Anti-IL5 nanobodies and their applications

Abstract

The present invention provides an IL5 nanobody and its applications. Specifically, the present invention provides an IL5 nanobody, a coding sequence encoding the nanobody, a corresponding expression vector, and a host cell capable of expressing the nanobody, as well as a method for producing the nanobody. The nanobody can specifically recognize human and cynomolgus monkey IL5 but cannot recognize mouse IL5. It has good binding activity and good IL5 / IL5R blocking activity, the blocking activity of which is significantly superior to that of the control antibody Nucala. It can effectively inhibit IL5-induced TF-1 cell proliferation with a more potent inhibitory activity than the control antibody Nucala. The expression yield in Pichia pastoris reaches 13 g / L, and the purity of the target protein expression supernatant is high.

Description

[Technical field]

[0001] The present invention relates to the technical field of biomedicine or biopharmaceuticals, more specifically to anti-IL5 nanobodies and their applications. [Background technology]

[0002] Eosinophils play an important role in defending the body against infection. However, in some individuals, elevated eosinophil levels may contribute to inflammation and may be involved in the development of certain inflammatory diseases. IL5 is the most selective for eosinophils among known cytokines and can regulate eosinophil growth, differentiation, recruitment, activation and survival. Its receptor, IL5Rα, is highly expressed on eosinophils and plays an important role in the clearance of allergens in blood and tissues by eosinophils. IL5 is currently considered to be one of the main drivers of the Th2 pathway, and after binding to its receptor, IL5 further activates the downstream JAK-STAT signaling pathway.

[0003] In recent years, IL5, as an important target in the research and development of immunomodulatory drugs, has played an important role in the field of immunotherapy. Three monoclonal antibody drugs targeting IL5 / IL5Rα have been approved for sale worldwide, two of which are IL5 antibodies (Mepolizumab by GSK and Reslizumab by Teva) and one is an IL5Rα antibody (Benralizumab by AstraZeneca), bringing new treatment options to patients. IL5 humanized monoclonal antibody injection (610) from Chinese local pharmaceutical company 3S Guojian and IL5 antibody (SHR-1703) from HENGRUI have also been approved for clinical trials by the National Medical Products Administration. The world's first approved IL5 monoclonal antibody drug, i.e. Mepolizumab from GSK, has been approved in numerous clinical studies targeting indications such as moderate asthma, Eosinophilic Granulomatous Polyangiitis (EGPA), Chronic Obstructive Pulmonary Disease (COPD), Chronic Rhinosinusitis with Nasal Polyposis (CRSwNP), Severe Eosinophilic Syndrome, Severe Atopic Dermatitis, Severe Bilateral Nasal Polyps, etc.

[0004] To date, nanobody drugs targeting IL5 have not yet been commercially available, and nanobody (Nb), i.e. heavy chain nanobody VHH (variable domain of heavy chain of heavy-chain antibody) - a heavy chain antibody (HCAb) that naturally lacks light chains in the body of camels, and a nanobody consisting of only one heavy chain variable domain obtained by cloning its variable domain, are the smallest unit currently available with complete function and stable binding to antigen. Nanobody has characteristics such as high stability, good water solubility, easy humanization, high targeting, strong penetration, etc., and plays a greater role than imaginable in immune experiments, diagnosis and therapy. Nanobody is gradually emerging in the new generation of antibody diagnosis and therapy.

[0005] Therefore, the development of new anti-IL5 nanobodies is expected to have good clinical application prospects, fill the market gap of IL5 nanobody drugs, and benefit patients. Summary of the Invention [Problem to be solved by the invention]

[0006] It is an object of the present invention to provide anti-IL5 nanobodies and applications thereof. [Means for solving the problem]

[0007] A first aspect of the invention provides an anti-IL5 Nanobody, said Nanobody being capable of specifically binding to IL5, wherein the complementarity determining regions (CDRs) of the VHH chain in said Nanobody comprise: (1) CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:3; (2) CDR1 as shown in SEQ ID NO: 10, CDR2 as shown in SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12; (3) CDR1 as set forth in SEQ ID NO: 19, CDR2 as set forth in SEQ ID NO: 20, and CDR3 as set forth in SEQ ID NO: 21, and (4) One or more selected from the group consisting of CDR1 shown in SEQ ID NO:28, CDR2 shown in SEQ ID NO:29, and CDR3 shown in SEQ ID NO:30.

[0008] In another preferred example, any one of the amino acid sequences in the above amino acid sequences further includes a derivative sequence that optionally has at least one (e.g., 1 to 3, preferably 1 to 2, more preferably 1) amino acid added, deleted, modified and / or substituted and retains the ability to bind to IL5.

[0009] In another preferred embodiment, the VHH chain of said anti-IL5 Nanobody further comprises a framework region FR, said framework region FR comprising: (1) FR1 as set forth in SEQ ID NO:4, FR2 as set forth in SEQ ID NO:5, FR3 as set forth in SEQ ID NO:6, and FR4 as set forth in SEQ ID NO:7; (2) FR1 as set forth in SEQ ID NO: 13, FR2 as set forth in SEQ ID NO: 14, FR3 as set forth in SEQ ID NO: 15, and FR4 as set forth in SEQ ID NO: 16; (3) FR1 as set forth in SEQ ID NO: 22, FR2 as set forth in SEQ ID NO: 23, FR3 as set forth in SEQ ID NO: 24, and FR4 as set forth in SEQ ID NO: 25; (4) FR1 as set forth in SEQ ID NO: 31, FR2 as set forth in SEQ ID NO: 32, FR3 as set forth in SEQ ID NO: 33, and FR4 as set forth in SEQ ID NO: 34; (5) FR1 as set forth in SEQ ID NO: 37, FR2 as set forth in SEQ ID NO: 38, FR3 as set forth in SEQ ID NO: 39, and FR4 as set forth in SEQ ID NO: 40, and (6) One or more selected from the group consisting of FR1 shown in SEQ ID NO: 43, FR2 shown in SEQ ID NO: 44, FR3 shown in SEQ ID NO: 45, and FR4 shown in SEQ ID NO: 46.

[0010] In another preferred embodiment, said CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR4.

[0011] In another preferred example, the amino acid sequence of the VHH chain of said anti-IL5 Nanobody is selected from the group consisting of SEQ ID NO:8, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47, or a combination thereof.

[0012] In another preferred embodiment, said anti-IL5 Nanobody comprises a monomer, a bivalent (bivalent antibody), a tetravalent (tetravalent antibody) and / or a multivalent (multivalent antibody).

[0013] In another preferred embodiment, said anti-IL5 Nanobody comprises two VHH chains having the amino acid sequences as shown in SEQ ID NO:41 and / or SEQ ID NO:47, preferably connected by a connecting peptide between said VHH chains.

[0014] In another preferred embodiment, the connecting peptide is a S b ) x -(G m S n ) y where a, b, m, n, x, y=0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (preferably, a=4 and b=1, m=3 and n=1).

[0015] In another preferred embodiment, the sequence of the connecting peptide is (G4S)4.

[0016] In another preferred embodiment, said anti-IL5 Nanobody is capable of discriminating between two different IL5 epitopes.

[0017] In another preferred embodiment, said nanobody comprises a humanized antibody, a camelid antibody, or a chimeric antibody.

[0018] A second aspect of the invention provides an anti-IL5 antibody, which is an antibody directed against an epitope of Interleukin 5 (IL5), comprising an anti-IL5 Nanobody according to the first aspect of the invention.

[0019] In another preferred embodiment, the anti-IL5 antibody comprises a monomer, a bivalent entity (bivalent antibody), a tetravalent entity (tetravalent antibody), and / or a multivalent entity (multivalent antibody).

[0020] In another preferred embodiment, the anti-IL5 antibody comprises one or more VHH chains having an amino acid sequence as shown in SEQ ID NO:8, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:35, SEQ ID NO:41 or SEQ ID NO:47.

[0021] In another preferred embodiment, the anti-IL5 antibody comprises two VHH chains having the amino acid sequences as shown in SEQ ID NO:41 and / or SEQ ID NO:47.

