Anti-il-5 antibody, pharmaceutical composition thereof and use thereof

Abstract

The present invention belongs to the field of biomedicine, and relates to an anti-IL-5 antibody, a pharmaceutical composition thereof and the use thereof. Specifically, the present invention relates to an anti-IL-5 antibody or an antigen-binding fragment thereof. The anti-IL-5 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises complementary determining regions HCDR1-HCDR3, and the light chain variable region comprises complementary determining regions LCDR1-LCDR3, wherein an amino acid sequence of HCDR1 is set forth in SEQ ID NO: 3, an amino acid sequence of HCDR2 is set forth in SEQ ID NO: 4, and an amino acid sequence of HCDR3 is set forth in SEQ ID NO: 5; and an amino acid sequence of LCDR1 is set forth in SEQ ID NO: 6, an amino acid sequence of LCDR2 is set forth in SEQ ID NO: 7, and an amino acid sequence of LCDR3 is set forth in SEQ ID NO: 8. The antibody of the present invention can specifically bind to IL-5, exhibits a high affinity, and has a good potential for treating or preventing diseases caused by eosinophils above the normal level.

Description

Anti-IL-5 antibodies, their pharmaceutical compositions and uses Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an anti-IL-5 antibody, its pharmaceutical composition, and its uses. Specifically, this invention relates to an anti-IL-5 monoclonal antibody or its antigen-binding fragment thereof, and a pharmaceutical composition containing said antibody or its antigen-binding fragment. This invention also relates to a nucleic acid molecule encoding said antibody or its antigen-binding fragment and a host cell containing it. Background Technology

[0002] Eosinophils (EOS) are mature white blood cells derived from hematopoietic stem cells (HSCs) in the bone marrow. They undergo a series of differentiation processes into granulocyte-macrophage progenitor cells, ultimately maturing under the induction of interleukin-5 (IL-5). Allergic diseases such as asthma, allergic rhinitis, and atopic dermatitis, as well as parasitic infections and certain hematologic malignancies, can all cause an increase in the number of eosinophils in the body. This surge in eosinophil count can lead to tissue inflammation and organ damage, resulting in related inflammatory diseases.

[0003] IL-5 is involved in the differentiation, activation, proliferation, survival, and recruitment of eosinophils, playing a crucial role in eosinophilic inflammation. In allergen-induced adaptive immunity, IL-5 is mainly secreted by Th2 cells and mast cells. IL-5 selectively binds to the α subunit of its receptor IL-5R and induces the assembly of the dimer receptor complex composed of the IL-5R α and βc subunits, activating the intracellular signaling JAK2-STAT1 / 3 / 5, thereby initiating the transcription of a series of genes (Kotsimbos ATC, et al. Memórias do Instituto Oswaldo Cruz, 1997, 92:75-91.). JAK2 activates Lyn and RAF-1 kinases or induces the anti-apoptotic protein BCL-XL in a NF-κB-dependent manner, inhibiting eosinophil apoptosis (Dibbert B, et al. Blood, 1998, 92(3):778-783; Alam R, et al. Int Arch Allergy Immunol, 1995.). IL-5 participates in the maturation, activation, proliferation, and chemotaxis of eosinophils through the PI3K, MAPK, and NF-κB pathways (Alam R, et al. Int Arch Allergy Immunol, 1995; Takatsu, KK International Immunology, 2009, 21(12):1303.).

[0004] Preclinical studies in IL-5-deficient mice have shown that the absence of IL-5 fails to induce the eosinophilia phenotype induced by parasitic infection (Kopf M, et al. Immunity. 1996 Jan; 4(1):15-24.). Instead, IL-5 transgenic mice exhibit high levels of eosinophils in their blood compared to normal mice (Dent, LA Journal of Experimental Medicine, 1990, 172(5):1425-1431.).

[0005] Studies have shown that IL-5 levels are significantly elevated in bronchial biopsy tissues from asthmatic patients, and that IL-5 levels in bronchial mucus are closely correlated with disease severity (Minshall EM, et al. J Allergy Clin Immunol. 1998 Mar; 101(3):386-90; Humbert M, et al. Am J Respir Crit Care Med, 1997, 156(3Pt 1):704-708.). Anti-IL-5 drug treatment significantly reduces the number of eosinophils in the bronchial mucosa and peripheral blood of asthmatic patients. These results indicate that IL-5 plays a crucial role in eosinophil production and suggest its potential in treating eosinophilic diseases.

[0006] Mepolizumab (GSK) is a humanized anti-IL-5 monoclonal antibody that has been approved for the treatment of severe eosinophilic asthma. However, there is still a lack of fully human IL-5 antibodies with higher affinity and lower immunogenicity in this field. Summary of the Invention

[0007] Through in-depth research and creative work, the inventors have obtained an anti-IL-5 antibody. The inventors have surprisingly discovered that this antibody can specifically bind to IL-5, exhibiting high affinity and / or low immunogenicity, and possesses good potential for treating or preventing diseases caused by elevated eosinophil levels. Therefore, the following invention is provided:

[0008] One aspect of the present invention relates to an anti-IL-5 antibody or an antigen-binding fragment thereof, said anti-IL-5 antibody comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising complementarity-determining regions HCDR1 to HCDR3, and said light chain variable region comprising complementarity-determining regions LCDR1 to LCDR3, wherein:

[0009] The amino acid sequences of HCDR1 are shown in SEQ ID NO:3, HCDR2 in SEQ ID NO:4, and HCDR3 in SEQ ID NO:5.

[0010] The amino acid sequences of LCDR1 are shown in SEQ ID NO:6, LCDR2 in SEQ ID NO:7, and LCDR3 in SEQ ID NO:8.

[0011] In this invention, unless otherwise specified, the complementary determination regions (CDRs) HCDR1 to HCDR3 and LCDR1 to LCDR3 are defined according to the Kabat numbering system.

[0012] In some embodiments of the present invention, the anti-IL-5 antibody or its antigen-binding fragment, wherein,

[0013] The amino acid sequence of the heavy chain variable region of the anti-IL-5 antibody is shown in SEQ ID NO:1; and

[0014] The amino acid sequence of the light chain variable region of the anti-IL-5 antibody is shown in SEQ ID NO:2.

[0015] In some embodiments of the present invention, the anti-IL-5 antibody is a monoclonal antibody.

