A method for detecting biological activity of anti-il-11 monoclonal antibody

Abstract

The application relates to the field of biological medicine, and specifically provides a detection method for the biological activity of an anti-IL-11 monoclonal antibody, which comprises the following steps: constructing a cell strain; stimulating the expression of a reporter gene in the cell strain by using an IL-11 / IL-11RA complex; adding the anti-IL-11 monoclonal antibody into the cell strain for incubation; adding a chromogenic substrate to detect the signal value of the reporter gene. The reporter gene method provided by the application can quickly and accurately detect the biological activity of the anti-IL-11 monoclonal antibody, meanwhile, the application constructs a stable and passable cell strain BAF / 3-GP130-STAT3-Luc which is suitable for the anti-IL-11 monoclonal antibody, can guarantee the stability of the detection process, and improves the accuracy of the biological activity detection; the method is simple, fast, low in cost, short in cycle, stable and reliable in result, and high in accuracy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine activity detection, in particular to a method for detecting the biological activity of anti-IL-11 monoclonal antibody. BACKGROUND

[0002] Interleukin-11 (IL-11) is a pleiotropic cytokine produced by a variety of cell types, mainly from white blood cells, fibroblasts and epithelial cells. The factor is composed of 199 amino acids, with a molecular weight of about 20 KDa. The corresponding genomic sequence is 7 kb, including 5 coding exons, located on chromosome 19q13.3-19q13.4. IL-11 belongs to the interleukin 6 family, has anti-apoptotic, anti-inflammatory and hematopoietic potential and other biological activities. The various activities of IL-11 are mediated by its cell surface receptor complex, and the IL-11 receptor complex is formed through three independent events: first, IL-11 binds to the membrane-specific receptor alpha (IL-11RA) with low affinity; second, IL-11 / IL-11RA heterodimer binds to receptor gp130 with high affinity to form a heterotrimer; finally, the heterotrimer binds to form a hexamer complex, activates the Janus kinase / signal transduction and transcriptional activator (JAK / STAT) pathway, the mitogen-activated protein kinase (MAPK) pathway and the mToR / PI3K pathway, and stimulates the transformation of lung fibroblasts into fibroblasts through an extracellular signal-regulated kinase (ERK)-dependent post-transcriptional manner, and promotes organ fibrosis. Therefore, inhibiting the production of IL-11 as a new treatment strategy for treating fibrotic diseases.

[0003] Studies have shown that anti-IL-11 monoclonal antibody can significantly reduce the collagen fiber content of fibroblasts cultured in vitro by blocking the activation of downstream signaling pathways through binding to IL-11, and reduce the fibrosis area of fibrosis model mice. Therefore, anti-IL-11 monoclonal antibody drugs have great potential as new anti-fibrosis drugs. In the research of anti-IL-11 monoclonal antibody, the biological activity detection method of anti-IL-11 monoclonal antibody to IL-11RA and IL-11 binding signaling pathway is very important, which is related to the therapeutic efficacy of anti-IL-11 monoclonal antibody, and is an important indicator of the effectiveness of anti-IL-11 monoclonal antibody product.

[0004] The currently commonly used biological activity detection methods of antibody drugs mainly include cell proliferation inhibition method, ELISA, surface plasmon resonance (SPR) and reporter gene method, etc. Different methods have their advantages and disadvantages. In order to meet the research and quality control of anti-IL-11 monoclonal antibody, it is urgent to develop a detection method suitable for the biological activity of anti-IL-11 monoclonal antibody. SUMMARY

[0005] In order to realize the detection of biological activity of anti-IL-11 monoclonal antibody, meet the drug research and quality control, the application discloses a detection method of biological activity of anti-IL-11 monoclonal antibody.

[0006] The specific technical scheme of the application is as follows:

[0007] The application provides a detection method of biological activity of anti-IL-11 monoclonal antibody, and the detection method comprises the following steps:

[0008] S1, constructing a stable passaged cell strain BAF / 3-GP130-STAT3-Luc;

[0009] S2, stimulating and activating the expression of a reporter gene in the cell strain BAF / 3-GP130-STAT3-Luc by an IL-11 / IL-11RA complex;

[0010] S3, adding gradient-diluted anti-IL-11 monoclonal antibody to the cell strain BAF / 3-GP130-STAT3-Luc for incubation;

[0011] S4, adding a chromogenic substrate, determining a reporter gene signal value, fitting a four-parameter curve according to the concentration of the anti-IL-11 monoclonal antibody and the reporter gene signal value, and detecting the biological activity of the anti-IL-11 monoclonal antibody.

[0012] The detection method provided by the application has the following mechanism: the anti-IL-11 monoclonal antibody competes with IL-11RA to bind IL-11, blocks the trimeric signal pathway formed by GP130 on the surface of the cell strain BAF / 3-GP130-STAT3-Luc, IL-11 and IL-11RA, and reduces the expression of luciferase of the cell strain BAF / 3-GP130-STAT3-Luc, so that the biological activity is detected according to the reporter gene signal value; in the method, the concentration of the anti-IL-11 monoclonal antibody is inversely proportional to the expression amount of luciferase.

[0013] Further, the anti-IL-11 monoclonal antibody comprises three heavy chain complementarity determining regions denoted as HCDR1, HCDR2 and HCDR3 respectively, and three light chain complementarity determining regions denoted as LCDR1, LCDR2 and LCDR3 respectively, the amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown as SEQ ID No: 1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown as SEQ ID No: 2, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown as SEQ ID No: 3, the amino acid sequence of the light chain complementarity determining region LCDR1 is shown as SEQ ID No: 4, the amino acid sequence of the light chain complementarity determining region LCDR2 is shown as SEQ ID No: 5, and the amino acid sequence of the light chain complementarity determining region LCDR3 is shown as SEQ ID No: 6.

[0014] Further, the anti-IL-11 monoclonal antibody further comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No: 31, and the amino acid sequence of the light chain variable region is shown as SEQ ID No: 32.

[0015] Further, the anti-IL-11 monoclonal antibody further comprises 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 SEQ ID No: 27, SEQ ID No: 28 or SEQ ID No: 29, and the amino acid sequence of the human antibody light chain constant region is shown as SEQ ID No: 30.

[0016] Further, in step S1, constructing the stably passaged cell strain BAF / 3-GP130-STAT3-Luc comprises the following method:

[0017] S11, transfecting the plasmid GP130 into BAF / 3 cells to obtain the cell strain BAF / 3-GP130 after screening;

[0018] S12, transfecting the plasmid STAT3-Luc-NeoR into the cell strain BAF / 3-GP130 to obtain the stable cell strain BAF / 3-GP130-STAT3-Luc after screening.

[0019] Further, in step S2, the following steps are specifically included:

[0020] S21, inoculating the cell strain BAF / 3-GP130-STAT3-Luc into a 96-well plate at a cell inoculation density of 2×10 4 -1×10 5 cells per well;

[0021] S22, the IL-11 / IL-11RA complex is added to a 96-well plate to stimulate the expression of the reporter gene in the cell strain BAF / 3-GP130-STAT3-Luc.

[0022] Further, the IL-11 / IL-11RA complex comprises IL-11 antigen with a stimulating final concentration of 0.02-40 μg / mL and IL-11RA antigen with a stimulating final concentration of 0.0025-2.5 μg / mL, and the ratio of the stimulating final concentrations of the IL-11 antigen and the IL-11RA antigen is 1-16:1.

[0023] Further, in step S3, the initial concentration of the gradient-diluted anti-IL-11 monoclonal antibody is 500 μg / mL, and the ratio of the equi-proportional dilution is 0.2-0.5:1.

[0024] Further, in step S3, the incubation time is 2-6 hours.

[0025] The present application also provides a method for detecting the biological activity of the anti-IL-11 monoclonal antibody, which can be used for quality control in the production and research of anti-IL-11 monoclonal antibody drugs.

[0026] The present application has the following advantages: the reporter gene method provided by the present application can quickly and accurately detect the biological activity of anti-IL-11 monoclonal antibody, and the stable passaging cell strain BAF / 3-GP130-STAT3-Luc suitable for anti-IL-11 monoclonal antibody is constructed in the present application, which can ensure the stability of the detection process and improve the accuracy of the biological activity detection. The method is simple, fast, low-cost, short-cycle, stable and reliable, and has high accuracy. The luciferase substrate used does not contain DTT, which is more conducive to the safe operation of the experimenters. The method provided by the present application can be used for quality control and clinical application of anti-IL-11 monoclonal antibody drugs, and has high application value. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is the plasmid map of the pScFv-Disb-HS vector in Example 2 of the present application;

[0028] 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 application;

[0029] Figure 3 It is the map of the vector pTSE in Example 5 of the present application;

[0030] Figure 4 It is the denatured polyacrylamide gel electrophoresis chart of the murine antibody molecule in Example 5 of the present application;

[0031] Figure 5 Figure for comparing the binding ability of the mouse-derived antibody molecule in Example 6 of the present application to IL-11;

[0032] Figure 6 Figure for comparing the competitive inhibition experiment of the mouse-derived antibody to IL-11 receptor protein IL-11RA in Example 7 of the present application;

[0033] Figure 7 Figure for comparing the inhibition of IL-11 binding to IL-11RA receptor on the surface of BaF / 3-IL-11RA cells by the mouse-derived antibody in Example 8 of the present application;

[0034] Figure 8 Figure for comparing the inhibition of TIMP-1 secretion from embryonic lung fibroblast MRC-5 by the mouse-derived antibody in Example 9 of the present application;

[0035] Figure 9 Figure for denaturing polyacrylamide gel electrophoresis of the humanized antibody molecule of Example 14 of the present application;

[0036] Figure 10 Figure for comparing the binding ability of the humanized antibody molecule in Example 15 of the present application to IL-11;

[0037] Figure 11 Figure for comparing the inhibition of IL-11 binding to IL-11RA receptor on the surface of BaF / 3-IL-11RA cells by the humanized antibody molecule in Example 16 of the present application;

