Medicinal preparation of anti-IL-11 monoclonal antibody

Abstract

The invention relates to the field of biological medicine, and particularly provides a pharmaceutical preparation of an anti-IL-11 monoclonal antibody, the pharmaceutical preparation comprises the anti-IL-11 monoclonal antibody, a buffer salt, a protein protective agent and a surfactant, and the anti-IL-11 monoclonal antibody comprises three heavy chain complementary determining regions respectively represented by HCDR1, HCDR2 and HCDR3 and three light chain complementary determining regions respectively represented by LCDR1, LCDR2 and LCDR3. According to the present invention, the buffer salt, the protein protective agent and the surfactant are synergistically matched to provide the suitable storage condition for the high-concentration anti-IL-11 monoclonal antibody, such that the particle performance of the pharmaceutical preparation is significantly changed, and the aggregate generation rate of the anti-IL-11 monoclonal antibody during the production, transportation and use process is reduced; the stability of physicochemical properties of the anti-IL-11 monoclonal antibody is improved, the potential safety risk is reduced, and the stability of a medicinal preparation is ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a pharmaceutical preparation of an anti-IL-11 monoclonal antibody. Background Art

[0002] Fibrosis can occur in many organs, such as the lungs, liver, kidneys, blood vessels, pancreas, skin, etc. The main pathological changes after fibrosis occur are mostly manifested as an increase in fibrous connective tissue in organ tissues, and a significant decrease in parenchymal cells. The more severe the fibrosis, the more likely it is that the tissues and organs are gradually developing towards hardening. In more serious cases, the structure of the organs may be destroyed, the organ function may decline, and even the organ function may be lost. Clinically, fibrosis can cause tissue cell damage, degeneration, necrosis, etc. The more common ones are pulmonary fibrosis, liver fibrosis, heart disease, kidney disease, asthma, etc.

[0003] IL-11 (interleukin-11) is a pleiotropic cytokine and a member of the IL-6 cytokine family. It shares the same signal transduction receptor subunit GP130. This family plays a vital role in the occurrence, development and metastasis of tumors. Studies have found that IL-11 transmits signals to tumor cells through the GP130 signal transduction chain, allowing tumor cells to obtain proliferation and activation signals. Blocking this signal pathway can be an effective treatment for a variety of tumors, chronic fibrosis and inflammatory diseases. Therefore, the drug development of anti-IL-11 monoclonal antibodies has important clinical significance.

[0004] During the process of process development of anti-IL-11 monoclonal antibodies, it was found 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, resulting in aggregation, denaturation or degradation. The structural stability of antibodies will not only affect the biological activity of antibodies, but also the safety of biopharmaceuticals. In particular, some protein aggregates will stimulate the body's immune response, which will reduce the efficacy of biological drugs in mild cases and even cause the death of patients in severe cases. High-concentration antibody drugs not only need to obtain high-purity products during production, but also maintain structural stability during transportation, storage and use. For this reason, high-concentration proteins pose challenges to the performance and stability of aggregates and particles. In order to meet the needs of patients with fibrosis, it is urgent to develop anti-IL-11 monoclonal antibody preparations 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 preparation of anti-IL-11 monoclonal antibody.

[0006] The specific technical solutions of the present invention are as follows:

[0007] The present invention provides a pharmaceutical preparation of an anti-IL-11 monoclonal antibody, comprising an anti-IL-11 monoclonal antibody, a buffer salt, a protein protective agent and a surfactant, wherein the anti-IL-11 monoclonal antibody comprises three heavy chain complementary determining regions represented by HCDR1, HCDR2 and HCDR3, respectively, and three light chain complementary determining regions represented by LCDR1, LCDR2 and LCDR3, respectively, and the anti-IL-11 monoclonal antibody is:

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

[0009] The beneficial effects of the present invention are as follows: the present invention provides suitable storage conditions for high-concentration anti-IL-11 monoclonal antibodies through the synergistic combination of buffer salts, protein protectants and surfactants, significantly changes the particle performance of the pharmaceutical preparation, effectively reduces the generation rate of aggregates of the anti-IL-11 monoclonal antibodies during production, transportation and use, reduces production and transportation costs, and improves the physicochemical properties of the anti-IL-11 monoclonal antibodies, enabling the antibodies to maintain good biological activity, reducing potential safety risks, and ensuring the long-term stability of the pharmaceutical preparation; in addition, the anti-IL-11 monoclonal antibodies provided by the present invention have a high binding ability with the IL-11 antigen, can block the binding of the IL-11 antigen to its receptor, thereby effectively inhibiting the pro-fibrotic effect of IL-11, inhibiting or preventing the production or proliferation of fibroblasts, and can be effectively used to treat or prevent human fibrotic diseases, inflammation, cancer or autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0011] Figure 2 This is a comparison chart of the affinity of the anti-IL-11 phage monoclonal antibody in the gradient dilution ELISA in Example 3 of the present invention;

[0012] Figure 3is a map of the vector pTSE in Example 5 of the present invention;

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

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

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

[0017] Figure 8 This is a comparison diagram of the mouse antibody in Example 9 of the present invention inhibiting the secretion of TIMP-1 by embryonic lung fibroblasts MRC-5;

[0018] Fig. 9 This is a denaturing polyacrylamide gel electrophoresis diagram of the humanized antibody molecule of Example 14 of the present invention;

[0019] Fig.10 This is a comparison of the binding ability between the humanized antibody molecule and IL-11 in Example 15 of the present invention;

[0020] Fig.11 This is a comparison diagram of the inhibition of IL-11 binding to the IL-11RA receptor on the surface of BaF / 3-IL-11RA cells by the humanized antibody molecule in Example 16 of the present invention;

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

[0022] Fig.13 This is a comparison diagram of biological activity detection (reporter gene) of the humanized antibody molecules in Example 18 of the present invention;

[0023] Fig.14 This is a comparison diagram of the inhibition of TIMP-1 secretion by embryonic lung fibroblasts MRC-5 by the humanized antibody molecule in Example 19 of the present invention;

