Specific binding proteins for il-4r and methods of making and using the same

Abstract

The application belongs to the field of biological medicine, and particularly relates to a specific binding protein of IL-4R and a preparation method and application thereof. The application provides a specific binding protein of IL-4R, which has specific CDRs, can be combined with human IL4R alpha with high affinity, and can block the IL4R alpha-IL4 / IL13-IL13R alpha1 interaction and corresponding intracellular signal transduction and biological effects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a specific binding protein of IL-4R and a preparation method and application thereof. BACKGROUND

[0002] Human interleukin 4 receptor alpha (human IL-4R alpha) is a common receptor of IL-4 / IL-13, and is divided into two types: the type I receptor is composed of IL-4R alpha and gamma c subunit, and only binds with IL-4; the type II receptor is composed of IL-4R alpha and IL-13R alpha 1 subunit, and can bind with IL-4 or IL-13. IL-4 and IL-13 share the same receptor signaling system, and the functions are partially overlapped. IL-4 can promote the activation and chemotaxis of eosinophils and mast cells to the inflammation site; IL-4 / IL-13 can promote the activation of B cells and the secretion of IgE, and up-regulate the expression of IgE receptors of B cells, mast cells and the like; IL-13 can promote mucus secretion, cell proliferation and collagen deposition. The signal transduction mediated by IL-4R plays an important role in the occurrence and development of type 2 inflammatory diseases.

[0003] Compared with inhibiting IL-4 or IL-13 alone, IL-4R monoclonal antibody can simultaneously inhibit the signal transduction of IL-4 / IL-13 pathway by specifically binding with IL-4R alpha, and has better curative effect on treating type 2 inflammatory diseases mediated by Th2. At present, studies have shown that 80%-90% of patients with asthma, atopic dermatitis and chronic sinusitis with nasal polyps have type 2 inflammatory diseases. At present, the monoclonal antibody dupilumab has been approved to treat five indications, which are moderate to severe atopic dermatitis, moderate to severe asthma, chronic sinusitis with nasal polyps, eosinophilic esophagitis and nodular prurigo.

[0004] Therefore, in the face of the needs of patients for disease treatment, it is urgent to develop a new IL-4R antibody to improve the quality of life of patients with type 2 inflammatory diseases. SUMMARY

[0005] Based on this, an embodiment of the present application provides a specific binding protein of IL-4R and a preparation method and application thereof.

[0006] In one aspect, the present application provides a specific binding protein of IL-4R, wherein the specific binding protein of IL-4R has an IL-4R binding domain.

[0007] The IL-4R binding domain has heavy chain CDR1 to heavy chain CDR3 shown in SEQ ID NO. 1 to SEQ ID NO. 3, and light chain CDR1 to light chain CDR3 shown in SEQ ID NO. 4 to SEQ ID NO. 6.

[0008] In some embodiments, the heavy chain variable region is as set forth in SEQ ID NO. 7; and

[0009] In some embodiments, the light chain variable region is as set forth in SEQ ID NO. 8.

[0010] In some embodiments, the specific binding protein for IL-4R further comprises a heavy chain constant region and a light chain constant region.

[0011] In some embodiments, the specific binding protein for IL-4R independently has the sequence of any one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD constant region.

[0012] In some embodiments, the heavy chain constant region and the light chain constant region are independently selected from any one of human, murine, rabbit, sheep, bovine, equine, porcine, canine, feline, camel, donkey, deer, mink, chicken, duck and goose species origin.

[0013] Another aspect of the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the specific binding protein for IL-4R.

[0014] Another aspect of the present application provides a vector comprising the nucleic acid molecule.

[0015] Another aspect of the present application provides a cell comprising the nucleic acid molecule or the vector.

[0016] Another aspect of the present application provides a method for preparing the cell, the method comprising the step of introducing the nucleic acid molecule or the vector into a target cell.

[0017] In some embodiments, the method for preparing the cell comprises the steps of culturing the cell, and isolating the specific binding protein for IL-4R from the resulting culture.

