Targeting double-antigen bispecific antibody fusion protein and application thereof

Abstract

The invention discloses a bispecific antibody fusion protein targeting double antigens and application of the bispecific antibody fusion protein. Relates to the technical field of biology. The bispecific antibody fusion protein constructed by the invention can be specifically combined with human IL-5 protein and human IgE protein. Animal experiments show that the fusion protein can significantly reduce the level of key inflammatory factors in an allergic rhinitis model mouse body, effectively relieve nasal symptoms such as sneezing and rhinorrhea, and show a good treatment effect. A novel candidate protein drug is provided for targeted therapy of allergic diseases, especially allergic rhinitis, and the protein has wide development and application prospects and important social value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a bispecific antibody fusion protein targeting two antigens and its applications. Background Technology

[0002] Allergic rhinitis (AR) is a chronic inflammatory disease of the nasal mucosa caused by an "excessive" or "inappropriate" Th2 immune response from the immune system to common environmental allergens such as pollen and dust mites. Its main clinical manifestations include paroxysmal sneezing, clear nasal discharge, nasal itching, and nasal congestion. In severe cases, it can affect patients' learning, work efficiency, sleep quality, and mental health, placing a heavy disease burden on society. Currently, first-line clinical management strategies for allergic rhinitis mainly include avoiding allergens and using medication to control symptoms. Commonly used medications include nasal or oral corticosteroids, second-generation antihistamines, leukotriene receptor antagonists, and decongestants. However, these conventional therapies cannot fundamentally intervene in the abnormal immune response process, and long-term use may be accompanied by local or systemic side effects.

[0003] The pathogenesis of allergic rhinitis is a complex process involving multiple steps and factors, among which immunoglobulin E (IgE) and interleukin-5 (IL-5) are two key pivotal factors that run through the acute exacerbation and chronic inflammatory stages.

[0004] The core function of IgE: a "trigger switch" for allergic reactions. Upon initial exposure to an allergen in an atopic individual, the immune system tends to initiate a Th2 response, producing cytokines such as IL-4 and IL-13, inducing B cell activation and differentiation into plasma cells, and generating large amounts of specific IgE against that allergen. These IgE antibodies bind firmly to high-affinity receptors (FcεRI) on the surface of mast cells in the nasal mucosa and basophils in the blood via their Fc fragments, sensitizing the body. When the body is exposed to the same allergen again, the allergen "bridges" with two or more IgE molecules bound to effector cells, immediately triggering mast cell / basophil activation. This leads to the rapid degranulation and release of pre-synthesized inflammatory mediators (such as histamine, kinins, and proteases), causing a series of acute early symptoms. Therefore, IgE is an indispensable initiating factor and key effector molecule for initiating immediate-type hypersensitivity reactions.

[0005] The core role of IL-5: a "persistent engine" for chronic inflammation. Activated mast cells not only release pre-existing mediators but also synthesize and secrete various new cytokines, among which IL-5 plays a crucial role. IL-5 is a core cytokine regulating the production, activation, recruitment, survival, and function of eosinophils. Driven by IL-5, eosinophil production increases in the bone marrow, while eosinophils in the blood are chemotactically attracted to and activated at sites of inflammation in the nasal mucosa. Activated eosinophils release large amounts of toxic proteins (such as major basic protein), reactive oxygen species, and pro-inflammatory cytokines, leading to nasal mucosal epithelial damage, tissue remodeling, increased nerve sensitivity, and persistent nasal congestion. This is the main pathological basis for the chronicity and refractory nature of allergic rhinitis symptoms. Therefore, IL-5 is a core driver of chronic, persistent inflammation characterized by eosinophil infiltration.

[0006] Based on the above mechanisms, monoclonal antibodies targeting IgE (such as omalizumab) and IL-5 / IL-5R (such as mepolizumab and benalizumab) have become important biological agents for the treatment of moderate to severe allergic rhinitis. However, single-target therapy has its inherent limitations: Anti-IgE therapy: It can effectively reduce the level of free IgE, prevent mast cell sensitization and activation, and has a significant effect on controlling acute and rapid symptoms. However, its intervention effect on eosinophilic inflammation that has been recruited to tissues and is maintained by factors such as IL-5 is relatively indirect and limited.

[0007] Anti-IL-5 / IL-5R therapy: It can strongly inhibit the biological process of eosinophils and effectively reduce chronic inflammation and tissue damage. However, it is relatively slow to take effect and does not block the early acute symptoms caused by immediate mast cell degranulation mediated by IgE.

[0008] Furthermore, IgE and IL-5 exhibit upstream and downstream connections and synergistic amplification effects in the disease process: IgE-triggered mast cell activation is a significant source of IL-5; while IL-5-maintained eosinophilic inflammation exacerbates tissue damage and clinical symptoms. This complexity of the pathological network suggests that simultaneous intervention at these two key nodes, IgE and IL-5, may produce synergistic or additive effects, enabling comprehensive and multi-dimensional control of allergic rhinitis from acute symptoms to chronic inflammation.

