Anti-il-11 hybridoma antibodies and their use in inhibiting fibrosis

Abstract

The application discloses an anti-IL-11 hybridoma antibody and application thereof in inhibition of fibrosis, adopts recombinant human IL11 / recombinant monkey IL11 protein as an antigen, obtains hybridoma cell strains which are all specifically combined with human, mouse and monkey IL11, obtains hybridoma with good affinity of the antibody and the antigen, obtains an antibody variable region sequence, and amplifies expression of the obtained carrier to obtain a human-mouse chimeric antibody, and the anti-IL-11 human-mouse chimeric antibody 900910 has very strong affinity to human, mouse antigen and monkey antigen. Meanwhile, homologous 3D modeling method is adopted for humanization, finally, two humanized heavy chain sequences and two humanized light chain sequences are obtained, and the humanized molecule affinity can reach a picomolar level. In addition, the anti-IL-11 hybridoma antibody can inhibit expression of fibrosis related genes IL-11 and fibrosis related proteins FN induced by TGF beta, and the effect is obvious in the anti-IL-11 antibody experiment in a UUO chronic kidney fibrosis model, and the anti-IL-11 hybridoma antibody can be applied to treatment of inhibition of fibrosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of drug detection, and particularly relates to an anti-IL-11 hybridoma antibody and application thereof in inhibition of fibrosis. BACKGROUND

[0002] Interleukin-11 is a stromal cell-derived cytokine belonging to the IL-6 superfamily, which is secreted by various types of cells such as osteoblasts, synoviocytes, fibroblasts, chondrocytes and trophoblasts, and then activates the downstream JAK / STAT signaling pathway by binding to IL-11Ralpha and GP130 receptors.

[0003] More and more studies have found that IL-11 plays an extremely important role in cell protection, anti-inflammation and anti-fibrosis of organs such as lungs. IL-11 is secreted by polarized cells or fibroblasts after injury, and has autocrine and paracrine effects. In polarized cells, IL-11 causes cell dysfunction, initiates apoptotic cell death, and at the same time blocks regeneration. In stromal cells, IL-11 triggers extracellular matrix production, invasion and migration of myofibroblasts. IL-11-activated fibroblasts and myofibroblasts secrete cytokines and chemokines, which have strong pro-inflammatory effects. Inhibition of IL-11 can protect parenchyma, resist fibrosis, and reduce stromal-driven inflammation. In tissues with regenerative capacity, inhibition of IL-11 can promote the proliferation and regeneration of damaged cells (such as hepatocytes) and organ regeneration.

[0004] In practical applications, IL-11 achieves medical applications by obtaining monoclonal antibodies. For example, Boehringer Ingelheim develops a monoclonal antibody drug targeting the IL-11 / IL-11Ra homologous pathway to develop a first-in-class therapy for treating various fibro-inflammatory diseases. Lassen Therapeutics develops a monoclonal antibody drug LASN01 targeting IL-11Ra, which is expected to be a new treatment for fibrosis and tumor cancer. In 2021, a new drug research application was submitted to the FDA to start a phase I clinical trial. In the prior art, CN115975027A discloses an IL-11 humanized antibody and application thereof. The antibody includes a heavy chain variable region CDR as shown in SEQ ID NO:4-6, a light chain variable region CDR as shown in SEQ ID NO:37-39, a heavy chain framework region as shown in SEQ ID NO:67-70, and a light chain framework region as shown in SEQ ID NO:71-74. The antibody has low immunogenicity, can specifically target and combine with IL-11, and block the combination of IL-11 and IL-11 receptors. However, the affinity of the antigen obtained by humanization modification of the IL-11 antibody is relatively weak, and further improvement is still needed.

[0005] Based on this, the present application is proposed. SUMMARY

[0006] To solve the above problems, the present application provides an anti-IL-11 hybridoma antibody and its application in inhibiting fibrosis, the steps are as follows:

[0007] An anti-IL-11 hybridoma antibody, comprising a heavy chain sequence and a light chain sequence, the heavy chain sequence comprising 900910-VH1 and 900910-VH2, and the light chain sequence comprising 900910-VK1 and 900910-VK2.

[0008] The amino acid sequence of 900910-VH1 is

[0009] EVQLVESGGGLVQPGGSLRLSCAASGLTFSSFGMHWVRQAPGKGLEWVAYISGGSSITHYSDTVKGRFTISRDNAKNTLFLQMTSLRAEDTAVYYCALGFDYWGQGTTLTVSS.

[0010] The amino acid sequence of 900910-VH2 is

[0011] EVQLVESGGGLVQPGGSLRLSCAASGLTFSSFGMHWVRQAPGKGLEWVAYISGGSSITHYSDTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCALGFDYWGQGTTLTVSS.

[0012] The amino acid sequence of 900910-VK1 is

[0013] DIVMTQSPSSLSASVGDRVTITCKASQIVDTDVVWYQQKPGKAPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISSLQPEDFATYFCQQYNSYPFTFGSGTKLEIK.