[0018] Another aspect of the present application provides an IL-4R detection product, the detection product comprising the specific binding protein for IL-4R.

[0019] Another aspect of the present application provides a method for detecting IL-4R in a sample to be tested, the method using the specific binding protein for IL-4R as a detection antibody to determine the presence of IL-4R in the sample to be tested by an immunological binding reaction.

[0020] Another aspect of the present application provides a medicament comprising the specific binding protein for IL-4R.

[0021] The present application provides a specific binding protein of IL-4R, which has specific CDRs, can bind to human IL4Rα with high affinity, and blocks IL4Rα-IL4 / IL13-IL13Rα1 interaction and corresponding intracellular signal transduction and biological effects. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the drawings needed to be used in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0023] Figure 1 Figure for detecting the binding activity of anti-human IL-4R antibody to human IL-4R overexpressing cells in an embodiment of the present application;

[0024] Figure 2 Figure for detecting the activity of anti-human IL-4R antibody in blocking IL4 / IL-4R overexpressing cell binding in an embodiment of the present application;

[0025] Figure 3 Figure for detecting the activity of anti-human IL-4R antibody in inhibiting IL-4 induced STAT6 phosphorylation in HEK293-hSTAT6 / 5×STAT6-Luc cells in an embodiment of the present application. DETAILED DESCRIPTION

[0026] The present application will be further described in detail below in conjunction with the embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and not used to limit the scope of the present application, and the purpose of providing these embodiments and examples is to make the disclosure of the present application more thoroughly and comprehensively understood. It should also be understood that the present application can be realized in many different forms and is not limited to the embodiments and examples described herein, and those skilled in the art can make various modifications or changes without departing from the spirit of the present application, and the equivalent forms obtained thereby also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs.

[0028] Terminology

[0029] Unless otherwise indicated or contradictory, the terms or phrases used herein have the following meanings: 1. The term "antibody" refers to a polypeptide that specifically binds to an antigen, including but not limited to monoclonal, polyclonal, chimeric, humanized, single chain, and synthetic antibodies.

[0030] The selection scope of the terms "and / or", "or / and", "and / or" used herein includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, including any two relevant listed items, any more relevant listed items, or a combination of all relevant listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in this application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0031] In this application, "multiple", "various", "multiple times", "multiple" and the like are used without special limitation, which means more than two or equal to two in quantity. For example, "one or more" means one or more than two.

[0032] As used herein, "combinations thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0033] In this application, "suitable combination", "suitable manner", "any suitable manner" and the like are described as "suitable" which can implement the technical solutions of the application, solve the technical problems of the application, and achieve the expected technical effects of the application.

[0034] In this application, "further", "further", "special" and the like are used for description purposes, indicating differences in content, but should not be understood as limiting the scope of protection of the application.

[0035] In this application, "optionally", "optional", "optional" means optional, i.e. selected from two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, and there is no special description, and no contradictory relationship or mutual restriction, each "optional" is independent.

[0036] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0037] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0038] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0039] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0040] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0041] The term "IL-4R" stands for interleukin-4 receptor, which is the receptor for interleukin-4 (IL-4) and is mainly involved in immune regulation and inflammatory responses.

[0042] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof) through at least one antigen recognition site located within the variable region of an immunoglobulin molecule. As used herein, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibody-containing fusion proteins, and any other modified immunoglobulin molecule, provided that the antibody exhibits the desired biological activity. Antibodies may be any of the five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), designated α, δ, ε, γ, and μ, respectively, based on the characteristic of their heavy chain constant structure. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional conformations. Antibodies may be naked or conjugated to other molecules (such as toxins, radioisotopes, etc.).

[0043] The term "antibody fragment" refers to a portion of a complete antibody. "Antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to a portion of a complete antibody that specifically binds to an antigen. An antigen-binding fragment may contain an antigen recognition site of the complete antibody (e.g., a CDR region (complementarity-determining region) sufficient to specifically bind to an antigen). Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antigen-binding fragments of antibodies may be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or may be artificially generated.

