A pharmaceutical preparation of an anti-il-11 monoclonal antibody

Abstract

The application relates to the field of biological medicines, and specifically provides a pharmaceutical preparation of an anti-IL-11 monoclonal antibody, which comprises the anti-IL-11 monoclonal antibody, buffer salt, a protein protective agent and a surfactant, wherein 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. The application provides suitable storage conditions for the high-concentration anti-IL-11 monoclonal antibody through the synergistic cooperation of the buffer salt, the protein protective agent and the surfactant, significantly changes the particle performance of the pharmaceutical preparation, reduces the generation rate of the aggregate of the anti-IL-11 monoclonal antibody in the production, transportation and use process, improves the stability of the physical and chemical properties of the anti-IL-11 monoclonal antibody, reduces potential safety risks and guarantees the stability of the pharmaceutical preparation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a pharmaceutical preparation of anti-IL-11 monoclonal antibody. BACKGROUND

[0002] Fibrosis can occur on various organs, such as: lung, liver, kidney, blood vessels, pancreas, skin and other parts, the main pathological changes after fibrosis are mainly the increase of fibrous connective tissue in the organ tissue, and the parenchymal cells will be significantly reduced, the more serious the fibrosis, the more the tissue organ gradually develops towards sclerosis, and the more serious the disease, the more the structure of the organ may be destroyed, and the function of the organ may be reduced, and even the function of the organ may be lost. Fibrosis can cause tissue cell damage, degeneration, necrosis, etc. in clinic, and the more common ones are pulmonary fibrosis, liver fibrosis, heart disease, kidney disease, asthma, etc.

[0003] IL-11 (interleukin-11) is a pleiotropic cytokine, which is a member of the IL-6 cytokine family, sharing the same signaling receptor subunit GP130, which plays a crucial role in the occurrence, development and metastasis of tumors. Research has found that IL-11 transmits signals to tumor cells through the GP130 signaling chain, so that tumor cells obtain proliferation and activation signals, and blocking this signaling pathway can become an effective treatment for various tumors, chronic fibrosis and inflammatory diseases, so the development of anti-IL-11 monoclonal antibody drugs has important clinical significance.

[0004] In the process of developing anti-IL-11 monoclonal antibody, it is found that, like most protein molecules, the complex structure of the antibody molecule is easily affected by various factors and aggregated, denatured or degraded during production, transportation and use. The structural stability of the antibody not only affects the biological activity of the antibody, but also affects the safety of biopharmaceuticals, especially some protein aggregates can stimulate the immune response of the human body, which can reduce the efficacy of biological drugs, and even cause death of patients. High-concentration antibody drugs not only require high-purity products during production, but also require structural stability during transportation, storage and use. Therefore, high-concentration proteins have challenged the performance and stability of aggregates and particulate matter. In order to meet the needs of fibrosis patients, it is urgent to develop a pharmaceutical preparation of anti-IL-11 monoclonal antibody with better stability. SUMMARY

[0005] In order to ensure the stability of high-concentration anti-IL-11 monoclonal antibody drugs, the present application provides a pharmaceutical preparation of anti-IL-11 monoclonal antibody.

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

[0007] The present application provides a kind of anti-IL-11 monoclonal antibody pharmaceutical preparation, the pharmaceutical preparation includes anti-IL-11 monoclonal antibody, buffer salt, protein protective agent and surfactant, wherein, the anti-IL-11 monoclonal antibody includes 3 respectively indicated as HCDR1, HCDR2 and HCDR3 heavy chain complementarity determining region and 3 respectively indicated as LCDR1, LCDR2 and LCDR3 light chain complementarity determining region, the anti-IL-11 monoclonal antibody is as follows:

[0008] A-III: the amino acid sequence of the heavy chain complementarity determining region HCDR1 is as shown in SEQ ID No:8, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is as shown in SEQ ID No:9, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is as shown in SEQ ID No:10, the amino acid sequence of the light chain complementarity determining region LCDR1 is as shown in SEQ ID No:11, the amino acid sequence of the light chain complementarity determining region LCDR2 is as shown in SEQ ID No:12, and the amino acid sequence of the light chain complementarity determining region LCDR3 is as shown in SEQ ID No:13.

[0009] The beneficial effects of the present application are as follows: the present application provides suitable storage conditions for high-concentration anti-IL-11 monoclonal antibody through the synergistic cooperation of buffer salt, protein protective agent and surfactant, significantly changes the particle performance of the pharmaceutical preparation, effectively reduces the generation rate of aggregates of anti-IL-11 monoclonal antibody during production, transportation and use, reduces production and transportation costs, at the same time improves the physical and chemical properties of anti-IL-11 monoclonal antibody, can make the antibody maintain good biological activity, reduce potential safety risk, and ensure the long-term stability of the pharmaceutical preparation; in addition, the anti-IL-11 monoclonal antibody provided by the present application has high binding capacity with IL-11 antigen, can block the binding of IL-11 antigen and its receptor, and then effectively inhibit the fibrogenic effect of IL-11, inhibit or prevent the generation or proliferation of fibrocytes, and can be effectively used for treating or preventing human fibrosis diseases, inflammation, cancer or autoimmune diseases. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 It is the plasmid map of pScFv-Disb-HS vector in embodiment 2 of the present application;

[0011] Figure 2 It is the comparison chart of gradient dilution ELISA anti-IL-11 phage monoclonal antibody affinity in embodiment 3 of the present application;

[0012] Figure 3Figure for the map of the vector pTSE in Example 5 of the present application;

[0013] Figure 4 Figure for the denaturing polyacrylamide gel electrophoresis of the murine antibody molecule in Example 5 of the present application;

[0014] Figure 5 Figure for the comparison of the binding ability of the murine antibody molecule to IL-11 in Example 6 of the present application;

[0015] Figure 6 Figure for the comparison of the competitive inhibition experiment of the murine antibody to the IL-11 receptor protein IL-11RA in Example 7 of the present application;

[0016] Figure 7 Figure for the comparison of the inhibition of the binding of IL-11 to the IL-11RA receptor on the surface of BaF / 3-IL-11RA cells by the murine antibody in Example 8 of the present application;

[0017] Figure 8 Figure for the comparison of the inhibition of the secretion of TIMP-1 from the embryonic lung fibroblast MRC-5 by the murine antibody in Example 9 of the present application;

[0018] Figure 9 Figure for the denaturing polyacrylamide gel electrophoresis of the humanized antibody molecule in Example 14 of the present application;

[0019] Figure 10 Figure for the comparison of the binding ability of the humanized antibody molecule to IL-11 in Example 15 of the present application;

[0020] Figure 11 Figure for the comparison of the inhibition of the binding of IL-11 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 application;

[0021] Figure 12 Figure for the comparison of the inhibition of the binding of IL-11 to the GP130 receptor on the surface of BaF / 3-GP130 cells by the humanized antibody molecule in Example 17 of the present application;

[0022] Figure 13 Figure for the comparison of the biological activity detection (reporter gene) of the humanized antibody molecule in Example 18 of the present application;

[0023] Figure 14 Figure for the comparison of the inhibition of the secretion of TIMP-1 from the embryonic lung fibroblast MRC-5 by the humanized antibody molecule in Example 19 of the present application;

[0024] Figure 15 Figure for the comparison of the cross-binding experiment of the humanized antibody molecule to IL-11 of different species in Example 20 of the present application;

[0025] Figure 16 This is a bar chart showing the changes in the lung-to-body weight ratio in the mouse pulmonary fibrosis model of Example 21 of this invention;

[0026] Figure 17 Images of hematoxylin and eosin (HE) staining and marson staining of lung tissue sections from the mouse pulmonary fibrosis model in Example 21 of this invention;

[0027] Figure 18 This is a bar chart showing the change in the heart-to-body weight ratio in the mouse cardiac fibrosis model of Example 22 of the present invention;

[0028] Figure 19 Images of hematoxylin and eosin (HE) staining and marson staining of heart tissue sections from the mouse cardiac fibrosis model in Example 22 of this invention;

[0029] Figure 20 This is a bar chart showing the urinary protein content in the kidneys of a mouse renal fibrosis model in Example 23 of this invention.

