Stable antibody formulation

Abstract

The present invention belongs to the field of immunology and relates to a stable antibody formulation. The stable antibody formulation comprises a therapeutically effective amount of an antibody molecule, an isotonicity agent, a buffering agent, a bulking agent, and a surfactant, and has a pH of 4.5-5.5. The antibody molecule is capable of specifically binding to both IL-5 and IL-4Rα simultaneously, and comprises at least two immunoglobulin variable domains, the first immunoglobulin variable domain being capable of specifically binding to an IL-4Rα molecule and a second immunoglobulin variable domain being capable of specifically binding to IL-5. The antibody in the antibody formulation substantially retains its physical and chemical stability and integrity during storage.

Description

[Technical field]

[0001] The present disclosure relates to the field of medicine. More specifically, the present disclosure relates to the preparation of stable antibody formulations. [Background technology]

[0002] Advances in biotechnology have made it possible, using recombinant DNA techniques, to produce a wide variety of proteins for use in pharmaceutical applications. Protein formulation presents unique challenges, as proteins tend to be larger and more complex than traditional organic or inorganic drugs.

[0003] Liquid antibody products have a short shelf life and chemical and physical instability during storage can render the antibody biologically ineffective. Chemical instability is due to deamidation, racemization, hydrolysis, oxidation, β-elimination, or exchange of disulfide bonds, while physical instability is due to antibody denaturation, aggregation, precipitation, or adsorption. Of these, aggregation, deamidation, and oxidation are known to be the most common causes of antibody degradation (see Wang et al., J. of Parenteral Science & Technology 42 (Suppl), S4-S26, 1988, and Cleland et al., Critical Reviews in Therapeutic Drug Carrier Systems 10(4): 307-377, 1993). Thus, there is a need for stable antibody liquid formulations, especially high-concentration stable antibody liquid formulations. Summary of the Invention

[0004] An object of the present disclosure is to provide an antibody formulation that is stable upon storage and application. According to the present disclosure, a stable formulation refers to a formulation in which the antibody contained therein essentially retains its physical and chemical stability and integrity upon storage. It is a further object to provide a stable liquid antibody formulation suitable for administration, such as subcutaneous administration or intravenous infusion.

[0005] Generally, the use of small amounts of pharmaceutical formulations is preferred for subcutaneous injection. For antibody-containing formulations, for example, high-dose antibody therapy requires high concentrations of antibody formulations for subcutaneous administration. Due to the need for high antibody concentrations, antibody-containing formulations suffer from challenges related to the physical and chemical stability of antibodies, aggregate formation, manufacturing difficulties, storage, and delivery of antibody formulations.

[0006] Generally, the formation of by-products and degradants and biological inactivation are the primary factors determining shelf life, and the formulations of the present disclosure achieve these desired stability levels.

[0007] In addition to sufficient physical and chemical stability, the formulation must have an acceptable pH value and osmolality. Also, high antibody concentrations are known to increase the viscosity and aggregation of the formulation. A suitable pharmaceutical formulation according to the present disclosure has a desired and suitable viscosity.

[0008] It has been found that the present disclosure makes available particularly stable antibody formulations that have advantageous properties in terms of preserving the activity of the antibody during long-term storage, prevent aggregation, and have a suitable viscosity even for high antibody concentrations. In its broadest aspect, the present disclosure provides a pharmaceutical formulation (the formulation of the present disclosure) with an antibody as an active ingredient. The composition includes a buffer system, a stabilizer, and a surfactant. The formulation of the present disclosure is liquid (e.g., aqueous solution), but is also suitable for being lyophilized and then formed into liquid formulations with lower, equal, or higher antibody concentrations.

[0009] 1. Overview

[0010] 1.1 A first aspect of the present disclosure provides a stable formulation:

[0011] (1) A formulation comprising a therapeutically effective amount of at least one antibody molecule, at least one tonicity agent, at least one buffering agent, and at least one surfactant, wherein the composition (i.e., the formulation) has a pH of 4.5 to 5.5.

[0012] (2) A formulation comprising a therapeutically effective amount of at least one antibody molecule, at least one tonicity agent, at least one buffering agent, and at least one surfactant, wherein the composition has a pH of 4.7 to 5.3.

[0013] (3) A formulation consisting essentially of a therapeutically effective amount of at least one antibody molecule, at least one tonicity agent, at least one buffering agent, and at least one surfactant, wherein the composition has a pH of 5.0 to 5.2.

[0014] The antibody molecules of compositions (1) to (3) are capable of specifically binding to both IL-5 and IL-4Rα and comprise at least two immunoglobulin variable domains having a first immunoglobulin variable domain capable of specifically binding to an IL-4Rα molecule and a second immunoglobulin variable domain capable of specifically binding to IL-5.

[0015] "Treatment" refers to both curative and prophylactic treatment. Those in need of treatment include those who have a disorder as well as those who wish to prevent a disorder. "Mammal" for the purposes of treatment refers to any animal classified as a mammal, including, but not limited to, humans, farm and livestock animals such as dogs, horses, cats, and cows, zoo animals, sport animals, and pet animals.

[0016] In the context of this disclosure, a "therapeutically effective amount" of an antibody in the pharmacological sense refers to an amount that has a therapeutic or prophylactic effect against the disorder that the antibody is effectively able to treat. A "disorder" refers to any condition that may benefit from treatment with the antibody, including chronic and acute disorders and diseases, as well as pathological conditions that predispose a mammal to the disorder. In a preferred embodiment, a "disorder" refers to a disease associated with expression of IL-4R and / or IL-5.

[0017] Formulations according to the present disclosure have the advantage of stably supporting high concentrations of biologically active antibodies in solution and are suitable for oral administration, including intravenous, intramuscular, or subcutaneous injection.

[0018] In preferred embodiments of the present disclosure, the pH is within the range of 4.5 to 5.5, and desirably between a value close to pH 4.5 and a value close to any of pH 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5.

[0019] Preferably, the pH is selected from a range of pH values ​​between pH 4.6 and a value close to any of pH 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. More preferably, the pH is selected from a range of pH values ​​between a value close to pH 4.7 and a value close to any of pH 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. More preferably, the pH is selected from a range of pH values ​​between a value close to pH 4.8 and a value close to any of pH 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. More preferably, the pH is selected from a range of pH values ​​between a value close to pH 4.9 and a value close to any of pH 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. More preferably, the pH is selected from a pH value between close to pH 5.0 and close to any of pH 5.1, 5.2, 5.3, 5.4, or 5.5. More preferably, the pH is selected from a pH value between close to pH 5.1 and close to any of pH 5.2, 5.3, 5.4, or 5.5. In a preferred embodiment, the pH may range from close to pH 5.2 to close to any of pH 5.3, pH 5.4, or pH 5.5, from close to pH 5.3 to close to pH 5.4 or pH 5.5, or from pH 5.4 to pH 5.5.

[0020] More preferably, the pH can be selected from any of the following pH values: about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5, and most preferably the pH is pH 5.0±0.2. These pH ranges provide formulations with enhanced protection from antibody aggregation and fragmentation, and are closer to physiological pH (about 7.2-7.4) to reduce pain during injection and the risk of anaphylactoid side effects.

[0021] According to a further preferred embodiment of the present disclosure, the tonicity agent preferably comprises a polyol, a sugar, a carbohydrate, a salt such as sodium chloride, or a mixture thereof. Preferably, the molecular weight of the polyol is less than about 600 kD (e.g., in the range of about 120 to about 400 kD). Also, the polyol is preferably selected from mannitol, trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol, xylitol, glycerin, lactitol, propylene glycol, polyethylene glycol, inositol, or a mixture thereof. Preferably, the sugar or carbohydrate is selected from the group consisting of monosaccharides, disaccharides, polysaccharides, and mixtures thereof. Preferably, the sugar or carbohydrate is selected from the group consisting of fructose, glucose, mannose, sucrose, sorbose, xylose, lactose, maltose, sucrose, dextran, pullularan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, water-soluble glucan, and a mixture thereof. Preferably, the tonicity agent comprises a sugar selected from the group consisting of reducing sugars, non-reducing sugars and mixtures thereof. More preferably, the tonicity agent comprises a sugar that is a non-reducing sugar, preferably selected from the group consisting of sucrose, trehalose and mixtures thereof. Most preferably, the tonicity agent comprises sucrose.

[0022] The concentration of the tonicity agent ranges from about 1 mg / ml to about 300 mg / ml, from about 1 mg / ml to about 200 mg / ml, or from about 1 mg / ml to about 100 mg / ml. More preferably, the concentration of the isotonicity agent in the liquid composition is about 60 mg / ml, about 65 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 81 mg / ml, about 82 mg / ml, about 83 mg / ml, about 84 mg / ml, about 85 mg / ml, about 86 mg / ml, about 87 mg / ml, about 88 mg / ml, about 89 mg / ml, about 90 mg / ml, about 91 mg / ml, about 92 mg / ml, about 93 mg / ml, about 94 mg / ml, about 95 mg / ml, about 96 mg / ml, about 97 mg / ml, about 98 mg / ml, about 99 mg / ml, about 100 mg / ml, about 105 mg / ml, about 110 mg / ml, about 120 mg / ml, or about 130 mg / ml.

[0023] According to a preferred embodiment of the present disclosure, the buffering agent can be selected from the group consisting of acetate, succinate, gluconate, citrate, histidine, acetic acid, phosphate, phosphoric acid, ascorbic acid, tartaric acid, maleic acid, glycine, lactate, lactic acid, ascorbic acid, imidazole, bicarbonate and carbonic acid, succinic acid, sodium benzoate, benzoic acid, gluconate, edetic acid, acetate, malic acid, imidazole, tris, phosphoric acid, and mixtures thereof.

[0024] According to the present disclosure, the use of acetate buffer as a particularly preferred buffering agent provides the liquid composition with a pH close to physiological pH, thereby reducing pain upon injection and the risk of anaphylactoid side effects, and also improving antibody stability and resistance to aggregation, oxidation, and fragmentation.

[0025] The concentration of the buffering agent can range from about 0.1 millimolar (mM) to about 100 millimolar (mM). Preferably, the concentration of the buffering agent is from about 0.5 mM to about 50 mM, more preferably from about 1 mM to about 30 mM, more preferably from about 1 mM to about 20 mM, more preferably from about 10 mM to about 20 mM. Preferably, the concentration of the buffering agent is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 10 mM, about 18 mM, about 19 mM, or about 20 mM.

[0026] The term "surfactant" in this context refers to organic substances with amphiphilic structures, i.e., organic substances consisting of groups with opposing solubility tendencies, typically hydrocarbon chains that are soluble in oil and ionic groups that are soluble in water. Based on the charge of the surface active sites, surfactants can be classified into anionic surfactants, cationic surfactants, and dispersants for various pharmaceutical compositions and biomaterial formulations.

[0027] According to a further preferred embodiment of the present disclosure, surfactants suitable for use in the present disclosure include, but are not limited to, non-ionic surfactants, ionic surfactants, and zetterionic surfactants.Non-limiting examples of typical surfactants that may be used in the present disclosure include sorbitan fatty acid esters (e.g., sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate), sorbitan trioleate, glycerin fatty acid esters (e.g., glycerin monocaprylate, glycerin monomyristate, glycerin monostearate), polyglycerin fatty acid esters (e.g., decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate), polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitol fatty acid esters (e.g., polyoxyethylene sorbitol tetrastearate, polyoxyethylene sorbitol tetraoleate). , polyoxyethylene glycerin fatty acid esters (e.g., polyoxyethylene glyceryl monostearate), polyethylene glycol fatty acid esters (e.g., polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g., polyoxyethylene lauryl ether), polyoxyethylene polyoxypropylene alkyl ethers (e.g., polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkyl phenyl ethers (e.g., polyoxyethylene nonyl phenyl ether), polyoxyethylene hydrogenated castor oil (e.g., polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene beeswax derivatives (e.g., polyoxyethylene sorbitol beeswax), polyoxyethylene lanolin derivatives (e.g., polyoxyethylene lanolin), polyoxyethylene fatty acid amides (e.g., polyoxyethylene stearic acid amide), C. 10 -C 18Alkyl sulfates (e.g., sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene C with an average of 2 to 4 moles of ethylene oxide units added 10 -C 18 Alkyl ether sulfates (e.g., sodium polyoxyethylene lauryl sulfate), C 10 -C 18 Alkyl sulfosuccinates (e.g. sodium lauryl sulfosuccinate), and lecithin, glycerophospholipids, sphingophospholipids (such as sphingomyelin), C 12 -C 18 Natural surfactants, such as sucrose esters of fatty acids. The composition may contain one or more of these surfactants. Preferred surfactants are polyoxyethylene sorbitan fatty acid esters, such as polysorbate 20, 40, 60, or 80. Polysorbate 80 (Tween 80) is particularly easy to use. According to the present disclosure, surfactants, particularly polysorbate 80, improve the stability of antibodies in liquid compositions, conferring resistance to aggregation and fragmentation.

[0028] The concentration of the surfactant is generally from about 0.01 mg / ml to about 10 mg / ml, from about 0.01 mg / ml to about 5.0 mg / ml, from about 0.01 mg / ml to about 2.0 mg / ml, from about 0.01 mg / ml to about 1.5 mg / ml, from about 0.01 mg / ml to about 01.0 mg / ml, from about 0.01 mg / ml to about 0.5 mg / ml, from about 0.01 mg / ml to about 0.4 mg / ml, from about 0.01 mg / ml to about 0.3 mg / ml, from about 0.01 mg / ml to about 0.2 mg / ml, from about 0.01 mg / ml to about 0.15 mg / ml, from about 0.01 mg / ml to about 0.1 mg / ml, or from about 0.01 mg / ml to about 0.05 mg / ml. More preferably, the concentration of the surfactant is about 5 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 1.1 mg / ml, about 1.2 mg / ml, about 1.3 mg / ml, about 1.4 mg / ml, about 1.5 mg / ml, about 1.6 mg / ml, about 1.7 mg / ml, about 1.8 mg / ml, about 1.9 mg / ml, or about 2 mg / ml.

[0029] 1.2 In some embodiments of the present disclosure, the first immunoglobulin variable domain is a first single domain antibody molecule, which binds to IL-4Rα and comprises three complementarity determining regions (CDRs), wherein CDR1 has the amino acid sequence set forth in SEQ ID NO:1, CDR2 has the amino acid sequence set forth in SEQ ID NO:2, and CDR3 has the amino acid sequence set forth in SEQ ID NO:3, or a CDR having 3, 2 or less than 1 amino acid substitution (e.g. conservative substitution) compared to said plurality of CDRs. The second immunoglobulin variable domain is a second single domain antibody molecule, which binds IL-5 and comprises three complementarity determining regions (CDRs), wherein CDR1 has the amino acid sequence set forth in SEQ ID NO:4, CDR2 has the amino acid sequence set forth in SEQ ID NO:5 and CDR3 has the amino acid sequence set forth in SEQ ID NO:6, or a CDR having less than 3, 2 or 1 amino acid substitutions (e.g. conservative substitutions) compared to said plurality of CDRs.

[0030] 1.3 In some embodiments of the present disclosure, the antibody molecule is a polypeptide fusion that does not include a linker group between the first single domain antibody molecule and the second single domain antibody molecule.