[0044] The terms “anti-IL-4R antibody” and “antibody that binds to IL-4R” refer to antibodies that specifically bind to IL-4R with sufficient affinity, making them suitable for use as diagnostic and / or therapeutic agents targeting IL-4R. As used herein, the terms “specifically bind,” “immunely specifically bind,” “immunely specifically recognize,” and “specifically recognize” are similar terms in the context of an antibody or its antigen-binding fragment. These terms indicate that the antibody or its antigen-binding fragment binds to the epitope via its antigen-binding domain, and that binding requires a degree of complementarity between the antigen-binding domain and the epitope.

[0045] As used herein, the terms “variable region” or “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically about 110 to 120 or 110 to 125 amino acids from the amino terminus of the mature heavy chain and about 90 to 115 amino acids from the mature light chain, which differ in sequence between antibodies and are responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in those regions called CDRs, while more conserved regions within a variable domain are called FR regions (Framework regions). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region is a rodent or mouse variable region.

[0046] The term "heavy chain variable region" consists of 4 FR regions and 3 CDR regions. According to the IMGT database, the genes of the heavy chain variable region are defined as the V gene, the D gene, and the J gene.

[0047] The term "light chain variable region" consists of 4 FR regions and 3 CDR regions. According to the IMGT database, the genes of the light chain variable region are defined as the V gene and the J gene.

[0048] The term "heavy chain" generally includes one variable region and three constant regions (CH1 / CH2 / CH3).

[0049] The term "light chain" generally includes one variable region and one constant region (CL).

[0050] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of antibodies or their antigen-binding fragments. In some respects, CDRs can be determined according to the Kabat numbering system (see, for example, Kabat EA and Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within the antibody heavy chain molecule are typically located at amino acid positions 31 to 35 (which may optionally contain one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme)) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs within the antibody light chain molecule are typically located at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In one specific embodiment, the CDRs of the antibody described herein have been determined according to the Kabat numbering scheme.

[0051] As used herein, the terms “constant region” and “constant domain” are interchangeable and have their common meanings in the art. A constant region is an antibody portion that does not directly participate in antibody-antigen binding but can exhibit a variety of effector functions, such as an antibody portion interacting with an Fc receptor, or, for example, the carboxyl-terminal portion of the light chain and / or heavy chain. Compared to the variable domains of immunoglobulins, the constant regions of immunoglobulin molecules typically have a more conserved amino acid sequence. In some respects, antibody or antigen-binding fragments contain a constant region or a portion thereof sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0052] As used herein, based on the amino acid sequence of constant structural domains, the term "heavy chain" in relation to antibody use can refer to any different type, for example, α, δ, ε, γ, and μ that produce antibodies of the IgA, IgD, IgE, IgG, and IgM classes, including subclasses of IgG, such as IgG1, IgG2, IgG3, and IgG4. The amino acid sequence of the heavy chain is well known in the art. In a specific embodiment, the heavy chain is a human heavy chain. In a specific embodiment, the heavy chain is a rodent or mouse heavy chain.

[0053] As used herein, based on the amino acid sequence of a constant domain, the term "light chain" can refer to any different type, such as κ or λ, in relation to antibody use. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain. In a specific embodiment, the light chain is a rodent or mouse light chain.

[0054] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof whose amino acid sequence is derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of antibodies or antigen-binding fragments thereof derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability, while the constant regions are homologous to sequences in antibodies or antigen-binding fragments thereof derived from another species (usually human) to avoid triggering an immune response in said species.

[0055] The term "human" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin locus, wherein such antibody or antigen-binding fragment thereof is prepared using any technique known in the art. This definition of human antibody or antigen-binding fragment thereof includes complete or full-length antibodies and fragments thereof.

[0056] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or its antigen-binding fragment) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or its antigen-binding fragment and an antigen). The affinity of molecule X for its partner Y can generally be represented by a dissociation constant (K). Affinity can be measured and / or represented in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant.

