Antibody fragments consisting of heavy and light chain constant regions in which the gamma constant region (Cγ1) and the epsilon constant region (Cε2-4) are fused, and uses thereof

Abstract

The present invention relates to an antibody fragment consisting of only the constant region of an antibody, more specifically, a heavy chain (C) without the variable region of an antibody. H ) and light chain (C L The present invention relates to an antibody fragment composed only of the constant region of IgCw-γ1ε2-4 / κ. For example, the present invention relates to an antibody fragment (IgCw-γ1ε2-4 / κ) composed of heavy and light chain constant regions in which the gamma constant region (Cγ1) and the epsilon constant region (Cε2-4) are fused, a nucleic acid encoding the same, a kit containing the same, and uses thereof. To evaluate the efficacy of an antibody in the development of new antibody drugs, a control group, i.e., a reference antibody, is required. However, existing reference antibodies cannot completely eliminate off-target effects due to nonspecific binding, i.e., reactions with other antigens, and have low purification yields. On the other hand, IgCw-γ1ε2-4 / κ, a recombinant protein composed only of the constant region of the antibody of the present invention, can overcome the problems of existing reference antibodies and can also be used as an Fc epsilon receptor blocker to suppress allergic reactions. Therefore, the IgCw-γ1ε of the present invention 2-4 / κ can be used in a variety of ways in the field of medical and life sciences.

Description

[Technical field]

[0001] The present invention relates to an antibody fragment consisting of only the constant region of an antibody, and more particularly to an antibody fragment consisting of a heavy chain (C) without the variable region of an antibody. H ) and light chain (C L For example, the antibody fragments may be composed of heavy and light chain constant regions (Cγ1) and epsilon constant regions (Cε2-4) fused together. L The present invention relates to an antibody fragment (designated IgCw-γ1ε2-4 / κ and abbreviated as IgCw-γεκ) composed of the above-mentioned IgCw-γ1ε2-4 / κ, a nucleic acid encoding the same, a kit containing the same, and uses thereof. [Background technology]

[0002] To verify the in vitro and in vivo efficacy of recombinant antibodies, a reference antibody is needed, i.e., a control antibody that does not bind to the antibody's target molecule (antigen) for which efficacy is to be assessed. A reference antibody can be isolated from a polyclonal antibody or produced from a cell culture transfected with expression constructs encoding the heavy and light chain regions of the antibody.

[0003] Even if the reference antibody is not expected to bind to the target molecule to which the antibody for evaluating efficacy is bound, the reference antibody also has a variable region involved in binding to any antigen, so the possibility of cross-reactivity resulting in a noise positive signal, particularly the possibility of off-target effects due to reactions with other antigens, cannot be completely ruled out.

[0004] On the other hand, immunoglobulin E (IgE) is an antibody that exists in very low concentrations in the blood. Measuring the concentration of IgE in the blood is important for diagnosing IgE-related immune diseases and tracking the effectiveness of treatment, but reference IgE protein is essential for quantifying IgE. IgE protein is also required for various immunological qualitative experiments.

[0005] Currently, the human IgE proteins available on the market are as follows: 1) monoclonal IgE purified from the blood of patients with high IgE concentrations (e.g., IgE myeloma patients) (e.g., products from Abcam, Athens Research & Technology, MyBioSource, Fitzgerald Industries International, Molecular innovations, and Merck Millipore), 2) monoclonal IgE purified from the culture medium of hybridoma cells created by fusing B cells derived from healthy individuals with myeloma cells, and 3) monoclonal IgE purified from the culture medium of a cell line obtained by transfection with the IgE gene.

[0006] These IgE antibodies are more than 100 times more expensive than IgG, and contain variable regions (VH and VL), so they may exhibit non-specific binding to unknown antigens in IgE quantification experiments and various immunological experiments. There is a common purpose international standard IgE [WHO IgE International standard, the most recent is the 3rd International Reference Preparation (IRP), coded 11 / 234] that is used to quantify total IgE in blood. WHO IRP 11 / 234 is made by collecting plasma (or serum) from patients in many countries with high IgE concentrations in their blood, placing them in ampoules, and freeze-drying them. Since it contains blood components, it is considered a biohazard, so caution is required when using it in laboratories, and it is polyclonal, so the possibility of non-specific binding to unknown antigens cannot be excluded.

[0007] Therefore, the present inventors attempted to develop a new type of antibody fragment that can be substituted for the IgE reference antibody while avoiding the shortcomings of the existing IgE reference antibody. In addition, the present inventors attempted to develop a molecule that can be substituted for IgE not only in quantitative experiments to measure IgE concentration but also in qualitative experiments to inhibit FcεR-IgE interaction by being able to bind to the Fc epsilon receptor (FcεR) while having a high production yield.

