Human il-1 beta protein binding molecules, genes encoding same and uses thereof

Abstract

The application discloses a human IL-1beta protein binding molecule, a coding gene and application thereof. The human IL-1beta protein binding molecule has an antigen binding site which is specifically combined with a human IL-1beta protein, the antigen binding site contains at least one heavy chain variable region and at least one light chain variable region, the amino acid sequence of the light chain variable region is shown as SEQ ID No. 42, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 43. The human IL-1beta protein binding molecule has high affinity and high blocking activity, and has great disease treatment value.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 202210438106.4, filed on April 25, 2022, filed on March 7, 2019, entitled Human IL-1β protein binding molecule and its encoding gene and applications. Technical Field

[0002] This invention belongs to the field of biopharmaceutical technology, specifically relating to a human IL-1β protein-binding molecule, its encoding gene, and its applications. Background Technology

[0003] IL-1β is the most extensively studied member of the IL-1 family. Many cells, including immune cells (such as monocytes and macrophages), can produce and secrete it. The release of IL-1β generally involves three steps: i) the production of an inactive precursor; ii) the maturation and activation of the precursor; and iii) the secretion of the activated protein extracellularly. Initially, IL-1β is produced as a 31 kDa precursor, which is then cleaved and activated to produce a 17 kDa mature form, which can then bind to the IL-1 receptor. Caspase-1 is the main cleavage protein, capable of cleaving IL-1β at two sites (D26 and D116) to yield the 26 kDa precursor and the 17 kDa mature form. In addition, other proteases such as PR-3 and Neutrophil elastase can also cleave IL-1β (Andrei C, Margiocco P, Poggi A, et al. Phospholipases Cand A2 control lysosome-mediated IL-1βsecretion: Implications for inflammatory processes[J]. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(26): 9745-9750; Van Damme J, De Ley M, Opdenakker G, et al. Homogeneous interferon-inducing 22K factor is related to endogenous pyrogen and interleukin-1.[J]. Nature, 1985, 314(6008): 266-268; Zsebo KM, Wypych J, Yuschenkoff VN, et al. Effects of Hematopoietin-1 and Interleukin 1Activities on Early Hematopoietic Cells of the Bone Marrow[J].Blood,1988,71(4):962-968).

[0004] IL-1β has strong biological activities, including: (1) mediating inflammatory responses: IL-1β can not only induce inflammatory responses itself, but also induce the expression of other inflammatory factors such as cyclooxygenase-2 (COX-2), iNOS, and IL-6, thereby further activating stromal cells and immune cells to produce more IL-1β to participate in inflammatory responses; (2) immunomodulatory effects, synergistically stimulating I cells and inducing many cells to produce other lymphokines; (3) participating in the formation of cachexia, having a negative nitrogen balance effect, and stimulating skeletal muscle to decompose proteins; (4) inducing acute phase proteins and participating in acute phase responses; (5) inducing fibroblast proliferation, etc.

[0005] Interleukin IL-1β release is a key factor in the early inflammatory response. After binding to its receptor IL-1RI, IL-1β interacts with its co-receptor IL-1RAcP to form the IL-1beta / IL-1RI / IL-1RAcP ternary complex, activating the NF-κB and MAPK signaling pathways in target cells, thereby inducing the expression of a series of inflammation-related molecules. A large body of basic and clinical data shows that IL-1 secretion plays a crucial role in acute gout, and blocking IL-1 by binding to cytokines or their receptors is a strategy to reduce cytokine storms. Inhibiting IL-1 secretion is feasible, and many IL-1 inhibitors are available, thus complementing existing methods for alleviating acute gout attacks (So A, Dumusc A, Nasi S, et al. The role of IL-1ingout: from bench to bedside[J]. Rheumatology, 2018).