[0057] As used herein, "epitope" is a term in the art and refers to a localized region of an antigen to which an antibody or its antigen-binding fragment can specifically bind. An epitope can be, for example, a continuous amino acid of a polypeptide (linear or continuous epitope), or an epitope can be, for example, derived from two or more discontinuous regions of one or more polypeptides (conformal, nonlinear, discontinuous, or non-continuous epitopes). In some embodiments, the epitope to which the antibody or its antigen-binding fragment specifically binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, hydrogen / atmosphere exchange in combination with mass spectrometry (e.g., liquid chromatography-electrospray ionization mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody / its antigen-binding fragment: Antigen crystals can be studied using well-known X-ray diffraction techniques and can be improved using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, Meth Enzymo1 (1985) Vols. 114 and 115, edited by Wyckoff HW et al.; US2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt1): 37-60; Bricogne G (1997) Meth Enzymo276A: 361-423, edited by Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt10): 1316-1323). Mutagenesis mapping studies can be performed using any method known to those skilled in the art. For descriptions of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe M et al., (1995) JBiol Chem 270:1388-1394 and Cunningham BC and Wells JA (1989) Science 244:1081-108.

[0058] If an antibody binds to a given epitope or overlapping epitope of a reference antibody such that it partially blocks the binding of the reference antibody to the epitope, then it can be said that the antibody "competitively inhibits" the binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. It can be said that the antibody competitively inhibits the binding of the reference antibody to the given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0059] In the preferred embodiment of this application, the "sample" is a human sample, but animal samples may also be used in the practice of this application. Non-limiting sources of samples used in this application include, for example, solid tissues, biopsies, ascites, aspirates, fluid leachates, blood (including circulating tumor cells), plasma, serum, cerebrospinal fluid, lymph, external sections of skin, respiratory tract, intestinal and genitourinary tract, tears, saliva, breast milk, tumors, organs, cell cultures and / or cell culture components.

[0060] The term “label” as used herein refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody in order to produce a “labeled” antibody. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzyme labeling, may catalyze a chemical change in a detectable substrate compound or composition.

[0061] Terms such as “treatment,” “curative,” “treatment,” “relief,” and “relief” refer to therapeutic measures that can cure, slow down, alleviate, and / or stop the progression of a pathological disease or condition. Therefore, those requiring treatment include those already diagnosed with or suspected of having the stated condition. In some embodiments, a subject’s cancer is successfully diagnosed according to the method of this application if the patient exhibits one or more of the following conditions.

[0062] Based on this, this application provides an IL-4R specific binding protein, wherein the IL-4R specific binding protein has an IL-4R binding domain.

[0063] The IL-4R binding domain has heavy chain CDR1 to heavy chain CDR3 as shown in SEQ ID NO.1 to SEQ ID NO.3, and light chain CDR1 to light chain CDR3 as shown in SEQ ID NO.4 to SEQ ID NO.6.

[0064] The specific sequences are shown in Table 1:

[0065] Table 1

[0066]

[0067] The IL-4R-specific binding protein of this application may have the aforementioned CDRs or derivative fragments having the aforementioned CDRs. The derivative fragments are formed by replacing amino acids at no more than six sites relative to their corresponding CDRs (“conservative modification” or “conservative substitution”), retaining the biological activity consistent with their corresponding complementarity-determining regions. For example, the derivative fragments may replace one amino acid with another, or one amino acid with multiple amino acids (e.g., two), at sites 1, 2, 3, 4, 5, or 6 of their corresponding complementarity-determining regions.

[0068] In the CDRs provided in this application, the derived fragments (conserved variants) refer to polypeptides formed by replacing one, two, or three amino acids with amino acids of similar or related properties compared to the amino acid sequence of the antibody in this application. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table 2.

[0069] Table 2

[0070]

[0071] "Conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will understand that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, the substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.

[0072] The IL-4R-specific binding protein provided in this application can be an antibody, an antigen-binding fragment of an antibody, or a small modular immunodrug. Further, the binding protein can be a monoclonal antibody, an F(ab')2 fragment, a Fab' fragment, a Fab fragment, an Fv fragment, a ScFv fragment, a biclonal antibody, a multispecific antibody, a microantibody, a chelated recombinant antibody, an internal antibody, a nanobody, a binding domain immunoglobulin fusion protein, or a small modular immunodrug.