[0009] Therefore, how to provide a bispecific antibody fusion protein that can simultaneously target IL-5 and IgE dual antigens is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0010] In view of this, the present invention provides a bispecific antibody fusion protein targeting two antigens and its application, which successfully achieves dual targeting of IgE and IL-5, simultaneously blocking the two core pathways of allergic rhinitis, improving the therapeutic effect, and meeting the urgent clinical need for precise and long-term treatment of allergic rhinitis.

[0011] To achieve the above objectives, this application adopts the following technical solution: The primary objective of this application is to provide: a bispecific antibody fusion protein targeting two antigens, wherein the bispecific antibody fusion protein is an asymmetric immunoglobulin-like molecule comprising: (a) A light chain, consisting, from the N-terminus to the C-terminus, of a variable region of an antibody light chain targeting a first antigen and a constant region of an antibody light chain; and (b) A heavy chain fusion chain, which consists, from the N-terminus to the C-terminus, an antibody heavy chain variable region targeting a first antigen, an immunoglobulin constant region, a flexible linker, and a single chain variable region targeting a second antigen. The light chain and the heavy chain are assembled through disulfide bonds and non-covalent interactions.

[0012] Another object of this application is to provide: a bispecific antibody fusion protein targeting IL-5 and IgE antigens, said bispecific antibody fusion protein being an asymmetric immunoglobulin-like molecule comprising: (a) A light chain, consisting, from N-terminus to C-terminus, of a variable region and a constant region of an antibody light chain targeting human IL-5; and (b) A heavy chain fusion chain, which consists, from the N-terminus to the C-terminus, of an antibody heavy chain variable region targeting human IL-5, a human immunoglobulin constant region, a flexible linker, and a single chain variable region targeting human IgE. The light chain and the heavy chain are assembled through disulfide bonds and non-covalent interactions; The nucleotide sequence of the light chain is shown below: DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNVHSFPFTFGGGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, SEQ ID NO.1; The nucleotide sequence of the heavy chain fusion strand is shown below: , SEQ ID NO.2.

[0013] As a preferred technical solution, the variable region of the antibody light chain targeting human IL-5 is composed of a frame region FR-L1, a complementarity-determining region CDR-L1, a frame region FR-L2, a complementarity-determining region CDR-L2, a frame region FR-L3, a complementarity-determining region CDR-L3, and a frame region FR-L4 connected sequentially from the N-terminus to the C-terminus. in, The amino acid sequences of FR-L1, FR-L2, FR-L3, and FR-L4 are shown below: FR-L1: DIVMTQSPDSLAVSLGERATINCKSS, SEQ ID NO.3; FR-L2: LAWYQQKPGQPPKLLIY, SEQ ID NO.4; FR-L3: TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC, SEQ ID NO.5; FR-L4: FGGGTKLEIK, SEQ ID NO.6.

[0014] The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown below: CDR-L1: QSLLNSGNQKNY, SEQ ID NO.7; CDR-L2: GAS; CDR-L3: QNVHSFPFT, SEQ ID NO.8.

[0015] The amino acid sequence of the variable region of the light chain of the antibody targeting human IL-5 is shown below: DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNVHSFPFTFGGGTKLEIK, SEQ ID NO.9.

[0016] The amino acid sequence of the constant region of the antibody light chain is shown below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, SEQ ID NO.10.

[0017] As a preferred technical solution, the variable region of the antibody heavy chain targeting human IL-5 is composed of a frame region FR-H1, a complementarity-determining region CDR-H1, a frame region FR-H2, a complementarity-determining region CDR-H2, a frame region FR-H3, a complementarity-determining region CDR-H3, and a frame region FR-H4 connected sequentially from the N-terminus to the C-terminus. in, The amino acid sequences of FR-H1, FR-H2, FR-H3, and FR-H4 are shown below: FR-H1: QVTLRESGPALVKPTQTLTLYCTVS, SEQ ID NO.11; FR-H2: VHWVRQPPGKGLEWLGV, SEQ ID NO.12; FR-H3: DYNSALMSRLSISKDTSRNQVVLTTMTNMDPVDTATYYC, SEQ ID NO.13; FR-H4: WGRGTPVTVSS, SEQ ID NO.14.

[0018] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown below: CDR-H1: GFSLTSYS, SEQ ID No.15; CDR-H2: IWASGGT, SEQ ID No. 16; CDR-H3: ARDPPSSLLRLDY, SEQ ID No. 17.

[0019] The amino acid sequence of the variable region of the heavy chain of the antibody targeting human IL-5 is shown below: QVTLRESGPALVKPTQTLTLYCTVSGFSLTSYSVHWVRQPPGKGLEWLGVIWASGGTDYNSALMSRLSISKDTSRNQVVLTMTNMDPVDTATYYCARDPPSSLLRLDYWGRGTPVTVSS, SEQ ID NO. 18.