[0006] In COPD patients, airway IL-1β and systemic inflammation are associated with frequent acute exacerbations and may mediate past and future acute exacerbations through a vicious cycle. (Fu J, McDonald VM, Baines KJ, et al. Airway IL-1β and Systemic Inflammation as Predictors of Future Exacerbation Risk in Asthma and COPD[J]. Chest, 2015, 148(3):618-629). Furthermore, IL-1β supports metastasis by promoting cancer stem cell and epithelial-mesenchymal transition. IL-1β can also participate in Th17 differentiation and the production of Th17-related cytokines. (Tominaga K, Yoshimoto T, Torigoe K, et al. IL-12 synergizes with IL-18 or IL-1β for IFN-γ production from human T cells[J]. International Immunology, 2000, 12(2):151-160).

[0007] Overexpression of IL-1β in chronic local inflammation can also promote tumor transformation. IL-1β plays an important role in the late stages of cancer development. It can be produced in the tumor microenvironment and drive angiogenesis through its interaction with angiogenic factors (such as VEGF) in cell signaling, thereby achieving tumor metastasis and spread (Voronov E, Carmi Y, Apte RN, et al. The role IL-1 in tumor-mediated angiogenesis[J]. Frontiers in Physiology, 2014:114-114). In addition, foreign bodies such as silica or asbestos inhaled into the lungs can induce lung cancer by activating pro-inflammatory cytokines such as interleukin-1β, and IL-1β can induce tumor cells to secrete endogenous TNF-α, promoting tumor growth. Therefore, inhibiting IL-1β expression can also achieve the effect of preventing and treating tumors.

[0008] Blocking the release of inflammatory factors can be a relatively effective way to treat inflammation. Anti-IL-1β antibodies can block the binding of IL-1β to its receptor, thus blocking downstream signal transduction. Novartis' canakinumab (trade name Ilaris) is a fully human, high-affinity anti-IL-1β antibody, initially approved for the treatment of juvenile idiopathic arthritis in children aged 2 years and older. Based on two international randomized, placebo-controlled trials, the efficacy of canakinumab in juvenile idiopathic arthritis has been demonstrated. In Trial 1, 33% of patients experienced disease-free activity within 15 days after a single injection of canakinumab. Trial 2 confirmed these results, with 82% of patients showing sustained efficacy after 2 years of treatment (Vanoni F, Minoia F, Malattia C, et al. Biologics in juvenile idiopathic arthritis: a narrative review. [J]. European Journal of Pediatrics, 2017, 176(9): 1147-1153). With further research, cannabidiol has achieved clinical success in the treatment of gout, atherosclerosis, coronary heart disease, and lung cancer.

[0009] Considering the need for novel therapeutic antibody molecules, improved drug efficacy, and reduced patient costs, the development of new anti-IL-1β antibodies remains essential. Summary of the Invention

[0010] The purpose of this application is to provide a novel human IL-1β protein-binding molecule.

[0011] To achieve the above-mentioned objectives, the technical solution of this application is as follows:

[0012] A human IL-1β protein-binding molecule, wherein the human IL-1β protein-binding molecule has an antigen-binding site that specifically binds to human IL-1β protein, the antigen-binding site comprising at least one heavy chain variable region and at least one light chain variable region, wherein the heavy chain variable region has three heavy chain complementarity-determining regions, the amino acid sequences of the three heavy chain complementarity-determining regions being shown in SEQ ID No. 11, SEQ ID No. 12 and SEQ ID No. 13 respectively; wherein the light chain variable region has three light chain complementarity-determining regions, the amino acid sequences of the three light chain variable regions being shown in SEQ ID No. 28, SEQ ID No. 29 and SEQ ID No. 30 respectively.

[0013] This application uses commercially available recombinant human IL-1β protein (novoprotein, CG93) as an antigen to immunize mice. Spleen cells from immunized mice are fused with mouse myeloma cells to prepare hybridoma cells for expressing murine human IL-1β polyclonal antibodies. Murine human IL-1β monoclonal antibodies with excellent blocking function are screened from the murine human IL-1β polyclonal antibodies. Then, based on the amino acid sequences of the heavy chain and light chain variable regions of the murine human IL-1β monoclonal antibody, and combined with the constant region sequence of human antibodies, a human-mouse chimeric antibody is designed. After humanization, the human IL-1β protein binding molecule of this application is obtained. This human IL-1β protein binding molecule can specifically bind to human IL-1β protein, blocking the secretion level of IL-6, and has functional inhibitory activity comparable to canakinumab (trade name Ilaris). Compared with fully humanized canakinumab, the human IL-1β protein binding molecule of this application is a novel antibody molecule with high affinity and high blocking activity, and has great therapeutic value.

