ANTI-IgE ANTIBODIES

Abstract

To provide improved anti-IgE antibodies and antigen binding agents, and compositions thereof, which target IgE, for instance, for use in treating disorders caused by IgE (such as allergic responses, or certain autoimmune responses), in particular, disorders caused by the interaction of IgE with the FcεRI receptor.SOLUTION: The improved anti-IgE antibodies and antigen binding agents of the invention may have improved affinity for IgE and / or an improved interaction with the Cε2 domain of IgE and / or an improved modified epitope on IgE and / or the ability to dissociate IgE from the FcεRI receptor for instance at pharmaceutically-relevant concentrations. In one aspect, improved or novel treatments for IgE mediated disorders are disclosed in which IgE is targeted.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention is in the field of improved anti-IgE antibodies and antigen binding agents targeting IgE, for example for use in the treatment of disorders caused by IgE (such as allergic responses or certain autoimmune responses), in particular disorders caused by the interaction of IgE with FcεRI receptors, and compositions thereof. In particular, the present invention relates to improved anti-IgE antibodies and antigen binding agents associated with novel variants of Omalizumab (Xolair®). The improved anti-IgE antibodies and antigen binding agents of the present invention may have improved affinity for IgE and / or improved interaction with the Cε2 domain of IgE and / or improved modified epitopes on IgE (e.g., further comprising the Cε2 domain of IgE) and / or the ability to dissociate IgE from FcεRI receptors at pharma- tically relevant concentrations. In one aspect, improved or novel treatments of IgE-mediated disorders are disclosed, in which IgE is targeted (e.g., free IgE and / or IgE complexed with FcεRI receptors). 2. Background of the Invention

[0002] IgE is a member of the immunoglobulin family that mediates allergic responses such as asthma, food allergies, type 1 hypersensitivity, and the widely affected familial sinusitis. IgE is secreted by B cells and expressed on their surface. IgE synthesized by B cells is anchored in the B cell membrane by a transmembrane domain linked to the mature IgE sequence by a short membrane-binding region. IgE also binds to B cells (as well as monocytes, eosinophils, and platelets) via its Fc region to the low affinity IgE receptor (FcεRII). Upon exposure of a mammal to an allergen, B cells are clonally expanded that synthesize IgE that binds to the allergen. This IgE is in turn released into the circulation by the B cells to which it is bound (via FcεRII), and by mast cells and basophils via the so-called high affinity receptor (FcεRI) found on the surface of mast cells and basophils. Such mast cells and basophils are thereby sensitized to the allergen. Subsequent exposure to allergen crosslinks FcεRI on these cells, thus activating the release of histamine and other factors that contribute to clinical hypersensitivity and anaphylaxis.

[0003] Omalizumab (Xolair®) is a recombinant DNA-derived humanized IgG1κ monoclonal antibody that selectively binds to human immunoglobulin E (IgE) [Cε3 domain]. The antibody has a molecular weight of approximately 149 kD. Xolair® is produced by Chinese hamster ovary cell suspension cultures in a nutrient medium containing the antibiotic gentamicin. Xolair® is a sterile, white, preservative-free, lyophilized powder contained in single-use vials that is reconstituted with Sterile Water for Injection (SWFI), USP (or alternatively, as a liquid formulation in a sterile syringe) and administered as a subcutaneous (SC) injection [see EP 602126 (and SPC / GB06 / 005 based thereon); WO 93 / 04173; US 6,267,958 (and Xolair® PTE based thereon); WO 97 / 04807; WO 97 / 04801; Presta et al. (1993) J. Immunol. 151:2623-2632].

[0004] Omalizumab is currently indicated for the treatment of moderate to severe persistent asthma in patients with positive skin tests or in vitro reactivity to perennial aeroallergens and symptoms not adequately controlled by inhaled corticosteroids (from Xolair® prescribing information).

[0005] The problems with Omalizumab are: 1) it targets free IgE but does not target (or does not efficiently target) the pathogenic species of IgE / FcεRI complexes at pharmacologic relevant doses; 2) presumably because the pathogenic species of IgE / FcεRI complexes are not targeted, it will take "at least 12-16 weeks for Xolair treatment to show efficacy (Xolair® 150 mg solution - Summary of Product Characteristics 2014) or indeed to establish whether Xolair® works for a particular patient or whether a different treatment is needed"; 3) it is not for patients who exhibit high levels of IgE (e.g. considering the high levels of free IgE in patients, because the pathogenic species of IgE / FcεRI complexes are not targeted and do not disappear over time); 4) "Omalizumab (Xolair® 150 mg solution Summary of Product Characteristics 2014) Type I local or systemic reactions, including anaphylaxis and anaphylactic shock, may occur"; 5) its affinity for IgE is not particularly good (approximately 2 nM).

[0006] The objective of the present invention is to identify novel antibodies to ameliorate one or more of these problems. A further challenge is to identify antibodies against new epitopes (enhanced IgE Cε2 interaction compared to omalizumab) and / or antibodies based on new variants of omalizumab with improved affinity and / or improved ability to dissociate the IgE / FcεRI complex.

[0007] It is a further object of the present invention to identify novel compounds, methods and compositions for the treatment of IgE associated disorders, in particular disorders associated with the IgE / FcεRI complex, such as allergic disorders. Summary of the Invention

[0008] In one aspect of the invention there is provided an anti-IgE antibody or antigen binding agent which contacts an epitope comprising residues T373, W374, S375, R376, A377, S378, G379, P381, Q417, C418, R419, T421, P426, R427, A428 of the Cε3 domain and residues D278 and T281 of the Cε2 domain of human IgE. In a further aspect the epitope may further comprise one or more of residues K380 and / or M430 of the Cε3 domain of human IgE and / or one or more of residues D276, V277, L279, S280, A282 and / or T298 of the Cε2 domain of human IgE.

[0009] The present invention is based on the observation of the crystal structure of Example 1, which shows for the first time the interaction of an improved antibody (based on Omalizumab) with IgE-Fc, where significant interaction was observed with the IgE Cε2 domain in the mutated region. This may result in improved functional properties of the anti-IgE antibody or antigen-binding agent compared to Omalizumab and / or Omalizumab Fab. For example, the anti-IgE antibody or antigen-binding agent is capable of dissociating human IgE from FcεRI at concentrations (or peak serum concentrations) of less than 7, 3, 1, 0.66, 0.5 or 0.3 μM (e.g., performed by the method described in Example 2). For example, the anti-IgE antibody or antigen-binding agent has an improved / stronger affinity (lower K D ) (e.g., using IgE-Fc) (e.g., carried out by the method described in Example 6); and / or improved ability to dissociate IgE / FcεRI complexes compared to omalizumab and / or omalizumab Fab (e.g., as determined by the method described in Example 2); and / or capability to dissociate human IgE from FcεRI at lower concentrations (or peak serum concentrations) than omalizumab and / or omalizumab Fab (e.g., as determined by the method described in Example 2). Improved K DBy K is meant at least 5, 10, 20, 30, 40, or 50% lower than that of omalizumab and / or omalizumab Fab. D may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3 nM. Improved ability or capability to dissociate human IgE from FcεRI means at least 5, 10, 20, 30, 40, 50, or 100% improvement over omalizumab and / or omalizumab Fab (e.g., when measuring the % dissociation and / or apparent rate of dissociation of IgE / FcεRI complex as described in Examples 2 and 7), and / or achieving dissociation at concentrations where omalizumab and / or omalizumab Fab do not achieve dissociation.

[0010] For the avoidance of doubt, the anti-IgE antibody or antigen binding agent of the invention is not omalizumab or omalizumab Fab.

[0011] In one aspect, the epitope is determined crystallographically (e.g., as described in Example 1) by determining IgE residues within 4 or 5 Å of the anti-IgE antibody or antigen binding agent in the crystal structure of the complexed IgE-Fc / anti-IgE antibody or antigen binding agent. The IgE-Fc used may be as that of SEQ ID NO: 108 (with the additional N265Q & N371Q mutations).

[0012] In one embodiment, an anti-IgE antibody or antigen-binding agent contacts an epitope at a specific binding site where the Cε3 and Cε2 domain portions of the epitope are on different chains of human IgE, which has two chains in the Fc domain, each with a Cε3 and a Cε2 domain. In one embodiment, an anti-IgE antibody or antigen-binding agent contacts an epitope on the same chain of human IgE at a specific binding site where the Cε3 domain and the Cε2 domain portions of the epitope are in contact.

[0013] For the avoidance of doubt, two anti-IgE antibodies or antigen-binding agents of the present invention are capable of binding to human IgE, but only one of these requires interaction with an epitope of the present invention comprising the Cε3 and Cε2 domains (the other, for example, may only interact with the other Cε3 domain).

[0014] In one embodiment (optionally further employing features of the first aspect of the invention), the anti-IgE antibody or antigen binding agent is specific for said epitope comprising residues T373, W374, S375, R376, A377, S378, G379, P381, Q417, C418, R419, T421, P426, R427, A428 of the Cε3 domain of human IgE and residues D278 and T281 of the Cε2 domain of human IgE. Optionally, said epitope may further comprise one or more of residues K380 and / or M430 of the Cε3 domain of human IgE and / or one or more of residues D276, V277, L279, S280, A282 and / or T298 of the Cε2 domain of human IgE. For the avoidance of doubt, an anti-IgE antibody or antigen-binding agent is specific for said epitope if it recognises and binds to a specific human IgE structure which contains said epitope rather than human IgE in general.

[0015] In a further aspect (optionally further employing features of the first aspect of the invention), there is provided an anti-IgE antibody or antigen binding agent comprising a heavy chain variable region comprising complementarity determining region CDR-H3 having the amino acid sequence of SEQ ID NO: 18, and a light chain variable region comprising complementarity determining region CDR-L1 having the amino acid sequence of SEQ ID NO: 29, wherein the light chain variable region further comprises a framework region FR-L3 having an amino acid sequence selected from SEQ ID NO: 32 with 1, 2, 3, 4, 5, 6, 7 or more amino acid substitutions that enhance the interaction of the anti-IgE antibody or antigen binding agent with the Cε2 domain of human IgE.

[0016] In a further aspect (optionally further employing features of the previous aspects of the invention), there is provided an anti-IgE antibody or antigen binding agent comprising a heavy chain variable region comprising complementarity determining region CDR-H3 having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising complementarity determining region CDR-L1 having the amino acid sequence of SEQ ID NO: 29, wherein the light chain variable region further comprises a framework region FR-L1 having the amino acid sequence of SEQ ID NO: 28 with 1, 2, 3, 4, 5, 6, 7 or more amino acid substitutions that enhance the interaction of the anti-IgE antibody or antigen binding agent with the Cε2 domain of human IgE.

[0017] Where the CDR-H3 and CDR-L1 regions anchor and orient the anti-IgE antibody or antigen binder on the IgE Cε3 region (as in Omalizumab), the change(s) to the FR-L3 and / or FR-L1 sequence allows for stronger interaction with the Cε2 domain of human IgE. The stronger interaction of the variants compared to Omalizumab or Omalizumab Fab can be demonstrated by affinity measurements [lower K D ] (e.g., performed by the method described in Example 6) and / or improved dissociation characteristics of the IgE / FcεRI complex (determined, for example, by the method described in Example 2).

[0018] A stronger interaction of the anti-IgE antibody or antigen-binding agent with the Cε2 domain of human IgE can be characterized by improved functional properties of the anti-IgE antibody or antigen-binding agent compared to omalizumab and / or omalizumab Fab. For example, the anti-IgE antibody or antigen-binding agent can dissociate human IgE from FcεRI at concentrations (or peak serum concentrations) of less than 7, 3, 1, 0.66, 0.5 or 0.3 μM (e.g., performed by the method described in Example 2). For example, the anti-IgE antibody or antigen-binding agent can have improved / stronger affinity (lower K D) (e.g., using IgE-Fc) (e.g., carried out by the method described in Example 6); and / or improved ability to dissociate IgE / FcεRI complexes compared to omalizumab and / or omalizumab Fab (e.g., as determined by the method described in Example 2); and / or capability to dissociate human IgE from FcεRI at lower concentrations (or peak serum concentrations) than omalizumab and / or omalizumab Fab (e.g., as determined by the method described in Example 2). Improved K D By K is meant at least 5, 10, 20, 30, 40, or 50% lower than that of omalizumab and / or omalizumab Fab. D may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3 nM. Improved ability or capability to dissociate human IgE from FcεRI means at least 5, 10, 20, 30, 40, 50, or 100% improvement over omalizumab and / or omalizumab Fab (e.g., when measuring the % dissociation and / or apparent rate of dissociation of IgE / FcεRI complex as described in Examples 2 and 7), and / or achieving dissociation at concentrations where omalizumab and / or omalizumab Fab do not achieve dissociation.

[0019] For the avoidance of doubt, the anti-IgE antibody or antigen binding agent of the present invention is not omalizumab, or omalizumab Fab. In one aspect, the FR-L3 region is mutated to one of the other naturally occurring amino acids at one or more of positions S60, S63, S76, S77, and / or Q79 (Kabat) with reference to SEQ ID NO: 129.

[0020] For example, the FR-L3 region may be mutated at position S60 (Kabat) to one of the other naturally occurring amino acids, such as M, R, K, N, Q or T, in particular M. For example, the FR-L3 region may be mutated at position S63 (Kabat) to one of the other naturally occurring amino acids, such as W or Y, in particular Y. For example, the FR-L3 region may be mutated to one of the other natural amino acids, in particular to N, at position S76 (Kabat). For example, the FR-L3 region may be mutated at position S77 (Kabat) to one of the other naturally occurring amino acids, such as R or K, in particular R.

[0021] For example, the FR-L3 region may be mutated at position Q79 (Kabat) to one of the other naturally occurring amino acids, such as R or K, in particular R. For example, the FR-L1 region may be mutated at G16 and / or R18 (Kabat) to one of the other naturally occurring amino acids, with reference to SEQ ID NO:20. In certain embodiments, the amino acid sequence of the mutated FR-L3 region of the anti-IgE antibody or antigen-binding agent is selected from SEQ ID NOs: 43-49, 60-83, 131, or 138.

[0022] In a further embodiment, the FR-L3 region is selected from the group consisting of FR-L2 and FR-L3-L3-L2-associated FR-L1 regions, which are characterized by their affinity for human IgE (lower K D ) with reference to SEQ ID NO: 129. In this case, the mutation may enhance the interaction of the anti-IgE antibody or antigen-binding agent with the Cε3 domain of IgE. For example, the FR-L3 region may be mutated to M (in particular), E, ​​or D at position S67 (Kabat). In certain embodiments, the amino acid sequence of the mutated FR-L3 region of the anti-IgE antibody or antigen-binding agent is selected from SEQ ID NOs: 53-59, 84-107, 131 or 138. An anti-IgE antibody or antigen-binding agent of the invention may have a light chain variable region further comprising the complementarity determining region CDR-L2, having the amino acid sequence which is SEQ ID NO:31.

[0023] In one embodiment, the CDR-L2 region is selected from the group consisting of a nucleotide sequence that is specific for human IgE (lower K DIn one particular embodiment, the CDR-L2 region is mutated at position S52 (Kabat) to one of the other naturally occurring amino acids to improve the IgE Cε3 domain. In this case, the mutation may enhance the interaction of the anti-IgE antibody or antigen-binding agent with the Cε3 domain of IgE. For example, the CDR-L2 region may be mutated at position S52 (Kabat) to D (in particular), E, ​​Q or R with reference to SEQ ID NO: 129. In one particular embodiment, the amino acid sequence of the mutated CDR-L2 region is selected from SEQ ID NO: 50 or SEQ ID NO: 51. An anti-IgE antibody or antigen-binding agent of the invention may have a heavy chain variable region further comprising the complementarity determining region CDR-H1, having the amino acid sequence which is SEQ ID NO:14.

[0024] The anti-IgE antibody or antigen-binding agent of the invention may have a heavy chain variable region further comprising the complementarity determining region CDR-H2, having the amino acid sequence which is SEQ ID NO:16. An anti-IgE antibody or antigen-binding agent of the invention may have a light chain variable region further comprising the complementarity determining region CDR-L3, having the amino acid sequence which is SEQ ID NO:33. An anti-IgE antibody or antigen-binding agent of the invention may have a heavy chain variable region further comprising the framework region FR-H1, having the amino acid sequence that is SEQ ID NO:13.

[0025] An anti-IgE antibody or antigen-binding agent of the invention may have a heavy chain variable region further comprising the framework region FR-H2, having the amino acid sequence that is SEQ ID NO:15. An anti-IgE antibody or antigen-binding agent of the invention may have a heavy chain variable region further comprising the framework region FR-H3, having the amino acid sequence that is SEQ ID NO:17. An anti-IgE antibody or antigen-binding agent of the invention may have a heavy chain variable region further comprising the framework region FR-H4, having the amino acid sequence that is SEQ ID NO:19.

[0026] An anti-IgE antibody or antigen-binding agent of the invention may have a light chain variable region further comprising the framework region FR-L2 having the amino acid sequence that is SEQ ID NO:30. An anti-IgE antibody or antigen-binding agent of the invention may have a light chain variable region further comprising the framework region FR-L4, having the amino acid sequence that is SEQ ID NO:34. The anti-IgE antibody or antigen-binding agent of the invention may have a light chain variable region, VL, having an amino acid sequence selected from SEQ ID NO:35, SEQ ID NO:132 or SEQ ID NO:134 or SEQ ID NO:141 or SEQ ID NO:144 or SEQ ID NO:145 or SEQ ID NO:158 or SEQ ID NO:159. An anti-IgE antibody or antigen-binding agent of the invention may have a heavy chain variable region, VH, having the amino acid sequence which is SEQ ID NO:1.

[0027] The anti-IgE antibody or antigen-binding agent of the invention may further comprise a light chain constant region. The anti-IgE antibodies or antigen-binding agents of the invention may have a light chain constant region that is a kappa constant region. The anti-IgE antibodies or antigen-binding agents of the invention may have a light chain constant region that has a L154P (Kabat) mutation.

[0028] The anti-IgE antibody or antigen-binding agent of the invention may have a light chain variable region and a light chain constant region VL-CL having an amino acid sequence selected from SEQ ID NO:39, or SEQ ID NO:41, or SEQ ID NO:117, or SEQ ID NO:119, or SEQ ID NO:125, or SEQ ID NO:127, or SEQ ID NO:136, or SEQ ID NO:143, optionally including a signal sequence having the amino acid sequence which is SEQ ID NO:160. The anti-IgE antibody or antigen-binding agent of the invention may further comprise the heavy chain constant region CH1. An anti-IgE antibody or antigen-binding agent of the invention may have a heavy chain variable region and a heavy chain constant region VH-CH1 having the amino acid sequence which is SEQ ID NO:5. The anti-IgE antibodies or antigen-binding agents of the invention may further comprise a heavy chain Fc region, Fc. The anti-IgE antibodies or antigen-binding agents of the invention may have an Fc derived from human IgG1 or human IgG4. An anti-IgE antibody or antigen-binding agent of the invention may have a heavy chain variable region, a heavy chain constant region and a heavy chain Fc region, VH-CH1-Fc, having the amino acid sequence that is SEQ ID NO:9.

[0029] In a further aspect of the invention there is provided an anti-IgE antibody or antigen-binding agent comprising a heavy chain variable region comprising the complementarity determining region CDR-H3 having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising the complementarity determining region CDR-L1 having the amino acid sequence of SEQ ID NO: 29, a. The light chain variable region further comprises a framework region FR-L3 having an amino acid sequence of SEQ ID NO: 32, wherein the FR-L3 region enhances the affinity of the anti-IgE antibody or antigen binding agent for human IgE (lower K D ) is mutated to one of the other naturally occurring amino acids at position S67 (Kabat), with reference to SEQ ID NO: 129, in order to improve b. The light chain variable region further comprises a complementarity determining region CDR-L2 having an amino acid sequence of SEQ ID NO: 31, wherein the CDR-L2 region determines the affinity of the anti-IgE antibody or antigen binding agent for human IgE (lower K D ) is mutated to one of the other natural amino acids at position S52 (Kabat) with reference to SEQ ID NO: 129, in order to improve The above anti-IgE antibodies or antigen-binding agents are provided.

[0030] The inventors believe that either or both of these mutations may improve the affinity of omalizumab or omalizumab Fab-based anti-IgE antibodies or antigen-binding agents for human IgE (improved or lower K D It has now been found that the affinity of the IgE-Fc antibody can be surprisingly improved (e.g., using IgE-Fc) (e.g., as performed by the method described in Example 6). In particular, the affinity improvement is in comparison to omalizumab and / or omalizumab Fab. The mutations can improve the interaction with the Cε3 domain of IgE. An improved or lower K DBy K is meant at least 5, 10, 20, 30, 40, or 50% lower than that of omalizumab and / or omalizumab Fab. D may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3 nM.

[0031] For example, the FR-L3 region may be mutated to M (particularly), E, ​​or D at position S67 (Kabat), with reference to SEQ ID NO:129. In certain embodiments, the amino acid sequence of the mutated FR-L3 region of the anti-IgE antibody or antigen-binding agent is selected from SEQ ID NOs: 52-59, 84-107, 131, or 138.

[0032] For example, the CDR-L2 region may be mutated to D (in particular), E, ​​Q or R at position S52 (Kabat) with reference to SEQ ID NO:129. In certain embodiments, the amino acid sequence of the mutated CDR-L2 region of the anti-IgE antibody or antigen binding agent is selected from SEQ ID NO:50 (in particular) or SEQ ID NO:51. The anti-IgE antibody or antigen binding agent may have a heavy chain variable region further comprising the complementarity determining region CDR-H1, having the amino acid sequence that is SEQ ID NO:14.

[0033] The anti-IgE antibody or antigen binding agent may have a heavy chain variable region further comprising the complementarity determining region CDR-H2, having the amino acid sequence that is SEQ ID NO:16. The anti-IgE antibody or antigen binding agent may have a light chain variable region further comprising the complementarity determining region CDR-L3, having the amino acid sequence that is SEQ ID NO:33. The anti-IgE antibody or antigen binding agent may have a heavy chain variable region further comprising the framework region FR-H1 having the amino acid sequence that is SEQ ID NO:13. The anti-IgE antibody or antigen binding agent may have a heavy chain variable region further comprising the framework region FR-H2 having the amino acid sequence that is SEQ ID NO:15.

[0034] The anti-IgE antibody or antigen binding agent may have a heavy chain variable region further comprising the framework region FR-H3 having the amino acid sequence that is SEQ ID NO:17. The anti-IgE antibody or antigen binding agent may have a heavy chain variable region further comprising the framework region FR-H4, having the amino acid sequence that is SEQ ID NO:19. The anti-IgE antibody or antigen binding agent may have a light chain variable region further comprising the framework region FR-L2 having the amino acid sequence that is SEQ ID NO:30. The anti-IgE antibody or antigen binding agent may have a light chain variable region further comprising the framework region FR-L4, having the amino acid sequence that is SEQ ID NO:34.

[0035] The anti-IgE antibody or antigen binding agent may have a light chain variable region VL having the amino acid sequence of SEQ ID NO: 20, comprising the consecutive FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 regions, except that the CDR-L2 region has an amino acid sequence selected from SEQ ID NO: 50 (in particular) or SEQ ID NO: 51. The anti-IgE antibody or antigen-binding agent may have a light chain variable region VL having the amino acid sequence of SEQ ID NO: 20, comprising the contiguous FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 regions, except that the FR-L3 region has the amino acid sequence of SEQ ID NO: 52.

[0036] The anti-IgE antibody or antigen binding agent may have a light chain variable region VL comprising consecutive FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3 and FR-L4 regions and having the amino acid sequence of SEQ ID NO: 20, except that the CDR-L2 region has an amino acid sequence selected from SEQ ID NO: 50 (in particular) or SEQ ID NO: 51, and the FR-L3 region has an amino acid sequence selected from SEQ ID NO: 52, 131 or 138. An anti-IgE antibody or antigen-binding agent of the invention may have a heavy chain variable region VH having the amino acid sequence which is SEQ ID NO:1. The anti-IgE antibody or antigen binding agent may further comprise a light chain constant region. The anti-IgE antibody or antigen binding agent may have a light chain constant region that is a kappa constant region. The anti-IgE antibody or antigen binding agent may have a light chain variable region and a light chain constant region VL-CL having the amino acid sequence of SEQ ID NO:24, except that the CDR-L2 region has an amino acid sequence selected from SEQ ID NO:50 (in particular) or SEQ ID NO:51.

[0037] The anti-IgE antibody or antigen-binding agent may have a light chain variable region and a light chain constant region VL-CL having the amino acid sequence of SEQ ID NO:24, except that the FR-L3 region has the amino acid sequence of SEQ ID NO:52. The anti-IgE antibody or antigen binding agent may have a light chain variable region and a light chain constant region VL-CL having the amino acid sequence of SEQ ID NO: 24, except that the CDR-L2 region has an amino acid sequence selected from SEQ ID NO: 50 (in particular) or SEQ ID NO: 51, and the FR-L3 region has an amino acid sequence selected from SEQ ID NO: 52, 131 or 138.

[0038] In a further aspect, the invention provides an anti-IgE antibody or antigen-binding agent comprising a heavy chain variable region and a light chain variable region, a. the heavy chain variable region comprises CDR-H1 having an amino acid sequence of SEQ ID NO: 14, CDR-H2 having an amino acid sequence of SEQ ID NO: 16, and CDR-H3 having an amino acid sequence of SEQ ID NO: 18, and the light chain variable region comprises CDR-L1 having an amino acid sequence of SEQ ID NO: 29, CDR-L2 having an amino acid sequence of SEQ ID NO: 50, CDR-L3 having an amino acid sequence of SEQ ID NO: 33, and a framework region FW-L3 having an amino acid sequence of SEQ ID NO: 131 or 138; or b. the heavy chain variable region comprises an amino acid sequence that is SEQ ID NO:1, and the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:132 or 139; The above anti-IgE antibody or antigen-binding agent is provided.

[0039] In one aspect, the anti-IgE antibody or antigen binding agent comprises a light chain variable region and a light chain constant region, VL-CL, having an amino acid sequence selected from SEQ ID NO: 136 or 143, and optionally further comprising a light chain constant region comprising a signal sequence having the amino acid sequence of SEQ ID NO: 160. In all embodiments described herein, the anti-IgE antibody or antigen binding agent may further comprise the heavy chain constant region CH1. The anti-IgE antibody or antigen binding agent may have a heavy chain variable region and a heavy chain constant region, VH-CH1, having the amino acid sequence that is SEQ ID NO:5.

[0040] The anti-IgE antibody or antigen binding agent may further comprise a heavy chain Fc region, Fc. The anti-IgE antibody or antigen-binding agent may have an Fc derived from human IgG1 or human IgG4. The anti-IgE antibody or antigen-binding agent may have a heavy chain variable region, a heavy chain constant region and a heavy chain Fc region, VH-CH1-Fc, having the amino acid sequence of SEQ ID NO:9. The anti-IgE antibody or antigen binding agent of all aspects of the invention may be selected from the group consisting of an intact antibody molecule having full-length heavy and light chains, or a fragment thereof.

[0041] The anti-IgE antibodies or antigen-binding agents of the invention may be administered as Fab fragments, modified Fab' fragments, Fab' fragments, F(ab')2 fragments, Fv, scFv, scab, diabodies, bispecific antibodies, triabodies, FabFv, Fab-Fv-Fv, tribodies, or (Fab-Fv) 2 -Fc. Without being bound by theory, an anti-IgE antibody or antigen-binding agent of the invention may have a lower risk of anaphylaxis associated with it if it has only one anti-IgE antigen-binding site rather than multiple. In one embodiment, the anti-IgE antibody is a Fab fragment that is linked, directly or via a linker, to an scFv that binds to a serum carrier protein, such as human serum albumin.

[0042] In one aspect, the scFv may comprise a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 152, CDR-H2 having the amino acid sequence of SEQ ID NO: 153, and CDR-H3 having the amino acid sequence of SEQ ID NO: 154, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 155, CDR-L2 having the amino acid sequence of SEQ ID NO: 156, and CDR-L3 having the amino acid sequence of SEQ ID NO: 157, preferably linked via a linker having SEQ ID NO: 151.

[0043] In one embodiment, the scFv has an amino acid sequence that is SEQ ID NO: 150. In a preferred embodiment, the Fab fragment has a. the heavy chain variable region comprises CDR-H1 having an amino acid sequence of SEQ ID NO: 14, CDR-H2 having an amino acid sequence of SEQ ID NO: 16, and CDR-H3 having an amino acid sequence of SEQ ID NO: 18, and the light chain variable region comprises CDR-L1 having an amino acid sequence of SEQ ID NO: 29, CDR-L2 having an amino acid sequence of SEQ ID NO: 50, CDR-L3 having an amino acid sequence of SEQ ID NO: 33, and a framework region FW-L3 having an amino acid sequence of SEQ ID NO: 131 or 138; or b. the heavy chain variable region comprises an amino acid sequence that is SEQ ID NO:1, and the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:132 or 139; It contains a heavy chain variable region and a light chain variable region.

[0044] In another embodiment, the Fab fragment further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain variable region and heavy chain constant region VL-CH1 have the amino acid sequence of SEQ ID NO:5, and the light chain variable region and light chain constant region VL-CL have the amino acid sequence selected from SEQ ID NO:136 or 143, and optionally a signal sequence having the amino acid sequence of SEQ ID NO:160. In another embodiment, the scFv is linked to the CH1 of the Fab fragment via a linker having the amino acid sequence of SEQ ID NO:149. In one embodiment, the heavy chain variable and constant regions, linker and scFv have the amino acid sequence of SEQ ID NO:147, optionally including a signal sequence having the amino acid sequence of SEQ ID NO:160.