[0031] 1.4 In some embodiments of the present disclosure, the antibody molecule is a polypeptide fusion comprising a biocompatible polymer having 1-100 atoms as a linker group for ligation of the first and second single domain antibody molecules. The linker group may be any linker group apparent to one of skill in the art. In embodiments, the linker group comprises or consists of polyglycine, polyalanine, polyserine, polylysine, polyglutamic acid, polyisoleucine, polyarginine residues, or combinations thereof. For example, a polyglycine or polyserine linker may comprise at least 5, 7, 8, 9, 10, 12, 15, 20, 30, 35, 40 glycine and serine residues. Linkers that can be used include Gly-Ser repeats, such as 1, 2, 3, 4, 5, 6, 7, or more repeats of Gly)4-Ser. In many embodiments, the linker has the sequence: (GIy)4-Ser-(Gly)3-Ser or ((Gly)4-Ser)n, where n is 4, 5, or 6.

[0032] 1.5 In some embodiments, the first and second single domain antibody molecules are ligated via the human IgG Fc region. The antibody molecule, with a homodimeric structure and named 4E9V10-2B3V2, comprises two identical single chain Fc fusion proteins. Each single chain Fc fusion protein consists of a first immunoglobulin variable domain, a human IgG Fc region and a second immunoglobulin variable domain. Each single chain Fc fusion protein is abbreviated as each single chain.

[0033] The procedure for producing the above antibodies includes a procedure of introducing a gene sequence encoding an anti-IL-4Rα single domain antibody or a gene sequence encoding an anti-IL-5 single domain antibody into a vector (obtained by the method described in Chinese patent application CN202010576200.7 "ANTI-IL-4Rα SINGLE-DOMAIN ANTIBODY AS WELL AS APPLICATION AND DRUG") by sequence synthesis, and producing a double-chain antibody molecule by genetic recombination technology.

[0034] The first single domain antibody molecule and the second immunoglobulin variable domain can be derived from naturally occurring molecules without light chains (e.g. VHH, nanobody, camelid-derived antibody). The single domain antibody molecule can be derived from camelids such as camel, llama, dromedary, alpaca, or guanaco. In other embodiments, the SDAB molecule can comprise other naturally occurring single domain molecules, such as shark single domain polypeptides (shark immunoglobulin neoantigen receptor (IgNAR)) or single domain scaffolds (e.g. fibronectin scaffolds). The single domain molecule can be derived from sharks.

[0035] 1.6 In some embodiments of the present disclosure, the antibody molecule is a double-chain antibody molecule consisting of two identical single-chain Fc fusion proteins paired and ligated to each other, each single-chain Fc fusion protein consisting of a first immunoglobulin variable domain, a human IgG Fc region, and a second immunoglobulin variable domain.

[0036] In the present application, one of the antigens that the bispecific antibody can recognize is the IL-5 protein, and the other is the IL-4Rα protein.

[0037] 1.7 In some embodiments of the present disclosure, the human IgG Fc region is derived from human IgG1, IgG2, IgG3, or IgG4.

[0038] 1.8 In some embodiments of the present disclosure, the human IgG Fc region is an amino acid sequence derived from a human IgG4 region, wherein the Fc region has the amino acid sequence set forth in SEQ ID NO:13 or is located at positions 116 to 356 of SEQ ID NO:9.

[0039] 1.9 In some embodiments of the present disclosure, the first immunoglobulin variable domain has the amino acid sequence set forth in SEQ ID NO:7 and the second immunoglobulin variable domain has the amino acid sequence set forth in SEQ ID NO:8.

[0040] The first immunoglobulin variable domain according to SEQ ID NO:7 is a humanized single domain antibody molecule. The second immunoglobulin variable domain according to SEQ ID NO:8 is a humanized single domain antibody molecule. SEQ ID NO:7 (4E9V10) is derived from the humanization of the amino acid sequence of the single domain antibody 4E9 in Chinese patent application CN202010576200.7 "ANTI-IL-4RA SINGLE-DOMAIN ANTIBODY AS WELL AS APPLICATION AND DRUG". SEQ ID NO:8 (2B3V2) is derived from the humanization of the amino acid sequence of the single domain antibody 2B3 in Chinese patent application CN202010843501.1 "IL-5 BINDING MOLECULES AND PREPARATION METHODS AND APPLICATIONS THEREOF". The method for humanizing the single domain antibody 2B3 is described in Chinese patent application CN202010843501.1 "IL-5 BINDING MOLECULES AND PREPARATION METHODS AND APPLICATIONS THEREOF."

[0041] The humanization method for the single domain antibody 4E9 is based on the results of big data analysis and is based on high-throughput screening of the antibody framework region mutation library. The detailed steps are as follows:

[0042] (1) Sequence analysis of human / camelid antibody data: Amino acid preference analysis is performed on the sequences of 13,873 Nb cases (human) batch-downloaded from the NCBI website, and amino acid preference analysis is also performed on the sequences of the applicant's 2,000 single-domain antibodies to obtain amino-terminal ratio data of each site in the framework region.

[0043] (2) Comprehensive weighting analysis of human / camel antibodies: Number the above human / camel antibody sequences according to the IMGT numbering rules, match the sequences one-to-one, perform weighting analysis based on the weights of 90% of human antibodies and 10% of camel antibodies, calculate the weighted proportion of amino acids at each site, rank the proportions from high to low, and according to the final weighting result at a single site in the framework region, only retain more than 10% of amino acid species, and calculate the final weights of amino acids with a proportion of more than 10% based on the criterion that the overall retention proportion is 1, which serves as the basis for the subsequent design of customized amino acid libraries.

[0044] (3) Scheme design of customized amino acid library: For a single site to be mutated, the number of amino acids exceeding 10% is n, the ratio of the highest ratio and the lowest ratio of amino acids exceeding 10% is V, and the nature of the site to be mutated is determined. If V≧3 ​​and n≦2, the site is regarded as a "high concentration site", otherwise, the site is regarded as a "medium / low concentration site". According to this method, the customized amino acid library is divided into a "high / medium concentration library" and a "low concentration library", and both are used to construct the customized amino acid library. The final weight of (2) above is the basis for the type and ratio of amino acid species in the library.

[0045] (4) High-throughput screening of customized amino acid libraries

[0046] A humanized antibody library is constructed against non-humanized 4E9, and the constructed library is panned with the corresponding antigen to ultimately obtain antibody sequences with high affinity and a high degree of humanization.

[0047] Preferably, 4E9V10 has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 has the amino acid sequence of SEQ ID NO:14, FR2 has the amino acid sequence of SEQ ID NO:15, FR3 has the amino acid sequence of SEQ ID NO:16, and FR4 has the amino acid sequence of SEQ ID NO:17.

[0048] 2B3V2 has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 has the amino acid sequence of SEQ ID NO:18, FR2 has the amino acid sequence of SEQ ID NO:19, FR3 has the amino acid sequence of SEQ ID NO:20, and FR4 has the amino acid sequence of SEQ ID NO:21.

[0049] Here, the term "humanized antibody" refers to an antibody obtained by fusing the heavy chain variable domain of a target antibody (such as an animal antibody) with the constant region of a human antibody, an antibody obtained by grafting the complementarity determining regions (CDR1-3 sequences) of a target antibody into the variable region of a human antibody, or an antibody obtained by subjecting a target antibody to amino acid mutations according to the characteristics of the framework regions (FR1-4) of a human antibody. Humanized antibodies may be obtained by synthesis or site-directed mutagenesis.

[0050] The step of preparing the above-mentioned antibody in item 1.9 may include a step of introducing the gene sequence of the anti-IL-4Rα humanized single domain antibody (4E9V10) (nucleotides 1 to 345 of SEQ ID NO: 11 or 12) or the gene sequence of the anti-IL-5 humanized single domain antibody (designated as 2B3V2) (nucleotides 1069 to 1434 of SEQ ID NO: 11 or 12) into a vector by sequence synthesis (obtained by the method described in Chinese patent application CN202010576200.7 "ANTI-IL-4Rα SINGLE-DOMAIN ANTIBODY AS WELL AS APPLICATION AND DRUG") to obtain a genetically modified plasmid, and expressing the double-chain antibody from the plasmid.

[0051] 1.10 In some embodiments of the present disclosure, a stable antibody formulation comprises a therapeutically effective amount of an antibody molecule, an isotonicity agent, a buffering agent, and a surfactant, wherein the pH of the formulation is between 4.5 and 5.5, and the antibody molecule comprises a fusion protein having the amino acid sequence set forth in SEQ ID NO: 9 or 10. Preferably, the antibody molecule is a double-chain antibody, and each single chain of the double-chain antibody molecule has the amino acid sequence set forth in SEQ ID NO. 9 or SEQ ID NO. 10. The two single-chain Fc regions automatically combine to form the double-chain antibody molecule.

[0052] In some embodiments of the present disclosure, the antibody molecule designated "4E9V10-2B3V2" has an amino acid sequence set forth in SEQ ID NO:9 or 10, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence that is at least 85%, 90%, 95% or more identical, or has up to 20, 15, 10, 5, 4, 3, 2, 1 amino acid alterations (e.g., deletions, insertions or substitutions (e.g., conservative substitutions)) relative to the amino acid sequence set forth in SEQ ID NO:9 or 10.

[0053] 4E9V10-2B3V2 has an amino acid sequence set forth in SEQ ID NO: 9 or 10, and the corresponding nucleotide sequence is as set forth in SEQ ID NO: 11 or 12. 4E9V10-2B3V2 comprises three segments (4E9V10-Fc segment-2B3V2), where 4E9V10 is located at the amino terminus (positions 1 to 115 of SEQ ID NO. 9 or 10), the Fc segment is located at positions 116 to 356 of SEQ ID NO. 9 or 10, and 2B3V2 is located at the carboxyl terminus (positions 357 to 478 of SEQ ID NO. 9 or 10).

[0054] 4E9V10-2B3V2 includes 4E9V10-2B3V2-1 (i.e., having the amino acid sequence set forth in SEQ ID NO: 9 and the nucleotide sequence set forth in SEQ ID NO: 11) and 4E9V10-2B3V2-2 (i.e., having the amino acid sequence set forth in SEQ ID NO: 10 and the nucleotide sequence set forth in SEQ ID NO: 12).

[0055] 2. Prescription

[0056] 2.1 In some embodiments of the present disclosure, the preparation buffer agent of item 1.1 is sodium acetate-acetic acid buffer, the tonicity agent includes sucrose, and the surfactant includes polysorbate.

[0057] 2.2 In some embodiments of the present disclosure, the formulation further comprises a bulking agent comprising glycine and arginine hydrochloride.

[0058] 2.3 Acetic Acid-Sodium Acetate In some embodiments of the present disclosure, the formulation (optionally) comprises acetic acid-sodium acetate at a concentration of 10-20 mmol / L, e.g., about 10 mmol / L to 19 mmol / L, about 10 mmol / L to 18 mmol / L, about 10 mmol / L to 17 mmol / L, about 10 mmol / L to 16 mmol / L, about 10 mmol / L to 15 mmol / L, about 10 mmol / L to about 14 mmol / L, about 10 mmol / L to about 16 mmol / L, about 10 mmol / L to about 15 mmol / L, about 10 mmol / L to about 16 ... The acetic acid-sodium acetate concentration ranges from about 10 mmol / L to about 13 mmol / L, about 10 mmol / L to about 12 mmol / L, about 10 mmol / L to about 11 mmol / L, about 10 mmol / L, about 11 mmol / L, about 12 mmol / L, about 13 mmol / L, about 14 mmol / L, about 15 mmol / L, about 16 mmol / L, about 17 mmol / L, about 18 mmol / L, about 19 mmol / L, or about 20 mmol / L. The overall concentration of acetic acid and sodium acetate in the formulation is 10 to 20 mmol / L.

[0059] Preferably, the mass ratio of acetic acid to sodium acetate is (0.2-2):(1-10). More preferably, the mass ratio of acetic acid to sodium acetate is 2:9.

[0060] 2.4 Arginine Hydrochloride In some embodiments of the present disclosure, the formulation (optionally) comprises arginine hydrochloride at a concentration of 25-100 mmol / L, e.g., at a concentration of about 25 mmol / L to 90 mmol / L, about 25 mmol / L to 80 mmol / L, about 25 mmol / L to 70 mmol / L, about 25 mmol / L to 60 mmol / L, about 25 mmol / L to about 50 mmol / L, about 25 mmol / L to about 40 mmol / L, about 25 mmol / L to about 30 mmol / L, about 25 mmol / L, about 30 mmol / L, about 40 mmol / L, about 50 mmol / L, about 60 mmol / L, about 70 mmol / L, about 80 mmol / L, about 90 mmol / L, or about 100 mmol / L.

[0061] 2.5 Sucrose In some embodiments of the present disclosure, the formulation includes (optionally) sucrose as an isotonicity agent at a concentration of 80 to 120 mmol / L, e.g., about 80 mmol / L to about 118 mmol / L, about 80 mmol / L to about 115 mmol / L, about 80 mmol / L to about 110 mmol / L, about 80 mmol / L to about 105 mmol / L, about 80 mmol / L to about 100 mmol / L, about 80 mmol / L to about 95 mmol / L, about 80 mmol / L to about 90 mmol / L, about 80 mmol / L, about 85 mmol / L, about 90 mmol / L, about 95 mmol / L, about 100 mmol / L, about 110 mmol / L, about 115 mmol / L, or about 120 mmol / L.

[0062] 2.6 Glycine In some embodiments of the present disclosure, the formulation (optionally) includes glycine as a protein stabilizer at a concentration of 50 to 75 mmol / L, e.g., about 50 mmol / L to about 73 mmol / L, about 50 mmol / L to about 70 mmol / L, about 50 mmol / L to about 67 mmol / L, about 50 mmol / L to about 64 mmol / L, about 50 mmol / L to about 60 mmol / L, about 50 mmol / L to about 55 mmol / L, about 50 mmol / L, about 55 mmol / L, about 60 mmol / L, about 64 mmol / L, about 67 mmol / L, about 70 mmol / L, or about 75 mmol / L.

[0063] 2.7 Polysorbate 80 In some embodiments of the present disclosure, the formulation (optionally) comprises polysorbate 80 as a surfactant at a concentration of about 0.02% to 0.03% (w / v, g / ml), e.g., about 0.02% to about 0.03%, about 0.02% to about 0.04%, about 0.03% to about 0.04%, about 0.03% to about 0.05%, about 0.04% to about 0.05%, about 0.02%, about 0.03%, about 0.04%, or about 0.05%.

[0064] 2.8 In a preferred embodiment of the present disclosure, the formulation may further comprise an antioxidant. The antioxidant is preferably selected from the group consisting of methionine, sodium sulfite, catalase, and platinum. Most preferably, the antioxidant is methionine.

[0065] 2.9 The concentration of the antioxidant is typically in the range of about 0.01 mg / ml to about 50 mg / ml, about 0.01 mg / ml to about 10.0 mg / ml, about 0.01 mg / ml to about 5.0 mg / ml, about 0.01 mg / ml to about 1.0 mg / ml, or about 0.01 mg / ml to about 0.02 mg / ml. The concentration of the antioxidant is about 0.01 mg / ml, 0.02 mg / ml, 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, about 0.06 mg / ml, about 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, about 0.10 mg / ml, 0.11 mg / ml, 0.12 mg / ml, 0.13 mg / ml, about 0.14 mg / ml, about Desirably, the concentration is 0.15 mg / ml, about 0.16 mg / ml, about 0.17 mg / ml, 0.18 mg / ml, 0.19 mg / ml, about 0.20 mg / ml, about 0.25 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, or 1.0 mg / ml.