[0014] In the aforementioned human IL-1β protein-binding molecule, both the heavy chain variable region (heavy chain framework region) and the light chain variable region (light chain framework region) originate from human antibody germline gene sequences or gene sequences that have at least 90% homology with human antibody germline gene sequences.

[0015] Preferably, in the above-mentioned human IL-1β protein-binding molecule, the heavy chain variable region has four heavy chain framework regions, and the amino acid sequences of the four heavy chain framework regions are shown in SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10, respectively.

[0016] Preferably, in the above-mentioned human IL-1β protein-binding molecule, the light chain variable region has four light chain framework regions, and the amino acid sequences of the four light chain framework regions are shown in SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26 and SEQ ID No. 27, respectively.

[0017] In summary, the aforementioned human IL-1β protein-binding molecule has a heavy chain variable region as shown in SEQ ID No. 40 and a light chain variable region as shown in SEQ ID No. 41.

[0018] In addition, this invention provides two other human IL-1β protein-binding molecules, which are developed in the same manner as the aforementioned human IL-1β protein-binding molecules, differing only in their amino acid sequences. In one of the human IL-1β protein-binding molecules, the amino acid sequence of the light chain variable region is shown in SEQ ID No. 42, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 43.

[0019] In another human IL-1β protein-binding molecule, the amino acid sequence of the light chain variable region is shown in SEQ ID No. 44, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 45.

[0020] The human IL-1β protein-binding molecule of this application can be a humanized chimeric antibody, an antigen-binding fragment, or a variable region fragment of a single-chain antibody. Preferably, the antibody subtype of the chimeric antibody is IgG1, IgG2, or IgG4.

[0021] In addition to heavy chain variable regions and light chain variable regions, chimeric antibodies and antigen-binding fragments also have heavy chain constant regions and light chain constant regions (amino acid sequences as shown in SEQ ID No. 35). The difference is that the antigen-binding fragment only has heavy chain constant region 1, and heavy chain constant region 1 is linked to the light chain constant region by interchain disulfide bonds; while the chimeric antibody has a complete heavy chain constant region: heavy chain constant region 1 - hinge region - heavy chain constant region 2 - heavy chain constant region 3 (amino acid sequences as shown in SEQ ID No. 34); while in the variable region fragment of single-chain antibody, the heavy chain variable region and light chain variable region are directly linked by a -(GGGGS)3- short peptide.

[0022] This application also provides a nucleotide molecule encoding the human IL-1β protein-binding molecule, as well as an expression vector containing the nucleotide molecule and a recombinant cell. The recombinant cell can be a prokaryotic cell or a eukaryotic cell, such as CHO cells, 293 cells, Escherichia coli cells, and yeast cells. When constructing different recombinant cells, the codons of the human IL-1β protein-binding molecule can be optimized according to the codon preferences of different cells to obtain the corresponding nucleotide molecule.

[0023] This application also provides the application of the aforementioned human IL-1β protein-binding molecule in the preparation of IL-1-mediated disease therapeutics, wherein the IL-1-mediated diseases include autoimmune-related diseases such as juvenile idiopathic arthritis, gout, asthma, immune encephalomyelitis, inflammatory bowel disease, psoriasis, vitiligo, diabetes, atherosclerosis, systemic lupus erythematosus, scleroderma, dermatomyositis, pancreatitis, nephritis, chronic obstructive pulmonary disease, and pulmonary fibrosis; as well as cancers such as gastric cancer, liver cancer, pancreatic cancer, colon cancer, rectal cancer, lung cancer, bladder cancer, prostate cancer, cervical cancer, ovarian cancer, fallopian tube cancer, breast cancer, leukemia, lymphoma, myeloma, glioma, and osteosarcoma.