[0030] Figure 21 Images of hematoxylin and eosin (HE) staining and marson staining of kidney tissue sections from the mouse kidney fibrosis model in Example 23 of this invention;

[0031] Figure 22 This is a bar chart showing the changes in liver weight in the mouse liver fibrosis model of Example 24 of the present invention;

[0032] Figure 23 This is a bar chart showing the changes in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in a mouse liver fibrosis model in Example 24 of this invention.

[0033] Figure 24 Images of hematoxylin and eosin (HE) staining and marson staining of liver tissue sections from the mouse liver fibrosis model in Example 24 of this invention;

[0034] Figure 25 This is a graph showing the thermal stability evaluation of the anti-IL-11 monoclonal antibody HA-IA in Example 25 of the present invention. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the following embodiments.

[0036] Example 1

[0037] The embodiment 1 of the present application provides a pharmaceutical preparation of an anti-IL-11 monoclonal antibody, the pharmaceutical preparation comprising the anti-IL-11 monoclonal antibody, a buffer salt, a protein protective agent and a surfactant, wherein the anti-IL-11 monoclonal antibody comprises three heavy chain complementarity determining regions represented by HCDR1, HCDR2 and HCDR3 respectively and three light chain complementarity determining regions represented by LCDR1, LCDR2 and LCDR3 respectively, and the anti-IL-11 monoclonal antibody is selected from any one of the following.

[0038]

[0039] The anti-IL-11 monoclonal antibody provided by the present application is used for treating or preventing human fibrosis diseases, inflammation, cancer or autoimmune diseases, wherein the fibrosis diseases include but are not limited to fibrosis of heart, liver, kidney, lung, gallbladder, bladder, stomach, bone marrow, penis, breast, blood vessel, eye, pancreas, spleen, brain, intestine, muscle or skin, etc.; the inflammation includes but is not limited to hepatitis, myocarditis, nephritis, pneumonia, cholecystitis, cystitis, gastritis, osteomyelitis, prostatitis, mastitis, pancreatitis, enteritis, arthritis, polymyositis, dermatomyositis or dermatitis, etc.; the cancer includes but is not limited to leukemia, lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer or bladder cancer, etc.; and the autoimmune disease includes but is not limited to psoriasis, Crohn's disease, primary biliary cirrhosis, systemic lupus erythematosus or multiple sclerosis, etc.

[0040] Embodiment 2: Screening of mouse-derived antibody molecules

[0041] In the present application, mice are immunized with IL-11 antigen (the IL-11 protein, IL-11-Fc antigen and IL-11-mFc ligand protein in subsequent experiments are all human IL-11), the immunization method is optimized, and a phage display library is created, and the construction, screening and identification of the phage display library are as follows.

[0042] Step 1: Immunizing mice with IL-11 antigen

[0043] 1. Experimental animals: species and strain: BALB / c, female, mice; body weight: 18-20 g;

[0044] The experimental animals are provided by Beijing Huafukang Biotechnology Co., Ltd.

[0045] 2. Immunization: The mice are immunized with human IL-11 as the immunogen (synthetic gene by Nanjing Kingsway Biotech Co., Ltd., and the vector is constructed and expressed and purified by the present company).

[0046] Step two: Construction of phage antibody library: Take the mouse spleen cells with high titer, use Trizol reagent (purchased from Ambion, item number: 15596026) to extract total RNA in mouse spleen cells, obtain cDNA by RT-PCR, use degenerate primers (the degenerate primers used are referred to in the literature: Journal of Immunological Methods 233 (2000) 167-177) for PCR amplification, thereby obtaining the immune mouse antibody heavy chain variable region gene library (VH) and light chain variable region gene library (VL). The pScFv-Disb-HS vector is a vector pComb3 vector (purchased from China Plasmid Vector Strain Cell Strain Gene Preservation Center) modified by a series of gene cloning methods for the construction and expression of phage single-chain antibody library. The modified vector is named pScFv-Disb-HS vector, and its plasmid map is shown in Figure 1 , and based on this vector, a mouse immune phage antibody library is constructed. The light and heavy chain variable region gene libraries are double-digested and ligated to the vector pScFv-Disb-HS which has also been treated by stepwise digestion, to construct the pScFv-Disb-HS-VH-VL gene library.

[0047] Step three: Coat the immunotube with IL-11 as the antigen, the antigen coating amount is 5 μg / 500 μL / tube, coat overnight at 4°C, then use 4% skim milk / PBST to block the immunotube and the immune phage antibody library, respectively, block at room temperature for 1 h. After blocking, the immune phage antibody library is added to the immunotube for antigen-antibody binding, the phage input amount is about 10 9 ~ 10 12 After reaction at room temperature for 1 h, use PBST-PBS to wash away the unbound phage, elute by 0.1 M pH 2.2 Glycine-HCl, and finally use 1.5 M pH 8.8 Tris-HCl to neutralize the eluted phage antibody solution to about pH 7.0.

[0048] Step four: Infect 10 ml of the above neutralized phage with the TG1 bacteria liquid grown to the logarithmic phase, and incubate in a 37°C incubator for 30 min. Take part of the bacterial liquid for gradient dilution and spread on 2YTAG plates for calculation of phage output. Centrifuge the remaining bacterial liquid to discard the supernatant, resuspend the bacterial pellet in a small amount of culture medium, aspirate and spread on a 2YTAG large plate for preparation for the next round of screening.

[0049] Step five: the bacteria infected and plated as above were scraped from the large plate and inoculated into 2YTAG liquid medium, and after being shaken to the logarithmic phase, M13KO7 helper phage was added for superinfection. The phage was prepared by culturing overnight at 28°C and 220 rpm, and the phage was purified by PEG / NaCl precipitation for the next round of screening. One round of phage library enrichment screening was performed.

[0050] Step six: screening of IL-11 phage single-chain antibody positive clones: after one round of screening, single colonies with good separation were picked and inoculated into 2YTAG liquid medium added in a 96-well deep well plate. The plate was incubated at 37°C and 220 rpm until the logarithmic growth phase, about 10 10 μL of helper phage M13KO7 was added to each well, and the plate was incubated at 37°C for 30 min. After centrifugation at 4000 rpm for 15 min, the supernatant was discarded, and the bacteria were resuspended and precipitated with 2YTAK. The plate was incubated at 28°C and 220 rpm overnight. After centrifugation at 4000 rpm and 4°C for 15 min, the amplified phage supernatant was aspirated for ELISA identification. Four mouse antibody molecules with high affinity were finally screened, designated as MA-I, MA-II, MA-III and MA-IV. The above obtained monoclonal antibodies were subjected to gene sequencing to determine the correct antibody sequence. After sequencing, the sequences of the four monoclonal antibodies screened above were as follows:

[0051]

[0052] Specifically, SEQ ID No: 16 (amino acid sequence of the heavy chain variable region of MA-I and MA-II):

[0053] EVKLEESGGGLVKPGGSLKLSCAASGFTFSDYYMFWVRQTPEKRLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNNLYLQMTSLKSEDTAMYYCARDGGYVS SPEAMDYWGQGTSVTVSS;

[0054] SEQ ID No: 17 (amino acid sequence of the light chain variable region of MA-I and MA-IV):

[0055] DIVLTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPPTFGGGTKLEIK;

[0056] SEQ ID No: 18 (amino acid sequence of the variable region of the light chain of MA-II):

[0057] DIVLTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK;

[0058] SEQ ID No: 19 (amino acid sequence of the variable region of the heavy chain of MA-III):

[0059] EVKLEQSGAEVVKPGALVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIG VIDPSDSYTTYNQKFKGKATLTVDTSSSTGYMQLSSLTSEDSAVYYCSQYGYDVN WYFDVWGAGTTVTVSS;

[0060] SEQ ID No: 20 (amino acid sequence of the variable region of the light chain of MA-III):

[0061] DIVMTQTTLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLI YEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGT KLEIK;

[0062] SEQ ID No: 21 (amino acid sequence of the variable region of the heavy chain of MA-IV):

[0063] EVQLEESGGGLVKPGGSLKLSCVASGFTFSDYYMFWVRQTPEKRLEWVATI SDGGSYSYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTAMYYCARDGGYISS PEAMDYWGQGTSVTVSS.

