AU2012238233A1 - Binding members for IgE molecules - Google Patents

Abstract

C kNRPorbl\DCC\RBR\4631614_I DOC-3/10/2012 This invention relates to binding members, especially antibody molecules, for IgE. The binding members are useful for, inter alia, treatment of disorders mediated by IgE 5 including allergies and asthma.

Description

Australian Patents Act 1990 - Regulation 3.2A ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title "Binding members for IgE molecules" The following statement is a full description of this invention, including the best method of performing it known to us:- C NRPoRl\DCC\RBR\4651614_ I DOC-/1/2012 BINDING MEMBERS FOR IgE MOLECULES This application is a divisional application of Australian Application No. 2008216008 the specification and drawings of which as originally filed are incorporated 5 herein in their entirety by reference. Field of the Invention This invention relates to binding members, especially antibody molecules, for IgE. 10 The binding members are useful for, inter alia, treatment of disorders mediated by IgE including allergies and asthma. IgE is a member of the immunoglobulin family and mediates allergic responses such as asthma, food allergies, type I hypersensitivity and sinus inflammation. 15 IgE is secreted by, and expressed on the surface of, B-cells. Briefly, IgE is anchored in the B-cell membrane by a transmembrane domain that is linked to the mature IgE molecule through a short membrane-binding region. IgE may also be bound via its Fc region to B-cells, monocytes, eosinophils and platelets through a low affinity IgE receptor 20 (FcERll, also known as CD23). Upon exposure to an allergen, B-cells that produce allergen-specific IgE are clonally amplified. Allergen-specific IgE is then released into the circulation by the B-cells where it is in turn bound by B-cells through the FceRlI, as well as by mast cells and basophils through a high affinity receptor (FccRI). Such mast cells and basophils are thereby sensitized for allergen. Subsequent exposure to the allergen 25 cross-links the FceRI on mast cells and basophils thereby activating their release of histamine and other factors responsible for clinical hypersensitivity and anaphylaxis. Binding members that inhibit binding to and functional activity through FcERI with or without simultaneous inhibition of FcERII are useful for inhibiting IgE-mediated 30 disease conditions, such as allergies and asthma.

C:\NRPonbr\DCC\RBR\4651614 I DOC-3/11/2012 la It is generally understood that FcERI and FcERIl bind to recognition site(s) in the IgE constant (Fc) domain. Various studies have been undertaken to identify these recognition sites. For example, peptides corresponding to specific portions of the IgE molecule have been used as either competitive inhibitors of IgE-receptor binding (Burt et 5 al., Eur. J. Immun, 17:437-440 [1987]; Helm et al., Nature, 331:180-183 [1988]; Helm et al., Proc. Natl. Acad. Sci., 86:9465-9469 [1989]; Vercelli et al., Nature, 338:649-651 [1989]; Nio et al., Peptide 2 Chemistry, 203-208 [1990]), or to elicit anti-IgE antibodies that might block IgE-receptor interaction (Burt et al., Molec. Immun. 24:379-389 [1987]; Robertson et al., Molec. Immun., 25:103-113 [1988]; Baniyash et al., Molec. immun. 25:705-711 [1988]). 5 More recently, XolairO (Omalizumab) has been produced and marketed for treating asthma patients. Xolair@ is a humanized IgGlk monoclonal antibody that selectively binds to human IgE, thereby reducing the binding of IgE to at least FceRI on the surface of mast cells and basophils. By reducing surface-bound IgE on Fc&RI-bearing cells, Xolair@ reduces somewhat the degree of release of mediators of the allergic response. Xolair@ is disclosed in 10 International patent application publication numbers: WO 93/04173 and WO 97/04807. However, other binding members for IgE, such as those with a higher affinity and/or potency than Xolair@, are needed to improve this promising therapeutic strategy. The Invention 15 By utilising appropriately designed selection techniques and assays, we have developed binding members which inhibit binding to and functional activity through FcERI (the high-affinity IgE receptor present on mast cells) with or without simultaneous inhibition of FcERII. 20 A binding member of the invention inhibits binding to and functional activity through FcERI with or without simultaneous inhibition of FcERII. The inhibition of binding may be by direct inhibition, for example, by neutralizing IgE. The binding member of the invention typically neutralizes human IgE with an IC50 of less than about 10 tuM as determined by, for example, an RBL-ER51 calcium signalling assay. In certain embodiments, the binding 25 member of the invention neutralizes human IgE with an IC50 of less than about I nM, or less than about 0.5 nM, or less than about 0.2 nM as determined by an RBL-ER51 calcium signalling assay, for example. The binding members of the invention may also bind to and neutralize non-human 30 IgE, meaning IgE orthologs that occur naturally in species other than human.

3 Binding members of the invention are normally specific for IgE over other immunoglobulins, and thus bind IgE selectively. Such selectivity may be determined or demonstrated, for example, in a standard competition assay. 5 The binding members are useful for treating and/or preventing disorders that are mediated by IgE, especially allergies and asthma. The binding members are useful for reducing circulating free IgE in a mammal, and useful for inhibiting allergen-induced mast cell degranulation either in vivo or in vitro. 10 The binding members are further useful for inhibiting biological responses mediated by IgE bound to FcERI with or without simultaneous inhibition of biological responses mediated by IgE bound to FcERII, either in vivo or in vitro. 15 The binding members of the invention also have diagnostic utility, such as for detecting the presence or amount of IgE, or the presence or amount of allergen-specific IgE, in a sample of interest, such as a sample from an asthmatic or allergic patient. Any suitable method may be used to determine the sequence of residues bound by a 20 binding member. For example, a peptide-binding scan may be used, such as a PEPSCAN based enzyme linked immuno assay (ELISA) as described in detail elsewhere herein. In a peptide-binding scan, such as the kind provided by PEPSCAN Systems, short overlapping peptides derived from the antigen are systematically screened for binding to a binding member. The peptides may be covalently coupled to a support surface to form an array of 25 peptides. Peptides may be in a linear or constrained conformation. A constrained conformation may be produced using peptides having a terminal Cys residue at each end of the peptide sequence. The Cys residues can be covalently coupled directly or indirectly to a support surface such that the peptide is held in a looped conformation. Thus, peptides used in the method may have Cys residues added to each end of a peptide sequence corresponding to 30 a fragment of the antigen. Double looped peptides may also be used, in which a Cys residue is additionally located at or near the middle of the peptide sequence. The Cys residues can be covalently coupled directly or indirectly to a support surface such that the peptides form a double-looped conformation, with one loop on each side of the central Cys residue. Peptides 4 can be synthetically generated, and Cys residues can therefore be engineered at desired locations, despite not occurring naturally in the IgE sequence. Optionally, linear and constrained peptides may both be screened in a peptide-binding assay. A peptide-binding scan may involve identifying (e.g. using ELISA) a set of peptides to which the binding 5 member binds, wherein the peptides have amino acid sequences corresponding to fragments of IgE (e.g. peptides of about 5, 10 or 15 contiguous residues of IgE), and aligning the peptides in order to determine a footprint of residues bound by the binding member, where the footprint comprises residues common to overlapping peptides. 10 Alternatively or additionally the peptide-binding scan method may involve identifying peptides to which the binding member binds with at least a given signal:noise ratio. Details of a suitable peptide-binding scan method for determining binding are known in the art. Other methods that are well known in the art and that could be used to determine the residues bound by an antibody, and/or to confirm peptide-binding scan results, include site directed 15 mutagenesis, hydrogen deuterium exchange, mass spectrometry, NMR, and X-ray crystallography. A binding member of the invention may or may not bind and/or neutralise IgE variants. Thus, a binding member of the invention may or may not inhibit binding of IgE 20 variants to FcERI with or without simultaneous inhibition of FcERII. Linear epitope sequences of IgE, e.g. as isolated peptide fragments or polypeptides comprising them, may be employed to identify, generate, isolate and/or test binding members of the present invention. 25 As described in more detail below, binding members according to the invention have been shown to neutralise IgE with high potency. Neutralisation means inhibition of a biological activity of IgE. Binding members of the invention may neutralise one or more biological activities of IgE, but typically inhibit IgE binding to FcERI with or without 30 simultaneous inhibition of binding to FcERII.

5 Neutralisation of IgE binding to FcERI with or without simultaneous inhibition of FcERIL may optionally be measured as a function of the biological activity of the receptor, such as allergen-induced mast cell degranulation. 5 Suitable assays for measuring neutralisation of IgE by binding members of the invention include, for example, ligand receptor biochemical assays and surface plasmon resonance (SPR) (e.g., BIACORE). Inhibition of biological activity may be partial or total. Binding members may inhibit 10 an IgE biological activity, such as receptor binding or mast cell degranulation, by 100%, or alternatively by: at least 95 %, at least 90 %, at least 85 %, at least 80 %, at least 75 %, at least 70 %, at least 60 %, or at least 50 % of the activity in absence of the binding member. The neutralising potency of a binding member is normally expressed as an ICso value, 15 in nM unless otherwise stated. In functional assays, IC 50 is the concentration of a binding member that reduces a biological response by 50 % of its maximum. In ligand-binding studies, IC 50 is the concentration that reduces receptor binding by 50 % of maximal specific binding level. IC 50 may be calculated by plotting % of maximal biological response as a function of the log of the binding member concentration, and using a software program, such 20 as Prism (GraphPad) to fit a sigmoidal function to the data to generate ICso values. Potency may be determined or measured using one or more assays known to the skilled person and/or as described or referred to herein. The neutralising potency of a binding member can be expressed as a geomean. 25 Geomean (also known as geometric mean), as used herein means the average of the logarithmic values of a data set, converted back to a base 10 number. This requires there to be at least two measurements, e.g. at least 2, preferably at least 5, more preferably at least 10 replicate. The person skilled in the art will appreciate that the greater the number of replicates the more robust the geomean value will be. The choice of replicate number can be left to the 30 discretion of the person skilled in the art. Neutralisation of IgE activity by a binding member in an assay described herein, indicates that the binding member binds to and neutralises IgE. Other methods that may be 6 used for determining binding of a binding member to IgE include ELISA, Western blotting, immunoprecipitation, affinity chromatography and biochemical assays. In another embodiment of the invention there is provided an isolated binding member 5 specific for immunoglobulin E which binding member has an IC50 for the binding of said binding member to immunoglobulin E in serum at least 10 fold lower than XolairTM, or alternatively at least 20 fold lower, at least 50 fold lower, at least 75 folder lower, at least 100 fold lower, at least 125 fold lower, at least 150 fold lower, at least 200 fold lower, at least 300 fold lower, at least 400 fold lower or at least 500 fold lower. 10 Neutralising potency of a binding member as calculated in an assay using IgE from a first species (e.g. human) may be compared with neutralising potency of the binding member in a similar assay under similar conditions using IgE from a second species (e.g. cynomolgus monkey), in order to assess the extent of cross-reactivity of the binding member for IgE of the 15 two species. Alternatively, cross-reactivity may be assessed in a competition binding assay, as described in more detail elsewhere herein. A binding member of the invention may have a greater neutralising potency in a human IgE binding or biological assay than in a similar assay with IgE from a species other 20 than human. Thus, neutralising potency of a binding member in an assay with human IgE may be greater than in a similar assay with IgE from a species other than human. Potency in a human IgE binding or biological assay may, for example, be about 5-fold greater than in a similar assay employing IgE of cynomolgus monkey, or in another embodiment, may be about 15 or 20 fold greater. More specifically, potency in the human RBL-ER51 calcium 25 signalling assay may be determined for a concentration of human IgE of 25 nglml, and compared to the potency using lOOng/mI of cynomolgus IgE under otherwise similar conditions. Examples of data obtained in similar RBL-ER51 calcium signalling assays using human IgE and cynomolgus IgE are shown in Table 2b. 30 A binding member of the invention may have a stronger affinity for human IgE than for IgE of other species. Affinity of a binding member for human IgE may be, for example, about 5 or 10-fold stronger than for cynomolgus monkey IgE, or in another embodiment, may 7 be about 100-fold stronger. Examples of data obtained for both human and cynomolgus monkey IgE are shown in Table 2a and b. A binding member of the invention may have an IgE-neutralising potency or IC 50 of 5 about 10 nM or less, with a 25 ng/ml concentration of human IgE in, for example, an RBL ER51 calcium signalling assay. Alternatively, the IC 50 is less than about 3 nM. In other embodiments, the ICso is less than about 1 nM, or less than about 0.5 nM, or less than about 0.2 nM. 10 In another embodiment of the invention there is provided an isolated binding member specific for immunoglobulin E which binding member has an IC50 geomean for inhibition of calcium signalling induced by 25ng/ml IgE in RBL-ER51 cells of less than InM, or alternatively less than 0.6nM, less than 0.5nM, less than 0.4nM, less than 0.3nM, less than 0.2nM or less than 0.lnM. 15 Binding kinetics and affinity (expressed as the equilibrium dissociation constant KD) of IgE-binding members for human IgE may be determined, e.g. using surface plasmon resonance (BIACORE). Binding members of the invention normally have an affinity for human IgE (KD) of less than about 10 nM, and in some embodiments have a KD of less than 20 about 5 nM, in other embodiments have a KD of less than 2nM. Affinity for cynomolgus monkey IgE is normally less than about 20 nM, in some embodiments have a KD of less than about 10 nM. A number of methodologies are available for the measurement of binding affinity of an 25 antibody to its antigens, one such methodology is KinExA. The Kinetic Exclusion Assay (KinExA) is a general purpose immunoassay platform (basically a flow spectrofluorimeter) that is capable of measuring equilibrium dissociation constants, and association and dissociation rate constants for antigen/antibody interactions. Since KinExA is performed after equilibrium has been obtained, it is an advantageous technique to use for measuring the KD of 30 a multivalent antigen/mAb interaction. The binding of an antibody to an IgE molecule is an example of binding to a multivalent antigen. The use of KinExA is particularly appropriate where a multivalent antigen means that multimers of antibody and antigen are formed comprising more than one antibody and more than one antigen. In such complex interaction 8 models accurate KD estimation can be difficult. The KinExA methodology can be conducted as described in Drake et al (2004) Analytical Biochemistry 328, 35-43. As measured by the KinExA methodology Antibody 11 has a KD of 6.3pM substantially lower than XotairTM which has a KD of 353pM. 5 In another embodiment of the invention there is provided an isolated binding member specific for immunoglobulin E with a KD of 300pM or lower as measured using the KinExA methodology. Alternatively a KD of 200pM or lower, 100pM or lower, 5OpM or lower, 20pM or lower or 10pM or lower. 10 In vivo endogenous IgE may be glycosylated and therefore glycosylated human IgE is a therapeutic target for human therapy. While recombinant human IgE, which may be bacterially-derived and not glycosylated, may be used in assays described herein, binding members of the invention may bind glycosylated human IgE, such as IgE produced by a 15 myeloma cell line such as U266.Bl. This represents a significant advantage of binding members of the invention, since glycosylated human IgE is the target antigen for in vivo human applications. A binding member of the invention may comprise an antibody molecule, preferably a 20 human antibody molecule or a humanized antibody molecule. In one aspect of the invention, the antibody molecule is a monoclonal antibody. An antigen binding site is generally formed by the variable heavy (VH) and variable light (VL) immunoglobulin domains, with the antigen-binding interface formed by six 25 surface polypeptide loops, termed complimentarity determining regions (CDRs). There are three CDRs in each VH (HCDRI, HCDR2, HCDR3) and in each VL LCDR1, LCDR2, LCDR3), together with framework regions (FRs). The binding member of the invention normally comprises an antibody VH and/or VL 30 domain. A VH domain of the invention comprises a set of HCDRs, and a VL domain comprises a set of LCDRs. An antibody molecule may comprise an antibody VH domain comprising a VH CDR1, CDR2 and CDR3 and a framework. It may alternatively or also comprise an antibody VL domain comprising a VL CDRI, CDR2 and CDR3 and a 9 framework. Examples of antibody VH domains (SEQ ID NOS:2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 288, 300, and 306) and antibody VL domains (SEQ ID NOS: 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 5 364, 366, 368, 370, 372, 374, 376, 378 and 380) and CDRs (SEQ ID NOS:3-5, 8-10, 13-15, 18-20, 23-25, 28-30, 33-35, 38-40, 43-45, 48-50, 53-55, 58-60, 63-65, 68-70, 73-75, 78-80, 83-85, 88-90, 93-95, 98-100, 103-105, 108-110, 113-115, 118-120, 123-125, 128-130, 133 135, 138-140, 143-145, 148-150, 153-155, 158-160, 163-165, 168-170, 173-175, 178-180, 183-185, 188-190, 193-195, 198-200, 203-205, 208-210, 213-215, 218-220, 223-225, 228 10 230, 233-235, 238-240, 243-245, 248-250, 253-255, 258-260, 263-265, 268-270, 273-275, 278-280, 283-285, 296-298, 289-291, 296-298, 301-303, 307-309, and 314-316) according to the present invention are as listed in the appended sequence listing that forms part of the present disclosure (also see Table 3a). Further CDRs are disclosed below and in Table 1. All VH and VL sequences, CDR sequences, sets of CDRs and sets of HCDRs and sets of LCDRs 15 disclosed herein represent aspects and embodiments of the invention. As described herein, a "set of CDRs" comprises CDRI, CDR2 and CDR3. Thus, a set of HCDRs refers to HCDR1, HCDR2 and HCDR3, and a set of LCDRs refers to LCDRI, LCDR2 and LCDR3. Unless otherwise stated, a "set of CDRs" includes HCDRs and LCDRs. 20 Alternatively, a binding member of the invention may comprise an antigen-binding site within a non-antibody molecule, normally provided by one or more CDRs e.g. a set of CDRs in a non-antibody protein scaffold, as discussed further below. 25 As described herein, a parent antibody molecule was isolated having the set of CDR sequences as shown in Table 1 (see Antibody 1). Through a process of optimisation we generated a panel of antibody clones numbered 2-28, with CDR sequences derived from the parent CDR sequences and having modifications at the positions indicated in Table 1. Thus, for example, it can be seen from Table 1 that Antibody 2 has the parent HCDR1, HCDR2, 30 LCDR1, LCDR2, and LCDR3 sequences, and has a parent HCDR3 sequence in which: Kabat residue 96 is replaced with S, Kabat residue 97 is replaced with L, Kabat residue 99 is replaced with S, and Kabat residue 100 is replaced with A.

10 Described herein is a binding member comprising the parent set of CDRs as shown in Table 1 (Antibody 1), in which HCDRI is SEQ ID NO: 3 (Kabat residues 31-35), HCDR2 is SEQ ID NO: 4 (Kabat residues 50-65), HCDR3 is SEQ ID NO: 5 (Kabat residues 95-102), LCDRI is SEQ ID NO: 8 (Kabat residues 24-34), LCDR2 is SEQ ID NO: 9 (Kabat residues 5 50-56) and LCDR3 is SEQ ID NO: 10 (Kabat residues 89-97). The binding member according to the invention may also be the parent binding member as shown in Table 1, wherein one or more of the CDRs have one or more amino acid additions, substitutions, deletions, and/or insertions. In some embodiments, the binding member comprises a set of CDRs having from one to ten additions, substitutions, deletions and/or insertions relative to 10 the parent sequences of Antibody 11, In another embodiment one to ten substitutions relative to Antibody 11. In another embodiment form one to eleven additions, substitutions, deletions and/or insertions relative to the parent sequences of Antibody 1. In another embodiment one to ten substitutions relative to Antibody 1. 15 In certain embodiments the binding member of the invention comprises H4CDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; wherein the HCDR3 has the amino acid sequence of SEQ ID NO: 5 optionally having from I to 5 amino acid additions, substitutions, deletions and/or insertions; and the LCDR3 has the amino acid sequence of SEQ ID NO: 10 optionally having from 1 to 6 amino acid additions, substitutions, deletions and/or insertions. In such 20 embodiments, the HCDRI may have the amino acid sequence SEQ ID NO: 3; the HCDR2 may have the amino acid sequence SEQ ID NO: 4; the LCDRI may have the amino acid sequence SEQ ID NO: 8; and the LCDR2 may have the amino acid sequence SEQ ID NO: 9. Alternatively, the HCDR1, the HCDR2, the LCDR1, and the LCDR2 may also collectively have one or more amino acid additions, substitutions, deletions, and/or insertions relative to 25 the parent sequences (Antibody 1), such as from one to ten substitutions.

11 A binding member of the invention may comprise one or a combination of CDRs as described herein. For example, the binding member of the invention may comprise an HCDRl having the amino acid sequence of SEQ ID NO: 3; an HCDR2 having the amino acid sequence of SEQ ID NO: 4; an HCDR3 having an amino acid sequence selected from the 5 group consisting of SEQ ID NOS: 5, 15, 25, 65, 75, 85, 95, 145, 155, 175, and 255; an LCDRI having the amino acid sequence of SEQ ID NO: 8; an LCDR2 having the amino acid sequence SEQ ID NO: 9; and an LCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270 and 280. 10 In certain embodiments, the binding member or VH domain of the invention comprises the parent HCDR3 (SEQ ID NO:5) with one or more of the following substitutions: Kabat residue 96 replaced by S, M, or T; Kabat residue 97 replaced L or G; 15 Kabat residue 98 replaced by K; Kabat residue 99 replaced by S, W, A, T, or E; Kabat residue 100 replaced by A or 1. In some embodiments, a binding member, or a VL domain thereof may comprise the 20 parent LCDR3 (SEQ ID NO 10) with Kabat residue 94 replaced by T, R, D, P, E, N, H, Q, or A. In certain embodiments, the binding member or VL domain of the invention comprises the parent LCDR3 (SEQ ID NO 10) with one or more of the following 25 substitutions: Kabat residue 94 replaced by T, R, D, P, E, N, H, Q, or A; Kabat residue 95 replaced T, K, S, I, G, H, M, F, R, N, K or Q; Kabat residue 95A replaced by L, H, D, G, R, N, Q, K or E; Kabat residue 95B replaced by T, H, S, Y, L or N; 30 Kabat residue 96 replaced by G or A; Kabat residue 97 replaced by P, S or G.

12 In one embodiment, the invention is a binding member in which: HCDR1 has amino acid sequence SEQ ID NO: 103, HCDR2 has amino acid sequence SEQ ID NO: 104, HCDR3 has amino acid sequence SEQ ID NO: 105, LCDR1 has amino acid sequence SEQ ID NO: 108, LCDR2 has amino acid sequence SEQ ID NO: 109, and LCDR3 has amino acid 5 sequence SEQ ID NO: 110. For example, see Antibody II of Table 1. Still other embodiments of the invention are binding members, such as antibody molecules, capable of competing with antibodies of the invention such as Antibody 11 of Table 1 for binding to human IgE, said binding members neutralizing human IgE with an 10 IC50 of less than about I nM in an assay described herein, or with an IC50 of less than about 0.5 nM. In some embodiments, the IC50 is less than about 0.2 nM. The invention provides binding members comprising an HCDRI and/or HCDR2 and/or HCDR3 of any of antibodies I to 28 and/or an LCDR1 and/or LCDR2 and/or LCDR3 15 of any of antibodies I to 28, e.g. a set of CDRs of any of antibodies I to 28 shown in Table 1. The binding member may comprise a set of VH CDRs of one of these antibodies. Optionally it may also comprise a set of VL CDRs of one of these antibodies, and the VL CDRs may be from the same or a different antibody as the VH CDRs. A VH domain comprising a set of HCDRs of any of antibodies I to 28, and/or a VL domain comprising a set of LCDRs of any 20 of antibodies 1 to 28, are also provided by the invention. Typically, a VH domain is paired with a VL domain to provide an antibody antigen binding site, although as discussed further below a VH or VL domain alone may be used to bind antigen. The Antibody I VH domain (see Table 1) may be paired with the Antibody 1 25 VL domain, so that an antibody antigen-binding site is formed comprising both the antibody I VH and VL domains. Analogous embodiments are provided for the other VH and VL domains disclosed herein. In other embodiments, the Antibody 1 VH is paired with a VL domain other than the Antibody 1 VL. Light-chain promiscuity is well established in the art. Again, analogous embodiments are provided by the invention for the other VH and VL 30 domains disclosed herein. Thus, the VII of the parent or of any of antibodies 2 to 28 may be paired with the VL of the parent or of any of antibodies 2 to 28.

13 A binding member may comprise a set of H and/or L CDRs of the parent antibody or any of antibodies 2 to 28 with as many as twenty, sixteen, ten, nine or fewer, e.g. one, two, three, four or five, amino acid additions, substitutions, deletions, and/or insertions within the disclosed set of H and/or L CDRs. Alternatively, a binding member may comprise a set of H 5 and/or L CDRs of the parent antibody or any of antibodies 2 to 28 with as many as twenty, sixteen, ten, nine or fewer, e.g. one, two, three, four or five, amino acid substitutions within the disclosed set of H and/or L CDRs. Such modifications may potentially be made at any residue within the set of CDRs. For example, modifications may be made at the positions modified in any of Antibodies 2 to 28, as shown in Table 1. Thus, the one or more 10 modifications, may comprise one or more substitutions at the following residues: Kabat residues 96, 97, 98, 99, and 100 in the HCDRs; and Kabat residues 94, 95, 95A, 95B, 96, and 97 in the LCDRs. A binding member may comprise an antibody molecule having one or more CDRs, 15 e.g. a set of CDRs, within an antibody framework. For example, one or more CDRs or a set of CDRs of an antibody may be grafted into a framework (e.g. human framework) to provide an antibody molecule. The framework regions may be of human germline gene sequences, or be non-germlined. Thus, the framework may be germlined where.one or more residues within the framework are changed to match the residues at the equivalent position in the most similar 20 human germline framework. Thus, a binding member of the invention may be an isolated human antibody molecule having a VH domain comprising a set of HCDRs in a human germline framework, e.g. human germline IgG VH framework. The binding member also has a VL domain comprising a set of LCDRs, e.g. in a human germline IgG VL framework. 25 VH and/or VL framework residues may be modified as discussed and exemplified herein e.g. using site-directed mutagenesis. A VH or VL domain according to the invention, or a binding member comprising such a VL domain, preferably has the VH and/or VL domain sequence of an antibody of Table 3. 30 A non-germlined antibody molecule has the same CDRs, but different frameworks, compared to a germlined antibody molecule. Germlined antibodies may be produced by germlining framework regions of the VH and VL domain sequences shown herein for these antibodies.

14 A binding member of the invention may be one which competes for binding to IgE with any binding member which both binds IgE and comprises a binding member such as VH and/or VL domain, CDR e.g. HCDR3, and/or set of CDRs disclosed herein. Competition 5 between binding members may be assayed easily in vitro, for example using ELISA and/or by tagging a specific reporter molecule to one binding member which can be detected in the presence of one or more other untagged binding members, to enable identification of binding members which bind the same epitope or an overlapping epitopc. Such methods are readily known to one of ordinary skill in the art, and are described in more detail herein. Thus, a 10 further aspect of the present invention provides a binding member comprising a human antibody antigen-binding site that competes with an antibody molecule, for example especially an antibody molecule comprising a VH and/or VL domain, CDR e.g. HCDR3 or set of CDRs of the parent antibody or any of antibodies 1 to 28, for binding to human IgE. In one embodiment, the binding member of the invention competes with Antibody I I of Table 1. 15 Another embodiment of the invention provides binding members which bind to a specific region of IgE. Binding may be determined for example by detecting or observing specific interactions between the binding member and the residues of IgE, e.g. in a structure of the binding member:IgE complex which may be determined for example using X-ray 20 crystallography. A structure of Antibody 11 bound to human IgE CE3-Cs4 domains determined using X-ray crystallography provided the opportunity to study two interactions of antibody I I Fabs with IgE within the crystal. IgE is a bivalent antigen since there are two light chains and two heavy chains. X ray crystallographic studies showed that the Fab bound to an epitope spread across two IgE heavy chains. 25 The first interaction indicated that the interaction site of Antibody 11 comprises residues Glu390 through to Asn394 inclusive and sugar moieties GlcNAc1 and Man6 of one IgE heavy chain and Leu340, Arg342, Ala428 to Thr434 inclusive, Thr436, Ser437 and Glu472 and sugar moiety Man 5 in the other IgE heavy chain. 30 In one embodiment of the invention there is provided an isolated binding member specific for immunogbulin E wherein said binding member binds to an epitope in the immunoglobulin E comprising: 15 residues Glu390 through to Asn394 inclusive in a first IgE heavy chain and Leu340, Arg342, Ala428 to Thr434 inclusive, Thr436, Ser437 and Glu472 in a second IgE heavy chain; in a further embodiment said epitope further comprising sugar moieties GlcNAcI and Man6 of a first IgE heavy chain and sugar moiety Man 5 in a second IgE heavy chain. 5 The second interaction indicated that the interaction site of Antibody 11 comprises residues Glu390, GIn392 to Asn394 inclusive and sugar moieties GlcNAcI and Man6 in a first IgE heavy chain and Leu340, Arg342, Ala428 to Thr434 inclusive, Thr436, Ser437 and Glu472 in a second IgE heavy chain. 10 In a further embodiment of the invention there is provided an isolated binding member specific for immunogbulin E wherein said binding member binds to an epitope in the immunoglobulin E comprising: residues Glu390, Gln392 to Asn394 inclusive in a first IgE heavy chain and Leu340, Arg342, 15 Ala428 to Thr434 inclusive, Thr436, Ser437 and Glu472 in a second IgE heavy chain; in a further embodiment said epitope further comprising sugar moieties G1cNAc1 and Man6 in a first IgE heavy chain. In a further embodiment of the invention there is provided an isolated binding member 20 specific for immunogbulin E wherein said binding member binds to an epitope in the immunoglobulin E comprising: residues Glu390, Gln392 to Asn393 inclusive in a first IgE heavy chain and Leu340, Arg342, Ala428 to Thr434 inclusive, Thr436, Ser437 and Glu472 in a second IgE heavy chain; in a further embodiment said epitope further comprising sugar moieties GlcNAc1 and Man6 25 in a first IgE heavy chain. In a further embodiment of the invention there is provided an isolated binding member specific for immunogbulin E which binds an epitope which comprises elements from a first IgE heavy chain and elements from a second IgE heavy chain. 30 In further aspects the present invention provides a binding member comprising a human antibody antigen-binding site which competes with an antibody antigen-binding site for binding to human IgE, wherein the antibody antigen-binding site is composed of a VH 16 domain and a VL domain, and wherein the VH and VL domains comprise a set of CDRs of the parent (Antibody 1), or of any of antibodies 2 to 28, as disclosed herein. In further aspects, the invention provides an isolated nucleic acid which comprises a 5 sequence encoding a binding member, VH domain and/or VL domain according to the present invention. For example, SEQ ID NOS: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 287, 299, and 305 encode exemplary VH domains of the present invention, and SEQ ID NOS: 317 ,319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 10 359, 361, 363, 365, 367, 369, 371, 373, 375, 377 and 379 encode exemplary VL domains of the present invention. The invention also includes methods of preparing a binding member, a VH domain and/or a VL domain of the invention, which comprise expressing said nucleic acid under conditions to bring about production of said binding member, VH domain and/or VL domain, and recovering it by isolating or purifying the binding member. 15 Another aspect of the present invention provides nucleic acid, generally isolated, encoding a VH CDR or VL CDR sequence disclosed herein. A further aspect provides a host cell containing or transformed with nucleic acid of the 20 invention. Further aspects of the present invention provide for compositions containing binding members of the invention, and their use in methods of inhibiting and/or neutralising IgE, including methods of treatment of the human or animal body by therapy. 25 For example, binding members according to the invention may be used in a method of treatment and/or prevention, or used in a method of diagnosis, of a biological response, disease, disorder, or condition in the human or animal body (e.g. in a human patient), or in vitro. 30 The method of treatment and/or prevention may comprise administering to said patient a binding member of the invention in an amount sufficient to measurably neutralize IgE.