[0008] As a result, molecules that consist only of constant regions and do not have variable regions, in particular, heavy chains in which the gamma constant region (Cγ1)-hinge and the epsilon constant region (Cε2-4) are fused (Cγ1-hinge-Cε2-4) and light chain kappa constant region (C K The present inventors have developed an antibody fragment (IgCw-γεκ) composed of the IgE-specific IgE-binding domain and the IgE-specific IgE-binding domain. The inventors have confirmed that the IgCw-γεκ molecule can be used as an IgE substitute in IgE concentration measurement experiments and immunological research using FcεR-IgE interactions, and that it can suppress IgE-mediated hypersensitivity reaction diseases, thereby completing the present invention.

[0009] The information provided in this Background section is intended to enhance understanding of the background of the invention only and, as such, may not include information that constitutes prior art already known to those of ordinary skill in the art to which the invention pertains. Summary of the Invention [Problem to be solved by the invention]

[0010] The object of the present invention is to provide a method for the preparation of a fusion protein comprising the steps of: H ) and light chain (C L The object of the present invention is to provide an antibody fragment consisting only of the constant region of

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

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

[0013] An object of the present invention is to provide a composition for evaluating antibody efficacy, comprising the antibody fragment.

[0014] An object of the present invention is to provide a composition for measuring an antibody concentration, which comprises the antibody fragment.

[0015] An object of the present invention is to provide a composition for suppressing an allergic reaction, which comprises the antibody fragment.

[0016] An object of the present invention is to provide a composition for suppressing IgE-mediated autoimmune reactions, which comprises the antibody fragment. [Means for solving the problem]

[0017] To achieve the above-mentioned object, the present invention provides 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 linked to the heavy chain constant region fragment.

[0018] The present invention also provides a nucleic acid encoding the antibody fragment.

[0019] The present invention also provides a kit comprising the antibody fragment.

[0020] The present invention also provides a composition for evaluating antibody efficacy, comprising the antibody fragment.

[0021] The present invention also provides a composition for measuring an antibody concentration, comprising the antibody fragment.

[0022] The present invention also provides a composition for suppressing an allergic reaction, comprising the antibody fragment.

[0023] The present invention also provides a composition for suppressing IgE-mediated autoimmune reactions, comprising the antibody fragment. [Brief description of the drawings]

[0024] [Figure 1] 1 shows the structure of IgCw-γ1ε2-4 / κ (simply IgCw-γεκ) protein, depicting an antibody fragment (approximately 130 kDa) consisting of the Cγ1-hinge region and Cε2-Cε3-Cε4 of a human antibody. [Diagram 2] This figure shows the results of analyzing the purification purity and integrity of each purified antibody protein. The results show that IgCw-γεκ has a molecular weight of about 130 kDa as expected, as shown in the figure. A shows the results of Coomassie staining after SDS-PAGE, and B shows the results of size exclusion chromatography. [Diagram 3] Figure 1 shows the binding analysis of IgCw-γεκ to FcεR. Flow cytometry results showing that IgCw-γεκ antibody fragment protein can bind to the high affinity human Fcε receptor (FcεR1) expressed on the surface of RBL-2H3-FcεRIα cells as well as control IgE. [Figure 4] This is a diagram showing the results of confirming degranulation reaction due to cross-linking of IgCw-γεκ. The experimental results show that when RBL-2H3-FcεRIα cells were treated with IgCw-γεκ for sensitization and then treated with anti-Cκ antibody (induction of cross-linking of FcεR1), IgCw-γεκ was able to induce β-hexosaminidase release (degranulation reaction) at a level similar to that of the control full-size IgE. A shows the experimental design, and B shows the experimental results. [Diagram 5]This is a diagram showing the results of suppressing degranulation reaction caused by IgCw-γεκ in IgE-sensitized cells. This is an experimental result showing that IgCw-γεκ can suppress β-hexosaminidase release (degranulation reaction) caused by 6C407 IgE + protein-L-biotin + streptavidin in RBL-2H3-FcεRIα cells. A shows the experimental design, B shows the results of measuring the level of β-hexosaminidase release in RBL-2H3-FcεRIα cells treated with a mixture of 6C407 IgE and IgCw-γεκ, and C shows the results of measuring the level of β-hexosaminidase release in cells treated with IgCw-γεκ after pretreating RBL-2H3-FcεRI cells with 6C407 IgE. [Figure 6] Figure 1 shows the results of IgCw-γεκ as a reference molecule for the quantification of human IgE antibodies. ELISA results showing that IgCw-γεκ protein can be used as a reference for the quantification of IgE antibodies. A shows the standard curve generated with 6C407 IgE, and B shows the standard curve generated with IgCw-γεκ. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein is one that is well known and commonly used in the art.

[0026] We attempted to produce an IgCw-εκ antibody fragment consisting only of the constant region of a human IgE antibody, but IgCw-εκ, which is composed only of the constant regions of the epsilon heavy chain and the kappa light chain, does not have a disulfide bond between the heavy and light chain constant regions. Therefore, when purified with Kappa XP-Agarose, which binds to human kappa constant regions, it was unable to bind to the heavy chain constant region, and only the kappa light chain constant region was purified.

