IL-5 binding molecule and application thereof

Abstract

The invention belongs to the field of antibody drugs, and discloses an IL-5 binding molecule and application thereof. The IL-5 binding molecule comprises at least one VHH chain, and the CDR of the VHH chain is as shown in SEQ ID NO.1-12. The nano antibodies of some examples of the disclosure, such as AIL-C14, AIL-C506, AIL-C633 and the like, have human and monkey cross activity. The antibody molecule obtained through antibody humanized transformation and affinity maturation has a remarkable capability of blocking the combination of IL-5 and IL-5R alpha.

Description

Technical Field

[0001] The present disclosure belongs to the field of antibody drugs, and specifically relates to an IL-5 binding molecule and its application. Background Art

[0002] Interleukin-5 (IL-5) is a homodimeric molecule that plays an important role in the occurrence and development of asthma. IL-5 is produced by a variety of immune cells, including: Th2 cells (helper T cells type 2), type 2 innate lymphoid cells (ILC2), eosinophils, mast cells, etc. IL-5 is mainly mediated by the IL-5 receptor (IL-5R) and performs signal transduction through the JAK-STAT and Ras / Raf-ERK pathways. It is an important cytokine that maintains the survival and function of B cells and eosinophils. Overexpression of IL-5 significantly increases the number of eosinophils in the body. In contrast, the gene lacking IL-5 or its receptor α chain (IL-5Rα) causes mice to show significant obstacles in the development and function of B cells and eosinophils.

[0003] Asthma is a chronic disease affecting approximately 350 million people worldwide. Eosinophilic asthma is the most common asthma phenotype, and inflammation with eosinophils in the airways is closely associated with disease severity, risk of exacerbations, airway hyperresponsiveness, and symptom exacerbations. Although most patients with asthma are controlled with corticosteroid therapy, some patients are difficult to control and are accompanied by significant treatment-related side effects. Approximately 10% of adults and 2.5% of children have severe asthma, which is associated with a significant decline in quality of life and increased risk of fixed airway obstruction, exacerbations, hospitalization, and death. Patients with severe asthma usually require repeated use of inhaled corticosteroids or long-term oral corticosteroids in combination with long-acting β2-agonists and long-acting muscarinic antagonists for adequate treatment. Severe asthma is a heterogeneous syndrome with clinical manifestations that vary depending on the age of onset of asthma (childhood or adulthood), the presence of allergies and other coexisting symptoms, the severity of airflow limitation, the frequency of exacerbations, and the response to treatment. Severe asthma also has different biological characteristics, which are usually identified by biomarkers in blood or exhaled air.

[0004] One of the notable features of type 2 inflammatory asthma is eosinophilic airway inflammation, which is manifested by increased eosinophil counts in the blood or elevated nitric oxide levels in exhaled air. Approximately 50% of mild to moderate asthma cases and more than 50% of severe asthma cases are type 2 inflammatory asthma. In type 2 inflammatory asthma, when patients are exposed to stimuli such as allergens, pollutants, viruses or bacteria, airway epithelial cells release a series of epithelial cytokines, including interleukin-25 (IL-25), interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP). These epithelial cytokines promote the activation of dendritic cells, which present allergens to naive T-helper cells (Th0), activating these cells to transform into Th2 cells, thereby activating the type 2 inflammatory pathway. Th2 cells produce cytokines such as IL-4, IL-5 and IL-13, among which IL-5 is one of the key inflammatory mediators. IL-5 promotes the proliferation, maturation and release of eosinophils from the bone marrow mainly by binding to the IL-5 receptors on the surface of eosinophils. These eosinophils then migrate to the airway tissues and release chemokines, oxidases and toxic proteins locally. These substances trigger chronic inflammation, increased mucus secretion and tissue damage in the airways, further leading to airway remodeling, including airway smooth muscle hyperplasia and basement membrane thickening. Structural changes in the airways make them more sensitive to environmental stimuli and increase airway hyperresponsiveness, thereby exacerbating asthma symptoms such as wheezing, shortness of breath and chest tightness.

[0005] IL-5 is an important therapeutic target in the pathological process of asthma, and many therapeutic strategies targeting the IL-5 signaling pathway have been approved for clinical treatment. Currently, three biological agents have been approved for the treatment of severe asthma, including Mepolizumab (GSK) and Reslizumab (Teva) targeting IL-5, and Benralizumab (AstraZeneca) targeting IL-5Rα. In Phase III clinical studies, Mepolizumab (subcutaneous or intravenous injection) or Reslizumab (intravenous injection) as an additional treatment can significantly reduce the incidence of exacerbations by about 50% and improve the quality of life of patients, regardless of whether the asthma is allergic. Benralizumab has been shown in studies to reduce the eosinophil count in the airway mucosa and sputum by 90% or more, and completely inhibit the number of eosinophils in the blood.

[0006] Nanobody is a single-domain antibody (VHH) derived from camelids, which has the characteristics of small molecular weight, high specificity and strong stability. So far, there is no nanobody drug targeting IL-5 on the market. Due to its small molecular weight and unique structure, nanobody has better tissue penetration ability and lower immunogenicity, and can neutralize the activity of IL-5 more accurately. In addition, by designing IL-5 nanobody as an inhaled drug, it can act directly on the airway, achieve higher local drug concentrations, and reduce systemic side effects. This targeted drug delivery method not only improves the effectiveness of treatment, but also significantly improves patient compliance and quality of life.

[0007] Currently, all IL-5 targets on the market are IgG-type monoclonal antibody injections, and there are no IL-5 inhibitors in the form of nano-antibodies on the market. Asthma medications include intravenous, subcutaneous, oral, and inhaled, among which inhalation is the best way of administration. Nano-antibodies, due to their small molecular weight and stable physical and chemical properties, can be very effectively delivered to the respiratory tract, alveoli and other asthma patient lesions through atomization, which can effectively increase patient compliance and avoid high systemic exposure from injections.

[0008] Therefore, the development of IL-5 nanobodies, especially in the form of inhaled drugs, is a very promising therapeutic strategy that can provide more effective and safe treatment options for patients with asthma and other type II inflammatory diseases. Summary of the invention

[0009] The purpose of the present disclosure is to overcome at least one of the deficiencies of the prior art and to provide IL-5 binding molecules based on nanobodies and their uses.

[0010] In order to solve the above problems, the inventors of the present disclosure conducted a large number of experiments, constructed an alpaca immune library, screened from the immune library, recombinantly expressed, and identified in vitro activity, and successfully obtained three new IL-5-targeting nanoantibodies AIL-C14, AIL-C506, and AIL-C633, which have human-monkey cross-activity. The antibody molecules obtained by antibody humanization and affinity maturation have a significant ability to block the binding of IL-5 to IL-5Rα.

[0011] The technical solution adopted by this disclosure is:

[0012] The first aspect of the present disclosure provides: an IL-5 binding molecule capable of specifically binding to IL-5, and comprising at least one VHH chain, wherein the VHH chain comprises a framework region FR and a complementarity determining region CDR, wherein the complementarity determining region CDR comprises at least one group selected from the following groups:

[0013] Group (1), having CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.3;

[0014] Group (2), having CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5, and CDR3 shown in SEQ ID NO. 6;

[0015] Group (3), having CDR1 shown in SEQ ID NO.7, CDR2 shown in SEQ ID NO.8, and CDR3 shown in SEQ ID NO.9;

[0016] Group (4), having CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 11;

[0017] Group (5), having CDR1 shown in SEQ ID NO.12, CDR2 shown in SEQ ID NO.5, and CDR3 shown in SEQ ID NO.6;

[0018] The CDRs of group (4) are sequences obtained from group (1) after affinity maturation; and the CDRs of group (5) are sequences obtained from group (2) after affinity maturation.

[0019] In some examples of IL-5 binding molecules, they are chimeric VHH antibodies, humanized VHH antibodies, VHH fused to an Fc fragment.

[0020] In some examples of IL-5 binding molecules, the VHH comprises any one of the amino acid sequences in SEQ ID NO.13 to SEQ ID NO.43, or an amino acid sequence having at least 85%, preferably at least 90% sequence homology with any one of the amino acid sequences in SEQ ID NO.13-43.

[0021] In some examples of IL-5 binding molecules, two or more VHHs are connected by a linker element, and the VHH comprises any amino acid sequence in SEQ ID NO. 13-43 or an amino acid sequence having at least 85% sequence homology with any amino acid sequence in SEQ ID NO. 13-43. Preferably, the linker element includes but is not limited to (G4S)n, where n is an integer of 1 or more, preferably, n is an integer of 1 to 4.

[0022] In some examples of IL-5 binding molecules, two or more VHHs are connected by a linker element, and the VHH comprises any amino acid sequence in SEQ ID NO. 13-43 or an amino acid sequence having at least 85% sequence homology with any amino acid sequence in SEQ ID NO. 13-43. Preferably, the linker element includes but is not limited to (G4S)n, where n is an integer of 1 or more, preferably, n is an integer of 1 to 4.

