WO2022061240A1 - Heteromultimeric proteins for reducing ige-dependent allergic effector cell activation - Google Patents

Abstract

Compositions are provided for rapid disruption of interactions between IgE and high affinity FcεR1α, without anaphylactogenic activity.

Description

HETEROMULTIMERIC PROTEINS FOR REDUCING IGE-DEPENDENT ALLERGIC

EFFECTOR CELL ACTIVATION

CROSS REFERENCE TO REL TED APPLICATION

[0001] The present application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/081 ,193 filed September 21 , 2020, U.S. Provisional Patent Application No. 63/155,557 filed March 2, 2021 , and U.S. Provisional Application No. 63/181 ,005 filed April 28, 2021 , the entire disclosure of which is hereby.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with Government support under contract W81 XWH-14-1 -0460 awarded by the Department of Defense and under contract AI1 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, (STAN1713WO_SEQLIST_ST25.txt), created on September 20, 2021 , and having a size of 1003000 bytes. The contents of the text file are incorporated herein by reference in its entirety.

BACKGROU D

[0004] Anaphylaxis represents one of the most severe forms of allergic reactions and can occur after exposure to allergens in certain foods, drugs or animal venoms. Due to its rapid systemic onset and acute physiological impact, it often requires immediate therapeutic intervention to prevent a fatal outcome. Mechanistically, anaphylaxis, like most other allergic reactions, is caused by the activation of basophils and mast cells by allergen-specific IgE antibodies. The systemic release of soluble mediators of the inflammatory cascade rapidly induces symptoms with peak severity within minutes to hours after allergen encounter. Primary treatment for anaphylaxis consists of immediate intramuscular administration of adrenaline. While adrenaline alleviates life-threatening symptoms of an anaphylactic reaction, there are currently no available disease-modifying interventions that stop IgE-dependent allergic effector cell activation and the subsequent inflammatory cascade after it has been triggered.

[0005] The development of agents capable of rapidly disrupting the interaction of IgE complexes is of great clinical interest, and is addressed herein.

SUMMARY

[0006] Compositions and methods are provided that are useful in the rapid treatment of IgE- dependent allergic cell activation. The compositions disclosed herein provide for rapid disruption of interactions between IgE and high affinity FceRl a, without anaphylactogenic activity. These agents can rapidly strip IgE from pre-formed complexes on the surface of immune effector cells, in a clinically relevant time frame for alleviating anaphylactic and other allergic reactions. The multimeric agents comprise at least (i) a highly disruptive anti- Ig E moiety that disrupts the binding of IgE to FCERI CC; and (ii) a high affinity anti-lgE binding moiety, wherein (i) and (ii) are linked in a structurally constrained and spatially constrained format that prevents spontaneous activation of IgE-bearing allergic effector cells. Moieties (i) and (ii) may be the same or different. In some embodiments an additional element is provided, (iii) a moiety that binds FcyRllb, including without limitation, high affinity anti- FcyRII DARPin®s or single domain antibodies (sdAbs).

[0007] In some embodiments the agent is a therapeutic protein. In some embodiments the therapeutic protein is a heterodimer, comprising moiety (i) on a first polypeptide, and moiety (ii) on a second polypeptide. In some embodiments the first and second polypeptides comprise IgG constant region sequences, e.g. CH1 , CH2, CH3 and hinge; or CH2, CH3 and C-terminal linkers. In some embodiments the first and second polypeptides are associated, e.g. and without limitation, by a knob in hole (KIH) association. In some embodiments element (iii) is provided by the IgG constant region sequence. In some embodiments a C-terminal fusion of the anchoring and disruptive DARPinOs to IgG-Fc is utilized, which provides for improved safety relative to an N-terminal fusion. Alternatively an additional binding element for (iii) may be included in the therapeutic protein.

[0008] Moieties (i) and (ii) can be directly fused to IgG constant region sequences; or can be joined through a polypeptide or non-polypeptide linker. The length of the linker can be designed to optimize interactions, e.g. a linker of from about 4 - 20 amino acids in length, from about 4 to about 15 amino acids in length, from about 4 to about 10 amino acids in length, from about 4 to about 8 amino acids in length, and may be, for example, 4, 5, 6, 7, 8, 9, 10, 11 , 12 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length. Shorter linkers, e.g. from about 4 to about 8 amino acids in length may be flexible, e.g. serine residues, glycine residues, or a combination thereof. Longer linkers may provide for a non-flexible configuration.

[0009] The linking of moieties (i) and (ii) must prevent the multivalent anti-lgE agent from spontaneously crosslinking and activating FCERI on allergic effector cells. This is achieved in some embodiments by structurally and spatially constrained linking of moieties (i) and (ii) to heterodimeric IgG-Fc domains through short flexible polypeptide segments. In some embodiments a C-terminal fusion of the anchoring and disruptive DARPin®s to IgG-Fc is utilized for improved safety. In other embodiments moieties (i) and (ii) are linked by rigid nonpolypeptide linkers to structurally position moities (i) and (ii) relative to their binding sites on IgE.

[0010] Moiety (i) has a half-maximal disruptive concentrations (D0₅o) of less than about 10 |iM, less than about 5 p,M, less than about 2.5 gM. In some embodiments (i) is a DARPin® protein. In some embodiments (i) is E2_79 (previously published), for example SEQ ID NO:4; or a variant thereof, for example SEQ ID NO:7. Moiety (i) may be fused to the IgG constant region sequences of the first polypeptide. The fused sequence may include, without limitation, the polypeptide of

SEQ ID N0:1 , SEQ ID N0:6, or a variant thereof. [001 1] Moiety (ii) binds to IgE at a high affinity. In some embodiments it binds to IgE at an epitope distal to the FceRl a binding site. The binding surface may lay across the IgE CE3 and CE4 domains. The binding surface may also lay in the IgE Ce2 or Ce1 domains. In some embodiments moiety (ii) is a DARPin® protein. In some embodiments (ii) comprises E07 (SEQ ID NO:3) or a variant thereof.

[0012] Polypeptide compositions are also provided of the high affinity IgE binding proteins E07 (SEQ ID NO:3) and variants thereof. The proteins may be fused to other sequences, e.g. IgG sequences, linkers, to disruptive IgE binding proteins, and the like. The proteins have been in vitro affinity matured for binding to human IgE. In some embodiments variant proteins maintain the binding affinity and contact residues of E07, but comprise amino acid substitutions in framework sequences that do not alter the binding surface.

[0013] Moiety (iii) binds to FcyRII. The affinity may range from micromolar to nanomolar to encompass both high and low affinity targeting. In some embodiments, moiety (iii) is engineered to have differential binding affinities to different subsets of FcyRs. In some embodiments moiety (iii) is a DARPin® protein, such as D1 1 (previously published). In other embodiments, moiety (iii) is a mutant version of an IgG Fc domains known to interact with FcyRs with altered specificities and affinities.

[0014] Moiety (iii) can be delivered through the IgG constant region or can be joined through a polypeptide or non-polypeptide linker. The length of the linker can be designed to optimize interactions, e.g. a linker of from about 4 - 20 amino acids in length, from about 4 to about 15 amino acids in length, from about 4 to about 10 amino acids in length, from about 4 to about 8 amino acids in length, and may be, for example, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length. Shorter linkers, e.g. from about 4 to about 8 amino acids in length may be flexible, e.g. serine residues, glycine residues, or a combination thereof. Longer linkers may provide for a non-f lexible configuration.

[0015] In embodiments where the first and second polypeptides comprise IgG constant region sequences, e.g. CH1 , CH2, CH3 and hinge, the native lgG1 hinge (SEQ ID NO:8), may be replaced with a modified hinge, e.g. the mutated hinge C220S (SEQ ID NO:9), a truncated N- terminal lgG1 hinge (SEQ ID NO: 10), a G4S linker (Seq ID NO: 11 ), a (G4S)2 linker (SEQ ID NO: 12) can facilitate optimal spacing. The polypeptides of SEQ ID NO:13 and 14 are exemplary. In some embodiments, a fusion is used to provide sufficient space between anchoring and disruptive domains, for example as knob-in-hole heterodimers with symmetric hinges or asymmetric hinges, e.g. SEQ ID NO: 15 and 16.

[0016] C-terminal or N-terminal KIH-IgG fusions of anchoring anti-lgE domains (such as E3_53 and E07) or disruptive domains (such as E2_79 or E2_79_aGly) can be constructed with anchoring domains on the hole arm and disruptive domains on the knob arm (such as SEQ ID NO:1 , 2, and 6) or with anchoring domains on the knob arm and disruptive domains on the hole arm (SEQ ID NO: 13, 14, 15, 16, 17 and 18). In the absence of affinity tag purification schemes, placement of the disruptive domain on the hole arm of the knob-in-hole IgG can minimize potential reactive homodimer anchor species and minimize toxic off target species during KIH manufacturing.

[0017] In some embodiments, the glycosylation consensus sequence in E2_79 is mutated to an aglycosylated variant. Removal of this glycosylation consensus sequence can be accomplished by multiple mutations to E2_79 such as N62D, N62Q, and T64A.

[0018] In some embodiments the affinity of the anchoring E07 arm of a KiH heterodimer is modulated using related lower affinity variants such as E3_53, the DARPin® affinity matured into E07, and mutants of E3_53. For example, substitution of E3_53_T36N into the KiH heterodimer with E2_79_aGly (SEQ ID 6 and 20) to generate low affinity anchoring domains, which provide a broader safety window compared to other bivalent DARPin® constructs. Other E3_53 mutants that contain only partial sets of the affinity matured E07 mutations, e.g. E3_53_T36D (SEQ ID NO: 21 ), provide a range of anchor affinities to facilitate lower affinity anchoring.

[0019] In some embodiments, a truncated linker is used, e.g. the linkers in E2_79 and E07 KiH IgG-Fc fusion constructs (SEQ ID NO: 22, 23) that reduce the length of the C-terminal IgG-Fc fusion linker and removed the C-terminal IgG-Fc lysine. Truncated linker combinations with two short linkers in the KiH heterodimer (SEQ ID NO 22 and 23), a shortened linker only on the anchoring arm (SEQ ID NO: 6 and 23), or a shortened linker only on the disruptive arm (SEQ ID NO: 22 and 2) all retained disruptive potencies similar to the original KiH molecules KiH_E07_79 (SEQ ID NO: 1 and 2) and KiH_E07_79_aGly (SEQ ID NO: 6 and 2).

[0020] In some embodiment the affinity of E2_79 disruptive domain is modulated. The following series of mutations systematically alters the degree of hydrophobic interactions, polar interactions, and pi-pi stacking predicted to contribute to the IgE binding affinity of E2_79: E2_79_Y33A (SEQ ID NO: 24), E2_79_W34A (SEQ ID NO: 25), E2_79_Y33V (SEQ ID NO: 26), E2_79_W34V (SEQ ID NO: 27), E2_79_Y33L (SEQ ID NO: 28), E2_79_W34L (SEQ ID NO: 29), E2_79_Y33R (SEQ ID NO: 30), E2_79_W34R (SEQ ID NO: 31 ), E2_79_W34F (SEQ ID NO: 32), E2_79_aGly_Y33A (SEQ ID NO: 33), E2_79_aGly_W34A (SEQ ID NO: 34), E2_79_aGly_Y33V (SEQ ID NO: 35), E2_79_aGly_W34V (SEQ ID NO: 36), E2_79_aGly_Y33L (SEQ ID NO: 37), E2_79_aGly_W34L (SEQ ID NO: 38), E2_79_aGly_Y33R (SEQ ID NO: 39), E2_79_aGly_W34R (SEQ ID NO: 40), E2_79_aGly_W34F (SEQ ID NO: 41 ).

[0021] In some embodiments the Fc region is selected to reduce unwanted effector functions, including without limitation, unwanted binding to activating FcyRs (e.g. the FcyRI; FcyRIIA; FcyRIIBI; FcyRIIB2; FcyRIIIA) by selecting one of: KiH IgG 1 L234A/L235A “lgG1 -LALA” mutant (SEQ ID NO: 192 and 193); aglycosylated KiH lgG1 N297A “lgG1 -N297A” mutant (SEQ ID NO: 194 and 195); KiH lgG4 S228P/L234A/L235A “lgG4-PAA” mutant KiH (SEQ ID NO: 42 and 43).

[0022] In some embodiments, a therapeutic protein of the disclosure comprises a polypeptide of Table 1 and a polypeptide of Table 2, providing a knob-in-hole IgG heterodimeric antiJgE fusion proteins. [0023] In some embodiments a therapeutic protein of the disclosure comprises a heterodimer selected from the following combinations of modified anchoring domains, disruptive domains, linkers, and IgG-Fc fusions can represent functional disruptive anti-lg E KiH IgG heterodimers set forth in Table 3.

[0024] The invention further provides: isolated nucleic acid encoding the therapeutic proteins, binding moieties 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 protein, 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 proteins and a pharmaceutically acceptable carrier or diluent. This composition for therapeutic use is sterile and may be lyophilized, e.g. being provided as a prepack in a unit dose with diluent and delivery device, e.g. inhaler, syringe, etc.