[0038] Figure 12 Figure for comparing the inhibition of IL-11 binding to GP130 receptor on the surface of BaF / 3-GP130 cells by the humanized antibody molecule in Example 17 of the present application;

[0039] Figure 13 Figure for comparing the inhibition of TIMP-1 secretion from embryonic lung fibroblast MRC-5 by the humanized antibody molecule in Example 18 of the present application;

[0040] Figure 14 Figure for comparing the cross-binding experiment of the humanized antibody molecule to IL-11 of different species in Example 19 of the present application;

[0041] Figure 15 Figure for the change in the ratio of lung weight to body weight in the mouse pulmonary fibrosis model in Example 20 of the present application;

[0042] Figure 16 Figure for hematoxylin-eosin (HE) staining and Masson staining of lung tissue sections in the mouse pulmonary fibrosis model in Example 20 of the present application;

[0043] Figure 17The bar chart of the change of the ratio of heart weight to body weight in the mouse heart fibrosis model in Example 21 of the present application;

[0044] Figure 18 The hematoxylin-eosin (HE) staining and Masson staining diagrams of the heart tissue section in the mouse heart fibrosis model in Example 21 of the present application;

[0045] Figure 19 The bar chart of the urine protein content in the kidney in the mouse kidney fibrosis model in Example 22 of the present application;

[0046] Figure 20 The hematoxylin-eosin (HE) staining and Masson staining diagrams of the kidney tissue section in the mouse kidney fibrosis model in Example 22 of the present application;

[0047] Figure 21 The bar chart of the change of the liver weight in the mouse liver fibrosis model in Example 23 of the present application;

[0048] Figure 22 The bar chart of the change of the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the serum of the mouse in the mouse liver fibrosis model in Example 23 of the present application;

[0049] Figure 23 The hematoxylin-eosin (HE) staining and Masson staining diagrams of the liver tissue section in the mouse liver fibrosis model in Example 23 of the present application;

[0050] Figure 24 The diagram for evaluating the heat stability of the anti-IL-11 monoclonal antibody HA-I-A in Example 24 of the present application;

[0051] Figure 25 The four-parameter curve diagram of the fluorescence signal under the stimulation of the IL-11 / IL-11RA complex containing only different concentrations of IL-11 antigen in Example 36 of the present application;

[0052] Figure 26 The four-parameter curve diagram of the inhibitory activity of the anti-IL-11 monoclonal antibody HA-I-A under the stimulation of different concentrations of IL-11 antigen in the IL-11 / IL-11RA complex in Example 36 of the present application;

[0053] Figure 27 The four-parameter curve diagram of the inhibitory activity of the anti-IL-11 monoclonal antibody HA-I-A under the stimulation of different ratios of the final concentration of IL-11 antigen to IL-11RA antigen in Example 36 of the present application;

[0054] Figure 28 The four-parameter curve diagram of the cell line BAF / 3-GP130-STAT3-Luc under different cell inoculation densities in Example 36 of the present application;

[0055] Figure 29 Figure 17 is a four-parameter curve plot of the anti-IL-11 monoclonal antibody HA-I-A at different dilution ratios in Example 36 of the present application;

[0056] Figure 30 Figure 18 is a four-parameter curve plot of the cell line BAF / 3-GP130-STAT3-Luc at different incubation times in Example 36 of the present application;

[0057] Figure 31 Figure 19 is a dose-effect curve plot of different target antibodies in Example 37 of the present application;

[0058] Figure 32 Figure 20 is a linear fitting plot of the measured values and theoretical values of the relative biological activity of different potency antibodies in Example 37 of the present application;

[0059] Figure 33 Figure 21 is a dose-effect curve plot of the cell line BAF / 3-GP130-STAT3-Luc at different passages in Example 37 of the present application. DETAILED DESCRIPTION

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition, other terms not specifically defined herein have the meanings that would be given to them by a person skilled in the art with the benefit of the present disclosure. Furthermore, all patents and publications referenced herein are incorporated by reference in their entirety.

[0061] The term "antibody" as used herein includes whole antibodies and any antigen-binding fragment thereof, including murine, humanized, bispecific or chimeric antibodies, and fragments such as Fab, F(ab)2, Fv or ScFv (single chain antibody), etc. Antibodies can be naturally occurring antibodies or antibodies altered by changes such as mutations, deletions, substitutions, etc., as long as they exhibit binding to the relevant target molecule. The "antibody" herein includes fragments and derivatives thereof, including synthetic antibodies and fragments. As used herein, an antibody is a polypeptide that is capable of specifically binding to a relevant target molecule (i.e., the antigen to which the antibody is specific).

[0062] The terms "variable region" and "constant region" as used herein refer to the sequence regions of the heavy and light chains of antibodies that are proximal to the N-terminus as variable regions (V regions) and the remaining amino acid sequences proximal to the C-terminus as constant regions (C regions). The variable region includes 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 denoted as HCDR1, HCDR2 and HCDR3, and the three CDR regions of the light chain are denoted as LCDR1, LCDR2 and LCDR3.

[0063] The term "murine antibody molecule" as used herein refers to an antibody obtained from a mouse immunized with a human IL-11 antigen.

[0064] The term "chimeric antibody molecule" as used herein refers to an antibody in which the variable region of a murine antibody is fused to the constant region of a human antibody, which can reduce the immune response induced by the murine antibody in the human body. The chimeric antibody is obtained by inserting the light and heavy chain variable region genes of a murine monoclonal antibody into an expression vector containing the constant region of a human antibody, so that the light and heavy chain variable regions of the expressed antibody molecule are of murine origin, and the constant region is of human origin. The entire antibody molecule is about 2 / 3 of human origin, so that the antibody produced reduces the immunogenicity of the murine antibody while retaining the ability of the parent antibody to specifically bind to the antigen.

[0065] The term "humanized antibody molecule" as used herein refers to an antibody in which the CDR of a murine monoclonal antibody is transplanted to the variable region of a human antibody, replacing the CDR of the human antibody, so that the human antibody acquires the antigen binding specificity of the murine monoclonal antibody while reducing its heterogeneity.

[0066] The term "CHO cell" refers to a Chinese hamster ovary cell; the term "HEK293E cell" refers to a human embryonic kidney 293E cell, and the term "NS0 cell" refers to a mouse NS0 thymoma cell.

[0067] The term "IL-11" as used herein refers to interleukin 11 (leptin) from any species, and includes isoforms, fragments, variants or homologues of IL-11 from any species, including but not limited to leukocytes, fibroblasts and epithelial cells produced in humans, etc. As used herein, a "fragment", "variant" or "homologue" of a protein can optionally be 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 to the amino acid sequence of a reference protein. In some embodiments, the fragments, variants, isoforms and homologues of the reference protein can be characterized as being capable of having the functions possessed by the reference protein.

[0068] The term "IL-11RA" as used herein refers to interleukin 11 receptor alpha from any species, and includes isoforms, fragments, variants or homologues of IL-11Rα from any species.

[0069] The term "anti-IL-11 monoclonal antibody" as used herein refers to an antibody capable of inhibiting IL-11 pathway signal transduction by binding to IL-11, thereby blocking the interaction between IL-11 and IL-11RA, GP130, so that the IL-11 / IL-11RA / GP130 pathway signal transduction is inhibited.

[0070] The term "JAK / STAT" as used herein refers to the JAK / STAT signal transduction pathway activated by cytokine stimulation, which is involved in many important biological processes such as cell proliferation, differentiation, apoptosis, and immune regulation.

[0071] The term "biological activity" as used herein refers to the specific ability or potential of a biological product to achieve a certain biological effect, and the biological effect of a specific cell line can be used to evaluate the corresponding activity of the biological product.

[0072] The term "four-parameter curve" as used herein refers to a curve fitted according to the four-parameter equation Y = Bottom + (Top-Bottom) / (1+10^((LogIC50-X)*HillSlpoe)).

[0073] BAF / 3 cells, i.e. mouse pro-B cells, are antibody-forming cell precursors in lymphocytes of peripheral lymphoid system tissues such as spleen and lymph nodes.

[0074] The present application will be further described in detail below in conjunction with the following examples.

[0075] Example 1

[0076] The present application provides a method for detecting the biological activity of an anti-IL-11 monoclonal antibody, which comprises the following steps:

[0077] A stable passaged cell line BAF / 3-GP130-STAT3-Luc is constructed; an IL-11 / IL-11RA complex is used to stimulate the expression of a reporter gene in the cell line BAF / 3-GP130-STAT3-Luc; a gradient-diluted anti-IL-11 monoclonal antibody is added to the cell line BAF / 3-GP130-STAT3-Luc for incubation; a chromogenic substrate is added, and the reporter gene signal value is determined; according to the concentration of the anti-IL-11 monoclonal antibody and the reporter gene signal value, a four-parameter curve is fitted, i.e. the biological activity of the anti-IL-11 monoclonal antibody is detected.

[0078] The anti-IL-11 monoclonal antibody 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 specific sequences are as follows

[0079]

[0080] The anti-IL-11 monoclonal antibody provided by the application is used for treating or preventing human fibrosis diseases, inflammation, cancer or autoimmune diseases, wherein the fibrosis diseases include but are not limited to fibrosis of heart, liver, kidney, lung, gallbladder, bladder, stomach, bone marrow, penis, breast, blood vessel, eye, pancreas, spleen, brain, intestinal tract, muscle or skin, etc.; 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, etc.; the cancer includes but is not limited to 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, etc.; and the autoimmune disease includes but is not limited to psoriasis, Crohn's disease, primary biliary cirrhosis, systemic lupus erythematosus or multiple sclerosis, etc.