[0024] The aforementioned IL-1-mediated disease treatment drugs can be used alone or in combination with other symptomatic drugs, preferably in the form of injectable formulations.

[0025] This application also provides the application of the human IL-1β protein binding molecule in the preparation of bispecific antibodies. When used to prepare bispecific antibodies, the human IL-1β protein binding molecule should be in the form of a humanized antigen-binding fragment or a single-chain antibody variable region fragment as one of the binding arms of the bispecific antibody.

[0026] Compared with the prior art, the beneficial effects of this application are reflected in:

[0027] This application uses commercially available recombinant human IL-1β protein (novoprotein, CG93) as an antigen to immunize mice. Spleen cells from immunized mice are fused with mouse myeloma cells to prepare hybridoma cells for expressing murine human IL-1β polyclonal antibodies. Murine human IL-1β monoclonal antibodies with excellent blocking function are screened from the murine human IL-1β polyclonal antibodies. Then, based on the amino acid sequences of the heavy chain and light chain variable regions of the murine human IL-1β monoclonal antibody, and combined with the constant region sequence of human antibodies, a human-mouse chimeric antibody is designed. After humanization, the human IL-1β protein binding molecule of this application is obtained. This human IL-1β protein binding molecule can specifically bind to human IL-1β protein, blocking the secretion level of IL-6, and has functional inhibitory activity comparable to canakinumab (trade name Ilaris). The anti-human IL-1β protein antibody of this application is a novel antibody molecule sequence with high affinity and high blocking activity, and has great value for disease treatment. Attached Figure Description

[0028] Figure 1 Results of experiments on the inhibition of IL6 release by culture supernatant of various subclonal hybridoma cells;

[0029] Figure 2 Results of experiments on the inhibition of IL6 release by culture supernatant of various subclonal hybridoma cells;

[0030] Figure 1 and Figure 2 In this text, "Sample" refers to a subclonal hybridoma cell culture supernatant sample, and "inhibition %" indicates the inhibition rate (%) of the subclonal hybridoma cell culture supernatant in inhibiting the release of IL6 factor.

[0031] Figure 3 The IC50 assay results for the inhibition of IL6 factor release by the anti-IL-1β human-mouse chimeric antibody;

[0032] Figure 3In the text, “Ab conc.(nM)” represents the antibody concentration (nM), “IL-6(pg / ml)” represents the IL6 secretion level (pg / ml), A13-8, A13-60, B8-8, B27-4, and C20-2 represent different anti-IL-1β human-mouse chimeric antibodies, and CAN and IgG1 represent the positive control antibody and negative control antibody, respectively. Detailed Implementation

[0033] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0034] Example 1: Preparation of IL-1β mouse monoclonal antibody

[0035] To generate mouse antibodies against IL-1β, commercially available recombinant human IL-1β (novoprotein, CG93) was used as an antigen to immunize BALB / c mice. Plasma titers were then screened using ELISA, and mice with the highest titers of anti-recombinant human IL-1β immunoglobulin were selected for hybridoma fusion. The specific steps are as follows:

[0036] (1) Immunizing mice

[0037] First, each of five BALB / c mice was subcutaneously immunized with 50 μg of recombinant human IL-1β. Then, the immunizations were administered alternately. Gold Adjuvant liquid (Sigma, T2684) and Alum Adjuvant (Thermo, 77161) was subcutaneously immunized in BALB / c mice (total 5 immunizations).

[0038] (2) Plasma titer testing

[0039] Blood was collected from the tails of immunized mice, and plasma titers were screened using ELISA to detect the immune response of each immunized mouse and to screen out mice with the highest titer of anti-recombinant human IL-1β immunoglobulin.