17 Conditions treatable in accordance with the present invention include any in which IgE plays a role, such as allergies and asthma. These and other aspects of the invention are described in further detail below. 5 It is convenient to point out here that "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. 10 IgE is immunoglobulin E. The amino acid sequence of the human IgE constant region is publicly available. In some embodiments IgE may be human or cynomolgus monkey IgE. As described elsewhere herein, IgE may be recombinant, and/or may be either glycosylated or unglycosylated. IgE is expressed naturally in vivo in glycosylated form, e.g. in U266.B 1 15 cells. Glycosylated IgE may also be expressed in recombinant systems. A binding member generally refers to one member of a pair of molecules that bind one another. The members of a binding pair may be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface, or a cavity, which binds to and is therefore complementary to a particular spatial and polar 20 organization of the other member of the pair of molecules. Examples of types of binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The present invention is generally concerned with antigen-antibody type reactions. A binding member normally comprises a molecule having an antigen-binding site. 25 For example, a binding member may be an antibody molecule or a non-antibody protein that comprises an antigen-binding site. An antigen binding site may be provided by means of arrangement of CDRs on non antibody protein scaffolds, such as fibronectin or cytochrome B etc. [1, 2, 3), or by 30 randomising or mutating amino acid residues of a loop within a protein scaffold to confer binding specificity for a desired target. Scaffolds for engineering novel binding sites in proteins have been reviewed in detail by Nygren et aL. [3]. Protein scaffolds for antibody 18 mimics are disclosed in WO/0034784, which is herein incorporated by reference in its entirety, in which the inventors describe proteins (antibody mimics) that include a fibronectin type III domain having at least one randomised loop. A suitable scaffold into which to graft one or more CDRs, e.g. a set of HCDRs, may be provided by any domain member of the 5 immunoglobulin gene superfamily. The scaffold may be a human or non-human protein. An advantage of a non-antibody protein scaffold is that it may provide an antigen-binding site in a scaffold molecule that is smaller and/or easier to manufacture than at least some antibody molecules. Small size of a binding member may confer useful physiological properties, such as an ability to enter cells, penetrate deep into tissues or reach targets within other structures, 10 or to bind within protein cavities of the target antigen. Use of antigen binding sites in non antibody protein scaffolds is reviewed in Wess, 2004 [4]. Typical are proteins having a stable backbone and one or more variable loops, in which the amino acid sequence of the loop or loops is specifically or randomly mutated to create an antigen-binding site that binds the target antigen. Such proteins include the IgG-binding domains of protein A from S. aureus, 15 transferrin, tetranectin, fibronectin (e.g. 10th fibronectin type III domain), lipocalins as well as gamma-crystalline and other AffilinTM scaffolds (Scil Proteins). Examples of other approaches include synthetic "Microbodies" based on cyclotides - small proteins having intra molecular disulphide bonds, Microproteins (VersabodiesTM, Amunix) and ankyrin repeat proteins (DARPins, Molecular Partners). Such proteins also include small, engineered protein 20 domains such as, for example, immuno-domains (see for example, U.S. Patent Publication Nos. 2003082630 and 2003157561. Immuno-domains contain at least one complementarity determining region (CDR) of an antibody. In addition to antibody sequences and/or an antigen-binding site, a binding member 25 according to the present invention may comprise other amino acids, e.g. forming a peptide or polypeptide, such as a folded domain, or to impart to the molecule another functional characteristic in addition to ability to bind antigen. Binding members of the invention may carry a detectable label, or may be conjugated to a toxin or a targeting moiety or enzyme (e.g. via a peptidyl bond or linker). For example, a binding member may comprise a catalytic site 30 (e.g. in an enzyme domain) as well as an antigen binding site, wherein the antigen binding site binds to the antigen and thus targets the catalytic site to the antigen. The catalytic site may inhibit biological function of the antigen, e.g. by cleavage.

19 Although, as noted, CDRs can be carried by non-antibody scaffolds, the structure for carrying a CDR or a set of CDRs of the invention will generally be an antibody heavy or light chain sequence or substantial portion thereof in which the CDR or set of CDRs is located at a location corresponding to the CDR or set of CDRs of naturally occurring VH and VL 5 antibody variable domains encoded by rearranged immunoglobulin genes. The structures and locations of immunoglobulin variable domains may be determined by reference to Kabat, et al., 1987 [5], and updates thereof findable under "Kabat" using any internet search engine). By CDR region or CDR, it is intended to indicate the hypervariable regions of the 10 heavy and light chains of the immunoglobulin as defined by Kabat et al. 1991 [6], and later editions. An antibody typically contains 3 heavy chain CDRs and 3 light chain CDRs. The term CDR or CDRs is used here in order to indicate, according to the case, one of these regions or several, or even the whole, of these regions which contain the majority of the amino acid residues responsible for the binding by affinity of the antibody for the antigen or 15 the epitope which it recognizes. Among the six short CDR sequences, the third CDR of the heavy chain (HCDR3) has a greater size variability (greater diversity essentially due to the mechanisms of arrangement of the genes which give rise to it). It may be as short as 2 amino acids although the longest 20 size known is 26. CDR length may also vary according to the length that can be accommodated by the particular underlying framework. Functionally, HCDR3 plays a role in part in the determination of the specificity of the antibody (see references 7, 8, 9, 10, 11, 12, 13, 14].ln another embodiment of the invention there is provided an isolated binding member comprising a HCDR3 sequence selected from Table 3a. 25 Antibody molecule refers to an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein comprising an antibody antigen-binding site. It must be understood here that the invention does not relate to the antibodies in natural form, that is to say they are not in their natural environment but have 30 been isolated or obtained by purification from natural sources, or else obtained by genetic recombination, or by chemical synthesis, including modification with unnatural amino acids. Antibody fragments that comprise an antibody antigen-binding site include, but are not limited to, molecules such as Fab, Fab', Fab'-SH, scFv, Fv, dAb and Fd. Various other 20 antibody molecules including one or more antibody antigen-binding sites have been engineered, including for example Fab 2 , Fab 3 , diabodies, triabodies, tetrabodies and minibodies. Antibody molecules and methods for their construction and use are described in [15]. 5 It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules that bind the target antigen. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the CDRs, of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP-A-184187, GB 10 2188638A or EP-A-239400, and a large body of subsequent literature. A hybridoma or other cell producing an antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced. As antibodies can be modified in a number of ways, the term "antibody molecule" 15 should be construed as covering any binding member or substance having an antibody antigen-binding site with the required specificity and/or binding to antigen. Thus, this term covers antibody fragments and derivatives, including any polypeptide comprising an antibody antigen-binding site, whether natural or wholly or partially synthetic. Chimeric molecules comprising an antibody antigen-binding site, or equivalent, fused to another polypeptide (e.g. 20 derived from another species or belonging to another antibody class or subclass) arc therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0 120694 and EP-A-0125023, and a large body of subsequent literature. Further techniques available in the art of antibody engineering have made it possible 25 to isolate human and humanised antibodies. For example, human hybridomas can be made as described by Kontermann & Dubel [16]. Phage display, another established technique for generating binding members has been described in detail in many publications, such as Kontermann & Dubel [16] and W092/01047 (discussed further below), and US patents US 5,969,108, US,5,565,332, US 5,733,743, US 5,858,657, US 5,871,907, US 5,872,215, US 30 5,885,793, US 5,962,255, US 6,140,471, US 6,172,197, US 6,225,447, US 6,291,650, US 6,492,160 and US 6,521,404.

21 Transgenic mice in which the mouse antibody genes are inactivated and functionally replaced with human antibody genes while leaving intact other components of the mouse immune system, can be used for isolating human antibodies [17]. Humanised antibodies can be produced using techniques known in the art such as those disclosed in for example 5 W091/09967, US 5,585,089, EP592106, US 5,565,332 and W093/17105. Further, W02004/006955 describes methods for humanising antibodies, based on selecting variable region framework sequences from human antibody genes by comparing canonical CDR structure types for CDR sequences of the variable region of a non-human antibody to canonical CDR structure types for corresponding CDRs from a library of human antibody 10 sequences, e.g. germline antibody gene segments. Human antibody variable regions having similar canonical CDR structure types to the non-human CDRs form a subset of member human antibody sequences from which to select human framework sequences. The subset members may be further ranked by amino acid similarity between the human and the non human CDR sequences. In the method of WO2004/006955, top ranking human sequences are 15 selected to provide the framework sequences for constructing a chimeric antibody that functionally replaces human CDR sequences with the non-human CDR counterparts using the selected subset member human frameworks, thereby providing a humanized antibody of high affinity and low immunogenicity without need for comparing framework sequences between the non-human and human antibodies. Chimeric antibodies made according to the method are 20 also disclosed. Synthetic antibody molecules may be created by expression from genes generated by means of oligonucleotides synthesized and assembled within suitable expression vectors, for example as described by Knappik et aL. [18] or Krebs et aL. [19]. 25 It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, constant light chain domain (CL) and constant heavy chain domain I (CHI) domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of 30 the VL and VH domains of a single antibody; (iv) the dAb fragment [20, 21, 22], which consists of a VH or a VL domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two 22 domains to associate to form an antigen binding site [23, 24]; (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (W094/13804; [25]). Fv, scFv or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains [26]. 5 Minibodies comprising a scFv joined to a CH3 domain may also be made [27]. Other examples of binding fragments are Fab', which differs from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain, including one or more cysteines from the antibody hinge region, and Fab'-SH, which is a Fab' fragment in which the cysteine residue(s) of the constant domains bear a free thiol group. 10 Antibody fragments of the invention can be obtained starting from a parent antibody molecule or any of the antibody molecules I to 28, by methods such as digestion by enzymes e.g. pepsin or papain and/or by cleavage of the disulfide bridges by chemical reduction. In another manner, the antibody fragments comprised in the present invention can be obtained 15 by techniques of genetic recombination likewise well known to the person skilled in the art or else by peptide synthesis by means of, for example, automatic peptide synthesizers, such as those supplied by the company Applied Biosystems, etc., or by nucleic acid synthesis and expression. 20 Functional antibody fragments according to the present invention include any functional fragment whose half-life is increased by a chemical modification, especially by PEGylation, or by incorporation in a liposome. A dAb (domain antibody) is a small monomeric antigen-binding fragment of an 25 antibody, namely the variable region of an antibody heavy or light chain [22]. VH dAbs occur naturally in camelids (e.g. camel, llama) and may be produced by immunizing a camelid with a target antigen, isolating antigen-specific B cells and directly cloning dAb genes from individual B cells. dAbs are also producible in cell culture. Their small size, good solubility and temperature stability makes them particularly physiologically useful and 30 suitable for selection and affinity maturation. Camelid VH dAbs are being developed for therapeutic use under the name "nanobodiesTM". A binding member of the present invention may be a dAb comprising a VH or VL domain substantially as set out herein, or a VH or VL domain comprising a set of CDRs substantially as set out herein.

23 Bispecific or bifunctional antibodies form a second generation of monoclonal antibodies in which two different variable regions are combined in the same molecule [28]. Their use has been demonstrated both in the diagnostic field and in the therapy field from 5 their capacity to recruit new effector functions or to target several molecules on the surface of tumour cells. Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways [29], e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. These antibodies can be obtained by chemical methods [30, 31] or somatic 10 methods [32, 33] but likewise and preferentially by genetic engineering techniques which allow the heterodimerization to be forced and thus facilitate the process of purification of the antibody sought [34]. Examples of bispecific antibodies include those of the BiTEm technology in which the binding domains of two antibodies with different specificity can be used and directly linked via short flexible peptides. This combines two antibodies on a short 15 single polypeptide chain. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Bispecific antibodies can be constructed as entire lgG, as bispecific Fab'2, as Fab'PEG, as diabodies or else as bispecific scFv. Further, two bispecific antibodies can be 20 linked using routine methods known in the art to form tetravalent antibodies. Bispecific diabodies, as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides, such as antibody fragments) of appropriate binding 25 specificities can be readily selected using phage display (W094113804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against IgE, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. Bispecific whole antibodies may be made by alternative engineering methods as described in Ridgeway et al., 1996 [35]. 30 Various methods are available in the art for obtaining antibodies against IgE. The antibodies may be monoclonal antibodies, especially of human, murine, chimeric or 24 humanized origin, which can be obtained according to the standard methods well known to the person skilled in the art. In general, for the preparation of monoclonal antibodies or their functional fragments, 5 especially of murine origin, it is possible to refer to techniques which are described in particular in the manual "Antibodies" [36] or to the technique of preparation from hybridomas described by Kbhler and Milstein [37]. Monoclonal antibodies can be obtained, for example, from an animal cell immunized 10 against IgE, or one of its fragments containing the epitope recognized by said monoclonal antibodies. Suitable fragments and peptides or polypeptides comprising them may be used to immunise animals to generate antibodies against IgE. Said IgE, or one of its fragments, can especially be produced according to the usual working methods, by genetic recombination starting with a nucleic acid sequence contained in the cDNA sequence coding for IgE or 15 fragment thereof, by peptide synthesis starting from a sequence of amino acids comprised in the peptide sequence of the IgE and/or fragment thereof. The monoclonal antibodies can, for example, be purified on an affinity column on which IgE or one of its fragments containing the epitope recognized by said monoclonal 20 antibodies, has previously been immobilized. More particularly, the monoclonal antibodies can be purified by chromatography on protein A and/or G, followed or not followed by ion exchange chromatography aimed at eliminating the residual protein contaminants as well as the DNA and the LPS, in itself, followed or not followed by exclusion chromatography on Sepharose gel in order to eliminate the potential aggregates due to the presence of dimers or 25 of other multimers. In one embodiment, the whole of these techniques can be used simultaneously or successively. An antigen-binding site is the part of a molecule that binds to and is complementary to all or part of the target antigen. In an antibody molecule it is referred to as the antibody antigen-binding site, and comprises the part of the antibody that binds to and is 30 complementary to all or part of the target antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope. An antibody antigen-binding site may be provided by one or more antibody variable domains. An 25 antibody antigen-binding site may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Isolated refers to the state in which binding members of the invention, or nucleic acid encoding such binding members, will generally be in accordance with the present invention. 5 Thus, binding members, VH and/or VL domains, and encoding nucleic acid molecules and vectors according to the present invention may be provided isolated and/or purified, e.g. from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes of origin other than the sequence encoding a polypeptide with the required function. Isolated members and isolated 10 nucleic acid will be free or substantially free of material with which they are naturally associated, such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo. Members and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes 15 be isolated - for example the members will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy. Binding members may be glycosylated, either naturally or by systems of heterologous eukaryotic cells (e.g. CHO or NSO (ECACC 85110503) cells, or they may be (for example if produced by 20 expression in a prokaryotic cell) unglycosylated. Heterogeneous preparations comprising anti-IgE antibody molecules also form part of the invention. For example, such preparations may be mixtures of antibodies with full-length heavy chains and heavy chains lacking the C-terminal lysine, with various degrees of glycosylation and/or with derivatized amino acids, such as cyclization of an N-terminal 25 glutamic acid to form a pyroglutamic acid residue. As used herein, the phrase "substantially as set out" refers to the characteristic(s) of the relevant CDRs of the VH or VL domain of binding members described herein will be either identical or highly similar to the specified regions of which the sequence is set out 30 herein. As described herein, the phrase "highly similar" with respect to specified region(s) of one or more variable domains, it is contemplated that from I to about 6, e.g. from 1 to 5, 26 including I to 3, or I or 2, or 3 or 4, amino acid substitutions may be made in the CDR and/or VH or VL domain. Detailed Description As noted above, a binding member in accordance with the present invention modulates 5 and may neutralise a biological activity of IgE. As described herein, IgE-binding members of the present invention may be optimised for neutralizing potency. Generally, potency optimisation involves mutating the sequence of a selected binding member (normally the variable domain sequence of an antibody) to generate a library of binding members, which are then assayed for potency and the more potent binding members are selected. Thus selected 10 "potency-optimised" binding members tend to have a higher potency than the binding member from which the library was generated. Nevertheless, high potency binding members may also be obtained without optimisation, for example a high potency binding member may be obtained directly from an initial screen e.g. a biochemical neutralization assay. A "potency optimized" binding member refers to a binding member with an optimized potency of 15 neutralization of a particular activity or downstream function. Assays and potencies are described in more detail elsewhere herein. The present invention provides both potency optimized and non-optimized binding members, as well as methods for potency optimization from a selected binding member. The prcscnt invention thus allows the skilled person to generate binding members having high potency. 20 Although potency optimization may be used to generate higher potency binding members from a given binding member, it is also noted that high potency binding members may be obtained even without potency optimization. 25 In a further aspect, the present invention provides a method of obtaining one or more binding members able to bind the antigen, the method including bringing into contact a library of binding members according to the invention and said antigen, and selecting one or more binding members of the library able to bind said antigen. 30 The library may be displayed on particles or molecular complexes, e.g. replicable genetic packages, such as yeast, bacterial or bacteriophage (e.g. T7) particles, viruses, cells or covalent, ribosomal or other in vitro display systems, each particle or molecular complex 27 containing nucleic acid encoding the antibody VH variable domain displayed on it, and optionally also a displayed VL domain if present. Phage display is described in WO 92/01047 and e.g. US patents US 5,969,108, US 5,565,332, US 5,733,743, US 5,858,657, US 5,871,907, US 5,872,215, US 5,885,793, US 5,962,255, US 6,140,471, US 6,172,197, US 5 6,225,447, US 6,291,650, US 6,492,160 and US 6,521,404, each of which is herein incorporated by reference in their entirety. Following selection of binding members able to bind the antigen and displayed on bacteriophage or other library particles or molecular complexes, nucleic acid may be taken 10 from a bacteriophage or other particle or molecular complex displaying a selected binding member. Such nucleic acid may be used in subsequent production of a binding member or an antibody VH or VL variable domain by expression from nucleic acid with the sequence of nucleic acid taken from a bacteriophage or other particle or molecular complex displaying a said selected binding member. 15 An antibody VH variable domain with the amino acid sequence of an antibody VH variable domain of a said selected binding member may be provided in isolated form, as may a binding member comprising such a VH domain. 20 Ability to bind IgE may be further tested, also ability to compete with e.g. a parent antibody molecule or an antibody molecule 2 to 28 (e.g. in scFv format and/or IgG format, e.g. lgG 1) for binding to IgE. Ability to neutralize IgE may be tested, as discussed further elsewhere herein. 25 A binding member according to the present invention may bind IgE with the affinity of the parent or other antibody molecule, e.g. scFv, or one of antibodies 2 to 28, e.g. IgG1, or with an affinity that is better. A binding member according to the present invention may neutralise a biological 30 activity of IgE with the potency of the parent or other antibody molecule, one of antibodies 2 to 28 e.g. scFv, or IgGl, or with a potency that is better.

28 Binding affinity and neutralization potency of different binding members can be compared under appropriate conditions. Variants of the VH and VL domains and CDRs of the present invention, including 5 those for which amino acid sequences are set out herein, and which can be employed in binding members for IgE can be obtained by means of methods of sequence alteration or mutation and screening for antigen binding members with desired characteristics. Examples of desired characteristics include but are not limited to: . Increased binding affinity for antigen relative to known antibodies which are specific 10 for the antigen . Increased neutralization of an antigen activity relative to known antibodies which are specific for the antigen if the activity is known - Specified competitive ability with a known antibody or ligand to the antigen at a specific molar ratio 15 Ability to immunoprecipitate complex - Ability to bind to a specified epitope o Linear epitope, e.g. peptide sequence identified using peptide-binding scan as described herein, e.g. using peptides screened in linear and/or constrained conformation 20 o Conformational epitope, formed by non-continuous residues - Ability to modulate a new biological activity of IgE, or downstream molecule. Such methods are also provided herein. Variants of antibody molecules disclosed herein may be produced and used in the 25 present invention. Following the lead of computational chemistry in applying multivariate data analysis techniques to the structure/property-activity relationships [38] quantitative activity-property relationships of antibodies can be derived using well-known mathematical techniques, such as statistical regression, pattern recognition and classification [39, 40, 41, 42, 43, 44]. The properties of antibodies can be derived from empirical and theoretical models 30 (for example, analysis of likely contact residues or calculated physicochemical property) of antibody sequence, functional and three-dimensional structures and these properties can be considered singly and in combination.

29 An antibody antigen-binding site composed of a VH domain and a VL domain is typically formed by six loops of polypeptide: three from the light chain variable domain (VL) and three from the heavy chain variable domain (VH). Analysis of antibodies of known atomic structure has elucidated relationships between the sequence and three-dimensional 5 structure of antibody combining sites [45, 46]. These relationships imply that, except for the third region (loop) in VH domains, binding site loops have one of a small number of main chain conformations: canonical structures. The canonical structure formed in a particular loop has been shown to be determined by its size and the presence of certain residues at key sites in both the loop and in framework regions [45, 46]. 10 This study of sequence-structure relationship can be used for prediction of those residues in an antibody of known sequence, but of an unknown three-dimensional structure, which are important in maintaining the three-dimensional structure of its CDR loops and hence maintain binding specificity. These predictions can be backed up by comparison of the 15 predictions to the output from lead optimization experiments. In a structural approach, a model can be created of the antibody molecule [47] using any freely available or commercial package, such as WAM [481. A protein visualisation and analysis software package, such as Insight II (Accelrys, Inc.) or Deep View [49] may then be used to evaluate possible substitutions at each position in the CDR. This information may then be used to make 20 substitutions likely to have a minimal or beneficial effect on activity. The techniques required to make substitutions within amino acid sequences of CDRs, antibody VH or VL domains and binding members generally are available in the art. Variant sequences may be made, with substitutions that may or may not be predicted to have a 25 minimal or beneficial effect on activity, and tested for ability to bind and/or neutralize lgE and/or for any other desired property. Variable domain amino acid sequence variants of any of the VH and VL domains whose sequences are specifically disclosed herein may be employed in accordance with the 30 present invention, as discussed. A further aspect of the invention is an antibody molecule comprising a VH domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99 % amino acid sequence identity with a VH4 30 domain of any of antibodies I to 28 shown in Table 3 and the appended sequence listing, or with an HCDR (e.g., HCDRl, HCDR2, or HCDR3) shown in Table 1. The antibody molecule may optionally also comprise a VL domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99 % amino acid sequence identity with a VL domain of any of the antibodies I to 28, 5 or with an LCDR (e.g., LCDR1, LCDR2, or LCDR3) shown in Table I. Algorithms that can be used to calculate % identity of two amino acid sequences include e.g. BLAST [50], FASTA [51], or the Snith-Waterman algorithm [52], e.g. employing default parameters. Particular variants may include one or more amino acid sequence alterations (addition, 10 deletion, substitution and/or insertion of an amino acid residue). In certain embodiments, the variants have less than about 20 such alterations. Alterations may be made in one or more framework regions and/or one or more CDRs. The alterations normally do not result in loss of function, so a binding member 15 comprising a thus-altered amino acid sequence may retain an ability to bind and/or neutralize IgE. It may retain the same quantitative binding and/or neutralizing ability as a binding member in which the alteration is not made, e.g. as measured in an assay described herein. The binding member comprising a thus-altered amino acid sequence may have an improved ability to bind and/or neutralize IgE. 20 Alteration may comprise replacing one or more amino acid residue(s) with a non naturally occurring or non-standard amino acid, modifying one or more amino acid residue into a non-naturally occurring or non-standard form, or inserting one or more non-naturally occurring or non-standard amino acid into the sequence. Examples of numbers and locations 25 of alterations in sequences of the invention are described elsewhere herein. Naturally occurring amino acids include the 20 "standard" L-amino acids identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E by their standard single-letter codes. Non-standard amino acids include any other residue that may be incorporated into a polypeptide backbone or result from modification of an existing amino acid residue. Non-standard amino acids may 30 be naturally occurring or non-naturally occurring. Several naturally occurring non-standard amino acids are known in the art, such as 4-hydroxyproline, 5-hydroxylysine, 3 methythistidine, N-acetylserine, etc. [53]. Those amino acid residues that are derivatised at their N-alpha position will only be located at the N-terminus of an amino-acid sequence.