[0016] In some embodiments of the present invention, the anti-IL-5 antibody or its antigen-binding fragment, wherein,

[0017] The antibody includes a non-CDR region, and the non-CDR region is derived from a species other than rodents, such as from human antibodies;

[0018] Preferably, the anti-IL-5 antibody is a fully human antibody;

[0019] Preferably, the anti-IL-5 antibody is a fully human monoclonal antibody.

[0020] In some embodiments of the present invention, the anti-IL-5 antibody or its antigen-binding fragment, wherein,

[0021] The anti-IL-5 antibody is an IgG1, IgG2, IgG3, or IgG4 subtype;

[0022] Preferably, the heavy chain constant region of the anti-IL-5 antibody is a human Ig gamma-1 chain C region (e.g., SEQ ID NO:9 or SEQ ID NO:15) or a human Ig gamma-4 chain C region; the light chain constant region is a human Ig kappa chain C region (e.g., SEQ ID NO:10).

[0023] Preferably, the anti-IL-5 antibody is IgG1, and according to the EU numbering system, its heavy chain constant region contains L234A and L235A mutations;

[0024] Preferably, the amino acid sequence of the heavy chain constant region of the anti-IL-5 antibody is as shown in SEQ ID NO:9 or SEQ ID NO:15.

[0025] In this invention, according to the EU numbering system, the point mutations (L234A) of leucine at position 234 to alanine and (L235A) of leucine at position 235 in the constant region of the heavy chain are also called LALA mutations.

[0026] In some embodiments of the present invention, the anti-IL-5 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein...

[0027] The amino acid sequence of the heavy chain is shown in SEQ ID NO:16 or SEQ ID NO:17; and

[0028] The amino acid sequence of the light chain is shown in SEQ ID NO:18.

[0029] In some embodiments of the present invention, the anti-IL-5 antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody or chimeric antibody.

[0030] In some embodiments of the present invention, the anti-IL-5 antibody or its antigen-binding fragment, wherein,

[0031] The anti-IL-5 antibody or its antigen-binding fragment binds to human IL-5 protein in the EC. 50 Less than or equal to 0.095 nM, less than or equal to 0.090 nM, or less than or equal to 0.085 nM;

[0032] Preferably, the anti-IL-5 antibody or its antigen-binding fragment binds to the human IL-5 protein in an EC. 50Smaller EC than Mepolizumab binds to human IL-5 protein 50 ;

[0033] Preferably, the EC 50 This was measured using the ELISA method.

[0034] In some embodiments of the present invention, the anti-IL-5 antibody or its antigen-binding fragment, wherein,

[0035] The anti-IL-5 antibody or its antigen-binding fragment binds to the cynomolgus monkey IL-5 protein in EC. 50 Less than or equal to 0.025 nM;

[0036] Preferably, the anti-IL-5 antibody or its antigen-binding fragment binds to the cynomolgus monkey IL-5 protein in the EC. 50 Smaller EC than Mepolizumab binds to cynomolgus monkey IL-5 protein 50 ;

[0037] Preferably, the EC 50 This was measured using the ELISA method.

[0038] In some embodiments of the present invention, the anti-IL-5 antibody or its antigen-binding fragment, wherein,

[0039] The anti-IL-5 antibody or its antigen-binding fragment binds to mouse IL-5 protein in EC. 50 Less than or equal to 0.095 nM or less than or equal to 0.090 nM;

[0040] Preferably, the EC 50 This was measured using the ELISA method.

[0041] In some embodiments of the present invention, the anti-IL-5 antibody or its antigen-binding fragment, wherein,

[0042] The anti-IL-5 antibody or its antigen-binding fragment can specifically bind to human IL-5 protein, cynomolgus monkey IL-5 protein and mouse IL-5 protein.

[0043] In some embodiments of the present invention, the anti-IL-5 antibody or its antigen-binding fragment, wherein,

[0044] The anti-IL-5 antibody or its antigen-binding fragment blocks the IC50 of mouse IL-5-induced TF-1 cell proliferation. 50Less than or equal to 1 nM, less than or equal to 0.1 nM, less than or equal to 0.01 nM, less than or equal to 0.001 nM, less than or equal to 0.0009 nM, less than or equal to 0.0008 nM, less than or equal to 0.0007 nM, less than or equal to 0.0006 nM, less than or equal to 0.0005 nM, less than or equal to 0.0004 nM, less than or equal to 0.0003 nM, or less than or equal to 0.0002 nM;

[0045] Preferably, the IC 50 This was measured using the CCK8 method.

[0046] In some embodiments of the present invention, the anti-IL-5 antibody or its antigen-binding fragment is capable of specifically binding to human IL-5 protein, cynomolgus monkey IL-5 protein and mouse IL-5 protein.

[0047] In some embodiments of the present invention, the anti-IL-5 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity-determining regions HCDR1 to HCDR3, and the light chain variable region comprising complementarity-determining regions LCDR1 to LCDR3, wherein:

[0048] The amino acid sequences of HCDR1 are shown in SEQ ID NO:3, HCDR2 in SEQ ID NO:4, and HCDR3 in SEQ ID NO:5; and the amino acid sequences of LCDR1 in SEQ ID NO:6, LCDR2 in SEQ ID NO:7, and LCDR3 in SEQ ID NO:8; and

[0049] The heavy chain constant region of the anti-IL-5 antibody is shown in SEQ ID NO:9 or SEQ ID NO:15, and the light chain constant region is shown in SEQ ID NO:10.

[0050] In some embodiments of the present invention, the anti-IL-5 antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region of the anti-IL-5 antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the anti-IL-5 antibody is as shown in SEQ ID NO:2; and

[0051] The heavy chain constant region of the anti-IL-5 antibody is shown in SEQ ID NO:9 or SEQ ID NO:15, and the light chain constant region is shown in SEQ ID NO:10.

[0052] In some embodiments of the present invention, the anti-IL-5 antibody or its antigen-binding fragment is described, wherein the antibody is the antibody designated Alloy-031 of the present invention.

[0053] The anti-IL-5 antibody or its antigen-binding fragment according to any one of the present invention is used for the treatment or prevention of diseases caused by elevated eosinophil levels.

