Il-4r binding molecule and use thereof

Abstract

Provided are an interleukin 4 receptor (IL-4R) binding molecule and a use thereof. A heavy chain variable region of the molecule comprises a heavy chain complementarity-determining region 1 (HCDR1), a heavy chain complementarity-determining region 2 (HCDR2), and a heavy chain complementarity-determining region 3 (HCDR3), wherein HCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 1, 4, 6, 9, 12, or 15 or any variant thereof; HCDR2 comprises an amino acid sequence as shown in SEQ ID NO: 2, 7, 10, 13, or 111 or any variant thereof; and HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 3, 5, 8, 11, 14, or 112 or any variant thereof. The antibody can significantly block the binding of IL-4 to IL-4R, and exhibits IL-4R signal blocking activity significantly superior to that of the positive control antibody dupilumab.

Description

An IL-4R binding molecule and its applications

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application No. 2024119323836, filed on December 26, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of antibody technology, specifically to an interleukin-4 (IL-4R) binding molecule and its applications. Background Technology

[0004] Asthma is a heterogeneous disease with a complex pathogenesis. Based on the presence or absence of type 2 inflammation, asthma can be divided into type 2 and non-type 2 asthma. Airway inflammation in asthma is primarily mediated by cytokines such as IL-4, IL-5, and IL-13. Most of these cytokines originate from type 2 helper T cells (Th2 cells). Besides Th2 cells, other innate immune cells such as type 2 innate lymphoid cells (ILC2), basophils, and mast cells can also produce cytokines such as IL-4, IL-5, and IL-13. Therefore, IL-4, IL-5, and IL-13 are collectively referred to as type 2 cytokines. Among the globally approved biological targeted therapies for asthma, the main targets include IgE, IL-5, IL-5R, IL-4R, and TSLP.

[0005] Currently, a large number of asthma patients face unmet treatment needs in clinical practice. This is especially true for patients with moderate to severe asthma, who often respond poorly to traditional treatments (such as inhaled or oral corticosteroids), are prone to acute exacerbations and decreased lung function, severely impacting their daily lives and quality of life. This is mainly related to the failure to effectively target and control the pathophysiological mechanisms of asthma. Furthermore, reducing the rate of acute exacerbations and minimizing the use of oral corticosteroids to avoid side effects are urgent treatment challenges for asthma patients.

[0006] Drugs targeting IL-4R exhibit bispecificity, binding specifically to IL-4R while simultaneously blocking the signaling pathways of both IL-4 and IL-13. They demonstrate significant inhibitory effects on the proliferation of Th2 cells and downstream cells such as eosinophils, mast cells, and basophils. Specifically, IL-4 promotes the differentiation of naive CD4+ T cells into Th2 cells, maintaining T2 cell proliferation; it promotes the expression of MHC class II molecules in B cells, enhancing their antigen-presenting capacity; it promotes antibody class switching, producing immunoglobulin G1 (IgG1) and immunoglobulin E (IgE); and it participates in the recruitment of eosinophils (EOS) and the release of pro-inflammatory factors. IL-13 plays a crucial role in altering airway smooth muscle function, mucus hypersecretion, and airway remodeling. Therefore, IL-4R is considered an important target for the treatment of type 2 asthma.

[0007] Among biologics targeting IL-4R, the fully human monoclonal antibody dupilumab has been approved for marketing both domestically and internationally. Conoco's recombinant humanized monoclonal antibody sepachiba (CM310), which targets IL-4R, is in the application stage for marketing approval. At the same time, several pharmaceutical companies are also conducting research on monoclonal antibodies targeting IL-4R as a target for asthma.

[0008] To achieve breakthroughs in antibody design, pharmaceutical companies have begun exploring nanobody technology. Nanobodies are derived from heavy chain antibodies naturally occurring in camels or sharks that lack the light chain; they are the variable domains of heavy chain antibodies. Compared to traditional monoclonal antibodies, they offer advantages such as simple structure, high stability, strong tissue penetration, and low production costs, while retaining the affinity and specificity of full-length antibodies. Therefore, they exhibit superior efficacy, pharmacokinetics, and development potential. Based on these advantages, nanobodies provide a new strategy for the development of asthma drugs.

[0009] Due to the high cost of imported biologics for asthma treatment, patients face significant medical expenses and a heavy burden on medical insurance. Therefore, there is a continued need to develop novel, specific, and highly effective antibody drugs targeting IL-4R to provide asthma patients with more treatment options. Unmet clinical needs in asthma treatment and the limitations of existing drugs are compelling researchers to explore new treatment strategies. IL-4R-targeting nanobodies, with their higher tissue penetration and production efficiency, have become an innovative direction for future asthma treatment. Developing IL-4R-targeting nanobodies and inhaled formulations can not only provide more targeted and effective solutions for treating type 2 asthma but also reduce production costs, bringing greater benefits to patients and the healthcare system. Therefore, further advancing the research and development of IL-4R nanobodies and inhaled drugs is not only a scientific breakthrough but also a crucial response to unmet clinical needs. Summary of the Invention

[0010] Based on the nanobody discovery process, this disclosure describes the development of an IL-4R binding molecule and demonstrates its blocking effect on IL-4R signaling.

[0011] The inventors of this invention conducted extensive experiments, constructing an alpaca immune library. From this library, they screened, recombinantly expressed, and identified the activity of nanobodies, successfully obtaining novel nanobodies AIL-182, AIL-184, AIL-B19, and AIL-B200 capable of binding to human IL-4R. Through humanization and affinity maturation, the obtained nanobodies significantly blocked the binding of IL-4 to IL-4R. In addition, dual-epitope nanobodies and tetravalent nanobodies were constructed using a fusion antibody Fc. These IL-4R-binding molecules exhibited significantly superior blocking activity against IL-4R signaling compared to the positive control antibody Dupilumab.

[0012] On one hand, this disclosure provides an interleukin-4 receptor (IL-4R) binding molecule, wherein the heavy chain variable region (VH) comprises a heavy chain complementarity-determining region 1 (HCDR1), a heavy chain complementarity-determining region 2 (HCDR2), and a heavy chain complementarity-determining region (HCDR3), wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, 4, 6, 9, 12, or 15 or any variant thereof; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, 7, 10, 13, or 111 or any variant thereof; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, 5, 8, 11, 14, or 112 or any variant thereof.

[0013] In some embodiments, the aforementioned IL-4R binding molecules HCDR1, HCDR2, and HCDR3 have amino acid sequences that are more than 80% identical to the amino acid sequences shown in SEQ ID NOs: 1, 4, 6, 9, 12, or 15, SEQ ID NOs: 2, 7, 10, 13, or 111, and SEQ ID NOs: 3, 5, 8, 11, 14, or 112.

[0014] In some preferred embodiments, the aforementioned IL-4R binding molecule comprises a combination selected from the following HCDR1, HCDR2, and HCDR3:

[0015] (1) Containing HCDR1, HCDR2, and HCDR3 sequences as shown in SEQ ID NOs: 1, 2, and 3, respectively; or

[0016] (2) Containing HCDR1, HCDR2, and HCDR3 sequences as shown in SEQ ID NOs: 4, 2, and 5, respectively; or

[0017] (3) Containing HCDR1, HCDR2, and HCDR3 sequences as shown in SEQ ID NOs: 6, 7, and 8, respectively; or

[0018] (4) Containing HCDR1, HCDR2, and HCDR3 sequences as shown in SEQ ID NOs: 9, 10, and 11, respectively; or

[0019] (5) Containing HCDR1, HCDR2, and HCDR3 sequences as shown in SEQ ID NOs: 12, 13, and 14, respectively; or

[0020] (6) Each contains the HCDR1, HCDR2 and HCDR3 sequences as shown in SEQ ID NOs: 15, 111 and 112 respectively.

[0021] In some embodiments, the aforementioned IL-4R binding molecules HCDR1, HCDR2, and HCDR3 have the amino acid sequences of CDR1, CDR2, and CDR3 within the heavy chain variable region (VH) sequence shown in SEQ ID NO:16, respectively.

[0022] In some embodiments, the aforementioned IL-4R binding molecules HCDR1, HCDR2, and HCDR3 have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:17, respectively.

[0023] In some embodiments, the aforementioned IL-4R binding molecules HCDR1, HCDR2, and HCDR3 have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:18, respectively.

[0024] In some embodiments, the aforementioned IL-4R binding molecules HCDR1, HCDR2, and HCDR3 have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:25, respectively.

[0025] In some embodiments, the aforementioned IL-4R binding molecules HCDR1, HCDR2, and HCDR3 have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:29, respectively.

[0026] In some embodiments, the aforementioned IL-4R binding molecules HCDR1, HCDR2, and HCDR3 have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:36, respectively.

[0027] In some embodiments, the aforementioned IL-4R binding molecules HCDR1, HCDR2, and HCDR3 have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:39, respectively.

[0028] In some embodiments, the aforementioned IL-4R binding molecules HCDR1, HCDR2, and HCDR3 have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:46, respectively.

[0029] In some embodiments, the aforementioned IL-4R binding molecules HCDR1, HCDR2, and HCDR3 have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:49, respectively.

[0030] In some embodiments, the aforementioned IL-4R binding molecules HCDR1, HCDR2, and HCDR3 have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:56, respectively.

[0031] In some embodiments, the heavy chain variable region of the aforementioned IL-4R binding molecule comprises: (i) an amino acid sequence selected from SEQ ID NOs: 16-57; and / or (ii) an amino acid sequence having more than 80% identity with the amino acid sequence shown in SEQ ID NOs: 16-57.

[0032] In some implementations, the aforementioned IL-4R binding molecule is a full-length antibody, Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv) fragment, scFv-scFv, microantibody, diabody, or single-domain antibody.

[0033] In some embodiments, the amino acid sequence of the aforementioned IL-4R binding molecule comprises an amino acid sequence obtained by at least one of the following methods: addition, deletion, modification, and / or substitution of the amino acid sequence shown in SEQ ID NOs: 16-57; wherein the addition, deletion, modification, and / or substitution includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid differences.

[0034] In some preferred embodiments, the aforementioned additions, deletions, modifications, and / or substitutions do not alter the binding function of the IL-4R binding molecule to IL-4R.

[0035] In some preferred embodiments, the aforementioned additions, deletions, modifications, and / or substitutions occur in the framework region of the molecule.

[0036] On the other hand, this disclosure provides a tandem molecule comprising the aforementioned IL-4R binding molecule.

[0037] In some implementations, the aforementioned tandem molecule comprises a first single-domain antibody heavy chain antibody variable region (VHH region) and a second single-domain antibody VHH region.

[0038] In some embodiments, the first single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in groups (1), (2), (5) or (6) above, and the second single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (3) or (4) above.

[0039] In some embodiments, the first single-domain antibody VHH region of the tandem molecule contains an HCDR combination as described in group (5) or (6) above, and the second single-domain antibody VHH region of the tandem molecule contains an HCDR combination as described in group (3) or (4) above.

[0040] In some embodiments, the first single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (5) above, and the second single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (3) above.

[0041] In some embodiments, the first single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (6) above, and the second single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (4) above.

[0042] In some embodiments, the first single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (5) above, and the second single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (4) above.

[0043] In some embodiments, the first single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (6) above, and the second single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (3) above.

[0044] In some embodiments, the first single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 16-38 or sequences having more than 80% identity with them, and the second single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 39-57 or sequences having more than 80% identity with them.

[0045] In some embodiments, the first single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 16 or 17 or sequences having more than 80% identity with them, and the second single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 39-57 or sequences having more than 80% identity with them.

[0046] In some embodiments, the first single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 16, and the second single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 46.

[0047] In some embodiments, the first single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 17, and the second single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 56.

[0048] In some embodiments, the first single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 16, and the second single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 56.

[0049] In some embodiments, the first single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 17, and the second single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 46.

[0050] In some embodiments, the tandem molecule further comprises a third single-domain antibody VHH region and a fourth single-domain antibody VHH region.

[0051] In some embodiments, the third single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in groups (1), (2), (5) or (6) above, and the fourth single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (3) or (4) above.

