Monoclonal antibody capable of being specifically combined with IL-33 as well as preparation method and application of monoclonal antibody

Abstract

The invention belongs to the technical field of biological medicines, and relates to a monoclonal antibody capable of being specifically combined with IL-33 as well as a preparation method and application thereof. The monoclonal antibody capable of being specifically bound with the IL-33 can be specifically bound with the IL-33 in a high-affinity mode, and therefore binding of the IL-33 and a receptor ST2 of the IL-33 is effectively blocked. IL-33 is an important cell factor, and plays a key role in the occurrence and development of various diseases, such as asthma, specific reactive / allergic dermatitis, chronic sinusitis, chronic obstructive pulmonary disease (COPD) and the like. By blocking the combination of IL-33 and ST2, the antibody can inhibit inflammatory reactions mediated by IL-33, and a new strategy is provided for the treatment of these diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to monoclonal antibodies that can specifically bind to IL-33, their preparation methods, and applications. Background Technology

[0002] Interleukin-33 (IL-33) is an important member of the IL-1 family with significant immunomodulatory functions. As a tissue-derived nuclear cytokine, IL-33 plays a crucial role in homeostasis and inflammation. It is primarily produced by activated epithelial cells, endothelial cells, and fibroblasts, and transmits signals by binding to its specific receptor ST2. This interaction can trigger a series of downstream signaling pathways, thereby regulating immune and inflammatory responses.

[0003] The primary targets of IL-33 in the body are tissue immune cells, such as mast cells, group 2 innate lymphoid cells (ILC2), and regulatory T cells (Tregs). In addition, it can affect a variety of other immune cells, including Th2 cells, eosinophils, basophils, dendritic cells, Th1 cells, CD8+ T cells, NK cells, INKT cells, B cells, neutrophils, and macrophages. This makes IL-33 play an important role in a variety of physiological and pathological processes.

[0004] IL-33 is closely associated with a variety of inflammatory diseases, autoimmune diseases, and cancers. For example, in respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and allergic rhinitis, abnormal expression of IL-33 may lead to increased airway inflammation and airway remodeling. Furthermore, IL-33 is also involved in the pathogenesis of various diseases, including cardiovascular diseases, musculoskeletal disorders, and central nervous system diseases.

[0005] IL-33, as an alarm molecule for immune responses and cell damage, plays a crucial role in the pathogenesis of various diseases, including allergic diseases, chronic inflammatory diseases, malignant tumors, and cardiovascular diseases. Therefore, IL-33 is considered a potential target for drug development in allergic diseases and autoimmune diseases. Developing monoclonal antibodies that specifically bind to IL-33 has significant application value for the prevention and treatment of related diseases; however, there is currently limited research on monoclonal antibodies that specifically bind to IL-33 in the market. Summary of the Invention

[0006] To overcome the above-mentioned defects, the purpose of this invention is to provide a monoclonal antibody that can specifically bind to IL-33, its preparation method and application, wherein the monoclonal antibody has high affinity and functionality.

[0007] The technical solution provided by this invention is as follows:

[0008] A monoclonal antibody capable of specifically binding to IL-33, the antibody comprising a heavy chain variable region and a light chain variable region;

[0009] The heavy chain variable region includes VH-CDR1, VH-CDR2 and VH-CDR3, and the light chain variable region includes VL-CDR1, VL-CDR2 and VL-CDR3.

[0010] The amino acid sequence of VH-CDR1 is shown in SEQ ID NO: 1;

[0011] The amino acid sequence of the VH-CDR2 is shown in SEQ ID NO: 2;

[0012] The amino acid sequence of the VH-CDR3 is shown in SEQ ID NO: 3;

[0013] The amino acid sequence of VL-CDR1 is shown in SEQ ID NO: 6;

[0014] The amino acid sequence of the VL-CDR2 is shown in SEQ ID NO: 7;

[0015] The amino acid sequence of the VL-CDR3 is shown in SEQ ID NO: 8.

[0016] Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 4; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 9.

[0017] Preferably, the heavy chain amino acid sequence is as shown in SEQ ID NO: 5; and the light chain amino acid sequence is as shown in SEQ ID NO: 10.

