Single-domain antibodies against il-4ra and uses and medicaments

Abstract

This invention discloses a single-domain antibody against IL-4Rα, its applications, and pharmaceuticals, relating to the field of antibody technology. The single-domain antibody disclosed in this invention has the amino acid sequence of CDR1 as shown in any one of SEQ ID NO. 37-53, the amino acid sequence of CDR2 as shown in any one of SEQ ID NO. 54-68, and the amino acid sequence of CDR3 as shown in any one of SEQ ID NO. 69-85. This single-domain antibody can specifically bind to IL-4Rα and can be used for the prevention, diagnosis, and / or treatment of IL-4Rα-related diseases such as autoimmune diseases.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically, to single-domain antibodies against IL-4Rα, as well as their applications and pharmaceuticals. Background Technology

[0002] IL-4 and IL-4Rα are ideal drug targets, primarily used to treat human diseases associated with type 2 helper T (Th2) cell abnormalities. The human IL-4Rα subunit (Swiss Prot database accession number: P24394) is a type I membrane protein with a molecular weight of 140 kDa, exhibiting high affinity for human IL-4. The IL-4 / IL-4Rα complex can dimerize IL-4Rα with the CD132 or IL-13Rα1 subunits via domains on the IL-4 molecule, forming two distinct signaling molecule complexes corresponding to class I and class II receptors, respectively. Furthermore, the complex formed by IL-13 binding to IL-13Rα1 can recruit the IL-4Rα subunit, similarly forming a class II receptor complex. Therefore, IL-4Rα can regulate the biological activities associated with both IL-4 and IL-13 molecules (Gessner et al., Immunobiology, 201:285, 2000). In vitro studies have shown that the activating effects of IL-4 and IL-13 affect a variety of cells, including T lymphocytes, B lymphocytes, eosinophils, giant cells, basophils, airway smooth muscle cells, lung fibroblasts, and endothelial cells.

[0003] IL4Rα expression levels are low across various cell types, typically ranging from 100 to 5000 molecules per cell (Lowenthal et al., J Immunol, 140:456, 1988), such as peripheral blood T cells, monocytes, airway epithelial cells, B cells, and lung fibroblasts. Type I receptors show a dominant expression on the surface of hematopoietic cells, while type II receptors are expressed on both hematopoietic and non-hematopoietic cell surfaces.

[0004] The polymorphism of IL-4Rα in human populations has been discussed in previous studies (Gessner et al, Immunobiology, 201:285, 2000). Studies have shown that the polymorphism of this molecule is associated with IgE levels or clinical atopy in some populations. For example, V75R576 IL-4Rα is associated with allergic asthma and enhanced IL-4Rα function (Risma et al, J. Immunol. 169(3) 1604-1610, 2002).

[0005] Numerous pieces of evidence suggest that the IL-4 / IL-13 pathway plays a crucial role in asthma pathology (see Chatila, Trends in Molecular Med, 10:493, 2004 for a review) and in numerous other conditions listed elsewhere in this article. Increased secretion of IL-4 and IL-13 is currently believed to trigger and maintain this disease process. IL-13 is considered an important ligand for triggering airway hyperresponsiveness (AHR), excessive mucus secretion, and airway remodeling, while IL-4 is considered a major contributing factor to Th2 polarization and IgE production (Wynn, Annu Rev Immunol, 21:425, 2003).

[0006] Evidence from animal models further supports the role of IL-4Rα in asthma. During allergen stimulation with ovalbumin (OVA, a model allergen), IL-4R antagonists (IL-4 mutant; C118 deletion) inhibited OVA-sensitized functional mice from developing allergic airway eosinophilia and AHR (Tomkinson et al., J. Immunol. 166(9):5792-5800, 2001). Furthermore, multiple in vivo studies have confirmed the positive effects of blocking IL-13 or IL-14 in animal models of asthma. For example, in a chronic model of OVA-induced persistent airway inflammation, therapeutic doses of anti-IL-13 monoclonal antibodies inhibited AHR, halted the progression of subepithelial fibrosis and inflammation, and restored hypermucus secretion to basal levels (Yang et al., J. Pharmacol. Exp. Ther. 313(1):8-15, 2005). In a mouse OVA model, but when administered during immunization, inhibition of IL-4 with anti-IL-4 antibodies showed a significant reduction in eosinophil infiltration (Coyle et al., Am J Resp Cell Mol Biol, 13:54, 1995). In similar models, IL-4-deficient mice showed significantly fewer eosinophils in bronchoalveolar lavage fluid and much less peribronchial inflammation after OVA stimulation (Brusselle et al., Clin Exp Allergy, 24:73, 1994).

[0007] In humans, phase IIa clinical studies have shown that IL-4Rα antagonists (so-called IL-4 mutant proteins) reduce allergen-induced delayed asthma responses and attenuate resting-phase lung inflammation in asthmatic patients (Wenzel et al., Lancet, 370:1422, 2007). These clinical data further reinforce the claim that IL-4Rα antagonists have clinical applicability in asthma.

[0008] Besides its role in asthma, IL-4Rα is associated with many other pathological conditions, such as COPD, including patients with varying degrees of chronic bronchitis, small airway disease, and emphysema. The underlying causes of COPD remain unknown. The "Dutch hypothesis" suggests that COPD and asthma share a common susceptibility, and therefore, similar mechanisms may contribute to the development of these two conditions (Sluiter et al., Eur Respir J, 4(4): p. 479-89, 1991). Zheng et al. (J Clin Invest, 106(9): 1081-93, 2000) demonstrated that overexpression of IL-13 in mouse lungs causes emphysema, increased mucosal production, and inflammation, which can reflect many characteristics of human COPD. Furthermore, IL-13-dependent response (AHR) in a mouse model of allergic inflammation has been shown to predict decreased lung function in smokers (Tashkin et al., Am J Respir Crit Care Med, 153(6Pt1): 1802-11, 1996). Furthermore, the association between IL-13 promoter polymorphism and susceptibility to COPD has been established (VanDer Pouw Kraan et al., Genes Immun, 3(7):436-9, 2002). Therefore, it is the IL-4 / IL-13 pathway, especially IL-13, that plays an important role in the pathogenesis of COPD.