31 Normally in the present invention an amino acid is an L-amino acid, but it may be a D-amino acid. Alteration may therefore comprise modifying an L-amino acid into, or replacing it with, a D-amino acid. Methylated, acetylated and/or phosphorylated forms of amino acids are also known, and amino acids in the present invention may be subject to such modification. 5 Amino acid sequences in antibody domains and binding members of the invention may comprise non-natural or non-standard amino acids described above. Non-standard amino acids (e.g. D-amino acids) may be incorporated into an amino acid sequence during synthesis, or by modification or replacement of the "original" standard amino acids after synthesis of the 10 amino acid sequence. Use of non-standard and/or non-naturally occurring amino acids increases structural and functional diversity, and can thus increase the potential for achieving desired IgE-binding and neutralizing properties in a binding member of the invention. Additionally, D-amino 15 acids and analogues have been shown to have better pharmacokinetic profiles compared with standard L-amino acids, owing to in vivo degradation of polypeptides having L-amino acids after administration to an animal e.g. a human. Novel VH or VL regions carrying CDR-derived sequences of the invention may be 20 generated using random mutagenesis of one or more selected VH and/or VL genes to generate mutations within the entire variable domain. Such a technique is described by Gram et al. [54], who used error-prone PCR. In some embodiments one or two amino acid substitutions are made within an entire variable domain or set of CDRs. 25 Another method that may be used is to direct mutagenesis to CDR regions of VH or VL genes. Such techniques are disclosed by Barbas et at. [55] and Schier et aL. [56]. All the above-described techniques are known as such in the art and the skilled person will be able to use such techniques to provide binding members of the invention using routine 30 methodology in the art. A further aspect of the invention provides a method for obtaining an antibody antigen binding site for IgE, the method comprising providing by way of addition, deletion, 32 substitution or insertion of one or more amino acids in the amino acid sequence of a VH domain set out herein, optionally combining the VH domain thus provided with one or more VL domains, and testing the VH domain or VH/VL combination or combinations to identify a binding member or an antibody antigen-binding site for IgE and optionally with one or more 5 desired properties, e.g. ability to neutralize IgE activity. Said VL domain may have an amino acid sequence which is substantially as set out herein. An analogous method may be employed in which one or more sequence variants of a VL domain disclosed herein are combined with one or more VH domains. 10 As noted above, a CDR amino acid sequence substantially as set out herein may be carried as a CDR in a human antibody variable domain or a substantial portion thereof. The HCDR3 sequences substantially as set out herein represent embodiments of the present invention and each of these may be carried as a HCDR3 in a human heavy chain variable domain or a substantial portion thereof. 15 Variable domains employed in the invention may be obtained or derived from any germline or rearranged human variable domain, or may be a synthetic variable domain based on consensus or actual sequences of known human variable domains. A variable domain can be derived from a non-human antibody. A CDR sequence of the invention (e.g. CDR3) may 20 be introduced into a repertoire of variable domains lacking a CDR (e.g. CDR3), using recombinant DNA technology. For example, Marks et al. [57] describe methods of producing repertoires of antibody variable domains in which consensus primers directed at or adjacent to the 5' end of the variable domain area are used in conjunction with consensus primers to the third framework region of human VH genes to provide a repertoire of VH variable domains 25 lacking a CDR3. Marks et al. further describe how this repertoire may be combined with a CDR3 of a particular antibody. Using analogous techniques, the CDR3-derived sequences of the present invention may be shuffled with repertoires of VH or VL domains lacking a CDR3, and the shuffled complete VH or VL domains combined with a cognate VL or VH domain to provide binding members of the invention. The repertoire may then be displayed in a suitable 30 host system, such as the phage display system of W092/01047, which is herein incorporated by reference in its entirety, or any of a subsequent large body of literature, including Kay, Winter & McCafferty [58], so that suitable binding members may be selected. A repertoire may consist of from anything from 104 individual members upwards, for example at least 105, 33 at least 106, at least 107, at least 108, at least 109 or at least 1010 members or more. Other suitable host systems include, but are not limited to yeast display, bacterial display, T7 display, viral display, cell display, ribosome display and covalent display. 5 A method of preparing a binding member for IgE antigen is provided, which method comprises: (a) providing a starting repertoire of nucleic acids encoding a VH domain which either include a CDR3 to be replaced or lack a CDR3 encoding region; (b) combining said repertoire with a donor nucleic acid encoding an amino acid 10 sequence substantially as set out herein for a VH CDR3 such that said donor nucleic acid is inserted into the CDR3 region in the repertoire, so as to provide a product repertoire of nucleic acids encoding a VH domain; (c) expressing the nucleic acids of said product repertoire; (d) selecting a binding member for IgE; and 15 (e) recovering said binding member or nucleic acid encoding it. Again, an analogous method may be employed in which a VL CDR3 of the invention is combined with a repertoire of nucleic acids encoding a VL domain that either include a CDR3 to be replaced or lack a CDR3 encoding region. 20 Similarly, one or more, or all three CDRs may be grafted into a repertoire of VH or VL domains that are then screened for a binding member or binding members for IgE. For example, one or more of the parent or antibody 2 to 28 HCDRI, HCDR2 and 25 HCDR3 or the parent or antibody 2 to 28 set of HCDRs may be employed, and/or one or more of the parent or antibody 2 to 28 LCDRI, LCDR2 and LCDR3 or the parent or antibody 2 to 28 set of LCDRs may be employed. Similarly, other VH and VL domains, sets of CDRs and sets of HCDRs and/or sets of 30 LCDRs disclosed herein may be employed. A substantial portion of an immunoglobulin variable domain may comprise at least the three CDR regions, together with their intervening framework regions. The portion may also 34 include at least about 50 % of either or both of the first and fourth framework regions, the 50 % being the C-terminal 50 % of the first framework region and the N-terminal 50 % of the fourth framework region. Additional residues at the N-terminal or C-terminal end of the substantial part of the variable domain may be those not normally associated with naturally 5 occurring variable domain regions. For example, construction of binding members of the present invention made by recombinant DNA techniques may result in the introduction of N or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps. Other manipulation steps include the introduction of linkers to join variable domains of the invention to further protein sequences including antibody constant 10 regions, other variable domains (for example in the production of diabodies) or detectable/functional labels as discussed in more detail elsewhere herein. Although in some aspects of the invention, binding members comprise a pair of VH and VL domains, single binding domains based on either VH or VL domain sequences form 15 further aspects of the invention. It is known that single immunoglobulin domains, especially VH domains, are capable of binding target antigens in a specific manner. For example, see the discussion of dAbs above. In the case of either of the single binding domains, these domains may be used to 20 screen for complementary domains capable of forming a two-domain binding member able to bind IgE. This may be achieved by phage display screening methods using the so-called hierarchical dual combinatorial approach as disclosed in W092/01047, herein incorporated by reference in its entirety, in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H) and the 25 resulting two-chain binding member is selected in accordance with phage display techniques, such as those described in that reference. This technique is also disclosed in Marks et al, ibid. Binding members of the present invention may further comprise antibody constant regions or parts thereof, e.g. human antibody constant regions or parts thereof. For example, 30 a VL domain may be attached at its C-terminal end to antibody light chain constant domains including human Cic or CA chains. Similarly, a binding member based on a VH domain may be attached at its C-terminal end to all or part (e.g. a CHI domain) of an immunoglobulin heavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE and IgM and any of the 35 isotype sub-classes, particularly IgGI and IgG2. IgG1 is advantageous due to its ease of manufacture and stability, e.g., half-life. Any synthetic or other constant region variant that has these properties and stabilizes variable regions may also be useful in the present invention. 5 Binding members of the invention may be labelled with a detectable or functional label. Thus, a binding member or antibody molecule can be present in the form of an immunoconjugate so as to obtain a detectable and/or quantifiable signal. An irnmunoconjugate may comprise an antibody molecule of the invention conjugated with 10 detectable or functional label. A label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorescers, radiolabels, enzymes, chemiluminescers or photosensitizers. Thus, binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance. 15 Suitable labels include, by way of illustration and not limitation, - enzymes, such as alkaline phosphatase, glucose-6-phosphate dehydrogenase ("G6PDH"), alpha-D-galactosidase, glucose oxydase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase and peroxidase e.g. horseradish peroxidase; 20 - dyes; - fluorescent labels or fluorescers, such as fluorescein and its derivatives, fluorochrome, rhodamine compounds and derivatives, GFP (GFP for "Green Fluorescent Protein"), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine; fluorophores such as lanthanide cryptates and chelates e.g. Europium etc 25 (Perkin Elmer and Cis Biointernational), - chemoluminescent labels or chemiluminescers, such as isoluminol, luminol and the dioxetanes; - bio-luminescent labels, such as luciferase and luciferin; - sensitizers; 30 - coenzymes; - enzyme substrates; - radiolabels including but not limited to bromine77, carbonl4, cobalt57, fluorine8, gallium67, gallium 68, hydrogen3 (tritium), indiuml 11, indium 113m, iodinel23m, 36 iodine 125, iodine 126, iodine 131, iodine 133, mercury 107, mercury203, phosphorous32, rhenium99m, rheniuml01, rhenium105, ruthenium95, ruthenium97, rutheniuml03, ruthenium 105, scandium47, selenium75, sulphur35, technetium99, technetium99m, telluriuml21m, telluriuml22m, tellurium125m, thuliuml65, thuliuml67, thulium168, 5 yttrium199 and other radiolabels mentioned herein; - particles, such as latex or carbon particles; metal sol; crystallite; liposomes; cells, etc., which may be further labelled with a dye, catalyst or other detectable group; - molecules such as biotin, digoxygenin or 5-bromodeoxyuridine; - toxin moieties, such as for example a toxin moiety selected from a group of 10 Pseudomonas exotoxin (PE or a cytotoxic fragment or mutant thereof), Diptheria toxin or a cytotoxic fragment or mutant thereof, a botulinum toxin A, B, C, D, E or F, ricin or a cytotoxic fragment thereof e.g. ricin A, abrin or a cytotoxic fragment thereof, saporin or a cytotoxic fragment thereof, pokeweed antiviral toxin or a cytotoxic fragment thereof and bryodin 1 or a cytotoxic fragment thereof. 15 Suitable enzymes and coenzymes are disclosed in Litman, et al., US 4,275,149, and Boguslaski, et al., US4318980, each of which are herein incorporated by reference in their entireties. Suitable fluorescers and chemiluminescers are disclosed in Litman, et al., US4275149, which is incorporated herein by reference in its entirety. Labels further include 20 chemical moieties, such as biotin that may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin or streptavidin. Detectable labels may be attached to antibodies of the invention using conventional chemistry known in the art. Immunoconjugates or their functional fragments can be prepared by methods known 25 to the person skilled in the art. They can be coupled to enzymes or to fluorescent labels directly or by the intermediary of a spacer group or of a linking group, such as a polyaldehyde, like glutaraldchyde, ethylenediaminetetraacctic acid (EDTA), diethylene triaminepentaacetic acid (DPTA), or in the presence of coupling agents, such as those mentioned above for the therapeutic conjugates. Conjugates containing labels of fluorescein 30 type can be prepared by reaction with an isothiocyanate. The methods known to the person skilled in the art existing for coupling the therapeutic radioisotopes to the antibodies either directly or via a chelating agent, such as 37 EDTA, DTPA mentioned above can be used for the radioelements which can be used in diagnosis. It is likewise possible to perform labelling with sodiuml25 by the chloramine T method [59] or else with technetium99m by the technique of Crockford et al., (US4424200, herein incorporated by reference in its entirety) or attached via DTPA as described by 5 Hnatowich (US 4,479,930, herein incorporated by reference in its entirety). There are numerous methods by which the label can produce a signal detectable by external means, for example, by visual examination, electromagnetic radiation, heat, and chemical reagents. The label can also be bound to another binding member that binds the 10 antibody of the invention, or to a support. The label can directly produce a signal, and therefore, additional components are not required to produce a signal. Numerous organic molecules, for example fluorescers, are able to absorb ultraviolet and visible light, where the light absorption transfers energy to these 15 molecules and elevates them to an excited energy state. This absorbed energy is then dissipated by emission of light at a second wavelength. This second wavelength emission may also transfer energy to a labelled acceptor molecule, and the resultant energy dissipated from the acceptor molecule by emission of light for example fluorescence resonance energy transfer (FRET). Other labels that directly produce a signal include radioactive isotopes and 20 dyes. Alternately, the label may need other components to produce a signal, and the signal producing system would then include all the components required to produce a measurable signal, which may include substrates, coenzymes, enhancers, additional enzymes, substances 25 that react with enzymic products, catalysts, activators, cofactors, inhibitors, scavengers, metal ions, and a specific binding substance required for binding of signal generating substances. A detailed discussion of suitable signal producing systems can be found in Ullman, et al. US 5,185,243, which is herein incorporated herein by reference in its entirety. 30 The present invention provides a method comprising causing or allowing binding of a binding member as provided herein to IgE. As noted, such binding may take place in vivo, e.g. following administration of a binding member or encoding nucleic acid to a human or animal (e.g., a mammal), or it may take place in vitro, for example in ELISA, Western 38 blotting, immunocytochemistry, immunoprecipitation, affinity chromatography, and biochemical or cell-based assays. Generally, complexes between the binding member of the invention and IgE may be 5 detected by, inter alia, enzyme-linked immunoassay, radioassay, immunoprecipitation, fluorescence immunoassay, chemiluminescent assay, immunoblot assay, lateral flow assay, agglutination assay and particulate-based assay. The present invention also provides for measuring levels of antigen directly, by 10 employing a binding member according to the invention for example in a biosensor system. For instance, the present invention comprises a method of detecting and/or measuring binding to IgE, comprising, (i) exposing said binding member to IgE and (ii) detecting binding of said binding member to IgE, wherein binding is detected using any method or detectable label described herein. This, and any other binding detection method described herein, may be 15 interpreted directly by the person performing the method, for instance, by visually observing a detectable label. Alternatively, this method, or any other binding detection method described herein, may produce a report in the form of an autoradiograph, a photograph, a computer printout, a flow cytometry report, a graph, a chart, a test tube or container or well containing the result, or any other visual or physical representation of a result of the method. 20 The amount of binding of binding member to IgE may be determined. Quantitation may be related to the amount of the antigen in a test sample, which may be of diagnostic interest. Screening for IgE binding and/or the quantitation thereof may be useful, for instance, in screening patients for diseases or disorders referred to herein and/or any other disease or 25 disorder involving aberrant IgE production, expression and/or activity. A diagnostic method of the invention may comprise (i) obtaining a tissue or fluid sample from a subject, (ii) exposing said tissue or fluid sample to one or more binding members of the present invention; and (iii) detecting bound IgE as compared with a control 30 sample, wherein an increase in the amount of IgE binding as compared with the control may indicate an aberrant level of IgE production, expression or activity. Tissue or fluid samples to be tested include blood, serum, urine, biopsy material, rumours, or any tissue suspected of 39 containing aberrant IgE levels. Subjects testing positive for aberrant IgE levels or activity may also benefit from the treatment methods disclosed later herein. The diagnostic method of the invention may further comprise capturing a complex of 5 the binding member and IgE via an immobilized antigen. For example, an antigen may be immobilized on a lateral strip assay for capturing antigen-specific IgE in a sample of interest. Those skilled in the art are able to choose a suitable mode of determining binding of the binding member to an antigen according to their preference and general knowledge, in 10 light of the methods disclosed herein. The reactivities of binding members in a sample may be determined by any appropriate means. Radioimmunoassay (RIA) is one possibility. Radioactive labelled antigen is mixed with unlabelled antigen (the test sample) and allowed to bind to the binding 15 member. Bound antigen is physically separated from unbound antigen and the amount of radioactive antigen bound to the binding member determined. The more antigen there is in the test sample the less radioactive antigen will bind to the binding member. A competitive binding assay may also be used with non-radioactive antigen, using antigen or an analogue linked to a reporter molecule. The reporter molecule may be a fluorochrome, phosphor or 20 laser dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red, and lanthanide chelates or cryptates. Suitable chromogenic dyes include diaminobenzidine. Other reporters include macromolecular colloidal particles or particulate material, 25 such as latex beads that are colored, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules may be enzymes, which catalyze reactions that develop, or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy 30 states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed.

40 The signals generated by individual binding member-reporter conjugates may be used to derive quantifiable absolute or relative data of the relevant binding member binding in samples (normal and test). 5 A kit comprising a binding member according to any aspect or embodiment of the present invention is also provided as an aspect of the present invention. In the kit, the binding member may be labelled to allow its reactivity in a sample to be determined, e.g. as described further below. Further the binding member may or may not be attached to a solid support. Components of a kit are generally sterile and in sealed vials or other containers. Kits may be 10 employed in diagnostic analysis or other methods for which binding members are useful. A kit may contain instructions for use of the components in a method, e.g. a method in accordance with the present invention. Ancillary materials to assist in or to enable performing such a method may be included within a kit of the invention. The ancillary materials include a second, different binding member which binds to the first binding member and is conjugated 15 to a detectable label (e.g., a fluorescent label, radioactive isotope or enzyme). Antibody based kits may also comprise beads for conducting an immunoprecipitation. Each component of the kits is generally in its own suitable container. Thus, these kits generally comprise distinct containers suitable for each binding member. Further, the kits may comprise instructions for performing the assay and methods for interpreting and analyzing the data 20 resulting from the performance of the assay. The present invention also provides the use of a binding member as above for measuring antigen levels in a competition assay, that is to say a method of measuring the level of antigen in a sample by employing a binding member as provided by the present invention 25 in a competition assay. This may be where the physical separation of bound from unbound antigen is not required. Linking a reporter molecule to the binding member so that a physical or optical change occurs on binding is one possibility. The reporter molecule may directly or indirectly generate detectable signals, which may be quantifiable. The linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non-covalently. 30 Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule.

41 In various aspects and embodiments, the present invention extends to a binding member that competes for binding to IgE with any binding member defined herein, e.g. the parent antibody or any of antibodies 2 to 28, e.g. in IgGI format. Competition between binding members may be assayed easily in vitro, for example by tagging a specific reporter 5 molecule to one binding member which can be detected in the presence of other untagged binding member(s), to enable identification of binding members which bind the same epitope or an overlapping epitope. Competition may be determined for example using ELISA in which IgE is immobilized to a plate and a first tagged or labelled binding member along with one or more other untagged or unlabelled binding members is added to the plate. Presence of 10 an untagged binding member that competes with the tagged binding member is observed by a decrease in the signal emitted by the tagged binding member. For example, the present invention includes a method of identifying an IgE binding compound, comprising (i) immobilizing IgE to a support, (ii) contacting said immobilized IgE 15 simultaneously or in a step-wise manner with at least one tagged or labelled binding member according to the invention and one or more untagged or unlabelled test binding compounds, and (iii) identifying a new IgE binding compound by observing a decrease in the amount of bound tag from the tagged binding member. Such methods can be performed in a high throughput manner using a multiwell or array format. Such assays may be also be performed 20 in solution. See, for instance, U.S. 5,814,468, which is herein incorporated by reference in its entirety. As described above, detection of binding may be interpreted directly by the person performing the method, for instance, by visually observing a detectable label, or a decrease in the presence thereof. Alternatively, the binding methods of the invention may produce a report in the form of an autoradiograph, a photograph, a computer printout, a flow cytometry 25 report, a graph, a chart, a test tube or container or well containing the result, or any other visual or physical representation of a result of the method. Competition assays can also be used in epitope mapping. In one instance epitope mapping may be used to identify the epitope bound by an IgE-binding member which 30 optionally may have optimized neutralizing and/or modulating characteristics. Such an epitope can be linear or conformational. A conformational epitope can comprise at least two different fragments of [gE, wherein said fragments are positioned in proximity to each other when IgE is folded in its tertiary or quaternary structure to form a conformational epitope 42 which is recognized by an inhibitor of IgE, such as an IgE- binding member. In testing for competition a peptide fragment of the antigen may be employed, especially a peptide including or consisting essentially of an epitope of interest. A peptide having the epitope sequence plus one or more amino acids at either end may be used. Binding members 5 according to the present invention may be such that their binding for antigen is inhibited by a peptide with or including the sequence given. The present invention further provides an isolated nucleic acid encoding a binding member of the present invention. Nucleic acid may include DNA and/or RNA. In one, the 10 present invention provides a nucleic acid that codes for a CDR or set of CDRs or VH domain or VL domain or antibody antigen-binding site or antibody molecule, e.g. scFv or IgG1, of the invention as defined above. The present invention also provides constructs in the form of plasmids, vectors, 15 transcription or expression cassettes which comprise at least one polynucleotide as above. The present invention also provides a recombinant host cell that comprises one or more constructs as above. A nucleic acid encoding any CDR or set of CDRs or VH domain or VL domain or antibody antigen-binding site or antibody molecule, e.g. scFv or IgGI as 20 provided, itself forms an aspect of the present invention, as does a method of production of the encoded product, which method comprises expression from encoding nucleic acid therefor. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression a VH or VL domain, or binding member may be isolated and/or purified using any suitable 25 technique, then used as appropriate. Nucleic acid according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA 30 molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

43 A yet further aspect provides a method of production of an antibody VH variable domain, the method including causing expression from encoding nucleic acid. Such a method may comprise culturing host cells under conditions for production of said antibody VH variable domain. 5 Analogous methods for production of VL variable domains and binding members comprising a VH and/or VL domain are provided as further aspects of the present invention. A method of production may comprise a step of isolation and/or purification of the 10 product. A method of production may comprise formulating the product into a composition including at least one additional component, such as a pharmaceutically acceptable excipient. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, 15 filamentous fungi, yeast and baculovirus systems and transgenic plants and animals. The expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. For a review, see for example PlUckthun [60]. A common bacterial host is E. coli. Expression in eukaryotic cells in culture is also available to those skilled in the art as 20 an option for production of a binding member [61, 62, 63]. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonic retina cells and many others. 25 Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids e.g. phagemid, or viral e.g. 'phage, as appropriate [64]. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid 30 constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Ausubel et al. [65].

44 A further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. Such a host cell may be in vitro and may be in culture. Such a host cell may be in vivo. In vivo presence of the host cell may allow intra-cellular expression of the binding members of the present invention as "intrabodies" or intra-cellular antibodies. 5 Intrabodies may be used for gene therapy. A still further aspect provides a method comprising introducing nucleic acid of the invention into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE 10 Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. Introducing nucleic acid in the host cell, in particular a eukaryotic cell may use a viral or a plasmid based system. The plasmid system may be maintained episomally or may bc incorporated into the host cell or into an artificial chromosome. Incorporation may be either by random or targeted integration of one 15 or more copies at single or multiple loci. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene. The purification 20 of the expressed product may be achieved by methods known to one of skill in the art. Nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences that promote recombination with the genome, in accordance with standard techniques. 25 The present invention also provides a method that comprises using a construct as stated above in an expression system in order to express a binding member or polypeptide as above. 30 Binding members of the present invention may be used in methods of diagnosis or treatment in human or animal subjects, especially human. Binding members for IgE may be used to treat disorders characterized by biological effects mediated by IgE, particularly allergies and asthma. For example, binding members of the invention may be used to treat 45 allergic rhinitis, allergic contact dermatitis, atopic dermatitis, anaphylactic reaction, food allergy, urticaria, inflammatory bowel disease, eosinophilic gastroenteritis, drug-induced rash, allergic opthalmopathy, or allergic conjunctivitis. 5 Binding members for IgE may be used to inhibit allergen-induced mast-cell degranulation in vivo or in vitro, reduce FcERI -mediated biological responses in vivo or in vitro, as well as to reduce circulating IgE in a human or animal patient. Accordingly, the invention provides a method for inhibiting allergen-induced mast cell 10 degranulation in a mammal, comprising administering to said mammal a binding member, for examples an antibody, VH domain, or VL domain of the invention, in an amount sufficient to neutralize IgE. The invention further provides a method for reducing FcERI-biological responses with 15 or without simultaneous reduction of FcERII-mediated biological responses, comprising, contacting a cell expressing the FceRI and/or the FcsRII with a binding member, for examples an antibody, VH domain, or VL domain of the invention, in the presence of IgE. The invention further provides a method for reducing FcERI-mediated biological 20 responses with or without simultaneous reduction of FcERII-mediated biological responses, comprising, contacting a cell expressing the FcERI and/or the FcERII with a binding member, for example an antibody, VH domain, or VL domain of the invention, in the presence of IgE. 25 When test cells are contacted with the binding member of the invention in vitro, a control cell(s) may also be used for positive controls (e.g., reactions containing no binding member) and/or negative controls (e.g., reactions containing no IgE and/or antigen). When cells are contacted by the binding member in vivo, for example, by 30 administering the binding member of the invention to a mammal exhibiting FcERI- and/or FceRl-mediated biological responses, the binding member of the invention is administered in amounts sufficient to neutralize IgE.

46 Still further, the invention provides a method for reducing circulating IgE in a mammal, such as a human, comprising administering a binding member, such as an antibody, VH domain, or VL domain of the invention, in an amount sufficient to neutralize and reduce circulating free IgE. 5 Binding members of the invention may be used in the diagnosis or treatment of diseases or disorders including but not limited to any one or more of the following: allergic rhinitis, allergic contact dermatitis, atopic dermatitis, anaphylactic reaction, food allergy, urticaria, inflammatory bowel disease, eosinophilic gastroenteritis, drug-induced rash, allergic 10 opthalmopathy, rhino-conjunctivitis, allergic conjunctivitis, asthma bronchiale, airway hyperresponsiveness, cosmetic allergy, drug-induced allergy, drug-induced hypersensitivity syndrome, metal allergy, occupational hypersensitivity pneumonitis, chronic hypersensitivity pneumonitis, cold hypersensitivity, helminthic infection induced hypersensitivity, latex allergy and hay fever. 15 The data presented herein with respect to binding and neutralization of IgE thus indicate that binding members of the invention can be used to treat or prevent such disorders, including the reduction of severity of the disorders. Accordingly, the invention provides a method of treating or reducing the severity of at least one symptom of any of the disorders 20 mentioned herein, comprising administering to a patient in need thereof an effective amount of one or more binding members of the present invention alone or in a combined therapeutic regimen with another appropriate medicament known in the art or described herein such that the severity of at least one symptom of any of the above disorders is reduced. 25 Binding members of the invention may be used in appropriate animals and in animal models of disease, especially monkeys. Thus, the binding members of the present invention are useful as therapeutic agents in the treatment of diseases or disorders involving IgE, e.g. IgE production, expression and/or 30 activity, especially aberrant production, expression, or activity. A method of treatment may comprise administering an effective amount of a binding member of the invention to a patient in need thereof, wherein production, expression and/or activity of IgE is thereby decreased. A method of treatment may comprise (i) identifying a patient demonstrating increased IgE levels 47 or activity, for instance using the diagnostic methods described above, and (ii) administering an effective amount of a binding member of the invention to the patient, wherein increased production, expression and/or activity of IgE is decreased. An alternative method of treatment may comprise (i) identifying a patient who has no apparent increase in IgE levels 5 but who is believed to benefit from administration of a binding member of the invention, and (ii) administering an effective amount of a binding member of the invention to the patient. An effective amount according to the invention is an amount that decreases the increased production, expression and/or activity of IgE so as to decrease or lessen the severity of at Icast one symptom of the particular disease or disorder being treated, but not necessarily cure the 10 disease or disorder. The invention also provides a method of antagonising at least one effect of IgE comprising contacting with or administering an effective amount of one or more binding members of the present invention such that said at least one effect of IgE is antagonised. 15 Effects of IgE that may be antagonised by the methods of the invention include biological responses mediated by FcERI and/or FceRII, and any downstream effects that arise as a consequence of these binding reactions. Accordingly, further aspects of the invention provide the use of an isolated binding 20 member, such as an antibody, VH domain or VL domain of the invention for the manufacture of a medicament for treating a disorder associated with, or mediated by, IgE as discussed herein. Such use of, or methods of making, a medicament or pharmaceutical composition comprise formulating the binding member with a pharmaceutically acceptable excipient. 25 A pharmaceutically acceptable excipient may be a compound or a combination of compounds entering into a pharmaceutical composition not provoking secondary reactions and which allows, for example, facilitation of the administration of the active compound(s), an increase in its lifespan and/or in its efficacy in the body, an increase in its solubility in solution or else an improvement in its conservation. These pharmaceutically acceptable 30 vehicles are well known and will be adapted by the person skilled in the art as a function of the nature and of the mode of administration of the active compound(s) chosen.

48 Binding members of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the binding member. Thus pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active 5 ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, inhaled, intra-tracheal, topical, intra-vesicular or by injection, as discussed below. 10 Pharmaceutical compositions for oral administration, such as for example single domain antibody molecules (e.g. "nanobodiesTM") etc are also envisaged in the present invention. Such oral formulations may be in tablet, capsule, powder, liquid or semi-solid form. A tablet may comprise a solid carrier, such as gelatin or an adjuvant. Liquid 15 pharmaceutical compositions generally comprise a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol may be included. 20 For intra-venous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles, such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, 25 antioxidants and/or other additives may be employed as required including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3' 30 pentanol; and m-cresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagines, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating 49 agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions, such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants, such as TWEEN

T

', PLURONICSTM or polyethylene glycol (PEG). 5 Binding members of the present invention may be formulated in liquid, semi-solid or solid forms depending on the physicochemical properties of the molecule and the route of delivery. Formulations may include excipients, or combinations of excipients, for example: sugars, amino acids and surfactants. Liquid formulations may include a wide range of antibody concentrations and pH. Solid formulations may be produced by lyophilisation, spray 10 drying, or drying by supercritical fluid technology, for example. Formulations of anti-IgE will depend upon the intended route of delivery: for example, formulations for pulmonary delivery may consist of particles with physical properties that ensure penetration into the deep lung upon inhalation; topical formulations (e.g. for treatment of scarring, e.g. dermal scarring) may include viscosity modifying agents, which prolong the time that the drug is resident at the site 15 of action. A binding member may be prepared with a carrier that will protect the binding member against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such 20 formulations are known to those skilled in the art [66]. Anti-IgE treatment may be given orally (such as for example single domain antibody molecules (e.g. "nanobodiesTM")) by injection (for example, subcutaneously, intra-articular, intra-venously, intra-peritoneal, intra-arterial or intra-muscularly), by inhalation, intra 25 tracheal, by the intra-vesicular route (instillation into the urinary bladder), or topically (for example intra-ocular, intra-nasal, rectal, into wounds, on skin). The treatment may be administered by pulse infusion, particularly with declining doses of the binding member. The route of administration can be determined by the physicochemical characteristics of the treatment, by special considerations for the disease or by the requirement to optimize efficacy 30 or to minimize side-effects. One particular route of administration is intra-venous. Another route of administering pharmaceutical compositions of the present invention is subcutaneously. It is envisaged that anti-IgE treatment will not be restricted to use in the clinic. Therefore, subcutaneous injection using a needle-free device is also advantageous.

