Anti-IGE antibodies

Abstract

This application relates to a set of anti-IgE antibodies directed against a novel region of Cε (the β5 helix region of Cε2) and their use in inhibiting IgE to Fcε receptors (FcεRI and CD23) for the treatment of IgE-mediated diseases such as allergic airway diseases, including asthma. Notably, these anti-IgE antibodies not only bind to Fcε receptors but also remove pre-bound IgE from the Fcε receptors.

Description

[Background technology]

[0001] Immunoglobulin E (IgE) is a sensor molecule that plays a central role in inducing immediate allergic reactions. IgE binds to its high-affinity receptor FcεRI, endowing mast cells and basophils, which are the primary effector cells in the immediate allergic reaction, with versatile recognition capabilities. Upon binding to allergens, mast cells and basophils are rapidly activated, releasing a wide variety of inflammatory mediator molecules, including histamine, proteases, lipid mediators, cytokines, and chemokines. Summary of the Invention

[0002] According to one aspect of the present disclosure, an antigen-binding polypeptide is provided, wherein the polypeptide exhibits specific binding to IgE.

[0003] In some embodiments, binding of the antigen-binding polypeptide to IgE disrupts the interaction between IgE and at least one Fcε receptor.

[0004] In some embodiments, the disrupted interaction comprises blocking binding of unbound IgE to at least one Fcε receptor, hi some embodiments, the disrupted interaction comprises dissociating bound IgE from at least one Fcε receptor.

[0005] In some embodiments, the disrupted interaction results in the inhibition of degranulation.

[0006] In some embodiments, at least one Fcε receptor comprises FcεRI. In some embodiments, at least one Fcε receptor comprises CD23. In some embodiments, at least one Fcε receptor comprises FcεRI and CD23.

[0007] In some embodiments, the antigen-binding polypeptide specifically binds to at least one amino acid residue within the β5 helix region of Cε2, the β5 helix region being a combination of the helix, the β5 helix joint, and the bottom half of β5 that connects to the helix of Cε2.

[0008] In some embodiments, the antigen-binding polypeptide specifically binds to at least one amino acid residue in a helix of Cε2. In some embodiments, the antigen-binding polypeptide specifically binds to at least one amino acid residue in the β5 helix joint of Cε2. In some embodiments, the antigen-binding polypeptide specifically binds to at least one amino acid residue in the bottom half of β5 that connects to the helix of Cε2.

[0009] In some embodiments, the antigen-binding polypeptide does not bind to amino acid residue T298 of Cε2.

[0010] In some embodiments, the antigen-binding polypeptide binds to at least two amino acid residues within the β5-Cε2 helix region.

[0011] In some embodiments, the antigen-binding polypeptide does not bind to the β3 region of Cε2. In some embodiments, the antigen-binding polypeptide does not bind to the β4 region of Cε2. In some embodiments, the antigen-binding polypeptide does not bind to the β3 or β4 region of Cε2.

[0012] In some embodiments, the antigen-binding polypeptide comprises at least one amino acid sequence selected from SEQ ID NOs: 1-25. In some embodiments, the antigen-binding polypeptide comprises at least one amino acid sequence selected from SEQ ID NOs: 26-50.

[0013] In some embodiments, the antigen-binding polypeptide comprises at least one amino acid sequence selected from SEQ ID NOs: 51-200.

[0014] According to another aspect of the present disclosure, there is provided an antibody or fragment thereof, in some embodiments, the antibody or fragment has a Fab region that specifically binds to IgE.

[0015] In some embodiments, binding of the Fab to IgE disrupts the interaction between IgE and at least one Fcε receptor.

[0016] In some embodiments, the disrupted interaction comprises blocking binding of unbound IgE to at least one Fcε receptor, hi some embodiments, the disrupted interaction comprises dissociating bound IgE from at least one Fcε receptor.

[0017] In some embodiments, the disrupted interaction results in the inhibition of degranulation.

[0018] In some embodiments, at least one Fcε receptor comprises FcεRI. In some embodiments, at least one Fcε receptor comprises CD23. In some embodiments, at least one Fcε receptor comprises FcεRI and CD23.

[0019] In some embodiments, the Fab region specifically binds to at least one amino acid residue within the β5 helix region of Cε2, which is a combination of the helix, the β5 helix joint, and the bottom half of β5 that connects to the helix of Cε2.

[0020] In some embodiments, the Fab region specifically binds to at least one amino acid residue in a helix of Cε2. In some embodiments, the Fab region specifically binds to at least one amino acid residue within the β5 helix joint of Cε2. In some embodiments, the Fab region specifically binds to at least one amino acid residue in the bottom half of β5 that connects to the helix of Cε2.

[0021] In some embodiments, the Fab region does not bind to amino acid residue T298 of Cε2.

[0022] In some embodiments, the Fab region binds to at least two amino acid residues within the β5-Cε2 helix region.

[0023] In some embodiments, the Fab region does not bind to the β3 region of Cε2. In some embodiments, the Fab region does not bind to the β4 region of Cε2. In some embodiments, the Fab region does not bind to the β3 or β4 regions of Cε2.

[0024] In some embodiments, the antibody is a bispecific antibody or binding fragment thereof.

[0025] In some embodiments, the antibody comprises a monovalent Fab', a divalent Fab2, a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or binding fragment thereof.

[0026] In some embodiments, the Fab region comprises at least one heavy chain sequence selected from SEQ ID NOs: 1-25. In some embodiments, the Fab region comprises at least one light chain sequence selected from SEQ ID NOs: 26-50. In some embodiments, the Fab region comprises at least one complementarity determining region (CDR) selected from SEQ ID NOs: 51-200.

[0027] In some embodiments, the antibody exhibits a KD of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, or less than 30 nM.

[0028] In some embodiments, the antibody comprises a humanized antibody.

[0029] According to another aspect of the present disclosure, there is provided an antibody or fragment thereof having a Fab region comprising a particular heavy chain sequence, light chain sequence and / or CDR sequence.

[0030] In some embodiments, the Fab region comprises at least one heavy chain sequence selected from SEQ ID NOs: 1-25. In some embodiments, the Fab region comprises at least one light chain sequence selected from SEQ ID NOs: 26-50. In some embodiments, the Fab region comprises at least one heavy chain sequence selected from SEQ ID NOs: 1-25 and at least one light chain sequence selected from SEQ ID NOs: 26-50.

[0031] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:1 and the light chain sequence of SEQ ID NO:26.

[0032] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:2 and the light chain sequence of SEQ ID NO:27.

[0033] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:3 and the light chain sequence of SEQ ID NO:28.

[0034] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:4 and the light chain sequence of SEQ ID NO:29.

[0035] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:5 and the light chain sequence of SEQ ID NO:30.

[0036] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:6 and the light chain sequence of SEQ ID NO:31.

[0037] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:7 and the light chain sequence of SEQ ID NO:32.

[0038] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:8 and the light chain sequence of SEQ ID NO:33.

[0039] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:9 and the light chain sequence of SEQ ID NO:34.

[0040] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:10 and the light chain sequence of SEQ ID NO:35.

[0041] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:11 and the light chain sequence of SEQ ID NO:36.

[0042] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:12 and the light chain sequence of SEQ ID NO:37.

[0043] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:13 and the light chain sequence of SEQ ID NO:38.

[0044] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:14 and the light chain sequence of SEQ ID NO:39.

[0045] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:15 and the light chain sequence of SEQ ID NO:40.

[0046] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:16 and the light chain sequence of SEQ ID NO:41.

[0047] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:17 and the light chain sequence of SEQ ID NO:42.

[0048] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:18 and the light chain sequence of SEQ ID NO:43.

[0049] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:19 and the light chain sequence of SEQ ID NO:44.

[0050] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:20 and the light chain sequence of SEQ ID NO:45.

[0051] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:21 and the light chain sequence of SEQ ID NO:46.

[0052] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:22 and the light chain sequence of SEQ ID NO:47.

[0053] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:23 and the light chain sequence of SEQ ID NO:48.

[0054] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:24 and the light chain sequence of SEQ ID NO:49.

[0055] In some embodiments, the Fab region comprises the heavy chain sequence of SEQ ID NO:25 and the light chain sequence of SEQ ID NO:50.

[0056] In some embodiments, the Fab region comprises an HCDR1 selected from SEQ ID NOs: 51-75.

[0057] In some embodiments, the Fab region comprises an HCDR2 selected from SEQ ID NOs: 76-100.

[0058] In some embodiments, the Fab region comprises an HCDR3 selected from SEQ ID NOs: 101-125.

[0059] In some embodiments, the Fab region comprises an LCDR1 selected from SEQ ID NOs: 126-150.

[0060] In some embodiments, the Fab region comprises an LCDR2 selected from SEQ ID NOs: 151-175.

[0061] In some embodiments, the Fab region comprises an LCDR3 selected from SEQ ID NOs: 176-200.

[0062] In some embodiments, the antibody exhibits a KD of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, or less than 30 nM.

[0063] In some embodiments, the antibody comprises a humanized antibody. In some embodiments, the humanized antibody comprises a heavy chain variable region selected from SEQ ID NOs: 201-202. In some embodiments, the humanized antibody comprises a light chain variable region selected from SEQ ID NOs: 203-206. In some embodiments, the humanized antibody comprises a heavy chain variable region selected from SEQ ID NOs: 201-202 and a light chain variable region selected from SEQ ID NOs: 203-206.

[0064] In some embodiments, the humanized antibody comprises a heavy chain variable region selected from SEQ ID NO: 201 and a light chain variable region selected from SEQ ID NOs: 203-204. In some embodiments, the humanized antibody comprises a heavy chain variable region selected from SEQ ID NO: 202 and a light chain variable region selected from SEQ ID NOs: 205-206.

[0065] In some embodiments, the humanized antibody comprises a complete heavy chain sequence selected from SEQ ID NOs: 207-208. In some embodiments, the humanized antibody comprises a complete light chain sequence selected from SEQ ID NOs: 209-212. In some embodiments, the humanized antibody comprises a complete heavy chain sequence selected from SEQ ID NOs: 207-208, and a light chain variable region selected from SEQ ID NOs: 209-212.

