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

[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 anti-IL-11 monoclonal antibody aggregates during production, transportation and use, reduces production and transportation costs, improves the physical and chemical properties of anti-IL-11 monoclonal antibody, maintains good biological activity of the antibody, reduces potential safety risks, and ensures 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 to its receptor, and 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 3 It is the map of vector pTSE in embodiment 5 of the present application;

[0013] Figure 4 Figure 4 is a denaturing polyacrylamide gel electrophoresis chart of the mouse-derived antibody molecules in Example 5 of the present application;

[0014] Figure 5 Figure 5 is a comparison chart of the binding ability of the mouse-derived antibody molecules to IL-11 in Example 6 of the present application;

[0015] Figure 6 Figure 6 is a comparison chart of the competitive inhibition experiment of the mouse-derived antibody to IL-11 receptor protein IL-11RA in Example 7 of the present application;

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

[0017] Figure 8 Figure 8 is a comparison chart of the inhibition of the secretion of TIMP-1 by embryonic lung fibroblast MRC-5 by the mouse-derived antibody in Example 9 of the present application;

[0018] Figure 9 Figure 14 is a denaturing polyacrylamide gel electrophoresis chart of the humanized antibody molecules in Example 14 of the present application;

[0019] Figure 10 Figure 15 is a comparison chart of the binding ability of the humanized antibody molecules to IL-11 in Example 15 of the present application;

[0020] Figure 11 Figure 16 is a comparison chart of the inhibition of IL-11 binding to IL-11RA receptor on the surface of BaF / 3-IL-11RA cells by the humanized antibody molecules in Example 16 of the present application;

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

[0022] Figure 13 Figure 18 is a comparison chart of the biological activity detection (reporter gene) of the humanized antibody molecules in Example 18 of the present application;

[0023] Figure 14 Figure 19 is a comparison chart of the inhibition of the secretion of TIMP-1 by embryonic lung fibroblast MRC-5 by the humanized antibody molecules in Example 19 of the present application;

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

[0025] Figure 16Figure for the change of the ratio of lung weight to body weight in the lung fibrosis model of mice in Example 21 of the present application;

[0026] Figure 17 Figures for hematoxylin-eosin (HE) staining and Masson staining of lung tissue sections in the lung fibrosis model of mice in Example 21 of the present application;

[0027] Figure 18 Figure for the change of the ratio of heart weight to body weight in the heart fibrosis model of mice in Example 22 of the present application;

[0028] Figure 19 Figures for hematoxylin-eosin (HE) staining and Masson staining of heart tissue sections in the heart fibrosis model of mice in Example 22 of the present application;

[0029] Figure 20 Figure for the content of urinary protein in the kidney fibrosis model of mice in Example 23 of the present application;

[0030] Figure 21 Figures for hematoxylin-eosin (HE) staining and Masson staining of kidney tissue sections in the kidney fibrosis model of mice in Example 23 of the present application;

[0031] Figure 22 Figure for the change of liver weight in the liver fibrosis model of mice in Example 24 of the present application;

[0032] Figure 23 Figures for the change of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in serum of mice in the liver fibrosis model of mice in Example 24 of the present application;

[0033] Figure 24 Figures for hematoxylin-eosin (HE) staining and Masson staining of liver tissue sections in the liver fibrosis model of mice in Example 24 of the present application;

[0034] Figure 25 Figure for the thermal stability evaluation of anti-IL-11 monoclonal antibody HA-I-A in Example 25 of the present application. DETAILED DESCRIPTION

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

[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 Type (amino acid sequence as shown in SEQ ID NO:30). HEK293E cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number: GNHu43) were transiently transfected to express antibodies, yielding chimeric antibodies CA-I and CA-II.

[0116] Example 12 Humanization of mouse antibody molecules

[0117] First, the sequences of the murine antibody molecules MA-I and MA-II from Example 2 were compared with the human antibody lineage database (v-base) to identify human antibody light and heavy chain lineages with high homology as candidate sequences. Then, the CDR sequences of the murine antibody molecules MA-I and MA-II were transplanted onto the human candidate sequences for homology modeling. Next, three-dimensional structural simulations were used to calculate key framework amino acid residues that might play an important role in maintaining the CDR ring structure, thereby designing reversion mutations for humanized antibodies. The light and heavy chain variable region sequences of the designed humanized antibodies containing reversion mutations were optimized and synthesized by Nanjing Genscript Biotech Co., Ltd., and then ligated into a transient expression vector. Analysis of the humanized light and heavy chain combinations yielded the following humanized anti-IL-11 monoclonal antibody molecules: HA-IA, HA-IB, HA-IC, and HA-ID for MA-I; and the following humanized antibody molecules: HA-II-A, HA-II-B, HA-II-C, and HA-II-D for MA-II. The eight monoclonal antibody sequences selected above are as follows:

