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] This invention relates to the field of biomedical technology, and in particular to a pharmaceutical formulation of an anti-IL-11 monoclonal antibody. Background Technology

[0002] Fibrosis can occur in various organs, such as the lungs, liver, kidneys, blood vessels, pancreas, and skin. The main pathological changes after fibrosis occur are an increase in fibrous connective tissue and a significant decrease in parenchymal cells within the organ tissues. The more severe the fibrosis, the more likely it is that the tissues and organs are gradually hardening. In severe cases, it may lead to structural damage to the organ, decreased organ function, and even organ failure. Clinically, fibrosis can cause tissue cell damage, degeneration, and necrosis. Common examples include pulmonary fibrosis, liver fibrosis, heart disease, kidney disease, and asthma.

[0003] IL-11 (interleukin-11) is a pleiotropic cytokine and a member of the IL-6 cytokine family, sharing the same signal transduction receptor subunit GP130. This family plays a crucial role in the occurrence, development, and metastasis of tumors. Studies have found that IL-11 transmits signals to tumor cells through the GP130 signaling pathway, enabling tumor cells to acquire signals for proliferation and activation. Blocking this signaling pathway could be an effective treatment for various tumors, chronic fibrosis, and inflammatory diseases. Therefore, the development of anti-IL-11 monoclonal antibodies has significant clinical implications.

[0004] During the development of anti-IL-11 monoclonal antibodies, it was discovered that, like most protein molecules, the complex structure of anti-IL-11 monoclonal antibodies is easily affected by various factors during production, transportation, and use, leading to aggregation, denaturation, or degradation. The structural stability of antibodies not only affects their biological activity but also the safety of biopharmaceuticals. In particular, some protein aggregates can trigger immune responses in the human body, which may reduce the efficacy of biopharmaceuticals or even cause death in severe cases. High-concentration antibody drugs not only require high-purity products during production but also need to maintain structural stability during transportation, storage, and use. Therefore, high-concentration proteins pose challenges to the performance and stability of aggregates and particulate matter. To meet the needs of fibrosis patients, there is an urgent need to develop drug formulations of anti-IL-11 monoclonal antibodies with better stability. Summary of the Invention

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

[0006] The specific technical solution of this invention is as follows:

[0007] This invention provides a pharmaceutical formulation of an anti-IL-11 monoclonal antibody, comprising an anti-IL-11 monoclonal antibody, a buffer salt, a protein protectant, and a surfactant. The anti-IL-11 monoclonal antibody comprises three heavy chain complementarity-determining regions (CMRs) represented by HCDR1, HCDR2, and HCDR3, respectively, and three light chain CMRs represented by LCDR1, LCDR2, and LCDR3, respectively. The anti-IL-11 monoclonal antibody is:

[0008] A-Ⅳ: The amino acid sequence of the heavy chain complementarity-determining region HCDR1 is shown in SEQ ID No:1, the amino acid sequence of the heavy chain complementarity-determining region HCDR2 is shown in SEQ ID No:14, the amino acid sequence of the heavy chain complementarity-determining region HCDR3 is shown in SEQ ID No:15, the amino acid sequence of the light chain complementarity-determining region LCDR1 is shown in SEQ ID No:4, the amino acid sequence of the light chain complementarity-determining region LCDR2 is shown in SEQ ID No:5, and the amino acid sequence of the light chain complementarity-determining region LCDR3 is shown in SEQ ID No:6.

[0009] The beneficial effects of this invention are as follows: This invention provides suitable storage conditions for high-concentration anti-IL-11 monoclonal antibodies through the synergistic effect of buffer salts, protein protectants, and surfactants, significantly altering the particle appearance of the drug formulation, effectively reducing the aggregation rate of anti-IL-11 monoclonal antibodies during production, transportation, and use, thus reducing production and transportation costs. Simultaneously, it improves the physicochemical properties of the anti-IL-11 monoclonal antibodies, enabling them to maintain good biological activity, reducing potential safety risks, and ensuring the long-term stability of the drug formulation. Furthermore, the anti-IL-11 monoclonal antibody provided by this invention has a high binding capacity to the IL-11 antigen, blocking the binding of the IL-11 antigen to its receptor, thereby effectively inhibiting the pro-fibrotic effect of IL-11, inhibiting or preventing the production or proliferation of fibroblasts, and can be effectively used to treat or prevent human fibrotic diseases, inflammation, cancer, or autoimmune diseases. Attached Figure Description