[0040] ELISA plates were coated with 1 μg / ml recombinant human IL-1β, 100 μl per well, and incubated overnight at 4°C. The plates were washed with 200 μl / well of PBS / Tween (0.1%), followed by blocking with 3% bovine serum albumin in 200 μl / well of PBS / Tween (0.1%) for 1 hour. After washing, a diluent of recombinant human IL-1β-immunized mouse serum was added to each well, and the plates were incubated at 37°C for 2 hours, followed by washing. Then, the plates were incubated with diluted HRP-conjugated goat anti-mouse IgG (H+L) antibody (Beijing Biolong Immunotherapy Co., Ltd., BF03001) at 37°C for 1 hour. After washing, 100 μl of TMB chromogenic solution (Biopanda, TMB-S-002) was added to each well for color development. After color change, 50 μl of 2M sulfuric acid was added to terminate the reaction. The results were then analyzed using a microplate reader with an OD of 450nm-620nm. The titer results are shown in Table 1.

[0041] Table 1. Immunological titers of BALB / c mice

[0042]

[0043]

[0044] (3) Fusion of hybridoma cells

[0045] Based on the test results in Table 1, mice with high titers of anti-recombinant human IL-1β immunoglobulin were selected for fusion. Before fusion, the mice underwent intraperitoneal booster immunization with the antigen for three consecutive days. Afterward, the mice were sacrificed, and their spleens were removed. Then, mouse spleen cells isolated from BALB / c mice were fused with mouse myeloma cell lines using an electrofusion method (a total of three fusions) to obtain hybridoma cells capable of secreting and expressing mouse polyclonal antibodies against human IL-1β. The fusion methods included:

[0046] B cells from mouse spleen were mixed with sp2 / 0 cells at a ratio of 1:1 to 1:2, centrifuged, and the supernatant was discarded. The cells were then resuspended in 10 ml of Medium C (BTX, 47-0001) and washed twice. The cell density was then adjusted to 1×10⁻⁶ cells using Medium C. 7 Electrofusion was performed at / ml.

[0047] The fused cells were then fused at a rate of 2 × 10⁻⁶. 4The cells were seeded into 96-well plates and then incubated for 4-7 days in selective medium consisting of RPMI Medium 1640 (Gibco, 11875-093) with the following concentrations: 1×Penicillin-Streptomycin (Gibco, 15140122), 1×GlutaMax-100× (Gibco, 35050-061), 1×HAT (Gibco, 21060-017), 1×HyMax-20× (Gibco, 113004), and 20% fetal bovine serum. After 4-7 days, HT was used to replace HAT for further incubation.

[0048] (4) Subcloning screening

[0049] After culturing hybridoma cells for 10-14 days, the culture medium in each well of a 96-well plate was screened for IL-1β mouse polyclonal antibodies by ELISA (method steps refer to step (2)). Then, using the limiting dilution method, the parent clone (OD>0.2) in the corresponding positive well was plated as a subclone at a cell number of 0.8 / well. After 10 days, the supernatant of the hybridoma cell culture medium was detected, and the subclones (OD>0.2) in the corresponding positive wells were transferred to a 24-well plate to generate a small amount of antibody for further characterization.

[0050] (5) In vitro functional screening of anti-IL-1β hybridoma supernatant

[0051] Based on the principle that IL-1β can stimulate MRC-5 (human embryonic fibroblasts) to produce IL-6, the expression level of IL-6 is detected by ELISA to indirectly reflect the blocking function of the antibody.

[0052] When MRC-5 cells reached 60% confluence, they were digested with trypsin and then resuspended in culture medium to adjust the MRC-5 cell density to 2 × 10⁶ cells / year. 4 Add 50 μl of MRC-5 cells to a 96-well plate, followed by 25 μl of hybridoma cell supernatant (i.e., the subclones screened in step (4)) and control antibody (all samples were diluted 1:1 and 1:10). Add 25 μl of diluted hIL-1β and incubate at 37°C in a 5% CO2 incubator for 18-24 hours. Then collect the supernatant and use an ELISA kit to detect the IL-6 secretion level, reading the absorbance value at 540 nm. The detection results are shown in […]. Figure 1 and Figure 2 .

