Polypeptides targeting the cytokine il-4 receptor alpha and uses thereof

Abstract

This invention relates to a polypeptide targeting the cytokine IL-4 receptor α and its applications. The amino acid sequence of the polypeptide targeting IL-4 receptor α comprises the sequence shown in SEQ ID NO.2 or SEQ ID NO.5. The polypeptide targeting IL-4 receptor α provided by this invention can specifically and affinityly bind to the cytokine IL-4 receptor α, resulting in IL-4Rα enrichment, and exhibits good inhibitory effects on the IL-4 and IL-13 signaling pathways, and can be applied to the treatment of IL-4Rα-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a polypeptide that targets the cytokine IL-4 receptor α and its applications, particularly in the preparation of drugs for treating Th2-mediated atopic diseases, allergic diseases, and fibrotic diseases. Background Technology

[0002] Interleukin-4 (IL-4) and interleukin-13 (IL-13) are crucial cytokines in type II immune responses, playing a central role in immune regulation, inflammation, tissue repair, and fibrosis. They transmit signals by sharing a common receptor subunit—IL-4 receptor α (IL-4Rα)—forming two main pathways: the type I receptor is activated by IL-4 binding to the type I receptor, which is composed of the IL-4Rα subunit and a normal γ chain, mainly expressed on lymphocytes, and involved in Th2 cell differentiation, B cell activation, and class conversion; the type II receptor pathway is key to the functional overlap between IL-4 and IL-13 and its close association with various diseases. IL-4 can activate this pathway by binding to the type II receptor composed of IL-4Rα and IL-13Rα1. More importantly, IL-13 also activates downstream signaling by preferentially binding to IL-13α1 and then recruiting IL-4Ra to form a complex.

[0003] Both of these signaling pathways depend on IL-4Rα. Upon binding to the receptor, the ligand activates receptor-associated Janus kinases (JAKs), which in turn phosphorylate STAT6 (a signal transducer and activator of transcription). After dimerization, STAT6 translocates into the nucleus, regulating the expression of a series of target genes, ultimately leading to pathophysiological changes such as IgE production by B cells, excessive mucus secretion, airway hyperresponsiveness, impaired epithelial barrier function, fibroblast activation, and collagen deposition (Bachert C. et al. Front. Immunol. 2024, 15: 1356298).

[0004] Based on the above mechanisms, abnormal activation of the IL-4 / IL-13 signaling pathway has been proven to be a core driver of various Th2-mediated immune diseases, including: atopic diseases such as moderate to severe atopic dermatitis, asthma (especially the eosinophilic phenotype), and chronic sinusitis with nasal polyps; allergic diseases such as food allergies and allergic bronchitis; and fibrotic diseases such as idiopathic pulmonary fibrosis. Therefore, targeting the IL-4 / IL-13 signaling pathway, especially its key hub IL-4Rα, has become an important strategy for treating these diseases.

[0005] Currently, treatment strategies targeting this pathway mainly focus on using monoclonal antibodies to target cytokines or their receptors. Drugs targeting this pathway have already been successfully marketed and their clinical value has been validated. Dupilumab is a fully humanized monoclonal antibody targeting IL-4Rα, which can simultaneously block the signal transduction of IL-4 and IL-13. It has been approved for the treatment of atopic dermatitis, asthma, chronic sinusitis with nasal polyps, eosinophilic esophagitis, etc. (Simpson EL et al. N. Engl. J. Med. 2016, 375: 2335-2348; Bhatt SP et al. N. Engl. J. Med. 2024, 390(24): 2274-2283), demonstrating significant efficacy and making it a benchmark drug in this field. Tralocinumab and leblizumab have both been approved for the treatment of moderate to severe atopic dermatitis in adults, specifically blocking IL-13 (Danese S. et al. Gut 2015, 64(2): 243-249; Corren J. et al. N. Engl. J. Med. 2011, 365(12): 1088-1098). Pitrakel is an early-developed monoclonal antibody targeting IL-4, but its clinical development has been limited (Wenzel S. et al. Lancet 2007, 370(9596): 1422-1431).