[0018] Preferably, both the heavy chain and the light chain further include a constant region, which is a constant region of mouse IgG, preferably a constant region of IgG1.

[0019] The present invention further provides a nucleic acid molecule that encodes the monoclonal antibody that specifically binds to IL-33.

[0020] The present invention further provides an expression vector containing the aforementioned nucleic acid molecule.

[0021] The present invention further provides a host cell containing the expression vector described above.

[0022] Preferably, the host cell is a eukaryotic cell, and more preferably a mammalian cell.

[0023] This invention further provides a method for preparing a monoclonal antibody capable of specifically binding to IL-33, the method comprising the following steps:

[0024] Step 1: Prepare an expression vector containing a nucleic acid molecule expressing the monoclonal antibody that specifically binds to IL-33;

[0025] Step 2: Transfect eukaryotic host cells with the expression vector from Step 1;

[0026] Step 3: Culture the eukaryotic host cells transfected in Step 2;

[0027] Step 4: Separate and purify to obtain the monoclonal antibody.

[0028] The present invention also relates to antibody immunoconjugates, bispecific molecules, chimeric antigen receptors, or pharmaceutical compositions comprising the aforementioned monoclonal antibodies capable of specifically binding to IL-33.

[0029] Furthermore, the pharmaceutical composition comprises a therapeutically effective amount of the monoclonal antibody capable of specifically binding to IL-33, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0030] The present invention further provides the use of the monoclonal antibody that specifically binds to IL-33 in the preparation of a drug.

[0031] Preferably, the drug is used to treat asthma, arthritis, atopic / allergic dermatitis, chronic sinusitis, chronic obstructive pulmonary disease, systemic sclerosis, liver fibrosis, psoriasis, ulcerative colitis, Crohn's disease, multiple sclerosis, diabetic nephropathy, inflammatory bowel disease, psoriasis, eosinophilic esophagitis, diabetic macular edema, age-related macular degeneration, dry eye disease, and tumors.

[0032] Beneficial effects

[0033] The monoclonal antibody described in this invention, capable of specifically binding to IL-33, can bind to IL-33 with high affinity, thereby effectively blocking the binding of ST2 to IL-33. IL-33 is an important cytokine that plays a crucial role in the occurrence and development of various diseases, such as asthma, atopic / allergic dermatitis, chronic sinusitis, and chronic obstructive pulmonary disease (COPD). By blocking the binding of IL-33 to ST2, this antibody can inhibit the IL-33-mediated inflammatory response, providing a new strategy for the treatment of these diseases. Attached Figure Description

[0034] Figure 1 To capture the binding ability of antibodies to human IL-33 by ELISA;

[0035] Figure 2 To compete with ELISA for detecting the ability of antibodies to block the interaction between IL-33 and ST2;

[0036] Figure 3 The results are based on the functional assay of anti-IL-33 monoclonal antibodies against cells. Detailed Implementation

[0037] the term

[0038] IL-33, also known as IL-1F11, is a member of the IL-1 family, produced by Th2 cells, mast cells, and innate lymphocytes. The human and mouse IL-33 cDNA encodes polypeptides of 270 and 266 amino acids, respectively, with corresponding full-length protein molecular weights of 30 kDa and 29.9 kDa. IL-33 can induce and regulate various inflammatory responses, potentially playing a role in chronic inflammation and autoimmune diseases. IL-33 primarily participates in Th2 cell-mediated immune responses, regulates mast cell function, and, as an intracellular nuclear factor, modulates gene transcription. IL-33 is a specific ligand for ST2. The ST2 gene is expressed in several cell types, such as fibroblasts and mast cells, and is also specifically expressed in Th2 cells in mice and humans. IL-33 signaling depends on the expression of its receptor, ST2. IL-33 binds to ST2, activating NFκB and MAPK via downstream signaling molecules such as myeloid differentiation factor 88 (MyD88), IL-1-associated protein kinase (IRAK), and tumor necrosis factor receptor-associated factor 6 (TRAF6), thereby regulating gene transcription and leading to the production and subsequent biological functions of Th2 cytokines IL-4, IL-5, and IL-13. IL-33 is a potent chemotactic agent for Th2 cells both in vitro and in vivo. In vitro, recombinant IL-33 increases the proportion of Th2 cells, induces morphological changes in Th2 cells in an IL-33 concentration-dependent manner, and promotes their migration into the collagen matrix. In vivo, it attracts adoptive ST2(+) Th2 cells to the IL-33 injection site. Therefore, IL-33 plays a key role in maintaining chronic inflammation by actively recruiting Th2 cells to inflammatory sites.