[0066] In some embodiments, the humanized antibody comprises a heavy chain complete sequence selected from SEQ ID NO: 207 and a light chain variable region selected from SEQ ID NOs: 209-210. In some embodiments, the humanized antibody comprises a heavy chain complete sequence selected from SEQ ID NO: 208 and a light chain variable region selected from SEQ ID NOs: 211-212.

[0067] According to another aspect of the present disclosure, a complex comprising the disclosed antigen-binding polypeptide or the disclosed antibody or fragment is provided, the complex comprising the polypeptide or antibody bound to an IgE protein.

[0068] According to another aspect of the present disclosure, there is provided a method of disrupting the interaction of IgE with at least one Fcε receptor by contacting a cell expressing at least one Fcε receptor with an antigen-binding polypeptide that specifically binds to the β5 helix region of Cε2, the β5 helix region being a combination of the helix, the β5 helix joint, and the bottom half of β5 that connects to the helix of Cε2.

[0069] In some embodiments, the antigen-binding polypeptide specifically binds to at least one amino acid residue in a helix of Cε2. In some embodiments, the antigen-binding polypeptide specifically binds to at least one amino acid residue within the β5 helix joint of Cε2. In some embodiments, the antigen-binding polypeptide specifically binds to at least one amino acid residue in the bottom half of β5 that connects to the helix of Cε2.

[0070] In some embodiments, the disrupted interaction comprises blocking binding of unbound IgE to at least one Fcε receptor, hi some embodiments, the disrupted interaction comprises dissociating bound IgE from at least one Fcε receptor.

[0071] In some embodiments, the disrupted interaction results in the inhibition of degranulation.

[0072] In some embodiments, at least one Fcε receptor comprises FcεRI. In some embodiments, at least one Fcε receptor comprises CD23. In some embodiments, at least one Fcε receptor comprises FcεRI and CD23.

[0073] In some embodiments, the Fab region does not bind to amino acid residue T298 of Cε2.

[0074] In some embodiments, the Fab region binds to at least two amino acid residues within the β5-Cε2 helix region.

[0075] In some embodiments, the antigen-binding polypeptide does not bind to the β3 region of Cε2. In some embodiments, the antigen-binding polypeptide does not bind to the β4 region of Cε2. In some embodiments, the antigen-binding polypeptide does not bind to the β3 or β4 region of Cε2.

[0076] In some embodiments, the antigen-binding polypeptide is an antibody or binding fragment thereof. In some embodiments, the antibody comprises a monovalent Fab', a divalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a camelid antibody or binding fragment thereof.

[0077] In some embodiments, the Fab region comprises at least one heavy chain sequence selected from SEQ ID NOs: 1-25.

[0078] In some embodiments, the Fab region comprises at least one light chain sequence selected from SEQ ID NOs: 26-50.

[0079] In some embodiments, the Fab region comprises at least one complementarity determining region (CDR) selected from SEQ ID NOs: 51-200.

[0080] In some embodiments, the antibody exhibits a KD of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, or less than 30 nM.

[0081] In some embodiments, the antibody comprises a humanized antibody.

[0082] In some embodiments, the interaction between IgE and at least one Fcε receptor is associated with an allergic condition.

[0083] In some embodiments, the allergic condition is selected from asthma, chronic idiopathic urticaria, nasal polyps, hay fever, or food allergies.

[0084] Various aspects of the present disclosure are set forth with particularity in the appended claims. This patent document contains at least one drawing / photograph executed in color. Copies of this patent with color drawing(s) / photograph(s) will be provided by the Patent Office upon request and payment of the necessary fee. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description and accompanying drawings that set forth illustrative embodiments, in which the principles of the present disclosure are utilized. [Brief explanation of the drawings]

[0085] [Figure 1] FIG. 1 shows the binding of IgE to its high affinity receptor FcεRI, in particular the role of Cε2 and Cε3, according to some embodiments of the present disclosure. [Figure 2] FIG. 1 shows a process for generating an anti-human IgE Fab library according to some embodiments of the present disclosure. [Figure 3] FIG. 1 shows a competition and removal assay according to some aspects of the present disclosure. [Figure 4] 1 shows the binding of selected Fabs tested in ELISA, according to some embodiments of the present disclosure. Purified Fabs were serially diluted and tested against recombinant Cε2-4 protein coated at 2 μg / mL. [Figure 5]Figure 1 shows the blocking activity of selected Fabs tested by flow cytometry, according to some embodiments of the present disclosure. 0.5 μg / mL IgE was incubated with different molar ratios of Fab and IgG for 1 hour at 37°C. BaF / 3 cell line transduced with human FcεRI α, β, and γ chains (BaF3-hFcεRI) was incubated with Fab-treated IgE for 2 hours at 37°C. IgE binding to cells was assessed as the binding capacity of fluorescently labeled antigen. [Figure 6] Figure 1 shows the depletion of human IgE (A) and the inhibition of antigen-induced degranulation (B) on human FcεRIα-expressing mouse bone marrow-derived mast cells (BMMC-hFcεRI) according to some embodiments of the present disclosure. BMMC-hFcεRI were incubated with 0.5 μg / mL of human IgE overnight. After washing, the cells were incubated with various concentrations of the indicated Fab for 48 hours. After washing, the cells were split into two experiments. In one experiment, the cells were incubated with fluorescently labeled antigen on ice, and the fluorescence level, which represents the amount of IgE on the cells, was measured by flow cytometry (A). In another experiment, the cells were stimulated with antigen in the presence of an anti-human CD63 antibody, and CD63-positive cells were considered degranulated cells (B). Conc is concentration, and MFI is mean fluorescence intensity. [Figure 7]Figures 1A and 1B show the competitive activity (A) and depletion activity (B) of IgE binding to human CD23 (hCD23) according to some embodiments of the present disclosure. (A) 2 μg / mL IgE was incubated with 40 μg / mL of the indicated Fab for 1 hour at 37°C. The BaF / 3 cell line transduced with human CD23 (BaF3-hCD23) was incubated with Fab-treated IgE for 2 hours on ice. After washing, cells were incubated with fluorescently labeled antigen on ice, and fluorescence was measured by flow cytometry. MFI was normalized to the MFI of cells incubated with untreated IgE. (B) BaF-hCD23 cells were incubated overnight with 2 μg / mL human IgE. After washing, cells were incubated with 10 μg / mL of the indicated Fab or omalizumab (Xolair) for 1 hour at 37°C. After washing, cells were incubated with fluorescently labeled antigen, and fluorescence levels were measured by flow cytometry. In the case of Xolair, the same mass and molar concentrations were used for comparison. [Figure 8] Figures 1A and 1B show the binding regions of three Fabs assessed by ELISA, according to some embodiments of the present disclosure. (A, B) Recombinant his-tagged Cε2 protein fragments were treated with PNGaseF under denaturing (A) or native (B) conditions. Cε2 preparations were coated onto microplates, and Fab binding was assessed. (C) Human or mouse IgE was coated onto microplates and detected with the indicated Fabs or antibodies by ELISA. (D) Schematic of the Cε2 fragment of human IgE. Cε2 consists of seven beta-sheets (numbered), one alpha-helix, and a connecting hinge. (E) Recombinant Cε2 mutants with human-to-mouse substitutions in the indicated regions were coated onto microplates. Binding of the indicated Fabs and IgGs was assessed by ELISA. The readings were normalized to those of an anti-human IgE polyclonal antibody. [Figure 9]

[0023] Figure 1 shows the binding amino acids of three Fabs in the β5 helix region assessed by ELISA, according to some embodiments of the present disclosure. (A) Amino acid sequences of human and mouse Cε2 β5-helix regions. Amino acids boxed in gray are common to human and mouse IgE. Amino acids underlined in orange were replaced with amino acids from mouse IgE in the indicated mutant IgE. (B) Wild-type (WT) and mutant chimeric IgEs with human-to-mouse substitutions in the indicated regions were coated onto a microplate. The binding levels of Fabs to anti-human IgE antibodies were assessed by ELISA. [Figure 10]

[0023] Figure 1 shows binding and stability of mutant IgE to cell surface FcεRI assessed by flow cytometry, according to some embodiments of the present disclosure. BaF3-hFcεRI cells were incubated overnight with the indicated human IgE mutants. After washing, cells were placed on ice for the indicated times (A, 0 h; B, 3 h; C, 6 h). After washing, cells were incubated on ice with fluorescently labeled antigen and subjected to flow cytometry. [Figure 11]

[0023] Figure 1 shows the competitive activity of three Fabs against mutant IgE as assessed by flow cytometry, according to some embodiments of the present disclosure. Human IgE mutants were incubated with the indicated Fabs or IgG for 1 hour at 37°C. BaF3-hFcεRI cells were incubated with the pretreated IgE mutants for 2 hours at 37°C. IgE binding was assessed by fluorescence antigen binding. [Figure 12]

[0023] Figure 1 shows the depletion activity of three Fabs (A) or IgGs with Fab regions (B) against FcεRI-binding mutant IgE, as assessed by flow cytometry, according to some embodiments of the present disclosure. Cells were incubated with 0.5 μg / mL of IgE mutants overnight. After washing, cells were incubated with 10 μg / mL of Fab or IgG at 37°C. The amount of remaining IgE was assessed by flow cytometry as fluorescent antigen binding at the indicated time points. [Figure 13] FIG. 1 shows dose response of standards in a degranulation inhibition assay, according to some embodiments of the present disclosure. [Figure 14] 1 shows the dose response of Fab(BH3), control, and calibrator in a degranulation inhibition assay according to some embodiments of the present disclosure. Fab shows dose-dependent inhibition of degranulation. [Figure 15] FIG. 1 shows the dose response of omalizumab (Xolair) and a standard in a degranulation inhibition assay, according to some embodiments of the present disclosure. [Figure 16] FIG. 1 shows binding of rabbit Fab of BC48 and humanized Fab of BC48 to Cε2-4 by ELISA, according to some embodiments of the present disclosure. [Figure 17] FIG. 1 shows binding of BH3 rabbit Fab and BH3 humanized Fab to Cε2-4 by ELISA, according to some embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0086] IgE is produced by B cells and plasma cells at sites of inflammation, either centrally or peripherally, and is distributed throughout the human body in low concentrations via the circulation. Once picked up by mast cells, IgE persists on FcεRI for several weeks. Therefore, targeting unbound, free IgE cannot remove IgE already bound to FcεRI for long periods of time. However, due to its low production, it is not difficult to neutralize unbound IgE using antibodies such as omalizumab.