[0064] Example 3 Gradient dilution ELISA to compare the affinities of antibodies

[0065] The four murine antibody molecules (MA-I, MA-II, MA-III and MA-IV) obtained in Example 2 were subjected to monoclonal phage display and purification, and then subjected to phage gradient dilution ELISA to identify the affinities, according to the following method:

[0066] IL-11 antigen was coated with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, and coated overnight at 4℃. The ELISA plate was washed with PBST for three times, and the four phage single monoclonal antibodies screened in Example 2 were diluted with PBST in a five-fold gradient, 100 μL of the diluted sample was added to each well, and the plate was incubated at room temperature for 1 hour. The ELISA plate was washed with PBST, and HRP-anti-M13 (purchased from Bio-viewshine, item number: GE27-9421-01) monoclonal antibody diluted with 1% BSA-PBST was added to the ELISA plate, and the plate was incubated at room temperature for 1 hour. Color development was performed using a TMB color development kit (purchased from Kangwei Century, item number: CW0050S), and color development was performed at room temperature for 10 minutes. After termination with 2M H2SO4, the plate was read at 450nm / 630nm using a microplate reader, and the corresponding EC50 value was calculated. The specific data are as follows:

[0067]

[0068] According to the above data and as shown in Table 1, the four different murine antibody molecules screened in Example 2 can bind to IL-11. Therefore, it can be concluded that the monoclonal antibody provided by the present application has high affinity with IL-11. Figure 2

[0069] Example 4

[0070] In Example 4, the anti-IL-11 monoclonal antibody further comprises a heavy chain constant region and a light chain constant region. The amino acid sequence of the heavy chain constant region is one of SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25 or SEQ ID No: 26. The amino acid sequence of the light chain constant region is as shown in SEQ ID No: 22. The specific sequences are as follows:

[0071] SEQ ID No: 22 (amino acid sequence of the light chain constant region of murine IgG1 type): k

[0072] ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0073] SEQ ID No: 23 (amino acid sequence of the heavy chain constant region of murine IgG1 type):

[0074] ​​AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG

[0075] SEQ ID No: 24 (amino acid sequence of heavy chain constant region of mouse IgG2a type):

[0076] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0077] SEQ ID No: 25 (amino acid sequence of heavy chain constant region of mouse IgG2b type):

[0078] AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK;

[0079] SEQ ID No:26 (amino acid sequence of heavy chain constant region of mouse IgG3 type):

[0080] ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPELELNETCAEAQDGELDGLWTTITIFISLFLLSVCYSASVTLFKVKWIFSSVVQVKQTAIPDYRNMIGQGA.

[0081] Preparation of mouse-derived antibody molecules

[0082] Example 5 of the present application preferably defines the mouse-derived antibody molecules to include a heavy chain constant region of mouse IgG1 type (the amino acid sequence of which is shown as SEQ ID No: 23) and mouse C kThe light chain constant region of type [type] (its amino acid sequence is shown in SEQ ID No:22). The specific antibody preparation method is as follows:

[0083] 1. The encoding genes for the heavy chain VH and light chain VL of the four antibody molecules screened in Example 2 were cloned into the vector pTSE (e.g., ...) containing the heavy chain and light chain constant region genes, respectively. Figure 3 As shown in SEQ ID No: 23), the preferred heavy chain constant region is the mouse IgG1 type constant region (amino acid sequence shown in SEQ ID No: 23), and the light chain constant region is mouse C k Chain (amino acid sequence as shown in SEQ ID No: 22), pTSE vector structure as shown Figure 3 (For the preparation process of the pTSE vector, please refer to paragraph

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

[0084] 2. HEK293 cells were transiently transfected (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number GNHu43) for antibody expression. Four monoclonal antibodies were purified using an AKTA instrument via a protein A affinity column. Protein concentration was determined using a BCA kit (purchased from Beijing Huitian Oriental Technology Co., Ltd., catalog number BCA0020). Protein size was then identified by SDS-PAGE. The results are shown below. Figure 4 As shown, from left to right, the images represent non-reduced MA-I, MA-II, MA-III, and MA-IV, protein molecular weight Marker 1, protein molecular weight Marker 2, and reduced MA-I, MA-II, MA-III, and MA-IV murine anti-IL-11 monoclonal antibodies. The molecular weight of each band is consistent with the theoretical values.

[0085] Example 6: Binding experiment of mouse antibody molecules with IL-11

[0086] IL-11 antigen was coated with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, and coated overnight at a temperature of 4°C. Washed five times with 300 μL / well PBST, then added 1% BSA-PBST, 280 μL / well, blocked at a temperature of 37°C for 1 h, added different dilution concentrations of MA-I, MA-II, MA-III and MA-IV mouse antibody molecules, the starting highest concentration of the four antibody molecules was 5 μg / mL, respectively, 5-fold gradient dilution, a total of 8 gradients for each antibody, incubated at a temperature of 37°C for 1 h. Washed five times with 300 μL / well PBST, then added Goat Anti-Mouse IgG-HRP (purchased from solarbio, item number: SE131) diluted 1:2000 with 1% BSA-PBST, incubated at a temperature of 37°C for 1 h. Color development with TMB color development kit, 100 μL / well, color development at room temperature for 8 min, then color development was terminated with 2M H2SO4. Readings were taken at 450 nm / 630 nm with a microplate reader, and the corresponding EC50 values were calculated, and the specific data are as follows:

[0087]

[0088] From the above data and as shown in Figure 5 , the four different mouse antibody molecules screened can bind to IL-11 and have high affinity.

[0089] Example 7 Competition inhibition experiment of mouse antibody with IL-11 receptor protein IL-11RA

[0090] IL-11-Fc was coated with carbonate buffer at pH 9.6, 200 ng / well / 100 μL, coated overnight at 4°C. Washed five times with 300 μL / well PBST, then added 1% BSA-PBST, 280 μL / well, blocked at 37°C for 1 h, then added IL-11RA-Fc (IgG4 type) diluted to 0.5 μg / mL with 1% BSA-PBST, 50 μL / well, then added MA-I, MA-II, MA-III and MA-IV mouse antibodies at different dilution concentrations, 50 μL / well, the starting highest concentration of the five antibodies was 100 μg / mL, each antibody was diluted by 2-fold gradient, a total of 13 gradients for each antibody, incubated at 37°C for 3 h. Washed five times with 300 μL / well PBST, then added Anti-Human IgG4-HRP Mouse monoclonal antibody (purchased from Sigma, item number: SAB4200770) diluted 1:5000 with 2% BSA-PBST, incubated at 37°C for 1 h. Color development with TMB color development kit, 100 μL / well, color development at room temperature for 15 min, then color development was terminated with 2M H2SO4. Readings were taken at 450 nm / 630 nm with a microplate reader, and the corresponding IC50 values were calculated, and the specific data are as follows:

[0091]

[0092] From the above data and as shown in Figure 6 , the four different mouse antibodies screened can all compete with the receptor protein IL-11RA, indicating that they can effectively inhibit the binding of IL-11 to the receptor protein IL-11RA.

[0093] Example 8 Inhibition of IL-11 binding to IL-11RA receptors on the surface of BaF / 3-IL-11RA cells by mouse antibodies

[0094] The BaF / 3-IL-11RA cell line was counted, and a certain number of cells were centrifuged and resuspended with PBS buffer. The cell density was adjusted to 1E+6 cells / mL, 100 μL / well, and added to a 96-well plate. The IL-11-mFc ligand protein was diluted with PBS to a concentration of 18 μg / mL, 50 μL / well, and added to the corresponding position of the 96-well plate containing the BaF / 3-IL-11RA cells. After mixing gently, the 96-well plate was placed at 4°C and incubated for 1 h. The four mouse antibody molecules MA-I, MA-II, MA-III, and MA-IV were gradient diluted with PBS, with an initial concentration of 800 μg / mL, 3-fold gradient dilution, a total of 10 gradients, 50 μL / well, and added to the corresponding position of the 96-well plate containing the mixture of BaF / 3-IL-11RA cells and IL-11-mFc ligand protein. After mixing, the plate was incubated at 4°C for 2 h. After incubation, the cells were centrifuged at 3000 rpm, washed once with PBS buffer, and the cell pellet was collected. Goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) was added to the cell pellet, and incubated at 4°C for 30 min. After centrifugation at 3000 rpm, the cells were washed once with PBS buffer, resuspended with 100 μL of PBS buffer, and detected by flow cytometry. The fluorescence signal in the FL1-A channel was collected. The dose-effect curve was drawn, and the corresponding IC50 value was calculated. The specific data are as follows:

[0095]

[0096] From the above data and Figure 7 It can be seen that the four different mouse candidate molecules screened can effectively inhibit the binding of IL-11 ligand protein to the cell surface IL-11RA receptor.

