant region of the mouse Cκ-type light chain): ADAAPTVSIFPPSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC, Sequence ID: 23 (Amino acid sequence of the constant region of the mouse IgG1 heavy chain): AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVH TAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG, Sequence ID: 24 (Amino acid sequence of the constant region of the mouse IgG2a heavy chain): AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEEDDPDVQISWFVNNV EVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK, Sequence ID: 25 (Amino acid sequence of the constant region of the mouse IgG2b heavy chain): AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFV NNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK, Sequence ID: 26 (Amino acid sequence of the constant region of the mouse IgG3 heavy chain): ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGK EFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPELELNETCAEAQDGELDGLWTTITIFISLFLLSVCYSASVTLFKVKWIFSSVVQVKQTAIPDYRNMIGQGA. Example 5 Preparation of mouse-derived antibody molecules

[0058] Example 5 of the present invention is based on Example 4 and is preferably limited to the fact that the mouse-derived antibody molecule includes a mouse IgG1 heavy chain constant region (its amino acid sequence is shown in SEQ ID NO: 23) and a mouse Cκ light chain constant region (its amino acid sequence is shown in SEQ ID NO: 22). The antibody preparation method is specifically as follows.

[0059] 1. The heavy chain VH and light chain VL coding genes of the four antibody molecules screened in Example 2 were cloned into a vector pTSE (as shown in Figure 3) containing the heavy chain and light chain constant region genes, respectively. Preferably, the heavy chain constant region was the mouse IgG1 type constant region (amino acid sequence shown in SEQ ID NO: 23), and the light chain constant region was the mouse-derived Cκ chain (amino acid sequence shown in SEQ ID NO: 22). The pTSE vector structure was as shown in Figure 3 (see paragraph

[0019] on page 3 of specification CN103525868A for the pTSE vector manufacturing process).

[0060] 2. HEK293 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number: GNHu43) were transiently transfected to express antibodies. Using an AKTA instrument, the antibodies were purified using a protein A affinity column to obtain four monoclonal antibodies. Simultaneously, the protein concentration was measured using a BCA reagent kit (purchased from Beijing Huitian Oriental Technology Co., Ltd., catalog number: BCA0020), and the protein size was subsequently identified by SDS-PAGE. The results are shown in Figure 4. From left to right, the images show unreduced MA-I, MA-II, MA-III, MA-IV, protein molecular weight Marker 1, protein molecular weight Marker 2, and reduced MA-I, MA-II, MA-III, MA-IV mouse-derived anti-IL-11 monoclonal antibodies. The molecular weight sizes of each band were consistent with theoretical values. Example 6: Binding experiment between mouse-derived antibody molecule and IL-11

[0061] IL-11 antigen was coated at 100 ng / well / 100 μl using pH 9.6 carbonate buffer and coated overnight at 4°C. After washing five times with 300 μL / well PBST, 280 μL / well of 1% BSA-PBST was added and the mixture was blocked for 1 hour at 37°C. MA-I, MA-II, MA-III, and MA-IV mouse-derived antibody molecules at different dilutions were added, with the initial maximum concentration of all four antibody molecules being 5 μg / mL. Each antibody was then serially diluted five-fold, resulting in a total of eight serial dilutions, and incubated for 1 hour at 37°C. After washing five times with 300 μL / well PBST, Goat Anti-Mouse IgG-HRP (purchased from solarbio, catalog number: SE131) diluted 1:2000 with 1% BSA-PBST was added and incubated for 1 hour at 37°C. The TMB chromogenic reagent kit was added to 100 μL / well and allowed to develop at room temperature for 8 minutes, after which the development was terminated with 2 M H2SO4. A microplate reader was used to read the values ​​at 450 nm / 630 nm, and the corresponding EC50 values ​​were calculated. The specific data were as follows:

[0062] [Table 4]

[0063] As shown in the data above and Figure 5, all four different mouse-derived antibody molecules obtained through screening were able to bind to IL-11, and all exhibited high affinity. Example 7: Competitive inhibition experiment of mouse-derived antibody and IL-11 receptor protein IL-11RA

[0064] IL-11-Fc was coated with 200 ng / well / 100 μL of carbonate buffer at pH 9.6 and coated overnight at 4°C. After washing five times with 300 μL / well using PBST, 280 μL / well of 1% BSA-PBST was added and the mixture was blocked for 1 hour at 37°C. First, 50 μL / well of IL-11RA-Fc (IgG type 4) diluted to 0.5 μg / mL with 1% BSA-PBST was added, followed by 50 μL / well of MA-I, MA-II, MA-III, and MA-IV mouse-derived antibodies at different dilutions. The initial maximum concentration of all five antibodies was 100 μg / mL, and each was serially diluted 2-fold, resulting in a total of 13 serial dilutions for each antibody, which were incubated for 3 hours at 37°C. After washing five times with 300 μL / well PBST, Anti-Human IgG4-HRP Mouse monoclonal antibody (purchased from Sigma, catalog number: SAB4200770), diluted 1:5000 with 2% BSA-PBST, was added and incubated at 37°C for 1 hour. TMB chromogenic reagent kit was added at 100 μL / well, and after development at room temperature for 15 minutes, development was terminated with 2M H2SO4. Numerical values ​​were read at 450 nm / 630 nm using a microplate reader, and the corresponding IC50 values ​​were calculated. Specific data were as follows:

[0065] [Table 5]

[0066] As shown in the data above and Figure 6, all four different mouse-derived antibodies obtained through screening were able to compete with the receptor protein IL-11RA. From this, it was clear that all four different mouse-derived antibodies could effectively inhibit the binding of IL-11 to the receptor protein IL-11RA.

