WO2022061236A1 - High affinity anti-ige antibodies - Google Patents

Abstract

Humanized or chimeric anti-IgE monoclonal antibodies are provided. The antibodies bind to human IgE, and find use in various therapeutic methods, including without limitation the reduction or prevention of allergic conditions.Humanized or chimeric anti-IgE monoclonal antibodies are provided. The antibodies bind to human IgE, and find use in various therapeutic methods, including without limitation the reduction or prevention of allergic conditions.

Description

HIGH AFFINITY ANTI-IGE ANTIBODIES

CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/081 ,190 filed September 21 , 2020, U.S. Provisional Patent Application No. 63/129,227 filed December 22, 2020, and U.S. Provisional Patent Application No. 63/155,543 filed March 2, 2021 the entire disclosure of which is hereby.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with Government support under contract W81XWH-14-1 - 0460 awarded by the Department of Defense and under contract AH 15469 awarded by the National Institutes of Health. The Government has certain rights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

[0003] A Sequence Listing is provided herewith in a text file, (S20- 054_STAN1710WO_ST25.txt), created on September 20, 2021 , and having a size of 92000 bytes. The contents of the text file are incorporated herein by reference in its entirety.

BACKGROUND

[0004] Allergic diseases have become the most common immune system disorder, affecting 10-40% of the population in industrialized countries. While the primary function of IgE antibodies is to mediate immune response protection to foreign antigens, the overproduction of IgE antibodies to normally benign environmental stimuli, such as dust mites, pet dander, pollen, and mold, can result in inflammatory allergic reactions associated with asthma, allergic rhinitis, atopic dermatitis, and food allergies. The interaction of IgE antibodies with the high affinity IgE receptor, FCERI, is a critical step in most allergic reactions. The binding of polyvalent antigens to the receptor-bound IgE leads to the release of histamines in allergic effector cells including mast cells and basophils, followed by the synthesis and release of prostaglandins, leukotrienes, and cytokines, stimulating additional inflammatory responses.

[0005] IgE binds two principle receptors, the high affinity FCERI receptor found primarily on mast cells, basophils and dendritic cells, and the lower affinity FCERI I (CD23) receptor found primarily on B cells and dendritic cells. Many antibody isotypes bind their Fc-receptors weakly, and often depend on the formation of avid immune complexes for stable interactions, yet IgE and FCERI bind with high affinity (Ko~100pM) through the ectodomain of the FCERI a-chain (FCERIO), and mast cells and basophils are therefore primed for activation on antigen exposure. This allergen-lgE-FcsRI-basophil/mast cell axis is responsible for a range of pathological outcomes including anaphylaxis and death. Therapeutic blockade of IgE must therefore avoid aggregation of lgE:FcsRI complexes on mast cells and basophils given the catastrophic effects of signaling through this axis. Consequently, early anti-lgE therapeutic antibodies were selected that did not bind to IgE on the surface of basophils and mast cells. Because these antibodies compete with lgE:FcsRI interactions, but do not target preformed complexes with high affinity, their rate of action is limited by the ~20 hour half-life of lgE:FcsRI complexes. This limitation has not stopped the application of anti-lgE in the treatment of chronic allergic diseases or gradual allergen desensitization, yet no anti-lgE antibodies have been described that can rapidly desensitize effector cells within a matter of minutes to hours. [0006] Though no antibodies for rapid desensitization have been described, studies have identified non-antibody anti-lgE ligands that can rapidly disrupt preformed complexes (e.g. the designed ankyrin repeat proteins (DARPin E2_79) and a nanobody (sdAB 026). Furthermore, multiple groups have demonstrated that omalizumab can modestly accelerate lgE:FcsRI dissociation at supraphysiologic concentrations unlike other anti-lgE antibodies. We have therefore employed the term disruptive “efficiency” (K_D/ID₅O) to describe the relative half- maximal disruptive concentration (ID₅o) as compared to the affinity for free IgE (KD) to help classify disruptive agents. Although structural studies of disruptive molecules have led to proposed mechanisms for the accelerated dissociation of lgE:FcsRI complexes, our understanding of this process has been limited by the transient nature of the intermediate complexes that must form between the inhibitors and lgE:FcsRI complexes. No prior studies have developed a systematic experimental pathway to the evolution of more efficient disruptive agents or provided structural insights into how disruptive inhibitors can bind and dissociate stable lgE:FcsRI complexes.

[0007] The IgE-receptor interaction has been well studied and targeted in the search for treatments for allergic diseases, in particular with the anti-lgE antibody Omalizumab, which has proven that IgE is a viable therapeutic target in this cascade, yet lgE:FceRI complexes are very stable and no human antibody therapeutics can efficiently target pre-formed complexes and rapidly desensitize cells at therapeutic doses. Improved agents that can target pre-formed complexes of IgE and the high affinity IgE receptor are of great interest.

SUMMARY

[0008] Compositions and methods are provided relating to high affinity humanized anti-lgE monoclonal antibodies. The antibodies of the invention bind to human IgE. In some embodiments the anti-lgE antibodies bind with sufficient affinity that the interaction between IgE and the high affinity receptor is disrupted without anaphylaxis, at therapeutically relevant doses. These antibodies find use in various therapeutic methods in the treatment of allergic conditions. Embodiments of the invention include isolated antibodies and derivatives and fragments thereof, pharmaceutical formulations comprising one or more of the high affinity humanized anti-lgE monoclonal antibodies; and cell lines that produce these monoclonal antibodies. Also provided are amino acid sequences of the antibodies, and screening methods for the analysis and development of such antibodies.

[0009] In some embodiments a variant of omalizumab is provided, comprising one or more amino acid substitutions that modulate the dwell time of the antibody binding to antigen. These variants can improve the safety of the antibody. In some embodiments the amino acid variation is a substitution on the omalizumab light chain at residue D74, or other surface interacting residues. It is shown herein that conversion of the acidic residue D at VL position 74 to a small hydrophobic (G), a basic (H), or a polar (Y) residue induces an activating phenotype in antibody fragments incubated with allergic patient basophils, demonstrating an acidic D or E residue is critical to prevent stable non-disruptive interactions at novel interfaces identified herein. Surface interacting residues of the antibody light chain are disclosed herein, and include residues 5, 7, 8, 9, 10, 18, 24, 28, 30, 33, 53, 57, 69, 71 , 74, and 78, which residues can provide for amino acid substitutions.

[0010] In some embodiments the variant antibody sequence increases receptor complex disruption without causing spontaneous activation of IgE-bearing mast cells and basophils. In some embodiments the omalizumab sequence variants disrupt lgE:FceRla complexes with ID50s of 1-2 micro molar or below, and that do not spontaneously activate IgE bearing effector cells at these concentrations.

[0011 ] In some embodiments an antibody of the invention comprises the CDR regions of SEQ ID NO:2, 3, 4 and SEQ ID NO:6, 7, 8. In some such embodiments, the variable region sequences for the heavy and light chain are SEQ ID NO:1 where residues 1-121 comprise the VH domain, and SEQ ID NO:5, which antibody may be referred to in the examples as C02. An H2L2 formatted version is provided as SEQ ID NO:9 and 10. In some embodiments the antibody comprises a variant of SEQ ID NO:2 at the HCDR1 position, which variant removes the S28N mutation. Such variants may be selected, without limitation, from the HCDR1 sequences provided in any of SEQ ID NO:52-SEQ ID NO:61 . One of the HCDR1 sequences of SEQ ID NO:52-SEQ ID NO:61 can also be used as a substitute for the HCDR1 sequences in, for example, B3_4 antibody, B3_3 antibody, C06 antibody, etc.

[0012] In some embodiments an antibody of the invention comprises the CDR regions of SEQ ID NO:12, 13, 14 and SEQ ID NO:16, 17, 18. In some such embodiments, the variable region sequences for the heavy and light chain are SEQ ID NO: 11 and SEQ ID NO:15, referred to in the examples as B3_4. An H2L2 formatted version is provided as SEQ ID NO:19 and 20. [0013] In some embodiments an antibody of the invention comprises the CDR regions of SEQ ID NO:22, 23, 24 and SEQ ID NO:26, 27, 28. In some such embodiments, the variable region sequences for the heavy and light chain are SEQ ID NO:21 and SEQ ID NO:25, referred to in the Examples as B3_3. An H2L2 formatted version is provided as SEQ ID NO:29 and 30.

[0014] In some embodiments an antibody of the invention comprises the CDR regions of SEQ ID NO:32, 33, 34 and SEQ ID NO:36, 37, 38. In some such embodiments, the variable region sequences for the heavy and light chain are SEQ ID NO:31 and SEQ ID NO:35, referred to in the examples as C06. An H2L2 formatted version is provided as SEQ ID NO:39 and 40.

[0015] Antibodies of interest include the provided high affinity humanized antibodies, and variants thereof. The monoclonal antibodies of the invention find particular utility as reagents for the diagnosis and immunotherapy of disease associated with IgE in humans, particularly in allergy therapy.

[0016] Various forms of the antibodies are contemplated herein. For example, the anti-lgE antibody may be a full length chimeric or humanized antibody, e.g. having a human immunoglobulin constant (Fc) region of any isotype, e.g. IgG 1 , lgG2a, lgG2b, lgG3, lgG4, IgA, etc. or an antibody fragment, e.g. a F(ab')2 fragment, and F(ab) fragment, etc. The antibody Fc region can be modified to alter binding to Fc receptors, to reduce or enhance binding as desired. In some such embodiments, the Fc sequence is modified to enhace FcyRllb binding, for example by introducing the amino acid substititions G236D and S267E. In other embodiments the Fc sequence is modified at residues G236D/S267E/K322A/N434A (using the Eu numbering scheme), relative to the original Omalizumab-Fc sequence. The modified Fc is provided as SEQ ID NO:43. In some embodiments the antibody is an lgG1 antibody. Fragments comprising CDR regions are also of interest. Furthermore, the antibody may be labeled with a detectable label. The antibody may be immobilized on a solid phase and/or conjugated with a heterologous compound. The antibody may also be provided as a bi-specific or multispecific antibody reactive with a second antigen.

[0017] In one embodiment an antibody comprising a V_HV sequence disclosed herein is provided as an H2L2-lgG construct, with a flexible Gly-Gly linker at each Fab elbow. H2L2 constructs comprise a flexible glycine linker in the elbow of the Fab, retaining the native hinge sequence, e.g. VH-linker-CH1 -hinge-CH2-CH3; VL-linker-CL. The length of the linker can be designed to optimize interactions, e.g. a linker of from about 1 - 20 amino acids in length, from about 1 to about 15 amino acids in length, from about 1 to about 10 amino acids in length, from about 4 to about 8 amino acids in length, and may be, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length. Non-limiting examples are provided as SEQ ID NO:9 and 10; 19 and 20; 29 and 30; 39 and 40; 47 and 51 ; 48 and 50; 48 and 51 ; 49 and 51 . Specific embodiments include linkers of 1 or 2 glycines in one or both of the V_H and V sequences.

[0018] Embodiments of the invention include isolated antibodies and derivatives and fragments thereof that comprise at least one, usually at least 3 CDR sequences as provided herein, usually in combination with framework sequences from a human variable region or as an isolated CDR peptide. In some embodiments an antibody comprises at least one light chain comprising the 3 light chain CDR sequences provided herein situated in a variable region framework, which may be, without limitation, a human or mouse variable region framework, and at least one heavy chain comprising the 3 heavy chain CDR sequence provided herein situated in a variable region framework, which may be, without limitation, a human or mouse variable region framework. V_H sequences can be fused to an Fc region sequence to provide a full-length heavy chain.

[0019] The invention further provides: isolated nucleic acid encoding the antibodies and variants thereof; a vector comprising that nucleic acid, optionally operably linked to control sequences recognized by a host cell transformed with the vector; a host cell comprising that vector; a process for producing the antibody comprising culturing the host cell so that the nucleic acid is expressed and, optionally, recovering the antibody from the host cell culture (e.g. from the host cell culture medium). The invention also provides a composition comprising one or more of the human anti-lgE antibodies and a pharmaceutically acceptable carrier or diluent. This composition for therapeutic use is sterile and may be lyophilized, e.g. being provided as a pre-pack in a unit dose with diluent and delivery device, e.g. inhaler, syringe, etc.

[0020] Also provided are methods for the treatment of allergic conditions, the methods comprising administering an effective dose or doses of an anti-lgE antibody of the invention.

[0021] In other embodiments, compositions and methods are provided for screening and designing antibodies and other binding agents to the lgE:FcsRI complex. In one embodiment a disulfide stabilized lgE:FcsRI complex is provided, including for example the protein of SEQ ID NO:42. Also provided are structure data of omalizumab bound to a partially dissociated lgE:FcsRI complex. This structure represents an intermediate along the disruption pathway, and provides features to guide the design of safe disruptive anti-lgE antibodies and other high affinity protein complexes. The dwell time of disruptive agents is a critical parameter that differentiates engineered omalizumab variants that safely strip IgE from human effector cells vs those that are anaphylactogenic.

[0022] In some embodiments a polystyrene bead-based disruption assay is provided, in which candidate agents are bound to biotinylated-lgE-Fc_2-4 (blgE-Fc_2-4) and FcsRIa-conjugated polystyrene beads to rapidly screen the disruptive potency of anti-lgE candidate agents. In a yeast display selection assay, a candidate agent can be displayed on a yeast cell surface and bound to free biotinylated-lgE-Fc_2-4 (blgE-Fc_2-4), blgE-Fc_2.4 bound to FCERICL Alternatively a “two-color efficiency” stain composed of a mix of an AF-488 labeled lgE-Fc_3.4 mutant (e.g. G335C) that is unable to bind FcsRIa and blgE-Fc_2.4 bound to FCERICL The complexes may be for a short period of time, e.g. less than about 2 hours, less than about 1 hour, less than about 30 minutes. Using combinations of staining reagents in a single pool allows independent measurement of free-lgE-binding and lgE:FcsRla complex-binding/disrupting binding. This “two-color efficiency” stain improves discrimination over a range of complex concentrations as compared to staining with complex alone.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 . The disruptive potency of omalizumab can be modulated by altering affinity and conformational flexibility: (A) Disruption assay measuring removal of blgE-Fc_2.4 from FCERICF beads. (B) IDsowith 95% Cl from fits in (A) vs. KD, Kd, or K_a of each variant for IgE. (Bottom right) disruptive efficiency (ID₅O/K_D). (C) Schematic of scFv omalizumab variants.

[0024] Figure 2. Differentiation of disruptive, competitive, and non-competitive anti-lg E agents on yeast. (A) Yeast surface expression of cMyc-tagged E3_53, E2_79, and omalizumab-scFv. (B) Schematic with anti-lgE:lgE-Fc3-₄ structures with FCERIO binding site highlighted in red. E3_53 (blue), E2_79 (cyan), Omalizumab (VL: orange VH: blue). (C) Titrations of blgE-Fc_2.4 (left) or blgE-Fc_2-4 precomplexed with FcsRIa-Ova (right) on yeast displayed anti-lgE. (D) Schematic of two-color yeast-based efficiency screen. Pool 1 : blgE-Fc_2.4 preincubated with a molar excess of FcsRIa-Ova. Pool 2: G335C-lgE-Fc_3.4 labeled with AF-488. (E) Gating scheme for singlet-cMyc+ yeast. (F) (Left) singlet-cMyc+ anti-lgE yeast stained with “pool 1 ” and anti-cMyc at indicated concentrations. (Right) singlet-cMyc+ yeast stained with “pool 1 ” and “pool 2” at indicated concentrations. (G) Normalized histogram of singlet-cMyc+ yeast controls stained with FcsRIa-Ova (1 pM) or with blgE-Fc2-4 (100nM) in complexes with FcsRIa-Ova (1 pM) detected with anti-Ova FITC.

