Antibody drug

Abstract

To provide development means of new antibody drugs by elucidating how HMK-12 Fab dissociates IgE molecules bound to an IgE receptor (FcεRI) on mast cells.SOLUTION: A method for screening antibody drugs comprises selecting an antibody that binds to a regulatory site other than an active site of a target antigen, thereby changing ta structure of the active site and controlling the affinity between the target antigen and a target antigen receptor.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a screening method for an antibody drug and an antibody drug. [Background technology]

[0002] Antibody drugs are drugs that block the action of causative substances by administering specific antibodies against those substances. Ordinary antibodies recognize a specific site on the causative substance. For example, the recognition site is a specific site on the target antigen that reacts with a receptor on the body side. An example of this is explained using antibody drugs for the treatment of type I allergic diseases.

[0003] In recent years, various allergic diseases have been increasing remarkably in developed countries including Japan. In particular, it is considered extremely important to suppress the activity of IgE antibodies that recognize allergens in the treatment of type I (immediate type) allergies, such as hay fever and food allergies. Currently, an anti-IgE antibody preparation (Omalizumab: drug name: Xolair), which inhibits the binding of IgE to IgE receptors, is being used clinically as a treatment for intractable asthma and other conditions. However, it is slow-acting and takes 2 to 3 weeks for the therapeutic effect to appear, and it has been reported that administration is restricted when serum IgE levels are high and that there are side effects such as anaphylactic shock. From this perspective, the development of an anti-IgE antibody drug that can immediately and long-term prevent type I allergic diseases is an urgent task.

[0004] The present inventors have developed an anti-IgE antibody (HMK-12 Fab) that immediately blocks type I allergic reactions, and have reported that the mechanism is due to the dissociation of IgE molecules bound to the IgE receptor (FcεRI) on mast cells (Patent Document 1). Furthermore, through research into PCA reactions (passive anaphylactic reactions), they have demonstrated that administration of HMK-12 Fab prior to allergen stimulation detaches IgE from mast cells, suppressing type I allergic reactions (Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2008-203656 A Summary of the Invention [Problem to be solved by the invention]

[0006] However, it remained unclear how the HMK-12 Fab dissociates IgE molecules bound to the IgE receptor (FcεRI) on mast cells. Therefore, an object of the present invention is to provide a means for developing new antibody drugs by elucidating how HMK-12 Fab dissociates IgE molecules bound to the IgE receptor (FcεRI) on mast cells. [Means for solving the problem]

[0007] The present inventors found that the Fab fragment of an anti-lgE antibody (HMK-12 Fab) rapidly dissociates preformed lgE-FcεRI complexes even after allergen stimulation, and inhibits lgE-mediated anaphylactic reactions. Therefore, the present inventors analyzed the IgE-binding site of HMK-12 Fab and found that the binding epitope of HMK-12 Fab is present in the IgE F(ab')2 region. Furthermore, from an analysis of the crystal structure of the HMK-12 Fab / IgE F(ab')2 complex, they found that (1) HMK-12 Fab recognizes an epitope on the surface of the side where the carbonyl terminus of Cε2 of IgE F(ab')2 forming a homodimer is present, and that in IgE before it is converted to IgE F(ab')2 by enzymatic treatment, the epitope is present on the surface close to Cε3 of the constant region, and (2) HMK-12 Fab binds to IgE F(ab')2 and dissociates IgE bound to a receptor by changing the asymmetric structure of the Fc domain, which is another part of IgE (allosteric effect). Therefore, we have found that antibody drugs can be screened by selecting antibodies that bind to a regulatory site other than the active site of a target antigen, thereby changing the structure of the active site and controlling the affinity between the target antigen and the target antigen receptor.

[0008] That is, the present invention provides the following inventions [1] to [5]. [1] A method for screening antibody drugs, comprising selecting an antibody that binds to a regulatory site other than the active site of a target antigen, thereby changing the structure of the active site and controlling the affinity between the target antigen and a target antigen receptor. [2] The method for screening an antibody drug according to [1], wherein the antibody that controls the affinity between the target antigen and the target antigen receptor is an antibody that inhibits the binding between the target antigen and the target antigen receptor. [3] The screening method described in [2], in which a regulatory site other than the active site of the target antigen is identified by crystal structure analysis of the target antigen. [4] The screening method according to any one of [1] to [3], wherein the target antigen is IgE, the target antigen receptor is FcεRI, and the regulatory site other than the active site of the target antigen is the heavy chain of the Cε2 homodimer of IgE F(ab´)2. [5] An antibody drug selected by the screening method according to any one of [1] to [4]. Effect of the Invention

[0009] According to the screening method of the present invention, by selecting an antibody that binds to a regulatory site other than the active site of a target antigen and changes the structure of the active site to control the affinity between the target antigen and the target antigen receptor, it is possible to develop an antibody drug for a disease based on a target antigen with an action mechanism completely different from that of conventional antibodies. [Brief description of the drawings]