[0027] Finally, in the antibody fragment IgCw-γεκ, consisting of the heavy and light chain constant regions fused to the gamma constant region (Cγ1)-hinge and epsilon constant region (Cε2-4), disulfide bonds between the constant regions were normally formed, and the expression level and purification yield were similar to those of the control 6C407 IgE. Thus, the fusion of the gamma constant region (Cγ1) with the epsilon constant region (Cε2-4) confers structural stability and high purification yield to the IgCw-γεκ protein.

[0028] In addition, the present invention provides a novel antibody fragment (IgCw-γ1ε) that can be substituted for the IgE reference antibody while avoiding the shortcomings of the previously known IgE reference antibody. 2-4 We have developed an antibody fragment that consists of two Cγ1-hinge-Cε2-4 hybrid heavy chains and two Cκ light chains, and is a molecule that consists only of constant regions and does not have variable regions.

[0029] Based on this, in one aspect, the present invention relates to 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 linked to the heavy chain constant region fragment.

[0030] In the present invention, the "antibody fragment" is a novel antibody format that includes an IgG antibody heavy chain constant region fragment and an IgE antibody heavy chain constant region fragment that lack the antibody variable region; and a light chain constant region fragment linked to the heavy chain constant region fragment. It has been discovered that IgCw-γεκ having such a new structure has various advantages and can be widely used in clinical fields such as antibody efficacy evaluation.

[0031] An unfragmented antibody comprises two heavy chains and two light chains linked by disulfide bonds. Each single light chain is linked to one of the heavy chains by a disulfide bond. The heavy chain portion of an antibody contains at its N-terminus a variable domain (V H) and the following multiple constant domains (3 or 4 constant domains, C H 1. C H 2. C H 3 and C H Each light chain portion has a variable region (VL) at the N-terminus and a constant region (C) at the other end (C-terminus). L ), and the light chain constant region has the first heavy chain constant region (C H 1), and the light chain variable region (V L ) is the variable region of the heavy chain (V H ) are aligned.

[0032] The constant region refers to the entire domain of an antibody other than the variable region. The constant region is not directly involved in binding between the antibody and its target antigen, but is involved in various effector functions in the body. The constant regions of the heavy and light chains are used to include all of those derived from IgA, IgD, IgE, IgG, and IgM, as well as IgY, IgW, and IgNAR.

[0033] In one embodiment, the IgG may include subtypes IgG1, IgG2, IgG3, and IgG4.

[0034] In one embodiment, the IgG antibody heavy chain constant region fragment can include Cγ1. The IgE antibody heavy chain constant region fragment can include one or more selected from the group consisting of Cε2, Cε3, and Cε4.

[0035] In a specific embodiment, the antibody fragment of the present invention comprises an IgG antibody heavy chain constant region fragment Cγ1, and an IgE antibody heavy chain constant region fragment Cε2, Cε3 and Cε4, and the IgG antibody heavy chain constant region fragment and the IgE antibody heavy chain constant region fragment may be linked in the N-terminus to C-terminus direction in the form of Cγ1-Cε2-Cε3-Cε4.

[0036] The IgG antibody heavy chain constant region fragment and the IgE antibody heavy chain constant region fragment can be linked via a hinge.

[0037] In one embodiment, the light chain constant region can include Cκ or Cλ. The light chain constant region fragment linked to the heavy chain constant region fragment can be linked, for example, by a disulfide bond or a peptide linker.

[0038] Recombinant antibody formats have frequently been challenged by low purification yields that can impact utility and cost. The production yield of antibodies is determined by the amount of the variable region of the heavy chain (V H ) and the light chain variable region (V L ) (amino acid sequence). Thus, the production yield of recombinant antibodies can be highly variable even with well-established production processes, as it depends on the amino acid sequence of the variable domain (V domain). In one embodiment of the present invention, it has been demonstrated that an antibody fragment composed only of the constant region of an antibody according to the present invention can be produced in high yield.

[0039] In another aspect, the present invention relates to a nucleic acid encoding said antibody fragment. The nucleic acid can be isolated and the antibody fragment can be produced in recombinant form. The nucleic acid can be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or further expression. Based on this, in yet another aspect, the present invention relates to a vector comprising said nucleic acid.

[0040] The term "nucleic acid" refers to a nucleic acid that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and the basic building block of a nucleic acid is a nucleotide, which includes not only natural nucleotides but also analogues in which the sugar or base moiety is modified. The nucleic acid sequences encoding the heavy and light chain variable regions of the present invention can be modified. Such modifications include addition, deletion, non-conservative substitution, or conservative substitution of nucleotides.

[0041] DNA encoding the antibody fragment is readily isolated or synthesized using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to DNA encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally 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.

[0042] The term "vector" as used herein includes, as a means for expressing a gene of interest in a host cell, plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, and other viral vectors, in which a nucleic acid encoding an antibody fragment is operably linked to a promoter.