[0052] In some preferred embodiments, the first single-domain antibody VHH region of the tandem molecule contains an HCDR combination as described in group (5) or (6) above, the second single-domain antibody VHH region of the tandem molecule contains an HCDR combination as described in group (3) or (4) above, the third single-domain antibody VHH region of the tandem molecule contains an HCDR combination as described in group (5) or (6) above, and the fourth single-domain antibody VHH region of the tandem molecule contains an HCDR combination as described in group (3) or (4) above.

[0053] In some preferred embodiments, the first single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (5) above, the second single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (3) above, the third single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (5) above, and the fourth single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (3) above.

[0054] In some preferred embodiments, the first single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (6) above, the second single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (4) above, the third single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (6) above, and the fourth single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (4) above.

[0055] In some preferred embodiments, the first single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (5) above, the second single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (4) above, the third single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (5) above, and the fourth single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (4) above.

[0056] In some preferred embodiments, the first single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (6) above, the second single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (3) above, the third single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (6) above, and the fourth single-domain antibody VHH region of the tandem molecule contains the HCDR combination as described in group (3) above.

[0057] In some embodiments, the third single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 16-38 or sequences having more than 80% identity with them, and the fourth single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 39-57 or sequences having more than 80% identity with them.

[0058] In some embodiments, the first single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 16 or 17 or sequences having more than 80% identity with them; the second single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 39-57 or sequences having more than 80% identity with them; the third single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 16 or 17 or sequences having more than 80% identity with them; and the fourth single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 39-57 or sequences having more than 80% identity with them.

[0059] In some embodiments, the first single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 16, the second single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 46, the third single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 16, and the fourth single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 46.

[0060] In some embodiments, the first single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 17, the second single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 56, the third single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 17, and the fourth single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 56.

[0061] In some embodiments, the first single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 16, the second single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 56, the third single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 16, and the fourth single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 56.

[0062] In some embodiments, the first single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 17, the second single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 46, the third single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 17, and the fourth single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 46.

[0063] In some implementations, the amino acid sequences of the IL-4R binding molecule or the VHH region of the single-domain antibody in the aforementioned tandem molecules are directly linked or indirectly linked through a linker.

[0064] In some embodiments, the aforementioned linker comprises the amino acid sequence GGGGS (SEQ ID NO: 100) or has the general sequence formula (GGGGS)n, where n is an integer selected from 1 to 8. In some specific embodiments, n may be selected, for example, 1, 2, 3, 4, 5, 6, 7, or 8.

[0065] In some preferred embodiments, the amino acid sequence of the aforementioned linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 101).

[0066] In some embodiments, the IL-4R binding molecules or single-domain antibody VHH regions in the aforementioned tandem molecules are linked in an optional order.

[0067] In some implementations, the IL-4R binding molecules or single-domain antibody VHH regions in the aforementioned tandem molecules are linked in order from the N-terminus to the C-terminus.

[0068] In some implementations, the IL-4R binding molecules or single-domain antibody VHH regions in the aforementioned tandem molecules are linked in order from the C-terminus to the N-terminus.

[0069] On the other hand, this disclosure provides a recombinant protein comprising the aforementioned IL-4R binding molecule or tandem molecule.

[0070] In some embodiments, the aforementioned recombinant protein also includes bioactive proteins or functional fragments that assist in its expression and / or secretion and / or prolong its half-life in vivo.

[0071] In some preferred embodiments, the aforementioned bioactive protein or functional fragment is selected from at least one of the following: immunoglobulin Fc domain, serum albumin, albumin-binding peptide, prealbumin, carboxyl-terminal peptide, elastin-like peptide, histidine tag (His tag), GST tag, MBP tag, FLAG tag, and SUMO tag.

[0072] In some implementations, the aforementioned bioactive protein or functional fragment is the Fc domain of human immunoglobulin.

[0073] In some preferred embodiments, the aforementioned bioactive protein or functional fragment is the Fc domain of human IgG, including the Fc domains of human IgG1, IgG2, IgG3, and IgG4.

[0074] In some preferred embodiments, the aforementioned bioactive protein or functional fragment is the Fc domain of human IgG4.

[0075] In some preferred embodiments, the aforementioned bioactive protein or functional fragment is a His tag.

[0076] In some preferred embodiments, the aforementioned recombinant protein comprises, from the N-terminus to the C-terminus: VH1, a linker, VH2, and a His tag.

[0077] In some preferred embodiments, the aforementioned recombinant protein comprises, from the N-terminus to the C-terminus, a VH1, a linker, a VH2, and an Fc domain.

[0078] In some preferred embodiments, the aforementioned VH1 comprises an amino acid sequence selected from SEQ ID NOs: 16 or 17.

[0079] In some preferred embodiments, the aforementioned linker comprises the amino acid sequence shown in SEQ ID NO: 101.

[0080] In some preferred embodiments, the aforementioned VH2 comprises an amino acid sequence selected from SEQ ID NOs: 46 or 56.

[0081] In some preferred embodiments, the aforementioned Fc domain comprises the amino acid sequence shown in SEQ ID NO: 102.

[0082] In some preferred embodiments, the aforementioned recombinant protein comprises an amino acid sequence selected from those shown in SEQ ID NOs: 103-110.

[0083] On the other hand, this disclosure provides a nucleic acid comprising a polynucleotide encoding the aforementioned IL-4R, tandem molecule, or recombinant protein.

[0084] In some preferred embodiments, the nucleic acid encoding the aforementioned IL-4R binding molecule is selected from the nucleic acid sequences shown in SEQ ID NOs: 58-99.

[0085] In some preferred embodiments, the nucleic acid encoding the aforementioned tandem molecule or recombinant protein comprises a nucleic acid sequence selected from those shown in SEQ ID NOs: 58-99.

[0086] In some preferred embodiments, the nucleic acid encoding the aforementioned tandem molecule or recombinant protein comprises the nucleic acid sequences shown in SEQ ID NOs: 58 and 88.

[0087] In some preferred embodiments, the nucleic acid encoding the aforementioned tandem molecule or recombinant protein comprises the nucleic acid sequences shown in SEQ ID NOs: 59 and 88.

[0088] In some preferred embodiments, the nucleic acid encoding the aforementioned tandem molecule or recombinant protein comprises the nucleic acid sequences shown in SEQ ID NOs: 58 and 98.

[0089] In some preferred embodiments, the nucleic acid encoding the aforementioned tandem molecule or recombinant protein comprises the nucleic acid sequences shown in SEQ ID NOs: 59 and 98.

[0090] On the other hand, this disclosure provides a carrier that contains one or more of the aforementioned nucleic acids.

[0091] On the other hand, this disclosure provides a cell that contains the aforementioned nucleic acid or vector.

[0092] On the other hand, this disclosure provides a method for generating the aforementioned IL-4R binding molecule, tandem molecule, or recombinant protein, the method comprising culturing a host cell containing the aforementioned nucleic acid or expression vector.

[0093] On the other hand, this disclosure provides a pharmaceutical composition or pharmaceutical composition comprising the aforementioned IL-4R binding molecule, tandem molecule, recombinant protein, nucleic acid, carrier and / or cell.

[0094] In some preferred embodiments, the aforementioned composition or pharmaceutical combination further includes a second therapeutic agent.

[0095] In some more preferred embodiments, the aforementioned second therapeutic agent is selected from β-2-adrenergic receptor agonists (SABA), anticholinergic drugs, adrenergic agonists, glucocorticoids, long-acting β-2-adrenergic receptor agonists (LABA), leukotriene antagonists, and mast cell stabilizers.

[0096] On the other hand, this disclosure provides an antibody-drug conjugate comprising the aforementioned IL-4R binding molecule, the aforementioned tandem molecule, or the aforementioned recombinant protein covalently attached to the therapeutic portion.

[0097] In some preferred embodiments, the aforementioned therapeutic component is a cytotoxic component, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a cleavage peptide, or a radioisotope.

[0098] On the other hand, this disclosure provides a bispecific antibody or a multispecific antibody, wherein one of its antigen-binding domains comprises the aforementioned IL-4R binding molecule, tandem molecule, or recombinant protein.

[0099] On the other hand, this disclosure provides a chimeric antigen receptor whose extracellular recognition unit comprises the aforementioned IL-4R binding molecule, tandem molecule or recombinant protein.

[0100] On the other hand, this disclosure provides the use of the aforementioned IL-4R binding molecules, tandem molecules, recombinant proteins, nucleic acids, vectors, cells, pharmaceutical compositions or pharmaceutical compositions, antibody-drug conjugates, bispecific antibodies or multispecific antibodies and / or chimeric antigen receptors in the preparation of medicaments or kits for detecting, treating, preventing and / or alleviating IL-4R-related diseases.

[0101] On the other hand, this disclosure provides the use of the aforementioned IL-4R binding molecules, tandem molecules, recombinant proteins, nucleic acids, vectors, cells, pharmaceutical compositions or pharmaceutical compositions, antibody-drug conjugates, bispecific antibodies or multispecific antibodies and / or chimeric antigen receptors for the detection, treatment, prevention and / or mitigation of IL-4R-related diseases.

[0102] On the other hand, this disclosure provides the aforementioned IL-4R binding molecules, tandem molecules, recombinant proteins, nucleic acids, vectors, cells, pharmaceutical compositions or pharmaceutical compositions, antibody-drug conjugates, bispecific antibodies or multispecific antibodies and / or chimeric antigen receptors for detecting, treating, preventing and / or alleviating diseases associated with IL-4R.

[0103] On the other hand, this disclosure provides a method for detecting, treating, preventing, and / or alleviating IL-4R-related diseases, wherein the method comprises administering to a subject in need an effective amount of the aforementioned IL-4R binding molecule, tandem molecule, recombinant protein, nucleic acid, carrier, cell, pharmaceutical composition or pharmaceutical composition, antibody-drug conjugate, bispecific antibody or multispecific antibody and / or chimeric antigen receptor.

[0104] In some implementations, the aforementioned diseases include Th2-mediated diseases, IL-13-mediated diseases, IL-4-mediated diseases, and / or IL-4 / IL-13-mediated diseases.

[0105] In some implementations, the aforementioned diseases are selected from chronic obstructive pulmonary disease, chronic urticaria, eosinophilic esophagitis, nodular prurigo, asthma, chronic sinusitis with nasal polyps, atopic dermatitis, neurodermatitis, pruritus, allergic fungal sinusitis, moderate atopic dermatitis, severe atopic dermatitis, eosinophilic duodenitis, eosinophilic gastroenteritis, chronic sinusitis, bullous pemphigoid, hypersensitivity reaction, allergic asthma, house dust mite allergy, alopecia areata, keloids, ulcerative colitis, milk allergy, respiratory disorders, eosinophilic sinusitis, and allergic bronchopulmonary aspergillosis. Chronic liver disease, chronic eczema, urticaria, food allergy, lichen planus, seasonal allergic rhinitis, allergic conjunctivitis, conjunctivitis, keloid, aspirin-induced respiratory disease, scar tissue, allergic bronchopulmonary aspergillosis, pulmonary aspergillosis, allergic diseases, allergic skin diseases, dermatitis, adult chronic pruritus, eczema, allergic dermatitis, contact dermatitis, sinusitis, nasal polyps, allergic rhinitis, chronic obstructive airway disease, idiopathic pulmonary fibrosis, systemic sclerosis, tuberculosis, eosinophilia, inflammatory diseases, autoimmune diseases, eosinophilic granulocytes Ulcerative gastritis, moderate to severe active ulcerative colitis, duodenitis, arthritis, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, autoimmune uveitis, tuberculosis, nephropathy, Hodgkin's disease, scleroderma, renal cell carcinoma, Burkitt lymphoma, non-Hodgkin's disease, Cezari syndrome, toxic arthritis, herpetic dermatitis, hypertrophic scars, Whipple's disease, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Grave's disease, preeclampsia, Sjogren's syndrome, autoimmune lymphoproliferative syndrome Autoimmune hemolytic anemia, cystic fibrosis, pulmonary alveolar proteinosis, adult respiratory distress syndrome, sarcoidosis, myasthenia gravis, chronic fatigue syndrome, anaphylactic shock, rheumatoid arthritis, B-cell lymphoma, T-cell lymphoma, multiple myeloma, head and neck cancer, breast cancer, ovarian cancer, lymphoma, blastoma, atherosclerosis, idiopathic asthma, radiation-induced pulmonary fibrosis, systemic sclerosis, multiple sclerosis and inflammatory disorders of the central nervous system, nephrotic syndrome, lupus nephritis, viral infections, parasitic infections, fungal infections, sarcoma, leukemia, psoriasis, diabetes. Attached Figure Description

[0106] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this specification and, together with the specification, serve to explain the principles of this specification.