[0039] "Binding to IL-33" or "binding with IL-33" refers to the ability to interact with human IL-33. "Specific binding" refers to the ability to bind to human IL-33 protein (as well as IL-33 proteins that may come from one or more non-human species) but to non-IL-33 proteins.

[0040] "Antigen binding site" refers to one or more segments on an antibody that have the ability to specifically bind to an antigen (such as the IL-33 protein).

[0041] "Monoclonal antibody" refers to a preparation of an antibody molecule consisting of a single amino acid, exhibiting single binding specificity and affinity for a specific epitope. Monoclonal antibodies or their antigen-binding fragments can be prepared by, for example, hybridoma technology, recombinant DNA technology, phage display technology, synthetic techniques such as CDR grafting, or combinations of such or other techniques known in the art.

[0042] "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its bound ligand (e.g., an antigen). Unless otherwise stated, "binding affinity" herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between an antibody and an antigen. Affinity can be measured using methods commonly known in the art, including prior art and the methods described herein.

[0043] The term "competition" in the context of competing antigen-binding proteins (e.g., neutralizing antigen-binding proteins or neutralizing antibodies) for the same epitope refers to competition between antigen-binding proteins, as determined by an assay in which the antigen-binding protein to be detected (e.g., an antibody or its immunologically functional fragment) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., IL-33 or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein is competing with another. Competitive inhibition is measured by measuring the amount of a label bound to a solid surface or cell in the presence of the antigen-binding protein being tested. Typically, the antigen-binding protein being tested is present in excess. Antigen-binding proteins identified by competitive assays (competing antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as a reference antigen-binding protein; and antigen-binding proteins that bind to a neighboring epitope sufficiently close to the binding epitope of the reference antigen-binding protein, the two epitopes spatially preventing each other from binding.

[0044] The term "EC" 50 The half-maximal effect concentration (WMP) is the antibody concentration that elicits an intermediate response between the baseline and the maximum value after a specific exposure time.

[0045] The term "IC" 50 "Half-maximal inhibitory concentration" (WMC), also known as the antibody concentration that inhibits a specific biological or biochemical function by 50% relative to the absence of an antibody, is the concentration of an antibody that inhibits a specific biological or biochemical function by 50%.

[0046] Methods for producing and purifying antibodies and antigen-binding fragments are well-known and publicly available in the prior art, such as the Cold Spring Harbor Guide to Antibody Experimentation. For example, mice can be immunized with human IL-33 or fragments thereof, and the resulting antibodies can be refolded, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can also be prepared using conventional methods.

[0047] "Treatment" means administering an oral or topical therapeutic agent, such as a composition containing an IL-33 antibody or its antigen-binding fragment, to a patient who has symptoms of one or more diseases. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves symptoms of one or more diseases, whether by inducing the regression of such symptoms or inhibiting their progression to any clinically measurable extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also called the "therapeutic effective dose") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms.

[0048] An "effective dose" includes a dose sufficient to improve or prevent the symptoms or condition of a medical condition. An effective dose also means a dose sufficient to allow or facilitate diagnosis. The effective dose for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective dose can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0049] "Pharmaceutical composition" refers to a mixture containing one or more of the IL-33 antibodies or their antigen-binding fragments described herein, along with other pharmaceutical components such as physiological / pharmaceutical-grade carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0050] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Reagents not specifically named are commercially available, conventional reagents.

[0051] Example 1

[0052] A mouse monoclonal antibody specifically against IL-33 was obtained using fusion hybridoma technology.

[0053] 1. Animal immunization

[0054] Transfer an appropriate amount of Freund's adjuvant to a 1.5 ml EP tube and mix thoroughly with a shaker. Prepare a human IL-33-his antigen protein solution using PBS. Mix the adjuvant and protein antigen solution as needed, and emulsify the antigen thoroughly using a syringe to form a stable water-in-oil solution, then administer subcutaneous injection to mice. Based on serum titer results, administer 2 to 3 booster immunizations after the initial immunization to achieve a good immune response. For animals with good titers, administer a final booster immunization via intraperitoneal injection, followed by cell fusion.

