A method for purifying anti-il-11 monoclonal antibodies

Abstract

The application relates to the field of biological medicine, and specifically provides a purification method of an anti-IL-11 monoclonal antibody, which comprises affinity chromatography: primary purification and concentration of target proteins containing the anti-IL-11 monoclonal antibody, virus inactivation of protein products in an acid solution to obtain a protein sample; anion exchange chromatography: secondary purification of the protein sample to collect flow-through; and cation exchange chromatography: purification of the flow-through to obtain a protein solution of the anti-IL-11 monoclonal antibody. The application provides a suitable purification process for the anti-IL-11 monoclonal antibody, improves the purification efficiency and recovery rate of the anti-IL-11 monoclonal antibody, the flow-through collected through the anion exchange chromatography is directly introduced into the cation exchange chromatography without treatment, sample treatment or solution replacement in the middle, the purification time is saved, impurities are avoided to be introduced in the purification process, the purity of the purified protein is improved, and the method has high practicability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a purification method of anti-IL-11 monoclonal antibody. BACKGROUND

[0002] Fibrosis can occur in various organs, such as liver, lung, kidney, retina, intestine, heart and skin, and is the main cause of disability and death in many diseases. The main pathology is the increase of fibrous connective tissue in organ tissue, the decrease of parenchymal cells, the continuous progress can cause the destruction of organ structure and the decrease of function, and even the failure, which seriously threatens human health and life. According to the relevant statistical data of the United States, nearly 45% of the patients died of various diseases are due to tissue fibrosis.

[0003] Fibrosis diseases include pulmonary fibrosis, liver fibrosis, liver cirrhosis, liver disease, kidney fibrosis, heart fibrosis, asthma and various eye diseases. Among them, liver fibrosis is a pathophysiological process, which refers to the abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. Any liver damage has a liver fibrosis process in the process of liver repair and healing. If the damage factor cannot be removed for a long time, the process of fibrosis will continue for a long time and develop into cirrhosis. There are many causes of liver fibrosis, and viral hepatitis, alcoholic liver disease, fatty liver disease, autoimmune disease and the like are commonly seen in clinical practice. Globally, non-alcoholic fatty liver disease accounts for a high proportion, and if it is not treated in time, it will worsen into non-alcoholic hepatitis, and even cause liver inflammation, liver cell death, liver fibrosis, and eventually lead to the production of liver cirrhosis and liver cancer; in addition, pulmonary fibrosis is a terminal change of a large class of pulmonary diseases characterized by fibroblast proliferation and a large amount of extracellular matrix accumulation accompanied by inflammatory damage and tissue structure destruction. The vast majority of pulmonary fibrosis patients have unknown causes (idiopathic), which is a large category of interstitial pneumonia. The most common disease type with pulmonary fibrosis as the main manifestation in idiopathic interstitial pneumonia is idiopathic pulmonary fibrosis, which is a serious interstitial lung disease that can cause progressive loss of lung function. Pulmonary fibrosis seriously affects the respiratory function of the human body, and is manifested as dry cough and progressive dyspnea. With the aggravation of the disease and lung damage, the respiratory function of the patient is continuously deteriorated. The incidence and mortality of idiopathic pulmonary fibrosis are increasing year by year, and the average survival period after diagnosis is only 2.8 years, with a higher mortality rate than most tumors, which is called a "tumor-like disease". At present, there is no particularly effective drug for the treatment of fibrosis diseases of various organs, and the treatment effect is general. Therefore, the research and development of drugs for treating anti-fibrosis diseases are particularly urgent.

[0004] Interleukin-11 (IL-11) is a hematopoietic cytokine, which belongs to a kind of multifunctional interleukin 6 (IL6) family cytokines, sharing the same signaling receptor subunit (GP130). This family plays a crucial role in the occurrence, development and metastasis of tumors. Studies have found that the blockade of this pathway can become an effective treatment for a variety of tumor cancers, chronic fibrosis and inflammatory diseases. At present, there is no product on the market in the world targeting IL-11, therefore, in order to meet the urgent needs of patients, the process development of anti-IL-11 monoclonal antibody has important clinical significance.

[0005] At present, in the process development of anti-IL-11 monoclonal antibody, the conventional antibody purification method is used to purify the anti-IL-11 monoclonal antibody, and the degradation product and the target protein are often difficult to separate, and the sample is unstable in the process, and the purification efficiency, purity and recovery rate are low, therefore, the application provides a purification method specially suitable for anti-IL-11 monoclonal antibody. SUMMARY

[0006] In order to meet the urgent needs of fibrosis patients as soon as possible, improve the drug recovery rate and purity in the drug development process, and ensure the biological activity of anti-IL-11 monoclonal antibody in the purification process, the application provides a purification method specially suitable for anti-IL-11 monoclonal antibody.

[0007] The specific technical scheme of the application is as follows:

[0008] The application provides a purification method of anti-IL-11 monoclonal antibody, which comprises the following steps:

[0009] S1, affinity chromatography: the target protein containing anti-IL-11 monoclonal antibody is preliminarily purified and concentrated, and the collected protein product is inactivated in an acid solution, so as to obtain a crude purified protein sample;

[0010] S2, anion exchange chromatography: the protein sample is purified again, and the flow-through liquid is collected;

[0011] S3, cation exchange chromatography: the flow-through liquid is refined, and the protein solution of high-purity anti-IL-11 monoclonal antibody is obtained.

[0012] Furthermore, the anti-IL-11 monoclonal antibody comprises three heavy chain complementarity-determining regions (LCDs) denoted as HCDR1, HCDR2, and HCDR3, respectively, and three light chain complementarity-determining regions (LCDs) denoted as LCDR1, LCDR2, and LCDR3, respectively. The amino acid sequence of the heavy chain complementarity-determining region HCDR1 is shown in SEQ ID No:1, the amino acid sequence of the heavy chain complementarity-determining region HCDR2 is shown in SEQ ID No:14, the amino acid sequence of the heavy chain complementarity-determining region HCDR3 is shown in SEQ ID No:15, the amino acid sequence of the light chain complementarity-determining region LCDR1 is shown in SEQ ID No:4, the amino acid sequence of the light chain complementarity-determining region LCDR2 is shown in SEQ ID No:5, and the amino acid sequence of the light chain complementarity-determining region LCDR3 is shown in SEQ ID No:6.

[0013] The beneficial effects of this invention are as follows: First, this invention uses a continuous three-step purification method to screen the most suitable conditions and chromatographic packing for the anti-IL-11 monoclonal antibody provided by this invention, effectively improving the purification efficiency and recovery rate of the anti-IL-11 monoclonal antibody provided by this invention. Specifically, the flow-through collected by anion exchange chromatography can directly enter cation exchange chromatography without further treatment, eliminating the need for sample processing or solution replacement. This not only greatly saves purification time but also avoids the introduction of impurities during purification, improving the purity of the purified protein and making it highly practical. Second, the anti-IL-11 monoclonal antibody provided by this invention has a high binding capacity to the IL-11 antigen, blocking the binding of the IL-11 antigen to its receptor, thereby effectively inhibiting the pro-fibrotic effect of IL-11 and inhibiting or preventing the production or proliferation of fibroblasts. It can be effectively used to treat or prevent human fibrotic diseases, inflammation, cancer, or autoimmune diseases. Attached Figure Description

[0014] Figure 1 This is a plasmid map of the pScFv-Disb-HS vector in Example 2 of the present invention;

[0015] Figure 2 This is a comparison diagram of the affinity of serially diluted ELISA anti-IL-11 phage monoclonal antibodies in Example 3 of the present invention;

[0016] Figure 3 This is a spectrum of the carrier pTSE in Embodiment 5 of the present invention;

[0017] Figure 4 This is a denaturing polyacrylamide gel electrophoresis image of the mouse antibody molecule in Example 5 of the present invention;

[0018] Figure 5 This is a comparison diagram of the binding ability of the mouse antibody molecule to IL-11 in Example 6 of the present invention;

[0019] Figure 6 This is a comparison diagram of the competitive inhibition experiment between the mouse antibody and the IL-11 receptor protein IL-11RA in Example 7 of the present invention;

[0020] Figure 7 This is a comparative diagram showing the inhibition of IL-11 binding to the IL-11RA receptor on the cell surface of BaF / 3-IL-11RA by murine antibody in Example 8 of the present invention;

[0021] Figure 8 This is a comparative diagram showing the inhibition of TIMP-1 secretion by mouse-derived antibodies in embryonic lung fibroblasts MRC-5 in Example 9 of the present invention;

[0022] Figure 9 This is a denaturing polyacrylamide gel electrophoresis image of the humanized antibody molecule in Example 14 of this invention;

[0023] Figure 10 This is a comparison diagram of the binding ability of humanized antibody molecules to IL-11 in Example 15 of the present invention;

[0024] Figure 11 This is a comparative diagram showing the inhibition of IL-11 binding to the IL-11RA receptor on the cell surface of BaF / 3-IL-11RA by humanized antibody molecules in Example 16 of the present invention.

[0025] Figure 12 This is a comparative diagram showing the inhibition of IL-11 binding to the GP130 receptor on the surface of BaF / 3-GP130 cells by humanized antibody molecules in Example 17 of the present invention.