[0107] Figure 1 shows the FACS binding data of antibodies AIL-A182-Fc, AIL-A184-Fc, AIL-B19-Fc, and AIL-B200-Fc to huIL-4R-HEK293 cells.

[0108] Figure 2 shows the FACS blocking data of antibodies AIL-A182-Fc, AIL-A184-Fc, AIL-B19-Fc, and AIL-B200-Fc blocking the binding of IL-4 to IL-4R on the cell surface.

[0109] Figure 3 shows the activity of antibodies AIL-A182-Fc, AIL-A184-Fc, AIL-B19-Fc, and AIL-B200-Fc in inhibiting IL-4-induced STAT6 phosphorylation.

[0110] Figure 4 shows the inhibitory activity of antibodies AIL-A182-Fc, AIL-A184-Fc, AIL-B19-Fc, and AIL-B200-Fc on IL-4-His-induced TF-1 cell proliferation.

[0111] Figure 5 shows the binding data of antibodies AIL-A182-VHH7-Fc, AIL-A184-VHH7-Fc, AIL-B19-VHH7-Fc, and AIL-B200-VHH7-Fc to human and monkey IL-4R.

[0112] Figure 6 illustrates the activity of antibodies AIL-A182-VHH7-Fc, AIL-A184-VHH7-Fc, AIL-B19-VHH7-Fc, and AIL-B200-VHH7-Fc in blocking the binding of IL-4 to IL-4R.

[0113] Figure 7 shows the binding of antibodies AIL-A182-VHH7-Fc, AIL-A184-VHH7-Fc, AIL-B19-VHH7-Fc, and AIL-B200-VHH7-Fc to IL-4R protein overexpressed on the surface of HEK293-huIL-4R cells.

[0114] Figure 8 shows the activity of antibodies AIL-A182-VHH7-Fc, AIL-A184-VHH7-Fc, AIL-B19-VHH7-Fc, and AIL-B200-VHH7-Fc in blocking the binding of IL-4 to IL-4R on the cell surface.

[0115] Figure 9 shows the detection results of antibodies after partial affinity maturation.

[0116] Figure 10 illustrates the role of antibodies B19_13 and B200_57 in inhibiting IL-4-induced STAT6 phosphorylation.

[0117] Figure 11 illustrates the role of antibodies B19_13 and B200_57 in inhibiting IL-4-His-induced TF-1 cell proliferation.

[0118] Figure 12 illustrates the binding activity of antibodies A182-VHH7-B19_13-His, A182-VHH7-B200_57-His, A184-VHH7-B19_13-His, and A184-VHH7-B200_57-His on human and monkey IL-4R and their activity in blocking the binding of IL-4 to human IL-4R protein.

[0119] Figure 13 shows the phosphorylation inhibitory activity of antibodies A182-VHH7-B19_13-His, A182-VHH7-B200_57-His, A184-VHH7-B19_13-His and A184-VHH7-B200_57-His.

[0120] Figure 14 shows the FACS binding and blocking detection results of antibodies A182-VHH7-B19_13-His, A182-VHH7-B200_57-His, A184-VHH7-B19_13-His, and A184-VHH7-B200_57-His in huIL-4R-HEK293.

[0121] Figure 15 illustrates the inhibitory activity of antibodies A182-VHH7-B19_13-His, A182-VHH7-B200_57-His, A184-VHH7-B19_13-His, and A184-VHH7-B200_57-His on IL-4 and IL-13-induced TF-1 cell proliferation.

[0122] Figure 16 illustrates the binding activity of antibodies A182-VHH7-B19_13-Fc, A182-VHH7-B200_57-Fc, A184-VHH7-B19_13-Fc, and A184-VHH7-B200_57-Fc to human or monkey IL-4R protein and their activity in blocking the binding of IL-4 to human IL-4R protein.

[0123] Figure 17 shows the FACS binding and blocking detection results of antibodies A182-VHH7-B19_13-Fc, A182-VHH7-B200_57-Fc, A184-VHH7-B19_13-Fc, and A184-VHH7-B200_57-Fc.

[0124] Figure 18 shows the STAT6 phosphorylation inhibition detection results of antibodies A182-VHH7-B19_13-Fc, A182-VHH7-B200_57-Fc, A184-VHH7-B19_13-Fc, and A184-VHH7-B200_57-Fc.

[0125] Figure 19 illustrates the inhibitory activity of antibodies A182-VHH7-B19_13-Fc, A182-VHH7-B200_57-Fc, A184-VHH7-B19_13-Fc, and A184-VHH7-B200_57-Fc on IL-4 and IL-13-induced TF-1 cell proliferation.

[0126] Figure 20 shows the PBMC-STAT6 phosphorylation detection results of antibodies A182-VHH7-B19_13-Fc, A182-VHH7-B200_57-Fc, A184-VHH7-B19_13-Fc, and A184-VHH7-B200_57-Fc. Detailed Implementation

[0127] I. Definition

[0128] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0129] As used herein and unless otherwise stated, the terms “about” or “approximately” mean within 10% of a given value or range. Where an integer is required, the term means within 10% of a given value or range, rounded up or down to the nearest integer.

[0130] The term "sequence identity" or "identity" has a generally accepted meaning in the art, and the percentage of sequence similarity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence identity can be measured along the full length of the polynucleotide or polypeptide or along a region of the molecule. While many methods exist for measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art.

[0131] Regarding antibody chain polypeptide sequences, the phrase "substantially identical" can be understood as an antibody chain exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a reference polypeptide sequence. Regarding nucleic acid sequences, the term can be understood as a nucleotide sequence exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference nucleic acid sequence.

[0132] Regarding the variable domains of antibodies, the term "variable" refers to certain portions of related molecules with extensive sequence differences between antibodies, used for the specific recognition and binding of a particular antibody to its specific target. However, variability is not uniformly distributed throughout the entire variable domain of an antibody. Variability is concentrated in three segments known as complementarity-determining regions (CDRs; namely CDR1, CDR2, and CDR3) or hypervariable regions, all located within the variable domains of the light and heavy chains. More conserved portions within the variable domain are called framework (FR) regions or framework sequences. Each variable domain of the natural heavy and light chains includes four FR regions, primarily employing a β-sheet configuration, which link three CDRs together. These CDRs form loops that connect the β-sheet structure and, in some cases, partially form the β-sheet structure. The CDRs of each chain are typically linked in proximity by the FR regions, and the formation of antibody target binding sites (epitopes or determinants) is facilitated by CDRs from other chains. Identifying the CDR region of an antibody by analyzing its amino acid sequence is a well-known method, and many definitions of CDRs are commonly used, such as the Kabat definition based on sequence variability, the Chothia definition based on the location of structural loop regions, and the IMGT definition based on the standardized immunogenetic numbering scheme and database sequence alignment of the international ImMunoGeneTics information system. A CDR may have the ability to specifically bind to associated epitopes.

[0133] As used herein, the term "CDR" refers to the complementarity-determining region, and it is known that each heavy and light chain of an antibody molecule has three CDRs. CDRs, also called hypervariable regions, are present in the variable regions of each heavy and light chain of the antibody and are highly variable sites in the primary structure of the CDR. In this specification, the CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 from the amino-terminal sequence of the heavy chain, and the CDRs of the light chain are represented by CDR1, CDR2, and CDR3 from the amino-terminal sequence of the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds. The numbering of immunoglobulin amino acid residues in the embodiments of this disclosure is based on the IMGT numbering scheme, but the CDR region sequence of the antibody in this disclosure can be defined by various methods such as Kabat, Chothia, and IMGT.

[0134] As used herein, the terms “CDR”, “CDR1”, “CDR2”, “CDR3”, “HCDR”, “HCDR1”, “HCDR2”, “HCDR3”, etc., include CDRs defined by any of the methods described above, such as IMGT, Kabat, Chothia, etc., unless otherwise stated.

[0135] As used herein, an "antibody fragment" or "antigen-binding fragment" refers to any portion of a full-length antibody that is less than the full length but contains at least a portion of the variable region of the antibody that binds to the antigen (e.g., one or more CDRs and / or one or more antibody binding sites), and thus retains binding specificity and at least a portion of the specific binding ability of the full-length antibody. Therefore, an antigen-binding fragment refers to an antibody fragment containing an antigen-binding portion that binds to the same antigen as an antibody fragment derived from the antibody fragment. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinant derivatives. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives thereof, such as bispecific antibodies, single-chain antibodies, dimeric molecules, linear antibodies, and multispecific antibodies formed from antibody fragments. Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0136] As used herein, “single-domain antibody,” “nanobody,” or “heavy chain antibody variable region (VHH)” derives from two distinct IgG subclasses produced by camels (e.g., llamas): IgG2 and IgG3, also known as heavy chain antibodies. These antibodies consist of only two heavy chains; they lack the CH1 region but still possess an antigen-binding domain called a VHH (or nanobody) at their N-terminus. Conventional Ig requires association from the variable regions of both the heavy and light chains to allow for a high degree of diversity in antigen-antibody interactions. While isolated heavy and light chains still exhibit this ability, they show very low affinity compared to paired heavy and light chains. The unique characteristic of heavy chain antibodies is that their monomeric antigen-binding regions possess the ability to bind antigens with specificity, affinity, and, in particular, diversity comparable to conventional antibodies, without requiring pairing with another region. This characteristic is primarily due to several major mutations within the amino acid sequences of the variable regions of the two heavy chains, which induce profound conformational changes relative to conventional Ig. Major substitutions in the variable regions prevent the light chain from binding to the heavy chain but also prevent unbound heavy chains from being reclaimed by immunoglobulin-binding proteins. The individual variable domains of these antibodies (called VHH, sdAb, nanobodies, or heavy chain antibody variable domains) are the smallest antigen-binding domains generated by the adaptive immune system. The third complementarity-determining region (CDR3) of the variable domains of these antibodies is typically found to be twice as long as that of conventional antibodies. This leads to an increased diversity of interfaces for antigen-interaction and antigen-antibody interactions, compensating for the absence of light chains. Due to the long CDR3, VHHs can extend into cracks in proteins that are inaccessible to conventional antibodies, including functionally interesting sites such as enzyme active sites or receptor-binding canyons on viral surfaces. Furthermore, the additional cysteine ​​residues contribute to structural stability, thereby increasing the strength of interactions. Compared to conventional antibodies carrying the variable domains (VH and VL) of conventional antibodies, VHHs offer many other advantages, including higher stability, solubility, expression yield, and refolding ability, as well as better in vivo tissue penetration. Moreover, unlike the VH domain of conventional antibodies, VHHs do not exhibit an intrinsic tendency to bind to light chains. This facilitates the induction of heavy chain antibodies in the presence of functional light chain loci. Furthermore, because VHH does not bind to the VL domain, recombining VHH into multispecific antibody constructs is much easier than constructs containing conventional VH-VL pairs or single-domain constructs based on the VH domain. VHH can be obtained from a designed and synthesized llama VHH library. RNA can be isolated from pygmy bronchograms (PBMCs) derived from llamas to produce cDNA via reverse transcription. The VHH gene can then be amplified by PCR and cloned into a phage display vector to construct a primordial VHH library.Synthetic (e.g., humanized) VHH libraries can be prepared by incorporating shuffled VHH CDRs 1, 2, and 3 generated via overlap PCR into modified human VH scaffolds to produce enhanced diversity while maintaining low immunogenicity. The VHH library can then be screened against antigens to obtain VHHs with the desired binding affinity. Nanobodies can be classified into several different types, namely monovalent nanobodies, bivalent nanobodies, bispecific nanobodies, multivalent nanobodies, and fusion nanobodies. Because nanobodies have a monomeric structure, they are easily modified into different structures, such as bivalent, multivalent, and bispecific nanobodies. Bivalent or multivalent nanobodies have two or more VHH structures that recognize the same epitope of an antigen, exhibiting higher affinity than monovalent nanobodies without affecting their pharmacokinetics and targeting ability. Bispecific nanobodies have two distinct VHHs that can bind to two different antigens or different epitopes on the same antigen, exhibiting stronger antigen recognition ability than monovalent nanobodies.