[0023] In some examples of IL-5 binding molecules, two VHHs are connected by a linker element, and the VHHs are selected from the VHHs shown in SEQ ID NO: 39 (C633-VHH6), SEQ ID NO: 42 (N_PR_C14_22), and SEQ ID NO: 43 (N_PR_C506_34), and the VHHs used are the same or different. Preferably, the linker element includes but is not limited to (G4S)n, where n is an integer of 1 or more, and preferably, n is an integer of 1 to 4. In particular, the IL-5 binding molecule is one of N_PR_C14_22-(G4S)4-C633-VHH6-His, N_PR_C506_34-(G4S)4-C633-VHH6-His, C633-VHH6-(G4S)4-N_PR_N_PR_C14_22-His, C633-VHH6-(G4S)4-N_PR_C506_34-His, N_PR_C14_22-(G4S)4-N_PR_C14_22-His, N_PR_N_PR_C506_34-(G4S)4-N_PR_C506_34-His, C633-VHH6-(G4S)4-C633-VHH6-His.

[0024] In some examples of IL-5 binding molecules, two VHHs are connected by a linker element, and the VHH comprises any amino acid sequence in SEQ ID NO. 44-50 or an amino acid sequence having at least 85%, preferably at least 90% sequence homology with any amino acid sequence in SEQ ID NO. 44-50. Preferably, the linker element includes but is not limited to (G4S)n, where n is an integer of 1 or more, preferably, n is an integer of 1 to 4.

[0025] In some examples of IL-5 binding molecules, the antibody Fc region is directly or via a linker element connected to the C-terminus or N-terminus of the IL-5 binding molecule. Preferably, the linker element includes but is not limited to (G4S)n, where n is an integer of 1 or more, preferably, n is an integer of 1 to 4. By introducing the antibody Fc region, dimerization of the IL-5 binding molecule can be achieved. The introduction of the Fc region can also utilize the affinity of Fc to simplify the purification operation of the antibody. The introduction of Fc is also beneficial to prolong the half-life of the antibody and improve the stability of the antibody in vivo.

[0026] In some examples of IL-5 binding molecules, it is a tetravalent nanobody with Fc. Specifically, by connecting two IL-5 binding molecules directly or through a linker element to the two ends of Fc, the Fc region can be folded independently, so that the Fc fusion antibody is further connected by a disulfide bond in the Fc hinge region to form a stable dimer, thereby obtaining a tetravalent nanobody with Fc. In particular, the VHH used in the tetravalent nanobody with Fc is selected from the VHH shown in SEQ ID NO: 32 (C506-VHH6), SEQ ID NO: 39 (C633-VHH6), SEQ ID NO: 42 (N_PR_C14_22), and SEQ ID NO: 43 (N_PR_C506_34), and the VHH used is the same or different. Preferably, the linker element includes but is not limited to (G4S) n, n is an integer of 1 or more, preferably, n is an integer of 1 to 4. In particular, the IL-5 binding molecule used in the tetravalent Fc-bearing nanobody is selected from one of C633-VHH6-(G4S)4-C633-VHH6-Fc, N_PR_C506_34-(G4S)4-C633-VHH6-Fc, C633-VHH6-(G4S)4-N_PR_C506_34-Fc, N_PR_C14_22-(G4S)4-C633-VHH6-Fc, and N_PR_C506_34-(G4S)4-C506-VHH6-Fc. In particular, the Fc used in the tetravalent Fc-bearing nanobody is the YTE mutant shown in SEQ ID NO.52.

[0027] There are no special requirements for the antibody Fc region, and it is preferably the Fc region of a human antibody, the purpose of which is to further dimerize the IL-5 binding molecule. In some examples of IL-5 binding molecules, the antibody Fc region includes the Fc segment of human IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgM, IgE or its variants and modifications.

[0028] In some examples of IL-5 binding molecules, the antibody Fc region is directly or via a linker element connected to the C-terminus or N-terminus of the IL-5 binding molecule. Preferably, the IL-5 binding molecule forms a dimer via the antibody Fc region.

[0029] In some examples of IL-5 binding molecules, the antibody Fc region is directly or via a linker element connected to the C-terminus or N-terminus of the IL-5 binding molecule, and the antibody Fc region includes the Fc segment of human IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgM, IgE or its variants and modifications. Preferably, the Fc mutant includes a YTE mutant such as SEQ ID NO.52. Preferably, the IL-5 binding molecule forms a dimer through the antibody Fc region.

[0030] The linker element may be a commonly used linker element in the art, preferably a flexible linker element, which is well known to those skilled in the art, such as the connecting peptide disclosed in CN112225807A. In some examples of IL-5 binding molecules, the linker element includes but is not limited to (G4S)n, where n is an integer of 1 or more, preferably, n is an integer of 1 to 4.

[0031] In some examples of IL-5 binding molecules, to facilitate purification after expression, a purification tag is connected to the N-terminus or C-terminus thereof, such as a His tag, an Fc tag, or other purification tags commonly used in the art.

[0032] The second aspect of the present disclosure provides: a composition comprising the IL-5 binding molecule or a derivative thereof as described in the first aspect of the present disclosure, and a pharmaceutically acceptable diluent, carrier or adjuvant.

[0033] In some examples of the composition, the derivative includes but is not limited to an antibody conjugate, which is obtained by connecting the conjugate directly or through a linker to an IL-5 binding molecule.

[0034] In some examples of the composition, the conjugate is selected from a drug, a tracer, an immunoconjugate.

[0035] In some examples of the composition, the drug is a cytotoxic drug, including but not limited to anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof. Specific cytotoxic drugs include but are not limited to DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0036] In some examples of the composition, the tracer includes but is not limited to fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) or any form of nanoparticles.

[0037] The third aspect of the present disclosure provides: a nucleic acid molecule encoding the IL-5 binding molecule or a derivative thereof according to the first aspect of the present disclosure.

[0038] The fourth aspect of the present disclosure provides: an expression vector comprising the nucleic acid molecule described in the third aspect of the present disclosure.

[0039] In some examples of expression vectors, the expression vector includes but is not limited to DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the expression vector includes a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0040] The fifth aspect of the present disclosure provides: a host cell, which expresses the IL-5 binding molecule or its derivative described in the first aspect of the present disclosure, or contains the nucleic acid molecule described in the third aspect of the present disclosure, or contains the expression vector described in the fourth aspect of the present disclosure.

[0041] In some examples of host cells, they are eukaryotic cells and prokaryotic cells. The eukaryotic cells include, but are not limited to, mammalian cells, insect cells, and yeast; the prokaryotic cells include Escherichia coli and Bacillus subtilis. The mammalian cells specifically include, but are not limited to, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, COS cells, mouse NSO thymoma cells, and mouse myeloma SP2 / 0 cells.

[0042] The sixth aspect of the present disclosure provides: a method for preparing an IL-5 binding molecule, comprising using the nucleic acid molecule of the third aspect of the present disclosure, or using the expression vector of the fourth aspect of the present disclosure, or using the host cell of the fifth aspect of the present disclosure for expression under conditions suitable for expressing the IL-5 binding molecule described in the first aspect of the present disclosure.

[0043] The seventh aspect of the present disclosure provides: a detection kit for IL-5, comprising the IL-5 binding molecule or its derivative described in the first aspect of the present disclosure.

[0044] The eighth aspect of the present disclosure provides: use of the IL-5 binding molecule described in the first aspect of the present disclosure, the composition described in the second aspect of the present disclosure, the nucleic acid molecule described in the third aspect of the present disclosure, the expression vector described in the fourth aspect of the present disclosure, the host cell described in the fifth aspect of the present disclosure, or a combination thereof in the preparation of a medicament for preventing and / or treating diseases or conditions associated with IL-5 / IL-5R signaling.

[0045] In some examples of use, the disease or condition is selected from asthma, severe eosinophilic asthma, eosinophilia, Churg-Strauss syndrome, atopic dermatitis, eosinophilic granuloma, granulomatosis with polyangiitis, chronic sinusitis with nasal polyps, eosinophilic esophagitis with nasal polyps, and diseases associated with pulmonary eosinophilia.

[0046] The beneficial effects of the present disclosure are:

[0047] Nanobodies of some examples disclosed herein, such as AIL-C14, AIL-C506, AIL-C633, etc., have human-monkey cross-activity. Antibody molecules obtained by antibody humanization and affinity maturation have a significant ability to block the binding of IL-5 to IL-5Rα.

[0048] The bispecific antibodies (N_PR_C14_22-(G4S)4-C633-VHH6-His, N_PR_C506_34-(G4S)4-C633-VHH6-His, C633-VHH6-(G4S)4-N_PR_N_PR_C14_22-His, C633-VHH6-(G4S)4-N_PR_C506_34-His, N_PR_C14_22-(G4S)4-N_PR_C14_22-His, N_PR_N_PR_C506_34-(G4S)4-N_PR_C506_34-His, C633-VHH6-(G4S)4-C633-VHH6-His), as well as tetravalent nanobodies with Fc (C633-VHH6-(G4S)4-C633-VHH6-Fc, N_PR_C506_34-(G4S)4-C633-VHH6-Fc, C633-VHH6-(G4S)4-N_PR_C506_34-Fc, N_PR_C14_22-(G4S)4-C633-VHH6-Fc, N_PR_C506_34-(G4S)4-C506-VHH6-Fc), especially the tetravalent nanobodies, whose functions were significantly better than the positive control hu39D10. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 to Figure 3 These are the results of the binding test between AIL-C14-Fc, AIL-C506-Fc and AIL-C633-Fc and human IL-5.