[0067] Example 8: Inhibition of IL-11 binding to the BaF / 3-IL-11RA cell surface IL-11RA receptor by mouse-derived antibody.

[0068] BaF / 3-IL-11RA cell lines were counted, a certain number of cells were collected, centrifuged, resuspended in PBS buffer, and the cell density was adjusted to 1E+6 cells / mL. These cells were then added to a 96-well plate at 100 μL / well. IL-11-mFc ligand protein was diluted to a concentration of 18 μg / mL in PBS and added at 50 μL / well to the corresponding wells of the 96-well plate containing BaF / 3-IL-11RA cells. After light mixing, the 96-well plate was incubated at 4°C for 1 hour. Four mouse-derived antibody molecules, MA-I, MA-II, MA-III, and MA-IV, were serially diluted in PBS to an initial concentration of 800 μg / mL. These were then serially diluted 3-fold, for a total of 10 serial dilutions, and added at 50 μL / well to the corresponding wells of the 96-well plate containing the BaF / 3-IL-11RA cell and IL-11-mFc ligand protein mixture. After homogeneous mixing, the cells were incubated at 4°C for 2 hours. After incubation, the cells were centrifuged at 3000 rpm, washed once with PBS buffer, and the cell pellet was collected. The pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from Southern Biotech, catalog number: 1030-02) was added to the cell pellet, incubated at 4°C for 30 minutes, centrifuged at 3000 rpm, washed once with PBS buffer, resuspended in 100 μL of PBS buffer, and measured using a flow cytometer to collect the fluorescence signal in the FL1-A channel. Dose-effect curves were plotted, and the corresponding IC50 values ​​were calculated. The specific data were as follows.

[0069] [Table 6]

[0070] From the data above and Figure 7, it was found that all four different mouse-derived candidate molecules obtained through screening could effectively inhibit the binding of IL-11 ligand protein to the cell surface IL-11RA receptor.

[0071] Example 9: Inhibition of TIMP-1 secretion from fetal lung fibroblasts (MRC-5) using mouse-derived antibodies.

[0072] Fetal lung fibroblasts (MRC-5) were counted after trypsin treatment, a certain number of cells were collected, centrifuged, and resuspended in MEM complete medium (purchased from GIBCO, catalog number: 10370-021) to adjust the cell density to 2E+5 cells / mL. These cells were then added to 96-well plates at 100 μL / well. IL-11-mFc ligand protein was diluted to a concentration of 16 μg / mL in MEM complete medium and added to the corresponding 96-well plates at 50 μL / well. Four mouse-derived antibody molecules, MA-I, MA-II, MA-III, and MA-IV, were serially diluted in MEM complete medium to an initial concentration of 40 μg / mL. These were then 2-fold serially diluted, resulting in a total of eight serial dilutions. These dilutions were added to 96-well plates containing the cell suspension and IL-11-mFc ligand protein suspension at 50 μL / well, gently mixed, and incubated overnight at 37°C in a CO2 incubator for approximately 20 hours. Cell culture supernatants were collected and detected using the TIMP-1 ELISA reagent kit (purchased from Yikesai Biotechnology Co., Ltd., catalog number: EH021-96).

[0073] Human TIMP-1 detection reagent kit: Cell supernatant and standard were added to the sample wells at a rate of 100 μL / well. Immediately, 50 μL / well of biotinylated antibody working solution (1:100 dilution) was added, the plate was sealed with a sealing film, and incubated at room temperature with shaking for 2 hours. After incubation, the plate was washed four times with washing solution, and 100 μL / well of enzyme complex working solution (1:100 dilution) from the TIMP-1 detection reagent kit was added. The plate was sealed with a sealing film and incubated at room temperature with shaking for 1 hour. After incubation, the plate was washed four times with washing solution. 100 μL / well of TMB chromogenic solution was added, incubated at room temperature under light shielding for approximately 15 minutes, and then the reaction was stopped with 100 μL / well of Stop solution. Numerical values ​​were read at 450 nm using a microplate reader, and the corresponding IC50 values ​​were calculated. The specific data were as follows.

[0074] [Table 7]

[0075] From the data above and Figure 8, it was found that all four different mouse-derived candidate molecules obtained through screening could effectively inhibit the release of TIMP-1 from human fetal lung fibroblasts (MRC-5) stimulated by the IL-11 ligand protein. Example 10

[0076] Example 10 of the present invention is further limited to the fact that the antibody or its antigen-binding fragment is a chimeric antibody molecule, the chimeric antibody molecule further comprises a human-derived antibody constant region, the human-derived antibody constant region comprises a human-derived antibody heavy chain constant region and a human-derived antibody light chain constant region, the amino acid sequence of the human-derived antibody heavy chain constant region is one of those shown in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, and the amino acid sequence of the human-derived antibody light chain constant region is shown in SEQ ID NO: 30.

