Low-viscosity variants of antibodies

Abstract

The present invention relates to variants of an antibody that display low viscosity. Compositions, kits, methods of use (e.g., treating asthma, chronic rhinosinusitis with nasal polyps, chronic spontaneous / idiopathic urticaria, or food allergy), and methods of making thereof are also provided.

Description

LOW-VISCOSITY VARIANTS OF ANTIBODIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of US Provisional Patent Application No. 63 / 605,349, filed on December 1, 2023, the contents of which are hereby incorporated herein by reference in their entirety and for all purposes.FIELD OF THE INVENTION

[0002] The present invention relates to antibody variants that display low viscosity. Further provided herein are pharmaceutical compositions comprising the antibody variants, methods of treating a condition using the antibody variants, methods of producing thereof, and kits comprising the antibody variants.BACKGROUND OF THE INVENTION

[0003] For the last several decades antibody therapeutics have become increasingly prevalent for the treatments of a diverse array of serious human maladies including many different cancers, as well as inflammatory, autoimmune, ophthalmologic, hematologic, infectious and metabolic diseases. Over 100 antibodies are currently approved as therapeutics making antibodies one of the most clinically impactful class of drugs in the pharmaceutical armamentarium (e.g., Mullard A (2021) Nat Rev Drug Discov, 20:491-495).

[0004] The most common route of administration of antibody therapeutics to patients has been intravenous infusion (IV). However, subcutaneous (SC) delivery is becoming increasingly more common, offering greater convenience for patients and healthcare professionals as well as potentially reducing healthcare costs (e.g. , PJ Carter & A Rajpal (2022) Cell 185:2789-2805). The typical injection volume for SC administration is <2.0 mL, commonly necessitating high antibody concentration (100-200 mg / mL) to deliver the desired dose. Such high antibody concentrations pose technical challenges that can sometimes raise the cost and delay the development of antibody therapeutics.

[0005] Commonly, antibody high concentration properties, including viscosity, are first evaluated at a late stage in preclinical development when large quantities (>100 mg) of one or a few clinical candidates are available. High concentration problems identified at this stage candelay projects and be resource-intensive to fix through protein engineering, replacement of clinical lead candidates and / or formulation. Thus, there exists a great need for the creation of improved antibodies that display reduced or low viscosity.BRIEF SUMMARY OF THE INVENTION

[0006] The present invention provides variants of an antibody that display low viscosity. Compositions, kits, methods of use (e.g., treating asthma, chronic rhinosinusitis with nasal polyps, chronic spontaneous / idiopathic urticaria, or food allergy), and methods of making thereof are also provided.

[0007] In one aspect of the present invention, there is provided an antibody comprising one or more viscosity -reducing mutations, wherein the one or more viscosity -reducing mutations comprises: S254T; T256E; or S254T and T256E, wherein the residues are numbered according to the EU index. In some embodiments, the one or more viscosity-reducing mutations further comprise M252Y. In some embodiments, the one or more viscosity-reducing mutations is M252Y and S254T. In some embodiments, the one or more viscosity-reducing mutations is M252Y and T256E. In some embodiments, the one or more viscosity-reducing mutations is M252Y, S254T, and T256E. In some embodiments, the one or more viscosity-reducing mutations further comprise N297G. In some embodiments, the one or more viscosity-reducing mutations further comprises one or more mutations each selected from the group consisting of: D54N; D30N; E55Q; D30N and D55Q; D54N, D30N, and E55Q; E345Q and E380Q; D249N and D312N; and any combination thereof, wherein residues 249, 312, 345, and 380 are numbered according to the EU index, and residues 30, 54, and 55 are numbered according to Kabat.

[0008] In one aspect of the present invention, there is provided a method for engineering an antibody to both reduce the viscosity and increase the serum half-life compared to the parent antibody, wherein the antibody is engineered to incorporate the M428L and N434S mutations or the M252Y, S254T, and T256E mutations. In another aspect of the present invention, there is provided an antibody comprising one or more viscosity -reducing mutations, wherein the one or more viscosity -reducing mutations increases serum half-life of the antibody compared to the parent antibody and wherein the viscosity-reducing mutation comprises the M428L and N434S mutations or the M252Y, S254T, and T256E mutations.