[0014] The amino acid sequence of 900910-VK2 is

[0015] DIVMTQSPSSLSASVGDRVTITCKASQIVDTDVVWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYNSYPFTFGSGTKLEIK.

[0016] The preparation method of the anti-IL-11 hybridoma antibody is as follows:

[0017] Step 1: Inject mice with recombinant human IL11 / recombinant monkey IL11 protein as antigen for cross immunization, and finally obtain hybridoma cell strains that specifically bind to human, mouse and monkey IL11;

[0018] Step 2: Obtain supernatant antibodies of hybridoma cell strains and perform affinity screening;

[0019] Step 3: Obtain variable region sequences based on TAKARA 5' RACE technology;

[0020] Step 4: Obtain anti-human IL-11 human-mouse chimeric antibodies by variable region gene amplification and transfection expression;

[0021] Step 5: Design humanization of anti-human IL-11 human-mouse chimeric antibodies;

[0022] Obtain heavy chain variable region gene and light chain variable region gene based on TAKARA 5' RACE technology,

[0023] In the anti-IL-11 chimeric antibody, the heavy chain variable region and the light chain variable region are included, the heavy chain variable region is HCVR, VH-CDR1, VH-CDR2, VH-CDR3, and the light chain variable region is HCVR, VL-CDR1, VL-CDR2, VL-CDR3, the amino acid sequence of the heavy chain variable region HCVR is

[0024] DVQLVESGGGLVQPGGSRKLSCAASGLTFSSFGMHWVRQAPEKGLEWVAYISGGSSITHYSDTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCALGFDYWGQGTTLTVSS (SEQ ID NO: 1);

[0025] The amino acid sequence of the heavy chain variable region LCVR is DIVMTQSQKFMSTSVGDRVSVTCKASQIVDTDVVWYQKKPGHSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPFTFGSGTKLEIK (SEQ ID NO: 2);

[0026] Among them, the CDR sequence of the mouse-derived anti-IL-11 antibody is as follows:

[0027]

[0028] The anti-IL-11 hybridoma antibody has good blocking activity of IL-11 / IL-11Ra / GP130 ternary complex formation;

[0029] Humanization of the antigen binding protein was performed using 3D modeling methods, using PDB BLAST to call the sequence closest antibody crystal structure model to determine the human framework regions suitable for building CDR grafted heavy and light chains on.

[0030] Two humanized heavy chain sequences 900910-VH1, 900910-VH2, and two humanized light chain sequences 900910-VK1, 900910-VK2 were co-designed,

[0031] 900910 heavy chain humanized sequences:

[0032] 900910-VH1:

[0033] EVQLVESGGGLVQPGGSLRLSCAASGLTFSSFGMHWVRQAPGKGLEWVAYISGGSSITHYSDTVKGRFTISRDNAKNTLFLQMTSLRAEDTAVYYCALGFDYWGQGTTLTVSS

[0034] 900910-VH2:

[0035] EVQLVESGGGLVQPGGSLRLSCAASGLTFSSFGMHWVRQAPGKGLEWVAYISGGSSITHYSDTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCALGFDYWGQGTTLTVSS

[0036] 900910 light chain humanized sequences:

[0037] 900910-VK1:

[0038] DIVMTQSPSSLSASVGDRVTITCKASQIVDTDVVWYQQKPGKAPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISSLQPEDFATYFCQQYNSYPFTFGSGTKLEIK

[0039] 900910-VK2:

[0040] DIVMTQSPSSLSASVGDRVTITCKASQIVDTDVVWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYNSYPFTFGSGTKLEIK.

[0041] Anti-IL-11 hybridoma antibodies can inhibit the expression of fibrosis-related genes IL-11 induced by TGFβ.

[0042] Anti-IL-11 hybridoma antibodies can inhibit the expression of fibrosis-related protein FN induced by TGFβ.

[0043] Technical principles

[0044] Recombinant human IL11 / recombinant monkey IL11 protein is used as an antigen to prepare hybridoma cells that produce mouse-derived monoclonal antibodies, obtain hybridoma cell strains that specifically bind to human, mouse, and monkey IL11, and obtain hybridomas with good affinity of antibodies to antigens. The variable sequences 900910 HCVR and 900910 LCVR of the obtained hybridoma 6G5; based on the 5' RACE technical principle of TAKARA, the cDNA sequence of the variable region of the mouse antibody expressed by the hybridoma cell strain is cloned to obtain the antibody variable region sequence, wherein the CDR sequence of the mouse-derived anti-IL-11 antibody, the heavy chain variable region VH-CDR1, VH-CDR2, VH-CDR3, and the light chain variable region VL-CDR1, VL-CDR2, VL-CDR3, and the obtained vector is amplified and expressed to obtain a human-mouse chimeric antibody. The affinity of the anti-IL-11 human-mouse chimeric antibody 900910 to human and mouse IL-11 reaches 10-09 order of magnitude, and has extremely strong affinity to human, mouse, and monkey antigens. At the same time, homologous 3D modeling method is used for humanization, and finally 2 humanized heavy chain sequences and 2 humanized light chain sequences are obtained, and 4 900910 humanized antibodies I600025 (VH1+VK1), I600026 (VH2+VK1), I600029 (VH1+VK2), and I600030 (VH2+VK2) are obtained. The affinity of the humanized molecule can reach the picomolar level.