[0025] Also provided are methods for the treatment of allergic conditions, the methods comprising administering an effective dose or doses of a therapeutic protein of the invention. In some embodiments the methods provide for a rapid therapeutic effect, e.g. within about 6 hours, within about 4 hours, within about 2 hours, within about 1 hour, within about 30 minutes. The proteins may be administered in combination with another agent, e.g. epinephrine, anti-histamine, etc. The methods can provide for rapid desensitization of allergic effector cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0001 ] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0002] Fig. 1 shows the structure of E3_53 and E2_79 in complex with lgE-Fc3-4: a. IgE-Fcs- 4:E3_53:E2_79 complex, b. Relative positions of E2_79 and E3_53 DARPin® N/C-term capping and internal repeat (IR) domains, c. Detailed views of E3_53(green):lgE(grey) interface showing polar interactions or hydrogen bonds (black dashes), d. A single chain of lgE-Fc_3-4from the IgE- FC_3-4:E3_53:E2_79 complex (green) aligned to site-2-proximal Ce4 domain of the Fc£Rla:lgE-Fc₃- 4 complex (grey). (Inset) displacement of IgE-Ca residues at the E3_53:lgE interface (black arrows), e. Comparison of E3_53 and CD23 footprints on lgE-Fc_3.4. f. Relative position of CD23 to a single chain of lgE-Fc_3.4 from the CD23:lgE-Fc_3.4 complex (yellow) aligned to the site-2- proximal CE4 of the lgE-Fc_3-4:Fc£Rla complex (grey). (Inset) displacement of IgE-Ca residues at the CD23:lgE interface (black arrows), g. Binding kinetics of E3_53 to free IgE or IgE immobilized on FcsRIa as assessed by BLI. h. Stripping of blgE-Fc2-₄ immobilized on FCERIQ functionalized beads after a 30-minute incubation with indicated anti-lgE agent (error bars shown when larger than symbol).

[0003] Fig. 2 shows affinity maturation of E3_53: a. Schematic of the yeast-based selection scheme, b. Mutations observed in E07, and their position in (Y) or outside of (N) the E3_53:lgE interface. The N48D* mutation in E07 arose from the parental library T48N mutant, c. Binding kinetics of E07 to free IgE or IgE immobilized on FccRIa as assessed by BLI. d. Stripping of big E- FC2-4 complexes immobilized on FCERIQ functionalized beads after a 30-minute incubation with indicated anti-lgE (error bars shown when larger than symbol), e. SPR disruption assay, with IgE immobilized on FCERIQ exposed to anti-lgE agents at indicated concentrations, f-g. BMMCs loaded with JW8-lgE and treated for 30 minutes with anti-lgE agents at the indicated concentrations, assessed for (f) activation (CD107a⁺) (anaphylactogenicity) and (g) IgE stripping (desensitization), h. BMMCs after treatment cells were stimulated with NIP(7)-BSA for 30 minutes and then assessed for activation (CD107a⁺) (antigen activation).

[0004] Fig. 3 shows engineering novel linkers and fusions for bivalent disruptive inhibitors: a. Schematic of novel linkers and fusions, b. Disruption of blgE-Fc2-4 complexes immobilized on FCERIQ functionalized beads after a 30-minute incubation with anti-lgE agents (n=2 error bars shown when larger than symbol), c. SPR disruption assay, with IgE immobilized on FCERIQ exposed to anti-lgE agents at indicated concentrations, d. BMMCs loaded with JW8-lgE and treated for 30 minutes with anti-lgE agents at the indicated concentrations, assessed for activation (CD107a⁺) (anaphylactogenicity) or IgE stripping (desensitization). After treatment cells were stimulated with NIP(7)-BSA for 30 minutes and then assessed for activation (CD107a⁺) (antigen activation), e. Basophils from allergic donors were treated for 30 minutes with anti-lgE agents at the indicated concentrations, assessed for activation (CD63⁺) (anaphylactogenicity) or IgE stripping (desensitization). After treatment cells were stimulated with 6-grass allergen mix for 30 minutes and then assessed for activation (CD63⁺) (antigen activation). All assays were conducted with or without DARPin® D1 1 FcyRllb blockade, f. Allergen stimulated cells from (e) were lysed and analyzed for pFcyRllb.

[0005] Fig. 4 shows KIH_E07_79 can rapidly desensitize basophils and mast cells in vivo and interrupt anaphylaxis in mice and in human cells: a. hlgE⁺,hFccRla⁺ mice were sensitized with MC903+Ova for 13 days prior to treatment with each anti-lgE agent as indicated, b. Blood basophils were analyzed for surface IgE 6h post treatment at d14 and d15, and c. peripheral mast cells were analyzed 6 hours post treatment on d15. d. hFccRla⁺ mice were sensitized with JW8lgE, challenged with NIP(20)-BSA, and treated 5 minutes post-exposure with anti-lgE agents as indicated, e. Core body temperature of mice after challenge, f. Whole blood from allergic donors was stimulated with grass allergen mix and treated 5 minutes post exposure with anti-lgE agents as indicated, g. Percent activated basophils after challenge across grass allergen concentrations. [0006] Fig. 5 shows IgE DARPin® complexes: a. Size exclusion chromatography (SEC) of IgE- FC3-4, E3_53, E2_79, or E3_53:E2_79:lgE-Fc3-₄ complexes on Hiload Superdex 200 16/600. b. Non-reducing 4-20% gradient SDS-PAGE gel with E3_53: IgE- Fc_3-4 or E3_53:E2_79:lgE-Fc3-₄ complexes, c. Representative crystals of bi53_79: lgE-Fcs-₄ in acetate buffer pH 4.6, 43% ethylene glycol hanging drops, d. Stereoscopic image of iterative build omit 2mFo-DFc map contoured at 1o and centered at the lgE:E3_53 interface, e. Schematic of IgE. f. Surface rendering of IgE complexes as for FcsRla:lgE-Fc3-₄ complex (1 f6a), CD23:lgE-Fc3-₄ (4ezm), omalizumab:lgE-Fc3-₄ (5hys).

[0007] Fig. 6 shows analysis of high affinity E07 mutant: a. Dissociation of E3_53:blgE-Fc2-₄ complexes during competition assays by round of selection vs E3_53 T48N. b. Change in relative frequency of mutations seen in R5 shuffled library input as compared to sequences observed in clones after FACS sorting, c. Detailed view of E07-T48D mutation (orange), highlighting neighboring unmutated residues within 4A with lgE-P553 for reference, d. Detailed view of I89Q/D90Y mutation and proximal (^ 4A) intrachain lgE-Q535, and interchain Y123/Y56. The D90Y mutation (magenta), falls within 4A of Q89, and clashes with neighboring residues in all modeled rotamers (red-inset).

[0008] Fig. 7 shows high affinity biE07_79: a. Size exclusion chromatography (SEC) of bi53_79 and biE07_79 on Superdex S200 10/30GL column, b. Non-reducing 4-20% gradient SDS-PAGE gel with bi53_79 and biE07_79. c. Correlation between measured affinity of anchoring domains for IgE (grey) or lgE:Fc£Rla (black) and the I D₅o-

[0009] Fig. 8 shows linker truncation of biE07_79 enhances anaphylactogenicity and limits disruption: a. Schematic of truncated linker, b. BMMCs loaded with JW8-lgE and treated for 30 minutes with anti-lgE agents at the indicated concentrations, assessed for activation (CD107a⁺) (anaphylactogenicity), or IgE stripping (desensitization), c. Stripping of blgE-Fc2-₄ complexes immobilized on FcsRIa functionalized beads after a 30-minute incubation with indicated anti-lgE (error bars shown when larger than symbol).

[0010] Fig. 9 shows effect of low nanomolar biE07_79 and omalizumab treatments on human basophils: Basophils from allergic donors were treated for 30 minutes with anti-lgE agents at the indicated concentrations, assessed for activation (CD63⁺) (anaphylactogenicity) or IgE stripping (desensitization). After treatment cells were stimulated with 6-grass allergen mix for 30 minutes and then assessed for activation (CD63⁺) (antigen activation). All assays were conducted with or without DARPin® D11 FcyRllb blockade. Results for biE07_79 shown in a. and omalizumab shown in b.

[0011 ] Fig. 10 shows topical sensitization of hlgE⁺,hFc£Rla⁺ mice: a. Schematic of sensitization of hlgE⁺,hFc£Rla⁺ mice with MC903+Ova for 14 days. b. Following sensitization the total serum IgE and c. blood basophils surface IgE were assessed.

[0012] Fig. 11A-B. A. Sec elution profile of purified KIH_E07_79 non-glycosylated form (SEQ ID NO:6), produced in HEK-293 cells, b. 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 KIH variants at a concentration of 2 micromolar. IgE displacement was measured in loss of signal (nm) relative to baseline (black line) over the course of 1200s.

[0013] FIG 12A-D: a. Bead based disruption of biotin-lgE (blgE) from high affinity receptor at the indicated concentration of each DARPin® species, b. Bead based disruption of blgE from high affinity receptor at the indicated concentration of omalizumab scFv or E07 fused to the N- terminus of the VH of omalizumab scFv by a (G4S)6 linker. Flexible linkage disrupts IgE but activates mast cells in IgE dependent fashion in the absence of antigen, c. Schematic of all DARPin® termini on DARPin®:lgE structure aligned to lgE:receptor structure, d. Measurement of distance between all DARPin® termini on a chain of IgE.

[0014] FIG. 13A-B: a. Schematic of C-terminal KIH lgG1 -Fc fusions to N-terminus of DARPin®s. b. Schematic of N-terminal KIH lgG1 -Fc fusions to C-terminus of DARPin®s.

[0015] Figure 14: SEC analysis of nickel affinity purified C and N-terminal DARPin® KiH fusions. 2uL of each 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.

[0016] Figure 15: Safety and efficacy of DARPin® IgG-Fc fusions in human whole blood basophils, a. Schematic of experimental design. Human whole blood in stimulation buffer from Buhlmann Flow CAST® assay was exposed to a titration of anti-lg E compounds for 30 minutes. The percent CD63+ activated basophils were measured to assess spontaneous activation in the presence of each anti-lgE compound (safety) and the remaining cell surface IgE was simultaneously measured (efficacy), b. Safety profile (%CD63+ basophils) of each anti-lgE over the indicated concentration range, c. Efficacy profile (MFI of residual surface IgE) of each anti- lgE over the indicated concentration range. Note that in the setting of activation (CD63+ cells in panel b.) loss of surface IgE in panel c. can arise from removal of cell surface IgE or following crosslinking and internalization of cell surface IgE.

[0017] Figure 16: Disruptive and anchoring domains can be fused to either symmetric arm of KiH heterodimers, a. Schematic of experimental design. Human whole blood in stimulation buffer from Buhlmann Flow CAST® assay was exposed to a titration of anti-lgE compounds for 30 minutes. Basophils were then measured to assess d the remaining cell surface IgE (efficacy), b. Half maximal disruptive concentration (DC50) of two KiH DARPin® fusions with inverted knob and hole fusions were identical.

[0018] Figure 17: Biochemical potency of KiH variants. Biotinylated lgE-Fc_2-4 (blgE-Fc_2-4) was immobilized on FCERIO functionalized beads and treated with anti-lgE compounds for 30-minutes to assess IgE disruption (technical replicates, error bars shown when larger than symbol). All titrations were performed in parallel, separated by panel for clarity, with KiH-E07-E2_79_aGly and E2_79 titrations duplicated across panels for comparison, a. Disruption of blgE-Fc2-₄ by KiH with a E2_79_aGly disruptive domain as compared to KiH with native E2_79 domain and native monomeric bacterial expressed E2_79. b. Disruption of blgE-Fc2-₄ by KiH with low affinity anchor domain E3_53(T36N) as compared to KiH with a E2_79_aGly disruptive domain and monomeric E2_79. c. Disruption of blgE-Fc2-₄ by KiH with short linkers on both arms, or with short linkers to anchoring or disruptive domains as compared to KiH with original linkers and monomeric E2_79.

DETAILED DESCRIPTION

[0019] Before embodiments of the present disclosure are further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of embodiments of the present disclosure.

[0021] It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes not only a single compound but also a combination of two or more compounds, reference to "a substituent" includes a single substituent as well as two or more substituents, and the like.

[0022] In describing and claiming the present invention, certain terminology will be used in accordance with the definitions set out below. It will be appreciated that the definitions provided herein are not intended to be mutually exclusive. Accordingly, some chemical moieties may fall within the definition of more than one term.

[0023] As used herein, the phrases “for example,” “for instance,” “such as,” or “including” are meant to introduce examples that further clarify more general subject matter. These examples are provided only as an aid for understanding the disclosure, and are not meant to be limiting in any fashion.

[0024] "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower KD. In an embodiment, affinity is determined by surface plasmon resonance (SPR), e.g. as used by Biacore systems. The affinity of one molecule for another molecule is determined by measuring the binding kinetics of the interaction, e.g. at 25°C.

[0025] DARPinOs (designed ankyrin repeat proteins) are a class of small (14-21 kDa) binding proteins comprised of a varying number of stacked ankyrin repeat domains, which are common structural motifs involved in protein-protein interactions. For example, see in WO 2002/020565. Natural ankyrin repeats are 33 residue motifs comprised of two a-helical structures connected by a loop that stack one on top of the other to form ankyrin repeat domains. A single DARPin® module may be comprised of a 33 residue repeat of which seven residues are randomized and non-conserved. Typically, two to four library modules are genetically fused and flanked by N-cap and C-cap repeats that serve to provide a hydrophilic surface, the whole forming one protein domain. Binding of ankyrin repeat domains can affect stability and effector function of the target protein.

[0026] DARPin@s can provide high potency (<5-100 pM) activity at low concentrations. DARPin®s are soluble at >100 g/L, and their high stability and solubility are considered desirable properties for drug compounds.

[0027] The term "ankyrin repeat unit" refers to an ankyrin repeat as described, for example, by Forrer et al., 2003. Ankyrin repeats are well known to the person skilled in the art. The term "ankyrin repeat domain" refers to a repeat domain comprising two or more consecutive ankyrin repeat units (modules) as structural units, and may include an N-terminal and/or a C-terminal capping unit (or module).

[0028] The term "framework residues" relates to amino acid residues of the repeat units, or the corresponding amino acid residues of the repeat modules, that contribute to the folding topology, i.e. which contribute to the fold of said repeat unit (or module) or which contribute to the interaction with a neighboring unit (or module). Such contribution might be the interaction with other residues in the repeat unit (or module), or the influence on the polypeptide backbone conformation as found in a-helices or p-sheets, or amino acid stretches forming linear polypeptides or loops.