[0010] [Figure 1] FIG. 13 shows that HMK-12 Fab inhibits binding of SPE-7 IgE to PT18 cells in a dose-dependent manner. [Diagram 2] FIG. 1 shows the results of SDS-PAGE under reducing and non-reducing conditions for HMK-12 Fab, 6HD5 Fab, and anti-rat λ chain. [Diagram 3] FIG. 13 shows the results of SDS-PAGE after treatment with serial dilutions of HMK-12 Fab, 6HD5 Fab, and anti-rat λ chain with dithiothreitol (DTT: 5 to 20 mM). [Figure 4] FIG. 13 shows the results of SDS-PAGE under non-reducing conditions after digestion of SPE-7 IgE with pepsin, and Western blot analysis of the reactivity with HMK-12 Fab. [Diagram 5] FIG. 1 shows the results of purification of HMK-12 Fab and SPE-7 IgE F(ab')2 using a Superose 12 10 / 300 GL column (A), and the SDS-PAGE results of fractions 9 to 14 of the purified complex of HMK-12 Fab and IgE F(ab')2, and their molecular weights. [Figure 6] FIG. 1 shows the crystal structure of the HMK-12 Fab / lgE F(ab′)2 complex. [Figure 7] FIG. 1 shows epitope analysis of HMK-12 Fab (1). [Figure 8] FIG. 1 shows epitope analysis of HMK-12 Fab (2). [Figure 9] FIG. 1 shows epitope analysis of HMK-12 Fab (3). [Figure 10]FIG. 13 shows the temperature dependence of the dissociation efficiency of preformed lgE-FcERI complex by HMK-12 Fab. [Figure 11] FIG. 13 shows that the reduced intensity of SPE-7 lgE+ PT18 cells is due to dissociation of the lgE-FcERI complex. [Figure 12] The results of investigating the in vivo effect of HMK-12 Fab on IgE-mediated anaphylactic reactions using a passive cutaneous anaphylaxis (PCA) assay are shown (results before and after stimulation). [Figure 13] Shown is a superposition of Cε2 of lgE onto Cε2 of the receptor complex (A), and a superposition of Cε2 of lgE with the other Cε2 of the receptor complex (B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] One aspect of the present invention is a method for screening antibody drugs, which is characterized by selecting an antibody that binds to a regulatory site other than the active site of a target antigen, thereby changing the structure of the active site and controlling the affinity between the target antigen and a target antigen receptor.

[0012] An antigen is a substance that produces antibodies in an animal's body and reacts specifically with the antibodies, and typically includes foreign proteins, polysaccharides, nucleic acids, nuclear proteins, lipoproteins, synthetic polymers, etc. Antigens can be classified into those that originate from within the body (self-proteins) and those that originate from the external environment (non-self), and IgE, the causative agent of type I allergy, is a self-protein antigen. Among these antigens, the antigen against which an antibody is to be produced is the target antigen. Specific target antigens include IgE, which is the causative antigen of type I allergy, the surface or spike protein of a virus particle, a cancer cell surface antigen, etc. Among these, IgE, which is the causative antigen of type I allergy, the surface or spike protein of a virus particle, or a cancer cell surface antigen is preferred, and IgE, which is the causative antigen of type I allergy, is more preferred.

[0013] An epitope, also called an antigenic determinant, is a part of an antigen that is recognized by antibodies, B cells, and T cells, and is the site to which an antibody binds. The site to which the antibody obtained by the present invention binds, i.e., an epitope, is a regulatory site other than the active site of a target antigen. Here, the regulatory site is a site that controls the affinity between a target antigen and a target antigen receptor. As an antibody that controls the affinity between a target antigen and a target antigen receptor, an antibody that inhibits the binding between a target antigen and a target antigen receptor is preferred.

[0014] Methods for selecting antibodies that bind to regulatory sites other than the active site of a target antigen and change the structure of the active site to control the affinity between the target antigen and the target antigen receptor include epitope mapping analysis, analysis of the crystal structure of the antigen-antibody complex, SPR method, BLI method, and cryo-electron microscopy.

[0015] For example, an epitope mapping analysis method involves designing and synthesizing overlapping polypeptides, and arraying these polypeptides in triplicate on a glass slide along with a negative control (unrelated random peptide) and a positive control (full-length protein). After blocking the array, a primary antibody against the target protein and a biotin-labeled secondary antibody are added to the overlapping peptide array in sequence and incubated. After a wash step, the antibody bound to the epitope is detected by fluorescent Cy3-conjugated streptavidin. The fluorescence of each peptide spot is captured by a laser scanner, and positive peptides that give off strong signals can be further analyzed by software. Epitope mapping analysis makes it possible to select antibodies that control the affinity between a target antigen and a target antigen receptor, in particular, antibodies that inhibit the binding between a target antigen and a target antigen receptor.

