Antibody fragments of a gamma constant region (Cy1) and epsilon constant region (Cepsilon2-4) fusion consisting of a heavy chain constant region and a light chain constant region and uses thereof

Abstract

The present invention relates to an antibody fragment consisting of only the constant region of an antibody, and more particularly to an antibody fragment having no variable region of an antibody and consisting of only a heavy chain constant region (C H ) and a light chain constant region (C L ). For example, the present invention relates to an antibody fragment consisting of a heavy chain constant region and a light chain constant region (IgCw-gamma1epsilon2-4 / kappa), in which a gamma constant region (Cgamma1) and an epsilon constant region (Cepsilon2-4) are fused; a nucleic acid encoding the antibody fragment; a kit comprising the antibody fragment; and a use of the antibody fragment. In order to evaluate the efficacy of an antibody in the development of a new antibody drug or the like, a control, i.e., a reference antibody, is required, and the existing reference antibody cannot be completely free from non-specific binding, i.e., off-target effects due to reactions with other antigens, and the purification yield is also low. On the other hand, when the recombinant protein IgCw-gamma1epsilon2-4 / kappa consisting of only the constant region of an antibody according to the present invention is used, the problems of the existing reference antibody can be overcome, and the recombinant protein can also be used as an Fc epsilon receptor inhibitor that inhibits an allergic reaction. Therefore, the IgCw-gamma1epsilon 2‑4 / kappa according to the present invention can be used in various ways in the field of biomedical science.

Description

Technical Field

[0001] This invention relates to antibody fragments comprising only antibody constant regions; more specifically, to antibody fragments comprising only heavy chain constant regions (CH) and light chain constant regions (CL) without antibody variable regions, such as antibody fragments comprising constant regions (CL) of both heavy and light chains, wherein a γ constant region (Cγ1) is fused with an ε constant region (Cε2-4) (referred to as "IgCw-γ1ε2-4 / κ", abbreviated as IgCw-γεκ); nucleic acids encoding said antibody fragments; kits comprising said antibody fragments; and uses of said antibody fragments. Background Technology

[0002] To demonstrate the in vitro and in vivo efficacy of recombinant antibodies, a reference antibody is needed—a control antibody that does not bind to the target molecule (antigen) of the antibody used to evaluate efficacy. Reference antibodies can be generated from cultures of cells isolated from polyclonal antibodies or transfected with expression constructs encoding the heavy and light chain regions of the antibody.

[0003] Although the reference antibody is not expected to bind to the target molecules bound by the antibody used to evaluate efficacy, the possibility of cross-reactivity that produces noisy positive signals cannot be completely ruled out, especially the possibility of off-target effects due to reactions with other antigens, because the reference antibody has a variable region involved in binding with the antigen.

[0004] On the other hand, immunoglobulin E (IgE) is an antibody present in the blood at very low concentrations. Measuring blood IgE concentrations is crucial for diagnosing IgE-related immune diseases and for tracking and managing treatment efficacy. Reference IgE protein is absolutely necessary for quantifying IgE. Furthermore, IgE protein is indispensable for various qualitative immunological assays.

[0005] Currently, commercially available human IgE proteins include: 1) monoclonal IgE purified from the blood of patients with high levels of IgE in their blood (e.g., patients with IgE myeloma) (e.g., products from Abcam, Athens Research & Technology, MyBioSource, Fitzgerald Industries International, Molecular Innovations, and Merck Millipore); 2) monoclonal IgE purified from conditioned cultures of hybridoma cells formed by fusing B cells derived from healthy individuals with myeloma cells; and 3) monoclonal IgE purified from culture media of cell lines obtained by transfecting the IgE gene.

[0006] These IgE antibodies are at least 100 times more expensive than IgG and lack nonspecific binding affinity for unknown antigens in IgE quantification assays and various immunological experiments because they include variable regions (VH and VL). Internationally standardized IgE exists [WHO International Standard IgE, most recently the Third International Reference Preparation (IRP), coded 11 / 234] for public purposes, used to quantify total IgE in blood. WHO IRP 11 / 234 is a lyophilized ampoule containing plasma (or serum) collected from each patient in various countries who have high concentrations of IgE in their blood; it is considered biohazardous due to the blood components it contains and should therefore be used with caution in laboratories; it is polyclonal IgE; and it is problematic because the possibility of nonspecific binding to unknown antigens cannot be ruled out.

[0007] Therefore, the inventors sought to develop novel antibody fragments that could replace IgE reference antibodies while avoiding the drawbacks of previously known IgE reference antibodies. Furthermore, the inventors sought to develop molecules that could replace IgE in quantitative experiments to measure IgE concentration, and in qualitative experiments to inhibit FcεR-IgE interaction, because they have high production yields and can bind to the Fcε receptor (FcεR).

[0008] As a result, the inventors developed molecules that include only constant regions and no variable regions, particularly heavy chains (Cγ1-hinge-Cε) that fuse γ constant regions (Cγ1) and ε constant regions (Cε2-4). 2-4 The present invention is based on the discovery that this IgCw-γεκ molecule can be used as an IgE substitute in IgE concentration measurement experiments and immunological studies based on FcεR-IgE interaction, and can inhibit IgE-mediated hypersensitivity diseases.

[0009] The information disclosed in this background section is provided only to enhance the understanding of the background of the invention, and therefore may not include information that forms the prior art that is already clear to those skilled in the art. Summary of the Invention

[0010] Therefore, one object of the present invention is to provide a system that includes only the heavy chain constant region (C H ) and light chain constant region (C L Antibody fragments without antibody variable regions.

[0011] Another object of the present invention is to provide a nucleic acid encoding the antibody fragment.

[0012] Another object of the present invention is to provide a kit comprising the antibody fragment.

[0013] Another object of the present invention is to provide a composition containing the said antibody fragment for evaluating antibody efficacy.

[0014] Another object of the present invention is to provide a composition containing the said antibody fragment for measuring antibody concentration.

[0015] Another object of the present invention is to provide a composition containing the antibody fragment for inhibiting allergic reactions.

[0016] Another object of the present invention is to provide a composition containing the said antibody fragment for inhibiting IgE-mediated autoimmune responses.

[0017] According to one aspect of the invention, the above and other objectives can be achieved by providing an antibody fragment comprising a heavy chain constant region fragment of an IgG antibody and a heavy chain constant region fragment of an IgE antibody, and a light chain constant region fragment connected to the heavy chain constant region fragment.

[0018] According to another aspect of the invention, a nucleic acid encoding the antibody fragment is provided.