[0073] In some embodiments, the heavy chain variable region is as shown in SEQ ID NO.7.

[0074] QSLEESGGRLVTPGTPLTLTCTVSGFALTTYHMNWVRQAPGKGLEWVGVISAAGRTYYPSWVNGRFTISKTSTTVDLKMTSLTTEDTATYFCARDGSTYVFDRFDVWGQGTLVTVSS (SEQ ID NO. 7).

[0075] In some embodiments, the light chain variable region is as shown in SEQ ID NO.8.

[0076] AQVLTQTPSPVSAAVGGTVTINCQASQSVYNNNFLPWYQQKPGQPPKLLIYRASTLASGVSSRFKGSGSGTHFTLTISDVQCDDAATYYCAGVYSNGDNAFGGGTELEIL (SEQ ID NO. 8).

[0077] Alternatively, this application does not specifically limit the species origin of the heavy chain constant region and the light chain constant region, and may include, but is not limited to, any of the following species: human, mouse, rabbit, sheep, cow, horse, pig, dog, cat, camel, donkey, deer, mink, chicken, duck and goose.

[0078] This application does not specifically limit the types of heavy chain constant regions and light chain constant regions of the antibody, which may include, but are not limited to, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

[0079] This application also provides a nucleic acid molecule containing a specific binding protein for encoding the IL-4R.

[0080] This application also provides a vector comprising the aforementioned nucleic acid molecule. This application does not specifically limit the type of recombinant expression vector, which may include, but is not limited to, antibody expression vectors. This application does not specifically limit the antibody expression vector, which may include, but is not limited to, bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or combinations thereof.

[0081] This application also provides a cell comprising the aforementioned nucleic acid molecule or the aforementioned vector. This application does not specifically limit the type of host cell, including but not limited to CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0082] This application also provides a method for preparing the cells described above, the method comprising the step of introducing the nucleic acid molecule or the vector into the target cell.

[0083] In one example, the import method uses transfection.

[0084] The term “transfection” refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques used for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chuetal, 1981, Gene 13:197.

[0085] This application also provides a method for preparing the IL-4R specific binding protein, the method comprising the following steps: culturing the cells and isolating the IL-4R specific binding protein from the resulting culture.

[0086] This application also provides an IL-4R detection product, the detection product comprising the IL-4R specific binding protein.

[0087] This application does not specifically limit the type of detection product, such as diagnostic reagents, test strips, test plates, or kits. In the detection product, the IL-4R specific binding protein competitively binds to IL-4R in the analyte.

[0088] This application also provides a method for detecting IL-4R in a sample to be tested, wherein the method uses the specific binding protein of IL-4R as the detection antibody and determines the presence of IL-4R in the sample to be tested through an immune binding reaction.

[0089] The term "immunobinding" refers to a specific binding reaction that occurs between an antibody molecule and an antigen (for which the antibody is specific). The strength or affinity of the immunobinding interaction can be expressed by the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates a higher affinity. The properties of the immunobinding between two molecules can be quantified using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex.

[0090] This application relates to methods for immunodetection or determination of target antigens (e.g., IL-4R), reagents for immunodetection or determination of target antigens (e.g., IL-4R), methods for immunodetection or determination of cells expressing target antigens (e.g., IL-4R), and diagnostic agents for diagnosing diseases associated with target antigen (e.g., IL-4R) positive cells, comprising an antibody or antibody fragment that specifically recognizes the target antigen (e.g., IL-4R) and binds to the amino acid sequence or its three-dimensional structure in the extracellular region as an active ingredient.

[0091] In this application, the method for detecting or determining the amount of a target antigen (e.g., IL-4R) can be any known method. For example, it includes immunoassay or assay methods. Immunoassay or assay methods are methods that use labeled antigens or antibodies to detect or determine the amount of antibody or antigen. Examples of immunoassay or assay methods include radiolabeled antibody immunoassays (RIA), enzyme immunoassays (EIA or ELISA), fluorescence immunoassays (FIA), luminescent immunoassays, Western blotting, physicochemical methods, etc.