[0097] Example 9 Inhibition of TIMP-1 Secretion from Embryonic Lung Fibroblast MRC-5 by Mouse Antibodies

[0098] Embryonic lung fibroblast MRC-5 was counted after trypsin digestion, a certain amount of cells was taken, the cells were resuspended after centrifugation with MEM complete medium (purchased from GIBCO, item number 10370-021), and the cell density was adjusted to 2E+5 cells / mL, 100 μL / well, and added to a 96-well plate. The IL-11-mFc ligand protein was diluted with MEM complete medium, and the concentration was prepared to be 16 μg / mL, 50 μL / well was added to the corresponding 96-well plate. The four mouse-derived antibody molecules MA-I, MA-II, MA-III and MA-IV were gradient diluted with MEM complete medium, and the initial concentration was prepared to be 40 μg / mL, 2-fold gradient dilution, a total of 8 gradients, 50 μL / well, added to the 96-well plate containing the cell suspension and the IL-11-mFc ligand protein suspension, mixed gently, and incubated in a 37℃ CO2 incubator overnight for about 20 h. The cell culture supernatant was detected by TIMP-1 ELISA kit (purchased from Yikexie Biotechnology Co., Ltd., item number EH021-96).

[0099] Human TIMP-1 detection kit: add cell supernatant and standard to sample wells, 100 μL / well. Immediately add biotinylated antibody working solution (1:100 dilution), 50 μL / well, cover the plate with sealing film, shake and incubate at room temperature for 2 h. After incubation, wash the plate 4 times with washing solution, add enzyme conjugate working solution (1:100 dilution) in the TIMP-1 detection kit, 100 μL / well. Cover the plate with sealing film, shake and incubate at room temperature for 1 h. After incubation, wash the plate 4 times with washing solution. Add TMB color developing solution, 100 μL / well, avoid light, incubate at room temperature for about 15 minutes, add 100 μL / well Stop solution to terminate the reaction. Read the value at 450 nm on a microplate reader, and calculate the corresponding IC50 value, the specific data are as follows:

[0100]

[0101] From the above data and Figure 8 It can be seen that the four different mouse-derived candidate molecules screened can effectively inhibit the release of TIMP-1 from human embryonic lung fibroblast MRC-5 stimulated by IL-11 ligand protein.

[0102] Example 10

[0103] The anti-IL-11 monoclonal antibody of the embodiment 10 of the present application is a chimeric antibody molecule, and the chimeric antibody molecule further comprises a human antibody constant region, the human antibody constant region comprises a human antibody heavy chain constant region and a human antibody light chain constant region, the amino acid sequence of the human antibody heavy chain constant region is one of SEQ ID No: 27, SEQ ID No: 28 or SEQ ID No: 29; and the amino acid sequence of the human antibody light chain constant region is as shown in SEQ ID No: 30.

[0104] SEQ ID No: 27 (amino acid sequence of heavy chain constant region of human IgG1 type):

[0105] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0106] SEQ ID No: 28 (amino acid sequence of heavy chain constant region of human IgG2 type):

[0107] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0108] SEQ ID No: 29 (amino acid sequence of heavy chain constant region of human IgG4 type):

[0109] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0110] SEQ ID No:30 (human C k chain constant region amino acid sequence):

[0111] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C.

[0112] Preparation of chimeric antibody molecules of the antibodies of Example 11

[0113] Example 11 of the present application is further defined on the basis of Example 10, wherein the humanized antibody constant region comprises a human IgG1 type heavy chain constant region (the amino acid sequence of which is shown as SEQ ID No: 27) and a human C k type light chain constant region (the amino acid sequence of which is shown as SEQ ID No: 30).

[0114] Specific preparation method:

[0115] The heavy chain variable region VH (SEQ ID No: 16) of the murine antibody molecules MA-I and MA-II obtained by screening the phage antibody library in Example 2, and 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) are kept as murine sequences, and are respectively cloned into the vector pTSE (as shown in Figure 3 SEQ ID No: 28) carrying the heavy chain constant region and light chain constant region genes, wherein the heavy chain constant region is human IgG1 type (the amino acid sequence is shown as SEQ ID No: 27), and the light chain constant region is human Ck Chimeric antibodies CA-I, CA-II were expressed by transient transfection of HEK293E cells (purchased from: Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, item number: GNHu43) and purified by protein A affinity chromatography.

[0116] Example 12 Humanization of murine antibody molecules

[0117] First, the sequences of murine antibody molecules MA-I and MA-II in Example 2 were compared with the human antibody germline database (v-base) to find higher homologous human antibody light and heavy chain germlines as candidate sequences, and then the CDR sequences of murine antibody molecules MA-I and MA-II were transplanted onto the human candidate sequences for homology modeling. Then, the key framework amino acid residues that may play an important role in maintaining the CDR loop structure were designed by three-dimensional structure simulation calculation, so as to design the back mutation of humanized antibodies. The designed light and heavy chain variable region sequences of humanized antibodies containing back mutations were synthesized by Nanjing Kingsriver Biotechnology Co., Ltd., and then connected to the transient expression vector. The light and heavy chains obtained by humanization were analyzed, in which MA-I obtained the following humanized anti-IL-11 monoclonal antibody molecules: HA-I-A, HA-I-B, HA-I-C, HA-I-D; MA-II obtained the following humanized antibody molecules: HA-II-A, HA-II-B, HA-II-C, HA-II-D; the sequences of the above 8 monoclonal antibodies screened are as follows:

[0118]

[0119] Specifically, SEQ ID No: 31 (amino acid sequence of the heavy chain variable region of HA-I-A, HA-I-C, HA-II-A and HA-II-B):

[0120] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVSS PEAMDYWGQGTLVTVSS;

[0121] SEQ ID No: 32 (amino acid sequence of the light chain variable region of HA-I-A):

[0122] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;

[0123] SEQ ID No:33 (amino acid sequence of the heavy chain variable region of HA-I-B and HA-II-C):

[0124] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVST ISDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVS SPEAMDYWGQGTLVTVSS;

[0125] SEQ ID No:34 (amino acid sequence of the light chain variable region of HA-I-B):

[0126] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;

[0127] SEQ ID No:35 (amino acid sequence of the light chain variable region of HA-I-C):

[0128] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;

[0129] SEQ ID No:36 (amino acid sequence of the heavy chain variable region of HA-I-D and HA-II-D):

[0130] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAMYYCARDGGYVS SPEAMDYWGQGTSVTVSS;

[0131] SEQ ID No:37 (amino acid sequence of the light chain variable region of HA-I-D):

[0132] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;

[0133] SEQ ID No:38 (amino acid sequence of the light chain variable region of HA-II-A) :

[0134] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;

[0135] SEQ ID No:39 (amino acid sequence of the light chain variable region of HA-II-B and HA-II-C) :

[0136] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;

[0137] SEQ ID No:40 (amino acid sequence of the light chain variable region of HA-II-D) :

[0138] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGTVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPWTFGGGTKVEIK.

[0139] Embodiment 13

[0140] The embodiment 13 of the present application further limits the humanized antibody constant region to include a humanized antibody heavy chain constant region and a humanized antibody light chain constant region, the amino acid sequence of the humanized antibody heavy chain constant region is one of SEQ ID No: 27, SEQ ID No: 28 or SEQ ID No: 29; the amino acid sequence of the humanized antibody light chain constant region is shown in SEQ ID No: 30.

[0141] The specific sequence of the humanized antibody constant region is the same as that of embodiment 10.

[0142] Preparation of humanized antibody molecules

[0143] The embodiment 14 of the present application further defines the humanized antibody constant region to comprise a human IgG1 type heavy chain constant region (the amino acid sequence of which is shown as SEQ ID No: 27) and a human C k type light chain constant region (the amino acid sequence of which is shown as SEQ ID No: 30).