[0029] The term "contact residues" refers to amino acid residues of the repeat units, or the corresponding amino acid residues of the repeat modules, that contribute to the interaction with epitope, e.g. human IgE, by virtue of direct interaction with the epitope, or the influence on other directly interacting residues, e.g. by stabilizing the conformation of the polypeptide of a repeat unit (or module) to allow or enhance the interaction of directly interacting residues with the epitope. Such framework and target interaction residues may be identified by analysis of the structural data obtained by physicochemical methods, such as X-ray crystallography, NMR and/or CD spectroscopy, or by comparison with known and related structural information well known to practitioners in structural biology and/or bioinformatics.

[0030] The term "folding topology" refers to the tertiary structure of said repeat units or repeat modules. The folding topology will be determined by stretches of amino acids forming at least parts of a-helices or p-sheets, or amino acid stretches forming linear polypeptides or loops, or any combination of a-helices, p-sheets and/or linear polypeptides/loops. For example, an ankyrin repeat unit/module consists of a p-turn, followed by two antiparallel ex— helices and a loop that reaches the turn of the next repeat unit/module. [0031] The term "capping module" refers to a polypeptide fused to the N- or C-terminal repeat module of a domain, wherein the capping module forms tight tertiary interactions (i.e. tertiary structure interactions) with the repeat module, thereby providing a cap that shields the hydrophobic core of the repeat module at the side not in contact with the consecutive repeat module from solvent. The N- and/or C-terminal capping module may be, or may be derived from, a capping unit or other structural unit found in a naturally occurring repeat protein adjacent to a repeat unit. The term "capping unit" refers to a naturally occurring folded polypeptide, wherein said polypeptide defines a particular structural unit which is N- or C-terminally fused to a repeat unit, wherein the polypeptide forms tight tertiary structure interactions with the repeat unit thereby providing a cap that shields the hydrophobic core of said repeat unit at one side from the solvent. Capping modules and capping repeats are described in WO 2002/020565.

[0032] An IgE polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source, usually a human IgE protein. See, for example, Garman et al., Structure of the Fc fragment of human IgE bound to its high-affinity receptor Fc epsilonRI alpha. Nature 406, 259-266 (2000), herein incorporated by reference). Additional 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.

[0033] IgE binds two principle receptors, the high affinity FceRI receptor found primarily on mast cells and basophils, and the lower affinity FCERI I (CD23) receptor found primarily on B cells. IgE and FCERI bind with high affinity (KD~100pM) through the ectodomain of the FcsRI a-chain (FceRlo), 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.

[0034] The term “FceRla extracellular region” or the like refers to an extracellular domain of an FcsRIa protein that is the portion of the FcsRIa chain 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 “FceRla 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 FcsRIa, 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. [0035] The term “IgE-Fc C£3-C£4” or the like refers to a third and fourth C-terminal constant domain, C£3 and C£4, of an IgE heavy chain that mediates binding to an FCERIQ. For the nucleotide and amino acid sequence of a human FcaRla, see Kochan, J. et al. 1988 Nucleic Acids Res. 16:3584-3584. The term “IgE-Fc C£3-C£4” 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 FcaRla (preferably of mammalian origin, e.g., human), for example comprising an amino acid sequence of a naturally occurring mammalian IgE-Fc C£3-C£4 or a fragment thereof.

[0036] Moiety (i), as used herein, refers to a “disruptive” binding agent that disrupts the binding of IgE to FCERI OC. The binding moiety is selected to have a half-maximal disruptive concentrations (DC₅o) of less than about 10 jiM, less than about 5 jiM, less than about 2.5 gM. In some embodiments (i) is a DARPin® protein. In some embodiments (i) is E2_79 (previously published) or a variant thereof, including without limitation the non-glycosylated variant of SEQ ID NO:7. The monovalent DARPin® E2_79 is specific for IgE, and can disrupt IgE-FcaRI interactions. E2_79 significantly reduces surface expression of FcaRI on human isolated primary basophils, and inhibits FcaRI-induced activation and leukotriene C4 (LTC4) biosynthesis. Moiety (i) binds to IgE at a high affinity. High affinity anchoring domains refer to agents that bind preformed IgE receptor complexes with nanomolar or better affinity, and thereby can be linked to disruptive moieties to allow for the accelerated removal of IgE from receptor complexes at nanomolar concentrations. Variants of interest include those having been in vitro affinity matured for binding to human IgE. In some embodiments variant proteins maintain the binding affinity and contact residues of E2_79, but comprise amino acid substitutions in framework sequences that do not alter the binding surface. Variants of interest also include those modified so as to not comprise potential N-glycosylation sites.

[0037] Suitable polypeptides for use as moiety (i) are set forth in Table 1 .

Table 1 , Moiety i

[0038] The reported half-life of IgE high affinity receptor complexes in solution is at least 20 hours, and observed half-life of IgE in SPR assays and is measured in days. A disruptive moiety is an agent that can accelerate the dissociation of IgE from the high affinity receptor, and by doing so remove receptor bound IgE faster than it would otherwise freely dissociate. For an agent to be considered disruptive it must facilitate more rapid dissociation rate of IgE from the receptor than would be observed in and isolated system of IgE and the receptor alone. The disruptive agents that show a clear therapeutic advantage reduce the half-life dramatically in a dose dependent fashion and at high concentrations can completely dissociate complexes in minutes-hours rather than days, for example within less than about 24 hours, less than about 12 hours, less than about 6 hours, less than about 3 hours, less than about 90 minutes, less than about 45 minutes, less than about 30 minutes.

[0039] Moiety (ii) binds to IgE at a high affinity. High affinity anchoring domains refer to agents that bind preformed IgE receptor complexes with nanomolar or better affinity, and thereby can be linked to disruptive moieties to allow for the accelerated removal of IgE from receptor complexes at nanomolar concentrations. In some embodiments (ii) binds to IgE at an epitope distal to the FCERI CX binding site. The binding surface may lay across the IgE CE3 and CE4 domains. In some embodiments moiety (ii) is a DARPin® protein. In some embodiments (ii) comprises E_07 (SEQ ID NO:3) or a variant thereof, e.g. having been in vitro affinity matured for binding to human IgE. In some embodiments variant proteins maintain the binding affinity and contact residues of E_07, but comprise amino acid substitutions in framework sequences that do not alter the binding surface.

[0040] Suitable polypeptides for use as moiety (ii) include those set forth in Table 2.

Table 2, Moiety ii

[0041] Moiety (iii) binds to FcyRII. The affinity may range from LIM to nanomolar to encompass both high and low affinity targeting. In some embodiments, moiety (iii) is engineered to have differential binding affinities to different subsets of FcyRs. In some embodiments moiety (iii) is a DARPin® protein, such as D11 (previously published). In other embodiments, moiety (iii) is a variant of an IgG Fc domain known to interact with FcyRs with altered specificities and affinities. [0042] In some embodiments a therapeutic protein of the disclosure comprises a heterodimer selected from the following combinations of modified anchoring domains, disruptive domains, linkers, and IgG-Fc fusions can represent functional disruptive anti-lg E KIH IgG heterodimers set forth in Table 3.

TABLE 3

[0043] A “functional Fc region” possesses an “effector function” of a native-sequence Fc region. The Fc can be that of any antibody type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., lgG1 , lgG2, lgG3, lgG4, Ig A1 and Ig A2) or subclass, including engineered subclasses with altered Fc portions that provide for reduced or enhanced effector cell activity. The Fc can be derived from any species. In one aspect, the immunoglobulin is of largely human origin. Exemplary effector functions include C1 q binding; GDC; Fc-receptor binding; ADCC; ADCP; down-regulation of cellsurface receptors (e.g., B-cell receptor), etc. Such effector functions generally require the Fc region to be interact with a receptor, e.g. the FcyRI; FcyRIIA; FcyRIIBI ; FcyRIIB2; FcyRIIIA; FcyRIIIB receptors, and the low affinity FcRn receptor; and can be assessed using various assays as disclosed, for example, in definitions herein. A “dead” Fc is one that has been mutagenized to retain activity with respect to, for example, prolonging serum half-life, but which does not activate a high affinity Fc receptor. An Fc may also have decreased binding to complement.

[0044] A “native-sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native-sequence human Fc regions include a native-sequence human lgG1 Fc region (non-A and A allotypes); native-sequence human lgG2 Fc region; native-sequence human lgG3 Fc region; and native-sequence human lgG4 Fc region, as well as naturally occurring variants thereof.

[0045] A “variant Fc region” comprises an amino acid sequence that differs from that of a nativesequence 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.

[0046] 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 C1 q 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 ai., 1999 Eur J Immunol. 29(8):2613- 24; and Shields RL. et al., 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.

[0047] 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.

[0048] 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. CHO 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 non-glycosylated fractions, for example, using lectin affinity chromatography (See e.g., WO 07/1 17505). 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 C1 q part of the first complement component C1 and a decrease or loss in antibody-dependent cell- mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), 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 antiinflammatory 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)). [0049] Constructs can have an Fc sequence with enhanced effector functions, e.g. by increasing their binding capacities to FcyRI HA and increasing ADCC activity. For example, fucose attached to the /V-linked glycan at Asn-297 of Fc sterically hinders the interaction of Fc with FcyRI 11 A, and removal of fucose by glyco-engineering can increase the binding to FcyRI 11 A, 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 FcyRI 11 A and augmentation of ADCC capacity in vitro and in vivo. Other Fc variants identified by yeast display also showed the improved binding to FcyRIIIA. See, for example Liu et al. (2014) JBC 289(6):3571 -90, herein specifically incorporated by reference.

[0050] Determination of affinity for an IgE, IgE receptor, FcyR, etc. can be performed using methods known in the art, e.g. Biacore measurements, etc. DARPin®s may have an affinity for the cognate antigen with a Kd of from about 10'⁷ to around about 10 ", including without limitation: from about 10^-7 to around about 10 ^w; from about 10^-7 to around about 10^-9; from about 10⁷ to around about 10^-8; from about 10'⁸ to around about 10 " ; from about 10^-8 to around about 10"°; from about 10^-8 to around about 10^-9; from about 10'⁹ to around about 10 " ; from about 10^{_ 9} to around about 10^-10; or any value within these ranges. The affinity selection may be confirmed with a biological assessment for inhibition of allergic reactions, for example, and in vitro or pre- clinical model, and assessment of potential toxicity. The term “high affinity” may include, for example, less than about 10⁹; less than about 10¹⁰; less than about 10 " .

[0051] The terms “variant,” “analog” and “mutein” refer to biologically active derivatives of the reference molecule that retain desired activity, such as IgE binding activity. 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. The term also includes molecules comprising one or more N-substituted glycine residues (a “peptoid”) and other synthetic amino acids or peptides. (See, e.g., U.S. Pat. Nos. 5,831 ,005; 5,877,278; and U.S. Pat. No. 5,977,301 ; Nguyen et al., Chem Biol. (2000) 7:463-473; and Simon et al., Proc. Natl. Acad. Sci. USA (1992) 89:9367-9371 for descriptions of peptoids). Preferably, the analog or mutein has at least the same I g E binding activity as the native molecule. Methods for making polypeptide analogs and muteins are known in the art and are described further below.

[0052] As explained above, analogs generally include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: (1 ) acidic— aspartate and glutamate; (2) basic— lysine, arginine, histidine; (3) non-polar— alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar— glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. One of skill in the art may readily determine regions of the molecule of interest that can tolerate change by reference to Hopp/Woods and Kyte- Doolittle plots, well known in the art.

[0053] 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.

[0054] “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.

[0055] In general, “identity” refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules (the reference sequence and a sequence with unknown % identity to the reference sequence) by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the reference sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5 Suppl. 3:353- 358, National biomedical Research Foundation, Washington, D.C., which adapts the local homology algorithm of Smith and Waterman Advances in Appl. Math. 2:482-489, 1981 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.

[0056] Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects “sequence identity.” Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs are readily available.

[0057] Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.

[0058] A variant of, for example, moiety (i), (ii) or (iii), including the polypeptides E_07, E2_79, D11 , and non-glycosylated E2_79 may comprise an amino acid sequence with at least 70% amino acid sequence identity relative to the reference sequences, for example 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity. Variants of interest have substantially the same or improved function relative to the starting molecule.

[0059] With respect to DARPin polypeptides, variability from the provided sequences may be in the framework positions, for example, in the capping modules. An amino acid may be substituted by a homologous amino acid; i.e. an amino acid is exchanged by an amino acid having a side chain with similar biophysical properties.

[0060] The term "consensus sequence" refers to an amino acid sequence, wherein said consensus sequence is obtained by structural and/or sequence aligning of multiple repeat units. Using two or more structural and/or sequence aligned repeat units, and allowing for gaps in the alignment, it is possible to determine the most frequent amino acid residue at each position. The consensus sequence is that sequence which comprises the amino acids which are most frequently represented at each position. In the event that two or more amino acids are represented above-average at a single position, the consensus sequence may include a subset of those amino acids. Said two or more repeat units may be taken from the repeat units comprised in a single repeat protein, or from two or more different repeat proteins.

[0061] A "consensus amino acid residue" is the amino acid found at a certain position in a consensus sequence. If two or more, e.g. three, four or five, amino acid residues are found with a similar probability in said two or more repeat units, the consensus amino acid may be one of the most frequently found amino acids or a combination of said two or more amino acid residues.

[0062] 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, such as IgE activity, as defined herein.

[0063] “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.

[0064] 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.

[0065] The terms "active agent”, “antagonist”, "inhibitor", "drug" and "pharmacologically active agent" are used interchangeably herein to refer to a chemical material or compound which, when administered to an organism (human or animal) induces a desired pharmacologic and/or physiologic effect by local and/or systemic action.