[0118]

[0119] Specifically, SEQ ID No:31 (amino acid sequences of the heavy chain variable regions of HA-IA, HA-IC, HA-II-A, and HA-II-B):

[0120] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVSS PEAMDYWGQGTLVTVSS;

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

[0122] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK; SEQ ID No:33 (Amino acid sequences of the heavy chain variable regions of HA-IB and HA-II-C):

[0123] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVST ISDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVS SPEAMDYWGQGTLVTVSS;

[0124] SEQ ID No:34 (Amino acid sequence of the light chain variable region of HA-IB):

[0125] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;SEQ ID No:35 (Amino acid sequence of the light chain variable region of HA-IC):

[0126] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK; SEQ ID No:36 (Amino acid sequences of the heavy chain variable regions of HA-ID and HA-II-D):

[0127] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAMYYCARDGGYVS SPEAMDYWGQGTSVTVSS;

[0128] SEQ ID No:37 (Amino acid sequence of the light chain variable region of HA-ID):

[0129] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;SEQ ID No:38 (Amino acid sequence of the light chain variable region of HA-II-A):

[0130] DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK; SEQ ID No:39 (Amino acid sequences of the light chain variable regions of HA-II-B and HA-II-C):

[0131] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;

[0132] SEQ ID No:40 (Amino acid sequence of the light chain variable region of HA-II-D):

[0133] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGTVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPWTFGGGTKVEIK.

[0134] Example 13

[0135] Based on Example 12, Example 13 of the present invention further defines the human antibody constant region as including a human antibody heavy chain constant region and a human antibody light chain constant region. The amino acid sequence of the human antibody heavy chain constant region is one of SEQ ID No:27, SEQ ID No:28 or SEQ ID No:29; the amino acid sequence of the human antibody light chain constant region is shown in SEQ ID No:30.

[0136] The specific sequence of the constant region of the human antibody described above is the same as that in Example 10.

[0137] Example 14 Preparation of humanized antibody molecules

[0138] Example 14 of the present invention further defines the constant region of the human antibody, based on Example 13, as including the heavy chain constant region of human IgG1 (whose amino acid sequence is shown in SEQ ID No: 27) and human C k The light chain constant region of type (its amino acid sequence is shown in SEQ ID No:30).

[0139] The heavy chain VH and light chain VL encoding genes of the eight humanized anti-IL-11 monoclonal antibody molecules HA-IA, HA-IB, HA-IC, HA-ID, HA-II-A, HA-II-B, HA-II-C, and HA-II-D obtained in Example 12 were cloned into the vector pTSE (e.g., ...) containing the heavy chain constant region and light chain constant region genes, respectively. Figure 3 As shown), the heavy chain constant region is human IgG1 type (amino acid sequence as shown in SEQ ID NO:27), and the light chain constant region is C. k Chain (amino acid sequence as shown in SEQ ID NO:30).

[0140] Two chimeric antibodies, CA-I and CA-II, obtained in Example 11, and eight humanized antibody molecules, HA-IA, HA-IB, HA-IC, HA-ID, HA-II-A, HA-II-B, HA-II-C, and HA-II-D, obtained in Example 12, were transiently transfected into HEK293 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number GNHu43) for antibody expression. Monoclonal antibodies were purified using an AKTA instrument via a protein A affinity column. Protein concentration was determined using a BCA kit (purchased from Beijing Huitian Oriental Technology Co., Ltd., catalog number BCA0020). Protein size was then identified by SDS-PAGE. The results are shown below. Figure 9 As shown, from left to right, the molecular weights of the non-reduced proteins are HA-IA, HA-IB, HA-IC, HA-ID, the chimeric antibody CA-I prepared in Example 11, the molecular weight marker of the reduced protein, HA-II-A, HA-II-B, HA-II-C, HA-II-D, and the chimeric antibody CA-II. The molecular weight of each band is consistent with the theoretical value.