[0006] Despite their proven efficacy, antibody drugs face several limitations, including high production costs, the need for cryogenic storage and transportation, the typical requirement for subcutaneous or intravenous administration (posing challenges to patient compliance), potential immunogenicity risks, and the difficulty in penetrating certain tissue barriers due to their large molecular weight. To overcome these drawbacks, researchers are actively developing peptide antagonists targeting the IL-4 / IL-13 pathway. Peptides offer potential advantages such as relatively small molecular weight, the ability to be administered orally or locally, relatively low production costs, and lower immunogenicity risks. Current research primarily focuses on the following strategies: antagonistic peptides targeting the IL-4Rα binding interface. These peptides aim to mimic or interfere with the interaction between IL-4 or IL-13 and IL-4Rα. Researchers have designed a series of peptide fragments derived from IL-4 or its receptor-binding domain by analyzing the crystal structure of the IL-4 / IL-4Rα complex. For example, some studies have reported that short peptides based on the helical region of IL-4 can inhibit IL-4-induced cell proliferation in vitro (Yang L. et al. Mol. Cancer Ther. 2012, 11(1): 235-243). Other studies have used phage display peptide library technology to screen for novel peptide sequences that can specifically bind to IL-4Rα and block its function (Hong HY et al. J. Cell. Mol. Med. 2008, 12(5): 2003-2014). However, most of the reported peptides targeting this pathway are still in the early stages of research and generally suffer from some common problems, such as poor affinity.

[0007] In summary, the IL-4 / IL-13 signaling pathway is a key target for treating various Th2 inflammatory diseases. While monoclonal antibody drugs, exemplified by dupilumab, have achieved great success, the development of novel therapeutic molecules with differentiated advantages remains of significant clinical and commercial value. Peptide drugs, as a promising therapeutic approach, have seen preliminary exploration in targeting IL-4Rα; however, currently disclosed candidate peptides generally suffer from core defects such as weak affinity and poor stability, and no products have yet entered the clinical development stage. Summary of the Invention

[0008] The problem the invention aims to solve: In light of the aforementioned deficiencies in the prior art, the objective of this invention is to provide a novel, high-affinity antagonistic peptide targeting IL-4Rα. The peptide described in this invention can effectively block the IL-4 and IL-13-mediated type II receptor signaling pathway, providing a potential novel peptide candidate drug that can be administered locally or systemically for the treatment of diseases such as atopic dermatitis and asthma, thereby filling the gaps and deficiencies in existing treatment methods, particularly peptide drugs, in this field.

[0009] Solution for solving the problem: In a first aspect, the present invention provides a polypeptide targeting the cytokine IL-4 receptor α, said polypeptide having the amino acid sequence shown in (I): Aaa1-Aaa2-Aaa3-Aaa4-Aaa5-Aaa6-Aaa7-Aaa8-Aaa9-Aaa 10 -Aaa 11 -Aaa 12 Formula (I); in, Aaa1 is selected from any one of Ser, Glu, Asp, and Ala; Aaa2 is selected from any one of Phe, Pro, Thr, Val, Ala, and Asn; Aaa3 is selected from any one of Ser, Leu, Pro, Thr, and Ala; Aaa4 is selected from any one of Phe, Ser, Glu, Gly, and Ala; Aaa5 is selected from any one of Gly, Tyr, Gln, His, and Pro; Aaa6 is selected from any one of Val, Arg, Ser, Asp, Leu, and Pro; Aaa7 is selected from any one of Pro, Thr, Ser, Arg, Asn, and Ala; Aaa8 is selected from any one of Ser, Lys, Val, and Leu; Aaa9 is selected from any one of Glu, Ser, Phe, Asn, Leu, and Ala; Aaa 10 Choose from Lys, Gly, Pro, Ser, or Arg; Aaa 11 Choose from any one of Leu, Glu, His, Trp, Asn, and Cys; Aaa 12 Choose from Leu, Ile, Phe, Ser, His, or Pro.

[0010] In a second aspect, the present invention provides a polynucleotide that encodes the polypeptide described in any of the preceding claims.

[0011] Thirdly, the present invention provides an expression vector comprising the polynucleotides described above.

[0012] Fourthly, the present invention provides a host cell comprising the expression vector described above, or the host cell having the polynucleotides described above integrated into its genome.

[0013] Fifthly, the present invention provides a composition comprising the polypeptide described in any one of the preceding claims.