[0137] As used in this article, the term "tandem molecule" refers to a whole obtained by connecting two or more molecules together along a single path, with each connection point connecting at most two molecules.

[0138] As used herein, the term "quadrivalent nanobody" refers to a nanobody with four VHH structures modified from a monovalent nanobody, wherein each VHH can bind the same or different antigens, or the same or different epitopes on the same antigen; the quadrivalent nanobody constructed in the embodiments of this disclosure has two sets of VHH structures that bind different epitopes on the same antigen, wherein each set contains two identical VHH structures.

[0139] As used herein, the term "humanized" antibody refers to a non-human (e.g., mouse) antibody form that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues of the complementarity-determining region (CDR) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) with the desired specificity, affinity, and capability, such as mouse, rat, or rabbit. Furthermore, in humanization, amino acid residues in the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may be mutated, thereby improving one or more binding properties (e.g., affinity) of the antibody. Mutations may be introduced, for example, by PCR-mediated mutation, and their effects on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conserved mutations are introduced. Such mutations may be amino acid substitutions, additions, or deletions. Furthermore, mutations within the CDR typically do not exceed one or two. Therefore, the humanized antibodies described in this disclosure also cover antibodies containing one or two amino acid mutations within the CDR.

[0140] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which an antibody binds at its complementary site. Epitope determinants typically comprise chemically active surface subtypes of a molecule, such as amino acid or sugar side chains, and often possess specific three-dimensional structural features as well as specific charge characteristics.

[0141] As used herein, the terms “specific binding” and “immunospecific binding” for an antibody or its antigen-binding fragment are used interchangeably and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with the same antigen through a non-covalent interaction between the antibody and the antibody-binding site of the antigen. The antigen may be an isolated antigen or present in tumor cells. Typically, antibodies that immune-specifically bind (or specifically bind) an antigen bind to the antigen with an affinity constant Ka of about or 1 × 10⁷ M⁻¹ or 1 × 10⁸ M⁻¹ or greater (or a dissociation constant (Kd) of 1 × 10⁻⁷ M or 1 × 10⁻⁸ M or less). The affinity constant can be determined by standard kinetic methods of antibody reactions, such as immunoassay, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art; see U.S. Patent No. 7,229,619, which describes exemplary SPR and ITC methods for calculating the binding affinity of antibodies. Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available.

[0142] As used herein, the terms “polynucleotide” and “nucleic acid molecule” refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) typically linked together by phosphodiester bonds. As used herein, the term “nucleic acid molecule” is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0143] As used herein, isolated nucleic acid molecules are nucleic acid molecules isolated from other nucleic acid molecules present in natural sources of nucleic acid molecules. “Isolated” nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when prepared by recombinant technology, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding provided antibody or antigen-binding fragments.

[0144] As used herein, a "vector" is a reproducible nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can typically be introduced via restriction enzyme digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplification of nucleic acids or for expression / display of the polypeptide encoded by the nucleic acid. Vectors are typically kept free but can be designed to integrate genes or portions thereof into the chromosome of the genome. Vectors for artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes, are also considered. The selection and use of such vectors are well known to those skilled in the art.

[0145] As used in this article, vectors also include “viral vectors” or “vectors of viruses.” Viral vectors are engineered viruses that are operatively linked to a foreign gene to transfer (as a medium or shuttle) the foreign gene into cells.

[0146] As used herein, "expression vector" includes a vector capable of expressing DNA operatively linked to regulatory sequences, such as promoter regions, that influence the expression of such DNA fragments. These additional fragments may include promoter and terminator sequences and optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, when introduced into a suitable host cell, results in the expression of clonal DNA. Suitable expression vectors are well known to those skilled in the art and include reproducible expression vectors in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain free or are integrated into the host cell genome.

[0147] As used herein, the term "affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is typically expressed as the equilibrium dissociation constant (KD). The equilibrium dissociation constant is the ratio of the dissociation rate constant to the binding rate constant (kdis and kon, respectively). A smaller KD indicates less dissociation and a stronger affinity between the antibody and the antigen. Affinity can be measured using methods commonly known in the art, such as KD determined in a BIACORE instrument using surface plasmon resonance (SPR). Typically, antibodies have a KD not exceeding 1 × 10⁻⁶. -5 M, for example, is less than approximately 1 × 10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 The antigen dissociates from M or a smaller equilibrium dissociation constant (KD).

[0148] As used herein, the term "bispecific antibody" (BsAb) refers to an antibody and / or antigen-binding molecule that specifically binds to two different antigenic determinants. Typically, a bispecific antibody and / or antigen-binding molecule contains two antigen-binding sites, each specific to a different antigenic determinant. In some embodiments, the bispecific antibody and / or antigen-binding molecule is capable of binding to two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two different cells.

[0149] II. Examples

[0150] For the purpose of clarity and concise description, the features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of this disclosure may include some embodiments having a combination of all or some of the features described.

[0151] Example 1: Alpaca Immunization

[0152] For the initial immunization, Freund's complete adjuvant was used to emulsify the antigen, while subsequent immunizations used Freund's incomplete adjuvant. The antigen dosage was 500 μg per animal. Subcutaneous injections were administered at multiple sites. Alpaca NM020 and NM021 underwent cross-immunization using huI4R-his(293) and huI4R-hFc(293) proteins. After immunization, serum titers were measured. Phage display libraries were established for alpaca NM020 immunized after the 5th and 6th immunizations and for alpaca NM021 immunized after the 4th immunization.

[0153] Example 2: Establishment of an Alpaca Immunobank

[0154] (1) After the serum titer test is qualified, 100 mL of peripheral blood mononuclear cells (PBMCs) will be collected for bank construction;

[0155] (2) Total RNA was extracted from PBMCs and reverse transcribed to obtain cDNA, which was used as a template for subsequent amplification of the VHH fragment;

[0156] (3) The antibody sequence was amplified using primers designed using conventional methods;

[0157] (4) Use the corresponding restriction endonuclease to digest the vector and the amplified antibody fragment;

[0158] (5) The ligation product was constructed using T4 ligase and then transferred into SS320 competent cells for electroporation using electroporation technology.

[0159] (6) The volume of the alpaca immune library was determined by dilution plating and sequencing analysis, as shown in Table 1. The effective volume of NM020-5 was 8.50 × 10⁻⁶. 8 The effective storage capacity of CFU, NM020-6 is 3.30 × 10⁻⁶. 8 The effective storage capacity of CFU, NM021-4 is 9.00 × 10⁻⁶. 8 The CFU database was successfully constructed and has good abundance, as shown in Table 1.

[0160] Table 1. Results of Alpaca Immunity Bank Establishment

[0161] Example 3: Screening of alpaca immune library and recombinant expression and purification of nanobodies

[0162] Inoculate the *Cyclocarya* strain into 2YT(C) at a specific inoculum concentration.+ -T + In a culture medium, shake and incubate until OD600≈0.5. Add Helper Phage for 1h~1.5h, then centrifuge, discard the supernatant, and pour in 2YT(C + -K + The cells were cultured overnight in culture medium. The next day, the supernatant was collected by centrifugation, mixed with PEG6000, and placed on ice to precipitate. After 1 hour, the supernatant was discarded by centrifugation, and pre-chilled PBS was added. The phage suspension was prepared by pipetting and dissolving. The phage library was screened and single clones were selected according to Example 2 of patent CN111690058B. After initial screening with ELISA, 1424 positive clones with human-monkey cross-activity were obtained. Sequencing analysis yielded 54 molecules with unique sequences. Based on sequence diversity analysis, 29 molecules were selected for recombinant expression in ExpiCHO-S cells, and antibody purification was performed using a protein A purification column.

[0163] Sequencing of the candidate nanobodies obtained from recombinant expression and purification yielded the following VHH region sequences for candidate positive clones:

[0164] Table 2. Variable region sequence of nanobodies

[0165] AIL-B19 SEQ ID Nos: 1-3, 18, 60

[0166] VHH sequence

[0167] Nucleic acid sequence

[0168] AIL-B200 SEQ ID Nos: 4, 2, 5, 29, 71

[0169] VHH sequence

[0170] Nucleic acid sequence

[0171] AIL-182SEQ ID Nos:6-8, 39, 81

[0172] VHH sequence

[0173] Nucleic acid sequence

[0174] AIL-184SEQ ID Nos:9-11, 49, 91

[0175] VHH sequence

[0176] Nucleic acid sequence

[0177] Example 4: FACS Binding Detection of Recombinant Nanobody

[0178] The candidate nanobodies obtained from recombinant expression and purification were subjected to FACS binding detection using the HEK293 cell line huIL-4R-HEK293 overexpressing huIL-4R. The huIL-4R-HEK293 cells were prepared at a density of 1×10⁻⁶. 6 Cell suspension at 100 μL / mL was added to each well of a 96-well round-bottom plate. The plate was centrifuged at 300 g / min for 5 min, and the supernatant was discarded. The nanobodies to be tested were serially diluted with FACS staining buffer, and 100 μL was added to each well. The plate was incubated at 4 °C for 60 min, followed by washing twice with FACS staining buffer. A 1:300 dilution of the secondary antibody PE-labeled anti-human IgG Fc was added, and the plate was incubated at 4 °C for 30 min. After washing twice with FACS staining buffer, the samples were analyzed using Beckman flow cytometry. Figure 1 shows partial antibody FACS binding data, indicating that AIL-A182-Fc, AIL-A184-Fc, AIL-B19-Fc, and AIL-B200-Fc bound to huIL-4R-HEK293 cells.

[0179] Example 5: FACS blocking detection of recombinant nanobody

[0180] To detect the effect of recombinant nanobodies on blocking the binding of IL-4 to IL-4R on the surface of huIL-4R-HEK293 cells, huIL-4R-HEK293 cells were prepared at a cell density of 1×10⁻⁶. 6Cell suspension at cell / mL. Add 100 μL to each well of a 96-well round-bottom plate. Centrifuge at 300 g / min for 5 min, discarding the supernatant. Quantitatively dilute the antibody with FACS staining buffer, adding 100 μL to each well, incubate at 4°C for 60 min, and wash twice with FACS staining buffer. Dilute the ligand IL-4-hFc(293)-Biotin with FACS staining buffer to a final concentration of 0.1 μg / mL, adding 100 μL to each well, incubate at 4°C for 60 min, and wash twice with FACS staining buffer. Dilute the secondary antibody Streptavidin PE Conjugate 1:300 with FACS staining buffer, adding 100 μL to each well, incubate at 4°C for 30 min, and wash twice with FACS staining buffer. Detect using a Beckman flow cytometer. Figure 2 shows some FACS blocking data for the antibodies. The results indicate that AIL-A182-Fc, AIL-A184-Fc, AIL-B19-Fc, and AIL-B200-Fc have good activity in blocking the binding of IL-4 to IL-4R on the cell surface.