[0050] Figures 4 to 6 These are the results of the binding test between AIL-C14-Fc, AIL-C506-Fc and AIL-C633-Fc and monkey IL-5.

[0051] Figure 7 to Figure 9 They are the results of ELISA blocking activity detection of AIL-C14-Fc, AIL-C506-Fc and AIL-C633-Fc respectively.

[0052] Figure 10 to Figure 12 These are the results of FACS blocking activity detection of AIL-C14-Fc, AIL-C506-Fc and AIL-C633-Fc.

[0053] Fig.13 This is the result of AIL-C14-Fc cell proliferation inhibition test.

[0054] Fig.14 This is the result of the cell proliferation inhibition test of AIL-C506-Fc and AIL-C633-Fc.

[0055] Figure 15 to Figure 17 They are the results of ELISA binding tests of AIL-C14-Fc, AIL-C506-Fc and AIL-C633-Fc humanized antibodies.

[0056] Figures 18 to 20 They are the results of ELISA blocking tests of AIL-C14-Fc, AIL-C506-Fc and AIL-C633-Fc humanized antibodies.

[0057] Figure 21 to Figure 23 They are the results of FACS blocking detection of AIL-C14-Fc, AIL-C506-Fc and AIL-C633-Fc humanized antibodies.

[0058] Figure 24 to Figure 26 They are the results of proliferation inhibition assay of humanized antibodies AIL-C14-Fc, AIL-C506-Fc and AIL-C633-Fc.

[0059] Fig. 27 and Fig.28 They are the results of ELISA binding assays for N_PR_C14_22 and N_PR_C506_34, respectively.

[0060] Fig.29 and Fig.30 These are the results of ELISA blocking assays for N_PR_C14_22 and N_PR_C506_34, respectively.

[0061] Fig.31 and Fig.32 These are the results of FACS blocking assays for N_PR_C14_22 and N_PR_C506_34, respectively.

[0062] Fig.33 and Fig.34 These are the results of N_PR_C14_22 and N_PR_C506_34 cell proliferation inhibition assays.

[0063] Fig.35 It is the result of ELISA binding test of double antibodies.

[0064] Fig.36 This is the result of FACS blocking test with double antibodies.

[0065] Figures 37 to 40 It is the result of proliferation inhibition test of dual epitope antibody.

[0066] Figure 41-42 This is the result of FACS blocking detection of tetravalent Fc-tagged nanobody.

[0067] Figure 43 to Figure 47This is the result of the proliferation inhibition test of tetravalent Fc-tagged nanoantibody TF-1.

[0068] Fig.48 is the concentration of OVA-specific IgE in serum.

[0069] Fig.49 Inflammatory cell typing in BALF. (A) Total number of inflammatory cells, (B) Total number of eosinophils, (C) Total number of neutrophils, (D) Total number of macrophages, (E) Ratio of eosinophils in the total number of inflammatory cells, (F) Ratio of neutrophils in the total number of inflammatory cells, (G) Ratio of macrophages in the total number of inflammatory cells.

[0070] Fig.50 These are the results of a routine blood test. (A) The number of lymphocytes, (B) The number of basophils, (C) The number of eosinophils, (D) The percentage of lymphocytes in white blood cells, (E) The percentage of basophils in white blood cells, (F) The percentage of eosinophils in white blood cells.

[0071] Fig.51 This is the scoring result of mouse lung pathology detection. DETAILED DESCRIPTION

[0072] the term

[0073] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure belongs.

[0074] The three letter codes and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0075] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse / camelid antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse / camelid antibody.

[0076] The term "humanized antibody" refers to an antibody produced by transplanting the CDR sequence of camel-derived VHH into the human antibody variable region framework, that is, different types of human germline antibody framework sequences. This can overcome the heterologous response induced by chimeric antibodies due to the large amount of camel-derived protein components they carry. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (Internet www.mrccpe.com.ac.uk / vbase)In the literature, the present invention provides a method for synthesizing antibodies that are suitable for human antibodies. The present invention provides a method for synthesizing antibodies that are suitable for human antibodies. The present invention provides a method for synthesizing antibodies that are suitable for human antibodies. The present invention provides a method for synthesizing antibodies that are suitable for human antibodies. The present invention provides a method for synthesizing antibodies that are suitable for human antibodies. The present invention provides a method for synthesizing antibodies that are suitable for human antibodies. The present invention provides a method for synthesizing antibodies that are suitable for human antibodies.

[0077] The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, IL-5).

[0078] The term "antibody framework" as used herein refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loop (CDR) of the variable domain. In essence, it is a variable domain without CDRs.

[0079] The term "amino acid difference" refers to the difference between a polypeptide and its variants, at one or more amino acid positions on the polypeptide fragment, wherein the variant can be obtained by replacing, inserting or deleting amino acids at one or more positions on the polypeptide.

[0080] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on an IL-5 molecule). An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0081] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, the antibody binds with an affinity (KD) of less than about 10-8M, such as less than about 10-9M, 10-10M, 10-11M or less.

[0082] The term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibodies of the present disclosure bind to IL-5 with a dissociation equilibrium constant (KD) of less than about 10-7M, such as less than about 10-8M, 10-9M, or 10-10M or less, e.g., as determined in a BIACORE instrument using surface plasmon resonance (SPR) technology.

[0083] When the term "competes" is used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, it means competition between antigen binding proteins, which is determined by the following assay: the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., IL-5 antigen or fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, such as: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct label assays, solid phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Manual). Press); solid phase direct labeling RIA with I-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25: 7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176: 546-552); and directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically, the assay involves the use of purified antigen that binds to an unlabeled test antigen binding protein and a labeled reference antigen binding protein (the antigen is on a solid surface or on a cell surface). Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell in the presence of the test antigen binding protein. Typically, the test antigen binding protein is present in excess. Antigen binding proteins identified by competition 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 an epitope that is sufficiently proximal to the epitope bound by the reference antigen binding protein that the two epitopes sterically hinder binding to each other.

[0084] As used herein, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence.

[0085] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which other DNA segments can be connected. In another embodiment, the vector is a viral vector, in which other DNA segments can be connected to the viral genome. The vector disclosed herein can be autonomously replicated in the host cell introduced therein (e.g., bacterial vectors and additional mammalian vectors with a bacterial origin of replication) or can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome (e.g., non-additional mammalian vectors).

[0086] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as in Cold Spring Harbor Manual of Antibody Laboratory Techniques, Chapters 5-8 and 15.

[0087] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include microorganisms (e.g., bacteria), plants, or animal cells. Easily transformed bacteria include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary Cell Line) and NS0 cells.

[0088] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human IL-5. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified by conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as -70°C, or lyophilized.

[0089] "Administering," "applying," and "treating" when applied to an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administering," "applying," and "treating" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "applying," and "treating" also mean treating a cell in vitro and ex vivo by an agent, a diagnostic, a combination composition, or by another cell. "Treatment," when applied to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0090] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments thereof disclosed herein or a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measurable degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptom has been alleviated can be evaluated by any clinical test method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., treatment methods or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0091] "Conservative modification" or "conservative substitution or replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. Those skilled in the art know that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). In addition, replacement of amino acids with similar structure or function is unlikely to destroy biological activity.

[0092] An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's general health, the method, route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0093] "Exogenous" refers to substances produced outside the body of an organism, cell, or human body, depending on the situation. "Endogenous" refers to substances produced inside the body of a cell, organism, or human body, depending on the situation.

[0094] "Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if every position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in the two sequences match or are homologous, then the two sequences are 95% homologous. In general, comparison is made when the two sequences are aligned to obtain the maximum percentage of homology.

[0095] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It should also be understood that all progeny may not be exactly identical in terms of DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. Where different designations are intended, this is clear from the context.

[0096] As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which a trace amount of a specific portion of nucleic acid, RNA and / or DNA is amplified as described in, for example, U.S. Patent No. 4,683,195. In general, sequence information from or outside the target region is required so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strand of the template to be amplified. The 5' terminal nucleotides of the two primers can be consistent with the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cell RNA, etc. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51: 263; Erlich, ed., (1989) PCR TECHNOLOGY (Stockton Press, NY). PCR used herein is considered to be an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which includes using a known nucleic acid and a nucleic acid polymerase as a primer to amplify or generate a specific portion of a nucleic acid.

[0097] "Optional" or "optionally" means that the event or environment described later can but need not occur, and the description includes occasions where the event or environment occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of a specific sequence can but does not have to be present.

[0098] "Pharmaceutical composition" means a mixture containing one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0099] There is no limitation on the disease associated with IL-5 as long as it is a disease associated with IL-5, for example, the therapeutic response induced by the molecules of the present disclosure can be produced by binding to human IL-5 and then suppressing or inhibiting eosinophil stimulation. Thus, when in preparations and formulations suitable for therapeutic use, the molecules of the present disclosure are very useful for those who suffer from allergic and / or atopic reactions, or reactions involving eosinophils, such as, but not limited to, asthma, asthma exacerbations, asthma exacerbations, chronic pneumonia, allergic rhinitis, perennial allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, atopic dermatitis, onchocercal dermatitis, intermittent angioedema, eosinophilic myalgia syndrome, eosinophilic gastroenteritis, helminthic infections, Hodgkin's disease, nasal polyps, Loeffler's syndrome, urticaria, eosinophilic bronchitis, nodular arteritis, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis, allergic conjunctivitis, onchocercal dermatitis, endometriosis, steroid-dependent eosinophilic bronchitis, and the like. In a preferred embodiment, such treatment can inhibit or reduce eosinophilic lung tissue infiltration. The antibody or fragment thereof can be administered from three times a day to once every 6 months, and the route of administration can be intravenous, subcutaneous, intramuscular, parenteral or topical.