[0014] In a sixth aspect, the present invention provides a conjugate comprising the polypeptide described in any of the preceding claims.

[0015] In a seventh aspect, the present invention provides the use of the polypeptide, the polynucleotide, the expression vector, the host cell, the composition, or the conjugate according to any one of the preceding claims in at least one of the following: 1) Used to prepare drugs that target and bind to IL-4Rα; 2) Used for detecting IL-4Rα; 3) Used to prepare detection reagents for detecting IL-4Rα; 4) Used for the preparation or screening of drugs for the treatment of IL-4Rα-related diseases.

[0016] The effects of the invention: The polypeptide compound provided by this invention can bind to human IL-4Ra with high affinity and specificity, thereby effectively blocking the IL-4 / IL-13 mediated signaling pathway. In vivo pharmacodynamic studies fully demonstrate that the polypeptide compound of this invention can antagonize downstream Th2 responses by inhibiting the IL-4 and IL-13 signaling pathways. It can be used to prevent and / or treat diseases related to IL-4Rα abnormalities and Th2 inflammatory diseases, such as targeted therapy for atopic diseases (e.g., moderate to severe atopic dermatitis), asthma, allergic diseases, or fibrotic diseases, and has good application prospects and market value. Detailed Implementation

[0017] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0018] Through extensive and in-depth research, the inventors utilized phage screening technology, specifically screening the IL-4Rα protein using a Ph.D.-12 phage peptide library, to obtain six phages with specific affinity for the IL-4Rα receptor. These phages were then sequenced to obtain the amino acid sequences of their peptides. Enzyme-linked immunosorbent assays revealed that the six peptides exhibited high binding affinity to the IL-4Rα protein. Based on these findings, the present invention was completed.

[0019] As used herein, the terms “comprising,” “having,” “including,” or “containing” can mean inclusive or open-ended and do not exclude additional, uncited elements or method steps. At the same time, “comprising,” “having,” “including,” or “containing” can also mean closed-ended and exclude additional, uncited elements or method steps.

[0020] As used herein, the term "amino acid" can include natural amino acids, non-natural amino acids, amino acid analogs, and all their D and L stereoisomers. The amino acids, their abbreviations, and English abbreviations used herein are shown below: Histidine (His, H); Serine (S); Glutamine (Gln, Q); Glycine (Gly, G); Threonine (Thr, T); Phenylalanine (Phe, F); Aspartic acid (Asp, D); Tyrosine (Tyr, Y); Isoleucine (Ile, I); Arginine (Arg, R); Alanine (Ala, A); Glutamine (Gln, Q); Valine (Val, V); Tryptophan (Trp, W); Leucine (Leu, L); Methionine (Met, M); Asparagine (Asn, N); Cysteine ​​(Cys, C); Lysine (Lys, K); Proline (Pro, P).

[0021] In this article, when referring to peptides, "amino acid" and "amino acid residue" have the same meaning, referring to the amino acid residues that remain after some groups are lost due to their participation in the formation of the linking bond when amino acids are linked by chemical bonds.

[0022] As used herein, the terms “polypeptide” and “peptide” are used interchangeably to refer to a polymeric form of amino acids of any length, including encoded and non-coding amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a similar peptide backbone.

[0023] As used herein, the terms “IL4Rα,” “interleukin-4 receptor α,” and “cytokine IL-4 receptor α” are used interchangeably and refer to the interleukin-4 receptor α subunit. The term “IL4Rα” includes variants, subtypes, homologs, orthologs, and paralogs. For example, in some cases, antibodies specific to human IL4Rα protein may cross-react with IL4Rα protein from species other than humans (e.g., monkeys). In other embodiments, antibodies specific to human IL4Rα protein may be completely specific to human IL4Rα protein and may not cross-react with proteins from other species or other types, or may cross-react with IL4Rα from some other species but not all other species.

[0024] As used herein, the terms “human IL4Rα,” “hIL-4RA,” “human IL-4 receptor A,” and “human IL-4 receptor α subunit” are used interchangeably to refer to the IL4Rα protein having a human amino acid sequence, such as the amino acid sequence of human IL4Rα with Genbank accession number NP_001244335.1. Unless otherwise specified or clearly understood from the context in which the terms are used herein, “IL-4RA” refers to human IL-4RA.