[0043] "Operably linked" refers to a functional association between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, such that the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0044] When a prokaryotic cell is used as the host, it generally contains a strong promoter capable of transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for the initiation of translation, and a transcription / translation termination sequence. Furthermore, for example, when a eukaryotic cell is used as the host, promoters derived from the genome of a mammalian cell (e.g., metallothionine 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, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and they generally have a polyadenylation sequence as a transcription termination sequence.

[0045] In some cases, the vector may be fused to other sequences to facilitate purification of the antibody fragment expressed therefrom, such as glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).

[0046] The vectors contain antibiotic resistance genes commonly used in the art as selection markers, such as genes for resistance to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0047] In yet another aspect, the present invention relates to a cell transformed with the above-mentioned vector. The cells used to produce the antibodies of the present invention may be, but are not limited to, prokaryotic, yeast or higher eukaryotic cells.

[0048] Prokaryotic host cells such as Escherichia coli, strains of Bacillus, such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus) can be used.

[0049] However, of greatest interest are animal cells, and examples of useful host cell lines can be, 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, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0050] In yet another aspect, the present invention relates to a method for producing the antibody fragment, comprising: (a) culturing the cells; and (b) recovering the antibody fragment from the cultured cells.

[0051] The cells can be cultured in a variety of media. Any commercially available medium can be used as the culture medium without limitation. Any other necessary supplements known to those skilled in the art may be included at appropriate concentrations. The culture conditions, such as temperature, pH, etc., are already used with the host cell selected for expression and will be apparent to those skilled in the art.

[0052] The antibody fragment can be recovered by, for example, removing impurities by centrifugation or ultrafiltration, and purifying the resultant using, for example, affinity chromatography, etc. Additional purification techniques, for example, anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, etc., can be used.

[0053] In another aspect, the invention relates to a kit comprising the antibody fragment. The kit may include a container containing the antibody fragment, and containers containing other reagents or samples.

[0054] The kit is suitable for containing one or more containers, such as bottles, tubes, etc., each container containing an individual component used in the method of the present invention. With the present specification, a person skilled in the art can easily distribute the necessary formulations in the containers.

[0055] In another aspect, the present invention relates to a composition for evaluating antibody efficacy, comprising the antibody fragment.

[0056] In experiments to verify any effect of antigen-specific antibodies, reference (or irrelevant isotype control) antibodies are commonly used as negative controls to distinguish non-specific background signals from antigen-specific antibody signals. However, existing reference antibodies may still give undesired noise signals due to as yet unknown cross-reactivities.

[0057] The antibody fragment according to the invention may be a better reference antibody in many experimental settings and situations compared to an irrelevant IgE control, since it has no antigen-binding capacity, eliminating the possibility of unpredictable cross-reactivity.

[0058] In another aspect, the present invention relates to a composition for measuring an antibody concentration, comprising the antibody fragment.The antibody fragment according to the present invention can be used as a reference for measuring the concentration of immunoglobulin in a biological sample.

[0059] In another aspect, the present invention relates to a composition for suppressing an allergic reaction, which comprises an antibody fragment.

[0060] Most allergic diseases are caused by an excessive immune response of immunoglobulin E (IgE). IgE is an antibody that is normally present in serum at a very low concentration. IgE can be produced by generally harmless antigens, and IgE can increase without any particular stimulation, which can lead to allergic diseases. Abnormally increased IgE can bind to high affinity IgE Fc receptors (FcεRI) expressed on the surface of mast cells and basophils. When an antigen (mainly a harmless antigen) binds to many IgE molecules simultaneously while the IgE molecules are bound to FcεRI, cross-linking occurs between each FcεRI receptor, and a signal is transmitted to the mast cells and basophils, which are activated. As a result of such cell activation, mast cells and basophils release chemical mediators such as histamine, leukotrienes, prostaglandins, bradykinin, and platelet-activating factor. The release of these chemical mediators results in allergic symptoms. The antibody fragments of the present invention may be used as blockers of the Fcε receptor (FcεRI) to inhibit allergic reactions.

[0061] In another aspect, the present invention relates to a composition for suppressing an autoimmune reaction, which comprises an antibody fragment.

[0062] One of the pathogenic 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 can secrete 1,000 times more IFN-α than other cell types upon activation. When a complex of an IgE isotype autoantibody and a DNA antigen (immune complex) binds to FcεRI on the surface of pDCs, the immune complex enters the cell via FcεRI. The immune complex stimulates intracellular receptors such as Toll-like receptors (TLR)-7 and -9, activating pDCs and secreting large amounts of inflammatory cytokines, which is known to exacerbate the disease. The antibody fragments according to the invention may be used as blockers of the Fc epsilon receptor (FcεRI) to suppress autoimmune reactions mediated by IgE autoantibodies.

[0063] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0064] Example 1: Sample preparation Example 1-1: Plasmid vector To create the KV10-IgCw-γεκ vector in which two CMV promoters each regulate genes and simultaneously express human Cγ1 / ε2-4 and human Cκ with leader sequences, a DNA fragment encoding a hybrid of the human heavy chain constant regions Cγ1 and ε2-4 was cloned between the NcoI and BamHI restriction sites of the KV10-IgCWγκ vector.