[0022] In another preferred embodiment, the structure of the anti-IL5 antibody from N-terminus to C-terminus is as shown in formula I: A1-A2-LB(I), Where: "-" is a peptide bond or connecting peptide, A1 and A2 are each independently any one of the anti-IL5 Nanobodies according to the first aspect of the invention; B is the Fc fragment of IgG, and L is none or a flexible linker.

[0023] In another preferred embodiment, A1 and A2 are each independently a VHH chain having an amino acid sequence as shown in SEQ ID NO:41 or SEQ ID NO:47.

[0024] In another preferred embodiment, the A1 is a VHH chain having an amino acid sequence as shown in SEQ ID NO:41, and the A2 is a VHH chain having an amino acid sequence as shown in SEQ ID NO:47.

[0025] In another preferred embodiment, the A1 is a VHH chain having an amino acid sequence as shown in SEQ ID NO:47, and the A2 is a VHH chain having an amino acid sequence as shown in SEQ ID NO:41.

[0026] In another preferred embodiment, the flexible linker is a connecting peptide.

[0027] In another preferred embodiment, the VHH chains are connected by a connecting peptide.

[0028] In another preferred embodiment, the connecting peptide is a S b ) x -(G m S n ) y where a, b, m, n, x, y=0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (preferably, a=4 and b=1, m=3 and n=1).

[0029] In another preferred embodiment, the connecting peptidic sequence is (G4S)4.

[0030] In another preferred embodiment, the antibody-like amino acid sequence is as set forth in SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, or SEQ ID NO:59.

[0031] In another preferred embodiment, the antibody is capable of specifically binding to an IL5 protein having a precise spatial structure.

[0032] In another preferred embodiment, the antibody can effectively block the interaction between IL5 and IL5R.

[0033] In another preferred example, the antibody can recognize human and cynomolgus monkey IL5 but cannot recognize mouse IL5.

[0034] In another preferred embodiment, the affinity of the antibody for IL5 is less than 1 nM.

[0035] In another preferred embodiment, the antibody has good IL5 / IL5R blocking activity, which is significantly better than that of the control antibody Nucala, which is Mepolizumab, a commercial drug from GSK.

[0036] In another preferred embodiment, the antibody can effectively inhibit the proliferation of TF-1 cells induced by IL5, and its inhibitory activity is superior to that of the control antibody Nucala.

[0037] In another preferred embodiment, the antibody is a nanobody.

[0038] A third aspect of the invention provides an anti-IL5 Nanobody Fc fusion protein, the structure of said fusion protein from N-terminus to C-terminus is as shown in formula Ia or Ib, ALB(Ia), BLA(Ib), Where: "-" is a peptide bond, A is one or more anti-IL5 Nanobodies according to the first aspect of the invention, B is the Fc fragment of IgG, and L is none or a flexible linker.

[0039] In another preferred embodiment, the flexible linker is a connecting peptide.

[0040] In another preferred embodiment, the Fc fragment of IgG comprises an Fc fragment of human IgG.

[0041] In another preferred embodiment, the Fc fragment of IgG is selected from the group consisting of Fc fragments of IgG1, IgG2, IgG3, IgG4, or a combination thereof.

[0042] In another preferred embodiment, the Fc fragment of IgG is IgG4.

[0043] In another preferred embodiment, the amino acid sequence of the Fc fragment is as shown in SEQ ID NO:63.

[0044] In another preferred embodiment, the amino acid sequence of the fusion protein is as set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, or SEQ ID NO:59.

[0045] In another preferred embodiment, said fusion protein is a Nanobody-Fc fusion protein directed against an IL5 epitope.

[0046] A fourth aspect of the invention provides a polynucleotide, said polynucleotide encoding a protein selected from the group consisting of an anti-IL5 Nanobody according to the first aspect of the invention, an anti-IL5 antibody according to the second aspect of the invention, or an anti-IL5 Nanobody Fc fusion protein according to the third aspect of the invention.

[0047] In another preferred embodiment, the polynucleotide sequence is in a combined form, preferably the polynucleotide sequence comprises one or more of SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:27, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60 or SEQ ID NO:62.

[0048] In another preferred embodiment, the polynucleotide comprises DNA or RNA.

[0049] A fifth aspect of the invention provides an expression vector, said expression vector comprising a polynucleotide according to the fourth aspect of the invention.

[0050] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0051] Preferably, the expression vector comprises a viral vector, such as a lentivirus, an adenovirus, an AAV virus, a retrovirus, or a combination thereof.

[0052] A sixth aspect of the invention provides a host cell, said host cell comprising an expression vector according to the fifth aspect of the invention or having integrated in its genome a polynucleotide according to the fourth aspect of the invention.

[0053] In another preferred embodiment, the host cell comprises a prokaryotic or eukaryotic cell.

[0054] In another preferred embodiment, the host cell is selected from the group consisting of E. coli, a yeast cell, a mammalian cell, a phage, or a combination thereof.

[0055] In another preferred embodiment, the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, Lactobacillus acidophilus, Streptomyces, Proteus mirabilis, or a combination thereof.

[0056] In another preferred embodiment, the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces sembichae, Myzosaccharomyces, Trichoderma, or a combination thereof.

[0057] In another preferred embodiment, the host cell is Pichia pastoris.

[0058] A seventh aspect of the present invention is (a) culturing a host cell according to the sixth aspect of the invention under conditions suitable for producing the Nanobody or an Fc-fusion protein thereof, thereby obtaining a culture comprising said anti-IL5 Nanobody or an Fc-fusion protein thereof; (b) isolating or recovering the anti-IL5 Nanobody or an Fc fusion protein thereof from the culture; and and (c) optionally purifying and / or modifying to obtain the anti-IL5 Nanobody or Fc-fusion protein thereof obtained in step (b).

[0059] An eighth aspect of the present invention provides an immunoconjugate comprising: (a) an anti-IL5 Nanobody according to the first aspect of the invention, or an anti-IL5 antibody according to the second aspect of the invention, or an anti-IL5 Nanobody Fc fusion protein according to the third aspect of the invention, and (b) a coupling moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or VLP, or a combination thereof.

[0060] In another preferred embodiment, the radionuclide is (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or (ii) a therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

[0061] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0062] In another preferred embodiment, the drug is a cytotoxic drug.

[0063] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of an antitubulin drug, a DNA minor groove binding agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folate antagonist, antimetabolite drug, a chemotherapy sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.

[0064] In another preferred embodiment, examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, and 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.

[0065] In another preferred embodiment, the toxin is an auristatin (e.g., auristatin E, auristatin F, MMAE, and MMAF), aureomycin, maytancetol, ricin, ricin A-chain, combretastatin, docarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine. , colchicine, dihydroxyanthracine diketone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, lotus root toxin A chain, α-sarcina, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotonin, calicheamicin, Sapaonaria officinalis inhibitor, glucocorticoids, or combinations thereof.

[0066] In another preferred embodiment, the coupling moiety is a detectable marker.

[0067] In another preferred embodiment, the coupling moiety is selected from the group consisting of a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biotoxin, a cytokine (such as IL-2), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug activating enzyme (such as DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) or a nanoparticle of any form.

[0068] A ninth aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition comprising: (i) an anti-IL5 Nanobody according to the first aspect of the invention, or an anti-IL5 antibody according to the second aspect of the invention, or an anti-IL5 Nanobody Fc fusion protein according to the third aspect of the invention, or an immunoconjugate according to the eighth aspect of the invention, and (ii) A pharma- ceutically acceptable carrier.

[0069] In another preferred embodiment, the immunoconjugative coupling moiety is a drug, a toxin, and / or a therapeutic isotope.

[0070] In another preferred embodiment, the pharmaceutical composition comprises other drugs for treating asthma, atopic dermatitis, arthritis, allergic rhinitis and / or eczema, such as corticosteroids (TCS), nedocrosil sodium, cromolyn sodium, theophylline, leukotriene receptor antagonists, or combinations thereof.