[0066] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to an animal, including, but not limited to, human and non-human primates, including simians and humans; rodents, including rats and mice; bovines; equines; ovines; felines; canines; avians, and the like. "Mammal" means a member or members of any mammalian species, and includes, by way of example, canines; felines; equines; bovines; ovines; rodentia, etc. and primates, e.g., non-human primates, and humans. Non-human animal models, e.g., mammals, e.g. non-human primates, murines, lagomorpha, etc. may be used for experimental investigations.

[0067] As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.

[0068] The term "polypeptide linker" refers to an amino acid sequence, which is able to link, for example, two protein domains, such as a binding moiety and immunoglobulin constant region sequences. Particular examples of such linkers are glycine-serine- linkers and proline-threonine- linkers of variable lengths; preferably, said linkers have a length between 4 and 20 amino acids.

[0069] The term "moiety" refers to a chemical group that is providing a particular activity, as defined herein. In some embodiments a moiety is a polypeptide, e.g. a DARPin®. Such polypeptide moieties can be covalently attached to, for example, a linker or an immunoglobulin sequence. While various linkers may be used, a fusion protein is preferred. A moiety may be joined to a multimerization moiety, for example immunoglobulin heavy chain constant regions which pair to provide functional immunoglobulin Fc domains, and leucine zippers or polypeptides comprising a free thiol which forms an intermolecular disulfide bond between two such polypeptides. The single Cys residue may be used for conjugating other moieties to the polypeptide, for example, by using the maleimide chemistry well known to the person skilled in the art.

[0070] The terms "nucleic acid molecule" and “polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

[0071] A "therapeutically effective amount" or "efficacious amount" means the amount of a compound that, when administered to a mammal or other subject for treating a disease, condition, or disorder, is sufficient to effect such treatment for the disease, condition, or disorder. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0072] The term “unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a compound calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for unit dosage forms depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0073] A "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" means an excipient, diluent, carrier, and adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use. "A pharmaceutically acceptable excipient, diluent, carrier and adjuvant" as used in the specification and claims includes both one and more than one such excipient, diluent, carrier, and adjuvant.

[0074] As used herein, a "pharmaceutical composition" is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human. In general a “pharmaceutical composition” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, and the like.

Multimeric compositions

[0075] Multimeric proteins are provided for rapid disruption of interactions between IgE and high affinity FCERI CC, without anaphylactogenic activity. These agents can rapidly strip IgE from preformed complexes on the surface of immune effector cells, in a clinically relevant time frame for alleviating anaphylactic and other allergic reactions. The multimeric agents comprise at least (i) a highly disruptive anti-lgE moiety that disrupts the binding of IgE to FcsRI a; and (ii) a high affinity anti-lgE binding moiety, wherein (i) and (ii) are linked in a structurally constrained and spatially constrained format that prevents spontaneous activation of IgE-bearing allergic effector cells, linked in a structurally constrained format, wherein no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% basophils are activated, preferably less than 5%, less than 2.5%, less than 1 % basophiles are activated as measured by a conventional assay or as disclosed in the Examples.

[0076] Moieties (i) and (ii) may be the same or different. In some embodiments an additional element is provided, (iii) a moiety that binds FcyRllb, including without limitation, high affinity anti- FcyRII DARPinOs or single domain antibodies (sdAbs). The constructs of the invention are typically provided in structurally constrained, multi-specific configurations, which include without limitation bispecific, trifunctional, etc. Tables 1 , 2 and 3 provide suitable polypeptides and combinations for this purpose.

[0077] A large variety of methods and protein configurations are known and used. For example, see Suursa et al. (2019) Pharmacology & Therapeutics 201 :103-119; and Labrijn et al. (2019) Nature Reviews Drug Discovery 18:585-608, each herein specifically incorporated by reference. Recombinant proteins can force the correct association of heavy-light chains and the heavy chains by multiple means. Examples are the knob-in-holes approach where one heavy chain is engineered with a knob consisting of relatively large amino acids and the other heavy chain is engineered with a hole consisting of relatively small amino acids (A. M. Merchant et al., 1998). Other examples are the constructs with their fragments connected by peptide chains, such as bispecific T cell engagers (BiTE) molecules, thereby circumventing random association of the chains (Mack, Riethmuller, & Kufer, 1995). Other types of bispecifics include chemically linked bonding moieties, consisting only of the binding regions.

[0078] Other bispecific antibody-like proteins include DVD- (Wu, C. et al., Nature Biotechnology, 25, p 1290-1297, 2007). To construct the DVD-lg molecule, the binding moieties are fused in tandem by a short linker with the moiety at the N terminus, followed by the other binding moiety and the heavy chain constant domains to form the DVD-lg protein heavy chain (VH1/VL1 ).

[0079] The constant domains of the construct may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. The intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc constant region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1 q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors. Constant region variants include those that alter the effector profile, binding to Fc receptors, and the like.

[0080] Depending on the amino acid sequence of the constant domain, the Fc can be assigned to different “classes.” There are five major classes of intact immunoglobulin antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, IgA, and lgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called a, 5, £, y, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinge-modifications or hingeless forms (Roux et al (1998) J. Immunol. 161 :4083- 4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US 2005/0048572; US 2004/0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called K and A, based on the amino acid sequences of their constant domains.

[0081 ] The term "epitope tagged" when used herein refers to a protein 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 protein. 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)).

[0082] The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the protein. 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.

[0083] By "solid phase" is meant a non-aqueous matrix to which the protein 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.

[0084] The invention also provides isolated nucleic acids encoding the provided moieties and multimeric proteins, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the proteins. 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:1-3 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 binding moiety, which may be fused to any appropriate constant region sequence.

[0085] For recombinant production of the proteins, the nucleic acid encoding it is inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the protein is readily isolated and sequenced using conventional procedures. 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.

[0086] The proteins 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.

[0087] 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.

[0088] 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.

[0089] The protein 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 protein. Protein A can be used to purify proteins that comprise human y1 , 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)). For example, moieties on the knob arm can be purified with the N-terminal affinity tag APMAEGGGQN-HHHHHHHHGGENLYFQGGS and moieties on the hole arm can be purified with the N-terminal affinity tag APMAEGGGQNYPYDVPDYAGENLYFQGGS. 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 protein to be recovered.

Methods of Use

[0090] At least one therapeutically effective dose of a protein as described herein is administered. By "therapeutically effective dose or amount" is intended an amount that, when the protein 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 anaphylaxis, coughing, wheezing, nasal congestion, runny nose, red eyes, hives, swelling, rash, shortness of breath, bronchial inflammation, or other IgE-mediated inflammation. By "therapeutically effective dose or amount" is intended an amount that brings about a positive therapeutic response with respect to treatment of an individual for an IgE-mediated disorder.

[0091] “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.

[0092] In some embodiments the therapeutic dose is sufficient to provide for rapid resolution of an acute IgE-mediated disorder, including without limitation an allergic reaction capable of causing anaphylaxis. The therapeutic dose may be sufficient to substantially reduce pre-formed IgE-receptor complexes on the surface of immune effector cells, without triggering anaphylaxis. The period of time for response may be rapid, e.g. less than 24 hours, less than 12 hours, less than 6 hours, less than 3 hours, less than 45 minutes, less than 30 minutes, less than 15 minutes.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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, the therapeutic protein 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.

[0097] The therapeutic protein 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.

[0098] 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.

[0099] The pharmaceutical compositions comprising therapeutic proteins 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 SC, IP, IM, or IV. In some embodiments of the invention, the pharmaceutical composition is administered by IM or SC injection, particularly by EVI or SC injection locally to the region. [00100] 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.

[00101 ] 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.

[00102] As a matter of convenience, the therapeutic protein or combination of therapeutic proteins 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 therapeutic proteins may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.

[00103] 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).

[00104] 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.

[00105] 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 [00106] Therapeutic formulations comprising one or more therapeutic proteins of the invention are prepared for storage by mixing the therapeutic protein 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 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" to be administered will be governed by such considerations, and is the minimum amount necessary to prevent the IgE associated disease.

[00107] The therapeutic dose may be at least about 1 jig/kg body weight, at least about 5 ig/kg body weight; at least about 10 j g/kg body weight, at least about 50 jxg/kg body weight, at least about 100 ig/kg body weight, at least about 250 jxg/kg body weight, at least about 500 ig/kg body weight, and not more than about 10 mg/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 protein conjugates, e.g. pegylated proteins. 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.

[00108] The therapeutic protein 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.

[00109] 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.

[001 10] 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).

[001 11 ] For the prevention or treatment of disease, e.g. undesirable allergic reactions, the appropriate dosage of therapeutic protein 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.

[001 12] 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 therapeutic protein. 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.

[001 13] 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

[001 14] 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.

[00115] 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.

[00116] 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

Fast acting disruptive IgE inhibitors rapidly desensitize allergic effector cells and resolve IgE- mediated anaphylaxis

[00117] Here, we report the structure-based engineering of potent, rapidly-acting IgE inhibitors that terminate IgE-mediated signaling in pre-activated human blood basophils in vitro and shut down pre-initiated allergic reactions and anaphylaxis in mice in vivo. These inhibitors are based on anti-lgE designed ankyrin repeat proteins (DARPin®s) and can dissociate preformed IgE receptor complexes with low nanomolar potencies. The most effective variant (KIH_E07_79) integrates four key elements to achieve unprecedented kinetic inhibitory activity in terminating a pre-activated allergic cascade: (1 ) a high affinity anti-lgE anchoring module (E07), (2) a highly disruptive anti-lgE inhibitory module (E2_79), (3) the structurally constrained positioning of E07 and E2_79 through IgG-mediated heterodimerization and (4) an FcyRII inhibitory signaling module. These fast acting disruptive IgE inhibitors demonstrate the feasibility of developing kinetically optimized inhibitors for the treatment of anaphylaxis and the rapid desensitization of allergic individuals.

Results

[00118] While the number of annual hospitalizations due to anaphylactic reactions is difficult to track and varies considerably across geographic locations, a life-time prevalence of 0.05-2 % in the US and 0.3% in Europe has been estimated, and the frequency is on the rise. Every year in the US hundreds of patients die due to an unexpected episode of severe systemic anaphylaxis. Mechanistically, an overwhelming amount of clinical and experimental evidence indicates that the predominant form of human anaphylaxis is immunoglobulin E (IgE) dependent. Antigen- mediated cross-linking of allergen-specific IgE bound to the high-affinity IgE receptor (FceRI) on mast cells and basophils induces cellular degranulation and the immediate release of various soluble mediators. Clinical symptoms secondary to degranulation peak in severity within half an hour to two hours depending on the route of antigen exposure.

[001 19] The universal first-line therapy for anaphylaxis is the immediate intramuscular application of adrenaline (i.e. epinephrine). Second- and third-line treatments consist of beta-2 agonist, antihistamines or glucocorticoids. These interventions rapidly alleviate the potentially fatal symptoms of anaphylaxis, but they do not interfere with the underlying pathomechanism of the disease. There is no suitable disease-modifying treatment option that acts fast enough to rapidly resolve the IgE-mediated allergic cascade. The therapeutic monoclonal anti-lgE antibody omalizumab (Xolair®), which is approved for the treatment of allergic asthma and chronic spontaneous urticaria, neutralizes free IgE. But with an average peak response of 12-16 weeks in allergic asthma and 1 -4 weeks in chronic spontaneous urticaria, the onset of action is too slow to warrant its therapeutic use in acute anaphylaxis.

[00120] We have described a class of kinetically disruptive IgE inhibitors based on designed ankyrin repeat protein (DARPin®) scaffolds. These anti-lgE binders not only neutralize free IgE but actively remove pre-bound IgE from FcsRI on the surface of allergic effector cells. The fusion of a disruptive DARPin® (E2_79) to a non-inhibitory anchor DARPin® (E3_53) results in a biparatopic anti-lgE binder (bi53_79) with markedly enhanced disruptive efficacy. These observations led us to ask whether kinetically disruptive anti-lgE inhibitors could be developed with sufficient potency and activity that they could terminate pre-activated allergic cascades and anaphylaxis. Using a directed evolution and structure-guided approach, we have engineered multifunctional anti-lgE DARPin® inhibitors that rapidly desensitize pre-activated human allergic effector cells in vitro and resolve ongoing systemic IgE-dependent anaphylaxis in vivo.

[00121 ] Structural basis of loE-anchorinq by the non-competitive E3 53 DARPin®. To gain insight into the binding epitopes of the bi-paratopic anti-lgE DARPin® bi53_79, we crystallized IgE-Fcs- 4 complexes with E3_53 and E2_79 (Fig. 5a-d) and solved the structure to a resolution of 2.8A (Table 4). We found that E3_53 binds IgE distal to the FcsRIa binding site and parallel to E2_79, placing it proximal to the CD23 binding site (Fig.1a, Fig. 5e-f). Both DARPin®s bind IgE in similar N-C orientations (Fig. 1 b), and E3_53 fused to the E2_79 C-terminus interferes with disruption activity, while E3_53 fused to the E2_79 N-terminus markedly enhances disruption. This structure reveals that the nonfunctional C-terminal fusion (bi79_53) spans a longer solvent accessible surface distance (SASD) on a single IgE chain than N-terminal fusions, potentially accounting for its reduced activity relative to bi53_79 (Table 5).

[00122] The E3_53:lgE interface buries -1000 A² of surface area on each IgE chain across the CE3 and CE4 domains. The IgE CE4 domain F-G loop protrudes prominently into a pocket formed by the N-terminal capping domain, internal-repeat 1 (IR1 ), and internal-repeat 2 (IR2) of E3_53 (Fig. 1c). This positions lgE-P533 in a hydrophobic pocket adjacent to E3_53-T48, L53, Y56, and L86 (Fig. 1d). Internal-repeat 3 (IR3) makes extensive contacts with IgE, including a salt bridge between Ig E-R525 and E3_53-D1 11 , and multiple hydrogen bonds with both the AB and EF helices of CE3 (Fig. 1c). Interactions also occur within the invariant E3_53 C-terminal capping domain, including a salt bridge between lgE-K352 and D156 of E3_53, and interactions between E3_53-F145 and a hydrophobic patch formed in CE3 by F346, I350, and R351 (Fig. 1c). E2_79 interactions with IgE are similar to those observed in a previously determined structure.