[0054] Preferably, the disease is selected from one or more of the following: asthma, allergic rhinitis, chronic sinusitis, atopic dermatitis, eczema, chronic spontaneous urticaria, chronic obstructive pulmonary disease, eosinophilic granulomatous polyangiitis, eosinophilic esophagitis, arthritis, eosinophilic syndrome, Churg Strauss syndrome, bullous pemphigoid, and eosinophilic gastroenteritis.

[0055] In some embodiments of the present invention, the disease caused by eosinophil levels above normal is an allergic disease caused by eosinophil levels above normal.

[0056] In some embodiments of the present invention, the asthma is eosinophilic asthma, such as mild eosinophilic asthma, moderate eosinophilic asthma, or severe eosinophilic asthma.

[0057] Another aspect of the present invention relates to an isolated nucleic acid molecule that encodes any anti-IL-5 antibody or antigen-binding fragment thereof as described in any one of the present invention.

[0058] The present invention also relates to a recombinant vector comprising the isolated nucleic acid molecules of the present invention.

[0059] The present invention also relates to a host cell comprising the isolated nucleic acid molecules of the present invention, or comprising the recombinant vector of the present invention.

[0060] Another aspect of the present invention relates to an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein the antibody or antigen-binding fragment thereof is any one of the anti-IL-5 antibodies or antigen-binding fragments thereof described in the present invention; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is an anti-tumor chemotherapy drug.

[0061] In some embodiments of the present invention, the antibody-drug conjugate wherein the antibody or its antigen-binding fragment is linked to a small molecule drug via a linker; for example, the linker is a hydrazone bond, a disulfide bond, or a peptide bond;

[0062] Preferably, the molar ratio of the antibody or its antigen-binding fragment to the small molecule drug is 1:(2-4), 1:2, 1:3 or 1:4.

[0063] Another aspect of the present invention relates to a pharmaceutical composition comprising an effective amount of any one of the anti-IL-5 antibodies or antigen-binding fragments thereof, or any one of the antibody-drug conjugates of the present invention; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0064] Another aspect of the present invention relates to the use of any anti-IL-5 antibody or antigen-binding fragment thereof described in any one of the present invention in the preparation of a medicament for treating or preventing diseases caused by elevated eosinophil levels;

[0065] Preferably, the disease is selected from one or more of the following: asthma, allergic rhinitis, chronic sinusitis, atopic dermatitis, eczema, chronic spontaneous urticaria, chronic obstructive pulmonary disease, eosinophilic granulomatous polyangiitis, eosinophilic esophagitis, arthritis, eosinophilic syndrome, Churg Strauss syndrome, bullous pemphigoid, and eosinophilic gastroenteritis.

[0066] Another aspect of the invention relates to a method for treating or preventing diseases caused by elevated eosinophil levels, comprising the step of administering to a subject in need an effective amount of any one of the anti-IL-5 antibodies or antigen-binding fragments thereof of the invention.

[0067] Preferably, the disease is selected from one or more of the following: asthma, allergic rhinitis, chronic sinusitis, atopic dermatitis, eczema, chronic spontaneous urticaria, chronic obstructive pulmonary disease, eosinophilic granulomatous polyangiitis, eosinophilic esophagitis, arthritis, eosinophilic syndrome, Churg Strauss syndrome, bullous pemphigoid, and eosinophilic gastroenteritis.

[0068] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0069] As used in this article, the term EC 50 The half-maximal concentration (50% of maximal effect) refers to the concentration that produces a 50% maximal effect.

[0070] As used in this article, the term IC 50The half-maximal inhibitory concentration (WMC) is a drug concentration at which the activity of a biological process or a component of that process (such as an enzyme, receptor, or cell) is inhibited by 50% under certain conditions.

[0071] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of a "light" (L) chain and a "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and heavy chains also contain "D" regions of approximately 3 or more amino acids. Each heavy chain is further divided by a heavy chain variable region (V... H ) and heavy chain constant region (C H The heavy chain constant region consists of three structural domains (C). H1 C H2 and C H3 It consists of ) light chains. Each light chain is composed of a light chain variable region (V L ) and light chain constant region (C L It consists of a light chain constant region composed of a structural domain C. L Composition. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. V H and V L The region can be further subdivided into highly variable regions (called complementary determinant regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy chain / light chain pair... H and V LThese amino acids form antigen-binding sites. The allocation of amino acids to each region or domain follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda Md (1987 and 1991)), or the definitions in the IMGT numbering system, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 2009; 38(suppl_1):D301-D307. The term "antibody" is not limited to any specific method of antibody production. For example, these include, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0072] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technique first reported by Kohler et al. G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J].nature,1975;256(5517):495), but it can also be obtained using recombinant DNA technology (see USPatent 4,816,567).

[0073] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (receptor antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human antibody (e.g., mouse, rat, or rabbit) with the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the antibody's performance. For more detailed information on humanized antibodies, please refer to, for example, Jones et al., Nature 1986; 321:522-525; Reichmann et al., Nature 1988; 332:323-329; Presta, Curr. Op. Struct. Biol., 1992; 2:593-596; and Clark M. Antibody humanization: a case of the 'Emperor's new clothes'? [J]. Immunol. Today, 2000; 21(8):397-402.

[0074] As used herein, the terms “full-length antibody,” “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably to refer to an antibody having a structure substantially similar to that of a natural antibody or a heavy chain having an Fc region as defined herein.

[0075] As used in this invention, the term "Fc region" refers to an antibody fragment formed by the second and third constant regions of the first heavy chain of an antibody being bonded to the second and third constant regions of the second heavy chain via disulfide bonds.

[0076] As used herein, the term "light chain" includes full-length light chains and segments thereof with sufficient variable region sequences to confer binding specificity. Full-length light chains include a variable region domain (VL) and a constant region domain (CL). The variable region domain of a light chain is located at the amino terminus of the polypeptide. Light chains include κ chains and λ chains.

[0077] As used herein, the term "heavy chain" includes the full-length heavy chain and its segments having sufficient variable region sequences to confer binding specificity. The full-length heavy chain includes a variable region domain (VH) and three constant region domains (CH1, CH2, and CH3). The VH domain is located at the amino terminus of the polypeptide, and the CH domain is located at the carboxyl terminus, with CH3 being closest to the carboxyl terminus of the polypeptide. The heavy chain can have any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 isotypes), IgA (including IgA1 and IgA2 isotypes), IgM, and IgE.