[0026] Figure 13 This is a comparative graph showing the biological activity detection (reporter gene) of humanized antibody molecules in Example 18 of the present invention;

[0027] Figure 14 This is a comparative diagram showing the inhibition of TIMP-1 secretion by humanized antibody molecules in embryonic lung fibroblasts MRC-5 in Example 19 of the present invention;

[0028] Figure 15 This is a comparative diagram of the cross-binding experiments between humanized antibody molecules and IL-11 from different species in Example 20 of the present invention;

[0029] Figure 16 This is a bar chart showing the changes in the lung-to-body weight ratio in the mouse pulmonary fibrosis model of Example 21 of this invention;

[0030] Figure 17 Images of hematoxylin and eosin (HE) staining and marson staining of lung tissue sections from the mouse pulmonary fibrosis model in Example 21 of this invention;

[0031] Figure 18 This is a bar chart showing the change in the heart-to-body weight ratio in the mouse cardiac fibrosis model of Example 22 of the present invention;

[0032] Figure 19 Images of hematoxylin and eosin (HE) staining and marson staining of heart tissue sections from the mouse cardiac fibrosis model in Example 22 of this invention;

[0033] Figure 20 This is a bar chart showing the urinary protein content in the kidneys of a mouse renal fibrosis model in Example 23 of this invention.

[0034] Figure 21 Images of hematoxylin and eosin (HE) staining and marson staining of kidney tissue sections from the mouse kidney fibrosis model in Example 23 of this invention;

[0035] Figure 22 This is a bar chart showing the changes in liver weight in the mouse liver fibrosis model of Example 24 of the present invention;

[0036] Figure 23 This is a bar chart showing the changes in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in a mouse liver fibrosis model in Example 24 of this invention.

[0037] Figure 24 Images of hematoxylin and eosin (HE) staining and marson staining of liver tissue sections from the mouse liver fibrosis model in Example 24 of this invention;

[0038] Figure 25 This is a graph showing the thermal stability evaluation of the anti-IL-11 monoclonal antibody HA-IA in Example 25 of the present invention. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to the following embodiments.

[0040] Example 1

[0041] This invention provides a method for purifying anti-IL-11 monoclonal antibodies, the method comprising:

[0042] S1. Affinity chromatography: The target protein containing anti-IL-11 monoclonal antibody is initially purified and concentrated, and the collected protein product is inactivated by virus in acid solution to obtain crude pure protein sample.

[0043] S2, Anion exchange chromatography: The protein sample is purified a second time, and the flow-through is collected;

[0044] S3. Cation exchange chromatography: After purifying the flow-through solution, a high-purity protein solution of anti-IL-11 monoclonal antibody is obtained.

[0045] The anti-IL-11 monoclonal antibody includes three heavy chain complementarity-determining regions represented by HCDR1, HCDR2, and HCDR3, respectively, and three light chain complementarity-determining regions represented by LCDR1, LCDR2, and LCDR3, respectively. The anti-IL-11 monoclonal antibody is selected from any of the following.

[0046]

[0047] The anti-IL-11 monoclonal antibody provided by this invention is used to treat or prevent human fibrotic diseases, inflammation, cancer, or autoimmune diseases. Fibrotic diseases include, but are not limited to, fibrosis of the heart, liver, kidneys, lungs, gallbladder, bladder, stomach, bone marrow, penis, breast, blood vessels, eyes, pancreas, spleen, brain, intestines, muscles, or skin. Inflammation includes, but is not limited to, hepatitis, myocarditis, nephritis, pneumonia, cholecystitis, cystitis, gastritis, osteomyelitis, prostatitis, mastitis, pancreatitis, enteritis, arthritis, polymyositis, dermatomyositis, or dermatitis. Cancer includes, but is not limited to, leukemia, lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, or bladder cancer. Autoimmune diseases include, but are not limited to, psoriasis, Crohn's disease, primary biliary cirrhosis, systemic lupus erythematosus, or multiple sclerosis.

[0048] Example 2: Screening of mouse antibody molecules

[0049] This invention optimizes the immunization method by immunizing mice with IL-11 antigen (in subsequent experiments, IL-11 protein, IL-11-Fc antigen, and IL-11-mFc ligand protein were all human IL-11) and creates a phage display library. The specific construction, screening, and identification of the phage display library are as follows:

[0050] Step 1: Immunize mice with IL-11 antigen

[0051] 1. Laboratory animals:

[0052] Species / strain: BALB / c, female, mouse;

[0053] Weight: 18-20g;

[0054] Laboratory animal provider: Beijing Huafukang Biotechnology Co., Ltd.

[0055] 2. Immunization: Mice were immunized with human IL-11 (a gene synthesized by Nanjing Genscript Biotech Co., Ltd., whose vector was constructed and purified by our company).

[0056] Step 2: Construction of the phage antibody library

[0057] Mouse spleen cells with high titers were collected, and total RNA was extracted from the cells using Trizol reagent (purchased from Ambion, catalog number: 15596026). cDNA was obtained by RT-PCR. Using the cDNA as a template, PCR amplification was performed using degenerate primers (reference: Journal of Immunological Methods 233 (2000) 167-177) to obtain the heavy chain variable region gene library (VH) and light chain variable region gene library of immunized mice. The pScFv-Disb-HS vector was modified from the pComb3 vector (purchased from the China Plasmid Vector Strains Cell Line Gene Preservation Center) using a series of gene cloning methods to construct and express a phage single-chain antibody library. The modified vector was named pScFv-Disb-HS, and its plasmid map is shown below. Figure 1 As shown, a mouse immune phage antibody library was constructed based on this vector. The light and heavy chain variable region gene libraries were double-digested and ligated into the pScFv-Disb-HS vector, which had been digested in the same steps, to construct the pScFv-Disb-HS-VH-VL gene library.

[0058] Step 3: Coat the immunotubes with IL-11 antigen at a rate of 5 μg / 500 μL / tube, and incubate overnight at 4°C. Then, block the immunotubes and the immunophage antibody library separately with 4% skim milk powder / PBST at room temperature for 1 hour. Add the blocked immunophage antibody library to the immunotubes for antigen-antibody binding; the phage dosage is approximately 10 μg / 500 μL. 9 ~10 12 After reacting at room temperature for 1 hour, unbound phages were washed away with PBST-PBS, followed by elution with 0.1M pH 2.2 Glycine-HCl. Finally, the eluted phage antibody solution was neutralized to approximately pH 7.0 with 1.5M pH 8.8 Tris-HCl.

[0059] Step 4: Infect 10 ml of TG1 bacterial culture grown to the logarithmic phase with the neutralized phage, incubate at 37°C for 30 min, then take a portion of the bacterial culture and perform serial dilutions, spreading it onto 2YTAG plates to calculate phage yield. Centrifuge the remaining bacterial culture, discard the supernatant, resuspend the bacterial pellet in a small amount of culture medium, aspirate it, and spread it onto large 2YTAG plates to prepare for the next round of screening.

[0060] Step 5: Scrape the infected bacterial cells from the large plate and inoculate them into 2YTAG liquid medium. After shaking to the logarithmic phase, add M13KO7 helper phage for superinfection. Incubate overnight at 220 rpm at 28°C to prepare phages. Purify the phages by PEG / NaCl precipitation for the next round of screening. Perform one round of phage library enrichment screening.

[0061] Step Six: Screening of IL-11 phage single-chain antibody-positive clones: After one round of screening, well-separated single-clone colonies are picked and inoculated into 96-well deep-well plates containing 2 YTAG liquid medium. The plates are incubated at 37°C and 220 rpm until the logarithmic growth phase. Approximately 10 μL of the medium is added to each well. 10 Helper phage M13KO7 was used for static infection at 37°C for 30 min. After centrifugation at 4000 rpm for 15 min, the supernatant was discarded, and the bacterial cells were resuspended in 2YTAK solution and cultured overnight at 28°C and 220 rpm. After centrifugation at 4000 rpm and 4°C for 15 min, the amplified phage supernatant was subjected to ELISA identification. Four murine antibody molecules with high affinity were ultimately screened and named MA-I, MA-II, MA-III, and MA-IV, respectively. The obtained monoclonal antibodies were sequenced to confirm their correct antibody sequences. The sequencing sequences of the four selected monoclonal antibodies are as follows:

[0062]

[0063] Specifically, SEQ ID No:16 (amino acid sequence of the heavy chain variable region of MA-Ⅰ and MA-Ⅱ):

[0064] EVKLEESGGGLVKPGGSLKLSCAASGFTFSDYYMFWVRQTPEKRLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNNLYLQMTSLKSEDTAMYYCARDGGYVS SPEAMDYWGQGTSVTVSS;

[0065] SEQ ID No:17 (Amino acid sequence of the light chain variable region of MA-Ⅰ and MA-Ⅳ):

[0066] DIVLTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPPTFGGGTKLEIK;

[0067] SEQ ID No:18 (Amino acid sequence of the light chain variable region of MA-II):

[0068] DIVLTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK;

[0069] SEQ ID No:19 (Amino acid sequence of the heavy chain variable region of MA-III):

[0070] EVKLEQSGAEVVKPGALVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIG VIDPSDSYTTYNQKFKGKATLTVDTSSSTGYMQLSSLTSEDSAVYYCSQYGYDVN WYFDVWGAGTTVTVSS;

[0071] SEQ ID No:20 (Amino acid sequence of the light chain variable region of MA-III):

[0072] DIVMTQTTLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLI YEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGT KLEIK;

[0073] SEQ ID No:21 (Amino acid sequence of the heavy chain variable region of MA-Ⅳ):

[0074] EVQLEESGGGLVKPGGSLKLSCVASGFTFSDYYMFWVRQTPEKRLEWVATI SDGGSYSYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTAMYYCARDGGYISS PEAMDYWGQGTSVTVSS.