[0092] To detect cells expressing peptides, known immunoassay methods can be used, with immunoprecipitation, fluorescent cell staining, and immunohistochemical staining being preferred. Alternatively, fluorescent antibody staining using the FMAT8100HTS (Applied Biosystem) can be employed.

[0093] The application does not impose any particular restrictions on the test sample used to detect or measure the target antigen (e.g., IL-4R), as long as it has the potential to contain cells expressing the target antigen (e.g., IL-4R), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

[0094] Depending on the required detection method, the detection product may also contain reagents for performing antigen-antibody reactions or for detecting the reaction. Reagents for performing antigen-antibody reactions include buffers, salts, etc. Reagents for detection include those commonly used in immunoassay or assay methods, such as labeled second antibodies that recognize the monoclonal antibody, its antibody fragments, or conjugates, and substrates corresponding to the labeled antibodies.

[0095] This application also provides a medicament comprising the IL-4R specific binding protein described above.

[0096] This application also provides the use of the aforementioned drugs in diseases related to IL4, IL13, or IL4R.

[0097] Optionally, the relevant diseases include allergic diseases, autoimmune diseases, tumors, or infectious diseases.

[0098] Allergic diseases such as asthma, allergic rhinitis, and atopic dermatitis: Overactivation of IL-4R leads to enhanced type II inflammatory response, causing symptoms such as airway hyperresponsiveness and increased mucus secretion.

[0099] In autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, abnormal expression of IL-4R may be involved in the regulation of autoimmune responses.

[0100] Tumors: IL-4R is highly expressed in certain tumor cells and may be associated with tumor immune escape and progression. This application does not specifically limit the type of tumor, including but not limited to one or more of breast cancer, lung cancer, gastric cancer, colorectal cancer, and prostate cancer.

[0101] This application also provides a treatment method comprising administering to a subject an effective dose of the IL-4R specific binding protein or the drug.

[0102] In this context, "application," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Application," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Application," "giving," and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0103] "Treatment" means administering an oral or topical therapeutic agent, such as a composition containing any of the antibodies or antigen-binding fragments of this application, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves symptoms of one or more diseases to induce the regression of such symptoms or inhibit their progression to any clinically measured extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Studentt test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0104] "Effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcome. For prophylactic use, beneficial or desired outcomes include eliminating or reducing risk, mitigating severity, or delaying the onset of a condition, including the condition itself, its complications, and the biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes presented during the development of the condition. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various target antigen-related conditions of this application or improving one or more symptoms of said conditions, reducing the dosage of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the target antigen-related condition of this disclosure in patients.

[0105] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0106] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. For temperature and time parameters, acceptable deviations due to instrument testing accuracy or operational precision are permissible.

[0107] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0108] Example 1

[0109] 1. Immunization and screening of human IL-4R antibodies

[0110] New Zealand White rabbits (purchased from Qingdao Kangda Aibo Biotechnology Co., Ltd., female, 12-14 weeks old) were used for protein immunization with human IL4R (HumanIL4R Protein, HisTag, purchased from Acro Inc., catalog number: ILRH5221). For the initial immunization, a 1:1 mixture of complete Freund's adjuvant and the antigen protein (0.4 mg) was emulsified and injected subcutaneously at multiple sites. Subsequently, every two weeks, TiterMax (purchased from Sigma Inc., catalog number T2684) was emulsified with the antigen protein (0.2 mg) at a 1:1 ratio and injected subcutaneously at multiple sites, for a total of four immunizations. To obtain the best immunization effect, antiserum was collected 7 days after each of the 0th, 1st, 2nd, 3rd, and 4th immunizations to assess the immunization effect against the anti-antigen response.