[0025] Figure 3. Directed evolution of scFv variants with enhanced disruptive potency and efficiency. (A) Overview of selections with number of transformants per library in brackets. Full details of selections outlined in Figure 10. (B) Controls and freshly induced R1 -4 of EP1 library stained with stained with two-color efficiency stain. (C) Selected hits and controls stained with two-color efficiency stain. (D) Histogram of yeast hits and controls stained with 100nM blgE- FC_2.4 (dotted line) or precomplexed blgE-Fc_2-4:FcERIa-Ova (100nM:1 pM-solid line). (Bottom right inset) two color plots to asses correlation between blgE-Fc_2.4 and FcsRIa-Ova signals. (E) HAE yeast stained with two-color efficiency stain compared to omalizumab variants. For clarity a subset of G335C-lgE-Fc_3-4 positive cells were selected and the relative staining intensity of biotin-lgE-Fc_2-4 was displayed by histogram (right). (F) ID₅o of with 95% Cl from fits vs. K_D, K_d, or K_a of each variant for IgE, with prior anti- Ig E agents included for reference (grey). (G) disruptive efficiency (ID₅O/K_D) for hits in (f). (H) Amino acid mutations from clones 813 and C02, HAE, and omalizumab. (I) Distribution of mutations in (H) mapped onto topology of VH and VL domains.

[0026] Figure 4. Structure of high affinity disruptive omalizumab variants. (A) Ribbon diagram of all omalizumab:lgE complexes aligned relative to CE3 showing variation in Fab CH/CL, IgE CE4, VH and VL domains. (B) Schematic of measured “swing,” and “opening,” distances on scFv:lgE-Fc3-4 structures. (C) The swing and opening distances of the IgE-Fc in antibody cocomplexes and in complex with FcsRIa (1f6a) was plotted and color mapped by the disruptive potency of each antibody when possible. (D) Heat map of omalizumab contacts at interfaces across all structures. Shading reflects percentage of NOS related lgE:omalizumab interfaces at which a contact was identified. Magenta asterisks denote region in which lack of density precluded modeling/interface-identification. (E) Detailed views of VH mutations aligned by variant to the native omalizumab scFv. (F) Same as in (E) for VL mutations, with lgE-R419 highlighted for reference.

[0027] Figure 5. Production of a disulfide stabilized lgE-Fc_2.4:FcERIa “locked” complex. (A) lgE-Fc_2.4:FcERIa complex (2y7q), with the interface of FcsRIa and IgE at “site-2” revealed to display position of lgE-Fc_2.4 (G335C) and FcsRIa (W156C) mutations. (B) Octet binding studies with locked-complex on tips exposed to anti-lg E agents in a twofold serial dilution from 400nM-25nM. (C) Binding profiles of yeast-displayed omalizumab scFv and E3_53 to free blgE-Fc2-4 (left axis, black) or locked-complex (right axis, red). (D) Same as in (C) for yeast displayed C02 and HAE. (E) Same as in (C) for clone A4, 7, 16. (F) Yeast displayed omalizumab clones 7, A4, and 16 stained with blgE-Fc2-4 (100nM):FcERIa-Ova (1 pM) complexes relative to yeast expressing C02, HAE, and E3_53. (G) ID₅o with 95% Cl vs. K_D, Kd, or K_a for IgE of clones A4, 7, and 16, with prior anti-lgE agents included for reference (grey). (H) Disruptive efficiency (ID₅O/K_D) of clones in (G). (I) BLI binding studies of C02 and clone 7 with locked-complex on tips with anti-lgE agents in a twofold serial dilution from 1600nM-100nM

[0028] Figure 6. Cryo-EM structure of the disruption-intermediate. (A) Front view of model fit to Cryo-EM density map contoured at 5o, with cartoon schematic (right) (B) Side view of model fit to Cryo-EM density map contoured at 5o, with cartoon schematic (left). (C) Displacement of FcsRIa in disruption-intermediate (magenta) as compared to the native lgE:FcERIa structure (black) relative to the site-2 FcsRIa binding site. Quantification of angle (©) and distance (A) of displacement indicated. (D) Detailed view of the Cryo-EM density map at the site-1 FcsRIa binding site. (E) Displacement of FCERIQ in disruption-intermediate (magenta) as compared to the native lgE:FcsRla structure (black) relative to the site-1 FcsRIa binding site. Quantification of angle (©) and distance (A) of displacement indicated. (F) Relative positions of FcsRIa glycans compared to native lgE:FcsRla structure with density contoured at 5o. (G) Conformations of CE2 domains from disruption-intermediate (this publication) and omalizumab:lgE-Fc2-4 (5g64) relative to the native site-1 FcsRIa binding pose (2y7q). Yellow accent denotes region of steric clash between CE2 and FcsRIa, with inset depicting back view of relative CE2/3 positions in 5g64.

[0029] Figure 7. Therapeutic window of high affinity disruptive agents (A) I D₅o with 95% Cl for omalizumab IgG variants. (B) Anaphylactogenicity of IgG variants in BMMC as measured by percent CD107a⁺ cells with individual BMMC cultures shown. (C) Anaphylactogenicity of IgG variants in basophils from human allergic donors (n=4) as measured by % CD63⁺ cells, with individual donors shown. (D) SPR studies of IgG binding to intact lgE:FcsRla complexes, with schematic of assay (top). Titrations performed from 2.5-0.78 pM in two-fold serial dilutions. (E) BLI studies of IgG binding to intact lgE:FcsRla complexes, with schematic of assay (top). (F) BMMC inhibition assays following 6-hour treatment with IgG variants at indicated concentrations. Cultures were split and assayed for surface-lgE or activated with NP(7)-BSA. (G) Activated cells from (J) were assayed for activation (%CD107a⁺), and data was normalized to 100% activation for untreated controls. (H) Basophil inhibition assays from grass-allergic donors following 6-hour treatment with IgG variants at indicated concentrations. Cultures were split and assayed for surface IgE or activated with 6-grass allergen mix. (I) Activated cells from J were assayed for activation (%CD63⁺). (J) Average inhibition observed by IgG variants at 500nM after 6-hour incubation. One-way repeated measures ANOVA with Bonferroni post- hoc tests (N=3).

[0030] Figure 8. Expression and kinetic analysis of soluble recombinant omalizumab variants. (A) Omalizumab constructs tested, source, and construct design: SP1 = human VEGF signal peptide, VH= heavy chain variable domain, CH= heavy chain constant domain, TEV= tobacco etch virus cleavage site, 8xHis= 8x poly-histidine affinity tag, T2A= Thosea asigna 2A cleavage peptide, SP2= human Ig Kappa signal peptide, VL= light chain variable domain, CL= light chain constant domain. (B) Non-reducing SDS-PAGE gel of size exclusion chromatography (SEC) purified species, note partial dissociation of Fab species lacking hinge disulfide into VH/VL despite monomer peak by SEC. (C) Size exclusion chromatography (Superdex S200 10/300 GL) from all variants after concentration to 100pM for disruption assays. (D) Biotinylated full length recombinant IgE was immobilized on streptavidin (SA) tips and single cycle kinetic analysis (SCK) binding experiments were conducted on the Octet Red 96, exported, and fit in BiaEvaluation 3.0. Each SCK experiment contained five 100s association cycles, four 100s dissociation cycles, and a final 600s dissociation cycle and each experiment was conducted with two-fold serial dilutions from 50nM to 1 ,56nM, binding data was reference subtracted to parallel blank tips exposed to same serial analyte dilutions. (E) Tabulated kinetic data for curves fit above. (F) A schematic of CE2 domain relative to each omalizumab epitope across the lgE-Fc_2.4 dimer (2wqr).

[0031] Figure 9. Kinetic analysis of omalizumab IgE-fragment interactions. (A) Biotinylated recombinant lgE-Fc_3.4 or lgE-Fc_2-4 was immobilized on SA-tips as indicated and binding experiments were conducted with three fold serial dilutions from 100nM to 1.23nM on the Octet Red 96. Each experiment contained a 200s association cycle and a 600s dissociation cycle, and binding data was reference subtracted to parallel blank tips exposed to same serial analyte dilutions. (B) Tabulated kinetic data for curves fit above.

[0032] Figure 10. Directed evolution of omalizumab variants. (A) Schematic of selection rounds conducted on error prone library one (EP1). Clone selected for subsequent selections highlighted in green. (B) R4 library clones and yeast controls were stained with G335C-lgE- FC_3-4 (1.25nM) and blgE-Fc_2-4:FcERIa-Ova (100nM:500nM) for 30 minutes. Gates were established relative to control yeast, and four clones with binding profiles similar to E2_79, defined as percent yeast falling within the control gate, were selected for sequencing (arrows). (C) VH and VL mutations of selected clones from (B) as compared to omalizumab scFv. (D) Schematic of selection rounds conducted on StEP library one (StEP1 ). Clone selected for subsequent selections highlighted in green. (E) Pre and post-StEP1 mutation frequencies. (F) Schematic of selection rounds conducted on error prone library two (EP2). Clones selected for subsequent libraries highlighted in green. (G) R3 of EP2 stained with G335C-lgE-Fc_3.4 (1.25nM) and blgE-Fc2-4:FcERIa-Ova (100nM:500nM). (H) Schematic of selection rounds conducted on StEP library two (StEP2). Clones selected for evaluation highlighted in green. (I) R2 StEP2 library stained as in (G). (J) relative mutation frequencies from twenty EP2-R3 clones used in the StEP2 library, and fifteen post selection StEP2-R2 clones. (K) MFI of clones in (F) plotted versus controls. (L) VH and VL mutations of clones selected from (K) (M) Triplicate disruption assay with omalizumab scFv, E2_79, and evolved omalizumab hits.

[0033] Figure 11 . Expression and validation of omalizumab library hits, HAE, and C02/HAE hybrid. (A) Non-reducing SDS-PAGE gel of scFv variants. (B) Size exclusion chromatography (Superdex S200 10/300 GL or HiLoad 16/600 when noted) for all variants after concentration to 100pM for disruption assays. (C) Biotinylated full length recombinant IgE was immobilized on SA-tips and single cycle kinetic analysis (SCK) binding experiments were conducted on the Octet Red 96, exported, and fit in BiaEvaluation 3.0. Each SCK experiment contained five 100s association cycles, four 100s dissociation cycles, and a final 600s dissociation cycle. Experiments were conducted with two-fold serial dilutions from 50nM to 1.56nM, and binding data was reference subtracted to parallel blank tips exposed to same serial analyte dilutions. (D) Tabulated kinetic data for curves fit above. (E) Disruption curves showing loss of blgE- Fc2-4 bound to FcsRIa-Ova coated polystyrene beads after 30-minute treatment with omalizumab variants. Curve fits (dotted line) from two replicate assays, with error bars (SD) shown when larger than symbol.

[0034] Figure 12. Structural analysis of C02/HAE hybrid and VH:VL flexibility in omalizumab variants. (A) Ribbon diagram of all omalizumabJgE pairs aligned at CE3 highlighting relative positions of VH, VL, or scFVs across structures, with the relative displacement of domains measured as compared to omalizumab:Fab complex (5hys) chains H (VH), L (VL), or H+L (ScFV) using angle_between_domai ns from Pymol Script Collection. The resulting angles and displacements were plotted and color-mapped by the mean I D₅o of each variant in bead-based disruption assays. (B) Location of VH mutations of C02/HAE hybrid aligned to the native omalizumab scFv complex, with native residues depicted in grey, and mutants colored by chain. (C) Cartoon representation of the ABangle vector coordinate system and the names and descriptions of angles between vectors measured. (D) ABangle output for omalizumab variants plotted vs mean ID₅o in bead-based disruption assays.

[0035] Figure 13. Design and purification of lgE-Fc2-4(G335C):FcERIa(W156C) locked complex and selection of high affinity anti-locked complex variants. (A) Cartoon schematic of locked-complex with 8xHis tagged-FcERIa(W156C) and un-tagged lgE-Fc_2-4 (G335C). Calculated peptide mass, number of known N-linked glycans, and total masses of human proteins. (B) SEC traces of products post Ni-NTA purification (black) and post MonoQ anion exchange (green). (C) Non-reducing and reducing SDS-PAGE (left), anti-lgE western blot (middle), and anti-His western blot (right) of locked complex. (D) Denaturing PNGaseF deglycosylation of reduced locked complex or lgE-Fc2-4 (E) Schematic of selection rounds conducted on C02/HAE StEP library. (F) Mutations in clones selected from the C02/HAE StEP library relative to C02 sequence. Mutations from shuffled HAE light chain are shown in italics, sporadic mutations are shown in bold. (G) Biotinylated IgE was immobilized on SA-tips and single cycle kinetic analysis (SOK) binding experiments were conducted on the Octet Red 96, exported, and fit in BiaEvaluation 3.0. Each SOK experiment contained five 100s association cycles, four 100s dissociation cycles, and a final 600s dissociation cycle. Experiments were conducted with two-fold serial dilutions from 50nM to 1.56nM, binding data was reference to blank tips exposed to analyte dilutions. (H) Tabulated kinetic data for curves in (G). (I) Disruption curves showing loss of blgE-Fc2-4 bound to FcsRIa-Ova coated polystyrene beads after 30-minute treatment with indicated variants. Curve fits (dotted line) from two replicate assays, with error bars (SD) shown when larger than symbol. (J) Locked-complex was capture-coupled on Ni-NTA tips. (SCK) binding experiments were then conducted on the Octet Red 96, exported, and fit to a heterogenous binding model in Clamp XP. Each SCK experiment contained five 100s association cycles, four 100s dissociation cycles, and a final 1000s dissociation cycle. Experiments were conducted with two-fold serial dilutions of each scFv from 1 .6-0.1 jiM. (K) Tabulated kinetic data for curves in (J).

[0036] Figure 14: Disruptive-intermediate structural determination and analysis. (A) Clone 7 incubated locked-complex in 0:1 , 1 :0, 1 :1 , 2:1 , and 3:1 molar ratios and subject to SEC on Superdex S200 10/300 GL column. SDS-PAGE electrophoresis of -10-12 mL peak. (B) Representative micrograph, 2D averages with final particles, and overview of data processing. (C) Local resolution estimates for resulting maps. (D) Angular distribution of particle projections from cryoSPARC. (E) Gold-standard FSC (cryoSPARC) with blue line at 0.143 FSC cut-off. (F) Using the amino acid residues from disruptive intermediate model a low- resolution map was generated and subtracted from the experimental density maps to highlight regions not attributable to protein density. The resultant map was contoured at 5o and displayed with a surface representation of the model to highlight areas of unmodeled protein density (in red) adjacent to all known proximal N-linked glycosylation sites on FcsRIa (magenta), IgE (grey/green), VH (blue) and VL (yellow). (G) ABangle output for C02:lgE-Fc_3.4 vs. the disruptive intermediate structure (separated by site-1 or 2 proximal scFvs). (H) All copies of the scFvJgE interface in this publication were aligned to the site-1 proximal CE3 domain of the ternary complex to assess the relative degree of VH and VL displacement from free-lgE structures. (I) The relative positions of the VL domain from both scFvs in the ternary structure is displaced as compared the C02:lgE-Fc_3.4 complex. (J) Cryo-EM density map contoured at 5o and displacement of the clone 7 binding pose (blue: VH, yellow:VL) from the orientation observed in C02:lgE-Fc_3.4 complex (grey).

[0037] Figure 15. Bulk library sequencing data. Using structural data derived from the structure of the disruptive intermediate a C02 library with mutations in VH and VL domains was generated to isolate potent additional disruptive variants. The library was subjected to rounds of selection and the final library was sequenced to identify regions associated with improved binding affinity and disruptive capacity. Mutant position in VH or VL domain and original residue relative to C02 coding sequence (parentheses) indicated along with their relative abundance.

[0038] Figure 16. Bilayer interferometry data. Binding studies of scFv of variants B3_3, B3_3, B3_4, C06 to recombinant human IgE.

[0039] Figure 17. Bead-based lgE:receptor disruption data, scFV. ID₅o of variants in scFV format in bead based disruption assay vs parental C02 scFV (dotted line).

[0040] Figure 18. Bead based lgE:receptor disruption data. I D₅o of variants in H2L2-lgG format in bead based disruption assay vs parental C02-H2L2-lgG. [0041 ] Figure 19. Anaphylactogeneicity profile. Anaphylactogenicity of scFv variants in human FcsRIa transgenic BMMCs as measured by percent CD107a+ cells (mean and SD shown) vs negative control (Humira) and positive control (Le27).

[0042] Figure 20. In vitro activity. Basophil inhibition assays from grass-allergic donors following 6-hour treatment with IgG variants at indicated concentrations (0-2500nM). Post treatment cultures were activated with 6-grass allergen mix and cells were assayed for activation (%CD63+).