[0075] Example 3: Comparison of antibody affinity using gradient dilution ELISA

[0076] The four murine antibody molecules (MA-I, MA-II, MA-III, and MA-IV) obtained in Example 2 were displayed and purified as monoclonal phages, and then affinity was identified by phage gradient dilution ELISA experiments. The specific methods are as follows:

[0077] IL-11 antigen was coated with carbonate buffer (pH 9.6), 100 ng / well / 100 μL, and incubated overnight at 4°C. After washing three times with PBST, the four phage monoclonal antibodies selected in Example 2 were serially diluted five-fold with PBST, with 100 μL of diluted sample added to each well, and incubated at room temperature for 1 hour. The ELISA plate was washed with PBST, and HRP-anti-M13 monoclonal antibody (purchased from Bio-viewshine, catalog number: GE27-9421-01) diluted with 1% BSA-PBST was added to the ELISA plate and incubated at room temperature for 1 hour. A TMB chromogenic kit (purchased from Kangwei Century, catalog number: CW0050S) was used for color development, incubated at room temperature for 10 minutes, and stopped with 2M H2SO4. Readings were taken at 450 nm / 630 nm using a microplate reader, and the corresponding EC50 values ​​were calculated. Specific data are as follows:

[0078]

[0079] Based on the above data and as follows Figure 2 As shown, the four different murine antibody molecules screened in Example 2 were all able to bind to IL-11, which demonstrates that the monoclonal antibody provided by the present invention has a high affinity for IL-11.

[0080] Example 4

[0081] Example 4 of this invention further specifies, based on Example 2, that the anti-IL-11 monoclonal antibody also includes a heavy chain constant region and a light chain constant region. The amino acid sequence of the heavy chain constant region is one of SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, or SEQ ID No:26; the amino acid sequence of the light chain constant region is as shown in SEQ ID No:22, and the specific sequence is as follows:

[0082] SEQ ID No:22 (Rat C) k (Amino acid sequence of the light chain constant region):

[0083] ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; SEQ ID No:23 (Amino acid sequence of the heavy chain constant region of mouse IgG1 type):

[0084] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG;

[0085] SEQ ID No:24 (Amino acid sequence of the heavy chain constant region of murine IgG2a):

[0086] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK;

[0087] SEQ ID No:25 (Amino acid sequence of the heavy chain constant region of murine IgG2b):

[0088] AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFV NNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK;

[0089] SEQ ID No:26 (Amino acid sequence of the heavy chain constant region of mouse IgG3 type):

[0090] ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGK EFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSW LQGEIFTCSVVHEALHNHHTQKNLSRSPELELNETCAEAQDGELDGLWTTITIFISLFLLSVCYSASVTLFKVKWIFSSVVQVKQTAIPDYRNMIGQGA.

[0091] Example 5: Preparation of mouse antibody molecules

[0092] Example 5 of the present invention, based on Example 4, preferably specifies that the murine antibody molecule includes the heavy chain constant region of murine IgG1 (its amino acid sequence is shown in SEQ ID No: 23) and murine C kThe light chain constant region of type [type] (its amino acid sequence is shown in SEQ ID No:22). The specific antibody preparation method is as follows:

[0093] 1. The encoding genes for the heavy chain VH and light chain VL of the four antibody molecules screened in Example 2 were cloned into the vector pTSE (e.g., ...) containing the heavy chain and light chain constant region genes, respectively. Figure 3 As shown in SEQ ID No: 23), the preferred heavy chain constant region is the mouse IgG1 type constant region (amino acid sequence shown in SEQ ID No: 23), and the light chain constant region is mouse C k Chain (amino acid sequence as shown in SEQ ID No: 22), pTSE vector structure as shown Figure 3 As shown (for the preparation process of the pTSE vector, please refer to paragraph

[0019] on page 3 of the instruction manual CN103525868A).

[0094] 2. HEK293 cells were transiently transfected (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number GNHu43) for antibody expression. Four monoclonal antibodies were purified using an AKTA instrument via a protein A affinity column. Protein concentration was determined using a BCA kit (purchased from Beijing Huitian Oriental Technology Co., Ltd., catalog number BCA0020). Protein size was then identified by SDS-PAGE. The results are shown below. Figure 4 As shown, from left to right, the images represent non-reduced MA-I, MA-II, MA-III, and MA-IV, protein molecular weight Marker 1, protein molecular weight Marker 2, and reduced MA-I, MA-II, MA-III, and MA-IV murine anti-IL-11 monoclonal antibodies. The molecular weight of each band is consistent with the theoretical values.

[0095] Example 6: Binding experiment of mouse antibody molecules with IL-11

[0096] IL-11 antigen was coated with carbonate buffer (pH 9.6), 100 ng / well / 100 μL, and incubated overnight at 4°C. The cells were washed five times with 300 μL / well PBST, then blocked with 280 μL / well of 1% BSA-PBST at 37°C for 1 h. Different concentrations of MA-I, MA-II, MA-III, and MA-IV mouse antibodies were added, with an initial maximum concentration of 5 μg / mL for each antibody. Each antibody was serially diluted 5-fold, for a total of 8 dilutions, and incubated at 37°C for 1 h. The cells were then washed five times with 300 μL / well PBST, and Goat Anti-Mouse IgG-HRP (purchased from Solarbio, catalog number: SE131) diluted 1:2000 with 1% BSA-PBST was added, and the cells were incubated at 37°C for 1 h. The TMB chromogenic kit was used for color development. 100 μL / well was incubated at room temperature for 8 min, then the color development was stopped with 2 M H₂SO₄. Readings were taken at 450 nm and 630 nm using a microplate reader, and the corresponding EC50 values ​​were calculated. Specific data are as follows:

[0097]

[0098] Based on the above data and as follows Figure 5 As shown, all four different murine antibody molecules screened were able to bind to IL-11 and had high affinity.

[0099] Example 7: Competitive inhibition experiment between murine antibody and IL-11 receptor protein IL-11RA

[0100] IL-11-Fc was coated with carbonate buffer (pH 9.6), 200 ng / well / 100 μL, overnight at 4°C. The cells were washed five times with 300 μL / well PBST, then blocked with 280 μL / well of 1% BSA-PBST at 37°C for 1 h. First, IL-11RA-Fc (IgG4 type) diluted to 0.5 μg / mL with 1% BSA-PBST was added, 50 μL / well. Then, different concentrations of MA-Ⅰ, MA-Ⅱ, MA-Ⅲ, and MA-Ⅳ murine antibodies were added, 50 μL / well. The initial maximum concentration of all five antibodies was 100 μg / mL, and each antibody was serially diluted 2-fold, for a total of 13 dilutions. All cells were incubated at 37°C for 3 h. Wash five times with 300 μL / well PBST, then add Anti-Human IgG4-HRPMouse monoclonal antibody (purchased from Sigma, catalog number: SAB4200770) diluted 1:5000 with 2% BSA-PBST, and incubate at 37°C for 1 h. Develop color with a TMB chromogenic kit, 100 μL / well, at room temperature for 15 min, then stop the development with 2M H2SO4. Read the values ​​using a microplate reader at 450 nm / 630 nm and calculate the corresponding IC50 values. Specific data are as follows:

[0101]

[0102] Based on the above data and such Figure 6 As shown, all four different murine antibodies screened were able to compete with the receptor protein IL-11RA, indicating that they could effectively inhibit the binding of IL-11 to the receptor protein IL-11RA.

[0103] Example 8: Mouse antibody inhibits the binding of IL-11 to the IL-11RA receptor on the cell surface of BaF / 3-IL-11RA.

[0104] BaF / 3-IL-11RA cell lines were counted, and a certain number of cells were centrifuged, resuspended in PBS buffer, and the cell density was adjusted to 1E+6 cells / mL. 100 μL / well was added to each well of a 96-well plate. IL-11-mFc ligand protein was diluted with PBS to a concentration of 18 μg / mL, and 50 μL / well was added to each well of a 96-well plate containing BaF / 3-IL-11RA cells. After gentle mixing, the 96-well plates were incubated at 4°C for 1 h. Four murine antibody molecules, MA-I, MA-II, MA-III, and MA-IV, were serially diluted with PBS at an initial concentration of 800 μg / mL, followed by 3-fold serial dilutions (10 dilutions in total). 50 μL / well was added to each well of a 96-well plate containing the mixture of BaF / 3-IL-11RA cells and IL-11-mFc ligand protein. After thorough mixing, the plates were incubated at 4°C for 2 h. After incubation, cells were centrifuged at 3000 rpm, washed once with PBS buffer, and the cell pellet was collected. Pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) was added to the cell pellet, and the cells were incubated at 4°C for 30 min. After centrifugation at 3000 rpm, the cells were washed once with PBS buffer, resuspended in 100 μL of PBS buffer, and analyzed by flow cytometry, collecting the fluorescence signal in the FL1-A channel. A dose-response curve was plotted, and the corresponding IC50 value was calculated. Specific data are as follows:

[0105]

[0106] Based on the above data and Figure 7 It can be seen that the four different mouse-derived candidate molecules screened can effectively inhibit the binding of IL-11 ligand protein to the IL-11RA receptor on the cell surface.