[0045] In one other embodiment, the heavy chain of a Fab fragment linked to an scFv having SEQ ID NO:147 is paired with a light chain variable and constant region having SEQ ID NO:136 or 143. The anti-IgE antibodies or antigen-binding agents of the invention may have an effector or reporter molecule attached to it.

[0046] The anti-IgE antibodies or antigen-binding agents of the invention may be glycosylated (e.g., in the Fc domain) and / or conjugated to a polymer selected from starch, albumin, and polyethylene glycol (PEG). In one aspect, the conjugated PEG may have a molecular weight in the range of 5-50 kDa. The anti-IgE antibodies or antigen-binding agents of the invention may be humanized.

[0047] A further aspect of the invention relates to an isolated DNA sequence encoding the heavy and / or light chain(s) of the anti-IgE antibody or antigen-binding agent of the invention. Furthermore, a cloning or expression vector is provided which comprises one or more DNA sequences of the invention. For example, the cloning or expression vector may comprise one or more DNA sequences selected from SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:42, or SEQ ID NO:133, or SEQ ID NO:135, or SEQ ID NO:137, or SEQ ID NO:140, or SEQ ID NO:142, or SEQ ID NO:144, and may optionally further comprise one or more DNA sequences selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12, or SEQ ID NO:148.

[0048] A further aspect of the invention is a host cell comprising one or more cloning or expression vectors of the invention. The host cell of the invention may optionally further comprise one or more cloning or expression vectors comprising one or more DNA sequences selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12 or SEQ ID NO:148. Also provided is a process for producing an anti-IgE antibody or antigen-binding agent of the invention comprising culturing a host cell of the invention and isolating the anti-IgE antibody or antigen-binding agent.

[0049] A further aspect relates to a pharmaceutical composition comprising an anti-IgE antibody or antigen-binding agent of the invention together with one or more pharma- ceutically acceptable excipients, diluents or carriers. Suitably, the anti-IgE antibody or antigen-binding agent of the invention is present in a dose of 50-200, preferably about or exactly 150 mg per mL of diluent. In certain embodiments, the excipient comprises one or both of L-arginine, L-histidine. The excipient may comprise polysorbate 20, separately or together. The diluent may be water or an aqueous isotonic solution. The pharmaceutical compositions of the present invention may be provided in sterile vials as a powder for reconstitution prior to subcutaneous administration, or in sterile syringes for their immediate subcutaneous administration. A pharmaceutical composition of the invention may contain a total dose of 75 to 600 mg, for example about or exactly 100 or 150 mg, of anti-IgE antibody or antigen-binding agent.

[0050] The pharmaceutical composition of the present invention may further comprise other active ingredients, either contained together with the anti-IgE antibody, or antigen-binding agent, or for separate co-administration with the anti-IgE antibody, or antigen-binding agent. For example, the pharmaceutical composition of the present invention may be used in the context of allergy-based specific immunotherapy, where the anti-IgE antibody or antigen-binding agent of the present invention is separately co-administered (but may be co-packaged) with the allergen. Thus, the pharmaceutical composition of the present invention may be for use in allergy-based specific immunotherapy, where the patient receives the pharmaceutical composition of the present invention 7, 6, 5, 4, 3, 2, or 1 day before (or on the same day as) the therapeutic allergen. The anti-IgE antibody or antigen-binding agent or composition of the invention may be for use as a medicament. The anti-IgE antibody or antigen-binding agent, or composition of the invention may be for use in the treatment or prevention of disease.

[0051] The anti-IgE antibody or antigen-binding agent or composition of the invention may be for use in the treatment or prevention of a disorder associated with the complex of human IgE and FcεRI. The anti-IgE antibody or antigen-binding agent, or composition of the invention may be for use in treating or preventing disorders caused by dissociation of the complex between human IgE and FcεRI and binding of human IgE by the anti-IgE antibody or antigen-binding agent.

[0052] The anti-IgE antibody or antigen-binding agent or composition of the present invention is useful for treating allergies; allergic asthma; severe asthma; moderate asthma; chronic spontaneous urticaria; chronic idiopathic urticaria; perennial allergic rhinitis; seasonal allergic rhinitis; acute asthma exacerbation; acute bronchospasm; status asthmaticus; hyper-IgE syndrome; allergic bronchopulmonary aspergillosis; prevention of anaphylactic reactions; food allergies; atopic dermatitis; allergic rhinitis; bee venom hypersensitivity. The compound may be for use in the treatment or prevention of one or more of: allergic skin diseases; idiopathic anaphylaxis; peanut allergy; latex allergy; inflammatory skin diseases; urticaria (sun-, cold-induced, local heat-induced, and / or delayed pressure-induced); cutaneous mastocytosis; systemic mastocytosis; eosinophil-associated gastrointestinal disorders; bullous pemphigoid; interstitial cystitis; nasal polyps; idiopathic angioedema; or non-allergic asthma.

[0053] There is further provided a method for treating or preventing a disease in a human subject, comprising administering to the subject an effective amount of an anti-IgE antibody or antigen-binding agent, or composition of the present invention. The method may be for treating or preventing a disorder associated with the complex of human IgE and FcεRI. The method of the present invention can treat or prevent a disease by dissociation of the complex of human IgE and FcεRI and binding of human IgE by the anti-IgE antibody or antigen-binding agent of the present invention.

[0054] The method of the invention may be for the treatment or prevention of one or more of: allergy; allergic asthma; severe asthma; moderate asthma; chronic spontaneous urticaria; chronic idiopathic urticaria; perennial allergic rhinitis; seasonal allergic rhinitis; acute asthma exacerbation; acute bronchospasm; status asthmaticus; hyper-IgE syndrome; allergic bronchopulmonary aspergillosis; prevention of anaphylactic reactions; food allergies; atopic dermatitis; allergic rhinitis; bee venom hypersensitivity; idiopathic anaphylaxis; peanut allergy; latex allergy; inflammatory skin diseases; urticaria (sun, cold-induced, local heat-induced, and / or delayed pressure-induced); cutaneous mastocytosis; systemic mastocytosis; eosinophil-associated gastrointestinal disorders; bullous pemphigoid; interstitial cystitis; nasal polyps; idiopathic angioedema; or non-allergic asthma.

[0055] In the present invention, it has been elucidated that an antibody or antigen-binding agent against a first polypeptide, which elicits its physiological response due to binding to a second polypeptide (such as a receptor), can bind to both free and bound first polypeptides and stabilize the conformation of such first polypeptide, such a stabilized conformation having a weaker binding affinity for the second polypeptide than in the absence of the antibody or antigen-binding agent, and thus inducing a more rapid dissociation of the first polypeptide from the second polypeptide.

[0056] In this regard, the invention provides a further aspect relating to an antibody or antigen-binding agent capable of binding to free and FcεRI-bound human IgE and stabilizing the conformation of IgE. When IgE is in such a conformation, it has a weaker binding affinity for FcεRI than in the absence of the antibody or antigen-binding agent, whereby FcεRI bound to human IgE dissociates from FcεRI. Optionally, when IgE is in such a conformation, it has a lower binding affinity for omalizumab or a fragment thereof than the antibody or antigen-binding agent of the invention. For example, the antibody or antigen-binding agent is an antibody as described herein.

[0057] In a further aspect, the present invention relates to a process for selecting such an antibody or antigen-binding agent as described herein, the process comprising the steps of: a. contacting a test antibody or antigen-binding agent with a sample containing human IgE bound to human FcεRI; b. measuring the dissociation constant of the test antibody or antigen-binding agent for dissociation of human IgE from human FcεRI; c. comparing the dissociation constant determined in step b) with the dissociation constant of omalizumab or a fragment thereof for dissociation of human IgE from human FcεRI; d. Selecting an antibody or antigen-binding agent if said antibody or antigen-binding agent dissociates IgE from FcεRI faster than omalizumab or a fragment thereof. Includes.

[0058] Alternatively, a process for selecting an antibody or antigen-binding agent according to the present invention comprises: a. contacting a test antibody or antigen-binding agent with a sample containing human IgE bound to human FcεRI; b. measuring the binding affinity of the test antibody or antigen-binding agent to human IgE derived from human FcεRI; c. comparing the binding affinity measured in step b) with the binding affinity of human IgE for FcεRI; d. selecting an antibody or antigen-binding agent if it has a higher binding affinity for IgE than IgE for FcεRI. Includes.

[0059] Optionally, the selected antibody or antigen binding agent causes IgE to adopt a stabilized conformation while still binding to FcεRI, in which IgE in said conformation may dissociate from FcεRI faster than IgE bound to FcεRI in the presence of omalizumab or a fragment thereof; and / or may have a higher binding affinity for the antibody or antigen binding agent than for FcεRI.

[0060] In a final aspect, the present invention provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: a. a heavy chain variable region comprising SEQ ID NO:1; i. SEQ ID NO: 109; or ii. SEQ ID NO: 113; or iii. SEQ ID NO: 121; or iv. SEQ ID NO: 132; or v. SEQ ID NO: 139 or a light chain variable region comprising b. SEQ ID NO:5; i. S77 and S79 are replaced with Q, SEQ ID NO:24; ii. SEQ ID NO: 117; or iii. SEQ ID NO: 125; or iv. SEQ ID NO: 136; or v. SEQ ID NO: 143 and

[0061] In one embodiment of this last aspect of the invention, the anti-IgE antibody or antigen binding agent contacts, or is specific for, an epitope comprising residues T373, W374, S375, R376, A377, S378, G379, P381, Q417, C418, R419, T421, P426, R427, A428 of the Cε3 domain and residues D278 and T281 of the Cε2 domain of human IgE, with reference to SEQ ID NO: 108.

[0062] References and sequence numbers are found in the examples which refer to the figures. [Brief description of the drawings]

[0063] [Figure 1]FIG. 1 shows that Omalizumab Fab3 contains three point mutations. Omalizumab Fab3 is derived from Omalizumab and contains three point mutations distal to the antigen-binding CDRs, two in the VL domain framework regions (Ser81Arg, Gln83Arg) and one in the Cκ domain (Leu158Pro). The heavy and light chains are colored white and blue, respectively. To indicate the orientation of the Fab, the mutated residues are colored red and CRDL1 is colored green. [Diagram 2] Overall structure of IgE-Fc complexed with Omalizumab Fab3. (A) Omalizumab Fab3 binds IgE-Fc with a 2:1 stoichiometry. Fab1 (green) engages IgE-Fc chain B (pink) exclusively via the Cε3 domain. Fab2 (blue) interacts with IgE-Fc chain A (yellow) via the Cε3 domain and forms a weak interaction with the Cε2 domain from IgE-Fc chain B (pink). (B) The two Fabs form a pseudo-symmetric complex with an approximately two-fold axis of the Fcε3-4 region. For clarity, the Cε2 domain is not shown. (C) IgE-Fc is asymmetrically curved in the Omalizumab Fab3 complex. The Cε2 domain from chain B (pink) contacts Fab2 (blue). [Diagram 3] FIG. 1 shows the interface between Omalizumab Fab3 and IgE-Fc. The interface between Omalizumab Fab3 Fab2 (heavy and light chains colored green and yellow, respectively) and the Cε3 domain (pink) from IgE-Fc is shown. Omalizumab Fab3 and Cε3 domain residue labels are colored blue and black, respectively. The interface involves hydrogen bonds and van der Waals interactions. A prominent feature of the interface is the cation-π interaction between Arg419 (Cε3 domain) and Phe103 (Omalizumab Fab3 CDRH3). The Phe103 side chain is mostly buried in the pocket created by Thr373, Trp374, Ser375, Gln417 and Arg419 (Cε3 domain). [Figure 4]Figure 3: Omalizumab Fab3 and DARPin E2_79 bind to an overlapping interface. Both Omalizumab Fab3 and DARPin E2_7920 bind to the Cε3 domain. IgE-Fc residues that form only part of the Omalizumab Fab3 interface are coloured orange, whereas those that form only part of the DARPin E2_79 interface, including part of the Cε3-Cε4 linker, are coloured blue. IgE-Fc residues coloured pink, including Arg419 and Met430, are common to both the Omalizumab Fab3 and DARPin E2_79 interfaces. [Diagram 5] Figure 1 shows conformational flexibility in IgE-Fc. (A) Side view of free IgE-Fc8 showing its sharp asymmetric curvature. (B) Front view of free IgE-Fc (rotated 90° counterclockwise from the view shown in (A)). (C) Side view of IgE-Fc in Omalizumab Fab3 complex, showing the partially curved conformation. (D) Front view of IgE-Fc in Omalizumab Fab3 complex (rotated 90° counterclockwise from the view shown in (C)). (E) Side view of fully extended IgE-Fc captured by anti-IgE-Fc Fab (Omalizumab Fab3)16. (F) Front view of extended IgE-Fc (rotated 90° counterclockwise from the view shown in (E)). [Figure 6]Figure 1: Conformational flexibility in IgE-Fc. The flexibility of IgE-Fc and its decurling from a curved conformation to a fully extended conformation have been previously explored by molecular mechanics16. The decurling of IgE-Fc is represented as a free energy surface, as previously explored16. (A) The extended conformation of IgE-Fc captured in the crystal structure of the omalizumab Fab3 / IgE-Fc complex16. (B) The partially curved IgE-Fc conformation observed in the crystal structure of the omalizumab Fab3 / IgE-Fc complex. (C) The curved conformation of free IgE-Fc7,8. The curved conformation of IgE-Fc occupies the lowest energy basin, whereas the partially curved conformation observed in the omalizumab Fab3 / IgE-Fc complex occupies a distinct energy basin (B). [Figure 7] Disruption of the interaction of IgE-Fc with FcεRI. In the omalizumab Fab3 complex, the Cε3 domain adopts the most open conformation reported so far for IgE-Fc, disabling engagement with FcεRIα. The structure of IgE-Fc complexed with FcεRIα8 is coloured yellow and the two subsites of receptor engagement are indicated. The structure of omalizumab Fab3 complexed with IgE-Fc is superimposed on the Cε4 domain and the Cε3 domain is coloured blue. The positions of His424 and Pro426 in the two structures are shown to highlight the different positions adopted by the Cε3 domain. [Figure 8] Disruption of the IgE-Fc interaction with CD23. (A) Cε3 domain residues common to both the omalizumab Fab3 and CD23 interface are colored pink. (B) Superposition of the Cε3 domain (dark grey) from the omalizumab Fab3 / IgE-Fc complex and the CD23 / Fcε3-4 complex11 shows the clash between CD23 (yellow) and omalizumab Fab3 (pink). [Figure 9]Figure 1. Interaction study of Omalizumab Fab3 with IgE-Fc. (A) Binding of Omalizumab Fab3 captured by a C-terminal His tag to IgE-Fc; Omalizumab Fab3 was run over IgE-Fc at the following concentrations: 100 nM (black), 50 nM (red), 25 nM (green), 12.5 nM (blue), 6.2 nM (cyan), 3.1 nM (purple), 1.6 nM (magenta) and 0.8 nM (dark red). Standard double referencing was used36; each concentration was run in duplicate. (B) Binding of the second Omalizumab Fab3 binding site was characterized using SPR sandwich binding experiments. After IgE-Fc was captured on the omalizumab Fab3 surface, a second omalizumab Fab3 molecule was added to the IgE-Fc / omalizumab Fab3 complex at concentrations of 1000 nM (black), 500 nM (red), 250 nM (green), 125 nM (blue), 62.5 nM (cyan), 31.2 nM (purple), 15.6 nM (magenta), 7.8 nM (dark red) and 0 nM (navy). (C) Comparison of the ability of omalizumab Fab3 to bind to IgE-Fc captured by a C-terminal His tag (red) and IgE-Fc captured by binding to FcεRIα (blue); for each, a two-fold dilution series was tested with a top concentration of 1000 nM. The inset shows that omalizumab Fab3 still binds to the IgE-Fc / FcεRIα complex, but with a lower Bmax value. (D) Accelerated dissociation of the IgE-Fc / FcεRIα complex mediated by increasing concentrations of omalizumab Fab3. After capturing IgE-Fc on immobilized FcεRIα, a 1:1 IgE-Fc / FcεRIα complex was first established by binding to omalizumab Fab3 at the following concentrations: 5000 nM (magenta), 1000 nM (purple), 200 nM (cyan), 40 nM (blue), 8 nM (green), 1.6 nM (red) and 0 nM (black). The inset shows a magnified view of the accelerated dissociation process. All concentrations were run in duplicate. All binding experiments were carried out at 25°C, except for those characterizing the second omalizumab Fab3 binding site (Fig. 4B), which were performed at 5°C to minimize allosteric communication between the two sites. [Figure 10]Figure 1: Analysis of direct binding, competition experiments and accelerated dissociation. Direct binding to immobilized omalizumab Fab3 (A), omalizumab Fab (B) and intact omalizumab (C) was measured for IgE-Fc. Fab or intact antibody was covalently immobilized at low density using an amine coupling kit (GE Healthcare); IgE-Fc was flowed over these surfaces at various concentrations using a 2-fold dilution series with a top concentration of 100 nM. All concentrations were run in duplicate. (D) TR-FRET competition binding experiment between omalizumab Fab3 and αγ-fusion protein for IgE-Fc. Binding of terbium-labeled αγ-fusion protein to Alexa Fluor 647-labeled IgE-Fc was measured using increasing concentrations of unlabeled omalizumab Fab3 as inhibitor: 0 μM (black), 2.5 nM (blue), 5 nM (green), 10 nM (magenta), 20 nM (red). As an inhibitor, omalizumab Fab affected both the apparent KD and Bmax of the interaction of IgE-Fc with αγ-fusion protein, indicating some allosteric inhibitory properties. (E) Comparison of the accelerated dissociation of IgE-Fc / sFcεRIα complex mediated by intact omalizumab (black), omalizumab Fab (red) or omalizumab Fab3 (blue) at a concentration of 5 μM, respectively. [Figure 11] 1 shows representative electron density maps. A stereo view of the 2Fo-Fc electron density map, contoured at 1.1σ, is shown for a portion of the chain A Cε3 domain and the covalently N-linked oligosaccharide moiety at Asn394. [Figure 12] Figure 1 shows a Biacore sensorgram of the dissociation of IgE-Fc from immobilized sFcεRIα. Dissociation was monitored in the presence of running buffer (solid line) or IgE binding partner (all other sensorgrams). The assay was performed as described in Assay Method (1) Example 2. [Figure 13]Figure 1 shows Biacore sensorgrams of dissociation of IgE-Fc from immobilized sFcεRIα. Dissociation was monitored in the presence of control supernatant (solid line) or IgE binding partner (all other sensorgrams). The assay was performed as described in Assay Method (2) Example 2. [Figure 14] Figure 1 shows the analysis of dissociation of Alexa488-labeled IgE-Fc from the surface of RBL-SX38 cells: Measured binding data were normalized to 100% at t=0 and dissociation data were plotted as the change in the percentage of IgE-Fc that remains bound as a function of time. [Figure 15] FIG. 1 shows an analysis of the effect of therapeutic doses of wild type omalizumab Fab and omalizumab Fab3 in a 72 hour PCA model. [Figure 16] FIG. 1 shows residues 224-547 of the wild-type human IgE-Fc sequence (shown in SEQ ID NO:108), with residues 224 and 547 shown in bold. Numbering is according to Dorrington and Bennich (1978) Immunol. Rev. 41:3-25, where the L (Leu; leucine) after L253 is numbered as L235a (boxed), and the residue thereafter is C254. The remaining residues are numbered consecutively without further inscription. Epitope residues are indicated with an asterisk (*).

[0064] The numbering of antibody amino acids herein may be derived from the consecutive amino acid sequence of the antibody (e.g., omalizumab, which comprises the VH sequence of SEQ ID NO: 1 and the VL sequence of SEQ ID NO: 20 or SEQ ID NO: 129) - the so-called "pdb" numbering - or may use the common Kabat numbering scheme. When the common immunoglobulin parts (CDR-complementarity determining regions, or FR-framework regions) of the VH or VL sequences are described, they are linked in the standard order (VH=FR-H1.CDR-H1.FR-H2.CDR-H2.FR-H3.CDR-H3.FR-H4; VL=FR-L1.CDR-L1.FR-L2.CDR-L2.FR-L3.CDR-L3.FR-L4). For omalizumab, the "pdb" numbering of the VH (SEQ ID NO:1) portion is FR-H1(amino acids 1-25), CDR-H1(26-36), FR-H2(37-50), CDR-H2(51-66), FR-H3(67-98), CDR-H3(99-110), FR-H4(111-121), while the Kabat numbering is FR-H1(amino acids 1-25), CDR-H1(26-35), FR-H2(36-49), CDR-H2(50-65), FR-H3(66-94), CDR-H3(95-102), FR-H4(103-113). For omalizumab, the "pdb" numbering of the VL (SEQ ID NO:20) portion is FR-L1(amino acids 1-23), CDR-L1(24-38), FR-L2(39-53), CDR-L2(54-60), FR-L3(61-92), CDR-L3(93-101), FR-L4(102-111), while the Kabat numbering is FR-L1(amino acids 1-23), CDR-L1(24-34), FR-L2(35-49), CDR-L2(50-56), FR-L3(57-88), CDR-L3(89-97), FR-L4(98-107).

[0065] The numbering of IgE antibodies is as reported by Dorrington & Bennich (1978) Immunol. Rev. 41:3-25. Thus, the IgE-Fc polypeptide used in the present invention (see SEQ ID NO: 108) is derived from V224-K547 (including the C225A mutation). As shown in Figure 16, the following numbering is as described in Dorrington and Bennich (1978) Immunol. Rev. 41:3-25, where the L (Leu, leucine) after position 253 is numbered as L253a, and the remaining residues are numbered consecutively from L253a as C254, etc. In the crystallographic experiments, the following mutations were also inserted into IgE-Fc to simplify the glycosylation pattern: N265Q & N371Q. The Cε2 region of IgE-Fc is generally accepted to occupy the sequence S226-D330. References herein to IgE may be references to human IgE (and vice versa) and may also constitute references to IgE-Fc in the context of the assays and methods described herein. The sequence of the Fab arm of a full-length human IgE antibody is not included in this description, as it is not present in the crystal structure.

[0066] References herein to "omalizumab" are references to the commercially available Xolair® product; or to an IgG full-length antibody comprising a heavy chain comprising a VH amino acid sequence which is SEQ ID NO: 1 and a light chain comprising a VL amino acid sequence which is SEQ ID NO: 20; or to an IgG full-length antibody comprising a heavy chain comprising a VH-CH1 amino acid sequence which is SEQ ID NO: 5 and a light chain comprising a VL-CL amino acid sequence which is SEQ ID NO: 24; or to an IgG full-length antibody comprising a heavy chain comprising a VH-CH1-Fc amino acid sequence which is SEQ ID NO: 9 and a light chain comprising a VL-CL amino acid sequence which is SEQ ID NO: 24. References herein to "omalizumab Fab" are references to a Fab fragment comprising a heavy chain comprising a VH amino acid sequence which is SEQ ID NO: 1 and a light chain comprising a VL amino acid sequence which is SEQ ID NO: 20; or to a Fab fragment comprising (among other things) a heavy chain comprising a VH-CH1 amino acid sequence which is SEQ ID NO: 5 and a light chain comprising a VL-CL amino acid sequence which is SEQ ID NO: 24.

[0067] general definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references described herein are incorporated herein by reference in their entirety. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0068] Unless otherwise required by context, singular terms include plurals and plural terms include the singular. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms such as "includes" and "included" is not limiting. Also, terms such as "element" or "component" include both elements and components that contain one unit and elements and components that contain more than one subunit, unless specifically stated otherwise.

[0069] In general, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are well known in the art and are generally performed according to conventional methods described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients. In order that the present invention may be more readily understood, selected terms are defined below.

[0070] The term "host" as used herein typically refers to a human subject, especially when a human or humanized framework is used as the acceptor structure. It will be understood by those skilled in the art that when another host is treated, the antibody or antigen binding agent may need to be adjusted for that host to avoid rejection or to be more compatible. It is known how to use CDRs in the present invention and how to engineer them into appropriate frameworks or peptide sequences for the desired delivery and function for various hosts. Other hosts may include other mammalian or vertebrate species. Thus, the term "host" may alternatively refer to animals such as mice, monkeys, dogs, pigs, rabbits, domesticated porcines (pigs and wild boars), ruminants, horses, poultry, felines, murines, bovines, canines, etc., where the antibody or antigen binding agent is suitably designed for compatibility with the host, as required.

[0071] The term "polypeptide" as used herein refers to any polymeric chain of amino acids. The terms "peptide" and "protein" are used interchangeably with the term polypeptide and also refer to a polymeric chain of amino acids. The term "polypeptide" encompasses natural or artificial proteins, protein fragments, and polypeptide analogs of protein sequences. Polypeptides may be monomeric or polymeric.

[0072] As used herein, the term "recovering" refers to the process by which a species, such as a polypeptide, is rendered substantially free of naturally associated components, for example, by isolation using protein purification techniques well known in the art.

[0073] The terms "specific binding" or "specifically binding" as used herein refer to the interaction of an antibody, protein, or peptide with a second chemical species and mean that the interaction is dependent on the presence of a particular structure (e.g., an "antigenic determinant" or "epitope" as defined below) on the chemical species; for example, an antibody recognizes and binds to a particular protein structure rather than the entire protein. If an antibody is specific for epitope "A", then the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction involving labeled "A" and an antibody will reduce the amount of labeled A that binds to the antibody. When an epitope of the invention is referred to herein, the anti-IgE antibody or antigen-binding agent of the invention is specific for said epitope.

[0074] The term "antibody" as used herein refers to any immunoglobulin (Ig) molecule comprising four polypeptide chains, two heavy (H) and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof that retains at least some portion of the epitope binding characteristics of an Ig molecule that allows it to specifically bind IgE. Such mutant, variant, or derivative antibody formats are known in the art and are described below. Non-limiting aspects thereof are discussed below. An antibody is said to be "capable of binding" a molecule (or epitope) if it is capable of binding the molecule (or epitope) to the antibody by specifically reacting with the molecule (or epitope).

[0075] As used herein, "monoclonal antibody" is intended to refer to a preparation of antibody molecules sharing a common heavy chain and a common light chain amino acid sequence, or any functional fragment, mutant, variant, or derivative thereof that retains at least the light chain epitope binding characteristics of an Ig molecule, as opposed to "polyclonal" antibody preparations which contain a mixture of different antibodies. Monoclonal antibodies can be produced by several known techniques, such as phage, bacterial, yeast, or ribosome display, as well as classical methods exemplified by hybridoma-derived antibodies (e.g., antibodies secreted by hybridomas prepared by hybridoma technology, such as the standard Kohler and Milstein hybridoma method ((1975) Nature 256:495-497)).

[0076] In full-length antibodies, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region comprises either four domains - CH1, hinge, CH2, and CH3 (heavy chain gamma, alpha, and delta), or CH1, CH2, CH3, and CH4 (heavy chain μ and epsilon). Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region (CL). The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The immunoglobulin molecule may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

[0077] The term "antigen-binding agent" as used herein refers to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (e.g., IgE), or synthetic variants of an antibody fragment that retain the desired binding ability to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments or certain portions of a full-length antibody or variants thereof. Embodiments include bispecific, dual specific and multispecific formats that can specifically bind to two or more different antigens or several epitopes or discontinuous epitopic regions of an antigen. Non-limiting examples of antigen-binding agents include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; and (v) a dAb fragment comprising a single variable domain (Ward et al., (1989) Nature 341:544-546; Winter et al., PCT Publication No. WO 90 / 05144, both of which are incorporated by reference herein). A1); (vi) isolated complementarity determining regions (CDRs), (vii) fusions of antibody fragments, such as those that are immunoglobulin in nature, e.g., diabodies, scabs, bispecific antibodies, triabodies, Fab-Fv, Fab-Fv-Fv, tribodies, (Fab-Fv)2-Fc, and (viii) antibody portions, such as CDRs or antibody loops grafted onto non-immunoglobulin frameworks, such as fibronectin or leucine zippers (see Binz et al. (2005) Nat. Biotech. 23:1257-1268, incorporated herein by reference).Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic or natural linker that allows them to be produced, recombinantly or otherwise, as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term antigen-binding agent. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but are forced to pair with complementary domains on another chain, creating two antigen-binding sites, by using a linker that is too short to allow pairing between the two domains on the same chain (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123). Such antibody binding moieties are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790pp. (ISBN3-540-41354-5)).

[0078] The term "antibody construct" as used herein refers to a polypeptide comprising one or more antigen-binding moieties of the invention linked to a linker polypeptide or immunoglobulin constant domain. A linker polypeptide comprises two or more amino acid residues linked by peptide bonds and is used to link one or more antigen-binding moieties. Such linker polypeptides are well known in the art (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123). An immunoglobulin constant domain refers to a heavy or light chain constant domain, e.g., a human IgA, IgD, IgE, IgG or IgM constant domain. Heavy and light chain constant domain amino acid sequences are well known in the art. Non-limiting examples of Ig heavy chain gamma 1 constant regions and Ig light chain lambda and kappa chains are provided in Tables 8 and 6, respectively.

[0079] Additionally, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions to generate tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Additionally, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

[0080] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds IgE is substantially free of antibodies that specifically bind to antigens other than IgE). However, an isolated antibody that specifically binds, for example, to human IgE may have cross-reactivity to other antigens, such as IgE molecules from other species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0081] The term "CDR-grafted antibody" refers to an antibody that contains heavy and light chain variable region sequences derived from one species, but in which the sequences of one or more CDR regions of the VH and / or VL are replaced with CDR sequences from another species, such as an antibody having human heavy and light chain variable regions in which one or more human CDRs (e.g., CDR3) have been replaced with murine CDR sequences.