50 Examples of intravenous formulations include: Formulation (1) comprises 5 An isolated binding member of the invention (optionally 10, 50, 100 or 150mg/ml of said binding member, for example, an antibody) 50mM sodium acetate 100mM NaCl pH5.5 10 Formulation (2) comprises An isolated binding member of the invention (optionally 10, 50, 100 or 150mg/ml of said binding member, for example, an antibody) 20mM Succinate 15 105mM NaCI 10mM Arginine pH 6.00 A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. 20 A binding member for IgE may be used as part of a combination therapy in conjunction with an additional medicinal component. Combination treatments may be used to provide significant synergistic effects, particularly the combination of an anti-IgE binding member with one or more other drugs. A binding member for IgE may be administered 25 concurrently or sequentially or as a combined preparation with another therapeutic agent or agents, for the treatment of one or more of the conditions listed herein. A binding member of the invention may be formulated and/or used in combination with other available treatments for asthma and allergic disorders, or other disorders involving 30 IgE mediated effects. A binding member according to the present invention may be provided in combination or addition with one or more of the following agents: 51 - a cytokine or agonist or antagonist of cytokine function (e.g. an agent which acts on cytokine signalling pathways, such as a modulator of the SOCS system), such as an alpha-, beta- and/or gamma-interferon; insulin-like growth factor type I (IGF-1), its receptors and associated binding proteins; interleukins (IL), e.g. one or more of IL-I to -33, and/or an 5 interleukin antagonist or inhibitor, such as anakinra; inhibitors of receptors of interleukin family members or inhibitors of specific subunits of such receptors, a tumour necrosis factor alpha (TNF-a) inhibitor, such as an anti-TNF monoclonal antibodies (for example infliximab, adalimumab and/or CDP-870) and/or a TNF receptor antagonist, e.g. an immunoglobulin molecule (such as etanercept) and/or a low-molecular-weight agent, such as pentoxyfylline; 10 - a modulator of B cells, e.g. a monoclonal antibody targeting B-lymphocytes (such as CD20 (rituximab) or MRA-aILI6R) or T-lymphocytes (e.g. CTLA4-lg, HuMax 11-15 or Abatacept); - a modulator that inhibits osteoclast activity, for example an antibody to RANKIL; - a modulator of chemokine or chemokine receptor function, such as an antagonist of CCRI, CCR2, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR1O 15 and CCR 1I (for the C-C family); CXCRI, CXCR2, CXCR3, CXCR4 and CXCR5 and CXCR6 (for the C-X-C family) and CX 3 CRI for the C-X 3 -C family; - an inhibitor of matrix metalloproteases (MMPs), i.e. one or more of the stromelysins, the collagenases and the gelatinases as well as aggrecanase, especially collagenase-l (MMP 1), collagenase-2 (MMP-8), collagenase-3 (MMP-13), stromelysin-l (MMP-3), stromelysin-2 20 (MMP-10) and/or stromelysin-3 (MMP-1 1) and/or MMP-9 and/or MMP-12, e.g. an agent such as doxycycline; - a leukotriene biosynthesis inhibitor, 5-lipoxygenase (5-LO) inhibitor or 5 lipoxygenase activating protein (FLAP) antagonist, such as zileuton; ABT-761; fenleuton; tepoxalin; Abbott-79175; Abbott-85761; N-(5-substituted)-thiophene-2-alkylsulfonamides; 25 2,6-di-tert-butylphenolhydrazones; methoxytetrahydropyrans such as Zeneca ZD-2138; the compound SB-210661; a pyridinyl-substituted 2-cyanonaphthalene compound, such as L 739,0 10; a 2-cyanoquinoline compound, such as L-746,530; indole and/or a quinoline compound, such as MK-591, MK-886 and/or BAY x 1005; - a receptor antagonist for leukotrienes (LT) B4, LTC4, LTD4, and LTE4, selected from 30 the group consisting of the phenothiazin-3-ls, such as L-651,392; amidino compounds, such as CGS-25019c; benzoxalamines, such as ontazolast; benzenecarboximidamides, such as BIIL 284/260; and compounds, such as zafirlukast, ablukast, montelukast, pranlukast, verlukast (MK-679), RG-12525, Ro-245913, iralukast (CGP 45715A) and BAY x 7195; 52 - a phosphodiesterase (PDE) inhibitor, such as a methylxanthanine, e.g. theophylline and/or aminophylline; and/or a selective PDE isoenzyme inhibitor, e.g. a PDE4 inhibitor and/or inhibitor of the isoform PDE4D and/or an inhibitor of PDE5; - a histamine type 1 receptor antagonist, such as cetirizine, loratadine, desloratadine, 5 fexofenadine, acrivastine, terfenadine, astemizole, azelastine, levocabastine, chlorpheniramine, promethazine, cyclizine, and/or mizolastine (generally applied orally, topically or parenterally); - a proton pump inhibitor (such as omeprazole) or gastroprotective histamine type 2 receptor antagonist; 10 - an antagonist of the histamine type 4 receptor; - an alpha-1 /alpha-2 adrenoceptor agonist vasoconstrictor sympathomimetic agent, such as propylhexedrine, phenylephrine, phenylpropanolamine, ephedrine, pseudoephedrine, naphazoline hydrochloride, oxymetazoline hydrochloride, tetrahydrozoline hydrochloride, xylometazoline hydrochloride, tramazoline hydrochloride and ethylnorepinephrine 15 hydrochloride; - an anticholinergic agent, e.g. a muscarinic receptor (MI, M2, and M3) antagonist, such as atropine, hyoscine, glycopyrrrolate, ipratropium bromide, tiotropium bromide, oxitropium bromide, pirenzepine and telenzepine; - a beta-adrenoceptor agonist (including beta receptor subtypes 1-4), such as 20 isoprenaline, salbutamol, formoterol, salmeterol, terbutaline, orciprenaline, bitolterol mesylatc and/or pirbuterol, e.g. a chiral enantiomer thereof; - a chromone, e.g. sodium cromoglycate and/or nedocromil sodium; - a glucocorticoid, such as flunisolide, triamcinolone acetonide, beclomethasone dipropionate, budesonide, fluticasone propionate, ciclesonide, and/or mometasone furoate; 25 - an agent that modulate nuclear hormone receptors, such as a PPAR; - an immunoglobulin (Ig) or Ig preparation or an antagonist or antibody modulating Ig function, such as anti-IgE that binds to the same or a different epitope as the binding member of the invention; - other systemic or topically-applied anti-inflammatory agent, e.g. thalidomide or a 30 derivative thereof, a retinoid, dithranol and/or calcipotriol; - combinations of aminosalicylates and sulfapyridine, such as sulfasalazine, mesalazine, balsalazide, and olsalazine; and immunomodulatory agents, such as the thiopurines; and corticosteroids, such as budesonide; 53 - an antibacterial agent, e.g. a penicillin derivative, a tetracycline, a macrolide, a beta lactam, a fluoroquinolone, metronidazole and/or an inhaled aminoglycoside; and/or an antiviral agent, e.g. acyclovir, famciclovir, valaciclovir, ganciclovir, cidofovir; amantadine, rimantadine; ribavirin; zanamavir and/or oseltamavir; a protease inhibitor, such as indinavir, 5 nelfinavir, ritonavir and/or saquinavir; a nucleoside reverse transcriptase inhibitor, such as didanosine, lamivudine, stavudine, zalcitabine, zidovudine; a non-nucleoside reverse transcriptase inhibitor, such as nevirapine, efavirenz; - a cardiovascular agent, such as a calcium channel blocker, beta-adrenoceptor blocker, angiotensin-converting enzyme (ACE) inhibitor, angiotensin- 2 receptor antagonist; lipid 10 lowering agent, such as a statin and/or fibrate; a modulator of blood cell morphology, such as pentoxyfylline; a thrombolytic and/or an anticoagulant, e.g. a platelet aggregation inhibitor; - a CNS agent, such as an antidepressant (such as sertraline), anti-Parkinsonian drug (such as deprenyl, L-dopa, ropinirole, pramipexole; MAOB inhibitor, such as selegine and rasagiline; comP inhibitor, such as tasmar; A-2 inhibitor, dopamine reuptake inhibitor, 15 NMDA antagonist, nicotine agonist, dopamine agonist and/or inhibitor of neuronal nitric oxide synthase) and an anti-Alzheimer's drug, such as donepezil, rivastigmine, tacrine, COX 2 inhibitor, propentofylline or metrifonate; - an agent for the treatment of acute and chronic pain, e.g. a centrally or peripherally acting analgesic, such as an opioid analogue or derivative, carbamazepine, phenytoin, sodium 20 valproate, amitryptiline or other antidepressant agent, paracetamol, or non-steroidal anti inflammatory agent; - a parenterally or topically-applied (including inhaled) local anaesthetic agent, such as lignocaine or an analogue thereof; - an anti-osteoporosis agent, e.g. a hormonal agent, such as raloxifene, or a 25 biphosphonate, such as alendronate; - (i) a tryptase inhibitor; (ii) a platelet activating factor (PAF) antagonist; (iii) an interleukin converting enzyme (ICE) inhibitor; (iv) an IMPDH inhibitor; (v) an adhesion molecule inhibitors including VLA-4 antagonist; (vi) a cathepsin; (vii) a kinase inhibitor, e.g. an inhibitor of tyrosine kinases (such as Btk, ltk, Jak3 MAP examples of inhibitors might 30 include Gefitinib, Imatinib mesylate), a serine / threonine kinase (e.g. an inhibitor of MAP kinase, such as p3 8 , JNK, protein kinases A, B and C and IKK), or a kinase involved in cell cycle regulation (e.g. a cylin dependent kinase); (viii) a glucose-6 phosphate dehydrogenase inhibitor; (ix) a kinin-B.subl. - and/or B.sub2. -receptor antagonist; (x) an anti-gout agent, 54 e.g. colchicine; (xi) a xanthine oxidase inhibitor, e.g. allopurinol; (xii) a uricosuric agent, e.g. probenecid, sulfinpyrazone, and/or benzbromarone; (xiii) a growth hormone secretagogue; (xiv) transforming growth factor (TGFp); (xv) platelet-derived growth factor (PDGF); (xvi) fibroblast growth factor, e.g. basic fibroblast growth factor (bFGF); (xvii) granulocyte 5 macrophage colony stimulating factor (GM-CSF); (xviii) capsaicin cream; (xix) a tachykinin NK.subl. and/or NK.sub3. receptor antagonist, such as NKP-608C, SB-233412 (talnetant) and/or D-4418; (xx) an elastase inhibitor, e.g. UT-77 and/or ZD-0892; (xxi) a TNF-alpha converting enzyme inhibitor (TACE); (xxii) induced nitric oxide synthase (iNOS) inhibitor or (xxiii) a chemoattractant receptor-homologous molecule expressed on TH2 cells (such as a 10 CRTH2 antagonist); (xxiv) an inhibitor of a P38 (xxv) agent modulating the function of Toll like receptors (TLR) and (xxvi) an agent modulating the activity of purinergic receptors, such as P2X7; (xxvii) an inhibitor of transcription factor activation, such as NFkB, API, and/or STATS. 15 An inhibitor may be specific or may be a mixed inhibitor, e.g. an inhibitor targeting more than one of the molecules (e.g. receptors) or molecular classes mentioned above. The binding member could also be used in association with a chemotherapeutic agent such as a tyrosine kinase inhibitor in co-administration or in the form of an immunoconjugate. 20 Fragments of said antibody could also be use in bispecific antibodies obtained by recombinant mechanisms or biochemical coupling and then associating the specificity of the above described antibody with the specificity of other antibodies able to recognize other molecules involved in the activity for which IgE is associated. 25 For treatment of an inflammatory disease, e.g. rheumatoid arthritis, osteoarthritis, asthma, allergic rhinitis, chronic obstructive pulmonary disease (COPD), or psoriasis, a binding member of the invention may be combined with one or more agents, such as non steroidal anti-inflammatory agents (hereinafter NSAIDs) including non-selective cyclo oxygenase (COX)-I / COX-2 inhibitors whether applied topically or systemically, such as 30 piroxicam, diclofenac, propionic acids, such as naproxen, flurbiprofen, fenoprofen, ketoprofen and ibuprofen, fenamates, such as mefenamic acid, indomethacin, sulindac, azapropazone, pyrazolones, such as phenylbutazone, salicylates, such as aspirin); selective COX-2 inhibitors (such as meloxicam, celecoxib, rofecoxib, valdecoxib, lumarocoxib, parecoxib and 55 etoricoxib); cyclo-oxygenase inhibiting nitric oxide donors (CINODs); glucocorticosteroids (whether administered by topical, oral, intra-muscular, intra-venous or intra-articular routes); methotrexate, leflunonide; hydroxychloroquine, d-penicillamine, auranofin or other parenteral or oral gold preparations; analgesics; diacerein; intra-articular therapies, such as 5 hyaluronic acid derivatives; and nutritional supplements, such as glucosamine. A binding member of the invention and one or more of the above additional medicinal components may be used in the manufacture of a medicament. The medicament may be for separate or combined administration to an individual, and accordingly may comprise the 10 binding member and the additional component as a combined preparation or as separate preparations. Separate preparations may be used to facilitate separate and sequential or simultaneous administration, and allow administration of the components by different routes e.g. oral and parenteral administration. 15 In accordance with the present invention, compositions provided may be administered to mammals. Administration is normally in a "therapeutically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular mammal being 20 treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the type of binding member, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g. decisions on dosage ctc, is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of 25 a disease being treated. Appropriate doses of antibody are well known in the art [67, 68]. Specific dosages indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered may be used. A therapeutically effective amount or suitable dose of a binding member of the invention can be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for 30 extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the antibody is for diagnosis, prevention or for treatment, the size and location of the area to be treated, the precise nature of the antibody (e.g. whole antibody, fragment or diabody) and the nature of 56 any detectable label or other molecule attached to the antibody. A typical antibody dose will be in the range 100 ig to I g for systemic applications, and 1 pg to 1 mg for topical applications. An initial higher loading dose, followed by one or more lower doses, may be administered. Typically, the antibody will be a whole antibody, e.g. the IgGl isotype, IgG2 5 isotype, IgG3 isotype or IgG4 isotype. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted for other antibody formats in proportion to molecular weight. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. Treatments may be every two to four weeks for subcutaneous administration and every four to 10 eight weeks for intra-venous administration. Treatment may be periodic, and the period between administrations is about two weeks or more, e.g. about three weeks or more, about four weeks or more, or about once a month. Treatment may be given before, and/or after surgery, and/or may be administered or applied directly at the anatomical site of surgical treatment. 15 Brief Description of the Tables and Figures Table I lists the amino acid sequences of the heavy chain CDRs and the light chain CDRs of each of antibodies 1-28. Table 2a shows example potencies of clones identified from the targeted mutagenesis libraries when tested in the Receptor-ligand binding HTRF@ Assays. Table 2b 20 shows the binding affinity (KD) for exemplary binding members of the invention to human IgE and cynomolgus monkey IgE, using SPR (BIACORE). Table 2b further shows the potency, expressed as IC50, for exemplary binding members of the invention, in an RBL-ER51 calcium signalling assay (at 4 hours with 25 ng/mI human or 100ng/ml cynomolgus monkey IgE). 25 Table 3a shows the sequences of exemplary binding members of the invention are shown in the appended sequence listing, in which SEQ ID NOS correspond as shown in Table 3a. Table 3b shows the VL DNA and VL amino sequences of exemplary binding members of the invention from the provisional applicaton which are shown in the 30 appended sequence listing, in which SEQ ID NOS correspond as shown in Table 3b.

57 Table 4. Example binding affinity calculationa using BlAcore and potency measurements using RBL-ER51 calcium signalling assay for germlined antibodies. Table 5. shows the direct interactions between IgE C3-CE4 and Antibody 11 Fab in the 5 x-ray crystallographic studies for interaction 1. Table 6. shows the direct interactions between IgE CW3-CE4 and Antibody 1 Fab in the x-ray crystallographic studies for interaction 2. Table 7. shows Crystal Parameters and X-ray Data-Processing and Refinement Statistics from the x-ray crystallographic studies. 10 Table 8. shows the summary study design in the safety studies (Example 8). Figure 1: relates to Example 2.7 and shows the molar concentration of antibody expressed as a log on the x-axis and the peak height in an RBL-ER51 calcium signalling assay on the y axis. The open squares relate to antibody 11, the crosses an irrelevant igGl control antibody and the inverted open triangles a 15 anti-IgE cross-linking antibody (Biosource). Note that the open squares and crosses are superimposed on one another in this figure. Figure 2: show§'the sequence of Cynomolgus C&3-4 FLAG His10. Figure 3: shows the sequence of variable heavy chain that encodes human anti oestradiaol scFv (D12_VH) and one of cynomologous IgHE gene haplotype, 20 (cyigHE TQ). Figure 4: shows the sequence of variable heavy chain that encodes human anti oestradiaol scFv (D12_VH) and one of cynomologous IgHE gene haplotype,cyIgHE ME. Figure 5: shows the sequence of the variable light chain of Human anti-eostradiol scFv 25 (D12_VL) and cynomolgus lambda constant region genes cyIGLC 4, Sequence Range: I to 708. Figure 6: shows the sequence of the variable light chain of Human anti-eostradiol scFv (D 12VL) and cynomolgus lambda constant region genes D 12_VL cy[GLC 7. Figure 7: relates to Example 3 and shows the percentage inhibition of maximum IgE 30 expression in B cells not treated with blocking anti-IgE wherein the x axis is the concentration of Antibody I1 in nM and the y axis is percentage inhibition. The upper graph relates to Antibody 11 and the lower graph the control antibody.

58 Figure 8: relates to Example 4 and shows the percentage inhibition of total release of p hexosaminidase +/- SEM wherein the x axis is the concentration of Antibody L I in nM and the y axis is percentage inhibition. The upper graph relates to Antibody II and the lower graph to the control antibody. 5 Figure 9: Relates to Example 5. The figure shows the percentage of bound IgE in human sera with increasing concentrations of the anti-IgE antibody, Antibody I1. The x-axis measures concentraton of Antibody 11 in micrograms/ml and the y axis shows amount of bound IgE as a percentage of free IgE analysed and plotted as a function of total IgE. 10 Figure 10: Relates to Example 7 and shows a) A ribbon representation of IgE Ce3-Ce4 dimer, where the two monomers are denoted IgEl and IgE2 and interacts with two Antibody 11 Fab molecules. Glycosylations at Asn394 are shown as ball-and-stick models; and b) A 90 degrees rotated view looking down from the top showing that the 15 majority of the interactions to IgE from the Fab fragment is provided by the heavy chain. The figure was generated using the program PyMOL (DeLano Scientific LLC, San Carlos, California, U.S.A) Figure 11: Relates to Example 7 and shows the glycosylation at position Asn394 of IgE. Figure 12: Relates to Example 8 and shows mean toxicokinetics profiles of Antibody 11 20 [gG 1 , Antibody 11 IgG 2 and E48 following 1 mg/kg (Day 1). 30 mg/kg (Day 8), and 100 mg/kg (Day 16 and beyond) doses in cynomolgus monkeys. Error bars represent standard deviations. The y-axis is serum concentration of antibody and the x axis is time in days following the first dose. Group I (Antibody 11 IgGl) is shown as filled circles, Group 2 (Antibody 11 IgG) 25 is shown as open triangles and Group 3 (a different anti-IgE molecule E48) is shown as filed squares. Figure 13: Relates to Example 8 and shows mean free IgE profiles in cynomolgus monkeys receiving weekly doses of Antibody 11 IgGI, Antibody 11 IgG2 and E48 (I mg/kg on Day 1, 30 mg/kg on Day 8, and 100 mg/kg on Day 16 and 30 beyond). Error bars are standard deviations. The y-axis is IgE concentration in ng/ml and the x axis is time in days. Group I (Antibody 11 IgGI) is shown as filled circles, Group 2 (Antibody I IIgG2) is shown as open 59 triangles and Group 3 (a different anti-IgE molecule E48) is shown as filed squares. Figure 14: Relates to Example 8 an shows a plot of platelet numbers (x10^9/L) expressed as a percentage change from the mean of the 2 pre-dose values 5 versus plasma concentration from an animal in Group 1 (Antibody 11 treated). This plot is representative of the other 16 animals across the 3 groups that showed no significant effect on platelets. The x-axis shows time in hours, the left y axis the level of platelets as a percentage change from the mean level pre-treatment and the left y-axis the concentration of anti-IgE 10 antibody in nmol/L. The closed squares show the platelet concentration and the filled diamonds the concentration of anti-IgE antibody. The closed triangles show the dosing of the anti-IgE antibody in mg/kg. Figure 15 Relates to Example 8 and shows a plot of platelet numbers (x 10 9 /L) expressed as a percentage change from the mean of the 2 pre-dose values 15 vesus plasma concentration from an animal in Group 1 (Antibody 11 IgGi treated) that showed a transient significant drop (35% below baseline) in platelet numbers on day 29. The x-axis shows time in hours, the left y axis the level of platelets as a percentage change from the mean level pre treatment and the left y-axis the concentration of anti-IgE antibody in nmol/L. 20 The closed squares show the platelet concentration and the filled diamonds the concentration of anti-IgE antibody. The closed triangles show the dosing of the anti-IgE antibody in mg/kg. Figure 16 Relates to Example 9 and shows Antibody 11 inhibition of IgE/FceRI mediated cytotoxicity. The x axis is molar concentration of Antibody 11 and 25 the y axis is percentage cytotoxicity. In the graph the open triangle and solid circle relate to the Mov 18 IgE experiment wherein the open triangle is the isotype control and the solid circle is Antibody 11 and in the graph the bold open triangle (which is hidden under the points at the right hand side of the graph) and open circle related to the NIP IgE control wherein the open bold 30 triangle is the isotype control and the open circle is Antibody 11. Figure 17 Relates to Example 9 and shows Antibody I 1 inhibition of IgE/CD23 mediated phagocytosis. The x axis is molar concentration of Antibody 11 and the y axis is percentage phagocytosis. In the graph the triangle and solid 60 circle relate to the Mov18 IgE experiment wherein the open triangle is the isotype control and the solid circle is Antibody 11 and in the graph the bold open triangle and open circle related to the NIP IgE control wherein the open bold triangle is the isotype control and the open circle is Antibody 11. 5 Examples NaYve human single chain Fv (scFv) phage display libraries cloned in to a phagemid vector based on the filamentous phage M13 were used for selections [69, 70]). Anti-IgE specific scFv antibodies are isolated from the phage display libraries using a 10 series of selection cycles on recombinant human IgE. Selected scFv antibodies are optimized for binding to human IgE and/or for potency, and are reformatted as IgG antibodies. 15 SEQUENCES Sequences of exemplary binding members of the invention are shown in the appended sequence listing, in which SEQ ID NOS correspond as shown in Table 3a below wherein: i) where an antibody number is followed by GL, for example 8GL this refers to the antibody wherein one or more of the residues have been mutated back to the 20 germline configuration, in general where GL is used all non-germline residues which can be mutated back to germline without appreciable loss of activity have been germlined; and ii) where an antibody number is followed by PGL, for example I IPGL this refers to the antibody wherein one or more of the residues have been mutated back to the 25 germline configuration, in general where PGL is used more residues have been mutated back to germline than GL but resulting in some loss of activity over the non-germlined.

61 Table 3a Antibody SEQ ID No. Description I 1 VH/DNA 1 2 VH/amino acid 1 3 HCDR1 1 4 HCDR2 1 5 HCDR3 1 317 VL/DNA 1 318 VL/amino acid 1 8 LCDRI 1 9 LCDR2 1 10 LCDR3 2 11 VH/DNA 2 12 VH/amino acid 2 13 HCDR1 2 14 HCDR2 2 15 HCDR3 2 319 VL/DNA 2 320 VL/amino acid 2 18 LCDRI 2 19 LCDR2 2 20 LCDR3 3 21 VH/DNA 3 22 VH/amino acid 3 23 HCDRI 3 24 HCDR2 3 25 HCDR3 3 321 VL/DNA 3 322 VL/amino acid 3 28 LCDR1 62 Antibody SEQ ID No. Description 3 29 LCDR2 3 30 LCDR3 4 31 VH/DNA 4 32 VH/amino acid 4 33 HCDRI 4 34 HCDR2 4 35 HCDR3 4 323 VL/DNA 4 324 VLlamino acid 4 38 LCDRI 4 39 LCDR2 4 40 LCDR3 5 41 VH/DNA 5 42 VI-/amino acid 5 43 HCDRI 5 44 HCDR2 5 45 HCDR3 5 325 VL/DNA 5 326 VL/amino acid 5 48 LCDRI 5 49 LCDR2 5 50 LCDR3 6 51 VH/DNA 6 52 VH/amino acid 6 53 HCDR1 6 54 H CDR2 6 55 ICDR3 6 327 VL/DNA 6 328 VLlamino acid 6 58 LCDRL 63 Antibody SEQ ID No. Description 6 59 LCDR2 6 60 LCDR3 7 61 VH/DNA 7 62 VH/amino acid 7 63 HCDRI 7 64 HCDR2 7 65 HCDR3 7 329 VL/DNA 7 330 VL/amino acid 7 68 LCDR1 7. 69 LCDR2 7 70 LCDR3 8 71 VH/DNA 8 72 VH/amino acid 8 73 HCDR1 8 74 HCDR2 8 75 HCDR3 8 331 VL/DNA 8 332 VL/amino acid 8 78 LCDR1 8 79 LCDR2 8 80 LCDR3 9 81 VH/DNA 9 82 VH/amino acid 9 83 HCDR1 9 84 HCDR2 9 85 HCDR3 9 333 VL/DNA 9 334 VL/amino acid 9 88 LCDR1 64 Antibody SEQ ID No. Description 9 89 LCDR2 9 90 LCDR3 10 91 VH/DNA 10 92 VH/amino acid 10 93 HCDR1 10 94 HCDR2 10 95 HCDR3 10 335 VL/DNA 10 336 VL/amino acid 10 98 LCDRI 10 99 LCDR2 10 100 LCDR3 11 101 VH/DNA 11 102 VH/amino acid 11 103 HCDR1 11 104 HCDR2 11 105 HCDR3 11 337 VL/DNA 11 338 VL/amino acid 11 108 LCDR1 11 109 LCDR2 11 110 LCDR3 12 111 VH/DNA 12 112 VH/amino acid 12 113 HCDR1 12 114 HCDR2 12 115 HCDR3 12 339 VL/DNA 12 340 VL/amino acid 12 118 LCDR1 65 Antibody SEQ ID No. Description 12 119 LCDR2 12 120 LCDR3 13 121 VH/DNA 13 122 VH/amino acid 13 123 HCDRI 13 124 HCDR2 13 125 HCDR3 13 341 VL/DNA 13 342 VL/amino acid 13 128 LCDR1 13 129 LCDR2 13 130 LCDR3 14 131 VH/DNA 14 132 VH/amino acid 14 133 HCDRI 14 134 HCDR2 14 135 HCDR3 14 343 VL/DNA 14 344 VL/amino acid 14 138 LCDRI 14 139 LCDR2 14 140 LCDR3 15 141 VH/DNA 15 142 VI/amino acid 15 143 HCDR1 15 144 HCDR2 15 145 HCDR3 15 345 VL/DNA 15 346 VL/amino acid 15 148 LCDR1 66 Antibody SEQ ID No. Description 15 149 LCDR2 15 150 LCDR3 16 151 VH/DNA 16 152 VH/amino acid 16 153 HCDRI 16 154 HCDR2 16 155 HCDR3 16 347 VL/DNA 16 348 VL/amino acid 16 158 LCDR1 16 159 LCDR2 16 160 LCDR3 17 161 VH/DNA 17 162 VHlamino acid 17 163 HCDRI 17 164 HCDR2 17 165 HCDR3 17 349 VL/DNA 17 350 VL/amino acid 17 168 LCDRI 17 169 LCDR2 17 170 LCDR3 18 171 VH/DNA 18 172 VH/amino acid 18 173 HCDRI 18 174 HCDR2 18 175 HCDR3 18 351 VL/DNA 18 352 VL/amino acid 18 178 LCDRI 67 Antibody SEQ ID No. Description 18 179 LCDR2 18 180 LCDR3 19 181 VH/DNA 19 182 VH/amino acid 19 183 HCDRI 19 184 HCDR2 19 185 HCDR3 19 353 VL/DNA 19 354 VL/amino acid 19 188 LCDRI 19 189 LCDR2 19 190 LCDR3 20 191 VH/DNA 20 192 VHlamino acid 20 193 HCDR1 20 194 HCDR2 20 195 HCDR3 20 355 VL/DNA 20 356 VL/amino acid 20 198 LCDR1 20 199 LCDR2 20 200 LCDR3 21 201 VH/DNA 21 202 VH/amino acid 21 203 HCDR1 21 204 HCDR2 21 205 HCDR3 21 357 VL/DNA 21 358 VL/amino acid 21 208 LCDR1 68 Antibody SEQ ID No. Description 21 209 LCDR2 21 210 LCDR3 22 211 VH/DNA 22 212 VH/amino acid 22 213 HCDR1 22 214 HCDR2 22 215 HCDR3 22 359 VL/DNA 22 360 VL/amino acid 22 218 LCDRI 22 219 LCDR2 22 220 LCDR3 23 221 VH/DNA 23 222 VH/amino acid 23 223 HCDR1 23 224 HCDR2 23 225 HCDR3 23 361 VL/DNA 23 362 VL/amino acid 23 228 LCDR1 23 229 LCDR2 23 230 LCDR3 24 231 VH/DNA 24 232 VH/amino acid 24 233 HCDRI 24 234 HCDR2 24 235 HCDR3 24 363 VL/DNA 24 364 VL/amino acid 24 238 LCDR1 69 Antibody SEQ ID No. Description 24 239 LCDR2 24 240 LCDR3 25 241 VH/DNA 25 242 VH/amino acid 25 243 HCDR1 25 244 HCDR2 25 245 HCDR3 25 365 VL/DNA 25 366 VL/amino acid 25 248 LCDRI 25 249 LCDR2 25 250 LCDR3 26 251 VH/DNA 26 252 VI-/amino acid 26 253 HCDRI 26 254 HCDR2 26 255 HCDR3 26 367 VL/DNA 26 368 VL/amino acid 26 258 LCDR1 26 259 LCDR2 26 260 LCDR3 27 261 VH/DNA 27 262 VH/amino acid 27 263 HCDRI 27 264 HCDR2 27 265 HCDR3 27 369 VL/DNA 27 370 VL/amino acid 27 268 LCDR1 70 Antibody SEQ ID No. Description 27 269 LCDR2 27 270 LCDR3 28 271 VH/DNA 28 272 VH/amino acid 28 273 HCDRI 28 274 HCDR2 28 275 HCDR3 28 371 VL/DNA 28 372 VUamino acid 28 278 LCDR1 28 279 LCDR2 28 280 LCDR3 8GL 281 VH/DNA 8GL 282 VH/amino acid 8GL 283 HCDR1 8GL 284 HCDR2 8GL 285 HCDR3 8GL 373 VL/DNA 8GL 374 VL/amino acid 8GL 296 LCDRI 8GL 297 LCDR2 8GL 298 LCDR3 8PGL 287 VH/DNA 8PGL 288 VH/amino acid 8PGL 289 HCDR1 8PGL 290 HCDR2 8PGL 291 HCDR3 8PGL 375 VL/DNA 8PGL 376 VL/amino acid 8PGL 296 LCDR1 71 Antibody SEQ ID No. Description 8PGL 297 LCDR2 8PGL 298 LCDR3 11GL 299 VHIDNA I1GL 300 VH/amino acid IIGL 301 HCDRI 11GL 302 HCDR2 11GL 303 HCDR3 11GL 377 VL/DNA IIGL 378 VL/amino acid I1GL 314 LCDRI IIGL 315 LCDR2 IIGL 316 LCDR3 IIPGL 305 VH/DNA 11PGL 306 VH/amino acid i IPGL 307 HCDR1 IIPGL 308 HCDR2 1I IPGL 309 HCDR3 IIPGL 379 VL/DNA t IPGL 380 VL/amino acid I1PGL 314 LCDR1 1IPGL 315 LCDR2 IPGL 316 LCDR3 381 Cynomolgus Ce3-4 FLAG His10 nucleotide 382 Cynomolgus Ce3-4 FLAG His10 protein 383 D12_VHcylgHE TQ nucleotide D12 VH cylgHE TQ 384p~~ protein 72 Antibody SEQ ID No. Description D12_HE cylgHE ME 385~ nucleotide D12_VH cylgHE ME 386 protein D1 2_VL cylgLC 4 387~ nucleotide 388 D12_VL cyIgLC 4 protein D12_VL cyfgLC 7 389 nucleotide 390 DI2_VL cyIgLC 7 protein 391 FceRIFc (NSO) nucleotide 392 FceRI_Fc (NSO) protein In the sequence listing filed with the provisional application (US provisional application number: 60/901304) the sequences of the 3' ggt codon, and corresponding Glycine residue, shown in the nucleotide and amino acid sequence for the VL DNA and corresponding VL 5 amino acid were included in the expressed scFv and IgG sequences of this antibody. The C terminal Glycine residue of the sequence corresponds to Kabat residue 108. This terminal glycine is not part of the VL sequence and has been removed from the sequences listed in Table 3a. The sequences for VL DNA and VL amino acid from the provisional application are included with the sequence listing and are listed in Table 3b below. The origin of this 10 residue and its encoding triplet ggt is explained below. To express the light chain of the IgG, a nucleotide sequence encoding the antibody light chain was provided, comprising a first exon encoding the VL domain, a second exon encoding the CL domain, and an intron separating the first exon and the second exon. Under normal 15 circumstances, the intron is spliced out by cellular mRNA processing machinery, joining the 3' end of the first exon to the 5' end of the second exon. Thus, when DNA having the said nucleotide sequence was expressed as RNA, the first and second exons were spliced together. Translation of the spliced RNA produces a polypeptide comprising the VL domain and CL 73 domain. After splicing, the Gly at Kabat residue 108 is encoded by the last base (g) of the VL domain framework 4 sequence and the first two bases (gt) of the CL domain. Therefore, the Glycine residue at Kabat residue 108 was included in the sequence lisings of 5 the VL sequences in the provisional application but as described above it should not be considered to be the C terminal residue of the VL domain of the antibody molecule and thus has been deleted from sequence listings in Table 3a. Table 3b SEQ ID NO Antibody Description 6 1 VL/DNA 7 1 VL/amino acid 16 2 VL/DNA 17 2 VL/anino acid 26 3 VL/DNA 27 3 VL/amino acid 36 4 VL/DNA 37 4 VL/amino acid 46 5 VL/DNA 47 5 VL/amino acid 74 SEQ ID NO Antibody Description 56 6 VL/DNA 57 6 VL/amino acid 66 7 VL/DNA 67 7 VL/amino acid 76 8 VL/DNA 77 8 VL/amino acid 86 9 VL/DNA 87 9 VL/amino acid 96 10 VL/DNA 97 10 VL/amino acid 106 11 VL/DNA 107 11 VL/amino acid 116 12 VL/DNA 117 12 VL/amino acid 75 SEQ ID NO Antibody Description 126 13 VL/DNA 127 13 VL/amino acid 136 14 VL/DNA 137 14 VL/amino acid 146 15 VL/DNA 147 15 VL/amino acid 156 16 VL/DNA 157 16 VLlamino acid 166 17 VL/DNA 167 17 VL/amino acid 176 18 VL/DNA 177 18 VLlamino acid 186 19 VL/DNA 187 19 VUamino acid 76 SEQ ID NO Antibody Description 196 20 VL/DNA 197 20 VL/amino acid 206 21 VL/DNA 207 21 VL/amino acid 216 22 VL/DNA 217 22 VL/amino acid 226 23 VL/DNA 227 23 VL/amino acid 236 24 VL/DNA 237 24 VL/amino acid 246 25 VL/DNA 247 25 VL/amino acid 256 26 VL/DNA 257 26 VL/amino acid 77 SEQ ID NO Antibody Description 266 27 VL/DNA 267 27 Vlamino acid 276 28 VL/DNA 277 28 VL/amino acid 294 29(8 GL) VL/DNA 295 29 (8 GL) VLlamino acid 294 30 (8 PGL) VL/DNA 295 30 (8 PGL) VL/amino acid 312 32 (l GL) VL/DNA 313 32 (11 GL) VL/amino acid 312 33 (11 PGL) VL/DNA 313 33 (11 PGL) VL/amino acid In the sequence listing in the provisional application the sequences listed as Antibodies 29 34 are listed in Table 3a as Antibody 8GL, 8PGL, I IGL and 11 PGL. Some of these antibodies shares a common VL domain and as a result some sequences ID Nos in the 78 sequence lisitng provided in the provisional application are empty. The correct composition of the antibodies is as follows: Antibody 29 corresponds to 8GL VH domain Antibody 30 corresponds to 8PGL VH domain 5 Antibody 31 corresponds to the VL domain shared by 8GL and 8PGL. Antibody 32 corresponds to 1 lGL VH domain Antibody 33 corresponds to IlPGL VH domain Antibody 34 corresponds to the VL domain shared by 11 GL and I1 PGL. Sequence ID Nos 286, 292, 293, 304, 310 and 311 are empty. This has been corrected in 10 Table 3a The invention will now be exemplified by the following non-limiting examples: Example 1. Lead Isolation 1.1 Selections 15 NaYve human single chain Fv (scFv) phage display libraries cloned in to a phagemid vector based on the filamentous phage M13 were used for selections (Vaughan et al., Nature Biotechnology 14: 309-314 (1996), Hutchings, Antibody Engineering, R.Kontermann and S. Dubel, Editors. 2001, Springer Laboratory Manuals, Berlin. P93). Anti-IgE specific scFv antibodies were isolated from the phage display libraries using a series of selection cycles on 20 either plasma purified human IgEK (Calbiochem) or plasma purified human IgEX (Biodesign) essentially as previously described by Vaughan et al (Vaughan et al., Nature Biotechnology 14: 309-314 (1996). In brief, for panning selections, human IgE in PBS (Dulbecco's PBS, pH7.4) was adsorbed onto wells of a Maxisorp microtitre plate (Nunc) overnight at 4 0 C. Wells were washed with PBS then blocked for 1 h with PBS-Marvel (3% w/v). Purified 25 phage in PBS-Marvel (3% w/v) were added to the welts and allowed to bind coated antigen for I h. Unbound phage were removed by a series of wash cycles using PBS-Tween (0.1% v/v) and PBS. Bound phage particles were eluted, infected into bacteria and rescued for the next round of selection (Vaughan et al., Nature Biotechnology 14: 309-314 (1996)). Alternate rounds of selection were performed using the kappa and lambda forms of IgE.

