A method for purifying an anti-IL-11 monoclonal antibody

Abstract

The present invention relates to the field of biomedicine, and specifically provides a purification method for an anti-IL-11 monoclonal antibody, including affinity chromatography: initially purifying and concentrating the target protein containing the anti-IL-11 monoclonal antibody, inactivating the virus in the protein product in an acidic solution to obtain a protein sample; anion exchange chromatography: subjecting the protein sample to secondary purification and collecting the flow-through fraction; cation exchange chromatography: subjecting the flow-through fraction to fine purification to obtain a protein solution of the anti-IL-11 monoclonal antibody. The present invention provides a suitable purification process for the anti-IL-11 monoclonal antibody, improving the purification efficiency and recovery rate of the anti-IL-11 monoclonal antibody. Among them, the flow-through fraction collected by anion exchange chromatography directly enters cation exchange chromatography without treatment, without sample treatment or solution replacement in the middle, saving purification time, avoiding the introduction of impurities during the purification process, improving the purity of the purified protein, and having high practicability.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a method for purifying an anti-IL-11 monoclonal antibody. Background Art

[0002] Fibrosis can occur in multiple organs, such as the liver, lungs, kidneys, retina, intestines, heart, and skin. It is the main cause of disability and death in many diseases. The main pathology is the increase of fibrous connective tissue in organ tissues, the reduction of parenchymal cells, and continuous progression can lead to the destruction of organ structure and the decline of function, and even failure, seriously threatening human health and life. According to relevant statistical data in the United States, among the patients who died from various diseases in that country, nearly 45% were attributed to tissue fibrosis diseases.

[0003] Fibrosis diseases include various diseases such as pulmonary fibrosis, liver fibrosis, cirrhosis, liver diseases, renal fibrosis, cardiac fibrosis, asthma, and 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 injury has a process of liver fibrosis during the process of liver repair and healing. If the injury factor cannot be removed for a long time and the fibrosis process persists for a long time, it will develop into cirrhosis. There are many causes of liver fibrosis, and viral hepatitis, alcoholic liver disease, fatty liver, autoimmune diseases, etc. are common clinically. Non-alcoholic fatty liver disease, which accounts for a relatively high proportion globally, will deteriorate into non-alcoholic hepatitis if not treated in time, and even cause liver inflammation, hepatocyte death, and liver fibrosis, ultimately leading to the occurrence of cirrhosis and liver cancer. In addition, pulmonary fibrosis is the end-stage change of a large category of lung diseases characterized by fibroblast proliferation, massive extracellular matrix aggregation, accompanied by inflammatory damage and tissue structure destruction. Most patients with pulmonary fibrosis have unknown causes (idiopathic), and this group of diseases is called idiopathic interstitial pneumonia, which is a large category of interstitial lung diseases. The most common disease type in idiopathic interstitial pneumonia with pulmonary fibrosis as the main manifestation is idiopathic pulmonary fibrosis, which is a severe interstitial lung disease that can lead to progressive loss of lung function. Pulmonary fibrosis seriously affects the respiratory function of the human body, manifested as dry cough and progressive dyspnea. As the disease and lung damage worsen, the respiratory function of patients deteriorates continuously. The incidence and mortality of idiopathic pulmonary fibrosis increase year by year, and the average survival period after diagnosis is only 2.8 years. The mortality rate is higher than that of most tumors, and it is called a "quasi-tumor disease". Currently, there are no particularly effective drugs for the treatment of fibrosis diseases in various organs, and the treatment effect is generally average. Therefore, the research and development of drugs for treating anti-fibrosis diseases is particularly urgent.

[0004] Interleukin-11 (IL-11) is a hematopoietic cytokine and belongs to a kind of multifunctional interleukin-6 (IL6) family cytokines, sharing the same signal transduction receptor subunit (GP130). This family plays a crucial role in the occurrence, development, and metastasis of tumors. Research has found that the blockade of this pathway can become an effective treatment method for various tumor cancers, chronic fibrosis, and inflammatory diseases. Currently, there is no marketed product globally for drugs targeting IL-11. Therefore, in order to meet the urgent needs of patients, the process development of anti-IL-11 monoclonal antibodies has important clinical significance.

[0005] Currently, during the process research and development of anti-IL-11 monoclonal antibodies, when using conventional antibody purification methods to purify anti-IL-11 monoclonal antibodies, it often occurs that it is difficult to separate the degradation products from the target protein, and the samples are unstable during the process, with low purification efficiency, purity, and recovery rate. Therefore, the present invention provides a purification method specifically applicable to anti-IL-11 monoclonal antibodies. Summary of the Invention

[0006] In order to quickly meet the urgent needs of fibrosis patients, improve the drug recovery rate and purity during the drug research and development process, and ensure the biological activity of anti-IL-11 monoclonal antibodies during the purification process, the present invention provides a purification method specifically applicable to anti-IL-11 monoclonal antibodies.

[0007] The specific technical solution of the present invention is as follows:

[0008] The present invention provides a purification method for anti-IL-11 monoclonal antibodies, and the method includes:

[0009] S1. Affinity chromatography: Initially purify and concentrate the target protein containing anti-IL-11 monoclonal antibodies, and inactivate the virus in the collected protein product in an acid solution to obtain a roughly purified protein sample;

[0010] S2. Anion exchange chromatography: Perform secondary purification on the protein sample and collect the flow-through;

[0011] S3. Cation exchange chromatography: After purifying the flow-through, a protein solution of highly pure anti-IL-11 monoclonal antibodies is obtained.

[0012] Furthermore, the anti-IL-11 monoclonal antibody includes 3 heavy chain complementary determining regions respectively represented by HCDR1, HCDR2, and HCDR3, and 3 light chain complementary determining regions respectively represented by LCDR1, LCDR2, and LCDR3. The anti-IL-11 monoclonal antibody is selected from any one of the following:

[0013] A-I: The amino acid sequence of the heavy chain complementarity determining region HCDR1 is as shown in SEQ ID No:1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is as shown in SEQ ID No:2, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is as shown in SEQ ID No:3, the amino acid sequence of the light chain complementarity determining region LCDR1 is as shown in SEQ ID No:4, the amino acid sequence of the light chain complementarity determining region LCDR2 is as shown in SEQ ID No:5, and the amino acid sequence of the light chain complementarity determining region LCDR3 is as shown in SEQ ID No:6;

[0014] A-II: The amino acid sequence of the heavy chain complementarity determining region HCDR1 is as shown in SEQ ID No:1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is as shown in SEQ ID No:2, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is as shown in SEQ ID No:3, the amino acid sequence of the light chain complementarity determining region LCDR1 is as shown in SEQ ID No:4, the amino acid sequence of the light chain complementarity determining region LCDR2 is as shown in SEQ ID No:5, and the amino acid sequence of the light chain complementarity determining region LCDR3 is as shown in SEQ ID No:7;

[0015] A-III: The amino acid sequence of the heavy chain complementarity determining region HCDR1 is as shown in SEQ ID No:8, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is as shown in SEQ ID No:9, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is as shown in SEQ ID No:10, the amino acid sequence of the light chain complementarity determining region LCDR1 is as shown in SEQ ID No:11, the amino acid sequence of the light chain complementarity determining region LCDR2 is as shown in SEQ ID No:12, and the amino acid sequence of the light chain complementarity determining region LCDR3 is as shown in SEQ IDNo:13;

[0016] A-IV: The amino acid sequence of the heavy chain complementarity determining region HCDR1 is as shown in SEQ ID No:1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is as shown in SEQ ID No:14, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is as shown in SEQ ID No:15, the amino acid sequence of the light chain complementarity determining region LCDR1 is as shown in SEQ ID No:4, the amino acid sequence of the light chain complementarity determining region LCDR2 is as shown in SEQ ID No:5, and the amino acid sequence of the light chain complementarity determining region LCDR3 is as shown in SEQ ID No:6.

[0017] The beneficial effects of the present invention are as follows: First, the present invention screens the most suitable conditions and chromatography packing materials for the anti-IL-11 monoclonal antibody provided by the present invention through a continuous three-step purification method, effectively improving the purification efficiency and recovery rate of the anti-IL-11 monoclonal antibody provided by the present invention. Among them, the flow-through solution collected by anion exchange chromatography can directly enter cation exchange chromatography without treatment, and there is no need for sample treatment or solution replacement in the middle, which not only greatly saves the purification time, but also avoids the introduction of impurities during the purification process, improves the purity of the purified protein, and has high practicability. Second, the anti-IL-11 monoclonal antibody provided by the present invention has a high binding ability with the IL-11 antigen, can block the binding of the IL-11 antigen to its receptor, and thus effectively inhibits the profibrotic effect of IL-11, inhibits or prevents the generation or proliferation of fibroblasts, and can be effectively used for the treatment or prevention of human fibrotic diseases, inflammation, cancer or autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the plasmid map of the pScFv-Disb-HS vector in Example 2 of the present invention;

[0019] Figure 2 It is the comparison diagram of the affinity of gradient-diluted ELISA anti-IL-11 phage monoclonal antibody in Example 3 of the present invention;

[0020] Figure 3 It is the map of the vector pTSE in Example 5 of the present invention;

[0021] Figure 4 It is the denaturing polyacrylamide gel electrophoresis map of the murine antibody molecule in Example 5 of the present invention;

[0022] Figure 5 It is the comparison diagram of the binding ability between the murine antibody molecule and IL-11 in Example 6 of the present invention;

[0023] Figure 6 It is the comparison diagram of the competitive inhibition experiment of the murine antibody and the IL-11 receptor protein IL-11RA in Example 7 of the present invention;