[0043] Figure 21 : Nucleotides encoding VH positions 28-29 were randomized using NNK mutagenesis and position 31 was mutated via VVC mutagenesis. The predicted starting amino acids by position are highlighted in blue (left panel). After three rounds of efficiency maturation selection, and screening for co-binding to intact lgE:FcsRla complexes, 13 hits were sequenced to identify favorable sequence profiles. Amino acids retained in the post selection pool are colored light blue, or dark blue if found in multiple instances (with the number of instances noted).

[0044] Figures 22A-C: A. SEC elution profile of C02_N28D_mutant-Fc-G2 (comprising the modified Fc sequence of SEQ ID NO:53) from a HiLoad 16/600 Superdex 200 column post protein A purification. B. Displacement of biotinylated IgE from polystyrene beads coated with the high affinity IgE receptor over thirty minutes at the indicated concentrations, with disruption measured by a loss in signal from AF647 labeled streptavidin. C. The high affinity IgE receptor was covalently immobilized to Octet tips and then loaded with recombinant human IgE. The preformed complexes were then exposed to C02 antibody variants at a concentration of 5 micromolar. IgE displacement was measured in loss of signal (nm) relative to baseline (black dotted line) over the course of 1200s.

[0045] Figure 23: C02 and B3_4 variants without HCDR1 glycosylation are safe, potent, and disruptive anti- Ig E antibodies, a. Schematic of experimental design. Mouse bone marrow mast cells (BMMC) transgenic for the human high affinity IgE receptor (huFcsRIa) were sensitized with NIP reactive JW8-lgE. Cells were then treated with anti-lgE compounds over the indicated concentration range for 0.5-20.5 h. At 0.5 h a fraction of cells were taken and assessed for spontaneous activation (%CD107+), at 20 h a fraction of cells were assed for surface IgE removal. The remainder of the cells were then stimulated with NIP₂₄-BSA and assessed for anti-lgE mediated inhibition of antigen induced activation after 0.5 h. b. Spontaneous activation of BMMCs after anti-lgE treatment, with positive activation control (Le27+) shown in right panel, c. IgE removal from BMMCs after anti-lgE treatment, d. Inhibition curves of antigen challenged BMMCs treated with anti-lgE compounds.

[0046] Figure 24: B3_4 and B3_4 elbow variants are monomeric and stable during multiple freeze thaw cycles. B3_4_N28D and elbow variants were purified by protein A affinity chromatography, subjected to 4 freeze thaw cycles, and 2uL of sample was injected into an ACQUITY UPLC Protein BEH SEC200 ,1 ,7pm, 4.6x150 mm column with a flow of 0.3 mL/min for 10 minutes using a mobile phase of 50 mM Sodium Phosphate, 500 mM NaCI, pH 6.2.

[0047] Figure 25: Thermal stability of B3_4 and B3_4 elbow variants. Samples were assayed using the UNcle system (Unchained Labs) for analysis. A temperature ramp of 1 °C/min was performed while being monitored from 25 °C to 95 °C for DSF and SLS at 266 nm and 473 nm respectively. Tm and Tagg were analyzed by using the UNcle Analysis Software. The Tm as determined is indicated by a solid dropline in DSF graph. Assays were performed in 20 mM Histidine 150 mM NaCI pH 6.0

[0048] Figure 26: B3_4 and B3_4 elbow variants are safe potent disruptive anti-lgE antibodies: a. Schematic of experimental design, mouse bone marrow mast cells (BMMC) transgenic for the human high affinity IgE receptor (huFcsRIa) were sensitized with NIP reactive JW8-lgE. Cells were then treated with anti-lgE compounds over the indicated concentration range for 20h. At 20h a fraction of cells were taken and assessed for spontaneous activation (%CD107+) and surface IgE removal, b. JW8 sensitized BMMCs were challenged with NIP₂₄-BSA and assessed for activation, c. Spontaneous activation of BMMCs after anti-lgE treatment, d. IgE removal from BMMCs treated after treatment, e. Curve fits of data in (b), with bottom constraint set to MFI of no IgE control.

[0049] Figure 27. B3_4 and B3_4 elbow variants are safe, potent, and disruptive anti-lgE antibodies: a. Schematic of experimental design, mouse bone marrow mast cells (BMMC) transgenic for the human high affinity IgE receptor (huFcsRIa) were sensitized with NIP reactive JW8-lgE. Cells were then treated with anti-lgE compounds over the indicated concentration range for 20h. At 20h cells were stimulated with NIP₂₄-BSA to assess anti-lgE mediated inhibition of antigen activation, b. Inhibition curves of antigen challenged BMMCs after anti-lgE treatment, c. Curve fits of data in (b) to calculate half maximal inhibition.

DETAILED DESCRIPTION OF THE INVENTION

[0050] The present invention relates to high affinity humanized monoclonal antibodies that are specific for IgE. Also disclosed is a nucleic acid, and amino acid sequence of such antibodies. The antibodies find use in therapeutic and diagnostic methods associated with IgE.

[0051] Omalizumab (Xolair®) is a recombinant DNA-derived humanized IgGI K monoclonal antibody that selectively binds to human immunoglobulin (IgE). The antibody has a molecular weight of approximately 149 kD. Omalizumab is presently indicated for the treatment of moderate to severe persistent asthma in patients with a positive skin test or in vitro reactivity to a perennial aeroallergen and symptoms that are inadequately controlled by inhaled corticosteroids; or for the treatment of chronic idiopathic urticaria (CIU; chronic hives without a known cause) in patients 12 years of age and older not controlled by H1 antihistamine treatment.

[0052] The sequence of the Omalizumab heavy chain (reference) is:

(SEQ ID NO:65)

EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITYDGSTNY ADSVKGRFTISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTV SSGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK

[0053] The sequence of Omalizumab light chain (reference) is:

(SEQ ID NO:66)

DIQLTQSPSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASYLESG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNR

[0054] An IgE polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide as used herein refers to a molecule derived from any source. The molecule need not be physically derived from an organism, but may be synthetically or recombinantly produced. A number of IgE nucleic acid and protein sequences are known. Representative IgE sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. P01854, P01855, and P06336; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference.

[0055] The term “FcsRIa extracellular region” or the like refers to an extracellular domain of a FcsRIa protein that is the portion of the FcsRIa chain that is exposed to the environment outside the cell and that binds to an IgE-Fc. For the nucleotide and amino acid sequence of a human IgE-Fc, see Flanagan, J.G. and Rabbitts, T. H. 1982 EMBO J. 1 :655-660. The term “FcsRIa extracellular region” or the like refers also to a polypeptide (preferably of mammalian origin, e.g., human) or, as context requires, a polynucleotide encoding such a polypeptide, that is capable of interacting with an IgE-Fc (preferably of mammalian origin, e.g., human), including, for example, an amino acid sequence of a naturally occurring mammalian FcsRIa extracellular region or a fragment thereof, e.g., an amino acid sequence that starts at amino acid 1 and ends at amino acid 176 of a human FCERIQ, using the numbering -25 to 232, and representative sequence, according to Kochan, J. et al. 1988 Nucleic Acids Res. 16:3584- 3584, or a fragment thereof.

[0056] The term “IgE-Fc CE3-CE4” or the like refers to a third and fourth C-terminal constant domain, CE3 and CE4, of an IgE heavy chain that mediates binding to a FCERICL For the nucleotide and amino acid sequence of a human FCERIO, see Kochan, J. et al. 1988 Nucleic Acids Res. 16:3584-3584. The term “IgE-Fc CE3-CE4” or the like refers also to a polypeptide (preferably of mammalian origin, e.g., human) or, as context requires, a polynucleotide encoding such a polypeptide, that is capable of interacting with a FCERIO (preferably of mammalian origin, e.g., human), for example comprising an amino acid sequence of a naturally occurring mammalian IgE-Fc CE3-CE4 or a fragment thereof.

[0057] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. "Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.

[0058] As used in this invention, the term "epitope" means any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0059] "Native antibodies and immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V_H) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains (Clothia et al., J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A. 82:4592 (1985)). [0060] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a [3-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the [3-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991 )). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0061] The CDR sequences of exemplary anti-lgE heavy and light chains combinations are set forth in the sequence listing and figures. In some embodiments the CDR sequences are maintained in a combination, i.e. a humanized antibody will comprise both heavy chain CDR sequences and light chain CDR sequences.

[0062] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')₂ fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0063] “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv species (scFv), one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. For a review of scFv see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0064] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1 ) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0065] There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgGi, lgG₂, lgG₃, lgG₄, IgAi, lgA₂. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called a, 8, E, y, and g, respectively. The subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known.

[0066] A “variant Fc region” comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native-sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein may possess at least about 80% homology with a native-sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

[0067] Fc variants include without limitation monomeric Fc variants, variants in which a region capable of forming a disulfide bond is deleted, or in which certain amino acid residues are eliminated at the N-terminal end of a native Fc form or a methionine residue is added thereto. Thus, in one embodiment of the invention, one or more Fc portions of the scFc molecule can comprise one or more mutations in the hinge region to eliminate disulfide bonding. In yet another embodiment, the hinge region of an Fc can be removed entirely. In still another embodiment, the scFc molecule can comprise an Fc variant.

[0068] Further, an Fc variant can be constructed to remove or substantially reduce effector functions by substituting, deleting or adding amino acid residues to effect complement binding or Fc receptor binding. For example, and not limitation, a deletion may occur in a complementbinding site, such as a C1q-binding site. Techniques of preparing such sequence derivatives of the immunoglobulin Fc fragment are disclosed in International Patent Publication Nos. WO 97/34631 and WO 96/32478. In addition, the Fc domain may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.

[0069] Variant Fc sequences may include three amino acid substitutions in the CH2 region to reduce FcyRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement C1q binding site at EU index positions 330 and 331 reduce complement fixation (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991 )). Substitution into human lgG1 of lgG2 residues at positions 233-236 and lgG4 residues at positions 327, 330 and 331 greatly reduces ADCC and CDC (see, for example, Armour KL. et al., 1999 Eur J Immunol. 29(8):2613-24; and Shields RL. etal., 2001 . J Biol Chem. 276(9):6591 -604). Other Fc variants are possible, including without limitation one in which a region capable of forming a disulfide bond is deleted, or in which certain amino acid residues are eliminated at the N-terminal end of a native Fc form or a methionine residue is added thereto. Thus, one or more Fc portions of the molecule can comprise one or more mutations in the hinge region to eliminate disulfide bonding. In yet another embodiment, the hinge region of an Fc can be removed entirely. In still another embodiment, the molecule can comprise an Fc variant.

[0070] In another variant, the Fc sequence is modified at residues G236D/S267E/K322A/N434A (using the Eu numbering scheme), relative to the original Omalizumab-Fc sequence. The modified Fc is provided as SEQ ID NO:43. These modifications selectively block C1 q binding and enhance half-life without interfering with the enhanced disruption mediated by the G236D/S267E Fc-mutations. The modified sequence finds use with any of the variable regions identified herein.

[0071] Further, an Fc variant can be constructed by substituting, deleting or adding amino acid residues to effect complement binding or Fc receptor binding. Techniques of preparing such sequence derivatives of the immunoglobulin Fc fragment are disclosed in International Patent Publication Nos. WO 97/34631 and WO 96/32478. In addition, the Fc domain may be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.

[0072] The Fc may be in the form of having native sugar chains, increased sugar chains compared to a native form or decreased sugar chains compared to the native form, or may be in an aglycosylated or deglycosylated form. The increase, decrease, removal or other modification of the sugar chains may be achieved by methods common in the art, such as a chemical method, an enzymatic method or by expressing it in a genetically engineered production cell line. Such cell lines can include microorganisms, e.g. Pichia Pastoris, and mammalians cell line, e.g. OHO cells, that naturally express glycosylating enzymes. Further, microorganisms or cells can be engineered to express glycosylating enzymes, or can be rendered unable to express glycosylation enzymes (See e.g., Hamilton, et al., Science, 313:1441 (2006); Kanda, et al, J. Biotechnology, 130:300 (2007); Kitagawa, et al., J. Biol. Chem., 269 (27): 17872 (1994); Ujita-Lee et al., J. Biol. Chem., 264 (23): 13848 (1989); Imai- Nishiya, et al, BMC Biotechnology 7:84 (2007); and WO 07/055916). As one example of a cell engineered to have altered sialylation activity, the alpha-2, 6-sialyltransferase 1 gene has been engineered into Chinese Hamster Ovary cells and into sf9 cells. Constructs expressed by these engineered cells are thus sialylated by the exogenous gene product. A further method for obtaining Fc molecules having a modified amount of sugar residues compared to a plurality of native molecules includes separating said plurality of molecules into glycosylated and nonglycosylated fractions, for example, using lectin affinity chromatography (See e.g., WO 07/117505). The presence of particular glycosylation moieties has been shown to alter the function of Immunoglobulins. For example, the removal of sugar chains from an Fc molecule results in a sharp decrease in binding affinity to the C1q part of the first complement component C1 and a decrease or loss in antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (GDC), thereby not inducing unnecessary immune responses in vivo. Additional important modifications include sialylation and fucosylation: the presence of sialic acid in IgG has been correlated with anti-inflammatory activity (See e.g., Kaneko, et al, Science 313:760 (2006)), whereas removal of fucose from the IgG leads to enhanced ADCC activity (See e.g., Shoj-Hosaka, et al, J. Biochem., 140:777 (2006)).

[0073] Constructs can have an Fc sequence with enhanced effector functions, e.g. by increasing their binding capacities to FcyRIIIA and increasing ADCC activity. For example, fucose attached to the AZ-linked glycan at Asn-297 of Fc sterically hinders the interaction of Fc with FcyRIIIA, and removal of fucose by glyco-engineering can increase the binding to FcyRIIIA, which translates into >50-fold higher ADCC activity compared with wild type lgG1 controls. Protein engineering, through amino acid mutations in the Fc portion of lgG1 , has generated multiple variants that increase the affinity of Fc binding to FcyRIIIA. Notably, the triple alanine mutant S298A/E333A/K334A displays 2-fold increase binding to FcyRIIIA and ADCC function. S239D/I332E (2X) and S239D/I332E/A330L (3X) variants have a significant increase in binding affinity to FcyRIIIA and augmentation of ADCC capacity in vitro and in vivo. Other Fc variants identified by yeast display also showed the improved binding to FcyRIIIA and enhanced tumor cell killing in mouse xenograft models. See, for example Liu et al. (2014) JBC 289(6):3571 -90, herein specifically incorporated by reference. [0074] “Antibody fragment”, and all grammatical variants thereof, as used herein are defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody, wherein the portion is free of the constant heavy chain domains (i.e. CH2, CH3, and CH4, depending on antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single chain polypeptide"), including without limitation (1 ) single-chain Fv (scFv) molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific or multivalent structures formed from antibody fragments. In an antibody fragment comprising one or more heavy chains, the heavy chain(s) can contain any constant domain sequence (e.g. CH1 in the IgG isotype) found in a non-Fc region of an intact antibody, and/or can contain any hinge region sequence found in an intact antibody, and/or can contain a leucine zipper sequence fused to or situated in the hinge region sequence or the constant domain sequence of the heavy chain(s).

[0075] Unless specifically indicated to the contrary, the term "conjugate" as described and claimed herein is defined as a heterogeneous molecule formed by the covalent attachment of one or more antibody fragment(s) to one or more polymer molecule(s), wherein the heterogeneous molecule is water soluble, i.e. soluble in physiological fluids such as blood, and wherein the heterogeneous molecule is free of any structured aggregate. A conjugate of interest is PEG. In the context of the foregoing definition, the term "structured aggregate" refers to (1) any aggregate of molecules in aqueous solution having a spheroid or spheroid shell structure, such that the heterogeneous molecule is not in a micelle or other emulsion structure, and is not anchored to a lipid bilayer, vesicle or liposome; and (2) any aggregate of molecules in solid or insolubilized form, such as a chromatography bead matrix, that does not release the heterogeneous molecule into solution upon contact with an aqueous phase. Accordingly, the term "conjugate" as defined herein encompasses the aforementioned heterogeneous molecule in a precipitate, sediment, bioerodible matrix or other solid capable of releasing the heterogeneous molecule into aqueous solution upon hydration of the solid.