[0141] Example 15: Experiment on the binding of humanized antibody molecules to IL-11

[0142] IL-11 antigen was coated with carbonate buffer (pH 9.6) at a concentration of 100 ng / well / 100 μL overnight at 4°C. The cells were washed five times with 300 μL / well PBST, and then blocked with 280 μL / well of 1% BSA-PBST at 37°C for 1 h. Humanized antibodies HA-IA, HA-IB, HA-IC, HA-ID, HA-II-A, HA-II-B, HA-II-C, HA-II-D and chimeric antibodies CA-I and CA-II prepared in Example 11 were diluted with 1% BSA-PBST. The initial concentration of all humanized antibodies was 10 μg / mL, and eight 5-fold serial dilutions were performed, incubating at 37°C for 1 h. Wash five times with 300 μL / well PBST, then add goat anti-human IgG Fab HRP (Invitrogen, catalog number: 31482) diluted 1:5000 with 1% BSA-PBST, and incubate at 37°C for 1 h. Develop color with a TMB chromogenic kit, 100 μL / well, at room temperature for 5 min, then stop the development with 2M H2SO4. Read the values ​​at 450 nm / 630 nm using a microplate reader and calculate the corresponding EC50 values. Specific data are as follows:

[0143]

[0144]

[0145] Based on the above data and experimental results, as follows: Figure 10 As shown, all eight different humanized antibody molecules can bind to IL-11. The EC50 values ​​of humanized antibody molecules HA-IA, HA-IB, HA-IC, and HA-ID are close to those of chimeric antibody CA-I, while the EC50 values ​​of humanized antibody molecules HA-II-A, HA-II-B, HA-II-C, and HA-II-D are close to those of chimeric antibody CA-II. This indicates that the humanized antibody molecules retain the high binding capacity of the murine parental antibodies MA-I and MA-II to IL-11.

[0146] Example 16: Humanized antibody molecules inhibit the binding of IL-11 to the IL-11RA receptor on the cell surface of BaF / 3-IL-11RA.

[0147] Four humanized antibody molecules (HA-IA, HA-IB, HA-IC, and HA-ID) with superior protein-level binding activity were selected for cell viability evaluation experiments. BaF / 3-IL-11RA cell lines were counted, and a certain number of cells were collected, centrifuged, resuspended in PBS buffer, and the cell density was adjusted to 1E+6 cells / mL. 100 μL / well was added to each well of a 96-well plate. IL-11-mFc ligand protein was diluted with PBS to a concentration of 18 μg / mL, and 50 μL / well was added to the corresponding well of the 96-well plate containing BaF / 3-IL-11RA cells. After gentle mixing, the 96-well plates were incubated at 4°C for 1 h. Four humanized antibody molecules, HA-IA, HA-IB, HA-IC, and HA-ID, were serially diluted with PBS buffer to prepare an initial concentration of 800 μg / mL. Ten 3-fold serial dilutions were performed, with 50 μL / well added to the corresponding wells of a 96-well plate containing a mixture of BaF / 3-IL-11RA cells and IL-11-mFc ligand protein. After thorough mixing, the plates were incubated at 4°C for 2 h. After incubation, the cells were washed once with PBS buffer at 3000 rpm, and the cell pellet was collected. Pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) was added to the cell pellet, and the plates were incubated at 4°C for 30 min. After washing once with PBS buffer at 3000 rpm, the cells were resuspended in 100 μL of PBS and analyzed by flow cytometry, collecting the fluorescence signal in the FL1-A channel. Dose-response curves were plotted, and the corresponding IC50 values ​​were calculated. Specific data are as follows:

[0148]

[0149] Based on the above data and Figure 11 It can be seen that all four humanized candidate molecules screened can inhibit the binding of IL-11 ligand protein to the IL-11RA receptor on the surface of BaF / 3-IL-11RA cells.

[0150] Example 17: Humanized antibody molecules inhibit the binding of IL-11 to the GP130 receptor on the surface of BaF / 3-GP130 cells.

[0151] BaF / 3-GP130 cell lines were counted, and a certain number of cells were centrifuged, resuspended in PBS buffer, and the cell density was adjusted to 1E+6 cells / mL. 100 μL / well was added to each well of a 96-well plate. IL-11-mFc ligand protein was diluted with PBS to a concentration of 12 μg / mL, and 50 μL / well was added to the corresponding well of a 96-well plate containing BaF / 3-GP130 cells. After gentle mixing, the 96-well plates were incubated at 4°C for 1 h. Four human antibody molecules, HA-IA, HA-IB, HA-IC, and HA-ID, were serially diluted with PBS buffer to an initial concentration of 2000 μg / mL. Ten 2-fold serial dilutions were performed, and 50 μL / well was added to the corresponding well of a 96-well plate containing BaF / 3-GP130 cells and IL-11-mFc ligand protein. After thorough mixing, the 96-well plates were incubated at 4°C for 2 h. After incubation, cells were washed once with PBS buffer at 3000 rpm and the cell pellet was collected. 100 μL / well of pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) was added to the cell pellet and incubated at 4°C for 30 min. After washing once with PBS buffer at 3000 rpm, cells were resuspended in PBS buffer at 100 μL / well and analyzed by flow cytometry, collecting fluorescence signals in the FL1-A channel. Dose-response curves were plotted, and the corresponding IC50 values ​​were calculated. Specific data are as follows:

[0152]

[0153] Based on the above data and Figure 12 It can be seen that all four humanized candidate molecules screened can block the binding of IL-11 ligand protein to the GP130 receptor on the surface of BaF / 3-GP130 cells.