[0100] In the present disclosure, the method for detecting or measuring the amount of IL-5 may be any known method. For example, it includes immunodetection or measurement methods.

[0101] Immunoassay or determination method is a method for detecting or determining the amount of antibody or antigen using labeled antigen or antibody. Examples of immunoassay or determination methods include radioactive material labeled immunoantibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, protein immunoblotting, physicochemical methods, etc.

[0102] The above-mentioned diseases associated with IL-5 can be diagnosed by detecting or measuring cells expressing IL-5 using the monoclonal antibodies or antibody fragments disclosed herein.

[0103] In order to detect cells expressing the polypeptide, known immunodetection methods can be used, and preferably immunoprecipitation, fluorescent cell staining, immunohistostaining, etc. In addition, fluorescent antibody staining using FMAT8100 HTS system (Applied Biosystem) can be used.

[0104] In the present disclosure, there is no particular limitation on the living sample used for detecting or measuring IL-5 as long as it has the possibility of containing cells expressing IL-5, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid or culture fluid.

[0105] The technical solution of the present disclosure is further illustrated below with reference to examples.

[0106] In the following examples, the abbreviations used have the common meanings in the art, and the meanings of some of the abbreviations or codes used are as follows:

[0107] hIL-5-His: human IL-5 protein with a His tag; hu39D10: refers to Reslizumab.

[0108] Example 1: Animal immunization and titer detection

[0109] Alpacas (NM018 and NM019) were immunized with recombinant protein hIL-5-hFc by subcutaneous injection to establish a phage display library. The dose for each immunization was 500 μg, and immunization was performed every two weeks. Blood was collected after the fourth and fifth immunizations for titer detection using ELISA, and the results are shown in Table 1.

[0110] Table 1 Antibody titer of immunized alpacas

[0111]

[0112] Example 2: Construction of phage display library and screening

[0113] 2.1 Construction of phage display library

[0114] Peripheral mononuclear cells (PBMC) were isolated from the blood of the immunized alpaca using Ficoll-Paque density gradient separation solution (GE). Total RNA from PBMC was extracted using the Trizol method and then reverse transcribed into cDNA for subsequent PCR amplification and library construction. The variable region sequence of the antibody was amplified by overlap PCR, and the DNA fragment was recovered from the gel and linked to the phage display library vector. The V-containing HHThe vector of the fragment was transformed into E. coli SS320. After the transformation was completed, the bacterial solution was spread on a solid medium containing ampicillin resistance. The storage capacity of the two antibody libraries was determined by the dilution spot plate method to be 3.70E+08 and 4.37E+08 respectively. Single clones were randomly selected for sequencing analysis, and the correct insertion rate of the antibody gene was greater than 80% and the abundance of the CDR region was high.

[0115] 2.2 Phage display library screening

[0116] After the library is built, the alpaca immune library constructed by our company is screened by using an immune tube and a magnetic bead screening instrument in a solid phase, liquid phase and solid phase, liquid phase cross-screening manner (the screening method refers to Example 3 in CN115746132B), and the specific phage pools for hIL-5-hFc (human interleukin-5), cyIL-5-hFc (monkey interleukin-5), mIL-5-mFc (mouse interleukin-5), and ratIL-5-mFc (rat interleukin-5) are enriched by trypsin elution. The enrichment of the enriched phage pool is detected by the ELISA method.

[0117] 2.3 Monoclonal screening

[0118] According to the enrichment of the phage pool, a total of 1,800 monoclones were selected to prepare phage supernatants in 96-well plates. Positive clones targeting hIL-5-hFc and cyIL-5-hFc proteins were selected by phage ELISA detection, and a total of 1,007 positive clones with human-monkey cross-activity were screened. All positive clones were sequenced and analyzed, and the antibody sequences were analyzed through the IMGT website. According to the integrity, diversity and antibody modification sites of the sequences, 96 sequences were finally selected for full-length construction.

[0119] Example 3: In vitro recombinant expression and purification of nanobodies and detection of binding activity

[0120] For easy identification and distinction, in the following examples, the obtained recombinant antibodies are named as original antibody number-Fc. For example, if the original antibody number is AIL-C14, the obtained recombinant antibody is named AIL-C14-Fc.

[0121] In this example, the binding activity of three candidate antibodies AIL-C14, AIL-C506, and AIL-C633 to IL-5 was detected. The amino acid sequences of AIL-C14, AIL-C506, and AIL-C633 are shown below, and the underlined + italicized parts are CDR regions (according to the IMGT partitioning rules).

[0122] AIL-C14: (SEQ ID NO.13)

[0123] QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKERDWVACINSGGDIT NYANSVKGRFTISSRDNAKNTVYLEMNSLKIDDTALYYCAARQQKYFCSPDVVGYNYWGQG TQVTVSS;

[0124] AIL-C633: (SEQ ID NO.14)

[0125] EVQLVESGGGLVQPGGSLRLSCVASGFTFSTAPMSWYRQTPGKDHEWVGTIPADDGK TWYADSVKGRFTISRDNAKNTVYLQMNNLNAEDTAVYSCASPHYWGQGTQVTVSS;

[0126] AIL-C506: (SEQ ID NO.15)

[0127] EVQVQESGGGLVQPGGSLRLSCAATGFTLDGYAIGWFRQAPGKEREGISCISSTGEIAF YADSVKGRFAISRDGAKNTVYLEMNSLKPEDTALYYCASRRQKYFCSNDAIGYGNWGQGT QVTVSS.

[0128] 3.1 In vitro recombinant expression and purification of nanobodies

[0129] The 96 antibody sequences obtained by sequencing in Gene Synthesis Example 2 were fused with the same Fc tag at the C-terminus of the sequence and constructed in the eukaryotic expression vector pcDNA3.4, and recombinant expression was performed using Expi CHO-S cells. The expression supernatant was collected by centrifugation and the antibody was purified using a protein A protein purification column.

[0130] The Fc tag is the Fc of wild-type human IgG1 antibody, and the amino acid sequence is shown in SEQ ID NO.51:

[0131] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0132] Unless otherwise specified, the amino acid sequences of Fc used in the following examples are all shown in SEQ ID NO.51.

[0133] 3.2 Detection of the binding activity of nanoantibodies to human IL-5 protein

[0134] Dilute human IL-5-His to 2 μg / mL with PBS, add to the ELISA plate, 30 μL per well, coat overnight, wash the plate 5 times with PBST, block with 5% milk at 37°C for 1 hour, add gradient dilution of recombinant antibody, incubate at 37°C for 1 hour, wash the plate 5 times with PBST, add secondary antibody Anti-human-IgG-Fc-HRP (diluted with PBS 1:10000) and incubate at 37°C for 1 hour, wash 5 times with PBST, add 30 μL TMB colorimetric solution to each well, react at room temperature in the dark for 5 minutes, then add 2M sulfuric acid to terminate the reaction, and measure the absorbance at 450nm with an ELISA reader. The results are as follows Figure 1-3 As shown, the recombinant antibodies AIL-C14-Fc, AIL-C506-Fc, and AIL-C633-Fc have good binding to human IL-5, and the binding activity is much higher than that of the positive antibodies hu39D10 and Mepolizumab.

[0135] 3.3 Detection of the binding activity of nanoantibodies to monkey IL-5 protein

[0136] Monkey IL-5-His was diluted to 2 μg / mL with PBS and ELISA was performed according to the method in 3.2 of Example 3. The results are as follows: Figure 4-6 As shown, the recombinant antibodies AIL-C14-Fc, AIL-C506-Fc, and AIL-C633-Fc have good binding to monkey IL-5, and the binding activity is much higher than that of the positive antibodies hu39D10 and Mepolizumab.

[0137] Example 4: Detection of blocking activity of candidate antibodies based on ELISA and FACS

[0138] In this example, the ability of three candidate antibodies, AIL-C14-Fc, AIL-C506-Fc, and AIL-C633-Fc, to block the binding of IL-5 to its receptor IL-5Rα was tested.

[0139] 4.1 ELISA-based detection of blocking activity of candidate antibodies

[0140] Dilute hIL-5-hFc to 2μg / mL with PBS, take 30μL and add it to the ELISA plate, and incubate overnight at 4°C. Wash the plate 3 times with PBST and block it with 5% milk at room temperature for 2h. Wash the plate 3 times with PBST, dilute the recombinant nanoantibody to be tested with 1% milk, add it to the ELISA plate, 30μL per well, and incubate at room temperature for 1h. Add hIL-5Rα-cHis (4μg / mL) without washing the plate, and incubate at room temperature for 1h. Wash the plate 3 times with PBST, add 6×His-HRP (proteintech; HRP-66005) antibody diluted 1:4000, and incubate at room temperature for 1h. Add 30μL TMB colorimetric solution to each well, react at room temperature in the dark for 5min, then add 50μL 2M sulfuric acid to terminate the reaction, and measure the absorbance at 450nm with an ELISA reader. The results are as follows: Figure 7-9 As shown, AIL-C14-Fc, AIL-C506-Fc, and AIL-C633-Fc have good ELISA blocking effects, and their blocking performance is better than that of positive antibodies hu39D10 and Mepolizumab.