[0019] According to another aspect of the invention, a kit comprising the antibody fragment is provided.

[0020] According to another aspect of the invention, a composition for evaluating antibody efficacy is provided, containing the antibody fragment.

[0021] According to another aspect of the invention, a composition for measuring antibody concentration containing the antibody fragment is provided.

[0022] According to another aspect of the invention, a composition for inhibiting allergic reactions containing the antibody fragment is provided.

[0023] According to another aspect of the invention, a composition containing the antibody fragment for inhibiting IgE-mediated autoimmune responses is provided. Attached Figure Description

[0024] Figure 1 IgCw-γ1ε was demonstrated 2-4 The structure of the / κ (abbreviated as "IgCw-γεκ") protein is shown, and antibody fragments (approximately 130 kDa) including the Cγ1-hinge region and Cε2-Cε3-Cε4 of the human antibody are shown.

[0025] Figure 2 shows the results of the analysis of the purity and integrity of the purified antibody proteins, and shows the results of the analysis of the purity and integrity of the antibody proteins purified from conditioned cultures of HEK293f cells transfected with each antibody gene expression vector. IgCw-γεκ was identified as having a molecular weight of approximately 130 kDa as expected. A shows the results of Coomassie Brilliant Blue staining after SDS-PAGE, and B shows the results of size exclusion chromatography.

[0026] Figure 3 shows the analysis results of the binding between IgCw-γεκ and FcεR, and more specifically the results of flow cytometry, which elucidate that the IgCw-γεκ antibody fragment protein can bind to the high-affinity human Fcε receptor (FcεR1) expressed on the surface of RBL-2H3-FcεRIα cells, comparable to the control IgE.

[0027] Figure 4 shows the identification results of the degranulation reaction induced by IgCw-γεκ crosslinking, and clarifies that when RBL-2H3-FcεRIα cells were sensitized by treatment with IgCw-γεκ and then treated with anti-Cκ antibody (to induce FcεR1 crosslinking), IgCw-γεκ could induce β-aminohexosidase release (degranulation reaction) to a level similar to that of full-size IgE as a control. A shows the experimental design and B shows the experimental results.

[0028] Figure 5 shows the results of IgCw-γεκ inhibiting the degranulation reaction in IgE-sensitized cells and elucidates that IgCw-γεκ can inhibit the release of β-aminohexosidase (degranulation reaction) induced by 6C407 IgE + protein-L-biotin + streptavidin in RBL-2H3-FcεRIα cells. A shows the experimental design, B shows the measurement results of the degree of β-aminohexosidase release in RBL-2H3-FcεRIα cells treated with a mixture of 6C407 IgE and IgCw-γεκ, and C shows the results of the degree of β-aminohexosidase release in RBL-2H3-FcεRI cells pretreated with 6C407 IgE and then treated with IgCw-γεκ.

[0029] Figure 6 shows IgCw-γεκ as a reference molecule for quantifying human IgE antibodies. More specifically, it shows the results of ELISA, illustrating that the IgCw-γεκ protein can be used as a reference for quantifying IgE antibodies. A represents the standard curve generated using 6C407 IgE, and B represents the standard curve generated using IgCw-γεκ. Detailed Implementation

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well-known and commonly used in the field.

[0031] An attempt was made to generate an IgCw-εκ antibody fragment that includes only the constant region of a human IgE antibody. However, IgCw-εκ, which includes only the constant regions of the ε heavy chain and κ light chain, does not form disulfide bonds between the heavy chain and light chain constant regions, and when purified with bound Kappa XP-agarose that binds to the human κ light chain constant region, only the κ light chain constant region is purified without binding to the heavy chain constant region.

[0032] Finally, the γ constant region (Cγ1)-hinge, which includes the constant regions of heavy and light chains, and the ε constant region (Cε) 2-4 The fused antibody fragment IgCw-γεκ possesses disulfide bonds that normally form between constant regions and exhibits expression levels and purification yields similar to those of the control 6C407 IgE. Therefore, the γ constant region (Cγ1) and the ε constant region (Cε) 2-4 The fusion of ) can achieve structural stability and high purification yield of IgCw-γεκ protein.

[0033] Furthermore, the inventors have developed a novel antibody fragment (called "IgCw-γ1ε") 2-4 IgCw-γεκ (abbreviated as "IgCw-γεκ") can be used as an alternative to previously known IgE reference antibodies while avoiding their drawbacks. IgCw-γεκ consists of two Cγ1-hinge-Cε... 2-4 An antibody fragment molecule consisting of a hybrid heavy chain and two Cκ light chains, which includes only constant regions and no variable regions.

[0034] Accordingly, in one aspect, the present invention relates to antibody fragments comprising a heavy chain constant region fragment of an IgG antibody and a heavy chain constant region fragment of an IgE antibody, and a light chain constant region fragment linked to the heavy chain constant region fragment.

[0035] The antibody fragment according to the invention is obtained by developing an antibody fragment as a novel antibody form, comprising an IgG antibody heavy chain constant region fragment and an IgE antibody heavy chain constant region fragment without an antibody variable region and a light chain constant region fragment linked to the heavy chain constant region fragment, and identifying that IgCw-γεκ having this novel structure has various advantages and can be widely used in clinical fields such as antibody efficacy testing.

[0036] Unfragmented antibodies consist of two heavy chains and two light chains linked by disulfide bonds. Each light chain is linked to one of the heavy chains by a disulfide bond. The antibody heavy chain portion has a variable domain (VH) at the N-terminus, followed by multiple constant domains (3 or 4 constant domains, i.e., C10, C20, C30, C40, C50, C60, C70, C80, C9 ... H 1. C H 2. C H 3 and C H 4. (Depending on the type of antibody). Each light chain moiety has a variable region (VL) at its N-terminus and a constant region (CL) at its other end (C-terminus). The light chain constant region is aligned with the first constant region (CH1) of the heavy chain, and the light chain variable region (VL)... L ) and heavy chain variable region (V H Alignment.

[0037] The constant region refers to all antibody domains except for the variable region. Constant regions do not directly participate in the binding of the antibody to its target antigen, but rather participate in various in vivo effector functions. Heavy chain and light chain constant regions encompass constant regions derived from IgA, IgD, IgE, IgG, and IgM, as well as constant regions derived from IgY, IgW, and IgNAR.

[0038] In one implementation, IgG may include IgG1, IgG2, IgG3 and IgG4 as subtypes.