[0016] The analysis of the crystal structure of an antigen-antibody complex can be carried out by the following steps: preparation of the antigen-antibody complex, crystallization, X-ray diffraction experiment of the antigen-antibody complex crystals produced by crystallization, determination of the structural coordinates by analyzing the X-ray diffraction intensity data, and visualization of the analyzed structural coordinates. The preparation of the antigen-antibody complex can be carried out by subjecting a solution containing a mixture of antibodies at a molar ratio of at least twice that of the antigen to a gel filtration column equilibrated with an appropriate buffer solution, and isolating the elution fraction with the retention time corresponding to the antigen-antibody complex. Crystallization can be carried out by mixing the antigen-antibody complex solution concentrated to a concentration of about 10 mg / mL after preparation with an appropriate precipitant solution, and then vapor equilibrating with a separately prepared precipitant solution. If the composition of the appropriate precipitant solution is unknown, a crystallization condition screening kit available on the market can be used. In the X-ray diffraction experiment of the antigen-antibody complex crystals produced by crystallization, X-rays are irradiated to the grown crystals attached to an X-ray diffractometer, and the diffracted X-rays from the crystals are recorded by an X-ray detector to obtain X-ray diffraction intensity data. The determination of structural coordinates from the analysis of X-ray diffraction intensity data can be carried out using molecular replacement techniques that take advantage of the similarity of antibody structures to each other. Visualization of structural coordinates can be carried out using appropriate molecular modeling software. X-ray crystallography of the target antigen-antibody complex can identify regulatory sites other than the active site of the target antigen.

[0017] Means for selecting an antibody that changes the function of the target antigen include means for detecting a change in the crystal structure of an antigen-antibody complex, and means for detecting a change in the characteristics of a target antigen when it binds to multiple epitopes.

[0018] The HMK-12 Fab is an IgE whose target antigen is IgE and whose target antigen receptor is FcεRI. That is, in an antibody whose target antigen is IgE and whose target antigen receptor is FcεRI, the regulatory site other than the active site of the IgE target antigen is an IgE epitope recognized by the antibody, and is preferably Cε2 (heavy chain) of IgE F(ab')2 forming a homodimer. These results demonstrated that, by binding to IgE F(ab´)2, HMK-12 Fab induces a conformational change in the asymmetric structure of the IgE Fc domain that is necessary for the target antigen IgE to bind to the target antigen receptor FcεRI, dissociating IgE from the receptor complex in an allosteric manner.

[0019] According to the present invention, it is possible to screen for antibody drugs against diseases based on target antigens. In the present invention, examples of diseases based on target antigens include type I allergies, viral infections, and cancers. EXAMPLES

[0020] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0021] Materials and Methods (1) Animals and cells Female Sprague-Dawley rats were purchased from SLC, Japan. Animals were housed under specific pathogen-free (SPF) conditions in the animal facility. To ensure that the experiments were performed under the same conditions without bias, all SPF animals were acclimated for 7 days before use. All animal studies were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Mouse mast cells (PT-18) were provided by Dr. C. Ra (Department of Immunology, Nihon University, Tokyo, Japan). PT-18 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA) supplemented with 10% heat-inactivated FCS, 3 mM L-glutamine, 1 mM sodium pyruvate, penicillin (100 U / ml), and streptomycin (100 μg / ml) and maintained in a humidified incubator (5% CO2) at 37 °C.

[0022] (2) Monoclonal antibodies SPE-7 IgE (anti-DNP mouse IgE antibody) and SPE-7 IgE F(ab´)2 were purchased from Sigma-Aldrich Co. LLC (USA) and Immuno-Biological Laboratories Co. (Gunma, JAPAN), respectively. Rat monoclonal antibody HMK-12 (mouse IgE) was described in Int Arch Allergy Appl Immunol. 85, 47-54 (1988) and Int Arch Allergy Appl Immunol. 128, 24-32 (2002). Rat IgG anti-mouse K antibody and HRP-labeled goat anti-rat IgG were purchased from BioLegend (CA, USA) and Jackson ImmunoResearch (PA, USA), respectively. Highly purified Fab fragments of HMK-12 were prepared using ficin, a cysteine ​​protease isolated from fig latex. Briefly, HMK-12 was purified stepwise from rat ascites using a combination of caprylic acid and ammonium sulfate methods. The purified HMK-12 was dialyzed against 0.1 M citrate buffer (pH 6.0), and then n-octyl-13-D-glucoside (DOTITE) and L-cysteine ​​(Wako) were added to increase the concentration to 0.5 mM and 25 mM, respectively. HMK-12 (1 mg / ml) was digested twice with 0.5 ml of immobilized ficin resin slurry (Thermo scientific prod. # 44881) in the presence of 25 mM cysteine / citrate buffer. The sample was then centrifuged and purified with Vivaspin (1,000,000) (Sartorius AG) to remove resin or undigested IgG. The resulting HMK-12 Fab fragment was further purified with a Protein G column (Thermo Fisher Scientific Inc.).

[0023] (3) PCA reaction Two rats were injected intradermally at several sites in freshly shaved skin with 1 μg / ml SPE-7 IgE and 2 days later with 5 μg / ml HMK-12 Fab or anti-κ Fab before being challenged with DNP-BSA (1 mg / ml) and 0.5% Evans blue in saline. Two days after intradermal injection of SPE-7 IgE, rats were given 0.5% Evans Blue in saline and 1 mg / ml DNP-OVA intravenously, and then rapidly injected with 10 μg / ml HMK-12 Fab or anti-κ Fab. In both cases, leakage of Evans Blue from blood vessels into tissues was quantified by absorbance measurement at 620 nm.