[0141] 4.2 FACS-based detection of blocking activity of candidate antibodies

[0142] The CHO stable cell line co-expressing hIL-5Rα and hIL-5Rβ proteins was constructed. The cells were collected by centrifugation at 1000 rpm for 5 min, washed once with 30 mL PBS, and then resuspended in FACS staining buffer to prepare a cell suspension. The cell density was adjusted to 1×10 6cells / mL, add 100μL to each well of a 96-well plate, centrifuge and discard the supernatant. Dilute the antibody to a gradient concentration with FACSstaining Buffer. Use FACS Buffer to dilute the ligand hIL-5-hFc protein to a concentration of 0.1μg / mL. Mix the antibody and ligand at a ratio of 1:1 and incubate at 4℃ for 1h. Add 100μL of the antibody and hIL-5-hFc incubation liquid to the 96-well cell plate containing cells, resuspend the cells, and incubate at 4℃ for 60min. After incubation, wash the cells with 200μL FACS staining Buffer, centrifuge at 1000rpm for 5min, and discard the supernatant. Use FACS staining Buffer to dilute the secondary antibody at a ratio of 1:300, add 100μL / well to the 96-well plate, and incubate at 4℃ for 30min. Wash twice with FACS staining buffer, 200 μL each time, and finally resuspend in 250 μL FACS staining buffer, transfer to flow tube, and detect signal value with flow cytometer. The result is as follows Figure 10-12 As shown in Table 2, AIL-C14-Fc, AIL-C506-Fc, and AIL-C633-Fc can significantly block the binding of IL-5 to cell surface IL-5Rα at the FACS level, and the blocking activity is equivalent to that of the positive antibody hu39D10.

[0143] Table 2. FACS blocking assay

[0144]

[0145] Example 5: Detection of the proliferation inhibition of TF-1 cells by recombinant nanobodies

[0146] Press 1×10 4 TF-1-hIL-5Rα-FL-E6075-A17 cells were plated in 96-well plates at a cell density of 1:1 / well. Premix IL-5-hFc (final concentration 45 ng / mL) and the recombinant antibody to be tested and incubate at room temperature for 30 min. Add the mixture to the cell plate and incubate at 37°C for 48 h. Add MTS reagent and incubate in the dark for 90 min. Measure OD using a microplate reader 492 nm. The results are as follows Figure 13-14 As shown in Table 3, AIL-C14-Fc, AIL-C506-Fc, and AIL-C633-Fc can inhibit the proliferation of TF-1-hIL-5Rα-FL-E6075-A17 cells stimulated by IL-5, IC 50 It is lower than the positive antibody hu39D10 and has a better inhibitory effect on cell proliferation than the positive antibody.

[0147] Table 3 TF-1 proliferation inhibition test results

[0148]

[0149] Example 6. Antibody affinity detection and epitope grouping

[0150] In this example, affinity determination and epitope grouping of three candidate antibodies AIL-C14-Fc, AIL-C506-Fc, and AIL-C633-Fc were performed based on bio-layer interferometry (BLI) technology.

[0151] 6.1 Affinity determination of candidate antibodies

[0152] The candidate antibody to be tested was fixed on the sensor chip through Protein A Sensor and equilibrated in buffer for 30 seconds. hIL-5-His was added to bind to the antibody on the chip. After the binding was completed, it was dissociated in Q buffer [PBS (10mM PH7.4) + 0.02% Tween20 + 0.2% BSA] with a dissociation time of 300 seconds. The antibody affinity kinetic constant (KD) was obtained by fitting the binding and dissociation data.

[0153] The test results are shown in Table 4. The affinities of the candidate antibodies AIL-C14-Fc and AIL-C506-Fc are comparable to that of hu39D10, and the affinity of AIL-C633-Fc is superior to that of the positive antibody hu39D10.

[0154] Table 4 Antibody affinity determination data table

[0155] Antibody Name KD(M) Ka(1 / Ms) Kd(1 / s) <![CDATA[R 2 ]]> Rmax(nm) hu39D10 2.48E-10 2.77E+05 6.87E-05 1 0.882 AIL-C14-Fc 3.16E-10 4.88E+05 1.54E-04 1 0.837 AIL-C506-Fc 2.30E-10 7.19E+05 1.66E-04 1 0.701 AIL-C633-Fc 1.48E-11 7.71E+05 1.14E-05 0.999 0.742

[0156] 6.2 Grouping of candidate antibody epitopes

[0157] GATOR (ProbeLife) was used to group the epitopes of candidate antibodies, and hIL-5-cHis was used as the detection protein to detect antibodies in pairs to determine whether the antibodies competed to bind to the same site on the antigen. The antigen hIL-5-His was fixed on the sensor chip by the His tag capture method, hu39D10 and Mepolizumab were used as saturated antibodies, and the antibody to be tested was used as a competing antibody, and the inhibition rate was used to reflect the epitope competition. The results are shown in Table 5. The positive antibody Mepolizumab has an epitope overlap with AIL-C633-Fc; it does not compete with antibodies AIL-C506-Fc and AIL-C14-Fc at all. The positive antibody hu39D10 recognizes the same epitope as AIL-C506-Fc and AIL-C14-Fc; it does not compete with antibody AIL-C633-Fc at all.

[0158] Table 5 Grouping of candidate antibody epitopes

[0159]

[0160] Example 7. Humanization of Nanobodies

[0161] Through flow cytometry antibody blocking experiment and proliferation inhibition experiment, AIL-C14-Fc, AIL-C506-Fc, and AIL-C633-Fc with better blocking effect and better proliferation inhibition effect were selected for subsequent antibody humanization.

[0162] Candidate antibodies AIL-C14, AIL-C506, and AIL-C633 were partitioned according to the IMGT partitioning rules. The human germline with the highest similarity to the parent antibody was searched using the Igblast database, and the antibody was humanized using the CDR transplantation method. According to the differential sites in the Framework region (FR region), the key residues of the hydrophobic core, the key residues of the hydrogen bond network in the functional region, the residues in the corner structure, etc. were analyzed, and appropriate amino acids were selected for back mutation. Multiple humanized antibodies were designed for each parent antibody, and recombinant expression was performed according to the method of Example 3 to obtain humanized antibody proteins. The humanized antibody sequences are shown in SEQ ID NO.16 to SEQ ID NO.41.

[0163] AIL-C14-VHH1: (SEQ ID NO.16)

[0164] QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKERDWVACINSGGDIT NYANSVKGRFTISRDNAKNTVYLEMNSLRADDTALYYCAARQQKYFCSPDVVGYNYWGQGTLVTVSS

[0165] AIL-C14-VHH2: (SEQ ID NO.17)

[0166] QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKERDWVACINSGGDITNYANSVKGRFTISRDNAKNTVYLEMNSLRAEDTALYYCAARQQKYFCSPDVVGYNYWGQGTQVTVSS

[0167] AIL-C14-VHH3: (SEQ ID NO.18)

[0168] QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKERDWVACINSGGDITNYANSVKGRFTISRDNAKNTLYLQMNSLKIEDTALYYCAARQQKYFCSPDVVGYNYWGQGTLVTVSS

[0169] AIL-C14-VHH4:(SEQ ID NO.19)

[0170] QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKERDWVACINSGGDITNYANSVKGRFTISRDNAKNTLYLQMNSLRAEDTALYYCAARQQKYFCSPDVVGYNYWGQGTQVTVSS

[0171] AIL-C14-VHH5:(SEQ ID NO.20)

[0172] QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKERDWVACINSGGDITNYANSVKGRFTISRDNAKNTVYLQMNSLRAEDTALYYCAARQQKYFCSPDVVGYNYWGQGTLVTVSS

[0173] AIL-C14-VHH6:(SEQ ID NO.21)

[0174] QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKGLDWVACINSGGDITNYANSVKGRFTISRDNAKNTLYLEMNSLRADDTALYYCAARQQKYFCSPDVVGYNYWGQGTLVTVSS

[0175] AIL-C14-VHH7:(SEQ ID NO.22)

[0176] QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKERDWVACINSGGDITNYANSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAARQQKYFCSPDVVGYNYWGQGTLVTVSS

[0177] AIL-C14-VHH8:(SEQ ID NO.23)

[0178] QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKGLEWVACINSGGDITNYANSVKGRFTISRDNAKNTVYLQMNSLRAEDTALYYCAARQQKYFCSPDVVGYNYWGQGTLVTVSS

[0179] AIL-C14-VHH9:(SEQ ID NO.24)

[0180] QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKGLDWVACINSGGDITNYANSVKGRFTISRDNAKNTLYLQMNSLRAEDTALYYCAARQQKYFCSPDVVGYNYWGQGTLVTVSS

[0181] AIL-C14-VHH10:(SEQ ID NO.25)

[0182] QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWVRQAPGKGLEWVACINSGGDITNYANSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAARQQKYFCSPDVVGYNYWGQGTLVTVSS

[0183] AIL-C506-VHH1:(SEQ ID NO.26)