[0009] IL-13 may play a role in the pathogenesis of inflammatory bowel disease. Heller et al. (Heller et al., Immunity, 17(5):629-38, 2002) reported that administration of soluble IL-13Rα2 to neutralize IL-13 improved colonic inflammation in a mouse model of human ulcerative colitis. Correspondingly, IL-13 expression was higher in rectal biopsy samples from patients with ulcerative colitis compared with controls (Inoue et al., Am J Gastroenterol, 94(9) JMl-6, 1999).

[0010] Besides asthma, the IL-4 / IL-13 pathway is also associated with other fibrotic conditions, such as systemic scleroderma (Hasegawa et al., J Rheumatol, 24(2):328-32, 1997), pulmonary fibrosis (Hancock et al., Am J Respir Cell MolBiol, 18(1):60-5, 1998), parasite-induced liver fibrosis (Fallon et al., J Immunol, 164(5):2585-91, 2000; Chiaramonte et al., J Clin Invest, 104(6):777-85, 1999; Chiaramonte, Hepatology 34(2):273-82, 2001), and bladder fibrosis (Hauber et al., J. Cyst Fibr, 2 189, 2003).

[0011] IL-4 is crucial for B cell-mediated activities such as B cell proliferation, immunoglobulin secretion, and FcεR expression. Clinical applications of IL-4Rα inhibitors include, for example, inhibiting IgE synthesis in allergy treatment (e.g., atopic dermatitis and food allergies), preventing transplant rejection in transplant therapy, and suppressing delayed-type anaphylactic or contact anaphylactic reactions.

[0012] IL-4R antagonists can also be used as adjuvants in allergic immunotherapy and as vaccine adjuvants. Antibodies against IL-4Rα have been described. Two examples are the neutralizing mouse anti-IL-4Rα monoclonal antibodies MAB230 (clone 25463) and 16146 (clone 25463.11), provided by R&D Systems and Sigma, respectively. These antibodies belong to the IgG2a subtype and were obtained via a mouse hybridoma method, developed from mice immunized with purified recombinant human IL-4Rα (baculovirus-derived). Two other neutralizing mouse anti-IL-4Rα antibodies, M57 and X2 / 45-12, were provided by BDBiosciences and eBioscience, respectively. These are IgG1 antibodies, also obtained via mouse hybridoma technology from mice immunized with recombinant soluble IL-4Rα.

[0013] However, there is still a need in the field for anti-IL-4Rα antibodies that can bind with high affinity to IL-4Rα and block the binding of IL-4 to IL-4Rα, especially anti-IL-4Rα heavy chain single-domain antibodies.

[0014] In view of this, the present invention is proposed. Summary of the Invention

[0015] The purpose of this invention is to provide a single-domain antibody against IL-4Rα, as well as its applications and pharmaceutical uses. This single-domain antibody can specifically bind to IL-4Rα, has high affinity, is easy to prepare, and has low production costs. Furthermore, this single-domain antibody exhibits low immunogenicity, and when used as a drug, it will not produce a strong immune response, thus showing broad application prospects.

[0016] This invention is implemented as follows:

[0017] In a first aspect, the present invention provides a single-domain antibody against IL-4Rα, comprising the following complementarity-determining regions: CDR1, CDR2 and CDR3;

[0018] The amino acid sequence of CDR1 is shown in any one of SEQ ID NO.37-53, the amino acid sequence of CDR2 is shown in any one of SEQ ID NO.54-68, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NO.69-85.

[0019] The single-domain antibody against IL-4Rα provided by this invention has CDR1, CDR2, and CDR3 that can vary or be selected within the sequence ranges of SEQ ID NO.37-53, SEQ ID NO.54-68, and SEQ ID NO.69-85, respectively. Regardless of the sequence combination of CDR1, CDR2, and CDR3, the resulting single-domain antibody can specifically bind to IL-4Rα and has good affinity. It can be used to prepare treatments for IL-4Rα-mediated diseases and reagents for detecting IL-4Rα. This single-domain antibody has a wide range of applications.

[0020] In addition, the single-domain antibody provided by this invention has a simple structure, is easy to prepare, has low production cost, and due to its low immune heterogeneity, it is not easy to produce an immune response when used as a drug to treat IL-4Rα-mediated diseases, and has good application prospects.

[0021] It should be noted that, based on the structure of the complementarity-determining region disclosed in this invention, those skilled in the art can easily conceive of replacing or deleting one or more amino acids to obtain single-domain antibody mutants with approximately the same or improved binding activity or affinity for IL-4Rα. This is easily achieved by those skilled in the art, and therefore, such single-domain antibody mutants also fall within the scope of protection of this invention.

[0022] Optionally, in some embodiments of the present invention, the complementarity-determining region of the single-domain antibody is shown in any one of the following (1)-(18):

[0023] (1): CDR1 is shown in SEQ ID NO.53, CDR2 is shown in SEQ ID NO.61, and CDR3 is shown in SEQ ID NO.73;

[0024] (2): CDR1 is shown in SEQ ID NO.37, CDR2 is shown in SEQ ID NO.61, and CDR3 is shown in SEQ ID NO.72;

[0025] (3): CDR1 is shown in SEQ ID NO.39, CDR2 is shown in SEQ ID NO.58, and CDR3 is shown in SEQ ID NO.78;

[0026] (4): CDR1 is shown in SEQ ID NO.53, CDR2 is shown in SEQ ID NO.63, and CDR3 is shown in SEQ ID NO.72;

[0027] (5): CDR1 is shown in SEQ ID NO.42, CDR2 is shown in SEQ ID NO.64, and CDR3 is shown in SEQ ID NO.74;

[0028] (6): CDR1 is shown in SEQ ID NO.46, CDR2 is shown in SEQ ID NO.66, and CDR3 is shown in SEQ ID NO.69;

[0029] (7): CDR1 is shown in SEQ ID NO.50, CDR2 is shown in SEQ ID NO.68, and CDR3 is shown in SEQ ID NO.85;

[0030] (8): CDR1 is shown in SEQ ID NO.48, CDR2 is shown in SEQ ID NO.55, and CDR3 is shown in SEQ ID NO.80;

[0031] (9): CDR1 is shown in SEQ ID NO.47, CDR2 is shown in SEQ ID NO.60, and CDR3 is shown in SEQ ID NO.83;

[0032] (10): CDR1 is shown as SEQ ID NO.49, CDR2 is shown as SEQ ID NO.67, and CDR3 is shown as SEQ ID NO.81;

[0033] (11): CDR1 is shown in SEQ ID NO.38, CDR2 is shown in SEQ ID NO.58, and CDR3 is shown in SEQ ID NO.77;