[0087] Omalizumab (Xolair®) was developed to neutralize free IgE in serum and block its binding to FcεRI and the low-affinity IgE receptor CD23. Omalizumab is a recombinant humanized IgG1κ monoclonal antibody that primarily binds to the Cε3 domain of human IgE. The efficacy of omalizumab has been reported for many allergic diseases, including allergic asthma, chronic urticaria, nasal polyposis, and hay fever. Omalizumab is approved for the treatment of moderate to severe persistent allergic asthma, chronic idiopathic urticaria (CIU), and nasal polyps in the United States. It is also approved for severe hay fever in Japan. Furthermore, its benefits as an adjunct to immunotherapy have been reported. One of the major drawbacks of omalizumab is its slow action. Because omalizumab does not affect FcεRI-bound IgE, the reduction of IgE on mast cells takes longer (approximately 70 days) than on basophils (approximately 7 days), depending on their respective half-lives. 12, 13 Therefore, improvement of allergic disease requires more than 2 weeks after initiation of treatment. 13

[0088] To improve efficacy, several IgE-associated molecules have been developed, including ligelizumab14-18, aεFab19, MEDI421220,21, DARPins (designed ankyrin repeat proteins)22-25, single-domain antibodies (sdab)02626, clizumab27-29, AIMAb7195 (formerly known as XmAb7195)30, bispecific IgE / CD3 antibodies (bsc-IgE / CD3)31, and DNA aptamers321,33,34. Because IgE binds to FcεRI and CD23 using the Cε3 moiety, most substances that directly target IgE Cε3 block its binding to the IgE receptor. However, the mode of action varies depending on the substance. Steric hindrance, allosteric hindrance, and facilitated dissociation mechanisms have been proposed for blocking and scavenging activity35. For example, omalizumab binding blocks IgE binding to CD23 through steric hindrance caused by direct overlap of its footprint with human CD23. 36, 37 Omalizumab blocks IgE binding to FcεRI in a different way. Binding of omalizumab alters the 3D conformation of IgE, rendering it unable to bind to FcεRI. This mode is called allosteric hindrance. 37 The best example of accelerated dissociation is DARPin E2_79. DARPin E2_79 shares a small portion of its footprint with FcεRI on the IgE molecule, and competition for this small portion is thought to accelerate FcεRI dissociation. 23 Therefore, IgE blockers may have different blocking and / or clearing activities depending on the binding site.

[0089] In addition to Cε3, the Cε2 portion of human IgE plays a role in stabilizing IgE binding to the FcεRI. 38 A modified version of the omalizumab Fab, called FabXo13, contacts some of the Cε2 amino acids. 37 The Fab portion of an antibody against mouse Cε2 (clone 6HD5) reduces mast cell activation in vivo and in vitro. 39 However, the binding epitope for Fab-6HD5 within Cε2 has not been described. Furthermore, the amino acid sequence homology between human and mouse IgE Cε2-Cε4 is very low (55% for Cε2-Cε4 and 63% for Cε2), and human IgE does not bind to mouse FcεRI. Therefore, this cannot be directly extrapolated to human IgE, and it is unclear whether antibodies targeting human Cε2 can block and / or eliminate IgE binding to its receptor.

[0090] In this invention, we have devised several Fab clones that primarily bind to Cε2 of human IgE. The Fab clones not only blocked IgE binding to FcεRI and CD23 but also removed previously bound IgE from these receptors. These Fabs possessed a previously undescribed common binding epitope within the Cε2 moiety. This invention adds another strategy for targeting IgE in a faster manner than omalizumab.

[0091] IgE plays a central role in immediate allergic reactions. Furthermore, the successful application of the anti-human IgE antibody omalizumab to human diseases has demonstrated the essential role of IgE in various allergic disorders, including asthma, chronic idiopathic urticaria, nasal polyps, hay fever, and food allergies.

[0092] The Cε2 region of human IgE is thought to be involved in the stability of IgE on FcεRI. However, it was unclear whether Fab or IgG targeting Cε2 could inhibit the binding of human IgE to FcεRI and CD23.

[0093] In this study, we first prepared a Fab library against human IgE produced from rabbits immunized with recombinant human IgE Cε2-4 fragments and selected by phage display. Fab fragments from selected clones against the Cε2 region were purified and subjected to competition, depletion, and degranulation inhibition assays. The binding sites of the three selected Fabs were investigated using partial chimeric fragments of human and mouse Cε2 and further narrowed down using chimeric IgE. Their competition and depletion activities against human CD23 on the cell surface were also evaluated.

[0094] Twenty-four clones against Cε2 were isolated from a rabbit Fab library. All of these Fab clones competitively inhibited human IgE binding to FcεRI. The clearing activity was highly correlated with the competitive inhibitory activity. Among these, three highly active clones were selected. These clones did not bind to mouse IgE. Using recombinant human Cε2, whose amino acid sequence was partially substituted with that of mouse Cε2, the β5 helix region was found to be the common binding region for these Fabs. Further investigation revealed that the latter half of the β5 helix region was particularly essential for inhibitory binding. These Fabs competitively inhibited human IgE binding to human CD23 and cleared CD23-bound IgE. Using mouse bone marrow-derived mast cells expressing human FcεRIα, it was shown that removal of IgE from the mast cell surface reduced mast cell activation. In summary, Fab clones against the Cε2 region of human IgE were isolated that elicited inhibitory activity against human IgE binding to its receptor.

[0095] antibody production In some embodiments, anti-IgE antibodies are produced by standard protocols by injecting an antigenic composition into a production animal. See, for example, Harlow and Lane, *Antibodies: A Laboratory Manual*, Cold Spring Harbor Laboratory, 1988. When using the entire protein or a larger portion of the protein, antibodies can be produced by immunizing a production animal with the protein and an appropriate adjuvant (e.g., Freund's, Freund's complete, oil-in-water emulsion, etc.). When using smaller peptides, it is advantageous to conjugate the peptide to a larger molecule to create an immunostimulatory conjugate. Commonly used conjugated proteins commercially available for such use include bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). To generate antibodies against specific epitopes, peptides derived from the complete sequence can be used. Alternatively, to generate antibodies against relatively short peptide portions of a protein target, conjugation of the polypeptide to a carrier protein, such as ovalbumin, BSA, or KLH, can elicit a superior immune response.

[0096] Polyclonal or monoclonal anti-IgE antibodies can be produced from animals genetically modified to produce human immunoglobulins. Transgenic animals can be produced by first creating a "knockout" animal that does not produce its own natural antibodies, and then stably transforming this animal with human antibody loci (e.g., by using human artificial chromosomes). In such cases, only human antibodies are produced in the animal. Techniques for generating such animals and deriving antibodies therefrom are described in U.S. Patent Nos. 6,162,963 and 6,150,584, which are incorporated herein by reference in their entirety. Such antibodies can be referred to as human xenogenous antibodies.

[0097] Alternatively, anti-IgE antibodies can be produced from phage libraries containing human variable regions. See U.S. Patent No. 6,174,708, which is incorporated herein by reference in its entirety.

[0098] In some aspects of any of the embodiments disclosed herein, the anti-IGE antibody is produced by a hybridoma.

[0099] In the case of monoclonal anti-IgE antibodies, hybridomas can be formed by isolating stimulated immune cells, such as cells from the spleen of the inoculated animal. These cells can then be fused to immortalized cells, such as myeloma cells or transformed cells, that can replicate indefinitely in cell culture, thereby producing immortal immunoglobulin-secreting cell lines. The immortal cell lines used can be selected to be deficient in enzymes required for the utilization of specific nutrients. Many such cell lines (such as myelomas) are known to those skilled in the art, and include, for example, thymidine kinase (TK) or hypoxanthine-guanine phosphoriboxyltransferase (HGPRT). These deficiencies allow the fused cells to be selected according to their ability to grow, for example, on hypoxanthine aminopterin thymidine medium (HAT).

[0100] Additionally, anti-IgE antibodies can be produced by genetic engineering.

[0101] The anti-IgE antibodies disclosed herein may have a reduced tendency to induce undesirable immune responses in humans, such as anaphylactic shock, and may also exhibit a reduced tendency to prime immune responses, which can prevent repeated administration of antibody therapeutics or imaging agents (e.g., human-anti-mouse-antibody "HAMA" responses). Such anti-IgE antibodies include, but are not limited to, humanized, chimeric, or xenogeneic human anti-IgE antibodies.

[0102] To generate antibodies with predominantly human domains, chimeric anti-IgE antibodies can be made by recombinant means, for example, by combining mouse variable light and heavy chain regions (VK and VH) obtained from a mouse (or other animal-derived) hybridoma clone with human constant light and heavy chain regions. The production of such chimeric antibodies is well known in the art and can be accomplished by standard means (e.g., as described in U.S. Pat. No. 5,624,659, incorporated herein by reference in its entirety).

[0103] The term "humanized," as applied to non-human (e.g., rodent or primate) antibodies, refers to hybrid immunoglobulins, immunoglobulin chains, or fragments thereof that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, rabbit, or primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance and to minimize immunogenicity when introduced into the human body. In some instances, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0104] Humanized antibodies contain human-like immunoglobulin domains and can be engineered to incorporate only the complementarity-determining regions of an animal-derived antibody. This can be achieved by carefully examining the sequences of the hypervariable loops of the variable regions of a monoclonal antigen-binding unit or monoclonal antibody and adapting them to the structure of a human antigen-binding unit or human antibody chain. See, e.g., U.S. Patent No. 6,187,287, which is incorporated herein by reference in its entirety.

[0105] Methods for humanizing non-human antibodies are well known in the art. A "humanized" antibody is one in which at least a portion of its sequence has been altered from its original form to make it more human immunoglobulin-like. In some versions, the constant (C) regions of the heavy (H) and light (L) chains are replaced with human sequences. This may be a fusion polypeptide comprising a variable (V) region and a heterologous immunoglobulin C region. In some versions, the complementarity-determining regions (CDRs) comprise non-human antibody sequences, with the V framework regions also converted to human sequences. See, e.g., EP 0 329 400. In some versions, the V regions are humanized by designing consensus sequences of human and mouse V regions and converting residues outside the CDRs that differ between the consensus sequences.