[0010] Figure 1 This is a plasmid map of the pScFv-Disb-HS vector in Example 2 of the present invention;

[0011] Figure 2 This is a comparison diagram of the affinity of serially diluted ELISA anti-IL-11 phage monoclonal antibodies in Example 3 of the present invention;

[0012] Figure 3This is a spectrum of the carrier pTSE in Embodiment 5 of the present invention;

[0013] Figure 4 This is a denaturing polyacrylamide gel electrophoresis image of the mouse antibody molecule in Example 5 of the present invention;

[0014] Figure 5 This is a comparison diagram of the binding ability of the mouse antibody molecule to IL-11 in Example 6 of the present invention;

[0015] Figure 6 This is a comparison diagram of the competitive inhibition experiment between the mouse antibody and the IL-11 receptor protein IL-11RA in Example 7 of the present invention;

[0016] Figure 7 This is a comparative diagram showing the inhibition of IL-11 binding to the IL-11RA receptor on the cell surface of BaF / 3-IL-11RA by murine antibody in Example 8 of the present invention;

[0017] Figure 8 This is a comparative diagram showing the inhibition of TIMP-1 secretion by mouse-derived antibodies in embryonic lung fibroblasts MRC-5 in Example 9 of the present invention;

[0018] Figure 9 This is a denaturing polyacrylamide gel electrophoresis image of the humanized antibody molecule in Example 14 of this invention;

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

[0020] Figure 11 This is a comparative diagram showing the inhibition of IL-11 binding to the IL-11RA receptor on the cell surface of BaF / 3-IL-11RA by humanized antibody molecules in Example 16 of the present invention.

[0021] Figure 12 This is a comparative diagram showing the inhibition of IL-11 binding to the GP130 receptor on the surface of BaF / 3-GP130 cells by humanized antibody molecules in Example 17 of the present invention.

[0022] Figure 13 This is a comparative graph showing the biological activity detection (reporter gene) of humanized antibody molecules in Example 18 of the present invention;

[0023] Figure 14 This is a comparative diagram showing the inhibition of TIMP-1 secretion by humanized antibody molecules in embryonic lung fibroblasts MRC-5 in Example 19 of the present invention;

[0024] Figure 15 This is a comparative diagram of the cross-binding experiments between humanized antibody molecules and IL-11 from different species in Example 20 of the present invention;

[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] Example 1 of the present invention provides a pharmaceutical formulation of an anti-IL-11 monoclonal antibody. The pharmaceutical formulation includes 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 represented by HCDR1, HCDR2, and HCDR3, respectively, and three light chain complementarity-determining regions represented by LCDR1, LCDR2, and LCDR3, respectively. The anti-IL-11 monoclonal antibody is selected from any of the following.

[0038]

[0039] The anti-IL-11 monoclonal antibody provided by this invention is used to treat or prevent human fibrotic diseases, inflammation, cancer, or autoimmune diseases. Fibrotic diseases include, but are not limited to, fibrosis of the heart, liver, kidneys, lungs, gallbladder, bladder, stomach, bone marrow, penis, breast, blood vessels, eyes, pancreas, spleen, brain, intestines, muscles, or skin. Inflammation includes, but is not limited to, hepatitis, myocarditis, nephritis, pneumonia, cholecystitis, cystitis, gastritis, osteomyelitis, prostatitis, mastitis, pancreatitis, enteritis, arthritis, polymyositis, dermatomyositis, or dermatitis. Cancer includes, but is not limited to, leukemia, lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, or bladder cancer. Autoimmune diseases include, but are not limited to, psoriasis, Crohn's disease, primary biliary cirrhosis, systemic lupus erythematosus, or multiple sclerosis.

[0040] Example 2: Screening of mouse antibody molecules

[0041] This invention optimizes the immunization method by immunizing mice with IL-11 antigen (in subsequent experiments, IL-11 protein, IL-11-Fc antigen, and IL-11-mFc ligand protein were all human IL-11) and creates a phage display library. The specific construction, screening, and identification of the phage display library are as follows:

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

[0043] 1. Laboratory animals: Species and strain: BALB / c, female, mouse; weight: 18-20g;

[0044] Laboratory animal provider: Beijing Huafukang Biotechnology Co., Ltd.