[0066] 2.10 In some embodiments of the present disclosure, the formulation comprises methionine at a concentration of 10 mmol / L.

[0067] 2.11 In an embodiment of the disclosure, the antibody molecule is present in an amount of less than 120 mg / ml, preferably in an amount of 100 mg / ml or less.

[0068] In a preferred embodiment of the present disclosure, the concentration of the antibody molecule may range from about 0.1 to about 120 mg / ml. Preferably, the concentration of the antibody is about 0.5 mg / ml, about 1 mg / ml, about 2 mg / ml, about 2.5 mg / ml, about 3 mg / ml, about 3.5 mg / ml, about 4 mg / ml, about 4.5 mg / ml, about 5 mg / ml, about 5.5 mg / ml, about 6 mg / ml, about 6.5 mg / ml, about 7 mg / ml, about 7.5 mg / ml, about 8 mg / ml, about 8.5 mg / ml, about 9 mg / ml, about 9.5 mg / ml. , about 10mg / ml, about 11mg / ml, about 12mg / ml, about 13mg / ml, about 14mg / ml, about 15mg / ml, about 16mg / ml, about 17mg / ml, about 18mg / ml, about 19mg / ml, about 20mg / ml, about 21mg / ml, about 22mg / ml, about 23mg / ml, about 24mg / ml, about 25mg / ml, about 26mg / ml, about 27mg / ml, about 28mg / ml, about 2 9mg / ml, about 30mg / ml, about 31mg / ml, about 32mg / ml, about 33mg / ml, about 34mg / ml, about 35mg / ml, about 36mg / ml, about 37mg / ml, about 38mg / ml , about 39mg / ml, about 40mg / ml, about 41mg / ml, about 42mg / ml, about 43mg / ml, about 44mg / ml, about 45mg / ml, about 46mg / ml, about 47mg / ml, about 48mg / ml, about 49 mg / ml, about 50 mg / ml, about 51 mg / ml, about 52 mg / ml, about 53 mg / ml, about 54 mg / ml, about 55 mg / ml, about 56 mg / ml, about 57 mg / ml, about 58 mg / ml, about 59 mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 110 mg / ml, or about 120 mg / ml.

[0069] 2.12 In some embodiments of the present disclosure, the concentration of the anti-IL-4Rα / IL-5 bispecific antibody is 100 mg / ml.

[0070] 2.13 In some embodiments of the present disclosure, the formulation comprises 100 mg / mL anti-IL-4Rα / IL-5 bispecific antibody, 20 mmol / L sodium acetate, 50 mmol / L glycine, 50 mmol / L arginine hydrochloride, 100 mmol / L sucrose, and 0.05 w / v% polysorbate 80, with a pH of 5.0 to 5.2.

[0071] 2.14 In some embodiments of the present disclosure, the formulation comprises 100 mg / mL anti-IL-4Rα / IL-5 bispecific antibody, 20 mmol / L sodium acetate, 50 mmol / L glycine, 50 mmol / L arginine hydrochloride, 100 mmol / L sucrose, 10 mmol / L methionine, and 0.05 w / v% polysorbate 80, with a pH of 5.0 to 5.2.

[0072] 2.15 In some embodiments of the present disclosure, the formulation comprises 100 mg / mL anti-IL-4Rα / IL-5 bispecific antibody, 10 mmol / L sodium acetate, 50 mmol / L glycine, 50 mmol / L arginine hydrochloride, 120 mmol / L sucrose, and 0.05 w / v% polysorbate 80, with a pH of 5.0 to 5.2.

[0073] 2.16 In some embodiments of the present disclosure, the formulation comprises 100 mg / mL anti-IL-4Rα / IL-5 bispecific antibody, 10 mmol / L sodium acetate, 75 mmol / L glycine, 25 mmol / L arginine hydrochloride, 120 mmol / L sucrose, and 0.05 w / v% polysorbate 80, with a pH of 5.0 to 5.2.

[0074] 2.17 In some embodiments of the present disclosure, the formulation comprises 100 mg / mL anti-IL-4Rα / IL-5 bispecific antibody, 20 mmol / L sodium acetate, 100 mmol / L arginine hydrochloride, 80 mmol / L sucrose, and 0.02 w / v% polysorbate 80, with a pH of 5.0 to 5.2.

[0075] 2.18 In some embodiments of the present disclosure, the formulation further comprises one or more selected from a preservative and / or an additive.

[0076] 2.19 In some embodiments of the present disclosure, the formulation may include a preservative. Preferably, the preservative is selected from phenol, m-cresol, benzyl alcohol, benzalkonium chloride, benzalthonium chloride, phenoxyethanol, and methylparaben. Typically, the concentration of the preservative ranges from about 0.001 mg / ml to about 50 mg / ml, from about 0.005 mg / ml to about 15.0 mg / ml, from about 0.008 mg / ml to about 12.0 mg / ml, or from about 0.01 mg / ml to about 10.0 mg / ml. Preferably, the concentration of the preservative is 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1.0 mg / ml, about 2.0 mg / ml, about 3.0 mg / ml, about 4.0 mg / ml, about 5.0 mg / ml, about 6.0 mg / ml, about 7.0 mg / ml, about 8.0 mg / ml, about 9.0 mg / ml, about 9.1 mg / ml, about 9.2 mg / ml, about 9.3 mg / ml, about 9.4 mg / ml, about 9.5 mg / ml, about 9.6 mg / ml, about 9.7 mg / ml, about 9.8 mg / ml, about 9.9 mg / ml, or about 10.0 mg / ml. Most preferably, the concentration of the preservative is about 0.1 mg / ml or 9.0 mg / ml.

[0077] In some embodiments of the present disclosure, the formulation does not include an antioxidant.

[0078] In some embodiments of the present disclosure, the formulation does not contain a preservative.

[0079] 3.1 Viscosity

[0080] Increased viscosity of protein formulations has a negative impact on processability, for example, in the administration of the drug to patients in liquid form. For example, high viscosity makes it difficult for liquid formulations to pass through the gauge of a syringe needle, causing discomfort to the patient. Furthermore, antibody formulations with relatively high concentrations but reasonably low viscosity are desired as a prerequisite for ease of manufacture, storage, and administration. The term "viscosity" as used herein refers to "kinetic viscosity" or "absolute viscosity". Generally, kinetic viscosity is expressed in centistokes (cSt). The SI unit of kinetic viscosity is mm 2 / s, which is equivalent to 1 cSt. The unit of absolute viscosity is centipoise (cP). The SI unit of absolute viscosity is millipascal second (mPa-s), where 1 cP = 1 mPa.s.

[0081] Suitable pharmaceutical formulations according to the present disclosure have a viscosity of less than 16 mPa.s, more preferably less than 7 mPa.s, preferably between 5.7 and 6.1 mPa.s.

[0082] 3.2 Osmotic pressure

[0083] In some embodiments of the present disclosure, the formulations of the present disclosure are isotonic.

[0084] The term "isotonic" means that the subject formulation has approximately the same osmotic pressure as human blood. In embodiments, isotonic formulations of the present disclosure generally have an osmotic pressure of 299 to 331 mOsm. For example, isotonicity can be measured using vapor pressure or freezing point measurement.

[0085] 3.3 Stability

[0086] In some embodiments of the present disclosure, the formulations of the present disclosure are stable for at least 3, 6, 9, 12 months (e.g., at least 24, 30, 36 months) at a temperature of about 2 degrees Celsius to about 25 degrees Celsius (e.g., about 4 degrees Celsius or about 25 degrees Celsius). In some embodiments, the antibody molecule remains intact after storage in the formulation for at least 3, 6, 9, 12 months (e.g., at least 24, 30, 36 months) at a temperature of about 2 degrees Celsius to about 25 degrees Celsius (e.g., about 4 degrees Celsius or about 25 degrees Celsius). For example, the antibody in the formulation retains at least 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or up to 100% of the biological activity, e.g., binding activity, of the antibody molecule after storage at a temperature of about 2 degrees Celsius to about 25 degrees Celsius (e.g., about 4 degrees Celsius or about 25 degrees Celsius). In some embodiments, the formulation comprises no more than 10%, no more than 9%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% high molecular weight (HMW) species after storage at a temperature of about 2 degrees Celsius to about 25 degrees Celsius (e.g., about 4 degrees Celsius or about 25 degrees Celsius) for at least 3, 6, 9, 12 months (e.g., at least 24, 30, 36 months). In another embodiment, the formulation comprises no more than 10%, no more than 9%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% low molecular weight (LMW) species after storage at a temperature of about 2 degrees Celsius to about 25 degrees Celsius (e.g., about 4 degrees Celsius or about 25 degrees Celsius) for at least 3, 6, 9, 12 months (e.g., at least 24, 30, 36 months).

[0087] In yet another embodiment, the formulation contains no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% of acidic species after storage at a temperature of about 2 degrees Celsius to about 25 degrees Celsius (e.g., about 4 degrees Celsius or about 25 degrees Celsius) for at least 3, 6, 9, 12 months (e.g., at least 24, 30, 36 months). In yet another embodiment, the formulation contains no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% of base species after storage at a temperature of about 2 degrees Celsius to about 25 degrees Celsius (e.g., about 4 degrees Celsius or about 25 degrees Celsius) for at least 3, 6, 9, 12 months (e.g., at least 24, 30, 36 months). The HMW, LMW, acidic and basic species in the formulations can be detected using standard techniques such as size exclusion-high performance liquid chromatography (SEC-HPLC) as disclosed herein.

[0088] In some embodiments, upon reconstitution of the lyophilized antibody formulation, the formulation retains at least 80%, 90%, 95% or more of the structure of the antibody molecules compared to the formulation before lyophilization, as derived, for example, by binding assays, bioassays, or the ratio of HMW to LMW species.

[0089] In some embodiments of the present disclosure, the monomer content of the formulation solution does not decrease by more than 5% after storage for 14 days at about 37 degrees Celsius. In the present disclosure, monomer refers to a double-chain antibody molecule.

[0090] In some embodiments of the present disclosure, the monomer content in a sample does not vary by more than 1.5% under conditions of -20 degrees Celsius, 5 degrees Celsius, rocking, freezing and thawing.

[0091] 4.Product

[0092] The liquid formulation may be contained in an article of manufacture such as a device, syringe, vial, etc., with instructions for use. In certain embodiments, the syringe or vial is made of glass, plastic, or a polymeric material such as a cyclic olefin polymer or copolymer. In other embodiments, the formulation may be present in an injectable device.

[0093] For example, the syringe is a pre-filled syringe.The syringe can be adapted for individual administration, for example, as an auto-injector (e.g., a pen-type injector) and / or a single vial system with instructions.Subject (e.g., patient) can be administered this formulation by injection (e.g., peripheral injection).

[0094] In some embodiments of the present disclosure, a container is provided comprising the stable bispecific antibody formulation.

[0095] In some embodiments of the present disclosure, the container comprises a syringe or a bottle made of either glass, plastic, or polymeric materials.

[0096] In some embodiments of the present disclosure, a sealed package having either the stable bispecific antibody formulation or a container is provided.

[0097] In some embodiments of the present disclosure, a delivery system is provided that includes the stable antibody formulation and is selected from a disposable syringe.

[0098] In some embodiments of the present disclosure, a medicament for the treatment of a disease is provided comprising the stable antibody formulation.

[0099] 5. Application to diseases and treatment methods

[0100] Some embodiments of the present disclosure provide for the use of a stable antibody formulation in the preparation of a medicament for the treatment of diseases, including asthma, allergic dermatitis, eczema, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scars, chronic obstructive pulmonary disease, atopic dermatitis, idiopathic pulmonary fibrosis, Kawasaki disease, sickle cell disease, Graves' disease, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, kidney disease, autoimmune diseases, adult eosinophilic granulomatosis with polyangiitis, severe eosinophilic asthma, Includes hypereosinophilic syndrome, chronic sinusitis with nasal polyps, chronic obstructive pulmonary disease, uncontrolled chronic sinusitis without nasal polyps, allergic fungal sinusitis, prurigo nodularis, familial cold urticaria, bullous pemphigoid, allergic fungal rhinosinusitis, chronic cold urticaria, peanut allergy, pollen allergy, eosinophilic esophagitis, keloids, pruritus, allergic bronchopulmonary aspergillosis, and seasonal allergic rhinitis.

[0101] In some embodiments of the present disclosure, the medicament is a medicament administered via intravenous or subcutaneous route.

[0102] Some embodiments of the present disclosure provide methods for treating diseases using any of the antibody formulations, including but not limited to asthma, allergic dermatitis, eczema, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scars, chronic obstructive pulmonary disease, atopic dermatitis, idiopathic pulmonary fibrosis, Kawasaki disease, sickle cell disease, Graves' disease, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, kidney disease, autoimmune diseases, treatment of adult eosinophilic granulomatosis with polyangiitis, severe eosinophilic asthma, Includes hypereosinophilic syndrome, chronic sinusitis with nasal polyps, chronic obstructive pulmonary disease, uncontrolled chronic sinusitis without nasal polyps, allergic fungal sinusitis, prurigo nodularis, familial cold urticaria, bullous pemphigoid, allergic fungal rhinosinusitis, chronic cold urticaria, peanut allergy, pollen allergy, eosinophilic esophagitis, keloids, pruritus, allergic bronchopulmonary aspergillosis, and seasonal allergic rhinitis.

[0103] Some embodiments of the present disclosure provide the use of glycine in the preparation of a stable antibody formulation, wherein the antibody molecule is stable even when the concentration of the antibody molecule reaches 100 mg / ml in the antibody formulation.

[0104] Some embodiments of the present disclosure provide the use of antibody formulations that improve the stability of high concentrations of antibodies in liquid formulations.

[0105] 6. Preparation Process

[0106] The present disclosure provides a method for preparing a stable antibody formulation, comprising step 1 of preparing an antibody molecule, and step 2 of mixing a therapeutically effective amount of the antibody molecule with a tonicity agent, a buffering agent, a surfactant, and / or other necessary ingredients to form a stable solution. In another aspect, a method or process for preparing the formulation described in the present disclosure is described. The method or process includes expressing the antibody molecule in cell culture, purifying the antibody molecule, e.g., by passing the antibody molecule through at least one of a chromatography or other similar purification step, an ultrafiltration / diafiltration step, and adjusting the concentration of the antibody molecule to, e.g., about 10-100 mg / mL using a formulation buffer agent and adjusting the pH of the formulation to about 4.5-5.3, e.g., 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, or 5.3.

[0107] definition

[0108] The term "protein formulation" or "antibody formulation" refers to a formulation in which the biological activity of the active ingredient is demonstrably effective and which does not contain additional ingredients that are toxic to the subject with which the formulation is administered.

[0109] A "medically acceptable" excipient (vehicle, additive) is one that can be reasonably administered to a mammal to obtain an effective amount of the active ingredient. For example, acceptability for injection is also related to the concentration of the excipient.

[0110] A "stable" formulation refers to one in which the protein contained therein substantially retains its physical and / or chemical stability and / or biological activity during storage. Various analytical techniques for measuring protein stability available to those skilled in the art are described, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. Furthermore, the formulation is stable after freezing (e.g., to -80°C) and thawing the product.