[0045] In addition, the anti-IL-11 hybridoma antibody 900910 can inhibit the expression of fibrosis-related genes IL-11 and fibrosis-related protein FN induced by TGFβ, and has obvious effect in the anti-IL-11 antibody experiment in the UUO chronic kidney fibrosis model, reduces the fibrosis area in the fibrosis-related tissue staining, and can be suitable for clinical experiments, and has broad medical prospects. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 Activation of anti-human IL-11 human-mouse chimeric antibody 900910 on hIL11 Effector Report;

[0047] Figure 2 Blocking activity of anti-human IL-11 human-mouse chimeric antibody 900910 on IL-11 / IL-11Ra / GP130 ternary complex;

[0048] Figure 3 Data are expressed as means ± SEM, n = 3. *P < 0.05, as determined by one-way ANOVA;

[0049] Figure 4 Figure 6 is a picture of immunofluorescence detection;

[0050] Figure 5 Figure 7 is a picture of UUO experimental tissue staining;

[0051] Figure 6 Figure 8 is a result of analyzing chronic fibrosis of UUO model. DETAILED DESCRIPTION

[0052] The present application will be further described by the following description of the drawings and specific examples. It is understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the scope of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Unless otherwise required by context, the use herein of the singular is also to be read as the plural and vice versa. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The materials, methods, and examples provided herein are illustrative only and in no way limit the scope of the application. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of materials, conditions, reaction times, and so forth are to be understood as being modified in all instances by the term "about". Unless otherwise indicated, the examples provided herein are not intended to be limiting.

[0053] EXAMPLE

[0054] I. Preparation of Anti-IL-11 Hybridoma Antibodies

[0055] Step 1: Preparation of hybridoma cells producing murine monoclonal antibodies

[0056] The method for preparing the murine monoclonal antibody employs the hybridoma preparation technology invented by Kohler and Milstein in 1975 (Nature, 1975, 256: 495-497). The recombinant human IL11 / recombinant monkey IL11 protein is used as the antigen, and the Balb / C, CD1, and other strains of mice are immunized by subcutaneous injection of multiple points after the antigen is fully emulsified with Freund's adjuvant (Sigma, Cat No: F5881 / F5506), and the recombinant human IL11 / recombinant monkey IL11 protein is cross-immunized. After each immunization for three times, the blood is collected, and the ELISA method is used to measure the titer of the mouse serum. When the titer of the serum reaches the target value, the pre-fusion immunization is performed. The spleen cells of the mouse with the highest titer are selected to be fused with the SP2 / 0 myeloma cells. The hybridoma cell strain is screened by using the culture medium containing HAT, and the supernatant of the hybridoma cells is screened by the ELISA method, and the hybridoma clone that combines with the human IL11 is selected. Then, the ELISA method is used to detect the specific binding of the supernatant of the hybridoma to the monkey and mouse IL11, and the hybridoma cell strain that specifically binds to the human, mouse, and monkey IL11 is obtained. The selected hybridoma cells are subjected to supernatant antibody affinity detection. Finally, the sequence analysis is performed on the hybridoma cell strain expressing the Hu-IL11 antibody.

[0057] Step 2: Antibody affinity screening of the supernatant of the hybridoma cell culture medium

[0058] Step 2: The SPR method is used to determine the affinity of the antibody in the supernatant of the hybridoma to the antigen, and the required test materials and instruments are as follows:

[0059]

[0060] The experimental method used in Step 2:

[0061] The sample cell and flow cell temperature of the Biacore 8K were set to 25 °C and the data collection frequency was 10 Hz. Anti-mouse antibodies were amino coupled to a CM5 chip following the use of mouse anti-capture kit instructions (Cytiva, Cat# BR100838). Antibodies in culture supernatant were captured as ligand using the anti-mouse antibody coupled CM5 chip. Human, monkey and mouse IL-11 antigens were diluted to 50 nM in running buffer (1 mg / mL CM-Dextran sodium salt, 1 mg / mL Bovine Serum Albumin, HBS-EP+, pH 7.4 buffer) as analyte and running buffer as 0 concentration for background subtraction. The Kinetic screening method was used with an analyte flow rate of 30 μL / min, an association time of 120 s, a dissociation time of 180 s, 10 mM glycine (pH 1.5) 50 μL / min regeneration for 60 s. Data was analyzed using a 1:1 binding model, FitLocal Kinetics model. Results are shown in Table 1.