[0106] In principle, framework sequences from a humanized antibody can serve as a template for CDR grafting. However, it has been demonstrated that linear replacement of CDRs into such frameworks can lead to a significant loss of binding affinity for the antigen. See Glaser et al. (1992) J. Immunol. 149:2606; Tempest et al. (1992) Biotechnology 9:266; and Shalaby et al. (1992) J. Exp. Med. 17:217. The more homologous a human antibody (HuAb) is to its original murine antibody (muAb), the less likely it is that the human framework will introduce distortions to the murine CDRs that could reduce affinity. Based on sequence homology searches against antibody sequence databases, HuAbIC4 offers good framework homology to muM4TS.22, but other highly homologous HuAbs are similarly suitable, particularly kappa light chains from human subgroup I or heavy chains from human subgroup III. See Kabat et al. (1987). Various computer programs, such as ENCAD (Levitt et al. (1983) J. Mol. Biol. 168:595), are available for predicting ideal sequences for V regions. Thus, the present invention encompasses HuAbs with different variable (V) regions. Determining suitable V region sequences and optimizing these sequences is within the skill of one of ordinary skill in the art. Methods for obtaining antibodies with reduced immunogenicity are also described in U.S. Pat. No. 5,270,202 and EP 699,755.

[0107] Humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences so that desired antibody characteristics, such as increased affinity for the target antigen(s), are achieved.

[0108] The process for humanizing a target antigen-binding unit can be as follows: Optimal germline receptor heavy and light chain variable regions are selected based on homology, canonical structure, and physical properties of the human antibody germline to be grafted. Computer modeling of the mVH / VL versus the grafted hVH / VL is performed to generate a prototype humanized antibody sequence. If modeling indicates the need for framework backmutations, a second variant with the indicated FW changes is generated. DNA fragments encoding the selected germline framework and mouse CDRs are synthesized. The synthesized DNA fragments are subcloned into an IgG expression vector, and the sequence is confirmed by DNA sequencing. The humanized antibody is expressed in cells such as 293F, and the protein is tested, for example, in MDM phagocytosis assays and antigen-binding assays. The humanized antigen-binding unit is compared to the parent antigen-binding unit in antigen-binding affinity, for example, by FACS on cells expressing the target antigen. If the affinity is more than two-fold lower than the parent antigen-binding unit, a second round of humanized variants can be generated and tested as described above.

[0109] As mentioned above, anti-IgE antibodies can be either "monovalent" or "multivalent." The former have one binding site per antigen-binding unit, while the latter contain multiple binding sites that can bind to multiple antigens of the same or different types. Depending on the number of binding sites, an antigen-binding unit can be bivalent (having two antigen-binding sites), trivalent (having three antigen-binding sites), tetravalent (having four antigen-binding sites), etc.

[0110] Multivalent anti-IgE antibodies can be further classified based on their binding specificity. A "monospecific" anti-IgE antibody is a molecule capable of binding to one or more antigens of the same type. A "multispecific" anti-IgE antibody is a molecule that has binding specificities for at least two different antigens. While such molecules usually bind only two different antigens (i.e., bispecific anti-IgE antibodies), antibodies with additional specificities, such as trispecific antibodies, are encompassed by this term as used herein. The present disclosure further provides multispecific anti-IgE antibodies. Multispecific anti-IgE antibodies are multivalent molecules capable of binding to at least two different antigens, for example, bispecific and trispecific molecules that exhibit binding specificities for two and three different antigens, respectively.

[0111] Polynucleotides and Vectors In some embodiments, the present disclosure provides isolated nucleic acids encoding any of the anti-IgE antibodies disclosed herein. In another embodiment, the present disclosure provides vectors comprising nucleic acid sequences encoding any of the anti-IgE antibodies disclosed herein. In some embodiments, the present invention provides isolated nucleic acids encoding the light chain CDRs and heavy chain CDRs of the anti-IgE antibodies disclosed herein.

[0112] The subject anti-IgE antibodies can be prepared by recombinant DNA technology, synthetic chemical technology, or a combination thereof. For example, sequences encoding the desired components of an anti-IgE antibody, including the light chain CDRs and heavy chain CDRs, are typically assembled and cloned into an expression vector using standard molecular techniques known in the art. These sequences may be assembled from PCR-generated fragments using respective template nucleic acids from other vectors encoding the desired protein sequences, or by assembly of synthetic oligonucleotides encoding the desired sequences. Expression systems can be created by transfecting appropriate cells with an expression vector containing the desired anti-IgE antibody.

[0113] Nucleotide sequences corresponding to various regions of the light or heavy chains of existing antibodies can be readily obtained and sequenced using conventional techniques, including but not limited to hybridization, PCR, and DNA sequencing. Hybridoma cells producing monoclonal antibodies serve as a preferred source of antibody nucleotide sequences. A vast number of hybridoma cells producing an array of monoclonal antibodies are available from public and private repositories. The largest depository is the American Type Culture Collection (atcc.org), which provides a diverse collection of well-characterized hybridoma cell lines. Alternatively, antibody nucleotides can be obtained from immunized or non-immunized rodents or humans and form organs such as spleen and peripheral blood lymphocytes. Specific techniques applicable to the extraction and synthesis of antibody nucleotides are described in Orlandi et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837, Larrick et al. (1989) Biochem. Biophys. Res. Commun. 160:1250-1255, Sastry et al. (1989) Proc. Natl. Acad. Sci., USA 86:5728-5732, and U.S. Pat. No. 5,969,108.

[0114] The nucleotides encoding the anti-IgE antibody can also be modified by substituting the coding sequences for human heavy and light chain constant regions in place of the homologous non-human sequences, thus preparing chimeric antibodies that retain the binding specificity of the original anti-IgE antibody.

[0115] host cell In some embodiments, the present disclosure provides a host cell that expresses any one of the anti-IgE antibodies disclosed herein. The subject host cell typically comprises a nucleic acid encoding any one of the anti-IgE antibodies disclosed herein.

[0116] The present invention provides host cells transfected with the above-described polynucleotides, vectors, or libraries of vectors. The vectors can be introduced into suitable prokaryotic or eukaryotic cells by any of several suitable means, including electroporation, microprojectile bombardment, lipofection, infection (in which the vector is coupled to an infectious agent), transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other agents. The choice of means for introducing the vector often depends on the characteristics of the host cell.

[0117] For most animal cells, any of the above methods is suitable for vector delivery. Preferred animal cells are vertebrate cells, preferably mammalian cells, that can express exogenously introduced gene products in large amounts, for example, at milligram levels. Non-limiting examples of preferred cells include NIH3T3 cells, COS, HeLa, and CHO cells.

[0118] After introduction into a suitable host cell, expression of the anti-IgE antibody can be determined using any nucleic acid or protein assay known in the art. For example, the presence of transcribed mRNA of the light chain CDR or heavy chain CDR, or the anti-IgE antibody, can be detected and / or quantified by conventional hybridization assays (e.g., Northern blot analysis), amplification procedures (e.g., RT-PCR), SAGE (U.S. Pat. No. 5,695,937), and array-based technologies (see, e.g., U.S. Pat. Nos. 5,405,783, 5,412,087, and 5,445,934) using probes complementary to any region of the polynucleotide encoding the anti-IgE antibody.

[0119] Expression of the vector can also be determined by examining the expressed anti-IgE antibodies. A variety of techniques are available in the art for protein analysis, including, but not limited to, radioimmunoassays, ELISA (enzyme-linked immunoradiometric assays), "sandwich" immunoassays, immunoradiometric assays, in situ immunoassays (using, for example, colloidal gold, enzyme, or radioisotope labels), Western blot analysis, immunoprecipitation assays, immunofluorescence assays, and SDS-PAGE.

[0120] Non-limiting embodiments The present disclosure is also described and exemplified by the following non-limiting embodiments. However, the use of these and other examples anywhere in the specification is exemplary only and in no way limits the scope and meaning of the disclosure or the scope and meaning of any exemplified term. Likewise, the present disclosure is not limited to any particular preferred embodiment or aspect described herein. Indeed, appropriate modifications and variations may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the spirit or scope of the invention.

[0121] 1. An antigen-binding polypeptide that exhibits specific binding to IgE.

[0122] 2. The antigen-binding polypeptide of embodiment 1, wherein said binding of said antigen-binding polypeptide to said IgE disrupts the interaction between said IgE and at least one Fcε receptor.

[0123] 3. The antigen-binding polypeptide of embodiment 2, wherein said disrupted interaction comprises blocking the binding of unbound IgE to said at least one Fcε receptor.

[0124] 4. The antigen-binding polypeptide of embodiments 2-3, wherein said disrupted interaction comprises dissociating bound IgE from said at least one Fcε receptor.

[0125] 5. The antigen-binding polypeptide of embodiments 2-4, wherein said disrupted interaction results in the inhibition of degranulation.

[0126] 6. The antigen-binding polypeptide of any one of embodiments 2 to 5, wherein said at least one Fcε receptor comprises FcεRI.

[0127] 7. The antigen-binding polypeptide of any one of embodiments 2 to 5, wherein said at least one Fcε receptor comprises CD23.

[0128] 8. The antigen-binding polypeptide of any one of embodiments 2 to 5, wherein said at least one Fcε receptor comprises FcεRI and CD23.

[0129] 9. The antigen-binding polypeptide of embodiments 1-8, wherein the antigen-binding polypeptide specifically binds to at least one amino acid residue within the β5 helix region of Cε2, and wherein the β5 helix region is a combination of the helix, the β5 helix joint, and the bottom half of the β5 that connects to the helix of Cε2.

[0130] 10. The antigen-binding polypeptide of embodiment 9, wherein said antigen-binding polypeptide specifically binds to at least one amino acid residue in the helix of Cε2.

[0131] 11. The antigen-binding polypeptide of embodiments 9-10, wherein said antigen-binding polypeptide specifically binds to at least one amino acid residue in the β5 helix joint of Cε2.

[0132] 12. The antigen-binding polypeptide of embodiments 9-11, wherein said antigen-binding polypeptide specifically binds to at least one amino acid residue in the bottom half of β5 that connects to the helix of Cε2.