[0076] The term "monoclonal antibody" (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Each mAb is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made in an immortalized B cell or hybridoma thereof, or may be made by recombinant DNA methods.

[0077] The monoclonal antibodies herein include hybrid and recombinant antibodies produced by splicing a variable (including hypervariable) domain of an anti-lgE antibody with a constant domain (e.g. "humanized" antibodies), or a light chain with a heavy chain, or a chain from one species with a chain from another species, or fusions with heterologous proteins, regardless of species of origin or immunoglobulin class or subclass designation, as well as antibody fragments (e.g., Fab, F(ab')₂, and Fv), so long as they exhibit the desired biological activity.

[0078] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0079] An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody will be purified (1 ) to greater than 75% by weight of antibody as determined by the Lowry method, and most preferably more than 80%, 90% or 99% by weight, or (2) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0080] The term "epitope tagged" when used herein refers to an anti-lgE antibody fused to an "epitope tag". The epitope tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the IgE antibody. The epitope tag preferably is sufficiently unique so that the antibody specific for the epitope does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues and usually between about 8-50 amino acid residues (preferably between about 9-30 residues). Examples include the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Mol. Cell. Biol. 5(12):3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering 3(6):547-553 (1990)).

[0081] The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody. The label may itself be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

[0082] By "solid phase" is meant a non-aqueous matrix to which the antibody of the present invention can adhere. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g. controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g. an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.

[0083] The term "derived from" is used herein to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means.

[0084] The terms "variant," "analog" and "mutein" refer to biologically active derivatives of the reference molecule that retain desired activity, such as as disrupting IgE complexes in the treatment of IgE-mediated disorders as described herein. In general, the terms "variant" and "analog" refer to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy biological activity and which are "substantially homologous" to the reference molecule as defined below. In general, the amino acid sequences of such analogs will have a high degree of sequence homology to the reference sequence, e.g., amino acid sequence homology of more than 50%, generally more than 60%-70%, even more particularly 80%-85% or more, such as at least 90%-95% or more, when the two sequences are aligned. Often, the analogs will include the same number of amino acids but will include substitutions, as explained herein. The term "mutein" further includes polypeptides having one or more amino acid-like molecules including but not limited to compounds comprising only amino and/or imino molecules, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic), cyclized, branched molecules and the like. Preferably, the analog or mutein has at least the same IgE activity as the native molecule.

[0085] By "derivative" is intended any suitable modification of the native polypeptide of interest, of a fragment of the native polypeptide, or of their respective analogs, such as glycosylation, phosphorylation, polymer conjugation (such as with polyethylene glycol), or other addition of foreign moieties, as long as the desired biological activity of the native polypeptide is retained. Methods for making polypeptide fragments, analogs, and derivatives are generally available in the art.

[0086] By "fragment" is intended a molecule consisting of only a part of the intact full-length sequence and structure. The fragment can include a C-terminal deletion an N-terminal deletion, and/or an internal deletion of the native polypeptide. Active fragments of a particular protein will generally include at least about 5-10 contiguous amino acid residues of the full- length molecule, preferably at least about 15-25 contiguous amino acid residues of the full- length molecule, and most preferably at least about 20-50 or more contiguous amino acid residues of the full-length molecule, or any integer between 5 amino acids and the full-length sequence, provided that the fragment in question retains biological activity, as defined herein.

[0087] "Substantially purified" generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90- 95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0088] By "isolated" is meant, when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro-molecules of the same type. The term "isolated" with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome. [0089] "Homology" refers to the percent identity between two polynucleotide or two polypeptide molecules. Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50%, preferably at least about 75%, more preferably at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence.

[0090] “IgE-mediated disorders” include IgE-mediated allergic diseases, inflammation, and asthma, such as, but not limited to, chronic spontaneous urticaria, allergic and atopic asthma, atopic dermatitis and eczema, allergic rhinitis, allergic conjunctivitis and rhinoconjunctivitis, allergic encephalomyelitis, allergic vasculitis, anaphylactic shock, allergies, such as, but not limited to, an animal allergy (e.g., cat), a cockroach allergy, a tick allergy, a dust mite allergy, an insect sting allergy (e.g. (bee, wasp, and others), a food allergy (e.g., strawberries and other fruits and vegetables, peanuts, soy, and other legumes, walnuts and other treenuts, shellfish and other seafood, milk and other dairy products, wheat and other grains, and eggs), a latex allergy, a medication allergy (e.g., penicillin, carboplatin), mold and fungi allergies (e.g., Alternaria alternata, Aspergillus and others), a pollen allergy (e.g., ragweed, Bermuda grass, Russian thistle, oak, rye, and others), and a metal allergy. The term is meant to encompass any IgE-mediated allergic reaction or allergen-induced inflammation, such as caused by any ingested or inhaled allergen, occupational allergen, environmental allergen, or any other substance that triggers a harmful IgE-mediated immune reaction.

[0091] The term "treatment" or "treating" as used herein refers to the ability to ameliorate, suppress, mitigate, or eliminate the clinical symptoms of an IgE-mediated disorder. The effect may be prophylactic in terms of completely or partially preventing IgE-mediated disorders (e.g., preventing or reducing the severity of an allergic reaction or asthmatic attack when administered before exposure to an allergen) and/or may be therapeutic in terms of partially or completely suppressing IgE-mediated disorders.

[0092] By "therapeutically effective dose or amount" of a high affinity omalizumab variant is intended an amount thatbrings about a positive therapeutic response with respect to treatment of an individual for an IgE-mediated disorder.

[0093] By “positive therapeutic response” is intended that the individual undergoing treatment exhibits an improvement in one or more symptoms of the IgE-mediated disorder for which the individual is undergoing therapy, such as a reduction in coughing, wheezing, nasal congestion, runny nose, red eyes, hives, swelling, rash, shortness of breath, bronchial inflammation, or other IgE-mediated inflammation. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, mode of administration, and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

[0094] “Pharmaceutically acceptable excipient or carrier” refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[0095] “Pharmaceutically acceptable salt” includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethyl succinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0096] The terms “subject”, “individual”, and “patient”, are used interchangeably herein and refer to any mammalian subject for whom diagnosis, prognosis, treatment, or therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; primates, and transgenic animals.

Polypeptides

[0097] In one aspect, the present invention is directed to humanized or chimeric monoclonal antibodies that are specifically reactive with IgE, and cell lines that produce such antibodies. Variable regions of exemplary antibodies are provided. Antibodies of interest include these provided combinations, as well as fusions of the variable regions to appropriate constant regions or fragments of constant regions, e.g. to generate F(ab)’ antibodies. Variable regions of interest include at least one CDR sequence of the provided anti-lg E antibody, where a CDR may be 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or more amino acids. Alternatively, antibodies of interest include a variable region as set forth in the provided antibodies, or pairs of variable regions (V_HVL) sequences as set forth herein, each variable region comprising a CDR1 , CDR2 and CDR3 sequence, including those sequences set forth in any of SEQ ID NO:1 and 5; 9 and 10; 11 and 15; 19 and 20; 21 and 25; 29 and 30; 31 and 35; 39 and 40; 45 and 5; 46 and 5; 47 and 51 ; 48 and 50; 48 and 51 ; 49 and 51 ; 62 and 25, 63 and 64.

[0098] Variable regions of interest include at least one CDR sequence from the variable regions provided herein, usually at least 2 CDR sequences, and more usually 3 CDR sequences. Exemplary CDR designations are shown in the Examples, corresponding to the underlined residues as stated, however one of skill in the art will understand that a number of definitions of the CDRs are commonly in use, including the Kabat definition (see “Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions.” Mol Immunol. 2010;47:694-700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of the structural loop regions (Chothia et al. “Conformations of immunoglobulin hypervariable regions.” Nature. 1989;342:877-883). Alternative CDR definitions of interest include, without limitation, those disclosed by Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001 ;309:657-670; Ofran et al. “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.” J Immunol. 2008;181 :6230-6235; Almagro “Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires.” J Mol Recognit. 2004;17:132-143; and Padlanet al. “Identification of specificity-determining residues in antibodies.” Faseb J. 1995;9:133-139., each of which is herein specifically incorporated by reference.

[0099] In some embodiments a polypeptide of interest has a contiguous sequence of at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, up to the complete provided variable region set forth in any of SEQ ID NO:1 and 5; 9 and 10; 11 and 15; 19 and 20; 21 and 25; 29 and 30; 31 and 35; 39 and 40; 45 and 5; 46 and 5; 47 and 51 ; 48 and 50; 48 and 51 ; 49 and 51 ; 62 and 25, 63 and 64. Polypeptides of interest also include variable regions sequences that differ by up to one, up to two, up to 3, up to 4, up to 5, up to 6 or more amino acids as compared to the amino acids sequence set forth herein. In other embodiments a polypeptide of interest is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identical to the amino acid sequence set forth herein.

[00100] In addition to Fabs, smaller antibody fragments and epitope-binding peptides having binding specificity for at least one epitope of IgE are also contemplated by the present invention and can also be used in the methods of the invention. For example, single chain antibodies can be constructed according to the method of U.S. Pat. No. 4,946,778 to Ladner et al, which is incorporated herein by reference in its entirety. Single chain antibodies comprise the variable regions of the light and heavy chains joined by a flexible linker moiety. Yet smaller is the antibody fragment known as the single domain antibody, which comprises an isolate VH single domain. Techniques for obtaining a single domain antibody with at least some of the binding specificity of the intact antibody from which they are derived are known in the art. For instance, Ward, et al. in "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted from Escherichia coli," Nature 341 : 644-646, disclose a method for screening to obtain an antibody heavy chain variable region (H single domain antibody) with sufficient affinity for its target epitope to bind thereto in isolate form.

[00101 ] The invention also provides isolated nucleic acids encoding the humanized or chimeric anti-lgE antibodies, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibody. Nucleic acids of interest may be at least about 80% identical to the provided nucleic acid sequences, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or identical. In some embodiments a contiguous nucleotide sequence encoding a polypeptide of any one of SEQ ID NO:1 and 5; 9 and 10; 11 and 15; 19 and 20; 21 and 25; 29 and 30; 31 and 35; 39 and 40; 45 and 5; 46 and 5; 47 and 51 ; 48 and 50; 48 and 51 ; 49 and 51 ; 62 and 25, 63 and 64. of at least about 20 nt., at least about 25 nt, at least about 50 nt., at least about 75 nt, at least about 100 nt, and up to the complete provided sequence may be used. Such contiguous sequences may encode a CDR sequence, or may encode a complete variable region. As is known in the art, a variable region sequence may be fused to any appropriate constant region sequence.

[00102] For recombinant production of the antibody, the nucleic acid encoding it is inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[00103] The anti-lgE antibody of this invention may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous or homologous polypeptide, which include a signal sequence or other polypeptide having a specific cleavage site at the N- terminus of the mature protein or polypeptide, an immunoglobulin constant region sequence, and the like. A heterologous signal sequence selected preferably may be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native antibody signal sequence, the signal sequence is substituted by a prokaryotic signal sequence selected. [00104] An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.

[00105] Suitable host cells for cloning or expressing the DNA are the prokaryote, yeast, or higher eukaryote cells. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/- DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51 ); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1 .982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). Host cells are transformed with the above-described expression or cloning vectors for anti-lgE antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[00106] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human yl , y2, or y4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1 -13 (1983)). Protein G is recommended for human y3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH₃ domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[00107] Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).

Methods of Use

[00108] At least one therapeutically effective dose of an antibody as described herein is administered. By "therapeutically effective dose or amount" is intended an amount that, when the antibody is administered, brings about a positive therapeutic response with respect to treatment of an individual for an IgE-mediated disorder. By "positive therapeutic response" is intended the individual undergoing the treatment according to the invention exhibits an improvement in one or more symptoms of the IgE-mediated disorder for which the individual is undergoing therapy, such as a reduction in coughing, wheezing, nasal congestion, runny nose, red eyes, hives, swelling, rash, shortness of breath, bronchial inflammation, or other IgE-mediated inflammation.

[00109] In certain embodiments, multiple therapeutically effective doses are administered according to a daily dosing regimen, or intermittently. For example, a therapeutically effective dose can be administered, one day a week, two days a week, three days a week, four days a week, or five days a week, and so forth. By "intermittent" administration is intended the therapeutically effective dose can be administered, for example, every other day, every two days, every three days, once a week, once every two weeks, once every three weeks, once a month, and so forth. For example, in some embodiments, an antibody is administered once every two to four weeks for an extended period of time, such as for 1 , 2, 3, 4, 5, 6, 7, 8, 10, 15, 24 months, and so forth. By "twice-weekly" or "two times per week" is intended that two therapeutically effective doses of the agent in question is administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses. By "thrice weekly" or "three times per week" is intended that three therapeutically effective doses are administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present invention, this type of dosing is referred to as "intermittent" therapy. In accordance with the methods of the present invention, a subject can receive intermittent therapy for one or more weekly or monthly cycles until the desired therapeutic response is achieved. The agents can be administered by any acceptable route of administration as noted herein below.

[00110] The antibody can be administered prior to, concurrent with, or subsequent additional therapies for treatment of IgE disorders. Agents can be provided in the same or in a different composition. Thus, the two agents can be presented to the individual by way of concurrent therapy. By "concurrent therapy" is intended administration to a human subject such that the therapeutic effect of the combination of the substances is caused in the subject undergoing therapy. Administration of separate pharmaceutical compositions can be at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day, or on different days), as long as the therapeutic effect of the combination of these substances is caused in the subject undergoing therapy.

[00111] In other embodiments of the invention, the pharmaceutical compositions comprising the agent or combination of agents are a sustained-release formulation, or a formulation that is administered using a sustained-release device. Such devices are well known in the art, and include, for example, transdermal patches, and miniature implantable pumps that can provide for drug delivery over time in a continuous, steady-state fashion at a variety of doses to achieve a sustained-release effect with a non-sustained-release pharmaceutical composition.

[00112] The pharmaceutical compositions comprising antibodies may be administered using the same or different routes of administration in accordance with any medically acceptable method known in the art. Suitable routes of administration include parenteral administration, such as subcutaneous (SC), intraperitoneal (IP), intramuscular (HVI), intravenous (IV), or infusion, oral and pulmonary, nasal, topical, transdermal, and suppositories. Where the composition is administered via pulmonary delivery, the therapeutically effective dose is adjusted such that the soluble level of the agent is equivalent to that obtained with a therapeutically effective dose that is administered parenterally, for example topical, SC, IP, IM, or IV. In some embodiments of the invention, the pharmaceutical composition is administered topically, e.g. by inhalation, eye drops, etc.

[00113] Factors influencing the respective amount of the various compositions to be administered include, but are not limited to, the mode of administration, the frequency of administration (i.e., daily, or intermittent administration, such as once every 2 to 4 weeks), the particular disease undergoing therapy, the severity of the disease, the history of the disease, whether the individual is undergoing concurrent therapy with another therapeutic agent, and the age, height, weight, health, and physical condition of the individual undergoing therapy. Generally, a higher dosage of this agent is preferred with increasing weight of the subject undergoing therapy. [00114] Where a subject undergoing therapy in accordance with the previously mentioned dosing regimens exhibits a partial response or a relapse following a prolonged period of remission, subsequent courses of concurrent therapy may be needed to achieve complete remission of the disease. Thus, subsequent to a period of time off from a first treatment period, a subject may receive one or more additional treatment periods. Such a period of time off between treatment periods is referred to herein as a time period of discontinuance. It is recognized that the length of the time period of discontinuance is dependent upon the degree of response (e.g., complete or partial recovery from an IgE-mediated disorder, such as an allergic disease, inflammation, or asthma) achieved with any prior treatment periods of concurrent therapy with these therapeutic agents.

[00115] As a matter of convenience, the antibody or combination of antibodies of the present invention can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for therapeutic use. In addition, other additives may be included such as stabilizers, buffers and the like. Particularly, the antibodies may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.

[00116] Compositions can be in liquid form or can be lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).

[00117] The kit can further comprise a second container comprising a pharmaceutically- acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery devices. The delivery device may be pre-filled with the compositions.