79 1.2 Inhibition ofIgE binding to FceRI by wipurfled scFv A representative number of individual scFv from the second round of selections were grown up in 96-well plates. ScFvs were expressed in the bacterial periplasm and screened for their inhibitory activity in a homogeneous FRET (Fluorescence resonance energy transfer) based 5 human IgE/human FcERI-binding assay. In this assay, samples competed for binding to human IgE (Calbiochem 401152) labelled with Europium Chelate (Perkin Elmer 1244-302), with human FesRI-Fc (in house NSO cell produced). The detailed assay method is provided in the Materials and Methods section. 1.3 Inhibition ofIgE binding to FceRI by purified scFv 10 ScFv which showed a significant inhibitory effect on the IgE:FcERI interaction as unpurified periplasmic extracts, were subjected to DNA sequencing (Vaughan et at. 1996, Nature Biotechnology 14: 309-314), (Osbourn 1996;Lmmunotechnology. 2, 181-196). Unique scFvs were expressed again in bacteria and purified by affinity chromatography (as described by Bannister et al (2006) Biotechnology and bioengineering, 94. 931-937). The potencies of 15 these samples were determined by competing a dilution series of the purified preparation against FcERI (in house NSO cell produced), for binding to human IgE (Calbiochem 401152) labelled with Europium Chelate (Perkin Elmer 1244-302). Purified scFv preparations e.g Antibody 1 were capable of inhibiting the IgE-FcsRI interaction. Detailed protocols are provided in Materials and Methods section. 20 1.4 Reformatting of scFv to IgGI Clones were converted from scFv to IgG format by sub-cloning the VH and VL domains into vectors expressing whole antibody heavy and light chains respectively. The VH domain was cloned into a vector (pEU 15.1 or pEU9.2) containing the human heavy chain constant domains and regulatory elements to express whole IgG1 or IgG2 heavy chain in mammalian 25 cells respectively. Similarly, the VL domain was cloned into either vector pEU3.4 for the expression of the human kappa light chain or pEU4.4 for the expression of the human lambda light chain constant domains, with regulatory elements to express whole IgG light chain in mammalian cells. Vectors for the expression of heavy chains and light chains were originally described by Persic et al. (Persic, L., et al. (1997) Gene 187, 9-18). Cambridge Antibody 30 Technology vectors have been engineered to include an EBV OriP element which, in 80 combination with the EBNAl protein, allows for episomal replication of the plasmid. To obtain IgGs, the heavy and light chain IgG expressing vectors were transfected into EBNA HEK293 mammalian cells. IgGs were expressed and secreted into the medium. Harvests were pooled and filtered prior to purification. The IgG was purified using Protein A 5 chromatography. Culture supematants were loaded on a Ceramic Protein A column (BioSepra) and washed with 50 mM Tris-HCl pH 8.0, 250 mM NaCl. Bound IgG was eluted from the column using 0.1 M Sodium Citrate (pH 3.0) and neutralised by the addition of Tris HCl (pH 9.0). The eluted material was buffer exchanged into PBS using NaplO columns (Amersham, #17-0854-02) and the concentration of IgG was determined 10 spectrophotometrically using an extinction coefficient based on the amino acid sequence of the IgG (Mach et al Anal. Biochem. 200(1): 20-26, 1992). The purified [gG were analysed for aggregation or degradation using SEC-HPLC and by SDS-PAGE. 1.5 Inhibition of calcium signalling in RBL-ER51 cells by purified scFv and IgG The neutralisation potency of purified scFv and IgG preparations against human IgE 15 bioactivity mediated through FcsRI was assessed using an RBL-ER5I calcium-signalling assay. RBL-2H3 cells (a rat basophilic cell line) were stably transfected with the human FceRI (RBL-ER5I cells). Free IgE in the vicinity of the cells binds to the FceRI on the cell surface and subsequent cross-linking of receptor-bound IgE leads to a calcium mobilisation that can be detected using a Fluorometric Imaging Plate Reader (FLIPR). A detailed 20 description of the protocol is provided in the Materials and Methods section. Purified scFv preparations of Antibody I were capable of inhibiting the IgE induced calcium signalling of the RBL-ER51 cells at the maximum concentration tested. When tested as a purified IgG, the IC 50 for Antibody I was calculated as being 34nM. 25 1.6 Selectivity and species cross reactivity of antibodies in DELFIA @ epitope competition assays The species cross reactivity and selectivity of antibodies to IgE and structurally related molecules; IgA, IgM, IgD and IgG, was established using DELFIA@ epitope competition assays. The assay determines relative cross reactivity by measuring inhibition of biotinylated 30 IgE (plasma purified, BIODESIGN International), binding each immobilised anti-IgE antibody.

81 Titrations of purified IgA, IgM, IgD, and IgG (all Calbiochem) were tested in each assay to establish the specificity profile for each structurally related protein, as measured by IC50 values in the assay. 5 Titrations of IgE species including cynomolgus IgE CE3-CE4 domain (in house HEK-EBNA derived), human IgE Cs3-CE4 domain (in house HEK-EBNA derived) and human IgE lambda (BIODESIGN International) were tested in each assay to establish the species cross-reactivity of the antibodies. Full-length human IgEX, along with human and cynomolgus [gE C63-Cs4 10 domains, produced inhibition curves. No inhibition was observed for any of the structurally related proteins. These data demonstrate that Antibody I binds to human IgEk, the C3-Ce4 domain of IgE and is cross reactive to cynomolgus IgE. However Antibody 1 does not bind to any of the most related human proteins to IgE. Details of the protocol are provided in the Materials and Methods section. 15 1.7 Inhibition of IgE binding to CD23 by purified IgG IM9 cells (a human B cell line) were shown to express CD23 but not FccRI under basal conditions. IgE binds to CD23 on the surface of IM9 cells. CD23-bound IgE can then be bound with anti-IgE- Phycoerythrin (Caltag) and detected by flow cytometry (FACSCalibur, BD Biosciences). 20 Antibodies were evaluated for inhibition of the IgE / CD23 interaction. A detailed protocol for this procedure is provided in Materials and Methods. In brief, titrations of the test IgG were mixed with IgE prior to incubation with [M9 cells. Following a 1 hour incubation, cells were washed and bound IgE was detected with anti-IgE-Phycoerythrin (Caltag). Antibody 1 25 inhibited the IgE/CD23 interaction with an IC50 of 16nM (n=3). 1.8 Cross-linking of FcERI-bound IgE Antibodies were evaluated for potential to cross-link FceRI-bound IgE using an RBL-ER51 calcium-signalling assay. RBL-ER51 cells, described in materials and methods, were loaded with IgE. Antibodies were incubated with the IgE-loaded cells and assessed for their ability to 30 stimulate a calcium response. Antibody I was not able to induce a detectable calcium response.

82 Example 2. Antibody optimisation 2.1 Optimisation of parent clone by targeted mutagenesis Antibody I was optimised using a targeted mutagenesis approach with affinity-based phage display selections. For the targeted mutagenesis approach, 5 large scFv phage libraries derived from the lead clone were created by oligonucleotide directed mutagenesis of the variable heavy (VH) and light (VL) chain complementarity determining regions 3 (CDR3) as described by Clackson and Lowman 2004 (A Practical Approach, 2004. Oxford University Press). 10 The libraries were subjected to affinity-based phage display selections in order to select variants with higher affinity for IgE. In consequence, these should show an improved inhibitory activity for IgE binding FcR1. The selections were performed essentially as described previously (Thompson 1996; 15 Journal of Molecular Biology. 256. 77-88). In brief, the scFv phage particles were incubated in solution with biotinylated human IgE X( U266 derived [lkeyama et. al. 1986. Molecular Immunology 23 (2); p 15 9

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16 7 ] and modified in house). ScFv-phage bound to antigen were then captured on streptavidin-coated paramagnetic beads (Dynabeads* M280) following the manufacturer's recommendations. The selected scFv 20 phage particles were then rescued as described previously (Vaughan et al., Nature Biotechnology 14: 309-314 (1996)), and the selection process was repeated in the presence of decreasing concentrations of bio-human IgE (250 nM to 25pM over 5 rounds). Upon completion of 5 rounds of selection, the VH and VL randomised libraries were 25 recombined to form a single library in which clones contained randomly paired individually randomised VH and VL sequences. Selections were then continued as previously described in the presence of decreasing concentrations of bio-human IgE (100pM to 1 pM over a finther 3 rounds).

83 2.2 Identification of improved clones from the targeted mutagenesis using an antibody ligand biochemical assay ScFv from the targeted mutagenesis selection outputs were expressed in bacterial periplasm and screened in an epitope competition HTRF@ (Homogeneous Time-Resolved 5 Fluorescence) assay format for inhibition of human IgE (U266-derived [Ikeyama et. al. 1986. Molecular Immunology 23 (2); pl 5 9

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167 ]) labelled with europium cryptate (CIS bio International 62EUSPEA), binding to anti human-IgE (Antibody 1, isolated in example 1). The detailed assay method is provided in the Materials and Methods section. ScFv that showed a significant inhibitory effect were subjected to DNA sequencing and unique scFv 10 were prepared as purified preparations. 2.3 Inhibition of IgE binding to FceRI by purled scFv Purified scFv were tested in a receptor-ligand binding HTRF@ (Homogeneous Time Resolved Fluorescence) assay format for inhibition of either human IgE (U266-derived [Ikeyama et. al. 1986. Molecular Immunology 23 (2); p159-167]) or cyno IgE (recombinant, 15 see materials and niethods) labelled with europium cryptate (CIS bio International 62EUSPEA), binding to human FcEsR-Fc (in house NSO cell produced). Example scFv potency data is included in Table 2a 84 Table 2a: Example potencies of clones identified from the targeted mutagenesis libraries when tested in the Receptor-ligand binding HTRF® Assays Clone scFv Geomean (95% CI) ICso (nM) (non-germlined) Human IgE assay Cynomolgus [gE assay Antibody 1 475 (399-565) Weak/Incomplete Antibody 2 28 (n=1) 317 (nl) Antibody 3 5 (n=1) 18 (n=1) Antibody 4 2 (0.4-14) 5 (2-17) Antibody 5 3(0.2-34) 6 (1-30) Antibody 6 3 (n=2) I (n=2) Antibody 7 9 (n=1) 186 (n=1) Antibody 8 5 (2-9) 12 (8-20) Antibody 9 9 (n=1) 132 (n=l) Antibody 10 10 (n=1) 116 (n=l) Antibody 11 2 (0.5-7) 7 (3-15) Antibody 12 3 (n=2) 7 (n=2) Antibody 13 2 (n=2) 7 (n=2) Antibody 14 8 (n=l) 15 (n--) Antibody 15 7 (n=1) 172 (n=1) Antibody 16 5 (2-11) 63 (46-87) Antibody 17 6 (n1l) 109 (n=1) Antibody 18 11 (n=1) 110 (n=1) Antibody 21 6 (n=1) 65 (n=1) Antibody 22 6 (n=1) 68 (n=1) Antibody 23 1 (n=1) 6 (n=1) Antibody 24 9 (n=1) 111 (n=1) Antibody 25 8 (n=1) 86 (n-1) Antibody 26 12 (n=1) 121 (n=1) Antibody 27 9 (n=1) 117 (n=1) Antibody 28 1 (n=1) 7 (n=1) 85 2.4 Inhibition of calcium signalling in RBL-ER5 cells by purifed IgG After re-formatting as IgG, potencies of optimised clones were determined using a modified RBL-ER51 calcium signalling assay. This assay was adapted from the method used during lead isolation to improve sensitivity for detection of more potent antibodies. A detailed 5 description of the protocol is provided in the Materials and Methods section. IC 50 potency data against human and cynomolgus IgE are given in Table 2b. Table 2b: Binding affinity Calculation using BlAcore and Potency measurement using RBL ER51 calcium signalling assay for optimised antibodies. ~ RBL-ER51 calcium signalling IC 5 o (nM) Biacore K(D (nM)Gema Geomean Antibody (Geomean) (95% CI) Human Cynomolgus Human IgE Cynomolgus IgE IgE IgE 0.088 0.151 4 2.1 8.0 (0.039-0.199) (0.086-0.265) 0.091 5 2.4 7.9 0.181 (0.005-1.79) 6 2.1 3.7 0.096 0.168 0.112 0.188 8 2.6 6.3 (0.02-0.62) (0.103-0.340) 0.069 0.153 11 1.6 9.3 (0.042-0.12) (0.068-0.34) 12 2.3 230 0.134 0.334 0.088 0.244 13 2.3 9.9 (0.038-0.02) (0.134-0.43) 0.292 16 4.6 62 4.38 (0.10-0.85) 18 8.0 0.532 2.97 0.095 0.111 19 2.5 7.9 (0.043-0.21) (0.008-1.55) 20 3.3 10.5 0.191 0.31 86 RBL-ER51 calcium signalling IC 50 (nM) Biacore KD (nM)Geoean Antibody (Geomean) (95% CI) Human Cynomolgus Human IgE Cynomolgus IgE IgE IgE 21 0.306 3.58 22 0.262 2.66 23 2.7 4.3 0.109 0.523 26 0.398 5.1 0.099 28 3.2 7.2 0.253 (0.017-0.586) 2.5. Germnlining The amino acid sequences of the VH and VL domains of the optimised anti-IgE antibodies were aligned to the known human germline sequences in the VBASE database (Tomlinson 1997; Journal of Molecular biology. 224. 487-499), and the closest germline was identified by 5 sequence similarity. For the VH domains of the Antibody 1 lineage this was Vhl DP-3 (1-f). For the VL domains it was VXI DPL8 (le). Without considering the Vernier residues (Foote & Winter 1992), which were left unchanged, there were 10 changes from germline in the frameworks of the VH domain and 2 in the VL domain of Antibody 1. Five of the changes in the VH domain and both the changes in the VL 10 domain were reverted to the closest germline sequence to identically match human antibodies. Changes at Kabat numbers 1, 20, 82a, 83 and 89 of the VH domain were left unchanged to retain potency (Antibody 8 GL and Antibody 11 GL). Germlining of these amino acid residues was carried out using standard site directed mutagenesis techniques with the appropriate mutagenic primers. Germlined IgG were then re-evaluated to confirm there had 15 not been a reduction in affinity or potency. Example affinities and potencies for germlined (GL) antibodies are provided in Table 4.

87 Table 4: Example binding affinity Calculation using BIAcore andPotency measurement using RBL-ER5 calcium signalling assay for gerinlined antibodies. RBL-ER51 calcium signalling ICs Biacore KD (nM) (nM) Antibody (germlined) (eoenHuaIg (Geomnean) Human IgE Human IgE Geomean (95% CI) 0.085 Antibody 8 GL 2.5 (0.057-0.13) 0.084 Antibody I1 GL 1.5 (0.063-0.11) 5 2.6 Inhibition of IgE binding to CD23 by purified IgG Some optimised antibodies were evaluated for inhibition of the IgE / CD23 interaction using the LM9 binding assay as previously described. Antibodies tested in this system were found to inhibit the IgE/CD23 interaction. The ICso values for Antibody 8 and antibody II were 10 16.5nM and 23nM respectively. 2.7 Cross-linking of FceRI-bound IgE Some optimised antibodies were evaluated for potential to cross-link FcERl-bound IgE using an RBL-ER51 calcium-signalling assay. RBL-ER5 1 cells, described in materials and methods, were maximally loaded with IgE. Optimised antibodies were incubated with the 15 IgE-loaded cells and assessed for their ability to stimulate a calcium response. No signalling could be detected (Figure 1).

88 2.8. Selectivity and species cross reactivity of optimised antibodies in DELFIA @ epitope competition assays The selectivity and species cross reactivity of the lead antibodies was re-evaluated using the DELFIA@ epitope competition assay as previously described (see section 1.6 and Materials 5 and Methods). Titrations of purified IgA, IgM, IgD, and IgG (all Calbiochem) were tested in each assay to establish the specificity profile for each structurally related protein, as measured by IC50 values in the assay. 10 Titrations of IgE species including human IgEX (U266 derived), human IgEK (Calbiochem), cynomolgus IgE Cs3-C4 domain (in house HEK-EBNA derived) and human IgE Cs3-Cs4 domain (in house HEK-EBNA derived) were tested in each assay to establish the species cross-reactivity of the antibodies. Full-length human IgEX and i, along with human and 15 cynomolgus IgE CE3-C84 domains, produced inhibition curves. No inhibition was observed for any of the structurally related human proteins (IgA, IgM, IgD and IgG). These data demonstrate that the panel of antibodies tested bind to human IgEX and K, the Cs3-CE4 domain of IgE and are cross reactive to cynomolgus IgE. However the antibodies do not bind to the proteins most related to IgE. 20 2.9 Binding affinity Calculation of affinity data for optimised clones using BIAcore The binding affinity of purified IgG samples of a representative number of clones to human and cynomolgus IgE was determined by surface plasmon resonance using BlAcore 2000 biosensor (BIAcore AB) essentially as described by Karlsson et al 1991; Joumal of Immunological Methods 145 (1-2) 229-240. In brief, Protein G' (Sigma Aldrich, P4689) was 25 covalently coupled to the surface of a CM5 sensor chip using standard amine coupling reagents according to manufacturer's instructions (BlAcore). This protein G' surface was used to capture purified anti-IgE antibodies via the Fc domain to provide a surface density of 50RU. Human IgE% or cynomolgus IgE prepared in HBS-EP buffer (BlAcore AB), at a range of concentrations, between 125 nM and 7.6 nM, were passed over the sensor chip surface. The 30 surface was regenerated using 10mM Glycine, pH 1.75 between each injection of antibody.

89 The resulting sensorgrams were evaluated using BIA evaluation 3.1 software and fitted to a bivalent analyte model, to provide relative binding data. Example affinities for the IgG tested are shown in Table 2b and Table 4. Materials and Methods - Example 1 and 2 5 Inhibition of IgE binding to FceRI by unpurifled scFv Selection outputs were screened in a receptor-ligand binding homogeneous FRET (Fluorescence resonance energy transfer) based assay format for inhibition of human IgE (Calbiochem 401152) labelled with Europium Chelate (Perkin Elmer 1244-302) binding to human FceRi-Fc (in house NSO cell produced). 10 Outputs during lead isolation were screened as undiluted, periplasmic extracts containing unpurified scFv, prepared in: 50mM MOPS buffer pH7.4, 0.5 mM EDTA and 0.5 M sorbitol. 15 pl of unpurified scFv sample was added to a 384 well assay plate (Perkin Elmer 6006280). 15 This was followed by the addition of 15 pl of I nM human FcERI-Fc (based on a MW of 260kDa), 15 pl of 40 nM anti human Fc IgG labelled with XL665 (CIS Bio International 61HFCXLA), and then 15 pl of 0.75 nM europium labelled human IgE. Non-specific control binding was defined using 300nM human IgE (Calbiochem). All dilutions were perfonned in 50 mM Tris-HCl (pH 7.8) containing 250 mM sodium chloride and 0.05% Tween20 (assay 20 buffer). Assay plates were then incubated for 1.5 hours at room temperature, prior to reading time resolved fluorescence at 615 nm and 665 nm emission wavelengths sequentially using a VICTOR2 plate reader (Perkin Elmer). 25 Data was normalised by VICTOR2 software to calculate counts per second (CPS). CPS values were subsequently used to calculate % specific binding as described in equation 1.

90 Equation 1: % specific binding = 5 (CPS of sample - CPS of non-specific binding control) X 100 (CPS of total binding control - non-specific binding control) Inhibition of IgE binding to FceRI by purified scFv Purified scFv from positive clones identified from screening were tested in receptor-ligand binding homogeneous FRET (Fluorescence resonance energy transfer) based assay format for 10 inhibition of human IgE (Calbiochem 401152) labelled with Europium Chelate (Perkin Elmer 1244-302), binding to human FcsRl-Fc (in house NSO cell produced). A titration of scFv concentrations was used in order to establish the scFv potency as measured by [C 50 values in the assay. IS pl of titration of purified scFv sample was added to a 384 well 15 assay plate (Perkin Elmer 6006280). This was followed by the addition of 15 p1 of IlnM human FccRI-Fc (based on a MW of 260kDa), 15 p1 of 40 nM anti human Fe IgG labelled with XL665 (CIS Bio International 61HFCXLA), and then 15 p1 of 0.75 nM europium labelled human IgE. Non-specific control binding was defined using 300nM human IgE (Calbiochem). All dilutions were performed in 50 mM Tris-HC1 (pH 7.8) containing 250 mM 20 sodium chloride and 0.05 % Tween20 (assay buffer). Assay plates were then incubated for 1.5 hours at room temperature, prior to reading time resolved fluorescence at 615 nm and 665 nm emission wavelengths sequentially using a VICTOR2 plate reader (Perkin Elmer). 25 Data was normalised by VICTOR2 software to calculate counts per second (CPS). CPS values were subsequently used to calculate % specific binding as described in equation 1.