[0071] In another preferred embodiment, the pharmaceutical composition is used for the preparation of a medicament for preventing and / or treating a disease or condition associated with IL5 / IL5R signaling.

[0072] A tenth aspect of the invention provides the use of an anti-IL5 Nanobody according to the first aspect of the invention, an anti-IL5 antibody according to the second aspect of the invention, an anti-IL5 Nanobody Fc fusion protein according to the third aspect of the invention, or an immunoconjugate according to the eighth aspect of the invention: (a) for the preparation of a medicament for the prevention and / or treatment of a disease or condition associated with IL5 / IL5R signalling; and (b) for the preparation of a reagent, detection plate or kit for detecting IL5.

[0073] In another preferred embodiment, the disease or condition includes, but is not limited to, asthma, atopic dermatitis, arthritis, allergic rhinitis, eczema, sinusitis, nasal polyps, chronic obstructive pulmonary disease, eosinophilic granulomatous polyangiitis, hypereosinophilic syndrome, or a combination thereof.

[0074] In another preferred embodiment, the IL5 is human IL5.

[0075] In another preferred embodiment, the reagent is a diagnostic reagent.

[0076] In another preferred embodiment, the diagnostic reagent is an imaging agent.

[0077] In another preferred embodiment, the reagent is used to detect IL5 protein or a fragment thereof in a sample.

[0078] An eleventh aspect of the invention provides a multispecific antibody, said multispecific antibody comprising an anti-IL5 Nanobody according to the first aspect of the invention, or an anti-IL5 antibody according to the second aspect of the invention.

[0079] In another preferred example, the multispecific antibody comprises a second antigen-binding region that targets a target selected from the group consisting of IL-4R, IL-4Rα, IL-13, IL-13R, IL-11, IL-11R, or a combination thereof.

[0080] In another preferred embodiment, the second antigen-binding region is a nanobody.

[0081] In another preferred embodiment, the multispecific antibody comprises one or more second antigen-binding regions.

[0082] In another preferred embodiment, the multispecific antibody further comprises an antibody Fc segment.

[0083] A twelfth aspect of the present invention provides a recombinant protein, the recombinant protein comprising: (i) an anti-IL5 Nanobody according to the first aspect of the invention, or an anti-IL5 antibody according to the second aspect of the invention, or an anti-IL5 Nanobody Fc fusion protein according to the third aspect of the invention, and (ii) With an optional tag sequence to aid in expression and / or purification.

[0084] In another preferred embodiment, the tag sequences include an Fc tag, an HA tag, and a 6His tag.

[0085] In another preferred embodiment, the recombinant protein specifically binds to the IL5 protein.

[0086] A thirteenth aspect of the present invention provides the use of an anti-IL5 Nanobody according to the first aspect of the invention, or an anti-IL5 antibody according to the second aspect of the invention, or an anti-IL5 Nanobody Fc fusion protein according to the third aspect of the invention, or an immunoconjugate according to the eighth aspect of the invention, for use in the detection of IL5 protein in a sample or for the treatment and / or prevention of a disease or condition associated with IL5 / IL5R signalling.

[0087] In another preferred embodiment, the disease or condition includes, but is not limited to, asthma, atopic dermatitis, arthritis, allergic rhinitis, eczema, sinusitis, nasal polyps, chronic obstructive pulmonary disease, eosinophilic granulomatous polyangiitis, hypereosinophilic syndrome, or a combination thereof.

[0088] In another preferred embodiment, the detection includes flow cytometry detection, cell immunofluorescence detection.

[0089] In another preferred embodiment, the use is diagnostic and / or non-diagnostic and / or therapeutic and / or non-therapeutic.

[0090] A fourteenth aspect of the present invention provides a method for detecting IL5 protein in a sample, the method comprising the steps of: (1) contacting a sample with an anti-IL5 Nanobody according to the first aspect of the invention, or an anti-IL5 antibody according to the second aspect of the invention, or an anti-IL5 Nanobody-Fc fusion protein according to the third aspect of the invention, or an immunoconjugate according to the eighth aspect of the invention; (2) detecting whether an antigen-antibody complex is formed, where the formation of the complex indicates the presence of IL5 protein in the sample.

[0091] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0092] A fifteenth aspect of the present invention provides an IL5 protein detection reagent, the detection reagent comprising: (i) an anti-IL5 Nanobody according to the first aspect of the invention, or an anti-IL5 antibody according to the second aspect of the invention, or an anti-IL5 Nanobody Fc fusion protein according to the third aspect of the invention, or an immunoconjugate according to the eighth aspect of the invention, and (ii) A detectably acceptable vector.

[0093] In another preferred embodiment, the coupling moiety of the immunoconjugate is a diagnostic isotope.

[0094] In another preferred embodiment, the detectably acceptable vector is a non-toxic, inert aqueous vector medium.

[0095] In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of an isotope tracer, a contrast agent, a flow cytometry detection reagent, a cellular immunofluorescence detection reagent, a nanomagnetic particle, and an imaging agent.

[0096] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0097] In another preferred embodiment, the detection reagent is in a liquid or powder form (eg, an aqueous solution, an injection, a lyophilized powder, a tablet, an oral preparation, or an aerosol preparation).

[0098] A sixteenth aspect of the present invention provides a kit for detecting IL5 protein, said kit comprising an immunoconjugate according to the eighth aspect of the present invention or a detection reagent according to the fifteenth aspect of the present invention, and instructions.

[0099] In another preferred embodiment, the instructions state that the kit is used for non-invasively detecting IL5 expression in a subject.

[0100] A seventeenth aspect of the present invention provides the use of an immunoconjugate according to the eighth aspect of the present invention for use in preparing an imaging agent for detecting IL5 protein in vivo.

[0101] In another preferred embodiment, the detection is used for the diagnosis or prognosis of asthma, atopic dermatitis, arthritis, allergic rhinitis, eczema, sinusitis, nasal polyps, chronic obstructive pulmonary disease, eosinophilic granulomatous polyangiitis, hypereosinophilic syndrome, and the like. Effect of the Invention

[0102] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be repeated here due to space limitations. [Brief description of the drawings]