[0077] Sequence ID: 27 (Amino acid sequence of the constant region of the human IgG1 heavy chain): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, Sequence ID: 28 (Amino acid sequence of the constant region of the human IgG2 heavy chain): ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, Sequence ID: 29 (Amino acid sequence of the constant region of the human IgG4 heavy chain): ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK, Sequence ID: 30 (Amino acid sequence of the constant region of the human Cκ chain light chain): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. Example 11 Preparation of Chimeric Antibody Molecules

[0078] Example 11 of the present invention is based on Example 10 and further limits the human-derived antibody constant region to include a human IgG1 heavy chain constant region (its amino acid sequence is shown in SEQ ID NO: 27) and a human Cκ light chain constant region (its amino acid sequence is shown in SEQ ID NO: 30). Specific preparation method:

[0079] In Example 2, the heavy chain variable region VH (SEQ ID NO: 16), the light chain variable region VL gene (SEQ ID NO: 17) of the mouse-derived antibody molecules MA-I and MA-II obtained from the immunophage antibody library screening were preserved in their mouse-derived sequences and cloned into vector pTSEs (as shown in Figure 3) containing the heavy chain constant region and light chain constant region genes, respectively. The heavy chain constant region was human IgG1 type (amino acid sequence shown in SEQ ID NO: 27), and the light chain constant region was human Cκ type (amino acid sequence shown in SEQ ID NO: 30). HEK293E cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number: GNHu43) were transiently transfected to express antibodies and obtain chimeric antibodies CA-I and CA-II. Example 12 Humanization of mouse-derived antibody molecules

[0080] First, the sequences of mouse-derived antibody molecules MA-I and MA-II in Example 2 were compared with the human antibody germline database (v-base) to identify candidate light and heavy chain germline sequences of human antibodies with high homology. Next, the CDR sequences of mouse-derived antibody molecules MA-I and MA-II were transplanted into human-derived candidate sequences, and homology modeling was performed. Subsequently, three-dimensional structural simulation was used to calculate important framework amino acid residues that may play a crucial role in maintaining the CDR loop structure, thereby designing recovery mutations for humanized antibodies. The light and heavy chain variable region sequences of humanized antibodies, including the designed recovery mutations, were optimized and synthesized by Nanjing Jinsirui Biotechnology Co., Ltd., and then ligated into transient expression vectors. The combinations of light and heavy chains obtained after humanization were analyzed, and from MA-I, humanized antibody molecules HA-IA, HA-IB, HA-IC, and HA-ID were obtained. From MA-II, humanized antibody molecules HA-II-A, HA-II-B, HA-II-C, and HA-II-D were obtained. The eight monoclonal antibody sequences obtained from the above screening were as follows:

[0081] [Table 8]

[0082] Specifically, Sequence ID: 31 (amino acid sequence of the heavy chain variable region of HA-IA, HA-IC, HA-II-A, and HA-II-B): QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATISDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVSSPEAMDYWGQGTLVTVSS, Sequence ID: 32 (amino acid sequence of the light chain variable region of HA-IA): DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK, Sequence ID: 33 (amino acid sequence of the heavy chain variable regions of HA-IB and HA-II-C): QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVSTISDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVSSPEAMDYWGQGTLVTVSS, Sequence ID: 34 (amino acid sequence of the light chain variable region of HA-IB): DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK, Sequence ID: 35 (Amino acid sequence of the light chain variable region of HA-IC): DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK, Sequence ID: 36 (amino acid sequence of the heavy chain variable region of HA-ID and HA-II-D): QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATISDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAMYYCARDGGYVSSPEAMDYWGQGTSVTVSS, Sequence ID: 37 (amino acid sequence of the light chain variable region of HA-ID): DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK, Sequence ID: 38 (amino acid sequence of the light chain variable region of HA-II-A): DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK, Sequence ID: 39 (amino acid sequence of the light chain variable regions of HA-II-B and HA-II-C): DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK, Sequence ID: 40 (amino acid sequence of the light chain variable region of HA-II-D): DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPWTFGGGTKVEIK. Example 13

[0083] Example 13 of the present invention is based on Example 12 and further limits the fact that the constant region of the human-derived antibody comprises a constant region of the heavy chain of the human-derived antibody and a constant region of the light chain of the human-derived antibody, the amino acid sequence of the constant region of the heavy chain of the human-derived antibody is one of those shown in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, and the amino acid sequence of the constant region of the light chain of the human-derived antibody is shown in SEQ ID NO: 30.

[0084] The specific sequence of the constant region of the human-derived antibody described above was the same as in Example 10. Example 14 Preparation of humanized antibody molecules

[0085] Example 14 of the present invention is based on Example 13 and further limits the human-derived antibody constant region to include a human IgG1 heavy chain constant region (its amino acid sequence is shown in SEQ ID NO: 27) and a human Cκ light chain constant region (its amino acid sequence is shown in SEQ ID NO: 30).

[0086] The heavy chain VH and light chain VL coding genes of the eight humanized antibody molecules HA-IA, HA-IB, HA-IC, HA-ID, HA-II-A, HA-II-B, HA-II-C, and HA-II-D obtained by humanization in Example 12 were cloned into vector pTSE (as shown in Figure 3) containing the heavy chain constant region and light chain constant region genes, respectively. The heavy chain constant region was human IgG1 type (amino acid sequence shown in SEQ ID NO: 27), and the light chain constant region was the Cκ chain (amino acid sequence shown in SEQ ID NO: 30).