[0111] In a biopharmaceutical formulation, a protein "retains physical stability" means that aggregation, precipitation, and / or denaturation do not change significantly as measured by visual observation of color and / or clarity, by UV light scattering (to measure visible aggregates), or by size exclusion chromatography (SEC). SEC can be used to measure soluble aggregates that are not necessarily precursors to visible aggregates. A protein "retains chemical stability" in a biopharmaceutical formulation if the chemical stability of the protein at a given time is such that the protein is considered to retain biological activity, as defined below. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Chemical changes may include size changes (e.g., clipping). This can be assessed using SEC, SDS-PAGE, and / or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Other types of chemical changes include charge changes (e.g., occurring as a result of deamidation). This can be assessed, for example, by ion exchange chromatography.

[0112] An antibody "retains its biological activity" in a pharmaceutical formulation if the change in the biological activity of the antibody at any given time is within about 10% (within assay error) of the biological activity exhibited when the pharmaceutical formulation was prepared as derived, for example, in an antigen binding assay. Additional assays for the "biological activity" of antibodies are described in more detail below.

[0113] Single domain antibodies (sdAbs, also called Nanobodies or VHHs by its developer Ablynx) are well known to those skilled in the art. Single domain antibodies are antibodies whose complementarity determining regions are part of a single domain polypeptide. Single domain antibodies therefore have a single complementarity determining region (CDR)1, a single CDR2 and a single CDR3. Examples of single domain antibodies include antibodies with only heavy chains (i.e., antibodies that do not naturally contain light chains), single domain antibodies derived from traditional antibodies, and engineered antibodies.

[0114] Single domain antibodies can be derived from any species, including mouse, human, camel, llama, goat, rabbit, and cow. For example, naturally occurring VHH molecules can be derived from camelids, such as camel, llama, dromedary, alpaca, and guanaco. Like intact antibodies, single domain antibodies can selectively bind to a specific antigen. Single domain antibodies may contain only the variable domain of an immunoglobulin chain with CDR1, CDR2, CDR3, and framework regions.

[0115] The term "sequence homology" in the present disclosure refers to the degree to which two (nucleic acid or amino acid) sequences have identical residues at the same positions in an alignment, often expressed as a percentage. Preferably, the homology is derived over the entire length of the sequences being compared. Thus, two copies of the exact same sequence have 100% homology.

[0116] In the present disclosure, bispecific antibodies can also be obtained from sequences having high homology to the sequences of CDRs 1 to 3 shown in the present disclosure. In some embodiments, sequences having "at least 80% homology," "at least 85% homology," "at least 90% homology," "at least 95% homology," or "at least 98% homology" to the sequences set forth in SEQ ID NOs: 1 to 6 can also achieve the objectives of the present disclosure.

[0117] In some embodiments, the object of the present disclosure can be achieved even with a sequence in which only one or several amino acids are replaced by other amino acids (e.g., including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid replacements) compared to the above sequence. These mutation forms include, but are not limited to, deletion, insertion, and / or substitution of one or more amino acids (typically 1 to 50, preferably 1 to 30, more preferably 1 to 20, and most preferably 1 to 10), and addition of one or several amino acids (typically 20 or less, preferably 10 or less, more preferably 5 or less) at the C-terminus and / or N-terminus. In fact, a person skilled in the art may consider so-called "conservative" amino acid replacements when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2, and CDR3 of a single domain antibody. In the case of replacement, it is preferable that the replacement is as conservative as possible. Conservative amino acid substitution can generally be described as an amino acid residue that is replaced by another amino acid residue that has a similar chemical structure and has little or no effect on the function, activity, or other biological properties of a polypeptide. Conservative amino acid substitution is commonly used in the art, for example, conservative amino acid substitution is a substitution in which one or several amino acids in the following groups (a) to (d) are replaced by another or several amino acid residues in the same group: (a) polar, negatively charged residues and their uncharged amides: Asp, Asn, Glu and Gln, (b) polar, positively charged residues: His, Arg, Lys, (c) aromatic residues: Phe, Tyr, Trp, (d) small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. Particularly preferred conservative amino acid substitutions are as follows:Asp to Glu, Asn to Gln or His, Glu to Asp, Gln to Asn, His to Asn or Gln, Arg to Lys, Lys to Arg, Gln, Phe to Met, Leu, Tyr, Trp to Tyr, Tyr to Phe, Trp, Ala to Gly or Ser, Ser to Thr, Thr to Ser, Gly to Ala or Pro, Met to Leu, Tyr or Ile, Leu to Ile or Val, Ile to Leu or Val, Val to Ile or Leu, Cys to Ser. Furthermore, those skilled in the art know that the inventiveness of single domain antibodies is reflected in the CDR1-3 regions, and the framework sequences FR1-4 are not invariant. Conservative sequence variants of the sequences disclosed in this disclosure can be adopted for the sequences of FR1-4.

[0118] The present disclosure relates to the following aspects:

[0119] 1. A stable antibody formulation comprising a therapeutically effective amount of an antibody molecule, an isotonicity agent, a buffering agent, and a surfactant, wherein the pH of the formulation is between 4.5 and 5.5, and the antibody molecule is capable of specifically binding to both IL-5 and IL-4Rα, the stable antibody formulation comprising at least two immunoglobulin variable domains having a first immunoglobulin variable domain capable of specifically binding to the IL-4Rα molecule and a second immunoglobulin variable domain capable of specifically binding to IL-5.

[0120] 2. The antibody formulation according to item 1, wherein the pH of the formulation is 4.7 to 5.3.

[0121] 3. The antibody formulation of item 1, wherein the first immunoglobulin variable domain is a first single domain antibody molecule having CDR1 set forth in SEQ ID NO:1, CDR2 set forth in SEQ ID NO:2, and CDR3 set forth in SEQ ID NO:3, and the second immunoglobulin variable domain is a second single domain antibody molecule having CDR1 set forth in SEQ ID NO:4, CDR2 set forth in SEQ ID NO:5, and CDR3 set forth in SEQ ID NO:6.

[0122] 4. The antibody formulation according to item 3, wherein the antibody molecule is a polypeptide fusion without a linker group between the first and second single domain antibody molecules.

[0123] 5. The antibody formulation according to item 3, wherein the antibody molecule is a polypeptide fusion, and the first and second single domain antibody molecules of the polypeptide fusion are ligated via a biocompatible polymer as a linker group.

[0124] 6. The antibody preparation according to item 3, wherein the first and second single domain antibody molecules are ligated via the human IgG Fc region, the antibody molecules having a homodimeric structure and designated 4E9V10-2B3V2, and each single-chain Fc fusion protein consists of a first immunoglobulin variable domain, a human IgG Fc region, and a second immunoglobulin variable domain.

[0125] 7. The antibody formulation according to item 6, wherein the human IgG Fc region is derived from human IgG1, IgG2, IgG3, or IgG4.

[0126] 8. The antibody formulation according to item 6, wherein the human IgG Fc region is derived from a human IgG4 region, and the Fc region has the amino acid sequence set forth in SEQ ID NO: 13 or is located at positions 116 to 356 of SEQ ID NO: 9.

[0127] 9. The antibody formulation according to item 1, wherein the first immunoglobulin variable domain has the amino acid sequence set forth in SEQ ID NO:7 and the second immunoglobulin variable domain has the amino acid sequence set forth in SEQ ID NO:8.

[0128] 10. A stable antibody formulation comprising a therapeutically effective amount of an antibody molecule, an isotonicity agent, a buffering agent, and a surfactant, wherein the pH of the formulation is 4.5 to 5.5, and the antibody molecule comprises a fusion protein having an amino acid sequence set forth in SEQ ID NO: 9 or 10.

[0129] 11. The antibody formulation according to item 1, wherein the buffering agent is acetic acid-sodium acetate buffer, the isotonicity agent is sucrose, and the surfactant is polysorbate.

[0130] 12. The antibody formulation according to item 11, further comprising a bulking agent comprising glycine and arginine hydrochloride.

[0131] 13. The antibody formulation according to item 11, wherein the concentration of acetic acid-sodium acetate is 10 to 20 mmol / L, and / or the concentration of arginine hydrochloride is 25 to 100 mmol / L, and / or the concentration of sucrose is 80 to 120 mmol / L, and / or the concentration of glycine is 50 to 75 mmol / L, and / or the concentration of polysorbate 80 is 0.02 to 0.05 w / v%.

[0132] 14. The antibody formulation according to item 1, further comprising methionine.

[0133] 15. The antibody formulation according to item 14, further comprising methionine at a concentration of 10 mmol / L.

[0134] 16. The antibody formulation of item 1, wherein the antibody molecules are present in an amount of less than 120 mg / mL.

[0135] 17. The antibody formulation according to item 1, wherein the concentration of the antibody molecule is from about 0.01 to about 100 mg / ml.

[0136] 18. The antibody formulation according to item 1, comprising 100 mg / mL anti-IL-4Rα / IL-5 bispecific antibody, 20 mmol / L sodium acetate, 50 mmol / L glycine, 50 mmol / L arginine hydrochloride, 100 mmol / L sucrose, and 0.05 w / v% polysorbate 80, and having a pH of 5.0 to 5.2.

[0137] 19. The antibody formulation according to item 2, comprising 100 mg / mL anti-IL-4Rα / IL-5 bispecific antibody, 20 mmol / L sodium acetate, 50 mmol / L glycine, 50 mmol / L arginine hydrochloride, 100 mmol / L sucrose, 10 mmol / L methionine, and 0.05 w / v% polysorbate 80, and having a pH of 5.0 to 5.2.

[0138] 20. The antibody formulation according to item 1, comprising 100 mg / mL of anti-IL-4Rα / IL-5 bispecific antibody, 10 mmol / L of sodium acetate, 50 mmol / L of glycine, 50 mmol / L of arginine hydrochloride, 120 mmol / L of sucrose, and 0.05 w / v% polysorbate 80, and having a pH of 5.0 to 5.2.

[0139] 21. The antibody formulation according to item 1, comprising 100 mg / mL anti-IL-4Rα / IL-5 bispecific antibody, 10 mmol / L sodium acetate, 75 mmol / L glycine, 25 mmol / L arginine hydrochloride, 120 mmol / L sucrose, and 0.05 w / v% polysorbate 80, and having a pH of 5.0 to 5.2.

[0140] 22. The antibody formulation according to item 1, comprising 100 mg / mL of anti-IL-4Rα / IL-5 bispecific antibody, 20 mmol / L of sodium acetate, 100 mmol / L of arginine hydrochloride, 80 mmol / L of sucrose, and 0.02 w / v% polysorbate 80, and having a pH of 5.0 to 5.2.

[0141] 23. The antibody formulation according to item 1, further comprising one or more selected from preservatives and / or additives.

[0142] 24. The antibody formulation according to item 1, wherein the monomer content of the formulation does not decrease by more than 5% after storage for 14 days at about 37 degrees Celsius.

[0143] 25. The antibody preparation according to item 1, wherein the monomer content in the sample does not change by more than 1.5% under conditions of -20 degrees Celsius, 5 degrees Celsius, rocking, or freezing and thawing.

[0144] 26. A container comprising a stable antibody formulation according to items 1 to 25.

[0145] 27. The container according to item 26, having a syringe or bottle made of any one or more of glass, plastic, or polymeric materials.

[0146] 28. A sealed package having the antibody formulation according to any of items 1 to 25 or the container according to item 26.

[0147] 29. A kit comprising the antibody preparation according to any one of items 1 to 25 and a container containing the antibody preparation according to any one of items 1 to 25.

[0148] 30. An antibody preparation according to any one of items 1 to 25 or a kit according to item 29, which is used for preparing a medicament for the prevention and / or treatment of an autoimmune disease.

[0149] 31. Use of an antibody preparation according to any one of items 1 to 25 for preparing a medicine for treating a disease, wherein the disease is asthma, allergic dermatitis, eczema, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scar, chronic obstructive pulmonary disease. The use of a stable antibody preparation is for the treatment of atopic dermatitis, idiopathic pulmonary fibrosis, Kawasaki disease, sickle cell disease, Graves' disease, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, kidney disease, autoimmune disease, adult eosinophilic granulomatosis with polyangiitis, severe eosinophilic asthma, hypereosinophilic syndrome, chronic sinusitis with nasal polyps, chronic obstructive pulmonary disease, uncontrolled chronic sinusitis without nasal polyps, allergic fungal sinusitis, prurigo nodularis, familial cold urticaria, bullous pemphigoid, allergic fungal rhinosinusitis, chronic cold urticaria, peanut allergy, pollen allergy, eosinophilic esophagitis, keloid, pruritus, allergic bronchopulmonary aspergillosis, and seasonal allergic rhinitis.

[0150] 32. The use according to item 31, wherein the medicament is a medicament administered intravenously or subcutaneously.

[0151] 33. A method for treating a disease using the antibody preparation according to any one of items 1 to 25, wherein the disease is selected from the group consisting of asthma, allergic dermatitis, eczema, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scar, chronic obstructive pulmonary disease, atopic dermatitis, idiopathic pulmonary fibrosis, Kawasaki disease, sickle cell disease, Graves' disease, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, kidney disease, autoimmune disease, and adult eosinophilic polyangiitis. The method is for treating granulomatous disease, severe eosinophilic asthma, hypereosinophilic syndrome, chronic sinusitis with nasal polyps, chronic obstructive pulmonary disease, uncontrolled chronic sinusitis without nasal polyps, allergic fungal sinusitis, prurigo nodularis, familial cold urticaria, bullous pemphigoid, allergic fungal rhinosinusitis, chronic cold urticaria, peanut allergy, pollen allergy, eosinophilic esophagitis, keloid, pruritus, allergic bronchopulmonary aspergillosis, and seasonal allergic rhinitis.

[0152] 34. A delivery system comprising the stable antibody formulation according to any one of claims 1 to 25, wherein the delivery system is selected from a disposable syringe.

[0153] 35. A method for preparing an antibody formulation according to any one of items 1 to 25, comprising step 1 of preparing an antibody molecule, and step 2 of mixing a therapeutically effective amount of the antibody molecule with a tonicity adjusting agent, a buffer agent, a surfactant and / or other necessary ingredients to form a stable solution.

[0154] 36. A pharmaceutical product for treating a disease, comprising the stable antibody formulation according to any one of items 1 to 25, the disease being selected from the group consisting of asthma, allergic dermatitis, eczema, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scar, chronic obstructive pulmonary disease, atopic dermatitis, idiopathic pulmonary fibrosis, Kawasaki disease, sickle cell disease, Graves' disease, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, kidney disease, autoimmune diseases, adult polyangiitis, and idiopathic pulmonary fibrosis. This medicine is for the treatment of eosinophilic granulomatosis, severe eosinophilic asthma, hypereosinophilic syndrome, chronic sinusitis with nasal polyps, chronic obstructive pulmonary disease, uncontrolled chronic sinusitis without nasal polyps, allergic fungal sinusitis, prurigo nodularis, familial cold urticaria, bullous pemphigoid, allergic fungal rhinosinusitis, chronic cold urticaria, peanut allergy, pollen allergy, eosinophilic esophagitis, keloid, pruritus, allergic bronchopulmonary aspergillosis, and seasonal allergic rhinitis.

[0155] 37. Use of glycine in the preparation of an antibody formulation according to any one of items 1 to 25, wherein the antibody molecule is stably present even when the concentration of the antibody molecule reaches 100 mg / ml in the antibody formulation.