[0009] In one aspect of the present invention, there is provided an antibody comprising one or more viscosity -reducing mutations, wherein the one or more viscosity-reducing mutations are each selected from the group consisting of: H310N; H310N, H433N, and H435N; H97N, HlOOaN, and HlOOcN; and any combination thereof. In some embodiments, the antibody comprises M252Y, S254T, T256E, E345Q and E380Q; M252Y, S254T, T256E, D249N and D312N; M252Y, S254T, T256E, D54N, D30N, and E55Q; M252Y, S254T, T256E, andD54N; M252Y, S254T, T256E, and D30N; M252Y, S254T, T256E, and E55Q; or M252Y, S254T, T256E, D54N, and D30N. In these embodiments, residues 249, 252, 254, 256, 310, 312, 345, 380, 433, and 435 are numbered according to the EU index, and residues 30, 54, 55, 97, 100a, and 100c are numbered according to Kabat.

[0010] In one aspect of the present invention, there is provided an anti-IgE antibody comprising one or more viscosity-reducing mutations, wherein the anti-IgE antibody comprises: a heavy chain variable domain (VH) comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO:5; CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and CORED comprising the amino acid sequence of SEQ ID NO: 7; a light chain variable domain (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO:8; CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; and the one or more viscosity-reducing mutations comprising: S254T; T256E; or S254T and T256E, wherein the residues are numbered according to the EU index. In some embodiments, the one or more viscosity-reducing mutations further comprise M252Y. In some embodiments, the one or more viscosity-reducing mutations is M252Y and S254T. In some embodiments, the one or more viscosity -reducing mutations is M252Y and T256E. In some embodiments, the one or more viscosity-reducing mutations is M252Y, S254T, and T256E. In some embodiments, the one or more viscosity-reducing mutations further comprise N297G. In some embodiments, the one or more viscosity-reducing mutations further comprises one or more mutations each selected from the group consisting of: D54N; D30N; E55Q; D30N and D55Q; D54N, D30N, and E55Q; E345Q and E380Q; D249N and D312N; and any combination thereof, wherein residues 249, 312, 345, and 380 are numbered according to the EU index, and residues 30, 54, and 55 are numbered according to Kabat.

[0011] The present invention in another aspect provides an antibody comprising one or more viscosity-reducing mutations, wherein the one or more viscosity-reducing mutations are each selected from the group consisting of: N297G; F243L, R292P, Y300L, V305I, and P396L; S239D and I332E; K326W and E333S; S298A, E333A, and K334A; L234F, L235E, andD265A; E233P, L234V, L235A, and delta G236; S267E, H268F, and S324T; P238D, E233D, G237D, H268D, P271G, and A330R; G236A, S239D, and I332E; L235V, F243L, R292P, Y300L, and P396L; L234A, L235A, and P329G; S239D, I332E, and A330L; L234A and L235A; N325S and L328F; S267E and L328F; T366W (knob) and T366S, L368A, and Y407V (hole); and any combination thereof, wherein the residues are numbered according to the EU index.

[0012] The present invention in another aspect provides an anti-IgE antibody comprising one or more viscosity-reducing mutations, wherein the anti-IgE antibody comprises: a heavy chain variable domain (VH) comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO:5; CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; a light chain variable domain (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO:8; CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; the one or more viscosity -reducing mutations are each selected from the group consisting of: N297G; F243L, R292P, Y300L, V305I, and P396L; S239D and I332E; K326W and E333S; S298A, E333A, and K334A; L234F, L235E, and D265A; E233P, L234V, L235A, and delta G236; S267E, H268F, and S324T; P238D, E233D, G237D, H268D, P271G, and A330R; G236A, S239D, and I332E; L235V, F243L, R292P, Y300L, and P396L; L234A, L235A, and P329G; S239D, I332E, and A330L; L234A and L235A; N325S and L328F; S267E and L328F; T366W (knob) and T366S, L368A, and Y407V (hole); and any combination thereof, wherein the residues are numbered according to the EU index.

[0013] The present invention in another aspect provides an anti-IgE antibody comprising one or more viscosity-reducing mutations, wherein the anti-IgE antibody comprises: a heavy chain variable domain (VH) comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO:5; CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; a light chain variable domain (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO:8; CDR-L2 comprising the amino acid sequence of SEQ ID NOV; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; the one or more viscosity -reducing mutations are each selected from the group consisting of: H310N; H435N; H310N, H433N, and H435N; H97N, HlOOaN, and HlOOcN; and any combination thereof, wherein residues 310, 433, and 435 are numbered according to the EU index, and residues 97, 100a, and 100c are numbered according to Kabat.

