Method for purifying anti-IL-11 monoclonal antibody

Abstract

The invention relates to the field of biological medicines, and particularly provides a purification method of an anti-IL-11 monoclonal antibody, which comprises the following steps: affinity chromatography: preliminarily purifying and concentrating a target protein containing the anti-IL-11 monoclonal antibody, and carrying out virus inactivation on a protein product in an acid solution to obtain a protein sample; anion exchange chromatography: carrying out secondary purification on the protein sample, and collecting flow-through liquid; and cation exchange chromatography: carrying out refined purification on the flow-through liquid to obtain a protein solution of the anti-IL-11 monoclonal antibody. A proper purification process is provided for the anti-IL-11 monoclonal antibody, the purification efficiency and the recovery rate of the anti-IL-11 monoclonal antibody are improved, flow-through liquid collected by anion exchange chromatography directly enters cation exchange chromatography without treatment, sample treatment or solution replacement is not needed in the process, the purification time is saved, and the purification efficiency and the recovery rate of the anti-IL-11 monoclonal antibody are improved. Impurities are prevented from being introduced in the purification process, the purity of purified protein is improved, and high practicability is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for purifying an anti-IL-11 monoclonal antibody. Background Art

[0002] Fibrosis can occur in many organs, such as liver, lung, kidney, retina, intestine, heart and skin, etc. It is the main cause of disability and death in many diseases. The main pathology is the increase of fibrous connective tissue and the decrease of parenchymal cells in organ tissues. Continuous progression can lead to organ structure destruction and functional impairment, and even failure, which seriously threatens human health and life. According to relevant statistics in the United States, nearly 45% of patients who died from various diseases in the country were attributed to tissue fibroproliferative diseases.

[0003] Fibrotic diseases include pulmonary fibrosis, liver fibrosis, cirrhosis, liver disease, renal fibrosis, cardiac fibrosis, asthma, eye diseases and many other diseases. Among them, liver fibrosis is a pathophysiological process, which refers to the abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. Any liver damage will undergo a process of liver fibrosis during the process of liver repair and healing. If the damaging factors cannot be removed for a long time, the fibrosis process will continue for a long time and develop into cirrhosis. There are many causes of liver fibrosis, and the most common ones in clinical practice are viral hepatitis, alcoholic liver disease, fatty liver disease, autoimmune diseases, etc. Non-alcoholic fatty liver disease accounts for a relatively high proportion of the world's cases. If not treated in time, it will deteriorate into non-alcoholic hepatitis, and may even cause liver inflammation, liver cell death, liver fibrosis, and ultimately lead to cirrhosis and liver cancer. In addition, pulmonary fibrosis is a terminal change of a large class of lung diseases characterized by fibroblast proliferation and accumulation of a large amount of extracellular matrix accompanied by inflammatory damage and tissue structure destruction. The cause of most pulmonary fibrosis patients is unknown (idiopathic). This group of diseases is called idiopathic interstitial pneumonia, which is a large category of interstitial lung disease. The most common type of disease with pulmonary fibrosis as the main manifestation among idiopathic interstitial pneumonia is idiopathic pulmonary fibrosis, a serious 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, and as the disease and lung damage worsen, the patient's respiratory function continues to deteriorate. The incidence and mortality of idiopathic pulmonary fibrosis are increasing year by year. The average survival period after diagnosis is only 2.8 years, and the mortality rate is higher than that of most tumors. It is called a "tumor-like disease". At present, there is no particularly effective drug for the treatment of fibrotic diseases of various organs, and the treatment effect is general. Therefore, the development of drugs for the treatment of anti-fibrotic diseases is particularly urgent.

[0004] Interleukin-11 (IL-11) is a hematopoietic cytokine that belongs to the multifunctional interleukin 6 (IL6) family of cytokines and shares the same signal transduction receptor subunit (GP130). This family plays a vital role in the occurrence, development and metastasis of tumors. Studies have found that blocking this pathway can be an effective treatment for a variety of tumor cancers, chronic fibrosis and inflammatory diseases. At present, there are no drugs targeting IL-11 on the market worldwide. Therefore, in order to meet the urgent needs of patients, the process development of anti-IL-11 monoclonal antibodies has important clinical significance.

[0005] At present, in the process of process development of anti-IL-11 monoclonal antibodies, conventional antibody purification methods are used to purify anti-IL-11 monoclonal antibodies, and degradation products and target proteins are often difficult to separate. In addition, the samples are unstable during the process, and the purification efficiency, purity and recovery rate are all low. Therefore, the present invention provides a purification method specifically suitable for anti-IL-11 monoclonal antibodies. Summary of the invention

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

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

[0008] The present invention provides a method for purifying an anti-IL-11 monoclonal antibody, the method comprising:

[0009] S1. Affinity chromatography: preliminary purification and concentration of the target protein containing anti-IL-11 monoclonal antibody, and inactivation of the collected protein product by virus in an acid solution to obtain a crude pure protein sample;

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

[0011] S3. Cation exchange chromatography: After the flow-through liquid is purified, a high-purity protein solution of anti-IL-11 monoclonal antibody is obtained.

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

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

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

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

[0016] A-IV: The amino acid sequence of the heavy chain complementary determining region HCDR1 is shown in SEQ ID No: 1, the amino acid sequence of the heavy chain complementary determining region HCDR2 is shown in SEQ ID No: 14, the amino acid sequence of the heavy chain complementary determining region HCDR3 is shown in SEQ ID No: 15, the amino acid sequence of the light chain complementary determining region LCDR1 is shown in SEQ ID No: 4, the amino acid sequence of the light chain complementary determining region LCDR2 is shown in SEQ ID No: 5, and the amino acid sequence of the light chain complementary determining region LCDR3 is 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 chromatographic fillers for the anti-IL-11 monoclonal antibody provided by the present invention through a continuous three-step purification method, thereby effectively improving the purification efficiency and recovery rate of the anti-IL-11 monoclonal antibody provided by the present invention, wherein the flow-through liquid collected by the anion exchange chromatography can directly enter the cation exchange chromatography without treatment, and no sample treatment or solution replacement is required 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 practicality. Secondly, 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, thereby effectively inhibiting the pro-fibrotic effect of IL-11, inhibiting or preventing the production or proliferation of fibroblasts, and can be effectively used to treat or prevent human fibrotic diseases, inflammation, cancer or autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0019] Figure 2 This is a comparison chart of the affinity of the anti-IL-11 phage monoclonal antibody in the gradient dilution ELISA in Example 3 of the present invention;

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

[0021] Figure 4 This is a denaturing polyacrylamide gel electrophoresis diagram of the mouse antibody molecule in Example 5 of the present invention;

[0022] Figure 5 This is a comparison chart of the binding ability between the mouse antibody molecule and IL-11 in Example 6 of the present invention;

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

[0024] Figure 7 This is a comparison diagram of the inhibition of IL-11 binding to the IL-11RA receptor on the surface of BaF / 3-IL-11RA cells by the mouse antibody in Example 8 of the present invention;

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

[0026] Fig. 9 This is a denaturing polyacrylamide gel electrophoresis diagram of the humanized antibody molecule of Example 14 of the present invention;

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

[0028] Fig.11 This is a comparison diagram of the inhibition of IL-11 binding to 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] Fig.12 This is a comparison diagram of the inhibition of IL-11 binding to the GP130 receptor on the surface of BaF / 3-GP130 cells by the humanized antibody molecule in Example 17 of the present invention;

[0030] Fig.13 This is a comparison diagram of biological activity detection (reporter gene) of the humanized antibody molecules in Example 18 of the present invention;

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

[0032] Fig.15 This is a comparison diagram of the cross-binding experiment between the humanized antibody molecule and IL-11 of different species in Example 20 of the present invention;

[0033] Fig.16 A bar graph showing changes in the ratio of lung to body weight in the mouse pulmonary fibrosis model in Example 21 of the present invention;