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[0068] To construct a plasmid vector for expressing chimeric IgE (6C407, 3D8), a V-linker was inserted between the MluI and NhEI restriction sites of the KV12-HL vector and between the DraIII and BstWI restriction sites. H and V K A DNA fragment encoding the domain was cloned.

[0069] To construct the KV10-IgCw-εκ vector, which simultaneously expresses human Cε1-4 and human Cκ with leader sequences regulated by two CMV promoters, a DNA fragment encoding the human heavy chain constant region (Cε1-4) was cloned between the NcoI and BamHI restriction sites of the KV10-IgCWγκ vector.

[0070] To generate a lentiviral vector expressing human FcεRIα, a DNA fragment encoding human FcεRIα was cloned between the EcoI and BamHI restriction sites of the pCDH-CMV-MCS-EF1-Puro vector.

[0071] Example 1-2: Preparation of immunoglobulin proteins using HEK293F cells The FreeStyle™ 293-F cell line (Thermo Fisher Scientific), adapted to grow in suspension culture in serum-free conditions, was used for antibody production.

[0072] 2 × 10 at the time of transfection 6 24 hours prior to transfection, 1 × 10 FreeStyle HEK293F cells were seeded into a 500 ml flask (Corning Cat# 431145) to have a cell density of 1 × 10 cells / ml.6 The cells were cultured at 100 ml per ml at 1000 x 1000 cells / ml. FreeStyle 293-F cells were cultured in serum-free FreeStyle 293 medium (Invitrogen Cat# 12338) in an incubator at 8% CO2 and 37°C with shaking at 130 rpm. To express 6C407 IgE, IgCw-εκ, and IgCw-γεκ proteins, 200 μg of KV plasmid encoding each gene and 400 μg of polyethyleneimine (PEI) reagent (Polyscience Cat# 23966-2) were added to 5 ml of FreeStyle 293 medium and incubated at room temperature for 10 minutes. The 5 ml of medium containing DNA was then filtered through a 0.22 μm syringe filter (Millipore Cat# SLGV033RB), mixed with 5 ml of medium containing polyethyleneimine, and incubated at room temperature for 10 minutes. Finally, 100 ml of Freestyle 293-F cells were transiently transfected with polyethyleneimine at a concentration of 4 μg / ml. After 7 days, the culture was centrifuged at 4°C and 400 g for 20 minutes to obtain the supernatant, which was then filtered through a 0.45 μm cellulose acetate filter (Sartorius Cat# 11106-47-N) and purified using the CaptureSelect TM KappaXP (Thermo Fisher Scientific; Cat# 2943212005) columns or CaptureSelect TM The IgG-CH1 (Thermo Fisher Scientific; Cat# 194320005) column was passed through. 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 (4°C, 1500g) using Vivaspin 20 (molecular cut-off 50,000, Sartorius Cat# VS2032).

[0073] The concentrations of the purified proteins were determined using the absorbance at 280 nm and the extinction coefficients, which were 1.49 for 6C407 IgE, 1.34 for IgCw-εκ, and 1.17 for IgCw-γεκ, and were calculated from the amino acid sequences at http: / / web.expasy.org / protparam / .

[0074] Example 1-3: Cell culture RBL-2H3 (ATCC® number: CRL-2256) derived from rat basophilic leukemia cells TM ) cell line and human embryonic kidney 293 cell line HEK293T (ATCC® number: CRL-3216 TM ) were cultured in DMEM (Dulbecco's Modified Eagle's Medium, Welgene Inc.) medium 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 an incubator with 5% CO2 at 37°C.

[0075] Examples 1-4: Cellular construction The human embryonic kidney 293 cell line HEK293T was used for production of supernatants containing lentivirus expressing FcεRIα.

[0076] 3 × 10 at the time of transfection 6 24 h before transfection, 60 mm 2 HEK293T cells were cultured at 1.5 × 10 6The cells were cultured in 4 ml of DMEM medium at 1000 cells / well. After 24 hours, the medium was discarded, and the cells were cultured in Opti-MEM medium containing 4 μg of pCDH plasmid, which contains a gene encoded to express FcεRIα, 3 μg of gag / pol (addgene cat# 14887), 1 μg of VSV-G (addgene cat# 14888), and 16 μg of polyethyleneimine (PEI) reagent (Polyscience Cat# 23966-2) in 3.8 ml of DMEM medium. TM (Thermo Fisher Scientific; Cat# 31985070) 200μl were mixed. After culturing for 16 hours at 37℃ with 5% CO2, the medium was discarded, and 4ml of DMEM was added and cultured for another 24 hours. After 24 hours, the medium was collected and centrifuged at 2000rpm for 3 minutes to obtain the supernatant, which was then filtered through a 0.45μm syringe filter (Millipore Cat# SLHV033RS) to obtain the supernatant containing the lentivirus.

[0077] A supernatant containing lentivirus was prepared in RBL-2H3 cells to express a receptor that recognizes human IgE.