[0020] The amino acid sequence of the constant region of the human immunoglobulin is shown below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV SEQ ID NO.19.

[0021] The amino acid sequence of the flexible linker is shown below: GGGGSGGGGSGGGGS, SEQ ID NO. 20.

[0022] As a preferred technical solution, the single-chain variable region targeting human IgE is composed of a variable region of the antibody heavy chain targeting human IgE, a flexible linker, and a variable region of the antibody light chain targeting human IgE, connected sequentially from the N-terminus to the C-terminus. in, The amino acid sequence of the variable region of the heavy chain of the human IgE antibody is shown below: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITYDGSTNYNPSVKGRLTISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSS, SEQ ID NO. 21.

[0023] The amino acid sequence of the variable region of the light chain of the human IgE antibody is shown below: DIQLTQSPSSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIK, SEQ ID NO. 22.

[0024] The amino acid sequence of the flexible linker is shown in SEQ ID NO.20; The amino acid sequence of the single-chain variable region targeting human IgE is shown below: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITYDGSTNYNPSVKGRLTISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSGGGG SGGGGSGGGGSDIQLTQSPSSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIK, SEQ ID NO.23.

[0025] As a preferred technical solution, the variable region of the heavy chain of the target human IgE antibody is composed of a frame region FR-H1, a complementarity-determining region CDR-H1, a frame region FR-H2, a complementarity-determining region CDR-H2, a frame region FR-H3, a complementarity-determining region CDR-H3, and a frame region FR-H4 connected sequentially from the N-terminus to the C-terminus. in, The amino acid sequences of FR-H1, FR-H2, FR-H3, and FR-H4 are shown below: FR-H1: EVQLVESGGGLVQPGGSLRLSCAVS, SEQ ID NO.24; FR-H2: WNWIRQAPGKGLEWVAS, SEQ ID NO.25; FR-H3: NYNPSVKGRLTISRDDSKNTFYLQMNSLRAEDTAVYYC, SEQ ID NO.26; FR-H4: WGQGTLVTVSS, SEQ ID NO. 27.

[0026] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown below: CDR-H1: GYSITSGYS, SEQ ID NO.28; CDR-H2: ITYDGST, SEQ ID NO.29; CDR-H3: ARGSHYFGHWHFAV, SEQ ID NO. 30.

[0027] As a preferred technical solution, the variable region of the light chain of the target human IgE antibody is composed of a frame region FR-L1, a complementarity-determining region CDR-L1, a frame region FR-L2, a complementarity-determining region CDR-L2, a frame region FR-L3, a complementarity-determining region CDR-L3, and a frame region FR-L4 connected sequentially from the N-terminus to the C-terminus. in, The amino acid sequences of FR-L1, FR-L2, FR-L3, and FR-L4 are shown below: FR-L1: DIQLTQSPSSLSSASVGDRVTITCRAS, SEQ ID NO.31; FR-L2:MNWYQQKPGKAPKLLIY, SEQ ID NO.32; FR-L3: YLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC, SEQ ID NO.33; FR-L4: FGQGTKVEIK, SEQ ID NO. 34.

[0028] The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown below: CDR-L1: QSVDYDGDSY, SEQ ID NO.35; CDR-L2: AAS; CDR-L3: QQSHEDPYT, SEQ ID NO. 36.

[0029] Another object of this application is to provide the use of the fusion protein in the preparation of reagents for the detection or treatment of IL-5, IgE targeting-related diseases and / or medicaments for allergic diseases.

[0030] Another object of this application is to provide: an isolated polynucleotide encoding a bispecific antibody fusion protein as described above.

[0031] Another object of this application is to provide: a biomaterial, said biomaterial being any of the following: 1) An expression cassette containing the polynucleotide; 2) A recombinant vector containing the polynucleotide, or a recombinant vector containing the expression cassette described in 1); 3) Recombinant bacteria containing the polynucleotide, or recombinant bacteria containing the expression cassette of 1), or recombinant bacteria containing the recombinant vector of 2); 4) Host cells containing the polynucleotide, or host cells containing the expression cassette of 1), or host cells containing the recombinant vector of 2), or host cells containing the recombinant bacteria of 3).

[0032] Another aspect of this application is to provide: a method for preparing the bispecific antibody fusion protein, culturing the host cells described above under conditions suitable for expression, and recovering the bispecific antibody fusion protein from the culture.

[0033] Another object of this application is to provide a pharmaceutical composition for the prevention and / or treatment of allergic diseases, comprising the bispecific antibody fusion protein described above.