[0025] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D () binds to the antigen.

[0026] As used herein, the terms “nucleic acid molecule,” “polynucleotide,” “polynucleotide,” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-restricted examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules can be linear or circular.

[0027] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, cosmids, etc.

[0028] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of *Escherichia coli* or *Salmonella*; Bacillus family members such as *Bacillus subtilis*; *Streptococcus pneumoniae*; and *Haemophilus influenzae*. Suitable microorganisms include *Saccharomyces cerevisiae* and *Pichia pastoris*. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.

[0029] As used in this article, the term "IC" 50 "Hyper-maximal inhibitory concentration" (HMC), also known as the half-maximal inhibitory concentration, refers to the concentration of a polypeptide that inhibits specific biological or biochemical functions by 50% relative to the absence of an antibody.

[0030] As used herein, the term "IL-4Rα-related disease" includes diseases and / or symptoms associated with activation of the IL-4Rα signaling pathway. Exemplary IL-4Rα-related diseases or conditions include immune-mediated inflammatory responses, such as allergic diseases, asthma, etc.

[0031] Peptides targeting IL-4Rα This invention provides a polypeptide that targets the cytokine IL-4 receptor α, the amino acid sequence of which is shown in formula (I): Aaa1-Aaa2-Aaa3-Aaa4-Aaa5-Aaa6-Aaa7-Aaa8-Aaa9-Aaa 10 -Aaa 11 -Aaa 12 Formula (I); in, Aaa1 is selected from any one of Ser, Glu, Asp, and Ala; Aaa2 is selected from any one of Phe, Pro, Thr, Val, Ala, and Asn; Aaa3 is selected from any one of Ser, Leu, Pro, Thr, and Ala; Aaa4 is selected from any one of Phe, Ser, Glu, Gly, and Ala; Aaa5 is selected from any one of Gly, Tyr, Gln, His, and Pro; Aaa6 is selected from any one of Val, Arg, Ser, Asp, Leu, and Pro; Aaa7 is selected from any one of Pro, Thr, Ser, Arg, Asn, and Ala; Aaa8 is selected from any one of Ser, Lys, Val, and Leu; Aaa9 is selected from any one of Glu, Ser, Phe, Asn, Leu, and Ala; Aaa 10 Choose from Lys, Gly, Pro, Ser, or Arg; Aaa 11 Choose from any one of Leu, Glu, His, Trp, Asn, and Cys; Aaa 12 Choose from Leu, Ile, Phe, Ser, His, or Pro.

[0032] In some implementations, Aaa1 is Ser, Glu, or Ala; Aaa2 is either Pro or Ala; Aaa3 is Ser, Leu, or Ala; Aaa4 is either Ser or Gly; Aaa5 is Tyr or Gln; Aaa6 is either Arg or Leu; Aaa7 represents Thr or Asn; Aaa8 represents Ser, Lys, or Leu; Aaa9 is either Ser or Leu; Aaa 10 For Gly or Arg; Aaa 11 For Glu or Asn; Aaa 12 For Ile or His.

[0033] In some implementations, Aaa1 is Glu or Ala; Aaa2 is either Pro or Ala; Aaa3 is either Leu or Ala; Aaa4 is either Ser or Gly; Aaa5 is Tyr or Gln; Aaa6 is either Arg or Leu; Aaa7 represents Thr or Asn; Aaa8 represents Lys or Leu; Aaa9 is either Ser or Leu; Aaa 10 For Gly or Arg; Aaa 11 For Glu or Asn; Aaa 12 For Ile or His.

[0034] In some embodiments, the amino acid sequence of the polypeptide comprises the sequence shown in any one of SEQ ID NO. 1 to 6.

[0035] In some embodiments, the amino acid sequence of the polypeptide comprises the sequence shown in SEQ ID NO.1 (SFSFGVPSEKLL).

[0036] In some embodiments, the amino acid sequence of the polypeptide comprises the sequence shown in SEQ ID NO.2 (EPLSYRTKSGEI).

[0037] In some embodiments, the amino acid sequence of the polypeptide comprises the sequence shown in SEQ ID NO.3 (STPEQSSSFPHF).