[0087] The two chimeric antibodies CA-I and CA-II obtained in Example 11, and the eight humanized antibody molecules HA-IA, HA-IB, HA-IC, HA-ID, HA-II-A, HA-II-B, HA-II-C, and HA-II-D obtained in Example 12 were transiently transfected into HEK293 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number: GNHu43) to induce antibody expression. Monoclonal antibodies were obtained by purification using a protein A affinity column with an AKTA instrument. Simultaneously, protein concentrations were measured using a BCA reagent kit (purchased from Beijing Huitian Oriental Technology Co., Ltd., catalog number: BCA0020), and protein sizes were subsequently identified by SDS-PAGE. The results are shown in Figure 9. From left to right, the non-reducing protein molecular weights are HA-IA, HA-IB, HA-IC, HA-ID, the chimeric antibody CA-I prepared in Example 11, the reduced protein molecular weights are Marker, HA-II-A, HA-II-B, HA-II-C, HA-II-D, and the chimeric antibody CA-II. The molecular weight sizes of each band were consistent with the theoretical values. Example 15

[0088] Example 15 limits the antibody or antigen-binding fragment disclosed in the present invention to one or more combinations of Fab, F(ab')2, Fv, and ScFv, and is not limited to the monoclonal antibodies disclosed in the present invention. Example 16

[0089] Example 16 of the present invention also provides a nucleic acid molecule encoding an antibody that binds to IL-11 or an antigen-binding fragment thereof.

[0090] The present invention also further provides a recombinant DNA expression vector comprising the nucleic acid molecule.

[0091] The present invention also relates to host cells transfected with the recombinant DNA expression vector, The host cells include prokaryotic cells, yeast cells, insect cells, or mammalian cells. More preferably, the host cell is a mammalian cell, and the mammalian cell is a HEK293 cell, a CHO cell, or an NS0 cell.

[0092] The present invention also provides a drug comprising an antibody or antigen-binding fragment thereof that binds to IL-11.

[0093] The present invention also provides a detection reagent used to detect an antibody or its antigen-binding fragment that binds to IL-11.

[0094] The present invention also provides a combination drug composition comprising an antibody or antigen-binding fragment that binds to IL-11, and a second composition selected from an antibody or antigen-binding fragment that binds to PD-1, PD-L1, VEGF, VEGFR, TNF-α, or TSLP.

[0095] Preferably, the antibody or antigen-binding fragment that binds to PD-1 is selected from DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12, DFPD1-13, Nivolumab, Pembrolizumab, Toripalimab, Sintilimab, Tislelizumab, Camrelizumab, Penpulimab, or Zimberelimab, where the heavy chain constant region and light chain constant region sequences of DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12, and DFPD1-13 are the same as the heavy chain constant region and light chain constant region of the anti-PD-1 monoclonal antibody provided in patent application no. CN201510312910.8.

[0096] Preferably, the antibody or antigen-binding fragment that binds to VEGF is selected from aflibercept, bevacizumab, or ranibizumab. Example 17

[0097] Example 17 of the present invention provides the use of an antibody or antigen-binding fragment that binds to IL-11 in the manufacture of a drug for treating or preventing human fibrotic diseases, inflammation, cancer, or autoimmune diseases.

[0098] Preferably, fibrotic diseases include, but are not limited to, fibrosis of the heart, liver, kidneys, lungs, gallbladder, bladder, stomach, bone marrow, penis, mammary glands, blood vessels, eyes, pancreas, spleen, brain, intestines, muscles, or skin.

[0099] Preferably, inflammation includes, but is not limited to, hepatitis, myocarditis, nephritis, pneumonia, cholecystitis, cystitis, gastritis, osteomyelitis, prostatitis, mastitis, pancreatitis, enteritis, arthritis, polymyositis, dermatomyositis, or dermatitis.

[0100] Preferably, cancer includes, but is not limited to, leukemia, lung cancer, stomach cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, or bladder cancer.

[0101] Preferably, autoimmune diseases include, but are not limited to, psoriasis, Crohn's disease, primary biliary cirrhosis, systemic lupus erythematosus, or multiple sclerosis.

[0102] The present invention also provides a method for treating or preventing a subject of human fibrotic disease, inflammation, cancer, or autoimmune disease, the method comprising administering to the subject a therapeutic or prophylactic effective amount of an antibody or antigen-binding fragment that binds to IL-11. Example 18: Binding experiment between humanized antibody molecule and IL-11

[0103] IL-11 antigen was coated with 100 ng / well / 100 μL of carbonate buffer at pH 9.6 and coated overnight at 4°C. After washing five times with 300 μL / well of PBST, 280 μL / well of 1% BSA-PBST was added and the mixture was blocked for 1 hour at 37°C. Humanized antibodies HA-IA, HA-IB, HA-IC, HA-ID, HA-II-A, HA-II-B, HA-II-C, HA-II-D and the chimeric antibodies CA-I and CA-II prepared in Example 11 were diluted with 1% BSA-PBST. The initial concentration of each humanized antibody was 10 μg / mL, and these were serially diluted five-fold to a total of eight levels, which were incubated for 1 hour at 37°C. After washing five times with 300 μL / well PBST, goat anti-human IgG Fab HRP (purchased from Invitrogen, catalog number: 31482), diluted 1:5000 with 1% BSA-PBST, was added and incubated at 37°C for 1 hour. The samples were then colored using a TMB chromogenic reagent kit, with 100 μL / well added at room temperature for 5 minutes, followed by 2M H2SO4 to complete the color development. Numerical values ​​were read at 450 nm / 630 nm using a microplate reader, and the corresponding EC50 values ​​were calculated. The specific data were as follows:

[0104] [Table 9]

[0105] As shown in the data above and the experimental results in Figure 10, all eight different humanized antibody molecules were able to bind to IL-11. Furthermore, the EC50 values ​​of the HA-IA, HA-IB, HA-IC, and HA-ID humanized antibody molecules were all close to those of the chimeric antibody CA-I, and the EC50 values ​​of the HA-II-A, HA-II-B, HA-II-C, and HA-II-D humanized antibody molecules were all close to those of the chimeric antibody CA-II. This indicates that the humanized antibody molecules maintain the high binding ability of the mouse-derived parental antibodies MA-I and MA-II to IL-11.