[0184] EVQVQESGGGLVQPGGSLRLSCAATGFTLDGYAIGWFRQAPGKEREGISCISSTGEIAFYADSVKGRFAISRDGAKNTVYLEMNSLRAEDTALYYCASRRQKYFCSNDAIGYGNWGQGTLVTVSS

[0185] AIL-C506-VHH2:(SEQ ID NO.27)

[0186] EVQVQESGGGLVQPGGSLRLSCAATGFTLDGYAIGWFRQAPGKEREGISCISSTGEIAFYADSVKGRFAISRDGAKNTLYLQMNSLRAEDTALYYCASRRQKYFCSNDAIGYGNWGQGTLVTVSS

[0187] AIL-C506-VHH3:(SEQ ID NO.28)

[0188] EVQLVESGGGLVQPGGSLRLSCAATGFTLDGYAIGWFRQAPGKEREGISCISSTGEIAFYADSVKGRFTISRDGAKNTLYLEMNSLRAEDTALYYCASRRQKYFCSNDAIGYGNWGQGTQVTVSS

[0189] AIL-C506-VHH4:(SEQ ID NO.29)

[0190] EVQVQESGGGLVQPGGSLRLSCAATGFTLDGYAIGWFRQAPGKEREGISCISSTGEIAFYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTALYYCASRRQKYFCSNDAIGYGNWGQGTLVTVSS

[0191] AIL-C506-VHH5:(SEQ ID NO.30)

[0192] EVQLVESGGGLVQPGGSLRLSCAATGFTLDGYAIGWFRQAPGKEREGISCISSTGEIAFYADSVKGRFAISRDNAKNTVYLQMNSLRAEDTALYYCASRRQKYFCSNDAIGYGNWGQGTLVTVSS

[0193] AIL-C506-VHH6:(SEQ ID NO.31)

[0194] EVQLVESGGGLVQPGGSLRLSCAASGFTLDGYAIGWFRQAPGKEREGISCISSTGEIAFYADSVKGRFAISRDNAKNTLYLQMNSLRAEDTALYYCASRRQKYFCSNDAIGYGNWGQGTLVTVSS

[0195] AIL-C506-VHH7:(SEQ ID NO.32)

[0196] EVQLQESGGGLVQPGGSLRLSCAASGFTLDGYAIGWFRQAPGKEREGISCISSTGEIAFYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCASRRQKYFCSNDAIGYGNWGQGTLVTVSS

[0197] AIL-C506-VHH8:(SEQ ID NO.33)

[0198] EVQLVESGGGLVQPGGSLRLSCAASGFTLDGYAIGWFRQAPGKGLEGISCISSTGEIAFYADSVKGRFTISRDNAKNTVYLQMNSLRAEDTALYYCASRRQKYFCSNDAIGYGNWGQGTLVTVSS

[0199] AIL-C633-VHH1:(SEQ ID NO.34)

[0200] EVQLVESGGGLVQPGGSLRLSCVASGFTFSTAPMSWYRQTPGKDHEWVGTIPADDGKT WYADSVKGRFTISRDNAKNTVYLQMNNLRAEDTAVYSCASPHYWGQGTLVTVSS

[0201] AIL-C633-VHH2:(SEQ ID NO.35)

[0202] EVQLVESGGGLVQPGGSLRLSCVASGFTFSTAPMSWYRQTPGKDHEWVGTIPADDGKT WYADSVKGRFTISRDNAKNTVYLQMNNLRAEDTAVYYCASPHYWGQGTLVTVSS

[0203] AIL-C633-VHH3:(SEQ ID NO.36)

[0204] EVQLVESGGGLVQPGGSLRLSCVASGFTFSTAPMSWYRQTPGKDHEWVGTIPADDGKT WYADSVKGRFTISRDNAKNTLYLQMNNLRAEDTAVYSCASPHYWGQGTLVTVSS

[0205] AIL-C633-VHH4:(SEQ ID NO.37)

[0206] EVQLVESGGGLVQPGGSLRLSCAASGFTFSTAPMSWYRQTPGKDHEWVGTIPADDGK TWYADSVKGRFTISRDNAKNTVYLQMNNLRAEDTAVYYCASPHYWGQGTLVTVSS

[0207] AIL-C633-VHH5: (SEQ ID NO.38)

[0208] EVQLVESGGGLVQPGGSLRLSCAASGFTFSTAPMSWYRQTPGKDHEWVGTIPADDGKTWYADSVKGRFTISRDNAKNTLYLQMNNLRAEDTAVYYCASPHYWGQGTLVTVSS

[0209] AIL-C633-VHH6: (SEQ ID NO.39)

[0210] EVQLVESGGGLVQPGGSLRLSCAASGFTFSTAPMSWYRQAPGKDHEWVGTIPADDGKTWYADSVKGRFTISSRDNAKNTLYLQMNNLRAEDTAVYSCASPHYWGQGTLVTVSS

[0211] AIL-C633-VHH7: (SEQ ID NO.40)

[0212] EVQLVESGGGLVQPGGSLRLSCAASGFTFSTAPMSWYRQTPGKGLEWVGTIPADDGKT WYADSVKGRFTISRDNAKNTLYLQMNNLRAEDTAVYYCASPHYWGQGTLVTVSS

[0213] AIL-C633-VHH8: (SEQ ID NO.41)

[0214] EVQLVESGGGLVQPGGSLRLSCAASGFTFSTAPMSWYRQAPGKGLEWVGTIPADDGK TWYADSVKGRFTISSRDNAKNTLYLQMNNLRAEDTAVYYCASPHYWGQGTLVTVSS.

[0215] Example 8: Humanized Antibody Functional Detection

[0216] In this example, the humanized antibodies of AIL-C14-Fc, AIL-C506-Fc, and AIL-C633-Fc were subjected to functional assays, including ELISA binding assay, ELISA blocking assay, FACS blocking assay, and TF-1 cell proliferation inhibition assay.

[0217] 8.1 Humanized Antibody ELISA Binding Detection

[0218] Using hIL-5-His as an antigen, the ELISA method was used to detect the binding ability of AIL-C14-Fc, AIL-C506-Fc and AIL-C633-Fc humanized antibodies to human IL-5. hIL-5-His was diluted to 2 μg / mL and added to the ELISA plate, and the detection was performed in the manner described in Example 3. The results are shown in FIG. Figure 15-17 As shown in Tables 6-8, all humanized antibodies strongly bound to human IL-5.

[0219] Table 6 Functional detection of AIL-C14-Fc humanized antibody

[0220] Sample name <![CDATA[ELISA combined with EC 50 (nM)]]> <![CDATA[ELISA Blocking IC 50 (nM)]]> <![CDATA[FACS Blocking IC 50 (nM)]]> hu39D10 0.1166 0.108 0.145 AIL-C14-Fc 0.04402 0.1297 0.4038 AIL-C14-VHH1-Fc 0.05295 0.05376 0.4141 AIL-C14-VHH2-Fc 0.04532 0.1748 0.3703 AIL-C14-VHH3-Fc 0.04501 0.1991 0.4827 AIL-C14-VHH4-Fc 0.06211 ～0.4673 0.4007 AIL-C14-VHH5-Fc 0.05024 0.1427 0.4093 AIL-C14-VHH6-Fc 0.04695 ～1.838e-005 0.5562 AIL-C14-VHH7-Fc 0.04268 0.2002 0.412 AIL-C14-VHH8-Fc 0.07996 0.137 0.4103 AIL-C14-VHH9-Fc 0.04197 0.2263 0.608

[0221] Table 7 Functional detection of AIL-C506-Fc humanized antibody

[0222] Antibody Name <![CDATA[ELISA combined with EC 50 (nM)]]> <![CDATA[ELISA blocking IC 50 (nM)]]> <![CDATA[FACS blocking IC 50 (nM)]]> hu39D10 0.1264 0.2266 0.1531 AIL-C506-Fc 0.06772 0.2846 0.3022 AIL-C506-VHH1-Fc 0.06465 0.2418 0.2753 AIL-C506-VHH3-Fc 0.07689 0.491 0.3302 AIL-C506-VHH4-Fc 0.09887 0.5836 0.9034 AIL-C506-VHH5-Fc 0.05553 0.2741 0.233 AIL-C506-VHH6-Fc 0.07315 0.3411 0.3986 AIL-C506-VHH7-Fc 0.08048 0.1624 0.2581 AIL-C506-VHH8-Fc 0.06593 0.3872 0.2471

[0223] Table 8 Functional detection of AIL-C633-Fc humanized antibody

[0224] Antibody Name <![CDATA[ELISA combined with EC 50 (nM)]]> <![CDATA[ELISA blocking IC 50 (nM)]]> <![CDATA[FACS Blocking IC 50 (nM)]]> hu39D10 0.6705 0.7435 0.2133 AIL-C633-Fc 0.5593 0.1712 0.5095 AIL-C633-VHH1-Fc 0.5299 ～0.0001018 0.5046 AIL-C633-VHH2-Fc 0.3657 ～1.675 0.602 AIL-C633-VHH3-Fc 0.5609 0.4548 0.5184 AIL-C633-VHH4-Fc 0.3048 ～0.5325 0.63 AIL-C633-VHH5-Fc 0.3304 0.626 0.5887 AIL-C633-VHH6-Fc 0.4647 ～1.080 0.4935 AIL-C633-VHH7-Fc 0.3355 0.9553 0.7043 AIL-C633-VHH8-Fc 0.3786 0.5613 0.6279

[0225] 8.2 Humanized Antibody ELISA Blocking Test

[0226] hIL-5-hFc was diluted with PBS to 2 μg / mL, 30 μL was added to the ELISA plate, and the humanized antibodies of AIL-C14-Fc, AIL-C506-Fc and AIL-C633-Fc were subjected to ELISA blocking test according to the method in Example 4. The results are as follows: Figure 18-20 As shown in Tables 6-8, the humanized antibodies all have the effect of blocking the binding of IL-5 to IL-5Rα in ELISA.