[0024] Fig.15 This is a comparison diagram of the cross-binding experiment between the humanized antibody molecule and IL-11 of different species in Example 20 of the present invention;

[0025] Fig.16 A bar graph showing changes in the ratio of lung to body weight in the mouse pulmonary fibrosis model in Example 21 of the present invention;

[0026] Fig.17 2 are hematoxylin and eosin (HE) staining and Masson's staining images of lung tissue sections in the mouse pulmonary fibrosis model in Example 21 of the present invention;

[0027] Fig.18 A bar graph showing changes in the ratio of heart to body weight in the mouse cardiac fibrosis model in Example 22 of the present invention;

[0028] Fig.19 2 are hematoxylin and eosin (HE) staining and Masson's staining images of cardiac tissue sections in the mouse cardiac fibrosis model in Example 22 of the present invention;

[0029] Fig. 20 A bar graph showing the urine protein content in the kidney of the mouse renal fibrosis model in Example 23 of the present invention;

[0030] Fig.21 The diagram is a hematoxylin and eosin (HE) staining and Masson's staining diagram of kidney tissue sections in the mouse kidney fibrosis model in Example 23 of the present invention;

[0031] Fig. 22 A bar graph showing changes in liver weight in the mouse liver fibrosis model in Example 24 of the present invention;

[0032] Fig.23 A bar graph showing changes in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mouse serum in the mouse liver fibrosis model in Example 24 of the present invention;

[0033] Fig.24 2. HE staining and Masson staining of liver tissue sections in the mouse liver fibrosis model in Example 24 of the present invention;

[0034] Fig.25 This is a graph for evaluating the thermal stability of the anti-IL-11 monoclonal antibody HA-IA in Example 25 of the present invention. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below with reference to the following examples.

[0036] Example 1

[0037] Example 1 of the present invention provides a pharmaceutical preparation of an anti-IL-11 monoclonal antibody, comprising an anti-IL-11 monoclonal antibody, a buffer salt, a protein protectant 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, 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 invention is used to treat or prevent human fibrotic diseases, inflammation, cancer or autoimmune diseases, wherein fibrotic diseases include but are not limited to fibrosis of the heart, liver, kidney, lung, gallbladder, bladder, stomach, bone marrow, penis, breast, blood vessels, eyes, pancreas, spleen, brain, intestine, muscle or skin; inflammation includes but is not limited to hepatitis, myocarditis, nephritis, pneumonia, cholecystitis, cystitis, gastritis, osteomyelitis, prostatitis, mastitis, pancreatitis, enteritis, arthritis, polymyositis, dermatomyositis or dermatitis; cancer includes but is not limited to leukemia, lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer or bladder cancer; autoimmune diseases include but are not limited to psoriasis, Crohn's disease, primary biliary cirrhosis, systemic lupus erythematosus or multiple sclerosis.

[0040] Example 2 Screening of mouse antibody molecules

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

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

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

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

[0045] 2. Immunization: Mice were immunized with human IL-11 as the immunizing antigen (synthesized gene by Nanjing GenScript Biotechnology Co., Ltd., and the vector was constructed and expressed and purified by our company).

[0046] Step 2: Construction of phage antibody library: Take mouse spleen cells with high titer, use Trizol reagent (purchased from Ambion, catalog number: 15596026) to extract total RNA from mouse spleen cells, obtain cDNA by RT-PCR, use cDNA as template, use degenerate primers (reference for degenerate primers used: Journal of Immunological Methods 233 (2000) 167-177) for PCR amplification, thereby obtaining the heavy chain variable region gene library (VH) and light chain variable region gene library (VL) of immune mouse antibodies. The pScFv-Disb-HS vector is a vector pComb3 (purchased from the China Plasmid Vector Strain Cell Line Gene Collection Center) modified by a series of gene cloning methods to be used 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 obtained as shown below. Figure 1 As shown, based on this vector, a mouse immune phage antibody library was constructed. The light and heavy chain variable region gene libraries were double-digested and connected to the vector pScFv-Disb-HS that was also digested in steps to construct the pScFv-Disb-HS-VH-VL gene library.

[0047] Step 3: Use IL-11 as antigen to coat the immunotubes, with an antigen coating volume of 5 μg / 500 μL / tube, coat overnight at 4°C, and then use 4% skim milk powder / PBST to block the immunotubes and immune phage antibody library respectively, and block at room temperature for 1 hour. The blocked immune phage antibody library is added to the immunotube for antigen-antibody binding, and the phage input volume is about 10 9 ~10 12 After reacting at room temperature for 1 h, unbound phages were washed away with PBST-PBS, eluted with 0.1 M Glycine-HCl at pH 2.2, and finally neutralized with 1.5 M Tris-HCl at pH 8.8 to a pH of about 7.0.

[0048] Step 4: Infect 10 ml of TG1 bacterial solution grown to the logarithmic phase with the neutralized phages above, place in a 37°C incubator for 30 minutes, take out part of the bacterial solution for gradient dilution, and spread on 2YTAG plates for calculating the phage output. Centrifuge the remaining bacterial solution and discard the supernatant, resuspend the bacterial pellet in a small amount of culture medium, aspirate it and spread it on a 2YTAG large plate to prepare for the next round of screening.

[0049] Step 5: Scrape the infected bacteria from the large plate, inoculate into 2YTAG liquid culture medium, shake to the logarithmic phase, add M13KO7 helper phage for superinfection, culture overnight at 28°C and 220rpm to prepare phage, PEG / NaCl precipitation to purify phage for the next round of screening, and conduct one round of phage library enrichment screening.