[0081] Example 2: Screening of mouse-derived antibody molecules

[0082] The application optimizes the immunization method by immunizing mice with IL-11 antigen (the IL-11 protein, IL-11-Fc antigen and IL-11-mFc ligand protein in subsequent experiments are all human IL-11), and creates a phage display library. The construction and screening identification of the specific phage display library are as follows:

[0083] Step one: Immunize mice with IL-11 antigen

[0084] 1. Experimental animals: species and strain: BALB / c, female, mice; body weight: 18-20 g; provider of experimental animals: Beijing Huafukang Biotechnology Co., Ltd.

[0085] 2. Immunization: Immunize the mice, and the immunization antigen is human IL-11 antigen (synthetic gene by Nanjing Jinssw Biological Technology Co., Ltd., and the vector is constructed and expressed and purified by the company).

[0086] Step two: Construction of phage antibody library: Take the mouse spleen cells with high titer, use Trizol reagent (purchased from Ambion, item number: 15596026) to extract total RNA in mouse spleen cells, obtain cDNA by RT-PCR, use degenerate primers (the degenerate primers used are referred to in the literature: Journal of Immunological Methods 233 (2000) 167-177) for PCR amplification, thereby obtaining the immune mouse antibody heavy chain variable region gene library (VH) and light chain variable region gene library (VL). The pScFv-Disb-HS vector is a vector pComb3 vector (purchased from China Plasmid Vector Strain Cell Strain Gene Preservation Center) modified by a series of gene cloning methods for the construction and expression of phage single-chain antibody library. The modified vector is named pScFv-Disb-HS vector, and its plasmid map is shown in Figure 1 , and based on this vector, a mouse immune phage antibody library is constructed. The light and heavy chain variable region gene libraries are double-digested and ligated to the vector pScFv-Disb-HS which has also been treated by stepwise digestion, to construct the pScFv-Disb-HS-VH-VL gene library.

[0087] Step three: Coat the immunotube with IL-11 as the antigen, the antigen coating amount is 5 μg / 500 μL / tube, coat overnight at 4°C, then use 4% skim milk / PBST to block the immunotube and the immune phage antibody library, respectively, block at room temperature for 1 h. After blocking, the immune phage antibody library is added to the immunotube for antigen-antibody binding, the phage input amount is about 10 9 ~ 10 12 After reaction at room temperature for 1 h, use PBST-PBS to wash away the unbound phage, elute by 0.1 M pH 2.2 Glycine-HCl, and finally use 1.5 M pH 8.8 Tris-HCl to neutralize the eluted phage antibody solution to about pH 7.0.

[0088] Step four: Infect 10 ml of the TG1 bacterial liquid grown to the logarithmic phase with the neutralized phage described above, and incubate in a 37°C incubator for 30 min. Take part of the bacterial liquid for gradient dilution and spread on 2YTAG plates for calculation of phage output. Centrifuge the remaining bacterial liquid to discard the supernatant, resuspend the bacterial pellet in a small amount of culture medium, aspirate and spread on a 2YTAG large plate for preparation for the next round of screening.

[0089] Step five: the bacteria infected and plated as above were scraped from the large plate and inoculated into 2YTAG liquid medium, and after being shaken to the logarithmic phase, M13KO7 helper phage was superinfected, and the phage was prepared by culturing overnight at 28°C and 220 rpm. The phage was purified by PEG / NaCl precipitation for the next round of screening. One round of phage library enrichment screening was performed.

[0090] Step six: screening of IL-11 phage single-chain antibody positive clones: after one round of screening, single colonies with good separation were picked and inoculated into 2YTAG liquid medium added to a 96-well deep well plate. After being cultured at 37°C and 220 rpm until the logarithmic growth phase, about 10 10 μL of helper phage M13KO7 was added to each well, and the culture was incubated at 37°C for 30 min. After centrifugation at 4000 rpm for 15 min, the supernatant was discarded, and the bacteria were resuspended and precipitated with 2YTAK. The culture was incubated overnight at 28°C and 220 rpm. After centrifugation at 4000 rpm and 4°C for 15 min, the amplified phage supernatant was aspirated for ELISA identification. Four mouse antibody molecules with high affinity were finally screened, designated as MA-I, MA-II, MA-III, and MA-IV. The above obtained monoclonal antibodies were subjected to gene sequencing to determine the correct antibody sequence. After sequencing, the sequences of the four monoclonal antibodies screened above are as follows:

[0091]

[0092]

[0093] Specifically, SEQ ID No: 16 (amino acid sequence of the heavy chain variable region of MA-I and MA-II):

[0094] EVKLEESGGGLVKPGGSLKLSCAASGFTFSDYYMFWVRQTPEKRLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNNLYLQMTSLKSEDTAMYYCARDGGYVS SPEAMDYWGQGTSVTVSS;

[0095] SEQ ID No: 17 (amino acid sequence of the light chain variable region of MA-I and MA-IV):

[0096] DIVLTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPPTFGGGTKLEIK;

[0097] SEQ ID No: 18 (amino acid sequence of the variable region of the light chain of MA-II):

[0098] DIVLTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK;

[0099] SEQ ID No: 19 (amino acid sequence of the variable region of the heavy chain of MA-III):

[0100] EVKLEQSGAEVVKPGALVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIG VIDPSDSYTTYNQKFKGKATLTVDTSSSTGYMQLSSLTSEDSAVYYCSQYGYDVN WYFDVWGAGTTVTVSS;

[0101] SEQ ID No: 20 (amino acid sequence of the variable region of the light chain of MA-III):

[0102] DIVMTQTTLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLI YEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGT KLEIK;

[0103] SEQ ID No: 21 (amino acid sequence of the variable region of the heavy chain of MA-IV):

[0104] EVQLEESGGGLVKPGGSLKLSCVASGFTFSDYYMFWVRQTPEKRLEWVATI SDGGSYSYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTAMYYCARDGGYISS PEAMDYWGQGTSVTVSS.

[0105] Example 3 Gradient dilution ELISA to compare the affinity of antibodies

[0106] The four 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 gradient dilution ELISA experiment of phage was performed to identify the affinity, and the specific method was as follows: IL-11 antigen was coated with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, and coated overnight at 4°C. After washing with PBST for three times, the four phage monoclonal antibodies screened in Example 2 were diluted with PBST at five gradient dilutions, 100 μL of the diluted sample was added to each well, and the mixture was incubated at room temperature for 1 hour. The ELISA plate was washed with PBST, and HRP-anti-M13 (purchased from Bio-viewshine, item number: GE27-9421-01) monoclonal antibody diluted with 1% BSA-PBST was added to the ELISA plate, and the mixture was incubated at room temperature for 1 hour. The TMB color developing kit was used for color development (purchased from Kangwei Century, item number: CW0050S), and the color development was carried out at room temperature for 10 minutes. After termination with 2M H2SO4, the reading was performed at 450nm / 630nm by using a microplate reader, and the corresponding EC50 value was calculated, and the specific data were as follows:

[0107]

[0108] According to the above data and as shown in Figure 2 , the four different murine antibody molecules screened in Example 2 can bind to IL-11, and therefore it can be illustrated that the monoclonal antibody provided by the application has high affinity with IL-11.

[0109] Example 4

[0110] In Example 4, the anti-IL-11 monoclonal antibody further comprises a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region is one of SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25 or SEQ ID No: 26, and 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:

[0111] SEQ ID No: 22 (amino acid sequence of the light chain constant region of murine C k type) :

[0112] ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; SEQ ID No: 23 (amino acid sequence of the heavy chain constant region of murine IgG1 type):

[0113] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG;

[0114] SEQ ID No:24 (Amino acid sequence of the heavy chain constant region of murine IgG2a):

[0115] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK;

[0116] SEQ ID No:25 (Amino acid sequence of the heavy chain constant region of murine IgG2b):

[0117] AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK;

[0118] SEQ ID No:26 (amino acid sequence of heavy chain constant region of mouse IgG3 type):

[0119] ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPELELNETCAEAQDGELDGLWTTITIFISLFLLSVCYSASVTLFKVKWIFSSVVQVKQTAIPDYRNMIGQGA.

[0120] Preparation of mouse-derived antibody molecules

[0121] Example 5 of the present application preferably defines the mouse-derived antibody molecules to include a heavy chain constant region of mouse IgG1 type (the amino acid sequence of which is shown as SEQ ID No: 23) and mouse C klight chain constant region (the amino acid sequence of which is shown as SEQ ID No: 22). The antibody preparation method is as follows:

[0122] 1. The genes encoding the heavy chain VH and light chain VL of the four antibody molecules screened in Example 2 were respectively cloned into the vector pTSE (as shown in Figure 3 k Figure 3

[0123] 2. The HEK293 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, with the item number GNHu43) were transiently transfected for antibody expression. The four monoclonal antibodies were purified by protein A affinity column using AKTA instrument, and the protein concentration was determined using BCA kit (purchased from Beijing Huitian Dongfang Science and Technology Co., Ltd., with the item number BCA0020). Then the protein size was identified by SDS-PAGE, and the results are shown in Figure 4

[0124] Example 6 Binding experiment of murine antibody molecules with IL-11

[0125] ​​​​IL-11 antigen was coated with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, and coated overnight at a temperature of 4°C. Washed five times with 300 μL / well PBST, then added 1% BSA-PBST, 280 μL / well, blocked at a temperature of 37°C for 1 h, added different dilution concentrations of MA-I, MA-II, MA-III and MA-IV mouse antibody molecules, the starting highest concentration of the four antibody molecules was 5 μg / mL, respectively, 5-fold gradient dilution, a total of 8 gradients for each antibody, incubated at a temperature of 37°C for 1 h. Washed five times with 300 μL / well PBST, then added Goat Anti-Mouse IgG-HRP (purchased from solarbio, item number: SE131) diluted 1:2000 with 1% BSA-PBST, incubated at a temperature of 37°C for 1 h. Color development with TMB color development kit, 100 μL / well, color development at room temperature for 8 min, then color development was terminated with 2M H2SO4. Readings were taken at 450 nm / 630 nm with a microplate reader, and the corresponding EC50 values were calculated, and the specific data are as follows:

[0126]

[0127] From the above data and as shown in Figure 5 , the four different mouse antibody molecules screened can bind to IL-11 and have high affinity.