[0227] 8.3 Humanized Antibody FACS Blocking Detection

[0228] FACS blocking detection was performed using a CHO stable cell line co-expressing hIL-5Rα and hIL-5Rβ proteins. The humanized antibodies of AIL-C14-Fc, AIL-C506-Fc and AIL-C633-Fc were graded diluted and flow blocking detection was performed according to Example 4. The results are shown in FIG. Figure 21-23 As shown in Tables 6-8, the humanized antibodies all have the effect of blocking the binding of IL-5 to IL-5Rα in FACS.

[0229] 8.4 Humanized antibody TF-1 proliferation inhibition assay

[0230] TF-1 proliferation inhibition was detected using the TF-1 cell line TF-1-hIL-5Rα-FL-E6075-A17 that overexpresses IL-5Rα. The cells can rely on IL-5 for cell proliferation. The cells were diluted and plated on a 96-well cell culture plate. The premixed IL-5-hFc (final concentration 45 ng / mL) and the humanized antibody to be tested were added to the cell culture plate. The proliferation inhibition test was performed according to the steps of Example 5. Figure 24-26 Table 9 is the proliferation inhibition results of humanized antibodies AIL-C14-VHH7-Fc, AIL-C506-VHH7-Fc, and AIL-C633-VHH6-Fc, indicating that the IC 50 It is lower than that of the positive antibody hu39D10, and has a better effect on inhibiting cell proliferation than the positive antibody hu39D10.

[0231] Table 9 Humanized antibody proliferation inhibition results

[0232]

[0233] Example 9, affinity maturation of nanobodies

[0234] The results of flow blocking experiments and proliferation inhibition experiments of humanized candidate molecules determined that the most preferred molecules were AIL-C14-VHH7-Fc, AIL-C506-VHH7-Fc, and AIL-C633-VHH6-Fc. Multiple mutation strategies were adopted to construct multiple large-capacity mutation libraries based on humanized antibodies AIL-C14-VHH7-Fc and AIL-C506-VHH7-Fc. The library capacity of the antibody library was determined by the dilution point plate method after electrotransfer, and the correct insertion rate of the antibody gene was verified by monoclonal sequencing analysis. Immunotubes and magnetic bead screening instruments were used to screen the phage mutation library in two ways, solid phase and liquid phase screening, and VHH antibody molecules that specifically bound to the hIL-5 antigen and had a higher affinity than the parent were screened at the ELISA level. The antibodies of affinity maturation candidates were expressed, and the affinity of the candidate antibodies was ranked by ELISA. The humanized AIL-C14-VHH7-Fc and AIL-C506-VHH7-Fc camel immune library molecules were modified by affinity maturation, and a total of 37 AIL-C14-VHH7-Fc affinity matured candidate molecules and 42 AIL-C506-VHH7-Fc affinity matured candidate molecules were constructed. After protein expression, purification and activity detection analysis, the affinity matured molecules N_PR_C14_22 and N_PR_C506_34, especially the N_PR_C506_34 molecule, have better functions than the parent antibody and positive antibody. The antibody sequences are shown below, and the underlined + italic marked parts are CDR regions (according to the IMGT partitioning rules). Figure 27-34 Table 10 shows the results of affinity matured candidate molecules N_PR_C14_22 and N_PR_C506_34 in ELISA binding, ELISA antibody blocking, flow cytometry antibody blocking, and TF-1 cell proliferation inhibition.

[0235] N_PR_C14_22: (SEQ ID NO.42)

[0236]

[0237] N_PR_C506_34: (SEQ ID NO.43)

[0238]

[0239] Table 10 Affinity maturation antibody function test

[0240]

[0241] Example 10. Preparation and functional testing of bivalent nanobodies

[0242] Based on the above results, the preferred molecules determined are N_PR_C14_22, N_PR_C506_34, and AIL-C633-VHH6. The above molecules were combined into bivalent VHH antibodies, and the two VHH sequences were connected by a (G4S)4 linker element, and further recombinantly expressed in ExpiCHO-S cells and purified. The bivalent antibody molecules were subjected to affinity kinetics detection, ELISA affinity detection, FACS antibody blocking detection, and proliferation inhibition detection.

[0243] Table 11 Evaluation of bivalent antibody molecules

[0244]

[0245] Note: In the table, (G4S) is the linker element unit, (G4S)n means there are n linker element units, His means His tag, and C633-VHH6 etc. are the VHHs with the same name defined previously.

[0246] 10.1 Kinetics of affinity testing of bivalent nanoantibodies

[0247] Biacore T200 (Cytiva) was used to detect the affinity kinetics of bivalent nanoantibodies. The anti-His antibody was diluted to 50 μg / mL with sodium acetate solution at pH 4.5 for capture on the CM5 chip. The antibody sample to be tested was diluted to 30 nM with 1×HBS-P+ buffer solution as a ligand, and the flow rate was set to 10 μL / min for ligand capture. The antigen hIL-5-hFc was diluted to 300, 150, 75, 37.5, 18.75, 9.38, 4.69, 2.34, and 1.17 nM with 1×HBS-EP+ (pH 7.4). Parameter settings: flow rate 30 μL / min, antigen capture 30 s, binding to the antibody 180 s, dissociation in buffer 1500 s, reaction temperature 25°C. The affinity results are shown in Table 11.

[0248] 10.2 ELISA binding detection of bivalent nanobodies

[0249] hIL-5-His was used as an antigen, and the binding ability of the above dual antibody combination to human IL-5 was detected by ELISA. hIL-5-His was diluted to 2 μg / mL and added to the ELISA plate, and the detection was performed in the manner described in Example 3. The results are shown in FIG. Fig.35 As shown in Table 11, except for C633-VHH6-(G4S)4-N_PR_C14_22-His, the other bivalent and bi-epitope proteins all had strong binding to human IL-5.

[0250] 10.3 FACS blocking assay of bivalent nanobodies

[0251] FACS blocking test was performed using a CHO stable cell line co-expressing hIL-5Rα and hIL-5Rβ proteins. The above dual antibodies were diluted in a gradient manner and FACS blocking test was performed according to Example 4. The results are shown in Fig.36 As shown in Table 11, both antibodies have the effect of blocking the binding of IL-5 to IL-5Rα at the FACS level.

[0252] 10.4 Bivalent Nanobodies in TF-1 Proliferation Inhibition Assay

[0253] TF-1 proliferation inhibition was detected using the TF-1 cell line TF-1-hIL-5Rα-FL-E6075-A17 that overexpresses IL-5Rα. The cells can rely on IL-5 for cell proliferation. The cells were diluted and plated on a 96-well cell culture plate. The premixed IL-5-hFc (final concentration 45 ng / mL) and the dual antibody to be tested were added to the cell culture plate. The proliferation inhibition test was performed according to the steps of Example 5. Figure 37-40 Table 11 shows the proliferation inhibition results of the dual antibodies. The results show that the IC 50 The level was equivalent to or lower than that of the more positive antibody hu39D10.

[0254] Example 11. Preparation and functional testing of tetravalent nanobodies containing Fc

[0255] 11.1 FACS blocking assay with tetravalent nanobody

[0256] FACS blocking assay was performed using a CHO stable cell line co-expressing hIL-5Rα and hIL-5Rβ proteins. The tetravalent Fc-tagged Nanobodies shown in Table 12 were graded diluted and FACS blocking assay was performed according to Example 4. The results are shown in Table 12. Figure 41-42 As shown in Table 12, the tetravalent Fc-tagged nanoantibodies have the effect of blocking the binding of IL-5 to IL-5Rα at the FACS level, and are significantly better than the positive control hu39D10.

[0257] Table 12. FACS blocking function detection

[0258]

[0259] Note: in the table, (G4S) is a linker element unit, (G4S)n indicates that there are n linker element units, His indicates a His tag, C633-VHH6 and the like are VHHs of the same name defined previously, and the YTE mutant sequence shown in SEQ ID NO.52 is used as Fc. The specific sequence is: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0260] 11.2 Proliferation inhibition assay of tetravalent nanoantibody TF-1 with Fc

[0261] The TF-1 proliferation inhibition was detected using the TF-1 cell line TF-1-hIL-5Rα-FL-E6075-A17 overexpressing IL-5Rα, and the test antibody was gradiently diluted and the proliferation inhibition test was performed according to the steps of Example 5. The results are shown in FIG. Figure 43-47 As shown in Table 13, all tetravalent Fc-tagged molecules had significantly better proliferation inhibition effects than the positive control hu39D10.

[0262] Table 13. Proliferation inhibition assay of tetravalent molecules with Fc

[0263]

[0264] Note: In the table, (G4S) is the linker element unit, (G4S)n means there are n linker element units, His means His tag, and C633-VHH6 etc. are the VHHs with the same name defined previously.