[0107] Example 9: Mouse-derived antibody inhibits TIMP-1 secretion from MRC-5 embryonic lung fibroblasts.

[0108] MRC-5 embryonic lung fibroblasts were digested with trypsin and counted. A certain number of cells were collected, centrifuged, and resuspended in MEM complete medium (purchased from GIBCO, catalog number 10370-021). The cell density was adjusted to 2E+5 cells / mL, and 100 μL / well was added to 96-well plates. IL-11-mFc ligand protein was diluted in MEM complete medium to a concentration of 16 μg / mL, and 50 μL / well was added to the corresponding 96-well plates. Four murine antibody molecules, MA-I, MA-II, MA-III, and MA-IV, were serially diluted in MEM complete medium to an initial concentration of 40 μg / mL. Eight 2-fold serial dilutions were performed, and 50 μL / well was added to each well of the 96-well plates containing cell suspension and IL-11-mFc ligand protein suspension. After gentle mixing, the plates were incubated overnight at 37°C in a CO2 incubator for approximately 20 hours. Cell culture supernatant was collected and tested using the TIMP-1 ELISA kit (purchased from Ecosai Biotechnology Co., Ltd., catalog number EH021-96).

[0109] Human TIMP-1 Detection Kit: Add cell supernatant and standards to sample wells, 100 μL / well. Immediately add biotinylated antibody working solution (1:100 dilution), 50 μL / well, cover with sealing film, and incubate at room temperature with shaking for 2 h. After incubation, wash the plate 4 times with wash buffer, add enzyme conjugate working solution (1:100 dilution) from the TIMP-1 detection kit, 100 μL / well. Cover with sealing film and incubate at room temperature with shaking for 1 h. After incubation, wash the plate 4 times with wash buffer. Add TMB chromogenic solution, 100 μL / well, incubate at room temperature in the dark for about 15 minutes, and stop the reaction with 100 μL / well stop solution. Read the values ​​using a microplate reader at 450 nm and calculate the corresponding IC50 values. Specific data are as follows:

[0110]

[0111] Based on the above data and Figure 8 It was found that the four different mouse-derived candidate molecules screened out could effectively inhibit the release of TIMP-1 from human embryonic lung fibroblasts MRC-5 stimulated by IL-11 ligand protein.

[0112] Example 10

[0113] Example 10 of the present invention further specifies that the anti-IL-11 monoclonal antibody is a chimeric antibody molecule. The chimeric antibody molecule also includes a human antibody constant region, which includes 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 the sequences shown in SEQ ID No:27, SEQ ID No:28 or SEQ ID No:29; the amino acid sequence of the human antibody light chain constant region is shown in SEQ ID No:30.

[0114] SEQ ID No:27 (Amino acid sequence of the heavy chain constant region of human IgG1):

[0115] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0116] SEQ ID No:28 (Amino acid sequence of the heavy chain constant region of human IgG2 type):

[0117] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0118] SEQ ID No:29 (Amino acid sequence of the heavy chain constant region of human IgG4):

[0119] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0120] SEQ ID No:30 (human C k (Amino acid sequence of the light chain constant region):

[0121] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C.

[0122] Example 11 Preparation of chimeric antibody molecules

[0123] Example 11 of the present invention further defines the constant region of the human antibody, based on Example 10, as including the heavy chain constant region of human IgG1 (whose amino acid sequence is shown in SEQ ID No: 27) and human C k The light chain constant region of type (its amino acid sequence is shown in SEQ ID No:30).

[0124] Specific preparation method:

[0125] The heavy chain variable region VH (SEQ ID No: 16) and light chain variable region VL gene (SEQ ID No: 17) of the murine antibody molecules MA-I and MA-II obtained from the screening of the phage antibody library in Example 2, as well as the light chain variable region VL gene (SEQ ID No: 18) of MA-I and MA-II, were cloned into the vector pTSE (e.g., [example of a vector]) containing the heavy chain constant region and light chain constant region genes, respectively, while keeping their murine sequences unchanged. Figure 3 As shown in the figure, the heavy chain constant region is human IgG1 type (amino acid sequence as shown in SEQ ID NO:27), and the light chain constant region is human C.k Type (amino acid sequence as shown in SEQ ID NO:30). HEK293E cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number: GNHu43) were transiently transfected to express antibodies, yielding chimeric antibodies CA-I and CA-II.

[0126] Example 12 Humanization of mouse antibody molecules

[0127] First, the sequences of the murine antibody molecules MA-I and MA-II from Example 2 were compared with the human antibody lineage database (v-base) to identify human antibody light and heavy chain lineages with high homology as candidate sequences. Then, the CDR sequences of the murine antibody molecules MA-I and MA-II were transplanted into the human candidate sequences for homology modeling. Next, three-dimensional structural simulations were used to calculate key framework amino acid residues that might play an important role in maintaining the CDR ring structure, thereby designing reversion mutations for humanized antibodies. The light and heavy chain variable region sequences of the designed humanized antibodies containing reversion mutations were optimized and synthesized by Nanjing Genscript Biotech Co., Ltd., and then ligated into a transient expression vector. Analysis of the humanized light and heavy chain combinations yielded the following humanized anti-IL-11 monoclonal antibody molecules: HA-IA, HA-IB, HA-IC, and HA-ID for MA-I; and the following humanized antibody molecules: HA-II-A, HA-II-B, HA-II-C, and HA-II-D for MA-II. The eight monoclonal antibody sequences selected above are as follows:

[0128]

[0129]

[0130] Specifically, SEQ ID No:31 (amino acid sequences of the heavy chain variable regions of HA-IA, HA-IC, HA-II-A, and HA-II-B):

[0131] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVSS PEAMDYWGQGTLVTVSS;

[0132] SEQ ID No:32 (Amino acid sequence of the light chain variable region of HA-IA):

[0133] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;

[0134] SEQ ID No:33 (Amino acid sequence of the heavy chain variable region of HA-IB and HA-II-C):

[0135] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVST ISDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVS SPEAMDYWGQGTLVTVSS;

[0136] SEQ ID No:34 (Amino acid sequence of the light chain variable region of HA-IB):

[0137] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;

[0138] SEQ ID No:35 (Amino acid sequence of the light chain variable region of HA-IC):

[0139] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;

[0140] SEQ ID No:36 (Amino acid sequence of the heavy chain variable region of HA-ID and HA-II-D):

[0141] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAMYYCARDGGYVS SPEAMDYWGQGTSVTVSS;

[0142] SEQ ID No:37 (Amino acid sequence of the light chain variable region of HA-ID):

[0143] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;

[0144] SEQ ID No:38 (Amino acid sequence of the light chain variable region of HA-II-A):

[0145] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;

[0146] SEQ ID No:39 (Amino acid sequence of the light chain variable region of HA-II-B and HA-II-C):

[0147] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;

[0148] SEQ ID No:40 (Amino acid sequence of the light chain variable region of HA-II-D):

[0149] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGTVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPWTFGGGTKVEIK.

[0150] Example 13

[0151] Based on Example 12, Example 13 of the present invention further defines the human antibody constant region as including 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; the amino acid sequence of the human antibody light chain constant region is shown in SEQ ID No:30.

[0152] The specific sequence of the constant region of the human antibody described above is the same as that in Example 10.

[0153] Example 14 Preparation of humanized antibody molecules

[0154] Example 14 of the present invention further defines the constant region of the human antibody, based on Example 13, as including the heavy chain constant region of human IgG1 (whose amino acid sequence is shown in SEQ ID No: 27) and human C k The light chain constant region of type (its amino acid sequence is shown in SEQ ID No:30).

[0155] The heavy chain VH and light chain VL encoding genes of the eight humanized anti-IL-11 monoclonal antibody molecules HA-IA, HA-IB, HA-IC, HA-ID, HA-II-A, HA-II-B, HA-II-C, and HA-II-D obtained in Example 12 were cloned into the vector pTSE (e.g., ...) containing the heavy chain constant region and light chain constant region genes, respectively. Figure 3 As shown), the heavy chain constant region is human IgG1 type (amino acid sequence as shown in SEQ ID NO:27), and the light chain constant region is C. k Chain (amino acid sequence as shown in SEQ ID NO:30).

[0156] Two chimeric antibodies, CA-I and CA-II, obtained in Example 11, and eight humanized antibody molecules, HA-IA, HA-IB, HA-IC, HA-ID, HA-II-A, HA-II-B, HA-II-C, and HA-II-D, obtained in Example 12, were transiently transfected into HEK293 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number GNHu43) for antibody expression. Monoclonal antibodies were purified using an AKTA instrument via a protein A affinity column. Protein concentration was determined using a BCA kit (purchased from Beijing Huitian Oriental Technology Co., Ltd., catalog number BCA0020). Protein size was then identified by SDS-PAGE. The results are shown below. Figure 9 As shown, from left to right, the molecular weights of the non-reduced proteins are HA-IA, HA-IB, HA-IC, HA-ID, the chimeric antibody CA-I prepared in Example 11, the molecular weight marker of the reduced protein, 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 theoretical value.