[0133] 13. The antigen-binding polypeptide of embodiments 9-12, wherein said antigen-binding polypeptide does not bind to amino acid residue T298 of Cε2.

[0134] 14. The antigen-binding polypeptide of embodiments 9-13, wherein said antigen-binding polypeptide binds to at least two amino acid residues within said β5-Cε2 helix region.

[0135] 15. The antigen-binding polypeptide of any one of embodiments 1 to 14, wherein said antigen-binding polypeptide does not bind to the β3 region of Cε2.

[0136] 16. The antigen-binding polypeptide of any one of embodiments 1 to 14, wherein said antigen-binding polypeptide does not bind to the β4 region of Cε2.

[0137] 17. The antigen-binding polypeptide of embodiment 1-14, wherein said antigen-binding polypeptide does not bind to the β3 or β4 region of Cε2.

[0138] 18. The antigen-binding polypeptide of embodiments 1 to 17, comprising at least one amino acid sequence selected from SEQ ID NOs: 1 to 25.

[0139] 19. The antigen-binding polypeptide of embodiments 1 to 18, comprising at least one amino acid sequence selected from SEQ ID NOs: 26 to 50.

[0140] 20. The antigen-binding polypeptide of embodiments 1 to 19, comprising at least one amino acid sequence selected from SEQ ID NOs: 51 to 200.

[0141] 21. An antibody or fragment thereof comprising a Fab region that specifically binds to IgE.

[0142] 22. The antibody or fragment of embodiment 21, wherein the binding of the Fab to the IgE disrupts the interaction between the IgE and at least one Fcε receptor.

[0143] 23. The antibody or fragment of embodiment 22, wherein said disrupted interaction comprises blocking binding of unbound IgE to said at least one Fcε receptor.

[0144] 24. The antibody or fragment of embodiments 22-23, wherein said disrupted interaction comprises dissociating bound IgE from said at least one Fcε receptor.

[0145] 25. The antibody or fragment according to embodiments 22 to 24, wherein the disrupted interaction results in the inhibition of degranulation.

[0146] 26. The antibody or fragment according to embodiments 22 to 25, wherein the at least one Fcε receptor comprises FcεRI.

[0147] 27. The antigen-binding polypeptide of any one of embodiments 22 to 25, wherein the at least one Fcε receptor comprises CD23.

[0148] 28. The antibody or fragment according to any one of embodiments 22 to 25, wherein the at least one Fcε receptor comprises FcεRI and CD23.

[0149] 29. The antibody or fragment of any one of embodiments 21 to 28, wherein the Fab region specifically binds to at least one amino acid residue within the β5 helix region of Cε2, and the β5 helix region is a combination of the helix, the β5 helix joint, and the lower half of β5 that connects to the helix of Cε2.

[0150] 30. The antibody or fragment of embodiment 29, wherein the Fab region specifically binds to at least one amino acid residue in the helix of Cε2.

[0151] 31. The antibody or fragment of embodiment 29-30, wherein the Fab region specifically binds to at least one amino acid residue in the β5 helix joint of the Cε2.

[0152] 32. The antibody or fragment according to embodiments 29 to 31, wherein the Fab region specifically binds to at least one amino acid residue in the lower half of β5 that connects to the helix of Cε2.

[0153] 33. The antibody or fragment according to embodiments 29 to 32, wherein the Fab region does not bind to amino acid residue T298 of Cε2.

[0154] 34. The antibody or fragment of any one of embodiments 29 to 33, wherein the Fab region binds to at least two amino acid residues within the β5-Cε2 helix region.

[0155] 35. The antibody or fragment according to any one of embodiments 21 to 34, wherein the Fab region does not bind to the β3 region of Cε2.

[0156] 36. The antibody or fragment according to any one of embodiments 21 to 34, wherein the Fab region does not bind to the β4 region of Cε2.

[0157] 37. The antibody or fragment according to any one of embodiments 21 to 34, wherein the Fab region does not bind to the β3 or β4 region of Cε2.

[0158] 38. The antibody or fragment of embodiments 21 to 37, wherein the antibody is a bispecific antibody or a binding fragment thereof.

[0159] 39. The antibody or fragment of embodiments 21-38, wherein the antibody comprises a monovalent Fab', a divalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or binding fragment thereof.

[0160] 40. The antibody or fragment of any one of embodiments 21 to 39, wherein the Fab region comprises at least one heavy chain sequence selected from SEQ ID NOs: 1 to 25.

[0161] 41. The antibody or fragment according to any one of embodiments 21 to 40, wherein the Fab region comprises at least one light chain sequence selected from SEQ ID NOs: 26 to 50.

[0162] 42. The antibody or fragment according to any one of embodiments 21 to 41, wherein the Fab region comprises at least one complementarity determining region (CDR) selected from SEQ ID NOs: 51 to 200.

[0163] 43. The antibody has a K of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, or less than 30 nM. D 43. The antibody or fragment according to any one of embodiments 21 to 42, which exhibits:

[0164] 44. The antibody or fragment of any one of embodiments 21 to 43, wherein the antibody comprises a humanized antibody.

[0165] 45. An antibody or fragment thereof having a Fab region comprising at least one heavy chain sequence selected from SEQ ID NOs: 1 to 25.

[0166] 46. ​​An antibody or fragment thereof having a Fab region comprising at least one light chain sequence selected from SEQ ID NOs: 26 to 50.

[0167] 47. An antibody or fragment thereof having a Fab region comprising at least one heavy chain sequence selected from SEQ ID NOs: 1 to 25 and at least one light chain sequence selected from SEQ ID NOs: 26 to 50.

[0168] 48. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 26.

[0169] 49. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 2 and the light chain sequence of SEQ ID NO: 27.

[0170] 50. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 28.

[0171] 51. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 4 and the light chain sequence of SEQ ID NO: 29.

[0172] 52. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 5 and the light chain sequence of SEQ ID NO: 30.

[0173] 53. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 6 and the light chain sequence of SEQ ID NO: 31.

[0174] 54. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 7 and the light chain sequence of SEQ ID NO: 32.

[0175] 55. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 8 and the light chain sequence of SEQ ID NO: 33.

[0176] 56. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 34.

[0177] 57. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 35.

[0178] 58. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 11 and the light chain sequence of SEQ ID NO: 36.

[0179] 59. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 12 and the light chain sequence of SEQ ID NO: 37.

[0180] 60. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 13 and the light chain sequence of SEQ ID NO: 38.

[0181] 61. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 14 and the light chain sequence of SEQ ID NO: 39.

[0182] 62. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 15 and the light chain sequence of SEQ ID NO: 40.

[0183] 63. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 16 and the light chain sequence of SEQ ID NO: 41.

[0184] 64. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 17 and the light chain sequence of SEQ ID NO: 42.

[0185] 65. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 18 and the light chain sequence of SEQ ID NO: 43.

[0186] 66. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 19 and the light chain sequence of SEQ ID NO: 44.

[0187] 67. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 20 and the light chain sequence of SEQ ID NO: 45.

[0188] 68. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 21 and the light chain sequence of SEQ ID NO: 46.

[0189] 69. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 22 and the light chain sequence of SEQ ID NO: 47.

[0190] 70. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 23 and the light chain sequence of SEQ ID NO: 48.

[0191] 71. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 24 and the light chain sequence of SEQ ID NO: 49.

[0192] 72. An antibody or fragment thereof having a Fab region comprising the heavy chain sequence of SEQ ID NO: 25 and the light chain sequence of SEQ ID NO: 50.

[0193] 73. An antibody or fragment thereof having a Fab region comprising an HCDR1 selected from SEQ ID NOs: 51 to 75.

[0194] 74. The antibody or fragment of embodiment 73, having a Fab region comprising an HCDR2 selected from SEQ ID NOs: 76-100.

[0195] 75. The antibody or fragment according to embodiments 73-74, having a Fab region comprising an HCDR3 selected from SEQ ID NOs: 101-125.

[0196] 76. The antibody or fragment of embodiments 73-75, having a Fab region comprising an LCDR1 selected from SEQ ID NOs: 126-150.

[0197] 77. The antibody or fragment of embodiments 73-76, having a Fab region comprising an LCDR2 selected from SEQ ID NOs: 151-175.

[0198] 78. The antibody or fragment of embodiments 73-77, having a Fab region comprising an LCDR3 selected from SEQ ID NOs: 176-200.

[0199] 79. The antibody has a K of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, or less than 30 nM. D The antibody or fragment according to any one of embodiments 45 to 78, which exhibits the following:

[0200] 80. The antibody or fragment of any of embodiments 45 to 79, wherein the antibody comprises a humanized antibody.

[0201] 81. A humanized antibody or fragment thereof comprising a heavy chain variable region selected from SEQ ID NOs: 201-202.

[0202] 82. A humanized antibody or fragment thereof comprising a light chain variable region selected from SEQ ID NOs: 203 to 206.

[0203] 83. A humanized antibody or fragment thereof, comprising a heavy chain variable region selected from SEQ ID NOs: 201 to 202, and a light chain variable region selected from SEQ ID NOs: 203 to 206.

[0204] 84. A humanized antibody or fragment thereof, comprising a heavy chain variable region selected from SEQ ID NO: 201 and a light chain variable region selected from SEQ ID NOs: 203-204.

[0205] 85. A humanized antibody or fragment thereof, comprising a heavy chain variable region selected from SEQ ID NO: 202 and a light chain variable region selected from SEQ ID NOs: 205 to 206.

[0206] 86. A humanized antibody or fragment thereof comprising a heavy chain complete sequence selected from SEQ ID NOs: 207-208.

[0207] 87. A humanized antibody or fragment thereof comprising a light chain complete sequence selected from SEQ ID NOs: 209-212.

[0208] 88. A humanized antibody or fragment thereof, comprising a heavy chain complete sequence selected from SEQ ID NOs: 207-208, and a light chain variable region selected from SEQ ID NOs: 209-212.

[0209] 89. A humanized antibody or fragment thereof comprising a heavy chain complete sequence selected from SEQ ID NO: 207 and a light chain variable region selected from SEQ ID NOs: 209-210.

[0210] 90. A humanized antibody or fragment thereof comprising a heavy chain complete sequence selected from SEQ ID NO: 208, and a light chain variable region selected from SEQ ID NOs: 211-212.