[0154] Example 18: Detection of the biological activity of humanized antibody molecules (reporter gene)

[0155] The BaF / 3-IL-11RA-GP130-STAT3-Luc engineered cell line was counted. The cell density was adjusted to 2E+6 cells / mL using sample diluent (containing 90% IMDM, 10% FBS, and 10 ng / mL mouseIL-3). After gentle mixing, the cell culture was added to 96-well plates at 50 μL / well. Four humanized antibody molecules, HA-IA, HA-IB, HA-IC, and HA-ID, were diluted to an initial concentration of 200 μg / mL using sample diluent. A 5-fold serial dilution was performed, resulting in 10 serial dilutions, at 100 μL / well, and added to the corresponding wells of the 96-well plates containing the engineered cell line. Two replicates were set for each sample concentration. IL-11 protein was prepared at a concentration of 10 μg / mL using sample diluent, at 50 μL / well, and added to the 96-well plates containing the engineered cell line and humanized antibody molecules. The cell culture plates were gently mixed and incubated at 37°C in a CO2 incubator for 6 hours. Centrifuge and discard the supernatant. Add lysis buffer, 10 μL / well to a 384-well plate, and add an equal volume of luciferase reaction substrate (purchased from Promega Biotechnology Co., Ltd., catalog number E2610). Incubate at room temperature for 5 min, read the fluorescence values ​​using a microplate reader, and calculate the corresponding IC50 values. Specific data are as follows:

[0156]

[0157] Based on the above data and Figure 13 As shown, the four humanized antibody molecules screened can block the binding of IL-11 to IL-11RA and GP130 receptors, and inhibit the transduction of the signaling pathway.

[0158] Example 19: Humanized antibody molecules inhibit TIMP-1 secretion by MRC-5 embryonic lung fibroblasts.

[0159] After trypsin digestion, MRC-5 embryonic lung fibroblasts were counted. A certain number of cells were collected, centrifuged, and resuspended in MEM complete medium. The cell density was adjusted to 2E+5 cells / mL, 100 μL / well, and added to 96-well plates. IL-11-mFc ligand protein was diluted in MEM complete medium to a concentration of 16 μg / mL, 50 μL / well, and added to the corresponding 96-well plates. Four humanized antibody molecules, HA-IA, HA-IB, HA-IC, and HA-ID, were serially diluted in MEM complete medium at an initial concentration of 40 μg / mL, with 3-fold serial dilutions for a total of 8 gradients, 50 μL / well, and added to 96-well plates containing cell suspension and IL-11-mFc ligand protein suspension. The mixture was gently mixed and incubated overnight at 37°C in a CO2 incubator (approximately 20 h). The cell culture supernatant was then used for detection using a TIMP-1 ELISA kit (method as in Example 9). The microplate reader was used to read the values ​​at 450 nm, and the corresponding IC50 values ​​were calculated. The specific data are as follows:

[0160]

[0161] Based on the above data and Figure 14 It can be seen that the four humanized antibody molecules screened can effectively inhibit the release of TIMP-1 from human embryonic lung fibroblasts MRC-5 stimulated by IL-11 ligand protein.

[0162] Example 20: Cross-binding experiment of humanized antibody molecules with IL-11 from different species

[0163] Humanized antibody HA-IA, which exhibits superior protein level and functional activity, was selected for cross-binding assays with IL-11 from different species. Human IL-11, mouse IL-11 (purchased from Beijing Sinocare Medical Technology Co., Ltd., catalog number: 50117-MNCE), rat IL-11 (purchased from Kanglang Biotechnology, catalog number: KL40001Ra), and cynomolgus monkey IL-11 (purchased from Sinocare Medical Technology, catalog number: 90925-CNCE) were coated with 100 ng / well / 100 μL of pH 9.6 carbonate buffer and incubated overnight at 4°C. After washing five times with 300 μL / well PBST, 280 μL / well of 1% BSA-PBST was added, and the mixture was blocked at 37°C for 1 h. Humanized antibody HA-IA was diluted with 1% BSA-PBST to an initial concentration of 50 μg / mL. A five-fold serial dilution was performed for a total of nine dilutions, with two replicates per dilution. 100 μL / well was added to each well of a 96-well plate and incubated at 37°C for 1 h. The plate was then washed five times with 300 μL / well of PBST. Goat anti-Human IgG Fab HRP (purchased from Invitrogen, catalog number: 31482) was diluted with 1% BSA-PBST to a working solution concentration of 1:5000. 100 μL / well was added to each well of a 96-well plate and incubated at 37°C for 1 h. The plate was then washed five times with 300 μL / well of PBST. The plate was then developed using a TMB chromogenic assay kit, 100 μL / well, at room temperature in the dark for 5 min, followed by termination of the development with 2M H₂SO₄. Readings were taken at 450 nm and 630 nm using a microplate reader, and the corresponding EC50 values ​​were calculated. Specific data are as follows:

[0164]

[0165] Based on the above data and as follows Figure 15 As shown, the humanized antibody molecule HA-IA can bind to human IL-11, mouse IL-11, rat IL-11, and cynomolgus monkey IL-11, and the affinity is high.

[0166] Example 21: Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody against pulmonary fibrosis

[0167] The therapeutic effect of the anti-IL-11 monoclonal antibody HA-IA on pulmonary fibrosis was studied using bleomycin (bLF) modeling.

[0168] Animal species: C57BL / 6J mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.)

[0169] Number, sex and age of mice: 6 mice / group, male, 6-8 weeks old;

[0170] The control group received only saline injections;

[0171] In the treatment group, HA-IA antibody molecules were injected twice a week for 4 weeks.

[0172] Animal weight was measured weekly, and any abnormalities were observed. Organ weight detection: lung organs were collected, their weight was measured, and the lung-to-body weight ratio was calculated. Lung pathological examination: lung sections were examined and the degree of pulmonary fibrosis was observed using hematoxylin and eosin (HE) and Masson staining.

[0173] The results are as follows Figure 16 As shown, after administration of HA-IA antibody molecules, the lung-to-body weight ratio of mice in the treatment group was significantly lower than that in the control group; the results are as follows. Figure 17 As shown, compared with the control group, the lung slices of the treatment group showed a significant reduction in pulmonary fibrosis, which indicates that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the production of pulmonary fibrosis.

[0174] Example 22: Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody against cardiac fibrosis

[0175] The therapeutic effect of the anti-IL-11 monoclonal antibody HA-IA on cardiac fibrosis was studied using isoproterenol modeling.

[0176] Animal species: C57BL / 6J mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.)

[0177] Number, sex and age of mice: 6 mice / group, male, 6-8 weeks old;

[0178] The control group received only saline injections;

[0179] The treatment group received two injections of HA-IA antibody molecules per week for four weeks.

[0180] Animal weight was measured weekly, and any abnormalities were observed. Organ weight assessment: Hearts were collected, their weight was measured, and the heart-to-body weight ratio was calculated. Cardiac pathology assessment: Heart sections were prepared and stained with hematoxylin and eosin (HE) and Masson's solution to observe the degree of cardiac fibrosis.

[0181] The results are as follows Figure 18 As shown, the heart-to-body weight ratio of mice in the treatment group was significantly smaller than that in the control group; the results are as follows. Figure 19 As shown, compared with the control group, the heart slices of the treatment group showed a significant reduction in cardiac fibrosis, which indicates that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the production of cardiac fibrosis.

[0182] Example 23: Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody against renal fibrosis

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

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

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

[0186] The control group received only saline injections;

[0187] The treatment group received two injections of HA-IA antibody molecules per week for four weeks.

[0188] Animal weight was measured weekly, and any abnormalities were observed. Organ weight testing included collecting the heart, measuring the weight of the kidneys, and testing for urinary protein levels. Kidney pathology testing involved preparing kidney sections and staining them with hematoxylin and eosin (HE) and Masson's solution to observe the degree of kidney fibrosis.

[0189] The results are as follows Figure 20 As shown, compared with the control group, the urinary protein content in the kidneys of mice in the treatment group was significantly lower than that in the control group; the results are as follows. Figure 21 As shown, compared with the control group, the kidney slices of the treatment group showed a significant reduction in renal fibrosis, which indicates that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the production of renal fibrosis.

[0190] Example 24: Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody against liver fibrosis

[0191] The therapeutic effect of anti-IL-11 monoclonal antibody HA-IA on liver fibrosis was studied using CCl4 modeling.

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

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

[0194] The control group received only saline injections;

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

[0196] Weight monitoring: Animal weight was measured weekly, and any abnormalities were observed; Liver pathology examination: Liver sections were stained with hematoxylin and eosin (HE) and masson stain to observe the degree of liver fibrosis; Organ weight detection: Kidneys were collected, liver weight was measured, and HE staining was performed; Serum detection: Serum was collected, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum were detected.