91 Equation 1: % specific binding 5 (CPS of sample - CPS of non-specific binding control) X 100 (CPS of total binding control - non-specific binding control)

IC

5 0 values were determined using GraphPad Prism software by curve fitting using a four 10 parameter logistic equation (Equation 2). Equation 2: Y=Bottom + (Top-Bottom)/(l +1 0^((LogEC50-X)*HillSlope)) 15 X is the logarithm of concentration. Y is specific binding Y starts at Bottom and goes to Top with a sigmoid shape. Inhibition of calcium signalling by purified scFv and IgG in RBL-2H3 cells stably transfected with the human FceR1 (RBL-ER5 cells) 20 The neutralisation potency of purified scFv and IgG preparations against human igE bioactivity mediated through FcsRI was assessed using a RBL-ER51 calcium-signalling assay. Human FesRi was cloned from human peripheral blood lymphocytes into the pcDNA3.1 vector and transfected, using a standard electroporation method, into RBL-2H3 cells (a rat basophilic cell line). Transfected cells were cloned by limiting dilution and 25 analysed for surface FcERI expression. The resulting RBL-ER51 cells were maintained in media containing G418 (Invitrogen 10131-027) to maintain stable receptor expression. Free IgE in the vicinity of the cells binds to the FccRI and subsequent cross-linking of reccptor-bound IgE leads to a calcium mobilisation that can be detected using a Fluorometric Imaging Plate Reader (FLIPR). 30 RBL-ER51 cells were seeded at 5x10 4 /100pl/well in culture media [DMEM (Invitrogen 41966)with 9% v/v FBS Non-Heat Inactivated (Invitrogen 10100-147)and 400ug/mL G418 92 (Invitrogen 10131-027)] into 96 well black-walled, flat-bottomed, tissue culture-treated plates (Costar) and incubated at 37*C, 5% CO 2 for 18-24 hours. After this time, media was aspirated, leaving cell monolayer intact, and replaced with IOOuL/well of FLUO-4AM loading buffer [DMEM with 0.1% FBS, 20mM HEPES, 2.5mM probenicid and 2ug/mL 5 FLUO-4AM (Teff Labs)] for 1-2 hours at 37"C, 5% CO 2 . Loading buffer was aspirated and cells washed 3 times with 200uL/well of PBS. The final wash was aspirated and replaced with 70uL/well of FLIPR buffer [125mM NaC 2 , 5nM KCI, ImM MgCl 2 , 1.5mM CaCI 2 , 30mM Hepes, 2.5mM Probenicid, 5mM glucose, 0.01% v/v FCS]. Plates were incubated at 37*C, 5%

CO

2 for 5-45 minutes. 10 Test solutions of purified scFv or IgG (in duplicate) were diluted to the desired concentration in FLLPR buffer in V-bottom plates (Greiner). An irrelevant antibody not directed at IgE was used as negative control. IgE (Calbiochem or U266-derived [Ikeyama et. al. 1986. Molecular Immunology 23 (2); p159-167]) was prepared in FLIPR buffer and mixed with appropriate 15 test antibody to give a final IgE concentration of 3.33pg/mL in a total volume of 40il/well. The concentration of IgE used in the assay was selected as the dose that at final assay concentration gave approximately 80% of maximal calcium response. All samples were incubated for 30 mins at room temperature, prior to transfer of 30RI of IgE / antibody mixture to the dye-loaded cells prepared above. Assay plates were incubated at 37"C for 10 minutes to 20 allow free IgE to bind to the RBL-ER51 cells. To measure calcium mobilisation following addition of cross-linking anti-IgE, the FLIPR (Molecular Devices) was calibrated for suitable exposure according to manufacturers instructions. Anti-IgE (Biosource AH10501), diluted in FLIPR buffer, was added to the assay 25 plates to a final concentration ofI 1Oug/mL. Fluorescence of the FLUO-4AM dye was recorded at 1-second intervals for 80 measurements followed by 8-second intervals for 40 measurements. The peak response from each well was exported and data was then analysed using Graphpad Prism software. Measurement of anti-IgE cross-linking in RBL-ER5 cells 30 To measure ability of purified IgGs to cross-link FcERl-bound IgE, RBL-ER51 cells were prepared and dye-loaded as described in the inhibition assay. Cells were incubated for 10 minutes in 1OOuL of lug/mL human IgE (Calbiochem or U266-derived (Ikeyama et. al. 1986.

93 Molecular Immunology 23 (2); p159-167]), diluted in FLIPR buffer, to allow IgE to bind to FcERI on the cell surface. The concentration of IgE used in the assay was selected as the dose that gave approximately 80% of maximal calcium response. To measure calcium mobilisation following addition of cross-linking anti-IgE, the FLIPR (Molecular Devices) was calibrated 5 for suitable exposure according to manufacturers instructions. 30uL of test antibodies, diluted to appropriate concentrations in FLIPR buffer were added to the IgE loaded assay plates. Anti-IgE (Biosource AH1050 1) was used as a positive control. Fluorescence of the FLUO 4AM dye (Teff Labs) was recorded at 1-second intervals for 80 measurements followed by 8 second intervals for 40 measurements. The peak response from each well was exported and 10 data was then analysed using Graphpad Prism software. Selectivity and species cross reactivity of antibodies in DEL FIA @ epitope competition assays Purified IgG were adsorbed onto 96-well Maxisorp microtitre plates (Nunc) in PBS at a concentration which gave a significant signal when biotinylated human IgE was added at approximately its estimated KD for that particular IgG. Excess IgG was washed away with 15 PBS-Tween (0.1% v/v) and the wells were blocked with PBS-Marvel (3% w/v) for 1 hour. A dilution series of each of the following competitors was prepared in PBS, starting at a concentration of approximately 1000-fold the KD value of the interaction between biotinylated human IgE and the respective IgG; human IgE lambda (U266 derived [Ikeyama et. al. 1986. Molecular Immunology 23 (2); p159-167]), human IgE kappa (Calbiochem), 20 human IgE CE3-C64 domain (in house HEK-EBNA derived), cynomolgus IgE CE3-CE4 domain (in house HEK-EBNA derived), human IgA, IgM, lgD, and lgD (all Calbiochem). To this series, an equal volume of biotinylated human IgE at a concentration of approximately the KD was added (resulting in a series starting at a ratio of competitor antigen:biotinylated human IgE of approximately 1000:1). These mixtures were then transferred onto the blocked 25 IgG and allowed to equilibrate for I hour. Unbound antigen was removed by washing with PBS-Tween (0.1% v/v), while the remaining biotinylated human IgE was detected by streptavidin-Europium3+ conjugate (DELFIA@ detection, PerkinElmer). Time-resolved fluorescence was measured at 620nm on an EnVision plate reader (PerkinElmer). Fluorescence data were analysed using either Graphpad Prism or Microsoftm Excel software.

94 Identification of improved clones using an antibody-ligand biochemical assay Selection outputs were screened in epitope competition HTRF@ (Homogeneous Time Resolved Fluorescence) assay format for inhibition of cryptate labelled human IgE (U266 derived [Ikeyama et. al. 1986. Molecular Immunology 23 (2); p159-167]) labelled with 5 europium cryptate (CIS bio International 62EUSPEA), binding to anti human IgE antibody (Antibody 1). During lead optimisation, selection outputs were screened as undiluted or diluted periplasmic extracts, containing unpurified scFv, prepared in; 50mM MOPS buffer pH7.4, 0.5 mM EDTA 10 and 0.5 M sorbitol. 4 nM anti human IgE antibody was pre-mixed with 20 nM anti human Fc lgG labelled with XL665 (CIS Bio International 61HFCXLA). 10 pl of unpurified scFv sample was added to a 384 well low volume assay plate (Costar 3676). This was followed by the addition of 5 p1 of 15 the anti human IgE antibody anti Fc-XL665 mix, and then 5 p1 of a 1/245 dilution of cryptate labelled human IgE (approximately 2.3nM cryptate labelled human IgE). Non-specific control binding was defined using 300nM human IgE (U266-derived [Ikeyama et. al. 1986. Molecular Immunology 23 (2); p 159 -1 6 7 ). All dilutions were performed in phosphate buffered saline (PBS) containing 0.4 M potassium fluoride and 0.1% BSA (assay buffer). 20 Assay plates were thcn centrifuged at 1000rpm at room temperature for 1 minute, and incubated for 3 hours at room temperature, prior to reading time resolved fluorescence at 620 nm and 665 nm emission wavelengths using an EnVision plate reader (Perkin Elmer). 25 Data was analysed by calculating % Delta F values for each sample. Delta F was determined according to equation 1. Equation 1: 30 % Delta F = (sample 665nm/620nm ratio value) -(non-specific control 665nm/620nm ratio valuc) X 100 non-specific control 665nm/620nm ratio value) 95 % Delta F values were subsequently used to calculate % specific binding as described in equation 2. Equation 2: 5 % specific binding = % Delta F of sample X 100 % Delta F of total binding control Inhibition of IgE binding to FcsRI by improved scFv (purified) Purified scFv were tested in a receptor-ligand binding HTRF@ (Homogeneous Time 10 Resolved Fluorescence) assay format for inhibition of either human IgE (U266-derived [lkeyama et. al. 1986. Molecular Immunology 23 (2); p 159-167]) or cyno IgE (recombinant, see materials and methods) labelled with europium cryptate (CIS bio International 62EUSPEA), binding to human FcsRl-Fc (in house NSO cell produced). 15 A titration of scFv concentrations was used in order to establish the scFv potency as measured by IC 50 values in the assay. 1.9nM human FcER1-Fc (based on MW of 260kDa) was pre mixed with 20 nM anti human Fe IgG labelled with XL665 (CIS Bio International 61HFCXLA). 10 pl of titration of purified scFv sample was added to a 384 well low volume assay plate (Costar 3676). This was followed by the addition of 5 0 of the FcERl -Fc anti Fc 20 XL665 mix, and then 5 pl of a 1/197 dilution of cryptate labelled human or cyno IgE (approximately 2.9nM cryptate labelled human or cyno IgE). Non-specific control binding was defined using 300nM of human or cynomolgus IgE (in house derived). All dilutions were performed in phosphate buffered saline (PBS) containing 0.4 M potassium fluoride and 0.1% BSA (assay buffer). 25 Assay plates were then centrifuged at 1000rpm at room temperature for I min, and incubated for 3 h at room temperature, prior to reading time resolved fluorescence at 620 nrm and 665 nm emission wavelengths using an EnVision plate reader (Perkin Elmer). 30 Data was analysed by calculating % Delta F values for each sample. Delta F was determined according to equation 1.

96 Equation 1: % Delta F (sample 665nm/620nm ratio value) - (non-specific control 665nm/620nm ratio value) X 100 5 (non-specific control 665nm/620nm ratio value) % Delta F values were subsequently used to calculate % specific binding as described in equation 2. 10 Equation 2: % specific binding = % Delta F of sample X 100 % Delta F of total binding control 15 IC 50 values were determined using GraphPad Prism software by curve fitting using a four parameter logistic equation (Equation 3). Equation 3: 20 Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HilJSlope)) X is the logarithm of concentration. Y is specific binding Y starts at Bottom and goes to Top with a sigmoid shape. 25 Identification of improved clones in the RBL-ER51 calcium signalling assay The neutralisation potency of purified IgG preparations from improved antibodies was assessed in a modified version of the RBL-ERS I calcium-signalling assay described for lead isolation. 30 RBL-ER51 cells were seeded at 5x10 4 /100ul/well in culture media [DMEM (Invitrogen 41966)with 9% v/v FBS Non-Heat Inactivated (Invitrogen 10100-147)and 400ug/mL G418 (Invitrogen 10131-027)] into 96 well black-walled, flat-bottomed, tissue culture-treated plates 97 (Costar) and incubated at 37*C, 5% CO 2 for 18-24 hours. After this time, media was aspirated and replaced with 50uL/well dilutions of test antibodies (6.67nM to 1.33pM) in assay media [DMEM (Invitrogen 41966)with 9% v/v FBS Non-Heat Inactivated (Invitrogen 10100-147), 400ug/mL G418 (Invitrogen 10131-027) and 1.6% Penicillin / Streptomycin (Invitrogen 5 15140-122)]followed by addition of IgE [human (U266-derived [Ikeyama et. al. 1986. Molecular Immunology 23 (2); p159-1 6 7 ]) or cynomolgus (recombinant, see materials and methods)] diluted in assay media to give a final IgE concentration of 25ng/mL and 1Ong/mi respectively. Assay plates were incubated for 4 hours at 37 0 C, 5% Co 2 . 10 After this time, antibody/IgE mixture was aspirated, leaving cell monolayer intact, and replaced with 1OuL/well of FLUO-4AM loading buffer [DMEM with 0.1% FBS, 20mM HEPES, 2.5mM probenicid and 2ug/mL FLUO-4AM (Invitrogen)]for 1-2 hours at 37*C, 5% CO2. Loading buffer was aspirated and cells washed 3 times with 200uL/well of PBS. The final wash was aspirated and replaced with IOuL/well of FLIPR buffer [125mM NaCI 2 , 5nM 15 KCI, ImM MgCl2, 1.5mM CaCl 2 , 30mM Hepes, 2.5mM Probenicid, 5mM glucose, 0.01% v/v FCS]. Plates were incubated at 37*C, 5% CO 2 for 5-45 minutes. To measure calcium mobilisation following addition of cross-linking anti-IgE, the FLIPR (Molecular Devices) was calibrated for suitable exposure according to manufacturers 20 instructions. Anti-IgE (Biosource AHI050 I), diluted in FLIPR buffer, was added to the assay plates to a final concentration of 2.3ug/mL (to cross-link human IgE) or 20ug/mL (to cross link cynomolgus IgE). Fluorescence of the FLUO-4AM dye was recorded at 1-second intervals for 80 measurements followed by 3-second intervals for 40 measurements. The peak response from each well was exported and data was then analysed using Graphpad Prism 25 software. Measurement of cross-linking of FceRI-bound IgE by optimised antibodies To measure ability of purified IgGs to cross-link FccRI-bound IgE, RBL-ER51 cells were prepared as described in the inhibition assay for assessment of improved antibodies. RBL 30 ER51 cells were seeded at 5x10 4 /100ul/well in culture media [DMEM (Invitrogen 41966) with 9% v/v FBS Non-Heat Inactivated (Invitrogen 10100-147)and 400ug/mL G418 (Invitrogen 10131-027)] into 96 well black-walled, flat-bottomed, tissue culture-treated plates 98 (Costar) and incubated at 37 0 C, 5% CO 2 for 18-24 hours. After this time, media was aspirated and replaced with IOuL/well of human IgE (U266-derived [Ikeyama et. al. 1986. Molecular Immunology 23 (2); p15 9

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1 67 ]) diluted to lug/mL in assay media [DMEM (Invitrogen 41966)with 9% v/v FBS Non-Heat Inactivated (Invitrogen 10 100-147), 400ug/mL G418 5 (Invitrogen 10131-027) and 1.6% Penicillin / Streptomycin (Invitrogen 15140-122)]. The IgE concentration was chosen to give maximal loading of the RBL-ER51 cells. Assay plates were incubated for 4 hours at 37*C, 5% CO 2 . After this time, the IgE solution was aspirated, leaving cell monolayer intact, and replaced 10 with OOuL/well of FLUO-4AM loading buffer [DMEM with 0.1% FBS, 20mM HEPES, 2.5mM probenicid and 2ug/mL FLUO-4AM (Invitrogen)] for 1-2 hours at 37 0 C, 5% CO 2 . Loading buffer was aspirated and cells washed x3 with 200uL/well of PBS. The final wash was aspirated and replaced with IOuL/well of FLIPR buffer [125mM NaCl 2 , 5nM KCI, 1mM MgC 2 , 1.5mM CaCI 2 , 30mM Hepes, 2.5mM Probenicid, 5mM glucose, 0.0 1% v/v 15 FCS]. Plates were incubated at 37"C, 5% CO 2 for 5-45 minutes. To measure calcium mobilisation following addition of cross-linking anti-IgE (1.53uM to 2.33nM), the FLIPR (Molecular Devices) was calibrated for suitable exposure according to manufacturers instructions. 30uL of test antibodies, diluted to appropriate concentrations in 20 FL[PR buffer, were added to the assay plates. Anti-IgE (Biosource AH10501) was used as a positive control. Fluorescence of the FLUO-4AM dye (Invitrogen) was recorded at I-second intervals for 80 measurements followed by 3-second intervals for 40 measurements. The peak response from cach well was exported and data was then analysed using Graphpad Prism software. 25 Inhibition of IgE binding to CD23 on IM9 cells by purfiedIgG Antibodies were evaluated for inhibition of the IgE / CD23 interaction using the IM9 cell binding assay. IM9 cells (a human B cell line) were maintained in culture media [RPMI 1640 glutamax (Invitrogen 61870-010); 9% v/v heat-inactivated FBS (Invitrogen 10 100-147)] using standard tissue culture procedures. 30 To test optimised IgG, the IM9 cells were pre-treated with 25ng/ml human IL-4 (Peprotech, 200-04) for 3 days at 37"C/5%CO 2 in order to up-regulate CD23 expression.

99 IM9 cells were harvested and resuspended in Flow buffer [PBS with 1% Goat serum (Sigma) and 0.1% BSA fraction V (Sigma) at Ix10 6 cells/mL. Fc receptor blocking was performed by addition of Fc fragments (TEBU-bio) to a final concentration of 5ug/mL. This cell suspension was plated at 1OuL/well in U-bottomed polypropylene plates (Greiner) and incubated on ice 5 for 30 minutes. Antibody dilutions (667nM to I nM) were prepared in U-bottomed polypropylene plates (Greiner) and mixed with IgE (U266-derived [Ikeyama et. al. 1986. Molecular Immunology 23 (2); p159-16 7 ]) to a final igE concentration of 1Oug/mL for 30 minutes at room 10 temperature. Cell plates were spun at 2000rpm for 2 minutes and supernatant was aspirated, leaving the cell pellet intact. Cells were resuspended in IOuL/well antibody/IgE mix and incubated on ice for 1 hour. Cell plates were centrifuged at 2000rpm for 2 minutes and antibody/IgE supernatants were aspirated. Cells were washed by resuspending in 200uL/well of Flow buffer and centrifuging as above. 15 IgE bound to the cell surface was detected with anti-IgE-phycoerythin (Caltag) diluted 1/30, v/v, 1O0uL/well. Assay plates were incubated on ice for 20 minutes in the dark before centrifuging at 2000rpm for 2 minutes and washing with 2 x 200uL of Flow buffer as described above. Cells were resuspended in 10OuL Cell Fix (BD biosciences) and analysed 20 using a FACSCalibur (BD Biosciences) to detect FL2 staining. Data was analysed using CellQuest Software (BD biosciences). FL2 Geomean fluorescence was exported and data was then analysed using Microsoft Excel and Graphpad Prism software. 25 Generation Human IgE Ce3-4-C-terminally tagged with FLAG His] 0 The fragment of human IgE CE3-4 was as described previously in Wurzburg et. al. (2000) Structure of the Human IgE-Fc CE3-C4 Reveals Conformational Flexibility in the Antibody Effector Domains. A cDNA fragment that encompassed nucleotides 2135 - 2868 (GenBank 30 accession number J00222) was amplified using RT-PCR from total RNA of ILl3 stimulated human PBMC. This PCR product was cloned into pCR2.1 TA (Invitrogen). To allow secretion of the expressed protein and generate a sequence that incorporated an 100 inframe C-terminal FLAG epitope and polyhistidine tag (His 10), the IgE Ce3-4 fragment was PCR amplified with primers that incorporated a 5' BssHUl site, and 3' FLAG epitope, polyhistidine tag (His 10) and Xbal site and subsequent insertion into pEU8.2 vector. The modified pEU8.2 vector contains an EF-1 promoter, the genomic sequence for murine IgGI 5 leader peptide, oriP origin of replication to allow episomal plasmid replication upon transfection into cell lines expressing the EBNA-l gene product (such as HEK293-EBNA cells). Protein was purified from conditioned media using IMAC chromatography followed by Size 10 Exclusion chromatography (SEC). Generation of Cynomolgus IgE Ce3-4 C-terminally tagged with FLAG His 10 The cynomolgus IgE constant region was determined by direct sequencing of PCR products amplified from genomic DNA using primers that encompass nucleotides 1174 - 2989 of the 15 human IgHE (heavy chain of IgE)locus (GenBank Accession J00222). The exons were identified by homology with the human sequence, and thus it was possible to predict the cDNA sequence for the cynomolgus IgE heavy chain constant region. A cDNA encoding the sequence for murine IgGI leader peptide, cynomolgus, Ce3-4 (Figure 20 2), and C-terminal FLAG epitope and polyhistidine tag was synthesised (DNA2.0) and cloned into pDONR221 (Invitrogen). Then using LR Gateway* reaction (Invitrogen) the gene of interest was transferred to the expression vector pDEST12.2 (Invitrogen) modified by the insertion of the oriP origin of replication from the pCEP4 vector (Invitrogen) to allow episomal plasmid replication upon transfection into cell lines expressing the EBNA- 1 gene 25 product (such as HEK293-EBNA cells). Protein was purified from conditioned media using IMAC chromatography followed by Size Exclusion chromatography. 30 Generation chimaeric D12 variable region and cynomolgus IgE constant region A cDNA encoding the variable heavy chain region that encoded the Human anti-eostradiol scFv (D 12_VH) and one of either two different haplotypes cynomolgus IgHE gene (cyIGHE TQ and cylGHE ME) were synthesised (DNA2.0) and cloned into pDONR221 (Invitrogen).

101 Also a cDNA representing the variable light chain Human anti-eostradiol scFv (D 2_VL) and one of either two cynomolgus lambda constant region genes (cyLGLC4 and cyIGLC7) were synthesised (DNA2.0) and cloned into pDONR221 (Invitrogen). 5 Then using LR Gateway* reaction (Invitrogen) the gene of interest was transferred to the expression vector pDEST12.2 (Invitrogen) modified by the insertion of the oriP origin of replication from the pCEP4 vector (Invitrogen) to allow episomal plasmid replication upon transfection into cell lines expressing the EBNA-l gene product (such as HEK293-EBNA cells). 10 Recombinant chimaeric IgE protein representing the variable heavy chain region of the Human anti-costradiol scFv fused to cynomolgus IgE CE1-4 (Figures 3 and 4), and variable light chain region of the Human anti-eostradiol scFv fused to cynomolgus Lambda constant region (Figures 5 and 6), expressed from HEK293 EBNA cells was purified using the method 15 as described in [keyam et. al. (1986) Mol Immunol 23 p 15 9

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6 7 . human FcERI-Fc (in house NSO cell produced) The FCERI encompassing the nucleotides 67- 711 (GenBank Accession number NM_00200 1) was cloned up stream of the genomic region of the human IgG 1 Fc as from pEU 1.2 first 20 described in Persic et al. (1997) Gene 187; 9-18. This was cloned into pcDNA3.1 EcoRI Xbal (SEQ ID NO: 391 and 392). Expression of the recombinant fusion protein FcRI_Fc was achieved by stable transfection of NSO cells with the pcDNA3.1 FcsRI_Fc construct. Stable expression was established by selection with G418, isolation of clones via limiting dilution and identification of the clones with the high expression level. The FcERIFc fusion 25 protein was then purified from the conditioned medium using Protein A affinity chromatography, followed by preparative Size Exclusion Chromatography. Example 3: Human B cells - inhibition of intracellular IgE 30 Peripheral Blood Mononuclear Cells (PBMC) were isolated from human heparinised whole blood by centrifugation on a Ficoll-Paque gradient (Pharmacia). B cells were subsequently isolated from the PBMC population using positive anti-CD 19 selection with magnetic beads (Miltenyi). Both the positive and the negative B cell fractions were collected as the cells were 102 passed over the magnetic column. The cells from the negative B cell fraction containing all PBMC cells except B cells, were treated with Mitomycin C to prevent proliferation. The cells were incubated with 50 pg/mL Mitomycin C for 30 min, then washed with tissue culture media (RPMI 1640 with Glutamax (Gibco) / 10% FCS (Gibco) / 50 U/mL Penicillin 5 /50pg/mL Streptomycin (Gibco)) and further incubated with PBS for 70 min before a final wash to ensure all Mitomycin C was removed. To induce the differentiation of the B cells, 4 x 10 4 B cells and 9.2x 10 5 cells from the B cell negative fraction were plated in a 96-well plate in tissue culture media supplemented with 3.5pM beta-mercaptoethanol (Sigma) and 20 pg/mL transferrin (Chemicon), and pre-incubated with anti-IgE monoclonal antibodies or a 10 control antibody at 0.001-1OOnM for 30 min before addition of human interleukin-4 (IL-4) at 1 Ong/mL. The cells were subsequently incubated in a humidified CO 2 incubator for 12-14 days. At day 12-14, the plates were given a brief spin, supernatants collected and the cells stained for intracellular IgE using the following protocol. 15 The cells were first incubated with PBS with 1% human serum for 10 minutes to block Fc Receptor binding. Cells were then fixed and permeabilised on ice using Cytofix/Cytoperm kit from Becton Dickinson. The cells were then washed before addition of a polyclonal rabbit anti-human lgE-FITC antibody (DAKO) at 1:6 final dilution and a monoclonal mouse anti human CD19 - RPE/Cy5 antibody (DAKO) at 1:10 final dilution. It is important that the cells 20 are thoroughly washed to avoid interference from residual anti-IgE MAb's (inonoclonal antibodies). After 30 min incubation the cells were washed and samples were analysed on a FACS Calibur using a HTS 96-well plate loader device. The percentage of cells in the CD 19+ population that co-express IgE were then recorded, and the expression of intracellular IgE is presented as % inhibition of maximum IgE expression in cells not treated with blocking anti 25 IgE monoclonal antibody. Antibody 11 inhibited the induction of IgE positive cells with an IC50 of 1.6 nM (Figure 7 - upper graph). An irrelevant antibody of the same format was used as negative control (CAT-002), and did not inhibit the induction of IgE postive B cells (Figure 7 - lower graph). 30 Example 4: Mast cell line (LAD2) - inhibition of p-hexosaminidase release LAD2 cells [A. S. Kirshenbaurm et al. Leukemia research 27 (2003)] were cultured at a cell density of 0.25-0.6 x 106 cells/mL in serum-free media (StemPro-34, Life Technologies) 103 supplemented with StemPro-34 nutrient supplement, 2 mM L-Glutamine and 100 ng/mL recombinant human stem cell factor (rhSCF, R&D). For the p-hexosaminidase assay, the cells were seeded at a density of 2.5 x 104 cells/well, and pre-incubated in a 96-well polypropylene plate together with blocking anti-IgE MAb's in a 5 concentration range of 0.0001-100 nM. Cells were incubated at 37'C for 30 minutes, before IgE at a concentration of 0.15nM was added, and the cells incubated for an additional 4 hours. After the incubation with IgE, cells were washed with buffer to remove any excess IgE, and subsequently IgE bound to the FceR's on the LAD2 cells was cross-linked with adgE (600 jg/mL goat-anti human IgE, Sigma) for 30 minutes at 37*C. The incubation was stopped 10 by cold centrifugation and the cell supernatants collected and transferred to a 96-well plate. P -hexosaminidase content was analysed by using a slightly modified version of the method published by Smith et al [Smith J et al. Biochem. J. (1997)323, 321-328). In brief, 2 mM p nitrophenyl-N-acetyl-D-glucosaminide in 0.2 M citrate-buffer, pH 4.5 was used as a substrate for the hexosaminidase. The reaction was stopped by addition of IM Tris-buffer pH 9.0. 15 Optical density was measured spectrophotometrically at 405nm (minus the values at 570nm) using a Spectramax reader from Molecular Devices. The effect of the anti-IgE MAb's to inhibit the release of p-hexosaminidase was calculated, and presented as percentage inhibition of total release +/- SEM. Antibody 11 inhibited p1-hexosaminidase with an IC50 of 0.04 nM (Figure 8 - upper graph), whereas an irrelevant MAb of the same format (CAT-002) did not 20 inhibit the p-hexosaminidase (Figure 8 - lower graph). Example 5: Anti-IgE Antibody binding of IgE in serum usina ELISA Assay description 25 Serum samples were prepared from blood samples from human donors. 96 well ELISA plates (Nunc Maxisorp, No. 442404) were coated with 150 l1well of 1 ig/ml FcERI-Fe-His diluted in PBS, and incubated at 4*C overnight. After the overnight incubation, the plates were washed three times with PBS containing 0.05% Tween 20 (PBST, Medicago 09-9401-100). To reduce background binding, plates were subsequently incubated with 200 Pl /well of block 30 buffer consisting of PBS containing 0.5% BSA, incubated at room temperature for 2 hours, and washed three times with PBST as described above. The samples (human serum or plasma with varied amounts of anti-IgE antibody, Antibody 11) and standards (ImmunoCAP Total IgE (human) calibrator, Phadia, Uppsala) were diluted in PBS containing 0.05% Tween 20, 104 and kept on ice until they were applied to the assay plates in a volume of 150 pl /well. The plates were sealed and samples incubated at room temperature for 2 hours. To remove unbound sample, plates were washed three times with PBST as described. Subsequently, 150 jil /well of rabbit anti-human IgE (DE1,30-1917-00, 420036-02, 841204, 9911302 from 5 Phadia, Uppsala) at a concentration of 0.25 ig/ml diluted in PBST, was added to detect bound human IgE. The plates were then sealed again, and incubated for I hour at room temperature. To remove unbound rabbit anti-human IgE antibodies, plates were washed three times with PBST as described above. A HRP-conjugated secondary antibody (Goat-anti-rabbit IgG, HRP conjugated. Pierce. 0.8 mg/ml) was used to detect the rabbit anti-human IgE. The conjugate 10 was diluted 1:25000 in PBST, 150 p.1 added per well, the plates sealed, and incubated for 1 hour at room temperature. The plates were then washed three times with PBST as described. TMB-substrate solution (DAKO Substrate-Chromogen, No. S1599), 150plwell, was then added to each well and the plates incubated for 10 minutes at room temperature. The reaction was stopped by adding 150pl/well of Stop solution (2M H 2 S0 4 ) and the 450nm absorbance 15 read on a Tecan SAFIRE instrument. Since the measurement of IC50 is dependent on the concentration of ligand (i.e. IgE) in an assay, in the present assay the IC 50 will vary depending on the amount of IgE ligand present in the human serum sample. In a representative experiment Antibody 11 had an IC 50 of 202 pM [Figure 9). In the same experiment XolairTM had an IC50 of 57nM. 20 Example 6: Measurement of complex formation between IgE and purified IgG. Characterisation of the immune complexes formed between purified human IgE and purified anti-IgE IgG (antibody 11) was performed by high-performance size exclusion liquid 25 chromatography. In addition, on line multi-angle light scattering (MALS) was used to estimate complex size. Complexes were formed by incubating IgE and IgG together at three different molar ratios (3:1, 1:1 and 1:3 respectively) in Dulbecco's PBS at 18*Cfor one hour. For the 1:1 molar ratio, the concentration of each protein was 2.5 pM. The higher ratios were achieved by increasing the concentration of the relevant protein to 7.5 piM. These samples 30 were analysed on two Bio-Sep-SEC-S 4000 columns (300 x 7.8mm) arranged in tandem. The columns were equilibrated and samples analysed in Dulbecco's PBS at a flow rate of 1 ml/min on an Agilent HP I100 HPLC system. Peaks were detected at 220 and 280nm using a 105 diode array detector and the eluate was also directed through a Wyatt Technologies DAWN EOS (MALS) and Optilab rEX(refractive index) detectors. Chromatography of the 1:1 molar ratio sample gave a doublet of peaks (detected by UV 5 absorbance) which were not completely resolved and corresponded to retention times of 13.88 minutes and 14.9 minutes. These retention times indicate the formation of non-covalent complexes of IgE with IgG. MALS analysis gave molecular masses of 1,085kDa (13.88min peak) and 702kDa(14.9min peak). These masses are consistent with complexes corresponding to a heterotetramer (predicted mass 674kDa, 2lgE:2IgG) and a heterohexamer (predicted mass S101O0kDa, 3IgE:3lgG). Chromatographic and MALS analysis of both the 3:1 and 1:3 (IgE:IgG) molar ratio samples gives a similar profile to the 1:1 sample with peaks corresponding to heterotetramer and heterohexamer detectable by UV absorbance. Additional peaks were detected corresponding to the excess IgE or IgG in the samples. 15 Example 7: Determination of the epitope bound by germlined Antibody 11 Use of X-ray crystallography to determine the precise 3-dimensional structure of proteins at atomic resolution is well known to those in the art and has been used to visualise in detail the parts of proteins that interact with antibodies (Padavattan et al, 2007; Karpusas et el., 2001). 20 This is the most dcfnitive epitope mapping technique, but requires considerable effort and relies on being able to obtain crystals of sufficient quality, which in turn depends on purity and quality of protein sample and expertise in being able to find the appropriate crystallisation conditions. Once crystals of the protein-antibody complex are obtained, they are irradiated with X-rays to give a diffraction pattern, which depends on the exact atomic distribution. The 25 diffraction pattern can be analysed by crystallographers to determine the three dimensional positional coordinates of the atoms in the structure. This allows a detailed inspection of the interaction sites between protein and antibody. 7.1 X-ray crystal structure determination of the IgE Ce3-Ce4 antibody complex 30 IgE domain Ce2-Ce3 was cloned and expressed and purified for the purpose of structure determination. Similarly a Fab fragment was prepared by digestion and purification of the full Antibody I1 developed to bind to IgE. The complex was formed by mixing and purified by size exclusion chromatography to remove non-complexed IgE domains and Fab molecules.