[0034] (12): CDR1 is shown as SEQ ID NO.45, CDR2 is shown as SEQ ID NO.54, and CDR3 is shown as SEQ ID NO.70;

[0035] (13): CDR1 is shown as SEQ ID NO.41, CDR2 is shown as SEQ ID NO.59, and CDR3 is shown as SEQ ID NO.84;

[0036] (14): CDR1 is shown as SEQ ID NO.43, CDR2 is shown as SEQ ID NO.57, and CDR3 is shown as SEQ ID NO.82;

[0037] (15): CDR1 is shown as SEQ ID NO.40, CDR2 is shown as SEQ ID NO.56, and CDR3 is shown as SEQ ID NO.76;

[0038] (16): CDR1 is shown as SEQ ID NO.44, CDR2 is shown as SEQ ID NO.58, and CDR3 is shown as SEQ ID NO.75;

[0039] (17): CDR1 is shown as SEQ ID NO.52, CDR2 is shown as SEQ ID NO.62, and CDR3 is shown as SEQ ID NO.71;

[0040] (18): CDR1 is shown as SEQ ID NO.51, CDR2 is shown as SEQ ID NO.65, and CDR3 is shown as SEQ ID NO.79.

[0041] Single-domain antibodies with the CDR structures shown in items (1)-(18) above can specifically bind to IL-4Rα and have high affinity, especially the CDR structures shown in items (1), (3) and (6).

[0042] Optionally, in some embodiments of the present invention, the single-domain antibody includes the following backbone regions: FR1, FR2, FR3 and FR4;

[0043] The amino acid sequences of FR1 are shown in any one of SEQ ID NO. 86-97; the amino acid sequences of FR2 are shown in any one of SEQ ID NO. 98-109; the amino acid sequences of FR3 are shown in any one of SEQ ID NO. 110-127; and the amino acid sequences of FR4 are shown in SEQ ID NO. 128.

[0044] It should be noted that the function of single-domain antibodies to specifically bind to IL-4Rα mainly depends on the CDR region. Based on this, while the single-domain antibody backbone region and CDR region sequences are disclosed in this invention, those skilled in the art will readily conceive of using sequences similar to the backbone region sequences disclosed in this invention, for example, sequences with homology of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher, as the backbone region sequence of the single-domain antibody, provided that the CDR region sequence remains unchanged or is slightly modified. This would allow the obtained single-domain antibody to have the same or similar function as the single-domain antibody described above in this invention, which is also within the scope of protection of this invention.

[0045] Optionally, in some embodiments of the present invention, the backbone regions FR1, FR2, and FR3 of the single-domain antibody are shown in any one of the following (a)-(r):

[0046] (a) FR1 is shown in SEQ ID NO.88, FR2 is shown in SEQ ID NO.100, and FR3 is shown in SEQ ID NO.115;

[0047] (b) FR1 is shown in SEQ ID NO.88, FR2 is shown in SEQ ID NO.100, and FR3 is shown in SEQ ID NO.114;

[0048] (c) FR1 is shown in SEQ ID NO.95, FR2 is shown in SEQ ID NO.102, and FR3 is shown in SEQ ID NO.123;

[0049] (d) FR1 is shown in SEQ ID NO.86, FR2 is shown in SEQ ID NO.100, and FR3 is shown in SEQ ID NO.117;

[0050] (e) FR1 is shown in SEQ ID NO. 94, FR2 is shown in SEQ ID NO. 105, and FR3 is shown in SEQ ID NO. 113;

[0051] (f) FR1 is shown in SEQ ID NO.87, FR2 is shown in SEQ ID NO.99, and FR3 is shown in SEQ ID NO.110;

[0052] (g) FR1 is shown in SEQ ID NO.91, FR2 is shown in SEQ ID NO.109, and FR3 is shown in SEQ ID NO.111;

[0053] (h)FR1 is shown in SEQ ID NO.87, FR2 is shown in SEQ ID NO.100, and FR3 is shown in SEQ ID NO.119;

[0054] (i) FR1 is shown in SEQ ID NO.89, FR2 is shown in SEQ ID NO.101, and FR3 is shown in SEQ ID NO.124;

[0055] (j) FR1 is shown in SEQ ID NO.93, FR2 is shown in SEQ ID NO.98, and FR3 is shown in SEQ ID NO.112;

[0056] (k)FR1 is shown in SEQ ID NO.94, FR2 is shown in SEQ ID NO.106, and FR3 is shown in SEQ ID NO.125;

[0057] (l) FR1 is shown in SEQ ID NO.92, FR2 is shown in SEQ ID NO.107, and FR3 is shown in SEQ ID NO.127;

[0058] (m)FR1 is shown in SEQ ID NO.87, FR2 is shown in SEQ ID NO.103, and FR3 is shown in SEQ ID NO.120;

[0059] (n)FR1 is shown in SEQ ID NO.87, FR2 is shown in SEQ ID NO.108, and FR3 is shown in SEQ ID NO.126;

[0060] (o)FR1 is shown in SEQ ID NO.97, FR2 is shown in SEQ ID NO.105, and FR3 is shown in SEQ ID NO.121;

[0061] (p)FR1 is shown in SEQ ID NO.90, FR2 is shown in SEQ ID NO.105, and FR3 is shown in SEQ ID NO.122;

[0062] (q)FR1 is shown in SEQ ID NO.88, FR2 is shown in SEQ ID NO.100, and FR3 is shown in SEQ ID NO.116;

[0063] (r)FR1 is shown in SEQ ID NO.96, FR2 is shown in SEQ ID NO.104, and FR3 is shown in SEQ ID NO.118.

[0064] Optionally, in some embodiments of the present invention, the amino acid sequence of the single-domain antibody is shown in any one of SEQ ID NO. 1-18.

[0065] In a second aspect, the present invention provides a fusion protein comprising a functional domain capable of specifically binding to IL-4Rα, said functional domain being composed of a single-domain antibody against IL-4Rα as described above.

[0066] The single-domain antibody provided by this invention can be fused with any other protein or substance to achieve different purposes. For example, it can bind to fluorescent proteins, enzymes, or radioactive elements to facilitate detection, or it can be fused with drug molecules for treating IL-4Rα-mediated diseases to achieve better therapeutic effects. The type of protein fused with this single-domain antibody can be rationally selected by those skilled in the art according to actual needs or purposes. Regardless of the type of substance fused, it falls within the protection scope of this invention.