[0197] 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.

[0198] Example 25: Thermostability Assessment of Anti-IL-11 Monoclonal Antibody HA-IA

[0199] 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.

[0200]

[0201] 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.

[0202] Example 26

[0203] 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.

[0204] Example 27

[0205] 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;

[0206] 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.

[0207] Example 28

[0208] Based on Example 1, Example 28 of the present invention provides a pharmaceutical formulation of an anti-IL-11 monoclonal antibody, wherein the content of the protein protectant is 150-350 mM;

[0209] It should be further noted that protein protectants include, but are not limited to, one or more combinations of sucrose, mannitol, trehalose, sorbitol, glycine, proline, methionine, lysine hydrochloride, arginine hydrochloride, or sodium chloride.

[0210] Example 29

[0211] Based on Example 1, Example 29 of the present invention provides a pharmaceutical formulation of an anti-IL-11 monoclonal antibody, wherein the content of surfactant is 0.005%-0.04% w / v;

[0212] It should be further noted that the surfactant is selected from polysorbate 80, polysorbate 20 or poloxamer.

[0213] Example 30

[0214] Based on Example 1, Example 30 of the present invention provides a pharmaceutical preparation of an anti-IL-11 monoclonal antibody, wherein the pH value of the pharmaceutical preparation is 5.5-6.5.

[0215] Example 31 Selection of pH range for pharmaceutical preparations

[0216] Based on Example 1, the anti-IL-11 monoclonal antibody HA-IA was selected as the pharmacologically active molecule to prepare the drug formulation. The preparation method included: preparing 20mM acetate-sodium acetate buffer (pH 4.5, 5.5) and 20mM phosphate buffer (pH 6.5, 7.5, and 8.5), respectively; ultrafiltration of different concentrations of anti-IL-11 monoclonal antibody HA-IA into buffers of different pH values; sterilization filtration using a 0.22μm filter; and aliquoting into 2ml vials, 1ml / vial. After aliquoting, the protein thermostability under different buffer conditions was tested. Then, the samples were placed in a stability test chamber at 40±2℃ for 2 weeks, and the stability was investigated for appearance, pH, protein concentration, purity, charge isomers, subvisible particles, viscosity, and particle size.

[0217] The specific design groups are as follows:

[0218]

[0219] The thermal stability test results are as follows:

[0220] 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

[0221] The data above show that the samples at pH 4.5 have a low Tm and do not exhibit the typical aggregation initiation temperature Tagg. The Tm of the samples at pH 5.5 is around 70℃, and the Tm of the samples at pH 6.5 to 8.5 is around 75℃. This indicates that the anti-IL-11 monoclonal antibody HA-IA is relatively stable in drug formulations at concentrations (10 mg / ml or 80 mg / ml).

[0222] Accelerated stability test at 40±2℃: The stability test conditions are set as follows:

[0223]

[0224] The accelerated stability test at 40±2℃ yielded the following data:

[0225]

[0226] Note: In terms of appearance, A indicates "colorless and clear liquid with no visible foreign matter"; B indicates "slight opalescence"; C indicates "no opalescence or slight particles"; and D indicates "slight opalescence or slight particles". The same meaning applies to subsequent tables.

[0227] The data above show that after acceleration at 40±2℃ for 1 week, the purity of each sample (SEC-HPLC) decreased more significantly with increasing pH. The purity of the 80 mg / ml sample was lower than that of the 10 mg / ml sample at the same pH. The samples with pH 5.5 and pH 6.5 had higher purity of the charge isomer main peak.

[0228] Based on the combined results of thermal stability and accelerated stability tests at 40±2℃, the anti-IL-11 monoclonal antibody HA-IA exhibits good stability at around pH 5.5 to pH 6.5.

[0229] Example 32 Screening of buffer salts

[0230] Based on Example 31, within the pH range of 5.0-6.5, an anti-IL-11 monoclonal antibody HA-IA with a protein concentration of 40 mg / ml was selected as the pharmacodynamic molecule. Different buffer salts were selected to prepare anti-IL-11 monoclonal antibody drug formulations. Thermostability and accelerated stability at 40±2℃ were tested under the same testing conditions as in Example 31, as follows:

[0231]

[0232] The thermal stability test results are as follows:

[0233]

[0234] The accelerated stability test results at 40±2℃ are as follows:

[0235]

[0236] Note: In terms of appearance, A indicates "colorless and clear liquid with no visible foreign matter"; B indicates "slight opalescence"; C indicates "no opalescence or slight particles"; and D indicates "slight opalescence or slight particles". The same meaning applies to subsequent tables.