[0211] 91. A complex comprising an antigen-binding polypeptide according to any one of embodiments 1 to 20 or an antibody or fragment according to any one of embodiments 21 to 90, wherein the complex comprises a polypeptide or antibody bound to an IgE protein.

[0212] 92. A method for disrupting the interaction between IgE and at least one Fcε receptor, comprising: contacting a cell expressing the at least one Fcε receptor with an antigen-binding polypeptide that specifically binds to the β5 helix region of the Cε2, wherein the β5 helix region is a combination of a helix, a β5 helix joint, and the bottom half of β5 that connects to the helix of Cε2.

[0213] 93. The method of embodiment 92, wherein the antigen-binding polypeptide specifically binds to at least one amino acid residue in the helix of Cε2.

[0214] 94. The method of embodiment 92-93, wherein the antigen-binding polypeptide specifically binds to at least one amino acid residue in the β5 helix joint of the Cε2.

[0215] 95. The method of embodiments 92-94, wherein said antigen-binding polypeptide specifically binds to at least one amino acid residue in the bottom half of β5 that connects to the helix of Cε2.

[0216] 96. The method of embodiments 92-95, wherein said disrupted interaction comprises blocking binding of unbound IgE to said at least one Fcε receptor.

[0217] 97. The method of embodiments 92-96, wherein said disrupted interaction comprises dissociating bound IgE from said at least one Fcε receptor.

[0218] 98. The method of embodiments 96-97, wherein the disrupted interaction results in the inhibition of degranulation.

[0219] 99. The method of embodiments 92 to 98, wherein the at least one Fcε receptor comprises FcεRI.

[0220] 100. The method of embodiments 92-98, wherein the at least one Fcε receptor comprises CD23.

[0221] 101. The method of embodiments 92 to 98, wherein the at least one Fcε receptor comprises FcεRI and CD23.

[0222] 102. The method of embodiments 92-101, wherein the Fab region does not bind to amino acid residue T298 of Cε2.

[0223] 103. The method of embodiments 92-102, wherein the Fab region binds to at least two amino acid residues within the β5-Cε2 helix region.

[0224] 104. The method of embodiments 92-103, wherein the antigen-binding polypeptide does not bind to the β3 region of Cε2.

[0225] 105. The method of embodiments 92-103, wherein the antigen-binding polypeptide does not bind to the β4 region of Cε2.

[0226] 106. The method of embodiments 92-103, wherein the antigen-binding polypeptide does not bind to the β3 or β4 region of Cε2.

[0227] 107. The method of embodiments 92 to 106, wherein the antigen-binding polypeptide is an antibody or a binding fragment thereof.

[0228] 108. The method of embodiment 107, wherein the antibody comprises a monovalent Fab', a divalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a camelid antibody or binding fragment thereof.

[0229] 109. The method of embodiments 107-108, wherein the Fab region comprises at least one heavy chain sequence selected from SEQ ID NOs: 1-25.

[0230] 110. The method of embodiments 107-109, wherein the Fab region comprises at least one light chain sequence selected from SEQ ID NOs: 26-50.

[0231] 111. The method of embodiments 107-110, wherein the Fab region comprises at least one complementarity determining region (CDR) selected from SEQ ID NOs: 51-200.

[0232] 112. The antibody has a K of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, or less than 30 nM. D The method according to any one of embodiments 107 to 111, wherein

[0233] 113. The method of embodiments 107-112, wherein the antibody comprises a humanized antibody.

[0234] 114. The method according to embodiments 92 to 113, wherein said interaction of IgE with at least one Fcε receptor is associated with an allergic condition.

[0235] 115. The method of embodiment 114, wherein the allergic condition is selected from asthma, chronic idiopathic urticaria, nasal polyps, hay fever, or food allergies.

[0236] Non-limiting Examples The present disclosure is also described and demonstrated by the following non-limiting examples. However, the use of these and other examples anywhere in the specification is exemplary only and in no way limits the scope and meaning of the disclosure or the scope and meaning of any exemplified term. Likewise, the present disclosure is not limited to any particular preferred examples or embodiments described herein. Indeed, appropriate modifications and variations may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the spirit or scope of the invention.

[0237] Example 1 - Development of anti-IgE antibodies from immunized rabbits Zealand White rabbits were immunized with recombinant IgE Cε2-4 protein. Phage display libraries were constructed from bone marrow and spleen and selected against the recombinant IgE Cε2-4 protein. Clones that showed specific binding to IgE Cε2-4 protein by ELISA were sequenced and their complementarity-determining regions (CDRs) were analyzed (Figures 1-4). These clones were expressed as Fabs with a 6xHis tag in HEK293 cells and purified using Ni-NTA column chromatography.

[0238] Example 2 - Three Fabs inhibit the binding of human IgE to human FcεRI We investigated whether the newly invented Fab could inhibit the binding of human IgE to human FcεRI, a heterotetrameric protein composed of one α chain, one β chain, and two γ chains. Using the BaF / 3 cell line retrovirally transduced with the α, β, and γ chains of human FcεRI, we assessed the binding of human IgE preincubated with three Fabs (21626B-C4-8 (BC48) (SEQ ID NOs: 24, 49, 74, 99, 124, 149, 174, and 199), 21626B-H3 (BH3) (SEQ ID NOs: 20, 45, 70, 95, 120, 145, 170, and 195), and 21626B-A8 (BA8) (SEQ ID NOs: 21, 46, 71, 96, 121, 146, 171, and 196)) or Xolair to cell-surface human FcεRI. To avoid potential interference of Fab with the detection of bound IgE, we measured the amount of IgE using fluorescently labeled antigen. Interestingly, all three Fabs inhibited IgE binding to cell surface human FcεRI at lower molar ratios compared to commercial Xolair (Fig. 5 ).

[0239] Example 3 - Three Fabs remove human IgE and inhibit antigen-stimulated degranulation of human FcεRIα-transgenic mouse bone marrow-derived mast cells (BMMCs) Furthermore, we evaluated whether our Fabs could remove IgE already bound to human FcεRI on the cell surface. The three Fabs, but not a control Fab that does not target human IgE, were able to dose-dependently remove IgE from the cell surface of human FcεRIα-transgenic BMMCs (Figure 6A). Furthermore, this removal coincided with a reduction in antigen-dependent activation of BMMCs (Figure 6B), indicating that the three Fabs can suppress mast cell responses via removal of bound IgE.

[0240] Example 4 - Three Fabs exhibit inhibitory and scavenging activity against IgE binding to the low affinity receptor CD2 To investigate the effect of Fabs on IgE binding to CD23, a low-affinity IgE receptor, competition and depletion assays were performed using a BaF / 3 cell line retrovirally transduced with human CD23. The amount of IgE on the cell surface was measured by its ability to bind fluorescently labeled antigen. We found that preincubation of human IgE with the three Fabs strongly inhibited its binding to cell surface CD23 (Figure 7A). Furthermore, the Fabs exhibited variable depletion activity on pre-bound IgE within 1 h (Figure 7B).

[0241] Example 5 - Three Fabs bind to the β5 helix region of Cε2 in a tertiary structure-dependent manner To identify the binding sites of the three Fabs, we prepared recombinant His6-tagged Cε2 proteins of human IgE and assessed the binding of the Fabs to native Cε2, boiled Cε2, and boiled Cε2 treated with PNGaseF by ELISA. Fab binding was abolished by boiling, suggesting that the tertiary structure is important for Fab binding to Cε2 (Figure 8A). Furthermore, treatment of recombinant Cε2 with PNGaseF did not affect Fab binding to Cε2, suggesting that N-glycan chains accessible to PNGaseF are not involved in the binding site (Figure 8B). To further narrow down the Fab binding site on Cε2, we attempted to replace portions of human Cε2 with the corresponding portions of mouse Cε2. Importantly, none of the three Fabs bound to mouse IgE (Figure 8C). Because these two Cε2 proteins are composed of seven β-sheets and one α-helix (Fig. 8D), we expressed seven chimeric Cε2 proteins in HEK293T cells and evaluated the binding of Fabs to them. Interestingly, replacing the β5 helix region of human Cε2 with that of mouse β5 helix abolished the binding of all three Fabs (Fig. 8E). These results indicate that the three Fabs share a critical binding site in the β5 helix region.

[0242] Example 6 - Detailed binding site within the β5 helix region The β5 helix region of human and mouse Cε2 differed by nine amino acids (Figure 9). Therefore, we prepared a chimeric human IgE in which part of the β5 helix region was replaced with that of mouse IgE (Figure 9A). Binding of three Fabs was abolished by mutations in the β5 helix region from the center to the C-terminus (Figure 9B). Furthermore, binding of two Fabs was partially reduced by mutations in β5 (Figure 9B). These results indicate that the important binding site within the β5 helix region involves at least the α-helical region, but the β5 region may also be involved.

[0243] Example 7 - Mutations in the β5 helix region did not affect IgE function Because binding of the three Fabs removed human IgE from the cell surface FcεRI and human IgE did not bind to the mouse FcεRI, we tested whether replacing the human β5 helix region of Cε2 with a mouse sequence affected the binding function of human IgE. All human chimeric IgEs evaluated in Figure 9 bound to the cell surface FcεRI and captured different doses of fluorescently labeled antigen at levels similar to those of wild-type IgE (Figure 10A). Antigen binding levels were similar 3 and 6 hours after washing away the IgE, indicating that the stability of IgE on the cell surface FcεRI was also unaffected by the introduced mutations (Figures 10B and C). These results suggest that the β5 helix substitutions do not affect the intrinsic binding function of IgE bound to the FcεRI.

[0244] Example 8 - Inhibition of IgE binding to FcεRI is Fab binding dependent Using these IgEs, we tested the ability of the Fabs to inhibit binding to cell surface FcεRI. As expected, mutation of the critical binding site of the Fab abolished competitive inhibition of IgE binding to cell surface FcεRI (Figure 11). Furthermore, for β5 mutant IgEs, competitive activity correlated with binding affinity (Figures 9B and 11). These results indicated that the effect of binding inhibition was Fab binding-dependent.