[0024] (4) Flow cytometry analysis To evaluate the effect of HMK-12 Fab on IgE binding to FcεRI, we first prepared Alexa 488-labeled SPE-7 IgE. First, various concentrations of Alexa 488-labeled SPE-7 IgE were premixed with an excess of HMK-12 Fab or IgG2a Fab (2 μM) and incubated at 37°C for 15 min. The mixture was added to PT-18 cells (mouse mast cells) and incubated at 37°C for 20 min. Then, the cells were washed twice with PBS and analyzed using a FACSCelesta flow cytometer (Becton Dickenson, CA, USA). To examine the efficiency of dissociation of preformed IgE-FcεRI complexes by HMK-12 Fab, 1x10 7 PT18 cells were preincubated with 0.2 μM Alexa 488-labeled SPE-7 IgE for 15 min at 37° C. The cells were washed twice to remove excess unbound antibodies and incubated with HMK-12 Fab or IgG2a Fab at various temperatures (2, 25, and 37° C.) for 5, 15, and 75 min. The cells were then washed and analyzed using a FACSCelesta flow cytometer. To further confirm the dissociation of the IgE-FcεRI complex, 1x10 7PT18 cells were preincubated with 0.2 μM Alexa 488-labeled SPE-7 IgE for 15 min at 37°C. After a washing step, cells were incubated with HMK-12 Fab or IgG2a Fab for 15 and 60 min at 37°C, and the frequency of SPE-7 IgE+PT18 cells was measured using a FACSCelesta flow cytometer. To measure the level of dissociated SPE-7 IgE, culture supernatants were added to DNP-BSA-coated plates to capture SPE-7 IgE / HMK-12 Fab immune complexes. Immune complexes were measured by ELISA using HRP-labeled anti-rat IgG(Fab´)2 secondary antibody.

[0025] (5) Western blot analysis SPE-7 IgE (1 μg) was reduced with 2.5% 2-mercaptoethanol (2-ME), electrophoresed in 7.5% SDS-PAGE sample buffer, and transferred to a PVDF membrane (Millipore corp.). The membrane was probed with HMK-12 Fab, 6HD5 Fab, and anti-rat λ chain, followed by secondary antibodies (HRP-conjugated anti-rat IgG for HMK-12 Fab and 6HD5 Fab, and HRP-conjugated anti-goat IgG for anti-rat λ chain). Signals were detected using Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific). Immunoreactivity was detected with an ImageQuant LAS 4000 (GE Healthcare). To examine how HMK-12 Fab reacts with IgE during the IgE reduction process, SPE-7 IgE was treated with serial dilutions of dithiothreitol (DTT: 5–20 mM) for 60 min at room temperature. Samples were then incubated with 5 μl of 1.5 M Tris-HCL (pH 8.8) and 10 μl of iodoacetamide (IAA) for 30 min on ice. After dialysis, SDS-PAGE was performed under non-reducing conditions, followed by Western blot analysis. To clarify the epitope on the IgE dimer recognized by HMK-12 Fab, SPE-7 IgE digested with pepsin at pH 3.8 or pH 4.5 was analyzed for reactivity of HMK12 Fab with pepsin-digested SPE-7 IgE by SDS-PAGE under non-reducing conditions and Western blot analysis.

[0026] (6) Crystallization The peak fraction containing the HMK-12 Fab / IgE F(ab´)2 complex was used for crystallographic studies. After adding sodium azide to a final concentration of 0.001% (w / v), the peak fraction was concentrated to 13.3 mg / ml using a VivaSpin with Mwco 10,000. Crystallization was performed by the hanging drop vapor diffusion method. Crystallization droplets were prepared by mixing 0.1 μl of concentrated complex sample with 0.1 μl of crystallization solution (14% (w / v) PEG3350, 0.09 M MES-NaOH (pH 6.5), 0.09 M magnesium acetate, 0.09% (w / v) β-octylglucoside, 0.1 M NDSB-256). The droplets were equilibrated in 50 μl of crystallization solution at 4 °C. Prior to freezing crystals for X-ray diffraction experiments, 0.2 μl cryoprotectant solution (15% (w / v) PEG3350, 0.1 M MES-NaOH (pH 6.5), 0.1 M magnesium acetate, 0.1% (w / v) β-octylglucoside, 0.1 M NDSB-256, 40% (v / v) ethylene glycol) was added to a crystallization drop containing needle-shaped crystallites with dimensions of tens of μm in length and a few μm in thickness.

[0027] (7) Diffraction experiments and structure analysis X-ray diffraction experiments and diffraction image processing were performed automatically using the multi-crystal data collection mode of the ZOO system at BL32XU of SPring-8. The X-ray wavelength used in the diffraction experiments was 1.0 Å. Diffraction images were collected from 348 crystals held in a nitrogen gas stream at 100 K. The diffraction images were processed and CC images were obtained in the best resolution shell from 3.00 Å to 2.90 Å. 0.5 Diffraction intensity data was obtained at a resolution of 2.9 Å with an average diffraction intensity of 0.899. The initial phase calculations were performed using the molecular replacement method with the program phenix.phaser, and the initial models used in the molecular replacement method were PDB ID 1i9i for IgG Fab, PDB ID 2vxq for IgE Fab, and PDB ID 1o0v for IgE Cε2. Structural refinement with the programs phenix.refine and phenix.rosetta_refine and manual model correction with the program COOT were performed iteratively until the crystallographic R-factor and Rfree factor converged to 0.226 and 0.267, respectively.