[0038] In some embodiments, the amino acid sequence of the polypeptide comprises the sequence shown in SEQ ID NO.4 (DVTGHDRVNSWS).

[0039] In some embodiments, the amino acid sequence of the polypeptide comprises the sequence shown in SEQ ID NO.5 (AAAGQLNLLRNH).

[0040] In some embodiments, the amino acid sequence of the polypeptide comprises the sequence shown in SEQ ID NO.6 (ANSAPPASAGCP).

[0041] In some embodiments, the amino acid sequence of the polypeptide is any one of SEQ ID NO. 1 to 6.

[0042] In some embodiments, the amino acid sequence of the polypeptide is the sequence shown in SEQ ID NO.1.

[0043] In some embodiments, the amino acid sequence of the polypeptide is the sequence shown in SEQ ID NO.2.

[0044] In some embodiments, the amino acid sequence of the polypeptide is the sequence shown in SEQ ID NO.3.

[0045] In some embodiments, the amino acid sequence of the polypeptide is the sequence shown in SEQ ID NO.4.

[0046] In some embodiments, the amino acid sequence of the polypeptide is the sequence shown in SEQ ID NO.5.

[0047] In some embodiments, the amino acid sequence of the polypeptide is the sequence shown in SEQ ID NO.6.

[0048] The IL-4Rα-targeting polypeptide provided by this invention has a small molecular weight, consisting of only 12 amino acids, is simple to synthesize, has stable properties, and exhibits excellent IL-4Rα affinity and targeting.

[0049] Polynucleotides, expression vectors, host cells, and preparation methods The present invention also provides a polynucleotide encoding the polypeptide described in any of the preceding claims.

[0050] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

[0051] The polynucleotide encoding the polypeptide of the present invention includes: a coding sequence that encodes only the polypeptide; a coding sequence of the polypeptide and various additional coding sequences; a coding sequence of the polypeptide (and optional additional coding sequences) and a non-coding sequence. Wherein, "polynucleotide encoding the polypeptide" may include a polynucleotide encoding the polypeptide, or it may include a polynucleotide that also includes additional coding and / or non-coding sequences.

[0052] The present invention also provides an expression vector comprising the polynucleotides described above. The expression vector further comprises a suitable promoter or control sequence, and can be used to transform a suitable host cell to enable it to express the protein.

[0053] In some embodiments, the expression vector of the present invention includes a plasmid vector or a viral vector; for example, the plasmid vector includes pET series vectors, and the viral vector includes bacteriophages.

[0054] The present invention also provides a host cell comprising the expression vector described above, or the host cell having the polynucleotides described above integrated into its genome.

[0055] In some embodiments, the host cells of the present invention include eukaryotic cells or prokaryotic cells; for example, the eukaryotic cells include yeast, CHO cells or HEK293 cells, and the prokaryotic cells include Escherichia coli.

[0056] The present invention also provides a method for preparing the aforementioned polypeptide, comprising the following steps: culturing the host cells described above under conditions suitable for expressing the polypeptide, thereby expressing the polypeptide, and purifying and isolating the polypeptide.

[0057] Couplings, Compositions and Uses The present invention also provides a composition comprising the polypeptide described in any of the preceding embodiments. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0058] The present invention also provides a conjugate comprising the polypeptide described in any of the preceding embodiments. In some embodiments, the conjugate comprises a therapeutic agent conjugated to the polypeptide. In other embodiments, the conjugate comprises a detection agent or diagnostic agent conjugated to the polypeptide. The conjugation may be performed via a linker or may be a direct conjugation. In some embodiments, in the conjugate, the IL-4Rα-targeting polypeptide is linked directly or via an amino acid linker to a heteropeptide or polypeptide molecule. Heteropeptides or polypeptides that may be mentioned include, but are not limited to, proteins or polypeptides that impart another functional activity to the conjugate. In some embodiments, in the conjugate, the IL-4Rα-targeting polypeptide is conjugated to a therapeutic agent, detection agent, or diagnostic agent. In some embodiments, the therapeutic agent, detection agent, or diagnostic agent is a protein or polypeptide. In other embodiments, the therapeutic agent, detection agent, or diagnostic agent is a non-protein chemical compound. Linkers may be used to covalently link the entities to which the conjugation occurs. Suitable linkers include chemical linkers or peptide linkers. For example, depending on the application requirements, you can choose to use a "cleavable connector", an acid-instable connector, a peptidase-sensitive connector, a light-instable connector, a dimethyl connector, or a connector containing a disulfide.