106 Crystals of the IgE Ce3-Ce4 / Fab complex were obtained that belong to the trigonal space group P3 2 21. They were analysed at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Complete diffraction data to 2.85 A resolution were obtained. The structure could be solved by Molecular Replacement (Rossman, 1972) using the variable and constant 5 part of a Fab fragment as separate search models, thereby orienting and positioning the Fab fragments in the crystallographic asymmetric unit. In total, three Fab fragments were identified in the asymmetric unit. Subsequently three IgE Ce3-Ce4moleculescould be placed in the asymmetric unit. Each IgE dimer binds two Fab molecules and thus, in total the asymmetric unit comprises one and a half complete IgE /Fab complexes. 10 7.1.1 Overall description of the IgE Ce3-Ce4 lAntibody 11 Fab complex The crystal structure shows that each IgE Ce3-Ce4 dimer, is bound in a symmetric or near symmetric fashion to two Fab fragments (Figure 10). Since the asymmetric unit of the crystal comprises one and a half IgE/Fab complexes, the incomplete complex forms a dimer with a 15 symmetry related partner in the neighbouring asymmetric unit, via a two-fold axis. Both molecules of the IgE dimer, denoted IgEl and IgE2, interacts with the Fab fragment of Antibody 11. The majority of the interactions are provided by the Fab Heavy chain, which interacts with both IgEl and IgE2 whereas the Light chain is only observed to interact with 20 IgEI. The epitope of the antigen is situated mainly in domain CW3, with contribution from one amino acid located close to the hinge in domain CE4. The three interaction sites between the IgE Ce3-Ce4 and the Fab in the asymmetric unit of the crystal are very similar. However, after refinement it was clear that one of the Fab molecules 25 is considerable less ordered than the other two Fab molecules. This is commonly seen in crystal structures and is explained by the fact that the particular region is flexible and adopts different orientations throughout the crystal such that the electron density is less well defined. This Fab molecule and the interaction it makes with the IgE Ce3-Ce4 molecule were therefore not considered in the analysis. 30 IgE is known to be glyscosylated in the Fc region at residue Asn394 (Wurzburg et al). Characterization of the Fc glycosylation, performed by mass spectrometry analysis after trypsin digestion, showed three different glycan variants bound to Asn394, consisting of the 107 core structure Man 3 GlcNAc2 with the extension of 2, 3 or 4 hexoses, probably all mannoses (Figure I1). Indeed from residue Asn394 in all three IgE Ce3 domains an extended electron density protrudes into the cavity between the two IgE molecules in the dimer. The electron density suggests a high-mannose-type structure, with two N-acetyl-glucosamine (GleNAc) 5 and three or four mannose units visible in each chain, consistent with the mass spectrometry analysis. Only one of the hexoses outside the core structure, Man6 which is coupled to Man4, is visible in the electron density indicating that the remaining 1-3 hexoses are flexible. 7.1.2 Describing the epitope and paratope 10 This crystal structure allows the epitope interactions between IgE Ce3-Ce4 and Fab to be examined in atomic detail. There are two independent IgE/Fab interactions in the solved structure, excluding the third Fab molecule due to its badly defined electron density map, which will be described. They are very similar indicated by an root-mean-square deviation between the two equivalent Fab Variable chain fragments of 0.31 A calculated using Cs 15 positions (superpose, CCP4 1994) and between the equivalent IgE monomers of 0.82 and 0.96 A respectively. Despite this high similarity the two interactions will be described separately and will be denoted IgE/Fab I and IgE/Fab2. Details of the interactions are captured in Table 5 and Table 6, where the residue number contains a chain indicator (HC: Fab Heavy chain, LC: Fab Light chain, IgEl, igE2). The numbering of the amino-acid residues of Antibody 11 is 20 according to the Kabat system (Kabat et al 1991). The distances were obtained using the CCP4 program CONTACT (CCP4, 1994). Both interactions involve the complementarity determining regions (CDRs) from both the Heavy and the Light chain of the antibody fragment, residues from the framework (the region 25 outside the CDRs of the Fab) and amino acid residues from both monomers in the IgE Ce3 CE4 monomer. The antibody Light chain interacts with IgEl in the IgE CW3-CE4 dimer, while the Heavy chain interacts with both monomers. The majority of the contacts are, however, between the Heavy chain and monomer IgE2 of the antigen. The two interactions are described in detail below. 30 7.1.3 Detailed description of the interaction between FabI and IgE. interaction I The interaction site defining the epitope of IgE CE3-CE4 covers an area of 1100 A 2 (calculated using the program areaimol, see reference CCP4, 1994) and is made up by amino acid residues Glu390 through to Asn394 inclusive of IgEI and Leu340, Arg342, Ala428 to Thr434 108 inclusive, Thr436, Ser437 and Glu472 in IgE2 of the antigen. In addition the sugar moieties GIcNAcI and Man6 in IgEl and Man5 in IgE are in contact with the Fab molecules. Amino acid residues from the Heavy chain interacting with the antigen, including the sugar moieties, are from CDR1: Tyr32, from CDR2: Asp53 and Asn54, from CDR3: Va195, Met96, lie 100, 5 Gly100b, Gly100c, Asp101 and Tyr102 and from the framework: Glul, Lys23, Thr30, Ala7l to Arg77 inclusive and Tyr79. Residues contributing from the Fab Light chain are Asp50 and Ser56 from CDR2 and Leu 46 and Tyr49 from the framework. The interaction include 19 hydrogen bonds in addition to non-polar van der Waals contacts. 10 7.1.4 Detailed description of the interaction between FabI and l2E, interaction 2 The interaction site defining the epitope of IgE CE3-C84 covers an area of 1165 A 2 (calculated using the program areaimol, see reference CCP4, 1994) and is made up by amino acid residues Glu390, Gln392 to Asn394 inclusive of IgEl and Leu340, Arg342, Ala428 to Thr434 inclusive, Thr436, Ser437 and Glu472 in IgE2 of the antigen. In addition the sugar moieties 15 GIcNAc l and Man6 in IgEl are in contact with the Fab Heavy chain. Amino acid residues from the Heavy chain interacting with the antigen, including the sugar moieties, are from CDRl: Tyr32, from CDR2: Pro52a, Asp53 and Asn54, from CDR3: Val95, Met96, IleI1O, Gly1OOb, GlyOOc, AsplOl and Tyr102 and from the framework: Glul, Lysl9, Lys23, Thr30, Ala7l to Ser75 inclusive, Arg77 and Tyr79. Residues contributing from the Fab Light chain 20 are Ser56 from CDR2 and Tyr49 from the framework. The interaction include 19 hydrogen bonds in addition to non-polar van der Waals contacts. Table 5: Direct interactions between IgE CW3-CE4 and Antibody 11 Fab, interaction 1 Monomer Distance Chain Fab Residue Fab Residue IgE IgE (A) Hydrogen bonds HC Tyr 32 OH IgEl GlcNAc 1 07 2.58 HC Met 96 0 IgEI Asn 394 ND2 3.08 HC Gly 100b 0 IgEI Arg 393 NH 1 2.60 HC Gly 100c 0 IgEI Arg 393 NE 2.80 HC Tyr 102 OH IgEl GlcNAc 106 3.08 HC Asp 53 0 IgE2 Met 430 N 2.83 HC Asp 53 0 IgE2 Arg 431 NH1 2.67 109 Monomer Distance Chain Fab Residue Fab IgE Residue IgE (A) HC Asp 53 ODI IgE2 Arg 431 NHI 2.77 HC Asp 53 OD I IgE2 Arg 431 NH2 2.60 HC Ala 710 IgE2 Ser 432 OG 2.64 HC Asp 72 OD2 IgE2 Arg 342 NH1 3.06 HC Asp 72 OD2 IgE2 Thr 434 N 3.02 HC Asp 72 OD2 IgE2 Thr 434 0 2.97 HC Thr 73 OGI IgE2 Ser 432 N 2.79 HC Thr 73 OGI IgE2 Ser 432 0 3.05 HC Ser 74 N IgE2 Ser 432 0 3.08 HC Ser 74 OG IgE2 Arg 342 NH 3.10 HC Ser 74 OG IgE2 Thr 433 OG l 2.87 HC Arg 77 NH 1 IgE2 Glu 472 OE2 2.56 Non-polar contacts < 4 A LC Leu 46 IgEl Arg 393 3.79 LC Tyr 49 IgEl Gln 392 3.85 LC Tyr 49 IgEl Arg 393 3.66 LC Asp 50 IgEl Arg 393 3.71 LC Ser 56 IgEl Glu 390 3.47 LC Ser 56 IgEl Lys 391 3.87 HC Glu 1 IgEl Man 6 3.40 HC Va195 IgEl Arg 393 3.49 HC Ile 100 IgEl Arg 393 3.55 HC Asp 101 IgEl Arg 393 3.50 HC Met 96 IgEl GIcNAc 1 3.67 HC Lys 23 IgE2 Glu 472 3.43 HC Thr 30 IgE2 Arg 431 3.80 HC Asp 53 IgE2 Leu 429 3.34 HC Asn 54 IgE2 Ala 428 3.45 HC Asp 72 IgE2 Ser 432 3.22 HC Asp 72 IgE2 Thr 433 3.53 110 Monomer Distance Chain Fab Residue Fab IgE Residue IgE (A) HC Thr 73 IgE2 Met 430 3.98 H C Thr 73 IgE2 Arg 431 3.22 HC Ser 74 IgE2 Leu 340 3.33 HC Ser 75 IgE2 Arg 342 3.32 HC Asp 76 IgE2 Man 5 3.27 HC Arg 77 IgE2 Thr 436 3.84 HC Tyr 79 IgE2 Thr 436 3.43 HC Tyr 79 IgE2 Ser 437 3.27 The distance cut-off used for hydrogen bonds is 3.2A, for non-polar interactions 4.OA Table 6: Direct interactions between IgE C3-C4 and Antibody 11 Fab, Interaction 2 Monom Distance Chain Fab Redidue Fab er Residue IgE A IgE Hydrogen bonds LC Ser 56 OG IgEl Glu 390 OE1 2.44 LC Ser 56 OG IgEl Glu 390 OE2 2.99 HC Tyr 32 OH IgEl GlcNAc 107 2.41 HC Gly 100b O IgEl Arg 393 NH2 2.58 HC Gly 100c O IgEl Arg 393 NE 3.01 HC Tyr 102 OH IgEl GlcNAc 1 06 2.73 HC Asp 53 0 IgE2 Met 430 N 2.74 HC Asp 53 0 IgE2 Arg 431 NH 1 2.62 HC Asp 53 ODI IgE2 Arg 431 NH1 2.88 HC Asp 53 ODI IgE2 Arg 431 NH2 2.65 HC Ala 710 IgE2 Ser 432 OG 2.87 HC Asp 72 ODI IgE2 Ser 432 0 3.13 HC Asp 72 OD2 IgE2 Arg 342 NH 1 3.17 111 Monom Distance Chain Fab Redidue Fab er Residue IgE IgE HC Asp 72 OD2 IgE2 Thr 434 0 3.11 HC Thr 73 OGI IgE2 Ser 432 N 2.94 HC Ser 74 N IgE2 Ser 432 0 3.17 HC Ser 74 OG IgE2 Arg 342 NHI 3.00 HC Ser 74 OG IgE2 Thr 433 OGI 2.77 HC Tyr 79 OH IgE2 Ser 437 N 3.09 Non-polar contacts < 4 A LC Tyr 49 IgEl Gln 392 3.76 LC Tyr 49 IgEl Arg 393 3.63 LC Ser 56 IgEl Gln392 3.89 HC Glu 1 IgEl Man 6 3.25 HC Val 95 IgE1 Arg 393 3.48 HC Met 96 IgEI GIcNAc 1 3.68 HC Met 96 IgEI Asn 394 3.31 HC Ile 100 IgEI Arg 393 3.44 HC Asp 101 IgEI Arg 393 3.81 HC Lys 19 IgE2 Ser 437 3.53 HC Lys 23 IgE2 Glu 472 3.70 HC Thr 30 IgE2 Arg 431 3.88 HC Pro 52a IgE2 Met 430 3.95 HC Asp 53 IgE2 Leu 429 3.51 HC Asn 54 IgE2 Met 430 3.90 HC Asn 54 IgE2 Ala 428 3.47 HC Asp 72 IgE2 Thr433 3.61 HC Thr 73 IgE2 Arg 431 3.26 HC Ser 74 IgE2 Leu 340 3.30 HC Ser 75 IgE2 Arg 342 3.55 HC Arg 77 IgE2 Thr436 3.88 HC Arg 77 IgE2 Glu 472 3.27 112 Monom Distance Chain Fab Redidue Fab er Residue IgE (A) IgE HC Tyr 79 IgE2 Thr436 3.37 The distance cut-off used for hydrogen bonds is 3.2A, for non-polar interactions 4.OA Material and Methods for Experiment 7 5 Over expression of IgE CE3-CE4. Cell lines and culture medium. In this work the original adherent cell line HEK293-EBNA (Invitrogen, Stockholm, Sweden) stably expressing the Epstein Barr virus Nuclear Antigen-I gene were used. Cells were adapted to suspension growth before transferred into DHI medium by stepwise medium 10 replacement (Davies et al. 2005). The DHI medium used deviated from the original description slightly by being CA2+-free. After adaptation a working cell bank was made and both cell lines were grown routinely in CA2+-free-DHI medium supplemented with 4 mM Glutamine, 2% v/v ultra-low IgG foetal bovine serum, 250 gg/ml G41 8 (all from Invitrogen, Stockholm, Sweden) and 0.1% w/v Pluronic F68 (Sigma-Aldrich, Stockholm, Sweden) to a 15 maximum of 20 passages. Transfection procedure The I mg/ml stock solution of linear 25 kDa polyethylenimin (Polysciences Europe, Eppenheim, Germany) was prepared in water, pH adjusted to 7.0, sterile filtered and stored in 20 small aliquots at -80*C until use. The transfection cocktail was prepared shortly before transfection in non-supplemented DHI media in a volume equivalent to one-tenth of the transfection volume. For preparing the transfection cocktail the DHI media was divided into two halves. 0,8 pg DNA per ml transfection volume was added to one half of the DHI medium and into the other half 2 pg PEI per ml transfection volume was added. After shaking 25 the two solutions briefly and incubating them for 5 minutes the DNA solution was slowly added to the PEI solution. The transfection cocktail was incubated for 20-30 minutes at room temperature before addition to the Wave bioreactor (Wave Biotech AG, Tagelswangen, 113 Switzerland). Four hours post transfection the culture was fed to the final production volume with supplemented DHI medium and HypPep1510 (Kerry Bio-Sciences, Almere, the Netherlands) to a final concentration of 0.3% (w/v). 5 Seeding cultures For expansion of the seeding culture the cells were grown in plastic shake bottles at 37*C in 5% CO 2 atmosphere placed in an orbital shaker incubator (Infors AG, Bottmingen, Switzerland). The cells were routinely passaged twice a week reaching approximately 2 x 106 cells/mI before splitting. Cell density and viability were assessed using a Cedex automatic cell 10 counter (Innovatis AG, Bielefeld, Germany). For Wave cultures the cells were split to 1x106 cells/ml one day before transfection to ensure that they were in logarithmic growth phase at the start of the experiment. Wave cultures were inoculated directly from shakers. All seeding cultures were concentrated by centrifugation and resuspended in fresh culture medium before addition to the bioreactors. 15 Wave cultures Expression was performed in Wave bioreactors (Wave Biotech AG, Tagelswangen, Switzerland) at a working volume of 10 L. The wave bioreactors were seeded to I x 106 cells/ml in 4.5 L supplemented DHI medium. After a 2 hours adaptation phase the culture was 20 transfected with 0.5 L transfection cocktail. Four hours post transfection the culture was fed to 10 L total volume with supplemented DHI medium and HyPep1510 to a final concentration of 0.3% (w/v). Samples were taken daily to determine cell density, viability and protein concentration. 25 Expression vector The vector expressing the human IgE Ce3-Cs4, with C-terminal Flag tag and 10-histidine tag, was derived from a vector described by Persic et al. (1997). The system is under the control of the EF I-a promotor. 30 Purification of IgE CO3-CE4 20 L of cell supernatant were concentrated five times and diafiltered to 2xPBS (308 mM NaCl, 20 mM phosphate, p 1 H 7.4) with a 10 kDa molecular weight cut-off cross-flow membrane (Pellicon 2, Millipore). The medium was batch bound with 30 mL NiSepharose 114 (GE Healthcare) for two hours at 4 *C, washed with five volumes 2xPBS and packed into an XK26 column. The column was then washed with five column volumes 40 mM imidazole in 2xPBS to wash away contaminating proteins. IgE was finally eluted with 400 mM imidazole in 2xPBS. The pool contained IgE with high purity and was concentrated about four times (to 5 -5 mg/mL) before it was run over a Superdex 200 50/60 SEC-column (1200 mL, GE Healthcare) with 2xPBS used as running buffer. Some larger proteins were separated out and IgE was found in the main peak. Only the main peak fractions were pooled because of contamination in the other two fractions. This step increased the purity of the sample to -99%. The total amount produced was 42 mg and the purified IgE had a concentration of 2 10 mg/mL. Analysis of glycosylation of IgE CF3-CE4 In-solution digestion with Trypsin Human IgE minimal domain, IgE Ce3-Ce4, 2 mg/ml, in 2 x PBS (composition 308 mM NaCl, 15 20 mM phosphate, pH 7.4) was mixed with 100 pl trypsin 0.02 mg/ml in 25 mM NH 4 HCO3. Digestion proceeded overnight in 37*C and was stopped with 2 1A formic acid (67%) in H 2 0. Nano-LC MS/MS: Analysis was performed on a 20 cm x 50 im i.d. fused silica column packed with ReproSil 20 Pur C 18 -AQ 3 im porous particles, connected to an LTQ-Orbitrap mass spectrometer (Thermo). 8 ptl sample injection was made (Agilent autosampler) and the peptides were trapped on a precolumn, 4.5 cm x 100 pim i.d., before separation. After 5 minutes linear run with 0.1% formic acid, the gradient was 10-50% acetonitril during 5-30 min (Agilent), 200 n/rmin, and the eluent was electrosprayed from the emitter tip. The instrument was operated in 25 data-dependent mode to switch between Orbitrap (FT-MS) survey scan and ion trap (IT MS/MS) of the three most abundant multiply protonated ions. Static electrospray MS/MS: To verify the charge state of the glycopeptide fragments, selected precursors were analyzed 30 with ESI needle at 1.6 kV, fragmented and detected in the Orbitrap opposed to the linear ion trap in the nano-LC analysis.

115 Glycopeptide data analysis: the calculated MH+ masses of possible glycopeptides were examined for the presence of glycosylation by use of the GlycoMod tool (http:expasy.org/tools/glycomod) (Cooper et al 5 2001). The protein sequence and a mass tolerance of 10 ppm was entered. All suggested glycopeptides were checked for the presence of glycan containing fragments. Production of Antibody 11 Fab. 10 IgG purification Antibody I I was purified from CHO-EBNA transient material using MabSelect SuRe (GE Healthcare) protein A chromatography media. The protein A eluate was buffer exchanged into PBS, pH7.2 using PD-10 columns (GE Healthcare) then 0.22gm filtered using a Millex-GP syringe tip filter (Millipore). 15 Fab digest and purification A digest buffer of 30mM DL-cysteine hydrochloride dissolved in GIBCO PBS (Invitrogen) was prepared. Papain from papaya latex (Sigma) was reconstituted in digest buffer to give a 1 Omg/mL solution and kept at room temperature for a minimum of 30 minutes before use. 20 Cysteine was added to Antibody 11 IgG to give a 30mM solution and papain was added at a ratio of 1mg papain to 100mg IgG. The digest was terminated after 90 minutes by the addition of 0.5M iodoacetamide (Sigma) to give 50mM iodoacetamide in the final digest mixture. The Fab was purified from the digest mixture using MabSelect SuRe (GE Healthcare) protein A chromatography media in a non-binding mode. The Fab fraction from the MabSelect SuRe 25 step was buffer exchanged into 50mM sodium acetate/1OOmM NaCI, pH5.5 using PD-10 columns (GE Healthcare) and then concentrated to 1 Omg/mL using Amicon Ultra-I5 5kDa MWCO centrifugal filter devices (Millipore). The final product was further purified using a Mustang Q acrodisc (Pall) and then 0.22pm filtered using a Millex-GP syringe tip filter (Millipore). 30 116 Generation of the IgE Ce3-Cc4 Antibody 11 Fab complex. A solution containing 2 mg/mL of IgE C63-Ce4 in buffer 308 mM NaCl and 20 mM 5 phosphate, pH 7.4, was mixed with a solution containing 10.6 mg/mL of Antibody 11 Fab in 50mM sodium acetate, 100mM NaCl, pH5.5 at a stoichiometric ratio of I to 1.1 of IgE Fc3-4 homodimer and Antibody 11 Fab heterodimer respectively. The mix was left in ice over night followed by gel filtration on a HiLoad Superdex200 16/60 column (GE Healthcare) equilibrated in 20 mM Tris HCI pH 7.6 and 0.15 M NaCl. The main peak containing the 10 complex was collected and concentrated to 10.4 mg/mL before used in crystallisation experiments. Crystallisation of the IgE Ce3-Ce4 Antibody 11 Fab complex. Crystallisations were carried out according to the method of sitting drop vapour diffusion. The 15 drops contained an equal volume of protein and reservoir solution (150+150 nL) and were set up in a Crystal Quick 96 well plate (Greiner Bio-one) with reservoir volumes of 80 uL. The complex crystals grew in drops with a reservoir solution of 100 mM MgCl2, 100 mM sodium citrate pH 5.0 and 15% PEG 4000 over a period of 2-3 weeks at 4*C. The crystals were harvested into a cryoprotectant solution (100 mLv MgCl 2 , 100 mM sodium citrate pH 5.0 and 20 15% PEG 4000 made 20% glycerol by addition of 100% glycerol) and cooled rapidly in liquid nitrogen. Data collection and Structure solution of the IgE Cs3-Cs4 Antibody II Fab complex. Diffraction data were collected from single crystals at the European Synchrotron Radiation 25 Facility (ESRF) in Grenoble, France at beam line ID-29. An initial dataset (data set 1, table 7) was recorded to 3 A resolution and later a higher resolution data set was collected to 2.85 A resolution, both of which belong to space group P3 2 21. The data was processed with autoPROC (Global Phasing Limited GPhL, Cambridge, UK). Statistics from the data processing is presented in table 7. The asymmetric unit contains three Fab molecules and 30 three molecules of IgE Ce3-Ce4 corresponding to a solvent content of 54 %. The structure was solved by the method of molecular replacement using the program PHASER (Read 2001, Storoni et al 2004, McCoy et at 2005). Initial models for the Fab fragment and the IgE Fc 117 domain was generated from the previously reported structures IAQK(Faber et al 1998) and IFP5 (Wurzburg et al 2000). Use of the entire Fab as the search model failed, due to variations of angles in the hinge region between the variable domain and the constant domains. Instead two separate search 5 models consisting of the variable domains and constant domains respectively were prepared and identified in the initial run of PHASER(Read 2001, Storoni et al 2004, McCoy et al 2005). These were later joined to complete Fab fragments. In total two Fab fragments and one variable domain was identified in this fashion. Subsequent runs of molecular replacement located three IgE Ce3-Ce4 molecules, of which one had to be trimmed back to comprise only 10 domain 4. At this stage better quality data had been collected (dataset 2, table 7) and this model was refine against the new data using the program autoBUSTER (Global Phasing Limited GPhL, Cambridge, UK). Subsequently the amino acids of the Fab molecules were manually altered to the correct sequence of Antibody 11 using the graphical program COOT (Emsley & Cowtan 2004). After a second round of restrained maximum-likelihood refinement 15 using isotropic B factors refinement in Refmac (CCP4 1994) the remaining domains of the last Fab and IgE molecules were manually fitted into the electron density. Two additional features of elongated electron density was observed protruding from amino acid residue Asn394 into the cavity between the IgE molecules. This was interpreted as glycosylations and therefore two N-acetyl-glucosamine and three to four mannose units were added to the IgE 20 models. Further rounds of refinement included manual rebuilding of loop regions in COOT (Emsley & Cowtan 2004) intervened by refinement in either autoBUSTER (Global Phasing Limited GPhL, Cambridge, UK) or Refmac5 (Murshudov et al 1997) applying TLS for individual domains and non-crystallographic symmetry (NCS) restrains for the IgE molecules. In total 213 waters were built in using the water picking option in Refmac5 25 (Murshudov et al 1997) followed by manual inspection. In the final refinement round the NCS restraints were released resulting in a final model with R=20.0 % and Rfree=27.0o.