[0181] Example 6: Detection of STAT6 phosphorylation inhibition of recombinant nanobody

[0182] To detect the inhibitory effect of antibody STAT6 phosphorylation, an overexpression cell line Stat6-HEK293-huIL-4R was constructed, which responded to IL-4 and IL-13. The nanobody to be tested was diluted with DMEM medium containing 10% FBS at concentrations of 100.0000, 33.3333, 11.1111, 3.7037, 1.2346, 0.4115, 0.1372, and 0.0457 μg / mL and added to 96-well cell culture plates, 25 μL per well. The Stat6-HEK293-huIL-4R cells in the culture flasks were transferred to centrifuge tubes, centrifuged, and the supernatant was discarded. After resuspending the cells in DMEM medium containing 10% FBS and counting, the cell density was adjusted to 3 × 10⁶ cells / well. 5Cells / mL were added to cell culture plates, 50 μL per well. Incubation was performed at 37°C, 5% CO2 for 30 min. The ligand IL-4-his was diluted to 1.6 ng / mL with DMEM + 10% FBS, and added to cell culture plates, 25 μL per well. Incubation was performed at 37°C, 5% CO2 for 6 h. 50 μL of Bright-Lite assay buffer was added to each well. Detection was performed using a Molecular Devices microplate reader. As shown in Figure 3, AIL-A182-Fc, AIL-A184-Fc, AIL-B19-Fc, and AIL-B200-Fc exhibited inhibitory activity against IL-4-induced STAT6 phosphorylation.

[0183] Example 7: Detection of the inhibition of IL-4-His-induced TF-1 cell proliferation by recombinant nanobodies

[0184] Transfer TF-1 cells in the logarithmic growth phase to 50 mL centrifuge tubes, centrifuge at 300 g for 5 min, and discard the supernatant. Resuspend the cells in 15 mL of 1640 complete medium and mix well. Incubate overnight for 12 h in a T75 culture flask. Resuspend the TF-1 cells in 1640 medium containing 5% FBS and adjust the cell density to 2 × 10⁶ cells / mL. 5 Cells / mL were collected in a 96-well plate, with 50 μL added to each well. Antibody was diluted with 1640 medium containing 5% FBS to create eight concentration gradients: 1000, 200, 66.6, 22.2, 7.407, 2.469, 0.823, and 0.082 nM. 25 μL of each diluted antibody was added to each well, and the plate was incubated at 37°C for 30 min. IL-4-His was diluted with 1640 medium containing 5% FBS to a concentration of 160 ng / mL. 25 μL of each diluted antibody was added to each well, and the plate was incubated at 37°C for 72 h. 50 μL of CellTiter-Glo assay solution was added to each well, and the cells / mL were measured using a Molecular Devices microplate reader. As shown in Figure 4, AIL-A182-Fc, AIL-A184-Fc, AIL-B19-Fc, and AIL-B200-Fc all inhibited IL-4-His-induced TF-1 cell proliferation.

[0185] Example 8: Affinity kinetics detection and epitope grouping experiment of recombinant nanobodies

[0186] The affinity of the recombinant antibody for huI4R-his was determined using GATOR (ProbeLife) biomembrane interference (BLI) technology. The recombinant antibody was immobilized on a sensor chip using the Fc capture method, and the antigen protein was used as the analyte. The results are shown in Table 3, indicating that the recombinant antibody bound well to the huI4R-his antigen protein.

[0187] GATOR (ProbeLife) was used to detect the epitope grouping of recombinant antibodies against huI4R-his, detecting antibodies in pairs to determine whether antibodies competitively bind to the same sites on the antigen. The test antigen huI4R-his was immobilized on the sensor chip using the His capture method, with CM310 as the saturating antibody and the test antibody as the competing antibody. Inhibition rate was used to reflect epitope competition. The results are shown in Table 4. AIL-B19-Fc, AIL-B200, CM310, and Dupilumab (provided by Sanyou Biotechnology) shared the same epitopes. The epitopes of AIL-A182-Fc and AIL-B19-Fc / AIL-B200-Fc partially overlap. AIL-A184-Fc has different epitopes from AIL-B19-Fc and AIL-B200-Fc. We further performed sequence analysis and humanization on AIL-B19, AIL-B200, AIL-A182, and AIL-A184.

[0188] Table 3. Affinity kinetics detection

[0189] Table 4. Candidate antibody epitope grouping

[0190] Example 9: Humanization and Functional Detection of Nanobodies

[0191] To reduce the immunogenicity of candidate antibodies and improve the safety and efficacy of clinical applications, AIL-A182, AIL-A184, AIL-B19, and AIL-B200 were humanized. Homology modeling was used based on the parent sequences. Camel-derived antibody sequences were aligned with human Germline sequences. Nine molecules were designed for AIL-A182 humanization, eight for AIL-A184, ten for AIL-B19, and nine for AIL-B200. The sequences were constructed into Fc-tagged protein expression vectors and then recombinantly expressed in CHO cells. Expression levels were detected using Gator assays. After protein purification, SDS-PAGE, SEC, ELISA, and affinity kinetics were performed. The purity of humanized antibodies AIL-A184-VHH7-Fc, AIL-B19-VHH7-Fc, and AIL-B200-VHH7-Fc is greater than 95%, and the SEC monomer ratio of both humanized antibodies and parent antibodies is over 95%.

[0192] The obtained humanized antibody was sequenced, and the VHH region sequences of the humanized antibody were as follows:

[0193] AIL-B19-VHH1

[0194] VHH sequence SEQ ID No:19

[0195] Nucleic acid sequence SEQ ID No:61

[0196] AIL-B19-VHH2

[0197] VHH sequence SEQ ID No:20

[0198] Nucleic acid sequence SEQ ID No:62

[0199] AIL-B19-VHH3

[0200] VHH sequence SEQ ID No:21

[0201] Nucleic acid sequence SEQ ID No:63

[0202] AIL-B19-VHH4

[0203] VHH sequence SEQ ID No:22

[0204] Nucleic acid sequence SEQ ID No:64

[0205] AIL-B19-VHH5

[0206] VHH sequence SEQ ID No:23

[0207] Nucleic acid sequence SEQ ID No:65

[0208] AIL-B19-VHH6

[0209] VHH sequence SEQ ID No:24

[0210] Nucleic acid sequence SEQ ID No:66

[0211] AIL-B19-VHH7

[0212] VHH sequence SEQ ID No:25

[0213] Nucleic acid sequence SEQ ID No:67

[0214] AIL-B19-VHH8

[0215] VHH sequence SEQ ID No:26

[0216] Nucleic acid sequence SEQ ID No:68

[0217] AIL-B19-VHH9

[0218] VHH sequence SEQ ID No:27

[0219] Nucleic acid sequence SEQ ID No:69

[0220] AIL-B19-VHH10

[0221] VHH sequence SEQ ID No:28

[0222] Nucleic acid sequence SEQ ID No:70

[0223] AIL-B200-VHH1

[0224] VHH sequence SEQ ID No:30

[0225] Nucleic acid sequence SEQ ID No:72

[0226] AIL-B200-VHH2

[0227] VHH sequence SEQ ID No:31

[0228] Nucleic acid sequence SEQ ID No:73

[0229] AIL-B200-VHH3

[0230] VHH sequence SEQ ID No:32

[0231] Nucleic acid sequence SEQ ID No:74

[0232] AIL-B200-VHH4

[0233] VHH sequence SEQ ID No:33

[0234] Nucleic acid sequence SEQ ID No:75

[0235] AIL-B200-VHH5

[0236] VHH sequence SEQ ID No:34

[0237] Nucleic acid sequence SEQ ID No:76

[0238] AIL-B200-VHH6

[0239] VHH sequence SEQ ID No:35

[0240] Nucleic acid sequence SEQ ID No:77

[0241] AIL-B200-VHH7

[0242] VHH sequence SEQ ID No:36

[0243] Nucleic acid sequence SEQ ID No:78

[0244] AIL-B200-VHH8

[0245] VHH sequence SEQ ID No:37

[0246] Nucleic acid sequence SEQ ID No:79

[0247] AIL-B200-VHH9

[0248] VHH sequence SEQ ID No:38

[0249] Nucleic acid sequence SEQ ID No:80

[0250] AIL-182-VHH1

[0251] VHH sequence SEQ ID No:40

[0252] Nucleic acid sequence SEQ ID No:82

[0253] AIL-182-VHH2

[0254] VHH sequence SEQ ID No:41

[0255] Nucleic acid sequence SEQ ID No:83

[0256] AIL-182-VHH3

[0257] VHH sequence SEQ ID No:42

[0258] Nucleic acid sequence SEQ ID No:84

[0259] AIL-182-VHH4

[0260] VHH sequence SEQ ID No:43

[0261] Nucleic acid sequence SEQ ID No:85

[0262] AIL-182-VHH5

[0263] VHH sequence SEQ ID No:44

[0264] Nucleic acid sequence SEQ ID No:86

[0265] AIL-182-VHH6

[0266] VHH sequence SEQ ID No:45

[0267] Nucleic acid sequence SEQ ID No:87

[0268] AIL-182-VHH7

[0269] VHH sequence SEQ ID No:46

[0270] Nucleic acid sequence SEQ ID No:88

[0271] AIL-182-VHH8

[0272] VHH sequence SEQ ID No:47

[0273] Nucleic acid sequence SEQ ID No:89

[0274] AIL-182-VHH9

[0275] VHH sequence SEQ ID No:48

[0276] Nucleic acid sequence SEQ ID No:90

[0277] AIL-184-VHH1

[0278] VHH sequence SEQ ID No:50

[0279] Nucleic acid sequence SEQ ID No:92

[0280] AIL-184-VHH2

[0281] VHH sequence SEQ ID No:51

[0282] Nucleic acid sequence SEQ ID No:93

[0283] AIL-184-VHH3

[0284] VHH sequence SEQ ID No:52

[0285] Nucleic acid sequence SEQ ID No:94

[0286] AIL-184-VHH4

[0287] VHH sequence SEQ ID No:53

[0288] Nucleic acid sequence SEQ ID No:95

[0289] AIL-184-VHH5

[0290] VHH sequence SEQ ID No:54

[0291] Nucleic acid sequence SEQ ID No:96

[0292] AIL-184-VHH6

[0293] VHH sequence SEQ ID No:55

[0294] Nucleic acid sequence SEQ ID No:97

[0295] AIL-184-VHH7

[0296] VHH sequence SEQ ID No:56

[0297] Nucleic acid sequence SEQ ID No:98

[0298] AIL-184-VHH8

[0299] VHH sequence SEQ ID No:57

[0300] Nucleic acid sequence SEQ ID No:99

[0301] 9.1. Humanized Antibody ELISA Binding Detection

[0302] Dilute the antigen samples hIL-4R-His and cyno IL-4R-His to 2 μg / mL with 1×PBS, and add 30 μL / well to each well of a 96-well ELISA plate. Incubate overnight (or more than 12 h) at 4°C. Wash the plate three times with PBST, add blocking buffer (5% PBS-milk), and block for 2 h at room temperature. Wash the plate three times with PBST, add 30 μL / well of antibody diluted with 1% PBS-milk, and incubate for 60 min at room temperature. Wash the plate three times with PBST, add diluted secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab97225) at a 1:8000 dilution ratio, diluted with 1% PBS-milk, and incubate for 60 min at room temperature. Wash the plate three times with PBST, and add 30 μL of TMB to each well. Stop the reaction by adding 2M stop solution and measure OD. 450 As shown in Figure 5, AIL-A182-VHH7-Fc, AIL-A184-VHH7-Fc, AIL-B19-VHH7-Fc, and AIL-B200-VHH7-Fc bind to human and monkey IL-4R.

[0303] 9.2. Humanized Antibody ELISA Blocking Detection

[0304] Dilute the antigen sample huI4R-hFc to 8 μg / mL with 1×PBS, and add 30 μL / well to a 96-well ELISA plate. Incubate overnight (or more than 12 h) at 4°C. Wash the plate three times with PBST, add blocking buffer (5% PBS-milk), and block at room temperature for 2 h. Wash the plate again with PBST, add 30 μL / well of antibody diluted with 1% PBS-milk, and incubate at room temperature for 60 min. Without washing, dilute the ligand sample IL-4-hFc-Biotin to 0.0625 μg / mL with 1% PBS-milk, and add 30 μL / well to a 96-well ELISA plate. Incubate at room temperature for 60 min. Wash the plate three times with PBST, add diluted secondary antibody NeutrAvidin-HRP (Thermo Fisher; 31001) at a 1:2000 dilution ratio, diluted with 1×PBS, and incubate at room temperature for 60 min. Wash the plate three times with PBST, and add 30 μL of TMB to each well. Stop the reaction by adding 2 M stop solution and measure the OD. 450 As shown in Figure 6, AIL-A182-VHH7-Fc had no blocking effect, AIL-A184-VHH7-Fc had a weak blocking effect, but AIL-B19-VHH7-Fc and AIL-B200-VHH7-Fc showed better activity in blocking IL-4 binding to IL-4R than the parent strain.