[0050] Step 6: Screening of positive clones of IL-11 phage single-chain antibody: After one round of screening, well-separated monoclonal colonies were picked and inoculated into 96-well deep-well plates with 2YTAG liquid medium. They were cultured at 37°C and 220 rpm until the logarithmic growth phase. About 10 10 The helper phage M13KO7 was statically infected at 37°C for 30 minutes. Centrifuge at 4000rpm for 15 minutes, discard the supernatant, resuspend the precipitate with 2YTAK, and culture overnight at 28°C and 220rpm. After centrifugation at 4000rpm and 4°C for 15 minutes, the amplified phage supernatant was aspirated for ELISA identification, and finally four mouse antibody molecules with high affinity were screened, named MA-I, MA-II, MA-III and MA-IV, and the monoclonal antibodies obtained above were sequenced to determine the correct antibody sequence. After sequencing, the sequences of the four monoclonal antibodies screened above are as follows:

[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 by gradient dilution ELISA

[0065] The four mouse antibody molecules (MA-I, MA-II, MA-III and MA-IV) obtained in Example 2 were displayed and purified by monoclonal phage, and then the affinity was identified by phage gradient dilution ELISA experiment. The specific method is as follows:

[0066] Coat the IL-11 antigen with a carbonate buffer solution of pH 9.6, 100 ng / well / 100 μL, and coat overnight at 4°C. Wash three times with PBST, dilute the four phage monoclonal antibodies screened in Example 2 with PBST in a five-fold gradient, add 100 μl of the diluted sample to each well, and let stand at room temperature for 1 hour. Wash the ELISA plate with PBST, add the HRP-anti-M13 (purchased from Bio-viewshine, item number: GE27-9421-01) monoclonal antibody diluted with 1% BSA-PBST to the ELISA plate, and let it stand at room temperature for 1 hour. Use the TMB color development kit (purchased from Kangwei Century, item number: CW0050S) for color development at room temperature for 10 minutes. After terminating with 2M H2SO4, read the plate at 450nm / 630nm with the microplate reader, and calculate the corresponding EC50 value. The specific data are as follows:

[0067]

[0068] Through the above data and Figure 2 As shown, the four different mouse antibody molecules screened in Example 2 can all bind to IL-11, which indicates that the monoclonal antibodies provided by the present invention have a high affinity for IL-11.

[0069] Example 4

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

[0071] SEQ ID No:22 (mouse C k The amino acid sequence of the light chain constant region of the type:

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

[0083] 1. The heavy chain VH and light chain VL coding genes of the four antibody molecules screened in Example 2 were cloned into the vector pTSE (such as Figure 3 The preferred heavy chain constant region is a mouse IgG1 type constant region (amino acid sequence is shown in SEQ ID No: 23), and the light chain constant region is a mouse C k chain (amino acid sequence as shown in SEQ ID No: 22), pTSE vector structure as shown in Figure 3 As shown (for the preparation process of pTSE vector, see the specification of CN103525868A, page 3, paragraph

[0019] ).

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

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

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

[0087]

[0088] Through the above data and Figure 5 As shown, the four different mouse antibody molecules screened out can bind to IL-11 with high affinity.

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

[0090] IL-11-Fc was coated with carbonate buffer at pH 9.6, 200 ng / well / 100 μL, overnight at 4°C. Washed five times with 300 μL / well PBST, then blocked at 37°C for 1h with 1% BSA-PBST, 280 μL / well, first added IL-11RA-Fc (IgG4 type) diluted to 0.5 μg / mL with 1% BSA-PBST, 50 μL / well, then added MA-Ⅰ, MA-Ⅱ, MA-Ⅲ and MA-Ⅳ mouse antibodies at different dilution concentrations, 50 μL / well, the starting maximum concentration of the five antibodies was 100 μg / mL, and each antibody was diluted 2-fold, with a total of 13 gradients, and incubated at 37°C for 3h. Wash five times with 300 μL / well PBST, then add Anti-Human IgG4-HRPMouse monoclonal antibody (purchased from Sigma, catalog number: SAB4200770) diluted 1:5000 with 2% BSA-PBST, and incubate at 37°C for 1 hour. TMB colorimetric kit was used for color development, 100 μl / well, and color development was performed at room temperature for 15 minutes, and then 2M H2SO4 was used to terminate the color development. The microplate reader was read at 450nm / 630nm, and the corresponding IC50 value was calculated. The specific data are as follows:

[0091]

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

[0093] Example 8 Mouse Antibodies Inhibit the Binding of IL-11 to the IL-11RA Receptor on the Surface of BaF / 3-IL-11RA Cells

[0094] Count the BaF / 3-IL-11RA cell line, take a certain number of cells, centrifuge and resuspend in PBS buffer, adjust the cell density to 1E+6 cells / mL, 100μL / well, and add to the 96-well plate. Dilute the IL-11-mFc ligand protein with PBS, prepare the concentration to 18μg / mL, 50μL / well, and add it to the corresponding position of the 96-well plate containing BaF / 3-IL-11RA cells. After gently mixing, place the 96-well plate at 4℃ and incubate for 1h. PBS gradient dilution of 4 mouse antibody molecules MA-I, MA-II, MA-III and MA-IV, with an initial concentration of 800μg / mL, 3-fold gradient dilution, a total of 10 gradients, 50μL / well, add to the corresponding position of the 96-well plate containing BaF / 3-IL-11RA cells and IL-11-mFc ligand protein mixture. After mixing evenly, place it at 4℃ and incubate for 2h. After the incubation, centrifuge at 3000rpm, wash the cells once with PBS buffer, and collect the cell pellet. Add the pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) to the cell pellet, incubate at 4°C for 30min, centrifuge at 3000rpm, wash once with PBS buffer, resuspend in 100μL PBS buffer, and detect on a flow cytometer to collect the fluorescence signal in the FL1-A channel. Draw a dose-effect curve and calculate the corresponding IC50 value. The specific data are as follows:

[0095]

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

[0097] Example 9 Mouse Antibodies Inhibit TIMP-1 Secretion by Embryonic Lung Fibroblasts MRC-5

[0098] Embryonic lung fibroblasts MRC-5 were digested with trypsin and counted. A certain number of cells were taken, centrifuged and resuspended in MEM complete medium (purchased from GIBCO, catalog number 10370-021), and the cell density was adjusted to 2E+5 cells / mL, 100 μL / well, and added to a 96-well plate. MEM complete medium was used to dilute the IL-11-mFc ligand protein to a concentration of 16 μg / mL, and 50 μL / well was added to the corresponding 96-well plate. MEM complete medium was used to gradient dilute the four mouse antibody molecules MA-I, MA-II, MA-III, and MA-IV to an initial concentration of 40 μg / mL, 2-fold gradient dilution, a total of 8 gradients, 50 μL / well, added to the 96-well plate containing the cell suspension and IL-11-mFc ligand protein suspension, gently mixed, and placed in a 37°C CO2 incubator for overnight incubation for about 20 hours. The cell culture supernatant was taken for detection using an ELISA kit for TIMP-1 (purchased from Ekosai Biotechnology Co., Ltd., catalog number EH021-96).