[0034] Fig.17 2 are hematoxylin and eosin (HE) staining and Masson's staining images of lung tissue sections in the mouse pulmonary fibrosis model in Example 21 of the present invention;

[0035] Fig.18 A bar graph showing changes in the ratio of heart to body weight in the mouse cardiac fibrosis model in Example 22 of the present invention;

[0036] Fig.19 2 are hematoxylin and eosin (HE) staining and Masson's staining images of cardiac tissue sections in the mouse cardiac fibrosis model in Example 22 of the present invention;

[0037] Fig. 20 A bar graph showing the urine protein content in the kidney of the mouse renal fibrosis model in Example 23 of the present invention;

[0038] Fig.21 The diagram is a hematoxylin and eosin (HE) staining and Masson's staining diagram of kidney tissue sections in the mouse kidney fibrosis model in Example 23 of the present invention;

[0039] Fig. 22A bar graph showing changes in liver weight in the mouse liver fibrosis model in Example 24 of the present invention;

[0040] Fig.23 A bar graph showing changes in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mouse serum in the mouse liver fibrosis model in Example 24 of the present invention;

[0041] Fig.24 2 are hematoxylin and eosin (HE) staining and Masson's staining images of liver tissue sections in the mouse liver fibrosis model in Example 24 of the present invention;

[0042] Fig.25 This is a graph for evaluating the thermal stability of the anti-IL-11 monoclonal antibody HA-IA in Example 25 of the present invention. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below with reference to the following examples.

[0044] Example 1

[0045] The present invention provides a method for purifying an anti-IL-11 monoclonal antibody, the method comprising:

[0046] S1. Affinity chromatography: preliminary purification and concentration of the target protein containing anti-IL-11 monoclonal antibody, and inactivation of the collected protein product by virus in an acid solution to obtain a crude pure protein sample;

[0047] S2, anion exchange chromatography: perform secondary purification on the protein sample and collect the flow-through;

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

[0049] The anti-IL-11 monoclonal antibody comprises three heavy chain complementary determining regions represented by HCDR1, HCDR2 and HCDR3, and three light chain complementary determining regions represented by LCDR1, LCDR2 and LCDR3, respectively. 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 to treat or prevent human fibrotic diseases, inflammation, cancer or autoimmune diseases, wherein fibrotic diseases include but are not limited to fibrosis of the heart, liver, kidney, lung, gallbladder, bladder, stomach, bone marrow, penis, breast, blood vessels, eyes, pancreas, spleen, brain, intestine, muscle or skin; inflammation includes but is not limited to hepatitis, myocarditis, nephritis, pneumonia, cholecystitis, cystitis, gastritis, osteomyelitis, prostatitis, mastitis, pancreatitis, enteritis, arthritis, polymyositis, dermatomyositis or dermatitis; cancer includes but is not limited to leukemia, lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer or bladder cancer; autoimmune diseases include but are not limited to psoriasis, Crohn's disease, primary biliary cirrhosis, systemic lupus erythematosus or multiple sclerosis.

[0052] Example 2 Screening of mouse antibody molecules

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

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

[0055] 1. Experimental animals:

[0056] Species and strains: BALB / c, female, mouse;

[0057] Weight: 18-20g;

[0058] Experimental animal provider: Beijing Huafukang Biotechnology Co., Ltd.

[0059] 2. Immunization: Mice were immunized with human IL-11 as the immunizing antigen (synthesized gene by Nanjing GenScript Biotechnology 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 high titer were taken, and total RNA from mouse spleen cells was extracted using Trizol reagent (purchased from Ambion, catalog number: 15596026). cDNA was obtained by RT-PCR, and PCR amplification was performed using degenerate primers (reference for degenerate primers used: Journal of Immunological Methods 233 (2000) 167-177) with cDNA as template, thereby obtaining the heavy chain variable region gene library (VH) and light chain variable region gene library (VL) of immune mouse antibodies. pScFv-Disb-HS vector was modified by a series of gene cloning methods to transform the vector pComb3 vector (purchased from China Plasmid Vector Strain Cell Line Gene Collection Center) for use in the construction and expression of phage single-chain antibody library. The modified vector was named pScFv-Disb-HS vector, and its plasmid map was obtained as shown in the following figure. Figure 1 As shown, based on this vector, a mouse immune phage antibody library was constructed. The light and heavy chain variable region gene libraries were double-digested and connected to the vector pScFv-Disb-HS that was also digested in steps to construct the pScFv-Disb-HS-VH-VL gene library.

[0062] Step 3: Use IL-11 as antigen to coat the immunotubes, with an antigen coating volume of 5 μg / 500 μL / tube, coat overnight at 4°C, and then use 4% skim milk powder / PBST to block the immunotubes and immune phage antibody library respectively, and block at room temperature for 1 hour. The blocked immune phage antibody library is added to the immunotube for antigen-antibody binding, and the phage input volume is about 10 9 ~10 12 After reacting at room temperature for 1 h, unbound phages were washed away with PBST-PBS, eluted with 0.1 M Glycine-HCl at pH 2.2, and finally neutralized with 1.5 M Tris-HCl at pH 8.8 to a pH of about 7.0.

[0063] Step 4: Infect 10 ml of TG1 bacterial solution grown to the logarithmic phase with the neutralized phages above, place in a 37°C incubator for 30 minutes, take out part of the bacterial solution for gradient dilution, and spread on 2YTAG plates for calculating the phage output. Centrifuge the remaining bacterial solution and discard the supernatant, resuspend the bacterial pellet in a small amount of culture medium, aspirate it and spread it on a 2YTAG large plate to prepare for the next round of screening.

[0064] Step 5: Scrape the infected bacteria from the large plate, inoculate into 2YTAG liquid culture medium, shake to the logarithmic phase, add M13KO7 helper phage for superinfection, culture overnight at 28°C and 220rpm to prepare phage, PEG / NaCl precipitation to purify phage for the next round of screening, and conduct one round of phage library enrichment screening.

[0065] Step 6: Screening of positive clones of IL-11 phage single-chain antibody: After one round of screening, well-separated monoclonal colonies were picked and inoculated into 96-well deep-well plates with 2YTAG liquid medium. They were cultured at 37°C and 220 rpm until the logarithmic growth phase. About 10 10 The helper phage M13KO7 was statically infected at 37°C for 30 minutes. Centrifuge at 4000rpm for 15 minutes, discard the supernatant, resuspend the precipitate with 2YTAK, and culture overnight at 28°C and 220rpm. After centrifugation at 4000rpm and 4°C for 15 minutes, the amplified phage supernatant was aspirated for ELISA identification, and finally four mouse antibody molecules with high affinity were screened, named MA-I, MA-II, MA-III and MA-IV, and the monoclonal antibodies obtained above were sequenced to determine the correct antibody sequence. After sequencing, the sequences of the four 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 Comparison of antibody affinity by gradient dilution ELISA

[0080] The four mouse antibody molecules (MA-I, MA-II, MA-III and MA-IV) obtained in Example 2 were displayed and purified by monoclonal phage, and then the affinity was identified by phage gradient dilution ELISA experiment. The specific method is as follows:

[0081] Coat the IL-11 antigen with a carbonate buffer solution of pH 9.6, 100 ng / well / 100 μL, and coat overnight at 4°C. Wash three times with PBST, dilute the four 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 (purchased from Bio-viewshine, item number: GE27-9421-01) monoclonal antibody diluted with 1% BSA-PBST to the ELISA plate, and let it stand at room temperature for 1 hour. Use the TMB color development kit (purchased from Kangwei Century, item number: CW0050S) for color development at room temperature for 10 minutes. After terminating with 2M H2SO4, read the plate at 450nm / 630nm with the microplate reader, and calculate the corresponding EC50 value. The specific data are as follows:

[0082]

[0083] Through the above data and Figure 2 As shown, the four different mouse antibody molecules screened in Example 2 can all bind to IL-11, which indicates that the monoclonal antibodies provided by the present invention have a high affinity for IL-11.