[0024] Figure 7 It is the comparison diagram of the murine antibody inhibiting the binding of IL-11 to the IL-11RA receptor on the surface of BaF / 3-IL-11RA cells in Example 8 of the present invention;

[0025] Figure 8 It is the comparison diagram of the murine antibody inhibiting the secretion of TIMP-1 by embryonic lung fibroblasts MRC-5 in Example 9 of the present invention;

[0026] Figure 9 It is the denaturing polyacrylamide gel electrophoresis map of the humanized antibody molecule in Example 14 of the present invention;

[0027] Figure 10 This is a comparison graph of the binding ability between the humanized antibody molecule and IL-11 in Example 15 of the present invention;

[0028] Figure 11 This is a comparison graph of the inhibition of the binding between IL-11 and the IL-11RA receptor on the surface of BaF / 3-IL-11RA cells by the humanized antibody molecule in Example 16 of the present invention;

[0029] Figure 12 This is a comparison graph of the inhibition of the binding between IL-11 and the GP130 receptor on the surface of BaF / 3-GP130 cells by the humanized antibody molecule in Example 17 of the present invention;

[0030] Figure 13 This is a comparison graph of the biological activity detection (reporter gene) of the humanized antibody molecule in Example 18 of the present invention;

[0031] Figure 14 This is a comparison graph of the inhibition of TIMP-1 secretion by MRC-5, embryonic lung fibroblasts, by the humanized antibody molecule in Example 19 of the present invention;

[0032] Figure 15 This is a comparison graph of the cross-binding experiment of the humanized antibody molecule with IL-11 of different species in Example 20 of the present invention;

[0033] Figure 16 This is a bar graph of the change in the ratio of lung to body weight in the mouse pulmonary fibrosis model in Example 21 of the present invention;

[0034] Figure 17 This is the hematoxylin and eosin (HE) staining and Masson staining diagrams of the lung tissue section in the mouse pulmonary fibrosis model in Example 21 of the present invention;

[0035] Figure 18 This is a bar graph of the change in the ratio of heart to body weight in the mouse cardiac fibrosis model in Example 22 of the present invention;

[0036] Figure 19 This is the hematoxylin and eosin (HE) staining and Masson staining diagrams of the heart tissue section in the mouse cardiac fibrosis model in Example 22 of the present invention;

[0037] Figure 20 This is a bar graph of the urinary protein content in the kidney in the mouse renal fibrosis model in Example 23 of the present invention;

[0038] Figure 21 This is the hematoxylin and eosin (HE) staining and Masson staining diagrams of the kidney tissue section in the mouse renal fibrosis model in Example 23 of the present invention;

[0039] Figure 22It is a bar graph showing the change in liver weight in the mouse liver fibrosis model in Example 24 of the present invention;

[0040] Figure 23 It is a bar graph showing the change in the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of mice in the mouse liver fibrosis model in Example 24 of the present invention;

[0041] Figure 24 It is a hematoxylin-eosin (HE) staining and Masson staining diagram of liver tissue sections in the mouse liver fibrosis model in Example 24 of the present invention;

[0042] Figure 25 It is a thermal stability evaluation diagram of anti-IL-11 monoclonal antibody HA-I-A in Example 25 of the present invention. Detailed implementation manners

[0043] The present invention will be further described in detail below in conjunction with the following examples.

[0044] Example 1

[0045] The present invention provides a purification method for anti-IL-11 monoclonal antibody, and the method includes:

[0046] S1. Affinity chromatography: Initially purify and concentrate the target protein containing anti-IL-11 monoclonal antibody, and inactivate the virus in the collected protein product in an acid solution to obtain a crudely purified protein sample;

[0047] S2. Anion exchange chromatography: Further purify the protein sample and collect the flow-through fraction;

[0048] S3. Cation exchange chromatography: After purifying the flow-through fraction, a protein solution of high-purity anti-IL-11 monoclonal antibody is obtained.

[0049] Among them, the anti-IL-11 monoclonal antibody includes three heavy-chain complementarity-determining regions respectively represented by HCDR1, HCDR2, and HCDR3, and three light-chain complementarity-determining regions respectively represented by LCDR1, LCDR2, and LCDR3, and the anti-IL-11 monoclonal antibody is selected from any one of the following.

[0050]

[0051] The anti-IL-11 monoclonal antibody provided by the present invention is used for treating or preventing human fibrotic diseases, inflammation, cancer or autoimmune diseases. Among them, fibrotic diseases include, but are not limited to, fibrosis of the heart, liver, kidney, lung, gallbladder, bladder, stomach, bone marrow, penis, breast, blood vessel, eye, pancreas, spleen, brain, intestine, muscle or skin, etc.; 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.; 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, etc.; autoimmune diseases include, but are not limited to, psoriasis, Crohn's disease, primary biliary cirrhosis, systemic lupus erythematosus or multiple sclerosis, etc.

[0052] Example 2 Screening of murine antibody molecules

[0053] In the present invention, mice were immunized with IL-11 antigen (subsequent experiments: IL-11 protein, IL-11-Fc antigen, and IL-11-mFc ligand protein are all human IL-11), the immunization method was optimized, and a phage display library was created. The construction, screening, and identification of the specific phage display library are as follows:

[0054] Step 1: Immunization of mice with IL-11 antigen

[0055] 1. Experimental animals:

[0056] Species and strain: BALB / c, female, mice;

[0057] Body weight: 18 - 20 g;

[0058] Provider of experimental animals: Beijing Huafukang Biotechnology Co., Ltd.

[0059] 2. Immunization: The mice were immunized with human IL-11 (synthetic gene by Nanjing Genscript Biotech Co., Ltd., and the vector was constructed and expressed and purified by our company).

[0060] Step 2: Construction of phage antibody library

[0061] Mouse spleen cells with higher potency were taken, and total RNA in the mouse spleen cells was extracted using Trizol reagent (purchased from Ambion, catalog number: 15596026). cDNA was obtained by RT-PCR. Using the cDNA as a template, degenerate primers (the degenerate primers used refer to the reference: Journal of Immunological Methods 233 (2000) 167 - 177) were used for PCR amplification to obtain the immunized mouse heavy chain variable region gene library (VH) and light chain variable region gene library (VL). The pScFv-Disb-HS vector was constructed by modifying the vector pComb3 vector (purchased from the China Plasmid Vector Strain Cell Line Gene Preservation Center) using a series of gene cloning methods for the construction and expression of the phage single-chain antibody library. The modified vector was named pScFv-Disb-HS vector, and its plasmid map is as shown in Figure 1 and based on this vector, a mouse immunized phage antibody library was constructed. The light and heavy chain variable region gene libraries were double digested with restriction enzymes and ligated to the vector pScFv-Disb-HS that had also been digested step by step to construct the pScFv-Disb-HS-VH-VL gene library.

[0062] Step 3: Coat the immunization tubes with IL-11 as the antigen. The antigen coating amount is 5 μg / 500 μL / tube, and coat overnight at 4°C. Then, use 4% skim milk / PBST to block the immunization tubes and the immunized phage antibody library respectively, and block at room temperature for 1 h. The blocked immunized phage antibody library was added to the immunization tubes for antigen-antibody binding. The phage input amount is about 10 9 ~10 12 phages. After reacting at room temperature for 1 h, unbound phages were washed away using PBST-PBS, eluted with 0.1 M Glycine-HCl at pH 2.2, and finally the eluted phage antibody solution was neutralized to about pH 7.0 using 1.5 M Tris-HCl at pH 8.8.

[0063] Step 4: Infect the above neutralized phages into 10 ml of TG1 bacterial solution that has grown to the logarithmic phase, let it stand in a 37°C incubator for 30 min, take out part of the bacterial solution for gradient dilution, and spread it on a 2YTAG plate for calculating the phage output. The remaining bacterial solution was centrifuged to discard the supernatant, the bacterial cell pellet was resuspended in a small amount of medium, aspirated and spread on a large 2YTAG plate to prepare for the next round of screening.

[0064] Step 5: Scrape the infected bacteria plated above from the large plate, inoculate them into 2YTAG liquid medium, shake until the logarithmic phase, then add M13KO7 helper phage for superinfection, and culture overnight at 220 rpm under the condition of 28 °C to prepare phage. Use PEG / NaCl to precipitate and purify the phage for the next round of screening. A total of one round of phage library enrichment screening is carried out.