[0014] In some embodiments according to any of the antibodies described above, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized or chimeric antibody.

[0015] In some embodiments according to any of the antibodies described above, the antibody is an anti-IgE antibody which comprises a heavy chain variable region (VH) of SEQ ID NO:3, or an amino acid sequence having at least about 95% sequence identity therewith, and a light chain variable region (VL) of SEQ ID NO:4, or an amino acid sequence having at least about 95% sequence identity therewith. In some embodiments, the anti-IgE antibody comprises a full-length heavy chain of SEQ ID NO: 1, or an amino acid sequence having at least about 95% sequence identity therewith. In some embodiments, the full-length heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-15. In some embodiments, the full-length heavy chain further comprises a C-terminal lysine residue (i.e., “K”). In some embodiments, the anti-IgE antibody comprises a full-length light chain of SEQ ID NO:2, or an amino acid sequence having at least about 95% sequence identity therewith.

[0016] In some embodiments according to any of the antibodies described above, the antibody is a full-length IgGl antibody. In some embodiments, the antibody binds human IgE with an affinity of at least about KA ~ 109M’1. In some embodiments, the antibody has a viscosity of about 180 cP or less at 180 mg / mL in 20 mM histidine acetate, pH 5.5. In some embodiments, the antibody has a viscosity of about 100 cP or less at 180 mg / mL in 20 mM histidine acetate, pH 5.5. In some embodiments, the antibody is an anti-IgE antibody as described herein. In other embodiments, the antibody is a low-viscosity variant of omalizumab.

[0017] Also provided are isolated nucleic acids encoding any of the antibodies described herein, vectors comprising such nucleic acids, and host cells comprising such nucleic acids or vectors.

[0018] Also provided are methods of making any of the antibodies described above, comprising: i) culturing a host cell comprising any of the isolated nucleic acids or the vectors described above, or any of the host cells described above, under a condition suitable for the expression of the antibody; and ii) obtaining the expressed antibody from said host cell.

[0019] Also provided are pharmaceutical compositions comprising any of the antibodies described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, there is provided a syringe comprising the pharmaceutical composition.

[0020] Also provided are methods of treating an individual having a condition. In some embodiments, the method comprises administering an antibody wherein the antibody is an anti-IgE antibody as described herein and the condition is selected from the group consisting of an asthma, chronic rhinosinusitis with nasal polyps, chronic spontaneous or idiopathic urticaria, and food allergy. In some embodiments, the method comprising administering to the individual an effective amount of any of the anti-IgE antibodies described above or any of the pharmaceutical compositions described above. In some embodiments, the asthma is moderate-to-severe asthma. In some embodiments, the anti-IgE antibody or the pharmaceutical composition is administered subcutaneously. In some embodiments, the individual is a human.

[0021] In some aspects, provided herein is an anti-IgE antibody comprising one or more viscosity-reducing mutations, the anti-IgE antibody comprising: a heavy chain variable domain (VH) comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO:5; CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; a light chain variable domain (VL) comprising: CDR- L1 comprising the amino acid sequence of SEQ ID NO:8; CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; and the one or more viscosity-reducing mutations comprising: S254T; T256E; or S254T and T256E, wherein the residues are numbered according to the EU index. In some embodiments, the one or more viscosity-reducing mutations further comprise M252Y. In some embodiments, the one or more viscosity-reducing mutations is M252Y and S254T. In some embodiments, the one or more viscosity-reducing mutations is M252Y and T256E. In some embodiments, the one or more viscosity-reducing mutations is M252Y, S254T, and T256E. In some embodiments, the one or more viscosity-reducing mutations further comprise N297G. In some embodiments, the one or more viscosity-reducing mutations further comprise one or more mutations each selected from the group consisting of: D54N; D30N; E55Q; D30N and D55Q; D54N, D30N, and E55Q; E345Q and E380Q; D249N and D312N; and any combination thereof, wherein residues 249, 312, 345, and 380 are numbered according to the EU index, and residues 30, 54, and 55 are numbered according to Kabat.