[0067] Thirdly, the present invention provides an antibody against IL-4Rα, wherein the antibody is a conventional antibody or a functional fragment thereof, and the heavy chain variable region of the antibody is composed of a single-domain antibody against IL-4Rα as described in any of the preceding claims.

[0068] Traditional antibodies are structurally composed of two identical heavy chains and two identical light chains. The light chains have a variable region (VL) and a constant region (CL); the heavy chains have a variable region (VH) and a constant region (CH1, CH2, CH3, and / or CH4). Given that this invention discloses the structure of a single-domain antibody capable of specifically binding IL-4Rα, those skilled in the art will readily conceive of modifying traditional antibodies using the single-domain antibody of this invention. For example, applying the CDR region structure of the single-domain antibody of this invention to a traditional antibody can yield a traditional antibody capable of specifically binding IL-4Rα. Such traditional antibodies also fall within the scope of protection of this invention. Furthermore, based on the structure of traditional antibodies, some of their structures, such as Fab, Fab', (Fab')2, Fv, scFv, or sdFv structures, also possess IL-4Rα binding specificity, and these also fall within the scope of protection of this invention.

[0069] Optionally, in some embodiments of the present invention, the functional fragment is the Fab, Fab', (Fab')2, Fv, scFv or sdFv structure of the conventional antibody.

[0070] The use of the single-domain antibody against IL-4Rα as described above, the fusion protein as described above, or the antibody as described above in the preparation of a medicament targeting IL-4Rα to treat a disease.

[0071] Optionally, in some embodiments of the present invention, the disease is selected from asthma, allergic dermatitis, eczema, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scars, chronic obstructive pulmonary disease (COPD), atopic dermatitis, idiopathic pulmonary fibrosis, Kawasaki disease, sickle cell disease, Graves' disease, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and nephropathy.

[0072] The antibody of the present invention can be used to treat diseases by neutralizing IL-4Rα, which targets IL-4Rα, thereby achieving the effect of treating diseases. Such diseases include, but are not limited to, the diseases mentioned above. Its use in the treatment of other diseases is also within the scope of protection of the present invention.

[0073] Fourthly, the present invention provides a medicament for treating a disease, comprising a single-domain antibody against IL-4Rα as described in any of the preceding claims, a fusion protein as described above, or an antibody as described above, and pharmaceutically acceptable excipients.

[0074] The pharmaceutically acceptable excipients mentioned above refer to pharmaceutical excipients used in the pharmaceutical field, such as diluents, fillers, binders, humectants, absorption enhancers, surfactants, disintegrants, adsorbent carriers, lubricants, etc. Other excipients, such as flavoring agents and sweeteners, may also be added. That is, the excipients mentioned are one or more of the following: diluents, fillers, binders, humectants, absorption enhancers, surfactants, disintegrants, adsorbent carriers, lubricants, flavoring agents, and sweeteners.

[0075] The dosage form of the drug provided by this invention is not strictly limited and can be prepared into various dosage forms according to existing methods in the pharmaceutical field, and administered to patients in need of treatment via oral, nasal, rectal, parenteral, or transdermal administration.

[0076] Fifthly, the present invention provides an isolated nucleic acid molecule that encodes a single-domain antibody as described in any of the preceding claims.

[0077] It should be noted that, based on the content disclosed in this invention, those skilled in the art can easily obtain the polynucleotide molecules encoding the above-mentioned single-domain antibodies and fusion proteins using conventional techniques in the field. Based on the degeneracy of codons, the polynucleotide molecules are variable, and their specific base sequences have multiple possibilities. Therefore, regardless of how the polynucleotide molecules change, as long as they can encode the single-domain antibodies or fusion proteins of this invention, they fall within the protection scope of this invention.

[0078] Optionally, in some embodiments of the present invention, the base sequence of the nucleic acid molecule is as shown in any one of SEQ ID NO.19-36.

[0079] In a sixth aspect, the present invention provides a carrier containing the nucleic acid molecules described above.

[0080] In a seventh aspect, the present invention provides recombinant cells containing the vector described above.

[0081] Eighthly, the present invention provides a method for preparing a single-domain antibody as described in any of the preceding claims, comprising: culturing recombinant cells as described above, and isolating and purifying the single-domain antibody from the culture product.

[0082] It should be noted that the single-domain antibodies, fusion proteins and antibodies of the present invention can be prepared by chemical synthesis, genetic engineering technology or other methods. Regardless of the method used to prepare the aforementioned single-domain antibodies, fusion proteins or antibodies of the present invention, they are all within the protection scope of the present invention. Attached Figure Description

[0083] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0084] Figure 1 Figure showing the purification results of human recombinant protein IL-4Rα.

[0085] Figure 2 The binding activity of the company's self-produced IL-4Rα recombinant protein with the tool antibody was determined. The IL-4Rα recombinant protein purchased from nearshore was designated as the control (Novo), the self-produced IL-4Rα recombinant protein was designated as RJK, the tool antibody was designated as Tab, and the human IgG was used as the negative isotype control antibody.

[0086] Figure 3 The enrichment results after screening a single-domain antibody library targeting the recombinant IL-4Rα protein are shown. P / N = number of monoclonal bacteria grown from phages eluted from positive wells in the biopanning process after infecting TG1 bacteria / number of monoclonal bacteria grown from phages eluted from negative wells after infecting TG1 bacteria. This parameter gradually increases after enrichment occurs. I / E = total number of phages added to positive wells in each round of biopanning / total number of phages eluted from positive wells in each round of biopanning. This parameter gradually approaches 1 after enrichment occurs. In the third round of enrichment, P / N = 1000 and I / E = 2.

[0087] Figure 4 The results of SDS-PAGE analysis of single-domain antibodies after one-step affinity chromatography on a nickel column are shown.

[0088] Figure 5 The affinity-efficacy curves of the purified single-domain antibody with IL-4Rα are shown.

[0089] Figure 6 The dose-response curve of the neutralization experiment of the purified Fc fusion single-domain nanobody in the IL-4-induced TF-1 cell proliferation assay.

[0090] Figure 7 The dose-response curve of the neutralization experiment of the purified Fc fusion single-domain nanobody in the IL-13-induced TF-1 cell proliferation assay.