[0106] Example 19: Inhibition of IL-11 binding to the BaF / 3-IL-11RA cell surface IL-11RA receptor by a humanized antibody molecule.

[0107] Four humanized antibody molecules with superior protein-level binding activity—HA-IA, HA-IB, HA-IC, and HA-ID—were selected, and cell activity evaluation experiments were conducted. BaF / 3-IL-11RA cell lines were counted, a certain number of cells were collected, centrifuged, resuspended in PBS buffer, and the cell density was adjusted to 1E+6 cells / mL. These cells were then added to a 96-well plate at 100 μL / well. IL-11-mFc ligand protein was diluted to a concentration of 18 μg / mL in PBS and added at 50 μL / well to the corresponding wells of the 96-well plate containing BaF / 3-IL-11RA cells. After light mixing, the 96-well plate was incubated at 4°C for 1 hour. Four humanized antibody molecules, HA-IA, HA-IB, HA-IC, and HA-ID, were serially diluted in PBS buffer to an initial concentration of 800 μg / mL. This was then 3-fold serially diluted, resulting in a total of 10 serial dilutions. 50 μL / well of each solution was added to the corresponding wells of a 96-well plate containing BaF / 3-IL-11RA cells and an IL-11-mFc ligand protein mixture. After homogeneous mixing, the mixtures were incubated at 4°C for 2 hours. After incubation, the cells were centrifuged at 3000 rpm, washed once with PBS buffer, and the cell pellet was collected. A pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from Southern Biotech, catalog number: 1030-02) was added to the cell pellet. After incubation at 4°C for 30 minutes, the cells were centrifuged at 3000 rpm, washed once, and resuspended in 100 μL of PBS. The fluorescence signal within the FL1-A channel was collected by flow cytometry. A dose-effect curve was plotted, and the corresponding IC50 value was calculated. The specific data was as follows:

[0108] [Table 10]

[0109] From the above data and Figure 11, it was found that all four humanization candidate molecules obtained through screening can inhibit the binding of IL-11 ligand protein to the BaF / 3-IL-11RA cell surface IL-11RA receptor. Example 20: Inhibition of IL-11 binding to the BaF / 3-GP130 cell surface GP130 receptor by a humanized antibody molecule.

[0110] BaF / 3-GP130 cell lines were counted, a certain number of cells were collected, centrifuged, resuspended in PBS buffer, and the cell density was adjusted to 1E+6 cells / mL. These cells were then added to a 96-well plate at 100 μL / well. IL-11-mFc ligand protein was diluted in PBS to a concentration of 12 μg / mL and added to the corresponding wells of the 96-well plate containing BaF / 3-GP130 cells at 50 μL / well. After light mixing, the 96-well plate was incubated at 4°C for 1 hour. Four human-derived antibody molecules, HA-IA, HA-IB, HA-IC, and HA-ID, were serially diluted in PBS buffer to an initial concentration of 2000 μg / mL. These were then serially diluted 2-fold, resulting in a total of 10 serial dilutions. These dilutions were then added to the corresponding wells of the 96-well plate containing BaF / 3-GP130 cells and IL-11-mFc ligand protein at 50 μL / well. After homogeneous mixing, the 96-well plate was incubated at 4°C for 2 hours. After incubation, the cells were centrifuged at 3000 rpm, washed once with PBS buffer, and the cell pellet was collected. 100 μL / well of pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from Southern Biotech, catalog number: 1030-02) was added to the cell pellet and incubated at 4°C for 30 minutes. After centrifugation at 3000 rpm and washing once with PBS buffer, the cells were resuspended in 100 μL / well of PBS buffer and measured using a flow cytometer to collect the fluorescence signal in the FL1-A channel. Dose-effect curves were plotted and the corresponding IC50 values ​​were calculated. The specific data were as follows.

[0111] [Table 11]

[0112] From the data above and Figure 12, it was found that all four humanization candidate molecules obtained through screening could inhibit the binding of IL-11 ligand protein to the GP130 receptor on the surface of BaF / 3-GP130 cells. Example 21: Biological activity assay of humanized antibody molecules (reporter gene)