[0045] 2. Immunization: Mice were immunized with human IL-11 (a gene synthesized by Nanjing Genscript Biotech Co., Ltd., whose vector was constructed and purified by our company).

[0046] Step 2: Construction of the phage antibody library: Mouse spleen cells with high titers were collected, and total RNA was extracted from the mouse spleen cells using Trizol reagent (purchased from Ambion, catalog number: 15596026). cDNA was obtained by RT-PCR. Using the cDNA as a template, PCR amplification was performed using degenerate primers (reference: Journal of Immunological Methods 233(2000)167-177) to obtain the heavy chain variable region gene library (VH) and light chain variable region gene library (VL) of the immunized mouse antibody. The pScFv-Disb-HS vector was modified from the pComb3 vector (purchased from the China Plasmid Vector Strains Cell Line Gene Preservation Center) using a series of gene cloning methods to construct and express the phage single-chain antibody library. The modified vector was named pScFv-Disb-HS, and its plasmid map is shown below. Figure 1 As shown, a mouse immune phage antibody library was constructed based on this vector. The light and heavy chain variable region gene libraries were double-digested and ligated into the pScFv-Disb-HS vector, which had been digested in the same steps, to construct the pScFv-Disb-HS-VH-VL gene library.

[0047] Step 3: Coat the immunotubes with IL-11 antigen at a rate of 5 μg / 500 μL / tube, and incubate overnight at 4°C. Then, block the immunotubes and the immunophage antibody library separately with 4% skim milk powder / PBST at room temperature for 1 hour. Add the blocked immunophage antibody library to the immunotubes for antigen-antibody binding; the phage dosage is approximately 10 μg / 500 μL. 9 ~10 12 After reacting at room temperature for 1 hour, unbound phages were washed away with PBST-PBS, followed by elution with 0.1M pH 2.2 Glycine-HCl. Finally, the eluted phage antibody solution was neutralized to approximately pH 7.0 with 1.5M pH 8.8 Tris-HCl.

[0048] Step 4: Infect 10 ml of TG1 bacterial culture grown to the logarithmic phase with the neutralized phage, incubate at 37°C for 30 min, then take a portion of the bacterial culture and perform serial dilutions, spreading it onto 2YTAG plates to calculate phage yield. Centrifuge the remaining bacterial culture, discard the supernatant, resuspend the bacterial pellet in a small amount of culture medium, aspirate it, and spread it onto large 2YTAG plates to prepare for the next round of screening.

[0049] Step 5: Scrape the infected bacterial cells from the large plate and inoculate them into 2YTAG liquid medium. After shaking to the logarithmic phase, add M13KO7 helper phage for superinfection. Incubate overnight at 220 rpm at 28°C to prepare phages. Purify the phages by PEG / NaCl precipitation for the next round of screening. Perform one round of phage library enrichment screening.

[0050] Step Six: Screening of IL-11 phage single-chain antibody-positive clones: After one round of screening, well-separated single-clone colonies are picked and inoculated into 96-well deep-well plates containing 2 YTAG liquid medium. The plates are incubated at 37°C and 220 rpm until the logarithmic growth phase. Approximately 10 μL of the medium is added to each well. 10 Helper phage M13KO7 was used for static infection at 37°C for 30 min. After centrifugation at 4000 rpm for 15 min, the supernatant was discarded, and the bacterial cells were resuspended in 2YTAK solution and cultured overnight at 28°C and 220 rpm. After centrifugation at 4000 rpm and 4°C for 15 min, the amplified phage supernatant was subjected to ELISA identification. Four murine antibody molecules with high affinity were ultimately screened and named MA-I, MA-II, MA-III, and MA-IV, respectively. The obtained monoclonal antibodies were sequenced to confirm their correct antibody sequences. The sequencing sequences of the four selected monoclonal antibodies are as follows:

[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 light chain variable region of MA-II):

[0057] DIVLTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK;

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

[0059] EVKLEQSGAEVVKPGALVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIG VIDPSDSYTTYNQKFKGKATLTVDTSSSTGYMQLSSLTSEDSAVYYCSQYGYDVN WYFDVWGAGTTVTVSS;

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

[0061] DIVMTQTTLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLI YEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGT KLEIK;

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

[0063] EVQLEESGGGLVKPGGSLKLSCVASGFTFSDYYMFWVRQTPEKRLEWVATI SDGGSYSYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTAMYYCARDGGYISS PEAMDYWGQGTSVTVSS.