[0034] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) This invention provides a novel universal structure for bispecific antibodies (light chain + heavy chain - Fc - linker - scFv), which effectively avoids chain mismatch, ensures product homogeneity, and retains the long-term cycling function of natural antibodies. Specifically applied to targeting IL-5 and IgE, this fusion protein can synergistically block the immediate symptoms and chronic inflammation of allergic rhinitis, achieving comprehensive treatment superior to single-target drugs, and providing a highly efficient and stable novel drug candidate for disease treatment.

[0035] (2) The fusion protein described in this invention provides a new candidate for the treatment of allergic rhinitis and has important economic and social significance. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0037] Figure 1 The diagram shows the bispecific antibody fusion protein 35 / 36 targeting IL-5 and IgE antigens provided by this invention.

[0038] Figure 2 The image shows the enzyme digestion identification results of the fusion protein expression plasmid in Example 1 of this invention. Lane 1 is the nucleic acid marker, lane 2 is the pcDNA3.4-35 band, lane 3 is the BamHI and EcoRV double digestion band of plasmid pcDNA3.4-35, lane 4 is the nucleic acid marker, lane 54 is the pcDNA3.4-36 band, and lane 6 is the BamHI and EcoRV double digestion band of plasmid pcDNA3.4-36.

[0039] Figure 3 The image shows the expression and purification results of the fusion protein prepared in Example 1 of this invention. Figure 3 In the equation, A represents the elution volume and OD. 280 Correspondence diagram of pH; Figure 3 In the table, B represents the SDS-PAGE result; M represents the protein marker; 1 represents the denatured and reduced sample after purification at 35 / 36°C; and 2 represents the non-denatured and non-reduced sample after purification at 35 / 36°C.

[0040] Figure 4 The image shows the ELISA results of the binding of fusion proteins 35 / 36 to recombinant human IL-5 and IgE proteins in Example 2 of this invention.

[0041] Figure 5The figure shows the results of the in vitro experiment in Example 3 of this invention, in which the fusion protein 35 / 36 inhibited the expression of LPS-induced inflammatory factors in PBMCs. The left figure shows the concentration of TNF-α in the cell supernatant of different treatment groups, and the right figure shows the concentration of IL-6 in the cell supernatant of different treatment groups.

[0042] Figure 6 The image shows the results of the Disease Activity Index (DAI) score in the mouse model experiment of allergic rhinitis in Example 4 of this invention.

[0043] Figure 7 The image shows the ELISA results of the mouse serum IgE level in the mouse model of allergic rhinitis in Example 4 of this invention.

[0044] Figure 8 Figure 4 shows the ELISA results of histamine levels in the mouse serum during the allergic rhinitis mouse model experiment of Example 4 of this invention.

[0045] Figure 9 The following is a Luminex result graph showing the serum IL-5, IL-6, and IL-13 levels in a mouse model of allergic rhinitis in Example 4 of this invention. The left graph shows the concentration of IL-5 in different treatment groups, the middle graph shows the concentration of IL-6 in different treatment groups, and the right graph shows the concentration of IL-13 in different treatment groups. Detailed Implementation

[0046] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0047] Example 1 Plasmid construction, protein expression and purification (1) The aforementioned sequences SEQ ID No.1 (numbered 35) and SEQ ID No.2 (numbered 36) were synthesized by gene (with restriction endonucleases BamHI and EcoRV restriction sites added to both ends of the sequences), and then constructed into the pcDNA3.4 plasmid expressing the fusion protein using pcDNA3.4-TOPOTA cloning kit (purchased from Invitrogen (Shanghai) Trading Co., Ltd.), that is, constructed as pcDNA3.4-35 and pcDNA3.4-36.

[0048] (2) The pcDNA3.4-35 and pcDNA3.4-36 plasmids from the previous step were transformed into E. coli TOP10, amplified by shaking in LB medium, extracted using a plasmid large-scale extraction kit (Beijing Polymer Biotechnology Co., Ltd.), and then digested with restriction endonucleases BamHI and EcoRV for identification. Figure 2 Lane 1 is the nucleic acid marker, lane 2 is the pcDNA3.4-35 band (this is the plasmid (nucleic acid) band, the bottom is in a supercoiled state and the top is in a linear state), lane 3 is the BamHI and EcoRV double digestion band of plasmid pcDNA3.4-35, lane 4 is the nucleic acid marker, lane 5 is the pcDNA3.4-36 band of plasmid (this is the plasmid (nucleic acid) band, the bottom is in a supercoiled state and the top is in a linear state), and lane 6 is the BamHI and EcoRV double digestion band of plasmid pcDNA3.4-36. The upper side of the double digestion band is the linearized band of the empty plasmid, and the lower side is the target band, and the position is consistent with the designed size and position.

[0049] (3) The expression plasmid was co-transfected into 293F cells using PEI transfection reagent for protein expression.