[00118] The kit can also comprise a package insert containing written instructions for methods of treating an IgE-mediated disorder, such as an allergic disease, inflammation, or asthma. The package insert can be an unapproved draft package insert or can be a package insert approved by the Food and Drug Administration (FDA) or other regulatory body

[00119] Therapeutic formulations comprising one or more antibodies of the invention are prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. The antibody composition will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The "therapeutically effective amount" of the antibody to be administered will be governed by such considerations, and is the minimum amount necessary to prevent the IgE associated disease.

[00120] The therapeutic dose may be at least about 0.01 j g/kg body weight, at least about 0.05 j g/kg body weight; at least about 0.1 j g/kg body weight, at least about 0.5 j g/kg body weight, at least about 1 j g/kg body weight, at least about 2.5 j g/kg body weight, at least about 5 j g/kg body weight, and not more than about 100 j g/kg body weight. It will be understood by one of skill in the art that such guidelines will be adjusted for the molecular weight of the active agent, e.g. in the use of antibody fragments, or in the use of antibody conjugates. The dosage may also be varied for localized administration, e.g. intranasal, inhalation, etc., or for systemic administration, e.g. i.m., i.p., i.v., and the like.

[001 1] The antibody need not be, but is optionally formulated with one or more agents that potentiate activity, or that otherwise increase the therapeutic effect. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.

[00122] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethylbenzyl 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-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating 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™, PLURONICS™ or polyethylene glycol (PEG). Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. [00123] The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[00124] The anti-lgE antibody is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the anti-lgE antibody is suitably administered by pulse infusion, particularly with declining doses of the antibody.

[00125] For the prevention or treatment of disease, the appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[00126] In another embodiment of the invention, an article of manufacture containing materials useful for the treatment of the disorders described above is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is the anti-lgE antibody. The label on, or associated with, the container indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[00127] The invention now being fully described, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention. EXPERIMENTAL

[00128] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[00129] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[00130] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims.

Example 1

Directed evolution of and structural insights into the facilitated dissociation of IgE high affinity receptor complexes.

[00131] Allergies develop from pathologic adaptive immune responses to benign foreign antigens and result in the production of allergen reactive antibodies of multiple isotypes. The effects of these antibodies are translated through a diverse collection of cell-surface and soluble immunoglobulin receptors. Antibodies of the IgE isotype were discovered following efforts to isolate reaginic antibodies responsible for type-l hypersensitivity. Today IgE is a proven therapeutic target in a growing list of allergic diseases.

[00132] We have previously demonstrated that non-antibody agents can act through facilitated dissociation to disrupt lgE:FceRI complexes, therefore we developed a high-throughput selection scheme to isolate omalizumab variants that can disrupt complexes at therapeutic doses. To probe the transition state that must exist during disruption of lgE:FceRI complexes, we engineered a stabilized lgE:FceRI complex and solved the structure of the complex bound to an omalizumab variant, revealing insights into the mechanism of facilitated dissociation. Using these insights, we further characterized our most potent omalizumab variants to identify clones capable of safely disarming allergic effector cells in in hours at nanomolar concentrations. These studies have important immediate therapeutic implications and provide detailed structural insights into the mechanism of facilitated dissociation that may be applicable to disrupting other stable macromolecular complexes.

[00133] We developed a yeast display screen to evolve the disruptive activity of omalizumab and sought to provide a biophysical and structural foundation for understanding its mechanism of action. We isolated and crystallized efficient and high-affinity omalizumab variants and evaluated the relationship between their biophysical characteristics and disruptive activity. To provide a structural foundation for understanding facilitated dissociation, we designed a disulfide stabilized lgE:FcsRI complex, engineered omalizumab variants that target this complex, and solved the structure of omalizumab bound to a partially dissociated lgE:FcsRI complex by cryo:EM. This structure represents an intermediate along the disruption pathway, reveals important features to guide the design of safe disruptive anti-lgE antibodies and provides insights into the phenomenon of facilitated dissociation which may be generalizable to other high affinity protein complexes. We then demonstrated that the dwell time of disruptive agents is a critical parameter that differentiates engineered omalizumab variants that safely strip IgE from human effector cells vs those that are anaphylactogenic. At therapeutically relevant doses our best non-anaphylactogenic antibodies completely desensitize basophils from allergic donors in hours, where omalizumab has no inhibitory effect. These studies demonstrate that antibody-based inhibitors can be selected and re-engineered to efficiently dissociate stable proteimprotein complexes, providing a new opportunity to use kinetically- active anti-lgE antibodies in allergy treatments and a framework for applying these approaches to the disruption of other macromolecular complexes.

[00134] The disruptive potency of omalizumab can be modulated by altering affinity and conformational flexibility: To facilitate yeast-display of omalizumab we produced two single open reading frame constructs: a soluble Fab construct linked by a 2A peptide, and a single chain variable fragment (scFv) construct (Figure 8A). We then developed a polystyrene beadbased disruption assay using biotinylated-lgE-Fc_2-4 (blgE-Fc_2.4) and FcsRIa-conjugated polystyrene beads to rapidly screen the disruptive potency of anti-lgE agents (Figure 1A). Surprisingly the omalizumab scFv exhibited higher affinity, slower dissociation rate (Kd), and a twofold improvement in I D₅o for complex disruption as compared to the Fab (Figure 1 B and 8 D and E). Functional differences between Fabs and scFvs are common, as each format can impose restrictions on the relative orientation of variable heavy (VH) and variable light (VL) domains. We hypothesized that changes in relative VH and VL orientations drove changes in K_D and ID₅o, so we expressed two additional constructs, a constrained scFv with a disulfide bond at the VH:VL interface (scFv_Cc) and a Fab construct with two glycine insertions at the elbow of the Fab heavy and light chains (FabH2L2) (Figure 1 C). Consistent with our hypothesis the scFvcc mutant exhibited a K_D and ID50 similar to the Fab, whereas the FabH2L2 construct showed improvements in KD and ID50 consistent with the scFv (Figure 1A and B). These changes do not seem to relate to solubility as constructs remain soluble after concentration to 100 |iM as assayed by size exclusion chromatography (SEC), with only the more potent native scFv showing signs of aggregation (Figure 8C).

[00135] Despite substantial changes in K_D and I D₅o across these omalizumab variants, their efficiency of disruption remains similar, and substantially worse than E2_79 (Figure 1 B). Because many of these agents also demonstrate some non-1 :1 binding to IgE, this behavior complicates the interpretation of efficiency as related to a single KD (Figure 8D-E). Prior work suggested that this deviation from 1 :1 binding could be due to the non-equivalence of epitopes within the IgE-Fc homodimer, because of the asymmetric arrangement of CE2 domains (Figure 8F) (Davies et al., 2017). However, deviation from 1 :1 binding occurred with omalizumab scFv, Fab, and E2_79 on IgE, lgE-Fc_2-4, and lgE-Fc_3-4 surfaces irrespective of the CE2 domain (Figure 9). Together these data support the hypothesis that disruptive potency and efficiency can be modulated by changes in inhibitor binding affinity, binding kinetics and intrinsic structural features (e.g. conformational flexibility).

[00136] Differentiation of disruptive, competitive, and non-competitive anti-lgE agents on yeast:

While selecting for improved affinity by yeast display is well established, selecting for disruptive efficiency has not been explored. Therefore, to develop a selection strategy for disruption, we first displayed three anti-lgE reagents as Aga2p fusions on S. cerevisiae'. the weak disruptor omalizumab scFv, the efficient disruptor E2_79, and the non-FcsRIa- competing DARPin E3_53 (Figure 2A-B). These three yeast-based controls showed distinct binding profiles to free biotinylated-lgE-Fc_2-4 (blgE-Fc_2.4) and blgE-Fc_2.4 bound to FcsRIa (Figure 2C). We reasoned that preformed complexes could serve as a surrogate for lgE:Fc£Rla complex disruption (lgE:Fc£Rla + anti-lgE-yeast-> lgE:anti-lgE-yeast + FcsRIa) or non-competitive binding (lgE:Fc£Rla + anti-lgE-yeast-> lgE:FcERIccanti-lgE-yeast). While this approach is similar to a competitive binding screen, we only exposed the yeast to lgE:Fc£Rla complexes for 30 minutes, a small fraction of the complex half-life (20 hours). Notably this approach only allowed partial discrimination of E2_79- and omalizumab-expressing yeast (Figure 2C). [00137] To allow for simultaneous screening of both free-lgE affinity and lgE:FcsRla complex disruption, we employed an AF-488 labeled lgE-Fc_3.4 mutant that is unable to bind FcsRIa (G335C-lgE-Fc_3.4). With this reagent, we can simultaneously compare the relative ability of yeast to bind/disrupt blgE-Fc₂₄:FcERIa-Ova complexes (IgE “pool 1 ”) and G335C-lgE-Fc_3.4 (IgE “pool 2”) in a single tube (Figure 2D). This combination of yeast staining reagents independently measures a free-lgE-binding signal as well as an lgE:FcsRla complex- binding/disrupting signal and is conceptually related to the efficiency of disruption (KD/I D₅O). This approach, referred to as the “two-color efficiency” stain, dramatically improved discrimination of E2_79 and omalizumab on the surface of yeast over a range of blgE-Fc₂- 4:FcERIa-Ova complex concentrations as compared to staining with complex alone (Figure 2F). While the stain is not a perfect representation of “efficiency” (G335C-lgE-Fc_3.4 is conformationally constrained, multivalent yeast binding is not excluded, and IgE species compete for free binding sites) the stain separates weak disruptive agents (omalizumab) from potent disruptive agents (E2_79). However, the screen does not distinguish agents that bind and disrupt complexes from those that non-competitively bind intact complex. Therefore, in a second assay, the ovalbumin-tagged FcsRIa (FcsRIa-Ova) was monitored with an anti-Ova antibody to discriminate non-competitive binders (E3_53) from competitive agents (E2_79 and omalizumab) (Figure 2G).

[00138] Directed evolution of scFv variants with enhanced disruptive potency and efficiency: Given that the two-color efficiency stain discriminates our control anti-lgE yeast, we next sought to use it for the selection of improved variants of omalizumab. We constructed a series of error-prone (EP) omalizumab libraries and shuffled mutations from promising clones using staggered extension PGR (StEP) (Figure 3A). During preliminary rounds of selection, libraries were enriched for IgE binding or lgE:Fc£Rla complex binding by magnetic activated cell sorting (MACS). These selections enriched for both high affinity and more disruptive variants. Once libraries decreased sufficiently in predicted diversity, they were selected by fluorescent activated cell sorting (FACS) using the two-color efficiency screen. Concentrations of lgE:Fc£Rla complex and free G335C-lgE-Fc_3.4 were titrated prior to sorting and conditions providing best visual separation were used.

[00139] The first error-prone library (EP1 ) progressed rapidly by our two-color disruptive efficiency screen during four rounds of selection (Figure 3B), and individual clones from round 4 (R4) were analyzed for similarity to E2_79 by our two-color disruptive efficiency screen (Figure 10B). The best clones were sequenced and demonstrated convergence (Figure 10C) and then shuffled with native omalizumab scFv to generate the StEP1 library. This library was subjected to two additional rounds of selection (Figure 10D), and three mutations were positively selected yielding clone 813 (VH:G15D, VH: I52V, and VLY31S) (Figure 11 E). Given that the first EP library was small (1.6 E7 transformants) a second EP library (EP2) was constructed from clone 813 with 4.0 E8 transformants and subjected to three rounds of selection (Figure 10F). Despite showing improvement after one round of FACS sorting (Figure 10G), no visible progress could be achieved in subsequent rounds despite titration of components of the two-color screen. Twenty clones were then sequenced yet revealed little library convergence. We therefore shuffled these 20 clones in a large StEP library (1 .0E8 transformants). The theoretical size of all permutations and combinations within this StEP library (8.8 E11 ) was far larger than the resultant library and there would be a strong bias against deleterious or neutral mutations as most mutations were found in only 1 of 20 shuffled plasmids. Nevertheless, after two rounds of selection (Figure 10H), all clones improved as compared to previous rounds (Figure 101). Sequencing of post selection clones revealed modest enrichment of several mutations relative to the 20 parental clones (Figure 10J). Therefore, a subset of 48 clones was screened for disruptive efficiency and three clones (B03, E04, C02) were selected for further characterization based on their relative staining intensities in the two-color efficiency stain (Figure 10K). The parental variant 813 and clones B03, E04, and C02 showed marked improvement in ID50 as compared to omalizumab scFv in small scale soluble expression trials, yet clone C02 showed the most improvement and was selected for further characterization.

[00140] Novel omalizumab variants exhibit enhanced affinity and disruptive efficiency. Clone 813 and C02 outperform omalizumab scFv in the yeast-based screen for disruptive efficiency (Fig. 3C). Despite multiple rounds of selection with lgE:FcsRla complexes clone 813 also did not bind preformed blgE-Fc2-₄:Fc£Rla-Ova complexes (Figure 3D), while C02 showed a modest amount of binding to intact blgE-Fc_2-4:Fc£:Rla-Ova complexes (Figure 3D). Efforts to affinity mature omalizumab by phage display were undertaken shortly after FDA approval yielding a high-affinity anti- Ig E variant HAE, the sequence of which is disclosed in US Patent publication US 2002/0054878 A1 . We therefore produced a yeast-displayed and soluble scFv of HAE as a comparator for affinity maturation without explicit selection for disruptive efficiency. Analysis of the HAE scFv in the two-color yeast efficiency screen indicates that HAE has intermediate disruptive efficiency below C02 and comparable to 813 (Figure 3E).

[00141] Using purified scFv proteins in binding and disruption studies, clone 813 showed a modest improvement in affinity as compared to omalizumab scFv, an almost two-fold improvement in ID50, and an increase in disruptive efficiency (Figure 3F and G and Figure 11 C-E). Clone C02 further improved on these changes, showing a twofold improvement of affinity, an almost tenfold improvement in ID₅₀, and a five-fold improvement in efficiency (Figure 3F and G and Figure 11C-E). HAE scFv bound IgE with the highest affinity of clones tested and was the most potent disruptor (Figure 3F,G and Figure 11 C-E). However, consistent with the two-color efficiency screen in yeast, the disruptive efficiency of HAE was worse than C02, and similar to clone 813 (Figure 3G) suggesting that affinity maturation alone does not produce the dramatic changes in disruptive efficiency seen in clone C02.

[00142] Sequence comparisons show that 813, C02 and HAE contain two mutated VL HCDR1 residues in common (Q27K, Y31 S/G), but no overlapping VH mutations (Figure 3H-I). These common sites of mutation in VL may account for the observed efficiency improvement in HAE. In an attempt to synergize the affinity and efficiency improvement of HAE and 002 we made a hybrid of 002 and HAE. Although the C02/HAE hybrid showed the highest disruptive potency, the affinity gains of HAE were partially lost, and the hybrid exhibited an intermediate efficiency (Figure 3F,G and Figure 11 C-E). Of note, clone 002 contained a novel N-linked glycosylation site within the CDR1 loop of the omalizumab VH domain; however, PNGase digestion of this site (Figure 11 A) did not induce significant changes in affinity, ID₅o, or disruptive efficiency (Figure 3F and G and Figure 11 C-E).

[00143] Structure of high affinity disruptive omalizumab variants: T o identify structural corelates to improved disruptive-potency and efficiency we crystallized native omalizumab scFv, clone 813, clone 002, clone HAE, and C02/HAE-hybrid in complex with lgE-Fc3-4. Prior structural studies of the E2_79 and omalizumab in complex with IgE suggested that the degree of potential steric overlap between each inhibitor and FcsRIa correlated with their disruptive- potency and efficiency. We therefore aligned all omalizumab:lgE pairs relative to the CE3 domain to analyze conformational arrangements that may modulate steric strain across disruptive omalizumab variants. The alignment revealed that the binding pose of omalizumab is well conserved in CDR proximal regions in all structures, but conformational flexibility is present in regions distal to the biding interface (Figure 4A). To asses if these global rearrangements correlated with disruptive potency we quantified the relative pose of VH, VL, and VH+VL to IgE CE3 across all omalizumabJgE pairs, yet this analysis did not reveal any clear correlation (Figure 12A). Given that the conformational flexibility between VH/VL domains modulated the potency of several omalizumab variants (Figure 1 ) we next assessed the relative VH to VL conformations across structures using the ABangle server. This analysis demonstrated a correlation between disruptive potency and the “HL” and “LC2” angles of Fab and scFvs (Figure 12D and E). These angles represent the torsion between VH and VL domains and the inward rotation of the VL domain in reference to a central axis between the VH and VL domains respectively, however the range of conformations observed amongst scFvs was similar and could not explain all variance in disruptive potency.