[0237] Based on the above experimental data, after two weeks of accelerated testing at 40±2℃, Experiments 1 to 6 were all colorless and clear liquids without visible foreign matter, while Experiments 7 to 12 all showed slight opalescence. The purity of the anti-IL-11 monoclonal antibody HA-IA protein in histidine buffer was better than that in other buffer salts. The purity of the main peak of the charge isomer of the anti-IL-11 monoclonal antibody HA-IA decreased the least in citrate buffer (pH 6.5) and histidine buffer (pH 6.0). Therefore, considering the experimental results of thermal stability and accelerated stability at 40±2℃, the anti-IL-11 monoclonal antibody HA-IA showed better stability in histidine buffer at pH 6.0.

[0238] Example 33 Screening of protein protectants and surfactants

[0239] Based on the above embodiments 31 and 32, this invention further screens protein protectants and surfactants, with specific experimental examples as follows:

[0240]

[0241] Accelerated stability testing was conducted at 40±2℃, with experimental conditions and procedures as described in Examples 31 and 32. The results are as follows: Thermal stability test results are as follows:

[0242] 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

[0243] The accelerated stability test results at 40±2℃ are as follows:

[0244]

[0245] Note: In terms of appearance, A indicates "colorless and clear liquid with no visible foreign matter"; B indicates "slight opalescence"; C indicates "no opalescence or slight particles"; and D indicates "slight opalescence or slight particles". The same meaning applies to subsequent tables.

[0246] Thermal stability tests showed that the Tm values ​​of Experiments 7 to 11 were lower than those of the other Experiments. Accelerated stability tests at 40±2℃ showed that in Experiments 8-11, when lysine hydrochloride, arginine hydrochloride, and sodium chloride were used as protein protectants, slight opalescence was observed at 0°C, and a small number of particles appeared after two weeks of accelerated testing. In Experiment 6, when proline was used as the protein protectant, a small number of particles appeared starting in the second week. SEC-HPLC purity, charge isomer, and CE-SDS purity data showed that mannitol, sorbitol, or trehalose were significantly superior to sucrose and glycine in Experiments 4 and 5 when used as protein protectants. Therefore, mannitol, sorbitol, or trehalose provides better stability, and mannitol is preferred as the protein protectant in subsequent embodiments.

[0247] Furthermore, based on the data of total particles and particles larger than 25 μm in the subvisible microparticles, it can be concluded that after adding surfactants in Experiments 12-14, the total number of particles and particles larger than 25 μm in the subvisible microparticles were significantly less than in other Experiments. At the same time, among the surfactants, the polysorbate 20 provided in Experiment 13 is preferred over polysorbate 80 and poloxamer. Therefore, the preferred surfactant is polysorbate 20.

[0248] Example 34 Screening of surfactant content

[0249] Based on the screening in the above embodiments, the anti-IL-11 monoclonal antibody HA-IA with a protein concentration of 40 mg / ml was selected as the pharmacodynamic molecule, 20 mM histidine salt buffer (pH 6.0) was selected as the formulation buffer, sorbitol was selected as the protein protectant, and polysorbate 20 was selected as the surfactant. These were added to the formulation, and the surfactant content was screened through accelerated stability and photostability experiments at 40±2℃. The specific scheme is as follows:

[0250]

[0251] The accelerated stability test was conducted at 40±2℃ for 4 weeks. The experimental conditions and procedures were the same as in Examples 31 and 32. The results are as follows:

[0252]

[0253] The data above show that after acceleration at 40±2℃ for 4 weeks, Experiment 1 contained a small amount of particles, while the other experimental samples were colorless and clear liquids without visible foreign matter. The total number of sub-visible particles increased in all experimental samples. The number of particles ≥25µm in Experiment 1 increased significantly, while the number of particles ≥25µm in Experiments 2-6 did not change significantly. However, the purity of the charge isomer main peak in Experiment 6 decreased significantly. Therefore, the surfactant content is preferably 0.005%-0.04% w / v polysorbate 20. An appropriate amount of surfactant helps control the appearance and sub-visible particle performance of the formulation.

[0254] Example 35 Stability testing of pharmaceutical formulations

[0255] In Example 35, three identical batches of the anti-IL-11 monoclonal antibody formulation (prepared by mixing 20 mM histidine salt buffer, 250 mM mannitol, 0.02% w / v polysorbate 20, pH 6, and 40 mg / ml anti-IL-11 monoclonal antibody HA-IA) were prepared using the formulation obtained in the above examples. Long-term, accelerated, and influencing factor stability studies were conducted on the proposed formulation. The results showed that the proposed formulation effectively improved the appearance (opalescent) and sub-visible microparticle characteristics of the protein. All indicators met the requirements for drug-likeness and could maintain the formulation's stability during production, storage, transportation, and use.