[0113] The BaF / 3-GP130-STAT3-Luc engineered cell line (construction method of the BaF / 3-GP130-STAT3-Luc engineered cell line: plasmid GP130 (purchased from Sino Biological Inc., catalog number: HG10974-UT) and plasmid STAT3-Luc-NeoR (purchased from Genomeditech (Shanghai) Co., Ltd., catalog number: GM-021003) were introduced into BAF / 3 cells (purchased from the National Center for Experimental Cell Resources of China) by two electroporations, and the cells were screened to obtain the BaF / 3-GP130-STAT3-Luc engineered cell line) were counted, the cell density was adjusted to 2E+6 cells / mL using a sample diluent (containing 90% IMDM, 10% FBS, and 10 ng / mL mouse IL-3), lightly mixed, and then 50 μL / well of the cell solution was added to a 96-well plate. Four types of humanized antibody molecules, HA-IA, HA-IB, HA-IC, and HA-ID, were each diluted to an initial concentration of 200 μg / ml with sample diluent, then serially diluted 5-fold for a total of 10 serial dilutions. These dilutions were added at 100 μL / well to the corresponding wells of a 96-well plate containing the engineered cell line, with two duplicate wells set up for each sample concentration. IL-11 protein was prepared using the sample diluent, adjusted to a concentration of 10 μg / mL, and added at 50 μL / well to the 96-well plate containing the engineered cell line and humanized antibody molecules. The cell culture plates were gently mixed and incubated in a 37°C CO2 incubator for 6 hours. The supernatant was removed by centrifugation, lysis solution was added, and the mixture was added to a 384-well plate at 10 μL / well. An equal volume of luciferase reaction substrate (purchased from Promega Corporation, catalog number: E2610) was added, and after a 5-minute reaction at room temperature, fluorescence values ​​were read using a microplate reader, and the corresponding IC50 values ​​were calculated. The specific data was as follows:

[0114] [Table 12]

[0115] As shown in the data above and Figure 13, all four humanized antibody molecules obtained through screening inhibit the binding of IL-11 to IL-11RA and GP130 receptors, thereby inhibiting the transmission of signaling pathways.

[0116] Example 22: Inhibition of TIMP-1 secretion from fetal lung fibroblasts (MRC-5) by a humanized antibody molecule.

[0117] Fetal lung fibroblasts (MRC-5) were counted after trypsin treatment, a certain number of cells were collected, centrifuged, and resuspended in MEM complete medium to adjust the cell density to 2E+5 cells / mL. These cells were then added to 96-well plates at 100 μL / well. IL-11-mFc ligand protein was diluted in MEM complete medium to adjust the concentration to 16 μg / mL and added to the corresponding 96-well plates at 50 μL / well. Four humanized antibody molecules, HA-IA, HA-IB, HA-IC, and HA-ID, were serially diluted in MEM complete medium to adjust the initial concentration to 40 μg / mL. These were then 3-fold serially diluted, resulting in a total of 8 serial dilutions. These were added to 96-well plates containing the cell suspension and IL-11-mFc ligand protein suspension at 50 μL / well, gently mixed uniformly, and incubated overnight in a 37°C CO2 incubator for approximately 20 hours. Cell culture supernatants were collected and detected using the TIMP-1 ELISA reagent kit (the method was the same as in Example 9). Numerical values ​​were read at 450 nm using a microplate reader, and the corresponding IC50 values ​​were calculated. The specific data were as follows:

[0118] [Table 13]

[0119] From the data above and Figure 14, it was found that all four humanized antibody molecules obtained through screening could effectively inhibit the release of TIMP-1 from human fetal lung fibroblasts (MRC-5) stimulated by the IL-11 ligand protein. Example 23: Cross-binding experiment between humanized antibody molecules and IL-11 from different species.

[0120] Humanized antibody molecules HA-IA, exhibiting superior protein levels and functional activity, were selected, and cross-binding assays were performed with IL-11 from different species. Human IL-11, mouse IL-11 (purchased from Sino Biological Inc., catalog number: 50117-MNCE), rat IL-11 (purchased from Shanghai Kanglang Biological Technology Co., Ltd., catalog number: KL40001Ra), and cynomolgus monkey IL-11 (purchased from Sino Biological Inc., catalog number: 90925-CNCE) were coated with 100 ng / well / 100 μL of carbonate buffer, respectively, and coated overnight at 4°C. After washing five times with 300 μL / well of PBST, 280 μL / well of 1% BSA-PBST was added, and the samples were blocked for 1 hour at 37°C. Humanized antibody HA-IA was diluted with 1% BSA-PBST to an initial concentration of 50 μg / mL, then serially diluted 5-fold for a total of 9 serial dilutions. Each serial dilution was prepared in two duplicate wells, and 100 μL / well was added to a 96-well plate and incubated at 37°C for 1 hour. After washing 5 times with 300 μL / well PBST, goat anti Human IgG Fab HRP (purchased from invitrogen, catalog number: 31482) was diluted with 1% BSA-PBST to a working solution concentration of 1:5000, and 100 μL / well was added to a 96-well plate and incubated at 37°C for 1 hour. After washing 5 times with 300 μL / well PBST, the solution was developed using a TMB chromogenic reagent kit, and after development at room temperature and protected from light for 5 minutes with 100 μL / well, the development was terminated with 2 M H2SO4. A microplate reader was used to read values ​​at 450nm / 630nm, and the corresponding EC50 values ​​were calculated. The specific data was as follows:

[0121] [Table 14]

[0122] As shown in the data above and Figure 15, the humanized antibody molecules HA-IA were all able to bind to human IL-11, mouse IL-11, rat IL-11, and cynomolgus monkey IL-11, and all exhibited high affinity. Example 24: Experiment on the therapeutic effect of anti-IL-11 monoclonal antibody on pulmonary fibrosis

[0123] We created a model using bleomycin (bLF) and studied the therapeutic effect of the anti-IL-11 monoclonal antibody HA-IA on pulmonary fibrosis.

[0124] Animal species: C57BL / 6J mouse (purchased from GemPharmatech LLC.) Quantity, sex, and age: 6 individuals / group, male, 6-8 weeks old The control group received only saline injections. The treatment group received HA-IA antibody molecules by injection twice a week for four weeks. The animals' body weight was measured once a week, and any abnormalities were observed. Organ weight detection: Lung organs were collected, their weight was measured, and the ratio of lung weight to body weight was calculated. Pulmonary pathology detection: Lung sections were prepared, and the degree of pulmonary fibrosis was observed using hematoxylin-eosin (HE) staining and Masson staining.