[0305] 9.3. FACS binding detection of humanized candidate molecules on HEK293-huIL-4R cells

[0306] Following the method in Example 4, FACS binding detection was performed on the humanized molecules, and the results are shown in Figure 7: AIL-A182-VHH7-Fc, AIL-A184-VHH7-Fc, AIL-B19-VHH7-Fc, AIL-B200-VHH7-Fc and HEK293-huIL-4R all bound to the IL-4R protein overexpressed on the cell surface. The binding activity of the humanized molecules to IL-4R on the cell surface was comparable to that of the parent molecules.

[0307] 9.4. FACS blocking detection of humanized candidate molecules

[0308] Following the method described in Example 5, FACS blocking assays were performed on the humanized molecules A184-VHH7-Fc, B19-VHH7-Fc, and B200-VHH7-Fc. The results are shown in Figure 8. All three molecules—AIL-A182-VHH7-Fc, AIL-A184-VHH7-Fc, AIL-B19-VHH7-Fc, and AIL-B200-VHH7-Fc—can block the binding of IL-4 to IL-4R on the cell surface. The blocking effect of AIL-B19-VHH7-Fc is superior to that of the parent molecule AIL-B19-Fc, while the blocking effect of AIL-B200-VHH7-Fc is comparable to that of the parent molecule AIL-B200-Fc.

[0309] 9.5. Humanized Antibody Affinity Kinetics Detection

[0310] Referring to the method in Example 8, affinity kinetics of the humanized antibodies were tested, and the results are shown in Table 5: the humanized antibody A184-VHH7-Fc had strong affinity, AIL-B19-VHH7-Fc had better affinity than the parent antibody AIL-B19-Fc, AIL-182-VHH7-Fc had comparable affinity to the parent antibody, and the humanized molecule AIL-B200-VHH7-Fc had comparable affinity to the parent antibody.

[0311] Table 5. Results of humanized antibody affinity kinetics assay

[0312] Example 10: Antibody Affinity Maturation

[0313] Based on affinity testing results, AIL-B200-VHH7-Fc and AIL-B19-VHH7-Fc exhibited weak affinity; therefore, AIL-B200-VHH7 and AIL-B19-VHH7 were selected for affinity maturation. Phage display libraries were constructed using single-point and double-point combination mutations. The library size was determined by dilution plating, and single-clone sequencing analysis verified the correct insertion rate of the antibody genes. Compared with the parent amino acid profile, the mutated amino acid distribution in each library was uniform, and the effective library size covered the theoretical library size by 5–10 times or more. Specific screening methods are described in Example 2. For B200, 57 candidate clones were obtained. Based on sequence diversity and ELISA results, 46 molecules were selected for full-length construction and Fc tag fusion for protein expression. For B19, 72 candidate clones were obtained, and 63 molecules were selected for full-length construction and Fc tag fusion for protein expression.

[0314] 10.1. ELISA Detection of Antibody Molecules Binding and Blocking After Affinity Maturation

[0315] Referring to the detection method in Example 9, the antibodies after affinity maturation were subjected to ELISA binding and blocking detection. Figure 9 and Table 5 show the detection results of some of the antibodies after affinity maturation. B19_13 and B200_57 can bind to both human IL-4R (Figure 9-A) and monkey IL-4R (Figure 9-B), and at the ELISA level, they can block the binding of IL-4 and IL-4R (Figure 9-C).

[0316] Sequencing of the affinity-matured antibodies yielded the VHH region sequences of B19_13 and B200_57, respectively:

[0317] Table 6. Variable region sequence of nanobodies after affinity maturation

[0318] B19_13SEQ ID Nos:12-14, 16, 58

[0319] VHH sequence

[0320] Nucleic acid sequence

[0321] B200_57SEQ ID Nos:15, 111, 112, 17, 59

[0322] VHH sequence

[0323] Nucleic acid sequence

[0324] 10.2. Detection of STAT6 phosphorylation inhibition by affinity maturation antibody

[0325] Following the method in Example 6, the antibody after affinity maturation was subjected to STAT6 phosphorylation inhibition detection, and the results are shown in Figure 10: B19_13 and B200_57 both inhibited IL-4-induced STAT6 phosphorylation.

[0326] 10.3. Detection of cell proliferation inhibition by TF-1 nanobody with maturation affinity

[0327] Referring to the method in Example 6, the affinity maturation antibody was used to detect the inhibition of TF-1 cell proliferation. The results are shown in Figure 11: Candidate antibodies B19_13 and B200_57 can both inhibit IL-4-His-induced TF-1 cell proliferation.

[0328] 10.4. Affinity kinetics of antibodies after affinity maturation

[0329] Following the method in Example 7, affinity kinetics were tested on the affinity-matured nanobodies, and the results are shown in Table 7: the affinity of the affinity-matured antibodies to the parent molecules was significantly improved.

[0330] Table 7. Affinity kinetics tests for B19_13 and B200_57

[0331] Example 11: Preparation and Activity Detection of Biseptopy Histidine-Tagged Nanobodies

[0332] To further enhance antibody activity, a dual-epitope histidine-tagged nanobody was designed. The antibody sequence was constructed into an expression vector, and after recombinant expression and purification in Expi CHO-S cells, activity analysis was performed. The activity assay results are summarized in Table 8.

[0333] Table 8. Summary of Dual Epitope Nanobody Activity Detection

[0334] A182-VHH7-B19_13-His SEQ ID No:103

[0335] A182-VHH7-B200_57-His SEQ ID No:104

[0336] A184-VHH7-B19_13-His SEQ ID No:105

[0337] A184-VHH7-B200_57-His SEQ ID No:106

[0338] 11.1. ELISA Binding and Blocking Detection of Dual-Epitope Histidine-Tagged Nanobodies

[0339] Referring to Example 10, ELISA binding and blocking assays were performed on the dual-epitope histidine-tagged nanobodies. The results are shown in Figure 12: A182-VHH7-B19_13-His, A182-VHH7-B200_57-His, A184-VHH7-B19_13-His, and A184-VHH7-B200_57-His all showed good binding to human and monkey IL-4R. A182-VHH7-B19_13-His, A182-VHH7-B200_57-His, A184-VHH7-B19_13-His, and A184-VHH7-B200_57-His all blocked the binding activity of IL-4 to human IL-4R protein.

[0340] 11.2. Detection of phosphorylation inhibition of dual-epitope nanobody

[0341] Example 6 describes the detection of the dual epitope histidine-tagged nanobody, as shown in Figure 13: The activity of the Stat6-HEK293-huIL-4R luciferase reporter system induced by IL-4 and IL-13 was detected, and the phosphorylation inhibition activity of A182-VHH7-B19_13-His, A182-VHH7-B200_57-His, and A184-VHH7-B19_13-His was superior to that of the positive control Dupilumab.

[0342] 11.3. FACS Binding and Blocking Detection of Dual-Epitope Histidine-Tagged Nanobodies

[0343] The dual-epitope nanohistidine-tagged antibodies were tested according to Examples 4 and 5. As shown in Figure 14, the binding activity of A182-VHH7-B19_13-His, A182-VHH7-B200_57-His, and A184-VHH7-B19_13-His on huIL-4R-HEK293 cells was superior to that of the positive antibody Dupilumab. The blocking effects of A182-VHH7-B19_13-His and A182-VHH7-B200_57-His were weaker than those of the positive antibody Dupilumab, while the blocking effects of A184-VHH7-B19_13-His and A184-VHH7-B200_57-His were comparable to those of the positive antibody Dupilumab.

[0344] 11.4. Detection of the inhibitory activity of dual-epitope histidine-tagged nanobodies on IL-4 / IL-13-induced TF-1 cell proliferation.

[0345] Referring to Example 7, the inhibition of TF-1 cell proliferation was detected by dual epitope histidine-tagged nanobodies. The results are shown in Figure 15. A182-VHH7-B19_13-His, A182-VHH7-B200_57-His, A184-VHH7-B19_13-His and A184-VHH7-B200_57-His can all inhibit IL-4 (Figure 15-A) and IL-13 (Figure 15-B) induced TF-1 cell proliferation, and are superior to the positive control Dupilumab.

[0346] 11.5. Affinity Kinetics Detection of Dual-Epitope Histidine-Tagged Nanobodies

[0347] Example 9 tested the dual-epitope histidine-tagged nanobodies, and the results are shown in Table 9: The binding activities of A182-VHH7-B19_13-His, A182-VHH7-B200_57-His, A184-VHH7-B19_13-His, and A184-VHH7-B200_57-His with IL-4R were comparable to those of the positive control Dupilumab.

[0348] Table 9. Affinity kinetics detection results of dual epitope nanobodies

[0349] Example 12: Preparation and Activity Detection of Tetravalent Nanobodies

[0350] The polyhistidine tag of the dual-epitope nanobody was replaced with an Fc tag and constructed into an expression vector. Recombinant expression was performed in Expi CHO-S cells. The tetravalent nanobody was purified by a protein A purification column and its function was analyzed. The results are summarized in Table 10.

[0351] Table 10. Summary of tetravalent nanobody activity assays

[0352] A182-VHH7-B19_13-Fc SEQ ID No:107

[0353] A182-VHH7-B200_57-Fc SEQ ID No:108

[0354] VFSCSVMHEALHNHYTQKSLSLSLGK

[0355] A184-VHH7-B19_13-Fc SEQ ID No:109

[0356] A184-VHH7-B200_57-Fc SEQ ID No:110

[0357] 12.1. ELISA Binding and Blocking Detection

[0358] Referring to Example 10, ELISA binding and blocking assays were performed on the tetravalent nanobodies. The results are shown in Figure 16 and Table 8: A182-VHH7-B19_13-Fc, A182-VHH7-B200_57-Fc, A184-VHH7-B19_13-Fc, and A184-VHH7-B200_57-Fc exhibited superior or comparable binding activity to human or monkey IL-4R protein compared to the positive control Dupilumab; their blocking activity against human IL-4 and human IL-4R protein was comparable to that of the positive control Dupilumab. Combining nanobodies into biepisode antibodies or tetravalent antibodies can enhance the binding activity of antibodies to human or monkey IL-4R protein; and the Fc-tagged tetravalent nanobodies showed superior protein-level binding activity compared to histidine-tagged biepisode antibodies.

[0359] 12.2. Detection of FACS binding and blocking of tetravalent nanobodies

[0360] The tetravalent nanobodies were subjected to FACS binding and blocking assays according to Examples 4 and 5. The results are shown in Figure 17. All four tetravalent antibodies could bind to IL-4R on the cell surface and could block the binding of IL-4R to IL-4. Except for A182-VHH7-B200_57-Fc, the binding activity of the tetravalent antibodies at the FACS level was superior to that of the positive control Dupilumab; the blocking activities of A182-VHH7-B19_13-Fc, A182-VHH7-B200_57-Fc, A184-VHH7-B19_13-Fc, and A184-VHH7-B200_57-Fc were all superior to those of the positive control Dupilumab.

[0361] 12.3. Detection of STAT6 phosphorylation inhibition

[0362] Referring to Example 6, STAT6 phosphorylation inhibition of the tetravalent nanobody was detected using Stat6-HEK293-huIL-4R cells. Figure 18-A shows the detection results of the tetravalent nanobody inhibiting IL-4-induced STAT6 luciferase reporter gene activity, and Figure 18-B shows the detection results of the tetravalent nanobody inhibiting IL-13-induced STAT6 luciferase reporter gene activity. (A182-VHH7-B19_13-Fc, A182-VHH7-B200_57-Fc, A184-VH) H7-B19_13-Fc and A184-VHH7-B200_57-Fc significantly inhibited IL-4 and IL-13-induced STAT6 luciferase reporter gene activity better than the positive control Dupilumab, indicating that A182-VHH7-B19_13-Fc, A182-VHH7-B200_57-Fc, A184-VHH7-B19_13-Fc and A184-VHH7-B200_57-Fc can block the IL-4 and IL-13 signaling pathways.