[0078] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety.” See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody antibodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0079] As used in this invention, the term "Fab fragment" consists of a light chain and a CH1 group, as well as a variable region of a heavy chain. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0080] As used in this invention, the term "Fab' fragment" comprises a portion of a light chain and a portion of a heavy chain (containing a VH domain and a CH1 domain, as well as a portion of the region between the CH1 and CH2 domains) so that interchain disulfide bonds can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0081] As used in this invention, the term "F(ab')2 fragment" comprises two light chains and two heavy chains containing portions of a constant region between CH1 and CH2 domains, so as to form interchain disulfide bonds between the two heavy chains. The F(ab')2 fragment thus consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0082] As used in this invention, the term "Fv region" includes variable regions from both the heavy and light chains, but lacks constant regions.

[0083] As used in this invention, the term “Fd” fragment refers to an antibody fragment consisting of VH and CH1 domains (Ward et al., Nature 341:544-546 (1989)).

[0084] As used in this invention, the term “dAb” segment (Ward et al., Nature 341:544-546 (1989)) consists of a VH domain.

[0085] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" entity, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated one. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0086] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0087] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells, or human cells.

[0088] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D () binds to the antigen.

[0089] As used in this article, the term "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies have an equilibrium dissociation constant of less than approximately 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D K binds to antigens (e.g., IL-5 protein). K can be measured using methods known to those skilled in the art. D For example, measurements can be taken using a Fortebio molecular interaction analyzer.

[0090] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; and the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0091] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0092] As used herein, the term "effective amount" means an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for disease prevention means an amount sufficient to prevent, stop, or delay the onset of disease; an effective amount for disease treatment means an amount sufficient to cure or at least partially stop the disease and its complications in patients who already have the disease.

[0093] Beneficial effects of the invention

[0094] The monoclonal antibody of the present invention can specifically bind to IL-5 and can very effectively block the binding of human IL-5 and IL-5 receptor, effectively reducing the level of eosinophils. Attached Figure Description

[0095] Figure 1A: Curve of binding activity between anti-IL-5 monoclonal antibody and human IL-5 protein.

[0096] Figure 1B: Curve of binding activity between anti-IL-5 monoclonal antibody and cynomolgus monkey IL-5 protein.

[0097] Figure 1C: Curve of binding activity between anti-IL-5 monoclonal antibody and mouse IL-5 protein.

[0098] Figure 2A: Curve of the blocking activity of anti-IL-5 monoclonal antibody against the binding of human IL-5 protein to CHO-huIL-5Rα cells.

[0099] Figure 2B: Curve of blocking activity of anti-IL-5 monoclonal antibody against the binding of mouse IL-5 protein to CHO-muIL-5Rα cells.

[0100] Figure 3A: Curve of the blocking activity of anti-IL-5 monoclonal antibody against human IL-5-induced TF-1 cell proliferation.

[0101] Figure 3B: Curve of the blocking activity of anti-IL-5 monoclonal antibody against the proliferation of mouse IL-5-induced TF-1 cells.

[0102] Figure 4A: Bar chart showing the effect of anti-IL-5 monoclonal antibody on the number of CD45+ cells in mouse lung tissue in a mouse asthma model.

[0103] Figure 4B: Bar chart showing the effect of anti-IL-5 monoclonal antibody on the number of eosinophils in mouse lung tissue in a mouse asthma model.

[0104] Figure 4C: Bar chart showing the effect of anti-IL-5 monoclonal antibody on serum IgE levels in a mouse asthma model.

[0105] Figure 4D: Pathological changes after administration of anti-IL-5 monoclonal antibody in a mouse asthma model. The six images in each group represent the results of six mouse samples from the same group.

[0106] Information on some of the sequences involved in this invention is provided in Table 1 below. The CDRs are numbered according to the Kabat numbering system.

[0107] Table 1: Partial sequences involved in this invention Detailed Implementation

[0108] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0109] Example 1: Construction of antibody library

[0110] Five humanized mice (purchased from Alloy, USA) were immunized with an equal volume of 1 mg human IL-5 antigen (purchased from AcroBiosystems) and Freund's adjuvant, once a week for a total of four immunizations, stimulating B cells to express antigen-specific antibodies. After the four immunizations, the spleens of the mice were harvested, and total RNA was extracted using Trizol RNA extraction reagent (purchased from Invitrogen). Total cDNA from fully humanized mice was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen). The IgG sequence was amplified from the cDNA by the first round of PCR. The PCR products were subjected to agarose gel electrophoresis, and the target fragment at 400-500 bp was recovered by gel excision. The first round of PCR products were used as a template for the second round of sequence amplification, adding homologous arms to the heavy and light chain genes. Finally, the target fragment was recovered using a PCR purification kit (purchased from QIAGEN).

[0111] The linearized yeast display vector and the second-round PCR products were mixed and electroporated into *Saccharomyces cerevisiae* (purchased from ATCC) to construct a fully human anti-IL-5 antibody library from five animals. The library size was determined to be 3 × 10⁻⁶. 7 .

[0112] Example 2: Screening of anti-IL-5 monoclonal antibodies

[0113] Dissolve human IL-5 protein (purchased from AcroBiosystems) in an appropriate volume of double-distilled water. Following the instructions of the Biotin-labeled reagent kit (purchased from Thermo), dissolve biotin and mix it with the protein solution. Incubate at 4°C for 2 hours. Remove excess biotin using a desalting column (purchased from Thermo). Desalting column pretreatment and sample collection were performed according to the product instructions.

[0114] The fully human antibody library constructed in Example 1 was seeded in SD-CAA amplification medium (1 L of SD-CAA amplification medium contains 6.7 g YNB, 5 g casein amino acids, 13.62 g Na2HPO4·12H2O, 7.44 g NaH2PO4, and 2% glucose) and cultured overnight at 30°C and 225 rpm. An appropriate amount of yeast cells was taken, the medium was removed by centrifugation, and the yeast cells were resuspended in SD-CAA induction medium and induced overnight. The concentration of the induced library was measured. An appropriate amount of yeast cells was taken, the medium was removed by centrifugation, and the yeast cells were resuspended in 50 mL of washing buffer. The supernatant was removed by centrifugation. The yeast cells were resuspended in 10 mL of washing buffer.