[0144] The coding genes of the heavy chain VH and light chain VL of the 8 humanized anti-IL-11 monoclonal antibody molecules HA-I-A, HA-I-B, HA-I-C, HA-I-D, HA-II-A, HA-II-B, HA-II-C, HA-II-D obtained from the humanization of embodiment 12 were respectively cloned into the vector pTSE (as shown in Figure 3 , which is equipped with the heavy chain constant region and light chain constant region genes, the heavy chain constant region being human IgG1 type (the amino acid sequence of which is shown as SEQ ID NO: 27), and the light chain constant region being C k ( the amino acid sequence of which is shown as SEQ ID NO: 30).

[0145] The 2 chimeric antibodies CA-I and CA-II obtained from embodiment 11 and the 8 humanized antibody molecules HA-I-A, HA-I-B, HA-I-C, HA-I-D, HA-II-A, HA-II-B, HA-II-C, HA-II-D obtained from embodiment 12 were respectively transiently transfected into HEK293 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, item number GNHu43) for antibody expression, the monoclonal antibodies were purified by protein A affinity column using AKTA instrument, and the protein concentration was determined using BCA kit (purchased from Beijing Huitian Dongfang Science and Technology Co., Ltd., item number BCA0020), and then the protein size was identified by SDS-PAGE, the results are shown in Figure 9 , from left to right are non-reduced protein molecular weights HA-I-A, HA-I-B, HA-I-C, HA-I-D, chimeric antibody CA-I prepared in embodiment 11, reduced protein molecular weight Marker, HA-II-A, HA-II-B, HA-II-C, HA-II-D, chimeric antibody CA-II, the molecular weight of each band is consistent with the theory.

[0146] Example 15 Binding experiment of humanized antibody molecules with IL-11

[0147] IL-11 antigen was coated with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, coated overnight at 4°C. Washed five times with 300 μL / well PBST, then added 1% BSA-PBST 280 μL / well, blocked at 37°C for 1 h. The initial concentration of humanized antibodies HA-I-A, HA-I-B, HA-I-C, HA-I-D, HA-II-A, HA-II-B, HA-II-C, HA-II-D and the chimeric antibodies CA-I, CA-II prepared in Example 11, humanized antibodies were all 10 μg / mL, 5-fold dilution gradient, a total of 8 gradients, incubated at 37°C for 1 h. Washed five times with 300 μL / well PBST, then added goat anti Human IgG Fab HRP (purchased from invitrogen, item number: 31482) diluted 1:5000 with 1% BSA-PBST, incubated at 37°C for 1 h. Color development with TMB color development kit, 100 μL / well, color development at room temperature for 5 min, then color development was terminated with 2M H2SO4. Readings were taken at 450 nm / 630 nm on a microplate reader, and the corresponding EC50values were calculated, and the specific data are as follows:

[0148]

[0149]

[0150] As shown in the above data and experimental results as shown in Figure 10 , the eight different humanized antibody molecules can bind to IL-11, and the EC50values of the humanized antibody molecules HA-I-A, HA-I-B, HA-I-C, HA-I-D are close to the chimeric antibody CA-I, and the EC50values of the humanized antibody molecules HA-II-A, HA-II-B, HA-II-C, HA-II-D are close to the chimeric antibody CA-II, indicating that the humanized antibody molecules retain the high binding ability of the mouse parent antibodies MA-I, MA-II to IL-11.

[0151] Example 16 Humanized antibody molecules inhibit the binding of IL-11 to IL-11RA receptors on the surface of BaF / 3-IL-11RA cells

[0152] The four humanized antibody molecules HA-I-A, HA-I-B, HA-I-C and HA-I-D with better activity at the protein level were selected for cell activity evaluation experiment. The BaF / 3-IL-11RA cell line was counted, a certain amount of cells were centrifuged and resuspended with PBS buffer, and the cell density was adjusted to 1E+6 cells / mL, 100 μL / well, and added to a 96-well plate. The IL-11-mFc ligand protein was diluted with PBS buffer to a concentration of 18 μg / mL, 50 μL / well, and added to the corresponding position of the 96-well plate containing the BaF / 3-IL-11RA cells. After gentle mixing, the 96-well plate was placed at 4°C for incubation for 1 h. The four humanized antibody molecules HA-I-A, HA-I-B, HA-I-C and HA-I-D were gradient diluted with PBS buffer, and the initial concentration was prepared to be 800 μg / mL, 3-fold gradient dilution, a total of 10 gradients, 50 μL / well, added to the corresponding position of the 96-well plate containing the mixture of BaF / 3-IL-11RA cells and IL-11-mFc ligand protein. After mixing, it was placed at 4°C for 2 h of incubation. After incubation, the cells were washed once by centrifugation at 3000 rpm with PBS buffer, and the cell precipitate was collected. Goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) was added to the cell precipitate, and incubated at 4°C for 30 min, then centrifuged at 3000 rpm for washing once, resuspended with 100 μL of PBS, and then detected by flow cytometry. The fluorescence signal in the FL1-A channel was collected. The dose-effect curve was drawn, and the corresponding IC50 value was calculated. The specific data are as follows:

[0153]

[0154] From the above data and Figure 11 It can be seen that the four humanized candidate molecules selected can inhibit the binding of IL-11 ligand protein to the surface IL-11RA receptor of BaF / 3-IL-11RA cells.

[0155] Example 17 Humanized antibody molecules inhibit the binding of IL-11 to the surface GP130 receptor of BaF / 3-GP130 cells

[0156] The BaF / 3-GP130 cell line was counted, and a certain number of cells were centrifuged and resuspended with PBS buffer. The cell density was adjusted to 1E+6 cells / mL, 100 μL / well, and added to a 96-well plate. The IL-11-mFc ligand protein was diluted with PBS, and the concentration was adjusted to 12 μg / mL, 50 μL / well, and added to the corresponding position of the 96-well plate containing the BaF / 3-GP130 cells. After mixing gently, the 96-well plate was placed at 4°C and incubated for 1 h. The four human antibody molecules HA-I-A, HA-I-B, HA-I-C, and HA-I-D were gradient diluted with PBS buffer, and the initial concentration was adjusted to 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 the BaF / 3-GP130 cells and the IL-11-mFc ligand protein. After mixing, the 96-well plate was placed at 4°C and incubated for 2 h. After incubation, the cells were washed once with PBS buffer at 3000 rpm, and the cell precipitate was collected. Goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) was added to the cell precipitate, 100 μL / well, and incubated at 4°C for 30 min. After washing once with PBS buffer at 3000 rpm, the cells were resuspended with 100 μL / well of PBS buffer, and the fluorescence signal in the FL1-A channel was detected by flow cytometry. The dose-effect curve was drawn, and the corresponding IC50 value was calculated. The specific data are as follows:

[0157]

[0158] From the above data and Figure 12 It can be seen that the four humanized candidate molecules screened can block the binding of the IL-11 ligand protein to the GP130 receptor on the surface of the BaF / 3-GP130 cells.

[0159] Example 18 Biological activity detection of humanized antibody molecules (reporter gene)

[0160] The BaF / 3-IL-11RA-GP130-STAT3-Luc engineered cell strain was counted, and the cell density was adjusted to 2E+6 cells / mL using a sample diluent (which contains 90% IMDM, 10% FBS, and 10 ng / mL mouse IL-3). After gentle mixing, 50 μL of the cell solution was added to each well of a 96-well plate. Four humanized antibody molecules, HA-I-A, HA-I-B, HA-I-C, and HA-I-D, were diluted to an initial concentration of 200 μg / mL using the sample diluent, and 5-fold gradient dilution was performed, for a total of 10 gradients, 100 μL / well. The 96-well plate containing the engineered cell strain was added to the corresponding positions, and two replicate wells were set up for each sample concentration. The sample diluent was used to prepare IL-11 protein at a concentration of 10 μg / mL, and 50 μL was added to each well of the 96-well plate containing the engineered cell strain and the humanized antibody molecules. The cell culture plate was gently mixed and incubated in a 37°C CO2 incubator for 6 h. The supernatant was discarded by centrifugation, and 10 μL of lysis solution was added to each well of a 384-well plate. An equal amount of luciferase reaction substrate (purchased from Promega Biotechnology Co., Ltd., catalog number E2610) was added, and the reaction was allowed to proceed at room temperature for 5 min. The fluorescence value was read on an enzyme marker, and the corresponding IC50 value was calculated. The specific data are as follows:

[0161]

[0162] As shown by the above data and Figure 13 , the four humanized antibody molecules selected can block the binding of IL-11 to IL-11RA and GP130 receptors and inhibit the conduction of the signal pathway.