[0363] 12.4. Cell proliferation inhibition detection

[0364] Referring to Example 7, the Fc-tagged tetravalent nanobodies were used to detect the proliferation inhibition of TF-1 cells, as shown in Figure 19-A: A182-VHH7-B19_13-His, A182-VHH7-B200_57-His, A184-VHH7-B19_13-Fc and A184-VHH7-B200_57-Fc could all inhibit IL-4-induced TF-1 cell proliferation, and their cell proliferation inhibition effects were all better than those of the control antibody Dupilumab. As shown in Figure 19-B: A182-VHH7-B19_13-His, A182-VHH7-B200_57-His, A184-VHH7--B19_13-Fc and A184-VHH7-B200_57-Fc can all inhibit IL-13-induced TF-1 cell proliferation, and their cell proliferation inhibition effects are all superior to the control antibody Dupilumab.

[0365] 12.5. Detection of phosphorylation of tetravalent nanobody PBMC-STAT6

[0366] One day in advance, PBMC cells were cultured in T75 flasks at 37°C for 16 h. Antibody (final concentration: 200 nM) was serially diluted in serum-free 1640 medium to 50 μL per well. 100 μL of PBMC cells was mixed with serum-free 1640 medium and added to the wells at a cell density of 2E5 / well, and incubated at 37°C for 30 min. IL-4-His (final concentration: 30 nM) was serially diluted in serum-free 1640 medium to 50 μL per well, and incubated at 37°C for 20 min. 100 μL of 4% formaldehyde solution was added to each well, and incubated at room temperature for 20 min. 100 μL of 0.5% Triton X-100 was added to each well, and incubated at room temperature for 5 min. Cells were centrifuged and washed with FACS buffer; 100 μL of a mixture of PE-labeled anti-STAT6 phosphorylation antibody (dilution 1:100) and APC-labeled anti-human CD3 antibody (dilution 1:100) was added to the cells; the mixture was incubated at room temperature for 60 min; the cells were centrifuged and washed with FACS buffer; the cells were resuspended in FACS buffer; and the cells were analyzed by flow cytometry. The results are shown in Figure 20: the candidate antibodies A184-VHH7-B200_57-Fc, A184-VHH7-B19_13-Fc, A182-VHH7-B19_13-Fc, and A182-VHH7-B200_57-Fc showed comparable inhibitory effects on STAT6 phosphorylation to the positive antibody Dupilumab.

[0367] 12.6. Affinity Kinetics Detection of Tetravalent Nanobodies

[0368] Affinity kinetics were tested according to Example 7, and the results are shown in Table 11. The kd value of A184-VHH7-B19_13-Fc exceeded the instrument's detection limit. The affinity of A182-VHH7-B19_13-Fc, A182-VHH7-B200_57-Fc, A184-VHH7-B19_13-Fc, and A184-VHH7-B200_57-Fc for IL-4R was better than that of the control, Dupilumab.

[0369] Table 11. Affinity kinetics detection results of tetravalent nanobodies

Claims

1. An interleukin-4 receptor (IL-4R) binding molecule, wherein the heavy chain variable region (VH) comprises a heavy chain complementarity-determining region 1 (HCDR1), a heavy chain complementarity-determining region 2 (HCDR2), and a heavy chain complementarity-determining region (HCDR3), wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, 4, 6, 9, 12, or 15 or any variant thereof; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, 7, 10, 13, or 111 or any variant thereof; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, 5, 8, 11, 14, or 112 or any variant thereof.

2. The IL-4R binding molecule according to claim 1, wherein HCDR1, HCDR2 and HCDR3 have amino acid sequences that are more than 80% identical to the amino acid sequences shown in SEQ ID NOs: 1, 4, 6, 9, 12 or 15, SEQ ID NOs: 2, 7, 10, 13 or 111, and SEQ ID NOs: 3, 5, 8, 11, 14 or 112; Preferably, the IL-4R binding molecule comprises a combination selected from the following HCDR1, HCDR2, and HCDR3: (1) Containing HCDR1, HCDR2, and HCDR3 sequences as shown in SEQ ID NOs: 1, 2, and 3, respectively; or (2) Containing HCDR1, HCDR2, and HCDR3 sequences as shown in SEQ ID NOs: 4, 2, and 5, respectively; or (3) Containing HCDR1, HCDR2, and HCDR3 sequences as shown in SEQ ID NOs: 6, 7, and 8, respectively; or (4) Containing HCDR1, HCDR2, and HCDR3 sequences as shown in SEQ ID NOs: 9, 10, and 11, respectively; or (5) Containing HCDR1, HCDR2, and HCDR3 sequences as shown in SEQ ID NOs: 12, 13, and 14, respectively; or (6) Each contains the HCDR1, HCDR2 and HCDR3 sequences as shown in SEQ ID NOs: 15, 111 and 112 respectively.

3. The IL-4R binding molecule according to claim 1 or 2, wherein HCDR1, HCDR2, and HCDR3 of the IL-4R binding molecule respectively have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:16; or The IL-4R binding molecule's HCDR1, HCDR2, and HCDR3 respectively have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:17; or The IL-4R binding molecule's HCDR1, HCDR2, and HCDR3 respectively have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:18; or The IL-4R binding molecule's HCDR1, HCDR2, and HCDR3 respectively have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:25; or The IL-4R binding molecule's HCDR1, HCDR2, and HCDR3 respectively have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:29; or The IL-4R binding molecule's HCDR1, HCDR2, and HCDR3 respectively have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:36; or The IL-4R binding molecule's HCDR1, HCDR2, and HCDR3 respectively have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:39; or The IL-4R binding molecule's HCDR1, HCDR2, and HCDR3 respectively have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:46; or The IL-4R binding molecule's HCDR1, HCDR2, and HCDR3 respectively have the amino acid sequences of CDR1, CDR2, and CDR3 within the VH sequence shown in SEQ ID NO:49; or The IL-4R binding molecules HCDR1, HCDR2 and HCDR3 have the amino acid sequences of CDR1, CDR2 and CDR3 within the VH sequence shown in SEQ ID NO:56, respectively.

4. The IL-4R binding molecule according to any one of claims 1-3, wherein the VH comprises: (i) an amino acid sequence selected from SEQ ID NOs: 16-57; and / or (ii) an amino acid sequence having more than 80% identity with the amino acid sequence shown in SEQ ID NOs: 16-57.

5. The IL-4R binding molecule of any of claims 1-4, characterized in that, The binding molecules are full-length antibodies, Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv) fragments, scFv-scFv, microantibodies, bispecific antibodies, or single-domain antibodies.

6. The IL-4R binding molecule according to any one of claims 1-5, wherein the amino acid sequence comprises an amino acid sequence obtained by at least one of addition, deletion, modification and / or substitution of the amino acid sequence shown in SEQ ID NOs: 16-57; wherein the addition, deletion, modification and / or substitution comprises 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid differences; preferably, the addition, deletion, modification and / or substitution does not change the binding function of the IL-4R binding molecule to IL-4R; preferably, the addition, deletion, modification and / or substitution occurs in the frame region of the molecule.

7. A tandem molecule comprising the IL-4R binding molecule according to any one of claims 1-6.

8. The tandem molecule of claim 7, wherein, The tandem molecule comprises a first single-domain antibody heavy chain antibody variable region (VHH region) and a second single-domain antibody VHH region; the first single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (1), (2), (5) or (6) of claim 2, and the second single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (3) or (4) of claim 2. Preferably, the first single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (5) or (6) of claim 2, and the second single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (3) or (4) of claim 2; More preferably, the first single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (5) of claim 2, and the second single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (3) of claim 2; More preferably, the first single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (6) of claim 2, and the second single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (4) of claim 2; More preferably, the first single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (5) of claim 2, and the second single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (4) of claim 2; More preferably, the first single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (6) of claim 2, and the second single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (3) of claim 2; Preferably, the first single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 16-38 or sequences having more than 80% identity with them, and the second single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 39-57 or sequences having more than 80% identity with them. More preferably, the first single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 16 or 17 or sequences having more than 80% identity with them, and the second single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 39-57 or sequences having more than 80% identity with them. More preferably, the first single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 16, and the second single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO:

46. More preferably, the first single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 17, and the second single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 56; More preferably, the first single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 16, and the second single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO:

56. More preferably, the first single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 17, and the second single-domain antibody VHH region of the tandem molecule comprises the heavy chain variable region of amino acid sequence SEQ ID NO:

46.

9. The tandem molecule of claim 8, wherein, It further comprises a third single-domain antibody VHH region and a fourth single-domain antibody VHH region; the third single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (1), (2), (5) or (6) of claim 2, and the fourth single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (3) or (4) of claim 2. Preferably, the first single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (5) or (6) of claim 2, the second single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (3) or (4) of claim 2, the third single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (5) or (6) of claim 2, and the fourth single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (3) or (4) of claim 2; More preferably, the first single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (5) of claim 2, the second single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (3) of claim 2, the third single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (5) of claim 2, and the fourth single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (3) of claim 2; More preferably, the first single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (6) of claim 2, the second single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (4) of claim 2, the third single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (6) of claim 2, and the fourth single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (4) of claim 2; More preferably, the first single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (5) of claim 2, the second single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (4) of claim 2, the third single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (5) of claim 2, and the fourth single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (4) of claim 2; More preferably, the first single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (6) of claim 2, the second single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (3) of claim 2, the third single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (6) of claim 2, and the fourth single-domain antibody VHH region of the tandem molecule comprises the HCDR combination as described in group (3) of claim 2; The third single-domain antibody VHH region of the tandem molecule contains a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 16-38 or sequences having more than 80% identity with them, and the fourth single-domain antibody VHH region of the tandem molecule contains a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 39-57 or sequences having more than 80% identity with them. Preferably, the first single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 16 or 17 or sequences having more than 80% identity with them; the second single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 39-57 or sequences having more than 80% identity with them; the third single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 16 or 17 or sequences having more than 80% identity with them; and the fourth single-domain antibody VHH region of the tandem molecule comprises a heavy chain variable region selected from amino acid sequences SEQ ID NOs: 39-57 or sequences having more than 80% identity with them. More preferably, the first single-domain antibody VHH region of the tandem molecule includes the heavy chain variable region of amino acid sequence SEQ ID NO: 16, the second single-domain antibody VHH region of the tandem molecule includes the heavy chain variable region of amino acid sequence SEQ ID NO: 46, the third single-domain antibody VHH region of the tandem molecule includes the heavy chain variable region of amino acid sequence SEQ ID NO: 16, and the fourth single-domain antibody VHH region of the tandem molecule includes the heavy chain variable region of amino acid sequence SEQ ID NO: 46; More preferably, the first single-domain antibody VHH region of the tandem molecule includes the heavy chain variable region of amino acid sequence SEQ ID NO: 17, the second single-domain antibody VHH region of the tandem molecule includes the heavy chain variable region of amino acid sequence SEQ ID NO: 56, the third single-domain antibody VHH region of the tandem molecule includes the heavy chain variable region of amino acid sequence SEQ ID NO: 17, and the fourth single-domain antibody VHH region of the tandem molecule includes the heavy chain variable region of amino acid sequence SEQ ID NO: 56; More preferably, the first single-domain antibody VHH region of the tandem molecule includes the heavy chain variable region of amino acid sequence SEQ ID NO: 16, the second single-domain antibody VHH region of the tandem molecule includes the heavy chain variable region of amino acid sequence SEQ ID NO: 56, the third single-domain antibody VHH region of the tandem molecule includes the heavy chain variable region of amino acid sequence SEQ ID NO: 16, and the fourth single-domain antibody VHH region of the tandem molecule includes the heavy chain variable region of amino acid sequence SEQ ID NO: 56; More preferably, the first single-domain antibody VHH region of the tandem molecule includes the heavy chain variable region of amino acid sequence SEQ ID NO: 17, the second single-domain antibody VHH region of the tandem molecule includes the heavy chain variable region of amino acid sequence SEQ ID NO: 46, the third single-domain antibody VHH region of the tandem molecule includes the heavy chain variable region of amino acid sequence SEQ ID NO: 17, and the fourth single-domain antibody VHH region of the tandem molecule includes the heavy chain variable region of amino acid sequence SEQ ID NO:

46.