[00144] Considerable variation in the relative positions of the CE4 domain to the CE3 domain are also evident in the global alignment of all omalizumabJgE pairs (Figure 4A), and “open,” and “closed,” conformations are known to modulate lgE:Fc£Rla interactions. We therefore analyzed Fc conformations across the omalizumabJgE pairs in which reference residues (336, 394, 497) were modeled as previously described. This analysis also revealed no correlation between the Fc conformational states and disruptive potency (Figure 4B and C).

[00145] The aforementioned analysis suggested that changes in disruption could not be explained by large conformational rearrangements in scFv variants. We therefore evaluated changes to the interface in all available structures (Figure 4D). Overall the interface was well conserved with minor changes near epitope proximal mutations, but most other differences were either only found in a fraction of NCS copies, or at regions proximal to solvent exposed side chains that could not be modeled. For example R427 of IgE was absent in the C02:lgE- FC_3-4 model, and related contacts with VL residues 69-71 were therefore not identified (Figure 4B asterisks). Although the residue positions involved at the interface did not change dramatically, mutations and novel contacts were identified throughout the scFvJgE interface in each variant.

[00146] During selections C02 accumulated mutations within the heavy chain CDR3 which reside at the core of the omalizumabJgE interface and form novel polar contacts (S100 and H101 ) or in neighboring residues that form contacts within the CDR3 loop (R98K) (Figure 4E). C02 also acquired mutations in the CDRI Ioop which introduced a N-linked glycan, yet the sugar does not appear to form stable contacts with IgE and does not produce a dramatic reorganization of adjacent residues (Figure 4E). In contrast HAE contains a cluster of nonoverlapping mutations within CDR2 of the VH domain, and only a single mutation extends to the omalizumab interface (T53K) (Figure 4E). The remainder of HAE mutations throughout the CDR2 are distal to the interface, yet surround Y54, a key interface residue found in all omalizumabJgE complexes, and form intrachain contacts throughout the region. Surprisingly the C02/HAE structure reveals that residues from both C02 and HAE adopt similar conformations in the hybrid molecule (Figure 12C), yet they do not act synergistically when combined.

[00147] Within the VL domain, 813, C02, and HAE converged on a Y31 S or G substitution that reduces steric bulk proximal to residue 32 (Figure 4F). This substitution facilitates the formation of intrachain hydrogen bonds between VL position 32 and Y36 which in turn forms the posterior edge of the binding pocket for R419 of IgE, a residue shown to be critical for omalizumab binding (Pennington et al., 2016). Adjacent to these core mutations, C02 and HAE both converged on an upstream Q27K mutation at the base of the CDR1 , and HAE contains additional mutations in and proximal to the VL CDR1 (S28P, M37L) which are not directly in contact with IgE (Figure 4F). These data demonstrate that alterations to the VL- CDR1 loop are critical to affinity and disruptive potency enhancement in two omalizumab variants isolated through distinct selection methods. [00148] T ogether these structural studies do not provide evidence for dramatic reorientation of scFv conformations or changes in IgE-Fc conformational dynamics that explain the range of disruptive potency across variants. However, they do highlight conformational changes between VH/VL domains across Fab and scFv formats that correlate with potency. The structures also demonstrate how multiple mutations potentially stabilize critical CDR loop conformations and change the chemical nature of the antibody:antigen interface. Nevertheless these structures do not reveal the transient lgE:FcsRI complexes along the omalizumab disruption pathway and thus cannot fully describe the potential effect of each mutation during disruption.

[00149] Production of a disulfide “locked” lgE-Fc₂-4:FcsRla complex to trap disruption intermediates: In order to target and trap transient intermediates along the omalizumab disruption pathway, we employed the disulfide by design server to generate a disulfide engineered lgE-Fc2-₄:FcERIa “locked” complex. The covalently stabilized locked complex should be resistant to full dissociation and would allow us to trap partially disrupted intermediates for structural studies. A favorable disulfide bond was predicted between IgE residue G335 and FcsRIa residue W156. In the smaller lgE-Fc3-₄ fragment the G335C mutation forms an interchain disulfide bond and traps the IgE-Fc in a closed conformation, but in the lgE-Fc_2.4 fragment the distance between G335 residues is larger and constrained by adjacent CE2 domains. We therefore hypothesized that when co-expressed with the FcERIa(W156C) mutant a fraction of lgE-Fc2-₄(G335C) might form the lgE:FcsRla disulfide bond, covalent linking the two proteins proximal to site-two of the asymmetric lgE:FcsRla complex (Figure 5A).

[00150] Using a two-step affinity and ion-exchange purification scheme a homogenous complex of the appropriate molecular weight containing a His-tagged FcsRIa and unlabeled lgE-Fc_2.4 was isolated (Figure 13A-C). This species was stable in SDS, yet reducible to monomeric components in the presence of DTT (Figure 13C). PNGaseF treatment of the complex revealed the presence of the expected 22.5kDa band associated with deglycosylated FcsRIa (Figure 13D). The locked complex exhibited binding characteristics similar to wild type lgE-Fc2-₄:FcERIa complexes with robust binding to the non-competitive inhibitor E3_53 and weak binding to omalizumab scFv, Fab, and E2_79 at concentrations -50-100 fold higher than their respective K_D for free lgE-Fc_2-4 (Figure 5B). We next assessed the binding affinity of yeast displayed anti-lgE agents for biotinylated locked-complex as compared to free blgE-Fc_2.4. In agreement with BLI studies E3_53 displays similar binding affinity for both the locked-complex and free-lgE, while omalizumab scFv binds free-lgE well, but not the locked-complex (Figure 5C). The disruptive variants C02 and HAE display an intermediate profile with robust free-lgE binding and enhanced locked-complex binding as compared to omalizumab scFv, further indicating that the locked complex can segregate agents by their disruptive potency (Figure 5D).

[00151] Isolation of omalizumab variants that form stable complexes with the locked lgE:FcsRla complex: To improve the binding of omalizumab scFv variants to the locked complex for structural studies, we produced a small shuffled library of C02 and HAE. After five rounds of selection the majority of clones converged on variants employing C02-VH and HAE- VL sequences with sporadic additional mutations (Figure 13E-F), yet several clones with the tightest locked-complex binding in yeast (Figure 5E) shared a sporadic mutation distal to the binding interface in the E-strand of the VL chain (clone A4:D74G, clone 7:D74H, and clone 16:D74Y) (Figure 13F). Unlike the other clones, Clone 7 was identical to C02 beyond the D74H mutation, a mutation independently identified in other selections (clone F04, D74H Figure 10C). In other words, Clone 7 comprises the variable region sequences of SEQ ID NO:1 and SEQ ID NO:5 with a D74H amino acid substitution in the light chain.

[00152] We then screened each clone in yeast for non-disruptive binding to wildtype blgE-Fc2- 4:FcsRla-ova complexes to identify clones that would best stabilize intermediates along the disruptive pathway. While both clones A4 and 16 showed a modest increase in co-binding of FcsRIa-ova compared to C02 or HAE, clone 7 displayed significantly greater co-binding with an intermediate phenotype to C02 and the non-competitive binder E3_53 (Figure 5F). Consistent with these observations soluble scFvs of clone A4 and 16 showed a disruptive I D₅o similar to C02 or HAE, yet the ID₅oof clone 7 increased five-fold (Figure 5G and 13G-I) despite displaying a higher affinity for free-lgE (Figure 5G). The resulting disruption efficiency of clone 7 is lower than any other omalizumab variant (Figure 5H) and suggests that the D74H mutation stabilizes an intermediate state along the disruption pathway. This conclusion is further supported by BLI binding studies of the scFvs to the locked complex. The single E-strand mutation (D74H) at the putative scFv to FcsRIa interface dramatically increases the affinity of clone 7 for the locked-complex as compared to C02 (Figure 6I). Notably, the binding profiles of the scFvs to the locked complex were poorly fit by 1 :1 models, indicating the presence of multiple epitopes within the complex, or induced conformational changes at a single epitope. The deviation from 1 :1 binding was most dramatic for clone C02, yet both C02 and HAE appear to have one high and one low affinity binding site within the locked-complex (56.9nM- 113nM and 2.86nM-25.8nM respectively) (Figure 13J and K). Binding models for clones harboring the VL E-strand mutation (A4, 7, and 16) predict extremely slow dissociation rates in a fraction of binding events, and insufficient dissociation for fitting occurred during the 1000s dissociation window (K_d range from 5.88E-6 s^-1 to 0.00s^-1 Figure 13J and K). The kinetics of these binding studies suggest several unexpected features of these disruptive variants, namely that they can likely bind and form complexes with lgE:FcsRla complexes at low concentrations prior to disruption, and that multiple omalizumab epitopes existing in the intact lgE:FcsRla complex. These observations raise the possibility of diverse binding events with distinct outcomes.

[00153] Cryo-EM structure of a partially disrupted lgE:FcsRI complex: We selected clone 7 for further structural studies given that it varied from C02 by a single amino acid, was capable of disruption, and appeared to stabilize an intermediate of disruption. Stable clone-7:lgE-Fc₂- 4(G335C):FCERIQ(W156C) complexes formed with a 1 :1 to 2:1 stoichiometry could be detected by SEC (Figure 14A). The structure of the clone-7-scFv₂:lgE-Fc_2.4(G335C):FcERIa(W156C) complex was solved to a resolution range of -7.3A by single particle cryo-EM (Figure 6A) with regions proximal to antibody and receptor interfaces resolved at higher local resolution ( <7 A) (Figure 14B-E). Although size exclusion chromatography suggested a binding stoichiometry between 1 and 2 scFvs per complex, no particle classes with single scFvs were identified. Density for all domains within the complex was well resolved and even facilitated modeling of known sugars, such as the core IgE-Fc-glycans at N394 (Figure 6A). Most unmodeled density occurred proximal to known N-linked glycosylation sites (Figure 14F).

[00154] In this disruption-intermediate, clone-7 scFvs occupy both site-1 and site-2 epitopes of the IgE-Fc homodimer in a pseudosymmetric manner. One of the scFvs is oriented with its VL domain adjacent to the lgE-Cs2 domains (site-1 ), while the other scFv VL domain is adjacent to FcsRIa (site-2). Both of these novel VL interfaces involve the unique D74H mutation of the E-strand of clone-7 (Figure 6B), providing a structural explanation for the increased binding affinity of clone 7 to the locked complex. In their native binding poses on IgE, omalizumab and FcsRIa would physically overlap each other, suggesting that one or both molecules must be displaced in this intermediate state.

[00155] Consistent with this observation rearrangements of the scFv VHVL conformations are observed relative to the native-C02 structure. We quantified the VH to VL domain orientations in the disruption-intermediate using the Abangle server and found they were distinct from those observed in the C02:lgE-Fc_3-4 structure, and discordant across site-1 and site-2 (Figure 14G). The low resolution of the EM structure precludes detailed models of these conformational rearrangements, but the density and alignment to existing structures supports the following gross structural observations: 1 . The VH domain binding pose remains relatively unperturbed 2. The VL-CDR1 loop, and the site of the most convergent scFv mutations, is displaced from the free-lgE binding pose at both sites (Figure 14H-J). Therefore, the adjacent FcsRIa and Cs2-domains must partially block omalizumab binding and impose distortions on the VH/VL conformation.

[00156] Despite rearrangements of the scFv binding pose, alignment of the site-2 scFv to the native lgE:FcsRla structure by the proximal lgE-Cs3 domain demonstrates that steric clashes between the native FcsRIa position persist (Figure 6C), indicating that the complex represents a strained, higher energy state that is driven by clone 7 binding energy. To measure FcsRIa displacement from the native binding pose at site-2 or site-1 , we aligned the disruptionintermediate structure to the native lgE:FcsRla structure at both site-2 and site-1 CE3 domains (Figure 6C-E). After alignment we visualized the relative displacement of FcsRIa across models and quantified the displacement (using angle_between_domains from Pymol Script Collection (PSICO)). This analysis suggests that FcsRIa is displaced relative to both CE3 binding sites in the disruption-intermediate, with more pronounced displacement at site-2 (0=16.5°, displacement=5.5A), where steric conflicts between the clone 7 scFv and FcsRIa need to be resolved. Additional predicted site-2 clashes occur between FcsRIa N-linked glycans (N42 and N166), which retain similar orientations across all published lgE:FcsRla and FcsRIa structures. Notably, in the disruption-intermediate, density at these glycan sites suggests that they also reorient and adopt alternate conformations to accommodate clone 7 binding (Figure 6F).

[00157] Although no major displacement of FcsRIa appears required for omalizumab engagement at site 1 , the CE2 domains in the complex adopt a novel arrangement as compared to other IgE structures to accommodate binding. Alignment of the disruptionintermediate complex to site-1 of the native lgE:FcsRla structure reveals that this conformation could accommodate the simultaneous binding of FcsRIa and omalizumab (Figure 6G). This CE2 conformation is distinct from that observed in omalizumab:lgE-Fc2-₄ complexes, where the CE2 domains pack between the tips of the extremely open CE3 domains, overlapping the native FcsRIa position (Davies et al., 2017) (Figure 6G). While the novel conformation does not directly clash with FcsRIa, CE2 interactions with CE3 and CE4 domains in the FcsRIa contribute to the stability of the complex, and the observed displacement could contribute to destabilization.

[00158] Therapeutic potential of high affinity disruptive agents'. Our structural and functional studies of omalizumab variants show that diverse binding events can occur with intact complexes, some of which may be non-disruptive. Although we evolved our disruptive omalizumab variants to avoid stable co-binding of FcsRIa, residual interactions with these hidden intermediate states could potentially lead to receptor crosslinking and activation. We therefore produced the most promising variants as assessed by I D₅o on beads as a full lgG1 antibodies and tested each for anaphylactogenicity in transgenic human FCERI⁺ mouse bone marrow mast cells (BMMC) and human basophils. Given the role of VH/VL flexibility in the ID50 and affinity of omalizumab, we also produced H2L2-lgG1 constructs with flexible Gly-Gly linkers at each Fab elbow. The IgG reagents displayed a similar trend to scFv variants in beadbased disruption assays, yet the C02_H2L2_lgG variant was modestly improved as compared to the scFv format and showed similar potency to HAEJgG (Figure 7A). Similar to omalizumab, most variants showed no evidence of anaphylactogenicity in either BMMCs or human basophils; however, despite showing robust disruption in bead based assays, clone 16 displayed dose dependent anaphylactogenicity in BMMC cultures, and pronounced activation in all human basophil samples (Figure 7B-C). Notably the modification of HAEJgG to the HAE_H2L2JgG format also induced anaphylactogenicity in some donors, suggesting that even small changes in VH/VL domain flexibility can alter the balance of non-disruptive to disruptive binding events (Figure 70).

[00159] Given the observation that some clones were anaphylactogenic, we next measured IgG binding to intact lgE:FcsRla complexes by SPR (Figure 7D). In these experiments, a loss in signal relative to the baseline could occur following removal of IgE from lgE:FcsRla complexes. However, if disruption occurs simultaneously with non-disruptive binding events, this could produce net-positive or net-neutral SPR binding signals. Strikingly both anaphylactogenic and non-anaphylactogenic variants show pronounced complex formation over a short timeframe (200s), while omalizumab shows no detectable association. To explore differences in the kinetics of non-disruptive and disruptive binding events on the time scale of bead-based assays, we next assayed the effects of higher antibody concentrations over thirty minutes using BLI (Figure 7E). In these BLI assays the stability of nondisruptive binding events correlates well with the activation profile in BMMCs and human basophils, and the anaphylactogenic clone 16 has the most prolonged net-positive signal as compared to non- anaphylactogenic clones. These results suggest that the balance of disruptive vs. non- disruptive binding events, and thus the dwell time on the receptor complexes, are critical parameters for non-activating disruptive antibodies.