[0062] Table 1: Hybridoma affinity screening data listing

[0063]

[0064] Step 3: Variable region gene cloning of anti-human IL-11 antibodies

[0065] Based on the principle of 5' RACE technology of TAKARA, the cDNA sequence of the variable region of mouse antibody expressed by hybridoma cell line was cloned. Briefly, the SMARTer 5' RACE Synthesis Kit (TAKARA, Cat. No. 634859) was used to synthesize the variable region gene-specific cDNA of heavy chain and light chain according to the instructions. The 5' and 3' ends of the cDNA sequence were modified by PCR primers designed to add appropriate leader sequences to the heavy chain and light chain variable region cDNA, respectively, so that the resulting PCR products could be cloned into the existing recombinant antibody expression heavy chain vector pHB-Fc and light chain vector pHB-Ck by the method of seamless cloning. The pHB-Fc expression vector contains the human IgG1 heavy chain constant region gene sequence, with L234A and L235A (Eunumbering) mutations on CH2 to weaken the ADCC effect of the antibody; the pHB-Ck vector contains the human kappa light chain constant region gene sequence. The heavy chain and light chain variable region PCR amplification products were cloned into the expression vector by In-fusion cloning reagent (TAKARA, Cat. No. 639650) to obtain a human-mouse chimeric antibody expression vector, and transformed into Stellar TM Competent Cells E. coli competent cells (TAKARA, Cat. No. 636763). Single colonies were selected for Sanger sequencing, and the antibody variable region sequence was analyzed. The results obtained anti-IL-11 chimeric antibody (Huabio No. 900910) derived from hybridoma 6G5 in Example 1, the variable region sequence is as follows:

[0066] 900910 HCVR SEQ ID NO: 1

[0067] DVQLVESGGGLVQPGGSRKLSCAASGLTFSSFGMHWVRQAPEKGLEWVAYISGGSSITHYSDTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCALGFDYWGQGTTLTVSS

[0068] 900910 LCVR SEQ ID NO: 2

[0069] DIVMTQSQKFMSTSVGDRVSVTCKASQIVDTDVVWYQKKPGHSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPFTFGSGTKLEIK

[0070] Among them, the CDRs refer to Table 2.

[0071] Table 2 CDR sequences of mouse anti-IL-11 antibody

[0072]

[0073] Step 4: Expression of anti-human IL-11 human-mouse chimeric antibody

[0074] The expression vector obtained in Step 3 was amplified in E. coli, and a sufficient amount of plasmid was prepared using an endotoxin-free plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., Catalog No. DP117) for transient transfection to express the chimeric antibody. The host cells used for expression were CHO-S cells (Thermo, Catalog No. R80007). After mixing the prepared two heavy chain vectors with the light chain vector with polyetherimide (PEI, Polysciences, Catalog No. 24765-1) to form a liposome complex, the CHO-S cells were transfected and cultured in a carbon dioxide incubator for 7 days. The cell culture supernatant was collected by centrifugation, and the human-mouse chimeric antibody was purified by Protein A affinity chromatography column.

[0075] Detection of affinity of A human-mouse chimeric antibody to human and mouse IL-11

[0076] Purpose of the test: This test uses the SPR method to determine the binding kinetic constant of human and mouse IL-11 to the antibody.

[0077] Test materials and instruments:

[0078]

[0079] Experimental method:

[0080] The sample chamber and flow cell temperature of the Biacore 8K were set to 25°C, and the data collection frequency was 10 Hz. The Series S Protein A chip captured the antibody sample to 300 RU as the ligand. Human and mouse IL-11 antigens were diluted to 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.56 nM, 0.78 nM, and 0.39 nM as a series of gradient concentrations of analytes using HBS-EP+ buffer, pH 7.4, and HBS-EP+ buffer, pH 7.4, as the 0 concentration for background subtraction. Using the multi-cycle kinetics method, the analyte flow rate was set to 30 μL / min, the binding time was 120 s, the dissociation time was 600 s, the 10 mM glycine (pH 1.5) was 50 μL / min, and the regeneration time was 60 s. 1:1 binding mode, Fit Local Kinetics mode to analyze the data.

[0081] Experimental results:

[0082] Table 3. Results of detection of affinity of anti-human IL-11 human-mouse chimeric antibody 900910 to human and mouse IL-11

[0083] Sample Antigen ka (1 / Ms) kd (1 / s) KD (M) 900910 Human IL-11 3.18E+05 1.85E-03 5.82E-09 900910 Mouse IL-11 3.13E+05 1.97E-03 6.31E-09

[0084] The affinity constant (KD(M)) results show that the affinity of the anti-IL-11 human-mouse chimeric antibody 900910 of the present application to human and mouse IL-11 is of the order of 10-9, with very strong affinity to human and mouse antigens.