[00123] IgE exhibits conformational flexibility in its Cs3 domains relative to the stable CE4 dimer. The flexible CE3 domains adopt an open conformation when binding to the FCERIQ, while CD23- bound IgE adopts a closed conformation. These receptor interactions are mutually exclusive. Although E3_53 does not compete with FcsRIa, the structure reveals that it interacts with both CE3 and CE4 domains, raising the possibility that E3_53 could act as a partial allosteric modulator and enhance bi53_79 disruptive activity. Comparison of E3_53:lgE interface residues in the FcsRIa complex (open) and the E3_53 complex (closed) shows minor displacements in the E3_53 epitope within the Cs3 AB and EF helices (Fig. 1d). In contrast, a major portion CD23 interactions, a known allosteric modulator, occurs with the mobile CE3 CD loop (Fig. 1 e-f).

[00124] To experimentally examine the impact of IgE conformational changes on E3_53 interactions, we measured the kinetics of E3_53 binding to free IgE and lgE:FcsRla complexes (Fig. 1g and Table 6). E3_53 dissociates slightly faster from lgE:FccRla complexes, yet has a similar affinity for both free and receptor bound IgE, consistent with prior equilibrium binding assays. We also determined the half-maximal disruptive concentrations (DC50) of E3_53, E2_79, a mix of E2_79 and E3_53, and bi53_79. FCERIQ coupled beads were loaded with biotinylated- lgE-Fc_2-4 (blgE-Fc₂-4), washed, and treated with each agent or mix of agents and assayed for remaining blgE-Fc_2-4 (Fig. 1 h). The E2_79 DCso is modestly improved in the presence of soluble E3_53 but is dramatically improved when covalently fused to E3_53 in bi53_79. E3_53 also slightly enhances the loss of bead-bound IgE alone. These results confirm that E3_53 primarily acts by anchoring E2_79 to complexes in bivalent molecules, but it may also be a weak allosteric modulator and/or catalyst for E2_79 mediated disruption.

[00125] High affinity anchoring domains increase disruptive potency. Given the anchoring role of E3_53 in enhancing bi53_79 disruptive activity, we hypothesized that increasing the dwell time of E3_53 on lgE:Fc£Rla complexes could further improve the potency of bi53_79. We therefore engineered a high affinity E3_53 variant using yeast display (Fig. 2a). A -3.0 x 10⁸ transformant error prone library was generated from E3_53, using a T48N mutation to remove a potential N- linked glycosylation site. Libraries were incubated with preformed blgE-Fc₂-4:Fc£Rla complexes, washed, and then exposed to unlabeled IgE for 1 -4 hours to isolate slowly dissociating variants retaining blgE-Fc₂-4 over five rounds of selection (Fig. 2a). These selections dramatically slowed the effective off-rate of blgE-Fc₂-4:Fc£ la complexes in competitive binding experiments on yeast (Fig. 6a). After five rounds, a shuffled mutant library was generated to enrich for favorable and exclude unfavorable mutations (Fig. 6b). Following a further selection round, clone E07 was chosen for characterization based on the number of enriched mutations (Fig. 2b). E07 showed enhanced binding affinity to free IgE and lgE:Fc£Rla complexes, primarily driven by a 7-10-fold slower dissociation rate (Fig. 2c, Table 6). E07 maintains a similar preference for free IgE as E3_53, with ~2-fold weaker binding to intact lgE:Fc£Rla complexes.

[00126] We produced E07 N-terminal fusions to E2_79 (Fig. 7a, b) and the resulting biE07_79 showed significantly improved IgE disruption in bead-based and SPR IgE disruption assays (Fig. 2d,e). Although the half-maximal disruptive concentration (DC₅o) of biE07_79 was lowered, IgE release plateaued prior to achieving full dissociation obtained with E2_79 or bi53_79 in beadbased assays (Fig. 2d). In SPR experiments, a similar effect is observed despite the shorter contact times and continuous flow (Fig. 2e), although the SPR signals are confounded b concurrent formation of inhibitor:lgE:Fc£Rla complexes. However, the enhanced DC50 directly correlates with the increased affinity of the E07 anchoring domain (Fig. 7c).

[00127] We next assessed whether biE07_79 is superior to bi53_79 in desensitizing allergic effector cells. We loaded bone marrow derived mast cells from human FcsRIa transgenic mice (BMMC*⁹) with human NIP-specific JW8-lgE, treated them for 30 minutes with bi53_79 or biE07_79 and subsequently stimulated them with NIP-BSA. While biE07_79 reduced cell surface IgE levels and inhibited antigen-mediated cell activation more efficiently than bi53_79, it also induced spontaneous activation of the BMMCs (Fig. 2f-h). These results suggest that bi E07_79 induces cell activation in the absence of antigen by crosslinking lgE:Fc£Rla complexes. Since we did not observe anaphylactogenicity with bi53_79 this is directly related to the affinity of the E07 anchoring domain.

[00128] Linker redesign and FcyRllb targeting improves IgE disruptive activity and effecter cell inhibition, while eliminating intrinsic anaphylactogenicity. bi E07_79 anaphylactogenicity could be caused by the more stable dwell time of the E07 anchor enabling the binding and crosslinking of neighboring Ig E: FcsRI complexes through the E2_79 domain. We therefore reengineered the 20 AA long (G4S)4 linker to reduce the possibility of receptor crosslinking by truncating the linker, yet the truncation reduced disruptive activity and induced higher anaphylactogenicity compared to the original biE07_79 comprising a (G4S)4 linker (Fig. 8a-c). We next sought to geometrically constrain the relative positions of the anchoring and disruptive modules through a knobs-in-holes (KIH), heterodimeric IgGi-Fc fusion. For this second approach, short linkers were used to fuse E07 and E2_79 DARPinOs to the C-termini of the KIH IgG-Fc, which aligned well with the N- termini of the two DARPinOs (Fig. 3a). The KIH_E07_79 construct not only provides a more structured linker between the two DARPin®s, but it also provides the potential for the IgG-Fc to engage FcyRllb inhibitory receptors to further suppress anaphylactogenicity. As further comparators to these constructs, we generated two additional trivalent DARPin®s, by fusing the FcyRllb-specific DARPin® D1 1 to the N-terminus of bi53_79 and biE07_79 to generate trivalent variants (triD1 1_53_79 and triD1 1_E07_79) with the (G4S)4 linker (Figure 3a). KIH_E07_79 showed increased disruptive activity and lower plateau in bead- and SPR-based assays while both trivalent variants showed reduced disruptive efficacy (Fig. 3b, c) compared to their bivalent counterparts (Fig. 2d,e).

[00129] All three proteins (KIH_E07_79, triD1 1_53_79 and triD11_E07_79) were non- anaphylactogenic in BMMC’⁹, even though D11 has a 1000-fold lower affinity for murine FcyRllb compared to its ~1.9nM Kd for the human receptor (Fig. 3d). In cell-based (BMMC*⁹) assays, KIH_E07_79 stripped IgE with higher efficacy than tri11_53_79 or tri1 1_E07_79 (Fig. 3d), consistent with results from bead-based and SPR IgE stripping assays. KIH_E07_79 removed IgE from cells within 30 minutes and showed the highest inhibitory potency in blocking antigen- induced BMMC’⁹ activation at concentrations as low as 4-20 nM (Fig. 3d). Together, these data indicate that conformational positioning of the anchoring and disruptive domains on the lgG1 -Fc scaffold enhances intrinsic IgE disruption activity and eliminates anaphylactogenicity of bispecific anti-lgE binders. In addition, the loss in disruptive efficacy of the trivalent DARPin®s is significantly compensated for by FcyRllb engagement.

[00130] We next compared the activities of these constructs using primary human basophils isolated from grass-allergic patients. To examine the impact of human FcyRllb, basophils were incubated with tri11_53_79, tri1 1_07_79 or KIH_E07_79 with and without prior blocking of FcyRllb. As observed on BMMC’⁹ (Fig. 2f), biE07_79 was also anaphylactogenic on isolated primary human basophils (Fig. 9a). In contrast, KIH_E07_79, tri1 1_53_79 and tri1 1 _07_79 did not activate human basophils, even in the presence of FcyRllb blocking (Fig. 3e). The trivalent DARPin®s and KIH_E07_79 were remarkably more efficient in stripping IgE than biE07_79 or omalizumab (Fig. 9a, b) and blocking of FcyRllb partially reduced their potency (Fig. 3e). These results indicate that co-engagement of human FcyRllb with both high affinity (i.e. DARPin® D1 1 ) and low affinity (i.e. lgG1 -Fc) domains increases the disruptive efficacy of the inhibitors. Notably, the relatively weaker IgE stripping by the trivalent DARPin®s in the murine BMMC*⁹ is greatly improved in the presence of the high affinity D1 1 interaction with human FcyRllb in the basophils.

[00131 ] To assess the impact on basophil activation of the different variants, we stimulated basophils with a 6-grass allergen mix after 30 minutes of inhibitor treatment (Fig. 3e). Neither biE07_79 nor omalizumab showed any inhibition of basophil activation in this experimental setup (Fig. 9a-b). However, KIH_E07_79, tri11_53_79 and tri11_E07_79 all abrogated allergen- mediated basophil activation at low nanomolar concentrations (Fig. 3e). Blocking of FcyRllb partially reversed the inhibition, which could depend on enhanced, FcyRllb-dependent recruitment to the basophils surface or on inhibitory signaling through FcyRllb ITIMs. We examined intracellular FcyRI Ib-phosphorylation in the presence of the inhibitors with or without FcyRllb blocking (Fig. 3f). Both trivalent constructs induced an inhibitory signal through FcyRllb phosphorylation, but this was not the case for KIH_E07_79. All three constructs are highly efficient in removing IgE from human primary basophils, although KIH_E07_79 shows the greatest intrinsic biochemical activity. Furthermore, the data indicate that the trivalent inhibitors exhibit increased potency on human cells through activation of inhibitory FcyRllb signaling.

[00132] KIH E07 79 rapidly desensitizes allergic effector cells and resolves pre-tiooered anaphylaxis in mice. We selected KIH_E07_79 to assess in vivo efficacy in mice, because it showed the most potent activity in biochemical and mouse cell assays. In addition, the KIH_E07_79 lgG1 -Fc domains can interact with the murine neonatal Fc-receptor, extending serum half-life in mice. Omalizumab was used for comparison in these studies. Double transgenic mice expressing human IgE and FcsRIa were epicutaneously sensitized with ovalbumin by daily topical co-application with the vitamin D analogue MC903 (Fig. 4a). This induces a strong Th2 response leading to systemic sensitization with expanded eosinophil, basophil and mast cell pools. Sensitized mice received two injections of either KIH_E07_79, omalizumab or PBS on days 14 and 15. Compared to EtOH control treated mice, sensitization with MC903 and OVA significantly increased total serum IgE and cell surface IgE levels on blood basophils (Fig. 10a-c). These levels remained unaltered in both the PBS and omalizumab treated groups 6 hours after the first injection (Fig. 4b). Strikingly, KIH_E07_79 rapidly reduced cell surface IgE to baseline levels after the first injection. After the second injection on day 15, cell surface IgE levels remained at baseline for KIH_E07_79 treated mice and were also reduced in the omalizumab treated group, consistent with the rapid 2-day turnover of basophils and a reduction in available free IgE. We also quantified IgE levels on peripheral mast cells in the skin distant from the treatment injection site by flow cytometry. While omalizumab only showed an intermediate decrease in surface IgE compared to the PBS control group within the two days of treatment, KIH_E07_79 reduced IgE levels on peripheral skin mast cells to baseline levels (Fig. 4c). These data indicate that KIH_E07_79 is a fast acting disruptive IgE inhibitor that rapidly desensitizes allergic effector cells in circulation and peripheral tissues in vivo.

[00133] Given the remarkable efficacy of systemic desensitization with KIH_E07_79, we sought to test its immediate impact on an acute anaphylactic reaction. We used a passive systemic anaphylaxis model in human FcsRIa transgenic mice. Mice received one injection of recombinant human NIP-specific JW8-lgE to systemically sensitize allergic effector cells. The next day, they were challenged with NIP-BSA antigen to induce systemic anaphylaxis. KIH_E07_79 or omalizumab were applied 5 minutes post antigen stimulation and we monitored changes in body core temperature as a read-out for systemic anaphylaxis (Fig. 4d) . Compared to omalizumab, KIH_E07_79 reduced the maximal drop in body core temperature by 50% and shortened the recovery time (Fig. 4e).

[00134] Finally, to investigate whether KIH_E07_79 may also resolve an ongoing allergic reaction in human cells, we collected whole blood of three grass allergic patients and performed a basophil activation test ex vivo. Whole blood was incubated with two concentrations of grass allergen mix. After 5 minutes, we added either KIH_E07_79, omalizumab or PBS to the stimulated whole blood and analyzed the samples 20 minutes later by flow cytometry for basophil activation (Fig. 4f). While KIH_E07_79 suppressed 80-90% of basophil activation, omalizumab showed only marginal effects (Fig. 4g). These results further demonstrate the remarkable potency of KI H_E07_79 to rapidly terminate pre-initiated allergic effector cell signaling consistent with its ability to systemically block an acute anaphylactic reaction.