[0082] The terms "Kabat numbering", "Kabat definition" and "Kabat labeling" are used interchangeably herein. These terms, as recognized in the art, refer to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable regions range from amino acid positions 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3) according to the Kabat numbering system. However, according to Chothia (Chothia et al. (1987) J. Mol. Biol., 196, 901-917 (1987)), the loop corresponding to CDR-H1 extends from residue 26 to residue 32. Thus, unless otherwise indicated, "CDR-H1" as used herein is intended to refer to residues 26-35 as described by a combination of the Kabat numbering system and Chothia's topological loop definition. For the light chain variable region, the hypervariable region ranges from amino acid positions 24-34 for CDRL1, amino acid positions 50-56 for CDRL2, and amino acid positions 89-97 for CDRL3.

[0083] The terms "acceptor" and "acceptor antibody" as used herein refer to an antibody or nucleic acid sequence that provides or encodes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequence of one or more framework regions. In some aspects, the term "acceptor" refers to an antibody amino acid or nucleic acid sequence that provides or encodes the constant region(s). In yet other aspects, the term "acceptor" refers to an antibody amino acid or nucleic acid sequence that provides or encodes one or more framework regions and the constant region(s). In certain aspects, the term "acceptor" refers to a human antibody amino acid or nucleic acid sequence that provides or encodes at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequence of one or more framework regions. According to this embodiment, the acceptor may contain at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 amino acid residues that are not present in one or more specific portions of a human antibody. The acceptor framework region and / or the acceptor constant region(s) can be derived or obtained, for example, from germline antibody genes, mature antibody genes, functional antibodies (e.g., antibodies known in the art, antibodies in development, or commercially available antibodies).

[0084] The term "CDR" as used herein refers to the complementarity determining region in an antibody variable sequence. There are three CDRs in each variable region of the heavy and light chains, designated CDRH1, CDRH2 and CDRH3 for the heavy chain CDRs and CDRL1, CDRL2 and CDRL3 for the light chain CDRs. The term "CDR set" as used herein refers to a group of three CDRs present in a single variable region that can bind to an antigen. The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 199:101-102 (1991)) 342:877-883 (1989)) found that certain subportions within the Kabat CDRs adopt nearly identical peptide backbone conformations despite a high degree of diversity at the amino acid sequence level. These subportions were designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" designate the light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995) and MacCallum (J Mol Biol 262(5):732-45 (1996)).Still other CDR boundary definitions may not strictly follow one of the above systems, but may nevertheless overlap with the Kabat CDRs, but shorten or extend them in light of predictions or experimental findings that certain residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. The methods used herein may use CDRs defined according to any of these systems, although preferred embodiments use Kabat or Chothia defined CDRs, or a combination thereof.

[0085] The term "canonical" residues as used herein refers to residues in a CDR or framework that define certain canonical CDR structures defined by Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia et al., J. Mol. Biol. 227:799 (1992), both of which are incorporated herein by reference). According to Chothia et al., a significant portion of the CDRs of many antibodies have nearly identical peptide backbone conformations, despite high diversity at the level of amino acid sequence. Each canonical structure specifies a set of primarily peptide backbone torsion angles for a contiguous segment of amino acid residues that form a loop.

[0086] As used herein, the terms "donor" and "donor antibody" refer to an antibody that provides one or more CDRs. In a preferred embodiment, the donor antibody is an antibody from a different species than the antibody from which the framework regions are obtained or derived. In the context of a humanized antibody, the term "donor antibody" refers to a non-human antibody that provides one or more CDRs.

[0087] The term "framework" or "framework sequence" as used herein refers to the remaining sequence of the variable region, excluding the CDRs. The exact definition of the CDR sequence can be determined by different systems, so the meaning of the framework sequence is subject to different interpretations accordingly. The six CDRs (CDR-L1, -L2, and -L3 of the light chain and CDR-H1, -H2, and -H3 of the heavy chain) also divide the framework region on the light and heavy chain into four subregions (FR1, FR2, FR3, and FR4) on each chain, with CDR1 located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Although not specifying a particular subregion as FR1, FR2, FR3, or FR4, the framework region as referred to by others represents the combined FRs in the variable region of a single native immunoglobulin chain. As used herein, FR represents one of the four subregions, and FR(s) represents two or more of the four subregions that make up the framework region.

[0088] As used herein, the term "germline antibody gene" or "gene fragment" refers to an immunoglobulin sequence encoded by a non-lymphoid cell that has not undergone the maturation process that results in gene rearrangement and mutation for expression of a particular immunoglobulin. See, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3):183-200 (2002); Marchalonis et al., Adv Exp Med Biol. 484:13-30 (2001). One advantage provided by various aspects of the invention takes advantage of the recognition that germline antibody genes are more likely than mature antibody genes to preserve essential amino acid sequence structures characteristic of individuals in a species, and therefore are less likely to be recognized as being from a foreign source when used therapeutically in that species.

[0089] The term "important" residues as used herein refers to certain residues in the variable region that more significantly affect the binding specificity and / or affinity of an antibody, particularly a humanized antibody. Important residues include, but are not limited to, one or more of the following: residues adjacent to the CDR, potential glycosylation sites (which may be N- or O-glycosylation sites), rare residues, residues that can interact with the antigen, residues that can interact with the CDR, canonical residues, contact residues between the heavy and light chain variable regions, residues in the Vernier zone, and residues in the region of overlap between the Chothia definition of the variable heavy chain CDR1 and the Kabat definition of the first heavy chain framework.

[0090] The term "humanized antibody" generally refers to an antibody that contains heavy and light chain variable region sequences derived from a non-human species (e.g. rabbit, mouse, etc.), but in which at least a portion of the VH and / or VL sequences have been altered to make them more "human-like", i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences have been introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. Another type of humanized antibody is a CDR-grafted antibody, in which at least one non-human CDR has been inserted into a human framework. The latter is typically the focus of the present invention.

[0091] In particular, the term "humanized antibody" as used herein is an antibody or variant, derivative, analog or fragment thereof that immunospecifically binds to an antigen of interest and comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence that is at least 50, 55, 60, 65, 70, 75 or 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of the non-human antibody CDR. In one aspect, a humanized antibody has a CDR region that has one or more (e.g., 1, 2, 3 or 4) amino acid substitutions, additions and / or deletions compared to the non-human antibody CDR. Furthermore, the non-human CDR can be engineered to be more "human-like" or compatible with the human body using known techniques. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, F(ab')c, Fv), with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions being those of a human immunoglobulin consensus sequence. Preferably, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some aspects, the humanized antibody contains both a light chain and at least the variable domains of a heavy chain. The antibody may also include the CH1, hinge, CH2, and CH3, or the CH1, CH2, CH3, and CH4 of the heavy chain. In some aspects, the humanized antibody contains only a humanized light chain. In some aspects, the humanized antibody contains only a humanized heavy chain. In certain aspects, the humanized antibody contains only a humanized variable domain of the light chain and / or a humanized heavy chain. Although some of the mutations discussed herein may not generally be "human", these are insufficient for an anti-IgE antibody or antigen-binding agent of the invention to be "humanized".

[0092] The humanized antibody can be selected from any class of immunoglobulins, including IgY, IgM, IgG, IgD, IgA, and IgE, and any isotype, including but not limited to IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains can be selected to optimize desired effector functions using techniques well known in the art.

[0093] The framework and CDR regions of the humanized antibody need not exactly match the parental sequences, for example, the donor antibody CDR or consensus framework can be mutagenized by substitution, insertion and / or deletion of at least one amino acid residue such that the CDR or framework residue at that site does not exactly match either the donor antibody or the consensus framework. However, in preferred embodiments, such mutations will not be extensive. Usually, at least 50, 55, 60, 65, 70, 75 or 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%, 98% or 99% of the humanized antibody residues will match those of the parental FR and CDR sequences. In one embodiment, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions, additions and / or deletions may be present in the humanized antibody compared to the parental FR and CDR sequences (e.g., compared to the Omalizumab or Omalizumab Fab sequences). The term "consensus framework" as used herein refers to a framework region in a consensus immunoglobulin sequence. The term "consensus immunoglobulin sequence" as used herein refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In an immunoglobulin family, each position in a consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

[0094] As used herein, "Vernier" zone refers to a subset of framework residues that can adjust the CDR structure and fine-tune the fit to the antigen as described by Foote and Winter (1992, J. Mol. Biol. 224:487-499, incorporated herein by reference). The Vernier zone residues form a layer underlying the CDRs and can affect the structure of the CDR and the affinity of the antibody.

[0095] The term "neutralizing" as used herein refers to the neutralization of IgE biological activity when an anti-IgE antibody or antigen-binding agent of the invention described herein specifically binds to IgE protein. Neutralization may be the result of different modes of binding of said antibody to IgE. Preferably, a neutralizing antibody is an antibody whose binding to IgE results in neutralization of IgE biological activity. Preferably, a neutralizing binding protein binds to IgE and reduces IgE biological activity by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 80%, 85% or more. Neutralization of IgE biological activity by a neutralizing antibody can be assessed by measuring one or more indicators of IgE biological activity as described herein.

[0096] As used herein, "neutralizing monoclonal antibody" is intended to refer to a preparation of antibody molecules that, upon binding to IgE, is capable of partially or completely inhibiting or reducing the biological activity of IgE. As used herein, the terms "attenuate," "attenuate," and the like refer to a decrease or reduction in the severity of a symptom or condition caused by elevated serum IgE levels.

[0097] The term "epitope" or "antigenic determinant" encompasses any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitopic determinants include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and in certain embodiments may have specific three-dimensional structural characteristics, and / or specific charge characteristics. An epitope is the region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0098] As used herein, the term "k on " is intended to refer to the association rate constant for the binding of an antibody to an antigen to form an antibody / antigen complex as known in the art. As used herein, the term "k off " is intended to refer to the dissociation rate constant for dissociation of an antibody from the antibody / antigen complex as known in the art. As used herein, the term "k d " or "k D " is intended to refer to the dissociation constant of a particular antibody-antigen interaction as known in the art.

[0099] The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (k D or k d ) and k d A smaller k represents a greater or higher affinity. The immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method involves measuring the rates of formation and dissociation of antigen-binding site / antigen complexes, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, the "association rate constant" ("k on ") and "dissociation rate constant" ("k off") can be determined by calculating the concentration and the actual rates of binding and dissociation (Nature 361:186-87 (1993)). off / k on The ratio of d Davies et al. (1990) Annual Rev Biochem 59:439-473.

[0100] The term "antibody conjugate" refers to a binding protein, such as an antibody or antibody fragment or binding portion thereof, chemically linked to a second chemical moiety, such as a therapeutic or cytotoxic agent. As used herein, the term "agent" refers to a chemical compound, a mixture of compounds, a biological macromolecule, or an extract made from biological material.

[0101] The terms "crystal" and "crystallized" as used herein refer to an antibody or antigen-binding portion thereof that exists in the form of a crystal. A crystal is a form of matter in the solid state, distinct from other forms such as an amorphous solid state or a liquid crystalline state. A crystal is composed of regular, repeating three-dimensional arrays of atoms, ions, molecules (e.g., proteins such as antibodies), or molecular assemblies (e.g., antigen / antibody complexes). These three-dimensional arrays are arranged according to specific numerical relationships that are well understood in the art. The basic unit, or building block, that is repeated in a crystal is called the asymmetric unit. The repetition of the asymmetric unit in an arrangement according to a given well-defined crystallographic symmetry provides the "unit cell" of the crystal. The repetition of the unit cell by regular transformations in all three dimensions provides a crystal. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, NY, (1999).

[0102] The term "polynucleotide" as used herein refers to a polymeric form of two or more nucleotides, either ribonucleotides or deoxynucleotides, or a modified form of either type of nucleotide. The term encompasses single- and double-stranded forms of DNA, but preferably is double-stranded DNA.

[0103] The term "isolated polynucleotide," as used herein, refers to a polynucleotide that is not associated with all or a portion of a polynucleotide with which it is found in nature; is operably linked to a polynucleotide with which it is not naturally linked; or is not naturally occurring as part of a larger sequence, because of its origin (e.g., of genomic, cDNA, or synthetic origin, or some combination thereof).

[0104] The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be ligated. Another type of vector is a viral vector, into which additional DNA fragments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, whereby they are replicated along with the host genome. In addition, certain vectors can direct the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0105] The term "operably linked" refers to a juxtaposition in a relationship that allows the described components to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a manner that expression of the coding sequence is achieved under conditions compatible with the control sequences. "Operably linked" sequences include both expression control sequences contiguous with a gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. As used herein, the term "expression control sequences" refers to polynucleotide sequences necessary to effect the expression and processing of the coding sequence to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, where appropriate, sequences that enhance protein secretion. The nature of such control sequences differs depending on the host organism; in prokaryotes, such control sequences generally include promoters, ribosomal binding sites, and transcription termination sequences; in eukaryotes, generally, such control sequences include promoters and transcription termination sequences. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and may further include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0106] "Transformation" as defined herein refers to any process by which exogenous DNA enters a host cell. Transformation can occur under natural or artificial conditions using a variety of methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. The method is selected based on the host cell to be transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA can replicate as an autonomously replicating plasmid or as part of the host chromosome. They also include cells that transiently express the inserted DNA or RNA for a limited period of time.

[0107] The term "recombinant host cell" (or simply "host cell") as used herein is intended to refer to a cell into which exogenous DNA has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Such progeny may not actually be identical to the parent cell, since certain modifications may occur in subsequent generations due to mutations or environmental influences, but are still encompassed within the scope of the term "host cell" as used herein. Preferably, host cells include prokaryotic and eukaryotic cells selected from any kingdom of life. Preferred eukaryotic cells include protists, fungi, plant and animal cells. Most preferably, host cells include, but are not limited to, the prokaryotic cell lines E. coli; the mammalian cell lines CHO, HEK293 and COS; the insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae.

[0108] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures can generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for all purposes.

[0109] As used herein, the term "effective amount" refers to an amount of a therapy that is sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, prevent progression of a disorder, cause regression of a disorder, prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., a prophylactic or therapeutic agent).

[0110] Specific regions or epitope mapping of the human IgE protein provided herein can be identified by any suitable epitope mapping method known in the art with any one of the antibodies provided by the invention. An example of such a method includes screening peptides of various lengths derived from IgE for binding to an antibody of the invention using the smallest fragment capable of specifically binding to the antibody that contains the sequence of the epitope recognized by the antibody. IgE peptides can be generated synthetically or by proteolytic digestion of the IgE protein. Peptides that bind to the antibody can be identified, for example, by mass spectrometry. In another example, NMR spectroscopy or X-ray crystallography can be used to identify the epitope bound by an antibody of the invention. Crystallization and X-ray crystallography techniques are preferred to determine the structure of IgE and the epitope on IgE to which the anti-IgE antibody or antigen-binding agent of the invention binds.

[0111] Antibodies for use in the present invention can be produced using the single lymphocyte antibody technique by cloning and expressing immunoglobulin variable region cDNA generated from a single lymphocyte selected for the production of specific antibodies, for example, by methods described by Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-78481; WO92 / 02551; WO2004 / 051268 and International Patent Application WO2004 / 106377. Screening for antibodies can be performed using an assay to measure binding to human IgE and / or an assay to measure the ability to block IgE from binding to its natural receptor. An example of a binding assay is ELISA.

[0112] Humanized antibodies (including CDR-grafted antibodies) are antibody molecules having one or more complementarity determining regions (CDRs) derived from a non-human species (e.g. rabbit or mouse) and framework regions derived from a human immunoglobulin molecule (see, e.g., U.S. Pat. No. 5,585,089; WO 91 / 09967). It will be appreciated that it is only necessary to introduce the specificity determining residues of the CDRs, rather than the entire CDR (see, e.g., Kashmiri et al., 2005, Methods, 36, 25-34). Humanized antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs were derived. The latter are often referred to as donor residues. Antibody molecules of the invention suitably have a binding affinity (K D Affinity can be measured using any suitable method known in the art, including BIAcore, as described in the examples herein (see Example 6), using isolated native or recombinant IgE or a suitable fusion protein / polypeptide.

[0113] Those skilled in the art can determine the affinity of the antibodies or antigen binding agents of the invention, as well as the extent to which a binding agent (such as an antibody) inhibits binding, using conventional techniques, such as those described by Scatchard et al. (Ann. KY. Acad. Sci. 51:660-672 (1949)), or by surface plasmon resonance (SPR) using a system such as BIAcore. For surface plasmon resonance, the target molecule is immobilized on a solid phase and exposed to a ligand in a mobile phase that migrates with the flow cell. When ligand binding to the immobilized target occurs, the partial refractive index changes, resulting in a change in the SPR angle, which can be monitored in real time by detecting changes in the intensity of the reflected light. The rate of change of the SPR signal can be analyzed to obtain apparent rate constants for the association and dissociation phases of the binding reaction. The ratio of these values ​​gives the apparent equilibrium constant (affinity) (see, e.g., Wolff et al, Cancer Res. 53:2560-65 (1993)).

[0114] It will be appreciated that the affinity of the antibodies provided by the present invention can be altered using any suitable method known in the art.Thus, the present invention also relates to variants of the antibody molecules of the present invention, which have improved affinity for IgE. Such variants can be obtained by several affinity maturation protocols, including CDR mutations (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio / Technology, 10, 779-783, 1992), use of mutant strains of E. coli (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996) and sexual PCR (Crameri et al., Nature, 391, 288-291, 1998). Vaughan et al. (supra) discuss these methods of affinity maturation.

[0115] Humanized antibodies and antigen-binding agents In one aspect of the invention, humanized anti-IgE monoclonal antibodies and antigen-binding agents are provided herein. Humanized antibodies are antibodies in which the heavy and / or light chains contain one or more CDRs (optionally including one or more modified CDRs) from a donor antibody (e.g., a non-human antibody such as a mouse or rabbit monoclonal antibody) grafted into the heavy and / or light chain variable region framework of an acceptor antibody (e.g., a human antibody). For review, see Vaughan et al, Nature Biotechnology, 16, 535-539, 1998.

[0116] In one embodiment, rather than introducing an entire CDR, only one or more specificity determining residues from any one of the above CDRs are introduced into the human antibody framework (see, e.g., Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment, only the specificity determining residues from one or more CDRs described herein are introduced into the human antibody framework. In another embodiment, only the specificity determining residues from each CDR described herein are introduced into the human antibody framework. When grafting the CDRs or specificity determining residues, any suitable acceptor variable region framework sequence can be used, taking into account the class / type of the donor antibody from which the CDRs are derived, including mouse, rabbit, primate and human framework regions.

[0117] Suitably, a humanised antibody according to the invention has a variable domain comprising a human acceptor framework region and one or more CDRs as specifically provided herein.Thus, in one aspect, there is provided a humanised monoclonal antibody which binds human IgE, the variable domain comprising a human acceptor framework region (with any of the mutations described herein) and a non-human donor CDR.

[0118] The construction of CDR-grafted antibodies is generally described in European Patent Application EP-A-0239400, which discloses a process in which the CDRs of a mouse monoclonal antibody are grafted onto the framework regions of a human immunoglobulin variable domain by site-directed mutagenesis using long oligonucleotides, and is incorporated herein. CDRs are relatively short peptide sequences that determine the antigen-binding specificity of an antibody and are carried on the framework regions of the variable domain.

[0119] The earliest studies on humanization of monoclonal antibodies by CDR grafting were carried out on monoclonal antibodies that recognize synthetic antigens such as NP. However, examples of humanization by CDR grafting of a mouse monoclonal antibody that recognizes lysozyme and a rat monoclonal antibody that recognizes an antigen on human T cells are described in Verhoeyen et al. (Science, 239, 1534-1536, 1988) and Riechmann et al. (Nature, 332, 323-324, 1988), respectively. Antibody humanization is achieved by grafting the CDRs of a non-human antibody, such as a mouse, rat, goat, or rabbit antibody, onto a "similar" human framework (acceptor), and by selecting the minimum number of critical framework residues (back mutations) manually selected from the donor monoclonal antibody to be incorporated into the human acceptor framework to maintain the original CDR conformation.Such methods are known in the art and are described in detail in the following publications: Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science 239:1534 (1988); Sims et al., J. Immunol. 151:2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993); Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 28(4 / 5):489-498 (1991), which are incorporated herein by reference. 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCT Publications WO91 / 09967, PCT / :US98 / 16280, US96 / 18978, US91 / 09630, US91 / 05939, US94 / 01234, GB89 / 01334, GB91 / 01134, GB92 / 01755; WO90 / 14443, WO90 / 14424, WO90 / 14430, EP229246, EP592,106; EP519,596, EP239,400, U.S. Pat. 332, 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539, and 4,816,567.

[0120] Human variable heavy and light chain germline subfamily classifications can be derived from the Kabat germline subgroup designations: VH1, VH2, VH3, VH4, VH5, VH6, or VH7 for specific VH sequences and JH1, JH2, JH3, JH4, JH5, and JH6 for specific variable heavy chain joining groups for framework 4; VK1, VK2, VK3, VK4, VK5, and VK6 for specific VL kappa sequences for frameworks 1, 2, and 3. For VK3, VK4, VK5 or VK6 and for a particular kappa linkage group for framework 4, JK1, JK2, JK3, JK4 or JK5; or for a particular VL lambda sequence for frameworks 1, 2 and 3, VL1, VL2, VL3, VL4, VL5, VL6, VL7, VL8, VL9 or VL10 and for a particular lambda linkage group for framework 4, JL1, JL2, JL3 or JL7.

[0121] If present, the constant region domains of the antibody molecule of the present invention can be selected taking into account the proposed function of the antibody molecule, in particular the effector functions that may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. In certain embodiments, human IgG constant region domains, in particular of IgG1 and IgG3 isotypes, may be used when the antibody molecule is intended for therapeutic use and antibody effector functions are required. Alternatively, IgG2 and IgG4 isotypes may be used when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required. It will be understood that sequence variants of these constant region domains may also be used. For example, an IgG4 molecule in which serine at position 241 has been changed to proline, as described in Angal et al., Molecular Immunology, 1993, 30(1), 105-108, may be used. It will also be understood by the skilled artisan that antibodies may be subject to various post-translational modifications. The type and extent of these modifications often depend on the host cell line used to express the antibody as well as the culture conditions. Such modifications may include alterations in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. A common modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) due to the action of carboxypeptidases (described in Harris, RJ. Journal of Chromatography 705:129-134, 1995). Thus, the C-terminal lysine of the antibody heavy chain may be absent.

[0122] CDR and human framework modifications Riechmann et al., found that introduction of CDRs alone (as defined by Kabat (Kabat et al., supra, and Wu et al., J. Exp. Med., 132, 211-250, 1970)) was not sufficient to provide satisfactory antigen-binding activity in the CDR-grafted product. It was found that some framework residues needed to be altered so that they corresponded to those of the donor framework region. Proposed criteria for selecting which framework residues needed to be altered are described in International Patent Application WO 90 / 07861, which is incorporated herein.

[0123] Substitution of non-human CDRs into a human variable domain framework will most likely result in retention of the correct spatial orientation of the CDRs, if the human variable domain framework adopts the same or similar conformation as the non-human variable framework from which the CDRs originate. This is achieved by obtaining human variable domains from a human antibody whose framework sequences exhibit a high degree of sequence identity with the non-human variable framework domain from which the CDRs are derived. As mentioned above, the heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be those of naturally occurring human antibodies or can be consensus sequences of several human antibodies. See Kettleborough et al, Protein Engineering 4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993) and Carter et al, WO92 / 22653.

[0124] Having identified the complementarity determining regions of the non-human donor immunoglobulin and the appropriate human acceptor immunoglobulin, the next step is to determine which, if any, residues from these components should be replaced to optimize the properties of the resulting humanized antibody. In general, the replacement of human amino acid residues with non-human amino acid residues should be minimized, since the introduction of non-human residues increases the risk of the antibody eliciting a human anti-donor antibody (HADA) response in humans. Art-recognized methods of determining immune response can be implemented to monitor HADA responses in a particular host or during clinical trials. Hosts administered humanized antibodies can be given an immunogenicity assessment at the start of and throughout administration of the therapy. HADA responses are measured by detecting antibodies against the humanized therapeutic reagent in serum samples from the host using methods known to those skilled in the art, including, for example, surface plasmon resonance technology (BIACORE) and / or solid-phase ELISA analysis.

[0125] The selection of amino acid residues for substitution (also referred to herein as "mutation") is determined, in part, by computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are described herein. In general, starting from a solved structure of an immunoglobulin chain or domain thereof, a molecular model is generated. The chains to be modeled are compared for amino acid sequence similarity with the chains or domains of the solved three-dimensional structure, and the chains or domains showing the highest sequence similarity are selected as the starting point for construction of the molecular model. Chains or domains with at least 50% sequence identity are selected for modeling, preferably those with at least 60%, 70%, 80%, 90% or more sequence identity are selected for modeling. The solved starting structure is modified to allow for differences between the actual amino acids in the immunoglobulin chains or domains to be modeled and those in the starting structure. The modified structures are then assembled into composite immunoglobulins. Finally, the model is refined by energy minimization and by verifying that all atoms are at appropriate distances from each other and that bond lengths and angles are within chemically allowed limits.

[0126] The selection of amino acid residues for substitution can also be determined, in part, by examination of the characteristics of the amino acid at a particular position, or by empirical observation of the effect of substituting or mutagenesing a particular amino acid. For example, when an amino acid differs between the donor variable region framework residue and a selected human variable region framework residue, the human framework amino acid is typically selected such that the amino acid is: (1) Direct non-covalent binding to antigens; (2) adjacent to the CDR region, (3) otherwise interacts with the CDR regions (e.g., is within about 3-6 angstroms of the CDR regions as determined by computer modeling), or (4) Involved in the VL-VH interface If it is reasonably predicted that this will be the case, then substitutions should be made with the equivalent framework amino acids from the donor antibody.

[0127] Residues that are "directly non-covalently bound to an antigen" include amino acids at positions in the framework region that have a good probability of directly interacting with amino acids on the antigen according to established chemical forces, e.g., by hydrogen bonds, van der Waals forces, hydrophobic interactions, etc. CDRs and framework regions are defined by Kabat et al. or Chothia et al, supra. If framework residues as defined by Kabat et al., supra, constitute structural loop residues as defined by Chothia et al., supra, amino acids present in the donor antibody can be selected for substitution in the humanized antibody. Residues "adjacent to a CDR region" include amino acid residues at positions immediately adjacent to one or more CDRs in the primary sequence of the humanized immunoglobulin chain, e.g., CDRs as defined by Kabat or CDRs as defined by Chothia (see, e.g., Chothia and Lesk 1MB 196:901 (1987)). These amino acids are particularly likely to interact with amino acids in the CDRs and, if selected from the acceptor, distort the donor CDRs and reduce affinity. Additionally, adjacent amino acids may directly interact with the antigen (Amit et al, Science, 233:747 (1986), incorporated herein by reference), and it is desirable to select these amino acids from the donor to retain all of the antigen contacts that provide affinity in the original antibody. As described herein, FR sequences can also be substituted / mutated to improve the affinity of the anti-IgE antibody or antigen-binding agent of the invention for IgE (and / or extend its interaction or epitope on Cε2 of IgE).

[0128] Residues that "otherwise interact with the CDR regions" include those determined by secondary structure analysis that are in sufficient spatial orientation to provide the CDR regions. In one embodiment, residues that "otherwise interact with the CDR regions" are identified by analyzing a three-dimensional model (e.g., a computer-generated model) of the donor immunoglobulin. Typically, the three-dimensional model of the original donor antibody shows that certain amino acids outside the CDRs are close to the CDRs and have a good probability of interacting with amino acids in the CDRs by hydrogen bonds, van der Waals forces, hydrophobic interactions, etc. At these amino acid positions, donor immunoglobulin amino acids can be selected rather than acceptor immunoglobulin amino acids. Amino acids according to this criterion should generally contain atoms that have side chain atoms within about 3 angstrom units (A) of some atoms in the CDRs and can interact with CDR atoms according to established chemical forces, such as those listed above. For atoms that can form hydrogen bonds, the 3 Å is measured between their nuclei, while for atoms that do not form bonds, the 3 Å is measured between their van der Waals surfaces. Thus, in the latter case, the nucleus must be within about 6 Å (3A plus the sum of van der Waals radii) for the atoms to be considered capable of interacting. In many cases, the nucleus will be 4 or 5-6 Å apart. In determining whether an amino acid can interact with a CDR, it is preferred not to consider the last 8 amino acids of the heavy chain CDR2 as part of the CDR, since from a structural point of view, these amino acids behave more as part of the framework.

[0129] Amino acids that can interact with amino acids in CDRs (or FRs) can be identified in yet another way. The solvent accessible surface area of ​​each framework amino acid is calculated in two ways: (1) in the intact antibody, and (2) in a hypothetical molecule consisting of the antibody with the CDRs removed. A significant difference between these figures of about 10 square angstroms or more indicates that the access of the framework amino acid to the solvent is at least partially blocked by the CDR, and thus the amino acid is in contact with the CDR. The solvent accessible surface area of ​​an amino acid can be calculated based on a three-dimensional model of an antibody using algorithms known in the art (e.g., Connolly, J. Appl. Cryst. 16:548 (1983) and Lee and Richards, J. Mol. Biol. 55:379 (1971), both of which are incorporated herein by reference). Framework amino acids may also indirectly interact with CDRs, in some cases, by influencing the conformation of another framework amino acid that in turn contacts the CDR.

[0130] It is known that certain amino acids at some positions in the framework can interact with CDRs in many antibodies (Chothia and Lesk, supra; Chothia et al, supra; and Tramontano et al, J. Mol. Biol. 215:175 (1990), all incorporated herein by reference). Of note, amino acids at positions 2, 48, 64, and 71 of the light chain and 71 and 94 of the heavy chain (numbered according to Kabat) are known to be able to interact with CDRs in many antibodies. Amino acids at positions 35 in the light chain and 93 and 103 in the heavy chain are also likely to interact with CDRs. At all these numbered positions, it is preferred that the choice of donor amino acid rather than acceptor amino acid (if they are different) is made in the humanized immunoglobulin. On the other hand, certain residues that can interact with the CDR regions, such as the first 5 amino acids of the light chain, may be selected from the acceptor immunoglobulin without losing affinity in the humanized immunoglobulin.