[0128] Example 7 Competition inhibition experiment of mouse antibody with IL-11 receptor protein IL-11RA

[0129] IL-11-Fc was coated with carbonate buffer at pH 9.6, 200 ng / well / 100 μL, coated overnight at 4°C. Washed five times with 300 μL / well PBST, then added 1% BSA-PBST, 280 μL / well, blocked at 37°C for 1 h, then added IL-11RA-Fc (IgG4 type) diluted to 0.5 μg / mL with 1% BSA-PBST, 50 μL / well, then added MA-I, MA-II, MA-III and MA-IV mouse antibodies at different dilution concentrations, 50 μL / well, the starting highest concentration of the five antibodies was 100 μg / mL, each antibody was diluted by 2-fold gradient, a total of 13 gradients for each antibody, incubated at 37°C for 3 h. Washed five times with 300 μL / well PBST, then added Anti-Human IgG4-HRP Mouse monoclonal antibody (purchased from Sigma, product number: SAB4200770) diluted 1:5000 with 2% BSA-PBST, incubated at 37°C for 1 h. Color development with TMB color development kit, 100 μL / well, color development at room temperature for 15 min, then color development was terminated with 2M H2SO4. Readings were taken at 450 nm / 630 nm with a microplate reader, and the corresponding IC50 values were calculated, and the specific data are as follows:

[0130]

[0131] From the above data and as shown in Figure 6 , the four different mouse antibodies screened 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.

[0132] Example 8 Inhibition of the binding of IL-11 to IL-11RA receptors on the surface of BaF / 3-IL-11RA cells by mouse antibodies

[0133] The BaF / 3-IL-11RA cell line was counted, and a certain number of cells were centrifuged and resuspended with PBS buffer. The cell density was adjusted to 1E+6 cells / mL, 100 μL / well, and added to a 96-well plate. The IL-11-mFc ligand protein was diluted with PBS to a concentration of 18 μg / mL, 50 μL / well, and added to the corresponding position of the 96-well plate containing the BaF / 3-IL-11RA cells. After mixing gently, the 96-well plate was placed at 4°C and incubated for 1 h. The four mouse antibody molecules MA-I, MA-II, MA-III, and MA-IV were gradient diluted with PBS, with an initial concentration of 800 μg / mL, 3-fold gradient dilution, a total of 10 gradients, 50 μL / well, and added to the corresponding position of the 96-well plate containing the mixture of BaF / 3-IL-11RA cells and IL-11-mFc ligand protein. After mixing, the plate was incubated at 4°C for 2 h. After incubation, the cells were centrifuged at 3000 rpm, washed once with PBS buffer, and the cell pellet was collected. Goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) was added to the cell pellet, and incubated at 4°C for 30 min. After centrifugation at 3000 rpm, the cells were washed once with PBS buffer, resuspended with 100 μL of PBS buffer, and detected by flow cytometry. The fluorescence signal in the FL1-A channel was collected. The dose-effect curve was drawn, and the corresponding IC50 value was calculated. The specific data are as follows:

[0134]

[0135] From the above data and Figure 7 It can be seen that the four different mouse candidate molecules screened can effectively inhibit the binding of IL-11 ligand protein to the cell surface IL-11RA receptor.

[0136] Example 9 Inhibition of TIMP-1 Secretion from Embryonic Lung Fibroblast MRC-5 by Mouse Antibodies

[0137] Embryonic lung fibroblast MRC-5 was counted after trypsin digestion, a certain amount of cells was taken, the cells were resuspended after centrifugation with MEM complete medium (purchased from GIBCO, item number 10370-021), and the cell density was adjusted to 2E+5 cells / mL, 100 μL / well, and added to a 96-well plate. The IL-11-mFc ligand protein was diluted with MEM complete medium, and the concentration was prepared to be 16 μg / mL, 50 μL / well was added to the corresponding 96-well plate. The four mouse-derived antibody molecules MA-I, MA-II, MA-III and MA-IV were gradient diluted with MEM complete medium, and the initial concentration was prepared to be 40 μg / mL, 2-fold gradient dilution, a total of 8 gradients, 50 μL / well, added to the 96-well plate containing the cell suspension and the IL-11-mFc ligand protein suspension, mixed gently, and incubated in a 37℃ CO2 incubator overnight for about 20 h. The cell culture supernatant was detected by TIMP-1 ELISA kit (purchased from Yikexie Biotechnology Co., Ltd., item number EH021-96).

[0138] Human TIMP-1 detection kit: add cell supernatant and standard to sample wells, 100 μL / well. Immediately add biotinylated antibody working solution (1:100 dilution), 50 μL / well, cover the plate with sealing film, shake and incubate at room temperature for 2 h. After incubation, wash the plate 4 times with washing solution, add enzyme conjugate working solution (1:100 dilution) in the TIMP-1 detection kit, 100 μL / well. Cover the plate with sealing film, shake and incubate at room temperature for 1 h. After incubation, wash the plate 4 times with washing solution. Add TMB color developing solution, 100 μL / well, avoid light, incubate at room temperature for about 15 minutes, add 100 μL / well Stop solution to terminate the reaction. Read the value at 450 nm by enzyme-labeled instrument, and calculate the corresponding IC50 value, the specific data are as follows:

[0139]

[0140] From the above data and Figure 8 It can be seen that the four different mouse-derived candidate molecules screened can effectively inhibit the release of TIMP-1 from human embryonic lung fibroblast MRC-5 stimulated by IL-11 ligand protein.

[0141] Example 10

[0142] The anti-IL-11 monoclonal antibody of the embodiment 10 of the present application is further limited to a chimeric antibody molecule, and the chimeric antibody molecule further comprises a human antibody constant region, the human antibody constant region comprises 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 SEQ ID No: 27, SEQ ID No: 28 or SEQ ID No: 29; and the amino acid sequence of the human antibody light chain constant region is as shown in SEQ ID No: 30.

[0143] SEQ ID No: 27 (amino acid sequence of heavy chain constant region of human IgG1 type):

[0144] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0145] SEQ ID No: 28 (amino acid sequence of heavy chain constant region of human IgG2 type):

[0146] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0147] SEQ ID No: 29 (amino acid sequence of heavy chain constant region of human IgG4 type):

[0148] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0149] SEQ ID No:30 (human C k chain constant region amino acid sequence):

[0150] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C.

[0151] Preparation of chimeric antibody molecules

[0152] The embodiment 11 of the present application further limits the humanized antibody constant region to include a human IgG1 type heavy chain constant region (the amino acid sequence of which is shown as SEQ ID No: 27) and a human C k type light chain constant region (the amino acid sequence of which is shown as SEQ ID No: 30) based on the embodiment 10.

[0153] Specific preparation method: the heavy chain variable region VH (SEQ ID No: 16) and the light chain variable region VL gene (SEQ ID No: 17) of the murine antibody molecules MA-I and MA-II obtained by screening the immunized phage antibody library in embodiment 2 are kept as murine sequences, and are respectively cloned into the vector pTSE (as shown in Figure 3 SEQ ID No: 28) carrying the heavy chain constant region and the light chain constant region genes, the heavy chain constant region is a human IgG1 type (the amino acid sequence is shown as SEQ ID NO: 27), and the light chain constant region is a human C kChimeric antibodies CA-I, CA-II were obtained by transient transfection of HEK293E cells (purchased from: Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, item number: GNHu43) for antibody expression.

[0154] Example 12 Humanization of murine antibody molecules

[0155] First, the sequences of murine antibody molecules MA-I and MA-II in Example 2 were compared with the human antibody germline database (v-base) to find higher homologous human antibody light and heavy chain germlines as candidate sequences, and then the CDR sequences of murine antibody molecules MA-I and MA-II were transplanted onto the human candidate sequences for homology modeling. Then, through three-dimensional structure simulation calculation, the key framework amino acid residues that may play an important role in maintaining the CDR loop structure were designed for the back mutation of humanized antibodies. The designed light and heavy chain variable region sequences of humanized antibodies containing back mutations were synthesized by Nanjing Kingsriver Biotechnology Co., Ltd., and then connected to the transient expression vector. The light and heavy chains obtained by humanization were analyzed, in which MA-I obtained the following humanized anti-IL-11 monoclonal antibody molecules: HA-I-A, HA-I-B, HA-I-C, HA-I-D; MA-II obtained the following humanized antibody molecules: HA-II-A, HA-II-B, HA-II-C, HA-II-D; the sequences of the 8 monoclonal antibodies screened above are as follows:

[0156]

[0157]

[0158] 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):

[0159] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVSS PEAMDYWGQGTLVTVSS;

[0160] SEQ ID No: 32 (amino acid sequence of the light chain variable region of HA-I-A):

[0161] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;

[0162] SEQ ID No:33 (amino acid sequence of the heavy chain variable region of HA-I-B and HA-II-C):

[0163] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVST ISDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVS SPEAMDYWGQGTLVTVSS;

[0164] SEQ ID No:34 (amino acid sequence of the light chain variable region of HA-I-B):

[0165] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;

[0166] SEQ ID No:35 (amino acid sequence of the light chain variable region of HA-I-C):

[0167] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;

[0168] SEQ ID No:36 (amino acid sequence of the heavy chain variable region of HA-I-D and HA-II-D):

[0169] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAMYYCARDGGYVS SPEAMDYWGQGTSVTVSS;

[0170] SEQ ID No:37 (amino acid sequence of the light chain variable region of HA-I-D):

[0171] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;

[0172] SEQ ID No:38 (amino acid sequence of the light chain variable region of HA-II-A) :

[0173] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;

[0174] SEQ ID No:39 (amino acid sequence of the light chain variable region of HA-II-B and HA-II-C) :

[0175] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;

[0176] SEQ ID No:40 (amino acid sequence of the light chain variable region of HA-II-D) :

[0177] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGTVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPWTFGGGTKVEIK.

[0178] Embodiment 13

[0179] The embodiment 13 of the present application further limits the humanized antibody constant region to include a humanized antibody heavy chain constant region and a humanized antibody light chain constant region, the amino acid sequence of the humanized antibody heavy chain constant region is one of SEQ ID No: 27, SEQ ID No: 28 or SEQ ID No: 29; the amino acid sequence of the humanized antibody light chain constant region is shown in SEQ ID No: 30.

[0180] The specific sequence of the humanized antibody constant region is the same as that of embodiment 10.

[0181] Preparation of humanized antibody molecules

[0182] The embodiment 14 of the present application further defines the humanized antibody constant region to include a human IgG1 type heavy chain constant region (the amino acid sequence of which is shown as SEQ ID No: 27) and a human C k type light chain constant region (the amino acid sequence of which is shown as SEQ ID No: 30).

[0183] The coding genes of the heavy chain VH and light chain VL of the 8 humanized anti-IL-11 monoclonal antibody molecules HA-I-A, HA-I-B, HA-I-C, HA-I-D, HA-II-A, HA-II-B, HA-II-C, HA-II-D obtained in the embodiment 12 were respectively cloned into the vector pTSE (as shown in Figure 3 , which is equipped with the heavy chain constant region and light chain constant region genes), the heavy chain constant region is human IgG1 type (the amino acid sequence is shown as SEQ ID NO: 27), and the light chain constant region is C k (whose amino acid sequence is shown as SEQ ID NO: 30).

[0184] The 2 chimeric antibodies CA-I and CA-II obtained in the embodiment 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, HA-II-D obtained in the embodiment 12 were respectively transiently transfected into HEK293 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, with the item number GNHu43), and the antibodies were expressed. The monoclonal antibodies were purified by protein A affinity column using AKTA instrument, and the protein concentration was determined using BCA kit (purchased from Beijing Huitian Dongfang Science and Technology Co., Ltd., with the item number BCA0020). Then the protein size was identified by SDS-PAGE, and the results are shown in Figure 9 , from left to right, the non-reduced protein molecular weights of HA-I-A, HA-I-B, HA-I-C, HA-I-D, the chimeric antibody CA-I prepared in the embodiment 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.

[0185] Example 15 Binding experiment of humanized antibody molecules with IL-11

[0186] IL-11 antigen was coated with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, coated overnight at 4°C. Washed five times with 300 μL / well PBST, then added 1% BSA-PBST 280 μL / well, blocked at 37°C for 1 h. The initial concentration of 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 antibodies CA-I, CA-II prepared in Example 11, humanized antibodies were all 10 μg / mL, 5-fold dilution gradient, a total of 8 gradients, incubated at 37°C for 1 h. Washed five times with 300 μL / well PBST, then added goat anti Human IgG Fab HRP (purchased from invitrogen, item number: 31482) diluted 1:5000 with 1% BSA-PBST, incubated at 37°C for 1 h. Color development with TMB color development kit, 100 μL / well, color development at room temperature for 5 min, then color development was terminated with 2M H2SO4. Readings were taken at 450 nm / 630 nm on a microplate reader, and the corresponding EC50values were calculated, and the specific data are as follows:

[0187]

[0188]

[0189] As shown in the above data and experimental results as shown in Figure 10 , the 8 different humanized antibody molecules can bind to IL-11, and the EC50values of the humanized antibody molecules HA-I-A, HA-I-B, HA-I-C, HA-I-D are close to the chimeric antibody CA-I, and the EC50values of the humanized antibody molecules HA-II-A, HA-II-B, HA-II-C, HA-II-D are close to the chimeric antibody CA-II, indicating that the humanized antibody molecules retain the high binding ability of the mouse parent antibodies MA-I, MA-II to IL-11.

[0190] Example 16 Humanized antibody molecules inhibit the binding of IL-11 to IL-11RA receptors on the surface of BaF / 3-IL-11RA cells

[0191] The four humanized antibody molecules HA-I-A, HA-I-B, HA-I-C and HA-I-D with better activity at the protein level were selected for cell activity evaluation experiment. The BaF / 3-IL-11RA cell line was counted, a certain amount of cells were centrifuged and resuspended with PBS buffer, and the cell density was adjusted to 1E+6 cells / mL, 100 μL / well, and added to a 96-well plate. The IL-11-mFc ligand protein was diluted with PBS buffer to a concentration of 18 μg / mL, 50 μL / well, and added to the corresponding position of the 96-well plate containing the BaF / 3-IL-11RA cells. After gentle mixing, the 96-well plate was placed at 4°C for incubation for 1 h. The four humanized antibody molecules HA-I-A, HA-I-B, HA-I-C and HA-I-D were gradient diluted with PBS buffer, and the initial concentration was prepared to be 800 μg / mL, 3-fold gradient dilution, a total of 10 gradients, 50 μL / well, added to the corresponding position of the 96-well plate containing the mixture of BaF / 3-IL-11RA cells and IL-11-mFc ligand protein. After mixing, it was placed at 4°C for 2 h of incubation. After incubation, the cells were washed once by centrifugation at 3000 rpm with PBS buffer, and the cell precipitate was collected. Goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) was added to the cell precipitate, and incubated at 4°C for 30 min, then centrifuged at 3000 rpm for washing once, resuspended with 100 μL of PBS, and then detected by flow cytometry. The fluorescence signal in the FL1-A channel was collected. The dose-effect curve was drawn, and the corresponding IC50 value was calculated. The specific data are as follows:

[0192]

[0193] From the above data and Figure 11 It can be seen that the four humanized candidate molecules selected can inhibit the binding of IL-11 ligand protein to the surface IL-11RA receptor of BaF / 3-IL-11RA cells.

[0194] Example 17 Humanized antibody molecules inhibit the binding of IL-11 to the surface GP130 receptor of BaF / 3-GP130 cells

[0195] The BaF / 3-GP130 cell line was counted, and a certain number of cells were centrifuged and resuspended with PBS buffer. The cell density was adjusted to 1E+6 cells / mL, 100 μL / well, and added to a 96-well plate. The IL-11-mFc ligand protein was diluted with PBS, and the concentration was prepared to 12 μg / mL, 50 μL / well, and added to the corresponding position of the 96-well plate containing the BaF / 3-GP130 cells. After mixing gently, the 96-well plate was placed at 4°C and incubated for 1 h. The four human antibody molecules HA-I-A, HA-I-B, HA-I-C and HA-I-D were gradient diluted with PBS buffer, and the initial concentration was prepared to 2000 μg / mL, 2-fold gradient dilution, a total of 10 gradients, 50 μL / well, added to the corresponding position of the 96-well plate containing the BaF / 3-GP130 cells and the IL-11-mFc ligand protein. After mixing, the 96-well plate was placed at 4°C and incubated for 2 h. After incubation, the cells were washed once with PBS buffer at 3000 rpm, and the cell precipitate was collected. Goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) was added to the cell precipitate, 100 μL / well, and incubated at 4°C for 30 min. After washing once with PBS buffer at 3000 rpm, the cells were resuspended with 100 μL / well of PBS buffer, and the fluorescence signal in the FL1-A channel was detected by flow cytometry. The dose-effect curve was drawn, and the corresponding IC50 value was calculated. The specific data are as follows:

[0196]

[0197] From the above data and Figure 12 It can be seen that the four humanized candidate molecules screened can block the binding of the IL-11 ligand protein to the GP130 receptor on the surface of the BaF / 3-GP130 cells.

[0198] Example 18 Humanized antibody molecules inhibit the secretion of TIMP-1 by embryonic lung fibroblast MRC-5 cells

[0199] Embryonic lung fibroblast MRC-5 was counted after trypsin digestion, a certain amount of cells was taken, the cells were resuspended with MEM complete medium after centrifugation, and the cell density was adjusted to 2E+5 cells / mL, 100 μL / well, and added to a 96-well plate. The IL-11-mFc ligand protein was diluted with MEM complete medium, and the concentration was prepared to be 16 μg / mL, 50 μL / well, and added to the corresponding 96-well plate. The four humanized antibody molecules HA-I-A, HA-I-B, HA-I-C and HA-I-D were gradient diluted with MEM complete medium, and the initial concentration was prepared to be 40 μg / mL, 3 times gradient dilution, a total of 8 gradients, 50 μL / well, added to the 96-well plate containing the cell suspension and the IL-11-mFc ligand protein suspension, mixed gently and uniformly, incubated in a 37°C CO2 incubator overnight, about 20 h, and the cell culture supernatant was detected by TIMP-1 ELISA kit (method same as Example 9). The microplate reader was read at 450 nm, and the corresponding IC50 value was calculated, and the specific data were as follows:

[0200]

[0201] From the above data and Figure 13 It can be seen that the four humanized antibody molecules screened can effectively inhibit the release of TIMP-1 of human embryonic lung fibroblast MRC-5 stimulated by IL-11 ligand protein.

[0202] Example 19 Cross-binding experiment of humanized antibody molecules with different species of IL-11

[0203] The humanized antibody molecule HA-I-A with better protein level and function detection activity was selected for cross-binding detection with different species of IL-11. Human IL-11, mouse IL-11 (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., item number: 50117-MNCE), rat IL-11 (purchased from Kanglang Biology, item number: KL40001Ra), and cynomolgus monkey IL-11 (purchased from Yiqiao Shenzhou, item number: 90925-CNCE) were coated with 100 ng / well / 100 μL of carbonate buffer at pH 9.6 at 4°C overnight. Five washes with 300 μL / well PBST were performed, and 1% BSA-PBST was added at 280 μL / well for blocking at 37°C for 1 h. The humanized antibody HA-I-A was diluted with 1% BSA-PBST at an initial concentration of 50 μg / mL, and 5-fold gradient dilution was performed, with a total of 9 gradients, and each gradient was duplicated in two wells, 100 μL / well was added to the 96-well plate, and incubation was performed at 37°C for 1 h. Five washes with 300 μL / well PBST were performed, and goat anti-human IgG Fab HRP (purchased from invitrogen, item number: 31482) was diluted with 1% BSA-PBST at a working concentration of 1:5000, 100 μL / well was added to the 96-well plate, and incubation was performed at 37°C for 1 h. Five washes with 300 μL / well PBST were performed, and TMB color developing kit was used for color development, 100 μL / well was added at room temperature for color development in the dark for 5 min, and then color development was terminated with 2M H2SO4. The microplate reader was read at 450 nm / 630 nm, and the corresponding EC50 values were calculated, and the specific data are as follows:

[0204]

[0205] From the above data and as shown in Figure 14 , 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 high affinity.