[0050] (4) 120 h after transfection, the supernatant was harvested, centrifuged and filtered, and the protein A affinity column (purchased from Cytiva) was treated with 5 column volumes of equilibration buffer (5.6 mM NaH2PO4, 14.4 mM Na2HPO4, 0.15 M NaCl, pH 7.2). The supernatant was loaded onto the column. After the supernatant was loaded, the column was washed with buffer (5.6 mM NaH2PO4, 14.4 mM Na2HPO4, 0.5 M NaCl, pH 7.2) to bring the contaminating protein to baseline. The protein was then eluted with 50 mM citrate / sodium citrate buffer (pH 3.2), and samples with a concentration of 100 mAu or more were collected. The pH was then adjusted to 7.0 with 1 M Tris-Cl (pH 8.0), concentrated in a concentration tube, filtered and sterilized, and stored at 4 °C. 3 μg of the sample was then used for SDS-PAGE staining. Figure 3 ): Figure 3 A is the chromatography diagram, showing the elution volume versus OD. 280 Correspondence diagram of pH; Figure 3 B shows the SDS-PAGE results. From left to right, the lanes represent the protein marker, the purified denatured / reduced sample, and the purified non-denatured / non-reduced sample. The protein positions are consistent with the designed size and position. The protein obtained in this step is the bispecific antibody fusion protein 35 / 36 (see schematic diagram). Figure 1 ).

[0051] Example 2 Antigen-antibody binding ELISA assay (1) Commercial recombinant human IL-5 and IgE proteins were diluted with pH 9.6 NaHCO3 coating buffer, plated on ELISA plates, 100 ng / well, and incubated overnight at 4°C.

[0052] (2) Wash the ELISA plate coated yesterday three times with PBS.

[0053] (3) Block with PBST containing 3% BSA, at room temperature for 30 min.

[0054] (4) Dilute the purified fusion protein 35 / 36 with PBST containing 3% BSA. The initial concentration was 100 μg / mL. The protein was then serially diluted 10 times to 0.001 μg / mL for a total of 6 concentrations. The diluted protein was added to an ELISA plate and incubated at room temperature for 30 min.

[0055] (5) Wash 3 times with PBST, add 100 μL of mouse anti-human HRP secondary antibody (1:5000) to each well, and incubate at room temperature for 30 min.

[0056] (6) Wash 3 times with PBST, add 50 μL of TMB colorimetric solution to each well, and incubate for 5 min.

[0057] (7) Add 50 μL of 1 M HCl to each well and detect OD using a microplate reader. 450 Absorbance values, results as follows Figure 4 As shown.

[0058] The results show that the fusion protein 35 / 36 has binding activity to the target proteins IL-5 and IgE in a dose-dependent manner.

[0059] Example 3 PBMC in vitro inflammation model inhibition test (1) Adjust the density of human PBMCs in the logarithmic growth phase to 2 × 10⁻⁶ using cell culture medium. 6 After achieving a flow rate of 100 μL / mL, the cells were inoculated into 96-well plates at a density of 100 μL per well, i.e., 2 × 10⁻⁶ cells / mL. 5 One hole / hole.

[0060] (2) Set up control wells (Ctrl) in a 96-well plate, add an equal amount of RMPI 1640, and add lipopolysaccharide (LPS) with a final concentration of 1 μg / mL to the other wells. Stimulate at 5% CO2 and 37℃ for 2 h to establish an inflammation model; (3) After stimulation, wash three times with PBS buffer, replace with new RPMI-1640 medium, and add different concentrations of 35 / 36 protein (0.1, 1, 10 μg / mL) to each well. Add 100 μL to each well. At the same time, set up: blank control group (Ctrl, no LPS, no drug) and model group (LPS stimulation, no drug), as shown in Table 1 (5 replicates per group). (4) After the addition was completed, the 96-well plate was incubated at 37°C with 5% (v / v) CO2 for 22 h. After incubation, the cell culture supernatant was collected by centrifugation, and the cytokine levels in the cell culture supernatant were detected using the Luminex Multifactor Detection Kit.

[0061] Table 1 Sample Hole Settings

[0062] IL-6 and TNF-α are pro-inflammatory cytokines that induce LPS-induced expression of inflammatory factors, playing a role in initiating and promoting inflammation. The results show that ( Figure 5 After LPS treatment, the 35 / 36 treatment group showed that the 10 μg / mL concentration group significantly downregulated the expression levels of TNF-α (p < 0.05) and IL-6 (p < 0.01), the 1 μg / mL concentration group significantly downregulated the expression level of TNF-α (p < 0.05) and also downregulated the expression level of IL-6 (the difference was not significant), and the 0.1 μg / mL concentration group downregulated the expression levels of both factors (the difference was not significant).

[0063] Example 4 Establishment and treatment experiments of a mouse model of allergic rhinitis (1) Thirty female C57BL / 6 mice aged 6-8 weeks, weighing 18-22 g, were randomly divided into 5 groups: normal control group (Ctrl group), allergic rhinitis group (AR group), low dose group (35 / 36-low), medium dose group (35 / 36-medium), and high dose group (35 / 36-high) (n=6).