[0131] Residues "involved in the VL-VH interface" or "packing residues" include those residues at the VL and VH interface as defined, for example, by Novotny and Haber, Proc. Natl. Acad. Sci. USA, 82:4592-66 (1985) or Chothia et al., supra. In general, unusual packing residues should be retained in the humanized antibody if they differ from those in the human framework.

[0132] Generally, one or more amino acids that meet the above criteria are substituted. In some embodiments, all or many of the amino acids that meet the above criteria are substituted. In some cases, there is some uncertainty as to whether a particular amino acid meets the above criteria, and whether alternative variant immunoglobulins are produced, one with that particular substitution and the other without. The alternative variant immunoglobulins so produced can be tested for the desired activity in any of the assays described herein, and the preferred immunoglobulins can be selected.

[0133] Usually, the CDR regions in a humanized antibody are substantially identical, and more usually, identical to the corresponding CDR regions of the donor antibody. Although usually not desirable, in some cases it is possible to make one or more conservative amino acid substitutions of the CDR residues without appreciably affecting the binding affinity of the resulting humanized immunoglobulin. Conservative or analogous substitutions are contemplated, for example, combinations such as leucine substituted for isoleucine or valine. Other amino acids that can often be substituted for one another include, but are not limited to, phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains); Lysine, arginine and histidine (amino acids with basic side chains); Aspartic acid and glutamic acid (amino acids with acidic side chains); Asparagine and glutamine (amino acids with amide side chains); and Cysteine ​​and methionine (amino acids with sulfur-containing side chains) Examples include:

[0134] Further candidates for substitution are acceptor human framework amino acids that are unusual or "rare" for human immunoglobulins at that position. These amino acids can be replaced with amino acids from the equivalent position of the donor antibody or from the equivalent position of a more typical human immunoglobulin. For example, a substitution is desirable if an amino acid in the human framework region of the acceptor immunoglobulin is rare for that position and the corresponding amino acid in the donor immunoglobulin is common for that position in human immunoglobulin sequences; or if an amino acid in the acceptor immunoglobulin is rare for that position compared to other human sequences and the corresponding amino acid in the donor immunoglobulin is also rare. These criteria help ensure that non-standard amino acids in the human framework do not disrupt the antibody structure. Furthermore, the humanized antibody can be made less immunogenic by replacing unusual human acceptor amino acids with amino acids from the donor antibody that happen to be typical for human antibodies.

[0135] The term "rare" as used herein refers to an amino acid that occurs at that position in less than about 20% of the sequences in a representative sample of sequences, but usually in less than about 10%, and the term "common" as used herein refers to an amino acid that occurs in more than about 25% of the sequences in a representative sample, but usually in more than about 50%. For example, all human light and heavy chain variable region sequences are grouped into "subgroups" of sequences that are particularly homologous to one another and have the same amino acids at certain key positions, respectively (Kabat et al, supra). When determining whether an amino acid in a human acceptor sequence is "rare" or "common" among human sequences, it is often preferable to consider only those human sequences in the same subgroup as the acceptor sequence.

[0136] Further candidates for substitution are acceptor framework residues that correspond to rare or unusual donor framework residues. Rare or unusual donor framework residues are those that are rare or unusual (as defined herein) for the donor antibody at that position. For the donor antibody, subgroups can be determined according to Kabat to identify residue positions that differ from the consensus. These donor-specific differences may represent somatic mutations in the donor sequence that enhance activity. Unusual residues predicted to affect binding can be retained, while residues predicted not to be important for binding can be substituted.

[0137] Further candidates for substitution are non-germline residues present in the acceptor framework regions. For example, when an acceptor antibody chain (i.e., a human antibody chain that has significant sequence identity with the donor antibody chain) is aligned with a germline antibody chain (also having significant sequence identity with the donor antibody), residues that do not match between the acceptor and germline chain frameworks can be replaced with the corresponding residues from the germline sequence.

[0138] Other than the specific amino acid substitutions discussed above, the framework regions of humanized immunoglobulins are usually substantially identical, and more usually identical (except as described herein for the purposes of the present invention), to the framework regions of the human antibodies from which they are derived. Of course, many amino acids in framework regions make little or no direct contribution to the specificity or affinity of the antibody. Thus, many individual conservative substitutions of framework residues can be tolerated without appreciable change in the specificity or affinity of the resulting humanized immunoglobulin. Thus, in one embodiment, the variable framework regions of a humanized immunoglobulin have at least 65, 75 or 85% sequence similarity or identity to a human variable framework region sequence or a consensus of such sequences. In another embodiment, the variable framework regions of a humanized immunoglobulin have at least 90%, preferably 95%, more preferably 96%, 97%, 98% or 99% sequence similarity or identity to a human variable framework region sequence or a consensus of such sequences. In general, however, such substitutions are undesirable (except as described herein).

[0139] As used herein, the degree of identity and similarity may be measured using, for example, the methods described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and the BLAST™ software available from NCBI (Altschul, S. F. et al., Nature 354:131-135, 1997). al., 1990, J. Mol. Biol. 215:403-410; Gish, W. & States, DJ 1993, Nature Genet. 3:266-272. Madden, TL et al., 1996, Meth. Enzymol. 266:131-141; Altschul, SF et al., 1997, Nucleic Acids Res. 25:3389-3402; Zhang, J. & Madden, TL 1997, Genome Res. 7:649-656.

[0140] Several reviews discussing CDR-grafted antibodies have been published, including Vaughan et al. (Nature Biotechnology, 16, 535-539, 1998), which is incorporated herein by reference.

[0141] The anti-IgE antibodies of the invention may also include further additional binding domains, such as, for example, the DVD-Ig molecules disclosed in WO2007 / 024715 or the so-called (FabFv)2Fc described in WO2011 / 030107. Thus, antibody as used herein includes bivalent, trivalent or tetravalent full-length antibodies.

[0142] Antigen-binding agents Antigen binding agents include single chain antibodies (i.e., full length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH or VL or VHH), e.g., as described in WO2001090190, scFv, bivalent, trivalent or tetravalent antibodies, Bis-scFv, diabodies, tribodies, triabodies, tetrabodies and epitope-antigen binding agents of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and producing these antibody fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv format was first disclosed in WO2009 / 040562, and its disulfide-stabilized version, Fab-dsFv, was first disclosed in WO2010 / 035012. Other antibody fragments for use in the present invention include the Fab and Fab' fragments described in International Patent Applications WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171. Multivalent antibodies may include multiple specificities, e.g., bispecificity, or may be monospecific (see, for example, WO92 / 22583 and WO05 / 113605). One such example of the latter is Tri-Fab (or TFM) described in WO92 / 22583.

[0143] A typical Fab' molecule comprises a pair of heavy and light chains, where the heavy chain comprises a variable domain VH, a constant domain CH1 and a natural or modified hinge region, and the light chain comprises a variable domain VL and a constant domain CL.

[0144] In one aspect, dimers of Fab' according to the present disclosure are provided to create F(ab')2, e.g., dimerization may be via a native hinge sequence, or a derivative thereof, or a synthetic hinge sequence as described herein. An antibody binding domain will generally comprise six CDRs, three from the heavy chain and three from the light chain. In one embodiment, the CDRs are in a framework and together form a variable region. Thus, in one embodiment, the antigen binding agent comprises a binding domain specific for IgE comprising a light chain variable region and a heavy chain variable region.

[0145] It will be appreciated that one or more (e.g., 1, 2, 3 or 4) amino acid substitutions, additions and / or deletions can be made to the CDRs or other sequences (e.g., variable domains) provided by the invention described above or below without significantly altering the ability of the antibody to bind IgE. The skilled artisan can easily test the effect of any amino acid substitutions, additions and / or deletions, for example by using the methods described herein, in particular in the Examples.

[0146] In one aspect, the binding affinity (K D One or more (e.g., 1, 2, 3, or 4) amino acid substitutions, additions, and / or deletions may be made to the CDR or framework regions used in the antibodies or fragments provided by the invention such that the K DTo reduce the affinity of a given antibody to less than, for example, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3 nM, the antibody is provided with modified humanized antibodies in which modifications have been made to the CDRs, framework regions, or both, to reduce the affinity of a given antibody to less than, for example, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3 nM. Antibody fragments of the present invention include Fab, Fab', F(ab')2, scFv, diabodies, scFAb, dFv, single domain light chain antibodies, dsFv, and peptides containing the CDRs.

[0147] Fab is an antibody fragment obtained by treating IgG with the protease papain (cleaving at amino acid residue 224 of the H chain), in which approximately half of the N-terminal side of the H chain and the entire L chain are bound together via disulfide bonds. Fab has a molecular weight of approximately 50,000 and antigen-binding activity. The Fab of the present invention can be obtained by treating the humanized CDR-grafted antibody of the present invention, which specifically reacts with IgE, with the protease papain. Alternatively, the Fab can be produced by inserting DNA encoding the Fab of the antibody into an expression vector for prokaryotes or eukaryotes, and introducing the vector into a prokaryote or eukaryote to express the Fab.

[0148] F(ab')2 is an antibody fragment obtained by treating IgG with the protease pepsin, and is slightly larger than Fab, which is bound via disulfide bonds in the hinge region. It has a molecular weight of approximately 100,000 and antigen-binding activity.

[0149] The F(ab')2 of the present invention can be obtained by treating a human CDR-grafted antibody that specifically reacts with IgE with a protease, pepsin, or by binding the Fab' described below via a thioether bond or disulfide bond to produce the F(ab')2. Fab' is an antibody fragment which can be obtained by cleaving the disulfide bond in the hinge region of F(ab')2 and has a molecular weight of about 50,000 and antigen-binding activity.

[0150] The Fab' of the present invention can be obtained by treating F(ab')2 that specifically reacts with IgE with dithiothreitol, a reducing agent. The Fab' of the present invention can also be produced by inserting DNA encoding the Fab' of the human CDR-grafted antibody of the present invention that specifically reacts with IgE into an expression vector for prokaryotes or eukaryotes, and introducing the vector into a prokaryote or eukaryote to express the Fab'. An scFv is a VH-P-VL or VL-P-VH polypeptide in which one chain of VH and one chain of VL are linked using a suitable peptide linker (P) of 12 or more residues and which has antigen-binding activity.

[0151] The scFv of the present invention can be produced by obtaining cDNA encoding the VH and VL of a human CDR-grafted antibody that specifically reacts with IgE of the present invention, constructing DNA encoding the scFv, inserting the DNA into an expression vector for prokaryotes or eukaryotes, and then introducing the expression vector into a prokaryote or eukaryote to express the scFv.

[0152] The Fab fragment of the invention can be linked to the scFv directly or via a linker. As used herein, "single chain variable fragment" or "scFv" refers to a single chain variable fragment stabilized by a peptide linker between the VH and VL variable domains, e.g., a peptide linker having the amino acid sequence SEQ ID NO: 151. The linkage to the Fab fragment can be a chemical conjugation, but is most preferably a translational fusion, i.e., a genetic fusion, in which the respective sequences are encoded in sequence by an expression vector. Thus, the linker is typically an amino acid linker as described herein. The scFv of the invention linked to the Fab fragment can be bound to a serum carrier protein to extend the half-life of the antibody fusion protein in vivo. The extension of the half-life in such a way is independent of IgE binding and can be advantageous.

[0153] As used herein, "serum carrier protein" refers to any suitable plasma carrier protein to which the scFv can bind, in one example the serum carrier protein is selected from thyroxine binding protein, transthyretin, alpha 1-acid glycoprotein, transferrin, fibrinogen and albumin, or any fragment thereof. Typically, the scFv binds to albumin, preferably human serum albumin. Any suitable albumin binding scFv can be incorporated into the antibody fusion proteins of the invention. Suitable albumin binding domains have been previously described in the art. Diabodies are antibody fragments in which scFv's with the same or different antigen-binding specificities form dimers and have bivalent antigen-binding activity for the same antigen or two specific antigen-binding activities for different antigens.

[0154] Diabodies of the invention, for example, bivalent diabodies specifically reactive with IgE, can be produced by obtaining cDNA encoding the VH and VL of an antibody specifically reactive with IgE, constructing a DNA encoding an scFv having a polypeptide linker of 3-10 residues, inserting the DNA into an expression vector for a prokaryote or an expression vector for a eukaryote, and then introducing the expression vector into a prokaryote or eukaryote to express the diabody. dsFv can be obtained by linking polypeptides in which one amino acid residue in each of VH and VL is replaced with a cysteine ​​residue via a disulfide bond between the cysteine ​​residues. The amino acid residue to be replaced with a cysteine ​​residue can be selected based on the three-dimensional structure of an antibody according to the method described by Reiter et al. (Protein Engineering, 7, 697 (1994)).

[0155] The dsFv of the present invention can be produced by obtaining cDNA encoding the VH and VL of a human CDR-grafted antibody that specifically reacts with IgE of the present invention, constructing DNA encoding a dsFv, inserting the DNA into an expression vector for a prokaryote or an expression vector for a eukaryote, and then introducing the expression vector into a prokaryote or eukaryote to express the dsFv. A peptide containing CDRs is constructed by including at least one region of H-chain and L-chain CDRs. Multiple CDRs can be linked directly or via a suitable peptide linker.

[0156] The peptide containing the CDR of the present invention can be produced by obtaining cDNA encoding the CDRs of VH and VL of a human CDR-grafted antibody that specifically reacts with IgE, constructing DNA encoding the CDRs, inserting the DNA into an expression vector for a prokaryote or an expression vector for a eukaryote, and then introducing the expression vector into a prokaryote or eukaryote to express the peptide. Alternatively, the peptide containing the CDR can be produced by chemical synthesis such as the Fmoc method (fluorenylmethoxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method). The antibodies of the present invention include antibody derivatives in which radioisotopes, proteins, drugs, etc. are chemically or genetically conjugated to the antibodies of the present invention.

[0157] The antibody derivatives of the present invention can be produced by chemically conjugating a radioisotope, a protein, or a drug to the N-terminus or C-terminus of the H chain or L chain of an antibody or antibody fragment that specifically reacts with IgE, to a suitable substituent or side chain of the antibody or antibody fragment, or to a sugar chain in the antibody or antibody fragment (Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)).

[0158] Alternatively, the antibody or antibody fragment of the present invention that specifically reacts with IgE can be produced genetically by linking DNA encoding the antibody or antibody fragment to other DNA encoding the protein to be bound, inserting that DNA into an expression vector, and introducing the expression vector into a host cell. Radioisotopes include 131I, 125I, and the like, which can be conjugated to antibodies, for example, by the chloramine T method.

[0159] The drug is preferably a low molecular weight compound. Examples include anticancer drugs such as alkylating agents (e.g., nitrogen mustard, cyclophosphamide), metabolic antagonists (e.g., 5-fluorouracil, methotrexate), antibiotics (e.g., daunomycin, bleomycin, mitomycin C, daunorubicin, doxorubicin), plant alkaloids (e.g., vincristine, vinblastine, vindesine), and hormonal drugs (e.g., tamoxifen, dexamethasone) (Clinical Oncology, edited by Japanese Society of Clinical Oncology, published by Cancer and Chemotherapy (1996); anti-inflammatory agents such as steroids (e.g., hydrocortisone, prednisone), non-steroids (e.g., aspirin, indomethacin), immunomodulators (e.g., aurothiomalate, penicillamine), immunosuppressants (e.g., cyclophosphamide, azathioprine), and antihistamines (e.g., chlorpheniramine maleate, clemastine) (Inflammation and Anti-inflammatory Therapy, Ishiyaku Shuppan (1982)). Methods for conjugating daunomycin to an antibody include a method in which daunomycin is conjugated to an amino group of an antibody via glutaraldehyde, and a method in which the amino group of daunomycin is conjugated to a carboxyl group of an antibody via a water-soluble carbodiimide.

[0160] Toxins such as ricin and diphtheria toxin can also be used to directly inhibit cancer cells. For example, a fusion antibody with a protein can be produced by linking a cDNA encoding an antibody or an antibody fragment to another cDNA encoding a protein, constructing a DNA encoding the fusion antibody, inserting the DNA into an expression vector for a prokaryote or an expression vector for a eukaryote, and then introducing it into a prokaryote or eukaryote to express the fusion antibody.

[0161] Further contemplated herein are fusions of binding agents, e.g., antibody fragments or antigen binding agents including immunoglobulin-like fragments and agents such as diabodies, scAbs, bispecific fragments, triabodies, Fab-Fv-Fv, Fab-Fv, tribodies, (Fab-Fv)2-Fc, and antibody fragments or portions such as CDRs or antibody loops encompassing the CDRs, grafted onto non-Ig frameworks such as fibronectin or leucine zippers, as described in Binz et al., (2005) Nat. Biotech. 23:1257-1268, which is incorporated herein in its entirety.

[0162] Conjugated anti-IgE monoclonal antibodies and antigen-binding agents If desired, the antibody or antigen-binding agent for use in the present invention can be conjugated to one or more effector molecules. It will be understood that the effector molecule may comprise a single effector molecule or two or more such molecules linked to form a single moiety that can be bound to the antibody of the present invention. If it is desired to obtain an antibody fragment linked to an effector molecule, this can be prepared by standard chemical or recombinant DNA procedures in which the antibody fragment is linked to the effector molecule directly or via a coupling agent. Techniques for conjugating such effector molecules to antibodies are well known in the art (see Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Particular chemical procedures include, for example, those described in WO93 / 06231, WO92 / 22583, WO89 / 00195, WO89 / 01476 and WO03 / 031581. Alternatively, where the effector molecule is a protein or polypeptide, linkage can be achieved using recombinant DNA procedures, for example as described in WO86 / 01533 and EP0392745.

[0163] The term effector molecule as used herein includes, for example, anti-neoplastic agents, drugs, toxins, biologically active proteins, such as enzymes, other antibodies or antibody fragments, antigen binding agents, synthetic (including PEG) or natural polymers, nucleic acids and fragments thereof, such as DNA, RNA and fragments thereof, radionuclides, in particular radioactive iodine, radioisotopes, chelated metals, nanoparticles and reporter groups, such as fluorescent compounds or compounds that can be detected by NMR or ESR spectroscopy.

[0164] Examples of effector molecules may include cytotoxins or cytotoxic agents, which include any agent that is detrimental to (e.g., kills) cells. Examples include combrestatins, dolastatins, epothilones, staurosporines, maytansinoids, spongistatins, rhizoxins, halichondrins, roridin, hemiasterlin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof.

[0165] Effector molecules also include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepaclorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicin or duocarmycin), and antimitotic agents (e.g., vincristine and vinblastine).

[0166] Other effector molecules may include chelated radionuclides such as In and Y, Lu, 77, Bismuth, 213, Californium, 252, Iridium, 192 and Tungsten, 188 / Rhenium, 188; or drugs such as, but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

[0167] Other effector molecules include proteins, peptides and enzymes. Enzymes of interest include, but are not limited to, proteases, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides and peptides of interest include, but are not limited to, toxins such as immunoglobulins, abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin, proteins such as insulin, tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor, or tissue plasminogen activator, thrombotic or anti-angiogenic agents, such as angiostatin or endostatin, or biological response modifiers such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factors and immunoglobulins.

[0168] Other effector molecules may include, for example, detectable substances useful in diagnosis. Detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radionuclides, positron-emitting metals (for use in positron emission tomography), and non-radioactive paramagnetic metal ions. For metal ions that can be conjugated to antibodies for use as diagnostic agents, see generally U.S. Patent No. 4,741,900. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin, and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radionuclides include 125I, 131I, 111In, and 99Tc.

[0169] In another example, the effector molecule may increase the half-life of the antibody in vivo and / or reduce the immunogenicity of the antibody and / or enhance the delivery of the antibody across an epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin binding proteins or albumin binding compounds such as those described in WO05 / 117984. In one aspect, the half-life provided by an effector molecule that is independent of IgE or anti-human IgE antibodies is advantageous.

[0170] When the effector molecule is a polymer, it may generally be a synthetic or natural polymer, such as an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer or a branched or unbranched polysaccharide, such as a homo- or hetero-polysaccharide. Particular optional substituents which may be present on the above synthetic polymers include one or more hydroxy, methyl or methoxy groups.

[0171] Particular examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, in particular optionally substituted poly(ethylene glycol), such as methoxypoly(ethylene glycol) or derivatives thereof. Particular natural polymers include lactose, amylose, dextran, glycogen or derivatives thereof. In one embodiment, the polymer is albumin or a fragment thereof, such as human serum albumin or a fragment thereof, hi one embodiment, the polymer is a PEG molecule.

[0172] "Derivatives" as used herein with respect to conjugates is intended to include reactive derivatives, e.g., thiol-selective reactive groups such as maleimides. The reactive groups can be linked to the polymer directly or via a linker segment. It will be understood that the residue of such a group will in some instances form part of the product as the linking group between the antibody fragment and the polymer.

[0173] The size of the natural or synthetic polymer may vary as required, but will generally be in the range of average molecular weight of 500 Da to 50,000 Da, e.g., 5,000 to 40,000 Da, e.g., 20,000 to 40,000 Da. The size of the polymer can be selected based on, inter alia, the intended use of the product, e.g., its ability to localize to a certain tissue, such as a tumor, or its long circulating half-life (for a review, see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example, for use in the treatment of tumors, for example, where the product is intended to leave the circulation and penetrate into tissues, it may be advantageous to use a low molecular weight polymer, e.g., having a molecular weight of about 5,000 Da. For applications where the product remains in the circulation, it may be advantageous to use a higher molecular weight polymer, e.g., having a molecular weight in the range of 20,000 Da to 40,000 Da.

[0174] Suitable polymers include polyalkylene polymers such as poly(ethylene glycol) or, in particular, methoxypoly(ethylene glycol) or its derivatives, especially having a molecular weight in the range of about 15,000 Da to about 40,000 Da.

[0175] In one example, the antibody for use in the present invention is conjugated to a poly(ethylene glycol) (PEG) moiety. In one particular example, the antibody is an antibody fragment, and the PEG molecule can be attached via any available amino acid side chain or terminal amino acid functional group located in the antibody fragment, such as any free amino, imino, thiol, hydroxyl, or carboxyl group. Such amino acids may be naturally present in the antibody fragment or can be engineered into the fragment using recombinant DNA methods (see, for example, U.S. Pat. No. 5,219,996; U.S. Pat. No. 5,667,425; WO98 / 25971, WO2008 / 038024). In one example, the antibody molecule of the present invention is a modified Fab fragment, in which the modification is the addition of one or more amino acids to the C-terminus of its heavy chain that allows for the attachment of an effector molecule. Suitably, the additional amino acids form a modified hinge region that contains one or more cysteine ​​residues to which the effector molecule can be attached. Multiple sites can be used to attach two or more PEG molecules.

[0176] Preferably, the PEG molecule is covalently linked via the thiol group of at least one cysteine ​​residue located in the antibody fragment. Each polymer molecule attached to the modified antibody fragment can be covalently linked to the sulfur atom of a cysteine ​​residue located in the fragment. The covalent linkage will generally be a disulfide bond, or in particular a sulfur-carbon bond. When the thiol group is used as the attachment point of an appropriately activated effector molecule, thiol-selective derivatives such as, for example, maleimide and cysteine ​​derivatives can be used. The activated polymer can be used as a starting material in the preparation of the above polymer-modified antibody fragment.

[0177] The activated polymer may be any polymer containing a thiol-reactive group, such as an α-halo carboxylic acid or ester, e.g., iodoacetamide, imide, e.g., maleimide, vinyl sulfone, or disulfide. Such starting materials can be obtained commercially (e.g., from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA) or prepared from commercially available starting materials using conventional chemical procedures. Particular PEG molecules include 20K methoxy-PEG-amine (available from Nektar, formerly Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA (available from Nektar, formerly Shearwater).

[0178] In one aspect, the antibody is PEGylated, i.e., by the methods disclosed, for example, in EP 0948544 or EP 1090037 [Poly(ethylene glycol) Chemistry, Biotechnical and Biomedical Applications, 1992, J. Milton Harris (ed), Plenum Press, New York, Poly(ethylene glycol) Chemistry and Biological Applications, 1997, J. Milton Harris and S. Zalipsky (eds), American Chemical Society, Washington DC and Bioconjugation Protein Coupling Techniques for the Biomedical Sciences, 1998, M. Aslam and A. Dent, Grove Publishers, New York; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545]. In one example, PEG is attached to a cysteine ​​in the hinge region. In one example, the PEG-modified Fab fragment has a maleimide group covalently linked to a single thiol group in the modified hinge region. A lysine residue can be covalently linked to the maleimide group, and each of the amine groups on the lysine residue can be attached to a methoxypoly(ethylene glycol) polymer having a molecular weight of about 20,000 Da. The total molecular weight of the PEG attached to the Fab fragment can then be about 40,000 Da.

[0179] A particular PEG molecule is lysine modified with 2-[3-(N-maleimido)propionamido]ethylamide of N,N'-bis(methoxypoly(ethylene glycol)) MW 20,000, also known as PEG2MAL40K (Nektar, formerly available from Shearwater).

[0180] Alternative sources of PEG linkers include GL2-400MA3 (where m in the structure below is 5) and GL2-400MA (where m is 2) and n is about 450. [ka] In other words, each PEG is about 20,000 Da.

[0181] Thus, in one embodiment, the PEG is 2,3-bis(methylpolyoxyethylene-oxy)-1-{[3-(6-maleimido-1-oxohexyl)amino]propyloxy}hexane (2-branched PEG, -CH2)3NHCO(CH2)5-MAL, Mw 40,000, known as SUNBRIGHT GL2-400MA3.

[0182] Further alternative PEG effector molecules of the type: [ka] is available from Dr Reddy, NOF and Jenkem.

[0183] In one aspect, an antibody of the invention is provided that is PEGylated (e.g., using PEG as described herein) attached via a cysteine ​​amino acid residue at or near amino acid 226 in the chain, e.g., amino acid 226 (by sequential numbering) of the heavy chain. In one aspect, the disclosure provides a Fab'PEG molecule comprising one or more PEG polymers, for example one or two polymers, such as a 40 kDa polymer or multiple polymers.

[0184] Fab'-PEG molecules according to the present disclosure may be particularly advantageous in that they have a half-life independent of the Fc fragment. In one example, the invention provides a method of treating a disease ameliorated by modulating human IgE biological activity comprising administering a therapeutically effective amount of an anti-IgE antibody or antigen binding agent thereof having a half-life that is independent of Fc, whereby the antibody or antigen binding agent thereof binds to IgE. In one embodiment, a Fab' is provided that is conjugated to a polymer, such as a PEG molecule, a starch molecule, or an albumin molecule. In one aspect there is provided an scFv conjugated to a polymer, such as a PEG molecule, a starch molecule or an albumin molecule. In one embodiment, the antibody or fragment is conjugated to a starch molecule, e.g., to increase half-life. Methods for conjugating starch to proteins described in U.S. Patent No. 8,017,739 are incorporated herein by reference.

[0185] Polynucleotides The present invention also provides an isolated DNA sequence encoding the heavy and / or light chain(s) of the antibody molecule of the present invention. Suitably, the DNA sequence encodes the heavy or light chain of the antibody molecule of the present invention. The DNA sequence of the present invention may comprise synthetic DNA produced, for example, by chemical processing, cDNA, genomic DNA or any combination thereof.

[0186] DNA sequences encoding the antibody molecules of the present invention can be obtained by methods well known to those skilled in the art. For example, DNA sequences encoding part or all of the antibody heavy and light chains can be synthesized from determined DNA sequences as needed, or based on the corresponding amino acid sequences. DNA encoding acceptor framework sequences is widely available to those skilled in the art and can be readily synthesized based on their known amino acid sequences. Standard techniques of molecular biology can be used to prepare DNA sequences encoding the antibody molecules of the present invention. The desired DNA sequence can be synthesized in whole or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used, if desired.

[0187] The present invention also relates to a cloning or expression vector comprising one or more DNA sequences of the present invention. Thus, a cloning or expression vector comprising one or more DNA sequences encoding an antibody of the present invention is provided. Preferably, the cloning or expression vector comprises two DNA sequences encoding the light and heavy chains of the antibody molecule of the present invention, respectively, and a suitable signal sequence. In one example, the vector comprises an intergenic sequence between the heavy and light chains (see WO03 / 048208).

[0188] The general methods by which vectors can be constructed, transfection methods and culture methods are well known to those skilled in the art, see, in this regard, Current Protocols in Molecular Biology, 1999, FMAusubel (ed), Wiley Interscience, New York and the Maniatis Manual published by Cold Spring Harbour Publishing.

[0189] Host cells expressing anti-IgE antibodies or fragments thereof Also provided are host cells comprising one or more cloning or expression vectors comprising one or more DNA sequences encoding the antibody of the present invention. Any suitable host cell / vector system can be used for the expression of the DNA sequences encoding the antibody molecules of the present invention. Bacterial, e.g., E. coli, and other microbial systems can be used, or eukaryotic, e.g., mammalian, host cell expression systems can be used. Suitable mammalian host cells include CHO, myeloma or hybridoma cells.

[0190] Suitable types of Chinese hamster ovary (CHO) cells for use in the present invention may include CHO and CHO-K1 cells, including dhfr-CHO cells, such as CHO-DG44 cells and CHO-DXB11 cells, which may be used with a DHFR selection marker, or CHOK1-SV cells, which may be used with a glutamine synthetase selection marker. Other cell types for use in expressing antibodies include lymphocytic cell lines, such as NSO myeloma cells and SP2 cells, COS cells. Other suitable cells may include human embryonic kidney (hek) fibroblasts, such as hek293F and ExpiHek cells, which are known in the art. CHO is preferred for the full-length Abs of the invention given that it is the standard host for the production of omalizumab (in one embodiment contemplated for antibodies of the invention, the standard glycosylation pattern of omalizumab) [see also WO2013 / 181577].