[0103] [Figure 1] Figure 1 shows the screening and enrichment process of IL5 nanobodies. After five rounds of panning, IL5-specific nanobody phages in the two libraries were enriched by 120-fold and 14.6-fold, respectively. [Diagram 2] Figure 1 shows the results of a preliminary screening with nanobodies that block the interaction between IL5 and IL5R by flow cytometry. The results show that 11 antibodies out of 96 nanobodies have blocking activity. [Diagram 3]Figure 1 shows the results of ELISA detection of the cross-species activity of 11 blocking IL5 nanobodies. The results show that all 11 nanobodies can recognize human and cynomolgus IL5, but not mouse IL5. [Figure 4] Figure 2 shows the results of Fortebio detection of nanobody affinity using Biolayer Interference. The results show that the affinity of all 11 nanobodies for IL5 is below 1 nM. [Diagram 5] Figure 2 shows the results of ELISA detection of the binding activity of the nanobodies. The results show that the binding activity of all four nanobodies is superior to that of the control antibody Nucala. [Figure 6] Figure 1 shows the blocking activity of candidate IL5 nanobodies identified by flow cytometry. The results show that all four nanobodies have good IL5 / IL5R blocking activity, where the blocking activity of Nb21, Nb66 is significantly superior to that of the control antibody Nucala. [Figure 7] Figure 1 shows the results of ELISA detection of the identity of the antigen-recognition epitopes of blocking nanobodies. The results show that Nb21 and Nb66 are distinct antigen-recognition epitopes. [Figure 8] The blocking activity of humanized bivalent nanobodies detected by flow cytometry is shown. The results show that the activity of humanized bivalent antibodies is significantly increased compared to that of monovalent antibodies, where the blocking activity of HuNb21-HuNb66-Fc and HuNb66-HuNb21-Fc bivalent bi-epitope antibodies is the best, significantly superior to the control antibody Nucala. [Figure 9] The results show that the content of biepitope bivalent IL5 nanobody in the fermentation supernatant of Pichia pastoris detected by Fortebio. The results show that under the fermentation conditions, the expression level of biepitope antibody increases continuously with the extension of the culture time, and the antibody yield can reach 13g / L after 202 hours of fermentation culture. [Figure 10]Figure 1 shows the SDS-PAGE detection results of biepitope bivalent IL5 nanobody Pichia pastoris fermentation supernatant. The results show that the expression yield of biepitope IL5 nanobody HuNb66-HuNb21 in Pichia pastoris can reach 13g / L, and the purity of the target protein expression supernatant is high. [Figure 11] Figure 1 shows the results of flow cytometry detection of the blocking activity of bivalent biepitope IL5 nanobodies. The results show that the blocking activity of the yeast-expressed bivalent biepitope antibody HuNb66-HuNb21 (IC50=0.841 μg / mL) is superior to that of the control antibody Nucala (IC50=2.035 μg / mL). [Figure 12] The results of detecting the inhibitory effect of bivalent biepitope IL5 nanobody on the proliferation of TF1 cells are shown. The results show that the bivalent biepitope antibody expressed by yeast can effectively inhibit the proliferation of TF-1 cells induced by IL5, and its inhibitory activity on the proliferation of TF-1 cells (IC50 HuNb66-HuNb21=0.0512nM) is superior to the inhibitory effect of the control antibody (IC50 Nucala=0.1782nM). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0104] After extensive and thorough research and numerous screenings, the inventors have unexpectedly discovered a class of IL5 nanobodies for the first time, and as shown in the experimental results, the nanobodies of the present invention can specifically identify human and cynomolgus monkey IL5, but cannot identify mouse IL5, and have good specificity and binding activity; the nanobodies of the present invention have good IL5 / IL5R blocking activity, and the blocking activity is significantly superior to that of the control antibody Nucala; the nanobodies of the present invention can effectively inhibit the proliferation of TF-1 cells induced by IL5, and their inhibitory activity is superior to that of the control antibody Nucala; the expression yield of the nanobodies of the present invention in Pichia pastoris can reach 13g / L, and the purity of the target protein expression supernatant is high.

[0105] term As used herein, the terms "Nanobodies of the invention", "Nanobodies of the invention", "anti-IL5 Nanobodies of the invention", "IL5 Nanobodies of the invention", "anti-IL5 Nanobodies", "IL5 Nanobodies" have the same meaning, are used interchangeably, and all refer to Nanobodies that specifically recognize and bind IL5 (including human IL5).

[0106] As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H) with the same structural characteristics. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, where the constant region of the light chain faces the first constant region of the heavy chain, and the variable region of the light chain faces the variable region of the heavy chain. Certain amino acid residues form an interface between the variable regions of the light and heavy chains.

[0107] As used herein, the terms "single domain", "VHH", "nanobody", "heavy chain antibody" (sdAb or nanobody) have the same meaning and are used interchangeably and refer to cloning the variable region of an antibody heavy chain to construct a nanobody (VHH) consisting of only one heavy chain variable region, which is the smallest fully functional antigen-binding fragment. Typically, an antibody that naturally lacks the light and heavy chain constant region 1 (CH1) is obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.

[0108] As used herein, the term "variable" refers to the difference in sequence of certain parts of the variable region in an antibody, which forms the binding and specificity of various specific antibodies to a particular antigen. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments called the complementarity determining regions (CDRs) or hypervariable regions of the light and heavy chain variable regions. The more conserved parts of the variable regions are called framework regions (FRs). Natural heavy and light chain variable regions each contain four FR regions, which are mostly in a β-folded configuration connected by three CDRs that form a connecting ring, and can sometimes form a partial β-folded structure. The CDRs of each chain are closely juxtaposed by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions are involved directly in binding the antibody to an antigen, but they also exhibit various effector functions, such as those involved in antibody-dependent cellular toxicity of the antibody.

[0109] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention further includes cell surface markers or antigens that bind to said anti-IL5 antibodies or fragments thereof.

[0110] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

[0111] As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0112] In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions, CDR1, CDR2 and CDR3.

[0113] In a preferred embodiment of the invention, the heavy chain of the antibody comprises the above-described heavy chain variable region and heavy chain constant region.

[0114] In the present invention, "antibody of the invention", "protein of the invention" or "polypeptide of the invention" are used interchangeably and all refer to a polypeptide that specifically binds to IL5 protein, e.g., a protein or polypeptide having a heavy chain variable region, which may or may not contain a starting methionine.

[0115] The present invention further provides other proteins or fusion expression products comprising the antibodies of the present invention. In particular, the present invention includes any protein or protein conjugates and fusion expression products (i.e., immunoconjugates and fusion expression products) having a heavy chain that includes a variable region, so long as the variable region is the same as or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the present invention.

[0116] In general, the antigen-binding properties of an antibody can be described by three specific regions located in the heavy chain variable region, called the variable region (CDR), which is divided into four framework regions (FR), and the amino acid sequences of the four FRs are relatively conserved and are not directly involved in the binding reaction. These CDRs form a cyclic structure, and the β-fold formed by the FRs between them are close to each other in a spatial structure, and the CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. Which amino acids constitute the FR region or CDR region can be determined by comparing the amino acid sequences of the same type of antibody.

[0117] The variable regions of the heavy chains of the antibodies of the invention are of particular interest since at least some of them are involved in antigen binding. Thus, the invention includes molecules having antibody heavy chain variable regions with CDRs, so long as the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein.

[0118] The present invention includes not only complete antibodies, but also immunologically active fragments of antibodies or fusion proteins formed by antibodies and other sequences. Thus, the present invention further includes fragments, derivatives and analogs of said antibodies.

[0119] As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains essentially the same biological function or activity as the antibody of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide in which one or more conservative or non-conserved amino acid residues (preferably conservative amino acid residues) have been substituted (such substituted amino acid residues may or may not be encoded by the genetic code), or (ii) a polypeptide having a substitution at one or more amino acid residues, or (iii) a polypeptide formed by fusing the mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a fusion protein formed with a leader sequence or a secretory sequence or a sequence for purifying the polypeptide or a protein sequence or a 6His tag). Following the teachings of the present specification, these fragments, derivatives, and analogs are within the knowledge of those skilled in the art.

[0120] The antibody of the present invention refers to a polypeptide having IL5 binding activity and comprising the above CDR region. The term further includes mutant forms of the polypeptide comprising the above CDR region, which have the same function as the antibody of the present invention. These mutant forms include (but are not limited to) deletion, insertion and / or substitution of one or more amino acids (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) and addition of one or more amino acids (usually 20 or less, preferably 10 or less, more preferably 5 or less) at the C-terminus and / or N-terminus. For example, when substituted by amino acids having similar or similar performance in the art, it usually does not change the function of the protein. As another example, addition of one or more amino acids at the C-terminus and / or N-terminus also usually does not change the function of the protein. The term further includes active fragments and active derivatives of the antibody of the present invention.

[0121] Variant forms of the polypeptide include homologous sequences, conservative variants, allelic variants, naturally occurring mutants, induced mutants, proteins encoded by DNA capable of hybridizing to the coating DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained by using antisera against the antibody of the present invention.

[0122] The invention further provides other polypeptides, such as fusion proteins, comprising a Nanobody or a fragment thereof. In addition to substantially full-length polypeptides, the invention further includes fragments of the Nanobodies of the invention. Typically, the fragments comprise at least about 50 contiguous amino acids of an antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

[0123] In the present invention, "conservative variants of the antibodies of the invention" refer to polypeptides formed by substitution of up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids having the same or similar properties compared to the amino acid sequence of the antibodies of the invention. These conservative variant polypeptides are best generated by making amino acid substitutions according to Table A. [Table 1]

[0124] The present invention further provides a polynucleotide molecule encoding said antibody or fragment thereof or fusion protein thereof. The polynucleotide of the present invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be coding strand or non-coding strand.