[0156] 38. Use of an antibody formulation according to any one of items 1 to 25, which improves the stability of a high-concentration antibody in a liquid formulation.

[0157] The antibody formulation of the present disclosure can specifically bind to both IL-4Rα and IL-5, compared to existing technologies. The concentration of antibody molecules in the formulation during storage is about 0.01 mg / ml to 120 mg / ml. Within this concentration range, especially at high antibody concentrations (e.g., 100 mg / ml), the formulation maintains physical and chemical stability and remains intact. The monomer content of the formulation solution does not decrease by more than 5% after storage for 14 days at about 37 degrees Celsius. Under conditions of -20 degrees Celsius, 5 degrees Celsius, rocking, freezing, and thawing, the monomer content in the sample does not fluctuate by more than 1.5%, meeting the application requirements of high-concentration antibody formulations. [Brief description of the drawings]

[0158] [Figure 1] 1 shows the results of an assay (ELISA) for the IL-4Rα / IL-5 bispecific antibody 4E9V10-2B3V2-1, which blocks the binding of IL-4 from IL-4Rα.

[0159] [Diagram 2] 1 shows the results of an assay (ELISA) for the IL-4Rα / IL-5 bispecific antibody 4E9V10-2B3V2-1, which blocks binding of IL-5 from the IL-5R.

[0160] [Diagram 3] 1 shows the results of an assay for IL-4Rα / IL-5 bispecific antibody 4E9V10-2B3V2-1, which neutralizes IL-4-induced TF-1 proliferation.

[0161] [Figure 4] 1 shows the results of an assay for IL-4Rα / IL-5 bispecific antibody 4E9V10-2B3V2-1, which neutralizes IL-13-induced TF-1 proliferation.

[0162] [Diagram 5] 1 shows the results of an assay for IL-4Rα / IL-5 bispecific antibody 4E9V10-2B3V2-1, which neutralizes IL-5-induced TF-1 proliferation.

[0163] [Figure 6] Dose effect curve of sample B (4E9V10-2B3V2-2) neutralizing IL-4 and IL-5 (4:1) mixture induced TF-1 proliferation.

[0164] [Figure 7] Dose effect curve of sample B (4E9V10-2B3V2-2) neutralizing IL-4 and IL-5 (4:1) mixture induced TF-1 proliferation.

[0165] [Figure 8] Dose effect curve of sample B (4E9V10-2B3V2-2) neutralizing IL-4 and IL-5 (3:1) mixture induced TF-1 proliferation.

[0166] [Figure 9] Dose effect curve of sample B (4E9V10-2B3V2-2) neutralizing IL-4 and IL-5 (3:1) mixture induced TF-1 proliferation.

[0167] [Figure 10] Dose effect curve of sample B (4E9V10-2B3V2-2) neutralizing IL-4 and IL-5 (2:1) mixture induced TF-1 proliferation.

[0168] [Figure 11] Dose effect curve of sample B (4E9V10-2B3V2-2) neutralizing IL-4 and IL-5 (2:1) mixture induced TF-1 proliferation.

[0169] [Figure 12] Dose effect curve of sample B (4E9V10-2B3V2-2) neutralizing IL-4 and IL-5 (1:1) mixture induced TF-1 proliferation.

[0170] [Figure 13] Dose effect curve of sample B (4E9V10-2B3V2-2) neutralizing IL-4 and IL-5 (1:1) mixture induced TF-1 proliferation.

[0171] [Figure 14] Dose effect curve of sample B (4E9V10-2B3V2-2) neutralizing IL-4 and IL-5 (1:2) mixture induced TF-1 proliferation.

[0172] [Figure 15] Dose effect curve of sample B (4E9V10-2B3V2-2) neutralizing IL-4 and IL-5 (1:2) mixture induced TF-1 proliferation.

[0173] [Figure 16] Dose effect curve of sample B (4E9V10-2B3V2-2) neutralizing IL-4 and IL-5 (1:3) mixture induced TF-1 proliferation.

[0174] [Figure 17] Dose effect curve of sample B (4E9V10-2B3V2-2) neutralizing IL-4 and IL-5 (1:3) mixture induced TF-1 proliferation.

[0175] [Figure 18] Dose effect curve of sample B (4E9V10-2B3V2-2) neutralizing IL-4 and IL-5 (1:4) mixture induced TF-1 proliferation.

[0176] [Figure 19] Dose effect curve of sample B (4E9V10-2B3V2-2) neutralizing IL-4 and IL-5 (1:4) mixture induced TF-1 proliferation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0177] The present disclosure will be described in more detail below with reference to examples, so that those skilled in the art can practice the present disclosure according to the description.

[0178] Example 1

[0179] Construction of Fc-fusion antibody eukaryotic expression vector for anti-IL-4Rα / IL-5 bispecific single domain antibody

[0180] About 4E9V10-2B3V2-1

[0181] (1) The codon-optimized gene sequence (positions 1 to 345 of SEQ ID NO: 11) of anti-IL-4Rα humanized single domain antibody (4E9V10) and the codon-optimized gene sequence (positions 1069 to 1434 of SEQ ID NO: 11) of anti-IL-5 humanized single domain antibody (named 2B3V2) were synthesized by sequence synthesis and inserted into the vector RJK-V4-3 (obtained according to the method disclosed in Chinese patent application no. CN202010576200.7 "ANTI-IL-4Rα SINGLE-DOMAIN ANTIBODY AS WELL AS APPLICATION AND DRUG"), respectively.

[0182] The vector RJK-V4-3 was obtained by the Applicant by fusing the Fc segment of the heavy chain coding sequence of human IgG4 based on the commercial vector pCDNA3.4 by Invitrogen (information about this vector is available at the following website: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf), i.e. a vector containing the hinge region, CH2 and CH3 regions of the IgG4 heavy chain. The procedure was carried out as follows:

[0183] (1) The restriction sites XbaI and AgeI on pcDNA3.4 were selected.

[0184] (2) A multiple cloning site (MCS) and a 6×His tag were introduced at the 5′ and 3′ ends of the coding sequence of the Fc fragment, respectively, via overlap PCR.

[0185] (3) The above fragment was amplified by PCR using a primer pair containing XbaI and AgeI restriction sites.

[0186] (4) pcDNA3.4 and the recombinant DNA fragment of (3) were digested with restriction endonucleases XbaI and AgeI, respectively.

[0187] (5) The digested vector was ligated with the insert fragment under the action of T4 ligase, and the ligated product was transformed into E. coli and amplified, and sequence analysis was performed for verification to obtain the recombinant plasmid.

[0188] The Fc segment of the heavy chain coding sequence of the fusion human IgG4 has the nucleotide sequence as shown in positions 346 to 1068 of SEQ ID NO:11.

[0189] (2) The constructed recombinant eukaryotic expression vector was transformed into DH5α E. coli, cultured, and subjected to endotoxin removal by plasmid maxiprep extraction.

[0190] (3) The extracted plasmids were sequenced again to identify them.

[0191] (4) After identification, the eukaryotic expression vector of anti-IL-4Rα / IL-5 bispecific single domain antibody was constructed as follows: the eukaryotic expression vector where the sequence of anti-IL-4Rα antibody exists was digested by restriction endonucleases XbaI and BamHI, the digested anti-IL-4Rα antibody was ligated into the eukaryotic expression vector containing the sequence of anti-IL-5 antibody and with the sticky ends of the same restriction endonucleases, and the ligation product was transformed and sequenced for identification. The clones confirmed by sequencing were subjected to plasmid maxiprep extraction to remove endotoxins. The extracted plasmids were sequenced again for identification. The confirmed recombinant vectors were prepared for subsequent transfer and expression into eukaryotic cells.

[0192] The anti-IL-4Rα / IL-5 bispecific antibody designated 4E9V10-2B3V2 (particularly 4E9V10-2B3V2-1) (each single chain of the bispecific antibody has the amino acid sequence set forth in SEQ ID NO.9 and the corresponding gene sequence set forth in SEQ ID NO:11) comprises three segments (4E9V10-Fc segment-2B3V2), where 4E9V10 is located at the amino terminus (positions 1 to 115 of SEQ ID NO.9), the Fc segment is located at positions 116 to 356 of SEQ ID NO.9, and 2B3V2 is located at the carboxyl terminus (positions 357 to 478 of SEQ ID NO.9).

[0193] About 4E9V10-2B3V2-2

[0194] (1) The codon-optimized gene sequence (positions 1 to 345 of SEQ ID NO: 12) of an anti-IL-4Rα humanized single domain antibody (4E9V10) and the codon-optimized gene sequence (positions 1069 to 1434 of SEQ ID NO: 12) of an anti-IL-5 humanized single domain antibody (named 2B3V2) were synthesized by sequence synthesis and inserted into a vector (obtained according to the method disclosed in Chinese Patent Application No. CN202010576200.7 "ANTI-IL-4Rα SINGLE-DOMAIN ANTIBODY AS WELL AS APPLICATION AND DRUG"), respectively.

[0195] The vector here is essentially the same as the previously described RJK-V4-3, except that the nucleotide sequence of the Fc segment of the human IgG4 heavy chain coding sequence to be fused has been replaced with nucleotides at positions 346 to 1068 of SEQ ID NO. 12.

[0196] (2) The constructed recombinant eukaryotic expression vector was transformed into DH5α E. coli, cultured, and endotoxin was removed by plasmid maxiprep extraction.

[0197] (3) The extracted plasmids were sequenced again to identify them.

[0198] (4) After confirmation by identification, the eukaryotic expression vector of anti-IL-4Rα / IL-5 bispecific single domain antibody was constructed as follows: the eukaryotic expression vector where the sequence of anti-IL-4Rα antibody exists was digested by restriction endonucleases XbaI and BamHI, the digested anti-IL-4Rα antibody was ligated into the eukaryotic expression vector containing the sequence of anti-IL-5 antibody and with the sticky ends of the same restriction endonucleases, and the ligated vector was transformed and sequenced for identification. The clone confirmed by sequencing was subjected to plasmid maxiprep extraction to remove endotoxin. The extracted plasmid was sequenced again for identification. The confirmed recombinant vector was prepared for subsequent transfer and expression into eukaryotic cells.

[0199] The anti-IL-4Rα / IL-5 bispecific antibody designated 4E9V10-2B3V2 (particularly 4E9V10-2B3V2-2) (each single chain of the bispecific antibody has the amino acid sequence set forth in SEQ ID NO.10 and the corresponding gene sequence set forth in SEQ ID NO:12) comprises three segments (4E9V10-Fc segment-2B3V2), where 4E9V10 is located at the amino terminus (positions 1 to 115 of SEQ ID NO.10), the Fc segment is located at positions 116 to 356 of SEQ ID NO.10, and 2B3V2 is located at the carboxyl terminus (positions 357 to 478 of SEQ ID NO.10).

[0200] Example 2

[0201] Anti-IL-4Rα / IL-5 bispecific single domain antibodies were expressed in suspension ExpiCHO-S cells according to the assay method described in China Patent No. CN202010576200.7 "ANTI-IL-4RΑ SINGLE-DOMAIN ANTIBODY AS WELL AS APPLICATION AND DRUG". Bispecific antibody 4E9V10-2B3V2-1 (sample A) and bispecific antibody 4E9V10-2B3V2-2 (sample B) were expressed and obtained, respectively.

[0202] Example 3

[0203] Anti-IL-4Rα / IL-5 bispecific single-domain antibodies were expressed in suspension 293F cells based on the assay method described in China Patent No. CN202010576200.7 "Anti-IL-4Rα single-domain antibody as well as application and drug". Bispecific antibody 4E9V10-2B3V2-1 (sample A) and bispecific antibody 4E9V10-2B3V2-2 (sample B) were expressed respectively.

[0204] Example 4

[0205] Anti-IL-4Rα / IL-5 bispecific single domain antibodies were purified based on the assay method described in China Patent No. CN202010576200.7 "ANTI-IL-4Rα SINGLE-DOMAIN ANTIBODY AS WELL AS APPLICATION AND DRUG". Bispecific antibody 4E9V10-2B3V2-1 (sample A) and bispecific antibody 4E9V10-2B3V2-2 (sample B) were obtained, respectively.

[0206] Samples A and B used in the following examples were prepared by expressing the corresponding recombinant vectors in suspension 293F cells and purifying them as described in Example 4.

[0207] Example 5

[0208] Blocking assay of receptor-ligand binding by bispecific antibody 4E9V10-2B3V2-1 (Sample A)

[0209] (1) Receptor protein (IL-4Rα or IL-5R) was diluted to 1 μg / ml in protein dilution buffer and coated onto plates overnight at 4°C.

[0210] (2) The plates were washed and blocked with 5% skim milk at 37 degrees Celsius.

[0211] (3) Biotin-bound ligand protein (IL-4 or IL-5) was diluted 2-fold relative to its EC80 concentration, and the antibody was diluted 2-fold relative to the initial concentration, followed by 5-fold gradient dilution. The ligand protein and diluted antibody (dupilumab, 4E9V10-2B3V2-1, 4E9-V0 or 2B3, reslizumab) and hIgG were transferred 1:1 to a new dispensing plate, respectively, and mixed well. 4E9V0 is the antibody 4E9 described in Chinese patent application number CN202010576200.7 "ANTI-IL-4RΑ SINGLE-DOMAIN ANTIBODY AS WELL AS APPLICATION AND DRUG". Also, 2B3 is a non-humanized single domain antibody 2B3 described in Chinese Patent Application No. CN202010843501.1 "IL-5 BINDING MOLECULES AND PREPARATION METHODS AND APPLICATIONS THEREOF". The reslizumab and dupilumab used in this example and Examples 6 to 8 are total antibodies produced by the applicant. The reslizumab used in this example and Examples 6 to 8 was prepared based on the method described in Chinese Patent Application No. CN202010843501.1 "IL-5 BINDING MOLECULES AND PREPARATION METHODS AND APPLICATIONS THEREOF". The dupilumab used in this example and Examples 6 to 8 was prepared based on the method described in Chinese Patent Application No. CN202010576200.7 "ANTI-IL-4RΑ SINGLE-DOMAIN ANTIBODY AS WELL AS APPLICATION AND DRUG".

[0212] (4) The plate was washed and the diluted ligand protein and antibody mixture was transferred in duplicate to the ELISA plate and incubated at 37 degrees Celsius.

[0213] (5) The plate was washed, diluted streptavidin [HRP] was added, and the mixture was incubated at 37 degrees Celsius.

[0214] (6) The plates were washed and single component TMB was added for color development at room temperature in the dark.

[0215] (7) After adding the stopping solution, the OD450 was immediately read using a microplate reader and plotted to calculate the EC50. 4E9-V0 2B3 is a non-humanized anti-IL-5 single domain antibody, and dupilumab, reslizumab, and hIgG were used as controls. The results are shown in Tables 1 and 2 and Figures 5 and 6, respectively.

[0216] [Table 1] TIFF2025504063000002.tif15170

[0217] [Table 2] TIFF2025504063000003.tif15170

[0218] The results show that the humanized bispecific antibodies are non-attenuated and have a slight advantage over the non-humanized antibodies in blocking IL-4 / IL-4Rα or IL-5 / IL-5R receptor-ligand binding. Compared to the corresponding commercial drugs, the humanized bispecific antibodies have similar or slightly more advantageous blocking abilities.

[0219] Example 6

[0220] Assay of IL-4- or IL-13-induced TF1 cell proliferation with bispecific antibody 4E9V10-2B3V2-1 (Sample A)

[0221] (1) TF-1 cells were harvested and subcultured 3 to 4 times and plated at 10,000 cells / well in a 96-well plate.