[0091] Figure 8 The images show the affinity kinetics curves and curve fitting results for single-domain antibodies 1H1 and 4E9. Detailed Implementation

[0092] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0093] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0094] Example 1

[0095] Constructing an expression vector for human recombinant IL-4Rα protein

[0096] The method is as follows:

[0097] (1) The coding sequence of IL-4Rα was obtained by searching in NCBI, with accession number NM_000418.3. The amino acid sequence generated by this sequence has accession number NP_000409.1.

[0098] (2) The amino acid sequence corresponding to NP_000409.1 was analyzed for transmembrane region and extracellular terminus using TMHMM and SMART websites, respectively.

[0099] (3) The analysis results show that the extracellular terminus of the IL-4Rα protein consists of amino acids 1-232, of which 1-25 are the signal peptide of the protein.

[0100] (4) The nucleotide sequence encoding the IL-4Rα protein from amino acid position 26 to 232 was cloned into the vector pcDNA3.4 using gene synthesis.

[0101] (5) The constructed vector was subjected to Sanger sequencing. After comparing with the original sequence and confirming that there were no errors, the recombinant plasmid was extracted in batches, endotoxin was removed, and it was transfected into suspension 293F for expression and purification of the target protein. The SDS-PAGE and Western blot analysis results of the purified IL-4Rα recombinant protein are as follows: Figure 1 As shown.

[0102] Example 2: Determination of the binding activity of human recombinant protein

[0103] The experimental method for determining binding activity was performed according to the method disclosed in Chinese Invention Patent Application No. 202010166444.8, entitled "Single-domain Antibody Specifically Binding to EpCAM and Its Application Thereof." Recombinant IL-4Rα protein purchased from nearshore areas was used as a positive control recombinant protein, and human IgG was used as a negative isotype control for the tool antibody. Curves were plotted, and the results are shown below. Figure 2 As shown, although the color development of the self-produced protein was weaker at high concentrations compared to the control, its binding ability to the tool antibody (EC50) was comparable. Neither the human IgG isotype control nor the IL-4Rα recombinant protein purchased from nearshore and the company's own production bound the protein.

[0104] Example 3

[0105] Constructing a single-domain antibody library targeting the IL-4Rα protein

[0106] The method is as follows:

[0107] (1) 2 mg of the IL-4Rα recombinant protein purified in Example 1 was mixed with an equal volume of Freund's complete adjuvant and used to immunize a Bactrian camel from Alashan, Inner Mongolia. The immunization was performed once a week for a total of 7 consecutive immunizations. Except for the first immunization, the remaining six immunizations were performed by mixing 1 mg of IL-4Rα recombinant protein with an equal volume of Freund's incomplete adjuvant. This immunization process was intended to stimulate the camel to produce antibodies against IL-4Rα.

[0108] (2) After the animal immunization was completed, 100 mL of peripheral blood lymphocytes were extracted from the camel and RNA was extracted from the cells.

[0109] (3) Use the extracted total RNA to synthesize cDNA, and amplify VHH (heavy chain antibody variable region) using cDNA as a template by nested PCR reaction.

[0110] (4) The pMECS vector and VHH fragment were digested with restriction endonucleases, and then the digested fragments and vector were ligated.

[0111] (5) The ligated fragment was transformed into competent TG1 cells to construct a phage display library of IL-4Rα protein and the library size was measured. The library size was approximately 1 × 10⁻⁶.9 .

[0112] Example 4

[0113] Screening for single-domain antibodies targeting the IL-4Rα protein

[0114] The method is as follows:

[0115] (1) Take 200 μL of recombinant TG1 cells from Example 2 and inoculate them into 2×TY medium. During the culture, add 40 μL of helper phage VCSM13 to infect TG1 cells and culture overnight to amplify the phage. The next day, precipitate the phage with PEG / NaCl and collect the amplified phage by centrifugation.

[0116] (2) 500 μg of recombinant IL-4Rα protein diluted in 100 mM pH 8.3 NaHCO3 was coupled onto an ELISA plate and incubated overnight at 4°C. A negative control well was also set up.

[0117] (3) On the second day, add 200 μL of 3% skim milk and seal at room temperature for 2 hours.

[0118] (4) After blocking, add 100 μL of the amplified phage library (approximately 2 × 10⁻⁶). 11 (One phage particle), incubated at room temperature for 1 hour.

[0119] (5) After 1 hour of treatment, wash 5 times with PBS (containing 0.05% Tween-20) to remove unbound phages.

[0120] (6) Phages specifically bound to the IL-4Rα recombinant protein were dissociated using trypsin at a final concentration of 2.5 mg / mL and then used to infect *E. coli* TG1 cells in the logarithmic growth phase. The cells were cultured at 37°C for 1 hour to produce and collect phages for the next round of screening. This screening process was repeated once to gradually enrich the cells. When the enrichment factor reached 10-fold or more, the enrichment effect was as follows: Figure 3 As shown.

[0121] Example 5

[0122] Use phage enzyme-linked immunosorbent assay (ELISA) to screen for specific positive clones against IL-4Rα.

[0123] The method is as follows:

[0124] (1) The IL-4Rα recombinant protein was screened for 3 rounds according to the single-domain antibody screening method in Example 2. After the screening, the phage enrichment factor for the IL-4Rα recombinant protein reached more than 10. 400 single colonies were selected from the positive clones obtained by screening and inoculated into 96-well plates of TB medium containing 100 μg / mL ampicillin. A blank control was set up. After the culture reached the logarithmic phase at 37°C, IPTG with a final concentration of 1 mM was added and cultured overnight at 28°C.

[0125] (2) Crude antibody was obtained by osmotic rupture method; IL-4Rα recombinant protein was released into 100mM NaHCO3 at pH 8.3, and 100μg IL-4Rα recombinant protein was coated in an enzyme-labeled plate at 4℃ overnight;

[0126] (3) Take 100 μL of the crude antibody extract obtained in the above steps and transfer it to an ELISA plate containing the antigen, and incubate at room temperature for 1 h;

[0127] (4) Wash away unbound antibodies with PBST, add 100 μl of Mouse anti-HA tag antibody diluted 1:2000 (Thermo Fisher), and incubate at room temperature for 1 h.

[0128] (5) Wash away unbound antibody with PBST, add 100ul of Anti-Rabbit HRPconjugate (goat anti-rabbit horseradish peroxidase labeled antibody, purchased from Thermo Fisher) diluted 1:20000, and incubate at room temperature for 1h.

[0129] (6) Wash away unbound antibodies with PBST, add horseradish peroxidase colorimetric solution, react at 37°C for 15 min, add stop solution, and read the absorbance value at 450 nm wavelength on the microplate reader.