[0064] Example 3: Comparison of antibody affinity using gradient dilution ELISA

[0065] The four murine antibody molecules (MA-I, MA-II, MA-III, and MA-IV) obtained in Example 2 were displayed and purified as monoclonal phages, and then affinity was identified by phage gradient dilution ELISA experiments. The specific methods are as follows:

[0066] IL-11 antigen was coated with carbonate buffer (pH 9.6), 100 ng / well / 100 μL, and incubated overnight at 4°C. After washing three times with PBST, the four phage monoclonal antibodies selected in Example 2 were serially diluted five-fold with PBST, with 100 μL of diluted sample added to each well, and incubated at room temperature for 1 hour. The ELISA plate was washed with PBST, and HRP-anti-M13 monoclonal antibody (purchased from Bio-viewshine, catalog number: GE27-9421-01) diluted with 1% BSA-PBST was added to the ELISA plate and incubated at room temperature for 1 hour. A TMB chromogenic kit (purchased from Kangwei Century, catalog number: CW0050S) was used for color development, incubated at room temperature for 10 minutes, and stopped with 2M H2SO4. Readings were taken at 450 nm / 630 nm using a microplate reader, and the corresponding EC50 values ​​were calculated. Specific data are as follows:

[0067]

[0068] Based on the above data and as follows Figure 2 As shown, the four different murine antibody molecules screened in Example 2 were all able to bind to IL-11, which demonstrates that the monoclonal antibody provided by the present invention has a high affinity for IL-11.

[0069] Example 4

[0070] Example 4 of this invention further specifies, based on Example 2, that the anti-IL-11 monoclonal antibody also includes a heavy chain constant region and a light chain constant region. The amino acid sequence of the heavy chain constant region is one of SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, or SEQ ID No:26; the amino acid sequence of the light chain constant region is as shown in SEQ ID No:22, and the specific sequence is as follows:

[0071] SEQ ID No:22 (Rat C) k (Amino acid sequence of the light chain constant region):

[0072] ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL NSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC;

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

[0074] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG;

[0075] SEQ ID No:24 (Amino acid sequence of the heavy chain constant region of murine IgG2a):

[0076] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK;

[0077] SEQ ID No:25 (Amino acid sequence of the heavy chain constant region of murine IgG2b):

[0078] AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFV NNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK;

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

[0080] ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGK EFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSW LQGEIFTCSVVHEALHNHHTQKNLSRSPELELNETCAEAQDGELDGLWTTITIFISLFLLSVCYSASVTLFKVKWIFSSVVQVKQTAIPDYRNMIGQGA.

[0081] Example 5: Preparation of mouse antibody molecules

[0082] Example 5 of the present invention, based on Example 4, preferably specifies that the murine antibody molecule includes the heavy chain constant region of murine IgG1 (its amino acid sequence is shown in SEQ ID No: 23) and murine C kThe light chain constant region of type [type] (its amino acid sequence is shown in SEQ ID No:22). The specific antibody preparation method is as follows:

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

[0087]

[0088] Based on the above data and as follows Figure 5 As shown, all four different murine antibody molecules screened were able to bind to IL-11 and had high affinity.

[0089] Example 7: Competitive inhibition experiment between murine antibody and IL-11 receptor protein IL-11RA

[0090] IL-11-Fc was coated with carbonate buffer (pH 9.6), 200 ng / well / 100 μL, overnight at 4°C. The cells were washed five times with 300 μL / well PBST, then blocked with 280 μL / well of 1% BSA-PBST at 37°C for 1 h. First, IL-11RA-Fc (IgG4 type) diluted to 0.5 μg / mL with 1% BSA-PBST was added, 50 μL / well. Then, different concentrations of MA-Ⅰ, MA-Ⅱ, MA-Ⅲ, and MA-Ⅳ murine antibodies were added, 50 μL / well. The initial maximum concentration of all five antibodies was 100 μg / mL, and each antibody was serially diluted 2-fold, for a total of 13 dilutions. All cells were incubated at 37°C for 3 h. Wash five times with 300 μL / well PBST, then add Anti-Human IgG4-HRPMouse monoclonal antibody (purchased from Sigma, catalog number: SAB4200770) diluted 1:5000 with 2% BSA-PBST, and incubate at 37°C for 1 h. Develop color with a TMB chromogenic kit, 100 μL / well, at room temperature for 15 min, then stop the development with 2M H2SO4. Read the values ​​using a microplate reader at 450 nm / 630 nm and calculate the corresponding IC50 values. Specific data are as follows:

[0091]

[0092] Based on the above data and as follows Figure 6 As shown, all four different murine antibodies screened were able to compete with the receptor protein IL-11RA, indicating that they could effectively inhibit the binding of IL-11 to the receptor protein IL-11RA.