[0222] (2) A solution of 10 μg / mL total antibody or bispecific antibody was prepared and subjected to 5-fold gradient dilution.

[0223] (3) The total antibody (obtained by the method disclosed in Chinese Patent Application No. CN202010576200.7 “ANTI-IL-4Rα SINGLE-DOMAIN ANTIBODY AS WELL AS APPLICATION AND DRUG”) and gradient diluted sample A were mixed 1:1 with IL-4 or IL13 at the EC80 concentration obtained by the proliferation assay disclosed in Chinese Patent Application No. CN202010576200.7 “Anti-IL-4Rα single-domain antibody as well as application and drug” to prepare a mixed solution.

[0224] (4) The solution mixed in the previous step was added to the cell culture wells in the same volume as the cell culture medium.

[0225] (5) After 72 hours of culture, cell viability was detected using a luminescent cell viability assay kit.

[0226] (6) Based on the assay results, the EC50 concentrations of different antibodies for neutralizing IL-4- or IL-13-induced TF-1 cell proliferation were calculated.

[0227] The test results are shown in Tables 3 and 4, and in Figures 3 and 4.

[0228] [Table 3] TIFF2025504063000004.tif15170

[0229] [Table 4] TIFF2025504063000005.tif15170

[0230] Example 7

[0231] Assay of tool antibodies (total antibodies) neutralizing human recombinant IL-5 protein-induced TF1 cell proliferation and proliferation

[0232] A. Human recombinant IL-5 protein induced TF1 cell proliferation

[0233] (1) TF-1 cells were harvested and subcultured 3 to 4 times and plated at 10,000 cells / well in a 96-well plate.

[0234] (2) A solution of human IL-5 protein with a maximum concentration of 500 ng / mL was prepared and diluted 5-fold.

[0235] (3) The IL-5 protein solution was gradient diluted and then added to the cell culture wells in the same volume as the cell culture medium.

[0236] (4) After 72 hours of culture, cell viability was detected using a luminescent cell viability assay kit.

[0237] (5) The EC80 concentration for IL-5-induced TF-1 cell proliferation was calculated based on the assay results, and the calculated result was 2.96 ng / mL.

[0238] B. Assay of total antibody neutralizing human IL-5-induced TF1 cell proliferation

[0239] (1) TF-1 cells were harvested and subcultured 3 to 4 times and plated at 10,000 cells / well in a 96-well plate.

[0240] (2) 10 μg / mL total antibody was prepared and subjected to a 5-fold gradient dilution.

[0241] (3) The gradient-diluted total antibody was mixed 1:1 with IL-5 at the EC80 concentration obtained in the proliferation assay to obtain a mixed solution.

[0242] (4) The mixed solution was added to the cell culture wells in an amount equal to the volume of the cell culture medium.

[0243] (5) After 72 hours of culture, cell viability was detected using a luminescent cell viability assay kit.

[0244] (6) Based on the assay results, the EC50 concentration of total antibodies that neutralized IL-5-induced TF-1 cell proliferation was calculated.

[0245] Example 8

[0246] Assay of humanized bispecific single domain antibody 4E9V10-2B3V2-1 that neutralizes IL-5-induced TF1 cell proliferation

[0247] (1) TF-1 cells were harvested and subcultured 3 to 4 times and plated at 10,000 cells / well in a 96-well plate.

[0248] (2) A solution of 10 μg / mL of total antibody or the 4E9V10-2B3V2-1 antibody was prepared and subjected to 5-fold gradient dilution.

[0249] (3) The gradient-diluted total antibody and 4E9V10-2B3V2-1 antibody were mixed 1:1 with the IL-5 protein at the EC80 concentration obtained in Example 7 to obtain a mixed solution.

[0250] (4) The mixed solution was added to the cell culture wells in an amount equal to the volume of the cell culture medium.

[0251] (5) After 72 hours of culture, cell viability was detected using a luminescent cell viability assay kit.

[0252] (6) The EC50 concentrations of different antibodies neutralizing IL-5-induced TF-1 cell proliferation were calculated based on the assay results, which are shown in Table 5 and Figure 5.

[0253] [Table 5] TIFF2025504063000006.tif15170

[0254] The results show that the humanized bispecific antibodies are non-attenuating and have a slight advantage over the non-humanized antibodies in neutralizing IL-5-induced TF-1 cell proliferation. Compared to the corresponding commercial drugs, the humanized bispecific antibodies have almost the same blocking ability.

[0255] Example 9

[0256] Affinity kinetic assays of the humanized bispecific antibody 4E9V10-2B3V2-1 were carried out as follows.

[0257] (1) Preparation of SD buffer: Dissolve appropriate amounts of bovine serum albumin and Tween 20 in 1 x PBS (pH 7.4) so ​​that the mass (or volume) fractions of bovine serum albumin and Tween 20 were 0.1% and 0.02%, respectively. The above-mentioned 4E9V10-2B3V2-1 antibody was prepared in SD buffer at a concentration of 10 μg / mL.

[0258] (2) Preparation of antigen working solution: First, the antigen was prepared in SD buffer at a concentration of 200 nM, and then a 2-fold gradient dilution was performed. A total of five concentration gradients were set up, with SD buffer as the blank control.

[0259] (3) Preparation of regeneration solution: An appropriate amount of 0.1 M glycine stock solution was diluted 10 times with deionized water and mixed well to obtain the regeneration solution.

[0260] Assay procedure: Octet 96 and data acquisition software were run on a corresponding computer. The underside and sides of the acquisition probe were cleaned with lens tissue soaked with an appropriate amount of 75% ethanol, and the instrument was pre-warmed for more than 15 minutes. Sensor pre-wetting: Before the assay, the sensor was soaked in SD buffer for more than 10 minutes for later use. Then, the machine operation was set as baseline → antibody → baseline → antigen binding → antigen dissociation → sensor regeneration for the execution of the assay. The assay results are shown in Table 6.

[0261] [Table 6] TIFF2025504063000007.tif32170

[0262] The results show that the affinities of bispecific antibodies targeting IL4Rα and IL-5 were determined with the two antigens, respectively, and the corresponding affinities were both less than 1 nM.

[0263] Example 10

[0264] In this experiment, a proliferation neutralization assay was established to measure the activity of 4E9V10-2B3V2-2 (sample B) to neutralize mixed human IL-4 and IL-5 induced TF-1 cell proliferation. For convenience, 4E9V10-2B3V2-2 will be referred to as sample B in the following description.

[0265] [Table 7] TIFF2025504063000008.tif47170

[0266] [Table 8] TIFF2025504063000009.tif42170

[0267] [Table 9] TIFF2025504063000010.tif24170

[0268] How to create a sample

[0269] Test Buffer: Test medium, RPMI-1640 + 10% FBS, stored at 2-8 degrees Celsius for later use.

[0270] Preparation of human IL-4: Human IL-4 was dissolved in sterile ultrapure water at 100 μg / ml. The prepared protein was stored at -80 degrees Celsius for future use.

[0271] Preparation of human IL-5: Human IL-5 was dissolved in sterile ultrapure water at 100 μg / ml. The prepared protein was stored at -80 degrees Celsius for future use.

[0272] 3. Testing methods and procedures

[0273] 3.1 Cell culture (TF-1)

[0274] Cell culture medium: RPMI-1640+10%FBS+2ng / ml GM-CSF, passageway concentration is 3E4 From 7E 5 It was decided.

[0275] 3.2 Preparation of cell suspension: Add an appropriate amount of TF-1 cells in good growth condition to a suspension of 1 × 10 5 From 4 × 10 5 The cells were placed in a 15 ml centrifuge tube at a concentration of cells / ml and centrifuged at 1200 r for 5 min. The supernatant was discarded and the pellet was resuspended in 1-2 ml of assay buffer.

[0276] 3.3 Cell counting and seeding: Count the cells in the resuspended cell suspension and ensure a cell density of 2 × 10 5 Prepare 1000 cells / ml and distribute 50 μl / well or 1 x 10 cells / ml in a 96-well cell culture plate with clear bottom and white border. 4 Cells were seeded per well.

[0277] 3.4 IL-4 and IL-5 were mixed in the following ratios (4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4) to a total protein concentration of 100 μg / ml. The 100 μg / ml mixed protein was diluted in test buffer to 3.11 ng / ml, 2.6928 ng / ml, 3.1532 ng / ml, 5.184 ng / ml, 15.568 ng / ml, 6.84 ng / ml, and 3.9116 ng / ml for later use. Controls or samples were diluted to 4 × 10 μg / ml in test buffer, and then diluted 5-fold in a 9-step concentration gradient, with the 10th well being 0 μg / ml. Each concentration was set up in duplicate. The mixture of IL-4 and IL-5 in each ratio was mixed 1:1 with the diluted sample.

[0278] 3.5 50 μl / well of the above mixed sample was added to the 96-well plate where the cells were plated.

[0279] 3.6 The 96-well plate was placed in a cell culture incubator at 37 degrees Celsius and 5% CO2 and incubated for 72 hours.

[0280] 3.7 Remove the cell plate from the incubator, add 100 μl of CellTiter-Glo to each well, incubate at room temperature in the dark for 15 minutes, and measure the cell viability of each well using a multi-function microplate reader.

[0281] In this assay, dupilumab, mepolizumab, and dupilumab + mepolizumab (mixed 1:1) were used as positive controls, human IgG was used as negative control, and sample B (4E9V10-2B3V2-2) was used as the sample to be tested. Each sample was set up in duplicate. Samples with an initial concentration of 4*10 μg / ml were diluted to 9 concentrations by 5-fold gradient dilution. The last duplicate well of each group was at a concentration of 0 μg / ml. In the 96-well cell plate, the concentration in column 2 was 10 μg / ml, the concentration in column 3 was 2 μg / ml, the concentration in column 4 was 0.4 μg / ml, the concentration in column 5 was 0.08 μg / ml, the concentration in column 6 was 0.016 μg / ml, the concentration in column 7 was 0.0032 μg / ml, the concentration in column 8 was 0.00064 μg / ml, the concentration in column 9 was 0.000128 μg / ml, the concentration in column 10 was 0.0000256 μg / ml, and the concentration in column 11 was 0 μg / ml. IL-4 and IL-5 mixed in the following ratios (4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4) were prepared for later use at 3.11ng / ml, 2.6928ng / ml, 3.1532ng / ml, 5.184ng / ml, 15.568ng / ml, 6.84ng / ml, and 3.9116ng / ml and mixed 1:1 with the samples. Then, the mixture and cells were mixed in a 1:1 volume ratio and cultured in a CO2 incubator (37 degrees Celsius, 5% CO2) for 72 hours. Finally, readings were taken using a multifunctional microplate reader and the data were processed to draw conclusions.

[0282] Using GraphPad Prism, four-parameter nonlinear regression curve fitting was performed to obtain EC50 and dose-effect curves, with molar concentration on the horizontal axis and relative light units (RLU) on the vertical axis. Figures 6-19 illustrate the dose-effect curves.

[0283] 4. Main equipment and reagents

[0284] [Table 10] TIFF2025504063000011.tif33170

[0285] [Table 11] TIFF2025504063000012.tif47170

[0286] 5. Summary of Results

[0287] [Table 12] TIFF2025504063000013.tif216170

[0288] As the results show, when IL4:IL5=4:1, in plate 1, the test window of the positive control dupilumab was 1.77-fold, with an EC50 of 0.8127nM, the test window of mepolizumab was 1.02-fold, with an EC50 of 0.001298nM, and the test window of sample B bivalent antibody was 1.59-fold, with an EC50 of 0.4245nM. In plate 2, the test window of sample B was 1.78-fold, with an EC50 of 0.3212nM, the window of the positive control dupilumab + mepolizumab was 1.68-fold, with an EC50 of approximately 0.8250nM, and the window of the negative control human IgG was 1.01-fold, with an EC50 of approximately 0.1051nM. The fitting curves based on the above data are shown in Figures 6 and 7.

[0289] For IL4:IL5 = 3:1, in plate 3, the test window of the positive control dupilumab was 1.83x, the EC50 was 0.4481nM, the test window of mepolizumab was 1.05x, the EC50 was 0.001322nM, and the test window of the sample B bivalent antibody was 1.53x, the EC50 was 0.2429nM. In plate 4, the test window of the sample B bivalent antibody was 1.60x, the EC50 was 0.2186nM, the window of the positive control dupilumab + mepolizumab was 1.67x, the EC50 was about 0.9652nM, and the window of the negative control human IgG was 1.02x, the EC50 was about 0.0009147nM. The fitting curves based on the above data are shown in Figures 8 and 9.

[0290] For IL4:IL5=2:1, in plate 5, the test window of the positive control dupilumab was 1.60x, the EC50 was 0.4519nM, the test window of mepolizumab was 0.99x, the EC50 was 2.936nM, and the test window of the sample B bivalent antibody was 1.55x, the EC50 was 0.2402nM. In plate 6, the test window of the sample B bivalent antibody was 1.63x, the EC50 was 0.2127nM, the window of the positive control dupilumab + mepolizumab was 1.76x, the EC50 was about 1.279nM, and the window of the negative control human IgG was 0.99x, the EC50 was not calculable. The fitted curves based on the above data are shown in Figures 10 and 11.

[0291] At IL4:IL5=1:1, in plate 7, the test window of the positive control dupilumab was 1.77-fold, with an EC50 of 0.5701nM, the test window of mepolizumab was 1.11-fold, with an EC50 of 14.89nM, and the test window of the sample B bivalent antibody was 1.71-fold, with an EC50 of 0.3692nM. In plate 8, the test window of the sample B bivalent antibody was 1.70-fold, with an EC50 of 0.3709nM, the window of the positive control dupilumab + mepolizumab was 1.92-fold, with an EC50 of approximately 1.733nM, and the window of the negative control human IgG was 1.07-fold, with an EC50 of approximately 14.70nM. The fitting curves based on the above data are shown in Figures 12 and 13.

[0292] For IL4:IL5=1:2, in plate 9, the test window of the positive control dupilumab was 1.68-fold, with an EC50 of 0.8986nM, the test window of mepolizumab was 1.06-fold, with an EC50 of 0.001038nM, and the test window of the sample B bivalent antibody was 1.58-fold, with an EC50 of 0.5947nM. In plate 10, the test window of the sample B bivalent antibody was 1.64-fold, with an EC50 of 0.7358nM, the window of the positive control dupilumab + mepolizumab was 1.62-fold, with an EC50 of approximately 1.960nM, and the window of the negative control human IgG was 0.99-fold, with an EC50 of approximately 0.002641nM. The fitting curves based on the above data are shown in Figures 14 and 15.

[0293] For IL4:IL5=1:3, in plate 11, the test window of the positive control dupilumab was 1.53x, the EC50 was 0.3429nM, the test window of mepolizumab was 1.01x, the EC50 was 0.001468nM, and the test window of the sample B bivalent antibody was 1.50x, the EC50 was 0.1523nM. In plate 12, the test window of the sample B bivalent antibody was 1.74x, the EC50 was 0.1077nM, the window of the positive control dupilumab + mepolizumab was 1.76x, the EC50 was about 0.8805nM, and the window of the negative control human IgG was 0.95x, the EC50 was about 0.001926nM. The fitting curves based on the above data are shown in Figures 16 and 17.