[0065] Step 6: Screening of IL-11 phage single-chain antibody positive clones: After one round of screening, pick well-separated monoclonal colonies and inoculate them into a 96-well deep-well plate containing 2YTAG liquid medium. Culture at 220 rpm at a temperature of 37 °C until the logarithmic growth phase. Add about 10 10 helper phages M13KO7 to each well, and statically infect for 30 min at a temperature of 37 °C. Centrifuge at 4000 rpm for 15 min, discard the supernatant, resuspend the bacteria precipitate with 2YTAK, and culture overnight at 220 rpm under the condition of 28 °C. After centrifuging at 4000 rpm at 4 °C for 15 min, aspirate the amplified phage supernatant for ELISA identification. Finally, four murine antibody molecules with relatively high affinity are obtained, named MA-I, MA-II, MA-III, and MA-IV respectively. Sequence the monoclonal antibodies obtained above to determine the correct antibody sequence. After sequencing, the sequences of the 4 monoclonal antibodies screened above are as follows:

[0066]

[0067] Specifically, SEQ ID No:16 (amino acid sequence of the heavy chain variable region of MA-Ⅰ and MA-Ⅱ):

[0068] EVKLEESGGGLVKPGGSLKLSCAASGFTFSDYYMFWVRQTPEKRLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNNLYLQMTSLKSEDTAMYYCARDGGYVS SPEAMDYWGQGTSVTVSS;

[0069] SEQ ID No:17 (amino acid sequence of the light chain variable region of MA-Ⅰ and MA-Ⅳ):

[0070] DIVLTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPPTFGGGTKLEIK;

[0071] SEQ ID No:18 (Amino acid sequence of the light chain variable region of MA-II):

[0072] DIVLTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK;

[0073] SEQ ID No:19 (Amino acid sequence of the heavy chain variable region of MA-III):

[0074] EVKLEQSGAEVVKPGALVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIG VIDPSDSYTTYNQKFKGKATLTVDTSSSTGYMQLSSLTSEDSAVYYCSQYGYDVN WYFDVWGAGTTVTVSS;

[0075] SEQ ID No:20 (Amino acid sequence of the light chain variable region of MA-III):

[0076] DIVMTQTTLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLI YEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGT KLEIK;

[0077] SEQ ID No:21 (Amino acid sequence of the heavy chain variable region of MA-IV):

[0078] EVQLEESGGGLVKPGGSLKLSCVASGFTFSDYYMFWVRQTPEKRLEWVATI SDGGSYSYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTAMYYCARDGGYISS PEAMDYWGQGTSVTVSS。

[0079] Example 3 Gradient Dilution ELISA to Compare the Affinity of Antibodies

[0080] 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 the phage gradient dilution ELISA experiment was performed to identify the affinity. The specific method is as follows:

[0081] Coat the IL-11 antigen with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, and coat overnight at 4°C. Wash three times with PBST. Dilute the 4 phage monoclonal antibodies screened in Example 2 with PBST in a five-fold gradient. Add 100 μl of the diluted sample to each well and let stand at room temperature for 1 hour. Wash the ELISA plate with PBST. Add the HRP-anti-M13 monoclonal antibody (purchased from Bio-viewshine, catalog number: GE27-9421-01) diluted with 1% BSA-PBST to the ELISA plate and let stand at room temperature for 1 h. Color development was carried out using a TMB color development kit (purchased from Kangwei Century, catalog number: CW0050S). Color development was carried out at room temperature for 10 minutes. After termination with 2M H2SO4, readings were taken with an ELISA reader at 450 nm / 630 nm, and the corresponding EC50 values were calculated. The specific data are as follows:

[0082]

[0083] From the above data and as Figure 2 shown, the 4 different murine antibody molecules screened in Example 2 can all bind to IL-11, indicating that the monoclonal antibodies provided by the present invention all have a high affinity for IL-11.

[0084] Example 4

[0085] In Example 4 of the present invention, on the basis of Example 2, it is further defined 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 those shown in SEQ ID No:23, SEQ ID No:24, SEQ ID No:25 or SEQ ID No:26; the amino acid sequence of the light chain constant region is as shown in SEQ ID No:22. The specific sequences are as follows:

[0086] SEQ ID No:22 (amino acid sequence of the light chain constant region of murine C k type):

[0087] ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC;

[0088] SEQ ID No:23 (amino acid sequence of the heavy chain constant region of murine IgG1 type):

[0089] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG;

[0090] SEQ ID No:24 (Amino acid sequence of the heavy chain constant region of murine IgG2a):

[0091] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK;

[0092] SEQ ID No:25 (Amino acid sequence of the heavy chain constant region of murine IgG2b):

[0093] AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK;

[0094] SEQ ID No:26 (Amino acid sequence of the heavy chain constant region of murine IgG3):

[0095] ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPELELNETCAEAQDGELDGLWTTITIFISLFLLSVCYSASVTLFKVKWIFSSVVQVKQTAIPDYRNMIGQGA。

[0096] Example 5 Preparation of Murine Antibody Molecules

[0097] In Example 5 of the present invention, on the basis of Example 4, it is preferably defined that the murine antibody molecule comprises the heavy chain constant region of murine IgG1 (the amino acid sequence of which is shown in SEQ ID No: 23) and murine C kThe light chain constant region of the type (the amino acid sequence is shown in SEQ ID No: 22). The method for preparing the antibody is as follows:

[0098] 1. The coding genes of the heavy chain VH and light chain VL of the 4 antibody molecules screened in Example 2 were respectively cloned into the vector pTSE containing the heavy chain and light chain constant region genes (as Figure 3 shown). The preferred heavy chain constant region is the IgG1 type constant region of mouse (the amino acid sequence is shown in SEQ ID No: 23), and the light chain constant region is the murine C k chain (the amino acid sequence is shown in SEQ ID No: 22). The structure of the pTSE vector is as Figure 3 shown (see paragraph

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

[0099] 2. Transiently transfected HEK293 cells (purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences, catalog number: GNHu43) for antibody expression. Four monoclonal antibodies were purified by using an AKTA instrument through a protein A affinity column. At the same time, a BCA kit (purchased from: Beijing Huitian Dongfang Technology Co., Ltd., catalog number: BCA0020) was used to measure the protein concentration, and then the protein size was identified by SDS-PAGE. The results are as Figure 4 shown. From left to right are non-reduced MA-I, MA-II, MA-III and MA-IV, protein molecular weight Marker1, protein molecular weight Marker2, and reduced MA-I, MA-II, MA-III and MA-IV murine anti-IL-11 monoclonal antibodies. The molecular weight of each band is consistent with the theory.

[0100] Example 6 Binding experiment of murine antibody molecule to IL-11

[0101] Coat the IL-11 antigen with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, and coat overnight at 4°C. Wash five times with 300 μL / well PBST, then add 1% BSA-PBST, 280 μL / well, and block at 37°C for 1 h. Add mouse-derived antibody molecules MA-I, MA-II, MA-III, and MA-IV at different dilution concentrations. The initial highest concentration of the 4 antibody molecules is 5 μg / mL, and they are diluted by 5-fold gradients respectively. Each antibody is diluted for 8 gradients and incubated at 37°C for 1 h. Wash five times with 300 μL / well PBST, then add Goat Anti-MouseIgG-HRP diluted 1:2000 with 1% BSA-PBST (purchased from solarbio, catalog number: SE131) and incubate at 37°C for 1 h. Color development is carried out using a TMB color development kit, 100 μL / well, at room temperature for 8 min, and then the color development is terminated with 2M H2SO4. Read the absorbance with an enzyme-linked immunosorbent assay (ELISA) reader at 450 nm / 630 nm and calculate the corresponding EC50 value. The specific data are as follows:

[0102]

[0103] Based on the above data and as Figure 5 shown, the 4 different mouse-derived antibody molecules screened out can all bind to IL-11 and have relatively high affinities.

[0104] Example 7 Competitive Inhibition Experiment of Mouse-derived Antibody with IL-11 Receptor Protein IL-11RA

[0105] Coat IL-11-Fc with carbonate buffer at pH 9.6, 200 ng / well / 100 μL, and coat overnight at 4°C. Wash five times with 300 μL / well PBST, then add 1% BSA-PBST, 280 μL / well, and block at 37°C for 1 h. First, add IL-11RA-Fc (IgG4 type) diluted to 0.5 μg / mL with 1% BSA-PBST, 50 μL / well, then add murine antibodies MA-Ⅰ, MA-Ⅱ, MA-Ⅲ, and MA-Ⅳ at different dilution concentrations, 50 μL / well. The starting highest concentration of the 5 antibodies is 100 μg / mL, and they are diluted by 2-fold gradient respectively. Each antibody is diluted for 13 gradients in total, and incubate at 37°C for 3 h. Wash five times with 300 μL / well PBST, then add Anti-Human IgG4-HRP Mouse monoclonal antibody (purchased from Sigma, catalog number: SAB4200770) diluted 1:5000 with 2% BSA-PBST, and incubate at 37°C for 1 h. Color development is carried out using a TMB color development kit, 100 μl / well, at room temperature for 15 min, and then the color development is terminated with 2M H2SO4. Read the absorbance at 450 nm / 630 nm with an enzyme-linked immunosorbent assay reader and calculate the corresponding IC50 values. The specific data are as follows:

[0106]

[0107] Through the above data and as Figure 6 shown, the 4 different murine antibodies screened out can all compete with the receptor protein IL-11RA, indicating that they can effectively inhibit the binding of IL-11 to the receptor protein IL-11RA.

[0108] Example 8 Inhibition of the binding of murine antibody to IL-11RA receptor on the surface of BaF / 3-IL-11RA cells by IL-11

[0109] Count the BaF / 3-IL-11RA cell line, take a certain number of cells, resuspend them in PBS buffer after centrifugation, adjust the cell density to 1E+6 cells / mL, add 100 μL per well into a 96-well plate. Dilute the IL-11-mFc ligand protein with PBS to a concentration of 18 μg / mL, add 50 μL per well to the corresponding positions of the 96-well plate containing BaF / 3-IL-11RA cells. After gently mixing, place the 96-well plate at 4°C and incubate for 1 h. Gradiently dilute 4 murine antibody molecules MA-I, MA-II, MA-III, and MA-IV with PBS. The initial concentration is 800 μg / mL, and dilute them 10 gradients with a 3-fold dilution, add 50 μL per well to the corresponding positions of the 96-well plate containing the mixture of BaF / 3-IL-11RA cells and IL-11-mFc ligand protein. After mixing evenly, incubate at 4°C for 2 h. After incubation, centrifuge at 3000 rpm, wash the cells once with PBS buffer, and collect the cell pellet. Add the pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) to the cell pellet, incubate at 4°C for 30 min, then centrifuge at 3000 rpm, wash once with PBS buffer, resuspend in 100 μL of PBS buffer, and detect on a flow cytometer. Collect the fluorescence signal in the FL1-A channel. Plot the dose-effect curve and calculate the corresponding IC50 value. The specific data is as follows:

[0110]

[0111] From the above data and Figure 7 it can be seen that the 4 different murine candidate molecules screened can effectively inhibit the binding of IL-11 ligand protein to the IL-11RA receptor on the cell surface.