[0093] Example 8: Mouse antibody inhibits the binding of IL-11 to the IL-11RA receptor on the cell surface of BaF / 3-IL-11RA.

[0094] BaF / 3-IL-11RA cell lines were counted, and a certain number of cells were centrifuged, resuspended in PBS buffer, and the cell density was adjusted to 1E+6 cells / mL. 100 μL / well was added to each well of a 96-well plate. IL-11-mFc ligand protein was diluted with PBS to a concentration of 18 μg / mL, and 50 μL / well was added to each well of a 96-well plate containing BaF / 3-IL-11RA cells. After gentle mixing, the 96-well plates were incubated at 4°C for 1 h. Four murine antibody molecules, MA-I, MA-II, MA-III, and MA-IV, were serially diluted with PBS at an initial concentration of 800 μg / mL, followed by 3-fold serial dilutions (10 dilutions in total). 50 μL / well was added to each well of a 96-well plate containing the mixture of BaF / 3-IL-11RA cells and IL-11-mFc ligand protein. After thorough mixing, the plates were incubated at 4°C for 2 h. After incubation, cells were centrifuged at 3000 rpm, washed once with PBS buffer, and the cell pellet was collected. Pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) was added to the cell pellet, and the cells were incubated at 4°C for 30 min. After centrifugation at 3000 rpm, the cells were washed once with PBS buffer, resuspended in 100 μL of PBS buffer, and analyzed by flow cytometry, collecting the fluorescence signal in the FL1-A channel. A dose-response curve was plotted, and the corresponding IC50 value was calculated. Specific data are as follows:

[0095]

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

[0097] Example 9: Mouse-derived antibody inhibits TIMP-1 secretion from MRC-5 embryonic lung fibroblasts.

[0098] MRC-5 embryonic lung fibroblasts were digested with trypsin and counted. A certain number of cells were collected, centrifuged, and resuspended in MEM complete medium (purchased from GIBCO, catalog number 10370-021). The cell density was adjusted to 2E+5 cells / mL, and 100 μL / well was added to 96-well plates. IL-11-mFc ligand protein was diluted in MEM complete medium to a concentration of 16 μg / mL, and 50 μL / well was added to the corresponding 96-well plates. Four murine antibody molecules, MA-I, MA-II, MA-III, and MA-IV, were serially diluted in MEM complete medium to an initial concentration of 40 μg / mL. Eight 2-fold serial dilutions were performed, and 50 μL / well was added to each well of the 96-well plates containing cell suspension and IL-11-mFc ligand protein suspension. After gentle mixing, the plates were incubated overnight at 37°C in a CO2 incubator for approximately 20 hours. Cell culture supernatant was collected and tested using the TIMP-1 ELISA kit (purchased from Ecosai Biotechnology Co., Ltd., catalog number EH021-96).

[0099] Human TIMP-1 Detection Kit: Add cell supernatant and standards to sample wells, 100 μL / well. Immediately add biotinylated antibody working solution (1:100 dilution), 50 μL / well, cover with sealing film, and incubate at room temperature with shaking for 2 h. After incubation, wash the plate 4 times with wash buffer, add enzyme conjugate working solution (1:100 dilution) from the TIMP-1 detection kit, 100 μL / well. Cover with sealing film and incubate at room temperature with shaking for 1 h. After incubation, wash the plate 4 times with wash buffer. Add TMB chromogenic solution, 100 μL / well, incubate at room temperature in the dark for about 15 minutes, and stop the reaction with 100 μL / well stop solution. Read the values ​​using a microplate reader at 450 nm and calculate the corresponding IC50 values. Specific data are as follows:

[0100]

[0101] Based on the above data and Figure 8 It was found that the four different mouse-derived candidate molecules screened out could effectively inhibit the release of TIMP-1 from human embryonic lung fibroblasts MRC-5 stimulated by IL-11 ligand protein.