[0191] Production of anti-IgE antibodies or their fragments The present invention also provides a process for the production of an antibody molecule according to the invention comprising culturing a host cell containing a vector of the invention under conditions suitable to result in expression of protein from DNA encoding the antibody molecule of the invention, and isolating the antibody molecule. An antibody molecule may contain only heavy or light chain polypeptides, in which case only heavy or light chain polypeptide coding sequences need to be used to transfect the host cell. For production of products containing both heavy and light chains, two vectors can be transfected into a cell line, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector may be used that includes sequences encoding both light and heavy chain polypeptides.

[0192] A process is provided for culturing a host cell and expressing an antibody or fragment thereof, isolating the latter, and optionally purifying it to provide an isolated antibody or fragment. In one aspect, the process further comprises the step of conjugating an effector molecule to the isolated antibody or fragment, such as, in particular, to a PEG polymer as described herein. In one aspect, a process for purifying an antibody (in particular an antibody or fragment according to the invention) is provided, comprising performing anion exchange chromatography in a non-binding mode such that impurities are retained on the column and the antibody is eluted. In one embodiment, purification uses affinity capture on a Protein A column followed by titration. In one embodiment, purification uses affinity capture on a Protein G column followed by HPLC titration. In one embodiment, purification uses affinity capture on an IgE column followed by titration. In one embodiment, purification uses cibacron blue or similar for purification of albumin fusion or conjugated molecules.

[0193] Suitable ion exchange resins for use in the process include Q.FF resin (supplied by GE-Healthcare). This step may be carried out, for example, at a pH of about 8.

[0194] The process may further include an initial capture step using cation exchange chromatography, performed at a pH of about 4-5, such as 4.5. The cation exchange chromatography may use a resin such as CaptoS resin or SP Sepharose FF (supplied by GE-Healthcare). The antibody or fragment may then be eluted from the resin using an ionic salt solution, such as sodium chloride at a concentration of 200 mM. Thus, the chromatography step or steps may optionally include one or more washing steps. The purification process may also include one or more filtration steps, such as a diafiltration step or a HPLC filtration step.

[0195] Thus, in one aspect there is provided a purified anti-IgE antibody or fragment, such as a humanized antibody or fragment, in particular an antibody or fragment according to the invention, that is substantially purified, in particular free or substantially free, from endotoxins and / or host cell proteins or DNA. As used above, purified from is intended to refer to at least 90% purity, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% w / w or more pure. Substantially free of endotoxin is generally intended to refer to an endotoxin content of 1 EU per mg of antibody product or less than 1 EU per mg of product, such as 0.5 or 0.1 EU. Substantially free of host cell protein or DNA is generally intended to refer to a host cell protein and / or DNA content of less than 400 μg or less, such as 400 μg per mg of antibody product or, optionally, 100 μg per mg, in particular less than 20 μg per mg.

[0196] Pharmaceutical Compositions Since the antibodies of the invention are useful in the treatment and / or prevention of pathologies, the invention also provides pharmaceutical or diagnostic compositions comprising the antibodies or antigen-binding agents of the invention together with one or more pharma- ceutically acceptable excipients, diluents, or carriers. Thus, there is provided the use of the antibodies or antigen-binding agents of the invention for the manufacture of a medicament. The compositions will usually be supplied as part of a sterile pharmaceutical composition that typically includes a pharma- ceutically acceptable carrier. The pharmaceutical compositions of the invention may further comprise a pharma- ceutically acceptable excipient.

[0197] The invention also provides a process for the preparation of a pharmaceutical or diagnostic composition comprising adding and mixing an antibody or antigen-binding agent of the invention together with one or more pharma- ceutically acceptable excipients, diluents, or carriers. The antibody or antigen-binding agent may be the sole active ingredient in a pharmaceutical or diagnostic composition or may be associated with other active molecules, including other antibody or non-antibody components, such as steroids or other drug molecules, particularly those whose half-lives are independent of IgE binding.

[0198] The pharmaceutical composition preferably comprises a therapeutically effective amount of the antibody or antigen-binding agent of the present invention. As used herein, the term "therapeutically effective amount" refers to the amount of a therapeutic agent required to treat, ameliorate, or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventive effect. For any disclosed antibody or antigen-binding agent, the therapeutically effective amount can be estimated initially in cell culture assays or animal models, usually rodents, rabbits, dogs, pigs, or primates. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes of administration in humans.

[0199] The exact therapeutically effective amount for a human subject will depend on the severity of the disease state, the subject's overall health, the subject's age, weight and sex, diet, time and frequency of administration, drug combination(s), sensitivity and tolerance / response to therapy. This amount can be determined by routine experimentation and is within the physician's judgment. In general, a therapeutically effective amount will be 0.01 mg / kg to 500 mg / kg, e.g., 0.1 mg / kg to 200 mg / kg, e.g., 100 mg / kg. Pharmaceutical compositions can be conveniently provided in unit dosage forms containing a predetermined amount of the active agent of the invention per dose. Therapeutic doses of antibodies or antigen-binding agents according to the present disclosure do not exhibit obvious toxic effects in vivo. Advantageously, the level of IgE activity in vivo can be maintained at an appropriately reduced level by administration of successive doses of an antibody or binding agent according to the present disclosure. Compositions may be administered individually to a patient or may be administered in combination (eg, simultaneously, sequentially or separately) with other agents, drugs or hormones.

[0200] The pharmaceutical composition may also contain a pharma- ceutically acceptable carrier for administration of the antibody or antigen-binding agent. The carrier should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Pharmaceutically acceptable salts can be used, for example mineral acid salts such as hydrochlorides, hydrobromides, phosphates and sulfates, or organic acid salts such as acetates, propionates, malonates and benzoates.

[0201] Pharmaceutically acceptable carriers in therapeutic compositions may further contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances such as wetting or emulsifying agents or pH buffering substances may be present in such compositions. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions for ingestion by the patient.

[0202] Preferred forms for administration include, for example, forms suitable for parenteral administration by injection or infusion, for example, by bolus injection or continuous infusion. If the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and may contain formulatory agents such as suspending agents, preservatives, stabilizers and / or dispersing agents. Alternatively, the antibody molecule may be in a dry form for reconstitution before use with an appropriate sterile liquid. Once formulated, the compositions of the invention can be administered directly to the subject. The subject being treated can be an animal. However, it is preferred that the compositions are adapted for administration to human subjects.

[0203] The pharmaceutical composition of the present invention can be administered by any number of routes, including but not limited to oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transdermal (see, for example, WO98 / 20734), subcutaneous, intraperitoneal, intranasal, intraintestinal, topical, sublingual, intravaginal or rectal routes.The pharmaceutical composition of the present invention can also be administered using a hypodermic spray.Typically, the therapeutic composition can be prepared as an injectable, either as a liquid solution or suspension.Solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared.

[0204] Direct delivery of the composition will generally be accomplished by injection, subcutaneously (particularly), intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The composition may also be administered to a lesion. Dose treatment may be a single dose schedule or a multiple dose schedule.

[0205] It is understood that the active ingredient in the composition is an antibody molecule. As such, it will be susceptible to degradation in the digestive tract. Thus, when the composition is administered by a route that uses the digestive tract, the composition must contain an agent that protects the antibody from degradation but releases the antibody once it is absorbed from the digestive tract. A thorough discussion of pharma- ceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, NJ 1991).

[0206] Structure-functional properties In one aspect of the invention, the antibody or antigen binding agent binds to free and FcεRI-bound human IgE. When the antibody (or antigen binding agent) of the invention binds to FcεRI-bound human IgE, it stabilizes the conformation of IgE. In such stabilized conformation, IgE has a weaker binding affinity for FcεRI or omalizumab (or a fragment thereof) than in the absence of the antibody or antigen binding agent of the invention, and the FcεRI-bound human IgE dissociates from the FcεRI. Preferably, IgE remains bound to the antibody or antigen binding agent described herein upon dissociation from FcεRI. As shown hereinafter (e.g., in Example 1 and FIG. 2), the antibody or antigen binding agent of the invention binds to IgE in a conformation that is different from the conformation that IgE has when bound to omalizumab.

[0207] Without wishing to be bound by theory, it is believed that the antibodies of the present invention cause IgE to adopt a partially curved conformation (Figure 2C and Figure 5C & D) and are therefore decurved from the free or FcεRI-bound IgE structure. Such decurving impairs the ability of IgE to bind or retain its binding to FcεRI. As shown in the Examples section, it is believed that the antibodies of the present invention compete with a binding site on IgE for binding to FcεRI, so that the antibodies are able to form a complex with IgE bound to FcεRI, and when bound to FcεRI, they change the structure of IgE and dissociate it from the FcεRI. It is believed that antibodies according to the present invention having these properties: a. a heavy chain variable region comprising SEQ ID NO:1; i. SEQ ID NO: 109; or ii. SEQ ID NO: 113; or iii. SEQ ID NO: 121; or iv. SEQ ID NO: 132; or v. SEQ ID NO: 139 or a light chain variable region comprising b. SEQ ID NO:5; i. S77 and S79 are replaced with Q, SEQ ID NO:24; ii. SEQ ID NO: 117; or iii. SEQ ID NO: 125; or iv. SEQ ID NO: 136; or v. SEQ ID NO: 143 and

[0208] An anti-IgE antibody or antigen-binding agent with such properties contacts or is specific for an epitope comprising residues T373, W374, S375, R376, A377, S378, G379, P381, Q417, C418, R419, P426, R427, A428 of the Cε3 domain and residues D278 and T281 of the Cε2 domain of human IgE, with reference to SEQ ID NO: 108.

[0209] The antibodies of the present invention have methionine residues at several positions. Oxidation of methionine residues is among the most common proteolytic pathways. Antibodies of the present invention in which methionine residues have been introduced at positions S64 and S71 with reference to SEQ ID NO:20 can undergo complete oxidation without affecting the ability of the antibody to promote dissociation of the IgE-Fc:sFcRIα complex.

[0210] Accordingly, the present invention also provides an anti-IgE antibody or antigen-binding agent comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:1, and a light chain variable region comprising CDR-L2 and FW-L3 having the amino acid sequence of SEQ ID NO:20, except that CDR-L2 has the amino acid sequence of SEQ ID NO:50 and FW-L3 has the amino acid sequence of SEQ ID NO:131 or 138, and in which the methionine residues at positions 64 and / or 71 have been oxidized with reference to SEQ ID NO:20.

[0211] The invention also provides an anti-IgE antibody or antigen-binding agent comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:1 and a light chain variable region having the amino acid sequence of SEQ ID NO:132 or 139, wherein the methionine residues at positions 64 and / or 71, with reference to SEQ ID NO:132 or 139, have been oxidized. Additional methionine residues in the antibodies of the invention may be oxidized without affecting the ability of the antibody to promote dissociation of the IgE-Fc:sFcRIα complex.The invention will now be further described, by way of example, with reference to the embodiments illustrated in the accompanying drawings.

[0212] example (Example 1) Structure of a mutant of the therapeutic anti-IgE antibody omalizumab bound to IgE-Fc reveals its mechanism of action summary Immunoglobulin E and its interaction with the receptors FcεRI and CD23 play a central role in allergic diseases. Omalizumab, a clinically approved therapeutic antibody, inhibits the interaction of IgE with FcεRI, preventing activation of mast cells and basophils, and blocks IgE binding to CD23. We have solved the crystal structure of a 2:1 complex of omalizumab-derived Fab and IgE-Fc, with one Fab bound to each Cε3 domain (but only one of the Fabs bound to the Cε2 domain). Free IgE-Fc is primarily sharply curved in solution, but in the complex, it is only partially curved, making interaction with FcεRI impossible; CD23 binding is sterically hindered due to the overlap of binding sites on each Cε3 domain. Solution-state interaction analysis demonstrates an orthosteric and allosteric basis for the inhibition of both receptor interactions and, together with the structures, demonstrate how omalizumab (particularly the described omalizumab variants) can exploit the intrinsic mechanical and allosteric capabilities of IgE to promote dissociation of receptor-bound IgE from FcεRI.

[0213] Introduction Immunoglobulin E (IgE) antibodies play an important role in allergic diseases, binding to allergens via their Fab arms and exerting their effector functions by binding to receptors for their Fc regions. 1The two major IgE receptors are FcεRI and CD23 / FcεRII, commonly referred to as the high-affinity and low-affinity receptors, respectively. On mast cells and basophils, IgE binds tightly (approximately 10–20 μg / ml) to such an extent that such cells are sensitized with prebound IgE and require only the presence of allergen to crosslink the IgE / FcεRI complex and elicit an immediate response. -10 M -1 K D ), which binds to FcεRI. CD23 is a homotrimer, thus resulting in the intrinsically low affinity (approximately 10 -7 M -1 K D ) can be enhanced when binding to aggregated IgE in immune complexes by an avidity effect nearly identical to that of FcεRI for IgE. 2 CD23 expressed on B cells is involved in the regulation of IgE, and its expression on airway and intestinal epithelial cells mediates transcytosis of IgE / allergen complexes. 1、2 Both FcεRI and CD23 are also expressed on a range of antigen-presenting cells. Thus, IgE-receptor interactions are involved in multiple aspects of the allergic response, and IgE is a long-standing target for therapeutic intervention. 3 .

[0214] The Fc region of IgE contains a disulfide-linked dimer of three domains: Cε2, Cε3 and Cε4. Initial FRET studies of chimeric IgE 4、5 , and IgE-Fc X-ray solution scattering study 6 showed a compact curved structure, and the crystal structure of IgE-Fc later showed a sharply asymmetrically curved conformation, with the (Cε2)2 domain pair folded back on the Cε3 and Cε4 domains. 7 The curvature, defined as the angle between the partial two-fold axis of the (Cε2)2 domain pair and that of Fcε3-4 (the region containing only the Cε3 and Cε4 domains), was found to be even sharper (from 62° to 54°) in the crystal structure of IgE-Fc bound to sFcεRIα, the soluble extracellular domain of the IgE-binding α-chain of the receptor.8 Recent FRET studies using N- and C-terminally labeled IgE-Fc confirmed this enhanced bending upon sFcεRIα binding. 9 .

[0215] The FcεRI binding site spans both Cε3 domains in the Cε2-proximal region, 8、10 The Cε2 domain is not directly involved; engagement of both chains results in a 1:1 binding stoichiometry. In contrast, two CD23 molecules bind IgE-Fc, one in each chain and the other at the Cε4-proximal end of the Cε3 domain. 11、12、13 CD23 binding also induces a conformational change in IgE-Fc. 14 , does not significantly affect curvature 9 However, the relatively "closed" conformation of the Cε3 domains complexed with the soluble head domain of CD23 (sCD23) compared to free IgE-Fc is not compatible with the more "open" conformation of these domains required for FcεRI binding, which may partially explain the mutual exclusion of FcεRI and CD23 binding. 11、12 , other factors such as partial conformational changes and alterations in conformational dynamics. 15 may also contribute to allosteric communication between the two receptor binding sites. 2 .

[0216] A more extreme degree of IgE-Fc flexibility has been discovered recently through studies of its complex with anti-IgE-Fc Fab, termed aεFab. 16 The two aεFab molecules bound IgE-Fc in a symmetric manner, one on each Cε3 domain capturing a fully extended conformation in which the partial 2-fold axes of the (Cε2)2 domain and the Fcε3-4 region are substantially identical. Analysis of the complex formation in solution, together with molecular dynamics simulations of free IgE-Fc, suggested that the (Cε2)2 domain pair could be "flipped" from one side of the Fcε3-4 region of the other. 16This IgE-Fc conformation stabilized by anti-IgE antibodies is incompatible with FcεRI binding, explaining its inhibitory activity.

[0217] Omalizumab is an anti-IgE monoclonal IgG1 antibody (Xolair®, Novartis) approved for therapeutic use. 17 It binds to free IgE and inhibits both FcεRI and CD23 binding; the binding site has been mapped to the Cε3 domain by peptide inhibition and molecular modeling. 18、19 The mechanism of action is unclear. However, binding to FRET-labeled IgE-Fc results in a small degree of decurling. 9 and thus indicating allosteric rather than direct inhibition.

[0218] Recently, a type of inhibitor that actively disrupts preformed IgE / FcεRI complexes has been discovered: Designed Ankyrin Repeat Proteins (DARPins) were found to bind to the Cε3 domain of receptor-bound IgE and promote its dissociation from FcεRI. 20 The crystal structure of a 2:1 complex of DARPin E2_79 constrained by an engineered disulfide bond with an Fcε3-4 molecule revealed the nature and location of the binding site, but its mechanism of action remained unclear. It was subsequently reported that omalizumab can similarly facilitate dissociation of IgE bound to FcεRI, but only at very high concentrations, substantially higher than those achieved in therapeutic use. 21、22 .

[0219] We report here the crystal structure of a complex between IgE-Fc and a new antibody fragment, a Fab derived from Omalizumab (Omalizumab Fab3), that contains three point mutations distal to the antigen (IgE-Fc) binding complementarity determining regions (CDRs). The mutations are S81R, Q83R and L158P, with reference to SEQ ID NO: 125 (or S77R, Q79R and L154P, with reference to SEQ ID NO: 129). The structure of the complex shows the mechanism of action of Omalizumab, and solution studies demonstrate that this mechanism exploits the intrinsic dynamics of IgE.

[0220] result Despite extensive efforts, crystallization studies on IgE-Fc complexed with omalizumab Fab have resulted in selective crystallization of only the Fab fragment. Others have reported similar failures to crystallize this complex. 23 Therefore, we designed a novel antibody, a Fab derived from Omalizumab, with three point mutations, two in the VL domain framework regions (Ser81Arg, Gln83Arg) and one in the Cκ domain (Leu158Pro) (SEQ ID NO: 125, PDB numbering) (Figure 1), with the aim of disrupting the favorable crystal contacts observed in the Omalizumab Fab crystal structure (results reported elsewhere). We call this Omalizumab-derived Fab "Omalizumab Fab3".

[0221] Overall structure of the IgE-Fc / omalizumab Fab3 complex The present inventors have determined the crystal structure of the complex between IgE-Fc and omalizumab Fab3 at a resolution of 3.7 Å (FIG. 2A). 1 and Fab 2 ) bind to an asymmetric, partially curved IgE-Fc molecule in which each Fab engages one Cε3 domain (Figure 2B & C). 1 engages with the Cε3 domain of IgE-Fc chain B, whereas Fab 2 Fab engages with the Cε3 domain of IgE-Fc chain A. Due to the partially curved conformation of IgE-Fc in the complex,2 The light chain of also forms a small interaction with the Cε2 domain from IgE-Fc chain B (see later in this example for details regarding this interaction).

[0222] The interface between IgE-Fc and omalizumab Fab3 Each omalizumab Fab3 molecule engages one end of the exposed face of the Cε3 domain (strands C, C', F and G, and the base of the FcεRI receptor-binding FG loop). Both the heavy and light chains of omalizumab Fab3 are involved, the former at approximately 715 Å. 2 contributes approximately 60% to the interfacial area (Figures 2 & 3).

[0223] The Omalizumab Fab3 heavy chain (SEQ ID NO:5) contacts, which differ slightly between the two interfaces, can be summarized as follows: Gly32 and Tyr33 (CDRH1) form van der Waals interactions with Ala377 and Ser378 (Cε3) (IgE-Fc sequence shown in SEQ ID NO:108 and FIG. 16), whereas Tyr54 (CDRH2) contacts Gly379-Pro381 (Cε3). The CDRH3 residues contribute the largest contact area and undergo significant conformational changes upon complex formation when compared to the unbound Fab structure (unpublished results, 19、23 ). CDRH3 residues Ser100, His101, Tyr102, and Trp106 all form van der Waals interactions with Cε3 domain residues, including Ser375-Gly379, Gln417, and Arg419 (Cε3). However, the most surprising feature of this part of the interface is its interaction with Phe103 (CDRH3). Phe103 is largely buried in a pocket made by Thr373, the backbone of Trp374, Ser375, Gln417, and Arg419 (Cε3), and forms a cation / π-stacking interaction with Arg419 (Figure 3).

[0224] Arg419 (Cε3) also plays an important role in the interaction with the Omalizumab Fab3 light chain (SEQ ID NO: 125) (Figure 3). Arg419 (Cε3) is within hydrogen-bonding distance of Tyr31 (CDRL1) and Asp32 (CDRL1) main chain carbonyl oxygen atoms, and also contacts Asp32, Asp34, and Tyr36 side chains (forming hydrogen bonds with the hydroxyl group of Tyr36). Asp32 also forms van der Waals interactions with Thr373 and Thr421 (Cε3). In contrast, only two CDRL2 residues contribute to the interface: Tyr53 (CDRL2) contacts Gln417 (Cε3), and both Tyr53 and Tyr57 form van der Waals interactions with Met430 (Cε3); Tyr57 also forms hydrogen bonds with the Met430 backbone. With regard to heavy chain interactions, Fab 1 and Fab 2 There are slight differences in the light chain contacts for

[0225] CDR contact residues in omalizumab Fab3 numbering in the format (pdb / Kabat / Chothia) Heavy chain sequence: SEQ ID NO:5; Light chain sequence: SEQ ID NO:125 CDRH1:Ser(31 / 31 / 31), Gly(32 / 32 / 31a), Tyr(33 / 33 / 32) CDRH2:Thy(54 / 53 / 53) CDRH3:Ser(100 / 96 / 96), His(101 / 97 / 97), Tyr(102 / 98 / 98), Phe(103 / 99 / 99), Trp(106 / 101B / 101B) CDRL1:Asp(30 / 27C / 30), Tyr(31 / 27D / 30A), Asp(32 / 28 / 30B), Gly(33 / 29 / 30C), Asp(34 / 30 / 30D), Tyr(36 / 32 / 32) CDRL2:Tyr(53 / 49 / 49), Ser(56 / 52 / 52), Tyr(57 / 53 / 53), Ser(60 / 56 / 56) CDRL1 and CDRH3 contain most of the residues involved in the interaction and therefore characterize how Omalizumab binds and orients itself to IgE-Fc. CDRL3 is not involved in binding to IgE-Fc.

[0226] Comparison of the omalizumab Fab3 interface with other anti-IgE complexes Binding sites on the Cε3 domain of omalizumab Fab3 and the recently described DARPin E2_79 20 overlap (Fig. 4), respectively, about 715 Å. 2 and about 753 Å 2 The Cε3 domain residues shared between the two interfaces include Ser375-Gly379, Gln417, Arg419, Arg427 and Met430, with the omalizumab Fab3 making closer contacts with the receptor-binding Cε3 FG loop, whereas the DARPin E2_79 interface extends in the opposite direction to include the Cε3-4 domain linker.

[0227] The overlapping binding site of omalizumab Fab3 with DARPin E2_79 is strikingly different from the interface recently described for omalizumab Fab3, which captured IgE-Fc in a fully extended conformation 16 (Figure 5). The interface area of ​​omalizumab Fab3 is approximately 1400 Å. 2 Not only is the Fab3 of omalizumab approximately twice as abundant as that of DARPin E2_79, but Fab3 of omalizumab also engages IgE-Fc at a site centered on Arg393 in Cε3 and also makes contacts with residues in the Cε2 domain and the Cε2-Cε3 linker. 16 The crystal structure of another anti-IgE antibody Fab, MEDI4212, in a 2:1 complex with Fcε3-4 reveals additional sites for antibody engagement within the Cε3 domain, one containing an N-linked oligosaccharide moiety at Asn394. 24 .

[0228] IgE-Fc adopts a partially curved conformation when bound to omalizumab Fab3 IgE-Fc is primarily curved in solution. 5、6、9、25、26、27、28 The crystal structure of free IgE-Fc showed a highly curved (62°), asymmetric conformation in which the (Cε2)2 domain pair folds back over the Cε3 and Cε4 domains (Figure 5A & B), with the Cε2 domain of one chain (chain B) in contact with the Cε4 domain of the other chain (chain A). 7、8 IgE-Fc becomes even more curved (54°) upon FcεRIα engagement. 8、9 The associated conformational change involves the rotation of the Cε3 domain of chain A, together with the (Cε2)2 domain pair, as a rigid unit, distal to the Cε3 domain of chain B. 8 .

[0229] Omalizumab Fab3 complex in which IgE-Fc adopts a fully extended, linear conformation 16 In contrast, IgE-Fc was not shown to be responsive to omalizumab, as earlier FRET studies showed that omalizumab caused IgE-Fc decurving. 9 Consistent with this, omalizumab adopts a partially curved conformation in the Fab3 complex (Figure 2C and Figure 5C & D). 1 The site where Fab binds is exposed in free, highly curved IgE-Fc. 2 Further decurving of IgE-Fc by just over 90° is required to make accessible the site occupied by Cε3. This decurving of IgE-Fc in the omalizumab Fab3 complex is associated with the opening of both Cε3 domains, making the Fcε3-4 region nearly symmetrical (FIG. 2B). The (Cε2)2 domain pair is located between the Cε3 and Cε4 domains from each chain and is no longer as closely associated with the Cε3 domain from chain A.

[0230] Recent molecular dynamics simulations investigating the decurving of IgE-Fc into an extended structure showed that the highly curved conformation observed in the crystal structure of free IgE-Fc occupies the lowest energy bowl, but another distinct and well-defined energy bowl is observed, corresponding to the partially curved IgE-Fc conformation.16 The partially curved conformation adopted by IgE-Fc in the omalizumab Fab3 / IgE-Fc complex occupies this particular energy bowl (Figure 6).

[0231] The Cε3 domain adopts a significantly open conformation in the omalizumab Fab3 / IgE-Fc complex In the crystal structure of IgE-Fc and the Fcε3-4 subfragment, the Cε3 domain binds to its two major receptors, FcεRI and CD23. 8、11、12、14 adopt a range of different orientations, a property that is associated with allosteric regulation of IgE binding to 7、8、10、11、13、14、16、24、29 Both the distance between the Cε3 domains and their position relative to the Cε4 domain have been used to describe the various conformations observed for the Fcε3-4 region. 29 (A complete description of these measurements is provided later in this example.) In the omalizumab Fab3 / IgE-Fc complex, the Cε3 domains are positioned further away from each other and from the Cε4 domain than in any other crystal structure containing IgE-Fc or Fcε3-4, thus adopting the most open conformation observed so far (FIG. 5); this conformation is significantly more open than that for IgE-Fc bound to FcεRI (FIG. 7).

[0232] Effect of omalizumab Fab3 on FcεRI and CD23 receptor binding Omalizumab not only inhibits the interaction of IgE-Fc with FcεRI, but also with CD23. 30 Consistent with the latter, a comparison of omalizumab Fab3 / IgE-Fc and CD23 / Fcε3-4 complexes 11 indicates a clash between omalizumab Fab3 and CD23 at both sites of CD23 engagement on Fcε3-4. Furthermore, Cε3 domain residues Arg376, Ser378, and Lys380 are involved in both omalizumab Fab3 and CD23 binding. 11、31 .

[0233] In contrast to CD23 binding to IgE, FcεRIα binds across both Cε3 domains. However, in the omalizumab Fab3 / IgE-Fc complex, the Cε3 domain adopts a conformation that is too open to allow simultaneous engagement of both chains (Figure 8). Furthermore, when the omalizumab Fab3 / IgE-Fc and sFcεRIα / IgE-Fc complexes are superimposed on their respective Cε3 domains in turn, potential steric clashes arise in both cases. When superimposed on the Cε3 domain of chain A, the omalizumab Fab3 Fab 2 The Cε2-Cε3 linker from the omalizumab Fab3 complex may clash with the (Cε2)2 domain pair in the FcεRIα complex, and the Cε2-Cε3 linker from the omalizumab Fab3 complex may clash with FcεRIα. When superimposed on the Cε3 domain of chain B, the omalizumab Fab3 Fab 1 and the Cε2-Cε3 linker (of the omalizumab Fab3 complex) may clash with FcεRIα, but 1 and FcεRIα binding sites do not actually overlap.

[0234] However, the omalizumab Fab3 CDRL1 residues are located immediately adjacent to the Cε3 domain FG loop that binds FcεRIα. This loop in chain B contributes to a hydrophobic "proline sandwich" interaction in which Pro426 in Cε3 wraps between two tryptophan residues of FcεRIα. Asp32 (CDRL1) contacts Thr421, Gly33 (CDRL1) contacts Pro426, Arg427, and Ala428, and Asp34 (CDRL1) contacts Arg427 and Ala428. These interactions alter the position of the Cε3 domain FG loop, further impairing IgE binding to FcεRI. Recently, binding of omalizumab to FcεRIα-bound IgE has been reported. 21、32 However, omalizumab inhibits sFcεRIα 8Based on the static crystal structure of IgE-Fc in complex with omalizumab Fab3, it is difficult to see how it engages with FcεRI-bound IgE. Therefore, we tested the binding of omalizumab Fab3 to IgE-Fc and characterized the interaction of omalizumab Fab3 with the IgE-Fc / FcεRI complex. Our results provide insight into the mechanism of action of omalizumab.

[0235] Interaction of omalizumab Fab3 with IgE-Fc in solution We characterized the IgE-Fc / Omalizumab Fab3 interaction in two different ways: by directly immobilizing Omalizumab Fab3 on a surface and binding to IgE-Fc, or by binding Omalizumab Fab3 to His-tagged captured IgE-Fc on an SPR sensor surface. C-terminal His-tagged IgE-Fc constructs were captured using an anti-His tag antibody (GE Healthcare) and the binding properties of Omalizumab Fab3, intact Omalizumab and Omalizumab Fab were compared. Not surprisingly, in competitive binding experiments, all three molecules competed for the same binding site and showed broadly similar binding affinities (data not shown). The Omalizumab Fab3 construct shows slightly higher affinity compared to Omalizumab Fab3 and intact Omalizumab (Figure 8A-C). Consistent with the crystal structure, two omalizumab Fab3 molecules bind to IgE-Fc: binding is apparently biphasic, with a high affinity (~1 nM) interaction observed at low ligand concentrations and a second (weaker) binding site (~30 nM) observed at higher concentrations (Figure 9A).