[00160] We next assessed if treatment of BMMCs with the most potent non-anaphylactogenic clones (HAEJgG or C02_ H2L2JgG) could rapidly desensitize cells. Although omalizumab itself can accelerate IgE dissociation at high concentrations, at serum concentrations observed in clinical studies (ranging from nanomolar to low micromolar) this effect is minimal over the course of hours. In contrast the observed ID₅₀ of HAEJgGI and C02_H2L2JgG1 for stripping IgE from humanized BMMCs falls in the nanomolar range after a 6-hour treatment (216nM, 95%CI [174.2-263.2nm] and 316nM, 95%CI [243.4-394.1 nM] respectively) (Figure 7F). Furthermore, these agents are able to suppress activation of BMMC with half maximal inhibition in the mid nanomolar range (550.5nM, 95%CI [425-642.7nM] and 624.9nM, 95%CI [412.8-805.4nM] respectively) (Figure 7G).

[00161] We also isolated human basophils from three grass allergic donors to measure the inhibitory profile of each antibody in human cells. These experiments also confirmed that both agents were capable of completely stripping cell surface IgE and suppressing IgE dependent activation in the mid nanomolar range (Figure 7H and I). Interestingly, C02_H2L2 significantly outperformed HAE in both basophil stripping and inhibition experiments and almost completely desensitized cells at a concentration of 500nM in 6 hours (Figure 7J). In comparison, omalizumab little effect on basophil IgE levels and signaling even at the highest concentrations studied.

[00162] IgE mediated type-1 hypersensitivity remains a significant source of human morbidity and mortality despite our advanced understanding of the underlying molecular mechanisms. In this work we have developed high throughput screening methodologies to isolate efficient and potent antibodies that can completely strip cell bound IgE in a matter of hours. Furthermore, we have developed yeast-based and biophysical screening tools to monitor the evolution of disruptive potency, efficiency, and anaphylactogenicity. Using these tools we also isolated omalizumab variants that interact with transient receptor complex intermediates along the complex disruption pathway with a covalently locked lgE:FcsRla complex. These methods allowed us to visualize a holocomplex of omalizumabJgE: FcsRIa by cryo-EM, revealing the disruption-intermediate state and interactions of the omalizumab with FcsRIa and CE3 domains of IgE. Employing a diverse collection of variants developed through these methods we then defined the key parameters, including the dwell time of disruptive inhibitors on the lgE:FcsRla complex, which determine whether the disruptive inhibitors activate IgE-bearing cells or safely desensitize them. Finally, we demonstrate that variants isolated by these methodologies, such as the C02 antibody, strip IgE and desensitize human basophils from allergic donors, under conditions where omalizumab was completely inactive.

[00163] Since we first reported omalizumab mediated disruption of lgE:FcsRla complexes, other studies have demonstrated that omalizumab can exhibit allosteric competition for FcsRIa binding, primarily through conformational rearrangements of the CE2 domains. These studies suggested that omalizumab does not act solely as an orthosteric inhibitor, and that facilitated dissociation could require two omalizumab binding interactions. In contrast our structural and kinetic studies strongly support a model in which omalizumab competes for FcsRIa binding through both orthosteric and allosteric mechanisms. Across multiple co-complex structures we predict small but consistent steric clashes between the omalizumab-VL domain and FcsRIa, and we have demonstrated disruption of lgE-Fc3-4 from FcsRIa in the absence of CE2 domains. The role of the CE2 domain in facilitated dissociation has important implications for the generalizability of the phenomenon. If facilitated dissociation requires a receptor ligand pair with the extensive conformational flexibility of the lgE-Cs2 domains, fewer biological systems may prove to be susceptible. In contrast, if disruption can be driven by small rearrangements of receptorJigand interfaces that allow antibodies to wedge themselves between protein pairs, the phenomenon and the selection reagents outlined here may be applicable to almost any high affinity receptor ligand pair. In support of this model a class of “kick-off ,” antibodies have recently been described that target preformed SIRPa:CD47 complexes adjacent to the protein protein interface and accelerate the dissociation of the macromolecular complex. Together these studies support a model in which kinetically active inhibitors could be evolved to for almost any protein complexes, expanding the drugability of pathways that are less susceptible to competitive inhibition.

Additional Functional Data for Variants B3 2, B3 3, B3 4, C06:

[00164] Bulk Library Sequencing Data: A C02 library with mutations in VH and VL domains was generated to isolate potent additional disruptive variants. The library was subjected to rounds of selection and the final library was sequenced to identify regions associated with improved binding affinity and disruptive capacity. Mutant position in VH or VL domain and original residue (parentheses) indicated, shown in Figure 15. The sequences of these variants are provided as SEQ ID NO:11 and 15 (B3_4); SEQ ID NO:21 and 25 (B3_3); SEQ ID NO:62 and 25 (B3_2).

[00165] Biolayer Interferometry Kinetic Studies: Binding studies of scFv of variants B3_2, B3_3, B3_4, C06 to recombinant human IgE, shown in Figure 16.

[00166] Bead based lgE:receptor disruption data (scFv): ID₅o of variants in scFV format in bead based disruption assay vs parental C02 scFV (dotted line), Shown in Figure 17.

[00167] Bead based lgE:receptor disruption data (H2L2-lgG): I D₅o of variants in IgG format in bead based disruption assay vs parental C02-H2L2-lgG, shown in Figure 18.

[00168] Anaphylactogenicity profile of variants (scFv format): Anaphylactogenicity of scFv variants in BMMC as measured by percent CD107a+ cells (mean and SD shown) vs negative control (Humira) and positive control (Le27), shown in Figure 19.

[00169] In vitro activity human basophils (H2L2-lgG format): Basophil inhibition assays from grass-allergic donors following 6-hour treatment with IgG variants at indicated concentrations (0-2500nM). Post treatment cultures were activated with 6-grass allergen mix and cells were assayed for activation (%CD63+), shown in Figure 20.

[00170] Structural determinants of safe disruptive omalizumab IgGs: Structure of the disruption-intermediate and novel omalizumab mutants Clone 16 and Clone 7 demonstrate that previously non-appreciated omalizumab VL interfaces are critical to the safety profile of disruptive variants. The following novel interface residues of omalizumab regulate interactions with intact IgE-receptor complex.

[00171] Leads derived from the clone C02 (Including B3_4) contain a novel S28N mutation to the original omalizumab VH domain which introduces a N-linked glycan at position 28 within the HCDR-1 . To facilitate the isolation of functionally equivalent antibodies lacking a novel N- linked glycan a stretch of residues within the HCDR-1 spanning positions 28-31 were randomized and subjected to rounds of disruptive efficiency maturation and screening for cobinding to intact lgE:FcsRla complexes via yeast display. These selections identified the following sequence tolerances across positions 28-31 as shown in Figure 21 .

[00172] The sequence tolerance at each position without introducing free cysteines is as follows:

Parental C02 HCDR1 : GYN₂₈ l29T3oS₃iGYSWN

Tolerated HCDR1 : GYX28X29X30X31GYSWN

Where X₂₈=A, H, N, S , T X₂g=I , L X₃₈=A, D , F , I , R, S , T , Y X₃I=A, D , H, N, R, S

[00173] Hits were further evaluated based on the presence of novel sequence labilities, performance in the screening assay. Nine novel variants lacking the N-linked glycosylation consensus sequence (aGlyl- aGly9) were selected along with an N28D variant produced via enzymatic cleavage of N-linked glycans in the HCDR1 previously demonstrated to preserve the C02 antibody function (aGly_png).

Omali zumab HCDR1 : GYS ITSGYSWN

SEQ ID NO : 2 C02 parental HCDR1 : GYNITSGYSWN

SEQ ID NO : 52 aGly_png HCDR1 : GYDITSGYSWN

SEQ ID NO : 53 aGlyl HCDR1 : GYNIRHGYSWN

SEQ ID NO : 54 aGly2 HCDR1 : GYNIARGYSWN

SEQ ID NO : 55 aGly3 HCDR1 : GYS IFSGYSWN SEQ ID NO : 56 aGly4 HCDR1 : GYAIDAGYSWN

SEQ ID NO : 57 aGly5 HCDR1 : GYS IYAGYSWN

SEQ ID NO : 58 aGly6 HCDR1 : GYS I IAGYSWN

SEQ ID NO : 59 aGly7 HCDR1 : GYSLFNGYSWN

SEQ ID NO : 60 aGly8 HCDR1 : GYTISRGYSWN

SEQ ID NO : 61 aGly9 HCDR1 : GYHIFDGYSWN

[00174] Given the observation that multiple high affinity disruptive omalizumab variants are able to transiently bind intact lgE:FcsRla complexes, these transient interaction may facilitate bridging of complexes to the inhibitory FcyRllb and actively suppress signaling in activated cells. Such interactions could also enhance targeting of intact complexes on the cell surface via avidity effects mediated by high affinity disruptive omalizumab variants (e.g. C02 or B3_4) binding both lgE:FcsRla complexes via CDRs and the FcyRllb receptors via their Fc domain. To explore the functional consequences of the aforementioned inhibitory mechanisms we produced variants of high affinity disruptive omalizumab variants with mutations in the Fc to enhance FcyRllb binding (G236D/S267E). These variants dramatically enhance disruptive and inhibitory function of high affinity variants by greater than 10-fold.

[00175] Although the G236D/S267E mutations in the IgG-Fc enhanced the speed and potency of IgE inhibition, the G236D/S267E mutation is also known to decrease antibody half-life and enhance C1 q binding. These features may not be desirable in the context of long term IgE blockade. We sought to identify additional mutations that could selectively block C1 q binding and enhance half-life without interfering with the enhanced disruption mediated by the G236D/S267E Fc-mutations. We identified two such mutations distal to G236D/S267E mutation sites. The first mutation, K322A (see Hezareh et al. (2001 ) J. Virol., doi:10.1128/jvi.75.24.12161 -12168.2001), is a mutation known to abolish C1 q binding. The second mutation N434A (Petkova et al. Int. Immunol. (2006), doi:10.1093/intimm/dxl110) is known to enhance FcRn binding and enhance IgG serum half-life in transgenic mouse models and in cynomolgus monkeys.

[00176] The resulting modified Fc sequence, mutant-Fc-G2 (SEQ ID:43), contains the mutations G236D/S267E/K322A/N434A using the Eu numbering scheme relative to the native Omalizumab-Fc sequence (SEQ ID:44), and can be paired with any of the disruptive anti-lgE antibodies disclosed herein to enhance disruption. For example combination of the mutant- Fc-G2 with C02_N28D (SEQ ID:45) a non-glycosylated variant of the C02 (SEQ ID:2) produced a monomeric IgG species following protein A purification (Figure 22a), and yielded an antibody with superior disruptive potency in biochemical disruption assays as compared to the parental C02 antibody or the C02_N28D mutant with the native lgG1 -Fc (Figure 22b and 22c).

Craig, D.B., and Dombkowski, A. A. (2013). Disulfide by Design 2.0: A web-based tool for disulfide engineering in proteins. BMC Bioinformatics.

Davies, A.M., Allan, E.G., Keeble, A.H., Delgado, J., Cossins, B.P., Mitropoulou, A.N., Pang, M.O.Y., Ceska, T., Beavil, A.J., Graggs, G., et al. (2017). Allosteric mechanism of action of the therapeutic anti-lgE antibody omalizumab. J. Biol. Chem.

Holdom, M.D., Davies, A.M., Nettleship, J.E., Bagby, S.C., Dhaliwal, B., Girardi, E., Hunt, J., Gould, H.J., Beavil, A. J., McDonnell, J.M., et al. (2011 ). Conformational changes in IgE contribute to its uniquely slow dissociation rate from receptor FceRI. Nat. Struct. Mol. Biol. 18, 571-576.

Kim, B., Eggel, A., Tarchevskaya, S.S., Vogel, M., Prinz, H., and Jardetzky, T.S. (2012). Accelerated disassembly of IgE-receptor complexes by a disruptive macromolecular inhibitor. Nature 491, 613-617.

Pennington, L.F., Tarchevskaya, S., Brigger, D., Sathiyamoorthy, K., Graham, M.T., Nadeau, K.C., Eggel, A., Jardetzky, T.S., Gould, H.J., Sutton, B.J., et al. (2016). Structural basis of omalizumab therapy and omalizumab-mediated IgE exchange. Nat. Commun. 7, 11610.

Sim, J., Sockolosky, J.T., Sangalang, E., Izquierdo, S., Pedersen, D., Harriman, W., Wibowo, A.S., Carter, J., Madan, A., Doyle, L., et al. (2019). Discovery of high affinity, pan-allelic, and pan-mammalian reactive antibodies against the myeloid checkpoint receptor SIRPa. MAbs.

Wurzburg, B.A., and Jardetzky, T.S. (2009). Conformational flexibility in immunoglobulin E-Fc 3-4 revealed in multiple crystal forms. J. Mol. Biol. 393, 176-190.

Example 2

[00177] VH and VL domain orientation is a critical determinate for the disruptive function of C02. Insertion of glycine residues after the VH and VL domains of C02_lgG (SEQ ID NO: 1 and 5) yields the construct C02_H2L2_lgG (SEQ ID NO: 9 and 10). C02_H2L2_lgG has improved disruptive potential as compared to C02_lgG (Figure 7A), and reduces constraints imposed on VH/VL domains by the constant domains of the Fab. B3_4, an affinity matured variant of C02, shows enhanced stripping of IgE in bead-based assays and cells as a scFv (Figure 17 and 19) or as a B4_4_H2L2_lgG (SEQ ID NO: 19 and 20) (Figure 18 and 20).

[00178] Both C02 and B3_4 contain glycosylation consensus sequences in the HCDR1 , therefore we undertook a screening campaign (Figure 21 ) and site directed mutagenesis to identify C02 and B3_4 mutants that lack carbohydrates in the CDRs. These studies yielded C02_N28D_lgG (SEQ ID NO: 45 and 5), C02_aGly_lgG (SEQ ID NO: 46 and 5), and B3_4_N28D_lgG (SEQ ID NO: 47 and 15). Each of these mutants were assessed for IgE stripping, safety, and antigen inhibition in IgE sensitized mouse BMMCs transgenic for the human high affinity IgE receptor (huFcsRIa). All mutants retained the function of their parental antibody and showed differentiation from the existing anti-lgE antibodies omalizumab and ligelizumab in rapid desensitization (Figure 23).

[00179] To further improve potency, we identified B3_4_N28D as a clone that could tolerate glycine insertions in the antibody Fab elbow without compromising antibody stability. We therefore made a series of flexible elbow mutants containing between 0-2 glycine insertions after the VH and VL domains (SEQ ID NO: 47-51). These heavy and light chains were combined to produce variants with no glycine insertions in the HC and two in the LC (“HL2” - SEQ ID NO: 47 and 51 ), variants with a single insertion in the HC and LC (“H1 LT’- SEQ ID 48 and 50), a single insertion in the HC and two insertions in the LC (“H1 L2”- SEQ ID 48 and 51), and two insertions in each chain (“H2L2”- SEQ ID 49 and 51 ). This series of elbow variants produced homogenous products after protein A purification that were stable for at least 4 freeze/thaw cycles (Figure 24), and exhibited favorable melting and aggregation temperatures as assessed by DSF (Figure 25).

[00180] We then assessed the potential of each variant to safely remove IgE from BMMCs and to inhibit antigen challenge in BMMCs. All variants showed no evidence of spontaneous activation of BMMCs (Figure 26A-C). All B3_4_N28D variants were differentiated from omalizumab as assessed by IgE removal from the surface of cells, and all B3_4_N28D variants with elbow flexibility showed a further >2 fold enhanced IgE stripping potency as compared to rigid B3_4_N28D (Figure 26D and E). B3_4_N28D and B3_4_N28D elbow variants showed similar improvement in inhibition of antigen challenge, with all showing ~10 fold improvement as compared to omalizumab, and some flexible elbow variants exhibiting a >25 fold improvement as compared to omalizumab (e.g. B3_4_N28D_H1 L1 ). However, these comparisons are limited by the weak inhibition observed in omalizumab treated samples which limits the accuracy of omalizumab IC50 calculations at the concentrations tested.