[0115] Add biotin-labeled IL-5 protein (final concentration 100 nM), incubate at room temperature for 30 min, collect yeast cells by centrifugation, and wash the yeast three times with 50 mL wash buffer. Resuspend the yeast cells in 5 mL wash buffer and add 200 μL of SA magnetic beads (purchased from Miltenyi), incubate upside down for 10 min. Wash the yeast and magnetic bead mixture three times with wash buffer, and add the mixture to an LS column (purchased from Miltenyi). Place the LS column on a magnetic rack and wash with wash buffer to remove non-specifically bound yeast cells. Remove the column from the magnetic rack and add wash buffer to elute the yeast. Centrifuge the eluted yeast and transfer it to 200 mL of SD-CAA amplification medium for amplification.

[0116] Yeast cells enriched by MACS (Magnetic Activated Cell Sorting) were seeded in SD-CAA amplification medium and cultured overnight at 30°C and 225 rpm in shake flasks. The yeast cells were resuspended in induction medium and induced overnight. A 1:500 dilution of anti-c-Myc mouse antibody (Thermo) and 100 nM biotin-labeled IL-5 antigen were added, and the mixture was incubated at room temperature for 10 min. The yeast cells were washed three times with PBS, and a 1:500 dilution of goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 (Invitrogen) and streptavidin APC-conjugated fluorescent antibody (Invitrogen) were added, and the mixture was incubated at 4°C in the dark for 15 min. The cells were resuspended in PBS and sorted using a BD FACSAria III flow cytometer to obtain yeast cells with high binding capacity to the IL-5 antigen.

[0117] Yeast culture with high binding capacity to IL-5 antigen, obtained by enrichment with MACS and FACS, was cultured overnight at 30°C and 225 rpm in SD-CAA amplification medium. The amplified single clones were treated with 0.1% SDS, centrifuged, and the supernatant was used as a template for PCR amplification. The PCR products were sent for sequencing to obtain the gene sequence.

[0118] At least eight candidate anti-IL-5 monoclonal antibodies were obtained and named Alloy-027, Alloy-031, Alloy-034, Alloy-046, Alloy-057, Alloy-073, Alloy-080, and Alloy-081, respectively. The amino acid sequences of the heavy chain variable region VH, light chain variable region VL, the three complementarity-determining regions HCDR1-HCDR3 of the heavy chain variable region, and the three complementarity-determining regions LCDR1-LCDR3 of the light chain variable region of Alloy-031 are shown in Table 1 as SEQ ID NOs:1-8. The CDRs are labeled according to the Kabat numbering system.

[0119] Example 3: Construction, expression, and purification of anti-IL-5 monoclonal antibody

[0120] The nucleotide sequences encoding the eight anti-IL-5 antibody heavy chain variable regions were ligated with the nucleotide sequences encoding the human hIgG1 heavy chain constant region (containing the LALA mutation, SEQ ID NO:9), and constructed into the EcoRI / Not I double-digested linearized pCDNA3.1 vector using homologous recombinase (purchased from Vazyme). The nucleotide sequences encoding the light chain variable regions were ligated with the nucleotide sequences encoding the human kappa light chain constant region (SEQ ID NO:10), and constructed into the EcoRI / Xhol I double-digested linearized pCDNA3.1 vector. The homologous recombination products were transformed into Top10 competent cells, plated on ampicillin-resistant plates, cultured overnight at 37°C, and single clones were picked for sequencing and plasmid extraction.

[0121] Using ExpiCHO TMThe expression system kit (Thermo) was used to co-transfect the extracted heavy and light chain plasmids into Expi-CHO cells according to the product instructions. After 5 days of cell culture, the supernatant was collected and the target protein was purified using protein A magnetic beads (GenScript). The magnetic beads were resuspended in an appropriate volume of binding buffer (PBS + 0.1% Tween 20, pH 7.4) (1-4 times the volume of the magnetic beads) and added to the sample to be purified. The sample was incubated at room temperature for 1 hour with gentle shaking. The sample was then placed on a magnetic rack (Suzhou Beaver Biotechnology), the supernatant was discarded, and the magnetic beads were washed three times with binding buffer. Add elution buffer (0.1M sodium citrate, pH 3.2) at 3-5 times the volume of the magnetic beads, shake at room temperature for 5-10 min, place back on the magnetic rack, collect the elution buffer, and transfer it to a collection tube containing neutralization buffer (1M Tris, pH 8.54). Mix well to obtain the target proteins, namely the candidate anti-IL-5 monoclonal antibodies Alloy-027, Alloy-031, Alloy-034, Alloy-046, Alloy-057, Alloy-073, Alloy-080, and Alloy-081.

[0122] Example 4: Affinity assay of anti-IL-5 monoclonal antibody

[0123] ForteBio affinity assays were performed according to existing methods (Estep, P et al., Solution-based high-throughput measurement of antibody-antigen affinity and epitope grading, MAbs, 2013.5(2):p.270-8). In short, the AHC sensor was equilibrated offline in analytical buffer for 30 min, then online for 120 s to establish a baseline, and the purified antibody obtained as described above was loaded online onto the AHC sensor. The sensor was then incubated in 100 nM IL-5 antigen for 200 sec, followed by dissociation in PBS for 600 sec. Kinetic analysis was performed using a 1:1 binding model.

[0124] Table 2: Affinity of anti-IL-5 monoclonal antibodies

[0125] The results are shown in Table 2.

[0126] The results showed that the monovalent affinity of anti-IL-5 monoclonal antibodies Alloy-031, Alloy-080, and Alloy-081 for human IL-5 protein was comparable to that of the control antibody Mepolizumab. Clones Alloy-027 and Alloy-046 showed superior monovalent affinity for human IL-5 protein compared to Mepolizumab. The monovalent affinity of anti-IL-5 monoclonal antibody Alloy-046 for cynomolgus monkey IL-5 protein was comparable to that of Mepolizumab.

[0127] Example 5: Determination of the binding of anti-IL-5 monoclonal antibody to human IL-5 protein, cynomolgus monkey IL-5 protein, and mouse IL-5 protein.