[0064] (2) Basic sensitization treatment: The AR group, low-dose group, medium-dose group and high-dose group were injected intraperitoneally with 200 μL of physiological saline solution containing 25 µg chicken ovalbumin (OVA) and 2 mg Al(OH)3 adjuvant on days 1, 7 and 14, respectively. The Ctrl group was injected intraperitoneally with physiological saline and then treated.

[0065] (3) Enhanced treatment: From day 21 to day 27, the basally sensitized mice were further enhanced by bilateral nasal drops of 20 µL of 10 mg / mL OVA (physiological saline solution) (10 µL per side) one hour before the enhancement. Each group was given physiological saline, physiological saline, and physiological saline containing 1 µg, 0.5 µg, and 0.25 µg of 35 / 36 protein, respectively, once a day, 10 µL per nasal cavity.

[0066] (4) After the last OVA nasal instillation, the number of times the mice scratched their noses, sneezed, and had runny noses within 15 minutes was recorded. The Disease Activity Index (DAI) score was calculated. Then, blood was collected from the mice, serum was separated, and the levels of IgE and histamine in the serum were detected using a commercial kit. The levels of IL-5, IL-6 and IL-13 were measured using the Luminex multifactor assay.

[0067] Disease Activity Index (DAI) score: DAI results are as follows Figure 6 As shown, compared with the Ctrl control group, the DAI score of mice in the AR model group was significantly increased (p < 0.0001). After treatment with three dose groups of 35 / 36, the DAI score was significantly decreased compared with the AR model group (p < 0.001 compared with the 35 / 36-low and 35 / 36-medium groups, and p < 0.0001 compared with the 35 / 36-high group). These results indicate that 35 / 36 can alleviate the increase in DAI score induced by OVA in a mouse model of allergic rhinitis and significantly reduce symptoms such as itching, nose scratching, sneezing, and runny nose in mice.

[0068] Mouse serum IgE detection: (1) Equilibrate the kit at room temperature for 30 min.

[0069] (2) Add the sample or different concentration standards to the corresponding well at a rate of 100 μL / well and incubate at room temperature (RT) for 2 hours.

[0070] (3) Wash the board 5 times and pat it dry on absorbent paper.

[0071] (4) Add 100 μL of HRP-labeled mouse IgE antibody to each well and incubate at RT in the dark for 20 min.

[0072] (5) Wash the board 5 times and pat it dry on absorbent paper.

[0073] (6) Add 100 μL of TMB solution per well and incubate at RT in the dark for 15 min.

[0074] (7) Add 50 μL of stop solution per well, mix well and measure OD immediately. 450 .

[0075] The ELISA results of IgE in serum are as follows: Figure 7 As shown, compared with the Ctrl control group, the serum IgE expression level in the AR model group mice was significantly increased (p < 0.0001). After treatment with 35 / 36, all three doses significantly downregulated IgE expression levels (AR group compared with 35 / 36-low group, 35 / 36-medium group, and 35 / 36-high group, p < 0.0001). Allergic rhinitis is essentially an IgE-mediated type I hypersensitivity reaction. The above results indicate that 35 / 36 can alleviate the upregulation of IgE expression induced by OVA in the mouse allergic rhinitis model, thereby inhibiting a series of subsequent symptoms such as nasal congestion, runny nose, and sneezing at the source.

[0076] Mouse serum histamine detection: (1) Equilibrate the kit at room temperature for 30 min.

[0077] (2) Add 50 μL of standard at different concentrations to each well of the standard.

[0078] (3) Sample addition: First add 40 μL of sample diluent to the well of the enzyme-labeled plate, and then add 10 μL of the sample to be tested.

[0079] (4) Add enzyme: Add 100 μL of enzyme labeling reagent to each well, except for blank wells.

[0080] (5) Incubation: After sealing with a sealing film, incubate at 37°C for 1 h.

[0081] (6) Washing: Remove the sealing film, discard the liquid, spin dry, fill each well with washing liquid, discard after 30 seconds, repeat 5 times, and pat dry.

[0082] (7) Color development: Add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and develop color at 37°C in the dark for 15 min.

[0083] (8) Termination: Add 50 μL of stop solution to each well to terminate the reaction and measure OD. 450 .

[0084] ELISA results of histamine in serum are as follows Figure 8As shown, compared with the Ctrl control group, the serum histamine expression level in the AR model group mice was significantly increased (p < 0.0001). After treatment with 35 / 36, all three doses significantly downregulated histamine expression levels (AR group compared with 35 / 36-low group, 35 / 36-medium group, and 35 / 36-high group, p < 0.0001). Histamine is the most important mediator causing early symptoms of allergic rhinitis. The above results indicate that 35 / 36 can alleviate the serum histamine upregulation induced by OVA in the mouse allergic rhinitis model, thereby reducing the stimulation of nerve endings, reducing glandular nasal secretion, alleviating the possible vasodilation and increased permeability, and thus reducing the occurrence of nasal congestion.