[0130] (7) When the OD value of the sample well is more than 5 times that of the control well, it is judged as a positive clone well;

[0131] (8) The bacteria in the positive clone wells were transferred to LB medium containing 100 μg / μL ampicillin in order to extract plasmids and perform sequencing.

[0132] (9) The gene sequences of each clone were analyzed using the sequence alignment software Vector NTI. Clones with identical CDR1, CDR2, and CDR3 sequences were considered the same clone, while clones with different sequences were considered different clones. A single-domain antibody specifically targeting the IL-4Rα protein was obtained. The amino acid sequence of this antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, constituting the entire VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system to obtain a single-domain antibody targeting the IL-4Rα protein.

[0133] The serial numbers of each single-domain antibody and their corresponding amino acid sequence analysis results, i.e., the sequence identifiers (SEQ ID NO.), are shown in Table 1 below.

[0134] Table 1. Sequence identifiers (SEQ ID NO.) corresponding to the CDR and FR of each single-domain antibody in the examples.

[0135]

[0136] Example 5

[0137] Purification and expression of a specific single-domain antibody against IL-4Rα protein in the host bacterium *Escherichia coli*.

[0138] The method is as follows:

[0139] (1) The plasmids (pMECS-VHH) of different clones obtained from the above sequencing analysis were electroporated into Escherichia coli HB2151 and plated on LB+amp+glucose culture plates containing ampicillin and glucose, and incubated overnight at 37°C.

[0140] (2) Select a single colony and inoculate it into 5 mL of LB broth containing penicillin, and incubate overnight at 37°C in a shaker.

[0141] (3) Inoculate 1 mL of overnight cultured bacteria into 330 mL of TB culture medium, and culture on a shaker at 37°C. When the OD600nm value reaches 0.6-0.9, add 1 M IPTG and culture on a shaker at 28°C overnight.

[0142] (4) Centrifuge to collect E. coli and use osmotic rupture method to obtain crude antibody extract;

[0143] (5) The antibody was purified by nickel column affinity chromatography. The SDS-PAGE analysis results of the purified single-domain antibody are as follows: Figure 4 As shown in the figure, VHH1-18 correspond to the antibody numbers in Table 1.

[0144] Example 6

[0145] The preparation method for the Fc fusion antibody, which is an Fc fusion of the above-mentioned single-domain antibody, is based on the method disclosed in Chinese invention patent application No. 202010166444.8, entitled "Single-domain antibody that can specifically bind to EpCAM and its application".

[0146] Example 7

[0147] Determination of the binding dose-response curve of a specific single-domain antibody against IL-4Rα protein.

[0148] The experimental method was performed according to the method disclosed in Chinese invention patent application number 202010166444.8, entitled "Single-domain antibody that specifically binds to EpCAM and its application thereto." Curves were plotted, and EC50 was calculated. 50 The result is as follows Figure 5 As shown in Table 2 below.

[0149] Table 2 EC50 of single-domain antibody binding to recombinant IL-4Rα protein 50

[0150] Antibody number 1A9 1B1 1B2 1B4 1C4 1D9 1H1 2A7 2C10 <![CDATA[EC 50 (nM)]]> 2.724 0.850 5.816 0.404 2.686 0.103 1.132 3.060 4.150 Antibody number 2C9 2H2 3C11 3E10 3F9 3G10 4B3 4E9 4H11 <![CDATA[EC 50 (nM)]]> 26.29 2.168 1.379 1.838 1.602 5.207 1.364 2.135 16.290

[0151] It can be seen that in the binding detection of 18 purified single-domain antibodies, except for antibodies 4H11 and 2C9 which have a binding capacity of more than 10 nM, the binding capacity of the other antibodies is less than 10 nM, and the binding capacity of antibodies 1B4 and 1D9 is less than 1 nM, which proves that the overall binding capacity of the single-domain antibodies screened in this embodiment is good.

[0152] Example 8

[0153] The expression and purification of tool antibody (Tab) targeting human IL-4Rα: Tab1 (dupilumab) sequence is from IMGT, and Tab2 (L1012H1031) and Tab3 (L1020H1031) sequences are from the patent (patent number: CN107474134).

[0154] The method is as follows:

[0155] (1) The Tab sequence was commissioned to General Biosystems (Anhui) Co., Ltd. for codon optimization of mammalian cell expression system and cloned into pcDNA3.1 vector.

[0156] (2) After resistance screening, plasmid-positive bacteria were selected for amplification, and plasmids were extracted using a plasmid extraction kit (Macherey Nagel, Cat#740412.50).

[0157] (3) Add 100 μg of plasmid (50 μg heavy chain + 50 μg light chain) per 100 mL of cells and use PEI to transiently express the plasmid in 293F cells (culture medium: FreeStyle 293Expression medium, Thermo, Cat#12338026+F-68, Thermo, Cat#24040032).

[0158] (4) After transfection for 6-24 hours, add 5% volume of 10% Peptone (Sigma, Cat#P0521-100G), and incubate at 8% CO2 and 130 rpm for about 7-8 days.

[0159] (5) When the cell viability dropped to 50%, the expression supernatant was collected and purified using a Protein A (GE, Cat#17-5438-02) gravity column.

[0160] (6) After PBS dialysis, the concentration was determined using Nanodrop, the purity was identified by SEC, and the binding ability was verified by indirect ELISA.

[0161] (7) The Tab obtained by the above method has a concentration of not less than 2 mg / ml and a purity of more than 94%. The EC50 of the Tab binding to IL-4Rα (Novoprotein, Cat#CS33) and the self-produced IL-4Rα recombinant protein is 0.25±0.05 nM.

[0162] Example 9

[0163] Human IL-4-induced TF1 cell proliferation and tool antibody neutralization proliferation experiment.

[0164] 1. Experimental method for human IL-4-induced TF1 cell proliferation:

[0165] (1) Seed 10,000 TF-1 cells per well in a 96-well plate after 3-4 passages following resuscitation.

[0166] (2) Prepare a solution of human IL-4 protein with a maximum concentration of 500 ng / mL and perform serial dilutions of 5-fold;

[0167] (3) Add the serially diluted IL-4 solution to the cell culture wells in equal volumes to the cell culture medium;

[0168] (4) After incubation for 72 hours, cell viability was detected using a luminescent cell viability assay kit;

[0169] (5) Calculate the EC80 concentration of IL-4 to induce TF-1 cell proliferation based on the test results.