[0245] Example 9 - Removal of pre-bound IgE on cell surface FcεRI is Fab binding dependent We further determined whether the Fab removal effect was dependent on Fab binding. Again, mutation of the critical binding site of the Fab abolished the removal of pre-bound IgE on cell surface FcεRI (Figure 12A). A similar but stronger effect was observed when the IgG version of the Fab clone was used (Figure 12B). In both cases, the removal activity of the β5 region correlated with binding affinity (Figures 9B and 11). These results indicated that the removal effect was dependent on Fab binding.

[0246] Example 10 - Humanized anti-IgEs antibodies (BC48 and BH3) BC48 and BH3 were humanized by grafting the CDRs into human germline genes. Humanized Fabs were expressed in HEK293 cells as 6xHis-tagged proteins and purified using a Ni-NTA column. Recombinant Cε2-4 was coated at 1 μg / mL in PBS overnight at 4°C. Wells were washed three times with PBS and blocked with 1% BSA / PBS. The purified Fabs were serially diluted and incubated with antigen for 1 hour at room temperature. The wells were washed, and bound rabbit Fabs were detected with peroxidase-conjugated goat anti-rabbit IgG F(ab')2 (ThermoFisher Scientific 31461), and human Fabs were detected with peroxidase-conjugated goat anti-human IgG F(ab')2 (Jackson Immuno Research 109-035-097). As shown in Figures 16 and 17, the humanized Fabs showed comparable binding to Cε2-4.

[0247] Sequence Listing Heavy chain sequence Below are shown the heavy chain sequences of certain non-limiting anti-IgE antibodies, identified by name (eg, "21626S-D5") and ID number (eg, "SEQ ID NO: 1"). 21626S-D5 SEQ ID NO: 1 TIFF2025533898000002.tif14315021626B-B9 SEQ ID NO: 2 TIFF2025533898000003.tif15515321626B-H8 SEQ ID NO: 3 TIFF2025533898000004.tif14715221626S-E6 SEQ ID NO: 4 TIFF2025533898000005.tif14915621626S-E12 SEQ ID NO: 5 TIFF2025533898000006.tif13914421626B-F7 SEQ ID NO: 6 TIFF2025533898000007.tif14014521626B-F5 SEQ ID NO: 7 TIFF2025533898000008.tif13514521626B-C5 SEQ ID NO: 8 TIFF2025533898000009.tif14214921626S-G9 SEQ ID NO: 9 TIFF2025533898000010.tif14314821626S-F8 SEQ ID NO: 10 TIFF2025533898000011.tif14514321626B-F10 SEQ ID NO: 11 TIFF2025533898000012.tif13514121626B-G12 SEQ ID NO: 12 TIFF2025533898000013.tif16014621626B-A11 SEQ ID NO: 13 TIFF2025533898000014.tif13614121626B-B5 SEQ ID NO: 14 TIFF2025533898000015.tif13914321626B-C2 SEQ ID NO: 15 TIFF2025533898000016.tif13714521626B-H4 SEQ ID NO: 16 TIFF2025533898000017.tif13514121626B-C8 SEQ ID NO: 17 TIFF2025533898000018.tif14014421626B-E6 SEQ ID NO: 18 TIFF2025533898000019.tif14814621626B-G5 SEQ ID NO: 19 TIFF2025533898000020.tif13714221626B-H3 SEQ ID NO: 20 TIFF2025533898000021.tif13314321626B-A8 SEQ ID NO: 21 TIFF2025533898000022.tif13714121626B-D1 SEQ ID NO: 22 TIFF2025533898000023.tif14615321626B-G1 SEQ ID NO: 23 TIFF2025533898000024.tif15515421626B-C4-8 SEQ ID NO: 24 TIFF2025533898000025.tif14114921626S-A1 SEQ ID NO: 25 TIFF2025533898000026.tif143150

[0248] Light chain sequence Below are provided the light chain sequences of certain non-limiting anti-IgE antibodies, identified by name (eg, "21626S-D5") and ID number (eg, "SEQ ID NO: 26"). 21626S-D5 SEQ ID NO: 26 TIFF2025533898000027.tif15014921626B-B9 SEQ ID NO: 27 TIFF2025533898000028.tif13314021626B-H8 SEQ ID NO: 28 TIFF2025533898000029.tif14914721626S-E6 SEQ ID NO: 29 TIFF2025533898000030.tif14714721626S-E12 SEQ ID NO: 30 TIFF2025533898000031.tif13614421626B-F7 SEQ ID NO: 31 TIFF2025533898000032.tif13214221626B-F5 SEQ ID NO: 32 TIFF2025533898000033.tif13814521626B-C5 SEQ ID NO: 33 TIFF2025533898000034.tif13314121626S-G9 SEQ ID NO: 34 TIFF2025533898000035.tif14514721626S-F8 SEQ ID NO: 35 TIFF2025533898000036.tif13814521626B-F10 SEQ ID NO: 36 TIFF2025533898000037.tif13614121626B-G12 SEQ ID NO: 37 TIFF2025533898000038.tif14014521626B-A11 SEQ ID NO: 38 TIFF2025533898000039.tif13414121626B-B5 SEQ ID NO: 39 TIFF2025533898000040.tif13914521626B-C2 SEQ ID NO: 40 TIFF2025533898000041.tif14114621626B-H4 SEQ ID NO: 41 TIFF2025533898000042.tif969621626B-C8 SEQ ID NO: 42 TIFF2025533898000043.tif14114221626B-E6 SEQ ID NO: 43 TIFF2025533898000044.tif13914521626B-G5 SEQ ID NO: 44 TIFF2025533898000045.tif12813821626B-H3 SEQ ID NO: 45 TIFF2025533898000046.tif13814221626B-A8 SEQ ID NO: 46 TIFF2025533898000047.tif14114921626B-D1 SEQ ID NO: 47 TIFF2025533898000048.tif14614621626B-G1 SEQ ID NO: 48 TIFF2025533898000049.tif14215221626B-C4-8 SEQ ID NO: 49 TIFF2025533898000050.tif14314021626S-A1 SEQ ID NO: 50 TIFF2025533898000051.tif131146

[0249] CDR sequences Below is a listing of certain non-limiting anti-IgE antibody sequences, identified by name (eg, "21626S-D5...") and ID number (eg, "SEQ ID NO:51..."). TIFF2025533898000052.tif140147

[0250] Humanized heavy chain sequence (variable region) Below are provided the humanized heavy chain sequences (variable regions) of certain non-limiting anti-IgE antibodies, identified by name (e.g., "h-21626B-C4-8") and ID number (e.g., "SEQ ID NO: 201"). h-21626B-C4-8 SEQ ID NO: 201 TIFF2025533898000053.tif139146h-21626B-H3 Sequence number 202 TIFF2025533898000054.tif137144

[0251] Humanized light chain sequence (variable region) Below are provided the humanized light chain sequences (variable regions) of certain non-limiting anti-IgE antibodies, identified by name (e.g., "h-21626B-C4-8") and ID number (e.g., "SEQ ID NO: 203"). h-21626B-C4-8 SEQ ID NO: 203 TIFF2025533898000055.tif146152h-21626B-C4-8E SEQ ID NO: 204 TIFF2025533898000056.tif136142h-21626B-H3 Sequence number 205 TIFF2025533898000057.tif142142h-21626B-H3KQ Sequence number 206 TIFF2025533898000058.tif130137

[0252] Humanized heavy chain sequence (complete sequence) Below are provided the humanized heavy chain sequences (full sequences) of certain non-limiting anti-IgE antibodies, identified by name (e.g., "h-21626B-C4-8") and ID number (e.g., "SEQ ID NO: 207"). h-21626B-C4-8 SEQ ID NO: 207 TIFF2025533898000059.tif197126TIFF2025533898000060.tif57150h-21626B-H3 Sequence number 208 TIFF2025533898000061.tif171136TIFF2025533898000062.tif106149

[0253] Humanized light chain sequence (complete sequence) Below are provided the humanized light chain sequences (full sequences) of certain non-limiting anti-IgE antibodies, identified by name (e.g., "h-21626B-C4-8") and ID number (e.g., "SEQ ID NO: 209"). h-21626B-C4-8 SEQ ID NO: 209 TIFF2025533898000063.tif95148TIFF2025533898000064.tif157140h-21626B-C4-8E SEQ ID NO: 210 TIFF2025533898000065.tif65148TIFF2025533898000066.tif182136h-21626B-H3 SEQ ID NO: 211 TIFF2025533898000067.tif36153TIFF2025533898000068.tif198126h-21626B-H3KQ SEQ ID NO: 212 TIFF2025533898000069.tif229146