[0111] When the New Zealand White rabbits reached their optimal immune response, peripheral blood mononuclear cells (PBMCs) were extracted from the rabbits, and antibody-expressing B cells were harvested. Individual B cells were sorted using flow cytometry (purchased from BDI Inc., model FACSARIA III). The antibody heavy and light chain genes were retrieved and cloned into the pCDNA3.4 expression vector. The vector was transiently transfected into 293 cells to obtain the antibody expression supernatant. Monoclonal antibodies with the desired function were selected by detecting antigen-binding activity and the activity blocking the binding of hIL4 and hIL4R.

[0112] One type of human IL-4R antibody obtained by the aforementioned method was named 23G3.

[0113] 2. Construction of human IL-4R light and heavy chain expression plasmids and antibody preparation

[0114] Positive clones (containing PCR products of light and heavy chain variable regions, respectively) were cloned into vectors containing the human IgG1 heavy chain constant region and vectors containing the human Kappa light chain constant region, respectively, and sequenced. Then, plasmids were prepared and transfected into CHO-S cells. After culturing for 7-10 days, the culture supernatant was collected, and antibodies were obtained by Protein A affinity chromatography and replaced with 1×PBS by ultrafiltration.

[0115] 3. Binding activity of anti-human IL-4R antibody to human IL-4R overexpressing cells

[0116] The binding activity of the anti-human IL-4R antibody in this disclosure to human IL-4R expressed on the surface of HEK293-IL-4R cells was detected by flow cytometry (FACS). Briefly, HEK293-IL-4R cells were harvested from cell culture flasks, washed twice, and resuspended in FACS buffer (PBS buffer containing 4% FBS). Then, the cells were cultured in a solution containing 2 × 10⁻⁶ cells / mL. 5 100 μL / well of anti-IL-4R antibody, serially diluted (starting at 10 μg / mL, 3-fold serial dilution) in FACS buffer was added to each well of a 96-well plate containing 100 cells / well. The plate was incubated on ice for 30 min. After washing twice with FACS buffer, 100 μL / well of FITC Anti-human IgG (Merk, catalog number: F9512, diluted 1:400 in FACS buffer) was added. The plate was incubated at 4°C in the dark for 30 min. Cells were washed three times and resuspended in FACS buffer. The MFI value was measured using BD FACSLyric. The EC50 value was calculated from the analyzed data.

[0117] The results show (e.g.) Figure 1 As shown in the figure, the EC50 value of 23G3 is 0.08067 μg / mL.

[0118] 4. Anti-human IL-4R antibody blocks the binding of IL4 / IL-4R overexpressing cells.

[0119] The activity of the anti-human IL-4R antibody in this disclosure in blocking the binding of human IL-4 protein to cell surface IL-4R was detected by flow cytometry (FACS). Briefly, HEK293-IL-4R cells were harvested from cell culture flasks, washed twice, and resuspended in FACS buffer (PBS buffer containing 4% FBS). Then, the cells were cultured in a solution containing 2 × 10⁻⁶ cells / mL. 5 Add 50 μL / well of serially diluted (starting at 20 μg / mL, 3-fold serial dilution) anti-IL-4R antibody to each well of a 96-well plate containing 10 cells / well. Then add 50 μL / well of biotin-labeled human IL-4 (Acro, catalog number: IL4-H82E0) and incubate at 4°C in the dark for 30 min. After washing twice with FACS buffer, add 100 μL / well of PE Streptavidin (BD, catalog number 405204, diluted 1:400 in FACS buffer) and incubate at 4°C in the dark for 30 min. Wash the cells three times, resuspend in FACS buffer, and determine the MFI value using BD FACS Lyric. Calculate the IC50 value through data analysis.

[0120] The results show (e.g.) Figure 2 As shown in the figure, the IC50 value of 23G3 is 0.06628 μg / mL.

[0121] 5. Determination of the binding affinity between anti-human IL-4R antibody and IL-4R protein

[0122] The affinity of the antibody for binding to IL-4R protein was determined using OctetRH96. Candidate antibodies were captured using the AHC2 probe (Sartorius, 18-5142); they were bound to multiple concentrations of IL-4R protein (Acrobiosystems, ILR-H5221) for 300 s and dissociated for 600 s. The antigen-binding affinity was calculated using a 1:1 model fitted to the data using Octet Analysis Studio software. The results (as shown in Table 3) indicate that the KD value of 23G3 was 3.589E-10M.