Sequences

C02-lgG (SEQ ID NO:1 ) (VH, CDR sequences underlined) EVQLVESGGGLVQPDGSLRLSCAVSGYNITSGYSWNWIRQTPGKGLEWVASVTYDGSTN YNPSVKGRITISRDGSKNTFYLQMNSLRAEDTAVYYCAKGNNYFGHWHFAVWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK

HCDR1 , SEQ ID NO:2, GYNITSGYSWN

HCDR2, SEQ ID N0:3, SVTYDGSTNYNPSVKG

HCDR3, SEQ ID N0:4, KGNNYFGHWHFAV

SEQ ID N0:5, VL:

DIQLTQSPSSLSASVGDRVTITCRASKSVDSDGDSYMNWYQQKPGRAPKLLIYAASYLESG

VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIKRTVAAPSVFIFP

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL

TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

LCDR1 , SEQ ID N0:6, RASKSVDSDGDSYMNWY

LCDR2, SEQ ID N0:7, AASYLES

LCDR3, SEQ ID N0:8, QQSHEDPY

Novel VL interface from cryo-EM structure

C02-H2L2-lgG

VH, SEQ ID NO:9

EVQLVESGGGLVQPDGSLRLSCAVSGYNITSGYSWNWIRQTPGKGLEWVASVTYDGSTN

YNPSVKGRITISRDGSKNTFYLQMNSLRAEDTAVYYCAKGNNYFGHWHFAVWGQGTLVTV

SGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE

EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

VL, SEQ ID NO:10

DIQLTQSPSSLSASVGDRVTITCRASKSVDSDGDSYMNWYQQKPGRAPKLLIYAASYLESG

VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIKGGRTVAAPSVFI

FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Novel VL interface from cryo-EM structure

B3_4-lgG

VH, SEQ ID NO:11

EVQLVESGGGLVQPDGSLRLSCAVSGYNITDGYSWNWIRQTPGKGLEWVASVTYDGSTN

YNPSVKGRITISRDGSKNTFYLQMNSLRAEDTAVYYCAKGNNYFGHWHFAVWGQGTLVTV

SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT

KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK

HCDR1 , SEQ ID N0:12, GYNITDGYSWN

HCDR2, SEQ ID N0:13, SVTYDGSTNYNPSVKG

HCDR3, SEQ ID N0:14, KGNNYFGHWHFAV

VL, SEQ ID N0:15

DIQLTQSPSSLSASVGDRVTITCRASKSVDADGDSYMNWYQAKPGRHPKLLIYAASYLESG

VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIKRTVAAPSVFIFP

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL

TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

LCDR1 , SEQ ID N0:16, RASKSVDADGDSYMNWY

LCDR2, SEQ ID N0:17, AASYLES

LCDR3, SEQ ID N0:18, QQSHEDPY

Novel VL interface from cryo-EM structure

B3_4-H2L2-lgG

VH, SEQ ID NO:19

EVQLVESGGGLVQPDGSLRLSCAVSGYNITDGYSWNWIRQTPGKGLEWVASVTYDGSTN

YNPSVKGRITISRDGSKNTFYLQMNSLRAEDTAVYYCAKGNNYFGHWHFAVWGQGTLVTV

SGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE

EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

VL, SEQ ID NO:20

DIQLTQSPSSLSASVGDRVTITCRASKSVDADGDSYMNWYQAKPGRHPKLLIYAASYLESG

VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIKGGRTVAAPSVFI

FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Novel VL interface from cryo-EM structure

B3_3-lgG

VH, SEQ ID NO:21

EVQLVESGGGLVQPDGSLRLSCAVSGYNITDGYSWNWIRQTPGKGLEWVASVTYDGSTN

YNPSVKGRITISRDGSKNTFYLQMNSLRAEDTAVYYCAKGNNYFGHWHFAVWGQGTLVTV

SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT

KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK

HCDR1 , SEQ ID NO:22, GYNITDGYSWN

HCDR2, SEQ ID NO:23, SVTYDGSTNYNPSVKG

HCDR3, SEQ ID NO:24 KGNNYFGHWHFAV

VL, SEQ ID NO:25

DIQLTQSPSSLSASVGDRVTITCRASKSVDDDWDSYMNWYQQKPGRAPKLLIYAASYLES

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIKRTVAAPSVFIF

PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST

LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

LCDR1 , SEQ ID NO:26, RASKSVDDDWDSYMNWY

LCDR2, SEQ ID NO:27, AASYLES LCDR3, SEQ ID NO:28, QQSHEDPY

Novel VL interface from cryo-EM structure

B3_3-H2L2-lgG

VH, SEQ ID NO:29

EVQLVESGGGLVQPDGSLRLSCAVSGYNITDGYSWNWIRQTPGKGLEWVASVTYDGSTN

YNPSVKGRITISRDGSKNTFYLQMNSLRAEDTAVYYCAKGNNYFGHWHFAVWGQGTLVTV

SGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE

EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

VL, SEQ ID NQ:30 DIQLTQSPSSLSASVGDRVTITCRASKSVDDDWDSYMNWYQQKPGRAPKLLIYAASYLES

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIKGGRTVAAPSV

FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS

STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Novel VL interface from cryo-EM structure

C06-lgG

VH, SEQ ID NO:31

EVQLVESGGGLVQPDGSLRLSCAVSGYNITDGYSWNWIRQTPGKGLEWVASVTYDGSTN

YNPSVKGRITISRDGSKNTFYLQMNSLRAEDTAVYYCAKGNNYFGHWHFAVWGQGTLVTV

SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT

KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK HCDR1 , SEQ ID NO:32, GYNITDGYSWN

HCDR2, SEQ ID NO:33, SVTYDGSTNYNPSVKG

HCDR3, SEQ ID NO:34, KGNNYFGHWHFAV

VL, SEQ ID NO:35

DIQLTQSPSSLSASVGDRVTITCRASKSVDGDGDSYMNWYQTKPGRAPKLLIYAASYLESG

VPSRFSGSGSGTYFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIKRTVAAPSVFIFP

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL

TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

LCDR1 , SEQ ID NO:36, RASKSVDGDGDSYMNWY

LCDR2, SEQ ID NO:37, AASYLES

LCDR3, SEQ ID NO:38, QQSHEDPY

Novel VL interface from cryo-EM structure

C06-H2L2-lgG VH, SEQ ID NO:39

EVQLVESGGGLVQPDGSLRLSCAVSGYNITDGYSWNWIRQTPGKGLEWVASVTYDGSTN

YNPSVKGRITISRDGSKNTFYLQMNSLRAEDTAVYYCAKGNNYFGHWHFAVWGQGTLVTV

SGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE

EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

VL, SEQ ID NO:40

DIQLTQSPSSLSASVGDRVTITCRASKSVDGDGDSYMNWYQTKPGRAPKLLIYAASYLESG

VPSRFSGSGSGTYFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIKGGRTVAAPSVFI

FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Novel VL interface from cryo-EM structure

SEQ ID NO:41 , Locked_Alpha_(W156C)_Protein

APMAEGGGQNHHHHHHHHGGENLYFQGGSPKVSLNPPWNRIFKGENVTLTCNGNNFFEV

SSTKWFHNGSLSEETNSSLNIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQASA

EVVMEGQPLFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYYCTGK VCQLDYESEPLNITVIKA*

SEQ ID NO:42, Locked_lgE_Fc2-₄_(G335C)_Protein

ASFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQE

GELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRCVSAYLSRPSPFD

LFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRD

WIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNF

MPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHE

AASPSQTVQRAVSVNPGK*

SEQ ID NO:43 mutant-Fc-G2

TCPPCPAPELLDGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVH

NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREP

QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY

SKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPGK

(SEQ ID NO:44) Omalizumab-Fc

TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY

SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

C02_N28D_lgG_HC (SEQ ID NO: 45)

EVQLVESGGGLVQPDGSLRLSCAVSGYDITSGYSWNWIRQTPGKGLEWVASVTYDGSTN

YNPSVKGRITISRDGSKNTFYLQMNSLRAEDTAVYYCAKGNNYFGHWHFAVWGQGTLVTV

SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT

KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK C02_aGly_lgG_HC (SEQ ID NO: 46)

EVQLVESGGGLVQPDGSLRLSCAVSGYNIRHGYSWNWIRQTPGKGLEWVASVTYDGSTN

YNPSVKGRITISRDGSKNTFYLQMNSLRAEDTAVYYCAKGNNYFGHWHFAVWGQGTLVTV

SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT

KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK

B3_4_N28D_lgG_HC (SEQ ID NO: 47)

EVQLVESGGGLVQPDGSLRLSCAVSGYDITDGYSWNWIRQTPGKGLEWVASVTYDGSTN

YNPSVKGRITISRDGSKNTFYLQMNSLRAEDTAVYYCAKGNNYFGHWHFAVWGQGTLVTV

SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT

KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPG

B3_4_N28D_H1 _lgG_HC (SEQ ID NO: 48)

EVQLVESGGGLVQPDGSLRLSCAVSGYDITDGYSWNWIRQTPGKGLEWVASVTYDGSTN

YNPSVKGRITISRDGSKNTFYLQMNSLRAEDTAVYYCAKGNNYFGHWHFAVWGQGTLVTV

SGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG

GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE

MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPG

B3_4_N28D_H2_lgG_HC (SEQ ID NO: 49)

EVQLVESGGGLVQPDGSLRLSCAVSGYDITDGYSWNWIRQTPGKGLEWVASVTYDGSTN

YNPSVKGRITISRDGSKNTFYLQMNSLRAEDTAVYYCAKGNNYFGHWHFAVWGQGTLVTV

SGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE

EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

WQQGNVFSCSVMHEALHNHYTQKSLSLSPG

B3_4_L1 JgG_LC (SEQ ID NO: 50)

DIQLTQSPSSLSASVGDRVTITCRASKSVDADGDSYMNWYQAKPGRHPKLLIYAASYLESG

VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIKGRTVAAPSVFIF

PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST

LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

B3_4_L2_lgG_LC (SEQ ID NO: 51 )

DIQLTQSPSSLSASVGDRVTITCRASKSVDADGDSYMNWYQAKPGRHPKLLIYAASYLESG

VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIKGGRTVAAPSVFI

FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:52 aGly_png HCDR1 : GYDITSGYSWN

SEQ ID NO:53 aGlyl HCDR1 : GYNIRHGYSWN

SEQ ID NO:54 aGly2 HCDR1 : GYNIARGYSWN

SEQ ID NO:55 aGly3 HCDR1 : GYSIFSGYSWN

SEQ ID NO:56 aGly4 HCDR1 : GYAIDAGYSWN

SEQ ID NO:57 aGly5 HCDR1 : GYSIYAGYSWN

SEQ ID NO:58 aGly6 HCDR1 : GYSIIAGYSWN

SEQ ID NO:59 aGly7 HCDR1 : GYSLFNGYSWN

SEQ ID NO:60 aGly8 HCDR1 : GYTISRGYSWN

SEQ ID NO:61 aGly9 HCDR1 : GYHIFDGYSWN

B3-2 VH SEQ ID NO:62

EVQLVESGGGLVQPDGSLRLSCAVSGYNITEGYSWNWIRQTPGKGLEWVASVTYDGSTN

YNPSVKGRITISRDGSKNTFYLQMNSLRAEDTAVYYCAKGNNYFGHWHFAVWGQGTLVTV

SS

813 VH SEQ ID NO:63

EVQLVESGGGLVQPDGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASVTYDGSTN

YNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTV

SS 813 VL SEQ ID NO:64:

DIQLTQSPSSLSASVGDRVTITCRASQSVDSDGDSYMNWYQQKPGKAPKLLIYAASYLESG

VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIK

Claims

WHAT is CLAIMED is:

1 . A high affinity humanized anti-lg E antibody that disrupts the complex between IgE and FceRla without causing spontaneous activation of IgE-bearing mast cells or basophils.

2. The antibody of claim 1 , wherein the antibody is a variant of omalizumab comprising at least one amino acid substitution at light chain residue D74.

3. The antibody of claim 1 , wherein the antibody is a variant of omalizumab comprising one or more amino acid substitutions at one or more of light chain residues 5, 7, 8, 9, 10, 18, 24, 28, 30, 33, 53, 57, 69, 71 , 74, and 78.

4. The antibody of claim 1 , wherein the antibody is an omalizumab sequence variant that disrupts lgE:FceRla complexes with ID₅o of 1 -2 micro molar or below, and does not spontaneously activate IgE bearing effector cells at these concentrations.

5. An antibody of claim 1 , comprising heavy chain CDR sequences as set forth in any one of:

A. SEQ ID NO:2, 3, 4;

B. SEQ ID NO:12, 13, 14;

C. SEQ ID NO: 22,23,24;

D. SEQ ID NO: 32, 33, 34.

6. An antibody of claim 1 or claim 5, comprising light chain CDR sequences as set forth in any one of:

A. SEQ ID NO:6,7,8;

B. SEQ ID NO:16,17,18;

C. SEQ ID NO:26,27,28;

D. SEQ ID NO:36,37,38.

7. An antibody of any of claims 1 -4, comprising CDR regions of SEQ ID NO: 3, 4 and SEQ ID NO:6, 7, 8, where the HCDR1 sequence is selected from one of SEQ ID NO:52-61.

8. The antibody of any of claims 1-7, wherein the variable region sequences for the heavy and light chain are as set forth in one of SEQ ID NO:1 and 5; 9 and 10; 11 and 15; 19 and 20; 21 and 25; 29 and 30; 31 and 35; 39 and 40; 45 and 5; 46 and 5; 47 and 51 ; 48 and 50; 48 and 51 ; 49 and 51 ; 62 and 25, 63 and 64.

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9. The antibody of any of claims 1-8, wherein the antibody comprises an Fc region.

10. The antibody of claim 9, wherein the Fc region is an lgG1 sequence.

11 . The antibody of claim 9 or 10, wherein the Fc region is modified from a native Fc sequence to alter binding to a Fc receptor by amino acid substitution or enzymatic modification.

12. The antibody of claim 11 , wherein the Fc modification comprises the amino acid substititions G236D and S267E.

13. The antibody of claim 11 , wherein the Fc modification further comprises the amino acid substitutions K322A and N434A.

14. The antibody of any of claims 1 -13, comprising a flexible amino acid linker of from 1 -20 amino acids in length at each Fab elbow.

15. The antibody of claim 14, wherein the linker is comprised of glycine or serine residues.

16. The antibody of claim 14 or 15, comprising the amino acid sequence pairs of any of 9 and 10; 19 and 20; 29 and 30; 39 and 40; 45 and 5; 46 and 5; 47 and 51 ; 48 and 50; 48 and 51 ; 49 and 51 .

17. A polynucleotide encoding an antibody according to any of claims 1 -16.

18. A vector comprising a polynucleotide according to claim 17.

19. A host cell comprising a vector according to claim 18 or a polynucleotide of claim 17.

20. A method of producing an antibody comprising: culturing a host cell of claim 19 under conditions suitable for expressing the antibody, and recovering the antibody from the cell mass or the cell culture medium.

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21 . A pharmaceutical composition comprising an antibody according to any of claims 1 -16, and a pharmaceutical acceptable excipient.

22. A method for treatment of an allergic condition, the method comprising administering an effective dose or doses of an anti-lgE antibody according to any of claims 1 - 16, or a pharmaceutical composition of claim 21 .

23. An anti-lgE antibody according to any of claims 1 -16, or a pharmaceutical composition of claim 21 , for use in a method of treatment of an allergic condition.

24. A disulfide stabilized lgE:FcsRI complex.

25. The disulfide stabilized complex of claim 24, comprising a protein of SEQ ID NO:42.

26. A method of screening for candidate binding agents to disrupt high affinity lgE:FcsRI complex by evaluating the dwell time of the interaction between binding agent and antigen.

27. The method of claim 26, comprising a polystyrene bead-based disruption assay comprising; binding a candidate agent to biotinylated- lgE-Fc_2.4 (blgE-Fc_2-4) and FcsRIa-conjugated polystyrene beads to rapidly screen the disruptive potency of anti-lgE candidate agents.

28. The method of claim 26 or 27, wherein the candidate agent is displayed on a yeast cell surface and bound to free biotinylated- lgE-Fc_2.4 (blgE-Fc_2.4) or blgE-Fc_2.4 bound to FcsRIa for a period of time less than about 2 hours; and measuring independently free-lgE-binding and lgE:FcsRla complex-binding/disrupting binding.

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