[0112] Example 9 Inhibition of TIMP-1 Secretion by Murine Antibodies in Fetal Lung Fibroblasts MRC-5

[0113] After trypsin digestion of MRC-5 human embryonic lung fibroblasts, the cells were counted. A certain number of cells were taken, centrifuged, and resuspended in MEM complete medium (purchased from GIBCO, catalog number 10370-021), and the cell density was adjusted to 2E+5 cells / mL. 100 μL per well was added to a 96-well plate. IL-11-mFc ligand protein was diluted with MEM complete medium to a concentration of 16 μg / mL, and 50 μL per well was added to the corresponding 96-well plate. Four murine antibody molecules, MA-I, MA-II, MA-III, and MA-IV, were serially diluted with MEM complete medium. The initial concentration was 40 μg / mL, and it was diluted 8-fold with a 2-fold serial dilution. 50 μL per well was added to the 96-well plate containing the cell suspension and IL-11-mFc ligand protein suspension. After gently mixing, it was incubated overnight at 37 °C in a CO2 incubator for about 20 h. The cell culture supernatant was detected using an ELISA kit for TIMP-1 (purchased from EKSELL BIO-TECHNOLOGY CO., LTD., catalog number EH021-96).

[0114] Human TIMP-1 detection kit: The cell supernatant and standard were added to the sample wells, 100 μL per well. Immediately add the biotinylated antibody working solution (diluted 1:100), 50 μL per well, cover with a sealing film, and incubate with shaking at room temperature for 2 h. After incubation, wash the plate 4 times with washing solution, add the enzyme conjugate working solution in the TIMP-1 detection kit (diluted 1:100), 100 μL per well. Cover with a sealing film and incubate with shaking at room temperature for 1 h. After incubation, wash the plate 4 times with washing solution. Add the TMB chromogenic solution, 100 μL per well, incubate in the dark at room temperature for about 15 minutes, and terminate the reaction with 100 μL per well of Stop solution. Read the absorbance at 450 nm with an ELISA reader and calculate the corresponding IC50 value. The specific data is as follows:

[0115]

[0116] From the above data and Figure 8 it can be seen that the four different murine candidate molecules screened can effectively inhibit the release of TIMP-1 from human embryonic lung fibroblasts MRC-5 stimulated by IL-11 ligand protein.

[0117] Example 10

[0118] In Example 10 of the present invention, the anti-IL-11 monoclonal antibody is further defined as a chimeric antibody molecule, and the chimeric antibody molecule further includes a human antibody constant region. The human antibody constant region includes a human heavy chain antibody constant region and a human light chain antibody constant region. The amino acid sequence of the human heavy chain antibody constant region is one of those shown in SEQ ID No: 27, SEQ ID No: 28, or SEQ ID No: 29; the amino acid sequence of the human light chain antibody constant region is as shown in SEQ ID No: 30.

[0119] SEQ ID No: 27 (Amino acid sequence of the heavy chain constant region of human IgG1):

[0120] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0121] SEQ ID No: 28 (Amino acid sequence of the heavy chain constant region of human IgG2):

[0122] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0123] SEQ ID No: 29 (Amino acid sequence of the heavy chain constant region of human IgG4):

[0124] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0125] SEQ ID No:30 (Human C k chain light chain constant region amino acid sequence):

[0126] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C.

[0127] Example 11 Preparation of Chimeric Antibody Molecule Antibodies

[0128] Example 11 of the present invention further defines on the basis of Example 10 that the human antibody constant region includes the heavy chain constant region of human IgG1 type (its amino acid sequence is shown in SEQ ID No:27) and human C k type light chain constant region (its amino acid sequence is shown in SEQ ID No:30).

[0129] Specific preparation method:

[0130] Keep the heavy chain variable regions VH (SEQ ID No:16) of murine antibody molecules MA-I and MA-II and the light chain variable region VL gene (SEQ ID No:17) of MA-I and the light chain variable region VL gene (SEQ ID No:18) of MA-II screened from the immunized phage antibody library in Example 2 unchanged in murine sequence, and clone them respectively onto the vector pTSE (as Figure 3 shown) with heavy chain constant region and light chain constant region genes. The heavy chain constant region is of human IgG1 type (amino acid sequence is shown in SEQ ID NO:27), and the light chain constant region is human Ck Type (amino acid sequence as shown in SEQ ID NO: 30). Transiently transfect HEK293E cells (purchased from: Institute of Basic Medicine, Chinese Academy of Medical Sciences, catalog number: GNHu43) for antibody expression to obtain chimeric antibodies CA-I and CA-II.

[0131] Example 12 Humanization of Murine Antibody Molecules

[0132] First, select the sequences of murine antibody molecules MA-I and MA-II in Example 2 and compare them with the human antibody germline database (v-base) to find the human antibody light and heavy chain germlines with relatively high homology as candidate sequences. Then, transplant the CDR sequences of murine antibody molecules MA-I and MA-II onto the human candidate sequences for homology modeling. Then, calculate the key framework amino acid residues that may play an important role in maintaining the CDR loop structure through three-dimensional structure simulation, so as to design the back mutations of the humanized antibody. The light and heavy chain variable region sequences of the designed humanized antibody containing back mutations are respectively optimized and synthesized by Nanjing Genscript Biotech Co., Ltd., and then ligated onto the transient expression vector. Analyze the combinations of the humanized light and heavy chains. Among them, the following humanized anti-IL-11 monoclonal antibody molecules are obtained from MA-I: HA-I-A, HA-I-B, HA-I-C, HA-I-D; the following humanized antibody molecules are obtained from MA-II: HA-II-A, HA-II-B, HA-II-C, HA-II-D; the sequences of the 8 monoclonal antibodies screened above are as follows:

[0133]

[0134] 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):

[0135] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVSS PEAMDYWGQGTLVTVSS;

[0136] SEQ ID No: 32 (amino acid sequence of the light chain variable region of HA-I-A):

[0137] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;

[0138] SEQ ID No:33 (Amino acid sequence of the heavy chain variable region of HA-I-B and HA-II-C):

[0139] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVST ISDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVS SPEAMDYWGQGTLVTVSS;

[0140] SEQ ID No:34 (Amino acid sequence of the light chain variable region of HA-I-B):

[0141] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;

[0142] SEQ ID No:35 (Amino acid sequence of the light chain variable region of HA-I-C):

[0143] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;

[0144] SEQ ID No:36 (Amino acid sequence of the heavy chain variable region of HA-I-D and HA-II-D):

[0145] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAMYYCARDGGYVS SPEAMDYWGQGTSVTVSS;

[0146] SEQ ID No:37 (Amino acid sequence of the light chain variable region of HA-I-D):

[0147] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;

[0148] SEQ ID No:38 (Amino acid sequence of the light chain variable region of HA-II-A):

[0149] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;

[0150] SEQ ID No:39 (Amino acid sequence of the light chain variable region of HA-II-B and HA-II-C):

[0151] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;

[0152] SEQ ID No:40 (Amino acid sequence of the light chain variable region of HA-II-D):

[0153] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGTVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPWTFGGGTKVEIK.

[0154] Example 13

[0155] In Example 13 of the present invention, on the basis of Example 12, the human antibody constant region is further defined to include a human antibody heavy chain constant region and a human antibody light chain constant region. The amino acid sequence of the human antibody heavy chain constant region is one of those shown in SEQ ID No:27, SEQ ID No:28 or SEQ ID No:29; the amino acid sequence of the human antibody light chain constant region is as shown in SEQ ID No:30.

[0156] The specific sequences of the above human antibody constant regions are the same as those in Example 10.

[0157] Preparation of Humanized Antibody Molecule in Example 14

[0158] In Example 14 of the present invention, on the basis of Example 13, the human antibody constant region is further defined to include the heavy chain constant region of human IgG1 type (the amino acid sequence of which is shown in SEQ ID No: 27) and human C k type light chain constant region (the amino acid sequence of which is shown in SEQ ID No: 30).

[0159] 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 humanized in Example 12 were respectively cloned into the vector pTSE containing the heavy chain constant region and light chain constant region genes (as Figure 3 shown), the heavy chain constant region is of human IgG1 type (the amino acid sequence is shown in SEQ ID NO: 27), and the light chain constant region is C k chain (the amino acid sequence is shown in SEQ ID No: 30).

[0160] The 2 chimeric antibodies CA-I and CA-II obtained in Example 11 and the 8 humanized antibody molecules HA-I-A, HA-I-B, HA-I-C, HA-I-D, HA-II-A, HA-II-B, HA-II-C, HA-II-D obtained in Example 12 were respectively transiently transfected into HEK293 cells (purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences, catalog number: GNHu43) for antibody expression. Monoclonal antibodies were purified using an AKTA instrument through a protein A affinity column, and at the same time, a BCA kit (purchased from: Beijing Huitian Dongfang Technology Co., Ltd., catalog number: BCA0020) was used to measure the protein concentration. Then, the protein size was identified by SDS-PAGE. The results are as Figure 9 shown. From left to right, they are non-reduced protein molecular weight HA-I-A, HA-I-B, HA-I-C, HA-I-D, the chimeric antibody CA-I prepared in Example 11, reduced protein molecular weight Marker, HA-II-A, HA-II-B, HA-II-C, HA-II-D, chimeric antibody CA-II. The molecular weight size of each band is consistent with the theory.