[0053] Depend on Figure 1 and Figure 2As can be seen, the antibodies secreted by each subclonal hybridoma cell exhibit different blocking functions. In this embodiment, subclones A13-8, A13-60, B8-8, B27-4 and C20-2 with better blocking functions were selected for gene sequencing and construction of anti-IL-1β human-mouse chimeric antibodies.

[0054] Example 2: Construction, Expression, and Purification of Anti-IL-1β Human-Mouse Chimeric Antibody

[0055] Subclonal hybridoma cells were collected and cultured in expanded quantities. mRNA was extracted, reverse transcribed into cDNA, and then amplified by PCR, cloned, and sequenced. The variable region sequences of the anti-IL-1β mouse monoclonal antibodies expressed by subclones A13-8, A13-60, B8-8, B27-4, and C20-2 were obtained, as shown in Table 2.

[0056] Table 2. Variable region sequences of various anti-IL-1β mouse monoclonal antibodies.

[0057]

[0058] A signal peptide sequence (as shown in SEQ ID No. 48) was added before each variable region sequence, and a heavy chain constant region sequence (as shown in SEQ ID No. 34) was added after the heavy chain variable region sequence, and a light chain constant region sequence (as shown in SEQ ID No. 35) was added after the heavy chain constant region sequence and the light chain constant region sequence. Then, a TGA stop codon was added after the heavy chain constant region sequence and the light chain constant region sequence. The corresponding gene sequences were synthesized according to the designed heavy chain and light chain amino acid sequences, and the synthesized gene sequences were constructed into a eukaryotic cell expression vector.

[0059] The heavy and light chain amino acid sequences of the positive control antibody CAN are derived from US Patent US20040063913, specifically shown in SEQ ID No. 36 and SEQ ID No. 37. Similarly, a signal peptide was added before the CAN amino acid sequence, and a TGA stop codon was added after the amino acid sequence before the corresponding gene sequence synthesis and eukaryotic cell expression vector construction. Gene synthesis and vector construction were completed by Suzhou Hongxun Biotechnology Co., Ltd.

[0060] The vector plasmid combination (i.e., the vector plasmid containing the antibody heavy chain and the vector plasmid containing the antibody light chain) was transiently transfected into 293 cells. After culturing for 7 days, the antibodies in the cell supernatant were purified and enriched by affinity chromatography.

[0061] After amplification, the vector plasmids were extracted using a plasmid extraction kit purchased from Tiangen Biotech (Beijing) Co., Ltd. The obtained vector plasmids were used for the following antibody combination expression. The light chain expression vector and heavy chain expression vector were mixed in a 1:1 ratio, and then 3 times the volume of diluted PEI transfection reagent (the volume of DNA added) was added. After forming the DNA-PEI complex, it was added to 293 cells. 24 hours after transfection, 10% of growth medium was added. Seven days later, the cell supernatant was collected, centrifuged at 4000 rpm for 20 minutes, and then filtered through a 0.22 μm filter for Protein A affinity chromatography purification. After purification, the cells were dialyzed against PBS overnight at 4°C.

[0062] Example 3: Determination of affinity and cross-species reaction of anti-IL-1β human-mouse chimeric antibody

[0063] This embodiment utilizes Biacore analysis to verify the binding affinity and binding kinetics of the anti-IL-1β human-mouse chimeric antibody, as well as its binding affinity and binding kinetics with monkey IL-1β (Sino Biological Inc, 90010-CNAE).

[0064] The chimeric antibody was linked to a Protein A chip, and binding was measured by flowing hIL-1β and monkey IL-1β into HBS-EP buffer at concentrations of 5 nm and 50 nm and a flow rate of 30 μl / min. Binding and dissociation curves were fitted to a 1:1 Langmuir binding model using BIAevaluation software. The measured KD, Ka, and Kd values ​​are shown in Table 3.

[0065] Table 3. Biacore binding data of anti-IL-1β human-mouse chimeric antibody.

[0066]

[0067] As shown in the table above, the anti-IL-1β human-mouse chimeric antibody expressed and purified in Example 2 has high affinity for both human IL-1β protein and monkey IL-1β protein.