[0059] In embodiments conjugated with therapeutic agents, suitable therapeutic agents include, but are not limited to, cytotoxins (e.g., cell growth inhibitors or cell killers), antitumor drugs, or radioisotopes. In embodiments conjugated with diagnostic or detection agents, suitable detection and diagnostic agents include, but are not limited to, various enzymes, such as horseradish peroxidase; prosthetic groups, such as streptavidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metal and non-radioactive paramagnetic metal ions used in various positron emission tomography (PET) imaging techniques.

[0060] This invention provides the use of the polypeptide, polynucleotide, or expression vector, host cell, composition, or conjugate described above in at least one of the following: 1) Used to prepare drugs that target and bind to IL-4Rα; 2) Used for detecting IL-4Rα; 3) Used to prepare detection reagents for detecting IL-4Rα; 4) Used for the preparation or screening of drugs for the treatment of IL-4Rα-related diseases.

[0061] In some embodiments, the IL-4Rα-related diseases are immune-mediated inflammatory responses or immune-mediated inflammatory diseases, including but not limited to: asthma, allergies (such as food allergies, allergic bronchitis), atopic dermatitis (including moderate to severe atopic dermatitis), chronic sinusitis, eosinophilic esophagitis, nasal polyps, psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, fibrotic diseases (such as idiopathic pulmonary fibrosis), multiple sclerosis, uveitis, Behcet's uveitis, dry eye syndrome, chronic spontaneous urticaria, etc.

[0062] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0063] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0064] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in this field (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions.

[0065] In addition to the specific methods, equipment, and materials used in the embodiments, based on the knowledge of those skilled in the art and the description of this invention, any prior art methods, equipment, and materials similar to or equivalent to those described, used, and materials in the embodiments of this invention can be used to implement this invention. Unless otherwise stated, parts and percentages are parts by weight and weight percentages.

[0066] Example 1: Screening of IL-4Rα-binding peptides using phage display peptide library To obtain peptides that specifically bind to human IL-4Ra, phage display technology was used for in vitro screening. The peptide library used was Ph.D.-12 (NEB, cat#E8111L). The screening strategy combined negative and positive screening to enrich high-affinity phage clones targeting the extracellular region of human IL-4Ra and reduce non-specific binding to unrelated proteins (Zhongding Biotechnology, cat#ZD20200004).

[0067] The screening process was as follows: In each round of screening, the phage peptide library was first co-incubated with wells coated with unrelated proteins, and the unbound supernatant was collected. This supernatant was then transferred to wells coated with human IL-4Ra for specific binding. After thorough washing to remove unbound and non-specifically bound phages, the specifically bound phages were eluted with acidic glycine-hydrochloric acid buffer (pH 2.5) and immediately neutralized with Tris-HCl buffer (pH 9.0). The eluted phages were then amplified by infecting *E. coli* and used for the next round of screening or subsequent analysis. The specific conditions and enrichment results for the two rounds of screening are shown in Table 1, where the enrichment factor in Table 1 is calculated as (number of phages produced / number of phages input) * dilution factor.

[0068] Table 1. Phage panning input-output and enrichment data

[0069] To identify single positive clones from the enriched library, phage ELISA screening was performed. The phage peptide library obtained from the second round of screening was screened using a blue-white screening method on plates containing IPTX / X-gal. 376 blue single clones were randomly selected and inoculated into 96-well plates containing 1 mL of 2*YT medium (with appropriate antibiotics). The plates were incubated at 37°C with shaking at 200 rpm for 5 hours. After centrifugation at 5000 rpm for 10 minutes, 600 mL of the supernatant was collected as the sample for testing.