[0078] 2×10 when infected 6 24 hours before infection, 60 mm 2 In a culture dish, place 1 x 10 RBL-2H3 cells. 6The cells were cultured in 4 ml of DMEM medium at 100 cells / well. After 24 hours, the medium was removed, the cells were washed once with PBS (phosphate-buffered saline, pH 7.4), and 3 ml of DMEM was mixed with 1 ml of the lentivirus-containing supernatant, and polybrene (Sigma-Aldrich; Cat# H9268) was added to a concentration of 10 μg / ml. After 24 hours of culture, the medium was discarded, 4 ml of DMEM medium was added, and the cells were cultured again for 12 hours. Then, the cells were cultured in the presence of puromycin (Sigma-Aldrich; Cat# 540411) at a concentration of 5 μg / ml.

[0079] Example 2: Analytical Method Example 2-1: Flow cytometry To confirm whether the human Fcε receptor was expressed in RBL-2H3-hFcεRIα cells, cells (1 × 10 6 After washing the cells with cold PBS, the cells were fixed by treating them with 4% paraformaldehyde diluted in PBS at room temperature for 20 minutes. The detection antibody, APC-conjugated mouse anti-human Fc epsilon RIAb (Abcam; Cat# 155369), was used after diluting it in buffer S (0.5% BSA, 2 mM EDTA, pH 8.5, diluted in PBS). The resultant was left at 4°C for 1 hour and then washed three times with cold PBS. Each sample was analyzed using a FACS CantoII analyzer (BD Biosciences).

[0080] To confirm whether IgCw-γεκ binds to Fcε receptors expressed on the cell surface, FcεR-RBL-2H3, FcεR+RBL-2H3-hFcεRIα cells (1 × 10 6RBL-2H3 and RBL-2H3-hFcεRIα cells were treated with immunoglobulin protein at a final concentration of 1 μM for 3 hours at 37°C. After washing the cells with cold PBS, the cells were fixed with 4% paraformaldehyde diluted in PBS for 20 minutes at room temperature. For RBL-2H3 and RBL-2H3-hFcεRIα cells, goat anti-human IgE (ε-chain specific) antibody (Sigma-Aldrich; Cat# I6284) was used as the primary antibody, and PE-conjugated donkey anti-goat IgG antibody (Abcam; Cat# Ab7004) was used as the secondary antibody, diluted in buffer S. After each fixation step (4°C, 1 hour), the cells were washed three times with cold PBS. Each sample was analyzed using a FACS CantoII analyzer (BD Biosciences).

[0081] Example 2-2: Enzyme-linked immunosorbent assay (ELISA) for measuring IgE concentration To quantify human IgE, we performed an enzyme immunoassay to generate a standard curve for full-size IgE and IgCw-γεκ using the Human IgE ELISA Ready-SET-Go (Invitrogen; Cat# 88-50610) kit included in the kit. The experimental method followed the kit's instructions.

[0082] The wells of a 96-well polystyrene plate were treated with the coating antibody and coated at 4°C for 16 hours. After that, the plate was washed four times with TBS (TRIS-buffered saline; 50 mM TRIS-Cl, 50 mM NaCl, pH 7.4) containing 0.05% Tween-20 at each settling step (room temperature). To block nonspecific antibody binding, the plate was treated with 3% bovine serum albumin (BSA) at room temperature for 2 hours. Human polyclonal IgE and IgCw-γεκ were serially diluted two-fold from a starting concentration of 250 ng / ml, and the plate was treated with the detection antibody at room temperature for 2 hours. 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.).

[0083] To measure the concentration of unknown antibodies, wells of a 96-well polystyrene plate were coated with the coating antibodies for 16 hours at 4°C. Monoclonal 6C407 IgE or 3D8 IgE were treated followed by detection antibodies (both for 1 hour at room temperature). The concentration of IgE samples was determined by interpolation of the Y-axis values ​​in two different standard curves using known concentrations of human IgE and IgCw-γεκ, respectively.

[0084] Example 2-3: Degranulation assay: beta hexosaminidase release assay To confirm whether the IgE-Fcε receptor complex exhibited degranulation, β-hexosaminidase release was measured. RBL-2H3-hFcεRIα cells were cultured at 5 × 10 5 To adjust the cells / well, 2.5 × 10 5Cells / well were cultured in 400 μl of DMEM medium in a 24-well plate (SPL; Cat# 30024). After 24 hours, IgE was added to 400 μl of DMEM medium without fetal bovine serum, penicillin, and streptomycin at a concentration of 10 nM for IgE sensitization, and then cultured for 3 hours. After removing IgE, the cells were 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 resulting material was placed in 160 μl of Siraganian buffer in an incubator at 5% CO2 and 37°C for 10 minutes. Goat anti-human kappa light chain antibody (Invitrogen; Cat# 31129) was mixed with Shiraganian buffer to a final concentration of 10 μg / ml, and 20 μl of the mixture was added to each well and incubated for 20 minutes. 0.1% Triton X-100, used as a control, was incubated in an incubator at 5% CO2 and 37°C for 1 hour. After incubation in an incubator at 5% CO2 and 37°C for 20 minutes, 50 μl of the mixture was added to each well of a 96-well polystyrene plate, followed by 50 μl of 1 mM p-NAG (p-nitrophenyl N-acetyl-beta-D-glucosamine in 0.1M 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 was measured at 405 nm using a microplate reader (Molecular Devices Inc.).