[0157] Example 15: Experiment on the binding of humanized antibody molecules to IL-11

[0158] IL-11 antigen was coated with carbonate buffer (pH 9.6) at a concentration of 100 ng / well / 100 μL overnight at 4°C. The cells were washed five times with 300 μL / well PBST, and then blocked with 280 μL / well of 1% BSA-PBST at 37°C for 1 h. Humanized antibodies HA-IA, HA-IB, HA-IC, HA-ID, HA-II-A, HA-II-B, HA-II-C, HA-II-D and chimeric antibodies CA-I and CA-II prepared in Example 11 were diluted with 1% BSA-PBST. The initial concentration of all humanized antibodies was 10 μg / mL, and eight 5-fold serial dilutions were performed, incubating at 37°C for 1 h. Wash five times with 300 μL / well PBST, then add goat anti-Human IgG Fab HRP (Invitrogen, catalog number: 31482) diluted 1:5000 with 1% BSA-PBST, and incubate at 37°C for 1 h. Develop color with a TMB chromogenic kit, 100 μL / well, at room temperature for 5 min, then stop the development with 2M H2SO4. Read the values ​​at 450 nm / 630 nm using a microplate reader and calculate the corresponding EC50 values. Specific data are as follows:

[0159]

[0160]

[0161] Based on the above data and experimental results, as follows: Figure 10 As shown, all eight different humanized antibody molecules can bind to IL-11. The EC50 values ​​of humanized antibody molecules HA-IA, HA-IB, HA-IC, and HA-ID are close to those of chimeric antibody CA-I, while the EC50 values ​​of humanized antibody molecules HA-II-A, HA-II-B, HA-II-C, and HA-II-D are close to those of chimeric antibody CA-II. This indicates that the humanized antibody molecules retain the high binding capacity of the murine parental antibodies MA-I and MA-II to IL-11.

[0162] Example 16: Humanized antibody molecules inhibit the binding of IL-11 to the IL-11RA receptor on the cell surface of BaF / 3-IL-11RA.

[0163] Four humanized antibody molecules (HA-IA, HA-IB, HA-IC, and HA-ID) with superior protein-level binding activity were selected for cell viability evaluation experiments. BaF / 3-IL-11RA cell lines were counted, and a certain number of cells were collected, centrifuged, resuspended in PBS buffer, and the cell density was adjusted to 1E+6 cells / mL. 100 μL / well was added to each well of a 96-well plate. IL-11-mFc ligand protein was diluted with PBS to a concentration of 18 μg / mL, and 50 μL / well was added to the corresponding well of the 96-well plate containing BaF / 3-IL-11RA cells. After gentle mixing, the 96-well plates were incubated at 4°C for 1 h. Four humanized antibody molecules, HA-IA, HA-IB, HA-IC, and HA-ID, were serially diluted with PBS buffer to prepare an initial concentration of 800 μg / mL. Ten 3-fold serial dilutions were performed, with 50 μL / well added to the corresponding wells of a 96-well plate containing a mixture of BaF / 3-IL-11RA cells and IL-11-mFc ligand protein. After thorough mixing, the plates were incubated at 4°C for 2 h. After incubation, the cells were washed once with PBS buffer at 3000 rpm, and the cell pellet was collected. Pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) was added to the cell pellet, and the plates were incubated at 4°C for 30 min. After washing once with PBS buffer at 3000 rpm, the cells were resuspended in 100 μL of PBS and analyzed by flow cytometry, collecting the fluorescence signal in the FL1-A channel. Dose-response curves were plotted, and the corresponding IC50 values ​​were calculated. Specific data are as follows:

[0164]

[0165] Based on the above data and Figure 11 It can be seen that all four humanized candidate molecules screened can inhibit the binding of IL-11 ligand protein to the IL-11RA receptor on the surface of BaF / 3-IL-11RA cells.

[0166] Example 17: Humanized antibody molecules inhibit the binding of IL-11 to the GP130 receptor on the surface of BaF / 3-GP130 cells.

[0167] BaF / 3-GP130 cell lines were counted, and a certain number of cells were centrifuged, resuspended in PBS buffer, and the cell density was adjusted to 1E+6 cells / mL. 100 μL / well was added to each well of a 96-well plate. IL-11-mFc ligand protein was diluted with PBS to a concentration of 12 μg / mL, and 50 μL / well was added to the corresponding well of a 96-well plate containing BaF / 3-GP130 cells. After gentle mixing, the 96-well plates were incubated at 4°C for 1 h. Four human antibody molecules, HA-IA, HA-IB, HA-IC, and HA-ID, were serially diluted with PBS buffer to an initial concentration of 2000 μg / mL. Ten 2-fold serial dilutions were performed, and 50 μL / well was added to the corresponding well of a 96-well plate containing BaF / 3-GP130 cells and IL-11-mFc ligand protein. After thorough mixing, the 96-well plates were incubated at 4°C for 2 h. After incubation, cells were washed once with PBS buffer at 3000 rpm and the cell pellet was collected. 100 μL / well of pre-prepared 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 washing once with PBS buffer at 3000 rpm, cells were resuspended in PBS buffer at 100 μL / well and analyzed by flow cytometry, collecting fluorescence signals in the FL1-A channel. Dose-response curves were plotted, and the corresponding IC50 values ​​were calculated. Specific data are as follows:

[0168]

[0169] Based on the above data and Figure 12 It can be seen that all four humanized candidate molecules screened can block the binding of IL-11 ligand protein to the GP130 receptor on the surface of BaF / 3-GP130 cells.

[0170] Example 18: Detection of the biological activity of humanized antibody molecules (reporter gene)

[0171] The BaF / 3-IL-11RA-GP130-STAT3-Luc engineered cell line was counted. The cell density was adjusted to 2E+6 cells / mL using sample diluent (containing 90% IMDM, 10% FBS, and 10 ng / mL mouseIL-3). After gentle mixing, the cell culture was added to 96-well plates at 50 μL / well. Four humanized antibody molecules, HA-IA, HA-IB, HA-IC, and HA-ID, were diluted to an initial concentration of 200 μg / mL using sample diluent. A 5-fold serial dilution was performed, resulting in 10 serial dilutions, with 100 μL / well added to the corresponding wells of the 96-well plates containing the engineered cell line. Two replicates were set for each sample concentration. IL-11 protein was prepared at a concentration of 10 μg / mL using sample diluent, at 50 μL / well, and added to 96-well plates containing the engineered cell line and humanized antibody molecules. The cell culture plates were gently mixed and incubated at 37°C in a CO2 incubator for 6 hours. Centrifuge and discard the supernatant. Add lysis buffer, 10 μL / well to a 384-well plate, and add an equal volume of luciferase reaction substrate (purchased from Promega Biotechnology Co., Ltd., catalog number E2610). Incubate at room temperature for 5 min, read the fluorescence values ​​using a microplate reader, and calculate the corresponding IC50 values. Specific data are as follows:

[0172]

[0173] Based on the above data and Figure 13 As shown, the four humanized antibody molecules screened can block the binding of IL-11 to IL-11RA and GP130 receptors, and inhibit the transduction of the signaling pathway.

[0174] Example 19: Humanized antibody molecules inhibit TIMP-1 secretion by MRC-5 embryonic lung fibroblasts.

[0175] After trypsin digestion, MRC-5 embryonic lung fibroblasts were counted. A certain number of cells were collected, centrifuged, and resuspended in MEM complete medium. The cell density was adjusted to 2E+5 cells / mL, 100 μL / well, and added to 96-well plates. IL-11-mFc ligand protein was diluted in MEM complete medium to a concentration of 16 μg / mL, 50 μL / well, and added to the corresponding 96-well plates. Four humanized antibody molecules, HA-IA, HA-IB, HA-IC, and HA-ID, were serially diluted in MEM complete medium at an initial concentration of 40 μg / mL, with 3-fold serial dilutions for a total of 8 gradients, 50 μL / well, and added to 96-well plates containing cell suspension and IL-11-mFc ligand protein suspension. The mixture was gently mixed and incubated overnight at 37°C in a CO2 incubator (approximately 20 h). The cell culture supernatant was then used for detection using a TIMP-1 ELISA kit (method as in Example 9). The microplate reader was used to read the values ​​at 450 nm, and the corresponding IC50 values ​​were calculated. The specific data are as follows:

[0176]

[0177] Based on the above data and Figure 14 It can be seen that the four humanized antibody molecules screened can effectively inhibit the release of TIMP-1 from human embryonic lung fibroblasts MRC-5 stimulated by IL-11 ligand protein.