[0084] Example 4

[0085] Example 4 of the present invention is further defined on the basis of Example 2, that the anti-IL-11 monoclonal antibody further includes a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region is one of SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25 or SEQ ID No: 26; the amino acid sequence of the light chain constant region is as shown in SEQ ID No: 22, and the specific sequence is as follows:

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

[0087] ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL NSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC;

[0088] SEQ ID No: 23 (amino acid sequence of the heavy chain constant region of mouse 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] AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFV NNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK;

[0094] SEQ ID No: 26 (amino acid sequence of the heavy chain constant region of mouse IgG3 type):

[0095] ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGK EFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSW LQGEIFTCSVVHEALHNHHTQKNLSRSPELELNETCAEAQDGELDGLWTTITIFISLFLLSVCYSASVTLFKVKWIFSSVVQVKQTAIPDYRNMIGQGA.

[0096] Example 5 Preparation of mouse antibody molecules

[0097] Example 5 of the present invention is based on Example 4 and preferably defines that the mouse antibody molecule includes a mouse IgG1 type heavy chain constant region (whose amino acid sequence is shown in SEQ ID No: 23) and a mouse C kThe antibody preparation method is as follows:

[0098] 1. The heavy chain VH and light chain VL coding genes of the four antibody molecules screened in Example 2 were cloned into the vector pTSE (such as Figure 3 The preferred heavy chain constant region is a mouse IgG1 type constant region (amino acid sequence is shown in SEQ ID No: 23), and the light chain constant region is a mouse C k chain (amino acid sequence as shown in SEQ ID No: 22), pTSE vector structure as shown in Figure 3 As shown (for the preparation process of pTSE vector, see the specification of CN103525868A, page 3, paragraph

[0019] ).

[0099] 2. HEK293 cells were transiently transfected (purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences, catalog number GNHu43) for antibody expression. Four monoclonal antibodies were purified by protein A affinity column using an AKTA instrument. The protein concentration was determined using a BCA kit (purchased from Beijing Huitian Oriental Technology Co., Ltd., catalog number BCA0020). The protein size was then identified by SDS-PAGE. The results are as follows: Figure 4 As shown, from left to right are non-reduced MA-I, MA-II, MA-III and MA-IV, protein molecular weight Marker 1, protein molecular weight Marker 2, and reduced MA-I, MA-II, MA-III and MA-IV mouse anti-IL-11 monoclonal antibodies. The molecular weight of each band is consistent with the theory.

[0100] Example 6 Binding experiment of mouse antibody molecules and IL-11

[0101] IL-11 antigen was coated with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, and coated overnight at 4°C. Washed five times with 300 μL / well PBST, then added 1% BSA-PBST, blocked at 280 μL / well at 37°C for 1 hour, and added MA-I, MA-II, MA-III and MA-IV mouse antibody molecules of different dilution concentrations. The starting maximum concentration of the four antibody molecules was 5 μg / mL, and each antibody was diluted 5 times, with a total of 8 gradients, and incubated at 37°C for 1 hour. Washed five times with 300 μL / well PBST, then added Goat Anti-Mouse IgG-HRP (purchased from solarbio, item number: SE131) diluted 1:2000 with 1% BSA-PBST, and incubated at 37°C for 1 hour. TMB colorimetric kit was used for color development, 100 μL / well, at room temperature for 8 min, and then 2M H2SO4 was used to terminate the color development. The microplate reader was read at 450nm / 630nm, and the corresponding EC50 value was calculated. The specific data are as follows:

[0102]

[0103] Through the above data and Figure 5 As shown, the four different mouse antibody molecules screened out can bind to IL-11 with high affinity.

[0104] Example 7 Competitive inhibition experiment between mouse antibody and IL-11 receptor protein IL-11RA

[0105] IL-11-Fc was coated with carbonate buffer at pH 9.6, 200 ng / well / 100 μL, overnight at 4°C. Washed five times with 300 μL / well PBST, then blocked at 37°C for 1h with 1% BSA-PBST, 280 μL / well, first added IL-11RA-Fc (IgG4 type) diluted to 0.5 μg / mL with 1% BSA-PBST, 50 μL / well, then added MA-Ⅰ, MA-Ⅱ, MA-Ⅲ and MA-Ⅳ mouse antibodies at different dilution concentrations, 50 μL / well, the starting maximum concentration of the five antibodies was 100 μg / mL, and each antibody was diluted 2-fold, with a total of 13 gradients, and incubated at 37°C for 3h. Wash five times with 300 μL / well PBST, then add Anti-Human IgG4-HRPMouse monoclonal antibody (purchased from Sigma, catalog number: SAB4200770) diluted 1:5000 with 2% BSA-PBST, and incubate at 37°C for 1 hour. TMB colorimetric kit was used for color development, 100 μl / well, and color development was performed at room temperature for 15 minutes, and then 2M H2SO4 was used to terminate the color development. The microplate reader was read at 450nm / 630nm, and the corresponding IC50 value was calculated. The specific data are as follows:

[0106]

[0107] Through the above data and Figure 6 As shown, the four different mouse antibodies screened out can 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 Mouse Antibodies Inhibit the Binding of IL-11 to the IL-11RA Receptor on the Surface of BaF / 3-IL-11RA Cells

[0109] Count the BaF / 3-IL-11RA cell line, take a certain number of cells, centrifuge and resuspend in PBS buffer, adjust the cell density to 1E+6 cells / mL, 100μL / well, and add to the 96-well plate. Dilute the IL-11-mFc ligand protein with PBS, prepare the concentration to 18μg / mL, 50μL / well, and add it to the corresponding position of the 96-well plate containing BaF / 3-IL-11RA cells. After gently mixing, place the 96-well plate at 4℃ and incubate for 1h. PBS gradient dilution of 4 mouse antibody molecules MA-I, MA-II, MA-III and MA-IV, with an initial concentration of 800μg / mL, 3-fold gradient dilution, a total of 10 gradients, 50μL / well, add to the corresponding position of the 96-well plate containing BaF / 3-IL-11RA cells and IL-11-mFc ligand protein mixture. After mixing evenly, place it at 4℃ and incubate for 2h. After the incubation, centrifuge at 3000rpm, 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 30min, centrifuge at 3000rpm, wash once with PBS buffer, resuspend in 100μL PBS buffer, and detect on a flow cytometer to collect the fluorescence signal in the FL1-A channel. Draw a dose-effect curve and calculate the corresponding IC50 value. The specific data are as follows:

[0110]

[0111] Through the above data and Figure 7 It can be seen that the four different mouse candidate molecules screened out can effectively inhibit the binding of IL-11 ligand protein to the cell surface IL-11RA receptor.

[0112] Example 9 Mouse Antibodies Inhibit TIMP-1 Secretion by Embryonic Lung Fibroblasts MRC-5

[0113] Embryonic lung fibroblasts MRC-5 were digested with trypsin and 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 / well, and added to a 96-well plate. MEM complete medium was used to dilute the IL-11-mFc ligand protein to a concentration of 16 μg / mL, and 50 μL / well was added to the corresponding 96-well plate. MEM complete medium was used to gradient dilute the four mouse antibody molecules MA-I, MA-II, MA-III, and MA-IV to an initial concentration of 40 μg / mL, 2-fold gradient dilution, a total of 8 gradients, 50 μL / well, added to the 96-well plate containing the cell suspension and IL-11-mFc ligand protein suspension, gently mixed, and placed in a 37°C CO2 incubator for overnight incubation for about 20 hours. The cell culture supernatant was taken for detection using an ELISA kit for TIMP-1 (purchased from Ekosai Biotechnology Co., Ltd., catalog number EH021-96).