[0055] 2. Hybridoma fusion and screening

[0056] Prior to cell fusion, mouse myeloma cells were cultured in the logarithmic growth phase. Immunized mice were sacrificed, and their spleens were harvested under sterile conditions. The fused cells were seeded into 96-well cell culture plates. Viable hybridoma cells were typically observed under a microscope after 7 to 10 days. Two weeks after cell seeding, the culture supernatant from each well was collected, and hybridomas were screened using ELISA with the human IL-33-his protein antigen.

[0057] The procedure is briefly described below: ELISA plates were coated with 60 µl of PBS solution containing 1 µg / ml of affinity-purified F(ab')2 fragment-specific goat anti-mouse IgG (Jackson Immunoresearch Laboratories, Inc., Cat#115-005-072) and incubated overnight at 4 °C. The plates were then washed once with PBS solution containing 0.05% v / v Tween-20 (PBST), and 200 µl / well of PBST solution containing 5% w / v skim milk powder was added. The plates were then blocked at 37 °C for 2 hours. After washing again, 60 µl / well of hybridoma supernatant was added, and the plates were incubated at 37 °C for 40 minutes, followed by 4 washes. 100 µl / well of biotin-labeled human IL-33-his protein solution (1:5000 diluted in PBST containing 2.5% w / v skim milk powder) was added, and the plates were incubated at 37 °C for 40 minutes, washed 4 times, and then blotted dry. Then, 100 µl / well of horseradish peroxidase-labeled streptavidin (Jackson ImmunoResearch Laboratories, Inc., Cat#016-030-084) diluted 1:5000 in PBST solution containing 2.5% w / v skim milk powder was added. After incubation at 37 °C for 40 minutes, the plate was washed four times and patted dry. 100 µl / well of TMB (InnoReagents, Cat#TMB-S-002) chromogenic substrate was added, and the plate was incubated at room temperature for 5 to 15 minutes, then stopped with 1M sulfuric acid solution. The absorbance of each well at 450 nm was measured. Hybridoma cells that showed positive ELISA binding were selected and transferred to 24-well plates for further culture. A second round of screening was performed using ELISA to identify hybridomas that specifically recognized the IL-33 antigen. Subcloning was performed using limiting dilution to obtain mouse monoclonal hybridoma cells, followed by antibody purification. In short, the protein A agarose column was washed with 5-10 column volumes of PBS buffer. Monoclonal hybridoma cell supernatant was passed through the column, followed by washing with PBS buffer until the protein absorbance reached baseline. The column was eluted with acidic elution buffer (0.1 M glycine-HCl, pH 2.7) and immediately collected into 1.5 ml tubes containing alkaline buffer (1 M Tris-HCl, pH 9.0). The fraction containing immunoglobulins was mixed and dialyzed overnight in PBS at 4 °C. Subsequently, the functional activity of the purified monoclonal antibody was characterized in vitro as described below.

[0058] Example 2

[0059] Binding activity study of mouse-derived anti-IL-33 monoclonal antibody

[0060] The binding activity of mouse-derived anti-IL-33 monoclonal antibodies produced by hybridoma clones was further tested using the following method.

[0061] Antibody binding ability is determined based on capture ELISA:

[0062] Add 100 µl / well of affinity-purified Fc fragment-specific goat anti-mouse IgG or affinity-purified Fc fragment-specific goat anti-human IgG (Jackson ImmunoResearch Laboratories, Inc., Cat#115-005-071 or Cat#109-005-098) in PBS solution to 96-well plates and incubate at 37 °C for 2 hours. After washing once with PBST, add 200 µl / well of PBST solution containing 5% w / v skim milk and block at 37 °C for 2 hours.Wash the plate again, and add 100 µl / well of diluted antibody solution to the ELISA plate coated with goat anti-mouse IgG secondary antibody. Add the reference antibody or hIgG (starting concentration 66.7 nM, serially diluted 5-fold with PBST containing 2.5% w / v skim milk) to the ELISA plate coated with goat anti-human IgG secondary antibody. (The reference antibody is Etokimab monoclonal antibody (molecular code ANB020) from AnaptysBio Biotechnology, USA.) The following text uses BM to denote the heavy chain amino acid sequence as QVQLMQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGTIYPRNSNTDYNQKFKARVTMTRDTSTSTVYMELSSLRSEDTAVYYCARPLYYYLTSPPTLFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE SEQ ID NO: 11; The light chain amino acid sequence is DIQLTQSPSFLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQAKTYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12). The plate was incubated at 37 °C for 40 minutes, followed by washing 4 times.Add biotin-labeled human IL-33 protein solution (SinoBiological, Cat#10368-HNAE, 83 ng / mL, in PBST containing 2.5% w / v skim milk, 100 µl / well), incubate at 37 °C for 40 min, then wash 4 times. Add horseradish peroxidase-labeled streptavidin (1:10000 diluted in PBST buffer, Jackson ImmunoResearch Laboratories, Inc., Cat#016-030-084) to 100 µl / well, incubate at 37 °C for 40 min, then wash 4 times and blot dry. Add 100 µl / well of ELISA substrate TMB (InnoReagents, Cat#TMB-S-002), incubate at room temperature for 5 to 15 min, then stop the incubation with 1M sulfuric acid solution. The absorbance of each well was read using a microplate reader in dual-wavelength mode: 450 nm for TMB and 630 nm as the reference wavelength. OD (450-630) values ​​were plotted against antibody concentration. The data were analyzed using Graphpad Prism software to derive the EC (extracorporeal membrane oxygenation). 50 Value. Result as follows Figure 1 As shown.

[0063] from Figure 1 As can be seen from this, the C1B1D1B1 monoclonal antibody of the present invention can specifically recognize human IL-33 antigen, and its binding activity is superior to that of the reference antibody.

[0064] Example 3

[0065] Assay for the blocking activity of mouse-derived IL-33 antibody

[0066] The blocking ability of the anti-IL-33 antibody of this invention against the interaction between IL-33 and ST2 was detected using a competitive ELISA. In short, human IL-33 protein (SinoBiological, Cat#10368-HNAE) was coated at 200 ng / well in a 96-well ELISA plate and incubated overnight at 4°C. The next day, the ELISA plate was washed with washing buffer (PBS + 0.05% Tween-20, PBST), and then blocked at 37°C for 2 hours with PBST containing 5% w / v skim milk. The plate was then washed again with washing buffer. Then, 100 μL / well of serially diluted mouse-derived anti-IL-33 antibody (starting concentration 133.3 nM, serially diluted 5-fold with PBST containing 2.5% w / v skim milk) was added to the ELISA plate, incubated at 37°C for 40 minutes, and then washed four times with washing buffer. Then, human ST2-Fc protein solution (fixed concentration 300 ng / mL, diluted with PBST containing 2.5% w / v skim milk) was added, and the plate was incubated at 37°C for 40 minutes, followed by washing four times with washing buffer. Horseradish peroxidase-labeled goat anti-human IgG secondary antibody (GAH-HRP) was then added, and the plate was incubated at 37°C for 40 minutes, followed by washing with washing buffer again. Finally, TMB was added, and the reaction was terminated with 1M H₂SO₄. The absorbance of each well was read using a microplate reader in dual-wavelength mode, with 450 nm for TMB and 630 nm as the reference wavelength. OD (450-630) values ​​were plotted against antibody concentration. The data were analyzed using Graphpad Prism software to derive the IC₂ value. 50 Values, see details in the results. Figure 2 .

[0067] from Figure 2 As can be seen, the monoclonal antibody C1B1D1B1 of the present invention has strong blocking activity against the interaction between human IL-33 and ST2.

[0068] Example 4

[0069] Assay of anti-IL-33 monoclonal antibody functional activity based on cells

[0070] The inhibitory activity of mouse anti-IL-33 monoclonal antibody on IL-33-induced IL5 release from KU812 cells was detected.