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

[0100]

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

[0102] Example 10

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

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

[0105] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

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

[0107] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

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

[0109] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

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

[0111] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C.

[0112] Example 11 Preparation of chimeric antibody molecules

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

[0114] Specific preparation method:

[0115] The heavy chain variable region VH (SEQ ID No: 16) of the mouse antibody molecules MA-I and MA-II obtained by screening the immune phage antibody library in Example 2, the light chain variable region VL gene of MA-I (SEQ ID No: 17), and the light chain variable region VL gene of MA-II (SEQ ID No: 18) were cloned into the vector pTSE (such as Figure 3 The heavy chain constant region is human IgG1 type (amino acid sequence is shown in SEQ ID NO: 27), and the light chain constant region is human Ck Type (amino acid sequence as shown in SEQ ID NO: 30). HEK293E cells (purchased from: Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number: GNHu43) were transiently transfected to express antibodies and obtain chimeric antibodies CA-I and CA-II.

[0116] Example 12 Humanization of murine antibody molecules

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

[0118]

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

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

[0141] The specific sequence of the above human antibody constant region 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, on the basis of Example 13, that the human antibody constant region includes a human IgG1 heavy chain constant region (whose amino acid sequence is shown in SEQ ID No: 27) and a human C k The light chain constant region of the type (the amino acid sequence of which is shown in SEQ ID No: 30).

[0144] The heavy chain VH and light chain VL coding genes of the eight humanized anti-IL-11 monoclonal antibody molecules HA-IA, HA-IB, HA-IC, HA-ID, HA-II-A, HA-II-B, HA-II-C, and HA-II-D obtained by humanization in Example 12 were cloned into the vector pTSE (such as Figure 3 The heavy chain constant region is human IgG1 type (the amino acid sequence is shown in SEQ ID NO: 27), and the light chain constant region is C k chain (the amino acid sequence is shown in SEQ ID NO: 30).

[0145] The two chimeric antibodies CA-I and CA-II obtained in Example 11 and the eight humanized antibody molecules HA-IA, HA-IB, HA-IC, HA-ID, HA-II-A, HA-II-B, HA-II-C, and HA-II-D obtained in Example 12 were transiently transfected into HEK293 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, with the catalog number GNHu43) for antibody expression, and monoclonal antibodies were obtained by purification through a protein A affinity column using an AKTA instrument. The protein concentration was determined using a BCA kit (purchased from Beijing Huitian Oriental Technology Co., Ltd., with the catalog number BCA0020), and the protein size was then identified by SDS-PAGE. The results are as follows: Fig. 9 As shown, from left to right are the non-reduced protein molecular weights HA-IA, HA-IB, HA-IC, HA-ID, the chimeric antibody CA-I prepared in Example 11, the reduced protein molecular weight Marker, HA-II-A, HA-II-B, HA-II-C, HA-II-D, and the chimeric antibody CA-II. The molecular weight of each band is consistent with the theory.

[0146] Example 15 Humanized antibody molecule and IL-11 binding experiment

[0147] IL-11 antigen was coated with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, overnight at 4°C. Wash five times with 300 μL / well PBST, and then add 1% BSA-PBST 280 μL / well to block for 1 hour at 37°C. Humanized antibodies HA-IA, HA-IB, HA-IC, HA-ID, HA-II-A, HA-II-B, HA-II-C, HA-II-D and chimeric antibodies CA-I and CA-II prepared in Example 11 were diluted with 1% BSA-PBST. The initial concentration of humanized antibodies was 10 μg / mL, and 8 gradients were diluted 5 times. Incubate at 37°C for 1 hour. Wash five times with 300 μL / well PBST, then add goat anti Human IgG Fab HRP (purchased from Invitrogen, catalog number: 31482) diluted 1:5000 with 1% BSA-PBST, and incubate at 37°C for 1 hour. TMB colorimetric kit was used for color development, 100 μL / well, color development at room temperature for 5 minutes, and then 2M H2SO4 was used to stop the color development. The microplate reader was read at 450nm / 630nm, and the corresponding EC50 value was calculated. The specific data are as follows:

[0148]

[0149]

[0150] Through the above data and experimental results, Fig.10 As shown, 8 different humanized antibody molecules can bind to IL-11, and the EC50 values ​​of HA-IA, HA-IB, HA-IC, and HA-ID humanized antibody molecules are close to those of the chimeric antibody CA-I, and the EC50 values ​​of HA-II-A, HA-II-B, HA-II-C, and HA-II-D humanized antibody molecules are close to those of the chimeric antibody CA-II, indicating that the humanized antibody molecules retain the high binding ability of the mouse parent antibodies MA-I and MA-II to IL-11.