[0161] Experiment on the Binding of Humanized Antibody Molecule to IL-11 in Example 15

[0162] Coat the IL-11 antigen with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, and incubate overnight at 4 °C. Wash five times with 300 μL / well PBST, then add 280 μL / well of 1% BSA-PBST and block at 37 °C for 1 h. Dilute the humanized antibodies HA-I-A, HA-I-B, HA-I-C, HA-I-D, HA-II-A, HA-II-B, HA-II-C, HA-II-D and the chimeric antibody CA-I prepared in Example 11 with 1% BSA-PBST. The initial concentration of the humanized antibodies is 10 μg / mL, and they are diluted in a 5-fold gradient for a total of 8 gradients and incubated at 37 °C for 1 h. Wash five times with 300 μL / well PBST, then add goat anti-Human IgG Fab HRP diluted 1:5000 with 1% BSA-PBST (purchased from Invitrogen, catalog number: 31482) and incubate at 37 °C for 1 h. Develop the color with a TMB color development kit, 100 μL / well, develop the color at room temperature for 5 min, and then terminate the color development with 2M H2SO4. Read the absorbance at 450 nm / 630 nm with an enzyme-linked immunosorbent assay reader and calculate the corresponding EC50 value. The specific data is as follows:

[0163]

[0164] Based on the above data and experimental results as Figure 10 shown, all 8 different humanized antibody molecules can bind to IL-11, and the EC50 values of the humanized antibody molecules HA-I-A, HA-I-B, HA-I-C, HA-I-D are close to those of the chimeric antibody CA-I, and the EC50 values of the humanized antibody molecules HA-II-A, HA-II-B, HA-II-C, HA-II-D are close to those of the chimeric antibody CA-II, indicating that the humanized antibody molecules retain the high binding ability of the murine parental antibodies MA-I and MA-II to IL-11.

[0165] Example 16 Inhibition of the Binding of Humanized Antibody Molecules to the IL-11RA Receptor on the Surface of BaF / 3-IL-11RA Cells

[0166] Four humanized antibody molecules, HA-I-A, HA-I-B, HA-I-C, and HA-I-D, with relatively better protein-level binding activities were selected for cell activity evaluation experiments. The BaF / 3-IL-11RA cell line was counted, a certain number of cells were taken, resuspended in PBS buffer after centrifugation, and the cell density was adjusted to 1E+6 cells / mL, 100 μL / well, and added to a 96-well plate. 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 positions of the 96-well plate containing BaF / 3-IL-11RA cells. After gently mixing, the 96-well plate was placed at 4°C and incubated for 1 h. The four humanized antibody molecules, HA-I-A, HA-I-B, HA-I-C, and HA-I-D, were serially diluted with PBS buffer. The initial concentration was prepared to be 800 μg / mL, with a 3-fold serial dilution, a total of 10 gradients, 50 μL / well, and added to the corresponding positions of the 96-well plate containing the mixture of BaF / 3-IL-11RA cells and IL-11-mFc ligand protein. After mixing evenly, it was placed at 4°C and incubated for 2 h. After the incubation, the cells were washed once with PBS buffer by centrifugation at 3000 rpm, and the cell pellet was collected. The pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) was added to the cell pellet, incubated at 4°C for 30 min, then centrifuged and washed once at 3000 rpm, resuspended in 100 μL of PBS, and then detected by flow cytometry. The fluorescence signal in the FL1-A channel was collected. A dose-effect curve was plotted, and the corresponding IC50 value was calculated. The specific data is as follows:

[0167]

[0168] From the above data and Figure 11 it can be seen that all four selected humanized candidate molecules can inhibit the binding of the IL-11 ligand protein to the IL-11RA receptor on the surface of BaF / 3-IL-11RA cells.

[0169] Example 17 Inhibition of the Binding of Humanized Antibody Molecules to the GP130 Receptor on the Surface of BaF / 3-GP130 Cells by IL-11

[0170] Count the BaF / 3-GP130 cell line, take a certain number of cells, resuspend them in PBS buffer after centrifugation, adjust the cell density to 1E+6 cells / mL, 100 μL / well, and add them to a 96-well plate. Dilute the IL-11-mFc ligand protein with PBS to a concentration of 12 μg / mL, 50 μL / well, and add it to the corresponding positions of the 96-well plate containing BaF / 3-GP130 cells. After gently mixing, place the 96-well plate at 4°C and incubate for 1 h. Gradiently dilute 4 human antibody molecules HA-I-A, HA-I-B, HA-I-C, and HA-I-D with PBS buffer, prepare an initial concentration of 2000 μg / mL, and perform 2-fold serial dilution for a total of 10 gradients, 50 μL / well, and add them to the corresponding positions of the 96-well plate containing BaF / 3-GP130 cells and IL-11-mFc ligand protein. After mixing evenly, place the 96-well plate at 4°C and incubate for 2 h. After the incubation, centrifuge at 3000 rpm and wash the cells once with PBS buffer, and collect the cell pellet. Add the pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02), 100 μL / well, and incubate at 4°C for 30 min. After centrifuging at 3000 rpm and washing once with PBS buffer, resuspend the cells in 100 μL / well of PBS buffer, detect them on a flow cytometer, and collect the fluorescence signal in the FL1-A channel. Plot the dose-effect curve and calculate the corresponding IC50 value. The specific data are as follows:

[0171]

[0172] From the above data and Figure 12 it can be seen that the 4 selected humanized candidate molecules can all block the binding of the IL-11 ligand protein to the GP130 receptor on the surface of BaF / 3-GP130 cells.

[0173] Example 18 Detection of the Biological Activity of Humanized Antibody Molecules (Reporter Gene)

[0174] Count the BaF / 3-IL-11RA-GP130-STAT3-Luc engineered cell line. Use the sample diluent (its components include 90% IMDM, 10% FBS, 10 ng / mL mouse IL-3) to adjust the cell density to 2E+6 cells / mL. After gently mixing, add the cell suspension to a 96-well plate, 50 μL / well. Dilute the 4 humanized antibody molecules HA-I-A, HA-I-B, HA-I-C, and HA-I-D to an initial concentration of 200 μg / ml with the sample diluent, and perform 5-fold serial dilutions for a total of 10 gradients, 100 μL / well, and add them to the corresponding positions of the 96-well plate containing the engineered cell line. Set two replicates for each sample concentration. Prepare IL-11 protein with the sample diluent at a concentration of 10 μg / mL, 50 μL / well, and add it to the 96-well plate containing the engineered cell line and the humanized antibody molecules. Gently mix the cell culture plate and incubate it in a 37°C CO2 incubator for 6 h. Centrifuge to discard the supernatant, add lysis buffer, 10 μL / well to a 384-well plate, add an equal amount of luciferase reaction substrate (purchased from Promega Biotechnology Co., Ltd., catalog number E2610), react at room temperature for 5 min, read the fluorescence value under a microplate reader, and calculate the corresponding IC50 value. The specific data is as follows:

[0175]

[0176] From the above data and Figure 13 As shown, all 4 humanized antibody molecules screened can block the binding of IL-11 to the IL-11RA and GP130 receptors and inhibit the signal pathway transduction.

[0177] Example 19 Inhibition of TIMP-1 Secretion by Humanized Antibody Molecules in Fetal Lung Fibroblasts MRC-5

[0178] After trypsin digestion of MRC-5 human embryonic lung fibroblasts, the cells were counted. A certain number of cells were taken, centrifuged, and resuspended in MEM complete medium. The cell density was adjusted to 2E+5 cells / mL, and 100 μL / well was added to a 96-well plate. The IL-11-mFc ligand protein was diluted with MEM complete medium to a concentration of 16 μg / mL, and 50 μL / well was added to the corresponding 96-well plate. Four humanized antibody molecules, HA-I-A, HA-I-B, HA-I-C, and HA-I-D, were serially diluted with MEM complete medium. The initial concentration was 40 μg / mL, and the dilution was performed in 3-fold increments for a total of 8 gradients. 50 μL / well was added to the 96-well plate containing the cell suspension and the IL-11-mFc ligand protein suspension. The mixture was gently mixed and incubated overnight at 37°C in a CO2 incubator for approximately 20 h. The cell culture supernatant was taken and detected using the TIMP-1 ELISA kit (the method was the same as in Example 9). The microplate reader was used to read the absorbance at 450 nm, and the corresponding IC50 values were calculated. The specific data are as follows:

[0179]

[0180] From the above data and Figure 14 it can be seen that the 4 selected humanized antibody molecules can effectively inhibit the release of TIMP-1 from human embryonic lung fibroblasts MRC-5 stimulated by the IL-11 ligand protein.

[0181] Example 20 Cross-binding experiment of humanized antibody molecules with IL-11 of different species

[0182] The humanized antibody molecule HA-I-A with better activity in protein level and function detection was selected for cross-binding detection with IL-11 of different species. Human IL-11, mouse IL-11 (purchased from Sino Biological Inc., catalog number: 50117-MNCE), rat IL-11 (purchased from Kanglang Biotechnology, catalog number: KL40001Ra), and cynomolgus monkey IL-11 (purchased from Sino Biological Inc., catalog number: 90925-CNCE) were respectively coated at 100 ng / well / 100 μL with carbonate buffer at pH 9.6 and incubated overnight at 4°C. Washed five times with 300 μL / well PBST, then 1% BSA-PBST, 280 μL / well, was added and blocked at 37°C for 1 h. The humanized antibody HA-I-A was diluted with 1% BSA-PBST, with an initial concentration of 50 μg / mL, and serially diluted 5-fold, for a total of 9 gradients, with two replicates for each gradient. 100 μL / well was added to the 96-well plate and incubated at 37°C for 1 h. Washed five times with 300 μL / well PBST, goat anti-Human IgG Fab HRP (purchased from Invitrogen, catalog number: 31482) was diluted with 1% BSA-PBST, and the working solution concentration was 1:5000. 100 μL / well was added to the 96-well plate and incubated at 37°C for 1 h. Washed five times with 300 μL / well PBST, developed color with TMB color development kit, 100 μL / well, developed color in the dark at room temperature for 5 min, and then terminated color development with 2 M H2SO4. Read with an enzyme-linked immunosorbent assay (ELISA) reader at 450 nm / 630 nm and calculate the corresponding EC50 value. The specific data is as follows:

[0183]

[0184]

[0185] As shown by the above data and as Figure 15 shown, the humanized antibody molecule HA-I-A can bind to human IL-11, mouse IL-11, rat IL-11, and cynomolgus monkey IL-11, and has a relatively high affinity.