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

[0115]

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

[0117] Example 10

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

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

[0120] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

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

[0122] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0123] SEQ ID No: 29 (human IgG4 type heavy chain constant region amino acid sequence):

[0124] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

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

[0126] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C.

[0127] Example 11 Preparation of chimeric antibody molecules

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

[0129] Specific preparation method:

[0130] The heavy chain variable region VH (SEQ ID No: 16) of the mouse antibody molecules MA-I and MA-II obtained by screening the immune phage antibody library in Example 2, the light chain variable region VL gene of MA-I (SEQ ID No: 17), and the light chain variable region VL gene of MA-II (SEQ ID No: 18) were cloned into the vector pTSE (such as Figure 3 The heavy chain constant region is 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). HEK293E cells (purchased from: Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number: GNHu43) were transiently transfected to express antibodies and obtain chimeric antibodies CA-I and CA-II.

[0131] Example 12 Humanization of murine antibody molecules

[0132] First, the sequences of the mouse antibody molecules MA-I and MA-II in Example 2 were selected and compared with the human antibody germline database (v-base) to find the human antibody light and heavy chain germlines with higher homology as candidate sequences, and then the sequences of the CDRs of the mouse antibody molecules MA-I and MA-II were transplanted to the human candidate sequences for homology modeling. Then, the key framework amino acid residues that may play an important role in maintaining the CDR ring structure were calculated by three-dimensional structural simulation to design the back mutation of the humanized antibody. The light and heavy chain variable region sequences of the designed humanized antibodies containing back mutations were optimized and synthesized by Nanjing GenScript Biotechnology Co., Ltd., respectively, and then connected to the transient expression vector, and the light and heavy chain combinations obtained by humanization were analyzed, among which MA-I obtained the following humanized anti-IL-11 monoclonal antibody molecules: HA-IA, HA-IB, HA-IC, HA-ID; MA-II obtained the following humanized antibody molecules: HA-II-A, HA-II-B, HA-II-C, HA-II-D; the 8 monoclonal antibody sequences screened above are as follows:

[0133]

[0134] Specifically, SEQ ID No: 31 (amino acid sequence of the heavy chain variable region of HA-IA, HA-IC, 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-IA):

[0137] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;

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

[0139] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVST ISDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVS SPEAMDYWGQGTLVTVSS;

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

[0141] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;

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

[0143] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;

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

[0145] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAMYYCARDGGYVS SPEAMDYWGQGTSVTVSS;

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

[0147] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;

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

[0149] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;

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

[0151] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;

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

[0153] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGTVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPWTFGGGTKVEIK.

[0154] Example 13

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

[0156] The specific sequence of the above human antibody constant region is the same as that in Example 10.

[0157] Example 14 Preparation of humanized antibody molecules

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

[0159] The heavy chain VH and light chain VL coding genes of the eight humanized anti-IL-11 monoclonal antibody molecules HA-IA, HA-IB, HA-IC, HA-ID, HA-II-A, HA-II-B, HA-II-C, and HA-II-D obtained by humanization in Example 12 were cloned into the vector pTSE (such as Figure 3 The heavy chain constant region is 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 two chimeric antibodies CA-I and CA-II obtained in Example 11 and the eight humanized antibody molecules HA-IA, HA-IB, HA-IC, HA-ID, HA-II-A, HA-II-B, HA-II-C, and HA-II-D obtained in Example 12 were transiently transfected into HEK293 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, with the catalog number GNHu43) for antibody expression, and monoclonal antibodies were obtained by purification through a protein A affinity column using an AKTA instrument. The protein concentration was determined using a BCA kit (purchased from Beijing Huitian Oriental Technology Co., Ltd., with the catalog number BCA0020), and the protein size was then identified by SDS-PAGE. The results are as follows: Fig. 9 As shown, from left to right are the non-reduced protein molecular weights HA-IA, HA-IB, HA-IC, HA-ID, the chimeric antibody CA-I prepared in Example 11, the reduced protein molecular weight Marker, HA-II-A, HA-II-B, HA-II-C, HA-II-D, and the chimeric antibody CA-II. The molecular weight of each band is consistent with the theory.

[0161] Example 15 Humanized antibody molecule and IL-11 binding experiment

[0162] IL-11 antigen was coated with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, overnight at 4°C. Wash five times with 300 μL / well PBST, and then add 1% BSA-PBST 280 μL / well to block for 1 hour at 37°C. Humanized antibodies HA-IA, HA-IB, HA-IC, HA-ID, HA-II-A, HA-II-B, HA-II-C, HA-II-D and chimeric antibodies CA-I and CA-II prepared in Example 11 were diluted with 1% BSA-PBST. The initial concentration of humanized antibodies was 10 μg / mL, and 8 gradients were diluted 5 times. Incubate at 37°C for 1 hour. Wash five times with 300 μL / well PBST, then add goat anti Human IgG Fab HRP (purchased from Invitrogen, catalog number: 31482) diluted 1:5000 with 1% BSA-PBST, and incubate at 37°C for 1 hour. TMB colorimetric kit was used for color development, 100 μL / well, color development at room temperature for 5 minutes, and then 2M H2SO4 was used to stop the color development. The microplate reader was read at 450nm / 630nm, and the corresponding EC50 value was calculated. The specific data are as follows:

[0163]

[0164] Through the above data and experimental results, Fig.10 As shown, 8 different humanized antibody molecules can bind to IL-11, and the EC50 values ​​of HA-IA, HA-IB, HA-IC, and HA-ID humanized antibody molecules are close to those of the chimeric antibody CA-I, and the EC50 values ​​of HA-II-A, HA-II-B, HA-II-C, and HA-II-D humanized antibody molecules are close to those of the chimeric antibody CA-II, indicating that the humanized antibody molecules retain the high binding ability of the mouse parent antibodies MA-I and MA-II to IL-11.

[0165] Example 16 Humanized antibody molecules inhibit the binding of IL-11 to the IL-11RA receptor on the surface of BaF / 3-IL-11RA cells

[0166] Four humanized antibody molecules HA-IA, HA-IB, HA-IC and HA-ID with better binding activity at the protein level were selected for cell activity evaluation experiments. BaF / 3-IL-11RA cell lines were counted, a certain number of cells were taken, centrifuged and resuspended with PBS buffer, the cell density was adjusted to 1E+6 cells / mL, 100μL / well, and added to a 96-well plate. PBS was used to dilute the IL-11-mFc ligand protein to a concentration of 18μg / mL, 50μL / well, and added to the corresponding position 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 1h. PBS buffer gradient dilution of four humanized antibody molecules HA-IA, HA-IB, HA-IC and HA-ID, prepared an initial concentration of 800μg / mL, 3-fold gradient dilution, a total of 10 gradients, 50μL / well, added to the corresponding position of the 96-well plate containing BaF / 3-IL-11RA cells and IL-11-mFc ligand protein mixture. After mixing evenly, incubate at 4°C for 2h. After the incubation, centrifuge the PBS buffer at 3000rpm to wash the cells once, 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 30min, centrifuge at 3000rpm to wash once, resuspend in 100μL PBS, and detect on the flow cytometer to collect the fluorescence signal in the FL1-A channel. Draw a dose-effect curve and calculate the corresponding IC50 value. The specific data are as follows:

[0167]

[0168] Through the above data and Fig.11 It can be seen that the four humanized candidate molecules screened out can all inhibit the binding of IL-11 ligand protein to the IL-11RA receptor on the surface of BaF / 3-IL-11RA cells.