[0102] Example 10

[0103] Example 10 of the present invention further specifies that the anti-IL-11 monoclonal antibody is a chimeric antibody molecule. The chimeric antibody molecule also includes a human antibody constant region, which includes a human antibody heavy chain constant region and a human antibody light chain constant region. The amino acid sequence of the human antibody heavy chain constant region is one of the sequences shown in SEQ ID No:27, SEQ ID No:28 or SEQ ID No:29; the amino acid sequence of the human antibody light chain constant region is shown in SEQ ID No:30.

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

[0105] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

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

[0107] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

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

[0109] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0110] SEQ ID No:30 (human C k (Amino acid sequence of the light chain constant region):

[0111] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C.

[0112] Example 11 Preparation of chimeric antibody molecules

[0113] Example 11 of the present invention further defines the constant region of the human antibody, based on Example 10, as including the heavy chain constant region of human IgG1 (whose amino acid sequence is shown in SEQ ID No: 27) and human C k The light chain constant region of type (its amino acid sequence is shown in SEQ ID No:30).

[0114] Specific preparation method:

[0115] The heavy chain variable region VH (SEQ ID No: 16) and light chain variable region VL gene (SEQ ID No: 17) of the murine antibody molecules MA-I and MA-II obtained from the screening of the phage antibody library in Example 2, as well as the light chain variable region VL gene (SEQ ID No: 18) of MA-I and MA-II, were cloned into the vector pTSE (e.g., [example of a vector]) containing the heavy chain constant region and light chain constant region genes, respectively, while keeping their murine sequences unchanged. Figure 3 As shown in the figure, the heavy chain constant region is human IgG1 type (amino acid sequence as shown in SEQ ID NO:27), and the light chain constant region is human C.k Type (amino acid sequence as shown in SEQ ID NO:30). HEK293E cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number: GNHu43) were transiently transfected to express antibodies, yielding chimeric antibodies CA-I and CA-II.

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

[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] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;

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

[0124] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVST ISDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVS SPEAMDYWGQGTLVTVSS;

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

[0126] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;

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

[0128] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;

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

[0130] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAMYYCARDGGYVS SPEAMDYWGQGTSVTVSS;

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

[0132] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;

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

[0134] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;

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

[0136] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;

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

[0138] DIVLTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGTVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPWTFGGGTKVEIK.

[0139] Example 13

[0140] 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.

[0141] The specific sequence of the constant region of the human antibody described above is the same as that in Example 10.

[0142] Example 14 Preparation of humanized antibody molecules

[0143] 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).

[0144] 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).

[0145] 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.

[0146] Example 15: Experiment on the binding of humanized antibody molecules to IL-11

[0147] 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:

[0148]

[0149]

[0150] 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.

[0151] 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.

[0152] 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:

[0153]

[0154] 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.

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

[0156] 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:

[0157]

[0158] 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.

[0159] Example 18: Detection of the biological activity of humanized antibody molecules (reporter gene)

[0160] 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:

[0161]

[0162] 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.

[0163] Example 19: Humanized antibody molecules inhibit TIMP-1 secretion by MRC-5 embryonic lung fibroblasts.

[0164] 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:

[0165]

[0166] 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.

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

[0168] 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:

[0169]

[0170] 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.

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

[0172] The therapeutic effect of the anti-IL-11 monoclonal antibody HA-IA on pulmonary fibrosis was studied using bleomycin (bLF) modeling.

[0173] Animal species: C57BL / 6J mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.)

[0174] Number, sex and age of mice: 6 mice / group, male, 6-8 weeks old;

[0175] The control group received only saline injections;

[0176] In the treatment group, HA-IA antibody molecules were injected twice a week for 4 weeks.

[0177] 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.

[0178] 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 sections 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.

[0179] Example 22: Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody against cardiac fibrosis

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

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

[0182] Number, sex and age of mice: 6 mice / group, male, 6-8 weeks old;

[0183] The control group received only saline injections;

[0184] The treatment group received two injections of HA-IA antibody molecules per week for four weeks.

[0185] 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.

[0186] 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.

[0187] Example 23: Therapeutic efficacy experiment of anti-IL-11 monoclonal antibody against renal fibrosis

[0188] The therapeutic effect of the anti-IL-11 monoclonal antibody HA-IA on renal fibrosis was studied using doxorubicin (dKF) modeling.