[00135] We have applied structural, biophysical and immunological studies to engineer anti-lgE agents that can rapidly disarm effector cells in minutes and interrupt the allergic signaling cascade. Consistent with previous models for the facilitated dissociation of IgE: FceRI complexes, improving the affinity and dwell times of the anchoring domain of bivalent anti-lgE inhibitors correspondingly improved their disruptive activities. Importantly, we demonstrate approaches that eliminate anaphylactogenicity that emerged during affinity maturation of our bivalent anti- lgE inhibitors, which also enhance biochemical and therapeutic potency. The KIH_E07_79 inhibitor achieves its potent biochemical and therapeutic activities by incorporating a heterodimeric human lgG1 -Fc to restrict the positioning of its two anti-lgE DARPin® elements and to engage inhibitory FcyRllb receptors on effector cells. The lgG1 -Fc fusion should also enhance KIH_E07_79 serum half -life and tissue transport in vivo. To our knowledge KIH_E07_79 represents the first example of an anti-lgE agent that can act as a rescue therapy for acute allergic reactions, to terminate pathologic IgE-mediated inflammatory responses at their source, without the need to target the myriad of downstream inflammatory pathways.

Methods

[001361 Recombinant Proteins, Antibodies and Media. ICIE-SUS1 1 and JW8-lgE were purchased from NBS-C BioScience (Vienna, Austria). The anaphylactogenic monoclonal anti-lgE antibody Le27 was kindly provided by Dr. Monique Vogel. Monoclonal anti-human CD40 antibody was purchased (Enzo Life Sciences, NY, USA). Recombinant extracellular part of human FceRla were produced in our laboratory as previously described. Recombinant human CD23 was purchased (R&D Systems, Minneapolis, MN, USA). Recombinant human IL-3 and IL-4 were purchased from Peprotec (London, UK). Cells were cultured in RPMI^+/+ medium composed of RPMI 1640 medium (Biochrome, Cambridge, UK) complemented with 10 % Hyclone FCS (Fisher Scientific, NH, USA), Penicillin 100 U per mL, 100 pig per mL Streptomycin (100x Penicillin/Streptomycin, Merck, Darmstadt, Germany) and 10mM HEPES buffer (Stock-Solution 1 M, Life Technologies, CA, USA). For flow cytometry we used the following antibodies: antihuman IgE FITC (clone Ige21 , Thermo Fisher Scientific, MA, USA), monoclonal mouse antihuman FcsRIa APC (clone AER-37, Thermo Fisher Scientific, MA, USA) and the appropriate isotype controls monoclonal mouse lgG1 ,K Isotype control FITC (Thermo Fisher Scientific, MA, USA) and mouse lgG2b Isotype control APC (Thermo Fisher Scientific, MA, USA), monoclonal rat anti-mouse CD200R FITC (clone OX-1 10, Bio-Rad, CA, USA), monoclonal rat anti-mouse CD1 17 PE (clone 2B8, Thermo Fisher Scientific, MA, USA), monoclonal mouse anti-human CD19 APC (clone HIB19, BD Bioscience), monoclonal mouse anti-human CD23 PE (clone EBVCS-5, Biolegend, CA, USA). For basophil activation testing the anti-human CCR3 and antihuman CD63 antibody staining mix from the Flow CAST® kit was used (Buhlmann Laboratories AG, Schbnenbuch, CH).

[001371 Prote/ expression. DARPin®s were synthesized and cloned into the pQE-30 vector between BamHI Hindi II restriction sites in XL-1 blue cells. Cells were grown to an OD600 of 0.6 in dYT media, induced in with IPTG at 1 mM for 4-6h at 37°C, pelleted, and resuspended in lysis buffer (40 mM Tris-HCI, pH 7.5, 300 mM NaCI, 20 mM imidazole, 10% glycerol, and 1 mg/ml lysozyme). Resuspended cells were subjected to three freeze thaw cycles, and then sonicated with immersion sonicator. Lysates were treated with EDTA free protease inhibitors (Pierce Cat A32965) per manufactures instructions, clarified by centrifugation at 30,000xg for 20 minutes, and filtered through 0.45pM filter. Filtered lysates were then applied Ni-NTA resin column, washed with 10 column volumes of 25mM imidazole in PBS at pH 7.4, and eluted with 300mM imidazole in PBS at pH 7.4. Eluted supernatants were further polished with size exclusion chromatography (SEC) on a S200 10/300 GL or HiLoad 16/60 S200 columns with PBS as a running buffer. Samples used for cellular assays were also treated with endotoxin removal resin (Pierce), and sterile filtered through 0.2pM syringe filter. E07 and KIH_E07_79 fusion proteins were expressed and purified using pTT5 vectors and HEK-293-6E cells (NRC). Briefly constructs were cloned into pTT5 vectors and transiently expressed as soluble proteins in HEK-293-6E cells. Supernatants were applied to Ni-NTA resin columns, washed with 10 column volumes of 25mM imidazole in PBS at pH 7.4, and eluted with 300mM imidazole in PBS at pH 7.4. Eluted supernatants were further polished with SEC on a S200 10/300 GL or HiLoad 16/60 S200 columns with PBS as a running buffer. lgE-Fc3-4 used for crystallization was digested with TEV protease prior to SEC purification, subjected to reverse Ni-NTA chromatography, and polished by SEC as noted above.

[00138] Crystallization. E3_53, E2_79, and lgE-Fc3-4 were mixed in equimolar ratios with E3_53 and E2_79, and intact complexes were purified by SEC in 20 mM Tris-HCI, pH 8.0, 50mM NaCI and concentrated to 7mg/ml and mixed 1 :1 with acetate buffer pH 4.6, 43% ethylene glycol w/v in hanging drops at 18°C. Crystals formed overnight, were harvested in well solution, and were flash frozen in liquid nitrogen for data collection.

[00139] Crystallographic data collection and refinement. Data was collected at SLAG on beamline. Structures were solved by molecular replacement in phenix using isolated Ce3 and Ce4 domain models from 4GT7, and E2_79 model derived from 4GRG, and E3_53 model derived. The resulting model was then subjected to a round of automatic building followed by multiple rounds of manual building and refinement in PHENIX.

[00140] Yeast library construction. Fresh electroporated cells were produced using previously published protocols. Insert DNA was amplified using the GeneMorph II random mutagenesis kit (Agilent), band purified, and further amplified with Platinum II Hot Start Mastermix (ThermoFischer). 50ug of insert DNA was mixed with 10ug of triple-cut pCTCon2 vector (Nhel, BamHI, Sall) and electroporated into electrocompetent yeast at 2500V in 2mm gap cuvettes in BioRad MicroPulser. Transformed cells were rescued in 50/50 YPD/1 M Sorbitol for 1 hour and then transferred to SD-CAA.

[001411 Staining for flow cytometry and FACS. Staining was performed in PBE buffer (PBS with 0.1% w/v BSA, 2.5mM EDTA, with penicillin 100U/ml_ and Streptomycin 100ug/mL) with reagents as indicated. The following secondary reagents were used: Streptavidin-AF647 (S21374-ThermoFisher) at 1 :400, anti-cMyc-lgY (ACMYC Gallus Immunotech) at 1 :400, anti- chicken-PE (EMD Millipore AP503H). Compensation controls were made with single stained yeast or OneComp eBeads (ThermoFisher 01-1111-41 ).

[001421 MACS and FACS selections. Yeast were induced for 24 hours at 30°C in SG-CAA overnight prior to selections. Tenfold the estimated library diversity (or a minimum of 10O tl of saturated overnight culture in later rounds) were used for selections. During macs selections yeast were subject to negative selection with magnetic binder beads and LS columns and subsequently stained as described per round. Yeast were then washed once in 50mL PBE and resuspended in PBE with magnetic binder beads (Miltenyi Biotec) and positively selected with LS columns and one 5mL PBE wash. The remaining yeast were eluted and enumerated on an Accuri Cytometer to estimate library recovery and resuspended at an OD600<1 in SD-CAA media for expansion. MACS selections were repeated until the estimated diversity was sufficiently low to allow for FACS selections (1 E5-1 E6 estimated diversity). FACS selections were performed as indicated using the reagents and protocols as outlined above on FACS Jazz Sorter in the Stanford Shared Sorting Facility.

[00143] Protein interaction measurements with SPR. All SPR measurements were carried out on a GE Healthcare Biacore X100 device (IL, USA). HBS-EP⁺ was used as running buffer at a flowrate of 10 pl/min. The target proteins were immobilized on flow cell 2 (Fc2) of a CM5 sensor chip by standard amine coupling. A blank immobilization was performed with the reference flow cell 1 (Fc1 ). The sensorgrams reflect binding responses on Fc2 minus binding responses on the reference Fc1. To determine binding kinetics, we used the BIAevaluation software. Affinity constants were calculated using a 1 :1 langmuir curve fitting model.

[00144] To assess whether anti-lgE DARPinOs disrupt FcsRIccIgE complexes, 1000 RU of recombinant human FcsRIa was immobilized on flow cell 2 at pH 4.0. A concentration of 20 nM Susi 1 -lgE was subsequently captured for 120 seconds to reach a response >100 RU. Various concentrations (2.5pM-0.078pM) of anti-lgE DARPinOs were injected for 120 seconds with a dissociation time of 180 seconds between each injection under constant buffer flow. At the end of each run the chip surface was regenerated with 50 mM NaOH and reloaded with Susi 1-lgE.

[00145] Culture, characterization and differentiation of BMMC^t9. Bone marrow-derived mast cells (BMMC’⁹) from huFcsRIa-transgenic mice ((B6.Cg-Fcer1 atm1 Knt Tg(FCER1 A)1 Bhk/J) were obtained by culturing femoral bone marrow cells in I L-3-containing medium for 3 to 4 weeks in BMMC culture medium MC/9 supplemented with 1 mM Sodium Pyruvate, 200mM L-Glutamine, 1 x Non-essential Amino Acids, 50 M |3-Mercaptoethanol, 30ng/ml mouse recombinant IL-3). BMMCs were characterized as CD1 17+, human FCERICC+ cells by flow cytometry.

[00146] Anaphylactogenicity, disruptive efficacy and inhibitory potency with BMMC'⁹. First. BMMC were sensitized with 3nM of human monoclonal chimeric IgE NIP-specific (murine lambda chain), JW8 overnight at 37° C in 5% CO2 atmosphere. Upon sensitization cells were washed three times with phosphate buffer saline (PBS) pH 7.4. Subsequently disruptive anti-lg Es inhibitors were diluted in supplemented BMMC culture medium MC/9 and were added to the sensitized cells for 30min. Thereafter the cells were washed and spitted into two fractions. The first fraction was used to measure the remaining surface IgE with a monoclonal anti-mouse anti-murine lambda chain - PE and the anaphylactogenicity by an activation marker staining, which correlates to cell degranulation. The second fraction of treated BMMCs were stimulated with 100ng/mL NIP(7)-BSA (Biosearch Technologies, INC) in the presence of activation marker staining.

[00147] Blood and basophil donors. Human peripheral whole blood was obtained from gras- allergic volunteering donors, which were provided informed consent in accordance with the Helsinki Declaration. The study was approved by the local ethics committee.

[00148] Primary human basophil isolation and culture. Primary human basophils were obtained from whole blood by using Percoll density centrifugation of dextran-sedimented supernatants und basophils were further purified with negative selection using the Milteny basophil isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). Basophil culture medium (RPMI+/+) was composed with RPM1 1640 medium (Biochrome) complemented with 10 % Hyclone FCS (Fisher Scientific), Penicillin 100 U per mL, 100 pg per mL Streptomycin and 10mM HEPES buffer (Stock-Solution 1 M, Life Technologies). As basophil survival-factor, recombinant human IL-3 from Peprotec (United States) was added.

[00149] Anaphylactogenicity, disruptive efficacy and inhibitory potency with primary human basophils. To assess anaphylactogenicity, disruptive efficacy and inhibitory potency of disruptive anti- Ig E DARPin®s, we cultured isolated primary human basophils at a density of 1 x10⁶ cells/ml per well in a 96 well round bottom plate in RPMI+/+ supplemented with 10 ng/ml recombinant human IL-3 overnight (Falcon, Tewksbury, MA, USA). To assess the inhibitory potency of IgE- dependent basophil activation, we first determined the 6-grass allergen-mix concentration (Biihlmann Laboratories AG, Schonenbuch, CH) to be used to reach suboptimal activation (ECSubt.) for each donor individually. For this, we cultured 25’000 basophils dose-dependently with the 6-gras allergen mix on 25’000 cells in RPMI+/+ containing human 10 ng/ml recombinant human IL-3 and Flow CAST® kit antibody staining (activatin staining of CD63 and CCR3) and measured activated cells by flow cytometry. In next step, we pre-incubated 50,000 basophils with FcyRllb-blocking reagent for 15 minutes and added directly the disruptive anti-lgE DARPinOs for another 15 minutes. Thereafter, the cells were three times washed and separated into two equal fraction. The first fraction was used to measure the remaining surface IgE with an anti-human IgE-PE antibody (eBioscience ) and for the anaphylactogenicity with an anti-human LAMP3 FITC (CD63), an activation marker staining, which correlates to cell degranulation. The second fraction we used to stimulate the cells with the ECSubopt. of the 6-gras allergen mix in the presence of Flow CAST® kit antibody staining (activation staining of CD63). Finally we determined activated basophil frequencies and surface measurements by flow cytometry.

[001501 Whole Blood Basophil Activation Assays. Whole blood experiments with grass-allergic volunteers were carried out according to the manufacturer’s manual (Buhlmann Laboratories AG). EDTA-blood was incubated with 1 and 3 ng/mL 6— grass allergen mix (Buhlmann Laboratories AG) and immediately after 5min KIG_E07_79 and Omalizuamb were added for another 20 minutes. Activated basophils were identified as CCR3+ and CD63+ cells by flow cytometry.

[00151 ] Western Blot analysis. The experiment was performed similar to the set-up in the previous section, where we tested the functional potency with purified basophils, but we used 2.5 x 10⁵ isolated basophils per condition. Thereafter, proteins were precipitated out of cell suspension with 10% trichloroacetic acid, separated in NuPAGE 12% Bis-Tris gels from Thermo Fisher and transferred to Invitrolon PVDF membranes (Invitrogen, USA). Antibodies used for detection were anti-phospho-FcyRIIB (phosphoY292) antibody (Abeam, UK), and anti-GAPDH (Merck Millipore, USA). Secondary HRP-labelled goat anti-rabbit and goat anti-mouse were purchased from BioRad (Hercules, USA). Immunoreactive bands were visualized by enhanced chemiluminescence ECL-Kit (GE Healthcare).