[0254] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. 1.Strunk RC,Bloomberg GR. 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Maurer M, Rosen K, Hsieh HJ, Saini S, Grattan C, Gimenez-Arnau A, et al.Omalizumab for the treatment of chronic idiopathic or spontaneous urticaria.N Engl J Med 2013;368:924-35. [ PubMed ] [ Cross Ref ] 8. Gevaert P, Calus L, Van Zele T, Blomme K, De Ruyck N, Bauters W, et al. 9.Gevaert P,Omachi TA,Corren J,Mullol J,Han J,Lee SE,et al.Efficacy and safety of omalizumab in nasal polyposis: 2 randomized phase 3 trials.J Allergy Clin Immunol 2020;146:595-605. 10.Okubo K,Okano M,Sato N,Tamaki Y,Suzuki H,Uddin A,et al.Add-On Omalizumab for Inadequately Controlled Severe Pollinosis Despite Standard-of-Care:A Randomized Study.J Allergy Clin Immunol Pract 2020;8:3130-40 e2。 11.Dantzer JA,Wood RA. The use of omalizumab in allergen immunotherapy.Clin Exp Allergy 2018;48:232-40。 12.Beck LA,Marcotte GV,MacGlashan D,Togias A,Saini S.Omalizumab-induced reductions in mast cell Fce psilon RI expression and function.J Allergy Clin Immunol 2004;114:527-30。 13.Kaplan AP,Gimenez-Arnau AM,Saini SS.Mechanisms of action that contribute to efficacy of omalizumab in chronic spontaneous urticaria.Allergy 2017;72:519-33。 14.Trischler J,Bottoli I,Janocha R,Heusser C,Jaumont X,Lowe P,et al.Ligelizumab treatment for severe asthma:learnings from the clinical development programme.Clin Transl Immunology 2021;10:e1255。 15.Maurer M,Gimenez-Arnau AM,Sussman G,Metz M,Baker DR,Bauer A,et al.Ligelizumab for Chronic Spontaneous Urticaria.N Engl J Med 2019;381:1321-3 16.Gauvreau GM,Arm JP,Boulet LP,Leigh R,Cockcroft DW,Davis BE,et al.Efficacy and safety of multiple doses of QGE031 (ligelizumab) versus omalizumab and placebo in inhibiting allergen-induced early asthmatic responses.J Allergy Clin Immunol 2016;138:1051–9 17.Gasser P,Tarchevskaya SS,Guntern P,Brigger D,Ruppli R,Zbaren N,et al.The mechanistic and functional profile of the therapeutic anti-IgE antibody ligelizumab differs from omalizumab.Nat Commun 2020;11:1 18. Jensen RK, Jabs F, Miehe M, Molgaard B, Pfutzner W, Mobs C, et al.Allergy 2020;75:1956-6 19.Drinkwater N,Cossins B,Keeble AH,Wright M,Cain K,Hailu H,et al.Human immunoglobulin E flexes between acutely bent and extended conformations.Nat Struct Mol Biol 2014;21:397-404。 20.Cohen ES,Dobson CL,Kack H,Wang B,Sims DA,Lloyd CO,et al.A novel IgE-neutralizing antibody for the treatment of severe uncontrolled asthma.MAbs 2014;6:756-64。 21.Sheldon E,Schwickart M,Li J,Kim K,Crouch S,Parveen S,et al.Pharmacokinetics,Pharmacodynamics,and Safety of MEDI4212,an Anti-IgE Monoclonal Antibody,in Subjects with Atopy:A Phase I Study. Adv Ther 2016;33:225-51。 22.Baumann MJ,Eggel A,Amstutz P,Stadler BM,Vogel M.DARPins against a functional IgE epitope.Immunol Lett 2010;133:78-84。 23.Kim B,Eggel A,Tarchevskaya SS,Vogel M,Prinz H,Jardetzky TS. Accelerated disassembly of IgE-receptor complexes by a disruptive macromolecular inhibitor.Nature 2012;491:613-7。 24.Prinz H,Striessnig J.Ligand-induced accelerated dissociation of (+)-cis-diltiazem from L-type Ca2+ channels is simply explained by competition for individual attachment points.J Biol Chem 1993;268:18580-5。 25.Eggel A,Baravalle G,Hobi G,Kim B,Buschor P,Forrer P,et al.Accelerated dissociation of IgE-FcepsilonRI complexes by disruptive inhibitors actively desensitizes allergic effector cells.J Allergy Clin Immunol 2014;133:1709-19 e8。 26.Jabs F,Plum M,Laursen NS,Jensen RK,Molgaard B,Miehe M,et al.Trapping IgE in a closed conformation by mimicking CD23 binding prevents and disrupts FcepsilonRI interaction.Nat Commun 2018;9:7。 27.Harris JM,Cabanski CR,Scheerens H,Samineni D,Bradley MS,Cochran C,et al.A randomized trial of quilizumab in adults with refractory chronic spontaneous urticaria.J Allergy Clin Immunol 2016;138:1730-2。 28.Harris JM,Maciuca R,Bradley MS,Cabanski CR,Scheerens H,Lim J,et al.A randomized trial of the efficacy and safety of quilizumab in adults with inadequately controlled allergic asthma.Respir Res 2016;17:29。 29.Gauvreau GM,Harris JM,Boulet LP,Scheerens H,Fitzgerald JM,Putnam WS,et al.Targeting membrane-expressed IgE B cell receptor with an antibody to the M1 prime epitope reduces IgE production.Sci Transl Med 2014;6:243ra85。 30.Chu SY,Horton HM,Pong E,Leung IW,Chen H,Nguyen DH,et al.Reduction of total IgE by targeted coengagement of IgE B-cell receptor and FcgammaRIIb with Fc-engineered antibody.J Allergy Clin Immunol 2012;129:1102-15。 31.Kirak O,Riethmuller G.A novel,nonanaphylactogenic,bispecific IgE-CD3 antibody eliminates IgE(+) B cells.J Allergy Clin Immunol 2015;136:800-2 e3。 32.Wiegand TW,Williams PB,Dreskin SC,Jouvin MH,Kinet JP,Tasset D.High-affinity oligonucleotide ligands to human IgE inhibit binding to Fc epsilon receptor I.J Immunol 1996;157:221-30。 33.Mendonsa SD,Bowser MT.In vitro selection of high-affinity DNA ligands for human IgE using capillary electrophoresis.Anal Chem 2004;76:5387-92。 34.Poongavanam MV,Kisley L,Kourentzi K,Landes CF,Willson RC.Ensemble and single-molecule biophysical characterization of D17.4 DNA aptamer-IgE interactions.Biochim Biophys Acta 2016;1864:154-64。 35.Ando T,Kitaura J.Tuning IgE:IgE-Associating Molecules and Their Effects on IgE-Dependent Mast Cell Reactions.Cells 2021;10。 36.Pennington LF,Tarchevskaya S,Brigger D,Sathiyamoorthy K,Graham MT,Nadeau KC,et al.Structural basis of omalizumab therapy and omalizumab-mediated IgE exchange.Nat Commun 2016;7:11610。 37.Davies AM,Allan EG,Keeble AH,Delgado J,Cossins BP,Mitropoulou AN,et al.Allosteric mechanism of action of the therapeutic anti-IgE antibody omalizumab.J Biol Chem 2017;292:9975-87。 38.McDonnell JM,Calvert R,Beavil RL,Beavil AJ,Henry AJ,Sutton BJ,et al.The structure of the IgE Cepsilon2 domain and its role in stabilizing the complex with its high-affinity receptor FcepsilonRIalpha.Nat Struct Biol 2001;8:437-41。 39.Hirano T,Koyanagi A,Kotoshiba K,Shinkai Y,Kasai M,Ando T,et al.The Fab fragment of anti-IgE Cepsilon2 domain prevents allergic reactions through interacting with IgE-FcepsilonRIalpha complex on ratmast cells.Sci Rep 2018;8:14237。

Claims

1. An antibody or fragment thereof comprising a Fab region that specifically binds to IgE, wherein the binding of the Fab to the IgE disrupts the interaction between the IgE and at least one Fcε receptor.

2. 2. The antibody or fragment of claim 1, wherein the disrupted interaction comprises blocking the binding of unbound IgE to the at least one Fcε receptor.

3. 2. The antibody or fragment of claim 1, wherein the disrupted interaction comprises dissociating bound IgE from the at least one Fcε receptor.

4. The antibody or fragment of claim 1 , wherein the disrupted interaction results in the inhibition of degranulation.

5. The antibody or fragment of claim 1 , wherein the at least one Fcε receptor comprises FcεRI.

6. The antigen-binding polypeptide of claim 1 , wherein the at least one Fcε receptor comprises CD23.

7. The antibody or fragment of claim 1 , wherein the at least one Fcε receptor comprises FcεRI and CD23.

8. The antibody or fragment of claim 1, wherein the Fab region specifically binds to at least one amino acid residue within the β5 helix region of Cε2, and the β5 helix region is a combination of a helix, a β5 helix joint, and the lower half of β5 that connects to the helix of Cε2.

9. The antibody or fragment of claim 8, wherein the Fab region specifically binds to at least one amino acid residue in the Cε2 helix.

10. The antibody or fragment of claim 8, wherein the Fab region specifically binds to at least one amino acid residue in the β5 helix joint of the Cε2.

11. 9. The antibody or fragment of claim 8, wherein the Fab region specifically binds to at least one amino acid residue in the lower half of β5 that connects to the Cε2 helix.

12. The antibody or fragment of claim 8, wherein the Fab region does not bind to amino acid residue T298 of Cε2.

13. The antibody or fragment of claim 8, wherein the Fab region binds to at least two amino acid residues within the β5-Cε2 helix region.

14. The antibody or fragment of claim 8, wherein the Fab region does not bind to the β3 region of Cε2.

15. The antibody or fragment of claim 8, wherein the Fab region does not bind to the β4 region of Cε2.

16. The antibody or fragment of claim 8, wherein the Fab region does not bind to the β3 or β4 region of Cε2.

17. The antibody or fragment of claim 1 , wherein the antibody is a bispecific antibody or a binding fragment thereof.

18. 2. The antibody or fragment of claim 1, wherein the antibody comprises a monovalent Fab', a bivalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or binding fragment thereof.

19. The antibody or fragment of claim 1, wherein the Fab region comprises at least one heavy chain sequence selected from SEQ ID NOs: 1-25.

20. The antibody or fragment of claim 1, wherein the Fab region comprises at least one light chain sequence selected from SEQ ID NOs: 26-50.

21. 2. The antibody or fragment of claim 1, wherein the Fab region comprises at least one complementarity determining region (CDR) selected from SEQ ID NOs: 51-200.

22. the antibody has a K of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, or less than 30 nM Ｄ 2. The antibody or fragment of claim 1, wherein:

23. The antibody or fragment of claim 1 , wherein the antibody is a humanized antibody.

24. The antibody or fragment thereof according to claim 23, wherein the humanized antibody comprises a heavy chain variable region selected from SEQ ID NOs: 201-202.

25. The antibody or fragment thereof of claim 23, wherein the humanized antibody comprises a light chain variable region selected from SEQ ID NOs: 203 to 206.

26. The antibody or fragment thereof according to claim 23, wherein the humanized antibody comprises a heavy chain variable region selected from SEQ ID NOs: 201-202 and a light chain variable region selected from SEQ ID NOs: 203-206.

27. The antibody or fragment thereof of claim 23, wherein the humanized antibody comprises a complete heavy chain sequence selected from SEQ ID NOs: 207-208.

28. The antibody or fragment thereof of claim 23, wherein the humanized antibody comprises a complete light chain sequence selected from SEQ ID NOs: 209-212.

29. 24. The antibody or fragment thereof of claim 23, wherein the humanized antibody comprises a heavy chain complete sequence selected from SEQ ID NOs: 207-208 and a light chain variable region selected from SEQ ID NOs: 209-212.

30. A conjugate comprising the antibody or fragment of claim 1, wherein the conjugate comprises a polypeptide or antibody bound to an IgE protein.

Images