[0068] Example 4: In vitro IC50 determination of anti-IL-1β human-mouse chimeric antibody

[0069] Based on the principle that IL-1β can stimulate MRC-5 (human embryonic fibroblasts) to produce IL-6, this embodiment uses an ELISA kit to detect the secretion level of IL-6 to indirectly reflect the ability of the anti-IL-1β human-mouse chimeric antibody to bind hIL-1β.

[0070] When MRC-5 cells reached 60% confluence, they were digested with trypsin and then resuspended in culture medium to adjust their density to 2 × 10⁶ cells / year.4 Add 50 μl of MRC-5 cells to a 96-well plate, followed by 25 μl of 10-fold serially diluted chimeric antibody and control antibody, and then 25 μl of diluted hIL-1β. Incubate at 37°C in a 5% CO2 incubator for 18-24 hours. Collect the supernatant and detect IL-6 secretion levels using an ELISA kit, reading the absorbance at 540 nm. Results are shown below. Figure 3 As shown.

[0071] Depend on Figure 3 It is evident that B27-4 and C20-2 exhibited functional inhibitory activity comparable to that of the positive control antibody.

[0072] Example 5: Humanization of Anti-IL-1β Antibody

[0073] To further improve the spatial configuration stability of the B27-4 antibody, while increasing the degree of humanization and reducing immune side effects, this embodiment further modifies the B27-4 antibody.

[0074] There are many reported successful cases of antibody humanization, which can be used for reference (Kettleborough CA, Saldanha JW, Heath VJ, et al. Humanization of a mouse monoclonal antibody by CDR–grafting: the importance of framework residues on loop conformation[J]. Protein Engineering, 1991, 4(7): 773-783; Acqua WF, Damschroder M, Zhang J, et al. Antibody humanization by framework shuffling[J]. Methods, 2005, 36(1): 43-60). In this example, the variable region of the B27-4 antibody was humanized by antibody CDR transplantation and protein homology modeling. After expression, the affinity of the modified humanized antibody was analyzed by Biacore, and the affinity of the humanized antibody hB27-4 and the control antibody CAN were compared.

[0075] After homology modeling and bioinformatics analysis, the information of the humanized antibody Hb27-4 is shown in the table below.

[0076] Table 4. Amino acid sequence information of humanized anti-IL-1β human-mouse chimeric antibody

[0077]

[0078] (Note: SEQ ID No. 1-48 is the amino acid sequence, and its corresponding nucleotide sequences are shown in SEQ ID No. 49-96.)

[0079] Following the methods described in Examples 2 and 3, the humanized antibody underwent gene synthesis, vector construction, and antibody expression. The antibody supernatant was then analyzed for affinity using a Biacore instrument. The binding and dissociation curves were fitted to a 1:1 Langmuir binding model using BIAevaluation software. The measured KD, Ka, and Kd values ​​are shown in the table below:

[0080] Table 5. Biacore binding data of humanized antibody hB27-4 and CAN antibody.

[0081] hB27-4 rHuman IL-1β 8.29E+05 4.51E-04 5.45E-10 CAN rHuman IL-1β 3.93E+06 6.01E-05 1.53E-11

[0082] As shown in Table 5, the modified antibody retained its affinity for human IL-1β protein well, and the affinity level was comparable to that of the control antibody. The humanized antibody hB27-4 also exhibited lower immunogenicity and fewer side effects while maintaining its existing biological functions.

Claims

1. A human IL-1β protein-binding molecule, wherein the human IL-1β protein-binding molecule has an antigen-binding site that specifically binds to human IL-1β protein, and the binding molecule contains at least one heavy chain variable region and at least one light chain variable region, characterized in that, The human IL-1β protein binding molecule is a humanized chimeric antibody, antigen-binding fragment, or variable region fragment of a single-chain antibody. The amino acid sequence of the light chain variable region is shown in SEQ ID No. 42, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

43.

2. A nucleotide molecule encoding the human IL-1β protein-binding molecule as described in claim 1.

3. An expression vector containing a nucleotide molecule encoding a human IL-1β protein-binding molecule as described in claim 2.

Images