[0085] Detection of the affinity of the B humanized monoclonal antibody to monkey IL-11

[0086] Purpose of the experiment: The experiment uses the SPR method to determine the antigen-antibody binding kinetic constant of the monkey.

[0087] Experimental materials and instruments:

[0088]

[0089] Experimental method:

[0090] The sample chamber and flow cell temperature of the Biacore 8k were set to 25°C, and the data collection frequency was 10 Hz. Cynomolgus IL-11 was immobilized as a ligand on a Series S Sensor Chip CM5 chip using an Amine Coupling Kit. The sample was diluted into a series of gradient concentrations as an analyte using HBS-EP+ buffer, pH 7.4, and HBS-EP+ buffer, pH 7.4, was used as the 0 concentration for background subtraction. The single-cycle kinetic method was used, with an analyte flow rate of 30 μL / min, a binding time of 120 s, a dissociation time of 600 s, 10 mM glycine (pH 1.5) 50 μL / min regeneration for 60 s. 1:1 binding mode, FitLocal Kinetics mode to analyze the data.

[0091] The experimental results are shown in the following table.

[0092] Table 4. Results of the detection of the affinity of the anti-human IL-11 human-mouse chimeric antibody 900910 to monkey IL-11

[0093] Sample Antigen ka (1 / Ms) kd (1 / s) KD (M) 900910 Cynomolgus IL-11 3.21E+05 1.72E-03 5.37E-09

[0094] The affinity constant (KD(M)) results show that the affinity of the anti-IL-11 human-mouse chimeric antibody 900910 of the present application to monkey IL-11 is of the order of 10-9, with very strong affinity to monkey antigens.

[0095] C Anti-IL-11 antibody blocks IL-11-mediated activation of hIL11 Effector Reporter Cell

[0096] hIL11 EffectorReport (Nanjing Kebai, cat#CBP74114) in good growth condition were collected and counted, resuspended in medium (composition: DMEM+10%FBS) to 1.2E6 cells / mL cell suspension, 50μL / well plated in 96-well white plate (Jingan Biotech, cat#J09601); antibody 900910 was diluted with medium to 200nM, and then diluted by 3 times gradient to 10 concentrations, a total of 11 concentration gradients; the diluted antibody was added to the 96-well white plate at 25μL / well; IL11 protein (Jin'an, cat#C006) was diluted to 0.2ng / mL, and 25μL / well was added to the 96-well white plate; the edge well was sealed with PBS liquid, 100μL / well, and continued to culture at 37℃, 5%CO2 for 6 hours; after incubation, the plate was taken out and balanced for 15 minutes, 100μL / well was added UltraLuciferase Detection Kit reagent (Yakeway, cat#A2000301N), reaction for 5 minutes, full wavelength detection of microplate reader. Using GraphPad Prism 8 software, the sample working concentration-bioluminescence reading was used to analyze the data by four-parameter equation to obtain the sample dose-effect curve.

[0097] Table 5. Anti-human IL-11 human-murine chimeric antibody 900910 and biological activity detection detection results

[0098] Sample R2 Bioactivity (nM) 900910 0.9862 0.085

[0099] D Anti-IL-11 antibody blocking IL-11 / IL-11Ra / GP130 ternary complex formation blocking activity analysis

[0100] The blocking activity of anti-IL-11 antibody on the formation of IL-11 / IL-11Ra / GP130 ternary complex was analyzed by competitive ELISA method. Human IL-11Ra (Sino Biological Inc. Cat#: 10252-H08H) was added to the enzyme-linked plate and coated overnight; the next day, the sample was prepared, first 0.4 μg / mL human IL-11 (Sino Biological Inc. Cat#: 12225-HNCE) was prepared, then mixed with gradient-diluted anti-IL-11 antibody (dilution concentration: 300, 100, 33.33, 11, 11, 3.70, 1.23, 0.41, 0.14, 0.046, 0.015, 0.0051 μg / mL) at equal volume 1:1, then added to the IL-11Ra-coated enzyme-linked plate for co-incubation, finally 2 μg / mL of GP130 (Sino Biological Inc. Cat#: 10974-H05H) was added for co-incubation. After one hour, HRP-labeled anti-mouse Fc secondary antibody was added for color development, OD450 detection of absorbance. The final experimental results showed that 900910 had good blocking activity on the formation of IL-11 / IL-11Ra / GP130 ternary complex, with an IC50 of 3.3 nM.