[0125] The results are shown in Figure 16. After administration of the HA-IA antibody molecule, the ratio of lung weight to body weight in the treated mice was significantly lower compared to the control group. The results are shown in Figure 17. Compared to the control group, lung fibrosis was significantly reduced in lung sections of the treated group. This indicates that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the development of lung fibrosis. Example 25: Experiment on the therapeutic effect of anti-IL-11 monoclonal antibody on cardiac fibrosis

[0126] We created a model using isoproterenol and studied the therapeutic effect of the anti-IL-11 monoclonal antibody HA-IA on cardiac fibrosis.

[0127] Animal species: C57BL / 6J mouse (purchased from GemPharmatech LLC.) Quantity, sex, and age: 6 individuals / group, male, 6-8 weeks old The control group received only saline injections. The treatment group received HA-IA antibody molecules by injection twice a week for four weeks. Animal body weight was measured once a week to observe for any abnormalities. Organ weight detection: The heart was retrieved, its weight was measured, and the ratio of heart weight to body weight was calculated. Cardiac pathology detection: Cardiac sections were prepared, and the degree of cardiac fibrosis was observed using hematoxylin-eosin (HE) staining and Masson staining.

[0128] The results are shown in Figure 18. The ratio of heart weight to body weight in the treated mice was significantly lower compared to the control group. The results are shown in Figure 19. Compared to the control group, cardiac fibrosis was significantly reduced in cardiac sections of the treated mice. This indicates that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the development of cardiac fibrosis. Example 26: Experiment on the therapeutic effect of anti-IL-11 monoclonal antibody on renal fibrosis.

[0129] We created a model using doxorubicin (dKF) and studied the therapeutic effect of the anti-IL-11 monoclonal antibody HA-IA on renal fibrosis.

[0130] Animal species: BALB / c mouse (purchased from GemPharmatech LLC.) Quantity, sex, and age: 6 individuals / group, male, 6-8 weeks old The control group received only saline injections. The treatment group received HA-IA antibody molecules by injection twice a week for four weeks. Animal body weight was measured once a week to observe for any abnormalities. Organ weight detection: Kidneys were collected, their weight was measured, and urinary protein content was detected. Renal pathology detection: Kidney sections were prepared, and the degree of renal fibrosis was observed using hematoxylin-eosin (HE) staining and Masson staining.

[0131] The results are shown in Figure 20. Compared to the control group, the amount of urinary protein in the kidneys of mice in the treatment group was significantly lower. The results are shown in Figure 21. Compared to the control group, kidney sections of mice in the treatment group showed a significant reduction in renal fibrosis. This indicates that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the development of renal fibrosis. Example 27: Experiment on the therapeutic effect of anti-IL-11 monoclonal antibody on liver fibrosis.

[0132] We created a model using CCl4 and studied the therapeutic effect of the anti-IL-11 monoclonal antibody HA-IA on liver fibrosis.

[0133] Animal species: C57BL / 6J mouse (purchased from GemPharmatech LLC.) Quantity, sex, and age: 6 individuals / group, male, 6-8 weeks old The control group received only saline injections. The treatment group received injections of HA-IA antibody molecules twice a week for four weeks. Weight monitoring: Animal weight was measured once a week, and the presence or absence of abnormalities in the animals was observed. Liver pathology detection: Liver sections were prepared, and the degree of liver fibrosis was observed using hematoxylin-eosin (HE) staining and Masson staining. Organ weight detection: Liver was collected, its weight was measured, and HE staining was performed. Serum detection: Serum was collected, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum were detected.

[0134] The results are shown in Figure 22. Compared to the control group, the liver weight of mice in the treatment group was significantly lower. The results are shown in Figure 23. Serum ALT and AST levels in mice in the treatment group were significantly lower compared to the control group. The results are shown in Figure 24. Compared to the control group, liver fibrosis was significantly reduced in liver sections of the treatment group. This indicates that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the development of liver fibrosis. Example 28 Evaluation of the thermal stability of the anti-IL-11 monoclonal antibody HA-IA

[0135] The thermal stability of the anti-IL-11 monoclonal antibody HA-IA was evaluated using a multifunctional protein thermal stability analysis system (purchased from Unchained Labs). Changes in protein structure were detected by monitoring the temperature-dependent changes in the protein's intrinsic fluorescence (starting at 25°C and increasing to 95°C at a heating rate of 0.3°C / min). This allowed for the determination of the protein's melting temperature (Tm) and evaluation of its structural stability. When the sample aggregated, scattered light interfered, increasing the scattered light signal. The colloidal stability of the protein was measured by static light scattering (characterized by Tag), and the results are shown in the table and Figure 25 below.

[0136] [Table 15]

[0137] The anti-IL-11 monoclonal antibody HA-IA had a melting temperature (Tm) of 79.5°C and an average Tag temperature of 75.9°C, demonstrating good structural and colloidal stability.

[0138] The present invention is not limited to the preferred embodiments described above, and anyone may obtain various other forms of products based on the suggestions of the present invention, but any changes to their shape or structure are included within the scope of protection of the present invention as long as they are identical or similar to the technical solutions of the present application.