[0028] (result) (1) The binding epitope of HMK-12 Fab is located in the IgE F(ab´)2 region To explore the novel activity of HMK-12 Fab on IgE-FcεRI complex, molecular interaction studies were performed. To investigate the effect of HMK-12 Fab on IgE binding to FcεRI, various concentrations of Alexa 488-labeled SPE-7 IgE (anti-DNP mouse IgE antibody) were premixed with an excess of HMK-12 Fab or IgG2a Fab. The mixture was then added to PT18 cells (mouse mast cells) and incubated at 37°C for 20 minutes. Following a washing step, the cells were subjected to flow cytometry analysis. The results showed that HMK-12 Fab dose-dependently inhibited the binding of SPE-7 IgE to PT18 cells, whereas IgG2a Fab showed no inhibitory effect compared to untreated cells, as shown in Figure 1. The inventors previously showed that HMK-12 Fab interacts with preformed IgE-FcεRI complexes at low temperatures (Sci Rep. 8, 14237 (2018)). HMK-12 Fab prevented the binding of SPE-7 IgE to FcεRI on the surface of PT18 cells. One possible explanation for these results is that HMK-12 Fab induces certain changes in the IgE Fc domain that are necessary for interaction with FcεRI. To further address the question of how HMK-12 Fab prevents IgE binding to cell surface FcεRI, we decided to identify the binding epitope of HMK-12 Fab on IgE molecule. First, SPE-7 IgE protein was run on 7.5% SDS-PAGE under reducing and non-reducing conditions. The protein was then transferred to a PVDF membrane and detected with HMK-12 Fab, 6HD5 Fab and anti-rat λ chain followed by HRP goat anti-rat IgG (Figure 2). Under reducing conditions, 6HD5 Fab and anti-rat λ chain detected IgE heavy chain (70 kDa) and light chain (23 kDa), respectively. Surprisingly, however, when the membrane was probed with HMK-12 Fab, it clearly showed no visible bands. On the other hand, under non-reducing conditions, all antibodies detected IgE and its aggregates around 150 kDa. Based on the above findings, we investigated how these anti-IgE antibodies react with IgE depending on the reduction method. First, SPE-7 IgE protein was treated with serial dilutions of dithiothreitol (DTT: 5-20 mM) at room temperature for 60 min. After dialysis, SDS-PAGE was performed under non-reducing conditions, followed by Western blot analysis. As a result, as shown in Figure 3, 6HD5 Fab detected total IgE molecules (200 kDa) and partially reduced IgE heavy chains (70 kDa) in a DTT dose-dependent manner. Similarly, anti-rat λ chain detected whole IgE molecules, IgE heavy chains, and light chains (25 kDa). However, HMK-12 Fab reacted only with total IgE molecules, but not with reduced IgE heavy chains, indicating that the binding epitope of HMK-12 Fab exists on the dimeric IgE structure, but not on the monomer generated by reduction of disulfide bonds. To clarify the epitope on the IgE dimer recognized by HMK-12 Fab, samples of SPE-7 IgE digested with pepsin, the same enzyme used to generate the F(ab´)2 fragment, were subjected to SDS-PAGE under non-reducing conditions and analyzed for reactivity with HMK-12 Fab by Western blot analysis (Fig. 4). The results showed that only the anti-rat λ chain could detect Fab (50 kDa) when digested at pH 3.8. On the other hand, HMK-12 Fab could detect larger fragments between 75 kDa and 150 kDa when digested at pH 4.5. Notably, HMK-12 Fab reacted most strongly with the 150 kDa band corresponding to IgE F(ab´)2, indicating that HMK-12 Fab recognizes an epitope on IgE F(ab´)2. To further confirm these results, we first purified HMK-12 Fab and SPE-7 IgE F(ab´)2 using a Superose 12 10 / 300 GL column (Figure 5A). Purified HMK-12 Fab and IgE F(ab´)2 were mixed at a molar ratio of 2.1:1 and incubated at 4°C for 8 h. After incubation, the sample was applied to a HiPrep 16 / 60 Sephacryl S-300 HR to remove uncomplexed Fab and particles with unexpectedly short retention times (Figure 5B). Peak fractions were then run on SDS-PAGE under non-reducing conditions. The results clearly showed two bands at 150 kDa and 45 kDa corresponding to IgE F(ab´)2 and HMK-12 Fab, respectively (Figure 5C). These results provide strong evidence that the binding epitope of HMK-12 Fab resides in the IgE F(ab´)2 region.

[0029] (2) Crystal structure of the HMK-12 Fab / IgE F(ab´)2 complex Based on the knowledge that HMK-12 Fab binds to an epitope present on the IgE F(ab´)2 region, we determined the crystal structure of the HMK-12 Fab / IgE F(ab´)2 complex at 2.9 Å resolution with R and R values ​​of 0.228 and 0.267, respectively (Fig. 6). The crystallographic data are summarized in Table 1.