[0236] Sandwich SPR experiments allow the two IgE-Fc / Omalizumab Fab3 binding sites to be characterized separately. Using this technique, Omalizumab Fab3 was covalently immobilized onto a sensor surface and IgE-Fc was flowed over this surface. At low concentrations, under these conditions, the high affinity site dominates the interaction and the binding curve can be described by monophasic interaction kinetics (K of approximately 1 nM). D , about 1.2x10 6M -1 s -1 k on , about 8x10 -4 s -1 k off This 1:1 IgE-Fc / omalizumab Fab3 complex captured on the SPR biosensor surface was then used to detect a second IgE-Fc complex with binding significantly weaker than the first (K of approximately 30 nM). D , about 2x10 5 M -1 s -1 k on , about 6x10 -3 s -1 k off ), and binding of a second omalizumab Fab3 molecule can be measured (Figure 9B).

[0237] Competition between omalizumab Fab3 and FcεRIα binding site and formation of omalizumab Fab3 / IgE-Fc / FcεRIα complex We next investigated the ability of omalizumab Fab3 to affect the interaction of IgE-Fc with FcεRIα. In solution competitive binding experiments, increasing concentrations of omalizumab Fab3 inhibited the binding of IgE-Fc to FcεRIα (Figure 8D). Mechanistically, omalizumab Fab3 reduces the number of available binding sites (B max ) and the apparent K of the IgE-Fc / FcεRIα interaction. D This is characteristic of a mixed inhibition mechanism. 33 . B max A decrease in A value indicates an allosteric inhibition process, and a decrease in the apparent affinity of the interaction is most commonly associated with direct competition for a shared binding site (i.e., orthosteric inhibition), but can also be seen for some allosteric inhibitors. Considering the binding site observed in the crystal structure, it is likely that omalizumab Fab3 inhibits IgE-Fc binding to FcεRI using both orthosteric and allosteric mechanisms.

[0238] Competition between omalizumab and FcεRIα binding sites has been described in many publications, but has always been interpreted as direct competition for the same (or overlapping) binding site. This interpretation has often been used to explain why omalizumab cannot bind to IgE-FcεRI complexes on cells. However, we observed that omalizumab Fab3 can bind with high affinity to IgE-Fc prebound to FcεRIα (Figure 9C, inset). The existence of omalizumab / IgE-Fc / FcεRI complexes has been shown to be consistent with other studies. 21、32 Although implied by, this complex has not been experimentally characterized previously. The data show that binding of IgE-Fc to FcεRIα did not significantly change the affinity of omalizumab Fab3 for IgE-Fc, but it did markedly change the number of available binding sites for omalizumab Fab3 in the population of IgE-Fc molecules bound to FcεRIα. We have determined that the K D and B. max We compared the binding values ​​to those captured by sFcεRIα and found that IgE-Fc bound to FcεRIα had less than 10% of the omalizumab Fab3 binding sites compared to IgE-Fc captured with a His tag, which showed binding levels consistent with a 2:1 stoichiometry, as expected (Figure 9C). Thus, the commonly assumed assumption that omalizumab does not bind to IgE bound to mast cells, because the FcεRIα and omalizumab binding sites overlap, is not the case. Instead, FcεRIα acts allosterically on IgE-Fc, altering the dynamic equilibrium of different IgE-Fc conformations. 16 , resulting in a substantial reduction in the number of omalizumab binding sites in the population of IgE-Fc molecules bound to FcεRIα.

[0239] Mechanism of enhanced omalizumab Fab3-mediated dissociation of IgE-Fc / FcεRIα complexes Kim et al. 20reported that DARPin E2_79 can promote the disassembly of preformed IgE-FcεRI complexes. To follow up on this observation, Eggel et al. 21 showed later that omalizumab can also promote dissociation of IgE from FcεRI. Similar to these observations, we found that when omalizumab Fab3 binds to the IgE-Fc / FcεRIα complex, it promotes dissociation of IgE-Fc from FcεRIα (Figure 9D), and that omalizumab Fab3 does this more efficiently than omalizumab Fab3 and much more efficiently than intact omalizumab (Figure 10E). One Fab engages the IgE-Fc / FcεRIα complex but does not promote dissociation of IgE-Fc from FcεRIα. Surprisingly, promotion of dissociation appears to occur only after occupation of the second binding site (i.e., the low affinity site). The (omalizumab Fab3)2 / IgE-Fc / FcεRIα tetrad complex must alter the energy landscape of IgE-Fc in such a way as to significantly reduce the energy barrier for IgE-Fc / FcεRIα dissociation and result in rapid dissociation of this otherwise highly stable complex.

[0240] Details of the interaction of omalizumab Fab3 light chain with the Cε2 domain In the omalizumab Fab3 / IgE-Fc complex, one Cε2 domain is located approximately 260 Å apart with two mutated residues (Ser81Arg and Gln83Arg). 2 The small interaction (approximately 715 Å between omalizumab Fab3 and Cε3 domains) 2 (compared to the average interaction area of ​​100–150 nm). There is no contact between Pro158 and IgE-Fc.

[0241] Fab 2The Arg81 side chain from the light chain (one of the mutated residues in Omalizumab Fab3; (SEQ ID NO: 125, PDB numbering)) wraps against Val277 and Asp278 from the Cε2 domain from chain B (SEQ ID NO: 108). Ser80 (Omalizumab Fab3) wraps against Asp278, Leu279 and Thr281 (Cε2 domain), whereas Ser64 (Omalizumab Fab3) wraps against Asp276 and Asp278. Ser64 and Ser80 are identical in Omalizumab and Omalizumab Fab3.

[0242] In the omalizumab Fab3 / IgE-Fc complex, Arg83 (one of the mutated residues in omalizumab Fab3) does not apparently contact the Cε2 domain due to a disorder in the Asp278 (Cε2 domain) side chain. However, if the Asp278 side chain is correctly aligned, a hydrogen bond or salt bridge can form between Arg83 and Asp278.

[0243] Crystallographically determined contacts between the omalizumab Fab3 / IgE-Fc complex Contacts between an antibody and an antigen within 4 Å in the crystal structure typically represent the epitope / paratope interface. The IgE-Fc residues within 4 Å of the Omalizumab Fab3 heavy and light chain CDRs define the following epitope: T373, W374, S375, R376, A377, S378, G379, P381, Q417, C418, R419, P426, R427, A428 (on chain A).

[0244] Additionally, IgE-Fc residues within 4 Å of the omalizumab Fab3 light chain FR1 and FR3 residues map the epitope to D278, extending to T281 (on chain B - Cε2 domain) [which contacts R18, S64, S80 and R81 as well as R83 of the antibody as shown in the molecular dynamics simulation described in Example 5]. Antibody and antigen contacts within 5 Å in the crystal structure are also useful in defining the antibody / antigen interface. Additional IgE-Fc residues within 5 Å of the Omalizumab Fab3 heavy and light chain CDRs are K380, M430 (on chain A).

[0245] Additionally, additional IgE-Fc residues within 5 Å of the omalizumab Fab3 light chain FR1 and FR3 residues are D276, V277, L279, S280, A282 (on chain B-Cε2 domain) [additionally contacting G16 of FR1 and R65 of FR3].

[0246] Analysis of the orientation of the Cε3 domain in the omalizumab Fab3 / IgE-Fc complex In one method for analyzing the position of the Cε3 domain relative to the Cε4 domain, the interatomic distance between the Asn394 Cα atom from the Cε3 domain from one chain and the Lys497 Cα atom from the Cε4 domain of the other chain is used to describe the "openness" of the Cε3 domain. 29 The atomic distances between Val336 Cα are used to describe the "wobble," or how close the Cε3 domains are to each other. 29 .

[0247] For IgE-Fc bound to FcεRI, in which the Cε3 domain adopts an open conformation, and for Fcε3-4 bound to FcεRI, the “openness” values ​​range from 23.5 to 28.4 Å, whereas the “sway” values ​​average 23.3 Å. 8、10 The corresponding values ​​for the Omalizumab Fab3 / IgE-Fc complex are, on average, 29.5 Å for "openness" and 29.4 Å for "swaying". In the Omalizumab Fab3 complex, the Cε3 domains adopt the most open conformation (furthest apart from each other) as described so far.

[0248] Consideration We report the structure at 3.7 Å resolution of a complex between IgE-Fc and a Fab fragment derived from the therapeutic anti-IgE antibody omalizumab; we call this Fab fragment omalizumab Fab3, which contains three point mutations in the framework region distal to the antigen-binding site. The structure shows two omalizumab Fab3 molecules complexed with IgE-Fc, one bound to each of the two Cε3 domains (but only one Fab bound to the Cε2 domain), providing an explanation for omalizumab's ability to inhibit IgE binding to both FcεRI and CD23. IgE-Fc is also found to adopt a partially curved conformation in the omalizumab Fab3 complex, consistent with our earlier studies using FRET-labeled IgE-Fc, which showed only slight decurling compared to free IgE-Fc. 9 .

[0249] IgE-Fc is primarily curved in solution. 5、6、9、25、26、27、28 In the crystal structure of free IgE-Fc, the (Cε2)2 domain pair folds back onto the Cε3 and Cε4 domains. 7、8 Recently, our understanding of the conformational flexibility of IgE-Fc was greatly enhanced when we solved the structure of the fully extended conformation captured in complex with anti-IgE-Fc Fab (aεFab). 16 Molecular dynamics simulations investigating the decurving of IgE-Fc from a highly curved to an extended conformation showed an energy bowl corresponding to a partially curved conformation (Figure 6). The omalizumab Fab3 / IgE-Fc complex reported here, in which the curvature between the (Cε2)2 domain pair and the Fcε3-4 domain is approximately 90°, corresponds to a different energy bowl in this simulation. 16 Interestingly, the location of the omalizumab Fab3 binding site does not preclude further decurling into a fully extended conformation, and thus IgE-Fc can undergo substantial conformational changes even when complexed with omalizumab.

[0250] In addition to the curvature of the (Cε2)2 domain pair relative to the Cε3 and Cε4 domains, structures of various IgE-Fc, Fcε3-4, and receptor complexes have demonstrated that the Cε3 domain can adopt a range of relative orientations, from closed to open. 7、8、10、11、13、14、16、24、29 Opening and closing of the Cε3 domain contributes to the allosteric regulation of receptor binding in IgE-Fc 11、12 In the CD23 complex, they are relatively closed 11、13、14 However, in the FcεRI complex, they are more open. 8、10 Comparison of the structures of CD23 / Fcε3-4 and the omalizumab Fab3 / IgE-Fc complex indicates that the CD23 and omalizumab sites overlap, and competitive binding experiments indicate that the inhibition of IgE binding to CD23 by omalizumab is directly orthosteric.

[0251] However, inhibition of FcεRI binding is more complex. In the omalizumab Fab3 complex, the Cε3 domain adopts a more open conformation than seen in any previous structure, and so likely that the two subsites of interaction between IgE-Fc and FcεRI, one each involving a Cε3 domain, cannot be simultaneously engaged. Another contribution to this inhibition is the binding of omalizumab Fab3 (Fab 1 ) molecules and may directly affect the conformation of contact residues of FcεRIα. Finally, even though the omalizumab Fab3 and FcεRIα binding epitopes on IgE-Fc do not strictly overlap, there is a possibility of steric clashes when the two bind simultaneously. Thus, the crystal structure suggests that the inhibitory mechanism of omalizumab is primarily allosteric, but also has a possible orthosteric component.

[0252] SPR studies allowed us to evaluate the kinetics and affinity of the two omalizumab Fab3 binding sites. The two affinities are significantly different, with K values ​​of ∼1 nM and ∼30 nM. DThe former exhibits a faster binding rate constant (approximately 2x10 5 M -1 s -1 Compared to about 2x10 6 M -1 s -1 k on ) and a slightly slower dissociation rate constant (approximately 6x10 -3 s -1 Compared to about 2x10 -3 s -1 The higher affinity interaction is associated with the Fab fragment having unhindered access to the curved IgE-Fc molecule. 1 Although the lower affinity and slower binding kinetics correspond to the binding of Fab 2 Although the inventors cannot be certain on this point.

[0253] Further SPR experiments to investigate the mechanism of inhibition of IgE-Fc binding to FcεRIα by omalizumab Fab3 revealed a decrease in the number of available sites for omalizumab Fab3 on IgE-Fc when complexed with FcεRIα (B max Although inhibition of IgE binding to FcεRI by omalizumab has frequently been interpreted in terms of direct competition for overlapping sites, it has been reported that omalizumab can bind to receptor-bound IgE. 21、32 We have directly demonstrated here the ability of omalizumab Fab3 to bind IgE-Fc when it is already bound to FcεRIα to form a trimolecular complex. The effect of pre-binding IgE-Fc to FcεRIα is to reduce the number of omalizumab Fab3 binding sites on IgE-Fc to less than 10% of those available on free IgE-Fc; this effect can be attributed solely to allosteric regulation.

[0254] The nature of the interaction of omalizumab Fab3 with the IgE-Fc / FcεRI complex provides insight into the mechanism of dissociation promotion, a phenomenon first reported for DAPRin and then for omalizumab. 20、21However, the latter at concentrations substantially higher than those used therapeutically. 22 and is shown here for the Omalizumab Fab fragment. We further demonstrate that dissociation occurs only after the initial binding of a second (low affinity) Omalizumab Fab3 molecule. In other words, the tetrad complex -(Omalizumab Fab3)2 / IgE-Fc / FcεRIα must be formed for significant enhancement of dissociation to occur.

[0255] Based on our observations with omalizumab Fab3, IgE-Fc and sFcεRIα, we postulate the following mechanism occurring with omalizumab, IgE and FcεRI: IgE binds to FcεRI and under these conditions a small population of these bound IgE molecules adopt a conformation to which an omalizumab molecule can bind; when a second omalizumab molecule binds to form a tetrameric complex, the energy landscape of IgE changes such that its interaction with FcεRI is destabilized, leading to rapid dissociation of IgE from FcεRI. The key to understanding this mechanism is the complexity of the energy landscape for IgE and the understanding of the different conformational states that exist in dynamic equilibrium.

[0256] The inhibitory activity of omalizumab is thought to exploit the inherent flexibility of IgE and the dynamics of the IgE / FcεRI complex, at least for the facilitating process of dissociation. IgE has several unusual structural features compared to other antibody isotypes, including the presence of a Cε2 domain and the unique, conformationally dynamic, molten globule-like features of the Cε3 domain. 34 Together, these properties create an allosteric communication pathway that prevents simultaneous engagement of CD23 and FcεRI; this is essential to avoid allergen-independent mast cell activation by cross-linking of FcεRI-bound IgE by trimeric CD23 molecules. 12 Other functional advantages related to IgE dynamics have been proposed for the membrane-bound IgE B cell receptor. 16The observation that omalizumab does not use the expected orthosteric mechanism for inhibition of IgE / FcεRI interactions indicates that it also exploits the unusual mechanical properties of IgE, both in its capacity as a blocking antibody and in its ability to avoid cross-linking of IgE bound to mast cells. Finally, omalizumab is able to inhibit IgE at higher concentrations than those used therapeutically by using the allosteric and inherent flexibility of IgE, even when in complex with its receptor. 21 Nevertheless, it is possible to actively dissociate IgE from FcεRI.

[0257] method Cloning, protein expression and purification. Omalizumab human IgG1 Fab and Omalizumab Fab3 were 16 The IgE-Fc was cloned, expressed and purified as described previously. 35 The IgE-Fc was as set forth in SEQ ID NO: 108 (V224-K547 as set forth in Dorrington & Bennich (1978) Immunol. Rev. 41:3-25, but with the following mutations inserted into IgE-Fc to simplify the glycosylation pattern: N265Q & N371Q). Omalizumab was purchased from Novartis Europharm Limited. The 2:1 Omalizumab Fab3 / IgE-Fc complex was purified by size exclusion chromatography, eluted in 25 mM Tris-HCl pH 7.5, 20 mM NaCl and 0.05% (w / v) NaN3 and concentrated to 23 mg / mL.

[0258] Surface Plasmon Resonance. SPR experiments were performed on a Biacore T200 instrument (GE Healthcare). Specific surfaces were prepared by either covalently coupling proteins using a coupling density of less than 300 resonance units using the amine coupling protocol (GE Healthcare) or by capturing His-tagged proteins using an anti-His sensor surface. To capture His-tagged ligands, anti-His tag monoclonal antibodies were used and immobilized according to the manufacturer's instructions (Biacore His Capture Kit, GE Healthcare). In binding experiments, binding times of 180-240 s were typically used and dissociated components were monitored for at least 500 s. Binding was performed at 25 μL / min in a running buffer of 20 mM HEPES pH 7.4, 150 mM NaCl, and 0.005% (v / v) surfactant P-20 (GE Healthcare). -1 Injections were performed at a flow rate of 100 s. Most experimental measurements were performed at 25°C; some sandwich binding experiments were performed at 5°C to minimize accelerated dissociation phenomena. Standard double-referencing data subtraction methods were used. 36 ,Dynamic fitting was performed using Origin software (OriginLab).

[0259] TR-FRET. IgE-Fc was labeled with a donor fluorophore by reacting 4 mg / mL of protein in 100 mM sodium bicarbonate, 50 mM NaCl, pH 9.3 with a 5-fold molar excess of terbium chelate isothiocyanate (Invitrogen). After 3 h incubation at room temperature with stirring, excess unreacted fluorophore was removed by dialysis into PBS (20 mM phosphate-buffered saline, 150 mM NaCl, pH 7.4). sFcεRIα-IgG4-Fc fusion protein (α-γ) was labeled by reacting 3 mg / mL of protein with a 2.5-fold molar excess of Alexa Fluor 647 succinimidyl ester (Invitrogen) for 1 h at room temperature. 37 was labeled with an acceptor fluorophore. Excess fluorophore was removed by dialysis into PBS.

[0260] TR-FRET inhibition assays were performed by competing 1 nM terbium-labeled IgE-Fc and 0-20 nM Alexa Fluor 647-labeled sFcεRIα-IgG4-Fc with a range of concentrations of omalizumab Fab3. The assays were performed in 384-well hi-base white plates (Greiner BioOne) using Lanthascreen buffer (Invitrogen) as diluent. Plates were incubated statically overnight at room temperature and read on an Artemis plate reader (Berthold Technologies). The TR-FRET ratio was then calculated for each well as the acceptor emission at 665 nm divided by the donor emission at 620 nm multiplied by 10,000.

[0261] Crystallization. Crystals with rectangular morphology, up to 400 μm long, were grown at 18 °C using the sitting drop vapor diffusion method. The vessel contained 50 μL of 4% (w / v) PEG8000 and 0.03 M sodium fluoride, the droplet contained 100 nL of protein and the vessel 300 nL. Despite extensive efforts in optimization, the diffraction quality of the crystals could not be further improved beyond that used for this study. Crystals typically started to grow after a few days and often dissolved in the droplet, but could be stabilized in 4 M TMAO (trimethylamine N-oxide), which was successfully used as a cryoprotectant.

[0262] Data collection and processing. Data were collected at the Diamond Light Source (Harwell, UK) on beamlines I02 and I03. The xia2 package 39 XDS implemented in 38 Integration was performed using the CCP4 suite. The crystals diffracted anisotropically and data from multiple crystals were combined. 40、41The data were then scaled to 3.7 Å resolution using AIMLESS, derived from the UCLA Diffraction Anisotropy Server. 42 Using 3.7 Å (a * ), 3.9Å(b * ) and 4.2 Å (c * ) to the resolution limit of 100 kDa. Matthews coefficient calculations indicated a solvent content of approximately 62% for a single 2:1 omalizumab Fab3 / IgE-Fc complex in the asymmetric unit (molecular weight of approximately 170 kDa).

[0263] Structure determination, model building and refinement. Search model is PDB entry 2wqr 8 and the CCP4 suite using protein atoms from the 1.9 Å resolution Omalizumab Fab structure (unpublished results). 40 PHASER derived from 43 and MOLREP 44 The structure was elucidated by molecular replacement with . 45 Later, PHENIX 46 The experiment was carried out using Coot 47 We replaced manual model building in the 48 , POLYGON 49 , and PHENIX graphic interface 50 The data processing and refinement statistics are presented in Table 1. The regions of the electron density map are shown in Figure 11. The boundaries were modeled using PISA. 51 Analyze using PyMOL 52 The figures were prepared using [Table 1]

[0264] References TIFF2025011170000004.tif55164 TIFF2025011170000005.tif220164 TIFF2025011170000006.tif214164 TIFF2025011170000007.tif56159

[0265] (Example 2) Measurement of enhanced dissociation of IgE-Fc from immobilized sFcεRIα by Biacore Biacore technology measures interactions between biomolecules without the need for labeling. One interactor, called the ligand, is directly immobilized or captured on the sensor surface, while the other, called the analyte, flows in solution over the captured surface. The sensor detects changes in mass at the sensor surface when the analyte binds to the ligand and when the analyte dissociates from the ligand. These correspond to both binding and dissociation processes. In the accelerated dissociation assay, sFcεRIα is the ligand and is immobilized on the sensor surface. IgE-Fc is the analyte and is captured by sFcεRIα. Dissociation of IgE-Fc from sFcεRIα is monitored using a buffer flowed over the sensor surface or a solution of an IgE binding partner flowed over the sensor surface. The details of the method are as follows:

[0266] Equipment: Biacore 3000, GE Healthcare AB, Uppsala, Sweden. Sensor chip: CM5. Catalog number BR100399. BIAnormalising solution: 70% (w / w) glycerol. Part of the BIAmaintenance Kit. Catalog number BR100651. The BIAmaintenance Kit was stored at 4°C.

[0267] Amine Coupling Kit: Catalog No. BR100633. Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was made up to 75 mg / mL in distilled water and stored in 200 μL aliquots at −70° C. N-Hydroxysuccinimide (NHS) was made up to 11.5 mg / mL in distilled water and stored in 200 μL aliquots at −70° C. Ethanolamine hydrochloride-NaOH pH 8.5 was stored at 4° C.

[0268] Reagents for oxidation of sFcεRIα. Carbohydrazide (SigmaAldrich, Cat. No. C11006) was made to 5 mM in distilled water. Sodium cyanoborohydride (SigmaAldrich, Cat. No. 156159) was made to 100 mM in sodium acetate (BDH, Cat. No. S1104-500GM) 100 mM pH=4. Sodium m-periodate (SigmaAldrich, Cat. No. S-1878) was made to 50 mM in sodium acetate (BDH, Cat. No. S1104-500GM) 100 mM pH=5.5. sFcεRIα was diluted to 1 mg / ml in 0.1 M sodium acetate, pH 5.5. Then 4 μl of sodium periodate (50 mM, 1 / 50 dilution) was added to 200 μl of 1 mg / ml sFcεRIα solution. The mixture was left on ice for 20 min. Prior to immobilization, the solution of sFcεRIα was diluted to 7 μg / ml in 10 mM sodium acetate (GE Healthcare, Cat. No. BR100669), pH=4.0.

[0269] Buffers: Running buffer is HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20, reconstituted from 10x stock solution): Catalog number BR100669. Immobilization buffer is Acetate 4.0 (10 mM Sodium Acetate pH 4.0). Catalog number BR100349. Buffers were stored at 4°C. Ligand: sFcεRIα is the extracellular portion of the alpha chain of the human high affinity IgE receptor. It was expressed as a recombinant protein in CHO cells and purified.

[0270] Analyte: IgE-Fc is the Fc portion of human IgE, expressed as a recombinant protein in CHO cells and purified. Wild-type human IgE-Fc carrying the C225A mutation (Cε2-Cε4 domains including number V224-K547 according to Dorrington & Bennich (1978) Immunol. Rev. 41:3-25) was used (SEQ ID NO: 108).

[0271] Mutant nomenclature: Omalizumab Fab1: S81R, Q83R [S77R, Q79R according to Kabat numbering] (variable region light chain as SEQ ID NO: 24 with S77 and S79 replaced by Q + kappa constant region: variable region heavy chain as SEQ ID NO: 5 + CH1 constant region) Omalizumab Fab2: L158P [L154P according to Kabat numbering] (variable region light chain + kappa constant region as SEQ ID NO: 113; variable region heavy chain + CH1 constant region as SEQ ID NO: 5) Omalizumab Fab3: S81R, Q83R, L158P [S77R, Q79R, L154P according to Kabat numbering] (variable region light chain as SEQ ID NO: 121; variable region heavy chain + CH1 constant region as SEQ ID NO: 5).

[0272] IgE binding partners (1): Full-length omalizumab (Novartis); recombinant Fab fragment of omalizumab expressed in CHO cells and purified.

[0273] IgE binding partners (2): Recombinant Fab fragments of omalizumab and its variants expressed in HEK-293 cells and assayed as culture supernatants. Culture supernatants were concentrated 10-fold before analysis.

[0274] Assay method (1): sFcεRIα was coupled to the sensor surface by aldehyde coupling at a level of approximately 500 response units (RU). HBS-EP buffer was used as running buffer at a flow rate of 30 μL / min. IgE-Fc was diluted to 10 nM in HBS-EP and injected over the immobilized sFcεRIα for 290 s, followed by three injections of running buffer or of IgE binding partner diluted in running buffer, each for a period of 690 s. The capture level of IgE-Fc was approximately 90 RU. The surface was regenerated by two 60 s injections of 10 mM glycine-HCl, pH 2.5. The amount of dissociation of IgE-Fc from immobilized sFcεRIα was calculated as a function of the initial amount bound, and the dissociation rate was calculated as the amount of IgE-Fc lost from immobilized sFcεRIα, normalized for the initial amount bound as a function of time.

[0275] Assay method (2): sFcεRIα was coupled to the sensor surface by aldehyde coupling at a level of approximately 2000 response units (RU). HBS-EP buffer was used as running buffer at a flow rate of 30 μL / min. IgE-Fc was diluted to 10 nM in HBS-EP and injected over the immobilized sFcεRIα for 180 s, followed by one 180 s injection of running buffer or of IgE binding partner diluted in running buffer. The capture level of IgE-Fc was approximately 275 RU. The surface was regenerated by two 60 s injections of 10 mM glycine-HCl, pH 2.5. The amount of dissociation of IgE-Fc from immobilized sFcεRIα was calculated as a function of the initial amount of binding, and the dissociation rate was calculated as the amount of IgE-Fc lost from immobilized sFcεRIα, normalized for the initial amount of binding as a function of time. [Table 2]

[0276] Further mutagenesis was performed to determine whether further mutations could be identified that promote dissociation of IgE-Fc from sFcεRIα beyond those described in Fab3. This resulted in the S64M mutation (see SEQ ID NO: 20) that can further increase dissociation of IgE from sFcεRIα in the context of Fab3. These data are presented in Figure 13 and Table 3. [Table 3]

[0277] Conclusion: Taken together, these data indicate that mutated forms of Omalizumab Fab are able to promote dissociation of IgE from the fixed high affinity IgE receptor FcεRI by mutations in the light chain that allow this, including but not limited to S64M, S81R, Q83R and L158P with reference to SEQ ID NO:24, resulting in SEQ ID NO:39.

[0278] (Example 3) Measurement of accelerated dissociation of IgE-Fc from FcεRI by flow cytometry Equipment: FACSCanto II flow cytometer (Becton Dickinson).

[0279] Cell line: RBL-SX38 cells expressing human FcεRI were cultured in minimal essential medium supplemented with 10% fetal bovine serum, 2 mM GlutaMAX, and 500 μg / mL Geneticin (Life Technologies). On the day of the assay, cells were washed in PBS, incubated in Accutase until detached, and then cultured at 1x10 6 The cells were resuspended at 1000 cells / mL. All subsequence incubation steps were performed in culture medium.

[0280] Assay method: 1x10 6RBL-SX38 cells at 1000x100 cells / mL were incubated with 5 nM Alexa-488-labeled IgE-Fc for 1 h at 37° C. Cells were washed twice in culture medium to remove unbound IgE-488, and then 1x10 cells / mL were incubated with 1x10 cells / mL Alexa-488-labeled IgE-Fc for 1 h at 37° C. 6 Cells were either suspended at 100 μg / mL or diluted in culture medium with 100 μg / mL of IgE-binding agent. Cells were then incubated at 37° C. with constant rotation. At each time point, 0.5×10 5 Cells were removed, washed in ice-cold PBS, and then fixed by resuspension in 1% paraformaldehyde in PBS for 16 h at 4° C. The amount of cell-bound Alexa-488 fluorescence was determined using a FACSCanto II flow cytometer.

[0281] Flow cytometry: Fixed cells were washed twice in FACS buffer (0.1% w / v BSA, 0.01% w / v NaN3 in PBS, pH 7.4) and resuspended in 200 μL of FACS buffer. Flow cytometry was performed on a FACSCanto II cytometer using standard methods, and the geometric mean fluorescence intensity of Alexa-488 bound to intact cells was calculated using FlowJo software. The dissociation rate of Alexa-488-labeled IgE-Fc was calculated as the change in geometric mean fluorescence intensity as a function of time as a result of incubating cells in cell culture medium with excess unlabeled IgE-Fc or in the presence of an IgE-binding agent.

[0282] Mutant nomenclature: Omalizumab Fab1: S81R, Q83R [S77R, Q79R according to Kabat numbering] (variable region light chain as SEQ ID NO: 24 with S77 and S79 replaced by Q + kappa constant region: variable region heavy chain as SEQ ID NO: 5 + CH1 constant region) Omalizumab Fab2: L158P [L154P according to Kabat numbering] (variable region light chain + kappa constant region as SEQ ID NO: 113; variable region heavy chain + CH1 constant region as SEQ ID NO: 5) Omalizumab Fab3: S81R, Q83R, L158P [S77R, Q79R, L154P according to Kabat numbering] (variable region light chain + kappa constant region as SEQ ID NO: 121; variable region heavy chain + CH1 constant region as SEQ ID NO: 5). [Table 4]

[0283] Conclusion: Taken together, these data indicate that mutant omalizumab Fabs are able to promote dissociation of IgE from the high affinity IgE receptor FcεRI expressed on the cell surface by mutations in the light chain that allow this, including but not limited to S81R, Q83R and L158P.