[0178] Example 20: Cross-binding experiment of humanized antibody molecules with IL-11 from different species

[0179] Humanized antibody HA-IA, which exhibits superior protein level and functional activity, was selected for cross-binding assays with IL-11 from different species. Human IL-11, mouse IL-11 (purchased from Beijing Sinocare Medical Technology Co., Ltd., catalog number: 50117-MNCE), rat IL-11 (purchased from Kanglang Biotechnology, catalog number: KL40001Ra), and cynomolgus monkey IL-11 (purchased from Sinocare Medical Technology, catalog number: 90925-CNCE) were coated with 100 ng / well / 100 μL of pH 9.6 carbonate buffer and incubated overnight at 4°C. After washing five times with 300 μL / well PBST, 280 μL / well of 1% BSA-PBST was added, and the mixture was blocked at 37°C for 1 h. Humanized antibody HA-IA was diluted with 1% BSA-PBST to an initial concentration of 50 μg / mL. A five-fold serial dilution was performed for a total of nine dilutions, with two replicates per dilution. 100 μL / well was added to each well of a 96-well plate and incubated at 37°C for 1 h. The plate was then washed five times with 300 μL / well of PBST. Goat anti-Human IgG Fab HRP (purchased from Invitrogen, catalog number: 31482) was diluted with 1% BSA-PBST to a working solution concentration of 1:5000. 100 μL / well was added to each well of a 96-well plate and incubated at 37°C for 1 h. The plate was then washed five times with 300 μL / well of PBST. The plate was then developed using a TMB chromogenic assay kit, 100 μL / well, at room temperature in the dark for 5 min, followed by termination of the development with 2M H₂SO₄. Readings were taken at 450 nm and 630 nm using a microplate reader, and the corresponding EC50 values ​​were calculated. Specific data are as follows:

[0180]

[0181] Based on the above data and as follows Figure 15 As shown, the humanized antibody molecule HA-IA can bind to human IL-11, mouse IL-11, rat IL-11, and cynomolgus monkey IL-11, and the affinity is high.

[0182] Example 21: Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody against pulmonary fibrosis

[0183] The therapeutic effect of the anti-IL-11 monoclonal antibody HA-IA on pulmonary fibrosis was studied using bleomycin (bLF) modeling.

[0184] Animal species: C57BL / 6J mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.)

[0185] Number, sex and age of mice: 6 mice / group, male, 6-8 weeks old;

[0186] The control group received only saline injections;

[0187] In the treatment group, HA-IA antibody molecules were injected twice a week for 4 weeks.

[0188] Animal weight was measured weekly, and any abnormalities were observed. Organ weight detection: lung organs were collected, their weight was measured, and the lung-to-body weight ratio was calculated. Lung pathological examination: lung sections were examined and the degree of pulmonary fibrosis was observed using hematoxylin and eosin (HE) and Masson staining.

[0189] The results are as follows Figure 16 As shown, after administration of HA-IA antibody molecules, the lung-to-body weight ratio of mice in the treatment group was significantly lower than that in the control group; the results are as follows. Figure 17 As shown, compared with the control group, the lung slices of the treatment group showed a significant reduction in pulmonary fibrosis, which indicates that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the production of pulmonary fibrosis.

[0190] Example 22: Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody against cardiac fibrosis

[0191] The therapeutic effect of the anti-IL-11 monoclonal antibody HA-IA on cardiac fibrosis was studied using isoproterenol modeling.

[0192] Animal species: C57BL / 6J mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.)

[0193] Number, sex and age of mice: 6 mice / group, male, 6-8 weeks old;

[0194] The control group received only saline injections;

[0195] The treatment group received two injections of HA-IA antibody molecules per week for four weeks.

[0196] Animal weight was measured weekly, and any abnormalities were observed. Organ weight assessment: Hearts were collected, their weight was measured, and the heart-to-body weight ratio was calculated. Cardiac pathology assessment: Heart sections were prepared and stained with hematoxylin and eosin (HE) and Masson's solution to observe the degree of cardiac fibrosis.

[0197] The results are as follows Figure 18 As shown, the heart-to-body weight ratio of mice in the treatment group was significantly smaller than that in the control group; the results are as follows. Figure 19 As shown, compared with the control group, the heart slices of the treatment group showed a significant reduction in cardiac fibrosis, which indicates that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the production of cardiac fibrosis.

[0198] Example 23: Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody against renal fibrosis

[0199] The therapeutic effect of the anti-IL-11 monoclonal antibody HA-IA on renal fibrosis was studied using doxorubicin (dKF) modeling.

[0200] Animal species: BALB / c mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.)

[0201] Number, sex and age of mice: 6 mice / group, male, 6-8 weeks old;

[0202] The control group received only saline injections;

[0203] The treatment group received two injections of HA-IA antibody molecules per week for four weeks.

[0204] Animal weight was measured weekly, and any abnormalities were observed. Organ weight testing included collecting the heart, measuring the weight of the kidneys, and testing for urinary protein levels. Kidney pathology testing involved preparing kidney sections and staining them with hematoxylin and eosin (HE) and Masson's solution to observe the degree of kidney fibrosis.

[0205] The results are as follows Figure 20 As shown, compared with the control group, the urinary protein content in the kidneys of mice in the treatment group was significantly lower than that in the control group; the results are as follows. Figure 21 As shown, compared with the control group, the kidney slices of the treatment group showed a significant reduction in renal fibrosis, which indicates that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the production of renal fibrosis.

[0206] Example 24: Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody against liver fibrosis

[0207] The therapeutic effect of anti-IL-11 monoclonal antibody HA-IA on liver fibrosis was studied using CCl4 modeling.

[0208] Animal species: C57BL / 6J mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.)

[0209] Number, sex and age of mice: 6 mice / group, male, 6-8 weeks old;

[0210] The control group received only saline injections;

[0211] The treatment group received two injections of HA-IA antibody molecules per week for four weeks.

[0212] Weight monitoring: Animal weight was measured weekly, and any abnormalities were observed; Liver pathology examination: Liver sections were stained with hematoxylin and eosin (HE) and masson stain to observe the degree of liver fibrosis; Organ weight detection: Kidneys were collected, liver weight was measured, and HE staining was performed; Serum detection: Serum was collected, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum were detected.

[0213] The results are as follows Figure 22 As shown, the liver weight of mice in the treatment group was significantly lower than that in the control group; the results are as follows. Figure 23 As shown, the serum ALT and AST levels in the treatment group were significantly lower than those in the control group; the results are as follows. Figure 24 As shown, compared with the control group, the liver slices of the treatment group showed a significant reduction in liver fibrosis, which indicates that the anti-IL-11 monoclonal antibody HA-IA can effectively inhibit the production of liver fibrosis.

[0214] Example 25: Thermostability Assessment of Anti-IL-11 Monoclonal Antibody HA-IA

[0215] The thermal stability of the anti-IL-11 monoclonal antibody HA-IA was assessed using a multifunctional protein thermal stability analysis system (purchased from Unchained Labs). Protein conformational changes were detected by monitoring intrinsic fluorescence as a function of temperature (starting at 25°C and increasing to 95°C at a rate of 0.3°C / min) to determine the protein melting temperature (Tm) and assess conformational stability. When samples aggregate, interference occurs in the scattered light waves, increasing the scattered light signal. The colloidal stability of the protein was determined by static light scattering (characterized using Tagg). The results are shown in the table below and appendix. Figure 25 As shown.

[0216]

[0217] The melting temperature (Tm) of the anti-IL-11 monoclonal antibody HA-IA was 79.5℃, and the average Tagg value was 75.9℃, indicating good conformational and colloidal stability.

[0218] Example 26

[0219] The anti-IL-11 monoclonal antibody HA-IA obtained through the above examples was purified using the following specific purification method:

[0220] S1. Affinity Chromatography: S101. Equilibrate the affinity chromatography column with buffer A. The column packing material is MabSelect Sure LX, and buffer A is 30 mM HEPES buffer with a pH of 7 and a conductivity of 10 mS / cm. S102. Load the target protein containing the anti-IL-11 monoclonal antibody HA-IA onto the affinity chromatography column at a flow rate of 100 cm / h. The loading volume is 20 mg protein / ml packing material, and the column height is 18 cm. S103. After loading, reequilibrate with buffer A, and then wash at least once with 2CV buffer B until the UV absorbance curve stabilizes. Stop washing after this process. Then, a second elution was performed using buffer A. Buffer B was 30 mM HEPES buffer with a pH of 5. S104: The affinity chromatography column was eluted using buffer C. The protein product was collected after elution and set aside. Buffer C was 20 mM glycine-hydrochloride with a pH of 3. S105: The pH of the collected protein product was adjusted to 3 using an acid solution and incubated at room temperature for 0.5 h under this pH condition. Finally, the pH of the protein product was adjusted back to 5 using 1 M Tris buffer to obtain the crude pure protein sample. The acid solution was citric acid.

[0221] S2. Anion Exchange Chromatography: S201. Equilibrate the anion exchange chromatography column with 2CV buffer D. The packing material for the anion exchange chromatography is Capto Q, and buffer D is citrate buffer with a pH of 6.5. S202. Load the crudely purified protein sample onto the anion exchange chromatography column at a flow rate of 100 cm / h. The loading volume is 20 mg protein / ml packing material, and the column height is 18 cm. S203. After loading, equilibrate the anion exchange chromatography column again with buffer D, and collect the flow-through for later use.