[0170] 2. Experimental method for neutralizing human IL-4-induced TF1 cell proliferation with tool antibody (Tab):

[0171] (1) Seed 10,000 TF-1 cells per well in a 96-well plate after 3-4 passages following resuscitation.

[0172] (2) Prepare solutions of different Tabs at 10 μg / mL and perform serial dilutions of 5 times;

[0173] (3) Mix the serially diluted Tab with the EC80 concentration of IL-4 obtained in the proliferation experiment at a ratio of 1:1 to prepare a mixture;

[0174] (4) Add the mixture from the previous step to the cell culture wells in an equal volume to the cell culture medium;

[0175] (5) After incubation for 72 hours, cell viability was detected using a luminescent cell viability assay kit;

[0176] (6) The EC50 concentration of Tab that neutralizes IL-4-induced TF-1 cell proliferation was calculated based on the detection results, and the results are shown in Table 3.

[0177] Table 3. Results of TF-1 proliferation detection by tool antibody (Tab) neutralizing IL-4-induced proliferation.

[0178] Antibody name Tab1 Tab2 Tab3 <![CDATA[EC 50 (nM)]]> 0.18 0.067 0.098

[0179] Example 10

[0180] Human IL-13-induced TF1 cell proliferation and tool antibody neutralization proliferation experiment.

[0181] 1. Experimental method for human IL-13-induced TF1 cell proliferation:

[0182] (1) Seed 10,000 TF-1 cells per well in a 96-well plate after 3-4 passages following resuscitation.

[0183] (2) Prepare a solution of human IL-4 protein with a maximum concentration of 500 ng / mL and perform serial dilutions of 5-fold;

[0184] (3) Add the serially diluted IL-4 solution to the cell culture wells in equal volumes to the cell culture medium;

[0185] (4) After incubation for 72 hours, cell viability was detected using a luminescent cell viability assay kit;

[0186] (5) Calculate the EC80 concentration of IL-13 to induce TF-1 cell proliferation based on the test results.

[0187] 2. Experimental method for neutralizing human IL-13-induced TF1 cell proliferation with tool antibody (Tab):

[0188] (1) Seed 10,000 TF-1 cells per well in a 96-well plate after 3-4 passages following resuscitation.

[0189] (2) Prepare solutions of different Tabs at 10 μg / mL and perform serial dilutions of 5 times;

[0190] (3) Mix the serially diluted Tab with the EC80 concentration of IL-13 obtained in the proliferation experiment at a ratio of 1:1 to prepare a mixture;

[0191] (4) Add the mixture from the previous step to the cell culture wells in an equal volume to the cell culture medium;

[0192] (5) After incubation for 72 hours, cell viability was detected using a luminescent cell viability assay kit;

[0193] (6) The EC50 concentration of Tab that neutralizes IL-13-induced TF-1 cell proliferation was calculated based on the test results. The results are shown in Table 4.

[0194] Table 4. Results of TF-1 proliferation detection by tool antibody (Tab) neutralizing IL-13-induced proliferation.

[0195] Antibody name Tab1 Tab2 Tab3 <![CDATA[EC 50 (nM)]]> 0.073 0.097 0.047

[0196] Example 11

[0197] Detection of the neutralization of IL-4-induced TF1 cell proliferation by a single-domain antibody specifically targeting IL-4Rα.

[0198] The method is as follows:

[0199] (1) Seed 10,000 TF-1 cells per well in a 96-well plate after 3-4 passages following resuscitation.

[0200] (2) The single-domain antibodies with different Tab and Fc fusions from Example 8 (from Example 6) were prepared into 10 μg / mL solutions and then serially diluted 5 times.

[0201] (3) The serially diluted Tab and single-domain antibody were mixed with IL-4 at EC80 concentration obtained in the proliferation experiment at a ratio of 1:1 to prepare a mixture;

[0202] (4) Add the mixture from the previous step to the cell culture wells in an equal volume to the cell culture medium;

[0203] (5) After incubation for 72 hours, cell viability was detected using a luminescent cell viability assay kit;

[0204] (6) Based on the detection results, the EC50 concentrations of different single-domain antibodies that neutralize IL-4-induced TF-1 cell proliferation were calculated, and the results are as follows: Figure 6 As shown in Table 5 below.

[0205] Table 5. Results of dose-response curve detection in the single-domain antibody neutralization of IL-4-induced TF-1 proliferation assay.

[0206] Antibody name 1A9 1B1 1B2 1B4 1C4 1D9 1H1 2A7 2C10 <![CDATA[EC 50 (nM)]]> 0.099 0.302 0.363 0.190 0.055 0.086 0.072 0.119 28.1 Antibody name 2C9 2H2 3C11 3E10 3F9 3G10 4B3 4E9 4H11 <![CDATA[EC 50 (nM)]]> 2.375 0.149 0.083 0.175 0.133 0.507 0.231 0.071 16680000

[0207] It can be seen that among the 18 antibodies, compared with other single-domain antibodies in Table 5, 4H11 has the worst neutralizing effect, 2C10 has a moderate neutralizing effect, and although 2C9 has a good neutralizing effect, its EC50 is still above 1 nM. The remaining single-domain antibodies all have EC50 below 1 nM and have a good neutralizing effect. Among them, the single-domain antibodies 1C4, 4E9, and 1H1 have the best neutralizing effect, with 1C4 reaching an EC50 of 55 pM.

[0208] Example 12

[0209] Detection of the neutralization of IL-13-induced TF1 cell proliferation by a single-domain antibody specifically targeting IL-4Rα.

[0210] The method is as follows:

[0211] (1) Seed 10,000 TF-1 cells per well in a 96-well plate after 3-4 passages following resuscitation.

[0212] (2) The single-domain antibodies with different Tab and Fc fusions from Example 8 (from Example 6) were prepared into 10 μg / mL solutions and then serially diluted 5 times.

[0213] (3) The serially diluted Tab and single-domain antibody were mixed with IL-13 at EC80 concentration obtained in the proliferation experiment at a ratio of 1:1 to prepare a mixture;

[0214] (4) Add the mixture from the previous step to the cell culture wells in an equal volume to the cell culture medium;

[0215] (5) After incubation for 72 hours, cell viability was detected using a luminescent cell viability assay kit;

[0216] (6) Based on the detection results, the EC50 concentrations of different single-domain antibodies that neutralize IL-13-induced TF-1 cell proliferation were calculated, and the results are as follows: Figure 7 As shown in Table 6 below.