[0284] (Example 4) Demonstration that omalizumab Fab3 has superior efficacy to wild-type omalizumab Fab when administered therapeutically in a 72-hour PCA model A solution of 25 μg / mL human anti-DNP-IgE was prepared by adding 7.5 μL of the 6.68 mg / mL stock to 1992.5 μL of PBS. A 20 μL injection of this solution gives a 500 ng dose of IgE. Animals (hIgER Tg mice) were shaved on the flanks and then injected id into each flank at 2 pm on day 0. 20 μL of PBS was injected into the left flank of each animal as a negative control. Anti-NDP-IgE (20 μL) was injected into the right flank. A total of 40 mice were injected. Treatment with wild-type omalizumab Fab or omalizumab Fab3 (with S81R, Q83R, L158P mutations [S77R, Q79R, L154P by Kabat numbering]) was started 18 hours (8 am) after IgE. Two groups of mice (n=8 / group) received 100 mg / kg sc of either wild-type omalizumab Fab or omalizumab Fab3. An additional group of eight mice received PBS sc. Mice were dosed again 10 hours later (6 pm) as above. At this time, 28 hours after IgE, two more groups (n=8 / group) were also dosed with 100 mg / kg sc wild-type omalizumab Fab or omalizumab Fab3. All groups were dosed again at 8 am, 6 pm, and again at 8 am on the final day of the experiment. 72 hours after id dosing (2 pm), all animals were injected iv with 100 μL of 2.5% w / v Evans Blue made up in 1 mg / mL DNP-HSA, 100 IU / ml heparin. One hour later, animals were sacrificed by Schedule 1 methods. Skin was removed from the flank surrounding the id injection site and punch biopsies were taken. Skin samples were placed in 700 μL of formamide and digested overnight at 55° C. After digestion, 100 μL x 2 of liquid was removed from each sample and placed into a 96-well ELISA plate. Absorbance was then measured at 620 nm.

[0285] Conclusion: These data indicate that mutant omalizumab Fabs capable of promoting dissociation of IgE from the high affinity IgE receptor FcεRI can also reduce passive cutaneous anaphylaxis in a statistically significant manner when compared to wild type omalizumab Fab (as shown by inhibition of leakage of Evans Blue dye from the reaction site). Mutations in the light chain that enable this include, but are not necessarily limited to, S81R, Q83R and L158P with reference to SEQ ID NO:24, resulting in SEQ ID NO:39.

[0286] (Example 5) Molecular dynamics simulation of omalizumab Fab3 complexed with IgE Fc method: The crystal structure of omalizumab Fab3 in complex with the IgE Fc region (see Example 1) was prepared in Molecular Operation Environment (MOE) 2014.0901 (1) by completing the missing side chains in some residues and the missing loop between the Cε2 and Cε3 domains prior to molecular mechanics (MD) simulations using Amber 14 (2). The complex structure was hydrogenated and solvated using the TIP3P explicit water model using 0.15 M NaCl salt solute in a truncated octahedral box extending 10 Å from either end of the box to the protein atoms. The system was set up using Amber ff12B and the oligosaccharide GLYCAM_06j-1 (3) force field and minimized by conjugate gradient method for 50,000 steps with a cutoff of 10.0 Å set for Coulombic and van der Waals interactions and grid-based neighbor lists. The system was then heated gradually from 0 to 300 K in 125 ps at constant volume, and then equilibrated for 2.25 ns in the NPT ensemble with restraints on all solute heavy atoms (harmonic force restraints are 5.0). For electrostatics, we used fourth-order PME with a cutoff of 8.0 Å for Coulombic interactions with default Fourier space and tolerance settings. Temperature was controlled using a weak coupling algorithm applied to protein and solvent, respectively, with a time constant of 1.0 ps, ​​and pressure was controlled with a time constant of 1.0 ps and a pressure of 4.46x10 -5 bar-1 The dynamics of the Fab was controlled using an isotropic Berenson barostat applied to the whole system with a compression rate of 1000 ns. Finally, a 1000 ns generation simulation without any constraints was performed using the same parameters for equilibration. To allow a time step of 4 fs on the GPU infrastructure, the hydrogen mass of the protein and sugar was redistributed to 3.024 Daltons using ParmEd (4), while the masses of the atoms to which they are attached were adjusted by the amount required to keep the total mass unchanged. The structure of wild-type omalizumab Fab in complex with the IgE Fc region was modeled from the crystal complex structure of omalizumab variant 3 with IgE Fc by actually mutating R81 and R83 in the antibody light chain to wild-type serine and asparagine, respectively. MD simulations were performed using the same setup protocol as for variant 3.

[0287] The AmberTolls cpptraj module (7) was used for cluster analysis of the MD trajectories for both Omalizumab Fab3 and Omalizumab Fab in complex with IgE Fc. A hierarchical condensation algorithm was employed where the average bond distance between each cluster was less than 2.0 Å. The distance between frames was calculated by the best-fit coordinate RMSD between Cα atoms in the antibody light chain V region residues. Clustering was performed only for every 10th frame, and all other frames were added to the cluster based on how close they were to the center of mass of the cluster after clustering.

[0288] References TIFF2025011170000011.tif29163

[0289] result: To test how the S81R and Q83R mutations in the light chain of Omalizumab Fab3 affect the interaction with IgE, 1 microsecond molecular dynamics simulations were performed on Omalizumab Fab and Omalizumab Fab3, respectively, in complex with the IgE Fc structure. Clustering of trajectory snapshots and analysis of the top two most abundant cluster center structures clearly show that in both clusters, the two arginine mutations in mutant 3 form strong hydrogen bond networks with residues D278 and S280, respectively, in the adjacent IgE Cε2 domain. Visual inspection of the trajectories confirms that the R81-S280 and R83-D278 pairing interactions are highly conserved and stable during the simulations, thus confirming that the uncurved Cε2 conformation relative to the disclosed closer Cε3 domain in the crystal structure is frozen by antibody cross-linking. Interestingly, the double arginine mutation is spatially adjacent to Cε2 D278 and S280 in the crystal structure of Omalizumab Fab3, but there is no direct hydrogen bond as suggested in the MD simulations. For Omalizumab Fab, as expected, the hydrogen bond network observed in Mutant 3 is missing in the top two cluster center structures. Visual trajectory inspection indicates that the Cε2 domain becomes less anchored to the antibody light chain framework and thus its relative position to the closer Cε3 domain is more variable than that of Omalizumab Fab3. In addition to this, the interface between Omalizumab Fab3 and IgE-Fc in the same complex was subjected to IOTA analysis (IOTA is a statistical potential tool to determine the probability of a given contact atom type at a protein interface or binding site) in which light chain positions S56, S64 and S71 (see SEQ ID NO: 113) in Omalizumab Fab3 were mutated to predict that they would increase the affinity of Omalizumab for IgE-Fc, thus enhancing the effect predicted to be seen for the S81R and Q38R mutations detailed above.

[0290] Conclusion: Taken together, the MD simulations propose the hypothesis that the S81R and Q38R mutations facilitate localization of the IgE Cε2 domain to the antibody through direct electrostatic and hydrogen-bonding interactions with the neighboring D278 and S280 in Cε2, affecting IgE plastic Fc by confining the uncurved conformation of Cε2. This is combined with statistical methods that predict that the S64M mutation and S56 (changes to D, E, Q, or R) and S71 (changes to D, E, or M) have similar effects.

[0291] (Example 6) Measurement of the affinity of anti-IgE Fab to IgE-Fc by Biacore Biacore technology measures interactions between biomolecules without the need for labeling. One interactor, called the ligand, is directly immobilized or captured on the sensor surface, while the other, called the analyte, flows in solution over the captured surface. The sensor detects changes in mass at the sensor surface when the analyte binds to the ligand and when the analyte dissociates from the ligand. These correspond to both binding and dissociation processes. In the kinetic assay, anti-IgE Fab is the ligand and is captured on the sensor surface. IgE-Fc is the analyte and is captured by anti-IgE Fab. The binding and dissociation of IgE-Fc from the captured anti-IgE Fab is monitored by either flowing IgE-Fc over the sensor surface (binding phase) or flowing buffer over the sensor surface (dissociation phase). The details of the method are as follows: Equipment: Biacore 3000, GE Healthcare AB, Uppsala, Sweden Sensor chip: CM5. Catalog number BR100399. BIAnormalising solution: 70% (w / w) glycerol. Part of the BIAmaintenance kit. Catalog number BR100651. The BIAmaintenance kit was stored at 4°C.

[0292] Amine Coupling Kit: Catalog No. BR100633. Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was made up to 75 mg / mL in distilled water and stored in 200 μL aliquots at −70° C. N-Hydroxysuccinimide (NHS) was made up to 11.5 mg / mL in distilled water and stored in 200 μL aliquots at −70° C. Ethanolamine hydrochloride-NaOH pH 8.5 was stored at 4° C.

[0293] Buffers: Running buffer is HBS-EP (reconstituted from 10x stock solution, 10mM HEPES pH 7.4, 150mM NaCl, 3mM EDTA, 0.05% Surfactant P20): Catalog number BR100669. Immobilization buffer is Acetate 4.0 (10mM Sodium Acetate pH 4.0): Catalog number BR100349. Buffers were stored at 4°C.

[0294] Ligand: Anti-IgE Fab was the ligand. These were transiently expressed as recombinant proteins in HEK293 cells and used without further purification.

[0295] Analyte: IgE-Fc is the Fc portion of human IgE, expressed as a recombinant protein in CHO cells and purified. Wild-type human IgE-Fc carrying the C225A mutation (Cε2-Cε4 domains including number V224-K547 according to Dorrington & Bennich (1978) Immunol. Rev. 41:3-25) was used (SEQ ID NO: 108).

[0296] Fab2 fragment of goat anti-human IgG1 (Fab2 fragment specific) (Jackson Immunolabs, Cat. No. 109-006-097) was immobilized on the sensor surface by amine coupling using standard methods. Anti-IgE Fab was diluted in running buffer (HBS-EP) and approximately 200 resonance units were captured on the surface. IgE-Fc was diluted in running buffer and a serial dilution series from 2 nM to 125 pM was passed over the captured anti-IgE Fab. The association phase was 180 seconds and the dissociation phase was 300 seconds. The sensor surface was regenerated by exposure to 40 mM HCl for 60 seconds, then 10 mM NaOH for 60 seconds, then 40 mM HCl for another 60 seconds. All binding data was processed by double referencing using BiaEvaluation software according to standard procedures. [Table 5] [Table 6] [Table 7] [Table 8] [Table 9]

[0297] Conclusion: These data show that mutated Omalizumab Fab can increase the affinity of Omalizumab Fab to IgE-Fc. The best combination of mutations for improving affinity is S71M combined with S56D. This increase in affinity is principally caused by a decrease in the dissociation rate of Fab from IgE-Fc. Considering the improvement of the antibody-IgE-Fc Cε2 interaction compared to non-mutated Omalizumab Fab, the affinity of Omalizumab Fab1 (with S81R, Q83R mutations [S77R, Q79R according to Kabat numbering]) and Fab3 (with S81R, Q83R, L158P mutations [S77R, Q79R, L154P according to Kabat numbering], see SEQ ID NO: 125) is also improved.

[0298] (Example 7) Measurement of accelerated dissociation of IgE-Fc from immobilized sFcεRIα by Biacore The effect of anti-IgE Fab on dissociation of IgE-Fc from sFcεRIα was measured using the method outlined in Example 2 (assay method 2). All anti-IgE Fabs were expressed in HEK-29s Fab, purified by standard methods, and quantified by absorbance at 280 nm using molar extinction coefficient calculations. In this assay, the concentration of IgE-Fc was 2 nM and the dissociation time was 200 seconds. The amount of dissociation of IgE-Fc from immobilized sFcεRIα was calculated as a function of the initial amount bound, and the dissociation rate was calculated as the amount of IgE-Fc lost from immobilized sFcεRIα, normalized for the initial amount bound as a function of time.

[0299] S80N (pdb numbering) is thought to interact with D278 of Cε2 IgE, and S67W / Y (pdb numbering) is thought to interact with T298 of Cε2 IgE. [Table 10]

[0300] Conclusion: These data indicate that mutant omalizumab Fabs are able to promote dissociation of IgE from the fixed high affinity IgE receptor FcεRI by mutations in the light chain (SEQ ID NO:20) including, but not limited to, mutations at positions S56, S64, S67, S71, S80, S81, Q83&L158 (positions S52, S60, S63, S67, S76, S77, Q79&L154, respectively, according to Kabat numbering).

[0301] (Example 8) Forced oxidation of mutant omalizumab Fab Anti-IgE Fab samples were subjected to a forced oxidation regimen to determine the effect of oxidation of methionines in the light chain variable region on the affinity of the Fab for IgE-Fc and its ability to promote dissociation of the IgE-Fc:sFcεRIα complex. Mutant omalizumab Fabs were incubated with 0.1% and 1% (v / v) hydrogen peroxide at room temperature for up to 14 days. After incubation, samples were buffer exchanged back into PBS pH 7.4 and concentrations were determined by absorbance at 280 nm using calculated extinction coefficients. Mass spectrometry to determine the amount of oxidation of light chain variable region methionines was performed by reduction and alkylation of the material under denaturing conditions, followed by trypsin digestion (50 μg / mL trypsin for 180 min at 37° C., followed by quenching with TFA) and then analysis by LC-MS using a Thermo Orbitrap Q Exactive Plus mass spectrometer. The percentage of oxidized methionine is calculated relative to the assumption of no methionine oxidation during synthesis and compared to a reference material kept at 4°C. [Table 11]

[0302] Conclusion: These data indicate that it is possible to generate mutant omalizumab Fabs in which the critical light chain methionines (M64 and M71) are essentially fully oxidized. Based on these data, material from day 1, day 3, and day 7 samples was pooled and used to determine the effect of methionine oxidation on the affinity of mutant omalizumab Fabs for IgE-Fc and their ability to promote dissociation of the IgE-Fc:sFcεRIα complex.

[0303] (Example 9) Measurement of enhanced dissociation of IgE-Fc from sFcεRIα by FRET The effect of anti-IgE Fab on the dissociation of IgE-Fc from sFcεRIα was measured in a homogeneous FRET assay. All anti-IgE Fabs were expressed in HEK-293 cells, purified by standard methods, and quantified by absorbance at 280 nm using molar absorptivity calculations. The FRET assay used Tb-labeled IgE-Fc as the donor and Alexa488-labeled sFcεRIα as the acceptor. Both reagents were mixed and equilibrated at room temperature for 60 min at a final assay concentration of 1 nM. Anti-IgE Fab was added to the mixture at a final assay concentration of 500 nM, and fluorescence was read every 20 min for 800 min (excitation at 330 nm, emission at 495 and 520 nm). Fluorescence emission was plotted as a function of time, and the dissociation rate of IgE-Fc from sFcεRIα was calculated as the half-life of the complex. These data are reported in Tables 10 and 11. [Table 12] [Table 13]

[0304] Conclusion: These data indicate that the mutant omalizumab is able to dissociate IgE-Fc from sFcεRIα at a faster rate than the wild-type sequence. In particular, the mutations in the light chain that enable this (see SEQ ID NO:20) include, but are not necessarily limited to, mutations at positions S64 and S67. Furthermore, oxidation of methionines (M64 and M71, SEQ ID NO:20) has no significant effect on the ability of the Fab to promote dissociation of the IgE-Fc:sFcεRIα complex.

[0305] The specification has been described with reference to embodiments of the present invention. However, those skilled in the art will appreciate that various modifications and changes can be made without departing from the scope of the invention as set forth in the following claims. The specification is therefore to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of the present invention. JPEG2025011170000021.jpg241170 JPEG2025011170000022.jpg241170 JPEG2025011170000023.jpg241170 JPEG2025011170000024.jpg241170 JPEG2025011170000025.jpg241170 JPEG2025011170000026.jpg241170 JPEG2025011170000027.jpg241170 JPEG2025011170000028.jpg241170 JPEG2025011170000029.jpg241170 JPEG2025011170000030.jpg241170 JPEG2025011170000031.jpg241170 JPEG2025011170000032.jpg241170 JPEG2025011170000033.jpg241170 JPEG2025011170000034.jpg241170 JPEG2025011170000035.jpg241170 JPEG2025011170000036.jpg241170 JPEG2025011170000037.jpg241170 JPEG2025011170000038.jpg241170 JPEG2025011170000039.jpg241170 JPEG2025011170000040.jpg241170 JPEG2025011170000041.jpg241170 JPEG2025011170000042.jpg241170 JPEG2025011170000043.jpg241170 JPEG2025011170000044.jpg241170 JPEG2025011170000045.jpg241170 [Sequence List Free Text]

[0306] SEQ ID NOs: 1-2: <223> V area SEQ ID NOs: 3-4: <223> V region containing signal sequence SEQ ID NOs: 5-6: <223> V region + gamma 1 CH1 constant region SEQ ID NOs: 7-8: <223> V region + gamma 1 CH1 constant region, including signal sequence SEQ ID NOs: 9-10: <223> V region + gamma 1 full-length constant region SEQ ID NOs: 11 to 12: <223> V region + gamma 1 full-length constant region, including signal sequence SEQ ID NOs: 20-21: <223> V region light chain SEQ ID NOs:22-23: <223> V region light chain containing signal sequence SEQ ID NO:24: <223> V region light chain + kappa constant region SEQ ID NO:25: <223> V region + kappa constant region SEQ ID NOs:26-27: <223> V region + kappa constant region, including signal sequence SEQ ID NOs:35-36: <223> S60M_S77R_Q79R_V area SEQ ID NOs:37-38: <223> S60M_S77R_Q79R_V region including signal sequence SEQ ID NOs: 39 to 41: <223> Kappa constant region including S60M_S77R_Q79R_V region + L154P SEQ ID NO:42: <223> Kappa constant region encompassing the S60M_S77R_Q79R_V region + L154P, including the signal sequence SEQ ID NO:108: <223> Wild-type human IgE-Fc (CE2-CE4 domains numbered V224-K547 according to Dorrington & Bennich (1978)) SEQ ID NO:109: <223> S77R_Q79R_V area (Fab1) SEQ ID NO:110: <223> 77R_Q79R_V area (Fab1) SEQ ID NOs: 111 to 112: <223> S77R_Q79R_V region including signal sequence (Fab1) SEQ ID NOs: 113 to 114: <223> Fab2_V area SEQ ID NOs: 115 to 116: <223> Fab2_V region including signal sequence SEQ ID NOs: 117-118: <223> Fab2_V region + kappa constant region including L154P SEQ ID NOs: 119-120: <223> Kappa constant region encompassing Fab2_V region + L154P, including signal sequence SEQ ID NOs: 121-122: <223> Fab3_S77R_Q79R_V area SEQ ID NOs: 123-124: <223> Fab3_S77R_Q79R_V region, including the signal sequence SEQ ID NOs: 125-126: <223> Fab3_S77R_Q79R_V region + kappa constant region including L154P SEQ ID NOs: 127-128: <223> Kappa constant region encompassing Fab3_S77R_Q79R_V region + L154P, including signal sequence SEQ ID NOs: 129-130: <223> Omalizumab_V domain SEQ ID NOs: 132-133: <223> S60M_S52D_S67M_S77R_Q79R_V area SEQ ID NOs: 134 to 135: <223> S60M_S52D_S67M_S77R_Q79R_V region including signal sequence SEQ ID NOs: 136-137: <223> S60M_S52D_S67M_S77R_Q79R_V domain + kappa constant domain SEQ ID NOs: 139-140: <223> S60R_S52D_S67M_S77R_Q79R_V area SEQ ID NOs: 141-142: <223> S60R_S52D_S67M_S77R_Q79R_V region including signal sequence SEQ ID NOs: 143-144: <223> S60R_S52D_S67M_S77R_Q79R_V region + kappa constant region SEQ ID NO:145: <223> S60K_S52D_S67M_S77R_Q79R_V area SEQ ID NO:146: <223> S60Q_S52D_S67M_S77R_Q79R_V area SEQ ID NOs: 147-148: <223> V region + gamma 1 CH1 constant region + linker + CA645 gL4gH5 scFv, including signal sequence SEQ ID NO:149: <223> Linker between CH1 and CA645 gL4gH5 scFv SEQ ID NO:151: <223> Linker between CA645 gH5 and CA645 gL4 in scFv SEQ ID NO:158: <223> S60M_S52D_S67M_S77R_Q79R_S63W_S76N_V area SEQ ID NO:159: <223> S60M_S52D_S67M_S77R_Q79R_S63Y_S76N_V area SEQ ID NO:160: <223> Signal sequence

Claims

1. An anti-IgE antibody or antigen binding agent that contacts an epitope comprising residues T373, W374, S375, R376, A377, S378, G379, P381, Q417, C418, R419, T421, P426, R427, A428 of the Cε3 domain, and residues D278 and T281 of the Cε2 domain of human IgE in reference to SEQ ID NO:

108.

2. 2. The anti-IgE antibody or antigen-binding agent of claim 1 which contacts an epitope further comprising residue K380 of the Cε3 domain of human IgE.

3. 3. The anti-IgE antibody or antigen-binding agent of claim 1 or 2, which contacts an epitope further comprising residue M430 of the Cε3 domain of human IgE.

4. 4. An anti-IgE antibody or antigen-binding agent according to claim 1 which contacts an epitope further comprising residue D276 of the Cε2 domain of human IgE.

5. 5. An anti-IgE antibody or antigen-binding agent according to claim 1 which contacts an epitope further comprising residue V277 of the Cε2 domain of human IgE.

6. 6. An anti-IgE antibody or antigen-binding agent according to claim 1 which contacts an epitope further comprising residue L279 of the Cε2 domain of human IgE.

7. 7. An anti-IgE antibody or antigen-binding agent according to claim 1 which contacts an epitope further comprising residue S280 of the Cε2 domain of human IgE.

8. 8. An anti-IgE antibody or antigen-binding agent according to claim 1 which contacts an epitope further comprising residues A282 (and / or T298) of the Cε2 domain of human IgE.

9. 9. An anti-IgE antibody or antigen-binding agent according to claim 1 to 8, wherein the Cε3 and Cε2 domains are on different chains of human IgE.

10. 10. An anti-IgE antibody or antigen-binding agent according to claim 1 which is specific for an epitope.

11. An anti-IgE antibody or antigen-binding agent, optionally as defined in claims 1 to 10, A heavy chain variable region comprising a complementarity determining region CDR-H3 having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising a complementarity determining region CDR-L1 having the amino acid sequence of SEQ ID NO: 29, the light chain variable region further comprises a framework region FR-L3 having an amino acid sequence selected from SEQ ID NO: 32, and having 1, 2, 3, 4, 5, 6, 7 or more amino acid substitutions to enhance the interaction of the anti-IgE antibody or antigen binding agent with the Cε2 domain of human IgE; Anti-IgE antibodies or antigen-binding agents.

12. 12. The anti-IgE antibody or antigen-binding agent of claim 11, wherein the FR-L3 region is mutated at positions S60, S63, S76, S77, and / or Q79 (Kabat) with reference to SEQ ID NO: 129 to one of the other naturally occurring amino acids.

13. 13. The anti-IgE antibody or antigen-binding agent of claim 11 or 12, wherein the FR-L3 region is mutated at position S60 (Kabat) to one of the other naturally occurring amino acids.

14. 14. The anti-IgE antibody or antigen-binding agent of claims 11 to 13, wherein the FR-L3 region is mutated to M, R, K, N, Q or T at position S60 (Kabat).

15. 15. The anti-IgE antibody or antigen-binding agent of claim 11 to 14, wherein the FR-L3 region is mutated to M at position S60 (Kabat).

16. 16. The anti-IgE antibody or antigen-binding agent of claims 11 to 15, wherein the FR-L3 region is mutated at position S63 (Kabat) to one of the other naturally occurring amino acids.

17. 17. The anti-IgE antibody or antigen-binding agent of claims 11 to 16, wherein the FR-L3 region is mutated to W or Y at position S63 (Kabat).

18. 18. The anti-IgE antibody or antigen-binding agent of claim 11 to 17, wherein the FR-L3 region is mutated to Y at position S63 (Kabat).

19. 19. The anti-IgE antibody or antigen-binding agent of claims 11 to 18, wherein the FR-L3 region is mutated at position S76 (Kabat) to one of the other naturally occurring amino acids.

20. 20. The anti-IgE antibody or antigen-binding agent of claim 11 to 19, wherein the FR-L3 region is mutated to N at position S76 (Kabat).

21. 21. The anti-IgE antibody or antigen-binding agent of claims 11 to 20, wherein the FR-L3 region is mutated at position S77 (Kabat) to one of the other naturally occurring amino acids.

22. 22. The anti-IgE antibody or antigen-binding agent of claim 11 to 21, wherein the FR-L3 region is mutated to R or K at position S77 (Kabat).

23. 23. The anti-IgE antibody or antigen-binding agent of claim 11 to 22, wherein the FR-L3 region is mutated to R at position S77 (Kabat).

24. 24. The anti-IgE antibody or antigen-binding agent of claim 11 to 23, wherein the FR-L3 region is mutated at position S79 (Kabat) to one of the other naturally occurring amino acids.

25. 25. The anti-IgE antibody or antigen-binding agent of claim 11 to 24, wherein the FR-L3 region is mutated to R or K at position S79 (Kabat).

26. 26. The anti-IgE antibody or antigen-binding agent of claim 11 to 25, wherein the FR-L3 region is mutated to R at position Q79 (Kabat).

27. 27. The anti-IgE antibody or antigen-binding agent of claim 11 to 26, wherein the amino acid sequence of the mutated FR-L3 region is selected from SEQ ID NOs: 43-49, 60-83, 131 or 138.

28. The FR-L3 region determines its affinity for human IgE (lower K Ｄ 27. The anti-IgE antibody or antigen binding agent of claim 11 to 26, further mutated at position S67 (Kabat) to one of the other naturally occurring amino acids to improve IgE reactivity.

29. 29. The anti-IgE antibody or antigen-binding agent of claim 28, wherein the FR-L3 region is mutated to M, E or D at position S67 (Kabat).

30. 30. The anti-IgE antibody or antigen-binding agent of claim 29, wherein the amino acid sequence of the mutated FR-L3 region is selected from SEQ ID NOs: 53-59, 84-107, 131 or 138.

31. 31. An anti-IgE antibody or antigen-binding agent comprising a heavy chain variable region comprising a complementarity determining region CDR-H3 having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising a complementarity determining region CDR-L1 having the amino acid sequence of SEQ ID NO: 29, and optionally an anti-IgE antibody or antigen-binding agent according to claims 1 to 30, wherein the light chain variable region further comprises a framework region FR-L1 having the amino acid sequence of SEQ ID NO: 28 with 1, 2, 3, 4, 5, 6, 7 or more amino acid substitutions to enhance the interaction of the anti-IgE antibody or antigen-binding agent with the Cε2 domain of human IgE.

32. 32. The anti-IgE antibody or antigen-binding agent of claim 31 , wherein the FR-L1 region is mutated at positions G16 and / or R18 (Kabat) to one of the other naturally occurring amino acids with reference to SEQ ID NO:

20.

33. 33. An anti-IgE antibody or antigen-binding agent according to claim 11 to 32, wherein the light chain variable region further comprises a complementarity determining region CDR-L2 having the amino acid sequence which is SEQ ID NO:

31.

34. The CDR-L2 region determines its affinity for human IgE (lower K Ｄ 34. The anti-IgE antibody or antigen-binding agent of claim 33, wherein the amino acid at position S52 (Kabat) is mutated to one of the other naturally occurring amino acids with reference to SEQ ID NO: 129 to improve affinity to the IgE antibody or antigen-binding agent of claim 33.

35. 35. The anti-IgE antibody or antigen binding agent of claim 34, wherein the CDR-L2 region is mutated to E, D, Q or R at position S52 (Kabat).

36. 36. The anti-IgE antibody or antigen-binding agent of claim 35, wherein the CDR-L2 region is mutated to D (preferably) or E at position S52 (Kabat).

37. 37. The anti-IgE antibody or antigen-binding agent of claims 34 to 36, wherein the amino acid sequence of the mutated CDR-L2 region is selected from SEQ ID NO:50 or SEQ ID NO:

51.

38. 38. An anti-IgE antibody or antigen-binding agent according to claim 11 to 37, wherein the heavy chain variable region further comprises a complementarity determining region CDR-H1 having the amino acid sequence which is SEQ ID NO:

14.

39. 39. An anti-IgE antibody or antigen-binding agent according to claim 11 to 38, wherein the heavy chain variable region further comprises a complementarity determining region CDR-H2 having the amino acid sequence which is SEQ ID NO:

16.

40. 40. An anti-IgE antibody or antigen-binding agent according to claim 11 to 39, wherein the light chain variable region further comprises a complementarity determining region CDR-L3 having the amino acid sequence which is SEQ ID NO:

33.

41. 41. The anti-IgE antibody or antigen-binding agent of claim 11 to 40, wherein the heavy chain variable region further comprises a framework region FR-H1 having the amino acid sequence of SEQ ID NO:

13.

42. 42. The anti-IgE antibody or antigen-binding agent of claim 11 to 41, wherein the heavy chain variable region further comprises a framework region FR-H2 having the amino acid sequence of SEQ ID NO:

15.

43. 43. The anti-IgE antibody or antigen-binding agent of claim 11 to 42, wherein the heavy chain variable region further comprises a framework region FR-H3 having the amino acid sequence of SEQ ID NO:

17.