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

[0190] Number, sex and age of mice: 6 mice / group, male, 6-8 weeks old;

[0191] The control group received only saline injections;

[0192] The treatment group received two injections of HA-IA antibody molecules per week for four weeks.

[0193] 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.

[0194] 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.

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

[0196] The therapeutic effect of anti-IL-11 monoclonal antibody HA-IA on liver fibrosis was studied using CCl4 modeling.

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

[0198] Number, sex and age of mice: 6 mice / group, male, 6-8 weeks old;

[0199] The control group received only saline injections;

[0200] The treatment group received two injections of HA-IA antibody molecules per week for four weeks.

[0201] 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.

[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] 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;

[0214] 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.

[0215] Example 29

[0216] 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;

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

[0218] Example 30

[0219] 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.

[0220] Example 31 Selection of pH range for pharmaceutical preparations

[0221] 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.

[0222] The specific design groups are as follows:

[0223]

[0224] The thermal stability test 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] 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).

[0227] Accelerated stability test at 40±2℃: The stability test conditions are set as follows:

[0228]

[0229] The accelerated stability test at 40±2℃ yielded the following data:

[0230]

[0231] 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.

[0232] 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.

[0233] 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.

[0234] Example 32 Screening of buffer salts

[0235] 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:

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

[0240]

[0241] 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.

[0242] 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.

[0243] Example 33 Screening of protein protectants and surfactants

[0244] Based on the above embodiments 31 and 32, this invention further screens protein protectants and surfactants, with specific experimental examples as follows:

[0245]

[0246] 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:

[0247] Experimental Example Tm (°C) Tagg (°C) Instance 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] The accelerated stability test results at 40±2℃ are as follows:

[0249]

[0250] 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.

[0251] 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.

[0252] 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.

[0253] Example 34 Screening of surfactant content

[0254] 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:

[0255]

[0256] 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:

[0257]

[0258] 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.

[0259] Example 35 Stability testing of pharmaceutical formulations

[0260] In Example 35, three identical batches of the anti-IL-11 monoclonal antibody formulation obtained from the above examples (a 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. Long-term, accelerated, and influencing factor stability studies were conducted on the proposed formulation. The results showed that the proposed formulation could effectively improve the appearance (opalescent) and sub-visible microparticles of the protein. All indicators met the requirements for drugability and could maintain the formulation's tolerance to various influencing factors during production, storage, transportation, and use.

[0261]

[0262] The results of the stability tests after 12 months at 2-8℃ and accelerated stability tests at 25±2℃ are as follows:

[0263]

[0264] The stability test results under simulated transportation conditions of vibration and illumination are as follows:

[0265]

[0266] 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.

[0267] 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-Ⅳ: The amino acid sequence of the heavy chain complementarity-determining region HCDR1 is shown in SEQ ID No:1, the amino acid sequence of the heavy chain complementarity-determining region HCDR2 is shown in SEQ ID No:14, the amino acid sequence of the heavy chain complementarity-determining region HCDR3 is shown in SEQ ID No:15, the amino acid sequence of the light chain complementarity-determining region LCDR1 is shown in SEQ ID No:4, the amino acid sequence of the light chain complementarity-determining region LCDR2 is shown in SEQ ID No:5, and the amino acid sequence of the light chain complementarity-determining region LCDR3 is shown in SEQ ID No:

6.

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-Ⅳ: The amino acid sequence of the heavy chain variable region is shown in SEQ ID No:21, and the amino acid sequence of the light chain variable region is shown in SEQ ID No:

17.

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 which is shown in SEQ ID No:23; the amino acid sequence of which is shown in SEQ ID No:

22.

4. 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.

5. 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.

6. The pharmaceutical formulation of the anti-IL-11 monoclonal antibody as claimed in claim 5, characterized in that, The buffer salts include acetate buffer, phosphate buffer, histidine buffer, or citrate buffer.

7. 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.

8. The pharmaceutical formulation of the anti-IL-11 monoclonal antibody as described in claim 7, 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.

9. 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.

10. The pharmaceutical formulation of the anti-IL-11 monoclonal antibody as described in claim 9, characterized in that, The surfactant is selected from polysorbate 80, polysorbate 20, or poloxamer.

11. 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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