[0186] Example 21 Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody on pulmonary fibrosis

[0187] Bleomycin (bLF) was used to model the study of the therapeutic effect of anti-IL-11 monoclonal antibody HA-I-A on pulmonary fibrosis.

[0188] Animal species: C57BL / 6J mice (purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.)

[0189] Quantity, gender, and mouse age: 6 per group, male, 6 - 8 weeks old;

[0190] The control group was only injected with normal saline;

[0191] In the administration group, the HA-I-A antibody molecule was injected twice a week for 4 consecutive weeks.

[0192] The body weight of the animals was measured once a week, and the animals were observed for any abnormalities; Organ weight detection: The lung organs were collected, the weight of the lung organs was measured, and the ratio of lung to body weight was calculated; Lung pathology detection: Lung sections were made and hematoxylin-eosin (HE) and Masson staining were used to observe the degree of lung fibrosis.

[0193] The results are as Figure 16 shown. After administration of the HA-I-A antibody molecule, the ratio of lung to body weight in the mice of the administration group was significantly smaller than that of the control group; The results are as Figure 17 shown. Compared with the control group, the lung sections of the administration group showed significantly less pulmonary fibrosis. Therefore, it can be shown that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the generation of pulmonary fibrosis.

[0194] Example 22 Therapeutic Efficacy Experiment of Anti-IL-11 Monoclonal Antibody on Cardiac Fibrosis

[0195] Isoproterenol was used to establish a model to study the therapeutic effect of the anti-IL-11 monoclonal antibody HA-I-A on cardiac fibrosis.

[0196] Animal species: C57BL / 6J mice (purchased from Jiangsu GIBIO Biotechnology Co., Ltd.)

[0197] Quantity, gender and mouse age: 6 per group, male, 6-8 weeks old;

[0198] The control group was only injected with normal saline;

[0199] In the administration group, the HA-I-A antibody molecule was injected twice a week for 4 consecutive weeks.

[0200] The body weight of the animals was measured once a week, and the animals were observed for any abnormalities; Organ weight detection: The heart was collected, the weight of the heart was measured, and the ratio of heart to body weight was calculated; Heart pathology detection: Heart sections were made and hematoxylin-eosin (HE) and Masson staining were used to observe the degree of cardiac fibrosis.

[0201] The results are as Figure 18 shown. The ratio of heart to body weight in the mice of the administration group was significantly smaller than that of the control group; The results are as Figure 19 shown. Compared with the control group, the heart sections of the administration group showed significantly less cardiac fibrosis. Therefore, it can be shown that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the generation of cardiac fibrosis.

[0202] Example 23: Therapeutic Efficacy Experiment of Anti-IL-11 Monoclonal Antibody on Renal Fibrosis

[0203] Doxorubicin (dKF) was used to establish a model to study the therapeutic effect of anti-IL-11 monoclonal antibody HA-I-A on renal fibrosis.

[0204] Animal species: BALB / c mice (purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.)

[0205] Number, gender and mouse age: 6 mice / group, male, 6 - 8 weeks old;

[0206] The control group was only injected with normal saline;

[0207] The administration group was injected with HA-I-A antibody molecules twice a week for 4 consecutive weeks.

[0208] The body weight of the animals was measured once a week, and the animals were observed for any abnormalities; organ weight detection: the heart was collected, the weight of the kidney was measured, and the urine protein content was detected; renal pathology detection: the kidney was sectioned and stained with hematoxylin and eosin (HE) and Masson to observe the degree of renal fibrosis.

[0209] The results are as Figure 20 shown. Compared with the control group, the urine protein content in the kidneys of the mice in the administration group was significantly lower than that in the control group; the results are as Figure 21 shown. Compared with the control group, the kidney sections of the administration group showed significantly less renal fibrosis. Therefore, it can be shown that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the occurrence of renal fibrosis.

[0210] Example 24: Therapeutic Efficacy Experiment of Anti-IL-11 Monoclonal Antibody on Liver Fibrosis

[0211] CCl4 was used to establish a model to study the therapeutic effect of anti-IL-11 monoclonal antibody HA-I-A on liver fibrosis.

[0212] Animal species: C57BL / 6J mice (purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.)

[0213] Number, gender and mouse age: 6 mice / group, male, 6 - 8 weeks old;

[0214] The control group was only injected with normal saline;

[0215] The administration group was injected with HA-I-A antibody molecules twice a week for 4 consecutive weeks.

[0216] Body weight monitoring: Measure the body weight of the animals once a week and observe whether there are any abnormalities in the animals; Liver pathological detection: Cut the liver into sections and observe the degree of liver fibrosis using hematoxylin and eosin (HE) and Masson staining; Organ weight detection: Collect the kidneys, measure the weight of the liver, and perform HE staining; Serum detection: Collect the serum and detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the mouse serum.

[0217] The results are as Figure 22 shown. Compared with the control group, the liver weight of the mice in the administration group was significantly lower than that of the control group; The results are as Figure 23 shown. The levels of ALT and AST in the serum of the mice in the administration group were significantly lower than those of the control group; The results are as Figure 24 shown. Compared with the control group, the liver sections of the administration group showed significantly less liver fibrosis. Therefore, it can be shown that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the generation of liver fibrosis.

[0218] Example 25 Evaluation of the thermal stability of anti-IL-11 monoclonal antibody HA-I-A

[0219] Use a multifunctional protein thermal stability analysis system (purchased from Unchained Labs) to evaluate the thermal stability of the anti-IL-11 monoclonal antibody HA-I-A. By monitoring the change of the endogenous fluorescence of the protein with temperature (starting from 25 °C and increasing the temperature at a rate of 0.3 °C / min to 95 °C), the change of the protein conformation is detected, so as to determine the melting temperature Tm of the protein and evaluate the conformational stability of the protein. When the sample aggregates, it will cause interference of the scattered light waves and increase the scattered light signal. The colloidal stability of the protein is measured by static light scattering (characterized by Tagg), and the results are referred to the following table and appendix Figure 25 shown.

[0220]

[0221] The melting temperature Tm of the anti-IL-11 monoclonal antibody HA-I-A is 79.5 °C, and the average Tagg is 75.9 °C, showing good conformational stability and colloidal stability.

[0222] Example 26

[0223] Purify the anti-IL-11 monoclonal antibody HA-I-A obtained through the above examples. The specific purification method is as follows:

[0224] S1. Affinity Chromatography: S101. Equilibrate the affinity chromatography column with Buffer A. The packing material of the affinity chromatography column is MabSelect Sure LX, Buffer A is 30 mM HEPES buffer with a pH value of 7 and a conductivity of 10 mS / cm; S102. Load the target protein containing the anti-IL-11 monoclonal antibody HA-I-A onto the affinity chromatography column at a flow rate of 100 cm / h. The loading capacity is 20 mg protein / ml packing material, and the column bed height of the affinity chromatography column is 18 cm; S103. After loading, re-equilibrate with Buffer A, and then perform at least one elution with 2 CV of Buffer B until the UV absorption curve drops to a stable state, then stop flushing, and perform a second elution with Buffer A. Buffer B is 30 mM HEPES buffer with a pH value of 5; S104. Elute the affinity chromatography column with Buffer C, and collect the protein product for standby after elution. Buffer C is 20 mM glycine-hydrochloride with a pH of 3; S105. Adjust the pH value of the collected protein product to 3 with an acid solution, incubate at room temperature for 0.5 h under this pH condition, and finally adjust the pH of the protein product back to 5 with 1 M Tris buffer to obtain a crudely purified protein sample. The acid solution is citric acid.

[0225] S2. Anion Exchange Chromatography: S201. Equilibrate the anion exchange chromatography column with 2 CV of Buffer D. The packing material of the anion exchange chromatography is Capto Q, and Buffer D is citric acid buffer with a pH value 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 capacity is 20 mg protein / ml packing material, and the column bed height of the anion exchange chromatography column is 18 cm; S203. After loading, equilibrate the anion exchange chromatography column with Buffer D again, and collect the flow-through for standby.

[0226] S3. Cation Exchange Chromatography: S301. Equilibrate the cation exchange chromatography column with 2 CV of Buffer E. The packing material of the cation exchange chromatography column is Capto SP, and Buffer E is 30 mM citric acid buffer with a pH value of 5.0 and a conductivity of 2 mS / cm; S302. Load the flow-through after secondary purification onto the cation exchange chromatography column at a flow rate of 100 cm / h. The loading capacity is 20 mg protein / ml packing material, and the column bed height of the cation exchange chromatography column is 18 cm; S303. Elute the cation exchange chromatography column with Eluent F, and a protein solution of highly purified anti-IL-11 monoclonal antibody is obtained after elution. Buffer F is 30 mM citric acid buffer and 80 mM NaCl with a pH value of 5.0.