[0030] [Table 1]

[0031] The complex structure revealed that two HMK-12 Fab fragments and one IgE F(ab´)2 molecule form a complex particle, in which the HMK-12 Fab fragment has two sites for intermolecular interaction with the IgE F(ab´)2 molecule. One site was the general epitope recognition site of HMK-12 Fab that contacted the Cε2 homodimer domain of IgE F(ab´)2. The other site was a cleft in the hinge region of the heavy chain of HMK-12 Fab, which accommodated the loop structure of the light chain of IgE F(ab´)2. The former interaction via the epitope recognition site of HMK-12 Fab mainly contributed to the complex formation. The average value of the crystallographic atomic position displacement factor of the Cα atoms of HMK-12 Fab was almost the same as that of the Cε2 domain of IgE F(ab´)2 (45.6 Å, respectively). 2 and 47.5 Å 2 In contrast, the average Cα atom of the Fab portion of IgE F(ab´)2 is 81.6 Å. 2 Therefore, the almost identical values ​​of the atomic position displacement factors of HMK-12 Fab and the Cε2 domain of IgE F(ab´)2 indicate that the two particles, HMK-12 Fab and the Cε2 domain of IgE F(ab´)2, behave like a single particle in the crystal. Interactions via the epitope recognition site are considered to be the main factor in complex formation.

[0032] The interaction between the cleft in the hinge region of the heavy chain of HMK-12 Fab and the light chain of IgE F(ab´)2 is thought to be involved in crystal formation. The linker peptide chain connecting the Cε2 domain and Fab portion of IgE F(ab´)2 is a long peptide chain that has little interaction with other parts of IgE F(ab´)2, so there is thought to be diversity in the relative position between the Cε2 domain and Fab portion of uncomplexed IgE F(ab´)2. By forming a complex, HMK-12 Fab is thought to fix the relative position between the Cε2 domain and the Fab portion of IgE F(ab´)2, resulting in the formation of crystallizable particles with little structural diversity.

[0033] Taken together, the results demonstrated the presence of two binding sites on HMK-12 Fab for interacting with IgE F(ab´)2. One binding site was a common epitope recognition site that recognized Cε2 of IgE F(ab´)2 as an epitope. The other binding site was a cleft in the hinge region of the heavy chain of HMK-12 Fab that interacted with the light chain of IgE F(ab´)2.

[0034] (3) Epitope Recognition The epitope region recognized by HMK-12 Fab was formed by amino acid residues from both of the two Cε2 domains of IgE F(ab´)2. The amino acid residues of IgE F(ab´)2 located at a distance less than or equal to 6 Å from the atoms of amino acid residues in the complementarity determining regions (CDRs) of HMK-12 Fab constituted the epitope of HMK-12 Fab, and five regions (E219-L222, D230-L232, D230-L234, D230-L236, D230-L238, D230-L239 ... * -H235 * , D259-D260, E296-K302, and G307-R315) (Figure 7). Four of the five regions, except for the second region, were present on the same Cε2 monomer. The second region D230, shown in red in Figure 8 * -H235 * was a loop structure derived from another Cε2 monomer that formed a homodimer with the aforementioned Cε2 monomer. This crystallographic observation revealed that the epitope region was composed of amino acid residues derived from both monomers of the Cε2 homodimer, with the homodimer structure being reinforced by an interchain disulfide bond. The first and third CDR regions (G26-N37 and H100-A110) of the VH of HMK-12Fab are thought to play an important role in recognizing the representative amino acid residues in the epitope region of the Cε2 homodimer (Figure 9). The representative amino acid residue in the Cε2 domain of IgE F(ab´)2 that binds to HMK-12Fab is N232. *, D309, L311, and H313 (stick model with green carbon atoms in Fig. 9 ) are more abundant with neighboring HMK-12 Fab atoms than with other amino acids in the epitope region (Fig. 7 ). The first CDR region interacted with the first half of the last β-strand of the Cε2 domain. The third CDR region interacted with the second half of the last β-strand of the Cε2 domain and the tip of a loop from the other monomer of the Cε2 homodimer. More specifically, the side chain hydroxyl groups (Y27, S33, and Y34) of the first CDR region and the main chain carbonyl group of S32 form hydrogen bonds with the main chain amide and carbonyl groups of D309 and the main chain carbonyl group of L311. The third CDR region interacted with N232, D309, L311, and H313 (stick model with green carbon atoms in Fig. 9 ). * The main chain carbonyl groups of Y103 and S104 and the main chain amide group of R106 form many hydrogen bonds surrounding the * In addition, the side chain of H100 forms a hydrogen bond with the polar side chain of N232. * It forms a hydrogen bond with the main chain carbonyl group of N232. * In addition to binding to Cε2, the third CDR region forms hydrogen bonds through the main chain carbonyl groups of G101 and Y102 with the side chain of H313 in the last half of the β-strand of Cε2. In summary, HMK-12 Fab was an antibody that could recognize epitope regions derived from both monomers in the Cε2 homodimer through amino acid residues present in the first and third CDRs. These results were consistent with the inventors' findings above, which showed that HMK-12 Fab did not react with fully reduced IgE lacking the homodimeric structure of the Cε2 domain.