[0163] Example 19 Inhibition of TIMP-1 Secretion from Embryonic Lung Fibroblast MRC-5 by Humanized Antibody Molecules

[0164] Embryonic lung fibroblast MRC-5 was counted after trypsin digestion, a certain amount of cells was taken, the cells were resuspended with MEM complete medium after centrifugation, and the cell density was adjusted to 2E+5 cells / mL, 100 μL / well, and added to a 96-well plate. The IL-11-mFc ligand protein was diluted with MEM complete medium, and the concentration was prepared to be 16 μg / mL, 50 μL / well, and added to the corresponding 96-well plate. The 4 humanized antibody molecules HA-I-A, HA-I-B, HA-I-C and HA-I-D were gradient diluted with MEM complete medium, and the initial concentration was prepared to be 40 μg / mL, 3 times gradient dilution, a total of 8 gradients, 50 μL / well, added to the 96-well plate containing the cell suspension and the IL-11-mFc ligand protein suspension, mixed gently and uniformly, incubated in a 37°C CO2 incubator overnight, about 20 h, and the cell culture supernatant was detected by TIMP-1 ELISA kit (method same as Example 9). The microplate reader was read at 450 nm, and the corresponding IC50 value was calculated, and the specific data were as follows:

[0165]

[0166] From the above data and Figure 14 It can be seen that the 4 humanized antibody molecules screened can effectively inhibit the release of TIMP-1 of human embryonic lung fibroblast MRC-5 stimulated by IL-11 ligand protein.

[0167] Example 20 Cross-binding experiment of humanized antibody molecules with different species of IL-11

[0168] The humanized antibody molecule HA-I-A with better protein level and function detection activity was selected for cross-binding detection with different species of IL-11. Human IL-11, mouse IL-11 (purchased from Beijing Yikui God State Technology Co., Ltd., item number: 50117-MNCE), rat IL-11 (purchased from Kanglang Biology, item number: KL40001Ra), and cynomolgus monkey IL-11 (purchased from Yikui God State, item number: 90925-CNCE) were coated with 100 ng / well / 100 μL of carbonate buffer at pH 9.6 at 4°C overnight. Five washes with 300 μL / well PBST were performed, and 1% BSA-PBST was added at 280 μL / well, and incubated at 37°C for 1 h. The humanized antibody HA-I-A was diluted with 1% BSA-PBST, and the initial concentration was 50 μg / mL, and 5-fold gradient dilution was performed, a total of 9 gradients, and each gradient had two duplicate wells, 100 μL / well was added to the 96-well plate, and incubated at 37°C for 1 h. Five washes with 300 μL / well PBST were performed, and goat anti-human IgG Fab HRP (purchased from invitrogen, item number: 31482) was diluted with 1% BSA-PBST at a working concentration of 1:5000, 100 μL / well was added to the 96-well plate, and incubated at 37°C for 1 h. Five washes with 300 μL / well PBST were performed, and TMB color developing kit was used for color development, 100 μL / well, color development at room temperature for 5 min, and then color development was terminated with 2M H2SO4. The microplate reader was read at 450 nm / 630 nm, and the corresponding EC50 values were calculated, and the specific data are as follows:

[0169]

[0170] From the above data and as shown in Figure 15 , the humanized antibody molecule HA-I-A can bind to human IL-11, mouse IL-11, rat IL-11, and cynomolgus monkey IL-11, and has high affinity.

[0171] Example 21 Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody on pulmonary fibrosis

[0172] Bleomycin (bLF) was used to model to study the therapeutic effect of anti-IL-11 monoclonal antibody HA-I-A on pulmonary fibrosis.

[0173] Animal species: C57BL / 6J mice (purchased from Jiangsu Jizhu Pharmaceutical Biology Technology Co., Ltd.)

[0174] Number, gender, and mouse age: 6 per group, male, 6-8 weeks;

[0175] The control group was only injected with normal saline.

[0176] The administration group was administered with the HA-I-A antibody molecule twice a week for 4 weeks.

[0177] The animal weight was measured once a week, and the animals were observed for abnormalities; organ weight detection: the heart was collected, the weight of the heart was measured, and the ratio of the heart to the body weight was calculated; heart pathology detection: the heart was sectioned and observed for the degree of heart fibrosis using hematoxylin-eosin (HE) and Masson staining.

[0178] As shown in Table 2, the ratio of the heart to the body weight of the mice in the administration group was significantly smaller than that of the control group; as shown in Table 3, compared with the control group, the heart sections of the administration group showed a significant reduction in heart fibrosis, thus indicating that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the generation of heart fibrosis. Figure 16 Figure 17 As shown in Table 3, compared with the control group, the heart sections of the administration group showed a significant reduction in heart fibrosis, thus indicating that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the generation of heart fibrosis.

[0179] Example 22 Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody on heart fibrosis

[0180] The therapeutic effect of the anti-IL-11 monoclonal antibody HA-I-A on heart fibrosis was studied using isoproterenol modeling.

[0181] Animal species: C57BL / 6J mice (purchased from Jiangsu Jicui Yekang Biotechnology Co., Ltd.)

[0182] Number, gender, and mouse age: 6 per group, male, 6-8 weeks;

[0183] The control group was only injected with normal saline;

[0184] The administration group was administered with the HA-I-A antibody molecule twice a week for 4 weeks.

[0185] The animal weight was measured once a week, and the animals were observed for abnormalities; organ weight detection: the heart was collected, the weight of the heart was measured, and the ratio of the heart to the body weight was calculated; heart pathology detection: the heart was sectioned and observed for the degree of heart fibrosis using hematoxylin-eosin (HE) and Masson staining.

[0186] As shown in Table 2, the ratio of the heart to the body weight of the mice in the administration group was significantly smaller than that of the control group; as shown in Table 3, compared with the control group, the heart sections of the administration group showed a significant reduction in heart fibrosis, thus indicating that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the generation of heart fibrosis. Figure 18 Figure 19 As shown in Table 3, compared with the control group, the heart sections of the administration group showed a significant reduction in heart fibrosis, thus indicating that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the generation of heart fibrosis.

[0187] Example 23 Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody on kidney fibrosis

[0188] ​​To study the therapeutic effect of anti-IL-11 monoclonal antibody HA-I-A on renal fibrosis using adriamycin (dKF) modeling.

[0189] Animal species: BALB / c mice (purchased from Jiangsu Jucu Yaoke Biotechnology Co., Ltd.)

[0190] Number, gender and mouse age: 6 per group, male, 6-8 weeks;

[0191] The control group was only injected with normal saline;

[0192] The administration group was given injections of HA-I-A antibody molecules twice a week for 4 weeks.

[0193] Animal body weight was measured once a week, and animals were observed for abnormalities; organ weight detection: the heart was collected, the weight of the kidney was measured, and the urine protein content was detected; kidney pathology detection: kidney sections were stained with hematoxylin-eosin (HE) and Masson to observe the degree of renal fibrosis.

[0194] The results are shown in Figure 20 Compared with the control group, the urine protein content in the kidneys of the mice in the administration group was significantly lower than that in the control group; the results are shown in Figure 21 Compared with the control group, the kidney sections of the administration group showed significantly reduced renal fibrosis, thus indicating that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the production of renal fibrosis.

[0195] Example 24 Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody on liver fibrosis

[0196] To study the therapeutic effect of anti-IL-11 monoclonal antibody HA-I-A on liver fibrosis using CCl4 modeling.

[0197] Animal species: C57BL / 6J mice (purchased from Jiangsu Jucu Yaoke Biotechnology Co., Ltd.);

[0198] Number, gender and mouse age: 6 per group, male, 6-8 weeks;

[0199] The control group was only injected with normal saline;

[0200] The administration group was given injections of HA-I-A antibody molecules twice a week for 4 weeks.