[0222] S3. Cation Exchange Chromatography: S301. Equilibrate the cation exchange chromatography column using 2CV buffer E. The cation exchange chromatography column packing material is Capto SP, and buffer E is 30mM citrate buffer with a pH of 5.0 and a conductivity of 2mS / cm. S302. Load the secondary purified flow-through solution onto the cation exchange chromatography column at a flow rate of 100cm / h. The loading capacity is 20mg protein / ml packing material, and the column height is 18cm. S303. Elute the cation exchange chromatography column using buffer F. After elution, a high-purity protein solution of anti-IL-11 monoclonal antibody is obtained. Buffer F consists of 30mM citrate buffer and 80mM NaCl with a pH of 5.0.

[0223] Example 27

[0224] The anti-IL-11 monoclonal antibody HA-IA obtained through the above examples was purified using the following specific purification method:

[0225] S1. Affinity Chromatography: S101. Equilibrate the affinity chromatography column with buffer A. The column packing material is ATProtein A Diamond Plus, and buffer A is 50 mM Tris-HCl buffer containing 120-160 mM NaCl (preferably 150 mM NaCl in this example). The pH of buffer A is 7.4, and its conductivity is 18 mS / cm. S102. Load the target protein containing the anti-IL-11 monoclonal antibody HA-IA onto the affinity chromatography column at a flow rate of 150 cm / h. The loading volume is 55 mg protein / ml packing material, and the column height is 20 cm. S103. After loading, reequilibrate with buffer A, then elute at least once with 4 CV buffer B until the UV absorbance curve stabilizes. Stop rinsing, and then elute with buffer A again. A second elution was performed using buffer B, which was 50 mM acetate buffer with a pH of 5.5. S104: The affinity chromatography column was eluted using buffer C, and the protein product was collected after elution. Buffer C was 50 mM acetate buffer with a pH of 3.5. S105: The pH of the collected protein product was adjusted to 3.4-3.8 using an acid solution, and incubated at room temperature for 2 hours at this pH. Finally, the pH of the protein product was adjusted back to 6.2 using 1 M Tris buffer to obtain the crude pure protein sample. The acid solution was citric acid.

[0226] S2. Anion Exchange Chromatography: S201. Equilibrate the anion exchange chromatography column with 4CV buffer D. The packing material for the anion exchange chromatography is NanoGel-50Q, and buffer D is a phosphate buffer with a pH of 7. S202. Load the crudely purified protein sample onto the anion exchange chromatography column at a flow rate of 150 cm / h. The loading volume is 70 mg protein / ml packing material, and the column height is 20 cm. S203. After loading, equilibrate the anion exchange chromatography column again with buffer D, and collect the flow-through for later use.

[0227] S3. Cation Exchange Chromatography: S301. Equilibrate the cation exchange chromatography column using 3CV buffer E. The cation exchange chromatography column packing material is NanoGel-50SP, and buffer E is 50mM acetate buffer with a pH of 6 and a conductivity of 2.5mS / cm. S302. Load the secondary purified flow-through solution onto the cation exchange chromatography column at a flow rate of 150cm / h. The loading capacity is 47mg protein / ml packing material, and the column height is 20cm. S303. Elute the cation exchange chromatography column using buffer F. After elution, a high-purity protein solution of anti-IL-11 monoclonal antibody is obtained. Buffer F consists of 50mM acetate buffer and 115mM NaCl with a pH of 5.8.

[0228] Example 28

[0229] The anti-IL-11 monoclonal antibody HA-IA obtained through the above examples was purified using the following specific purification method:

[0230] S1. Affinity Chromatography: S101. Equilibrate the affinity chromatography column with buffer A. The column packing material is Eshmuno A, which can also be replaced with NMab Pro. Buffer A is 60 mM phosphate buffer with a pH of 8 and a conductivity of 30 mS / cm. S102. Load the target protein containing anti-IL-11 monoclonal antibody onto the affinity chromatography column at a flow rate of 300 cm / h. The loading volume is 60 mg protein / ml packing material. The column height is 22 cm. S103. After loading, reequilibrate with buffer A, then elute at least once with 6 CV buffer B until the UV absorbance curve stabilizes. Stop rinsing, then elute a second time with buffer A. The first wash uses buffer B, which is 60 mM Tris-HCl buffer. Alternatively, phosphate buffer or citrate buffer can be used, with a pH of 6. S104: Elute the affinity chromatography column using buffer C, which is 200 mM citrate buffer with a pH of 4. S105: Adjust the pH of the collected protein product to 5 using an acid solution and incubate at room temperature for 3 hours. Finally, adjust the pH of the protein product back to 8 using 1 M Tris buffer to obtain the crude, pure protein sample. The acid solution is hydrochloric acid.

[0231] S2. Anion Exchange Chromatography: S201. Equilibrate the anion exchange chromatography column with 6CV buffer D. The packing material for the anion exchange chromatography is Capto Adhesive, which can also be replaced with Eshmuno Q. Buffer D is an acetate buffer with a pH of 7.5. S202. Load the crudely purified protein sample onto the anion exchange chromatography column at a flow rate of 200 cm / h. The loading volume is 120 mg protein / ml packing material. The column height of the anion exchange chromatography column is 22 cm. S203. After loading, equilibrate the anion exchange chromatography column again with buffer D, and collect the flow-through for later use.

[0232] S3. Cation Exchange Chromatography: S301. Equilibrate the cation exchange chromatography column using 6CV buffer E. The packing material for the cation exchange chromatography column is Capto MMC, which can also be replaced with Eshmuno S. Buffer E is 60mM phosphate buffer with a pH of 6.0 and a conductivity of 5mS / cm. S302. Load the secondary purified flow-through solution onto the cation exchange chromatography column at a flow rate of 200cm / h. The loading capacity is 75mg protein / ml packing material. The column height of the cation exchange chromatography column is 22cm. S303. Elute the cation exchange chromatography column using buffer F. After elution, a high-purity protein solution of anti-IL-11 monoclonal antibody is obtained. Buffer F is 60mM phosphate buffer and 150mM NaCl with a pH of 6.0.

[0233] Example 29

[0234] Based on Example 27, Example 29 of the present invention further specifies that in step S105 of the purification method for anti-IL-11 monoclonal antibody, the acid solution used in the inactivation process is preferably acetic acid.

[0235] Examples 30-32

[0236] Based on Example 29, Examples 30-32 of this invention further specify the step S105 of the purification method for anti-IL-11 monoclonal antibody, in which the pH of the protein product is adjusted back to the following pH and conductivity range using 1M Tris buffer to obtain a crude pure protein sample. Other methods are the same as in Example 27, and the specific data are as follows.

[0237] Example pH value Sample Conductivity (mS / cm) Example 30 6.5 5 Example 31 7.5 5 Example 32 7.0 4

[0238] Examples 33-35

[0239] Based on Example 32, Examples 33-35 of this invention further specify the buffer F in step S303 of the purification method for anti-IL-11 monoclonal antibody. All other methods and parameters are the same as in Example 27, as detailed below.

[0240]

[0241] Compare with Example 1

[0242] The purification method provided in Comparative Example 1 is based on Example 35, except that the order of anion exchange chromatography in step S2 and cation exchange chromatography in step S3 is interchanged. All other methods and parameters are the same as in Example 35.

[0243] Compare with Example 2

[0244] Comparative Example 2 of this invention, based on Example 35, used the classic three-step purification process for monoclonal antibodies to purify the anti-IL-11 monoclonal antibody HA-IA. All packing materials used in the three chromatographic steps were manufactured by Cytiva. The packing material for the first step affinity chromatography column was MabSelectSuRe LX, the packing material for the second step anion exchange chromatography column was Capto Q, and the packing material for the third step cation exchange chromatography column was Capto SPImpAct. All other methods and parameters were identical to those in Example 35.

[0245] Experiment 1: Physicochemical detection and related impurity detection of anti-IL-11 monoclonal antibody

[0246] The anti-IL-11 monoclonal antibody HA-IA obtained by the purification methods provided in the above embodiments and comparative examples of the present invention was purified using gel chromatography to detect its purity and analyze the content of aggregates, monomers, and degradation products in the sample during the purification process. Furthermore, ion chromatography was used to analyze the content of acid-base peaks of charge isomers, and a dedicated kit was used to detect the content of process-related impurities. Simultaneously, the total recovery rate was calculated using the following formula: Total recovery rate = affinity chromatography protein yield (%) * anion exchange chromatography protein yield (%) * cation exchange chromatography protein yield (%). Specific data are as follows:

[0247]

[0248] Based on the above experimental data, it can be concluded that the purification method provided in the above embodiments of the present invention can purify the anti-IL-11 monoclonal antibody with a SEC purity of over 99% and a total protein yield of over 75%. The anti-IL-11 monoclonal antibody stock solution obtained by the purification method provided in Example 35 has the highest purity and yield, with a total protein recovery rate of over 81%. In antibody production, this recovery rate can significantly improve the yield and reduce production costs.

[0249] Compared with Examples 26 and 28, the composition and content of the packing material of the chromatography column and each buffer provided in Example 27 are more in line with the purification process conditions of the anti-IL-11 monoclonal antibody HA-IA.

[0250] Compared to Example 27, Example 29 uses acetic acid as the acid solution, resulting in a clearer appearance of the inactivated sample without precipitation and a higher protein recovery rate.