[0039] In one embodiment, the heavy chain constant region fragment of the IgG antibody may include Cγ1. The heavy chain constant region fragment of the IgE antibody may include at least one selected from Cε2, Cε3, and Cε4.

[0040] In one specific embodiment, the antibody fragment according to the present invention comprises an IgG antibody heavy chain constant region fragment Cγ1 and IgE antibody heavy chain constant region fragments Cε2, Cε3, and Cε4, and the IgG antibody heavy chain constant region fragment can be linked to the IgE antibody heavy chain constant region fragment, in the form of C from the N-terminus to the C-terminus. γ 1-Cε2-Cε3-Cε4.

[0041] The constant region of the IgG antibody heavy chain can be linked to the constant region of the IgE antibody heavy chain via hinges.

[0042] In one embodiment, the light chain constant region may include Cκ or Cλ. The light chain constant region segment linked to the heavy chain constant region segment may be attached thereto, for example, via a disulfide bond or via a peptide linker.

[0043] Recombinant antibody formulations often face challenges due to low purification yields, which can impact usability and cost. Antibody production yield depends on the heavy chain variable region (V0). H ) and light chain variable region (V LThe characteristics (amino acid sequence) of the recombinant antibody. Therefore, even in a mature manufacturing process, the production yield of recombinant antibodies can be quite variable depending on the amino acid sequence of the variable domain (V domain). In one embodiment of the invention, it has been found that antibody fragments comprising only the antibody constant region according to the invention can be produced in high yields.

[0044] In another aspect, the present invention relates to nucleic acids encoding antibody fragments. Antibody fragments can be generated through recombination. The nucleic acids are isolated and inserted into a replicative vector, followed by further cloning (DNA amplification) or further expression. Based on this, in another aspect, the present invention relates to vectors comprising nucleic acids.

[0045] The term "nucleic acid" is intended to encompass both DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acids, include naturally derived nucleotides as well as analogs with modified sugar or base portions. The sequences of nucleic acids encoding the heavy chain variable regions and light chain variable regions of this invention can vary. Such variations include the addition, deletion, or non-conserved or conserved substitution of nucleotides.

[0046] DNA encoding antibody fragments can be readily isolated or synthesized using standard procedures, such as oligonucleotide probes capable of specifically binding to the heavy and light chains of the DNA encoding the antibody. A variety of vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0047] As used herein, the term "vector" refers to a tool used to express a target gene in a host cell and includes plasmid vectors, granular vectors, and viral vectors (such as phage vectors, adenovirus vectors, retroviral vectors, and adeno-associated virus vectors). In the vector, a polynucleotide encoding an antibody is operatively linked to a promoter.

[0048] The term "operationally linked" refers to a functional link between a nucleic acid expression regulatory sequence (e.g., a promoter, signal sequence, or array of transcription regulator binding sites) and another nucleic acid sequence, enabling the regulatory sequence to regulate the transcription and / or translation of the other nucleic acid sequence.

[0049] When using prokaryotic cells as a host, it typically includes an effective promoter capable of transcription (such as the tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, or T7 promoter), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. In addition, for example, when using eukaryotic cells as the host, it includes promoters derived from mammalian cell genomes (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5k promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIVLTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter), and usually has a polyadenylated sequence as a transcription termination sequence.

[0050] Optionally, the vector can be fused with another sequence to facilitate the purification of the antibody expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), 6xHis (hexahistidine; Quiagen, USA), etc.

[0051] Vectors include antibiotic resistance genes commonly used in the art as selective markers, and examples include genes that confer resistance to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, genimycin, neomycin, and tetracycline.

[0052] In another aspect, the present invention relates to cells transformed using the vectors mentioned above. The cells used to generate the antibodies of the present invention can be prokaryotic cells, yeast, or higher eukaryotic cells, but are not limited thereto.

[0053] Prokaryotic host cells, such as *Escherichia coli*; *Bacillus* species, such as *Bacillus subtilis* and *Bacillus thuringiensis*; *Streptomyces* species; *Pseudomonas* species (e.g., *Pseudomonas putida*); *Proteus mirabilis*; and *Staphylococcus* species (e.g., *Staphylococcus carnosus*).

[0054] Examples of host cell lines of greatest interest to animal cells and of usefulness include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, and HT1080.

[0055] In another aspect, the present invention relates to a method for generating antibody fragments, the method comprising: (a) culturing cells; and (b) recovering antibody fragments from the cultured cells.

[0056] Cells can be cultured in a variety of media. Any commercially available media can be used without restriction. All other necessary supplements well known to those skilled in the art can be included at appropriate concentrations. Culture conditions (such as temperature and pH) are routinely used for selecting host cells for expression, as will be clear to those skilled in the art.

[0057] The recovery of antibody fragments can be performed, for example, by centrifugation or ultrafiltration to remove impurities, and by purifying the resulting product using, for example, affinity chromatography. Other additional purification techniques can be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite (HA) chromatography.

[0058] In another aspect, the present invention relates to a kit comprising antibody fragments. The kit may include a container holding the antibody fragments and a container holding another reagent or sample.

[0059] The kit suitably includes at least one container (such as a bottle or tube), and each container contains a separate component used in the method of the present invention. Those skilled in the art can readily dispense the desired formulation into the container.

[0060] In another aspect, the present invention relates to compositions containing antibody fragments for evaluating antibody efficacy.

[0061] In experiments demonstrating the role of antigen-specific antibodies, a reference (or unrelated isotype control) antibody is typically used as a negative control to distinguish nonspecific background signals from antigen-specific antibody signals. However, due to the lack of known cross-reactivity, conventional reference antibodies may still generate unwanted noise signals.

[0062] Compared to irrelevant IgE controls, the antibody fragments according to the present invention can be better reference antibodies in many experimental environments and conditions because they have no antigen-binding ability and can exclude the possibility of accidental cross-reactivity.

[0063] In another aspect, the present invention relates to compositions containing antibody fragments for measuring antibody concentrations. The antibody fragments according to the invention can be used as a reference for measuring immunoglobulin concentrations in biological samples.

[0064] In another aspect, the present invention relates to compositions containing antibody fragments for inhibiting allergic reactions.