[0294] For IL4:IL5 = 1:4, the test window of the positive control dupilumab was 1.58x, the EC50 was 0.1562nM, the test window of mepolizumab was 1.14x, the EC50 was 1.105nM, and the test window of the sample B bivalent antibody was 1.50x, the EC50 was 0.07883nM on plate 13. On plate 14, the test window of the sample B bivalent antibody was 1.67x, the EC50 was 0.04987nM, the window of the positive control dupilumab + mepolizumab was 1.79x, the EC50 was about 0.3949nM, and the window of the negative control human IgG was 1.03x, the EC50 was about 0. The fitting curves based on the above data are shown in Figures 18 and 19.

[0295] Conclusion: Under the assay conditions described above, in the assay of mixed IL-4 and IL-5 at different ratios, the window of bivalent antibody sample B was comparable to that of dupilumab and mixed dupilumab + mepolizumab, respectively, and the EC50 was significantly lower than that of dupilumab and mixed dupilumab + mepolizumab, respectively. Thus, the neutralizing activity of bivalent antibody sample B was similar to that of dupilumab and superior to that of mixed dupilumab + mepolizumab.

[0296] Example 11

[0297] formulation

[0298] 1. An aqueous solution formulation of the bispecific antibody (4E9V10-2B3V2-2) targeting IL-4Rα and IL-5 prepared in Example 4 was prepared as follows.

[0299] 1.1 Tween-free buffer (10 mM sodium acetate, 120 mM sucrose, 75 mM glycine, 25 mM arginine hydrochloride, pH 5.0) was prepared as follows:

[0300] 1) Sodium acetate, sucrose, glycine, and arginine hydrochloride were accurately weighed according to Table 13, dissolved in approximately 800 ml of ultrapure water, and mixed well.

[0301] 2) Prepare a 50 ml beaker, measure out acetic acid according to Table 13, wash repeatedly with ultrapure water, and transfer all the acetic acid to the solution obtained in step 1 above and mix well.

[0302] 3) The solution was transferred to a 1 L measuring flask and the volume was accurately adjusted, after which the flask was inverted repeatedly to mix well.

[0303] [Table 13] TIFF2025504063000014.tif41170

[0304] 1.2 High concentration Tween stock buffer (10 mM sodium acetate, 120 mM sucrose, 75 mM glycine, 25 mM arginine hydrochloride, 5% polysorbate 80, pH 5.0) was prepared as follows: Sodium acetate is sodium acetate trihydrate.

[0305] a) Sodium acetate, sucrose, glycine, and arginine hydrochloride were accurately weighed according to Table 14, dissolved in approximately 100 ml of ultrapure water, and mixed well.

[0306] b) Prepare a 50 ml beaker, measure out acetic acid according to Table 14, wash repeatedly with ultrapure water, and transfer all the acetic acid into the solution obtained in step a) above and mix well.

[0307] c) Prepare two 50 ml centrifuge tubes and accurately weigh out polysorbate 80 according to Table 14. Using 35 ml of the solution obtained in step b) above, dissolve the polysorbate 80 by vortexing, mix well, wash repeatedly with the solution obtained in step b) above, and transfer all the polysorbate 80 to the solution obtained in step b) above and mix well.

[0308] 3) The solution was transferred to a 250 ml measuring flask and the volume was accurately adjusted, after which the measuring flask was inverted repeatedly to mix well.

[0309] [Table 14] TIFF2025504063000015.tif56170

[0310] 1.3 Formulation buffer (10 mM acetate-sodium acetate, 120 mM sucrose, 75 mM glycine, 25 mM arginine hydrochloride, 0.05% polysorbate 80, pH 5.0) was prepared.

[0311] The following steps were carried out: 1 L of Tween-free buffer prepared in 1.1 and 10.101 ml of high-concentration Tween stock buffer prepared in 1.2 were mixed thoroughly.

[0312] NOTE: Each buffer can be prepared in larger or smaller volumes as needed.

[0313] To prepare the desired formulation, the antibody protein can be added to a formulation buffer to obtain the desired concentration of the formulation.

[0314] 2. The assay method is as follows.

[0315] 2.1 pH measurement

[0316] Measurements were performed according to General Chapter 0631 “Measurement of pH Value” of the Pharmacopoeia of the People’s Republic of China (2015 edition, Part IV).

[0317] 2.2 Protein concentration measurement

[0318] Measurements were performed using a UV spectrophotometer. Pure water was weighed and the samples were diluted. The absorbance of the diluted sample solutions was then measured at 280 nm. Two parallel solutions were prepared for each sample test. The protein content was calculated according to the following formula: C(mg / ml)=A×D / ε, where A is the absorbance value of the solution, D is the dilution factor, and ε is the extinction coefficient: 1.666 (mg / ml). -1 .cm -1 Protein content was expressed as the mean value of two parallel calculations.

[0319] 2.3 Osmolarity (molarity assay)

[0320] Measurements were performed according to General Chapter 0632 “Molar concentration assay for osmotic pressure” of the Pharmacopoeia of the People’s Republic of China (2015 edition, Part IV).

[0321] 2.4 Viscosity

[0322] Protein viscosity was measured using a RheoSense microVISC viscometer.

[0323] 2.5 Purity (SEC-HPLC)

[0324] Measurements were performed using high performance liquid chromatography. Separation was performed using a size exclusion chromatography column with a mobile phase of 50 mmol / L phosphate buffer + 300 mmol / L NaCl + 10% acetonitrile, pH 6.8 (7.8 g NaH2PO4·2H2O and 17.53 g NaCl were weighed and dissolved in 800 ml ultrapure water, mixed well, then 100 ml acetonitrile was added, pH was adjusted to 6.8 with NaOH, volume was increased to 1000 ml, filtered through a 0.22 μm filter membrane, and stored at room temperature). The flow rate was 0.5 ml / min, the detection wavelength was 280 nm, and the column temperature was 25 degrees Celsius. The test sample was diluted to 2 mg / ml with ultrapure water to prepare the test sample solution, and the working standard was diluted to 2 mg / ml with ultrapure water to prepare the system application solution. The sample buffer was diluted with ultrapure water at the same ratio, and the diluted sample buffer was used as the blank solution. 50 μl each of the blank solution, the system applicability solution, and the test sample solution were injected into the liquid chromatograph.

[0325] 2.6 Purity (nrCE-SDS)

[0326] The measurements were performed using capillary gel electrophoresis. Prior to electrophoresis, the capillary column was washed with 0.1 mol / L sodium hydroxide, 0.1 mol / L hydrochloric acid, ultrapure water, and the electrophoresis gel at 70 psi. An appropriate amount of the test sample (containing about 100 μg of protein) was weighed out, and 93 μl of pH 6.0 sample buffer (0.44 g of Na2HPO4·12H2O and 1.36 g of NaH2PO4·2H2O were weighed into a 50 ml centrifuge tube, 90 ml of ultrapure water was added to completely dissolve, and after mixing well, ultrapure water was added to make 100 ml to prepare phosphate buffer. 200 μl of phosphate buffer was accurately weighed out, 100 μl of 10% sodium dodecyl sulfate aqueous solution and 700 μl of ultrapure water were added and mixed well to obtain sample buffer), 2 μl internal standard (10 kDa), and 5 μl 250 mmol / L NEM (31 mg of N-ethylmaleimide was weighed and dissolved in 1 ml of ultrapure water) were added, mixed well, heated at 70 degrees Celsius for 10 minutes, cooled to room temperature, and transferred to a sample vial to prepare the test sample solution. A blank solution was prepared by using the same volume of sample buffer as the test sample and performing the same operation. Sample injection conditions: 5 kV, 20 seconds, separation voltage: 15 kV, 35 minutes. The temperature of the capillary column was controlled at 25 degrees Celsius, and the width of the test window was 100 × 800 μm.

[0327] 2.7 Charge variants (icIEF)

[0328] The measurements were performed using imaging capillary isoelectric focusing (iCIEF). The inner diameter of the capillary is 100 μm and the total length is 5 cm. Before electrophoresis of the sample, the capillary column needs to be washed with 0.5% MC (methylcellulose) solution and ultrapure water. The sample was loaded by vacuum injection for 55 seconds. The prefocus voltage was 1.5 KV for 1 minute, and the focusing voltage was 3 KV for 7 minutes. The temperature of the sample disk was 10 degrees Celsius, the temperature of the capillary column was room temperature, and the detection wavelength was 280 nm. A 500 mmol / L arginine solution was used as a cathode stabilizer, a 10 mol / L urea solution was used to improve the solubility of the protein, and a 0.5% MC solution was used to reduce the adhesion of the protein to the capillary. The concentrated and desalted test samples were diluted to 1.0 mg / ml with ultrapure water. The test sample solution was prepared by mixing 20 μl of diluted (1.0 mg / ml) sample with 35 μl of 1% MC solution, 4 μl of ampholytes (pH 3 to 10), 2 μl of cathodic stabilizer, 3 μl of 10 mol / L urea solution, 34 μl of ultrapure water, 1 μl of pI 6.82 manufacturer, and 1 μl of pI 9.91 manufacturer. Blank and standard solutions were prepared in a similar manner using the sample buffer and working standard solution, respectively.

[0329] 2.8 Binding activity (ELISA method)

[0330] IL-5 antigen at 400μg / ml was diluted to 0.5μg / ml in 1x ELISA coating solution and coated on 96-well plate at 50.0μl / well and left at 2-8 degrees Celsius overnight. The coated ELISA plate was removed and the coating solution was discarded. ELISA wash solution was added at 250μl / well and the plate was left for 30 seconds, after which the ELISA wash solution was discarded. The washes were repeated 4 times. ELISA working solution was added for blocking at 250μl / well and the plate was incubated at room temperature for 2 hours. After incubation, the gradient diluted samples were transferred in duplicate to 96-well plate at 50.0μl / well and incubated at room temperature for 1 hour. Diluted biotinylated human IL-4R at 0.5ug / ml was transferred to 96-well plate at 50.0μl / well and incubated at room temperature for 1 hour. The working solution of the secondary antibody was obtained by diluting streptavidin-HRP in the ELISA working solution 1:10000. The working solution of the secondary antibody was added at 50.0 μl / well and incubated at room temperature for 1 h. The reaction solution in the 96-well plate that reacted with the secondary antibody working solution was discarded. The ELISA washing solution was added at 250 μl / well and then discarded. The washing was repeated four times. The plate was tapped dry with clean absorbent paper. The TMB color development solution was added at 100 μl / well and left at room temperature for 10 min. After color development, 50 μl / well of 2 mol / l H2SO4 was added to the 96-well microplate to stop the color reaction. The absorbance value was read at 450 nm using a microplate reader, with 630 nm as the reference wavelength. The concentration is shown on the horizontal axis, and the OD 450-630 nm A four-parameter curve was fitted using Graphpad Prism to obtain the EC 50 and R 2 was obtained directly. The relative binding activity of the test samples can be calculated according to the following formula (the binding activity results are rounded to the nearest whole number):

[0331] [Number 1] TIFF2025504063000016.tif9170

[0332] 3. Optimizing the additive ratio

[0333] 3.1 Formulation design

[0334] A total of six groups of prescriptions were designed as shown in Table 15.

[0335] [Table 15] TIFF2025504063000017.tif64170

[0336] Protein 4E9V10-2B3V2-2 was concentrated and buffer exchanged into each formulation. After buffer exchange was completed, the concentration was adjusted to a target (4E9V10-2B3V2-2) concentration of approximately 100mg / ml (pH of each formulation with 4E9V10-2B3V2-2 added is listed in column TO of Table 18). After buffer exchange was completed, the remaining samples were placed in 2R medium borosilicate glass injection vials, except for the freeze-thaw test sample, which was placed in a 5ml Flexboy storage bag, and tested according to Table 16.

[0337] [Table 16] TIFF2025504063000018.tif47170

[0338] Note: -: No inspection point T0 is the starting point (zero point or starting point)

[0339] 3.2 Test results

[0340] In the results below, Agt means stirring (650 rpm). Ft-5cyc means five freeze-thaw cycles (-80 degrees Celsius / room temperature).

[0341] (1) Visual inspection results

[0342] Due to obvious stratification in D05 during buffer exchange using ultracentrifuge tubes, this group of formulations was excluded from the study. The appearance results of the other formulations are shown in Table 17. After five freeze-thaw cycles, all formulation samples were clear and colorless in appearance.

[0343] The sample was laid flat inside the bag, which diluted the slight yellow tint of the sample itself.

[0344] The formulations of samples D01, D02, and D03 were all milky white and slightly yellow at time T0 and at various temperatures. The formulation of sample D04 was clear and slightly yellow at time T0 and slightly milky white and slightly yellow at various temperatures. The formulation of sample D06 was strongly milky white and slightly yellow at time T0 and at various temperatures.

[0345] [Table 17] TIFF2025504063000019.tif37170

[0346] Note: 0: transparent and colorless (due to small amount, observed directly in cryopreservation bag without removing sample or placing in penicillin vial). +: transparent and slightly yellow. ++: slightly milky and slightly yellow. +++: milky and slightly yellow. ++++: very milky and slightly yellow.

[0347] (2) pH test results

[0348] The pH results are shown in Table 18. The pH of samples at each test point for each formulation group showed no significant difference compared to T0 (all within the tolerance of ±0.3).

[0349] [Table 18] TIFF2025504063000020.tif42170

[0350] (3) Protein concentration measurement results

[0351] The protein concentration results are shown in Table 19. The protein concentrations of the samples in each group did not show any significant changes compared to point T0 (all within the acceptable range of ±10%).

[0352] [Table 19] TIFF2025504063000021.tif64170

[0353] The viscosity and osmolality results are shown in Table 20. The viscosity for all formulations was in the range of 5.7 to 6.1 mPa.s and the osmolality was in the range of 299 mOsmol / kg to 331 mOsmol / kg.

[0354] [Table 20] TIFF2025504063000022.tif33169

[0355] (4) Purity (SEC-HPLC) test results

[0356] The results of SEC-HPLC (size exclusion high performance liquid chromatography) are shown in Tables 21 to 23. After 14 days of observation with 48 hours of rocking at 25 degrees Celsius, the monomer content decreased compared to T0, but the decrease rate was within 1.0%. Under conditions of -20 degrees Celsius, 5 degrees Celsius, or freeze-thaw testing, the monomer content of the samples in all formulations was essentially the same. Compared to T0, the monomer content changed within 0.2%. The purity measurement results (by SEC-HPLC) showed no significant differences between the components.

[0357] [Table 21]

[0358] TIFF2025504063000023.tif43169

[0359] [Table 22] TIFF2025504063000024.tif45169

[0360] [Table 23] TIFF2025504063000025.tif47169

[0361] (5) Purity (nrCE-SDS) test results

[0362] The results of nrCE-SDS (non-reducing capillary electrophoresis sodium dodecyl sulfate) are shown in Tables 24 to 26. Compared with T0, the main peak content of the formula of samples D01 to D04 under the test conditions fluctuated by less than 1.0%. The main peak content of the formula of sample D06 after the fluctuation test was significantly reduced.

[0363] [Table 24] TIFF2025504063000026.tif45170

[0364] [Table 25] TIFF2025504063000027.tif43170

[0365] [Table 26] TIFF2025504063000028.tif44170

[0366] (6) Charge variant (icIEF) test results

[0367] icIEF is the whole column imaging capillary isoelectric focusing. The charge variant (icIEF) results are shown in Table 27 to Table 29. According to the results in the table, it can be seen that the main peak content value of the formulation of the D04 sample is relatively less variable, so the formulation of D04 can be used as an alternative formulation.