[001521 Active sensitization mouse model. Mice double transgenic for the human epsilon heavy chain and the human alpha subunit of FCERI were purchased from Genoway (Lyon, France). These mice were bred and housed in specific pathogen-free conditions at the University of Bern. All animal experimentation was approved by the local ethics committee (authorization BE66/18). [00153] To induce active sensitization, we applied 20pL of 2nM of MC903 solved in 100% EtOH on both ears of the mice (calcipotriol; Tocris Bioscience, Bristol, United Kingdom). After drying, 100 pg of OVA (A5503, Sigma-Aldrich) in 20 pl of PBS was distributed to both ears. All mice were treated for 13 days. At day 12, we collected submandibular blood at an untreated timepoint. At day 14 and day 15, we treated the mice with 200pL of 6.66pM anti-lgE inhibitors (Omalizumab and KIH_E07_79) by intraperitoneal injection. Six hours after the first injection we collected peripheral blood, respectively, scarified the mice at day 15 whereby we collected skin (ears) and peripheral blood for further analysis. Single cells suspensions were prepared and analyzed by flow cytometry.

[00154] Single-cell suspensions of indicated tissues were incubated with DAPI and murine Fc- block for dead cell exclusion and stained with anti-mouse fluorochrome-conjugated mAbs against CD4 (RM4-5), CD8(53.6.7), CD19 (6D5), NK1 .1 (PK136), IgE (Ige21 ), CD45 (30-F1 1), CD49b (DX5), c-Kit (2B8), CD200R3(Ba13). Peritoneal mast cells were identified as live Lin' (CD19, NK1 .1 , CD4/CD8), CD45⁺, c-Kit⁺, CD200R3+. Skin and blood basophils were identified as live Lin", CD45⁺, and CD200R3⁺ cells coexpressing CD49b. All samples were acquired on an LSRII (BD Biosciences, San Diego, Calif) and analyzed with FlowJo software version 10.3.

[001551 Passive cutaneous anaphylaxis mouse model. Mice transgenic for human FcsRIa (huFceRla tg) on a mixed C57BL/6 J - C57BL/6 N background were obtained from Prof J.-P. Kinet. All animal experimentation was approved by the local ethics committee (authorization BE66/18). Seven days before the experiment, huFccRIa tg mice were subcutaneously implanted with an electronic temperature transponder (IPTT-300) from BMDS (Delaware, USA) to measure body-core temperature as instructed by the manufacturer. On day 0 mice were passively sensitized with 20 pg NIP-specific human JW8-lgE (in 200 pl) by intraperitoneal injection. On day 1 mice were challenged by intraperitoneal injection of 200 pg NIP20-BSA (Biosearch Technologies, Petaluma, CA, USA). After 5 minutes, the mice were treated with 200pL of 5pM omalizumab or KIH_E07_79 by intraperitoneal injection. Body-core temperature was measured before challenge (baseline) and every 10 min after antigen-challenge for 60 min and every 30 minutes until 120 min. Data is represented as measured temperature after challenge minus baseline temperature (A core body temperature) for each time point.

Table 4 | Data collection and refinement statistics (molecular replacement)

Data collection

Space group P 1 21 1

Cell dimensions a, b, c (A) 51-9O8, 137-793, 88.25 a, p, y (°) 90, 105.678, 90

Resolution (A) 37.8 - 2.8 (2.9 - 2.8)

Rmerge O.I948 (O.8242)

I/ oI 5.07 (0.66)

Completeness (%) 98.65 (98.37)

Redundancy 3.7 (3.7)

Refinement

Resolution (A) 37.8 - 2.8 (2.9 - 2.8)

No. reflections 29048 (2901)

Rwork / Rfree % 21-35 (27-75)/ 25-60

(34-66)

No. atoms

Protein 7371

Ligand/ion 116

B-factors

Protein 60.55

Ligand/ion 101.45

R.m.s. deviations

Bond lengths (A) 0.005

Bond angles (°) 0.94 Data collected from a single crystal at 100°K.

*Values in parentheses are for highest-resolution shell

Table 5 | Solvent Accessible Surface Distances for Bivalent DARPin®s

Anti-IgE r- SA , SD x N_T-C , Linker , le ,ngth . D _r i.sruptive

(+ unmodeled aa) bi79_53 85-90 A 77.9 A No* bi53_79 56-59 A 84.8 A Yes

*bi79_53 showed no enhancement in disruption as compared to monomeric E2_79.

Table 6 | Binding kinetics of ligelizumab and omalizumab variants for human IgE.

Anti-IgE Target Association k_a (M 's ¹) Dissociation k (s ¹) Affinity

E3_53 IgE 5.187 x 10⁵ 1.114 x IO'² 21.48

E3_53 IgE:Fc£RIa 4.499 x 10⁵ 2.197 x IO'² 48.82

E07 IgE 3.212 x lO⁵ 1.0921 x IO’³ 3.4

E07 IgE:FceRIa 4.060 x 10⁵ 3.127 x 10'³ 7.705

Example 2

Modification of Glycosylation Sites

[00156] Glycosylation sites external to the IgG-Fc pose a challenge during therapeutic development. Therefore a T296A mutation was introduced into the Knob_lgG1_E2_79 construct (SEQ ID NO:1 ) to remove potential N-linked glycosylation sites within the knobs in hole DARPin® fusion molecule KIH_E07_79. This mutation yielded the Knob_lgG1_E2_79_non-glycosylated construct (SEQ ID NO:6). The T296A mutation was chosen based on analysis of the crystal structure presented in Fig. 1 for its ability to preserve the DARPin® framework and binding interface of the DARPin® E2_79 with IgE.

[00157] The same amino acid substitutions provide for the production of a non-glycosylated form of DARPin® E2_79 (SEQ ID NO:4), provided herein as DARPin® E2_79_non-glycosylated (SEQ ID NO:7). The amino acid substitution may be noted as T64A when referencing E2_79, which can be produced as a non-glycosylated fusion protein or monomer in eukaryotic expression systems.

[00158] Co expression of the Knob_lgG1_E2_79_non-glycosylated (SEQ ID NO:6) with Hole_lgG1_E07 (SEQ ID NO:2) yielded a functional heterodimer of the expected mass as assessed by SEC (Fig. 1 1 a) that was able to disrupt preformed IgE receptor complexes to a similar degree as the parental glycosylated molecule (Fig. 11 b).

Example 3 [00159] The potency of disruptive anti-lgE binders (such as E2_79, or non-glycosylated E2_79 (E2_79_aGly)) can be dramatically increased via covalent fusion to anchoring modules (such as E3_53 or E07). This enhancement of disruption cannot be reproduced with a soluble mixture of disruptive and anchoring monomers (Figure 12a). This strategy is applicable to antibody-based disruptors as well (Figure 12b). Therefore, structural studies were undertaken to understand the relationship of anchoring domains to disruptive domains to define the parameters required for the interdomain linkages of bivalent disruptors (such as bi53_79 or knob-in-hole (KIH) lgG1 heterodimers containing disruptive and anchoring domains).

[00160] The structure of the IgE-Fc in complex with anchoring (E3_53) and disruptive (E2_79) DARPinOs revealed that both agents bind parallel along the IgE-Fc in the same N-to-C terminal orientation (Figure 12c). Measurement of the point-to-point distance of all possible terminal fusions of DARPinOs on an IgE chain was used to define the minimum possible linker length for each fusion (Figure 12d). For DARPin® N-to-N terminal fusions, no intervening portions of the IgE-Fc, DARPin®, or predicted FcsRIa position blocked linkage. In contrast all other linkages are restricted by intervening portions of the IgE-Fc and DARPin® domains and thus require additional linker lengths beyond the distance between termini (Figure 12d).

[00161 ] In fusions of the aforementioned DARPin® and antibody anti-lgE domains, linkers that afford between 30-100 A of spacing facilitate disruption. Any anchoring and disruptive anti-lgE binding domains (VHHs, scFvs, etc.) that bind these epitopes with this constrained spacing should also have improved disruptive function. Unstructured linkers (e.g. GS flexible linkers) that afford a distance of 30-100 A or a distance sufficient to join domains of moderate and high affinity anti-lgE domains (KD <20nM) produce non-specific crosslinking of lgE:receptor complexes and are a safety issue (Figure 2f, Figure 8b, Figure 12b inset).

[00162] We have shown that fusion of anchoring and disruptive domains to a KI H-lgG1 -Fc enhances disruption, and that part of this effect is mediated through lgG1 -Fc to FcR interactions (Figure 3e). Fusion to the lgG1 -Fc is also an established method to improve PK properties of therapeutic molecules such as half-life. We therefore employed to the E2_79:E3_53:lgE crystal structure to establish the parameters required for linking disruptive DARPin®s to IgG-Fc domains.

[00163] For C-terminal KIH-IgG fusions to the N-termini of anchoring and disruptive DARPin®s minimal linkers such as G4S (SEQ ID NOs: 1 , 2, 6, 17, and 18) are sufficient to mediate potent disruption (Figure 3). Further these relatively short linkers on the rigid IgG-Fc scaffold prevent off-target cellular activation (Figure 3d and e) seen with non-scaffolded flexible linkers of multiple lengths (Figure 2f, Figure 8b, Figure 12b inset).

[00164] For N-terminal fusion of disruptive and anchoring motifs, the native lgG1 hinge (SEQ ID NO:8), employed in fusion molecules such as etanercept, forms multiple interchain disulfide bonds restricting the length of the free N-terminus. Therefore, linkers with longer free N-termini are required. Mutants of the native hinge at position 220 (Eu numbering) abolish a cysteine residue responsible for HC to LC intrachain bonds in native lgG1 and HC-HC bonds in IgG-Fc fusions lacking a LC. This mutation eliminates disulfide bonds in the first ten residues of the hinge. Mutations such as C220S (SEQ ID NO:9) therefore facilitate up to 32.4 A of spacing as predicted by the length of the ten residues upstream of the HC-HC disulfide at position 226 of the lgG1 -Fc. Hinge mutants such as C220S can therefore span the distance between the C- terminus of anchoring and disruptive domains (Figure 12d). Such mutated hinges can be incorporated into N-terminal knob-in-hole heterodimers to produce N-terminal disruptive anti- Ig E agents (SEQ ID NO: 13 and 14).

[00165] Other formats, such as a fusion of a truncated N-terminal lgG1 hinge (SEQ ID NO: 10) to a G4S linker (Seq ID NO: 11 ) or to a (G4S)2 linker (SEQ ID NO: 12) also provide sufficient space between anchoring and disruptive domains. These can be generated as knob-in-hole heterodimers with symmetric hinges or asymmetric hinges such as (SEQ ID NO: 15 and 16).

[00166] While the aforementioned N-terminal lgG1 fusions are sufficient to link anchoring and disruptive domains as assessed in the crystal structure, they do not achieve the same exposed membrane proximal orientation of FcyR IgG-Fc binding domains as C-terminal IgG fusions (Figure 13a and b). We have demonstrated co-engagement of FcyRs by disruptive anti-lgE fusion molecules enhances potency (Figure 3e), and these structural observations demonstrate how C-terminal fusions position the IgG-Fc to enhanced potency.

[00167] All C-terminal or N-terminal KIH-IgG fusions of anchoring anti-lgE domains (such as E3_53 and E07) or disruptive domains (such as E2_79 or E2_79_aGly) can be constructed with anchoring domains on the hole arm and disruptive domains on the knob arm (such as SEQ ID NO:1 , 2, and 6) or with anchoring domains on the knob arm and disruptive domains on the hole arm (SEQ ID NO:13, 14, 15, 16, 17 and 18). During the production of KIH-IgGs hole-hole homodimers can constitute several percent of the final product yield, while knob-knob homodimers are rarely observed. Although off-target homodimers with disruptive domains (such as E2_79 or E2_79_aGly) will block and/or displace IgE from the high affinity receptor, homodimers of anchoring domains have the potential to crosslink lgE:receptor complexes and activate mast cells and basophils. In the absence of affinity tag purification schemes, placement of the disruptive domain on the hole arm of the knob-in-hole IgG will minimize potential reactive homodimer anchor species. Therefore such fusions (SEQ ID NO: 13, 14, 15, 16, 17 and 18) can minimize toxic off target species during KIH manufacturing.

Example 4

Optimization of knobs-in-holes IgG heterodimeric antiJgE fusion proteins:

[00168] Both C- and N-terminal fusions of disruptive (e.g. E2_79) and anchoring (e.g. E07) DARPin® domains to the IgG-Fc can facilitate bivalent binding to IgE. However, it is not obvious from structural studies if the Fc orientation or the spatial constraints of N-terminal fusions are compatible with potent IgE disruption (Figure 13). Therefore C-terminal (SEQ ID NO: 17 and 18) and N-terminal (SEQ ID NO: 13 and 14) KiH DARPin® fusions were produced and purified to homogeneity by nickel affinity chromatography. Chromatograms revealed that C-terminally fused material produced a single homogenous species, while N-terminal fusions exhibited a shoulder in the main peak (Figure 14).

[00169] To establish if these N-terminal fusions were functional we assayed N and C-terminal fusions for IgE stripping and spontaneous activation in human basophils using Buhlmann Flow CAST® basophil activation test (Figure 15). While C-terminal KiH_E07_79 fusions were safe and potent at IgE removal, N-terminal KiH fusions showed IgE removal and pronounced spontaneous activation of basophils (Figure 15). Because IgE crosslinking and activation on basophils is known to promote IgE internalization the extent of IgE removal in N-terminal treated samples cannot be interpreted. Therefore, these studies demonstrate that C-terminal fusion of the anchoring and disruptive DARPin®s to IgG-Fc is required for safety. Furthermore, DARPin® variants of E2_79 and E07 with similar epitopes and orientations should also require C-terminal fusion to the IgG-Fc.