[0256] The process involves withstanding the challenges of various influencing factors.

[0257]

[0258] The results of the stability tests after 12 months at 2-8℃ and accelerated stability tests at 25±2℃ are as follows:

[0259]

[0260] The stability test results under simulated transportation conditions of vibration and illumination are as follows:

[0261]

[0262] In summary, the results of accelerated stability at 25±2℃, long-term stability at 2-8℃, and stability under simulated shaking and light exposure during transportation show that, under the above stability conditions, no significant changes or changes were observed in any of the quality attributes within an acceptable range after a certain period of time. This indicates that the pharmaceutical formulation provided by this invention can maintain the physicochemical and biological characteristics of the anti-IL-11 monoclonal antibody, meeting the shelf life and clinical requirements of pharmaceutical formulations.

[0263] This invention is not limited to the preferred embodiments described above. Anyone can derive other products in various forms under the guidance of this invention. However, regardless of any changes in shape or structure, any technical solution that is the same as or similar to this application falls within the protection scope of this invention.

Claims

1. A pharmaceutical formulation of an anti-IL-11 monoclonal antibody, characterized in that, The pharmaceutical formulation comprises an anti-IL-11 monoclonal antibody, a buffer salt, a protein protectant, and a surfactant. The anti-IL-11 monoclonal antibody includes three heavy chain complementarity-determining regions (CMRs) represented by HCDR1, HCDR2, and HCDR3, and three light chain CMRs represented by LCDR1, LCDR2, and LCDR3, respectively. The anti-IL-11 monoclonal antibody is: A-II: The amino acid sequence of the heavy chain complementarity-determining region HCDR1 is shown in SEQ ID No:1, the amino acid sequence of the heavy chain complementarity-determining region HCDR2 is shown in SEQ ID No:2, the amino acid sequence of the heavy chain complementarity-determining region HCDR3 is shown in SEQ ID No:3, the amino acid sequence of the light chain complementarity-determining region LCDR1 is shown in SEQ ID No:4, the amino acid sequence of the light chain complementarity-determining region LCDR2 is shown in SEQ ID No:5, and the amino acid sequence of the light chain complementarity-determining region LCDR3 is shown in SEQ ID No:

7.

2. The pharmaceutical formulation of the anti-IL-11 monoclonal antibody as described in claim 1, characterized in that, The anti-IL-11 monoclonal antibody further includes a heavy chain variable region and a light chain variable region, and the anti-IL-11 monoclonal antibody is as follows: 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.

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

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

22.

4. The pharmaceutical formulation of the anti-IL-11 monoclonal antibody as described in claim 1, characterized in that, The anti-IL-11 monoclonal antibody further includes a heavy chain variable region and a light chain variable region, and the anti-IL-11 monoclonal antibody is selected from any one of the following: 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.

5. The pharmaceutical formulation of the anti-IL-11 monoclonal antibody as described in claim 4, characterized in that, The anti-IL-11 monoclonal antibody further 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.

6. The pharmaceutical formulation of the anti-IL-11 monoclonal antibody as claimed in claim 1, characterized in that, The protein concentration of the anti-IL-11 monoclonal antibody is 10-80 mg / ml.

7. The pharmaceutical formulation of the anti-IL-11 monoclonal antibody as claimed in claim 1, characterized in that, The content of the buffer salt is 10-40 mM.

8. The pharmaceutical formulation of the anti-IL-11 monoclonal antibody as claimed in claim 7, characterized in that, The buffer salts include acetate buffer, phosphate buffer, histidine buffer, or citrate buffer.

9. The pharmaceutical formulation of the anti-IL-11 monoclonal antibody as described in claim 1, characterized in that, The protein protectant content is 150-350 mM.

10. The pharmaceutical formulation of the anti-IL-11 monoclonal antibody as described in claim 9, characterized in that, The protein protectant is one or a combination of sucrose, mannitol, trehalose, sorbitol, glycine, proline, methionine, lysine hydrochloride, arginine hydrochloride, or sodium chloride.

11. The pharmaceutical formulation of the anti-IL-11 monoclonal antibody as described in claim 1, characterized in that, The surfactant content is 0.005%-0.04% w / v.

12. The pharmaceutical formulation of the anti-IL-11 monoclonal antibody as described in claim 11, characterized in that, The surfactant is selected from polysorbate 80, polysorbate 20, or poloxamer.

13. The pharmaceutical formulation of the anti-IL-11 monoclonal antibody as described in claim 1, characterized in that, The pH value of the pharmaceutical preparation is 5.5-6.5.

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