[0169] Example 17 Humanized antibody molecules inhibit the binding of IL-11 to the GP130 receptor on the surface of BaF / 3-GP130 cells

[0170] BaF / 3-GP130 cell lines were counted, a certain number of cells were taken, centrifuged and resuspended in PBS buffer, the cell density was adjusted to 1E+6 cells / mL, 100 μL / well, and added to a 96-well plate. IL-11-mFc ligand protein was diluted with PBS to a concentration of 12 μg / mL, 50 μL / well, and added to the corresponding position of the 96-well plate containing BaF / 3-GP130 cells. After gently mixing, the 96-well plate was placed at 4°C and incubated for 1 hour. 4 human antibody molecules HA-IA, HA-IB, HA-IC and HA-ID were gradiently diluted with PBS buffer to prepare an initial concentration of 2000 μg / mL, 2-fold gradient dilution, a total of 10 gradients, 50 μL / well, and added to the corresponding position of the 96-well plate containing BaF / 3-GP130 cells and IL-11-mFc ligand protein. After mixing evenly, the 96-well plate was incubated at 4°C for 2 hours. After the incubation, wash the cells once with PBS buffer by centrifugation at 3000rpm, 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, 100μL / well, and incubate at 4℃ for 30min. Wash once with PBS buffer by centrifugation at 3000rpm, resuspend the cells in 100μL / well PBS buffer, detect on the flow cytometer, and collect the fluorescence signal in the FL1-A channel. Draw the dose-effect curve and calculate the corresponding IC50 value. The specific data are as follows:

[0171]

[0172] Through the above data and Fig.12 It can be seen that the four humanized candidate molecules screened out can all block the binding of IL-11 ligand protein to the GP130 receptor on the surface of BaF / 3-GP130 cells.

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

[0174] The BaF / 3-IL-11RA-GP130-STAT3-Luc engineering cell line was counted, and the cell density was adjusted to 2E+6 cells / mL using sample diluent (its ingredients include 90% IMDM, 10% FBS, 10ng / mL mouseIL-3). After gently mixing, the cell solution was added to a 96-well plate at 50μL / well. The four humanized antibody molecules HA-IA, HA-IB, HA-IC and HA-ID were diluted to an initial concentration of 200μg / ml using sample diluent, 5-fold gradient dilution, a total of 10 gradients, 100μL / well, and added to the corresponding position of the 96-well plate containing the engineering cell line, and two replicates were set for each sample concentration. The sample diluent was used to prepare IL-11 protein at a concentration of 10μg / mL, 50μL / well, and added to the 96-well plate containing the engineering cell line and humanized antibody molecules. The cell culture plate was gently mixed and placed in a 37℃ CO2 incubator for 6h. After centrifugation, discard the supernatant, add lysis buffer, add 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 minutes, read the fluorescence value on a microplate reader, and calculate the corresponding IC50 value. The specific data are as follows:

[0175]

[0176] Through the above data and Fig.13 As shown, the four humanized antibody molecules screened out can block the binding of IL-11 to IL-11RA and GP130 receptors, inhibiting the conduction of signal pathways.

[0177] Example 19 Humanized antibody molecules inhibit the secretion of TIMP-1 by embryonic lung fibroblasts MRC-5

[0178] Embryonic lung fibroblasts MRC-5 were digested with trypsin and counted, a certain number of cells were taken, and the cells were resuspended in MEM complete medium after centrifugation, and the cell density was adjusted to 2E+5 cells / mL, 100 μL / well, and added to a 96-well plate. MEM complete medium was used to dilute the IL-11-mFc ligand protein to a concentration of 16 μg / mL, 50 μL / well, and added to the corresponding 96-well plate. MEM complete medium was used to gradiently dilute 4 humanized antibody molecules HA-IA, HA-IB, HA-IC and HA-ID, with an initial concentration of 40 μg / mL, 3-fold gradient dilution, a total of 8 gradients, 50 μL / well, and added to a 96-well plate containing a cell suspension and an IL-11-mFc ligand protein suspension, gently mixed, placed in a 37°C CO2 incubator and incubated overnight, about 20 hours, and the cell culture supernatant was taken and detected using an ELISA kit of TIMP-1 (the method is the same as in Example 9). The microplate reader reads at 450nm and calculates the corresponding IC50 value. The specific data are as follows:

[0179]

[0180] Through the above data and Fig.14 It can be seen that the four humanized antibody molecules screened out can effectively inhibit the release of TIMP-1 by human embryonic lung fibroblasts MRC-5 stimulated by IL-11 ligand protein.

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

[0182] The humanized antibody molecule HA-IA with better protein level and functional detection activity was selected for cross-binding detection with IL-11 of different species. Human IL-11, mouse IL-11 (purchased from Beijing Sino Biological Technology Co., Ltd., catalog number: 50117-MNCE), rat IL-11 (purchased from Kanglang Bio, catalog number: KL40001Ra), and cynomolgus monkey IL-11 (purchased from Sino Biological, catalog number: 90925-CNCE) were coated with pH 9.6 carbonate buffer at 100 ng / well / 100 μL, and coated 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 1h. Humanized antibody HA-IA was diluted with 1% BSA-PBST, the initial concentration was 50μg / mL, 5-fold gradient dilution, a total of 9 gradients, two replicates for each gradient, 100μL / well was added to a 96-well plate, and incubated at 37°C for 1h. Wash five times with 300μL / well PBST, dilute goat anti Human IgG Fab HRP (purchased from invitrogen, catalog number: 31482) with 1% BSA-PBST, the working solution concentration was 1:5000, 100μL / well was added to a 96-well plate, and incubated at 37°C for 1h. Wash five times with 300μL / well PBST, color with TMB color development kit, 100μL / well, color at room temperature in the dark for 5min, and then stop color development with 2M H2SO4. The microplate reader reads at 450nm / 630nm, and the corresponding EC50 value is calculated. The specific data are as follows:

[0183]

[0184]

[0185] Through the above data and Fig.15 As shown, the humanized antibody molecule HA-IA can bind to human IL-11, mouse IL-11, rat IL-11, and cynomolgus monkey IL-11 with high affinity.

[0186] Example 21 Experimental study on the therapeutic efficacy of anti-IL-11 monoclonal antibody on pulmonary fibrosis

[0187] The therapeutic effect of anti-IL-11 monoclonal antibody HA-IA on pulmonary fibrosis was studied using bleomycin (bLF) model.

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

[0189] Number, gender and age of mice: 6 / group, male, 6-8 weeks;

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

[0191] The drug administration group was injected with HA-IA antibody molecules twice a week for 4 weeks.

[0192] The animal body weight was measured once a week, and the animals were observed for any abnormalities. Organ weight detection: 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 sliced ​​and stained with hematoxylin and eosin (HE) and Masson's staining to observe the degree of lung fibrosis.

[0193] The results are as follows Fig.16 As shown in Figure 2, after administration of HA-IA antibody molecules, the ratio of lung to body weight of mice in the administration group was significantly smaller than that in the control group; Fig.17 As shown, compared with the control group, the lung sections of the drug-treated group showed a significant reduction in pulmonary fibrosis, thus indicating that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the occurrence of pulmonary fibrosis.

[0194] Example 22 Experimental study on the therapeutic efficacy of anti-IL-11 monoclonal antibody on cardiac fibrosis

[0195] The therapeutic effect of anti-IL-11 monoclonal antibody HA-IA on cardiac fibrosis was studied using isoproterenol modeling.

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

[0197] Number, gender and age of mice: 6 / group, male, 6-8 weeks;

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

[0199] The drug-treated group was injected with HA-IA antibody molecules twice a week for 4 weeks.

[0200] The animal body weight was measured once a week and the animals were observed for any abnormalities; organ weight detection: the heart was collected, the heart weight was measured, and the heart-body weight ratio was calculated; cardiac pathology detection: the heart was sectioned and the degree of cardiac fibrosis was observed using hematoxylin and eosin (HE) and Masson staining.

[0201] The results are as follows Fig.18 As shown in Figure 2, the heart-to-body weight ratio of mice in the drug-treated group was significantly smaller than that in the control group; Fig.19 As shown, compared with the control group, the heart sections of the drug-treated group showed a significant reduction in cardiac fibrosis, thus indicating that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the occurrence of cardiac fibrosis.