[0085] Luminex multifactor detection experiment: (1) Equilibrate the kit at room temperature for 30 min.

[0086] (2) Preparation of standards: The standards are thoroughly mixed with diluent and serially diluted. Take 100 μL of each of the three factors into the same EP tube and make up to 1 mL as the highest concentration of the standard curve. Serially dilute 3 times to make a total of 7 standards and 1 blank. Preparation of beads: Thoroughly mix the premixed beads mixture, take the corresponding volume according to the ratio, mix and set aside. Preparation of detection antibodies: Thoroughly mix the premixed detection antibody mixture, take the corresponding volume according to the ratio, mix and set aside. Preparation of PE-streptavidin: Thoroughly mix the PE-streptavidin reagent, take the corresponding volume according to the ratio, mix and set aside. Preparation of washing solution: Take 20 mL of 25× washing solution + 480 mL of deionized water, mix and set aside.

[0087] (3) Add 50 μL of diluted beads (capture antibody) to each well, and add 50 μL of standard or sample to each well. RT, 2 h, 800 rpm.

[0088] (4) Place the microplate on the magnetic rack for 1 min to ensure that the beads are adsorbed. Clean with cleaning solution, 100 μL per well, 3 times.

[0089] (5) Add 50 μL of diluted biotin-labeled detection antibody complex to each well, RT, 1 h, 800 rpm.

[0090] (6) Place the microplate on the magnetic rack for 1 min to ensure that the beads are adsorbed. Clean with cleaning solution, 100 μL per well, 3 times.

[0091] (7) Add 50 μL of diluted streptavidin-labeled PE to each well and incubate at room temperature for 0.5 h at 800 rpm.

[0092] (8) Place the microplate on the magnetic rack for 1 min to ensure that the beads are adsorbed. Clean with cleaning solution, 100 μL per well, 3 times.

[0093] (9) Resuspend the beads in 100 μL of washing solution, incubate for 2 min at 800 rpm, and then perform the test.

[0094] Luminex results for IL-5, IL-6, and IL-13 in serum are as follows: Figure 9 As shown, compared with the Ctrl control group, the expression levels of three cytokines in the serum of AR model mice were significantly increased (p < 0.0001). After treatment with 35 / 36, all three doses significantly downregulated the expression levels of the three cytokines. IL-5 is a key factor in eosinophil production, activation, and survival; eosinophil infiltration can be observed at the inflammatory sites of patients with allergic rhinitis. IL-6, as previously mentioned, is an important type of cytokine that promotes inflammation. IL-13 is a key cytokine secreted by Th2 cells after polarization, which can promote B cell activation and produce large amounts of IgE targeting specific allergens. IL-5 and IL-13 are also synthesized and released by activated mast cells in the late-phase reaction. These results indicate that 35 / 36 can alleviate the upregulation of key cytokines in serum induced by OVA in a mouse model of allergic rhinitis, thereby alleviating and treating allergic rhinitis symptoms.

[0095] The results above show that the fusion protein 35 / 36 can significantly alleviate and treat the symptoms of allergic rhinitis. By achieving precise blocking of the core pathology of allergic rhinitis through a dual-target mechanism, it not only represents an innovation in treatment technology, but will also generate profound economic and social value.

[0096] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0097] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A bispecific antibody fusion protein targeting two antigens, characterized in that, The bispecific antibody fusion protein is an asymmetric immunoglobulin-like molecule comprising: (a) A light chain, consisting, from the N-terminus to the C-terminus, of a variable region of an antibody light chain targeting a first antigen and a constant region of an antibody light chain; and (b) A heavy chain fusion chain, which consists, from the N-terminus to the C-terminus, an antibody heavy chain variable region targeting a first antigen, an immunoglobulin constant region, a flexible linker, and a single chain variable region targeting a second antigen. The light chain and the heavy chain are assembled through disulfide bonds and non-covalent interactions.

2. A bispecific antibody fusion protein targeting IL-5 and IgE antigens, characterized in that, The bispecific antibody fusion protein is an asymmetric immunoglobulin-like molecule comprising: (a) A light chain, consisting, from N-terminus to C-terminus, of a variable region and a constant region of an antibody light chain targeting human IL-5; and (b) A heavy chain fusion chain, which consists, from the N-terminus to the C-terminus, of an antibody heavy chain variable region targeting human IL-5, a human immunoglobulin constant region, a flexible linker, and a single chain variable region targeting human IgE. The light chain and the heavy chain are assembled through disulfide bonds and non-covalent interactions; The nucleotide sequence of the light chain is shown in SEQ ID NO.1; the nucleotide sequence of the heavy chain fusion chain is shown in SEQ ID NO.

2.