[0035] (4) HMK-12 Fab rapidly dissociates the IgE-FcεRI complex and inhibits anaphylactic reactions. IgE binds to FCεRI on the surface of mast cells and basophils with high affinity (dissociation constant, Kd = 10 -10M) (J Allergy 76, 3627-3641 (2021)). This affinity is thought to be at least several orders of magnitude higher than that of IgG for Fc gamma receptors (FcγRs) and IgE for the low affinity receptor CD23. Despite such a tight association between IgE and FcεRI, we showed that HMK-12 Fab has the unique property of being able to dissociate IgE from FcεRI. To gain further insight into these phenomena, we investigated the efficiency of dissociation of preformed IgE-FcERI complexes by HMK-12 Fab. For this purpose, PT18 cells were preincubated with 0.2 μM Alexa 488-labeled SPE-7 IgE for 15 min at 37 °C. The cells were washed twice to remove excess unbound antibodies and incubated with different concentrations of HMK-12 Fab or IgG2a Fab (0.02 μM, 0.2 μM, 2 μM) at various temperatures (2, 25, and 37° C.) for 5, 15, and 75 min. After washing at each temperature, the fluorescence intensity of SPE-7 IgE+PT18 cells was measured using a FACSCelesta flow cytometer. As a result, the fluorescence intensity of SPE-7 IgE+PT18 cells dramatically decreased by 46, 73, and 89%, respectively, after incubation with 2 μM HMK-12 Fab for 5, 15, and 75 minutes at 37°C (Figure 10). However, at 2°C, there was no change in the fluorescence intensity of SPE-7 IgE+PT18 cells even after incubation with HMK-12 Fab. To further confirm that the decrease in fluorescence intensity of SPE-7 IgE+PT18 cells was due to dissociation of IgE-FcERI complexes, culture supernatants were analyzed for the amount of dissociated SPE-7 IgE by ELISA. PT18 cells preincubated with Alexa 488-labeled SPE-7 IgE were washed and incubated with 2 μM HMK-12 Fab or IgG2a Fab at 37°C. After incubation with HMK-12 Fab for 15 and 60 min at 37°C, flow cytometric analysis showed a 76% and 85% decrease in the intensity of SPE-7 IgE+PT18 cells, respectively (Figure 11, top). In contrast, ELISA data after incubation with HMK-12 Fab for 15 and 60 min indicated that the concentrations of SPE-7 IgE / HMK-12 Fab immune complexes in culture supernatants were 1.5 nM and 2 nM, respectively (Figure 11, bottom). However, no immune complexes were detected when SPE-7 IgE+PT18 cells were incubated with IgG2a Fab. Taken together, our results clearly demonstrate that low amounts of HMK-12 Fab rapidly dissociate preformed IgE-FcERI complexes in a temperature-dependent manner. To further address the role of HMK-12 Fab fragments (HMK-12 Fab) in allergic reactions, we investigated the in vivo effect of HMK-12 Fab on IgE-mediated anaphylactic reactions using a passive cutaneous anaphylaxis (PCA) assay. First, 1 μg / ml of SPE-7 IgE was injected intradermally into rats. Two days later, 5 μg / ml of HMK-12 Fab or anti-κ Fab was injected into the same site. 15 min later, rats were intravenously injected with saline containing 0.5% Evans blue and 1 mg / ml DNP-BSA. The leakage of Evans blue from blood vessels into tissues was then quantified by absorbance measurement at 620 nm wavelength. The results shown in Figure 12 (before stimulation) indicate that a small amount of HMK-12 Fab could inhibit PCA reactions, but there was no inhibition of PCA reactions by anti-κ Fab. Then, 2 days after intradermal injection of SPE-7 IgE, rats were intravenously administered with saline containing 0.5% Evans blue and 1 mg / ml DNP-OVA. Immediately after administration, rats were intradermally injected with 5 μg / ml HMK-12 Fab or anti-κ Fab. Quantitative evaluation of leaked Evans blue revealed that HMK-12 Fab could inhibit PCA reaction, but anti-κ Fab (after stimulation) did not. All these results clearly indicate that HMK-12 Fab can inhibit anaphylactic reaction even after allergen stimulation.

[0036] (Consideration) We demonstrated that the Fab fragment of a monoclonal anti-IgE antibody, HMK-12 Fab, has novel actions in several respects. First, the binding epitope of HMK-12 Fab is located in the IgE F(ab´)2 region but not in the IgE Fc domain. Second, HMK-12 Fab can rapidly dissociate preformed IgE-FcεRI complexes and inhibit IgE-mediated anaphylactic reactions even after allergen stimulation. Finally, X-ray crystallographic studies revealed that HMK-12 Fab targets a distinct epitope on IgE F(ab´)2, thereby causing a decrease in the asymmetry of the IgE Fc domain and dissociating IgE from the receptor complex in an allosteric manner.