[0071] 5 x 10 5KU812 cells (human basophil-like cell line, ATCC number CRL-2099) were placed in 50 μL / well of RPMI 1640 medium (Gibco, catalog number A10491-01), supplemented with 10% fetal bovine serum (Gibco, catalog number 10099-141), and seeded in 96-well flat-bottom cell plates (Biofil, catalog number 170424-078). Add 50 μl / well of serially diluted anti-IL-33 antibody of this invention or negative control antibody (a self-made anti-CD22 antibody as an isotype negative control) to the cell plate (starting from 600 nM, serially diluted 3-fold in culture medium). Then add 50 μl of human IL-33 protein (SinoBiological, Cat#10368-HNAE) to the cell plate to a final concentration of 1.73 ng / ml to induce IL5 release from cells. Incubate the cell plate at 37°C in a 5% CO2 incubator for 24 hours. Afterward, collect the supernatant from each well and maintain it at 4°C until ELISA detection using a Human IL-5 DuoSet ELISA kit (R&D Systems, Cat#DY205). Analyze the data using Graphpad Prism software and report the IC50. 50 Value, result as Figure 3 As shown.

[0072] Depend on Figure 3 It can be seen that the mouse anti-IL-33 monoclonal antibody of the present invention can inhibit the release of IL5 induced by IL-33 stimulation in KU812 cells, and has a strong IL-33 blocking biological activity.

[0073] Example 5

[0074] Variable region sequencing of anti-IL-33 monoclonal antibody

[0075] Total RNA was extracted from cultured mouse monoclonal cell lines using the FastPure Cell / Tissue Total RNA Isolation Kit (Vazyme, Cat#RC101). The procedure is briefly described below: centrifugation was used to collect 1.5 x 10⁻⁶ RNA samples. 6Transfer the cells to a 1.5 ml centrifuge tube and aspirate the supernatant. Add 500 µl of Buffer RL1 to the cell pellet and mix gently by pipetting. Transfer the processed cells to gDNA-Filter Columns (already placed in a collection tube), centrifuge at 13,000 g at room temperature for 2 minutes, and retain the supernatant in the collection tube. Add 1.6 times the volume of Buffer RL2 and mix gently. Transfer the mixture to RNAPure Columns, centrifuge at 13,000 g at room temperature for 1 minute, and discard the waste liquid. Add 500 µl of Buffer RW1 to the RNAPure Columns, centrifuge at 13,000 g at room temperature for 1 minute, and discard the waste liquid. Add 700 µl of Buffer RW2, centrifuge at 13,000 g at room temperature for 1 minute, and discard the waste liquid. Add another 700 µl of Buffer RW2, centrifuge at 13,000 g at room temperature for 1 minute, and discard the waste liquid. Centrifuge at 13,000g for 2 minutes to completely remove residual Buffer RW2 from the RNAPure Columns. Transfer the adsorption column to a new RNase-free CollectionTubes 1.5 ml centrifuge tube and add 50 µl of RNase-free ddH2O dropwise to the center of the adsorption column. Incubate at room temperature for 2 minutes, then centrifuge at 13,000g for 1 minute at room temperature to elute the RNA.

[0076] Next, the total RNA was converted into cDNA using Takara's reverse transcription cDNA kit (Cat#6210A). The experimental system was prepared as follows: 5 µl total RNA + 0.5 µl Oligo(dT) Primer + 0.5 µl Random 6 mers + 1.0 µl ldNTP Mixture + 3.0 µl RNase-free ddH2O (total 10 µl). The mixture was first pre-denatured at 65°C for 5 minutes, then placed on ice for 2 minutes. Next, 4 µl 5x PrimeScript II Buffer + 0.5 µl RNase Inhibitor + 1.0 µl PrimeScript II RTase + 4.5 µl RNase-free ddH2O (total 20 µl) were added. After mixing, the mixture was run on a PCR instrument at 40°C for 50 minutes, then at 70°C for 10 minutes to complete cDNA synthesis.

[0077] Poly G was further added to the 3' end of the cDNA. The reaction system was prepared as follows: 5 µl cDNA + 33.5 µl ddH2O + 5 µl 10x TdT Buffer + 5 µl CoCl + 1 µl dGTP + 0.5 µl Terminal TransPerase (total 50 µl system). After mixing, the mixture was run at 37°C for 30 minutes, then at 70°C for 10 minutes to complete the Poly G tailing.