Claims

1. An antibody or antigen-binding fragment that binds to IL-11, comprising 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 The amino acid sequence of the heavy chain complementarity determination region HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain complementarity determination region HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the heavy chain complementarity determination region HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the light chain complementarity determination region LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of the light chain complementarity determination region LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain complementarity determination region LCDR3 is shown in SEQ ID NO:

6. A-I, The amino acid sequence of the heavy chain complementarity determination region HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain complementarity determination region HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the heavy chain complementarity determination region HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the light chain complementarity determination region LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of the light chain complementarity determination region LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain complementarity determination region LCDR3 is shown in SEQ ID NO:

7. A-II, The amino acid sequence of the heavy chain complementarity determination region HCDR1 is shown in SEQ ID NO: 8, the amino acid sequence of the heavy chain complementarity determination region HCDR2 is shown in SEQ ID NO: 9, the amino acid sequence of the heavy chain complementarity determination region HCDR3 is shown in SEQ ID NO: 10, the amino acid sequence of the light chain complementarity determination region LCDR1 is shown in SEQ ID NO: 11, the amino acid sequence of the light chain complementarity determination region LCDR2 is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain complementarity determination region LCDR3 is shown in SEQ ID NO: 13, A-III, and The amino acid sequence of the heavy chain complementarity determination region HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain complementarity determination region HCDR2 is shown in SEQ ID NO: 14, the amino acid sequence of the heavy chain complementarity determination region HCDR3 is shown in SEQ ID NO: 15, the amino acid sequence of the light chain complementarity determination region LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of the light chain complementarity determination region LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain complementarity determination region LCDR3 is shown in SEQ ID NO:

6. A-IV, An antibody that binds to IL-11 or an antigen-binding fragment thereof, characterized by being one selected from the group consisting of the following.

2. It includes heavy chain variable regions and light chain variable regions. The antibody or its antigen-binding fragment is The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17, MA-I, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18, MA-II, MA-III, in which the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 19 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 20, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 21, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17, MA-IV, An antibody or antigen-binding fragment thereof that binds to IL-11, characterized in that it is one selected from the group consisting of the following.

3. It further includes the heavy chain steady region and the light chain steady 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, and SEQ ID NO:

26. The antibody or antigen-binding fragment that binds to IL-11, according to claim 2, characterized in that the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

22.

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

5. It includes heavy chain variable regions and light chain variable regions. The antibody or its antigen-binding fragment is The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 32, HA-I-A, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 34, HA-I-B, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 35, HA-I-C, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 37, HA-I-D, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 38, HA-II-A, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 39, HA-II-B, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 39, HA-II-C, and The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 40, HA-II-D, An antibody or antigen-binding fragment thereof that binds to IL-11, characterized in that it is one selected from the group consisting of the following.

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

7. The human-derived antibody constant region includes a human-derived antibody heavy chain constant region and a human-derived antibody light chain constant region. The amino acid sequence of the constant region of the human-derived antibody heavy chain is one of those shown in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO:

29. The antibody or antigen-binding fragment that binds to IL-11 according to claim 4 or 6, characterized in that the amino acid sequence of the constant region of the human-derived antibody light chain is shown in SEQ ID NO:

30.

8. The antibody or antigen-binding fragment thereof that binds to IL-11, as described in claim 1, characterized in that the antibody or antigen-binding fragment thereof is a combination of one or more types from Fab, F(ab')2, Fv, and ScFv.

9. A nucleic acid molecule characterized by encoding an antibody that binds to IL-11 as described in claim 1, or an antigen-binding fragment thereof.

10. A recombinant DNA expression vector characterized by comprising the nucleic acid molecule described in claim 9.

11. A host cell transfected with the recombinant DNA expression vector described in claim 10, The host cells include prokaryotic cells, yeast cells, insect cells, or mammalian cells. Preferably, the host cell is a mammalian cell, and the mammalian cell is a HEK293 cell, a CHO cell, or an NS0 cell.

12. A drug comprising an antibody that binds to IL-11 as described in claim 1, or an antigen-binding fragment thereof.

13. A detection reagent characterized by being used to detect an antibody or antigen-binding fragment thereof that binds to IL-11 as described in claim 1.

14. A combination drug composition characterized by comprising an antibody or antigen-binding fragment that binds to IL-11 as described in claim 1, and a second composition selected from an antibody or antigen-binding fragment that binds to PD-1, PD-L1, VEGF, VEGFR, TNF-α, or TSLP.

15. Use of an antibody or antigen-binding fragment thereof that binds to IL-11 according to claim 1 in the manufacture of a drug for treating or preventing human fibrotic diseases, inflammation, cancer or autoimmune diseases.

16. The use according to claim 15, characterized in that the fibrotic disease includes fibrosis of the heart, liver, kidneys, lungs, gallbladder, bladder, stomach, bone marrow, penis, mammary glands, blood vessels, eyes, pancreas, spleen, brain, intestines, muscles, or skin.

17. The use according to claim 15, characterized in that the inflammation includes hepatitis, myocarditis, nephritis, pneumonia, cholecystitis, cystitis, gastritis, osteomyelitis, prostatitis, mastitis, pancreatitis, enteritis, arthritis, polymyositis, dermatomyositis, or dermatitis.

18. The use according to claim 15, characterized in that 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.

19. The use according to claim 15, characterized in that the autoimmune disease includes psoriasis, Crohn's disease, primary biliary cirrhosis, systemic lupus erythematosus, or multiple sclerosis.

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