[0206] Example 20 Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody on pulmonary fibrosis

[0207] Bleomycin (bLF) was used to model to study the therapeutic effect of anti-IL-11 monoclonal antibody HA-I-A on pulmonary fibrosis.

[0208] Animal species: C57BL / 6J mice (purchased from Jiangsu Jizhuangkang Biological Technology Co., Ltd.); number, gender, and mouse age: 6 / group, male, 6-8 weeks;

[0209] The control group was only injected with normal saline; the treatment group was given injections of HA-I-A antibody molecules twice a week for 4 weeks.

[0210] Animal body weight was measured once a week, and the animals were observed for abnormalities; organ weight detection: lung organs were collected, the weight of the lung organs was measured, and the lung to body weight ratio 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.

[0211] As shown in Figure 15 , the lung to body weight ratio of the mice in the administration group was significantly smaller than that of the control group after administration of the HA-I-A antibody molecule; as shown in Figure 16 , compared with the control group, the lung sections of the administration group showed a significant reduction in lung fibrosis, thus indicating that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the generation of lung fibrosis.

[0212] Example 21 Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody on cardiac fibrosis

[0213] Isoproterenol modeling was used to study the therapeutic effect of anti-IL-11 monoclonal antibody HA-I-A on cardiac fibrosis.

[0214] Animal species: C57BL / 6J mice (purchased from Jiangsu Jicui Yekang Biotechnology Co., Ltd.); number, gender and mouse age: 6 per group, male, 6-8 weeks;

[0215] The control group was only injected with normal saline; the administration group was given injections of HA-I-A antibody molecules twice a week for 4 weeks.

[0216] Animal body weight was measured once a week, and the animals were observed for abnormalities; organ weight detection: the heart was collected, the weight of the heart was measured, and the heart to body weight ratio was calculated; heart pathology detection: heart sections were made and hematoxylin-eosin (HE) and Masson staining were used to observe the degree of cardiac fibrosis.

[0217] As shown in Figure 17 , the heart to body weight ratio of the mice in the administration group was significantly smaller than that of the control group; as shown in Figure 18 , compared with the control group, the heart sections of the administration group showed a significant reduction in cardiac fibrosis, thus indicating that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the generation of cardiac fibrosis.

[0218] Example 22 Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody on kidney fibrosis

[0219] Doxorubicin (dKF) modeling was used to study the therapeutic effect of anti-IL-11 monoclonal antibody HA-I-A on kidney fibrosis.

[0220] Animal species: BALB / c mice (purchased from Jiangsu Jizhuangkang Biotechnology Co., Ltd.); number, gender and mouse age: 6 per group, male, 6-8 weeks.

[0221] The control group was only injected with normal saline; the administration group was injected with HA-I-A antibody molecules twice a week for 4 weeks.

[0222] Animal body weight was measured once a week, and animals were observed for abnormalities; organ weight detection: the heart was collected, the weight of the kidney was measured, and the urine protein content was detected; kidney pathology detection: kidney sections were stained with hematoxylin-eosin (HE) and Masson to observe the degree of kidney fibrosis.

[0223] The results are shown in Table 1. Figure 19 As shown in Table 2, the urine protein content in the kidneys of mice in the administration group was significantly lower than that in the control group; the results are shown in Table 3. Figure 20 As shown in Table 3, the kidney sections of the administration group showed significantly reduced kidney fibrosis compared with the control group, thus indicating that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the production of kidney fibrosis.

[0224] Example 23 Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody on liver fibrosis

[0225] The therapeutic effect of anti-IL-11 monoclonal antibody HA-I-A on liver fibrosis was studied using CCl4 modeling.

[0226] Animal species: C57BL / 6J mice (purchased from Jiangsu Jizhuangkang Biotechnology Co., Ltd.); number, gender and mouse age: 6 per group, male, 6-8 weeks.

[0227] The control group was only injected with normal saline; the administration group was injected with HA-I-A antibody molecules twice a week for 4 weeks.

[0228] Body weight monitoring: animal body weight was measured once a week, and animals were observed for abnormalities; liver pathology detection: liver sections were stained with hematoxylin-eosin (HE) and Masson to observe the degree of liver fibrosis; organ weight detection: the kidney was collected, the weight of the liver was measured, and HE staining was performed; serum detection: serum was collected, and the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the serum of mice were detected.

[0229] The results are shown in Table 4. Figure 21 As shown in Table 5, the liver weight of mice in the administration group was significantly lower than that in the control group; the results are shown in Table 6. Figure 22 As shown in Table 6, the ALT and AST levels in the serum of mice in the administration group were significantly lower than those in the control group; the results are shown in Table 7. Figure 23As shown, compared with the control group, the liver sections of the administration group showed a significant reduction in liver fibrosis, thus indicating that the anti-IL-11 monoclonal antibody HA-I-A molecule can effectively inhibit the generation of liver fibrosis.

[0230] Example 24 Thermal stability evaluation of anti-IL-11 monoclonal antibody HA-I-A

[0231] The thermal stability of the anti-IL-11 monoclonal antibody HA-I-A was evaluated using a multi-functional protein thermal stability analysis system (purchased from Unchained Labs). Changes in protein conformation were detected by monitoring the endogenous fluorescence of the protein as the temperature changed (from 25℃ to 95℃ at a temperature rise rate of 0.3℃ / min), and the protein melting temperature Tm was determined to evaluate the stability of the protein conformation. When the sample aggregates, it will cause interference of scattered light waves, and the scattered light signal will increase, and the colloidal stability of the protein (characterized by Tagg) was determined by static light scattering. The results are shown in the following table and the accompanying Figure 24

[0232]

[0233] The melting temperature Tm of the anti-IL-11 monoclonal antibody HA-I-A was 79.5℃, and the average Tagg was 75.9℃, showing good conformational stability and colloidal stability.

[0234] Example 25

[0235] The method provided in Example 25 of the present application detects the biological activity of the anti-IL-11 monoclonal antibody HA-I-A screened in the above-mentioned examples, and the specific method is as follows:

[0236] S1, construct a stable passaged cell strain BAF / 3-GP130-STAT3-Luc, and the specific method is as follows:

[0237] S11, transfect the plasmid GP130 (purchased from Yiqiao Shenzhou Biotechnology Co., Ltd., item number: HG10974-UT) into BAF / 3 cells (purchased from the National Experimental Cell Resource Center), and obtain the cell strain BAF / 3-GP130 after screening; S12, transfect the plasmid STAT3-Luc-NeoR into the cell strain BAF / 3-GP130, and obtain the stable cell strain BAF / 3-GP130-STAT3-Luc after screening, and the specific method is as follows:

[0238] ​(1) using Lonza electroporator to transfect plasmid GP130 into BAF / 3 cells, 48 hours after transfection, adding 1 μg / mL of Puromycin (purchased from Thermo Fisher Scientific) for screening, using flow cytometry to detect the expression of GP130 on the surface of surviving cells, obtaining positive clone cells; (2) the above obtained positive clone cells are transferred to 96-well plates at a density of 0.5 per hole, after incubation at 37℃, 5% CO2 for two weeks, selecting the clones with round cell masses, after amplification culture, using GP130 antibody (purchased from Thermo Fisher Scientific) to detect the expression of GP130 on the surface of cells, selecting the cell strain BAF / 3-GP130 with higher expression for standby; (3) using Lonza electroporator to transfect plasmid STAT3-Luc-NeoR into the above cell strain BAF / 3-GP130 with higher expression, 48 hours after transfection, adding 2 mg / mL of Geneticin (purchased from Thermo Fisher Scientific) for screening, adding IL-11 antigen (purchased from Yiqi God of the State Biotechnology Co., Ltd.) and IL-11RA antigen (purchased from Yiqi God of the State Biotechnology Co., Ltd.) to activate cells, detecting chemiluminescence value, determining the cells as positive clones, selecting the positive clone cell strain BAF / 3-GP130-STAT3-Luc with better response for standby; (4) the above obtained cell strain BAF / 3-GP130-STAT3-Luc is transferred to 96-well plates at a density of 0.5 per hole, after incubation at 37℃, 5% CO2 for two weeks, selecting the clones with round cell masses, after amplification culture, obtaining stable cell strain BAF / 3-GP130-STAT3-Luc for standby.

[0239] S2, stimulating the expression of reporter gene in cell strain BAF / 3-GP130-STAT3-Luc with IL-11 / IL-11RA complex, the specific method is as follows:

[0240] S21, transferring cell strain BAF / 3-GP130-STAT3-Luc to 96-well plates at a cell inoculation density of 2×10 4 -1×10 5 per hole, the optional cell inoculation density includes but is not limited to 2×10 4 per hole, 4×10 4 per hole, 6×10 4 per hole, 8×10 4 per hole or 1×10 5 per hole, as long as it is 2×10 4 -1×10 5The expression requirement can be met in the cell inoculation density range of 0.5-1000 cells per well; S22, the IL-11 / IL-11RA complex is added to the 96-well plate to stimulate the expression of the reporter gene in the activated cell strain BAF / 3-GP130-STAT3-Luc.