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

[0152] Four humanized antibody molecules HA-IA, HA-IB, HA-IC and HA-ID with better binding activity at the protein level were selected for cell activity evaluation experiments. BaF / 3-IL-11RA cell lines were counted, a certain number of cells were taken, centrifuged and resuspended with PBS buffer, the cell density was adjusted to 1E+6 cells / mL, 100μL / well, and added to a 96-well plate. PBS was used to dilute the IL-11-mFc ligand protein to a concentration of 18μg / mL, 50μL / well, and added to the corresponding position of the 96-well plate containing BaF / 3-IL-11RA cells. After gently mixing, the 96-well plate was placed at 4°C and incubated for 1h. PBS buffer gradient dilution of four humanized antibody molecules HA-IA, HA-IB, HA-IC and HA-ID, prepared an initial concentration of 800μg / mL, 3-fold gradient dilution, a total of 10 gradients, 50μL / well, added to the corresponding position of the 96-well plate containing BaF / 3-IL-11RA cells and IL-11-mFc ligand protein mixture. After mixing evenly, incubate at 4°C for 2h. After the incubation, centrifuge the PBS buffer at 3000rpm to wash the cells once, and collect the cell pellet. Add the pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) to the cell pellet, incubate at 4°C for 30min, centrifuge at 3000rpm to wash once, resuspend in 100μL PBS, and detect on the flow cytometer to collect the fluorescence signal in the FL1-A channel. Draw a dose-effect curve and calculate the corresponding IC50 value. The specific data are as follows:

[0153]

[0154] Through the above data and Fig.11 It can be seen that the four humanized candidate molecules screened out can all inhibit the binding of IL-11 ligand protein to the IL-11RA receptor on the surface of BaF / 3-IL-11RA cells.

[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, a certain number of cells were taken, centrifuged and resuspended in PBS buffer, the cell density was adjusted to 1E+6 cells / mL, 100 μL / well, and added to a 96-well plate. IL-11-mFc ligand protein was diluted with PBS to a concentration of 12 μg / mL, 50 μL / well, and added to the corresponding position of the 96-well plate containing BaF / 3-GP130 cells. After gently mixing, the 96-well plate was placed at 4°C and incubated for 1 hour. 4 human antibody molecules HA-IA, HA-IB, HA-IC and HA-ID were gradiently diluted with PBS buffer to prepare an initial concentration of 2000 μg / mL, 2-fold gradient dilution, a total of 10 gradients, 50 μL / well, and added to the corresponding position of the 96-well plate containing BaF / 3-GP130 cells and IL-11-mFc ligand protein. After mixing evenly, the 96-well plate was incubated at 4°C for 2 hours. After the incubation, wash the cells once with PBS buffer by centrifugation at 3000rpm, and collect the cell pellet. Add the pre-prepared goat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech, catalog number 1030-02) to the cell pellet, 100μL / well, and incubate at 4℃ for 30min. Wash once with PBS buffer by centrifugation at 3000rpm, resuspend the cells in 100μL / well PBS buffer, detect on the flow cytometer, and collect the fluorescence signal in the FL1-A channel. Draw the dose-effect curve and calculate the corresponding IC50 value. The specific data are as follows:

[0157]

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

[0159] Example 18 Biological Activity Detection of Humanized Antibody Molecules (Reporter Gene)

[0160] The BaF / 3-IL-11RA-GP130-STAT3-Luc engineering cell line was counted, and the cell density was adjusted to 2E+6 cells / mL using sample diluent (its ingredients include 90% IMDM, 10% FBS, 10ng / mL mouseIL-3). After gently mixing, the cell solution was added to a 96-well plate at 50μL / well. The four humanized antibody molecules HA-IA, HA-IB, HA-IC and HA-ID were diluted to an initial concentration of 200μg / ml using sample diluent, 5-fold gradient dilution, a total of 10 gradients, 100μL / well, and added to the corresponding position of the 96-well plate containing the engineering cell line, and two replicates were set for each sample concentration. The sample diluent was used to prepare IL-11 protein at a concentration of 10μg / mL, 50μL / well, and added to the 96-well plate containing the engineering cell line and humanized antibody molecules. The cell culture plate was gently mixed and placed in a 37℃ CO2 incubator for 6h. After centrifugation, discard the supernatant, add lysis buffer, add 10 μL / well to a 384-well plate, add an equal amount of luciferase reaction substrate (purchased from Promega Biotechnology Co., Ltd., catalog number E2610), react at room temperature for 5 minutes, read the fluorescence value on a microplate reader, and calculate the corresponding IC50 value. The specific data are as follows:

[0161]

[0162] Through the above data and Fig.13 As shown, the four humanized antibody molecules screened out can block the binding of IL-11 to IL-11RA and GP130 receptors, inhibiting the conduction of signal pathways.

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

[0164] Embryonic lung fibroblasts MRC-5 were digested with trypsin and counted, a certain number of cells were taken, and the cells were resuspended in MEM complete medium after centrifugation, and the cell density was adjusted to 2E+5 cells / mL, 100 μL / well, and added to a 96-well plate. MEM complete medium was used to dilute the IL-11-mFc ligand protein to a concentration of 16 μg / mL, 50 μL / well, and added to the corresponding 96-well plate. MEM complete medium was used to gradiently dilute 4 humanized antibody molecules HA-IA, HA-IB, HA-IC and HA-ID, with an initial concentration of 40 μg / mL, 3-fold gradient dilution, a total of 8 gradients, 50 μL / well, and added to a 96-well plate containing a cell suspension and an IL-11-mFc ligand protein suspension, gently mixed, placed in a 37°C CO2 incubator and incubated overnight, about 20 hours, and the cell culture supernatant was taken and detected using an ELISA kit of TIMP-1 (the method is the same as in Example 9). The microplate reader reads at 450nm and calculates the corresponding IC50 value. The specific data are as follows:

[0165]