[0101] Step 5 Humanization design of anti-IL-11 murine chimeric antibody 900910

[0102] The humanization of antigen binding proteins uses the method of 3D modeling: first, the three-dimensional structure model of the murine antigen binding protein is modeled, the optimal structure model is selected, and 5-10 optimal structure solutions are selected by homology modeling method. The Loop region is generally modeled by homology modeling method, such as the CDR amino acid sequence alignment result shows less than 50% Identity, then the CDR3 structure model is built by de novo modeling method. Use PDB BLAST to retrieve the 10 antibody crystal structure models closest to the sequence (structure resolution higher than 2.5 angstrom), compare the automatic modeling model, and select the optimal structure model. Then compare the variable region sequence of the antigen binding protein with the available sequences in the NCBI IgBlast database, identify and analyze, and finally determine the human framework region (FR region) suitable for constructing CDR grafting heavy chain and light chain thereon.

[0103] In the modification, according to the conserved amino acid residues of the human antibody FR region and the important amino acid residues in the antibody FR region, the modification sites are designed, and the variable regions of the heavy and light chains of the antigen binding protein 900910 are respectively subjected to humanization mutation design. The designed humanization sequence should meet the requirements of not affecting the stability of the antibody structure, not affecting the binding of the antigen binding protein to the antigen, not introducing glycosylation, phosphorylation and other protein modification sites, not introducing sites susceptible to oxidation, amination and the like, and enhancing the structural stability. Through analysis, two humanized heavy chain sequences and two humanized light chain sequences are designed for the murine antigen binding protein sequence of 900910, and the sequences are shown in the following table:

[0104] 900910 heavy chain humanization sequence:

[0105] 900910-VH1:

[0106] EVQLVESGGGLVQPGGSLRLSCAASGLTFSSFGMHWVRQAPGKGLEWVAYISGGSSITHYSDTVKGRFTISRDNAKNTLFLQMTSLRAEDTAVYYCALGFDYWGQGTTLTVSS

[0107] 900910-VH2:

[0108] EVQLVESGGGLVQPGGSLRLSCAASGLTFSSFGMHWVRQAPGKGLEWVAYISGGSSITHYSDTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCALGFDYWGQGTTLTVSS

[0109] 900910 light chain humanization sequence:

[0110] 900910-VK1:

[0111] DIVMTQSPSSLSASVGDRVTITCKASQIVDTDVVWYQQKPGKAPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISSLQPEDFATYFCQQYNSYPFTFGSGTKLEIK

[0112] 900910-VK2:

[0113] DIVMTQSPSSLSASVGDRVTITCKASQIVDTDVVWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYNSYPFTFGSGTKLEIK

[0114] Wherein the CDR sequences refer to Table 6, and the CDR sequence partition is according to Kabat format.

[0115] The above two 900910 heavy chain humanized sequences and two 900910 light chain humanized sequences are randomly combined to obtain four 900910 humanized antibodies, I600025 (VH1+VK1), I600026 (VH2+VK1), I600029 (VH1+VK2) and I600030 (VH2+VK2), which are respectively expressed and purified by CHO-S cells. The binding ability of 900910 humanized proteins to IL-11 antigen, the blocking activity of IL-11 and its receptor binding, and other indicators are screened by flow cytometry, Biacore and other detection methods. A plurality of humanized anti-IL-11 antigen binding proteins with excellent performance are obtained, and I600030 is the best molecule in the humanized molecules after analysis.

[0116] Table 6. Humanized sequences of anti-human IL-11 human-mouse chimeric antibody 900910

[0117]

[0118]

[0119] Table 7. Detection results of affinity and blocking activity of anti-human IL-11 human-mouse chimeric antibody 900910 humanized molecules

[0120]

[0121] II. Inhibition of fibrosis by IL-11 hybridoma antibodies in vitro

[0122] Detection of the ability of anti-IL-11 hybridoma antibody 900910 to inhibit TGFβ (transforming growth factor β) induced fibrosis in NICH0018 (healthy human skin fibroblasts).

[0123] qPCR detection:

[0124] NICH0018 cells in log phase in culture flask were washed twice with 10 mL PBS after removing the culture medium (89% DMEM (Dulbecco's medium) + 10% FBS (fetal bovine serum) + 1% P / S (penicillin, streptomycin)), and then 2 mL of 0.25% trypsin was added and digested in an incubator (37°C, 5% carbon dioxide) for 3-5 minutes. After stopping the digestion by adding the culture medium, a cell suspension was obtained, centrifuged (500g x 5 min) to discard the culture medium, resuspended to 1 mL with the culture medium, and then counted on a counting instrument. The cells were plated into a six-well plate at a cell number of 5 x 105 per well, supplemented with the culture medium to 2 mL per well, and cultured in an incubator for 8 h to adhere, and then the complete culture medium (DMEM without FBS) was replaced for 16 h of starvation treatment. The culture medium was prepared according to a final concentration of 10 ng / mL TGF-beta or 15 μg / mL 900910, and after removing the original culture medium, 2 mL of the ordinary culture medium was added according to the blank group, 2 mL of the culture medium of 10 ng / mL TGF-beta was added according to the stimulation group, and 2 mL of the culture medium of a final concentration of 10 ng / mL TGF-beta and 15 μg / mL 900910 was added according to the antibody group, with three repeats in each group. After incubation in an incubator for 24 h, the cells were collected into an RNase-free EP tube with a cell scraper after washing twice with PBS, and the cell precipitate was obtained by centrifugation (500g x 5 min).