118 Table 7: Crystal Parameters and X-ray Data-Processing and Refinement Statistics Data set 1 Data set 2 Space group P3 2 21 P3 2 21 Wavelength (A) 0.976 0.976 Cell constants a (A) 140.62 141.562 b (A) 140.62 141.562 c (A) 244.65 245.562 Resolution range (A) 2.93-35.16 2.85 -109.11 Resolution highest shell (A) 2.93-3.01 2.85 -2.92 Completeness overall (%) 99.9 100.0 Completeness highest shell (%) 100.0 100.0 Reflections, unique 60601 66338 Multiplicity 6.6 10.7 Multiplicity highest shell 6.8 11.0 Rmerge overall (%)1 0.133 0.099 Rmerge highest shell (%) Q.914 0.75 Mean(l)/sd(I) 13.1 20.2 Mean(l)/sd(l) highest shell 2.0 3.4 Rvalue ove (%) 2 N/A 20.0 Rvalue free(%) N/A 27.0 1 Rrerge =FZki [( i 1lh - (hi )/ E' 1I] 2 Rvaiuc =EIilk [Fob. - IFc-lrji / EIk IFob,i 5 Rrre is the cross-validation R factor computed for the test set of 5 % of unique reflections 119 References cited in Example 7 Davies A. Greene A. Lullau E. Abbott WM. Optimisation and evaluation of a high throughput mammalian protein expression system. Protein Expression & Purification. 5 42(1):111-21. (2005) CCP4 (Collaborative Computational Project, Number 4) (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr D 50: 760-763 10 Cooper C.A., Gasteiger E., Packer N. GlycoMod - A software Tool for Determining Glycosylation Compositions from Mass Spectrometric Data Proteomics 1:340-349 (2001). 15 Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr D60: 2126-2132 (2004) Faber, C., Shan, L., Fan, Z., Guddat, L.W., Furebring, C., Ohlin, M., Borrebaeck, C.A., Edmundson, A.B. Three-dimensional structure of a human Fab with high affinity for 20 tetanus toxoid. Immunotechnology 3 : 2 5 3 - 2 7 0 (1998) Kabat,E.A., Wu,T.T., Perry,H., Gottesman,K. and Foeller,C. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242. 25 Karpusas, M., Lucci, J., Ferrant, J., Benjamin, C., Taylor, F.R., Strauch, K., Garber, E., Hsu, Y.M. (2001) Structure of CD40 ligand in complex with the Fab fragment of a neutralizing humanized antibody. Structure 9, 321, (2001) Leslie A. (1991) Macromolecular data processing. In Moras,D., Podjarny,A.D. and 30 Thierry,J.C. (eds), Crystallographic Computing V. Oxford University Press, Oxford, UK, pp. 27-38 120 McCoy, A.J., Grosse-Kunstleve, R.W., Storoni, L.C. & Read, R.J. Likelihood-enhanced fast translation functions. Acta Cryst D61, 458-464 (2005) Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of Macromolecular 5 structures by the maximum-likelihood method, Acta Crystallogr D53: 240-255 Padavattan, S., Schirmer, T., Schmidt, M., Akdis, C., Valenta, R., Mittermann, I., Soldatova, L., Stater, J., Mueller, U. & Markovic-Housley, Z. Identification of a B-cell Epitope of Hyaluronidase, a Major Bee Venom Allergen, from its Crystal Structure in Complex with a 10 Specific Fab. JMoI Biol 368, 742-752. (2007) Persic L. Roberts A. Wilton J. Cattaneo A. Bradbury A. Hoogenboom HR. An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene. 187(1):9-18. (1997) 15 Read, R.J. Pushing the boundaries of molecular replacement with maximum likelihood. Acta Cryst. D57, 1373-1382 (2001) Rossmann, M.G. (edt): "The Molecular Replacement Method" Gordon & Breach, New York (1972) 20 Storoni, L.C., McCoy, A.J. & Read, R.J. Likelihood-enhanced fast rotation functions. Acta Cryst D60, 432-438 (2004) Wurzburg, B.A., Garman, S.C. and Jerdetzky, H.S. Structure of the human IgE-Fc C epsilon 3-C epsilon 4 reveals conformational flexibility in the antibody effector domains. Immunity, 13, 375-385 (2000). 25 Example 8: Assessment of the General Safety and Capacity of germlined anti-IgE mAbs to Induce Decreases in Platelet Numbers In Juvenile Cynomolgus Monkeys An investigative (non-GLP compliant) study was performed in juvenile cynomolgus monkeys 30 to assess the general safety and relative abilities of antibodies of the invention anti-IgE mAbs 121 Antibody 11 IgGi Antibody 11 IgG 2 and and another anti-IgE antibody E48 to cause decreases in numbers of blood platelets. The objectives of the study were 1) to determine the general safety and relative abilities of the 5 three candidate anti-IgE mAbs to induce a reduction in platelet counts/TCP and associated effects in juvenile cynomolgus monkeys 2) to determine preliminary pharmacokinetic parameters for the mAbs in monkeys 3) to assess the capacity of the three candidate mAbs to cause a reduction in free IgE and determine the (PK/PD) relationship between mAb concentraton and free IgE levels 10 Materials and Methods for Example 8 Eighteen purpose-bred cynomolgus monkeys (Macacafascicularis) were obtained from Bioculture, Mauritius. The animals were between 63 to 67 weeks old at the start of dosing. Monkeys were pre-selected (from a larger pool of 100 animals) to have high IgE levels (U/ml) which were normalised across 3 groups each containing 3 male and 3 female monkeys and 15 receiving either Antibody I1 IgG, (Group 1), Antibody 11 IgG 2 (Group 2) or E48 (Group 3). Each of the 3 mAbs were formulated in 50mM sodium acetate, 100mM NaCl, pH5.5 and adminstered to animals in a dose volume of 2 mL/kg by slow intravenous injection (using a motorised syringe/infusion pump) at a rate of 1 mL/min. 'The animals were dosed once weekly (for 5 weeks/5 doses) with rising dose levels of Img/kg, 30mg/kg and 100mg/kg (x3) on Days 20 1, 8, 16, 22 and 29 (Table 8). Additional doses of Antibody 11 IgGi and Antibody 11 IgG 2 were administered to Groups 1 and 2 respectively on Day 37. The 2 highest dose levels were predicted to achieve serum concentrations that have previously shown to result in thrombocytopenia (TCP) with Xolair in juvenile cynomolgus monkeys.The low dose was expected to allow a determination of the ability of the mAbs to effect a reduction in free IgE 25 levels 122 Table 8 Summary Study Design Dose level (mg/kg/day) on Day: Number of Animals Group Description 1 8 16 22 29 37 Male Female 1 Ab 1 IIgG, 1 30 100 100 100 100 3 3 2 Ab 1 IgG 2 1 30 100 100 100 100 3 3 3 E48 1 30 100 100 100 - 3 3 (Ab 11 = Antibody 11) 5 Animals were observed for 8 weeks post the Day 28 dose and examined for recovery from any toxicological effects. All animals were observed daily for signs of ill health or overt toxicity and body weights and food consumption recorded. In addition, each animal was given a detailed physical 10 examination daily during dosing periods and at least once weekly during non-dosing periods. All animals were also observed prior to each dose and at 0.5, 2, 6, 24, 48 and 168 hours post dose. Blood samples for analysis of standard haematology parameters (including platelet counts; 15 collected in EDTA) and coagulation parameters (collected in trisodium citrate) were taken from the femoral vein/artery twice pre-treatment (Weeks -2 and -1). Further samples for platelet counts and standard haematology were collected at 24 hours and 144 hours after each dosing occasion (Days 2, 7, 9, 14, 17, 22, 23, 26, 30 and 35; samples from Groups I and 2 only on Days 38 and 43) and every 2 weeks during the 8-week recovery period (Days 43, 57, 20 71 and 82). Samples for coagulation were collected at 144 hours after each dosing occasion (Days 7, 14, 22, 26 and 35) and at the end of the recovery period (Day 82). Samples for coagulation were also collected on Day 57. Blood samples for complement activation (C3a, C3b and BB fragments) were taken once pre-treatment (Week -1) and approximately 24 hours following completion of the treatment period (Day 30) 25 123 Serum samples for TK analysis were collected from all groups on day Day I at pre-dose, 0.5, 6, 12, 24, 48, 144 hours post-dose, on Days 8, 16, 22 and 29 at 0.5, 24 and 144 hours post dose, on Day 29 (Groups 3 & 4 only) at 336, 672, 1008, 1272 hours post-dose, on day 37(Groups I & 2 only) at 0.5, 24, 144, 480, 816, 1080 hours post-dose. Samples were 5 analysed for mAb using a generic sandwich immunoassay (using biotinylated human IgE for mAb and Alexa-647 labelled murine anti-human IgG detection reagent) and the Gyrolab Bioaffy platform (incorporating streptavidin bead columns). Further serum samples for IgE analysis were collected on Day 1 at pre-dose, 0.5, 6, 12, 24, 48, 144 hours post-dose, on Days 8, 16, 22 and 29 at 0.5, 144 hours post-dose and at the end of the study (Day 82) at 1272 10 hours (Groups 3 & 4) or 1080 hours (Groups I & 2) post-final dose. Samples were analysed for free IgE by immunoassay using the ImmunoCap system (Phadia AB, Uppsala,Sweden) with human IgG-FcERla for free IgE capture and Rabbit anti-human IgE (PCS-conjugate) for detection. 15 On termination of the animals on Day 85, a full macroscopic examination was performed under the general supervision of a pathologist and all lesions were recorded. Absolute organs weights and organ:body weight rations were determined. Tissues from a range of organs were collected and stored frozen but no microscopic examination was performed (except for macroscopic abnormalities or an unscheduled death, see below) 20 Results for Example 8 General Safety Observations 25 All 3 mAbs were generally well-tolerated with no clinical signs of ill-health throughout the study with the exception of a single animal receiving Antibody 11 that was sent to necropsy ahead of schedule due to deteriorating clinical condition and reduced bodyweight. Since this animal deteriorated well into the recovery period and there were no findings noted during the pathology or haematology review of these animals, the observed effects are not believed to be 30 mAb-related. Incidences of soft or liquid faeces were noted across all groups, however since these findings were not dose-related, were not seen in all animals or at all timepoints within the same animal and were seen as frequently during the dosing and recovery periods, they are unlikely to be mAb-related. Mean body weights and mean body weight gains showed some 124 individual variation in animals within each group throughout the study. However all animals gained weight as expected over the treatment period. (with the exception of the 1 animal discussed above) and there was no clear differences between the groups. No clear treatment related effects on absolute or relative organ weights were noted in any group. In gross 5 pathology and microscopic pathology examinations, no findings were noted in the limited range of tissues examined that would suggest an effect of mAb treatment. Toxicokinetics (TK) and IgE levels 10 No gender difference in TK was observed in this study. In general the exposure was similar for these 3 mAbs, and appeared linear with dose in the 1-100 mg/kg dose range. The mean TK profiles of Antibody 11 IgGj, Antibody 11 IgG 2 and E48 are shown in Figure 12. No apparent IgE-sink effect on TK was observed, even at the lowest dose level. The TK of these 3 antibodies appeared typical for an human IgG in cynomolgus monkeys. 15 The mean maximum observed concentration (Cmax) following the last 100 mg/kg dose was 18700, 15900 and 24000 nM for Antibody 11 IgGj, Antibody II IgG 2 and E48, respectively. The mean terminal TK half-life following the last 100 mg/kg dose was approximately 10-13 days. There was no evidence of reduced TK exposure due to the potential development of 20 primate anti-human antibodies in these animals. The mean free IgE profiles following weekly dosing of Antibody 11 IgGI, Antibody 11 IgG 2 and E48 at various dose levels in cynomolgus monkeys are shown in Figure 13. The average baseline IgE before the animals received the first dose was 514, 414 and 690 ng/mL for 25 Antibody I1 IgGi, Antibody 11 IgG 2 and E48 groups, respectively. On Day 1, 1 mg/kg dose induced a 75-80% reduction in free IgE at 1 hour after the dose. Due to the low exposure after the 1 mg/kg dose, free IgE returned to baseline level within I week. Higher doses resulted in consistent suppression of free IgE during the treatment period. Free IgE returned to baseline for the 2 Antibody II groups at the end of the study, while the free IgE in the E48 group 30 remained suppressed.

125 Effects on platelets None of the 3 mAbs (Antibody 11 IgG 1 , Antibody I I gG 2 nor E48) induced a significant decrease in platelet counts at any timepoint in any animal with the exception of a single 5 animal receiving Antibody 11 IgGi that had a reduction in platelets (34.9%) at a single timepoint on day 29 (24 hours following the third 100mg/kg dose on day 28). A 4 'h dose of Antibody II igG 1 and Antibody 11 IgG 2 on day 37 did not induce any further platelet reduction in this or any animal within these 2 groups. 10 Figure 14 shows a plot of platelet numbers (x10 9 /L) expressed as a percentage change from the mean of the 2 pre-dose values vesus plasma concentration from an animal in Group 1 (Antibody II LgGI-treated). This plot is is representative of the other 16 animals across the 3 groups that showed no significant effect on platelets [Change for Antibody 11 animal]. Figure 15 shows the same plot for the animal in Group 1 (Antibody 11 IgGi-treated) that showed a 15 transient significant drop (35% below baseline) in platelet numbers on day 29. Interestingly, the Antibody I I lgGI-treated monkey that showed a transient drop in platelet numbers after dosing on day 29 had the highest Cmax value (29400 nmol/L)(but not exposure) at this time. The plasma levels subsequently dropped sharply and the platelet counts 20 returned to pre-dose values. This hints to the possibility that a higher threshold of plasma concentration might be required to evoke decreases in platelet numbers. However a single E48-treated animal reached similar levels (28500nm/L) with no corresponding platelet effects (Figure 14). Other Haematological Effects. 25 With the exception of platelet counts (see below), no consistent effects of mAb treatment were noted on the majority of of haematological parameters (haemoglobin concentration, packed cell volume, mean cell volume, mean cell haemoglobin concentration, red cell distribution width, platelet crit, platelet distribution width, red blood cell count, mean cell haemoglobin, haemoglobin distribution width, mean platelet volume, reticulocyte count, total 30 and differential white cell count) and blood coagulation parameters (prothrombin time, activated partial thromboplastin time). An increase in the numbers of reticulocytes was observed in all groups however the changes were not dose/exposure-related, were not consistent within a group (animals within a group had higher, lower or unchanged levels from 126 pre-dose values) or within an animal (values within animals rose and fell between time-points independent of exposure) and, in the absence of a parallel control group, the relationship to mAb treatment cannot be fully determined at this time. Any such changes had generally reversed at the end of the recovery period. No significant treatment-related effects on 5 complement activation (C5a, C3a or BB fragments) were noted. Discussion and Conclusions This study has shown that anti-IgE mAbs Antibody I I IgGi, Antibody 11 IgG 2 and E48 were well-tolerated when administered at high repeated dose levels (up to 100mg/kg) to juvenile 10 cynomolgus monkeys with no significant adverse toxicological effects. Only 1 animal out of 18 monkeys showed a significant drop in platelet numbers at a single timepoint after dosing with 100mg/kg Antibody 11 IgG, when plasma concentrations of mAb reached almost 30000 nmoL/L. The plasma Cmax concentrations reached with all 3 mAbs in this study are expected to be far in excess of those that will be achieved in the clinic (e.g. 200nmol/L). 15 Example 9: Functional inhibition of bgE effects on FeeRI and CD23. The ability of the optimised Antibody 11 to inhibit functionally the interaction of IgE with FceRI and CD23 was evaluated in an IgE-mediated cell killing assay adapted from Bracher et 20 al (Journal Immunol. Methods 2007 323:160-171). U937 cells pre-treated with IL-4 were shown to express both FceRI and CD23. When co-cultured with the ovarian tumour cells IGROV1 in the presence of IgE specific to an antigen expressed on IGROVI cells, the U937 cells were able to kill the tumour cells. The killing was mediated both by cytotoxicity and phagocytosis mechanisms which were shown to be triggered through the interaction of IgE 25 with FeeRI and CD23 respectively on the U937 effector cells. Antibody 11 and an isotype control were evaluated in this assay for inhibition of IgE mediated killing through either FccRI or CD23. A detailed protocol for this procedure is provided in Materials and Methods. In brief, titrations of the test IgG were mixed with a target 30 specific (MOv18) or irrelevant (NIP) IgE prior to incubation with IL-4-stimulated U937 effector cells and labelled-IGROVI target cells. Following a 2.5 hours incubation, the cells were washed, stained with an anti-CD89-phycoerythrin antibody (BD Biosciences) and 127 propidium iodide (Molecular Probes). After washing, the cell fluorescence was analysed using a FACSCalibur flow cytometer (BD Biosciences). The fluorescent dyes above were used to differentiate live cells from cells killed by cytotoxicity and cells killed by phagocytosis. Conversely to the isotype control antibody, Antibody 11 was able to inhibit 5 both the IgE/FcERI-mediated cytotoxicity (figure 16) and the IgE/CD23-mediated phagocytosis (figure 17). Materials and Method - Example 9 10 Antibodies were evaluated for inhibition of IgE-mediated IGROVI tumour cell killing by the U937 cells. IGROVI cells (a human ovarian carcinoma cell line) and U937 cells (a human myelomonocytic cell line) were maintained in culture medium [RPMIl640, 10% v/v FCS, 2mM glutamine, 5000U/ml penicillin, 100ug/ml streptomycin (all from Invitrogen)] using standard tissue culture procedures. The MOv18 IgE directed against FBP (folate binding 15 protein) expressed on the IGROVI cells was used as a tumour specific antibody. The NIP (hapten 4-hydroxy-3-nitro-phenacetyl)-specific IgE was used as a control irrelevant antibody. The MOv18 and NIP antibodies were prepared as described in Gould et al, (1999) Eur. J. Immunol. 29:3527-3537. U937 cells were pre-treated for 4 days prior to the killing experiment with lOng/ml 20 recombinant human IL-4 (R&D Systems) in order to up-regulate the expression of CD23. The day before the killing experiment, the IGROV I target cells were labelled with the fluorescent dye CFSE (Carboxy-fluorescein diacetate succinimidyl ester, Molecular Probes). Briefly, the cells were trypsinised (Trypsin/EDTA, Gibco), washed in culture medium and resuspended in PBS at 5OxlO 6 cclls/mI. The cells were then incubated at 37*C for 10 minutes with CFSE at 25 0.01mM. After the labelling, the cells were washed once in ice-cold culture medium and then incubated overnight at 37 0 C, 5%C0 2 . To evaluate the inhibitory effect of Antibody 11, antibody dilutions were prepared in 12x75 mm tubes (Falcon, BD Biosciences) and 2ug of MOvI or NIP IgE were added given a final volume of 80ul. This mixture was incubated without cells for 30 minutes. IL-4-stimulated 30 U937 cells were washed once in medium and resuspended at 1.33x106 cells/ml. CFSE labelled IGROV1 cells were trypsinised, washed once in medium and resuspended at 4x10 5 cells/ml. The cells were added to the tubes containing the antibodies (120u1 for the U937 cells and 200ul for the IGROVI cells), mixed and incubated for 2.5 hours at 37 0 C, 5%C0 2 . 'ihe 128 cells were then washed in ice-cold FACS buffer (calcium/magnesium-free PBS, 5% normal goat serum) and incubated for 25 minutes with an anti-CD89-phycoerythrin antibody (BD Biosciences, 10ug/ml) to label the U937 effector cells. The cells were washed once more in ice-cold FACS buffer and dead cells were stained by adding 0.25ug/mi propidium iodide 5 (Molecular Probes). After 15 minutes at 4 0 C, the cells were washed in ice-cold FACS buffer, resuspended in 250ul ice-cold FACS buffer and the fluorescence was analysed using a FACSCalibur flow cytometer (BD Biosciences) according to manufacturer instructions. Cells with the relevant single staining were used to adjust the voltage and compensation of the detection channels (FL1, FL2 and FL3). 10 The combination of fluorescent dyes used in this assay allowed for the gating of different cell populations [live effector cells phycoerythrinn positive), phagocytosed IGROVI tumour cells (phycoerythrin and CFSE positive), live tumour cells (CFSE positive), dead tumour cells (CFSE and propidium iodide positive), dead effector cells (phycoerythrin and propidium iodide positive)]. These gates were used to calculate the percentage of target cells killed by 15 FceRI-mediated cytotoxicity (equation 1) and by CD23-mediated phagocytosis (equation 2). Equation 1: % cytotoxicity = {[(RISL control- RI) + R3] / RI SL} x 100 Where: 20 RI = total number of CFSE positive tumour cells R3 = number of killed but intact tumour cells (no fragmentation or phagocytosis) RlSL control = Average RI of 3 control samples of effector and target cells without antibody (RI Spontaneous Loss control). 25 Equation 2: % phagocytosis = (R2/RISL control) x 100 Where: R2 = number of tumour cells phagocytosed by effector cells RlSL control = Average R1 of 3 control samples of effector and target cells without 30 antibody (RI Spontaneous Loss control).

129 References All references cited anywhere in this specification, including those cited anywhere above, are incorporated herein by reference in their entirety and for all purposes. I Haan & Maggos (2004) BioCentury, 12(5): A l-A6 2 Koide et al. (1998) Journal of Molecular Biology, 284: 1141-1151. 3 Nygren et al. (1997) Current Opinion in Structural Biology, 7: 463-469 4 Wess, L. In: BioCentury, The Bernstein Report on BioBusiness, 12(42), Al-A7, 2004 5 Kabat, E.A. et al, Sequences of Proteins of Immunological Interest. 4 " Edition. US Department of Health and Human Services. 1987 6 Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition. US Department of Health and Human Services, Public Service, NIH, Washington 7 Segal et al., PNAS, 71:4298-4302, 1974 8 Amit et al., Science, 233:747-753, 1986 9 Chothia et al., J. Mol. Biol., 196:901-917, 1987 10 Chothia et al., Nature, 342:877- 883, 1989 11 Caton et al., J. 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ISBN: 0-8247 0487-8 45 Chothia C. et al. Journal Molecular Biology (1992) 227, 799-817 46 Al-Lazikani, et al. Journal Molecular Biology (1997) 273(4), 927-948 131 47 Chothia, et al. Science, 223,755-758 (1986) 48 Whitelegg, N.R.u. and Rees, A.R (2000). Prot. Eng., 12, 815-824 49 Guex, N. and Peitsch, M.C. Electrophoresis (1997) 18, 2714-2723 50 Altschul et al. (1990) J. Mol. Biol. 215: 405-410 51 Pearson and Lipman (1988) PNAS USA 85: 2444-2448 52 Smith and Waterman (1981) J. Mol Biol. 147: 195-197 53 Voet & Voet, Biochemistry, 2nd Edition, (Wiley) 1995. 54 Gram et al., 1992, Proc. NatI. Acad. Sci., USA, 89:3576-3580 55 Barbas et al., 1994, Proc. Natl. Acad. Sci., USA, 91:3809-3813 56 Schier et al., 1996, J. Mol. Biol. 263:551-567 57 Marks et al BiolTechnology, 1992, 10:779-783 58 Kay, B.K., Winter, J., and McCafferty, J. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, San Diego: Academic Press 59 Hunter W. M. and Greenwood F. C. (1962) Nature 194:495 60 PlIckthun, A. Bioflechnology 9: 545-551 (1991) 61 Chadd HE and Chamow SM (2001) Current Opinion in Biotechnology 12: 188-194 62 Andersen DC and Krummen L (2002) Current Opinion in Biotechnology 13: 117 63 Larrick JW and Thomas DW (2001) Current Opinion in Biotechnology 12:411-418 64 Sambrook and Russell, Molecular Cloning: a Laboratory Manual: 3rd edition, 2001, Cold Spring Harbor Laboratory Press 65 Ausubel et al. eds., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, John Wiley & Sons, 4 th edition 1999 66 Robinson, J. R. ed., Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York, 1978 67 Ledermann J.A. et al. (1991) Int. J. Cancer 47: 659-664 68 Bagshawe K.D. et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922 69 Vaughan, T. J., et al. (1996). Nature Biotechnology 14, 309-314. 70 Hutchings, C. Generation of NaYve Human Antibody Libraries, in Antibody Engineering, R. Kontermann and S. Dubel, Editors. 2001, Springer Laboratory Manuals, Berlin. p. 93 132 aoOL >. (100 0 0 66 w 96 L6 96 ca 96 S9 V9 u. 1,9 g 09 69 > Cu 99 II. 09 C.) v___ co .

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134 96 U)0 8396 U) x >. x = > V96 -j co u)0 z a o co 96 - c- - ZZ wc 0l 16 (L U) V)co c z co w- x x C6 c Z6 06 68 a - 9S I E S z Z9 U 09 a 1.c > ov - 3LZ VLZ c 9z C 0 0 0 0 0 co0 V,0 0 0 a 0 0 0 0 1, c ~. m 8 .0 . M, .0 -0 , m .0 m A . , 0 M 135 96 a D 996 - 4 Z 4 > 0 4 0 - VS6 Xi Cy W a c u C 96 I- Z C0~4 C Z U 0 76 I. z U) a .0 w 4 w a 0 C6, Z6 06 U) 68 o 99 C I.S z 09 6Z 8Lz 9z C I 00 "S o o~ 0 0 0 04 E _ C4 :esC C:NRPonbl\DCC\RBRW651614 I DOC-3/10/2012 136 The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general 5 knowledge in the field of endeavour to which this specification relates. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group 10 of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims (25)

1. An isolated binding member specific for immunoglobulin E which binding member has an IC50 geomean for inhibition of calcium signalling induced by 25ng/ml IgE in RBL 5 ER51 cells of less than InM.

2. An isolated binding member according to claim 1 wherein the IC50 geomean for inhibition of calcium signalling is less than 0.lnM. 10

3. An isolated binding member specific for immunoglobulin E which binding member has an IC50 for the binding of said binding member to immunoglobulin E in serum at least 10 fold lower than XolairTM.

4. An isolated binding member according to claim 3 wherein said binding member 15 has an IC50 at least 50 fold lower than XolairTM.

5. An isolated binding member specific for human immunoglobulin E comprising a set of CDRs: HCDRI, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 wherein the set of CDRs has 10 or fewer amino acid additions, substitutions, deletions and/or insertions from 20 a reference set of CDRs in which: HCDR1 has the amino acid sequence of SEQ. ID. NO: 103; HCDR2 has the amino acid sequence of SEQ. ID. NO: 104; HCDR3 has the amino acid sequence of SEQ. ID. NO: 105; LCDRI has the amino acid sequence of SEQ. ID. NO: 108; 25 LCDR2 has the amino acid sequence of SEQ. ID. NO: 109; LCDR3 has the amino acid sequence of SEQ. ID. NO: 110;

6. An isolated binding member specific for human immunoglobulin E according to Claim 5 wherein the amino acid substitutions comprises 6 or fewer amino acid 30 substitutions. C:\NRPonbl\DCC\BRW65 1614 1 DOC-3110/2012 138

7. An isolated binding member comprising a set of CDRs: HCDRI, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 wherein HCDR3 comprises:the amino acid sequence of SEQ. ID. NO: 105; 5

8. An isolated binding member according to Claim 5 comprising a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 wherein the set of CDRs compriss: HCDR1 has the amino acid sequence of SEQ. ID. NO: 103; HCDR2 has the amino acid sequence of SEQ. ID. NO: 104; 10 HCDR3 has the amino acid sequence of SEQ. ID. NO: 105; LCDRI has the amino acid sequence of SEQ. ID. NO: 108; LCDR2 has the amino acid sequence of SEQ. ID. NO: 109; LCDR3 has the amino acid sequence of SEQ. ID. NO: 110; 15

9. An isolated binding member or VH domain comprising the antibody I HCDR3 (SEQ ID NO:5) with one or more of the following substitutions: Kabat residue 96 replaced by S, M, or T; Kabat residue 97 replaced L or G; Kabat residue 98 replaced by K; 20 Kabat residue 99 replaced by S, W, A, T, or E; Kabat residue 100 replaced by A or I.

10. An isolated binding member or VL domain comprising the antibody I LCDR3 (SEQ ID NO 10) with one or more of the following substitutions: 25 Kabat residue 94 replaced by T, R, D, P, E, N, H, Q, or A; Kabat residue 95 replaced T, K, S, I, G, H, M, F, R, N, K or Q; Kabat residue 95A replaced by L, H, D, G, R, N, Q, K or E; Kabat residue 95B replaced by T, H, S, Y, L or N; Kabat residue 96 replaced by G or A; 30 Kabat residue 97 replaced by P, S or G. C NRPonbl)CC\RBR\4651614 I DOC-3/10/2012 139

11. An isolated binding member specific for immunogbulin E which binds an epitope which comprises elements from a first IgE heavy chain and elements from a second IgE heavy chain. 5

12. An isolated binding member specific for immunoglobulin E wherein said binding member binds to an epitope in immunoglobulin E comprising: residues Glu390 through to Asn394 inclusive in a first IgE heavy chain and Leu340, Arg342, Ala428 to Thr434 inclusive, Thr436, Ser437 and Glu472 in a second IgE heavy chain. 10

13. An isolated binding member specific for immunoglobulin E according to Claim 12 wherein the epitope further comprises sugar moieties GlcNAc1 and Man6 in a first IgE heavy chain and sugar moiety Man 5 in a second IgE heavy chain. 15

14. An isolated binding member specific for immunoglobulin E wherein said binding member binds to an epitope in immunoglobulin E comprising: residues Glu390, Gln392 to Asn394 inclusive in a first IgE heavy chain and Leu340, Arg342, Ala428 to Thr434 inclusive, Thr436, Ser437 and Glu472 in a second IgE heavy chain 20

15. An isolated binding member specific for immunologbulin E according to Claim 14 wherein the epitope further comprises sugar moieties GlcNAc I and Man6 in a first IgE heavy chain. 25

16. The binding member according to any one of Claims I to 15 wherein the binding member is a monoclonal antibody.

17. An isolated nucleic acid molecule encoding an isolated binding member according to any one of claims I to 16. 30

18. A host cell transformed with a nucleic acid molecule according to Claim 17. CANRPonblDCCRBR\4651614_1 DOC-3/10/2012 140

19. A method of producing an isolated binding member according to any one of claims 1 to 17 comprising culturing host cells according to Claim 18 under conditions for production of said binding member. 5

20. A pharmaceutical composition comprising a binding member according to any one of claims I to 16 and a pharmaceutically acceptable excipient.

21. A pharmaceutical composition according to Claim 20 comprising: 10 an isolated binding member according to any one of claims I to 16 20mM Succinate 105mM NaCI 10mM Arginine pH 6.00 15

22. The composition according to Claim 20 or 21 for use as a medicament.

23. Use of the composition of claim 22 for treating a disorder associated with IgE. 20

24. Use of the composition according to claim 20 or 21, wherein the disorder is one or more of an allergy, asthma, or bronchitis.

25. Use of the composition according to claim 20 or 21 wherein the disorder is one or more of allergic rhinitis, allergic contact dermatitis, atopic dermatitis, anaphylactic 25 reaction, food allergy, urticaria, inflammatory bowel disease, eosinophilic gastroenteritis, drug-induced rash, allergic opthalmopathy, allergic conjunctivitis, asthma bronchiale, airway hyperresponsiveness, cosmetic allergy, drug-induced allergy, drug-induced hypersensitivity syndrome, metal allergy, occupational hypersensitivity pneumonitis, chronic hypersensitivity pneumonitis, cold hypersensitivity, helminthic infection induced 30 hypersensitivity, latex allergy and hay fever.