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

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

[0168] The humanized antibody molecule HA-IA with better protein level and functional detection activity was selected for cross-binding detection with IL-11 of different species. Human IL-11, mouse IL-11 (purchased from Beijing Sino Biological Technology Co., Ltd., catalog number: 50117-MNCE), rat IL-11 (purchased from Kanglang Bio, catalog number: KL40001Ra), and cynomolgus monkey IL-11 (purchased from Sino Biological, catalog number: 90925-CNCE) were coated with pH 9.6 carbonate buffer at 100 ng / well / 100 μL, and coated overnight at 4°C. Wash five times with 300 μL / well PBST, then add 1% BSA-PBST, 280 μL / well, and block at 37°C for 1h. Humanized antibody HA-IA was diluted with 1% BSA-PBST, the initial concentration was 50μg / mL, 5-fold gradient dilution, a total of 9 gradients, two replicates for each gradient, 100μL / well was added to a 96-well plate, and incubated at 37°C for 1h. Wash five times with 300μL / well PBST, dilute goat anti Human IgG Fab HRP (purchased from invitrogen, catalog number: 31482) with 1% BSA-PBST, the working solution concentration was 1:5000, 100μL / well was added to a 96-well plate, and incubated at 37°C for 1h. Wash five times with 300μL / well PBST, color with TMB color development kit, 100μL / well, color at room temperature in the dark for 5min, and then stop color development with 2M H2SO4. The microplate reader reads at 450nm / 630nm, and the corresponding EC50 value is calculated. The specific data are as follows:

[0169]

[0170] Through the above data and Fig.15 As shown, the humanized antibody molecule HA-IA can bind to human IL-11, mouse IL-11, rat IL-11, and cynomolgus monkey IL-11 with high affinity.

[0171] Example 21 Experimental study on the therapeutic efficacy of anti-IL-11 monoclonal antibody on pulmonary fibrosis

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

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

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

[0175] The control group was injected with saline only;

[0176] The drug administration group was injected with HA-IA antibody molecules twice a week for 4 weeks.

[0177] The animal body weight was measured once a week, and the animals were observed for any abnormalities. Organ weight detection: lung organs were collected, the weight of the lung organs was measured, and the ratio of lung to body weight was calculated. Lung pathology detection: lung sections were sliced ​​and stained with hematoxylin and eosin (HE) and Masson's staining to observe the degree of lung fibrosis.

[0178] The results are as follows Fig.16 As shown in Figure 2, after administration of HA-IA antibody molecules, the ratio of lung to body weight of mice in the administration group was significantly smaller than that in the control group; Fig.17 As shown, compared with the control group, the lung sections of the drug-treated group showed a significant reduction in pulmonary fibrosis, thus indicating that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the occurrence of pulmonary fibrosis.

[0179] Example 22 Experimental study on the therapeutic efficacy of anti-IL-11 monoclonal antibody on cardiac fibrosis

[0180] The therapeutic effect of 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, gender and age of mice: 6 / group, male, 6-8 weeks;

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

[0184] The drug-treated group was injected with HA-IA antibody molecules twice a week for 4 weeks.

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

[0186] The results are as follows Fig.18 As shown in Figure 2, the heart-to-body weight ratio of mice in the drug-treated group was significantly smaller than that in the control group; Fig.19 As shown, compared with the control group, the heart sections of the drug-treated group showed a significant reduction in cardiac fibrosis, thus indicating that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the occurrence of cardiac fibrosis.

[0187] Example 23 Experimental study on the therapeutic efficacy of anti-IL-11 monoclonal antibody on renal fibrosis

[0188] The therapeutic effect of 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, gender and age of mice: 6 / group, male, 6-8 weeks;

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

[0192] The drug-treated group was injected with HA-IA antibody molecules twice a week for 4 weeks.

[0193] The animal body weight was measured once a week and the animals were observed for any abnormalities; organ weight detection: the heart was collected, the kidney weight was measured, and the urine protein content was detected; renal pathology detection: kidney sections were sliced ​​and the degree of renal fibrosis was observed using hematoxylin and eosin (HE) and Masson staining.

[0194] The results are as follows Fig. 20 As shown, compared with the control group, the urine protein content in the kidneys of the mice in the drug-treated group was significantly lower than that in the control group; Fig.21 As shown, compared with the control group, the kidney sections of the drug-treated group showed a significant reduction in renal fibrosis, which indicates that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the occurrence of renal fibrosis.

[0195] Example 24 Experimental study on the therapeutic efficacy of anti-IL-11 monoclonal antibody on liver fibrosis

[0196] CCl4 modeling was used to study the therapeutic effect of anti-IL-11 monoclonal antibody HA-IA on liver fibrosis.

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

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

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

[0200] The drug-treated group was injected with HA-IA antibody molecules twice a week for 4 weeks.

[0201] Body weight monitoring: The animal body weight was measured once a week, and the animals were observed for any abnormalities. Liver pathology detection: The liver was sectioned and the degree of liver fibrosis was observed using hematoxylin and eosin (HE) and Masson staining. Organ weight detection: The kidneys were collected, the liver weight was measured, and HE staining was performed. Serum detection: Serum was collected to detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum.

[0202] The results are as follows Fig. 22 As shown in Figure 2, the liver weight of mice in the drug-treated group was significantly lower than that in the control group; Fig.23 As shown in Figure 2, the levels of ALT and AST in the serum of mice in the drug-treated group were significantly lower than those in the control group. Fig.24 As shown, compared with the control group, the liver sections of the drug-treated group showed a significant reduction in liver fibrosis, which indicates that the anti-IL-11 monoclonal antibody HA-IA antibody molecule can effectively inhibit the occurrence of liver fibrosis.

[0203] Example 25 Evaluation of thermal stability of anti-IL-11 monoclonal antibody HA-IA

[0204] The thermal stability of the anti-IL-11 monoclonal antibody HA-IA was evaluated using a multifunctional protein thermal stability analysis system (purchased from Unchained Labs). The conformational changes of the protein were detected by monitoring the changes in the intrinsic fluorescence of the protein with temperature (starting from 25°C and heating to 95°C at a rate of 0.3°C / min), thereby determining the melting temperature Tm of the protein and evaluating the conformational stability of the protein. When the sample aggregates, it will cause interference of the scattered light waves and increase the scattered light signal. The colloidal stability of the protein (characterized by Tagg) was determined by static light scattering. The results are shown in the following table and attached. Fig.25 shown.