[0125] A polynucleotide encoding a mature polypeptide of the present invention includes a coding sequence encoding only the mature polypeptide, a coding sequence for the mature polypeptide and various additional coding sequences, a coding sequence for the mature polypeptide (and any additional coding sequences) and non-coding sequences.

[0126] The term "polynucleotide encoding a polypeptide" includes a polynucleotide that encodes the polypeptide, or further includes a polynucleotide that contains additional coding and / or non-coding sequences.

[0127] The present invention further relates to polynucleotides that hybridize with the above sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the above polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refers to (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60oC, or (2) addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42oC, or (3) hybridization that occurs only when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

[0128] The full-length nucleotide sequence of the antibody of the present invention or its fragments can usually be obtained by PCR amplification, recombinant methods or artificial synthesis.Especially when the length of the fragment is short, a feasible method is to use artificial synthesis to synthesize the relevant sequence.Generally, multiple small fragments are first synthesized, and then connected to obtain a very long fragment of sequence.In addition, the coding sequence of the heavy chain and an expression tag (e.g., 6His) can be fused to form a fusion protein.

[0129] Once the relevant sequence is obtained, recombinant methods can be used to obtain the relevant sequence in large quantities. Typically, this is achieved by cloning it into a vector, then transforming it into a cell, and then isolating the relevant sequence from the host cell that has been propagated by conventional methods. The biological molecules involved in the present invention (nucleic acids, proteins, etc.) include biological molecules that exist in isolated form.

[0130] Currently, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) can be obtained entirely by chemical synthesis. The DNA sequences can then be introduced into a variety of existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequences of the present invention by chemical synthesis.

[0131] The present invention further relates to vectors containing said appropriate DNA sequences and appropriate promoter or control sequences, which can be used to transform an appropriate host cell so as to express the protein.

[0132] The host cell may be a prokaryotic cell, such as, for example, a bacterial cell, or a lower eukaryotic cell, such as, for example, a yeast cell, or a higher eukaryotic cell, such as, for example, a mammalian cell. Representative examples include bacterial cells of E. coli, Streptomyces, Chinese hamster bacteria, fungal cells such as yeast, insect cells of Drosophila S2 or Sf9, animal cells such as CHO, COS7 and 293 cells, and the like.

[0133] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be obtained by treating with CaCl2 method after exponential growth phase, the steps used being well known in the art. Another method is to use MgCl2. Transformation can also be carried out by electroporation method if necessary. When the host is a eukaryote, DNA transfection methods such as calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection and electroporation, liposome packaging, etc. can be selected.

[0134] The obtained transformant can be cultured in a conventional manner to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used for the culture can be selected from a variety of conventional media. The culture is performed under conditions suitable for the growth of the host cell. After the host cell has grown to an appropriate cell density, the selected promoter is induced using an appropriate method (e.g., temperature change or chemical induction), and the cell is further cultured for a certain period of time.

[0135] The recombinant polypeptide in the method can be expressed intracellularly or in the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various isolation methods using physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation, treatment with protein precipitants (salting out), centrifugation, osmotic sterilization, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid phase chromatography (HPLC) and various other liquid chromatography techniques, as well as combinations of these methods.

[0136] The antibodies of the present invention can be used alone, can be conjugated or coupled to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modulating moiety, or any combination of these substances.

[0137] Detectable markers for diagnostic purposes include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0138] Therapeutic agents that can be conjugated or coupled to the antibodies of the present invention include, but are not limited to, 1. radionuclides, 2. biological toxins, 3. cytokines such as IL-2, 4. gold nanoparticles / nanorods, 5. viral particles, 6. liposomes, 7. nanomagnetic particles, 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), and the like.

[0139] Therapeutic agents that can be conjugated or coupled to the antibodies of the present invention include, but are not limited to, 1. radionuclides; 2. biotoxins; 3. cytokines, such as IL-2; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), and the like.

[0140] Interleukin-5 (IL5) Interleukin-5 (IL5) is the most selective for eosinophils among known cytokines and can regulate the growth, activation, survival and migration of eosinophils. Interleukin-5 exerts its proliferative and differentiation effects through receptors containing interleukin-5-specific α and general β-subunits. IL5 plays a key role in the migration of eosinophils from the bone marrow to the back and other organs. IL5 signaling maintains the survival and function of B cells and eosinophils through JAK-STAT, Btk and Ras / Raf-ERK signaling. Overexpression of IL5 in vivo significantly increases the number of eosinophils and B cells, whereas mice lacking functional genes for IL5 or IL5 receptor show numerous developmental and functional impairments in the B cell and eosinophil system. In humans, the biological effects of IL5 are the most favorable characteristics of eosinophils. IL5 is currently considered to be one of the major drivers of the Th2 pathway.

[0141] Interleukin-5 receptor alpha (IL5Rα) IL5Rα, the receptor for IL5, is highly expressed in eosinophils and plays an important role in eosinophils removing allergens from blood and tissues. IL5 receptor is composed of α and βc chains, and the α subunit is specific for IL5 molecules, while the βc subunit is also identified by interleukin 3 (IL3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of IL5Rα in activated B cells is regulated by various transcription factors, including E12, E47, Sp1, c / EBPβ, and Oct2.

[0142] Pharmaceutical Compositions The present invention further provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above-mentioned antibody or its active fragment or its fusion protein, and a pharma- ceutically acceptable carrier. Usually, these substances can be formulated in a non-toxic, inert, pharma- ceutically acceptable aqueous vector medium, where the pH is usually about 5-8, preferably about 6-8, and the pH value varies depending on the nature of the substance to be formulated and the disease to be treated. The formulated pharmaceutical composition can be administered by a conventional route, where it includes but is not limited to intraperitoneal, intravenous, or topical administration.

[0143] The pharmaceutical composition of the present invention can be directly used for binding of IL5 protein molecules, and therefore can be used for the treatment of asthma, atopic dermatitis, arthritis, allergic rhinitis, eczema, etc. Furthermore, other therapeutic agents can be used in combination.

[0144] The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the nanobody of the present invention (or a conjugate thereof) and a pharma- ceutically acceptable carrier or excipient. Such vectors include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The drug formulation should be consistent with the method of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection by conventional methods, for example, using saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be prepared under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight daily. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents.

[0145] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, where the safe and effective amount is usually at least about 10 μg / kg body weight and in most cases will not exceed about 50 mg / kg body weight, and preferably the dosage is from about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dosage must take into account factors such as the route of administration and the health condition of the patient, all of which are within the skill of a skilled physician.

[0146] Anti-IL5 nanobody In the present invention, the amino acid sequence of the VHH chain of said anti-IL5 Nanobody is selected from one or more of SEQ ID NO:8, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47.

[0147] In a preferred embodiment of the invention, said anti-IL5 Nanobodies comprise monomeric, bivalent (bivalent antibody), tetravalent (tetravalent antibody) and / or multivalent (multivalent antibody).

[0148] Typically, said anti-IL5 Nanobody comprises two VHH chains having the amino acid sequences as depicted in SEQ ID NO:41 and / or SEQ ID NO:47.

[0149] In another preferred embodiment, the VHH chains are connected by a connecting peptide.

[0150] In another preferred embodiment, the connecting peptide is a S b ) x -(G m S n ) y where a, b, m, n, x, y=0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (preferably, a=4 and b=1, m=3 and n=1).

[0151] In another preferred embodiment, the connecting peptidic sequence is (G4S)4.

[0152] Labeled nanobodies In a preferred embodiment of the invention, said Nanobody carries a detectable marker, more preferably said marker is selected from the group consisting of an isotope, a colloidal gold marker, a colored marker or a fluorescent marker.

[0153] Colloidal gold labeling can be performed by methods known to those skilled in the art. In a preferred embodiment of the invention, IL5 Nanobodies are labeled with colloidal gold to obtain colloidal gold labeled Nanobodies.