[0078] Furthermore, the tailed cDNA was used as a template for gene amplification of the antibody variable region. For amplifying the antibody heavy chain variable region sequence, the Novizan kit (Cat#P525-03) was used to prepare the PCR reaction system as follows: 25µl 2x Phanta MaxMaster Mix (Dye Plus) + 2.0µl BSJ-Pri3 Antisense P-mIgG1 + 2.0µl BSJ-Pri1 Universal C1 + 1.25µl poly G-tailed cDNA + 19.75µl ddH2O (total 50µl system). For amplifying the variable region sequence of the antibody light chain, first use Takara's kit (Cat#R010A) to prepare the PCR reaction system: 10µl 5xPrimeSTAR Buffer + 0.5µl PrimeSTAR + 1.25µl BSJ-Pri1 Universal C1+ + 1.25µl BSJ-Pri19+ + 4µl aberrant-L-R2+ + 4µl dNTP + 1.25µl cDNA with a Poly G tail + 27.75µl lddH2O (total 50 µl system).

[0079] The temperature cycling for antibody heavy chain variable region PCR amplification is as follows:

[0080] 98℃ * 5min + (98℃ *10s + 62℃ * 10s + 72℃ * 1min) * 2 Cycle +

[0081] (98℃*10s + 60℃ * 10s + 72℃ * 1min) * 4 Cycles +

[0082] (98℃*10s + 58℃ * 10s + 72℃ * 1min) * 10 Cycles +

[0083] (98℃*10s + 56℃ * 10s + 72℃ * 1min) * 20 Cycles +

[0084] 72℃ * 10min + 4℃ * ∞

[0085] The temperature cycling for antibody light chain variable region PCR amplification is as follows:

[0086] 98℃ * 5min + (98℃ * 10s + 64℃ * 10s + 72℃ * 1min) * 2 Cycle +

[0087] (98℃*10s + 62℃ * 10s + 72℃ * 1min) * 6 Cycles +

[0088] (98℃*10s + 60℃ * 10s + 72℃ * 1min) * 12 Cycles +

[0089] (98℃*10s + 58℃ * 10s + 72℃ * 1min) * 16 Cycles +

[0090] 72℃ * 10min + 4℃ * ∞

[0091] PCR products were analyzed by 1% agarose gel electrophoresis, and corresponding DNA bands (approximately 500 bp for VH and approximately 500 bp for the Vkappa light chain) were excised. DNA extraction was performed using a TIANGEN gel DNA extraction kit (Cat#DP209-03). The procedure is briefly described as follows: Weigh the gel, add an equal volume of PN solution, and incubate at 50°C for 10 minutes until the gel is completely dissolved. Transfer the resulting solution to a CA2 adsorption column (place the adsorption column in a collection tube), incubate at room temperature for at least 2 minutes, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid. Add 600 µl of PW wash buffer to the column, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid. Add another 600 µl of PW wash buffer to the column, incubate for 5 minutes, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid. Centrifuge again at 12,000 rpm for 2 minutes at room temperature to remove any residual liquid in the column, and allow to air dry completely for at least 2 minutes at room temperature. Transfer the adsorption column to a clean centrifuge tube, add 35 µl of elution buffer EB, and incubate at room temperature for at least 2 minutes. Centrifuge at 12,000 rpm for 2 minutes to obtain the prepared DNA sample. Sequencing of the purified PCR product yields the variable region sequence of the antibody.

[0092] The amino acid sequence information of the anti-IL-33 antibody C1B1D1B1 is as follows:

[0093] VH-CDR1

[0094] SYWMH (SEQ ID NO: 1);

[0095] VH - CDR2

[0096] EIHPSNGRTNYNEKFKS (SEQ ID NO: 2);

[0097] VH - CDR3

[0098] SGFDY (SEQ ID NO: 3);

[0099] VH (Heavy chain variable region)

[0100] QVQLQQPGAELVKPGASVKLSCKASGYTFT SYWMH WVKQRPGQGLEWIG EIHPSNGRTNYNEKFKS KATLTVDKSSSTAYMQLSSLTSEDSTVYYCAS SGFDY WGQGTTLTVSS (SEQ ID NO: 4);