[0227] Example 27

[0228] The anti-IL-11 monoclonal antibody HA-I-A obtained through the above embodiments is purified, and the specific purification method is as follows:

[0229] S1. Affinity chromatography: S101. Equilibrate the affinity chromatography column with buffer A. The packing material of the affinity chromatography column is ATProtein A Diamond Plus, buffer A is 50 mM Tris-HCl buffer, the Tris-HCl buffer contains 120 - 160 mM NaCl, preferably 150 mM NaCl in this embodiment, and the pH value 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-I-A onto the affinity chromatography column at a flow rate of 150 cm / h, and the loading capacity is 55 mg protein / ml packing material, and the column bed height of the affinity chromatography column is 20 cm; S103. After the loading is completed, re-equilibrate with buffer A, and then perform at least one washing and elution with 4 CV of buffer B until the curve of the ultraviolet absorption value drops to a stable state, then stop washing, and then perform a second washing with buffer A. Buffer B is 50 mM acetate buffer, and its pH value is 5.5; S104. Elute the affinity chromatography column with buffer C, and collect the protein product for standby after elution. Buffer C is 50 mM acetate buffer, and its pH is 3.5; S105. Adjust the pH value of the collected protein product to 3.4 - 3.8 with an acid solution, and incubate at room temperature for 2 h under this pH condition, and finally adjust the pH of the protein product back to 6.2 with 1 M Tris buffer to obtain a crudely purified protein sample; the acid solution is citric acid.

[0230] S2. Anion exchange chromatography: S201. Equilibrate the anion exchange chromatography column with 4 CV of buffer D. The packing material of the anion exchange chromatography is NanoGel-50Q, and buffer D is phosphate buffer with a pH value of 7; S202. Load the crudely purified protein sample onto the anion exchange chromatography column at a flow rate of 150 cm / h, and the loading capacity is 70 mg protein / ml packing material, and the column bed height of the anion exchange chromatography column is 20 cm; S203. After the loading is completed, equilibrate the anion exchange chromatography column with buffer D again, and collect the flow-through for standby.

[0231] S3. Cation exchange chromatography: S301. Equilibrate the cation exchange chromatography column with 3 column volumes (CV) of buffer E. The packing material of the cation exchange chromatography column is NanoGel-50SP, buffer E is 50 mM acetate buffer with a pH value of 6 and a conductivity of 2.5 mS / cm; S302. Load the flow-through solution after secondary purification onto the cation exchange chromatography column at a flow rate of 150 cm / h. The loading capacity is 47 mg protein / ml of packing material, and the column bed height of the cation exchange chromatography column is 20 cm; S303. Elute the cation exchange chromatography column with eluent F, and a protein solution of high-purity anti-IL-11 monoclonal antibody is obtained after elution. Buffer F is 50 mM acetate buffer and 115 mM NaCl with a pH value of 5.8.

[0232] Example 28

[0233] Purify the anti-IL-11 monoclonal antibody HA-I-A obtained through the above examples. The specific purification method is as follows:

[0234] S1. Affinity chromatography: S101. Equilibrate the affinity chromatography column with buffer A. The packing material of the affinity chromatography column is Eshmuno A, which can also be replaced with NMab Pro. Buffer A is 60 mM phosphate buffer with a pH value of 8 and a conductivity of 30 mS / cm; S102. Load the target protein containing the anti-IL-11 monoclonal antibody onto the affinity chromatography column at a flow rate of 300 cm / h. The loading capacity is 60 mg protein / ml of packing material, and the column bed height of the affinity chromatography column is 22 cm; S103. After loading, re-equilibrate with buffer A, and then perform at least one wash and elution with 6 CV of buffer B until the curve of the ultraviolet absorption value drops to a stable state, then stop washing, and perform a second wash with buffer A. Buffer B is 60 mM Tris-HCl buffer, which can also be selected to be replaced with phosphate buffer or citrate buffer with a pH value of 6; S104. Elute the affinity chromatography column with buffer C, and collect the protein product for standby after elution. Buffer C is 200 mM citrate with a pH of 4; S105. Adjust the pH value of the collected protein product to 5 with an acid solution, incubate at room temperature for 3 h under this pH condition, and finally adjust the pH of the protein product back to 8 with 1 M Tris buffer to obtain a crudely purified protein sample; the acid solution is hydrochloric acid.

[0235] S2. Anion exchange chromatography: S201. Equilibrate the anion exchange chromatography column with 6 CV of buffer D. The packing material for anion exchange chromatography is Capto adhere, 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 capacity is 120 mg of protein / ml of packing material, and the column bed 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 fraction for standby;

[0236] S3. Cation exchange chromatography: S301. Equilibrate the cation exchange chromatography column with 6 CV of buffer E. The packing material for cation exchange chromatography is Capto MMC, which can also be replaced with Eshmuno S. Buffer E is a 60 mM phosphate buffer with a pH of 6.0 and a conductivity of 5 mS / cm; S302. Load the flow-through fraction after secondary purification onto the cation exchange chromatography column at a flow rate of 200 cm / h. The loading capacity is 75 mg of protein / ml of packing material, and the column bed height of the cation exchange chromatography column is 22 cm; S303. Elute the cation exchange chromatography column with eluent F, and a protein solution of highly purified anti-IL-11 monoclonal antibody is obtained after elution. Buffer F is a 60 mM phosphate buffer and 150 mM NaCl with a pH of 6.0.

[0237] Example 29

[0238] In Example 29 of the present invention, on the basis of Example 27, it is further defined that in S105 of the purification method of anti-IL-11 monoclonal antibody, the acid solution in the inactivation treatment process is preferably acetic acid.

[0239] Examples 30 - 32

[0240] In Examples 30 - 32 of the present invention, on the basis of Example 29, it is further defined that in step S105 of the purification method of anti-IL-11 monoclonal antibody, the pH of the protein product is adjusted back to the following pH and conductivity range with 1 M Tris buffer to obtain a crudely purified protein sample. Other methods are the same as those in Example 27, and the specific data are as follows.

[0241] Example pH value Sample conductivity (mS / cm) Example 30 6.5 5 Example 31 7.5 5 Example 32 7.0 4

[0242] Examples 33 - 35

[0243] Example 33 - 35 of the present invention further defines the eluent F in step S303 of the purification method of the anti - IL - 11 monoclonal antibody on the basis of Example 32. Other methods and parameters are exactly the same as those in Example 27, as follows.

[0244]

[0245] Control Example 1

[0246] On the basis of Example 35, the purification method provided by Control Example 1 exchanges the order of the anion - exchange chromatography in step S2 and the cation - exchange chromatography in step S3. Other methods and parameters are exactly the same as those in Example 35.

[0247] Control Example 2

[0248] Control Example 2 of the present invention uses the classical three - step purification process of monoclonal antibodies to purify the anti - IL - 11 monoclonal antibody HA - I - A on the basis of Example 35. All the packing materials used in the three - step chromatography are produced by Cytiva. The packing material of the first - step affinity chromatography column is MabSelectSuRe LX, the packing material of the second - step anion - exchange chromatography column is Capto Q, and the packing material of the third - step cation - exchange chromatography column is Capto SP ImpAct. Other methods and parameters are exactly the same as those in Example 35.

[0249] Experiment 1. Physicochemical detection and related impurity detection of the anti - IL - 11 monoclonal antibody

[0250] For the anti - IL - 11 monoclonal antibody HA - I - A obtained by the purification methods provided by the above - mentioned examples and control examples of the present invention, gel chromatography technology is used to detect the purity, and the contents of aggregates, monomers and degradation products of the samples during the purification process are analyzed; in addition, ion chromatography technology is used to analyze the contents of acid - base peaks of charge isomers, and a special kit is used to detect the contents of process - related impurities. At the same time, the total recovery rate is calculated by the following formula: Total recovery rate = Affinity chromatography protein recovery rate (%) * Anion - exchange chromatography protein recovery rate (%) * Cation - exchange chromatography protein recovery rate (%). The specific data are as follows:

[0251]

[0252] From the above experimental data, it can be concluded that for the anti - IL - 11 monoclonal antibody purified by the purification method provided by the above - mentioned examples of the present invention, the SEC purity can reach more than 99%, and the total protein recovery rate can reach more than 75%; in the anti - IL - 11 monoclonal antibody stock solution obtained by the purification method provided in Example 35, the purity and recovery rate are the highest, and the total protein recovery rate reaches more than 81%. In antibody production, this recovery rate can significantly increase the yield and reduce the production cost.

[0253] Compared with Examples 26 and 28, in Example 27, the filler of the chromatography column and the composition and content of each buffer are more in line with the purification process conditions of the anti-IL-11 monoclonal antibody HA-I-A.

[0254] Compared with Example 27, in Example 29, the acid solution is acetic acid, the appearance of the inactivated sample is clear, there is no precipitation, and the protein recovery rate is higher.

[0255] It can be seen from the comparison of Examples 30 - 32 that in step S105, adjusting the pH of the protein product to 7.0 with 1M Tris buffer and the conductivity being 4 mS / cm can significantly improve the total protein recovery rate of the anti-IL-11 monoclonal antibody HA-I-A.

[0256] It can be seen from the comparison of Examples 33 - 35 that when the eluent F includes 50 mM acetate buffer and 100 mM NaCl and the pH value is 5.5, the protein recovery rate is higher.

[0257] Compared with Example 1, in Example 35, swapping the order of anion exchange chromatography in step S2 and cation exchange chromatography in step S3 is not conducive to the purification of the anti-IL-11 monoclonal antibody HA-I-A. The best three-step purification process for the purification of the anti-IL-11 monoclonal antibody HA-I-A provided in Example 35 is affinity chromatography, anion exchange chromatography, and cation exchange chromatography in sequence.