[0205]

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

[0207] Embodiment 26

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

[0209] Embodiment 27

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

[0211] It should be further explained that the buffer salt includes but is not limited to one or more combinations of acetate buffer, phosphate buffer, histidine buffer or citrate buffer.

[0212] Embodiment 28

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

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

[0215] Embodiment 29

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

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

[0218] Embodiment 30

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

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

[0221] On the basis of Example 1, the anti-IL-11 monoclonal antibody HA-IA was selected as the pharmacodynamic molecule to prepare a pharmaceutical preparation, and the preparation method included: preparing 20mM acetic acid-sodium acetate buffer (pH4.5, 5.5) and 20mM phosphate buffer (pH6.5, 7.5 and 8.5) respectively, ultrafiltration of different concentrations of anti-IL-11 monoclonal antibody HA-IA was exchanged into buffers of different pH, sterilized and filtered with a 0.22μm filter, and dispensed into 2ml vials, 1ml / bottle. After the dispensing was completed, the thermal stability of the protein under different buffer conditions was detected, and then the sample was placed in a stability test box, the temperature was set to 40±2°C, and placed for 2 weeks, and the stability of appearance, pH, protein concentration, purity, charge isomers, sub-visible particles, viscosity and particle size were investigated respectively.

[0222] The specific design groups are as follows:

[0223]

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

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

[0226] From the above data, it can be seen that the Tm of the sample at pH 4.5 is relatively low, and the typical aggregation initiation temperature Tagg is not observed. The Tm of the sample at pH 5.5 is around 70°C, and the Tm of the samples at pH 6.5 to 8.5 is around 75°C. This shows that the anti-IL-11 monoclonal antibody HA-IA is relatively stable in drug preparations of concentrations (10 mg / ml or 80 mg / ml).

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

[0228]

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

[0230]

[0231] Note: In the Appearance column, A stands for “colorless, clear liquid, no visible foreign matter”; B stands for “slight opalescence”; C stands for “no opalescence, slight particles”; D stands for “slight opalescence, slight particles”. The same is true in the following tables.

[0232] From the above data, it can be seen that after 1 week of acceleration at 40±2℃, as the pH increased, the purity (SEC-HPLC) of each experimental sample showed a more obvious downward trend, and the purity of the 80mg / ml sample was lower than that of the 10mg / ml sample at the same pH; the main peak purity of the charge isomers of the samples at pH5.5 and pH6.5 was higher.

[0233] Based on the experimental results of thermal stability and 40±2℃ accelerated stability, the anti-IL-11 monoclonal antibody HA-IA has good stability at around pH 5.5 to pH 6.5.

[0234] Example 32 Screening of buffer salts

[0235] On the basis of Example 31, in the pH range of 5.0-6.5, the anti-IL-11 monoclonal antibody HA-IA with a protein concentration of 40 mg / ml was selected as the pharmacodynamic molecule, and different buffer salts were selected to prepare anti-IL-11 monoclonal antibody pharmaceutical preparations, and the thermal stability and 40±2°C accelerated stability were tested. The test conditions were the same as in Example 31, as follows:

[0236]

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

[0238] Experimental example Tm(℃) Tagg(℃) Experimental example Tm(℃) Tagg(℃) 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 results of the 40±2℃ accelerated stability test are as follows:

[0240]

[0241] Note: In the Appearance column, A stands for “colorless, clear liquid, no visible foreign matter”; B stands for “slight opalescence”; C stands for “no opalescence, slight particles”; D stands for “slight opalescence, slight particles”. The same is true in the following tables.

[0242] The above experimental data show that after 2 weeks of acceleration at 40±2℃, Experimental Examples 1 to 6 were all colorless clear liquids without visible foreign matter, and Experimental Examples 7 to 12 all showed slight opalescence; the purity of the anti-IL-11 monoclonal antibody HA-IA protein in histidine salt buffer was better than that of other buffer salts; the purity decrease ratio of the main peak of the charge isomer of the anti-IL-11 monoclonal antibody HA-IA in citrate buffer (pH6.5) and histidine salt buffer (pH6.0) was the smallest. Therefore, based on the experimental results of thermal stability and 40±2℃ accelerated stability, the anti-IL-11 monoclonal antibody HA-IA had good stability in histidine salt buffer at pH6.0.

[0243] Example 33 Screening of protein protective agents and surfactants

[0244] Based on the above-mentioned Examples 31 and 32, the present invention further screens protein protective agents and surfactants. The specific experimental examples are as follows:

[0245]

[0246] 40±2℃ accelerated stability test, experimental conditions and process refer to Examples 31 and 32, the results are as follows: The thermal stability test results are as follows:

[0247] Experimental example Tm(℃) Tagg(℃) Instance Column Tm(℃) Tagg(℃) 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 results of the 40±2℃ accelerated stability test are as follows:

[0249]

[0250] Note: In the Appearance column, A stands for “colorless, clear liquid, no visible foreign matter”; B stands for “slight opalescence”; C stands for “no opalescence, slight particles”; D stands for “slight opalescence, slight particles”. The same is true in the following tables.

[0251] The results of thermal stability test show that the Tm of Experimental Examples 7 to 11 are lower than those of other Experimental Examples, and the results of accelerated stability test at 40±2°C show that in Experimental Examples 8-11, when lysine hydrochloride, arginine hydrochloride, and sodium chloride are selected as protein protectants, there is slight opalescence at 0 o'clock, and a small amount of particles begin to appear after two weeks of acceleration. When proline is selected as the protein protectant in Experimental Example 6, a small amount of particles begin to appear in the second week. The SEC-HPLC purity, charge isomers, and CE-SDS purity data show that when mannitol, sorbitol, or trehalose is selected as the protein protectant in Experimental Examples 1-3, it is significantly better than sucrose and glycine selected in Experimental Examples 4 and 5. Therefore, when mannitol, sorbitol, or trehalose is selected as the protein protectant, the stability is better. The present invention preferably uses mannitol as the protein protectant in subsequent embodiments.