[0128] Dilute human IL-5 (from AcroBiosystems), cynomolgus monkey IL-5 (from Sino Biological), and mouse IL-5 protein (from AcroBiosystems) to 1 μg / mL using 1x ELISA coating working solution, and coat 96-well ELISA plates with 100 μL / well. Incubate overnight at 4°C. Remove the coated ELISA plates and discard the coating solution. Add 250 μL / well of ELISA wash buffer, incubate for 30 seconds, discard the wash buffer, and repeat 3 times. Add 250 μL / well of ELISA blocking buffer and incubate at room temperature for 2 hours. Serially dilute the purified antibody with ELISA working solution, adding 100 μL / well to the blocked ELISA plates and incubate at room temperature for 1 hour. Dilute HRP anti-huIgG (from Southern Biotech) 1:10000 with ELISA working solution for later use. Discard the reaction solution from the ELISA plates after the binding reaction is complete. Add 250 μL / well of ELISA washing buffer, let stand for 30 seconds, discard the washing buffer, and repeat 3 times. Add 100 μL / well of HRP working solution and incubate at room temperature for 1 hour. For the ELISA plate after the reaction with HRP working solution, discard the reaction solution, add 250 μL / well of ELISA washing buffer, let stand for 30 seconds, discard the washing buffer, and repeat 3 times. Add 100 μL / well of TMB single-component colorimetric solution and react at room temperature for 1-3 minutes. For the ELISA plate after the colorimetric reaction, add 50 μL / well of ELISA stop solution to terminate the reaction. Take the ELISA plate after the reaction has been terminated and read the absorbance value at 450 nm within 5 minutes of termination, record the value, and perform data processing.

[0129] Table 3: Binding of anti-IL-5 monoclonal antibodies to human / cynomolgus monkey / mouse IL-5 protein

[0130] Notes: ND = Not detected.

[0131] The results are shown in Figures 1A to 1B and Table 3. The results indicate that the anti-IL-5 monoclonal antibodies Alloy-027, Alloy-031, Alloy-034, Alloy-046, Alloy-057, Alloy-073, Alloy-080, and Alloy-081 exhibited good binding activity to both human IL-5 protein and cynomolgus monkey IL-5 protein, and showed comparable or even superior binding activity compared to the control antibody Mepolizumab.

[0132] As shown in Figure 1C and Table 3, the results showed that the anti-IL-5 monoclonal antibodies Alloy-027, Alloy-031, Alloy-034, Alloy-046, Alloy-057, Alloy-073, Alloy-080, and Alloy-081 all exhibited good binding activity with mouse IL-5 protein.

[0133] Example 6: Anti-IL-5 monoclonal antibody blocks the binding of IL-5 to IL-5Rα-overexpressing CHO cells

[0134] Nucleotide sequences encoding the full-length human IL-5Rα amino acid sequence (SEQ ID NO:11) and the full-length mouse IL-5Rα amino acid sequence (SEQ ID NO:12) were inserted into the multiple cloning site of the pCHO1.0 vector (purchased from Thermo) using double digestion with AvrII and PacI, respectively. After plasmid linearization, the sequences were processed using Freestyle... TM MAX reagent (purchased from Invitrogen) was used to transfect CHO cells, and the transfection procedure was performed according to the manufacturer's instructions. After transfection, the cells were selected by adding methotrexate (purchased from Sigma) and puromycin (purchased from Invitrogen) under pressure to obtain CHO cells stably overexpressing human IL-5Rα (CHO-huIL-5Rα cells) and CHO cells overexpressing mouse IL-5Rα (CHO-muIL-5Rα cells).

[0135] The purified monoclonal antibody was diluted with 1×PBS, and 3-fold dilutions were performed at 400 nM for a total of 12 spots. 60 μL of the diluted sample was added to each well of a new 96-well flow cytometry plate. Then, 60 μL of biotinylated human IL-5 protein (purchased from AcroBiosystems) diluted with 1×PBS was added to each well, and the mixture was incubated at 4°C in the dark for 30 minutes. The CHO-huIL-5Rα cells were then expanded to a density of 2×10⁶ cells / well. 6Cells / mL, 100 μL / well added to a 96-well flow cytometry plate, centrifuged and the supernatant discarded. Add 100 μL / well of the incubated sample to the centrifuged cell wells, mix well, and incubate at 4°C in the dark for 30 minutes. Wash twice with 1×PBS, add 100 μL / well of SAPE antibody (Thermo) diluted 100-fold with 1×PBS, and incubate at 4°C for 30 minutes. Wash twice with 1×PBS, resuspend cells in 100 μL / well of 1×PBS, and analyze on a CytoFlex flow cytometer, calculating the corresponding median fluorescence intensity.

[0136] The purified monoclonal antibody was diluted with 1×PBS, and 3-fold dilutions were performed at 400 nM for a total of 12 spots. 60 μL of the diluted sample was added to each well of a new 96-well flow cytometry plate. Then, 60 μL of biotinylated mouse IL-5 protein (purchased from Kactus) diluted with 1×PBS was added to each well, and the mixture was incubated at 4°C in the dark for 30 minutes. The expanded CHO-muIL-5Rα cell density was adjusted to 2×10⁶ cells / well. 6 Cells / mL, 100 μL / well added to a 96-well flow cytometry plate, centrifuged and the supernatant discarded. Add 100 μL / well of the incubated sample to the centrifuged cell wells, mix well, and incubate at 4°C in the dark for 30 minutes. Wash twice with 1×PBS, add 100 μL / well of SAPE antibody (Thermo) diluted 100-fold with 1×PBS, and incubate at 4°C for 30 minutes. Wash twice with 1×PBS, resuspend cells in 100 μL / well of 1×PBS, and analyze on a CytoFlex flow cytometer, calculating the corresponding median fluorescence intensity.

[0137] Table 4: Anti-IL-5 monoclonal antibodies block the binding of IL-5 to IL-5Rα-overexpressing CHO cells Notes: ND = Not detected.

[0138] The results are shown in Figure 2A and Table 4. The results indicate that the anti-IL-5 monoclonal antibodies Alloy-027, Alloy-031, Alloy-034, Alloy-046, Alloy-057, Alloy-073, Alloy-080, and Alloy-081 can completely block the binding of human IL-5 protein to CHO-huIL-5Rα cells. Among them, Alloy-046 and Alloy-080 have comparable blocking activity to the control antibody MepoLizumab.