[0125] RT-PCR was used to detect the expression of fibrosis-related genes IL-11 (encoding IL-11 protein) and COL IV (encoding collagen type IV) in the cells. 350 μL Buffer RLT was added to the cell pellet to mix and lyse the cells, and 350 μL of 70% ethanol was added to the lysate and mixed evenly. The previous lysate was transferred to a 2 mL collection tube containing a RNeasy MinElute spin column, centrifuged at 8000 g for 15 s, and the filtrate was discarded. 350 μL Buffer RW1 was added to the RNeasy MinElute spin column, centrifuged at 8000 g for 15 s, and the filtrate was discarded. The RNeasy MinElute spin column was placed in a new 2 mL collection tube, 500 μL Buffer RPE was added, centrifuged at 8000 g for 15 s, and the filtrate was discarded. 500 μL of 80% ethanol was added to the RNeasy MinElute spin column, centrifuged at 8000 g for 2 min, and the collection tube was discarded. The RNeasy MinElute spin column was placed in a new 1.5 mL collection tube, and 14 μL of RNase-free water was added to the center of the adsorption column, and the highest speed of the centrifuge was used to centrifuge for 1 min. After eluting the RNA, the RNA concentration was detected. The reaction system and reaction conditions for removing the genome are as follows:

[0126] Table 8. Reaction system components

[0127] Reagent Name Volume (μL) 5X gDNA Eraser Buffer 2ul gDNA Eraser 1ul Total RNA 250 ng RNase_Free_dH2O up to 10 ul

[0128] Table 9. Reaction system reaction time

[0129] Temperature (°C) Time 42 2 min 4 ∞

[0130] After obtaining the reaction solution of the removal reaction group, a reverse transcription reaction was performed, and the reaction system and reaction conditions are as follows:

[0131] Table 10. Reaction system components

[0132]

[0133] Table 11 Reaction temperature and reaction time relationship

[0134]

[0135] Tables 8-11 reflect the relationship between different reaction systems and reaction times, and it is known that the reaction temperature of the system should be maintained at 37-42°C.

[0136] After cDNA was obtained, quantitative PCR was performed:

[0137] The reaction solution was prepared according to the following table, and the mixture was added to the corresponding reaction tube, 5.6 μL each. And 1 μL cDNA sample was mixed with 3.4 μL DEPC H2O to form Mix, and was added to the corresponding hole. At the same time, negative control hole was set, and the template was sterile water.

[0138] Table 12. Reaction solution system components

[0139] Reagent Volume 2x Fast SYBE Green Master Mix 5 μL Forward Primer (10 μM) 0.3 μL Reverse Primer (10 μM) 0.3 μL

[0140] Program setting: comparative CT (ΔΔCT) method was used, and software running program was set. PCR amplification program: denaturation 95 degrees for 10 min, 1 cycle; denaturation 95 degrees for 15 s, extension 60 degrees for 30 s, 40 cycles; denaturation 95 degrees for 15 s, extension 60 degrees for 1 min, 0.15 degrees gradient rise to 95 degrees. Running quantitative PCR.

[0141] Gene detection results are shown in Figure 3 The results show that anti-IL-11 hybridoma antibody 900910 can inhibit the increase of fibrosis-related genes IL-11 and COL IV induced by TGFβ. It is proved that 900910 can inhibit the fibrosis of NICH0018 cells induced by TGFβ.

[0142] Immunofluorescence detection:

[0143] After the NICH0018 cells in the logarithmic growth phase in the culture bottle were removed from the culture medium (89% DMEM (Dulbecco's medium) + 10% FBS (fetal bovine serum) + 1% P / S (penicillin, streptomycin)), they were washed twice with 10 mL PBS, and then 2 mL 0.25% trypsin was added and digested in the incubator (37°C, 5% carbon dioxide) for 3-5 min. After adding the culture medium to stop the digestion, the cell suspension was obtained, centrifuged (500g×5min) to discard the culture medium, resuspended to 1 mL with the culture medium, and counted on the counting instrument. 1×10 5Cells were seeded into 12-well plates pre-placed with treated coverslips, and culture medium was added to a final volume of 2 mL per well. After 8 hours of incubation, the cells adhered to the plates, and the medium was replaced with complete DMEM (FBS-free) for 16 hours of starvation. Culture medium was prepared to a final concentration of 10 ng / mL TGFβ or 15 μg / mL 900910. After removing the original medium, 2 mL of plain medium was added to the blank group, 2 mL of medium containing 10 ng / mL TGF-beta was added to the stimulation group, and 2 mL of medium containing both 10 ng / mL TGF-beta and 15 μg / mL 900910 was added to the antibody group. Each group was in triplicate. After 24 hours of incubation, the plates were removed, the culture medium was discarded, and the plates were washed three times with PBS. The slides were fixed with 4% paraformaldehyde at room temperature for 15 minutes, and then washed three times with PBS. Incubate the slide with Beyotime immunostaining blocking solution at room temperature for 10 min. Wash the slide three times with PBS. Dilute Fibronectin-AF488 (1:200) and TRITC-phalloidin (1:1000) with Beyotime immunostaining primary antibody dilution solution and incubate overnight at 4°C. Wash three times with Beyotime immunostaining washing solution for 3 min each time. Add one drop of Thermo Fisher Scientific ProLong immunostaining solution to the slide. TM Glass anti-quenching sealing agent (containing NucBlue) TM (Staining agent), remove the processed cell slides and mount them with the cell side down. Use an Olympus CKX53 fluorescence inverted microscope to acquire 420nm fluorescence signals (cell nuclei) excited by 340-390nm fluorescence, 510nm fluorescence signals (Fibronectin) excited by 460-495nm fluorescence, and 575nm fluorescence signals (phalloidin) excited by 530-550nm fluorescence. Use ImageJ software to merge the images from each channel.