[0065] Most allergic diseases are caused by an excessive immune response to immunoglobulin E (IgE). IgE is an antibody present in serum at very low concentrations under normal conditions. IgE is usually produced by harmless antigens and often increases even without specific stimulation. In this situation, allergic diseases may occur. Abnormally increased IgE can bind to high-affinity IgE Fc receptors (FcεRIs) expressed on the surface of mast cells and basophils. When an antigen (mainly a harmless antigen) binds to several IgE molecules simultaneously while IgE molecules are bound to FcεRIs, cross-linking occurs between FcεRI receptors, and signal transduction into mast cells and basophils leads to activation. Due to cellular activation, mast cells or basophils release chemical mediators such as histamine, leukotrienes, prostaglandins, bradykinin, and platelet-activating factor. The release of these chemical mediators causes allergic symptoms. The antibody fragment according to the invention can be used as an Fcε receptor (FcεRI) blocker to inhibit allergic reactions.

[0066] In another aspect, the present invention relates to compositions containing antibody fragments for inhibiting IgE-mediated autoimmune responses.

[0067] One of the pathogenesis mechanisms of autoimmune diseases such as systemic lupus erythematosus (SLE) is the secretion of large amounts of inflammatory cytokines (IFN-α, TNF, IL-6) by plasmacytoid dendritic cells (pDCs). Like mast cells, pDCs express FcεRI on their surface and, upon activation, can secrete 1,000 times more IFN-α than other cells. When a complex of IgE isotype autoantibodies and DNA antigens (immune complexes) binds to FcεRI on the pDC surface, the immune complexes enter the cell via FcεRI. The infiltrated immune complexes stimulate intracellular receptors such as Toll-like receptors (TLRs)-7 and TLR-9 to activate pDCs, leading to the secretion of large amounts of inflammatory cytokines and exacerbation of the disease. The antibody fragment according to the present invention can be used as an Fcε receptor (FcεRI) blocker to inhibit the autoimmune response mediated by IgE autoantibodies.

[0068] The invention will be described in more detail below with reference to the following embodiments. However, it will be apparent to those skilled in the art that the following embodiments are provided for illustrative purposes only and should not be construed as limiting the scope of the invention based on its subject matter.

[0069] Example 1: Sample Preparation

[0070] Example 1-1: Plasmid Vector

[0071] A DNA fragment (encoding the human heavy chain constant regions Cγ1 and ε2-4) was cloned between the restriction sites NcoI and BamHI in the KV10-IgCWγκ vector to form a vector simultaneously expressing human Cγ1 / ε2-4. 2-4 The KV10-IgCw-γεκ vector contains human Cκ (each with a leader sequence, and the two CMV promoters regulate genes through them respectively).

[0072]

[0073]

[0074] Encode V respectively H and V K DNA fragments of the domains were cloned between the MluI and NhEI restriction sites and between the DraIII and BstWI restriction sites in the KV12-HL vector to form a plasmid vector for chimeric IgE (6C407, 3D8) expression.

[0075] The DNA fragment encoding the human heavy chain constant region (Cε1-4) was cloned between the restriction sites NcoI and BamHI of the KV10-IgCWγκ vector to form the KV10-IgCw-εκ vector that simultaneously expresses human Cε1-4 and human Cκ (each with a leader sequence, and the two CMV promoters regulate genes through them respectively).

[0076] The DNA fragment encoding human FcεRIα was cloned between the restriction sites EcoI and BamHI of the pCDH-CMV-MCS-EF1-Puro vector to form a lentiviral vector expressing human FcεRIα.

[0077] Examples 1-2: Preparation of Immunoglobulins using HEK293F cells

[0078] Antibodies were produced using the FreeStyle™ 293-F cell line (ThermoFisher Scientific), which is suitable for growth by suspension culture under serum-free conditions.

[0079] To adjust the cell number to 2 × 10⁶ during transfection 6 Cells / ml, 24 hours before transfection, in a 500ml flask (Corning, catalog number 431145) at a concentration of 1×10⁻⁶ cells / ml. 6100 ml of FreeStyle HEK293F cells were cultured at 130 rpm in serum-free FreeStyle 293 medium (Invitrogen, catalog number 12338) at 37°C with shaking in an 8% CO2 incubator. To express 6C407 IgE, IgCw-εκ, and IgCw-γεκ proteins, 200 μg of KV plasmids encoding each gene and 400 μg of polyethyleneimine (PEI) reagent (Polyscience, catalog number 23966-2) were allowed to stand in 5 ml of FreeStyle 293 medium at room temperature for 10 minutes. Then, 5 ml of medium containing DNA was filtered through a 0.22 μm syringe filter (Millipore, catalog number SLGV033RB) and mixed with 5 ml of medium containing polyethyleneimine, and allowed to stand at room temperature for 10 minutes. Finally, 100 ml of Freestyle293-F cells were transiently transfected to adjust the polyethyleneimine concentration to 4 μg / ml. After 7 days, the culture medium was centrifuged at 400 g for 20 min at 4°C to obtain the supernatant, which was then filtered through a 0.45 μm cellulose acetate filter (Sartorius, catalog number 11106-47-N) and allowed to pass through a CaptureSelect™ Kappa XP (Thermo Fisher Scientific; catalog number 2943212005) column or a CaptureSelect™ IgG-CH1 (Thermo Fisher Scientific; catalog number 194320005) column. After washing the column with PBS (phosphate-buffered saline, pH 7.4), the protein was eluted with 0.1 M glycine solution (glycine-HCl, pH 3.0). The eluted protein was concentrated using a Vivaspin 20 (molecular weight cutoff 50,000, Sartorius, catalog number VS2032) (4°C, 1500 g).

[0080] The concentration of purified protein was determined using absorbance and extinction coefficients at 280 nm. The molar extinction coefficients at 280 nm were 1.49 (for 6C407 IgE), 1.34 (for IgCw-εκ), and 1.17 (for IgCw-γεκ), and were calculated from their respective amino acid sequences via the webpage http: / / web.expasy.org / protparam / .

[0081] Examples 1-3: Cell Culture

[0082] RBL-2H3 cells derived from rat basophilic leukemia were cultured in DMEM (Dürbeco Modified Eagle Medium, Welgene Inc.). Cell lines CRL-2256TM (number: CRL-2256TM) and HEK293T (human embryonic kidney 293 cell line) Cell number: CRL-3216TM. The DMEM used in this study was supplemented with 10% fetal bovine serum (Sigma Inc.), 100 U / ml penicillin (Welgene Inc.), and 100 μg / ml streptomycin (Welgene Inc.). All cells were cultured in a 5% CO2, 37°C incubator.

[0083] Examples 1-4: Cell Construction

[0084] The human embryonic kidney 293 cell line HEK293T was used to produce a supernatant containing a lentivirus expressing FcεRIα.