[0241] S3, the gradient-diluted anti-IL-11 monoclonal antibody HA-I-A is added to the cell strain BAF / 3-GP130-STAT3-Luc for incubation;

[0242] S4, the chromogenic substrate is added, the reporter gene signal value is determined, and a four-parameter curve is fitted according to the concentration of the anti-IL-11 monoclonal antibody and the reporter gene signal value, so as to detect the biological activity of the anti-IL-11 monoclonal antibody.

[0243] Embodiment 26

[0244] On the basis of embodiment 25, embodiment 26 of the present application further limits that in step S22, the IL-11 / IL-11RA complex comprises IL-11 antigen with a stimulation final concentration of 0.02 μg / mL and IL-11RA antigen with a stimulation final concentration of 0.02 μg / mL, and the stimulation final concentration ratio of the IL-11 antigen and the IL-11RA antigen is 1:1.

[0245] Embodiment 27

[0246] On the basis of embodiment 25, embodiment 27 of the present application further limits that in step S22, the IL-11 / IL-11RA complex comprises IL-11 antigen with a stimulation final concentration of 0.4 μg / mL and IL-11RA antigen with a stimulation final concentration of 0.05 μg / mL, and the stimulation final concentration ratio of the IL-11 antigen and the IL-11RA antigen is 8:1.

[0247] Embodiment 28

[0248] On the basis of embodiment 25, embodiment 28 of the present application further limits that in step S22, the IL-11 / IL-11RA complex comprises IL-11 antigen with a stimulation final concentration of 40 μg / mL and IL-11RA antigen with a stimulation final concentration of 2.5 μg / mL, and the stimulation final concentration ratio of the IL-11 antigen and the IL-11RA antigen is 16:1.

[0249] Embodiment 29

[0250] On the basis of embodiment 25, embodiment 29 of the present application further limits that in step S3, the initial concentration of the gradient-diluted anti-IL-11 monoclonal antibody is 500 μg / mL, and the ratio of the equi-ratio dilution is 1:2.

[0251] Embodiment 30

[0252] On the basis of embodiment 25, the embodiment 30 of the present application further limits that in step S3, the initial concentration of the gradient diluted anti-IL-11 monoclonal antibody is 500 μg / mL, and the ratio of the isometric dilution is 1:5.

[0253] Embodiment 31

[0254] On the basis of embodiment 25, the embodiment 31 of the present application further limits that in step S3, the initial concentration of the gradient diluted anti-IL-11 monoclonal antibody is 500 μg / mL, and the ratio of the isometric dilution is 1:4.

[0255] Embodiment 32

[0256] On the basis of embodiment 25, the embodiment 32 of the present application further limits that in step S3, the incubation time is 2 hours.

[0257] Embodiment 33

[0258] On the basis of embodiment 25, the embodiment 33 of the present application further limits that in step S3, the incubation time is 5 hours.

[0259] Embodiment 34

[0260] On the basis of embodiment 25, the embodiment 34 of the present application further limits that in step S3, the incubation time is 6 hours.

[0261] Embodiment 35

[0262] The embodiment 35 of the present application provides a method for detecting the biological activity of the anti-IL-11 monoclonal antibody on the basis of the above-mentioned embodiments 1-34, which is used for the quality control in the production and research of the anti-IL-11 monoclonal antibody drug.

[0263] Embodiment 36 Optimization of the method for detecting the biological activity of the anti-IL-11 monoclonal antibody

[0264] The anti-IL-11 monoclonal antibody HA-I-A selected from the above-mentioned embodiments is subjected to the optimization of the method for detecting the biological activity according to the method provided in the embodiment 25.

[0265] (1) Optimization of the final concentration of IL-11 antigen in the IL-11 / IL-11RA complex

[0266] First, the cell strain BAF / 3-GP130-STAT3-Luc is diluted to 8×10 4The cell strain BAF / 3-GP130-STAT3-Luc is added to the 96-well plate at a cell seeding density of 8×10 Figure 25 as shown.

[0267] Then, according to the above method, the cell strain BAF / 3-GP130-STAT3-Luc is added to the 96-well plate at a cell seeding density of 8×10 4 The anti-IL-11 monoclonal antibody HA-I-A is set to have an initial concentration of 500 μg / mL, and 10 concentration points are diluted at a gradient dilution ratio of 1:5. The IL-11 antigen is set to have two different final stimulation concentrations of 1 μg / mL and 0.4 μg / mL, and is diluted to 4 μg / mL and 0.16 μg / mL, respectively, and is then transferred to the above-mentioned cell plate. After incubation at 37°C in a 5% CO2 environment for 5 hours, the chemiluminescence value is detected by adding the Britelite Plus luciferase reagent kit (purchased from PerkinElmer Inc.), and a four-parameter curve of the inhibitory activity of the antibody under the action of different stimulation concentrations of the IL-11 antigen in the IL-11 / IL-11RA complex is drawn.

[0268] The detection results are shown in Table 1. Figure 26 As shown in Table 1, a better dose-effect curve can be obtained when the final stimulation concentration of the IL-11 antigen is 0.4 μg / mL, and the amount of the IL-11 antigen is also saved. Therefore, the optimal stimulation concentration of the IL-11 antigen in the present application is preferably 0.4 μg / mL.

[0269] (2) IL-11 / IL-11RA complex concentration optimization

[0270] On the basis of the optimal final stimulation concentration of the IL-11 antigen of 0.4 μg / mL obtained above, in order to determine the final stimulation concentration ratio of the IL-11 antigen and the IL-11RA antigen in the IL-11 / IL-11RA complex, the final stimulation concentration ratio of the IL-11 antigen and the IL-11RA antigen is set to be 1:1, 2:1, 4:1, 8:1 and 16:1. The final stimulation concentration of the IL-11 is set to be 0.4 μg / mL and is diluted to 0.16 μg / mL. According to the final stimulation concentration ratio, the concentration of the IL-11RA antigen is set to be 0.16 μg / mL, 0.08 μg / mL, 0.04 μg / mL, 0.02 μg / mL and 0.01 μg / mL. The cell strain BAF / 3-GP130-STAT3-Luc is added to the 96-well plate at a cell seeding density of 8×10 4The cell seeding density of 1 x 10 4 cells / well was added to the 96-well plate, and the initial concentration of the anti-IL-11 monoclonal antibody HA-I-A was set to 500 μg / mL, which was diluted by the ratio of 1:5 to dilute 8 concentration points, and then added to the above-mentioned cell plate. The IL-11 / IL-11RA complex was transferred to the above-mentioned cell plate, and the cell plate was cultured at 37°C in 5% CO2. After 5 hours of incubation, the color developing substrate Britelite Plus luciferase kit was added to measure the reporter gene signal value. According to the concentration of the anti-IL-11 monoclonal antibody and the reporter gene signal value, a four-parameter curve was fitted.

[0271] The data, as shown in Figure 27 , showed that when the final concentration ratio of IL-11 antigen to IL-11RA antigen was 8:1 and 16:1, a better dose-effect curve could be obtained.

[0272] (3) Optimization of cell strain BAF / 3-GP130-STAT3-Luc cell seeding density

[0273] On the basis of the above-mentioned optimized optimal stimulation concentration of IL-11 antigen of 0.4 μg / mL and the selection of the final concentration ratio of IL-11 antigen to IL-11RA antigen of 8:1, the cell strain BAF / 3-GP130-STAT3-Luc cell seeding density was further optimized, as follows:

[0274] First, five different cell seeding densities of the cell strain BAF / 3-GP130-STAT3-Luc were set, which were 2 x 10 4 cells / well, 4 x 10 4 cells / well, 6 x 10 4 cells / well, 8 x 10 4 cells / well, and 1 x 10 5 cells / well, which were added to the 96-well plate, respectively. Second, the IL-11 / IL-11RA complex (i.e., the IL-11 antigen with a final stimulation concentration of 0.4 μg / mL and the IL-11RA antigen with a final stimulation concentration of 0.05 μg / mL, and the final concentration ratio of IL-11 antigen to IL-11RA antigen was selected to be 8:1) was added, respectively. Finally, the initial working concentration of the anti-IL-11 monoclonal antibody HA-I-A was set to 500 μg / mL, which was diluted by the ratio of 1:5 to dilute 8 concentration points, and then transferred to the above-mentioned cell plate. After 5 hours of culture at 37°C in 5% CO2, the color developing substrate Britelite Plus luciferase kit was added to detect the chemiluminescence value, and a four-parameter curve was fitted according to the measured chemiluminescence value.

[0275] The detection results, as shown in Figure 28As shown in the figure, with the increase of the cell seeding density of the cell strain BAF / 3-GP130-STAT3-Luc, the RLU value increases, and when the cell seeding density is 8 x 10 4 cells / well, the RLU value is higher and the upper and lower platforms are better.

[0276] (4) Optimization of the dilution ratio of the anti-IL-11 monoclonal antibody HA-I-A

[0277] On the basis of the optimal stimulation concentration of the IL-11 antigen of 0.4 μg / mL and the ratio of the stimulation final concentrations of the IL-11 antigen and the IL-11RA antigen of 8:1, and the cell seeding density of the cell strain BAF / 3-GP130-STAT3-Luc of 8 x 10 4 cells / well, further optimization of the dilution ratio of the anti-IL-11 monoclonal antibody HA-I-A was carried out, as follows:

[0278] First, the cell strain BAF / 3-GP130-STAT3-Luc was added to the 96-well plate at a cell seeding density of 8 x 10 4 cells / well; second, the IL-11 / IL-11RA complex (i.e., the IL-11 antigen at a stimulation final concentration of 0.4 μg / mL and the IL-11RA antigen at a stimulation final concentration of 0.05 μg / mL, and the ratio of the stimulation final concentrations of the IL-11 antigen and the IL-11RA antigen was 8:1) was added; and finally, the starting working concentration of the anti-IL-11 monoclonal antibody HA-I-A was set to 500 μg / mL, and four different dilution ratios of 1:2, 1:3, 1:4 and 1:5 were set, and eight concentration gradients of each dilution were prepared, and the diluted antibody was transferred to the cell plate; and incubation was carried out at 37°C and 5% CO2 for 5 hours, and then the color developing substrate Britelite Plus luciferase kit was added, and the chemiluminescence value was detected, and a four-parameter curve was fitted according to the determined chemiluminescence value.