[0154] Detection Method The present invention also relates to a method for detecting IL5 protein, the steps of which are broadly as follows: obtaining a cell and / or tissue sample, lysing the sample in a medium and detecting the level of IL5 protein in the lysed sample.

[0155] In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.

[0156] kit The present invention further provides a kit comprising the antibody (or fragment thereof) or detection plate of the present invention, and in a preferred embodiment of the present invention, the kit further comprises a container, an instruction manual, a buffer, and the like.

[0157] The present invention further provides a detection kit for detecting IL5 levels, which includes an antibody that identifies IL5 protein, a dissolution medium for dissolving the sample, and generally necessary reagents and buffers for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit may be an in vitro diagnostic device.

[0158] Applicable As described above, the Nanobodies of the invention have broad biological and clinical application value, which relates to multiple areas such as diagnosis and treatment of IL5-related diseases, basic medical research, biological research, etc. Preferred applications are clinical diagnosis and targeted therapy against IL5.

[0159] The main advantages of the present invention are: (a) The Nanobodies of the invention are able to effectively block the interaction of IL5 with IL5R. (b) The nanobodies of the present invention can recognize human and cynomolgus monkey IL5, but cannot recognize mouse IL5. (c) The Nanobodies of the invention have stronger binding and blocking activity compared to the control antibody Nucala. (d) The Nanobodies of the invention can be expressed in Pichia pastoris, the expression yield can reach 13 g / L, and the purity of the target protein expression supernatant is high. (e) The Nanobody of the invention can effectively inhibit the proliferation of TF-1 cells induced by IL5, and the inhibitory effect is superior to that of the control antibody Nucala.

[0160] The present invention will be further described by the following specific examples. It should be understood that these examples are only used to describe the present invention and do not limit the scope of the present invention. The experimental methods in the following examples that do not show specific conditions usually follow general conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning-A Laboratory Manual (3rd Edition) (2001) CSHL Publishing), or those suggested by manufacturers. Unless otherwise stated, percentages and parts are calculated by weight.

[0161] Example 1: Screening of IL5-specific nanobodies by phage display technology In the initial stage, highly purified human IL5 protein was mixed with immune adjuvants and immunized into two Xinjiang Bactrian camels. After seven immunizations, peripheral blood was collected, from which lymphocytes were then extracted and RNA was isolated. After reverse transcription of RNA, VHH genes were isolated by nested PCR amplification, and the VHH fragments were cloned into pMECS vectors and electrotransformed into TG1 competent cells to establish a phage display library of anti-IL5 nanobodies. Measurement showed that the storage capacity of the two libraries was 6.4 × 10, respectively. 8 CFU and 1.3 × 10 8 CFU, and the insertion rate of the target fragment VHH of the library reaches 91.7% and 100%, respectively. Then, phage display technology is used to screen IL5-specific nanobodies, and the specific screening method refers to the scheme in Example 3 of patent CN110144011B. After five rounds of "binding-washing-elution" enrichment process, specific phage enrichment of 120 times and 14.6 times, respectively, is finally obtained (Figure 1). After PE-ELISA identification and sequencing analysis, 96 strains of IL5-specific nanobodies with different sequences are finally obtained.

[0162] Example 2: Screening for blocking IL5 nanobodies Inoculate IL5 nanobody clone strains of different sequences into 1mL of TB medium containing an appropriate concentration of ampicillin, add IPTG inducer when culturing in a thermostatic shaker at 37℃ until logarithmic growth phase, and induce at 28℃ for 16 hours. After 16 hours, disrupt the cells using osmotic shock to obtain nanobody crude extracts. For each sample, 1E6 HEK293F / IL5Ra cells are taken and resuspended in 0.5% BSA-PBS buffer, and 200μL of the above IL5 nanobody crude extract is added. At the same time, set up negative control (lysate). Add appropriate concentration of IL5-biotin to all samples, incubate at 4℃ for 20 minutes, wash twice with 1×PBS, add SA-PE, incubate at 4℃ for 20 minutes in the dark, wash the cells twice with PBS, and then measure the results using a flow cytometer (BD The results are shown in Figure 2. 11 IL5 nanobodies with good blocking effect were pre-screened, where the lower the percentage of positive cells, the better the blocking effect of the antibody, and the numbers of the 11 nanobodies are Nb6, Nb13, Nb20, Nb21, Nb25, Nb26, Nb50, Nb66, Nb71, Nb85, and Nb92, respectively. According to sequence analysis, the 11 blocking IL5 nanobody sequences can be divided into four families, and the main research subjects are as shown in Table 1. [Table 2]

[0163] Example 3: Species-specific detection of IL5 Nanobodies ELISA was used to detect whether the nanobodies of the 11 strains obtained in Example 2 could cross-react with other species IL5. 1 μg / mL human IL5, mouse IL5, cynomolgus IL5 and IgG1 proteins were added to a microtiter plate, respectively, and coated at 4°C, 100uL / well overnight. After washing five times with PBST, 300uL of 1% BSA was added to each well and sealed at room temperature for 2 hours. After washing five times with PBST, 100uL of 2μg / mL prokaryotic expressed nanobodies (Nb6, Nb13, Nb20, Nb21, Nb25, Nb26, Nb50, Nb66, Nb7) were added to each well. 1, Nb85, Nb92) was added, incubated at 37°C for 1 hour, washed 5 times with PBST, 100uL of diluted mouse anti-HA antibody (1:2000 dilution) was added, incubated at 37°C for 1 hour, washed 5 times with PBST, 100uL of diluted basic phosphatase modified anti-mouse antibody (1:2000 dilution) was added, incubated at 37°C for 1 hour, washed 5 times with PBST, a color development solution was added, and the absorbance value at 405nm wavelength was measured with a microplate reader. The results are shown in Figure 3, and all 11 nanobodies can identify human and cynomolgus monkey IL5, but cannot identify mouse IL5.

[0164] Example 4: Affinity measurements of IL5 Nanobodies The binding kinetics of the 11 nanobodies obtained in Example 2 to human IL5 is measured using a Fortebio Red96 instrument through bio-layer interferometry (BLI). For kinetics measurement, the IL5 antigen protein is diluted to 1.5 μg / mL with PBST buffer, and the 11 IL5 nanobodies are serially diluted 2-fold with PBST buffer in six concentration gradients (30 nM, 15 nM, 7.5 nM, 3.75 nM, 1.88 nM, 0.94 nM), and the operating conditions of the instrument are set as follows: temperature 30° C., shake speed 1000 rpm. The antibody is captured using a protein A-coated probe, the capture time is 180 seconds, and it is bound to the serially diluted antigen, the binding time is 300 seconds, the dissociation time is 360 seconds, and 10 mM glycine (PH1.7) is regenerated three times for 5 seconds each time. Analyze using FortebioAnalysis 9.0 version, fit with 1:1 binding model Global mode, and calculate binding rate (Kon), dissociation rate (Kdis) and dissociation constant KD. The results are shown in Figure 4, and the affinity of the 11 nanobody strains for IL5 is all less than 1 nM.

[0165] Example 5: Detection of binding activity of IL5 Nanobodies by ELISA Detect and compare the binding activity of four nanobodies (Nb21, Nb25, Nb66 and Nb92) with different sequences to IL5 by ELISA. Dilute all IL5 nanobodies to 1 μg / mL, take 100uL / well to coat, and leave at 4℃ overnight. Wash 5 times with PBST, then add 300uL of 1% BSA to each well, leave at 37℃ for 2 hours, and seal. Wash 5 times with PBST, add 100uL of serially diluted IL5-biotin, starting with a concentration gradient of 2 μg / mL and 12 points of 2-fold serial dilution, add samples and incubate at 37℃ for 1 hour. Wash 5 times with PBST, add 100uL of SA-HRP (diluted 1:5000 in PBS) and incubate at 37℃ for 1 hour. Wash five times with PBST, add 100uL of TMB coloring solution to each well, react for 5 minutes in the dark at room temperature, add 50uL of 2M sulfuric acid to stop the reaction, and measure the absorbance at 450nm with a microplate reader. The results are shown in Figure 5, and the binding activity of the four nanobodies is superior to that of the control antibody Nucala.