[0070] The direct ELISA method was used for detection: A 96-well plate was coated with 2 mg / mL human IL-4Ra protein (100 mL / well) and incubated overnight at 4°C. Blocked with 2% (w / v) skim milk powder-PBST (200 mL / well) and incubated at 37°C for 1 hour. Phage clone supernatant (100 mL / well) was added and incubated at 37°C for 1 hour. After washing three times with PBST, 1:15000 diluted HRP-labeled anti-M13 antibody (100 mL / well) was added and incubated at 37°C for 1 hour. After washing five times with PBST, 100 mL / well of TMB chromogenic buffer was added, and the reaction was carried out at 37°C in the dark for 15 minutes. The reaction was stopped with 2 M sulfuric acid (100 mL / well), and the absorbance (OD) of each well was measured at 450 nm. 450 Change factor = (OD) IL-4Ra -OD空白 ) / (OD 非相关蛋白 -OD 空白 The results are shown in Table 2.

[0071] Table 2. Partial absorbance (OD) of Ph.D.-12 peptide library 450 )result

[0072] As shown in Table 2, after two rounds of screening, phages that specifically bind to human IL-4Ra protein were effectively enriched.

[0073] The positive clones were subjected to single-stranded DNA extraction and sequencing. Double peaks and empty vectors in the sequencing results were removed. Six peptides containing stop codons that met the screening criteria were found in the Ph.D.-12 peptide library, and their sequences are shown in Table 3.

[0074] Table 3. Human IL-4Ra-specific binding peptides obtained by phage display screening

[0075] Example 2: Detection of the affinity between peptides and human IL-4Ra using surface plasmon resonance technology. Detection was performed using a surface plasmon resonance (SPR) analyzer (Biacore T200). The buffer was HBS-EP+ buffer containing 0.05% (v / v) Tween-20. The extracellular domain of human IL-4Ra protein was used as the ligand, and the analyte was used as the analyte. The CM5 chip was activated for 7 min with an equal volume mixture of N-hydroxysuccinimide and N-ethyl-N'-(dimethylaminopropyl)carbodiimide. Human IL-4Ra protein was diluted with sodium acetate buffer at pH 5.0 and injected into the designated channels of the chip. Ethanolamine solution was added for blocking. The analyte was serially diluted, and each concentration was injected into the test channel sequentially using a single-cycle or sequential injection method. After each binding-dissociation cycle, the chip surface was regenerated with 10 mM glycine-HCl buffer (pH 1.5). BSA was used as a negative control to verify binding specificity. The baseline drift of the detection results was deducted from the blank buffer, and the binding and dissociation phase data were globally fitted using a 1:1 binding model. The binding rate constant, dissociation rate constant, and equilibrium dissociation constant of each peptide screened in Example 1 with human IL-4Ra protein were calculated. The results are shown in Table 4.

[0076] Table 4. Affinity parameters of representative peptides to human IL-4Ra protein

[0077] The results are shown in Table 4. The KD values ​​of SEQ ID NO.2, SEQ ID NO.5 and human IL4Ra are all less than 1 μM. Among them, the KD value of SEQ ID NO.5 reached 108 nM, showing extremely high affinity, and can be used as the first choice candidate molecule for subsequent functional verification and drug development.

[0078] Example 3: Detection of the in vitro activity of peptides in blocking the IL-4 / IL-13 signaling pathway To verify the functional activity of the screened peptides, HEK-Blue was used. TM Cellular-level functional blockade experiments were conducted using the IL-4R / IL-13 reporter gene cell line (Invivogen). This cell line stably expresses human IL-4Ra and IL-13Ra1, as well as the SEAP reporter gene driven by NF-κB and STAT6 response elements.

[0079] Experimental methods: HEK-Blue IL-4R cells in logarithmic growth phase were collected and their concentration was adjusted to 2*10⁻⁶ cells / cells using phenol red-free RPMI 1640 complete medium. 5 Cells / mL were seeded into 96-well plates (100 μL / well) and incubated overnight at 37°C with 5% CO2. The next day, the supernatant was discarded, and serum-free medium containing different concentrations of the test peptide (0.1 nM-1000 nM, serially diluted) was added. IL-4 (10 ng / mL) or IL-13 (10 ng / mL) was added as a stimulating factor. Blank control (cells and medium only) and positive control (cells and IL-4 / IL-13 only, no peptide) were included. Each group had three replicates. After culturing at 37°C with 5% CO2 for 24 hours, 50 μL of the supernatant was collected, and an equal volume of colorimetric enzyme assay reagent (QUANTI-Blue™ solution, Invivogen) was added. The plate was incubated at 37°C for 1–2 hours, and the absorbance (OD) at 630 nm was measured using a microplate reader. 630 ).