[0368] [Table 27] TIFF2025504063000029.tif46170

[0369] [Table 28] TIFF2025504063000030.tif45170

[0370] [Table 29] TIFF2025504063000031.tif44170

[0371] The purity (SEC-HPLC, nrCE-SDS) results show that the monomer content (SEC-HPLC) and main peak content (nrCE-SDS) fluctuate within 1.0% compared to T0. Also, the charge variant (icIEF) results show that the main peak content of the D04 sample formulation has relatively little variation.

[0372] The formulation of formulation E01(DO4) (10 mmol / L acetic acid-sodium acetate, 75 mmol / L glycine, 25 mmol / L arginine hydrochloride, 120 mmol / L sucrose, 0.05% PS80, pH 5.0, protein concentration 100 mg / ml) was confirmed.

[0373] The validation procedure is shown in Table 30. The protein was concentrated and buffer exchanged into each formulation. After the buffer exchange was completed, the remaining samples were placed in 2R medium borosilicate glass injection vials, except for the freeze-thaw test sample, which was placed in a 5ml Flexboy storage bag, and tested according to Table 30.

[0374] [Table 30] TIFF2025504063000032.tif55170

[0375] Note: No inspection points

[0376] Experimental Results

[0377] (1) Viscosity and osmotic pressure test results

[0378] The viscosity and osmolality results are shown in Table 31. The viscosity was 4.590 mPa.s. The osmolality was 286 mOsmol / kg.

[0379] [Table 31] TIFF2025504063000033.tif16170

[0380] (2) Appearance, pH, and protein concentration test results

[0381] Table 32 shows the results for appearance, pH, and protein concentration. Under each test condition, the appearance was clear and yellow. Due to the difference in sample source, the appearance of the formulation at validation was better than that of the sample of formulation D04 at screening. No significant differences were observed in pH (within the acceptable range of ±0.3) and protein concentration (within the acceptable range of ±10%) under the various different test conditions.

[0382] [Table 32] TIFF2025504063000034.tif43170

[0383] (4) Purity (SEC-HPLC) test results

[0384] The results of the purity (SEC-HPLC) test are shown in Table 33. After 14 days of observation at 37 degrees Celsius, the monomer content decreased by 2.6%. Under conditions of -20 degrees Celsius, 5 degrees Celsius, rocking, freezing and thawing, the monomer content in the samples did not vary by more than 1.5%.

[0385] [Table 33] TIFF2025504063000035.tif46170

[0386] (4) Purity (nrCE-SDS) test results

[0387] The results of the purity (nrCE-SDS) test are shown in Table 34. After 14 days at 37 degrees Celsius, the monomer content decreased by 1.9%. Under conditions of -20 degrees Celsius, 5 degrees Celsius, rocking, freezing, and thawing, the monomer content in the samples did not vary by more than 1.0%.

[0388] [Table 34] TIFF2025504063000036.tif47170

[0389] (5) Charge variant (icIEF) test results

[0390] Table 35 shows the charge variant (icIEF) test results. After 14 days at 37 degrees Celsius, the main peak content decreased by 5.2% compared to T0. This indicates that the content of each component of the charge variant in the sample fluctuated under high temperature conditions. Under conditions of -20 degrees Celsius, 5 degrees Celsius, rocking, freezing, and thawing, the main peak content in the sample did not fluctuate by more than 2.0%.

[0391] [Table 35] TIFF2025504063000037.tif46170

[0392] (6) Binding activity test results

[0393] The results of the binding activity (ELISA) test are shown in Table 36. After the 4E9V10-2B3V2-2 protein was tested at -20 degrees Celsius and 5 degrees Celsius for 30 and 91 days, the binding activity (ELISA) results were all pass (criteria: 70%-130%).

[0394] [Table 36] TIFF2025504063000038.tif20170

[0395] (7) Overview

[0396] The results of all formulations showed that the components showed little change when tested under conditions of -20 degrees Celsius, 5 degrees Celsius, shaking, freezing and thawing, and the binding activity met the pass criteria after 91 days of testing at -20 degrees Celsius and 5 degrees Celsius. This shows that 4E9V10-2B3V2-2 in the formulation containing formulation E01 (D04) shows good stability under conditions of 5 degrees Celsius, -20 degrees Celsius, shaking, freezing and thawing.

[0397] The embodiments of the present disclosure disclosed in the above embodiments are not limited to the examples listed in the description and embodiments. The embodiments can be applied to various fields suitable for the present disclosure. Additional modifications can be easily implemented by those skilled in the art, and the present disclosure is not limited to specific details without departing from the general concept defined by the claims and the scope of equivalents thereof.

Claims

1. A stable antibody formulation comprising a therapeutically effective amount of an antibody molecule, an isotonicity agent, a buffering agent, and a surfactant, wherein the pH of the formulation is between 4.5 and 5.5, and wherein the antibody molecule is capable of specifically binding to both IL-5 and IL-4Rα, the stable antibody formulation comprising at least two immunoglobulin variable domains having a first immunoglobulin variable domain capable of specifically binding to an IL-4Rα molecule and a second immunoglobulin variable domain capable of specifically binding to IL-5.

2. 2. The stable antibody formulation of claim 1, wherein the pH of the formulation is from 4.7 to 5.

3.

3. 2. The stable antibody formulation of claim 1, wherein the first immunoglobulin variable domain is a first single domain antibody molecule having a CDR1 according to SEQ ID NO:1, a CDR2 according to SEQ ID NO:2, and a CDR3 according to SEQ ID NO:3, and the second immunoglobulin variable domain is a second single domain antibody molecule having a CDR1 according to SEQ ID NO:4, a CDR2 according to SEQ ID NO:5, and a CDR3 according to SEQ ID NO:

6.

4. 4. The stable antibody formulation of claim 3, wherein the antibody molecule is a polypeptide fusion that does not include a linker group between the first single domain antibody molecule and the second single domain antibody molecule.

5. The stable antibody formulation of claim 3, wherein the antibody molecule is a polypeptide fusion comprising a biocompatible polymer, and the first single domain antibody molecule and the second single domain antibody molecule of the polypeptide fusion are ligated together using the biocompatible polymer as a linker group.

6. 4. The stable antibody formulation of claim 3, wherein the first and second single domain antibody molecules are ligated via a human IgG Fc region, the antibody molecules having a homodimeric structure and designated 4E9V10-2B3V2, and each single-chain Fc fusion protein consists of a first immunoglobulin variable domain, a human IgG Fc region, and a second immunoglobulin variable domain.

7. The stable antibody formulation of claim 6 , wherein the human IgG Fc region is derived from human IgG1, IgG2, IgG3, or IgG4.

8. 7. The stable antibody formulation of claim 6, wherein the human IgG Fc region is derived from a human IgG4 region, and the Fc region has the amino acid sequence set forth in SEQ ID NO:13 or is located at positions 116 to 356 of SEQ ID NO:

9.

9. 2. The stable antibody formulation of claim 1, wherein the first immunoglobulin variable domain has the amino acid sequence set forth in SEQ ID NO:7 and the second immunoglobulin variable domain has the amino acid sequence set forth in SEQ ID NO:

8.

10. 1. A stable antibody formulation comprising a therapeutically effective amount of an antibody molecule, an isotonicity agent, a buffering agent, and a surfactant, wherein the pH of the formulation is between 4.5 and 5.5, and the antibody molecule comprises a fusion protein having an amino acid sequence as set forth in SEQ ID NO: 9 or 10.

11. The stable antibody formulation of claim 1 or 10, wherein the buffering agent is acetic acid-sodium acetate buffer, the isotonicity agent is sucrose, and the surfactant is polysorbate.

12. 12. The stable antibody formulation of claim 11, further comprising a bulking agent comprising glycine and arginine hydrochloride.

13. 12. The stable antibody formulation of claim 11, wherein the concentration of acetic acid-sodium acetate is 10 to 20 mmol / L, and / or the concentration of arginine hydrochloride is 25 to 100 mmol / L, and / or the concentration of sucrose is 80 to 120 mmol / L, and / or the concentration of glycine is 50 to 75 mmol / L, and / or the concentration of polysorbate 80 is 0.02 to 0.05 w / v%.

14. The stable antibody formulation of claim 1, further comprising methionine.

15. 15. The stable antibody formulation of claim 14, further comprising methionine at a concentration of 10 mmol / L.

16. 2. The stable antibody formulation of claim 1, wherein the antibody molecule is present in an amount of less than 120 mg / mL.

17. 2. The stable antibody formulation of claim 1, wherein the concentration of the antibody molecule is from about 0.01 to about 100 mg / ml.

18. 2. The stable antibody formulation of claim 1, comprising 100 mg / mL anti-IL-4Rα / IL-5 bispecific antibody, 20 mmol / L sodium acetate, 50 mmol / L glycine, 50 mmol / L arginine hydrochloride, 100 mmol / L sucrose, and 0.05 w / v % polysorbate 80, and having a pH of 5.0 to 5.

2.

19. 3. The stable antibody formulation of claim 2, comprising 100 mg / mL anti-IL-4Rα / IL-5 bispecific antibody, 20 mmol / L sodium acetate, 50 mmol / L glycine, 50 mmol / L arginine hydrochloride, 100 mmol / L sucrose, 10 mmol / L methionine, and 0.05 w / v % polysorbate 80, and having a pH of 5.0 to 5.

2.

20. 2. The stable antibody formulation of claim 1, comprising 100 mg / mL anti-IL-4Rα / IL-5 bispecific antibody, 10 mmol / L sodium acetate, 50 mmol / L glycine, 50 mmol / L arginine hydrochloride, 120 mmol / L sucrose, and 0.05 w / v % polysorbate 80, and having a pH of 5.0 to 5.

2.

21. 2. The stable antibody formulation of claim 1, comprising 100 mg / mL anti-IL-4Rα / IL-5 bispecific antibody, 10 mmol / L sodium acetate, 75 mmol / L glycine, 25 mmol / L arginine hydrochloride, 120 mmol / L sucrose, and 0.05 w / v % polysorbate 80, and having a pH of 5.0 to 5.

2.

22. 2. The stable antibody formulation of claim 1, comprising 100 mg / mL anti-IL-4Rα / IL-5 bispecific antibody, 20 mmol / L sodium acetate, 100 mmol / L arginine hydrochloride, 80 mmol / L sucrose, and 0.02 w / v % polysorbate 80, and having a pH of 5.0 to 5.

2.

23. The stable antibody formulation of claim 1 , further comprising one or more selected from a preservative and / or an additive.

24. 2. The stable antibody formulation of claim 1, wherein the monomer content of the formulation does not decrease by more than 5% after storage for 14 days at about 37 degrees Celsius.

25. 2. The stable antibody formulation of claim 1, wherein the monomer content in a sample does not change by more than 1.5% under conditions of -20 degrees Celsius, 5 degrees Celsius, rocking, or freezing and thawing.

26. A container comprising the stable antibody formulation according to claims 1 to 25.

27. 27. The container of claim 26, comprising a syringe or bottle made from any one or more of glass, plastic, or polymeric materials.

28. A sealed package comprising the stable antibody formulation of any one of claims 1 to 25 or the container of claim 26.

29. A kit comprising the antibody formulation of any one of claims 1 to 25 and a container containing the antibody formulation.

30. The antibody formulation according to any one of claims 1 to 25 or the kit according to claim 29, for use in the preparation of a medicament for the prevention and / or treatment of an autoimmune disease.

31. Use of the stable antibody formulation according to any one of claims 1 to 25 for preparing a medicament for treating a disease, wherein the disease is asthma, allergic dermatitis, eczema, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scar, chronic obstructive pulmonary disease, atopic dermatitis, idiopathic pulmonary fibrosis, Kawasaki disease, sickle cell disease, Graves' disease, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, kidney disease, autoimmune diseases, adult polyangiitis, Use of a stable antibody preparation for the treatment of eosinophilic granulomatosis, severe eosinophilic asthma, hypereosinophilic syndrome, chronic sinusitis with nasal polyps, chronic obstructive pulmonary disease, uncontrolled chronic sinusitis without nasal polyps, allergic fungal sinusitis, prurigo nodularis, familial cold urticaria, bullous pemphigoid, allergic fungal rhinosinusitis, chronic cold urticaria, peanut allergy, pollen allergy, eosinophilic esophagitis, keloid, pruritus, allergic bronchopulmonary aspergillosis, and seasonal allergic rhinitis.

32. 32. The use according to claim 31 , wherein the medicament is administered by intravenous or subcutaneous route.

33. A method for treating a disease using the stable antibody formulation according to any one of claims 1 to 25, wherein the disease is selected from the group consisting of asthma, allergic dermatitis, eczema, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scars, chronic obstructive pulmonary disease, atopic dermatitis, idiopathic pulmonary fibrosis, Kawasaki disease, sickle cell disease, Graves' disease, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, kidney disease, autoimmune diseases, adult polyangiitis eosinophilic ... The method is for the treatment of eosinophilic granulomatosis, severe eosinophilic asthma, hypereosinophilic syndrome, chronic sinusitis with nasal polyps, chronic obstructive pulmonary disease, uncontrolled chronic sinusitis without nasal polyps, allergic fungal sinusitis, prurigo nodularis, familial cold urticaria, bullous pemphigoid, allergic fungal rhinosinusitis, chronic cold urticaria, peanut allergy, pollen allergy, eosinophilic esophagitis, keloid, pruritus, allergic bronchopulmonary aspergillosis, or seasonal allergic rhinitis.

34. 26. A delivery system comprising the stable antibody formulation of any one of claims 1 to 25, wherein the delivery system is selected from a disposable syringe.

35. 26. A method for preparing a stable antibody formulation according to any one of claims 1 to 25, comprising the steps of: 1) preparing an antibody molecule; and 2) mixing a therapeutically effective amount of the antibody molecule with a tonicity agent, a buffer agent, a surfactant and / or other necessary ingredients to form a stable solution.

36. A pharmaceutical product comprising the stable antibody formulation according to any one of claims 1 to 25 and used to treat a disease, the disease being selected from the group consisting of asthma, allergic dermatitis, eczema, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scarring, chronic obstructive pulmonary disease, atopic dermatitis, idiopathic pulmonary fibrosis, Kawasaki disease, sickle cell disease, Graves' disease, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, kidney disease, autoimmune diseases, and adult polyangiitis. Medicines for the treatment of eosinophilic granulomatosis, severe eosinophilic asthma, hypereosinophilic syndrome, chronic sinusitis with nasal polyps, chronic obstructive pulmonary disease, uncontrolled chronic sinusitis without nasal polyps, allergic fungal sinusitis, prurigo nodularis, familial cold urticaria, bullous pemphigoid, allergic fungal rhinosinusitis, chronic cold urticaria, peanut allergy, pollen allergy, eosinophilic esophagitis, keloid, pruritus, allergic bronchopulmonary aspergillosis, and seasonal allergic rhinitis.

37. 26. Use of glycine in the preparation of a stable antibody formulation according to any one of claims 1 to 25, wherein the antibody molecule is stably present even when the concentration of the antibody molecule reaches 100 mg / ml in the antibody formulation.

38. Use of an antibody formulation according to any one of claims 1 to 25 for improving the stability of high-concentration antibodies in liquid formulations.

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