[0166] Example 6: Detection of blocking activity of IL5 antibodies by flow cytometry Stably transfected HEK293F cells highly expressing IL5R were centrifuged at 1000 rpm for 5 min and the supernatant was discarded. The cells were washed once with 5 mL of PBS, followed by adding 2 mL of PBS to resuspend the cells and counting to obtain 3 × 10 cells per well. 5The cells are dispensed into a 96-well plate at 1000x the number of cells. Four nanobodies (Nb21, Nb25, Nb66 and Nb92) and the control antibody Nucala are serially diluted 2-fold (20μg / ml, 10μg / ml, 5μg / ml, 2.5μg / ml, 1.25μg / ml, 0.625μg / ml, 0.31μg / ml, 0.16μg / ml, 0.08μg / ml, 0.04μg / ml, 0.02μg / ml, 0.01μg / ml), and the diluted antibodies are mixed with an equal volume of 2.5μg / ml of IL5-biotin, after which the cells are resuspended in the 96-well plate and incubated at 4℃ for 20 minutes. Incubate, centrifuge at 3000 rpm, 4 ° C, add 200uL PBS / well, resuspend and centrifuge at 3000 rpm, 4 ° C for 4 minutes, add diluted SA-PE antibody (diluted 0.3:100), incubate at 4 ° C for 20 minutes, centrifuge at 3000 rpm, 4 ° C for 4 minutes, discard the supernatant, add 200uL PBS / well to wash the cells, wash twice, add 200uL PBS to resuspend the cells, transfer to a flow tube, and detect the PE signal of each sample with a flow cytometer. The results are shown in Figure 6, and the four nanobodies all have good IL5 / IL5R blocking activity, where the blocking activity of Nb21 and Nb66 is significantly better than that of the control antibody Nucala.

[0167] Example 7: Antigen Recognition Epitope Analysis of IL5 Nanobodies Using ELISA, two preferred nanobodies (Nb21 and Nb66) with better blocking activity in Example 6 can detect different IL5 antigen epitopes. Specifically, 1 μg / mL IL5 antigen protein is coated overnight at 4°C, the enzyme plate is washed 5 times with PBST, and then 1% BSA is added and sealed at room temperature for 2 hours, the enzyme plate is washed 5 times with PBST, 100uL of diluted antibody mixture is added, and incubated at 37°C for 1 hour (the working concentration of nanobody is 20μg / mL, the working concentration of biotinylated nanobody is 3μg / mL, and the three groups of samples are 3μg / mL Nb21-biotin, 20μg / mL Nb6 ... 100uL of SA-HRP (diluted 1:5000) was added, incubated at 37℃ for 1 hour, washed 5 times with PBST, added 100uL of TMB color developing solution to each well, reacted for 5 minutes in the dark at room temperature, added 50uL of 2M sulfuric acid to stop the reaction, and measured the absorbance at 450nm with a microplate reader. The results are shown in Figure 7, and Nb21 and Nb66 have different antigen recognition epitopes.

[0168] Example 8: Construction and expression of bivalent humanized nanobodies In the above Example 6, two nanobodies Nb21 and Nb66 with good blocking activity were humanized in the framework region, and the modification scheme is shown in Example 3 of Patent CN110144010B. The antibody sequences after humanization are shown in Table 2. [Table 3]

[0169] The humanized antibodies are combined in pairs, fused with Fc, and expressed in pCDNA3.1+ vector. The fused sequences are shown in Table 3. [Table 4]

[0170] The constructed plasmid is introduced into HEK293F cells, and the expression method is described in Example 3 of Patent CN2018101517526.

[0171] Example 9: Detection of blocking activity of bivalent humanized IL5 nanobodies by flow cytometry The purified humanized bivalent antibody obtained in Example 8 is compared with the monovalent nanobody and the positive control Nucala for blocking activity at the cellular level. Specifically, stably transfected HEK293F cells highly expressing IL5R are centrifuged at 1000 rpm for 5 min and the supernatant is discarded. The cells are washed once with 5 mL of PBS, followed by adding 2 mL of PBS to resuspend the cells, and after counting, the cells are harvested at 3 × 10 per well. 5 Dispense into 96-well plates with cells. Each antibody to be tested is serially diluted 2-fold (80μg / ml, 40μg / ml, 20μg / ml, 10μg / ml, 5μg / ml, 2.5μg / ml, 1.25μg / ml, 0.625μg / ml, 0.31μg / ml, 0.16μg / ml, 0.08μg / ml, 0.04μg / ml), and the diluted antibody is mixed with IL5-biotin, and then resuspended and dispensed into 96-well plates. The cells are incubated at 4℃ for 20 minutes, centrifuged at 3000 rpm at 4℃, and then Add 200uL PBS / well, resuspend and centrifuge at 3000rpm at 4℃ for 4 minutes, add diluted SA-PE antibody (diluted 0.3:100), incubate at 4℃ for 20 minutes, centrifuge at 3000rpm at 4℃ for 4 minutes, discard the supernatant, add 200uL PBS / well to wash the cells, wash twice, resuspend the cells in 200uL PBS, transfer to flow tubes, and detect the PE signal of each sample by flow cytometer.

[0172] The results are shown in Figure 8, where the humanized bivalent antibodies have significantly increased activity compared to the monovalent antibodies, with the blocking activity of HuNb21-HuNb66-Fc and HuNb66-HuNb21-Fc heterobivalent antibodies being better and significantly superior to the control antibody Nucala.

[0173] Example 10: Expression of bivalent biepitope IL5 Nanobodies in Pichia pastoris Preferably, the humanized Nb66 and Nb21 are combined to form a bivalent bi-epitope nanobody, the antibody amino acid sequence is as shown in SEQ ID NO:61, and the base sequence after Pichia pastoris codon optimization is as shown in SEQ ID NO:62, which is cloned into pPICZaA vector and then expressed in Pichia pastoris. Briefly, the expression method is as follows: pPICZaA-HuNb66-HuNb21 is linearized with Sac I restriction endonuclease and then electrotransformed into X-33 competent cells. The electrotransformed samples are coated on YPD plate media containing different concentrations of bleomycin resistance and cultured in an incubator at 30 ° C for 3 to 4 days. For specific embodiments, refer to the instruction manual for the pPICZaA vector provided by Invitrogen. After the monoclones grow on the plate medium, the monoclones of different concentrations on the plate are picked and placed on BMGY medium. When the OD value of the BMGY culture reaches about 20, the bacteria are collected and then replaced with BMMY medium. Culture at 28 ° C and 250 rpm. Then, samples are taken every 24 hours. A final volume of 1% methanol is added to take the sample. The sample is centrifuged at 12000 rpm for 5 minutes, the supernatant is collected and stored at -20 ° C., and the induction is continued for 5 days. The culture is terminated, and the supernatant is collected to measure the content of the target protein. Then, high-yield clones were selected and cultured in a 7L fermenter, and the fermentation conditions were induction culture at 24°C, pH 6.5, and methanol feed rate of 8.5mL / L / h. The supernatant was collected at various times during the fermentation process to detect the content of the target protein, and the results were as shown in Figure 9. The expression yield of bi-epitope IL5 nanobody HuNb66-HuNb21 in Pichia pastoris can reach about 13g / L. The collected samples were diluted 13 times and subjected to SDS-PAGE detection, and the results were as shown in Figure 10. The target protein was transparent and the purity of the expressed supernatant was high.

[0174] Example 11: Detection of blocking activity of bivalent biepitope IL5 Nanobodies The bivalent biapitopic antibody expressed by yeast was purified by protein A affinity chromatography to obtain a relatively high purity antibody, and then the blocking activity was detected. The detection method was the same as in Example 9. The results are shown in Figure 11. The blocking activity (IC 50 = 0.841 μg / mL) was compared with the control antibody Nucala (IC 50 = 2.035 μg / mL).