[0085] To adjust the cell number to 3 × 10⁶ during transfection 6 Cells / ml, 24 hours before transfection at 60 mm 2 In a petri dish (SPL; catalog number 11060), 4 ml of DMEM was used with 1.5 × 10⁻⁶ ppm. 6 HEK293T cells were cultured at a density of 10 cells / well. After 24 hours, the culture medium was discarded, and 3.8 ml of DMEM was mixed with 200 μl of Opti-MEM™ (Thermo Fisher Scientific; catalog 31985070) supplemented with 4 μg of pCDH plasmid encoding the gene, 3 μg g g ag / pol (Addgene, catalog 14887), 1 μg VSV-G (Addgene, catalog 14888), and 16 μg polyethyleneimine (PEI) reagent (Polyscience, catalog 23966-2) to express FcεRIα. The product was cultured at 5% CO2 and 37°C for 16 hours, the culture medium was discarded, and the cells were cultured in 4 ml of fresh DMEM for 24 hours. After 24 hours, the culture medium was collected and centrifuged at 2,000 rpm for 3 minutes to obtain the supernatant, which was then filtered through a 0.45 μm syringe filter (Millipore, catalog SLHV033RS) to obtain a supernatant containing lentivirus.

[0086] Prepare a supernatant containing lentiviruses for expressing receptors that recognize human IgE in RBL-2H3 cells.

[0087] In order to adjust the cell number to 2×10 during infection 6 24 hours before infection, at 60mm 2In 4 ml of DMEM in a culture dish, with 1×10 6 RBL-2H3 cells were cultured at 10 cells / well. After 24 hours, the culture medium was removed, and the cells were removed once with PBS (phosphate-buffered saline, pH 7.4). The cells were then mixed with 3 ml of DMEM and 1 ml of supernatant containing lentivirus, and 10 μg / ml polybrene (Sigma-Aldrich; catalog number H9268) was added. After 24 hours of culture, the culture medium was removed, and 4 ml of DMEM was added, followed by another 12 hours of culture. The cells were then cultured at 5 μg / ml puromycin (Sigma-Aldrich; catalog number 540411).

[0088] Example 2: Analysis Method

[0089] Example 2-1: Flow Cytometry

[0090] To determine whether the receptor is expressed in RBL-2H3-hFcεRIα cells expressing the human Fcε receptor, cells (1×10⁻⁶) were... 6 Cells were washed with cold PBS and then fixed for 20 minutes at room temperature in 4% paraformaldehyde diluted in PBS. Mouse anti-human FcεRI Ab (Abcam; catalog number 155369) conjugated with the detection antibody APC was diluted in buffer S (0.5% BSA, 2 mM EDTA, pH 8.5 in PBS). The product was allowed to stand at 4°C for 1 hour, then washed three times with cold PBS. The product was analyzed using a FACS CantoII analyzer (BD Biosciences).

[0091] To determine whether IgCw-γεκ binds to the Fcε receptor expressed on the cell surface, FcεR-RBL-2H3 and FcεR+RBL-2H3-hFcεRIα cells (1×10⁻⁶) were used. 6 Cells were treated with 1 μM immunoglobulin at 37°C for 3 hours. Cells were washed with cold PBS and fixed for 20 minutes at room temperature with 4% paraformaldehyde diluted in PBS. For RBL-2H3 and RBL-2H3-hFcεRIα cells, goat anti-human IgE (ε-chain specific) antibody (Sigma-Aldrich; catalog number I6284) as the primary antibody and PE-conjugated donkey anti-goat IgG antibody (Abcam; catalog number Ab7004) as the secondary antibody were diluted in buffer S. Cells were washed three times with cold PBS each time they were allowed to stand (4°C, 1 hour). Each sample was analyzed using a FACSCantoII analyzer (BD Biosciences).

[0092] Example 2-2: Enzyme-linked immunosorbent assay (ELISA) for measuring IgE concentration

[0093] Enzyme-linked immunosorbent assay (ELISA) was performed using the Human IgE ELISA Ready-SET-Go (Invitrogen; catalog number 88-50610) kit for quantifying human IgE to generate standard curves for full-size IgE and IgCw-γεκ included in the kit. The experimental method followed the kit instructions.

[0094] The wells of a 96-well polystyrene plate were coated with the coating antibody at 4°C for 16 hours. Then, at each resting stage (room temperature), the cells were washed four times with TBS (TRIS buffered saline; 50 mM TRIS-Cl, 50 mM NaCl, pH 7.4) containing 0.05% Tween-20. To block the binding of non-specific antibodies, the cells were treated with 3% BSA (bovine serum albumin) at room temperature for 2 hours. Human polyclonal IgE and IgCw-γεκ were serially diluted 2-fold from an initial concentration of 250 ng / ml, and the wells were treated with the dilution buffer at room temperature for 2 hours and with the detection antibody at room temperature for 1 hour. Finally, the reaction substrate solution was added to each well, and the absorbance was measured at 450 nm using a microplate reader (Molecular Devices Inc.).

[0095] To measure the concentration of antibodies of unknown concentration, the wells of a 96-well polystyrene plate were coated with the antibody at 4°C for 16 hours. The wells were then treated with monoclonal 6C407 IgE or 3D8 IgE, followed by treatment with the detection antibody (both at room temperature for 1 hour). The concentration of the IgE sample was determined by interpolating the Y-axis values ​​from two different standard curves generated using known concentrations of human IgE and IgCw-γεκ, respectively.

[0096] Examples 2-3: β-Aminohexosidase release assay (degranulation assay)

[0097] The secretion of β-aminohexosidase was measured to determine whether the IgE-Fcε receptor-bound complex exhibited degranulation activity. This was done to adjust the number of RBL-2H3-hFcεRIα cells to 5 × 10⁻⁶. 5 Cells / well, cultured 2.5 × 10⁶ cells / well in 400 μl DMEM in a 24-well plate (SPL; catalog number 30024) 24 hours ago. 5Cells / well. After 24 hours, to sensitize with IgE, 10 nM IgE was cultured for 3 hours in 400 μl of DMEM free of fetal bovine serum, penicillin, and streptomycin. IgE was removed, and cells were then washed twice with 500 μl of Siraganian buffer (119 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.4 mM MgCl2, 25 mM PIPES, 40 mM NaOH, 1 mM CaCl2, 0.1% BSA, pH 7.2). The product was allowed to stand for 10 minutes in 160 μl of Siraganian buffer at 37°C with 5% CO2. Goat anti-human κ light chain antibody (Invitrogen; catalog number 31129) was mixed with Siraganian buffer to obtain a final concentration of 10 μg / ml, and 20 μl of the resulting mixture was added to each well, followed by incubation for 20 minutes. The 0.1% Triton X-100 used as a control group was allowed to stand for 1 hour in a 5% CO2, 37°C incubator. After incubation for 20 minutes in a 5% CO2, 37°C incubator, 50 μl of the product was added to each well of a 96-well polystyrene plate, along with 50 μl of 1 mM p-NAG (p-nitrophenyl N-acetyl-β-D-glucosamine in 0.1 M citrate buffer, pH 4.5). After treatment at 37°C for 1 hour, 200 μl of stop solution (0.1 M Na2CO3 / NaHCO3, pH 10.0) was added to each well, and the absorbance at 405 nm was measured using a microplate reader (Molecular Devices Inc.).