[0258] Example 35 is different from Example 2 only in the packing material of the chromatography column. The purification effect of the packing material provided in Example 35 is significantly higher than that of Control Example 2, with a higher total recovery rate and lower cost. It can effectively remove product-related impurities and process-related impurities. Therefore, it can be concluded that the purification of the anti-IL-11 monoclonal antibody HA-I-A is more suitable for the packing material provided in Example 35. Therefore, the preferred affinity chromatography of the present invention is AT Protein A Diamond Plus. As a packing material, AT Protein A Diamond Plus has excellent binding specificity, alkali resistance and pressure-flow characteristics, lower ligand shedding, can capture antibodies in cell supernatant, remove most impurities. In addition, it can significantly increase the loading capacity. It is an alkali-resistant Protein A affinity packing material, which can achieve more efficient cleaning and sterilization, avoid cross-contamination and increase the service life. The packing material of the anion exchange chromatography column is preferably NanoGel-50Q, and the packing material of the cation exchange chromatography column is preferably NanoGel-50SP. The NanoGel-50SP chromatography adopts a binding and elution mode, that is, the target protein flows through during sample loading and impurities are adsorbed on the chromatography column, effectively achieving the purification purpose. NanoGel-50Q and NanoGel-50SP belong to a rigid polymer matrix monodisperse matrix, with uniform particles, smaller particle size and high resolution and good separation effect. The combination of the above different chromatography column packing materials can not only effectively remove process-related impurities such as HCP, DNA, and Protein A, but also adsorb product-related impurities such as aggregates and production fragments, improve the purity of the product, ensure the purification of products that meet the quality requirements, and significantly reduce the purification process cost.

[0259] Experiment 2: Detection of the binding activity and biological activity of the anti-IL-11 monoclonal antibody

[0260] For the anti-IL-11 monoclonal antibody HA-I-A purified by the purification methods provided in the above-mentioned examples and Control 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-I-A to IL-11 to evaluate the activity of the purified antibody. The detection results are as follows:

[0261] Example Binding activity (%) Biological activity (%) Example 26 86 101 Example 27 103 72 Example 28 82 99 Example 29 79 82 Example 30 84 98 Example 31 92 104 Example 32 81 79 Example 33 96 88 Example 34 105 93 Example 35 76 84

[0262] As can be seen from the above table, the anti-IL-11 monoclonal antibody HA-I-A purified by the method provided in the above-mentioned examples of the present invention has good binding activity and biological activity, within the range of 90% ± 20%, indicating that the anti-IL-11 monoclonal antibody HA-I-A purified under the conditions defined by the method provided by the present invention through a large number of experimental conditions screening has good biological activity.

[0263] The present invention is not limited to the above-mentioned optimal embodiments. Any person can obtain other various forms of products under the inspiration of the present invention. However, no matter what changes are made in its shape or structure, as long as it has a technical solution that is the same as or similar to the present application, it falls within the protection scope of the present invention.

Claims

1. A purification method for an anti-IL-11 monoclonal antibody, characterized in that, The method includes: S1. Affinity chromatography: preliminarily purifying and concentrating the target protein containing the anti-IL-11 monoclonal antibody, and inactivating viruses in the collected protein product in an acid solution to obtain a crudely purified protein sample; S2. Anion exchange chromatography: subjecting the protein sample to secondary purification and collecting the flow-through fraction; S3. Cation exchange chromatography: after subjecting the flow-through fraction to fine purification, a protein solution of the anti-IL-11 monoclonal antibody with high purity is obtained. The anti-IL-11 monoclonal antibody includes three heavy chain complementarity determining regions respectively represented by HCDR1, HCDR2, and HCDR3, and three light chain complementarity determining regions respectively represented by LCDR1, LCDR2, and LCDR3. The anti-IL-11 monoclonal antibody is selected from any one of the following: A-I: The amino acid sequence of the heavy chain complementarity determining region HCDR1 is as shown in SEQ ID No:1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is as shown in SEQ ID No:2, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is as shown in SEQ ID No:3, the amino acid sequence of the light chain complementarity determining region LCDR1 is as shown in SEQ ID No:4, the amino acid sequence of the light chain complementarity determining region LCDR2 is as shown in SEQ ID No:5, and the amino acid sequence of the light chain complementarity determining region LCDR3 is as shown in SEQ ID No:

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

7.

2. The purification method of the anti-IL-11 monoclonal antibody according to 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 anti-IL-11 monoclonal antibody is selected from any one of the following: MA-I: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No:16, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No:

17. MA-II: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No:16, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No:

18.

3. The purification method of the anti-IL-11 monoclonal antibody according to claim 2, wherein, 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 those shown in SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, or SEQ ID No:26; the amino acid sequence of the light chain constant region is as shown in SEQ ID No:

22.

4. The purification method of the anti-IL-11 monoclonal antibody according to claim 1, characterized in that, The anti-IL-11 monoclonal antibody further comprises a heavy chain variable region and a light chain variable region, and the anti-IL-11 monoclonal antibody is selected from any one of the following: HA-I-A: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 32; HA-I-B: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 33, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 34; HA-I-C: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 35; HA-I-D: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 36, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 37; HA-II-A: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 38; HA-II-B: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 39; HA-II-C: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 33, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 39; HA-II-D: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 36, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No:

40.

5. The purification method of the anti-IL-11 monoclonal antibody according to claim 4, characterized in that, The anti-IL-11 monoclonal antibody further comprises a human heavy chain constant region and a human light chain constant region. The amino acid sequence of the human heavy chain constant region is one of those shown in SEQ ID No: 27, SEQ ID No: 28, or SEQ ID No: 29; the amino acid sequence of the human light chain constant region is as shown in SEQ ID No:

30.

6. The purification method of the anti-IL-11 monoclonal antibody according to claim 1, wherein In step S1, the affinity chromatography comprises the following steps: S101. Equilibrate the affinity chromatography column with buffer A; S102. Load the target protein containing the anti-IL-11 monoclonal antibody 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 bed height of the affinity chromatography column is 18 - 22 cm; S103. After the loading is completed, re-equilibrate with buffer A, and then perform at least one washing and elution with 2 - 6 CV of buffer B until the curve of the ultraviolet absorption value drops to a stable state, then stop the washing, and then perform a second washing with buffer A; S104. Elute the affinity chromatography column with buffer C, and collect the protein product for standby after elution; S105. Adjust the pH value of the collected protein product to 3 - 4 using the acid solution, incubate at room temperature for 0.5 - 3 h under this pH condition, and finally adjust the pH of the protein product back to 5 - 8 with 1 M Tris buffer to obtain the crudely purified protein sample; The buffer A is selected from Tris - HCl buffer, HEPES buffer or phosphate buffer, the concentration of the buffer A is 30 - 60 mM, and its pH value is 7 - 8, and its conductivity is 10 - 30 mS / cm; the buffer B is selected from Tris - HCl buffer, HEPES buffer, phosphate buffer, citrate buffer or acetate buffer, the concentration of the buffer B is 30 - 60 mM, and its pH value is 5 - 6; the buffer C is selected from glycine - hydrochloride, citrate or acetate buffer, the concentration of the buffer C is 20 - 200 mM, and its pH is 3 - 4.

7. The purification method of the anti-IL-11 monoclonal antibody according to claim 6, wherein, The filler of the affinity chromatography column is selected from MabSelect Sure LX, Eshmuno A, NMab Pro or AT Protein A Diamond Plus.

8. The purification method of the anti-IL-11 monoclonal antibody according to claim 1, characterized in that, In step S2, the anion - exchange chromatography includes the following steps: S201. Equilibrate the anion - exchange chromatography column with 2 - 6 CV of buffer D; S202. Load the crudely purified protein sample onto the anion - exchange chromatography column at a flow rate of 100 - 200 cm / h, the loading capacity is 20 - 120 mg protein / ml filler, and the column bed height of the anion - exchange chromatography column is 18 - 22 cm; S203. After the loading is completed, equilibrate the anion - exchange chromatography column again with the buffer D, and collect the flow - through fraction for standby; The buffer D is selected from citrate buffer, acetate buffer or phosphate buffer, and its pH value is 6.5 - 7.

5.

9. The purification method of the anti-IL-11 monoclonal antibody according to claim 8, wherein The filler of the anion - exchange chromatography is selected from Capto Q, Capto adhere, Eshmuno Q or NanoGel - 50Q.

10. The purification method of the anti-IL-11 monoclonal antibody according to claim 1, characterized in that, In step S3, the cation - exchange chromatography includes the following steps: S301. Equilibrate the cation - exchange chromatography column with 2 - 6 CV of buffer E; S302. Load the flow - through fraction after secondary purification onto the cation - exchange chromatography column at a flow rate of 100 - 200 cm / h, the loading capacity is 20 - 75 mg protein / ml filler, and the column bed height of the cation - exchange chromatography column is 18 - 22 cm; S303. Elute the cation - exchange chromatography column with eluent F, and after elution, a protein solution of high - purity anti - IL - 11 monoclonal antibody is obtained; The buffer E is selected from citrate buffer, acetate buffer or phosphate buffer, the concentration of the buffer E is 30 - 60 mM, its pH value is 5.0 - 6.0, and its conductivity is 2 - 5 mS / cm; the buffer F includes buffer E and 80 - 150 mM additive, and the additive is NaCl.

11. The purification method of the anti-IL-11 monoclonal antibody according to claim 10, characterized in that, The packing material of the cation exchange chromatography column is Capto SP, Capto MMC, Eshmuno S or NanoGel-50SP.

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