10. The tandem molecule according to claim 8 or 9, characterized in that, The amino acid sequences of the IL-4R binding molecule or the VHH region of the single-domain antibody in the tandem molecule are directly linked or indirectly linked through a linker.

11. The tandem molecule of claim 10, wherein, The linker contains the amino acid sequence GGGGS (SEQ ID NO: 100) or has the general sequence formula (GGGGS)n, where n is an integer selected from 1 to 8; preferably, the amino acid sequence of the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 101).

12. The tandem molecule according to any one of claims 7 to 11, characterized in that, The IL-4R binding molecules or single-domain antibody VHH regions in the tandem molecules are linked in an optional order; or The IL-4R binding molecules or single-domain antibody VHH regions in the tandem molecules are linked in order from the N-terminus to the C-terminus; or The IL-4R binding molecules or single-domain antibody VHH regions in the tandem molecules are linked in order from the C-terminus to the N-terminus.

13. A recombinant protein, characterized in that, It comprises the IL-4R-binding molecule as described in any one of claims 1-6 or the tandem molecule as described in any one of claims 7-12.

14. The recombinant protein according to claim 13, characterized in that, It also includes bioactive proteins or functional fragments that assist in their expression and / or secretion and / or prolong their half-life in vivo; Preferably, the bioactive protein or functional fragment is selected from at least one of the following: immunoglobulin Fc domain, serum albumin, albumin-binding polypeptide, prealbumin, carboxyl-terminal peptide, elastin-like polypeptide, His tag, GST tag, MBP tag, FLAG tag, and SUMO tag.

15. The recombinant protein according to claim 14, characterized in that, The bioactive protein or functional fragment is a human immunoglobulin Fc domain, preferably a human IgG Fc domain, including the Fc domains of human IgG1, IgG2, IgG3, and IgG4, more preferably the Fc domain of human IgG4, and / or the Fc domain contains an amino acid sequence as shown in SEQ ID NO: 102; or the bioactive protein or functional fragment is a His tag.

16. The recombinant protein according to any one of claims 13-15, comprising from the N-terminus to the C-terminus: (1) VH1, a linker, VH2, and a His tag; or (2) VH1, a linker, VH2, and an Fc domain; Preferably, VH1 comprises an amino acid sequence selected from SEQ ID NOs: 16 or 17; and / or the linker comprises an amino acid sequence selected from SEQ ID NO: 101; and / or VH2 comprises an amino acid sequence selected from SEQ ID NOs: 46 or 56; and / or the Fc domain comprises an amino acid sequence selected from SEQ ID NO:

102. More preferably, the recombinant protein comprises an amino acid sequence selected from the sequence shown in SEQ ID NOs: 103-110.

17. A nucleic acid comprising a polynucleotide encoding an IL-4R binding molecule according to any one of claims 1-6, a tandem molecule according to any one of claims 7-12, or a recombinant protein according to any one of claims 13-16; Preferably, the nucleic acid encoding the IL-4R binding molecule is selected from the nucleic acid sequences shown in SEQ ID NOs: 58-99; Preferably, the nucleic acid encoding the tandem molecule or recombinant protein comprises a nucleic acid sequence selected from those shown in SEQ ID NOs: 58-99; More preferably, the nucleic acid encoding the tandem molecule or recombinant protein comprises the nucleic acid sequences shown in SEQ ID NOs: 58 and 88; More preferably, the nucleic acid encoding the tandem molecule or recombinant protein comprises the nucleic acid sequences shown in SEQ ID NOs: 59 and 88; More preferably, the nucleic acid encoding the tandem molecule or recombinant protein comprises the nucleic acid sequences shown in SEQ ID NOs: 58 and 98; More preferably, the nucleic acid encoding the tandem molecule or recombinant protein comprises the nucleic acid sequences shown in SEQ ID NOs: 59 and 98.

18. A vector comprising one or more nucleic acids according to claim 17.

19. A cell comprising the nucleic acid according to claim 17 or the vector according to claim 18.

20. A method for generating an IL-4R binding molecule according to any one of claims 1-6, a tandem molecule according to any one of claims 7-12, or a recombinant protein according to any one of claims 13-16, the method comprising culturing a host cell according to claim 19 comprising the nucleic acid according to claim 17 or the expression vector according to claim 18.

21. A pharmaceutical composition or pharmaceutical composition comprising an IL-4R binding molecule according to any one of claims 1-6, a tandem molecule according to any one of claims 7-12, a recombinant protein according to any one of claims 13-16, a nucleic acid according to claim 17, a carrier according to claim 18, and / or a cell according to claim 19; Preferably, the composition or pharmaceutical combination further includes a second therapeutic agent; Preferably, the second therapeutic agent is selected from β-2-adrenergic receptor agonists (SABA), anticholinergic drugs, adrenergic agonists, glucocorticoids, long-acting β-2-adrenergic receptor agonists (LABA), leukotriene antagonists, and mast cell stabilizers.

22. An antibody-drug conjugate comprising an IL-4R binding molecule according to any one of claims 1-6, a tandem molecule according to any one of claims 7-12, or a recombinant protein according to any one of claims 13-16, covalently attached to a therapeutic portion; Preferably, the therapeutic component is a cytotoxic component, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a cleavage peptide, or a radioactive isotope.

23. A bispecific antibody or a multispecific antibody, wherein one antigen-binding domain comprises the IL-4R binding molecule according to any one of claims 1-6, the tandem molecule according to any one of claims 7-12, or the recombinant protein according to any one of claims 13-16.

24. A chimeric antigen receptor, wherein the extracellular recognition unit comprises an IL-4R binding molecule according to any one of claims 1-6, a tandem molecule according to any one of claims 7-12, or a recombinant protein according to any one of claims 13-16.

25. Use of the IL-4R binding molecule according to any one of claims 1-6, the tandem molecule according to any one of claims 7-12, the recombinant protein according to any one of claims 13-16, the nucleic acid according to claim 17, the vector according to claim 18, the cell according to claim 19, the pharmaceutical composition or pharmaceutical composition according to claim 21, the antibody-drug conjugate according to claim 22, the bispecific antibody or multispecific antibody according to claim 23, and / or the chimeric antigen receptor according to claim 24 in the preparation of medicaments or kits for detecting, treating, preventing, and / or alleviating IL-4R-related diseases; said diseases include Th2-mediated diseases, IL-13-mediated diseases, IL-4-mediated diseases, and / or IL-4 / IL-13-mediated diseases.

26. The use of claim 25, wherein, The diseases mentioned are selected from chronic obstructive pulmonary disease, chronic urticaria, eosinophilic esophagitis, nodular prurigo, asthma, chronic sinusitis with nasal polyps, atopic dermatitis, neurodermatitis, pruritus, allergic fungal sinusitis, moderate atopic dermatitis, severe atopic dermatitis, eosinophilic duodenitis, eosinophilic gastroenteritis, chronic sinusitis, bullous pemphigoid, hypersensitivity reaction, allergic asthma, house dust mite allergy, alopecia areata, keloids, ulcerative colitis, milk allergy, respiratory disorders, eosinophilic sinusitis, allergic bronchopulmonary aspergillosis, chronic liver disease, chronic eczema, urticaria, food allergy, lichen planus, seasonal allergic rhinitis, and allergic conjunctivitis.

27. Use of the IL-4R binding molecule according to any one of claims 1-6, the tandem molecule according to any one of claims 7-12, the recombinant protein according to any one of claims 13-16, the nucleic acid according to claim 17, the vector according to claim 18, the cell according to claim 19, the pharmaceutical composition or pharmaceutical composition according to claim 21, the antibody-drug conjugate according to claim 22, the bispecific antibody or multispecific antibody according to claim 23, and / or the chimeric antigen receptor according to claim 24 for the detection, treatment, prevention, and / or mitigation of IL-4R-related diseases; wherein The diseases include Th2-mediated diseases, IL-13-mediated diseases, IL-4-mediated diseases and / or IL-4 / IL-13-mediated diseases; Preferably, the disease is selected from chronic obstructive pulmonary disease, chronic urticaria, eosinophilic esophagitis, nodular prurigo, asthma, chronic sinusitis with nasal polyps, atopic dermatitis, neurodermatitis, pruritus, allergic fungal sinusitis, moderate atopic dermatitis, severe atopic dermatitis, eosinophilic duodenitis, eosinophilic gastroenteritis, chronic sinusitis, bullous pemphigoid, hypersensitivity reaction, allergic asthma, house dust mite allergy, alopecia areata, keloids, ulcerative colitis, milk allergy, respiratory disorders, eosinophilic sinusitis, allergic bronchopulmonary aspergillosis, chronic liver disease, chronic eczema, urticaria, food allergy, lichen planus, seasonal allergic rhinitis, and allergic conjunctivitis.

28. The IL-4R binding molecule according to any one of claims 1-6, the tandem molecule according to any one of claims 7-12, the recombinant protein according to any one of claims 13-16, the nucleic acid according to claim 17, the vector according to claim 18, the cell according to claim 19, the pharmaceutical composition or pharmaceutical composition according to claim 21, the antibody-drug conjugate according to claim 22, the bispecific antibody or multispecific antibody according to claim 23, and / or the chimeric antigen receptor according to claim 24, for the detection, treatment, prevention, and / or alleviation of IL-4R-related diseases; wherein The diseases include Th2-mediated diseases, IL-13-mediated diseases, IL-4-mediated diseases and / or IL-4 / IL-13-mediated diseases; Preferably, the disease is selected from chronic obstructive pulmonary disease, chronic urticaria, eosinophilic esophagitis, nodular prurigo, asthma, chronic sinusitis with nasal polyps, atopic dermatitis, neurodermatitis, pruritus, allergic fungal sinusitis, moderate atopic dermatitis, severe atopic dermatitis, eosinophilic duodenitis, eosinophilic gastroenteritis, chronic sinusitis, bullous pemphigoid, hypersensitivity reaction, allergic asthma, house dust mite allergy, alopecia areata, keloids, ulcerative colitis, milk allergy, respiratory disorders, eosinophilic sinusitis, allergic bronchopulmonary aspergillosis, chronic liver disease, chronic eczema, urticaria, food allergy, lichen planus, seasonal allergic rhinitis, and allergic conjunctivitis.

29. A method of detecting, treating, preventing, and / or ameliorating a disease associated with IL-4R, wherein, The method includes administering to a desired subject an effective amount of the IL-4R binding molecule according to any one of claims 1-6, the tandem molecule according to any one of claims 7-12, the recombinant protein according to any one of claims 13-16, the nucleic acid according to claim 17, the vector according to claim 18, the cell according to claim 19, the pharmaceutical composition or pharmaceutical composition according to claim 21, the antibody-drug conjugate according to claim 22, the bispecific antibody or multispecific antibody according to claim 23, and / or the chimeric antigen receptor according to claim 24; The diseases mentioned include Th2-mediated diseases, IL-13-mediated diseases, IL-4-mediated diseases, and / or IL-4 / IL-13-mediated diseases. Preferably, the disease is selected from chronic obstructive pulmonary disease, chronic urticaria, eosinophilic esophagitis, nodular prurigo, asthma, chronic sinusitis with nasal polyps, atopic dermatitis, neurodermatitis, pruritus, allergic fungal sinusitis, moderate atopic dermatitis, severe atopic dermatitis, eosinophilic duodenitis, eosinophilic gastroenteritis, chronic sinusitis, bullous pemphigoid, hypersensitivity reaction, allergic asthma, house dust mite allergy, alopecia areata, keloids, ulcerative colitis, milk allergy, respiratory disorders, eosinophilic sinusitis, allergic bronchopulmonary aspergillosis, chronic liver disease, chronic eczema, urticaria, food allergy, lichen planus, seasonal allergic rhinitis, and allergic conjunctivitis.

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