[0098] Examples 2-4: Measuring the secretion of β-aminohexosidase to determine the blocking effect of Fcε receptors

[0099] The secretion of β-aminohexosidase was measured to determine whether the IgE-Fcε receptor-binding complex acts as an Fcε receptor blocker, thereby preventing degranulation induced by other IgEs. To adjust the number of RBL-2H3-hFcεRIα cells to 5 × 10⁻⁶ cells... 5 Cells / well, cultured 2.5 × 10⁶ cells / well in 400 μl DMEM in a 24-well plate 24 hours ago. 5Cells / well. After 24 hours, to sensitize IgE, dilution buffer (obtained by serial dilution of 10 nM IgE as the blocking IgE starting at a concentration of 20 nM in 400 μl of DMEM free of fetal bovine serum, penicillin, and streptomycin) was added, followed by 3 hours of incubation. Cells were washed twice with 500 μl of Siraganian buffer and allowed to stand for 10 minutes in 160 μl of Siraganian buffer in a 5% CO2, 37°C incubator. 140 nM recombinant biotinylated protein L (ThermoFisher Scientific; catalog 29997) and 70 nM streptavidin-fluorescein (Vector Laboratories; catalog SA-5001) were mixed with Siraganian buffer, and 20 μl of the resulting mixture was added to each well, followed by 20 minutes of standing. 0.1% Triton X-100, used as a control, was incubated for 1 hour in a 5% CO2, 37°C incubator. After incubation at 37°C for 20 minutes in 5% CO2, 50 μl of the product was added to each well of a 96-well polystyrene plate, along with 50 μl of 1 mM p-NAG (p-nitrophenyl N-acetyl-β-D-glucosamine in 0.1 M citrate buffer, pH 4.5). After treatment at 37°C for 1 hour, 200 μl of stop solution (0.1 M Na2CO3 / NaHCO3, pH 10.0) was added to each well, and the absorbance at 405 nm was measured using a microplate reader (Molecular Devices Inc.).

[0100] Example 3: Confirmation of IgCw-γεκ purification yield

[0101] The yields of immunoglobulin molecules produced by transient transfection of HEK293F cells with KV10-IgCw-γεκ, KV12-6C407 IgE, KV10-IgCw-εκ, and plasmids followed by suspension culture were compared (Table 1). The predicted structures of the expressed proteins are shown in... Figure 1 Seven days post-transfection, all proteins were purified using Kappa XP-agarose gels that bind to the human κ constant region. IgCw-εκ, which contains only the ε heavy chain constant region and the κ light chain constant region, does not form disulfide bonds between the heavy and light chain constant regions, thus only the κ constant region was purified (Figure 2). On the other hand, the γ constant region (Cγ1), which includes both heavy and light chain constant regions, and the ε constant region (Cε)... 2-4The fused antibody fragment IgCw-γεκ possesses disulfide bonds that form normally between constant regions and exhibits expression levels similar to the control 6C407 IgE (Figure 2), with an average yield of 17 mg / L (same as 6C407 IgE), and an average yield of approximately 27 mg / L when purified on KappaXP-agarose (Table 1). This indicates that the IgCw-γεκ protein is structurally more stable and has a higher purification yield compared to the IgCw-εκ protein.

[0102] [Table 1]

[0103] Antibody (fragment) protein yield obtained from HEK293F cell culture

[0104]

[0105] Depending on the purification method, the purification yield of IgCw-γεκ protein is similar to or higher than that of IgE.

[0106] Example 4: Confirmation of human Fcε receptor expression in RBL-2H3 cells

[0107] Flow cytometry was performed to determine whether hFcεR was expressed in RBL-2H3-hFcεRIα cells expressing the human Fcε receptor.

[0108] Anti-Fcε antibodies do not bind to wild-type RBL-2H3 cells, but they do bind to RBL-2H3-hFcεRIα cells that express the Fcε receptor (Figure 3). This means that the human Fcε receptor is not expressed in wild-type RBL-2H3 cells, but is only present in RBL-2H3-hFcεRIα cells that express the human Fcε receptor.

[0109] Example 5: Confirmation of IgCw-γεκ binding to Fcε receptor and background signal

[0110] Flow cytometry was performed to determine whether IgCw-γεκ binds to the Fcε receptor on cells that express the human Fcε receptor on their surface.

[0111] IgCw-γεκ binds to RBL-2H3-hFcεRIα cells that express hFcεRIα (as with full-size IgE), but not to RBL-2H3 cells lacking any hFcεRIα (Figure 3). This means that, like full-size IgE, IgCw-γεκ can recognize the Fcε receptor.

[0112] Example 6: Using IgCw-γεκ as a reference for degranulation experiments using IgE

[0113] The β-aminohexosidase secretion assay was used to determine whether IgCw-γεκ could be used instead of full-size IgE in experiments using IgE (such as degranulation experiments).

[0114] Experiments were performed using polyclonal IgE and 6C407 IgE, as well as 6C407 IgG and PBS as controls. No secretion of β-aminohexosidase was observed in RBL-2H3-hFcεRIα cells in the negative controls 6C407 IgG and PBS, and β-aminohexosidase secretion was observed only in IgE-treated samples (Figure 4). IgCw-γεκ showed a secretion percentage similar to or higher than that of full-size IgE, indicating that IgCw-γεκ functions similarly to full-size IgE. This implies that IgCw-γεκ can be used as a reference molecule in place of human IgE in degranulation experiments.

[0115] Example 7: Using IgCw-γεκ as an Fcε receptor blocker

[0116] To determine whether IgCw-γεκ can be used as an inhibitor of Fcε receptors.