[0217] Table 6. Results of dose-response curve detection in the single-domain antibody neutralization of IL-13-induced TF-1 proliferation assay.

[0218] Antibody name 1A9 1B1 1B2 1B4 1C4 1D9 1H1 2A7 2C10 <![CDATA[EC 50 (nM)]]> 0.097 0.602 1.704 0.26 0.093 0.089 0.07 0.17 <![CDATA[2.2e+10 11 ]]> Antibody name 2C9 2H2 3C11 3E10 3F9 3G10 4B3 4E9 4H11 <![CDATA[EC 50 (nM)]]> 1.17 0.166 0.072 0.27 0.149 0.487 0.135 0.077 <![CDATA[1.1e+10 11 ]]>

[0219] It can be seen that among the 18 antibodies, compared with other single-domain antibodies in Table 5, 4H11 and 2C10 have poor neutralization effects. Although 2C9 and 1B2 have good neutralization effects, their EC50 is still above 1 nM. The EC50 of other single-domain antibodies is below 1 nM. Among them, the neutralization effects of single-domain antibodies 1H1, 3C11, and 4E9 rank in the top three, and the EC50 of 1H1 is 70 pM.

[0220] Example 13

[0221] Based on the experimental results of Examples 11 and 12, the single-domain antibodies 1H1 and 4E9 showed the best overall neutralizing effect. These two antibodies were selected for antigen-antibody affinity assays using an Octet RED96 instrument. The specific implementation plan is as follows:

[0222] (1) SD buffer preparation: Dissolve an appropriate amount of bovine serum albumin and Tween 20 in 1×PBS (pH 7.4) to make the mass (or volume) fractions of bovine serum albumin and Tween 20 0.1% and 0.02%, respectively.

[0223] (2) Preparation of antigen working solution: The antigen (IL-4Rα recombinant protein) was prepared in SD buffer to 10 μg / mL.

[0224] (3) Preparation of antibody working solution: The antibody is first prepared into 100nM with SD buffer, and then diluted in a 2-fold gradient, with a total of 5 concentration gradients. In addition, a zero concentration is set.

[0225] (4) System: Open Octet 96 and its accompanying computer with DataAcquisiton software, use lens paper to take an appropriate amount of 75% ethanol to clean the bottom and sides of the acquisition probe, and preheat the instrument for more than 15 minutes.

[0226] (5) Sensor pre-wetting: Before the experiment, the sensor is soaked in SD buffer for more than 10 minutes and ready for use.

[0227] (6) The binding and dissociation times are shown in Table 7:

[0228] Table 7. Computer Operation Steps

[0229] Experimental steps Time (s) sample Baseline1 60 SD Loading 300 antigen Baseline2 60 SD Association 300 Antibody Dissociation 300 SD

[0230] (7) After the analysis is complete, turn off the instrument and the computer.

[0231] (8) Data Analysis: Calculate KD, Ka, Kd, ​​and Full R in Data Analysis software. 2 Isocorrelation constants, results as follows Figure 8 And as shown in Table 8

[0232] Table 8 Summary of affinity kinetics data for clones 1H1 and 4E9

[0233] Clone name KD(M) Ka(1 / Ms) Kd(1 / s) 1H1 1.41E-11 5.32E+05 7.50E-06 4E9 2.39E-10 5.56E+05 1.33E-04

[0234] According to existing literature (patent CN107474134), the affinities of Tab2 and Tab3 are 1.04 × 10⁻⁶. -10 M and 1.83×10 -10 M, the affinity of the single-domain antibody 1H1 obtained in the embodiments of the present invention is significantly better than that of the control antibody, at 1.41 × 10⁻⁶. -11 4E9, on the other hand, is basically equivalent to the control antibody.

[0235] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A single-domain antibody against IL-4Rα, characterized in that, It includes the following complementary determinant regions: CDR1, CDR2, and CDR3; The complementarity-determining regions of the single-domain antibody are shown below: CDR1 is shown in SEQ ID NO.38, CDR2 is shown in SEQ ID NO.58, and CDR3 is shown in SEQ ID NO.77; or, CDR1 is shown in SEQ ID NO.47, CDR2 is shown in SEQ ID NO.60, and CDR3 is shown in SEQ ID NO.

83.

2. The single-domain antibody against IL-4Rα according to claim 1, characterized in that, The single-domain antibody includes the following backbone regions: FR1, FR2, FR3, and FR4; The amino acid sequence of FR4 is shown in SEQ ID NO.128; the backbone regions FR1, FR2, and FR3 of the single-domain antibody are shown below: FR1 is shown in SEQ ID NO. 94, FR2 is shown in SEQ ID NO. 106, and FR3 is shown in SEQ ID NO. 125; or, FR1 is shown in SEQ ID NO.89, FR2 is shown in SEQ ID NO.101, and FR3 is shown in SEQ ID NO.

124.

3. The single-domain antibody against IL-4Rα according to claim 2, characterized in that, The amino acid sequence of the single-domain antibody is shown in SEQ ID NO.11 or 9.

4. A fusion protein, characterized in that, It is obtained by fusing the anti-IL-4Rα single-domain antibody as described in any one of claims 1-3 with Fc.

5. The use of the single-domain antibody against IL-4Rα according to any one of claims 1-3 or the fusion protein according to claim 4 in the preparation of a medicament for treating diseases, characterized in that, The diseases mentioned are asthma, allergic dermatitis, chronic primary urticaria, or chronic obstructive pulmonary disease.

6. The application according to claim 5, characterized in that, The allergic dermatitis mentioned is atopic dermatitis.

7. A medicine for treating a disease, characterized in that, It includes a single-domain antibody against IL-4Rα as described in any one of claims 1-3 or a fusion protein as described in claim 4, and pharmaceutically acceptable excipients.

8. An isolated nucleic acid molecule, characterized in that, Its encoding is a single-domain antibody as described in any one of claims 1-3.

9. The isolated nucleic acid molecule according to claim 8, characterized in that, The base sequence of the nucleic acid molecule is shown in SEQ ID NO.29 or 27.

10. A carrier containing the nucleic acid molecule of claim 9.

11. Recombinant cells containing the vector of claim 10.

12. A method for preparing a single-domain antibody as described in any one of claims 1-3, characterized in that, It includes: The recombinant cells as described in claim 11 are cultured, and the single-domain antibody is isolated and purified from the culture product.

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