[00170] In parallel we sought to establish if the hole or knob arm of the KiH heterodimer should be fused to the anchoring DARPin® domain (e.g. E07). Given the known propensity of KiH constructs to form off target hole-hole homodimers, we produced homodimeric C-terminal IgG- Fc fusions of E07 and E2_79 and assessed each for stripping and spontaneous activation (Figure 15). While neither homodimer stripped IgE effectively, only homodimers of the anchoring E07 domain spontaneously activated basophils (Figure 15). Therefore, placement of the anchoring domain on the knob arm of the KiH heterodimer can minimize reactive E07 homodimers during protein production.

[00171 ] Finally, to verify that anchoring and disruptive domains can be fused to either symmetric arm of a KiH molecule without perturbing potency, we exchanged the anchor and disruptor position in the KiH molecule. Both KiH heterodimers exhibited similar potency in human cells (Figure 16).

[00172] To further define the critical biochemical and structural parameters of KiH_E07_79 we explored the effect of five additional variables on the potency of KiH_E07_79 mediated IgE disruption: 1 .) the presence of a glycosylation consensus site in the E2_79 domain on the disruptive arm of KiH, 2.) the affinity of the anchoring E07 arm in the KiH heterodimer, 3.) the length of the linker arm on each side of the KiH heterodimer, 4.) the affinity of the disruptive arm of E2_79, and 5.) the potential of the KiH lgG1 -Fc fusion to interact with and activate IgG-Fc receptors.

[00173] 1.) Glycosylation consensus sequence in E2_79: We have shown that mutation of glycosylation consensus site found in E2_79 (SEQ ID NO: 4) to an aglycosylated variant E2_79(T64A) or “E2_79_aGly” (SEQ ID NO: 7) yields functional KiH heterodimers (SEQ ID NO: 2 and 6) that strip IgE in BLI IgE disruption assays (Figure 1 1 ). Removal of this glycosylation consensus sequence can be accomplished by multiple mutations to E2_79 such as N62D, N62Q, and T64A. Quantitative comparisons of aglycosylated and glycosylated E2_79 in KiH molecules shows that each has a similar disruptive potency in bead-based IgE removal assays (Figure 17A and D), although loss of the E2_79 glycan partially reduces potency in the E2_79(T64A) mutant variant.

[00174] 2.) Modulation of the affinity of the anchoring E07 arm of the KiH heterodimer: To assess the role of anchor domain affinity in KiH mediated disruption we employed a mutant of E07’s parental DARPin® E3_53, E3_53_T36N (SEQ ID 19). E3_53_T36N, also referred to as “E3_53 NxT” in previous figures and text, is a mutant that abolishes a glycosylation site in the E3_53 : Ig E interface and was isolated during preliminary rounds of E3_53 affinity maturation in yeast. E3_53_T36N was used as the basis for the affinity maturation library used to select E07 (Figure 2A) and shows dramatically faster off rates from IgE as compared to evolved E3_53 variants such as E07 in binding assays (Figure 6A). Substitution of E3_53_T36N into the KiH heterodimer with E2_79_aGly (SEQ ID 6 and 20) yields a functional anti-lgE disruptor with modestly reduced potency as compared to KiH_E07_79_aGly (Figure 17 B and D). This demonstrates that low affinity anchoring domains, which provide a broader safety window in other bivalent DARPin® constructs (Figure 2F), retain function in KiH IgG-Fc fusions. Other E3_53 mutants which contain only partial sets of the affinity matured E07 mutations, e.g. E3_53_T36D (SEQ ID NO: 21 ), could provide a range of anchor affinities to facilitate lower affinity anchoring.

[00175] 3.) Modulation of the linker arm lengths in the KiH heterodimer: T o assess the role of linker length in the KiH heterodimer, we explored the effects of shortening linkers within KiH on the potency of IgE disruption. We have previously demonstrated that unstructured linkers can pose a safety risk (Figure 2F) that can be rescued with a structured scaffold (Figure 3D). We have also demonstrated that linker truncation can negatively impact potency and safety in unstructured DARPin® fusions (Figure 8A-C). We therefore constructed truncated linkers in E2_79 and E07 KiH IgG-Fc fusion constructs (SEQ ID NO: 22, 23) which reduce the length of the C-terminal IgG-Fc fusion linker and removed the C-terminal IgG-Fc lysine. We then assayed the linker variants for IgE disruption in bead-based assays (Figure 17 C and D). Truncated linker combinations with two short linkers in the KiH heterodimer (SEQ ID NO 22 and 23), a shortened linker only on the anchoring arm (SEQ ID NO: 6 and 23), or a shortened linker only on the disruptive arm (SEQ ID NO: 22 and 2) all retained disruptive potencies similar to the original KiH molecules KiH_E07_79 (SEQ ID NO: 1 and 2) and KiH_E07_79_aGly (SEQ ID NO: 6 and 2). Thus these linkers, or linker of similar amino acid lengths should facilitate bivalent binding and disruption when fused to the IgG-Fc.

[00176] 4.) Modulation of the affinity of E2_79 disruptive domain in the KiH heterodimer: To address the affinity of the anchoring arm we explored mutations at two critical sites of the E2_79:lgE interface previously shown to be critical for IgE binding: crystal structure 4GRG residues Y45 and W46 (corresponding to positions Y33 and Y34 in SEQ ID NO: 4) (2). Double mutation of Y33A/W34A abolishes IgE binding. Therefore, we identified a series of novel single mutations to Y33 or W34 predicted to partially reduce E2_79s affinity for IgE. In the DARPin® scaffold these two positions are predicted to tolerate diverse amino acid substitutions, however the following series of mutations systematically alters the degree of hydrophobic interactions, polar interactions, and pi-pi stacking predicted to contribute to the IgE binding affinity of E2_79: E2_79_Y33A (SEQ ID NO: 24), E2_79_W34A (SEQ ID NO: 25), E2_79_Y33V (SEQ ID NO: 26), E2_79_W34V (SEQ ID NO: 27), E2_79_Y33L (SEQ ID NO: 28), E2_79_W34L (SEQ ID NO: 29), E2_79_Y33R (SEQ ID NO: 30), E2_79_W34R (SEQ ID NO: 31 ), E2_79_W34F (SEQ ID NO: 32), E2_79_aGly_Y33A (SEQ ID NO: 33), E2_79_aGly_W34A (SEQ ID NO: 34), E2_79_aGly_Y33V (SEQ ID NO: 35), E2_79_aGly_W34V (SEQ ID NO: 36), E2_79_aGly_Y33L (SEQ ID NO: 37), E2_79_aGly_W34L (SEQ ID NO: 38), E2_79_aGly_Y33R (SEQ ID NO: 39), E2_79_aGly_W34R (SEQ ID NO: 40), E2_79_aGly_W34F (SEQ ID NO: 41 ).

[00177] 5.) Silencing of the IgG-Fc fusion in the in the KiH heterodimer: To address potential detrimental IgG-Fc signaling through Fc receptors present on basophils and mast cells, fusion of anchoring and disruptive domains to the C-terminus of silenced variant Fc regions can reduce unwanted effector functions. For example the following silenced variant Fc regions would reduce unwanted binding to activating FcyRs (e.g. the Fey Fl I: FcyRIIA; FcyRIIBI; FcyRIIB2; FcyRIIIA):

[00178] A KiH lgG1 L234A/L235A “lgG1 -LALA” mutant (SEQ ID NO: 192 and 193)

[00179] An aglycosylated KiH lgG1 N297A “lgG1 -N297A” mutant (SEQ ID NO: 194 and 195) [00180] A KiH lgG4 S228P/L234A/L235A “lgG4-PAA” mutant KiH (SEQ ID NO: 42 and 43) are all predicted to improve the safety profile of anti-lg E disruptive agents.

[00181 ] Optimized knobs-in-holes IgG heterodimeric antiJgE fusion proteins: Considering the studies to optimize the safety, potency, and biochemical properties of disruptive KiH variants the following combinations of modified anchoring domains, disruptive domains, linkers, and IgG-Fc fusions can represent functional disruptive anti-lg E KiH IgG heterodimers set forth in Table 3.

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Claims

WHAT is CLAIMED is:

1. A therapeutic protein comprising:

(i) a highly disruptive anti-lgE moiety that disrupts the binding of IgE to FceR1 a; and (ii) a high affinity anti-lgE binding moiety, wherein (I) and (II) are linked in a structurally constrained format; and wherein the protein can strip IgE from pre-formed complexes on the surface of immune effector cells in a clinically relevant time.

2. The protein of claim 1 , further comprising (iii) a moiety that binds FcyRllb.

3. The protein of claim 1 or claim 2, wherein the protein is a heterodimer, comprising moiety (i) on a first polypeptide, and moiety (ii) on a second polypeptide.

4. The protein of claim 3, wherein the first and second polypeptides each comprise IgG constant region sequences.

5. The protein of claim 3 or claim 4, wherein the first and second polypeptides are associated.

6. The protein of claim 5, wherein the first and second polypeptides are associated in a knob in hole (KIH) configuration.

7. The protein of any of claims 4-6, wherein moieties (i) and (ii) are directly fused to the IgG constant region sequences.

8. The protein of any of claims 4-6, wherein moieties (i) and (ii) are joined to the IgG constant region sequences through a polypeptide linker.

9. The protein of claim 8, wherein the polypeptide linker is from 4-8 amino acids in length.

10. The protein of any of claims 1 -9, wherein moiety (i) has a half-maximal disruptive concentration (DC50) of less than 5 mM.

11 . The protein of claim 10, wherein moiety (i) is a DARPin®.

12. The protein of any of claims 1 -1 1 , wherein moiety (i) comprises a polypeptide selected from Table 1 or a variant thereof.

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13. The protein of any of claims 1 -12, wherein moiety (ii) is a polypeptide that binds to IgE at a high affinity.

14. The protein of claim 13, wherein moiety (ii) binds to IgE at an epitope distal to the FCER1 a binding site.

15. The protein of claim 13 or 14, wherein moiety (ii) is a DARPin® protein.

16. The protein of any of claims 1 -15, wherein moiety (ii) comprises a polypeptide selected from Table 2 or a variant thereof.

17. The protein of any of claims 2-16, wherein moiety (iii) is provided by an immunoglobulin constant region sequence, optionally a variant IgG Fc with altered FcyR specificity or affinity.

18. The protein of claim 17, wherein moiety (i) and moiety (ii) are fused to the C-terminus of an IgG Fc.

19. The protein of any of claims 1 -18, where the protein is a heterodimer of polypeptides selected from the combinations set forth in Table 3.

20. The protein of any of claims 2-16, wherein moiety (iii) is an IgG Fc sequence in addition to the immunoglobulin constant region sequences of claim 8.

21 . The protein of any of claims 2-16, wherein moiety (iii) is a DARPin® protein.

22. The protein of claim 20, wherein moiety (iii) is DARPin® D11 (SEQ ID NO:5) or a variant thereof.

23. The protein of any of claims 17-22, wherein moiety (iii) is joined to the IgG constant region through a linker.

24. The protein of claim 23, wherein the linker is a polypeptide of from 2-20 amino acids in length.

25. The protein of claim 6, wherein moiety (i) is present on the hole and moiety (ii) is present on the knob; or wherein moiety (ii) is present on the hole and moiety (i) is present on the knob.

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26. The protein of claim 4 or 17-19, wherein the first and second polypeptides comprise IgG constant region sequences CH1 , CH2, CH3 and hinge.

27. The protein of claim 26, wherein the lgG1 hinge (SEQ ID NO:8) is replaced with a modified hinge selected from SEQ ID NO:9 and 10; or a linker selected from SEQ ID NO: 11 and SEQ ID NO: 12.

28. The protein of claim 6, 16-19 or 25, comprising a knob-in-hole heterodimer with symmetric hinges or asymmetric hinges.

29. A protein comprising the amino acid sequence of SEQ ID NO:3, or a variant thereof.

30. An isolated nucleic acid encoding the protein of any of claims 1 -29, or Table 1 or Table 2.

31 . A vector comprising the nucleic acid of claim 30.

32. A cell comprising the nucleic acid of claim 30 or the vector of claim 31 .

33. A method of producing a protein of any of claims 1 -29, the method comprising: culturing a cell of claim 32 under conditions suitable for expressing the protein, and recovering the protein from the cell mass or the cell culture medium.

34. A pharmaceutical composition comprising the protein of any of claims 1 -29, and a pharmaceutically acceptable excipient.

35. A method for the treatment of an allergic condition, the method comprising: administering an effective dose of the protein of any of claims 1 -29 or the pharmaceutical composition of claim 34, to an individual in need thereof.

36. The method of claim 35, wherein the allergic condition is an acute response to an allergen.

37. The method of claim 35 or 36, wherein a therapeutic effect is obtained within less than 6 hours.

38. The method of any of claims 35-37, wherein the protein is administered in combination with an additional therapeutic agent.

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39. The method of claim 38, wherein the additional therapeutic agent is epinephrine or antihistamine.

40. The protein of any of claims 1 -29 or the pharmaceutical composition of claim 33 for use in a method for the treatment of an allergic condition, the method comprising: administering an effective dose of, to an individual in need thereof.

41. The protein of any of claims 1-29 or the pharmaceutical composition of claim 33, wherein the allergic condition is an acute response to an allergen.

42. The protein of any of claims 1-29 or the pharmaceutical composition of claim 33, wherein a therapeutic effect is obtained within less than 6 hours.

43. The protein of any of claims 1-29 or the pharmaceutical composition of claim 33, wherein the protein is administered in combination with an additional therapeutic agent.

44. The protein of any of claims 1-29 or the pharmaceutical composition of claim 33, wherein the additional therapeutic agent is epinephrine or antihistamine.

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