[0144] The results of immunofluorescence staining are shown in the figure. Figure 5 The results showed that the anti-IL-11 hybridoma antibody 900910 could inhibit the expression of the fibrosis-associated protein FN induced by TGFβ. This demonstrates that 900910 can inhibit TGFβ-induced fibrosis in NICH0018 cells.

[0145] Pharmacodynamic evaluation of anti-IL-11 antibodies in a triple unilateral ureteroscopic ligation (UUO) chronic kidney injury model

[0146] The anti-IL-11 antibody was evaluated for pharmacodynamics in a UUO chronic kidney fibrosis model. Mice were randomly divided into 4 groups, 6 in each group. Among them, 6 in the normal control group, 6 in the IgG control group (given control IgG antibody, 30 mg / kg), 6 in the positive control group (valsartan, 30 mg / kg), and 6 in the 900910 group (30 mg / kg). On the day of surgery, the left ureter of the mice was ligated using inhalation anesthesia. One day after UUO surgery, dosing began, with 2 doses per week for 3 weeks (6 doses in total). Before sampling, the animal body weight was measured. Blood was taken, and serum was aliquoted and stored at -80°C. The surgical kidney was weighed; part was preserved in formalin (for tissue staining). The indicators included serum creatinine content, serum urea nitrogen content, kidney tissue PAS staining, Masson-trichrome and FN staining. Data analysis was performed using GraphPad Prism 8.0.

[0147] The analysis results showed that there was no obvious kidney fibrosis and impaired kidney function in the normal control group. In the model group (IgG control group), obvious tissue fibrosis occurred. The control drug and anti-IL-11 antibody drugs showed different degrees of inhibition of fibrosis, manifested as a decrease in the fibrosis area in the fibrosis-related tissue staining.

Claims

1. An anti-IL-1 1 antibody, characterized in that, The anti-IL-11 antibody comprises a heavy chain sequence comprising 900910-VH1, 900910-VH2, and a light chain sequence comprising 900910-VK1, 900910-VK2, The amino acid sequence of 900910-VH1 is EVQLVESGGGLVQPGGSLRLSCAASGLTFSSFGMHWVRQAPGKGLEWVAYISGGSSITHYSDTVKGRFTISRDNAKNTLFLQMTSLRAEDTAVYYCALGFDYWGQGTTLTVSS; The amino acid sequence of 900910-VH2 is EVQLVESGGGLVQPGGSLRLSCAASGLTFSSFGMHWVRQAPGKGLEWVAYISGGSSITHYSDTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCALGFDYWGQGTTLTVSS; The amino acid sequence of 900910-VK1 is DIVMTQSPSSLSASVGDRVTITCKASQIVDTDVVWYQQKPGKAPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISSLQPEDFATYFCQQYNSYPFTFGSGTKLEIK; The amino acid sequence of 900910-VK2 is DIVMTQSPSSLSASVGDRVTITCKASQIVDTDVVWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYNSYPFTFGSGTKLEIK; The anti-IL-11 antibody is any one of the combinations of 900910-VH1 and 900910-VK1, 900910-VH1 and 900910-VK2, 900910-VH2 and 900910-VK1, 900910-VH2 and 900910-VK2.

2. The anti-IL-11 antibody of claim 1, wherein The anti-IL-11 antibody has a blocking activity of the formation of an IL-11 / IL-11Ra / GP130 ternary complex.

3. The anti-IL-11 antibody of claim 1, wherein The anti-IL-11 antibody can inhibit the IL-11 related to fibrosis induced by TGFβ.

4. The anti-IL-11 antibody of claim 1, wherein The anti-IL-11 antibody can inhibit the expression of FN, a fibrosis related protein induced by TGFβ.

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