[0201] Body weight monitoring: animal body weight was measured once a week, and animals were observed for abnormalities; liver pathology detection: liver sections were stained with hematoxylin-eosin (HE) and Masson to observe the degree of liver fibrosis; organ weight detection: the kidney was collected, the weight of the liver was measured, and HE staining was performed; serum detection: serum was collected, and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the serum of mice were detected.

[0202] The results are as follows Figure 22 As shown, the liver weight of mice in the treatment group was significantly lower than that in the control group; the results are as follows. Figure 23 As shown, the serum ALT and AST levels in the treatment group were significantly lower than those in the control group; the results are as follows. Figure 24 As shown, compared with the control group, the liver slices of the treatment group showed a significant reduction in liver fibrosis, which indicates that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the production of liver fibrosis.

[0203] Example 25: Thermostability Assessment of Anti-IL-11 Monoclonal Antibody HA-IA

[0204] The thermal stability of the anti-IL-11 monoclonal antibody HA-IA was assessed using a multifunctional protein thermal stability analysis system (purchased from Unchained Labs). Protein conformational changes were detected by monitoring intrinsic fluorescence as a function of temperature (starting at 25°C and increasing to 95°C at a rate of 0.3°C / min) to determine the protein melting temperature (Tm) and assess conformational stability. When samples aggregate, interference occurs in the scattered light waves, increasing the scattered light signal. The colloidal stability of the protein was determined by static light scattering (characterized using Tagg). The results are shown in the table below and appendix. Figure 25 As shown.

[0205]

[0206] The melting temperature (Tm) of the anti-IL-11 monoclonal antibody HA-IA was 79.5℃, and the average Tagg value was 75.9℃, indicating good conformational and colloidal stability.

[0207] Example 26

[0208] Based on Example 1, Example 26 of the present invention provides a pharmaceutical preparation of an anti-IL-11 monoclonal antibody, wherein the protein concentration of the anti-IL-11 monoclonal antibody is 10-80 mg / ml.

[0209] Example 27

[0210] Based on Example 1, Example 27 of this invention provides a pharmaceutical formulation of an anti-IL-11 monoclonal antibody, wherein the content of buffer salt is 10-40 mM;

[0211] It should be further noted that the buffer salts include, but are not limited to, one or more combinations of acetate buffer, phosphate buffer, histidine buffer, or citrate buffer.

[0212] Example 28

[0213] Embodiment 28 provides a pharmaceutical preparation of anti-IL-11 monoclonal antibody based on embodiment 1, wherein the content of the protein protective agent is 150-350 mM;

[0214] It should be further explained that the protein protective agent includes but is not limited to one or a combination of sucrose, mannitol, trehalose, sorbitol, glycine, proline, methionine, lysine hydrochloride, arginine hydrochloride or sodium chloride.

[0215] Embodiment 29

[0216] Embodiment 29 provides a pharmaceutical preparation of anti-IL-11 monoclonal antibody based on embodiment 1, wherein the content of the surfactant is 0.005%-0.04% w / v;

[0217] It should be further explained that the surfactant is selected from polysorbate 80, polysorbate 20 or poloxamer.

[0218] Embodiment 30

[0219] Embodiment 30 provides a pharmaceutical preparation of anti-IL-11 monoclonal antibody based on embodiment 1, wherein the pH value of the pharmaceutical preparation is 5.5-6.5.

[0220] Selection of pH range of pharmaceutical preparation in embodiment 31

[0221] Based on embodiment 1, anti-IL-11 monoclonal antibody HA-I-A is selected as the pharmacodynamic molecule to prepare a pharmaceutical preparation, and the preparation method comprises: preparing 20 mM acetic acid-sodium acetate buffer (pH 4.5, 5.5) and 20 mM phosphate buffer (pH 6.5, 7.5 and 8.5) respectively, ultrafiltration of different concentrations of anti-IL-11 monoclonal antibody HA-I-A into different pH buffers, sterilization filtration with a 0.22 μm filter, and dispensing into 2 ml vials, 1 ml per vial. After dispensing, the protein thermal stability under different buffer conditions is detected, then the samples are placed in a stability test box, the temperature is set to 40±2℃, and the appearance, pH, protein concentration, purity, charge isomer, sub-visible particles, viscosity and particle size are observed after 2 weeks.

[0222] The specific design groups are as follows:

[0223]

[0224] The thermal stability detection results are as follows:

[0225] Experimental Example Tm (°C) Tagg (°C) Experimental Example Tm (°C) Tagg (°C) 1 66.2 NA 6 64.2 NA 2 70.4 78.7 7 71.2 79.1 3 77.7 75.3 8 78.4 78.0 4 76.0 73.7 9 75.7 73.5 5 75.8 73.8 10 75.5 73.1

[0226] From the above data, it can be seen that the Tm of the sample at pH 4.5 is lower, and no typical aggregation initiation temperature Tagg is observed. The Tm of the sample at pH 5.5 is about 70°C, and the Tm of the sample at pH 6.5 to 8.5 is about 75°C. Therefore, it can be seen that the anti-IL-11 monoclonal antibody HA-I-A is relatively stable in the pharmaceutical preparation at a concentration (10 mg / ml or 80 mg / ml).

[0227] 40±2°C accelerated stability test, the stability test conditions are set as follows:

[0228]

[0229] 40±2°C accelerated stability test, the data are as follows:

[0230]

[0231] Note: In appearance, A represents "colorless and clear liquid, no visible foreign matter"; B represents "slight opalescence"; C represents "no opalescence, slight particles"; D represents "slight opalescence, slight particles", and the same indications are used in the subsequent tables.

[0232] From the above data, it can be seen that after 40±2°C acceleration for 1 week, the purity (SEC-HPLC) of the sample of each experimental example shows a more obvious downward trend with the increase of pH, and the purity of the 80 mg / ml sample is lower than that of the 10 mg / ml sample at the same pH; the main peak purity of the charge isomer of the sample at pH 5.5 and pH 6.5 is higher.

[0233] Based on the results of thermal stability and 40±2°C accelerated stability test, the anti-IL-11 monoclonal antibody HA-I-A has better stability at pH 5.5 to pH 6.5.

[0234] Example 32: Screening of buffer salts

[0235] Based on Example 31, in the pH range of 5.0-6.5, the anti-IL-11 monoclonal antibody HA-I-A with a protein concentration of 40 mg / ml is selected as the pharmacodynamic molecule, different buffer salts are selected, and the anti-IL-11 monoclonal antibody pharmaceutical preparation is prepared, and the thermal stability and 40±2°C accelerated stability are detected. The detection conditions are the same as those in Example 31, and are as follows:

[0236]

[0237] The thermal stability test results are as follows:

[0238] Experimental Example Tm (°C) Tagg (°C) Experimental Example Tm (°C) Tagg (°C) 1 69.9 81.9 7 71.0 80.8 2 71.5 80.7 8 71.9 79.8 3 72.2 80.0 9 71.2 75.8 4 69.5 81.2 10 71.9 74.0 5 72.0 81.4 11 71.3 76.6 6 71.8 81.0 12 71.4 77.2

[0239] The 40±2°C accelerated stability test results are as follows:

[0240]

[0241] Note: A in appearance means "colorless and clear liquid, no visible impurities"; B means "slight opalescence"; C means "no opalescence, slight particles"; D means "slight opalescence, slight particles", and the same in the following table.

[0242] From the above experimental data, after 2 weeks of acceleration at 40±2℃, experimental example 1 to experimental example 6 are colorless and clear liquid, no visible impurities, and experimental example 7 to experimental example 12 all appear slight opalescence; the purity of anti-IL-11 monoclonal antibody HA-I-A protein in histidine salt buffer is better than that in other buffer salts; the purity of anti-IL-11 monoclonal antibody HA-I-A in citrate buffer (pH 6.5) and histidine salt buffer (pH 6.0) is the least, and therefore, according to the experimental results of thermal stability and 40±2℃ acceleration stability, the stability of anti-IL-11 monoclonal antibody HA-I-A in histidine salt buffer at pH 6.0 is better.