[0101] Heavy chain

[0102] QVQLQQPGAELVKPGASVKLSCKASGYTFT SYWMH WVKQRPGQGLEWIG EIHPSNGRTNYNEKFKS KATLTVDKSSSTAYMQLSSLTSEDSTVYYCAS SGFDY WGQGTTLTVSS

[0103] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 5);

[0104] VL - CDR1

[0105] RSSKSLLYRDGKTSLN (SEQ ID NO: 6);

[0106] VL-CDR2

[0107] LMSTRAS (SEQ ID NO: 7);

[0108] VL-CDR3

[0109] QQLVEFPLT (SEQ ID NO: 8);

[0110] VL (Variable region of light chain)

[0111] DIVITQDELSNPVSSGESVSISC RSSKSLLYRDGKTSLN WFLQRPGQSPQLLIY LMSTRAS GVSDRFSGSGSGTDFTLEISRVKAEDVGVYYC QQLVEFPLT FGAGTKLELK (SEQ ID NO: 9);

[0112] Light chain

[0113] DIVITQDELSNPVSSGESVSISC RSSKSLLYRDGKTSLN WFLQRPGQSPQLLIY LMSTRAS GVSDRFSGSGSGTDFTLEISRVKAEDVGVYYC QQLVEFPLT FGAGTKLELK

[0114] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRGEC (SEQ ID NO: 10);

[0115] Human IL-33-his protein:

[0116] EYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSETLEHHHHHH (SEQ ID NO: 13);

[0117] Human ST2-Fc protein:

[0118] (SEQ ID NO: 14).

[0119] In summary, the monoclonal antibody C1B1D1B1 of the present invention can specifically recognize human IL-33 with high affinity and can specifically block the binding of human IL-33 and human ST2. It has a novel sequence and is a new anti-IL-33 monoclonal antibody.

[0120] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A monoclonal antibody capable of specifically binding to IL-33, characterized in that, The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region includes VH-CDR1, VH-CDR2 and VH-CDR3, and the light chain variable region includes VL-CDR1, VL-CDR2 and VL-CDR3. The amino acid sequence of VH-CDR1 is shown in SEQ ID NO: 1; The amino acid sequence of the VH-CDR2 is shown in SEQ ID NO: 2; The amino acid sequence of the VH-CDR3 is shown in SEQ ID NO: 3; The amino acid sequence of VL-CDR1 is shown in SEQ ID NO: 6; The amino acid sequence of the VL-CDR2 is shown in SEQ ID NO: 7; The amino acid sequence of the VL-CDR3 is shown in SEQ ID NO:

8.

2. The monoclonal antibody capable of specifically binding to IL-33 according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 4; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

9.

3. The monoclonal antibody capable of specifically binding to IL-33 according to claim 1, characterized in that, The heavy chain amino acid sequence is shown in SEQ ID NO: 5; the light chain amino acid sequence is shown in SEQ ID NO:

10.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes a monoclonal antibody that can specifically bind to IL-33 as described in any one of claims 1 to 3.

5. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, The host cell contains the expression vector as described in claim 5.

7. The method for preparing a monoclonal antibody capable of specifically binding to IL-33 as described in any one of claims 1 to 3, characterized in that, It includes the following steps: Prepare an expression vector containing a nucleic acid molecule expressing a monoclonal antibody as described in any one of claims 1 to 3 that can specifically bind to IL-33; The obtained expression vector was transfected into eukaryotic host cells and cultured. The antibody was obtained by separation and purification.

8. An antibody immunoconjugate, a bispecific molecule, a chimeric antigen receptor or pharmaceutical composition comprising any one of claims 1 to 3 that is capable of specifically binding to a monoclonal antibody of IL-33.

9. The use of the monoclonal antibody capable of specifically binding to IL-33 as described in any one of claims 1 to 3 in the preparation of a drug.

10. The application according to claim 9, characterized in that, The drug is used to treat asthma, arthritis, atopic / allergic dermatitis, chronic sinusitis, chronic obstructive pulmonary disease, systemic sclerosis, liver fibrosis, psoriasis, ulcerative colitis, Crohn's disease, multiple sclerosis, diabetic nephropathy, inflammatory bowel disease, psoriasis, eosinophilic esophagitis, diabetic macular edema, age-related macular degeneration, dry eye disease, and tumors.

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