3. The bispecific antibody fusion protein according to claim 2, characterized in that, The variable region of the light chain of the antibody targeting human IL-5 is composed of, from the N-terminus to the C-terminus, the frame region FR-L1, the complementarity-determining region CDR-L1, the frame region FR-L2, the complementarity-determining region CDR-L2, the frame region FR-L3, the complementarity-determining region CDR-L3, and the frame region FR-L4 connected together. in, The amino acid sequences of FR-L1, FR-L2, FR-L3, and FR-L4 are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively. The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO.7, "GAS", and SEQ ID NO.8, respectively. The amino acid sequence of the variable region of the light chain of the antibody targeting human IL-5 is shown in SEQ ID NO.9; The amino acid sequence of the constant region of the antibody light chain is shown in SEQ ID NO.

10.

4. The bispecific antibody fusion protein according to claim 2, characterized in that, The variable region of the heavy chain of the antibody targeting human IL-5 is composed of, from the N-terminus to the C-terminus, a frame region FR-H1, a complementarity-determining region CDR-H1, a frame region FR-H2, a complementarity-determining region CDR-H2, a frame region FR-H3, a complementarity-determining region CDR-H3, and a frame region FR-H4 connected together. in, The amino acid sequences of FR-H1, FR-H2, FR-H3, and FR-H4 are shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14, respectively. The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID NO.15, SEQ ID NO.16, and SEQ ID NO.17, respectively. The amino acid sequence of the variable region of the heavy chain of the antibody targeting human IL-5 is shown in SEQ ID NO.18; The amino acid sequence of the constant region of the human immunoglobulin is shown in SEQ ID NO.19; the amino acid sequence of the flexible linker is shown in SEQ ID NO.

20.

5. The bispecific antibody fusion protein according to claim 2, characterized in that, The single-chain variable region targeting human IgE is composed of the variable region of the antibody heavy chain targeting human IgE, a flexible linker, and the variable region of the antibody light chain targeting human IgE, connected sequentially from the N-terminus to the C-terminus. in, The amino acid sequence of the variable region of the heavy chain of the target human IgE antibody is shown in SEQ ID NO.21; The amino acid sequence of the variable region of the light chain of the target human IgE antibody is shown in SEQ ID NO.22; The amino acid sequence of the flexible linker is shown in SEQ ID NO.20; The amino acid sequence of the single-chain variable region targeting human IgE is shown in SEQ ID NO.23; The variable region of the heavy chain of the target human IgE antibody is composed of, from the N-terminus to the C-terminus, a frame region FR-H1, a complementarity-determining region CDR-H1, a frame region FR-H2, a complementarity-determining region CDR-H2, a frame region FR-H3, a complementarity-determining region CDR-H3, and a frame region FR-H4 connected together. in, The amino acid sequences of FR-H1, FR-H2, FR-H3, and FR-H4 are shown in SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, and SEQ ID NO.27, respectively. The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID NO.28, SEQ ID NO.29, and SEQ ID NO.30, respectively. The variable region of the light chain of the target human IgE antibody is composed of, from the N-terminus to the C-terminus, a frame region FR-L1, a complementarity-determining region CDR-L1, a frame region FR-L2, a complementarity-determining region CDR-L2, a frame region FR-L3, a complementarity-determining region CDR-L3, and a frame region FR-L4 connected together. in, The amino acid sequences of FR-L1, FR-L2, FR-L3, and FR-L4 are shown in SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, and SEQ ID NO.34, respectively. The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO.35, "AAS", and SEQ ID NO.36, respectively.

6. The use of the fusion protein according to any one of claims 1-5 in the preparation of reagents for detecting or treating IL-5, IgE targeting-related diseases and / or drugs for allergic diseases.

7. An isolated polynucleotide, characterized in that, The polynucleotide encodes the bispecific antibody fusion protein as described in any one of claims 1-5.

8. A biomaterial, characterized in that, The biomaterial is any one of the following: 1) An expression cassette containing the polynucleotide of claim 7; 2) A recombinant vector containing the polynucleotide of claim 7, or a recombinant vector containing the expression cassette of claim 1); 3) Recombinant bacteria containing the polynucleotide of claim 7, or recombinant bacteria containing the expression cassette of claim 1), or recombinant bacteria containing the recombinant vector of claim 2; 4) A host cell containing the polynucleotide of claim 7, or a host cell containing the expression cassette of claim 1), or a host cell containing the recombinant vector of claim 2), or a host cell containing the recombinant bacteria of claim 3).

9. A method for preparing the bispecific antibody fusion protein according to any one of claims 1-5, characterized in that, The host cells of claim 8 are cultured under conditions suitable for expression, and the bispecific antibody fusion protein is recovered from the culture.

10. A pharmaceutical composition for the prevention and / or treatment of allergic diseases, comprising the bispecific antibody fusion protein as described in any one of claims 1-5.

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