[0085] Example 2-4: β-hexosaminidase release assay to confirm the role of Fcε receptor as a blocker To determine whether the IgE-Fcε receptor complex acts as a blocker of the Fcε receptor that prevents other IgE-induced degranulation events, β-hexosaminidase release was measured. RBL-2H3-hFcεRIα cells were cultured at 5 × 10 5 To prepare 2.5 × 10 cells / well, 24 hours before 5 The cells / well were cultured in 400 μl of DMEM medium in a 24-well plate. After 24 hours, IgE was sensitized by adding 10 nM IgE and blocker IgE, which were serially diluted in increments of 2 from the starting concentration of 20 nM, to 400 μl of DMEM medium without fetal bovine serum, penicillin, or streptomycin, and then cultured for 3 hours. The cells were then washed twice with 500 μl of Shiraganian buffer and placed in 160 μl of Shiraganian buffer for 10 minutes in a 5% CO2, 37°C incubator. Recombinant biotinylated Protein L (Thermo Fisher Scientific; Cat# 29997) and streptavidin-fluorescein (Vector Laboratories; Cat# SA-5001) were mixed in Shiraganian buffer at a final concentration of 140 nM, and 20 μl of each was added to each well for 20 min. 0.1% Triton X-100 was used as a control and incubated in a 5% CO2 and 37°C incubator for 1 h. The resulting mixture was incubated in a 5% CO2 incubator at 37°C for 20 minutes, and 50 μl of the mixture was added to each well of a 96-well polystyrene plate. 50 μl of 1 mM p-NAG (p-nitrophenyl N-acetyl-beta-D-glucosamine in 0.1M citrate buffer, pH 4.5) was then added to each well. The resulting mixture was treated at 37°C for 1 hour, and 200 μl of stop solution (0.1M Na2CO3 / NaHCO3, pH 10.0) was added to each well, and the absorbance was measured at 405 nm using a microplate reader (Molecular Devices Inc.).

[0086] Example 3: Confirmation of purification yield of IgCw-γεκ The yields of each immunoglobulin molecule produced by suspension culture of KV10-IgCw-γεκ, KV12-6C407 IgE, KV10-IgCw-εκ, and the plasmids transiently transfected into HEK293F cells were compared (Table 1). The predicted structures of the expressed proteins are shown in Figure 1. All proteins were purified using Kappa XP-Agarose, which binds to the human kappa constant region, 7 days after transfection. IgCw-εκ, which is composed only of the constant regions of the epsilon heavy chain and the kappa light chain, was able to purify only the kappa constant region because no disulfide bond is formed between the heavy and light chain constant regions (Figure 2). On the other hand, the antibody fragment IgCw-γεκ, which consists of the heavy and light chain constant regions in which the gamma constant region (Cγ1) and the epsilon constant region (Cε2-4) are fused, had normal disulfide bonds between the constant regions, and its expression level was similar to that of the control 6C407 IgE (Figure 2). The average yield was ~17 mg / L, the same as that of 6C407 IgE, when purified with KappaXP-agarose, and was IgG-C H When purified with 1-agarose, the concentration was 27 mg / L (Table 1). This indicates that the IgCw-γεκ protein is structurally more stable than the IgCw-εκ protein and has a superior purification yield.

[0087] [Table 1]

[0088] It has been shown that, depending on the purification method, the purification yield of IgCw-γεκ protein is similar to or even higher than that of IgE.

[0089] Example 4: Confirmation of expression of human Fcε receptor in RBL-2H3 cells To confirm the expression of hFcεR in RBL-2H3-hFcεRIα cells expressing human Fcε receptor, flow cytometry was performed.

[0090] Anti-Fcε antibody did not bind to wild-type RBL-2H3 cells, but did bind to RBL-2H3-hFcεRIα cells expressing Fcε receptors (Figure 3), indicating that human Fcε receptors are not expressed in wild-type RBL-2H3 cells, but are present only in RBL-2H3-hFcεRIα cells expressing human Fcε receptors.

[0091] Example 5: Confirmation of IgCw-γεκ binding to Fcε receptor and confirmation of background signal To confirm whether IgCw-γεκ binds to each Fcε receptor on cells expressing human Fcε receptors on their surface, flow cytometry was performed.

[0092] IgCw-γεκ binds to RBL-2H3-hFcεRIα cells that have been engineered to express hFcεRIα in the same way as full-sized IgE, but does not bind to RBL-2H3 cells that do not express any hFcεRIα (Figure 3), indicating that IgCw-γεκ can recognize Fcε receptors like full-sized IgE.