[0139] As shown in Figure 2B and Table 4, the results showed that the anti-IL-5 monoclonal antibodies Alloy-027, Alloy-031, Alloy-034, Alloy-046, Alloy-057, Alloy-073, and Alloy-080 could completely block the binding of mouse IL-5 protein to CHO-muIL-5Rα cells, among which antibodies Alloy-027, Alloy-031, and Alloy-034 showed strong blocking activity.

[0140] Example 7: Anti-IL-5 monoclonal antibody blocks the proliferation of human IL-5 and mouse IL-5-induced TF-1 cells.

[0141] TF-1 cells (purchased from ATCC) were centrifuged, the supernatant was discarded, and the cells were washed once with 50 mL of 1×PBS. The cells were then resuspended in RPMI 1640 containing 10% FBS and the cell density was adjusted to 2×10⁻⁶ cells / mL. 5 100 μL / well of antibody was added to a 96-well cell culture plate. 50 μL / well of antibody was diluted with RPMI 1640 medium containing 10% FBS. Human IL-5 protein (purchased from AcroBiosystems) and mouse IL-5 protein (purchased from AcroBiosystems) were diluted with RPMI 1640 medium containing 10% FBS, and 50 μL / well of each were added to the same 96-well plate. The plates were incubated at 37°C with 5% CO2 for 3 days. 20 μL of CCK8 solution (purchased from Dojindo) was added to each well, and the plates were incubated at room temperature for 8 hours. The absorbance at 450 nm was then measured.

[0142] Table 5: Anti-IL-5 monoclonal antibody blocks IL-5-induced TF-1 cell proliferation

[0143] The results are shown in Figure 3A and Table 5. The results indicate that the anti-IL-5 monoclonal antibodies Alloy-027, Alloy-031, Alloy-034, Alloy-046, Alloy-057, Alloy-073, Alloy-080, and Alloy-081 can completely block the proliferation of TF-1 cells induced by human IL-5 protein. Among them, clones Alloy-031, Alloy-046, and Alloy-073 have comparable blocking activity to the control antibody Mepolizumab, while clone Alloy-027 shows superior blocking activity compared to Mepolizumab.

[0144] As shown in Figure 3B and Table 5, the results indicate that the anti-IL-5 monoclonal antibodies Alloy-027, Alloy-031, Alloy-034, Alloy-046, Alloy-057, Alloy-073, and Alloy-080 can completely block the proliferation of mouse IL-5 protein-induced TF-1 cells. Among them, antibodies Alloy-027 and Alloy-031 have the strongest activity in blocking TF-1 cell proliferation.

[0145] Example 8: Efficacy of anti-IL-5 monoclonal antibody in an ovalbumin-induced mouse asthma model

[0146] The monoclonal antibody Alloy-031 exhibits good affinity for mouse IL-5 and can block the binding of mouse IL-5 to CHO-muIL-5Rα cells. Therefore, this study used an ovalbumin (purchased from Sigma) to induce an asthma model in C57BL / 6 wild-type mice (purchased from Biocytogen) to test the efficacy of the monoclonal antibody Alloy-031.

[0147] First, mice were sensitized by subcutaneous injection of 0.2 mL of 500 μg / mL ovalbumin or 1×PBS (vehicle) at multiple sites. Six mice were injected per group, once a week for three weeks. One day before nebulization challenge, mice were intraperitoneally injected with an equal volume of 1×PBS or 24 mg / kg Alloy-031. Starting in the fourth week, mice were challenged by nebulization with 2.5% m / v ovalbumin or 1×PBS for six consecutive days. On the seventh day, blood was collected from the orbital fossa, and the IgE content in the mouse serum was measured using an ELISA kit (Thermo). Then, the mice were anesthetized, and the trachea was exposed through the neck. The bronchoalveolar lavage was performed with an equal volume of pre-cooled PBS, and the bronchoalveolar lavage fluid was collected. The cell pellet from the centrifuged bronchoalveolar lavage fluid was analyzed by flow cytometry to detect the number of CD45-positive immune cells and eosinophils. Simultaneously, mouse lung tissue was collected, fixed with 4% paraformaldehyde, and then stained with H&E.

[0148] The flow cytometry results are shown in Figures 4A and 4B. The results showed that the antibody Alloy-031 significantly reduced the number of CD45+ leukocytes in the bronchoalveolar lavage fluid of mice sensitized by OVA. The number of eosinophils in the bronchoalveolar lavage fluid of mice in the OVA model group was abnormally high, while the anti-IL-5 antibody Alloy-031 of this invention significantly reduced the number of eosinophils.

[0149] The ELISA results are shown in Figure 4C. The results showed that, compared with the OVA model group, the concentration of total IgE in the serum of mice in the Alloy-031 antibody-treated group was significantly lower, and the IgE content was basically close to that of the blank control group.

[0150] The pathological staining results are shown in Figure 4D. The results showed that, compared with the OVA model group, the peribronchial inflammatory cell infiltration in the lung tissue of mice in the Alloy-031 antibody-treated group was significantly reduced, and the production of mucus near the bronchi was also significantly reduced.

[0151] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

Claims

1. An anti-IL-5 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising complementarity determining regions HCDR1 to HCDR3 and a light chain variable region comprising complementarity determining regions LCDR1 to LCDR3, wherein: the amino acid sequence of HCDR1 is set forth in SEQ ID NO: 3, the amino acid sequence of HCDR2 is set forth in SEQ ID NO: 4, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO: 5, and the amino acid sequence of LCDR1 is set forth in SEQ ID NO: 6, the amino acid sequence of LCDR2 is set forth in SEQ ID NO: 7, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:

8.

2. The anti-IL-5 antibody or antigen-binding fragment thereof of claim 1, wherein: the amino acid sequence of the heavy chain variable region of the anti-IL-5 antibody is set forth in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region of the anti-IL-5 antibody is set forth in SEQ ID NO:

2.

3. The anti-IL-5 antibody or antigen-binding fragment thereof of any one of claims 1-2, wherein: the antibody comprises a non-CDR region, and the non-CDR region is from a species other than murine, e.g., from a human antibody; preferably, the anti-IL-5 antibody is a fully human antibody; preferably, the anti-IL-5 antibody is a fully human monoclonal antibody.