44. 44. The anti-IgE antibody or antigen-binding agent of claim 11 to 43, wherein the heavy chain variable region further comprises a framework region FR-H4 having the amino acid sequence of SEQ ID NO:

19.

45. 45. The anti-IgE antibody or antigen-binding agent of claim 11 to 44, wherein the light chain variable region further comprises a framework region FR-L2 having the amino acid sequence of SEQ ID NO:

30.

46. 46. ​​The anti-IgE antibody or antigen-binding agent of claim 11 to 45, wherein the light chain variable region further comprises a framework region FR-L4 having the amino acid sequence of SEQ ID NO:

34.

47. 47. The anti-IgE antibody or antigen-binding agent of claim 11 to 46, wherein the light chain variable region VL has an amino acid sequence selected from SEQ ID NO:35, SEQ ID NO:132, or SEQ ID NO:134, or SEQ ID NO:139 or SEQ ID NO:141, or SEQ ID NOs:145-146, or SEQ ID NOs:158-159, optionally including a signal sequence having the amino acid sequence of SEQ ID NO:

160.

48. 48. An anti-IgE antibody or antigen-binding agent according to claim 11 to 47, wherein the heavy chain variable region VH has the amino acid sequence of SEQ ID NO:

1.

49. 49. The anti-IgE antibody or antigen-binding agent of claim 11 to 48, further comprising a light chain constant region.

50. 50. The anti-IgE antibody or antigen-binding agent of claim 49, wherein the light chain constant region is a kappa constant region.

51. 51. The anti-IgE antibody or antigen-binding agent of claim 49 or 50, wherein the light chain constant region has a L154P mutation (Kabat).

52. 52. The anti-IgE antibody or antigen binding agent of claim 49 to 51, wherein the light chain variable region and light chain constant region VL-CL have an amino acid sequence selected from SEQ ID NO:39, or SEQ ID NO:41, or SEQ ID NO:117, or SEQ ID NO:119, or SEQ ID NO:125, or SEQ ID NO:127, or SEQ ID NO:136, or SEQ ID NO:143, optionally including a signal sequence having the amino acid sequence of SEQ ID NO:

160.

53. 53. An anti-IgE antibody or antigen-binding agent according to claim 11 to 52, further comprising the heavy chain constant region CH1.

54. 54. The anti-IgE antibody or antigen-binding agent of claim 53, wherein the heavy chain variable region and the heavy chain constant region VH-CH1 have the amino acid sequence of SEQ ID NO:

5.

55. 55. The anti-IgE antibody or antigen-binding agent of claim 11 to 54, further comprising a heavy chain Fc region, Fc.

56. 56. The anti-IgE antibody or antigen-binding agent of claim 55, wherein the Fc is derived from human IgG1 or human IgG4.

57. 57. The anti-IgE antibody or antigen-binding agent of claim 55 or 56, wherein the heavy chain variable region, the heavy chain constant region and the heavy chain Fc region, VH-CH1-Fc, have the amino acid sequence of SEQ ID NO:

9.

58. An anti-IgE antibody or antigen-binding agent comprising a heavy chain variable region comprising a complementarity determining region CDR-H3 having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising a complementarity determining region CDR-L1 having the amino acid sequence of SEQ ID NO: 29, a. The light chain variable region further comprises a framework region FR-L3 having an amino acid sequence of SEQ ID NO: 32, wherein the FR-L3 region enhances the affinity of the anti-IgE antibody or antigen binding agent for human IgE (lower K Ｄ and / or the amino acid sequence of position S67 (see Kabat, SEQ ID NO: 129) is mutated to one of the other naturally occurring amino acids to improve the b. The light chain variable region further comprises a complementarity determining region CDR-L2 having an amino acid sequence of SEQ ID NO: 31, wherein the CDR-L2 region determines the affinity of the anti-IgE antibody or antigen binding agent for human IgE (lower K Ｄ ) is mutated at position S52 (see Kabat, SEQ ID NO: 129) to one of the other natural amino acids to improve The above anti-IgE antibody or antigen-binding agent.

59. 59. The anti-IgE antibody or antigen-binding agent of claim 58, wherein the FR-L3 region is mutated to M, E or D at position S67.

60. 60. The anti-IgE antibody or antigen-binding agent of claim 59, wherein the FR-L3 region is mutated to M at position S67 (Kabat).

61. 61. The anti-IgE antibody or antigen-binding agent of claim 60, wherein the amino acid sequence of the mutated FR-L3 region is selected from SEQ ID NOs: 52-59, 84-107, 131 or 138.

62. 62. The anti-IgE antibody or antigen binding agent of claims 58 to 61, wherein the CDR-L2 region is mutated to E, D, Q or R at position S52 (Kabat).

63. 63. The anti-IgE antibody or antigen-binding agent of claim 62, wherein the CDR-L2 region is mutated to D or E at position S52 (Kabat).

64. 64. The anti-IgE antibody or antigen binding agent of claim 63, wherein the CDR-L2 region is mutated to D at position S52 (Kabat).

65. 65. The anti-IgE antibody or antigen-binding agent of claim 63 or 64, wherein the amino acid sequence of the mutated CDR-L2 region is selected from SEQ ID NO: 50 (preferably) or SEQ ID NO:

51.

66. 66. An anti-IgE antibody or antigen-binding agent according to claims 58 to 65, wherein the heavy chain variable region further comprises a complementarity determining region CDR-H1 having the amino acid sequence which is SEQ ID NO:

14.

67. 67. An anti-IgE antibody or antigen-binding agent according to claims 58 to 66, wherein the heavy chain variable region further comprises a complementarity determining region CDR-H2 having the amino acid sequence which is SEQ ID NO:

16.

68. 68. An anti-IgE antibody or antigen-binding agent according to claims 58 to 67, wherein the light chain variable region further comprises a complementarity determining region CDR-L3 having the amino acid sequence which is SEQ ID NO:

33.

69. 69. The anti-IgE antibody or antigen-binding agent of claims 58 to 68, wherein the heavy chain variable region further comprises a framework region FR-H1 having the amino acid sequence of SEQ ID NO:

13.

70. 70. The anti-IgE antibody or antigen-binding agent of claims 58 to 69, wherein the heavy chain variable region further comprises a framework region FR-H2 having the amino acid sequence of SEQ ID NO:

15.

71. 71. The anti-IgE antibody or antigen-binding agent of claims 58 to 70, wherein the heavy chain variable region further comprises a framework region FR-H3 having the amino acid sequence of SEQ ID NO:

17.

72. 72. The anti-IgE antibody or antigen-binding agent of claims 58 to 71, wherein the heavy chain variable region further comprises a framework region FR-H4 having the amino acid sequence of SEQ ID NO:

19.

73. 73. The anti-IgE antibody or antigen-binding agent of claims 58 to 72, wherein the light chain variable region further comprises a framework region FR-L2 having the amino acid sequence of SEQ ID NO:

30.

74. 74. The anti-IgE antibody or antigen-binding agent of claims 58 to 73, wherein the light chain variable region further comprises a framework region FR-L4 having the amino acid sequence of SEQ ID NO:

34.

75. 75. The anti-IgE antibody or antigen-binding agent of claims 58 to 74, wherein the light chain variable region VL comprises the consecutive FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3 and FR-L4 regions and has an amino acid sequence which is SEQ ID NO:20, with the proviso that the CDR-L2 region has an amino acid sequence selected from SEQ ID NO:50 or SEQ ID NO:

51.

76. 75. The anti-IgE antibody or antigen-binding agent of claims 58 to 74, wherein the light chain variable region VL comprises the consecutive FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3 and FR-L4 regions and has the amino acid sequence of SEQ ID NO:20, with the proviso that the FR-L3 region has the amino acid sequence of SEQ ID NO:

52.

77. 75. The anti-IgE antibody or antigen-binding agent of claims 58 to 74, wherein the light chain variable region VL comprises consecutive FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3 and FR-L4 regions and has an amino acid sequence which is SEQ ID NO:20, with the proviso that the CDR-L2 region has an amino acid sequence selected from SEQ ID NO:50 or SEQ ID NO:51, and the FR-L3 region has an amino acid sequence selected from SEQ ID NO:52, 131 or 138.

78. 78. An anti-IgE antibody or antigen-binding agent according to claim 58 to 77, wherein the heavy chain variable region VH has the amino acid sequence of SEQ ID NO:

1.

79. 79. The anti-IgE antibody or antigen-binding agent of claims 58 to 78, further comprising a light chain constant region.

80. 80. The anti-IgE antibody or antigen-binding agent of claim 79, wherein the light chain constant region is a kappa constant region.

81. 81. The anti-IgE antibody or antigen-binding agent of claim 79 or 80, wherein the light chain variable region and light chain constant region VL-CL have the amino acid sequence of SEQ ID NO:24, with the proviso that the CDR-L2 region has an amino acid sequence selected from SEQ ID NO:50 or SEQ ID NO:

51.

82. 81. The anti-IgE antibody or antigen-binding agent of claim 79 or 80, wherein the light chain variable region and light chain constant region VL-CL have the amino acid sequence of SEQ ID NO:24, provided that the FR-L3 region has the amino acid sequence of SEQ ID NO:

52.

83. 81. The anti-IgE antibody or antigen-binding agent of claim 79 or 80, wherein the light chain variable region and light chain constant region VL-CL have the amino acid sequence of SEQ ID NO:24, with the proviso that the CDR-L2 region has an amino acid sequence selected from SEQ ID NO:50 or SEQ ID NO:51, and the FR-L3 region has an amino acid sequence selected from SEQ ID NO:52, 131 or 138.

84. 1. An anti-IgE antibody or antigen-binding agent comprising a heavy chain variable region and a light chain variable region, comprising: a. the heavy chain variable region comprises a CDR-H1 having an amino acid sequence of SEQ ID NO: 14, a CDR-H2 having an amino acid sequence of SEQ ID NO: 16, and a CDR-H3 having an amino acid sequence of SEQ ID NO: 18; the light chain variable region comprises a CDR-L1 having an amino acid sequence which is SEQ ID NO:29, a CDR-L2 having an amino acid sequence which is SEQ ID NO:50, a CDR-L3 having an amino acid sequence which is SEQ ID NO:33, and a framework region FW-L3 having an amino acid sequence which is SEQ ID NO:131 or 138; or b. the heavy chain variable region comprises an amino acid sequence that is SEQ ID NO: 1, and the light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 132 or 139.

85. 85. The anti-IgE antibody or antigen-binding agent of claim 84, wherein the light chain variable region and light chain constant region VL-CL further comprises a light chain constant region having an amino acid sequence selected from SEQ ID NO: 137 or 145, optionally including a signal sequence having the amino acid sequence of SEQ ID NO:

160.

86. 86. An anti-IgE antibody or antigen-binding agent according to claim 58 to 85, further comprising the heavy chain constant region CH1.

87. 87. The anti-IgE antibody or antigen-binding agent of claim 86, wherein the heavy chain variable region and the heavy chain constant region VH-CH1 have the amino acid sequence of SEQ ID NO:

5.

88. 88. The anti-IgE antibody or antigen-binding agent of claims 58 to 87, further comprising a heavy chain Fc region, Fc.

89. 89. The anti-IgE antibody or antigen-binding agent of claim 88, wherein the Fc is derived from human IgG1 or human IgG4.

90. 90. An anti-IgE antibody or antigen-binding agent according to claim 88 or 89, wherein the heavy chain variable region, the heavy chain constant region and the heavy chain Fc region VH-CH1-Fc have the amino acid sequence of SEQ ID NO:

9.

91. 91. The anti-IgE antibody or antigen-binding agent of claim 1 , selected from the group consisting of an intact antibody molecule having full-length heavy and light chains, or a fragment thereof.

92. Fab fragment, modified Fab' fragment, Fab' fragment, F(ab') ２ Fragment, Fv, scFv, scab, diabody, bispecific antibody, triabody, FabFv, Fab-Fv-Fv, tribody, or (Fab-Fv) ２ -Fc.

93. 93. The anti-IgE antibody of claim 92, which is a Fab fragment linked directly or via a linker to a scFv that binds to a serum carrier protein, such as human serum albumin.

94. The scFv comprises a heavy chain variable region and a light chain variable region, preferably linked via a linker having SEQ ID NO: 151; the heavy chain variable region comprises a CDR-H1 having an amino acid sequence of SEQ ID NO: 152, a CDR-H2 having an amino acid sequence of SEQ ID NO: 153, and a CDR-H3 having an amino acid sequence of SEQ ID NO: 154; The light chain variable region comprises CDR-L1 having an amino acid sequence of SEQ ID NO: 155, CDR-L2 having an amino acid sequence of SEQ ID NO: 156, and CDR-L3 having an amino acid sequence of SEQ ID NO:

157.

94. An anti-IgE antibody as described in claim 93.

95. 95. An anti-IgE antibody as described in claim 93 or 94, wherein the scFv has an amino acid sequence of SEQ ID NO:

150.

96. 95. The anti-IgE antibody of claim 93 or 94, wherein the Fab fragment comprises a heavy chain variable region and a light chain variable region, and comprises: a. the heavy chain variable region comprises a CDR-H1 having an amino acid sequence of SEQ ID NO: 14, a CDR-H2 having an amino acid sequence of SEQ ID NO: 16, and a CDR-H3 having an amino acid sequence of SEQ ID NO: 18; the light chain variable region comprises a CDR-L1 having an amino acid sequence which is SEQ ID NO:29, a CDR-L2 having an amino acid sequence which is SEQ ID NO:50, a CDR-L3 having an amino acid sequence which is SEQ ID NO:33, and a framework region FW-L3 having an amino acid sequence which is SEQ ID NO:131 or 138; or b. the heavy chain variable region comprises an amino acid sequence that is SEQ ID NO:1; The light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 132 or 139.

97. 97. The anti-IgE antibody of claim 96, wherein the Fab fragment further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain variable region and the heavy chain constant region VL-CH1 have the amino acid sequence of SEQ ID NO:5, and the light chain variable region and the light chain constant region VL-CL have the amino acid sequence selected from SEQ ID NO:136 or 143, and optionally a signal sequence having the amino acid sequence of SEQ ID NO:

160.

98. 98. An anti-IgE antibody as described in claim 93 or 97, wherein the scFv is linked to the CH1 of the Fab fragment via a linker having the amino acid sequence of SEQ ID NO:

149.

99. 99. An anti-IgE antibody as described in claim 93 or 98, wherein the heavy chain variable and constant regions, the linker and the scFv have the amino acid sequence of SEQ ID NO: 147, optionally including a signal sequence having the amino acid sequence of SEQ ID NO:

160.

100. 100. An anti-IgE antibody or antigen-binding agent according to claim 1 to 99, having an effector or reporter molecule attached thereto.

101. 101. The anti-IgE antibody or antigen-binding agent of claim 1 , wherein the anti-IgE antibody or antigen-binding agent is glycosylated and / or conjugated to a polymer selected from starch, albumin, and polyethylene glycol (PEG).

102. 102. The anti-IgE antibody or antigen-binding agent of claim 101, wherein the polymer is PEG having a molecular weight in the range of 5-50 kDa.

103. 103. The anti-IgE antibody or antigen-binding agent of claim 1 which is humanized.

104. 104. An isolated DNA sequence encoding the heavy and / or light chain(s) of an anti-IgE antibody or antigen-binding agent according to claims 1 to 103.

105. A cloning or expression vector comprising one or more DNA sequences according to claim 104.

106. A cloning or expression vector comprising one or more DNA sequences selected from SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:42, or SEQ ID NO:133, or SEQ ID NO:135, or SEQ ID NO:137, or SEQ ID NO:140, or SEQ ID NO:142, or SEQ ID NO:

144.

107. 107. The cloning or expression vector of claim 106, further comprising one or more DNA sequences selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12 or SEQ ID NO:

148.

108. 108. A host cell comprising one or more cloning or expression vectors according to claims 105 to 107.

109. The host cell of claim 108, further comprising one or more cloning or expression vectors comprising one or more DNA sequences selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12 or SEQ ID NO:

148.

110. 110. A method for the production of an anti-IgE antibody or antigen-binding agent according to claims 1 to 94, comprising culturing a host cell according to claim 108 or 109, and isolating the anti-IgE antibody or antigen-binding agent.

111. 104. A pharmaceutical composition comprising an anti-IgE antibody or antigen-binding agent according to claims 1 to 103 together with one or more pharma- ceutically acceptable excipients, diluents or carriers.

112. 112. The pharmaceutical composition of claim 111, wherein the anti-IgE antibody or antigen-binding agent is present in a dosage of 50-200 mg / mL of diluent, preferably 150 mg / mL.

113. 113. The pharmaceutical composition of claim 111 or 112, wherein the excipient comprises L-arginine.

114. 114. The pharmaceutical composition of claims 111-113, wherein the excipient comprises L-histidine.

115. 115. The pharmaceutical composition of claims 111 to 114, wherein the excipient comprises polysorbate 20.

116. 116. The pharmaceutical composition of claims 111 to 115, wherein the diluent is water.

117. 117. The pharmaceutical composition of claims 111 to 116, wherein the composition is contained in a sterile syringe for subcutaneous administration thereof.

118. 118. The pharmaceutical composition of claims 111 to 117, wherein the composition contains a total dose of 75 to 600 mg of the anti-IgE antibody or antigen-binding agent.

119. 119. The pharmaceutical composition of claims 111 to 118, further comprising other active ingredients together with the anti-IgE antibody, or antigen-binding agent, or for separate co-administration with the anti-IgE antibody, or antigen-binding agent.

120. 120. The pharmaceutical composition of claim 119, wherein the anti-IgE antibody or antigen-binding agent is co-administered separately with the allergy-based specific immunotherapy.

121. 112. An anti-IgE antibody or antigen-binding agent according to claims 1 to 103, or a composition according to claims 102 to 111 for use as a medicament.

122. 112. An anti-IgE antibody or antigen-binding agent according to claims 1 to 103, or a composition according to claims 102 to 111 for use in the treatment or prevention of disease.

123. An anti-IgE antibody or antigen-binding agent according to claims 1 to 103, or a composition according to claims 102 to 111, for use in the treatment or prevention of a disorder associated with the complex of human IgE and FcεRI.

124. An anti-IgE antibody or antigen-binding agent described in claims 1 to 103, or a composition described in claims 102 to 111, for use in treating or preventing disorders caused by dissociation of the complex between human IgE and FcεRI and binding of human IgE by the anti-IgE antibody or antigen-binding agent.

125. 121. An anti-IgE antibody, or antigen-binding agent, according to claims 1 to 103, or a composition for use according to claims 111 to 120, for use in the treatment or prevention of one or more of the following: Allergy; Allergic asthma; Severe asthma; Moderate asthma; Chronic spontaneous urticaria; Chronic idiopathic urticaria; Perennial allergic rhinitis; Seasonal allergic rhinitis; Acute asthma exacerbation; Acute bronchospasm; Status asthmaticus; Hyper IgE syndrome; Allergic bronchopulmonary aspergillosis; Prevention of anaphylactic reactions; Food allergies; Atopic dermatitis; allergic rhinitis; bee venom hypersensitivity; idiopathic anaphylaxis; peanut allergy; latex allergy; Inflammatory skin diseases; urticaria (sun-, cold-induced, local heat-induced, and / or delayed pressure-induced); cutaneous mastocytosis; systemic mastocytosis; eosinophil-associated gastrointestinal disorders; bullous pemphigoid; interstitial cystitis; nasal polyps; idiopathic angioedema; or non-allergic asthma.

126. A method for treating or preventing a disease in a human subject, comprising administering to the subject an effective amount of an anti-IgE antibody or antigen-binding agent described in claims 1 to 103, or a composition described in claims 111 to 120.

127. A method according to claim 126 for the treatment or prevention of a disorder associated with the complex of human IgE and FcεRI.

128. 128. The method of claim 126 or 127, wherein the treatment or prevention is mediated through dissociation of the complex between human IgE and FcεRI and binding of human IgE by an anti-IgE antibody or antigen-binding agent.

129. 129. The method of claims 126 to 128, for the treatment or prevention of one or more of the following: Allergy; Allergic asthma; Severe asthma; Moderate asthma; Chronic spontaneous urticaria; Chronic idiopathic urticaria; Perennial allergic rhinitis; Seasonal allergic rhinitis; Acute asthma exacerbation; Acute bronchospasm; Status asthmaticus; Hyper IgE syndrome; Allergic bronchopulmonary aspergillosis; Prevention of anaphylactic reactions; Food allergies; Atopic dermatitis; allergic rhinitis; bee venom hypersensitivity; idiopathic anaphylaxis; peanut allergy; latex allergy; Inflammatory skin diseases; urticaria (sun-, cold-induced, local heat-induced, and / or delayed pressure-induced); cutaneous mastocytosis; systemic mastocytosis; eosinophil-associated gastrointestinal disorders; bullous pemphigoid; interstitial cystitis; nasal polyps; idiopathic angioedema; or non-allergic asthma.

130. An anti-IgE antibody or antigen-binding agent according to claims 1 to 103, or a composition according to claims 111 to 120, or an anti-IgE antibody or antigen-binding agent for use according to claims 121 to 125, or a composition, or a method according to claims 126 to 129, wherein the anti-IgE antibody or antigen-binding agent is capable of dissociating human IgE from FcεRI at a concentration (or peak serum concentration) of less than 7, 3, 1, 0.66, 0.5 or 0.3 μM.

131. An anti-IgE antibody or antigen-binding agent according to claims 1 to 103, or a composition according to claims 111 to 120, or an anti-IgE antibody or antigen-binding agent for use according to claims 121 to 125, or a method according to claims 126 to 129, wherein the anti-IgE antibody or antigen-binding agent is capable of dissociating human IgE from FcεRI at a lower concentration (or peak serum concentration) than omalizumab (or alternatively, omalizumab Fab).

132. An anti-IgE antibody or antigen-binding agent according to claims 1 to 103, or a composition according to claims 111 to 120, or an anti-IgE antibody or antigen-binding agent for use according to claims 121 to 125, or a method according to claims 126 to 129, wherein the anti-IgE antibody or antigen-binding agent is capable of a higher percentage dissociation of human IgE from FcεRI than omalizumab (or alternatively, omalizumab Fab).

133. An anti-IgE antibody or antigen-binding agent according to claims 1 to 103, or a composition according to claims 111 to 120, or an anti-IgE antibody or antigen-binding agent for use according to claims 121 to 125, or a composition, or a method according to claims 126 to 129, wherein the anti-IgE antibody or antigen-binding agent is capable of effecting an apparent dissociation rate of human IgE from FcεRI that is higher than for omalizumab (or alternatively, omalizumab Fab).

134. The anti-IgE antibody or antigen-binding agent has an improved affinity (K) for human IgE that is at least 10% lower than that of omalizumab (or alternatively, omalizumab Fab). Ｄ 130. An anti-IgE antibody or antigen-binding agent according to claims 1 to 103, or a composition according to claims 111 to 120, or an anti-IgE antibody or antigen-binding agent or composition for use according to claims 121 to 125, or a method according to claims 126 to 129, having a specific binding domain (e.g. using IgE-Fc).

135. An anti-IgE antibody or antigen-binding agent according to claims 1 to 103, or a composition according to claims 111 to 120, or an anti-IgE antibody or antigen-binding agent for use according to claims 121 to 125, or a composition, or a method according to claims 126 to 129, wherein the anti-IgE antibody or antigen-binding agent is not omalizumab or omalizumab Fab.

136. An antibody or antigen-binding agent that binds to free human IgE and human IgE bound to FcεRI, wherein the antibody or antigen-binding agent binds to human IgE bound to FcεRI and changes the conformation of IgE such that human IgE bound to FcεRI in said conformation has a weaker binding affinity for FcεRI than in the absence of the antibody or antigen-binding agent, and human IgE bound to FcεRI dissociates from FcεRI.

137. 137. The antibody or antigen-binding agent of claim 136, wherein human IgE bound to FcεRI in a conformation has a lower binding affinity for omalizumab, or a fragment thereof, than for the antibody or antigen-binding agent.

138. 138. An antibody or antigen-binding agent according to claim 136 or 137, which is an antibody according to any one of claims 1 to 103, 130 to 135.

139. 139. An antibody or antigen-binding agent according to claims 136 to 138 for use as a medicament.

140. 139. An antibody or antigen-binding agent according to claims 136 to 138 for use in the treatment or prevention of a disorder associated with the complex of human IgE and FcεRI.

141. 139. An antibody or antigen-binding agent according to claims 136 to 138 for use in the treatment or prevention of a disorder mediated by dissociation of the complex between human IgE and FcεRI and binding of human IgE by the antibody or antigen-binding agent.

142. 139. The antibody or antigen-binding agent for use according to claims 136 to 138 for the treatment or prevention of one or more of the following: Allergy; Allergic asthma; Severe asthma; Moderate asthma; Chronic spontaneous urticaria; Chronic idiopathic urticaria; Perennial allergic rhinitis; Seasonal allergic rhinitis; Acute asthma exacerbation; Acute bronchospasm; Status asthmaticus; Hyper IgE syndrome; Allergic bronchopulmonary aspergillosis; Prevention of anaphylactic reactions; Food allergies; Atopic dermatitis; allergic rhinitis; bee venom hypersensitivity; idiopathic anaphylaxis; peanut allergy; latex allergy; Inflammatory skin diseases; urticaria (sun-, cold-induced, local heat-induced, and / or delayed pressure-induced); cutaneous mastocytosis; systemic mastocytosis; eosinophil-associated gastrointestinal disorders; bullous pemphigoid; interstitial cystitis; nasal polyps; idiopathic angioedema; or non-allergic asthma.

143. A method for the treatment or prevention of a disease in a human subject, comprising administering to the subject an effective amount of an antibody or antigen-binding agent described in claims 136 to 138.

144. A method according to claim 143 for the treatment or prevention of a disorder associated with the complex of human IgE and FcεRI.

145. 145. The method of claim 143 or 144, wherein the treatment or prevention is mediated through dissociation of the complex between human IgE and FcεRI and binding of human IgE by an antibody or antigen-binding agent.

146. 146. The method of claims 143 to 145, for the treatment or prevention of one or more of the following: Allergy; Allergic asthma; Severe asthma; Moderate asthma; Chronic spontaneous urticaria; Chronic idiopathic urticaria; Perennial allergic rhinitis; Seasonal allergic rhinitis; Acute asthma exacerbation; Acute bronchospasm; Status asthmaticus; Hyper IgE syndrome; Allergic bronchopulmonary aspergillosis; Prevention of anaphylactic reactions; Food allergies; Atopic dermatitis; allergic rhinitis; bee venom hypersensitivity; idiopathic anaphylaxis; peanut allergy; latex allergy; Inflammatory skin diseases; urticaria (sun-, cold-induced, local heat-induced, and / or delayed pressure-induced); cutaneous mastocytosis; systemic mastocytosis; eosinophil-associated gastrointestinal disorders; bullous pemphigoid; interstitial cystitis; nasal polyps; idiopathic angioedema; or non-allergic asthma.

147. 139. A method for selecting an antibody or antigen-binding agent according to claim 1 to 103 or 130 to 138, comprising the steps of: a. contacting a test antibody or antigen-binding agent with a sample containing human IgE bound to human FcεRI; b. Measuring the dissociation constant of the test antibody or antigen-binding agent for dissociation of human IgE from human FcεRI; c. Comparing the dissociation constant measured in step b) with the dissociation constant of omalizumab or a fragment thereof for dissociation of human IgE from human FcεRI; d. Selecting said antibody or antigen-binding agent if it dissociates IgE from FcεRI faster than omalizumab or a fragment thereof. The above method.

148. 139. A method for selecting an antibody or antigen-binding agent according to claims 1 to 103 or 130 to 138, comprising: a. contacting a test antibody or antigen-binding agent with a sample containing human IgE bound to human FcεRI; b. Measuring the binding affinity of the test antibody or antigen-binding agent for human IgE from human FcεRI; c. Comparing the binding affinity measured in step b) with the binding affinity of human IgE to human FcεRI; d. Selecting the antibody or antigen-binding agent if it has a higher binding affinity for IgE than IgE to FcεRI.

149. 149. The method of claim 147 or 148, comprising: The selected antibody or antigen binding agent causes IgE to adopt the following conformation while remaining bound to FcεRI and in a stabilized conformation: a. dissociates from FcεRI faster than IgE bound to FcεRI in the presence of omalizumab or a fragment thereof; and / or b. It has a higher binding affinity for the antibody or antigen-binding agent than for FcεRI.

150. Anti-IgE antibodies or antigen-binding agents, including: a. a heavy chain variable region comprising SEQ ID NO:1; i. SEQ ID NO: 109; or ii. SEQ ID NO: 113; or iii. SEQ ID NO: 121; or iv. SEQ ID NO: 132; or v. SEQ ID NO: 139 or a light chain variable region comprising b. SEQ ID NO:5; i. SEQ ID NO:24, in which S77 and S79 are replaced with Q; ii. SEQ ID NO: 117; or iii. SEQ ID NO: 125; or iv. SEQ ID NO: 136; or v. SEQ ID NO:

143.

151. The anti-IgE antibody or antigen binding agent of claim 150, wherein the antibody or antigen binding agent is in contact with or contacts and is specific for an epitope comprising: With reference to SEQ ID NO: 108, residues T373, W374, S375, R376, A377, S378, G379, P381, Q417, C418, R419, T421, P426, R427, A428 of the Cε3 domain of human IgE and residues D278 and T281 of the Cε2 domain.

152. an antibody or antigen-binding agent that binds to a first polypeptide, which elicits a physiological response by binding to a second polypeptide (such as a receptor); the antibody or antigen binding agent is capable of binding to both free and bound said first polypeptide and stabilizing a conformation of the first polypeptide, the first polypeptide having a lower binding affinity for a second polypeptide in the stabilized conformation in the presence of the antibody or antigen binding agent than in the absence of the antibody or antigen binding agent; The antibody or antigen-binding agent induces a more rapid dissociation of the first polypeptide from the second polypeptide.

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