[0202] Example 23 Experimental study on the therapeutic efficacy of anti-IL-11 monoclonal antibody on renal fibrosis

[0203] The therapeutic effect of anti-IL-11 monoclonal antibody HA-IA on renal fibrosis was studied using doxorubicin (dKF) modeling.

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

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

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

[0207] The drug-treated group was injected with HA-IA antibody molecules twice a week for 4 weeks.

[0208] The animal body weight was measured once a week and the animals were observed for any abnormalities; organ weight detection: the heart was collected, the kidney weight was measured, and the urine protein content was detected; renal pathology detection: kidney sections were sliced ​​and the degree of renal fibrosis was observed using hematoxylin and eosin (HE) and Masson staining.

[0209] The results are as follows Fig. 20 As shown, compared with the control group, the urine protein content in the kidneys of the mice in the drug-treated group was significantly lower than that in the control group; Fig.21 As shown, compared with the control group, the kidney sections of the drug-treated group showed a significant reduction in renal fibrosis, which indicates that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the occurrence of renal fibrosis.

[0210] Example 24 Experimental study on the therapeutic efficacy of anti-IL-11 monoclonal antibody on liver fibrosis

[0211] CCl4 modeling was used to study the therapeutic effect of anti-IL-11 monoclonal antibody HA-IA on liver fibrosis.

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

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

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

[0215] The drug-treated group was injected with HA-IA antibody molecules twice a week for 4 weeks.

[0216] Body weight monitoring: The animal body weight was measured once a week, and the animals were observed for any abnormalities. Liver pathology detection: The liver was sectioned and the degree of liver fibrosis was observed using hematoxylin and eosin (HE) and Masson staining. Organ weight detection: The kidneys were collected, the liver weight was measured, and HE staining was performed. Serum detection: Serum was collected to detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum.

[0217] The results are as follows Fig. 22 As shown in Figure 2, the liver weight of mice in the drug-treated group was significantly lower than that in the control group; Fig.23 As shown in Figure 2, the levels of ALT and AST in the serum of mice in the drug-treated group were significantly lower than those in the control group. Fig.24 As shown, compared with the control group, the liver sections of the drug-treated group showed a significant reduction in liver fibrosis, which indicates that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the occurrence of liver fibrosis.

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

[0219] The thermal stability of the anti-IL-11 monoclonal antibody HA-IA was evaluated using a multifunctional protein thermal stability analysis system (purchased from Unchained Labs). The conformational changes of the protein were detected by monitoring the changes in the intrinsic fluorescence of the protein with temperature (starting from 25°C and heating to 95°C at a rate of 0.3°C / min), thereby determining the melting temperature Tm of the protein and evaluating 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 (characterized by Tagg) was determined by static light scattering. The results are shown in the following table and attached. Fig.25 shown.

[0220]

[0221] The melting temperature Tm of anti-IL-11 monoclonal antibody HA-IA was 79.5℃, and the average Tagg was 75.9℃, showing good conformational stability and colloidal stability.

[0222] Embodiment 26

[0223] The anti-IL-11 monoclonal antibody HA-IA obtained in the above example was purified, and the specific purification method was as follows:

[0224] S1. Affinity chromatography: S101. Balance the affinity chromatography column with buffer A. The filler of the affinity chromatography column is MabSelect Sure LX. Buffer A is 30 mM HEPES buffer with a pH of 7 and a conductivity of 10 mS / cm. S102. Load the target protein containing anti-IL-11 monoclonal antibody HA-IA onto the affinity chromatography column at a flow rate of 100 cm / h. The loading capacity is 20 mg protein / ml filler. The column bed height of the affinity chromatography column is 18 cm. S103. After loading, rebalance with buffer A, and then rinse at least once with 2 CV of buffer B until the curve of the UV absorption value drops to a stable state, then stop rinsing. , and then use buffer A for the second elution, buffer B is 30mM HEPES buffer, and its pH value is 5; S104, use buffer C to elute the affinity chromatography column, and collect the protein product after elution for use, buffer C is 20mM glycine-hydrochloride, and its pH value is 3; S105, use acid solution to adjust the pH value of the collected protein product to 3, and incubate at room temperature for 0.5h under this pH condition, and finally use 1M Tris buffer to adjust the pH of the protein product to 5 to obtain a crude and pure protein sample; the acid solution is citric acid.

[0225] S2. Anion exchange chromatography: S201. Use 2CV of buffer D to balance the anion exchange chromatography column. The filler of the anion exchange chromatography is Capto Q. The buffer D is a citric acid buffer with a pH value of 6.5. S202. Load the crude protein sample onto the anion exchange chromatography column at a flow rate of 100 cm / h. The loading capacity is 20 mg protein / ml filler. The column bed height of the anion exchange chromatography column is 18 cm. S203. After loading, use buffer D to balance the anion exchange chromatography column again, collect the flow-through liquid, and set aside.

[0226] S3. Cation exchange chromatography: S301. Use 2CV of buffer E to balance the cation exchange chromatography column. The filler of the cation exchange chromatography column is Capto SP, and buffer E is 30mM citric acid buffer with a pH value of 5.0 and a conductivity of 2mS / cm. S302. Load the flow-through after the secondary purification onto the cation exchange chromatography column at a flow rate of 100cm / h. The loading capacity is 20mg protein / ml filler. The column bed height of the cation exchange chromatography column is 18cm. S303. Use eluent F to elute the cation exchange chromatography column. After elution, a high-purity protein solution of anti-IL-11 monoclonal antibody is obtained. Buffer F is 30mM citric acid buffer and 80mM NaCl with a pH value of 5.0.

[0227] Embodiment 27

[0228] The anti-IL-11 monoclonal antibody HA-IA obtained in the above example was purified, and the specific purification method was as follows:

[0229] S1. Affinity chromatography: S101. Equilibrate the affinity chromatography column with buffer A. The filler 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. In this embodiment, it is preferably 150 mM NaCl. The pH value of buffer A is 7.4 and its conductivity is 18 mS / cm. S102. Load the target protein containing anti-IL-11 monoclonal antibody HA-IA onto the affinity chromatography column at a flow rate of 150 cm / h. The loading capacity is 55 mg protein / ml filler. The column bed height of the affinity chromatography column is 20 cm. S103. After loading, re-equilibrate with buffer A, and then elute at least once with 4 CV of buffer B until the curve of the ultraviolet absorption value drops to a stable state, stop rinsing, and then use buffer A Perform a second elution, buffer B is 50mM acetate buffer, and its pH value is 5.5; S104, use buffer C to elute the affinity chromatography column, and collect the protein product after elution for use, buffer C is 50mM acetate buffer, and its pH value is 3.5; S105, use an acid solution to adjust the pH value of the collected protein product to 3.4-3.8, and incubate at room temperature for 2h under this pH condition, and finally use 1M Tris buffer to adjust the pH of the protein product to 6.2 to obtain a crude and pure protein sample; the acid solution is citric acid.