[0252] In addition, it can be concluded from the data of total particles in subvisible particles and particles larger than 25 um that after the addition of surfactants in Experimental Examples 12-14, the total number of particles in subvisible particles and particles larger than 25 um were significantly less than those in other experimental examples. At the same time, among the surfactants, polysorbate 20 provided in the preferred experiment 13 is better than polysorbate 80 and poloxamer, so the surfactant is preferably polysorbate 20.

[0253] Example 34 Screening of surfactant content

[0254] According to the screening of the above embodiment, the anti-IL-11 monoclonal antibody HA-IA with a protein concentration of 40 mg / ml was selected as the pharmacological molecule, 20 mM histidine salt buffer (pH 6.0) was used as the preparation buffer salt, sorbitol was used as the protein protective agent, and polysorbate 20 was used as the surfactant. The surfactant content was screened by 40±2°C accelerated stability and light stability experiments. The specific scheme is as follows:

[0255]

[0256] The accelerated stability test was carried out at 40±2°C for 4 weeks. The experimental conditions and process were referred to Examples 31 and 32. The results are as follows:

[0257]

[0258] From the above data, it can be concluded that after accelerated temperature of 40±2℃ for 4 weeks, there are a small amount of particles in Experimental Example 1, while the samples of other Experimental Examples are all colorless clear liquids without visible foreign matter; the total number of subvisible microparticles in the samples of each Experimental Example increases, the number of particles ≥25um in the sample of Experimental Example 1 increases significantly, and the number of particles ≥25um in the samples of Experimental Examples 2-6 does not change significantly. However, the purity of the main peak of the charge isomers in the sample of Experimental Example 6 decreases significantly. For this reason, the content of surfactant is preferably 0.005%-0.04% w / v polysorbate 20. An appropriate amount of surfactant helps to control the appearance and subvisible microparticle performance of the preparation.

[0259] Example 35: Stability test of pharmaceutical preparations

[0260] In this Example 35, the drug preparation of the anti-IL-11 monoclonal antibody screened in the above examples (a drug preparation prepared by mixing 20 mM histidine salt buffer, 250 mM mannitol, 0.02% w / v polysorbate 20, pH 6, and anti-IL-11 monoclonal antibody HA-IA at a concentration of 40 mg / ml) was used to prepare three identical batches of samples, and the proposed formulation was subjected to long-term, accelerated, and influencing factor stability investigations. The results showed that the proposed formulation could effectively improve the appearance (opalescence) of the protein and the performance of sub-visible particles, and all indicators met the requirements for the drugability of the formulation, and could maintain the formulation's tolerance to the challenges of various influencing factors during production, storage, transportation, and use.

[0261]

[0262] The results of the 12-month stability test at 2-8°C and 25±2°C are as follows:

[0263]

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

[0265]

[0266] In summary, the results of accelerated stability at 25±2°C, long-term stability at 2-8°C, shaking and light stability for simulated transportation showed that, after being placed under the above stability conditions for a certain period of time, each quality attribute did not change significantly or the changes were within an acceptable range, indicating that the pharmaceutical preparation provided by the present invention can maintain the physicochemical and biological properties of the anti-IL-11 monoclonal antibody and meet the shelf life and clinical needs of the pharmaceutical preparation.

[0267] The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that are the same or similar to those of the present application fall within the protection scope of the present invention.

Claims

1. A pharmaceutical preparation of an anti-IL-11 monoclonal antibody, characterized in that: The pharmaceutical preparation comprises an anti-IL-11 monoclonal antibody, a buffer salt, a protein protective agent and a surfactant, wherein the anti-IL-11 monoclonal antibody comprises three heavy chain complementary determining regions represented by HCDR1, HCDR2 and HCDR3 respectively and three light chain complementary determining regions represented by LCDR1, LCDR2 and LCDR3 respectively, and the anti-IL-11 monoclonal antibody is: A-IV: The amino acid sequence of the heavy chain complementary determining region HCDR1 is shown in SEQ ID No: 1, the amino acid sequence of the heavy chain complementary determining region HCDR2 is shown in SEQ ID No: 14, the amino acid sequence of the heavy chain complementary determining region HCDR3 is shown in SEQ ID No: 15, the amino acid sequence of the light chain complementary determining region LCDR1 is shown in SEQ ID No: 4, the amino acid sequence of the light chain complementary determining region LCDR2 is shown in SEQ ID No: 5, and the amino acid sequence of the light chain complementary determining region LCDR3 is shown in SEQ ID No:

6.

2. The pharmaceutical preparation of anti-IL-11 monoclonal antibody according to claim 1, characterized in that: The anti-IL-11 monoclonal antibody further comprises a heavy chain variable region and a light chain variable region, and the anti-IL-11 monoclonal antibody is: 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 preparation of anti-IL-11 monoclonal antibody according to claim 2, characterized in that: The anti-IL-11 monoclonal antibody also includes a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region is one of SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25 or SEQ ID No: 26; the amino acid sequence of the light chain constant region is as shown in SEQ ID No:

22.

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

5. The pharmaceutical preparation of anti-IL-11 monoclonal antibody according to claim 1, characterized in that The content of the buffer salt is 10-40mM; Preferably, the buffer salt comprises acetate buffer, phosphate buffer, histidine buffer or citrate buffer.

6. The pharmaceutical preparation of anti-IL-11 monoclonal antibody according to claim 1, characterized in that: The content of the protein protective agent is 150-350mM; Preferably, the protein protective agent is one of sucrose, mannitol, trehalose, sorbitol, glycine, proline, methionine, lysine hydrochloride, arginine hydrochloride or sodium chloride, or a combination thereof.

7. The pharmaceutical preparation of anti-IL-11 monoclonal antibody according to claim 1, characterized in that: The content of the surfactant is 0.005%-0.04% w / v; Preferably, the surfactant is selected from polysorbate 80, polysorbate 20 or poloxamer.

8. The pharmaceutical preparation of anti-IL-11 monoclonal antibody according to claim 1, characterized in that: The pH value of the pharmaceutical preparation is 5.5-6.5.

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