[0251] Compared with Examples 30-32, it can be seen that in step S105, the pH of the protein product is adjusted back to 7.0 with 1M Tris buffer and the conductivity is 4mS / cm, which can significantly improve the total recovery rate of anti-IL-11 monoclonal antibody HA-IA protein.

[0252] Compared with Examples 33-35, it can be seen that when buffer F includes 50mM acetate buffer and 100mM NaCl and the pH value is 5.5, the protein recovery rate is higher.

[0253] Compared with Example 1, Example 35 reverses the order of anion exchange chromatography in step S2 and cation exchange chromatography in step S3, which is not conducive to the purification of anti-IL-11 monoclonal antibody HA-IA. The three-step purification process provided in Example 35, which sequentially involves affinity chromatography, anion exchange chromatography and cation exchange chromatography, is the optimal purification process for anti-IL-11 monoclonal antibody HA-IA.

[0254] Compared to Example 2, Example 35 differs only in the packing material of the chromatography column. The packing material provided in Example 35 exhibits significantly higher purification efficiency than the control example 2, with a higher total recovery rate and lower cost. It effectively removes product-related and process-related impurities. Therefore, it can be concluded that the packing material provided in Example 35 is more suitable for the purification of the anti-IL-11 monoclonal antibody HA-IA. Thus, the preferred affinity chromatography material of this invention is AT Protein A Diamond Plus. AT Protein A Diamond Plus, as a packing material, possesses excellent binding specificity, alkali resistance, and pressure-flow rate characteristics, with low ligand shedding. It can capture antibodies in cell supernatants, removing most impurities. Furthermore, it can significantly increase the loading capacity and is an alkali-resistant protein... Affinity-based packing materials enable more efficient cleaning and sterilization, avoid cross-contamination, and extend service life. The preferred packing material for anion exchange chromatography columns is NanoGel-50Q, and for cation exchange chromatography columns, NanoGel-50SP. NanoGel-50SP chromatography employs a combined elution mode, where the target protein flows through the column and impurities are adsorbed onto the column during sample loading, effectively achieving purification. Both NanoGel-50Q and NanoGel-50SP are rigid polymer-based monodisperse matrices with uniform particle size, high resolution, and good separation. This combination of different chromatography column packing materials not only effectively removes process-related impurities such as HCP, DNA, and Protein A, but also adsorbs product-related impurities such as aggregates and fragments, improving product purity, ensuring the purified product meets quality requirements, and significantly reducing purification process costs.

[0255] Experiment 2: Detection of the binding activity and biological activity of anti-IL-11 monoclonal antibodies

[0256] Regarding the anti-IL-11 monoclonal antibody HA-IA purified by the purification methods provided in the above embodiments and Comparative Examples 1-2 of the present invention, the present invention uses ELISA to analyze the specific binding ability of the purified anti-IL-11 monoclonal antibody HA-IA to IL-11, in order to evaluate the activity of the purified antibody. The detection results are as follows:

[0257]

[0258]

[0259] As can be seen from the table above, the anti-IL-11 monoclonal antibody HA-IA purified by the method provided in the above embodiments of the present invention has good binding activity and biological activity, within the range of 90% ± 20%. This indicates that the anti-IL-11 monoclonal antibody HA-IA purified under the conditions specified by the method provided in the present invention has good biological activity after screening under a large number of experimental conditions.

[0260] This invention is not limited to the preferred embodiments described above. Anyone can derive other products in various forms under the guidance of this invention. However, regardless of any changes in shape or structure, any technical solution that is the same as or similar to this application falls within the protection scope of this invention.

Claims

1. A method for purifying an anti-IL-11 monoclonal antibody, characterized in that, The method includes: S1, affinity chromatography: preliminary purification and concentration of the target protein containing anti-IL-11 monoclonal antibody, and virus inactivation of the collected protein product in an acidic solution to obtain a crudely purified protein sample; S2, anion exchange chromatography: secondary purification of the protein sample, collection of the flow-through; S3, cation exchange chromatography: further purification of the flow-through to obtain a high-purity protein solution of anti-IL-11 monoclonal antibody, wherein the anti-IL-11 monoclonal antibody comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), respectively. The amino acid sequence of the heavy chain complementarity-determining region HCDR1 is shown in SEQ ID No:1, the amino acid sequence of the heavy chain complementarity-determining region HCDR2 is shown in SEQ ID No:14, the amino acid sequence of the heavy chain complementarity-determining region HCDR3 is shown in SEQ ID No:15, and the amino acid sequence of the light chain complementarity-determining region LCDR1 is shown in SEQ ID No:

15. As shown in No:4, the amino acid sequence of the light chain complementarity-determining region LCDR2 is shown in SEQ ID No:5, and the amino acid sequence of the light chain complementarity-determining region LCDR3 is shown in SEQ ID No:

6.

2. The purification method for the anti-IL-11 monoclonal antibody as described in claim 1, characterized in that, The anti-IL-11 monoclonal antibody further includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:21, and the amino acid sequence of the light chain variable region is shown in SEQ ID No:

17.

3. The purification method for the anti-IL-11 monoclonal antibody as described in claim 2, characterized in that, The anti-IL-11 monoclonal antibody further includes a heavy chain constant region and a light chain constant region. The amino acid sequence of the heavy chain constant region is one of SEQ ID No:23, SEQ ID No:24, SEQ ID No:25 or SEQ ID No:

26. The amino acid sequence of the light chain constant region is shown in SEQ ID No:

22.

4. The purification method for the anti-IL-11 monoclonal antibody as described in claim 1, characterized in that, In step S1, the affinity chromatography includes the following steps: S101. Equilibrate the affinity chromatography column using buffer A; S102. The target protein containing the anti-IL-11 monoclonal antibody is loaded onto the affinity chromatography column at a flow rate of 100-300 cm / h, with a loading capacity of 20-60 mg protein / ml packing material, and the column height of the affinity chromatography column is 18-22 cm. S103. After the sample loading is completed, use the buffer A to reequilibrate, and then rinse at least once with buffer B of 2-6CV until the UV absorbance curve drops to a stable value. Stop rinsing and then use the buffer A for a second rinse. S104. Elute the affinity chromatography column with buffer C, and collect the protein product after elution for later use. S105. Adjust the pH of the collected protein product to 3-4 using the acid solution, and incubate at room temperature for 0.5-3 hours under this pH condition. Finally, adjust the pH of the protein product back to 5-8 using 1M Tris buffer to obtain the crude pure protein sample. Buffer A is selected from Tris-HCl buffer, HEPES buffer, or phosphate buffer. The concentration of buffer A is 30-60 mM, its pH is 7-8, and its conductivity is 10-30 mS / cm. Buffer B is selected from Tris-HCl buffer, HEPES buffer, phosphate buffer, citrate buffer, or acetate buffer. The concentration of buffer B is 30-60 mM, and its pH is 5-6. Buffer C is selected from glycine-hydrochloride, citrate, or acetate buffer. The concentration of buffer C is 20-200 mM, and its pH is 3-4.

5. The purification method for the anti-IL-11 monoclonal antibody as described in claim 4, characterized in that, The packing material for the affinity chromatography column is selected from MabSelect Sure LX, Eshmuno A, NMab Pro, or AT Protein A Diamond Plus.

6. The purification method for the anti-IL-11 monoclonal antibody as described in claim 1, characterized in that, In step S2, the anion exchange chromatography includes the following steps: S201. Equilibrate the anion exchange chromatography column using 2-6 CV buffer D; S202. The crudely purified protein sample is loaded onto the anion exchange chromatography column at a flow rate of 100-200 cm / h, with a loading capacity of 20-120 mg protein / ml packing material, and the column height of the anion exchange chromatography column is 18-22 cm. S203. After the sample loading is completed, the anion exchange chromatography column is equilibrated again using the buffer D, and the flow-through solution is collected for later use. The buffer D is selected from citrate buffer, acetate buffer or phosphate buffer, and its pH value is 6.5-7.

5.

7. The purification method for the anti-IL-11 monoclonal antibody as described in claim 6, characterized in that, The packing material for the anion exchange chromatography is selected from Capto Q, Capto adhere, Eshmuno Q, or NanoGel-50Q.

8. The purification method for the anti-IL-11 monoclonal antibody as described in claim 1, characterized in that, In step S3, the cation exchange chromatography includes the following steps: S301. Equilibrate the cation exchange chromatography column using 2-6 CV buffer E. S302. The flow-through solution after secondary purification is loaded onto the cation exchange chromatography column at a flow rate of 100-200 cm / h, with a loading capacity of 20-75 mg protein / ml packing material, and the column height of the cation exchange chromatography column is 18-22 cm. S303. The cation exchange chromatography column is eluted using buffer F to obtain a high-purity protein solution of anti-IL-11 monoclonal antibody. Buffer E is selected from citrate buffer, acetate buffer, or phosphate buffer, with a concentration of 30-60 mM, a pH of 5.0-6.0, and a conductivity of 2-5 mS / cm. Buffer F comprises buffer E and 80-150 mM of additive, wherein the additive is NaCl.

9. The purification method for the anti-IL-11 monoclonal antibody as described in claim 8, characterized in that, The packing material for the cation exchange chromatography column is Capto SP, Capto MMC, Eshmuno S, or NanoGel-50SP.

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