[0175] Example 12: Inhibitory effect of bivalent biepitope IL5 nanobodies on the proliferation of TF1 cells Recovered and cultured TF-1 cells (IL5-induction treated) were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, resuspended in 5 mL of PBS, centrifuged at 1000 rpm for 5 min, resuspended and counted the cells in 20 mL of 1640 medium, and the cell solution concentration was adjusted to 6 × 10 5 1. Dilute the IL5 antibody to 25ng / mL and dispense 60uL / well into a 96-well plate. Mix 50uL of serially diluted IL5 antibody with 50uL of 25ng / mL IL5 protein, then take 40uL of the mixture and add it to the cell solution. At the same time, add 200uL of PBS to the wells around all the cells to prevent the solution in the cell wells from evaporating. Place in a 37℃, 5% CO2 incubator and culture for 72 hours. Remove the cell culture plate, add 10uL / well of CCK8 solution, place at 37℃, and allow to develop for 4 hours. After the color development is completed, read the OD450 value of each well with a microplate reader.

[0176] The results are shown in FIG. 12. The yeast-expressed bivalent biepitope antibody can effectively inhibit IL5-induced proliferation of TF-1 cells, and its inhibitory activity against TF-1 cell proliferation (IC 50 HuNb66-HuNb21 = 0.0512 nM) was the control antibody inhibitory effect (IC 50 Nucala =0.1782nM).

[0177] All documents mentioned in the present invention are incorporated by reference in this application as if each document was incorporated by reference individually. Furthermore, after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalents are also included in the scope defined by the appended claims of this application.

Claims

1. 1. An anti-IL5 nanobody, comprising: The Nanobody is capable of specifically binding to IL5, and the complementarity determining regions (CDRs) of the VH chain in the Nanobody comprise: (1) CDR1 as set forth in SEQ ID NO: 19, CDR2 as set forth in SEQ ID NO: 20, and CDR3 as set forth in SEQ ID NO: 21; (2) CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3; (3) CDR1 as set forth in SEQ ID NO: 10, CDR2 as set forth in SEQ ID NO: 11, and CDR3 as set forth in SEQ ID NO: 12; and (4) An anti-IL5 nanobody, characterized in that the CDR1 shown in SEQ ID NO: 28, the CDR2 shown in SEQ ID NO: 29 and the CDR3 shown in SEQ ID NO: 30 are selected from the group consisting of CDR1 shown in SEQ ID NO: 28, CDR2 shown in SEQ ID NO: 29 and CDR3 shown in SEQ ID NO:

30.

2. The VH chain of said anti-IL5 Nanobody further comprises a framework region FR, said framework region FR comprising: (1) FR1 as shown in SEQ ID NO: 4, FR2 as shown in SEQ ID NO: 5, FR3 as shown in SEQ ID NO: 6, and FR4 as shown in SEQ ID NO: 7; (2) FR1 as set forth in SEQ ID NO: 13, FR2 as set forth in SEQ ID NO: 14, FR3 as set forth in SEQ ID NO: 15, and FR4 as set forth in SEQ ID NO: 16; (3) FR1 as set forth in SEQ ID NO: 22, FR2 as set forth in SEQ ID NO: 23, FR3 as set forth in SEQ ID NO: 24, and FR4 as set forth in SEQ ID NO: 25; (4) FR1 as shown in SEQ ID NO: 31, FR2 as shown in SEQ ID NO: 32, FR3 as shown in SEQ ID NO: 33, and FR4 as shown in SEQ ID NO: 34; (5) FR1 as set forth in SEQ ID NO: 37, FR2 as set forth in SEQ ID NO: 38, FR3 as set forth in SEQ ID NO: 39, and FR4 as set forth in SEQ ID NO: 40; and (6) FR1 as shown in SEQ ID NO: 43, FR2 as shown in SEQ ID NO: 44, FR3 as shown in SEQ ID NO: 45, and FR4 as shown in SEQ ID NO:

46.

2. An anti-IL5 nanobody according to claim 1.

3. The amino acid sequence of the VH chain of said anti-IL5 Nanobody is selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 47, SEQ ID NO: 8, SEQ ID NO: 41, SEQ ID NO: 17, SEQ ID NO:

35.

2. An anti-IL5 nanobody according to claim 1.

4. An anti-IL5 antibody, An anti-IL5 antibody, characterized in that the antibody comprises one or more anti-IL5 nanobodies described in claim 1.

5. The antibodies are characterized in that they include monomeric, bivalent and / or multivalent antibodies. The antibody described in claim 4.

6. The anti-IL5 antibody is characterized in that it comprises one or more VHH chains having the amino acid sequence shown in SEQ ID NO: 26, SEQ ID NO: 47, SEQ ID NO: 8, SEQ ID NO: 41, SEQ ID NO: 17, or SEQ ID NO:

35. The antibody described in claim 4.

7. 1. An anti-IL5 Nanobody Fc fusion protein comprising: The structure of the fusion protein from N-terminus to C-terminus is as shown in formula Ia or Ib: A-L-B (Ia), B-L-A (Ib), Where: A is one or more anti-IL5 Nanobodies according to claim 1, B is an Fc fragment of IgG, L is none or a flexible linker, the flexible linker being (G ａ S ｂ ) ｘ - (G ｍ S ｎ ) ｙ wherein a, b, m, n=1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10, and x, y=0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10, and x and y are not simultaneously 0.

8. The amino acid sequence of the fusion protein is as shown in SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO:

59. The fusion protein of claim 7.

9. A polynucleotide comprising:

10. The polynucleotide, characterized in that it encodes a protein selected from the group consisting of an anti-IL5 Nanobody according to claim 1, an anti-IL5 antibody according to claim 4, or an anti-IL5 Nanobody Fc fusion protein according to claim 7.

10. An expression vector comprising: The expression vector, characterized in that it comprises the polynucleotide according to claim 9.

11. A host cell comprising: A host cell, characterized in that it comprises an expression vector according to claim 10 or has integrated into its genome a polynucleotide according to claim 9.

12. 1. A method for producing an anti-IL5 Nanobody, or an Fc-fusion protein of said anti-IL5 Nanobody, comprising the steps of: (a) culturing the host cell of claim 11, thereby obtaining a culture comprising said anti-IL5 Nanobody or an Fc-fusion protein of said anti-IL5 Nanobody; (b) isolating or recovering the anti-IL5 Nanobody or an Fc-fusion protein of the anti-IL5 Nanobody from the culture; and (c) purifying to obtain the anti-IL5 Nanobody or an Fc-fusion protein of said anti-IL5 Nanobody obtained in step (b).

13. 1. An immunoconjugate comprising: The immunoconjugate comprises: (a) an anti-IL5 Nanobody according to claim 1, or an anti-IL5 antibody according to claim 4, or an anti-IL5 Nanobody Fc-fusion protein according to claim 7, and (b) a coupling moiety selected from a detectable marker, a drug, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein, or a VLP, wherein the drug is a cytotoxic drug; An immunoconjugate comprising:

14. 1. A pharmaceutical composition comprising: The pharmaceutical composition comprises: (i) an anti-IL5 Nanobody according to claim 1, or an anti-IL5 antibody according to claim 4, or an anti-IL5 Nanobody Fc-fusion protein according to claim 7, or an immunoconjugate according to claim 13, and (ii) a pharma- ceutically acceptable carrier.

15. Use of an anti-IL5 Nanobody according to claim 1, an anti-IL5 antibody according to claim 4, an anti-IL5 Nanobody Fc-fusion protein according to claim 7, or an immunoconjugate according to claim 13, in (a) for use in the preparation of a medicament for the prevention and / or treatment of a disease or condition associated with IL5 / IL5R signaling, the disease or condition associated with IL5 / IL5R signaling comprising asthma, atopic dermatitis, arthritis, allergic rhinitis, eczema, sinusitis, nasal polyps, chronic obstructive pulmonary disease, eosinophilic granulomatous polyangiitis, hypereosinophilic syndrome, or (b) Use thereof for the preparation of a reagent, a detection plate or a kit for detecting IL5.

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