[0117] IgCw-γεκ cannot bind to protein L because it lacks a variable region. Therefore, activation signals in RBL-2H3-hFcεRIα cells can only be observed when full-size IgE binds to Fcε. When RBL-2H3-hFcεRIα cells are treated with a mixture of 6C407IgE and IgCw-γεκ... Figure 5B When RBL-2H3-hFcεRIα cells are pretreated with 6C407IgE and then treated with IgCw-γεκ, Figure 5C At the same concentration, the signal of 6C407 IgE decreased with increasing IgCw-γεκ concentration (Figure 5). This means that IgCw-γεκ interferes with the binding of full-size IgE to the Fcε receptor, indicating that IgCw-γεκ can be used as an Fcε receptor blocker.

[0118] Example 8: Using IgCw-γεκ as a reference for IgG concentration quantification

[0119] To determine whether IgCw-γεκ can be used as a substitute for full-size IgE, which is a reference for determining IgG concentrations.

[0120] Using known concentrations of human polyclonal IgE ( Figure 6A ) and IgCw-γεκ( Figure 6BTwo logarithmic standard curves were generated using enzyme-linked immunosorbent assay (ELISA). The same experiment was performed using two monoclonal IgE samples (6C407 and 3D8) with unknown concentrations. IgE concentrations were determined by interpolation in each curve (Table 2), and the linear interpolation formula used in this study (https: / / formulas.tutorvista.com / math / interpolation-formula.html) is x = x1 + (x2 - x1) × (y - y1) / (y2 - y1).

[0121] [Table 2]

[0122] IgE concentration measured using two standard curves

[0123]

[0124] *The IgE concentration determined by the IgCw-γεκ standard curve is corrected by multiplying it by the molecular weight ratio (the molecular weight ratio of IgE (approximately 190 kDa): IgCw-γεκ (approximately 130 kDa) = 1.462:1).

[0125] The normalized concentrations of 6C407 IgE and 3D8 IgE were 75.366 ng / ml and 24.737 ng / ml, respectively, which are very similar to the concentrations determined by standard curves using human polyclonal antibodies (6C407 IgE: 70.42 ng / ml, 3D8 IgE: 25.01 ng / ml). This indicates that IgCw-γεκ can be used as a reference molecule to determine human IgE concentrations.

[0126] Industrial applicability

[0127] The antibody fragment according to the invention can be widely used as a substitute for IgE reference antibodies used in various immunological studies. The antibody fragment can be used both as a reference molecule to measure the concentration of IgE antibodies in a sample and as an Fcε receptor (FcεRI) blocker to inhibit type 1 hypersensitivity reactions.

[0128] Furthermore, the antibody fragments according to the present invention can be used as blocking agents to inhibit autoimmune responses associated with autoantibodies of the IgE isotype.

[0129] Furthermore, the antibody fragments according to the present invention do not contain variable regions, thus completely avoiding nonspecific cross-reactivity, i.e., off-target effects due to reactions with other antigens, which is a drawback of conventional full-length isotype control antibodies. Therefore, they are more suitable for use in quantitative and qualitative experiments of IgE, as well as as isotype control antibodies to evaluate the activity of experimental / therapeutic antibodies.

[0130] Although specific configurations of the invention have been described in detail, those skilled in the art will understand that this specification is provided for illustrative purposes to illustrate preferred embodiments and should not be construed as limiting the scope of the invention. Therefore, the essential scope of the invention is defined by the appended claims and their equivalents.