4. The anti-IL-5 antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein: the anti-IL-5 antibody is of the IgGl, IgG2, IgG3, or IgG4 subtype; preferably, the heavy chain constant region of the anti-IL-5 antibody is a human Ig gamma-1 chain C region or a human Ig gamma-4 chain C region; the light chain constant region is a human Ig kappa chain C region; preferably, the anti-IL-5 antibody is of the IgGl subtype, and its heavy chain constant region contains L234A and L235A mutations according to the EU numbering system; preferably, the amino acid sequence of the heavy chain constant region of the anti-IL-5 antibody is set forth in SEQ ID NO: 9 or SEQ ID NO: 15, and / or the light chain constant region is set forth in SEQ ID NO:

10.

5. The anti-IL-5 antibody or antigen-binding fragment thereof of any one of claims 1-4, comprising a heavy chain and a light chain, wherein: the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 16 or SEQ ID NO: 17; and the amino acid sequence of the light chain is set forth in SEQ ID NO:

18.

6. The anti-IL-5 antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody is selected from the group consisting of a Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody, humanized antibody, or chimeric antibody. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 6. The anti-IL-5 antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein, ​ 7. The anti-IL-5 antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein, The EC50of the anti-IL-5 antibody or antigen-binding fragment thereof binding to human IL-5 protein is less than or equal to 0.095 nM, less than or equal to 0.090 nM, or less than or equal to 0.085 nM. 50 The EC50of the anti-IL-5 antibody or antigen-binding fragment thereof binding to human IL-5 protein is less than or equal to 0.095 nM, less than or equal to 0.090 nM, or less than or equal to 0.085 nM. Preferably, the anti-IL-5 antibody or its antigen-binding fragment binds to the human IL-5 protein in an EC. 50 Smaller EC than Mepolizumab binds to human IL-5 protein 50 ; Preferably, the EC 50 is measured by the ELISA method.

8. The anti-IL-5 antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein, EC50of the anti-IL-5 antibody or antigen-binding fragment thereof binding to cynomolgus IL-5 protein 50 less than or equal to 0.025 nM; Preferably, the anti-IL-5 antibody or its antigen-binding fragment binds to the cynomolgus monkey IL-5 protein in the EC. 50 Smaller EC than Mepolizumab binds to cynomolgus monkey IL-5 protein 50 ; Preferably, the EC 50 is measured by the ELISA method.

9. The anti-IL-5 antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein, The EC50of the anti-IL-5 antibody or antigen-binding fragment thereof binding to mouse IL-5 protein 50 less than or equal to 0.095 nM or less than or equal to 0.090 nM; Preferably, the EC 50 is measured by the ELISA method.

10. The anti-IL-5 antibody or antigen-binding fragment thereof of any one of claims 1-9, wherein, the anti-IL-5 antibody or antigen-binding fragment thereof is capable of specifically binding to a human IL-5 protein, a cynomolgus IL-5 protein, and a mouse IL-5 protein.

11. The anti-IL-5 antibody or antigen-binding fragment thereof of any one of claims 1-10, wherein, The anti-IL-5 antibody or antigen-binding fragment thereof blocks mouse IL-5-induced TF-1 cell proliferation IC 50 less than or equal to 1 nM, less than or equal to 0.1 nM, less than or equal to 0.01 nM, less than or equal to 0.001 nM, less than or equal to 0.0009 nM, less than or equal to 0.0008 nM, less than or equal to 0.0007 nM, less than or equal to 0.0006 nM, less than or equal to 0.0005 nM, less than or equal to 0.0004 nM, less than or equal to 0.0003 nM, or less than or equal to 0.0002 nM; Preferably, said IC 50 is measured by CCK8 method.

12. An isolated nucleic acid molecule encoding the anti-IL-5 antibody or antigen-binding fragment thereof of any one of claims 1-11.

13. A recombinant vector comprising the isolated nucleic acid molecule of claim 12.

14. A host cell comprising the isolated nucleic acid molecule of claim 12, or comprising the recombinant vector of claim 13.

15. An antibody drug conjugate comprising an antibody or antigen binding fragment thereof and a small molecule drug, wherein, the antibody or antigen-binding fragment thereof is the anti-IL-5 antibody or antigen-binding fragment thereof of any one of claims 1-11; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapeutic drug.

16. The antibody drug conjugate of claim 15, wherein, the antibody or antigen-binding fragment thereof is linked to the small molecule drug via a linker; for example, the linker is a hydrazone bond, a disulfide bond, or a peptide bond; preferably, the molar ratio of the antibody or antigen-binding fragment thereof to the small molecule drug is 1:(2-4).

17. A pharmaceutical composition comprising an effective amount of the anti-IL-5 antibody or antigen-binding fragment thereof of any one of claims 1-11 or the antibody-drug conjugate of any one of claims 15-16; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

18. Use of the anti-IL-5 antibody or antigen-binding fragment thereof of any one of claims 1-11 in the manufacture of a medicament for treating or preventing a disease caused by elevated levels of eosinophils; preferably, the disease is one or more selected from the group consisting of asthma, allergic rhinitis, chronic rhinosinusitis, atopic dermatitis, eczema, chronic spontaneous urticaria, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, eosinophilic esophagitis, arthritis, hypereosinophilic syndrome, Churg Strauss syndrome, bullous pemphigoid, and eosinophilic gastroenteritis.

19. The anti-IL-5 antibody or antigen-binding fragment thereof of any one of claims 1-11 for use in treating or preventing a disease caused by elevated levels of eosinophils. Preferably, the disease is one or more selected from asthma, allergic rhinitis, chronic rhinosinusitis, atopic dermatitis, eczema, chronic spontaneous urticaria, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, eosinophilic esophagitis, arthritis, hypereosinophilic syndrome, Churg Strauss syndrome, bullous pemphigoid, and eosinophilic gastroenteritis.

20. A method of treating or preventing a disease caused by elevated levels of eosinophils above normal, comprising the step of administering to a subject in need thereof an effective amount of an anti-IL-5 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11; Preferably, the disease is one or more selected from asthma, allergic rhinitis, chronic rhinosinusitis, atopic dermatitis, eczema, chronic spontaneous urticaria, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, eosinophilic esophagitis, arthritis, hypereosinophilic syndrome, Churg Strauss syndrome, bullous pemphigoid, and eosinophilic gastroenteritis.

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