[0265] Example 12: Evaluation of the efficacy of recombinant antibodies in the B-hIL-5 / hIL-5Rα double humanized mouse asthma model

[0266] The mouse sequences were replaced with human IL-5 and IL-5Rα sequences to obtain humanized mice that only expressed human IL-5 and IL-5Rα proteins, which were used to evaluate the efficacy of human IL-5 target antibodies. Animal grouping: 28 B-hIL-5 / hIL-5Rα humanized mice were randomly divided into 4 groups according to body weight, with 4 animals in group G1 and 6 animals in each group G2-G4, of which group G1 was the blank control group and G2-G4 was the test article treatment group. Asthma model was established: On days 0, 7, and 14, animals in group G1 were intraperitoneally injected with 200 μL PBS, and each animal in group G2-G4 was sensitized by intraperitoneal injection of 200 μL of OVA / AI(OH)3 mixture at a concentration of 200 μg / mL. On days 21-25, animals in group G1 were treated with PBS atomization every day, and animals in group G2-G4 were treated with 2% OVA atomization every day, 30 minutes each time, for 5 consecutive days. In the modeling group (G2), the proportion of eosinophils in BALF increased, the level of serum OVA-specific IgE increased, the lymphocytes and eosinophils in the blood increased significantly, the inflammatory cells and eosinophils infiltrated the lung tissue, and the bronchial mucus exudation increased (see Figure 48-51 ), indicating that the asthma model was successfully induced in this humanized mouse.

[0267] G2-G4 were administered to the airway three times a week, on days 20, 22 and 24, respectively. The specific grouping and administration are shown in Table 14. The experimental endpoint was reached on day 26, and samples were collected to test various biochemical indicators.

[0268] Table 14. Animal grouping

[0269]

[0270] Note: a: The administration volume of G2-G4 groups was 50 μL / mouse. C633-C633 is the abbreviation of C633-VHH6-(G4S)4-C633-VHH6 dual antibody.

[0271] 12.1 Changes in animal weight during the experiment

[0272] In this experiment, no obvious abnormal state was observed in the animals of each group. During the whole experiment, the weight of the animals of each group gradually increased, and no obvious toxic and side effects of the drug were observed, see Table 15.

[0273] Table 15. Body weight changes of B-hIL5 / hIL5R mice during the experiment

[0274]

[0275] 12.2 OVA-specific IgE levels in serum

[0276] The ELISA method was used to detect the level of OVA-specific IgE in the serum of mice on day 26. The results showed that compared with the G1 group, the level of OVA-specific IgE in the G2 group increased. Compared with the G2 control group, the levels of OVA-specific IgE in the G3 and G4 groups did not change significantly, as shown in Table 1. Fig.48 , Table 16.

[0277] Table 16. OVA-specific IgE content in mouse serum

[0278]

[0279] 12.3 Changes of Immune Cells in Bronchoalveolar Lavage Fluid

[0280] Eosinophil infiltration is one of the key indicators for the evaluation of asthma. The total number of inflammatory cells (mCD45+), eosinophils, neutrophils and macrophages in the BALF of mice on day 26 was detected by FACS. Fig.49 Compared with the G1 group, the mCD45 + The number of cells and eosinophils increased significantly ( Fig.49 A, 49B), compared with the G2 group, mCD45 in the G3-G4 group + The total number of eosinophils was significantly decreased ( Fig.49 A, 49B), compared with the G2 group, the proportion of eosinophils in the G3-G4 group was significantly reduced, and there was a statistical difference. Under the condition of the same dosing frequency, the efficacy of C633-C633-Fc (G4) was better than that of C633-C633-His (G3).

[0281] 12.4 Routine blood test

[0282] Blood routine tests were performed to detect lymphocytes, basophils, and eosinophils in the blood of mice on the 26th day. The results showed that compared with the G1 group, the number and proportion of lymphocytes and eosinophils in the G2 group increased significantly, and there was a statistical difference; compared with the G2 group, the number and proportion of lymphocytes and eosinophils in the G3 and G4 groups decreased significantly ( Fig.50 A, 50C, 50D, 50F). The trend of the eosinophil percentage results was consistent with the results of flow cytometry detection of eosinophils in BALF.

[0283] 12.5 Lung Tissue Pathology

[0284] In the OVA-induced mouse asthma model, the infiltration of inflammatory cells and the secretion of mucus are key indicators in pathological detection. Inflammatory cells include a variety of cells, among which eosinophils are one of the most critical inflammatory cells that can affect the severity of asthma.

[0285] At the end of the experiment, the lung tissues of the animals were collected for HE detection. In the G2 group, different degrees of mixed inflammatory cells and eosinophil infiltration around the blood vessels and bronchial tubes, as well as bronchial mucus exudation were observed under the microscope, indicating that the asthma model was successfully established. After the G3-G4 group mice were intervened with different test products, the infiltration of mixed inflammatory cells and eosinophils around the blood vessels and bronchial tubes in the lungs was significantly inhibited ( Fig.51 A, 51C), no significant improvement was observed on bronchial mucus formation ( Fig.51 B).

[0286] The above is a further detailed description of the present disclosure, which should not be regarded as a limitation on the specific implementation of the present disclosure. For ordinary technicians in the technical field to which the present disclosure belongs, simple deduction or replacement without departing from the concept of the present disclosure is within the protection scope of the present disclosure.

Claims

1. An IL-5 binding molecule, characterized in that Capable of specifically binding to IL-5, and comprising at least one VHH chain, wherein the VHH chain comprises a framework region FR and a complementarity determining region CDR, wherein the complementarity determining region CDR comprises at least one group selected from the following groups: Group (1), having CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.3; Group (2), having CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5, and CDR3 shown in SEQ ID NO. 6; Group (3), having CDR1 shown in SEQ ID NO.7, CDR2 shown in SEQ ID NO.8, and CDR3 shown in SEQ ID NO.9; Group (4), having CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 11; Group (5), having CDR1 shown in SEQ ID NO.12, CDR2 shown in SEQ ID NO.5, and CDR3 shown in SEQ ID NO.6; The CDRs of group (4) are sequences obtained from group (1) after affinity maturation; and the CDRs of group (5) are sequences obtained from group (2) after affinity maturation.

2. The IL-5 binding molecule according to claim 1, characterized in that The VHH is a humanized VHH.

3. The IL-5 binding molecule according to claim 1, characterized in that The VHH comprises any one of the amino acid sequences in SEQ ID NO.13 to SEQ ID NO.43, or an amino acid sequence having at least 85%, preferably at least 90% sequence homology with any one of the amino acid sequences in SEQ ID NO.13-43.

4. The IL-5 binding molecule according to any one of claims 1 to 3, characterized in that It is composed of two or more VHHs connected by a linker element, and the VHH comprises any amino acid sequence in SEQ ID NO.13-43 or an amino acid sequence having at least 85% sequence homology with any amino acid sequence in SEQ ID NO.13-43.

5. The IL-5 binding molecule according to claim 4, characterized in that The VHH comprises any one of the amino acid sequences in SEQ ID NOs. 44-50 or an amino acid sequence having at least 85%, preferably at least 90% sequence homology with any one of the amino acid sequences in SEQ ID NOs. 44-50.

6. The IL-5 binding molecule according to claim 1, characterized in that It also includes an antibody Fc region, wherein the antibody Fc region is directly or via a linker element connected to the C-terminus or N-terminus of the IL-5 binding molecule.

7. The IL-5 binding molecule according to claim 6, characterized in that The antibody Fc region includes the Fc segment of human IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgM, IgE or its variants and modifications.

8. The IL-5 binding molecule according to claim 6 or 7, characterized in that It forms dimers via the antibody Fc region.

9. A composition, characterized in that It comprises the IL-5 binding molecule or its derivative according to any one of claims 1 to 8, and a pharmaceutically acceptable diluent, carrier or adjuvant.

10. A nucleic acid molecule, characterized in that Encoding the IL-5 binding molecule or its derivative according to any one of claims 1 to 8.

11. An expression vector, characterized in that: Comprising the nucleic acid molecule of claim 10.

12. A host cell, characterized in that It expresses the IL-5 binding molecule or derivative thereof according to any one of claims 1 to 8, or comprises the nucleic acid molecule according to claim 10, or comprises the expression vector according to claim 11.

13. A method for preparing an IL-5 binding molecule, characterized in that: The method comprises using the nucleic acid molecule of claim 10 under conditions suitable for expressing the IL-5 binding molecule of any one of claims 1 to 8, or using the expression vector of claim 11, or using the host cell of claim 12 for expression.

14. A detection kit for IL-5, characterized in that It comprises the IL-5 binding molecule or a derivative thereof according to any one of claims 1 to 8.

15. Use of the IL-5 binding molecule of any one of claims 1 to 8, the composition of claim 9, the nucleic acid molecule of claim 10, the expression vector of claim 11, the host cell of claim 12, or a combination thereof in the preparation of a medicament for preventing and / or treating a disease or condition associated with IL-5 / IL-5R signaling.

16. The use according to claim 15, characterized in that The disease or condition is selected from asthma, severe eosinophilic asthma, hypereosinophilia, Churg-Strauss syndrome, atopic dermatitis, eosinophilic granuloma, granulomatosis with polyangiitis, chronic sinusitis with nasal polyps, eosinophilic esophagitis with nasal polyps, and pulmonary eosinophilia-related disease.

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