[0123] Table 3: Binding affinity of anti-human IL-4R antibody to IL-4R protein

[0124]

[0125] 6. Antibody inhibits IL-4-induced STAT6 phosphorylation activity in HEK293-hSTAT6 / 5×STAT6-Luc cells.

[0126] HEK293-hSTAT6 / 5×STAT6-Luc cells in logarithmic growth phase were resuspended in culture medium (DMEM + 10% FBS) and seeded at 60 μL / well into 96-well plates, with each well containing 4 × 10⁶ cells. 4 Cells were incubated overnight at 37°C in a 5% CO2 incubator. Then, 30 μL of serially diluted anti-IL-4Rα antibody (starting at 400 nM, 5-fold serial dilution) was added to each well. 30 μL of IL-4 protein (40 ng / mL, Sino Biological, catalog number: 11846-HNAE) was added to each well, and the cells were incubated at 37°C in a 5% CO2 incubator for 6 h. ONE-GLO assay reagent (Promega, catalog number: E6120) was added, and the cells were incubated at room temperature in the dark for 3-5 min. The luminescence value was read using SpectraMax i3x. The IC50 value was calculated through data analysis. Results showed (e.g.) Figure 3 As shown in the figure, the IC50 value of 23G3 is 6.835 nM.

[0127] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. An antibody against IL-4R, characterized in that, The anti-IL-4R antibody comprises heavy chain CDR1 to heavy chain CDR3 as shown in sequence as SEQ ID NO.1 to SEQ ID NO.3, and light chain CDR1 to light chain CDR3 as shown in sequence as SEQ ID NO.4 to SEQ ID NO.

6.

2. The antibody against IL-4R according to claim 1, characterized in that, The heavy chain variable region of the anti-IL-4R antibody is shown in SEQ ID NO.

7.

3. The antibody against IL-4R according to claim 1, characterized in that, The light chain variable region of the anti-IL-4R antibody is shown in SEQ ID NO.

8.

4. The antibody against IL-4R according to any one of claims 1 to 3, characterized in that, The antibody against IL-4R also includes a heavy chain constant region and a light chain constant region.

5. The antibody against IL-4R according to claim 4, characterized in that, The antibody against IL-4R satisfies one or more of the conditions shown in 1) and 2) below: 1) The anti-IL-4R antibody independently possesses the sequence of any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD; and, 2) The heavy chain constant region and the light chain constant region are independently selected from any one of the following species: human, mouse, rabbit, sheep, cow, horse, pig, dog, cat, camel, donkey, deer, mink, chicken, duck and goose.

6. A nucleic acid molecule, characterized in that, Used to encode an antibody against IL-4R as described in any one of claims 1 to 5.

7. A carrier, characterized in that, It includes the nucleic acid molecule as described in claim 6.

8. A cell, characterized in that, It comprises the nucleic acid molecule of claim 6 or the vector of claim 7.

9. A method for preparing the cell according to claim 8, the method comprising the step of introducing the nucleic acid molecule or the vector into the target cell.

10. A method for preparing the anti-IL-4R antibody according to any one of claims 1 to 5, characterized in that, The preparation method includes the following steps: Cultivating the cells according to claim 8, and; Antibodies against IL-4R were isolated from the resulting culture.

11. An IL-4R detection product, characterized in that, The detection product includes the antibody against IL-4R as described in any one of claims 1 to 5.

12. A method for detecting IL-4R in a sample for non-therapeutic and diagnostic purposes, characterized in that, The detection method uses the anti-IL-4R antibody as described in any one of claims 1 to 5 as the detection antibody, and determines the presence of IL-4R in the sample to be tested through an immune binding reaction.

13. A drug, characterized in that, The drug comprises an antibody against IL-4R as described in any one of claims 1 to 5.

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