[0230] S2. Anion exchange chromatography: S201. Use 4CV of buffer D to balance the anion exchange chromatography column. The filler of the anion exchange chromatography is NanoGel-50Q, and the buffer D is a phosphate buffer with a pH value of 7; S202. Load the crude and purified protein sample onto the anion exchange chromatography column at a flow rate of 150 cm / h, with a loading capacity of 70 mg protein / ml filler, and the column bed height of the anion exchange chromatography column is 20 cm; S203. After the loading is completed, use buffer D to balance the anion exchange chromatography column again, collect the flow-through liquid, and set aside;

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

[0232] Embodiment 28

[0233] The anti-IL-11 monoclonal antibody HA-IA obtained in the above example was purified, and the specific purification method was as follows:

[0234] S1. Affinity chromatography: S101. Balance the affinity chromatography column with buffer A. The filler of the affinity chromatography column is Eshmuno A, which can also be replaced by NMab Pro. Buffer A is 60 mM phosphate buffer, and its pH value is 8, and its conductivity is 30 mS / cm; S102. Load the target protein containing anti-IL-11 monoclonal antibody onto the affinity chromatography column at a flow rate of 300 cm / h, the loading capacity is 60 mg protein / ml filler, and the column bed height of the affinity chromatography column is 22 cm; S103. After the loading is completed, re-balance with buffer A, and then elute with 6CV of buffer B at least once until the curve of the ultraviolet absorption value drops to a stable state, stop rinsing, and then use buffer A for the second The first elution, buffer B is 60mM Tris-HCl buffer, which can also be replaced by phosphate buffer or citrate buffer, and its pH value is 6; S104, the affinity chromatography column is eluted with buffer C, and the protein product is collected after elution for later use, buffer C is 200mM citrate, and its pH is 4; S105, the pH value of the collected protein product is adjusted to 5 with an acid solution, and incubated at room temperature for 3h under this pH condition, and finally the pH of the protein product is adjusted back to 8 with 1M Tris buffer to obtain a crude and pure protein sample; the acid solution is hydrochloric acid.

[0235] S2. Anion exchange chromatography: S201. Use 6CV of buffer D to balance the anion exchange chromatography column. The filler of the anion exchange chromatography is Capto adhere, which can also be replaced by Eshmuno Q. Buffer D is an acetate buffer with a pH value of 7.5. S202. Load the crude protein sample onto the anion exchange chromatography column at a flow rate of 200 cm / h. The loading capacity is 120 mg protein / ml filler. The column bed height of the anion exchange chromatography column is 22 cm. S203. After the loading is completed, use buffer D to balance the anion exchange chromatography column again, collect the flow-through liquid, and set aside.

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

[0237] Embodiment 29

[0238] Example 29 of the present invention is based on Example 27, and further defines that in S105 of the method for purifying anti-IL-11 monoclonal antibodies, the acid solution in the inactivation treatment process is preferably acetic acid.

[0239] Examples 30-32

[0240] Based on Example 29, Examples 30-32 of the present invention further define step S105 in the purification method of anti-IL-11 monoclonal antibodies, using 1M Tris buffer to adjust the pH of the protein product to the following pH and conductivity range to obtain a crude and pure protein sample. Other methods are the same as Example 27, and the specific data are as follows.

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

[0242] Examples 33-35

[0243] Based on Example 32, Examples 33-35 of the present invention further define the eluent F in step S303 in the purification method of anti-IL-11 monoclonal antibodies, and other methods and parameters are the same as those in Example 27, as follows.

[0244]

[0245] Comparative Example 1

[0246] The purification method provided in Control Example 1 is based on Example 35, except that the anion exchange chromatography in step S2 and the cation exchange chromatography in step S3 are interchanged in order. Other methods and parameters are the same as those in Example 35.

[0247] Comparative Example 2

[0248] Comparative Example 2 of the present invention uses the classic three-step purification process of monoclonal antibodies on the basis of Example 35 to purify the anti-IL-11 monoclonal antibody HA-IA, and the fillers used in the three-step chromatography are all produced by Cytiva. The filler of the affinity chromatography column in the first step is MabSelectSuRe LX, the filler of the anion exchange chromatography column in the second step is Capto Q, and the filler of the cation exchange chromatography column in the third step is Capto SP ImpAct. Other methods and parameters are the same as those in Example 35.

[0249] Experiment 1: Physical and chemical testing of anti-IL-11 monoclonal antibodies and related impurity testing

[0250] The anti-IL-11 monoclonal antibody HA-IA obtained by the purification method provided by the above-mentioned embodiment and control example of the present invention was tested for purity by gel chromatography technology, and the content of aggregates, monomers and degradation products of the sample during the purification process was analyzed; in addition, the acid-base peak content of the charge isomer was analyzed by ion chromatography technology, and the content of process-related impurities was detected by a dedicated kit. At the same time, the total recovery rate was calculated by the following formula: total recovery rate = affinity chromatography protein yield (%) * anion exchange chromatography protein yield (%) * cation exchange chromatography protein yield (%), and the specific data are as follows:

[0251]

[0252] It can be concluded from the above experimental data that the anti-IL-11 monoclonal antibody purified by the purification method provided in the above embodiments of the present invention has a SEC purity of more than 99%, and the total protein recovery rate can reach more than 75%; the anti-IL-11 monoclonal antibody stock solution obtained by the purification method provided in Example 35 has the highest purity and yield, 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] Example 27 Compared with Examples 26 and 28, the components and contents of the filler of the chromatography column and each buffer provided in Example 27 are more in line with the purification process conditions of the anti-IL-11 monoclonal antibody HA-IA.

[0254] In Example 29, compared with Example 27, 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] As can be seen from Examples 30-32, in step S105, the pH of the protein product is adjusted back to 7.0 using 1 M Tris buffer, and the conductivity is 4 mS / cm, which can significantly improve the total recovery rate of the anti-IL-11 monoclonal antibody HA-IA protein.

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

[0257] In Example 35, compared with Example 1, the order of the anion exchange chromatography in step S2 and the cation exchange chromatography in step S3 is interchanged, which is not conducive to the purification of the anti-IL-11 monoclonal antibody HA-IA. The optimal three-step purification process for purifying the anti-IL-11 monoclonal antibody HA-IA provided in Example 35 is affinity chromatography, anion exchange chromatography and cation exchange chromatography in sequence.

[0258] Compared with Example 2, Example 35 only differs in the filler of the chromatography column. The purification effect of the filler provided in Example 35 is significantly higher than that of Control Example 2, with a high total recovery rate and lower cost, and 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-IA is more suitable for the filler provided in Example 35; therefore, the preferred affinity chromatography of the present invention is AT Protein A Diamond Plus. As a filler, AT Protein A Diamond Plus has excellent binding specificity, alkali resistance and pressure-flow rate characteristics, lower ligand shedding, can capture antibodies in the cell supernatant, and remove most impurities. In addition, it can significantly increase the loading capacity and is an alkali-resistant Protein. A affinity filler can achieve more efficient cleaning and sterilization, avoid cross contamination, and increase service life; the filler of the anion exchange chromatography column is preferably NanoGel-50Q, and the filler of the cation exchange chromatography column is preferably NanoGel-50SP. The NanoGel-50SP chromatography adopts a combined elution mode, that is, when the sample is added, the target protein flows through and the impurities are adsorbed on the chromatography column, which effectively achieves the purpose of purification. NanoGel-50Q and NanoGel-50SP are rigid polymer matrix monodisperse matrices with uniform particles, smaller particle size, high resolution and good separation effect. The combination of the above different chromatography column fillers can not only effectively remove process-related impurities such as HCP, DNA, and Protein A, but also can adsorb product-related impurities such as aggregates and product fragments, thereby improving the purity of the product, ensuring that the purified product meets the quality requirements, and significantly reducing the cost of the purification process.

[0259] Experiment 2: Binding activity and biological activity detection of anti-IL-11 monoclonal antibodies

[0260] For the anti-IL-11 monoclonal antibody HA-IA purified by the purification method provided in the above embodiments of the present invention and comparative examples 1-2, the present invention uses ELISA to analyze the specific binding ability of the purified anti-IL-11 monoclonal antibody HA-IA to IL-11 to evaluate the activity of the purified antibody. The test results are as follows:

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

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

[0263] The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that are the same or similar to those of the present application fall within the protection scope of the present invention.

Claims

1. A method for purifying an anti-IL-11 monoclonal antibody, characterized in that: The method comprises: S1, affinity chromatography: preliminarily purifying and concentrating the target protein containing anti-IL-11 monoclonal antibody, and inactivating the collected protein product in an acid solution to obtain a crude and pure protein sample; S2, anion exchange chromatography: performing secondary purification on the protein sample and collecting the flow-through liquid; S3, cation exchange chromatography: after purifying the flow-through liquid, a high-purity protein solution of anti-IL-11 monoclonal antibody is obtained.