[0037] Since the discovery of IgE, many monoclonal anti-IgE antibodies, such as omalizumab, ligelizumab, and kiruizumab, have been produced for therapeutic purposes. However, most of the binding epitopes of these antibodies were found within the Cε3 and Cε4 domains, which overlap with the FcεRI binding site on IgE. Thus, these antibodies can neutralize free IgE, but do not interact efficiently with preformed IgE / FcεRI complexes. On the other hand, HMK-12 Fab can interact with preformed IgE / FcεRI complexes, but competition experiments revealed that HMK-12 Fab effectively blocks the binding of IgE to FcεRI on mast cells in a dose-dependent manner. These observations facilitated the identification of the IgE epitope recognized by HMK-12 Fab. The following protein analysis under reducing and non-reducing conditions showed that the HMK-12 Fab binding epitope is present on IgE dimers but not on monomers. Furthermore, pepsin digestion studies surprisingly revealed that the HMK-12 Fab binding epitope is present on the IgE F(ab´)2 region but not on the Fc domain. This observation was confirmed by X-ray crystallographic studies showing that the HMK-12 Fab fragment provides two sites for interacting with one IgE F(ab´)2 molecule to form a composite particle. One binding site was the conventional epitope recognition site of HMK-12 Fab that contacted the Cε2 homodimer domain of IgE F(ab´)2, and the other binding site was a cleft in the hinge region of the heavy chain of HMK-12 Fab that interacted with the light chain of IgE F(ab´)2.

[0038] In addition to the inhibition of receptor complex formation, another surprising finding from the present study is the HMK-12 Fab-mediated removal of IgE from the receptor complex. To gain further insight into the role of HMK-12 Fab, we attempted to investigate the function of HMK-12 Fab by superimposing the HMK-12 Fab / IgE F(ab´)2 complex onto the IgE Fc / FcERI complex. The crystal structure of the HMK-12 Fab / IgE F(ab´)2 complex suggested that the binding of HMK-12 Fab to IgE reduces the diversity of the spatial arrangement between the Cε2 and Cε3 domains. This is most likely due to the fact that the binding epitope of HMK-12 Fab, located in the lower half of the Cε2 domain, is in close proximity to the Cε3 domain via a short linker region in full-length IgE. Figure 13 shows the superposition of the IgE Fc-FcεRI complex (PDB ID: 2Y7Q) and the crystal structure of the HMK-12 Fab / IgE F(ab´)2 complex determined in this study. In the IgE Fc-FcεRI complex structure, the two Fc fragments form a structurally asymmetric dimer, and the Cε domains of each have different spatial arrangements. As a result, the space formed between the two Cε3 domains is thought to accommodate FcεRI binding. In the superposition of IgE Cε2 on the Cε2 of the receptor complex in this study (Figure 13A), the Fv domain of the light chain of HMK-12 Fab is superimposed on the Cε3 domain of the receptor complex structure. In the superposition of IgE Cε2 on the other Cε2 of the receptor complex in this study (Figure 13B), the Fv domain of the heavy chain of HMK-12 Fab overlaps with the other Cε3 domain in the receptor complex structure. HMK-12 Fab binding to the Cε2 domain of IgE causes the Fc fragment of IgE to adopt a spatial configuration different from that observed in the crystal structure of the Fc and FcERI complex.

[0039] Crystal structure analysis revealed that the pair of IgE heavy chain Cε2 domains linked by disulfide bonds and HMK-12 Fab exhibit two-fold rotational symmetry. Although the crystal structure is affected by the crystal field, the two-fold rotational symmetry relationship would essentially be preserved in solution, since analysis of crystallographic atomic displacement factors showed a tightly bound complex of HMK-12 Fab and Cε2 domains. However, recent developments have demonstrated the importance of the asymmetric spatial arrangement of IgE Fc domains for the exceptionally slow dissociation rate of the IgE-FcεRI complex (12,35,36). Therefore, it is possible that the two-fold rotational symmetry of the IgE heavy chain induced by HMK-12 binding may cause a decrease in the binding affinity of IgE to FcεRI.

[0040] As mentioned above, because HMK-12 Fab was large enough to interact with the Cε3 domain of IgE, the two Cε3 domains adjacent to the two Cε2 / HMK-12 Fab moieties could be affected by the two-fold rotational symmetry relationship between the Cε2 domain and HMK-12 Fab, leading to a decrease in the asymmetric structure of the IgE Fc domain, a feature required for receptor binding. As a result, it could cause the removal of IgE from the receptor complex or the inhibition of receptor complex formation. Finally, we have shown that HMK-12 Fab, which binds to IgE F(ab´)2 at two sites, indirectly changes the structural features of the IgE Fc domain and rapidly dissociates the IgE-FcεRI complex. The characteristics of HMK-12 Fab may explain why this antibody can inhibit anaphylactic reactions even after allergen challenge. All these findings, known as allosteric regulation of IgE-FcεRI interaction, may provide better therapeutic options for the prevention and treatment of allergic diseases.

Claims

1. A screening method for antibody drugs, characterized by selecting antibodies that bind to a regulatory site other than the active site of a target antigen, thereby changing the structure of the active site and controlling the affinity between the target antigen and the target antigen receptor.

2. 2. The method for screening an antibody drug according to claim 1, wherein the antibody that controls the affinity between the target antigen and the target antigen receptor is an antibody that inhibits the binding between the target antigen and the target antigen receptor.

3. The screening method according to claim 1, wherein a regulatory site other than the active site of the target antigen is identified by crystal structure analysis of the target antigen.

4. 2. The screening method according to claim 1, wherein the target antigen is IgE, the target antigen receptor is FcεRI, and the regulatory site other than the active site of the target antigen is the heavy chain of the Cε2 homodimer of IgE F(ab')2.

5. An antibody drug selected by the screening method according to any one of claims 1 to 4.

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