[0093] Example 6: Use of IgCw-γεκ as a reference for degranulation experiments with IgE In experiments where IgE is used, such as in the degranulation assay, the use of IgCw-γεκ instead of full-sized IgE was confirmed by β-hexosaminidase release experiments.

[0094] Along with polyclonal IgE and 6C407 IgE, the experiments were also performed with 6C407 IgG and PBS used as controls. β-hexosaminidase release from RBL-2H3-hFcεRIα cells was not observed with the negative controls 6C407 IgG and PBS, and was observed only in the IgE-treated samples (Figure 4). IgCw-γεκ showed a secretion percentage similar to or higher than that of full-sized IgE, indicating that the function of IgCw-γεκ is similar to that of full-sized IgE. This indicates that IgCw-γεκ can be used as a reference molecule in degranulation experiments instead of human IgE.

[0095] Example 7: Use of IgCw-γεκ as a blocker of Fcε receptors We examined whether IgCw-γεκ could be used as a blocker of the Fcε receptor.

[0096] IgCw-γεκ cannot bind to Protein L because it does not have a variable region. Therefore, activation signals of RBL-2H3-hFcεRIα cells can be observed only when full-sized IgE binds to Fcε. Not only when a mixture of 6C407 IgE and IgCw-γεκ was treated with RBL-2H3-hFcεRIα cells (Figure 5B), but also when RBL-2H3-hFcεRIα cells were pretreated with 6C407 IgE and then treated with IgCw-γεκ (Figure 5C), the signal for 6C407 IgE decreased with increasing concentration of IgCw-γεκ (Figure 5). This means that IgCw-γεκ interfered with the binding of full-sized IgE to the Fcε receptor, indicating that IgCw-γεκ can be used as a blocker of the Fcε receptor.

[0097] Example 8: Use of IgCw-γεκ as a reference for quantification of IgG concentration We sought to determine whether IgCw-γεκ could be used as a surrogate for full-size IgE, the reference used to determine IgG concentrations.

[0098] Two logarithmic standard curves were constructed by enzyme-linked immunosorbent assay (ELISA) using known concentrations of human polyclonal IgE (Figure 6A) and IgCw-γεκ (Figure 6B), respectively. Identical experiments were performed using two monoclonal IgE specimens (6C407 and 3D8) of unknown concentrations. The IgE concentrations were determined by interpolation in each curve (Table 2), using the linear interpolation formula (https: / / formulas.tutorvista.com / math / interpolation-formula.html) x = x1 + (x2 - x1) × (y - y1) / (y2 - y1).

[0099] [Table 2] *The IgE concentration determined by the IgCw-γεκ standard curve was multiplied by the molecular weight ratio to correct the value [molecular ratio of IgE (~190 kDa):IgCw-γεκ (~130 kDa) = 1.462:1].

[0100] 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), indicating that IgCw-γεκ can be used as a reference molecule to determine the concentration of human IgE. [Industrial Applicability]

[0101] The antibody fragments according to the invention can be widely used as alternatives to IgE reference antibodies used in various immunological studies, can be used as reference molecules for the measurement of the concentration of IgE antibodies in a sample and can be used as blockers of the Fc epsilon receptor (FcεRI) to inhibit type 1 allergic reactions.

[0102] The antibody fragments according to the invention can also be used as blockers to inhibit autoimmune reactions associated with autoantibodies of the IgE isotype.

[0103] In addition, since the antibody fragment of the present invention does not contain a variable region, it is possible to completely eliminate non-specific cross-reaction, i.e., off-target effects due to reactions with other antigens, which is a drawback of previously used full-length isotype control antibodies, and therefore it can be more appropriately used in quantitative and qualitative experiments of IgE and can be used as an isotype control antibody in the effectiveness evaluation of the activity of experimental / therapeutic antibodies.

[0104] Although the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific description is merely a preferred embodiment and does not limit the scope of the present invention. Therefore, it can be said that the substantial scope of the present invention is defined by the appended claims and their equivalents. [Sequence List Free Text]

[0105] Electronic file attached.

Claims

1. An antibody fragment comprising: a heavy chain constant region fragment of an IgG antibody consisting of Cγ1 and a heavy chain constant region fragment of an IgE antibody consisting of Cε2, Cε3 and Cε4; and a light chain constant region fragment consisting of Cκ or Cλ linked to the heavy chain constant region fragment of the IgG antibody; the IgG antibody heavy chain constant region fragment and the IgE antibody heavy chain constant region fragment are linked in the form of Cγ1-Cε2-Cε3-Cε4 in the N-terminal to C-terminal direction; The antibody fragment does not contain the variable domains of the antibody.

2. The antibody fragment according to claim 1 , wherein the IgG antibody heavy chain constant region fragment and the IgE antibody heavy chain constant region fragment are linked via a hinge.

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

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

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

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

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

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

9. A composition for suppressing an autoimmune reaction, comprising the antibody fragment according to any one of claims 1 to 3.

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