2. The method for purifying an anti-IL-11 monoclonal antibody according to claim 1, characterized in that: The anti-IL-11 monoclonal antibody comprises three heavy chain complementary determining regions represented by HCDR1, HCDR2 and HCDR3, respectively, and three light chain complementary determining regions represented by LCDR1, LCDR2 and LCDR3, respectively, and the anti-IL-11 monoclonal antibody is selected from any one of the following: AI: the amino acid sequence of the heavy chain complementary determining region HCDR1 is shown as SEQ ID No: 1, the amino acid sequence of the heavy chain complementary determining region HCDR2 is shown as SEQ ID No: 2, the amino acid sequence of the heavy chain complementary determining region HCDR3 is shown as SEQ ID No: 3, the amino acid sequence of the light chain complementary determining region LCDR1 is shown as SEQ ID No: 4, the amino acid sequence of the light chain complementary determining region LCDR2 is shown as SEQ ID No: 5, and the amino acid sequence of the light chain complementary determining region LCDR3 is shown as SEQ ID No: 6; A-II: the amino acid sequence of the heavy chain complementary determining region HCDR1 is shown in SEQ ID No: 1, the amino acid sequence of the heavy chain complementary determining region HCDR2 is shown in SEQ ID No: 2, the amino acid sequence of the heavy chain complementary determining region HCDR3 is shown in SEQ ID No: 3, the amino acid sequence of the light chain complementary determining region LCDR1 is shown in SEQ ID No: 4, the amino acid sequence of the light chain complementary determining region LCDR2 is shown in SEQ ID No: 5, and the amino acid sequence of the light chain complementary determining region LCDR3 is shown in SEQ ID No: 7; A-III: the amino acid sequence of the heavy chain complementary determining region HCDR1 is shown in SEQ ID No: 8, the amino acid sequence of the heavy chain complementary determining region HCDR2 is shown in SEQ ID No: 9, the amino acid sequence of the heavy chain complementary determining region HCDR3 is shown in SEQ ID No: 10, the amino acid sequence of the light chain complementary determining region LCDR1 is shown in SEQ ID No: 11, the amino acid sequence of the light chain complementary determining region LCDR2 is shown in SEQ ID No: 12, and the amino acid sequence of the light chain complementary determining region LCDR3 is shown in SEQ ID No: 13; A-IV: The amino acid sequence of the heavy chain complementary determining region HCDR1 is shown in SEQ ID No: 1, the amino acid sequence of the heavy chain complementary determining region HCDR2 is shown in SEQ ID No: 14, the amino acid sequence of the heavy chain complementary determining region HCDR3 is shown in SEQ ID No: 15, the amino acid sequence of the light chain complementary determining region LCDR1 is shown in SEQ ID No: 4, the amino acid sequence of the light chain complementary determining region LCDR2 is shown in SEQ ID No: 5, and the amino acid sequence of the light chain complementary determining region LCDR3 is shown in SEQ ID No:

6.

3. The method for purifying an anti-IL-11 monoclonal antibody according to claim 2, 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: MA-Ⅰ: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 17; MA-Ⅱ: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 18; MA-III: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 19, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 20; MA-IV: The amino acid sequence of the heavy chain variable region is shown in SEQ ID No:21, and the amino acid sequence of the light chain variable region is shown in SEQ ID No:

17.

4. The method for purifying an anti-IL-11 monoclonal antibody according to claim 3, characterized in that: The anti-IL-11 monoclonal antibody also includes a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region is one of SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25 or SEQ ID No: 26; the amino acid sequence of the light chain constant region is as shown in SEQ ID No:

22.

5. The method for purifying anti-IL-11 monoclonal antibodies according to claim 2, 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-IA: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 32; HA-IB: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 33, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 34; HA-IC: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 35; HA-ID: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 36, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 37; HA-II-A: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 38; HA-II-B: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 39; HA-II-C: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 33, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 39; HA-II-D: The amino acid sequence of the heavy chain variable region is shown in SEQ ID No:36, and the amino acid sequence of the light chain variable region is shown in SEQ ID No:

40.

6. The method for purifying anti-IL-11 monoclonal antibodies according to claim 5, characterized in that: The anti-IL-11 monoclonal antibody also includes a human antibody heavy chain constant region and a human antibody light chain constant region, the amino acid sequence of the human antibody heavy chain constant region is one of 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.

7. The method for purifying an anti-IL-11 monoclonal antibody according to claim 1, characterized in that: In step S1, the affinity chromatography comprises the following steps: S101, balancing the affinity chromatography column with buffer A; S102, loading 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 filler, and the column bed height of the affinity chromatography column is 18-22 cm; S103, after the loading is completed, the buffer A is used for re-equilibrium, and then eluted at least once with 2-6CV of buffer B until the curve of the ultraviolet absorption value drops to a stable state, then the rinsing is stopped, and the buffer A is used for a second elution; S104, eluting the affinity chromatography column using buffer C, and collecting the protein product after elution for later use; S105, adjusting the pH value of the collected protein product to 3-4 using the acid solution, and incubating at room temperature for 0.5-3h under this pH condition, and finally adjusting the pH of the protein product back to 5-8 using 1M Tris buffer to obtain a crude and pure protein sample; Preferably, the filler of the affinity chromatography column is selected from MabSelect Sure LX, Eshmuno A, NMab Pro or ATProtein A Diamond Plus.

8. The method for purifying anti-IL-11 monoclonal antibodies according to claim 7, characterized in that: The buffer A is selected from Tris-HCl buffer, HEPES buffer or phosphate buffer, the concentration of the buffer A is 30-60mM, the pH value is 7-8, and the conductivity is 10-30mS / 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 30-60mM, and the pH value is 5-6; the buffer C is selected from glycine-hydrochloride, citrate or acetate buffer, the concentration of the buffer C is 20-200mM, and the pH value is 3-4.

9. The method for purifying an anti-IL-11 monoclonal antibody according to claim 1, characterized in that: In step S2, the anion exchange chromatography comprises the following steps: S201, equilibrate the anion exchange chromatography column using 2-6 CV of buffer D; S202, loading the crudely purified protein sample onto the anion exchange chromatography column at a flow rate of 100-200 cm / h, with a loading capacity of 20-120 mg protein / ml filler, and a column bed height of the anion exchange chromatography column of 18-22 cm; S203, after the sample loading is completed, the anion exchange chromatography column is equilibrated again with the buffer D, and the flow-through liquid is collected for standby use; Preferably, the filler of the anion exchange chromatography is selected from Capto Q, Capto adhere, Eshmuno Q or NanoGel-50Q.

10. The method for purifying anti-IL-11 monoclonal antibodies according to claim 9, characterized in that: The buffer D is selected from citric acid buffer, acetate buffer or phosphate buffer, and its pH value is 6.5-7.

5.

11. The method for purifying an anti-IL-11 monoclonal antibody according to claim 1, characterized in that: In step S3, the cation exchange chromatography comprises the following steps: S301, using 2-6 CV of buffer E to balance the cation exchange chromatography column; S302, loading the flow-through liquid after secondary purification onto the cation exchange chromatography column at a flow rate of 100-200 cm / h, with a loading capacity of 20-75 mg protein / ml filler, and the column bed height of the cation exchange chromatography column is 18-22 cm; S303, eluting the cation exchange chromatography column with an eluent F, to obtain a high-purity anti-IL-11 monoclonal antibody protein solution after elution; Preferably, the filler of the cation exchange chromatography column is Capto SP, Capto MMC, Eshmuno S or NanoGel-50SP; Preferably, the buffer E is selected from a citric acid buffer, an acetate buffer or a phosphate buffer, the concentration of the buffer E is 30-60 mM, the pH value is 5.0-6.0, and the conductivity is 2-5 mS / cm; the buffer F comprises buffer E and 80-150 mM of an additive, and the additive is NaCl.

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