[0131] Sequence List Free Text

[0132] Electronic documents are attached. sequence list <110> Fate Antibody Generation Company <120> Antibody fragments fused from a γ constant region (Cγ1) and an ε constant region (Cε2-4), consisting of a heavy chain constant region and a light chain constant region, and their applications. <130> 2683 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 436 <212> PRT <213> Artificial Sequence <220> <223> human Cgamma1 epsilon2-4 <400> 1 Met Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 1 5 10 15 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 20 25 30 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 35 40 45 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 50 55 60 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 65 70 75 80 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 85 90 95 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Val 100 105 110 Cys Ser Arg Asp Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser Ser 115 120 125 Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu 130 135 140 Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp 145 150 155 160 Gly Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu 165 170 175 Gly Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His 180 185 190 Trp Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His 195 200 205 Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn Pro Arg Gly 210 215 220 Val Ser Ala Tyr Leu Ser Arg Pro Ser Pro Phe Asp Leu Phe Ile Arg 225 230 235 240 Lys Ser Pro Thr Ile Thr Cys Leu Val Val Asp Leu Ala Pro Ser Lys 245 250 255 Gly Thr Val Asn Leu Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn 260 265 270 His Ser Thr Arg Lys Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr Val 275 280 285 Thr Ser Thr Leu Pro Val Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr 290 295 300 Tyr Gln Cys Arg Val Thr His Pro His Leu Pro Arg Ala Leu Met Arg 305 310 315 320 Ser Thr Thr Lys Thr Ser Gly Pro Arg Ala Ala Pro Glu Val Tyr Ala 325 330 335 Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu Ala 340 345 350 Cys Leu Ile Gln Asn Phe Met Pro Glu Asp Ile Ser Val Gln Trp Leu 355 360 365 His Asn Glu Val Gln Leu Pro Asp Ala Arg His Ser Thr Thr Gln Pro 370 375 380 Arg Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Ser Arg Leu Glu Val 385 390 395 400 Thr Arg Ala Glu Trp Glu Gln Lys Asp Glu Phe Ile Cys Arg Ala Val 405 410 415 His Glu Ala Ala Ser Pro Ser Gln Thr Val Gln Arg Ala Val Ser Val 420 425 430 Asn Pro Gly Lys 435 <210> 2 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> human Ckappa <400> 2 Met Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 1 5 10 15 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 20 25 30 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 35 40 45 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 50 55 60 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 65 70 75 80 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 85 90 95 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 3 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> 6C407 IgE Heavy chain <400> 3 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Ser Arg Ser Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 4 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> 6C407 IgE Light chain <400> 4 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met Leu Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Ala Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 5 <211> 429 <212> PRT <213> Artificial Sequence <220> <223> Human Cepsilon <400> 5 Met Ala Ser Thr Gln Ser Pro Ser Val Phe Pro Leu Thr Arg Cys Cys 1 5 10 15 Lys Asn Ile Pro Ser Asn Ala Thr Ser Val Thr Leu Gly Cys Leu Ala 20 25 30 Thr Gly Tyr Phe Pro Glu Pro Val Met Val Thr Trp Asp Thr Gly Ser 35 40 45 Leu Asn Gly Thr Thr Met Thr Leu Pro Ala Thr Thr Leu Thr Leu Ser 50 55 60 Gly His Tyr Ala Thr Ile Ser Leu Leu Thr Val Ser Gly Ala Trp Ala 65 70 75 80 Lys Gln Met Phe Thr Cys Arg Val Ala His Thr Pro Ser Ser Thr Asp 85 90 95 Trp Val Asp Asn Lys Thr Phe Ser Val Cys Ser Arg Asp Phe Thr Pro 100 105 110 Pro Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly His Phe 115 120 125 Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly 130 135 140 Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln Val Met Asp Val Asp 145 150 155 160 Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln 165 170 175 Ser Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp Arg Thr Tyr 180 185 190 Thr Cys Gln Val Thr Tyr Gln Gly His Thr Phe Glu Asp Ser Thr Lys 195 200 205 Lys Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg 210 215 220 Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr Cys 225 230 235 240 Leu Val Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu Thr Trp 245 250 255 Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu 260 265 270 Lys Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr Leu Pro Val Gly 275 280 285 Thr Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val Thr His 290 295 300 Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr Ser Gly 305 310 315 320 Pro Arg Ala Ala Pro Glu Val Tyr Ala Phe Ala Thr Pro Glu Trp Pro 325 330 335 Gly Ser Arg Asp Lys Arg Thr Leu Ala Cys Leu Ile Gln Asn Phe Met 340 345 350 Pro Glu Asp Ile Ser Val Gln Trp Leu His Asn Glu Val Gln Leu Pro 355 360 365 Asp Ala Arg His Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly Ser Gly 370 375 380 Phe Phe Val Phe Ser Arg Leu Glu Val Thr Arg Ala Glu Trp Glu Gln 385 390 395 400 Lys Asp Glu Phe Ile Cys Arg Ala Val His Glu Ala Ala Ser Pro Ser 405 410 415 Gln Thr Val Gln Arg Ala Val Ser Val Asn Pro Gly Lys 420 425 <210> 6 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Human Ckappa <400> 6 Met Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 1 5 10 15 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 20 25 30 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 35 40 45 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 50 55 60 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 65 70 75 80 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 85 90 95 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 7 <211> 552 <212> PRT <213> Artificial Sequence <220> <223> IgCwepsilonLkappa <400> 7 Met Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 1 5 10 15 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 20 25 30 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 35 40 45 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 50 55 60 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 65 70 75 80 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 85 90 95 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ala Ser Thr Gln 115 120 125 Ser Pro Ser Val Phe Pro Leu Thr Arg Cys Cys Lys Asn Ile Pro Ser 130 135 140 Asn Ala Thr Ser Val Thr Leu Gly Cys Leu Ala Thr Gly Tyr Phe Pro 145 150 155 160 Glu Pro Val Met Val Thr Trp Asp Thr Gly Ser Leu Asn Gly Thr Thr 165 170 175 Met Thr Leu Pro Ala Thr Thr Leu Thr Leu Ser Gly His Tyr Ala Thr 180 185 190 Ile Ser Leu Leu Thr Val Ser Gly Ala Trp Ala Lys Gln Met Phe Thr 195 200 205 Cys Arg Val Ala His Thr Pro Ser Ser Thr Asp Trp Val Asp Asn Lys 210 215 220 Thr Phe Ser Val Cys Ser Arg Asp Phe Thr Pro Pro Thr Val Lys Ile 225 230 235 240 Leu Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln 245 250 255 Leu Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr 260 265 270 Trp Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser 275 280 285 Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu 290 295 300 Ser Gln Lys His Trp Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr 305 310 315 320 Tyr Gln Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser 325 330 335 Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro Ser Pro Phe Asp 340 345 350 Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr Cys Leu Val Val Asp Leu 355 360 365 Ala Pro Ser Lys Gly Thr Val Asn Leu Thr Trp Ser Arg Ala Ser Gly 370 375 380 Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys Gln Arg Asn Gly 385 390 395 400 Thr Leu Thr Val Thr Ser Thr Leu Pro Val Gly Thr Arg Asp Trp Ile 405 410 415 Glu Gly Glu Thr Tyr Gln Cys Arg Val Thr His Pro His Leu Pro Arg 420 425 430 Ala Leu Met Arg Ser Thr Thr Lys Thr Ser Gly Pro Arg Ala Ala Pro 435 440 445 Glu Val Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys 450 455 460 Arg Thr Leu Ala Cys Leu Ile Gln Asn Phe Met Pro Glu Asp Ile Ser 465 470 475 480 Val Gln Trp Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg His Ser 485 490 495 Thr Thr Gln Pro Arg Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Ser 500 505 510 Arg Leu Glu Val Thr Arg Ala Glu Trp Glu Gln Lys Asp Glu Phe Ile 515 520 525 Cys Arg Ala Val His Glu Ala Ala Ser Pro Ser Gln Thr Val Gln Arg 530 535 540 Ala Val Ser Val Asn Pro Gly Lys 545 550

Claims

1. An antibody fragment consisting of: a heavy chain constant region segment of an IgG antibody consisting of a constant region gene segment gamma 1 (C gamma 1) γ 1) and a heavy chain constant region segment of an IgE antibody consisting of a constant region gene segment epsilon 2 (C epsilon 2), a constant region gene segment epsilon 3 (C epsilon 3) and a constant region gene segment epsilon 4 (C epsilon 4); and a light chain constant region fragment consisting of constant region gene segment kappa (CK) linked to a heavy chain constant region fragment of an IgG antibody, wherein the IgG antibody heavy chain constant region fragment is linked to the IgE antibody heavy chain constant region fragment, in the order from N-terminus to C-terminus C γ 1 - Cε2 - Cε3 - Cε4, and wherein the heavy chain constant region fragment of the IgE antibody is linked to the heavy chain constant region fragment of the IgG antibody by a hinge.

2. The antibody fragment of claim 1, wherein the antibody fragment does not comprise a variable domain.

3. The antibody fragment of claim 1, wherein the light chain constant region fragment linked to the heavy chain constant region fragment is bonded thereto by a disulfide bond or via a peptide linker.

4. A nucleic acid encoding the antibody fragment of any one of claims 1 to 3.

5. A kit comprising the antibody fragment of any one of claims 1 to 3.

6. A composition for evaluating antibody efficacy comprising the antibody fragment of any one of claims 1 to 3.

7. A composition for measuring antibody concentration comprising the antibody fragment of any one of claims 1 to 3.

8. A composition for inhibiting an allergic reaction comprising the antibody fragment of any one of claims 1 to 3.

9. A composition for inhibiting an IgE-mediated autoimmune reaction comprising the antibody fragment of any one of claims 1 to 3.

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