[0243] Example 33 Screening of protein protectants and surfactants

[0244] On the basis of the above-mentioned examples 31 and 32, the protein protectants and surfactants are further screened, and the specific experiments are as follows:

[0245]

[0246] 40±2℃ acceleration stability experiment, the experimental conditions and processes refer to examples 31 and 32, and the results are as follows:

[0247] Experimental Example Tm (°C) Tagg (°C) Example Column Tm (°C) Tagg (°C) 1 72.0 79.5 8 68.4 76.4 2 72.3 79.6 9 68.0 76.4 3 72.6 79.1 10 68.0 76.1 4 72.6 79.8 11 67.4 76.3 5 72.3 80.0 12 72.5 79.4 6 71.8 79.5 13 73.5 81.4 7 67.3 78.6 14 72.8 79.7

[0248] 40±2℃ acceleration stability detection results are as follows:

[0249]

[0250] Note: A in appearance means "colorless and clear liquid, no visible impurities"; B means "slight opalescence"; C means "no opalescence, slight particles"; D means "slight opalescence, slight particles", and the same in the following table.

[0251] The results of the thermal stability test show that the Tm of experimental examples 7 to 11 is lower than that of other experimental examples, and the results of the 40±2°C accelerated stability test show that in experimental examples 8-11, when the protein protective agent is selected to be lysine hydrochloride, arginine hydrochloride and sodium chloride, there are slight opalescence at 0 time, and a small amount of particles appear after two weeks of acceleration. In experimental example 6, when the protein protective agent is selected to be proline, a small amount of particles appear after two weeks. The SEC-HPLC purity, charge isomer and CE-SDS purity data show that the protein protective agent selected in experimental examples 1-3 is mannitol, sorbitol or trehalose, which is obviously better than sucrose and glycine selected in experimental examples 4 and 5. Therefore, when the protein protective agent is selected to be mannitol, sorbitol or trehalose, the stability is better, and the application preferably selects mannitol as the protein protective agent in the subsequent examples.

[0252] In addition, from the total particle and particle greater than 25um data in the sub-visible particles, it can be concluded that after the addition of surfactants in experimental examples 12-14, the number of total particles and particles greater than 25um in the sub-visible particles is significantly less than that of other experimental examples. Among the surfactants, the preferred polysorbate 20 in experimental example 13 is better than polysorbate 80 and poloxamer. Therefore, the preferred surfactant is polysorbate 20.

[0253] Example 34: Screening of surfactant content

[0254] According to the screening of the above examples, the anti-IL-11 monoclonal antibody HA-I-A with a protein concentration of 40mg / ml is selected as the pharmacodynamic molecule, 20mM histidine salt buffer (pH6.0) is selected as the formulation buffer salt, sorbitol is selected as the protein protective agent, and polysorbate 20 is selected as the surfactant. The surfactant content is screened by 40±2°C accelerated stability and light stability experiments, and the specific scheme is as follows:

[0255]

[0256] The 40±2°C accelerated stability experiment is placed for 4 weeks, and the experimental conditions and processes are referred to examples 31 and 32, and the results are as follows:

[0257]

[0258] From the above data, it can be concluded that the experimental example 1 has a small amount of particles, and the other experimental example samples are colorless and clear liquids without visible impurities; the total particle number of sub-visible particles of each experimental example sample increases, the particle number of the experimental example 1 sample ≥25 um increases significantly, and the particle number of the experimental example 2-6 sample ≥25 um does not change significantly, but the main peak purity of the charge isomer of the experimental example 6 sample decreases more. Therefore, the content of the surfactant is preferably 0.005%-0.04% w / v polysorbate 20, and an appropriate amount of surfactant is helpful to control the appearance and sub-visible particle performance of the preparation.

[0259] Stability test of pharmaceutical preparation of example 35

[0260] In this example 35, three batches of samples of the pharmaceutical preparation of the anti-IL-11 monoclonal antibody prepared by mixing 20 mM histidine salt buffer, 250 mM mannitol, 0.02% w / v polysorbate 20, anti-IL-11 monoclonal antibody HA-I-A with a pH value of 6 and a concentration of 40 mg / ml are prepared, and the long-term, accelerated and influencing factor stability of the prepared preparation prescription is investigated. The results show that the prepared preparation prescription can effectively improve the appearance (opalescence) and sub-visible particle performance of the protein, and each index meets the requirements of the preparation of the medicine, and can withstand the challenges of various influencing factors during production, storage, transportation and use.

[0261]

[0262] The results of the 25±2°C accelerated stability test and the 2-8°C long-term stability test are as follows:

[0263]

[0264] The results of the stability test under the simulated transportation shaking condition and the light condition are as follows:

[0265]

[0266] In summary, the results of the 25±2°C accelerated stability, the 2-8°C long-term stability, the shaking and light stability under the simulated transportation condition show that each quality attribute does not change significantly or changes within an acceptable range within a certain time under the above several stability conditions, indicating that the pharmaceutical preparation provided by the present application can maintain the physicochemical and biological properties of the anti-IL-11 monoclonal antibody, and meet the effective period of the pharmaceutical preparation and the clinical needs.

[0267] The present application is not limited to the above best mode, anyone under the inspiration of the present application can derive other various forms of products, but regardless of any changes in its shape or structure, as long as it has the same or similar technical solutions as this application, it falls within the scope of the present application.

Claims

1. A pharmaceutical preparation of an anti-IL-1 1 monoclonal antibody, characterized in that, The pharmaceutical preparation comprises an anti-IL-11 monoclonal antibody, a buffer salt, a protein protective agent and a surfactant, wherein the anti-IL-11 monoclonal antibody comprises three heavy chain complementarity determining regions represented by HCDR1, HCDR2 and HCDR3 respectively and three light chain complementarity determining regions represented by LCDR1, LCDR2 and LCDR3 respectively, and the anti-IL-11 monoclonal antibody is: A-III: the amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown as SEQ ID No: 8, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown as SEQ ID No: 9, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown as SEQ ID No: 10, the amino acid sequence of the light chain complementarity determining region LCDR1 is shown as SEQ ID No: 11, the amino acid sequence of the light chain complementarity determining region LCDR2 is shown as SEQ ID No: 12, and the amino acid sequence of the light chain complementarity determining region LCDR3 is shown as SEQ ID No:

13.

2. The pharmaceutical preparation of the anti-IL-11 monoclonal antibody according to claim 1, wherein 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: MA-III: the amino acid sequence of the heavy chain variable region is shown as SEQ ID No: 19, and the amino acid sequence of the light chain variable region is shown as SEQ ID No:

20.

3. The pharmaceutical preparation of the anti-IL-11 monoclonal antibody according to claim 2, wherein The anti-IL-11 monoclonal antibody further comprises a heavy chain constant region and a light chain constant region, and the amino acid sequence of the heavy chain constant region is one of SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25 or SEQ ID No: 26; and the amino acid sequence of the light chain constant region is shown as SEQ ID No:

22.

4. The pharmaceutical preparation of the anti-IL-11 monoclonal antibody according to claim 1, characterized by, The protein concentration of the anti-IL-11 monoclonal antibody is 10-80 mg / ml.

5. The pharmaceutical preparation of the anti-IL-11 monoclonal antibody according to claim 1, characterized by, The content of the buffer salt is 10-40 mM.

6. The pharmaceutical preparation of the anti-IL-11 monoclonal antibody according to claim 5, wherein The buffer salt comprises acetate buffer, phosphate buffer, histidine salt buffer or citrate buffer.

7. The pharmaceutical preparation of the anti-IL-11 monoclonal antibody according to claim 1, wherein The content of the protein protective agent is 150-350 mM.

8. The pharmaceutical preparation of the anti-IL-11 monoclonal antibody according to claim 7, wherein The protein protective agent is one or a combination of sucrose, mannitol, trehalose, sorbitol, glycine, proline, methionine, lysine hydrochloride, arginine hydrochloride or sodium chloride.

9. The pharmaceutical preparation of the anti-IL-11 monoclonal antibody according to claim 1, wherein The content of the surfactant is 0.005%-0.04% w / v.

10. The pharmaceutical preparation of the anti-IL-11 monoclonal antibody according to claim 9, wherein The surfactant is selected from polysorbate 80, polysorbate 20 or poloxamer.

11. The pharmaceutical preparation of the anti-IL-11 monoclonal antibody according to claim 1, wherein The pH value of the pharmaceutical preparation is 5.5-6.5.

Images