[0080] Data processing: OD of the positive control 630 The value is used as the 100% signal, with the OD value as the blank control. 630 The value was used as the 0% signal to calculate the inhibition rate of the signal by different concentrations of peptide. The IC was calculated using nonlinear regression analysis in GraphPad Prism software. 50 The values ​​are shown in Table 5.

[0081] Table 5. In vitro blocking efficacy of peptides against the IL-4 / IL-13 signaling pathway 50 value

[0082] As shown in Table 5, the high-affinity peptides screened in this invention can effectively block the IL-4 / IL-13-mediated signaling pathway.

[0083] Example 4: Evaluation of the anti-inflammatory activity of the peptide in vivo (OVA-induced mouse asthma model) To further evaluate the in vivo anti-inflammatory effects of the peptides, an ovalbumin (OVA)-induced mouse asthma model was used. SPF-grade BALB / c female mice (6-8 weeks old, weighing 18-22 grams) were acclimatized for one week before being divided into 5 groups of 8 mice each. (1) Normal control group: intraperitoneal injection and nebulized inhalation of normal saline; (2) Model control group: OVA sensitization and nebulization stimulation; (3) Positive control group: Dexamethasone (1 mg / kg) intraperitoneal injection; (4) Low-dose treatment group: intraperitoneal injection of SEQ ID NO.5 polypeptide (1 mg / kg); (5) High-dose treatment group: intraperitoneal injection of SEQ ID NO.5 polypeptide (10 mg / kg); Sensitization protocol: On days 0 and 14, mice were intraperitoneally injected with 100 μL of OVA solution (100 μg OVA, 1 mg aluminum hydroxide gel). Control group mice were intraperitoneally injected with an equal volume of physiological saline. Starting from day 21, mice were challenged for 7 consecutive days by nebulized inhalation of 0.5% OVA solution (30 minutes / day). Treatment group mice were intraperitoneally injected with the corresponding dose of the peptide or positive control drug 30 minutes before each nebulized challenge.

[0084] Evaluation indicators: (1) Lung tissue pathological examination: After euthanizing the mice, the lower lobe of the right lung was taken, fixed with 4% paraformaldehyde, routinely embedded in paraffin, sectioned, and stained with HE to observe the degree of airway inflammation; (2) Bronchoalveolar lavage fluid (BALF) cell classification and counting: BALF was collected, centrifuged, smeared, Wright-Gymsa stained, and eosinophils, lymphocytes and neutrophils were counted; (3) Serum IgE level: detected by ELISA; (4) Cytokine level in lung tissue: IL-4, IL-13 and IL-5 levels were detected by ELISA.

[0085] The experimental results showed that, compared with the model control group, the high-dose treatment group (10 mg / kg SEQ ID NO: 5) significantly reduced the number of eosinophils in BALF (from 78.5% to 12.3%), reduced the serum total IgE level (from 456.3 ng / mL to 187.5 ng / mL), and alleviated airway wall thickening and inflammatory cell infiltration, suggesting that the peptide has a good anti-inflammatory effect in vivo.

[0086] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A polypeptide targeting the cytokine IL-4 receptor α, characterized in that, The amino acid sequence of the polypeptide is the sequence shown in SEQ ID NO.2 or SEQ ID NO.

5.

2. A polynucleotide, characterized in that, The polynucleotide encodes the polypeptide of claim 1.

3. An expression carrier, characterized in that, The expression vector comprises the polynucleotide of claim 2.

4. A host cell, characterized in that, The host cell contains the expression vector of claim 3, or the host cell genome is integrated with the polynucleotide of claim 2.

5. A composition, characterized in that, The composition comprises the polypeptide of claim 1.

6. Use of the polypeptide of claim 1, the polynucleotide of claim 2, the expression vector of claim 3, the host cell of claim 4, or the composition of claim 5 in at least one of the following: 1) Used to prepare detection reagents for detecting IL-4Rα; 2) Used for the preparation or screening of drugs for the treatment of IL-4Rα-related diseases; The IL-4Rα-related diseases are immune-mediated inflammatory diseases, including asthma, allergies, and atopic dermatitis.

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