[0279] The detection results are shown in the figure. Figure 29 When the dilution ratio of the anti-IL-11 monoclonal antibody HA-I-A is 1:5, the eight selected concentration points can better cover the upper and lower platforms, and the linearity is better.

[0280] (5) Incubation time optimization

[0281] On the basis of the optimal stimulation concentration of the IL-11 antigen of 0.4 μg / mL and the ratio of the stimulation final concentrations of the IL-11 antigen and the IL-11RA antigen of 8:1, and the cell seeding density of the cell strain BAF / 3-GP130-STAT3-Luc of 8 x 10 4Based on the 1:5 serial dilution ratio of anti-IL-11 monoclonal antibody HA-IA, the incubation time was further optimized, as follows:

[0282] First, set the cell line BAF / 3-GP130-STAT3-Luc at 8×10⁻⁶. 4 Cells were seeded at a density of 10 cells / well into 96-well plates. Next, IL-11 / IL-11RA complex (i.e., IL-11 antigen at a final stimulation concentration of 0.4 μg / mL and IL-11RA antigen at a final stimulation concentration of 0.05 μg / mL, with a final stimulation concentration ratio of IL-11 antigen to IL-11RA antigen of 8:1) was added. Finally, the initial concentration of the anti-IL-11 monoclonal antibody HA-IA was set at 500 μg / mL, and it was serially diluted 1:5 in eight steps. The diluted antibody was then transferred to the cell plate. The cells were incubated at 37°C in a 5% CO2 incubator for 2, 3, 4, 5, and 6 hours. After each incubation, the Britelite Plus luciferase kit was added, and the chemiluminescence value was measured. A four-parameter curve was then fitted based on the measured chemiluminescence values.

[0283] Test results as follows Figure 30 As shown, the RLU increases with the increase of incubation time. In order to ensure the signal-to-noise ratio while reducing the incubation time, the present invention preferably has an incubation time of 5 hours.

[0284] In summary, through the above-mentioned methodological optimization, the preferred IL-11 / IL-11RA complex of this invention consists of an optimal stimulation concentration of 0.4 μg / mL for IL-11 antigen and an optimal stimulation concentration of 0.05 μg / mL for IL-11RA antigen, with a final stimulation concentration ratio of 8:1 for IL-11 antigen and 8 × 10⁻⁶ cells for IL-11RA antigen. 4 The initial concentration of the anti-IL-11 monoclonal antibody HA-IA was selected as 500 μg / mL, and the serial dilution ratio was 1:5. Meanwhile, the incubation time was preferably 5 hours.

[0285] Example 37: Validation of the method for detecting the biological activity of anti-IL-11 monoclonal antibody

[0286] (1) Suitability verification: The method is for the biological activity of anti-IL-11 monoclonal antibody, for this reason, the experimental group selects anti-IL-11 monoclonal antibody HA-I-A provided by the application, and the control group uses monoclonal antibody drugs of different target points: Nivolumab (target point is PD-1) and Rituximab (target point is CD20). According to the test conditions determined in Example 36, the biological activity of the antibodies in the experimental group and the control group is detected by using the detection method provided in Example 25, and the reactivity of the cell strain BAF / 3-GP130-STAT3-Luc to the above antibodies is determined.

[0287] The results are shown in Figure 31 As the concentration of anti-IL-11 monoclonal antibody HA-I-A increases, the RLU value decreases, showing a good dose-effect curve, and the control group has no obvious effect, indicating that the detection method of the biological activity of the antibody provided by the application has good suitability.

[0288] (2) Relative accuracy verification: The specific method includes: using 500 μg / mL of anti-IL-11 monoclonal antibody HA-I-A as the reference for the accuracy verification of the method, preparing 5 samples with different initial concentration titers, which are 250 μg / mL, 375 μg / mL, 500 μg / mL, 625 μg / mL and 750 μg / mL, and the corresponding theoretical relative biological activity values are 50%, 75%, 100%, 125% and 150% respectively, and each titer level is detected according to the test conditions determined in Example 36 and the detection method provided in Example 25, four times by two people, a total of eight times. According to the experimental results, a four-parameter curve is fitted to obtain the IC50 value, and finally the relative biological activity and recovery rate of each of the 5 sample groups are calculated according to the following formula.

[0289] Relative biological activity (%) = reference IC50 / sample IC50 x 100%;

[0290] Recovery rate (%) = measured value of relative activity / theoretical value of relative activity x 100%;

[0291]

[0292] Through the above data, the relative bias is within 20%, and the recovery rate is between 80% and 120%, as shown in Figure 32 The measured relative biological activity of the 5 sample groups is fitted with the theoretical value, and the R 2 value is 0.9914, the linear fitting result is good, and the average value of the relative biological activity and the corresponding theoretical value have good consistency, indicating that the method established in the present study has good accuracy.

[0293] (3) Cell passage stability verification

[0294] The specific method comprises: continuously culturing the cell strain BAF / 3-GP130-STAT3-Luc, freezing at the 5th, 10th, 15th, 20th and 25th generations respectively, then uniformly recovering the cells, detecting the biological activity of the cells of different generations according to the test conditions determined in Example 36 and the detection method provided in Example 25, fitting a four-parameter curve according to the experimental results, investigating the passage stability of the cells and obtaining the specific data as follows:

[0295]

[0296] As shown by the above data and Figure 33 , the variation coefficient (CV%) of the cells of different generations is 16.9%, which is less than 20%, indicating that the stability of the cells within 25 generations is good and can meet the needs of the application.

[0297] The application is not limited to the above best embodiment, and anyone can derive other various forms of products under the inspiration of the application, but regardless of any change in shape or structure, any technical solution with the same or similar technical solution as the application falls within the protection scope of the application.

Claims

1. A method for detecting the biological activity of an anti-IL-11 monoclonal antibody, characterized by, The detection method comprises the following steps: S1, constructing a stably passaged cell strain BAF / 3-GP130-STAT3-Luc; S2, stimulating expression of a reporter gene in the cell strain BAF / 3-GP130-STAT3-Luc by an IL-11 / IL-11RA complex; S3, adding gradient-diluted anti-IL-11 monoclonal antibodies to the cell strain BAF / 3-GP130-STAT3-Luc for incubation; S4, adding a chromogenic substrate, determining a reporter gene signal value, and fitting a four-parameter curve according to the concentration of the anti-IL-11 monoclonal antibodies and the reporter gene signal value, so as to detect biological activity of the anti-IL-11 monoclonal antibodies. The anti-IL-11 monoclonal antibodies comprise 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 amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown as SEQ ID No: 1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown as SEQ ID No: 2, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown as SEQ ID No: 3, the amino acid sequence of the light chain complementarity determining region LCDR1 is shown as SEQ ID No: 4, the amino acid sequence of the light chain complementarity determining region LCDR2 is shown as SEQ ID No: 5, and the amino acid sequence of the light chain complementarity determining region LCDR3 is shown as SEQ ID No:

6.

2. The method for detecting the biological activity of the anti-IL-11 monoclonal antibody according to claim 1, wherein The anti-IL-11 monoclonal antibodies further comprise a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No: 31, and the amino acid sequence of the light chain variable region is shown as SEQ ID No:

32.

3. The method for detecting the biological activity of the anti-IL-11 monoclonal antibody according to claim 2, wherein The anti-IL-11 monoclonal antibodies further comprise 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 SEQ ID No: 27, SEQ ID No: 28 or SEQ ID No: 29, and the amino acid sequence of the human antibody light chain constant region is shown as SEQ ID No:

30.

4. The method for detecting the biological activity of anti-IL-11 monoclonal antibody according to claim 1, wherein In step S1, constructing the stably passaged cell strain BAF / 3-GP130-STAT3-Luc comprises the following method: S11, transfecting a plasmid GP130 into BAF / 3 cells to obtain a cell strain BAF / 3-GP130 after screening; S12, transfecting a plasmid STAT3-Luc-NeoR into the cell strain BAF / 3-GP130 to obtain a stable cell strain BAF / 3-GP130-STAT3-Luc after screening.

5. The method of claim 1, wherein the biological activity of the anti-IL-11 monoclonal antibody is detected by the method of claim 1. In step S2, the following steps are specifically included: S21, the cell strain BAF / 3-GP130-STAT3-Luc is inoculated into a 96-well plate at a cell inoculation density of 2 x 10 4 -1 x 10 5 cells per well. S22, adding the IL-11 / IL-11RA complex into a 96-well plate to stimulate expression of a reporter gene in the cell strain BAF / 3-GP130-STAT3-Luc.

6. The method for detecting the biological activity of the anti-IL-11 monoclonal antibody according to claim 5, wherein The IL-11 / IL-11RA complex comprises IL-11 antigen with a stimulating final concentration of 0.02-40 μg / mL and IL-11RA antigen with a stimulating final concentration of 0.0025-2.5 μg / mL, and the ratio of the stimulating final concentration of the IL-11 antigen to the IL-11RA antigen is 1-16:

1.

7. The method of claim 1, wherein the biological activity of the anti-IL-11 monoclonal antibody is detected by the method of claim 1. In step S3, the initial concentration of the gradient-diluted anti-IL-11 monoclonal antibody is 500 μg / mL, and the ratio of the equi-proportional dilution is 0.2-0.5:

1.

8. The method for detecting the biological activity of the anti-IL-11 monoclonal antibody as described in claim 1, characterized in that, In step S3, the incubation time is 2-6 hours.

9. The method for detecting the biological activity of the anti-IL-11 monoclonal antibody according to any one of claims 1-8 is used for quality control in the production and research and development of anti-IL-11 monoclonal antibody drugs.

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