[0022] In other aspects, provided herein is an anti-IgE antibody comprising one or more viscosity-reducing mutations, the anti-IgE antibody comprising: a heavy chain variable domain (VH) comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO:5; CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; a light chain variable domain (VL) comprising: CDR-LI comprising the amino acid sequence of SEQ ID NO:8; CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; and the one or more viscosity-reducing mutations are each selected from the group consisting of: N297G; F243L, R292P, Y300L, V305I, and P396L; S239D and I332E; K326W and E333S; S298A, E333A, and K334A; L234F, L235E, and D265A; E233P, L234V, L235A, and delta G236; S267E, H268F, and S324T; P238D, E233D, G237D, H268D, P271G, and A330R; G236A, S239D, and I332E; L235V, F243L, R292P, Y300L, and P396L; L234A, L235A, and P329G; S239D, I332E, and A330L; L234A and L235A; N325S and L328F; S267E and L328F; T366W (knob) and T366S, L368A, and Y407V (hole); and any combination thereof, wherein the residues are numbered according to the EU index.

[0023] In other aspects, provided herein is an anti-IgE antibody comprising one or more viscosity-reducing mutations, the anti-IgE antibody comprising: a heavy chain variable domain (VH) comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO:5; CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; a light chain variable domain (VL) comprising: CDR- L1 comprising the amino acid sequence of SEQ ID NO:8; CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; and the one or more viscosity-reducing mutations are each selected from the group consisting of: H310N; H435N; H310N, H433N, and H435N; H97N, HlOOaN, and HlOOcN; and any combination thereof, wherein residues 310, 433, and 435 are numbered according to the EU index, and residues 97, 100a, and 100c are numbered according to Kabat.

[0024] In some embodiments, the anti-IgE antibody is a monoclonal antibody. In some embodiments, the anti-IgE antibody is a humanized or chimeric antibody.

[0025] In some embodiments, the anti-IgE antibody comprises a heavy chain variable region (VH) of SEQ ID NO:3, or an amino acid sequence having at least about 95% sequence identity therewith, and a light chain variable region (VL) of SEQ ID NO:4, or an amino acid sequence having at least about 95% sequence identity therewith.

[0026] In some embodiments, the anti-IgE antibody comprises a full-length heavy chain of SEQ ID NO: 1, or an amino acid sequence having at least about 95% sequence identity therewith. In some embodiments, the full-length heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-15.

[0027] In some embodiments, the anti-IgE antibody comprises a full-length light chain of SEQ ID NO:2, or an amino acid sequence having at least about 95% sequence identity therewith.

[0028] In some embodiments, the anti-IgE antibody is a full-length IgGl antibody.

[0029] In some embodiments, the anti-IgE antibody binds human IgE with an affinity of at least about KA ~ 109M'1.

[0030] In some embodiments, the anti-IgE antibody has a viscosity of about 180 cP or less at 180 mg / mL in 20 mM histidine acetate, pH 5.5. In some embodiments, the anti-IgE antibody has a viscosity of about 100 cP or less at 180 mg / mL in 20 mM histidine acetate, pH 5.5.

[0031] In other aspects, provided herein is an isolated nucleic acid encoding an anti-IgE antibody described herein.

[0032] In other aspects, provided herein is a vector comprising an isolated nucleic acid described herein.

[0033] In other aspects, provided herein is a host cell comprising a nucleic acid described herein or a vector described herein.

[0034] In other aspects, provided herein is a method of producing an anti-IgE antibody comprising culturing a host cell described herein under conditions suitable for the expression of the anti-IgE antibody. In some embodiments, the method further comprises recovering the anti-IgE antibody produced by the host cell.

[0035] In other aspects, provided herein is a pharmaceutical composition comprising an anti- IgE antibody described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration.

[0036] In other aspects, provided herein is a syringe comprising a pharmaceutical composition described herein.

[0037] In other aspects, provided herein is a method of treating an individual having a condition, wherein the condition is selected from the group consisting of an asthma, chronic rhinosinusitis with nasal polyps, chronic spontaneous or idiopathic urticaria, and food allergy, the method comprising administering to the individual an effective amount of an anti-IgE antibody described herein or a pharmaceutical composition described herein. In some embodiments, the asthma is moderate-to-severe asthma. In some embodiments, the individual is human.

[0038] In certain aspects, provided herein is a method of manufacturing an antibody having a reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: S254T; T256E; S254T and T256E; S254T andM252Y; T256E and M252Y; M252Y, S254T, and T256E; N297G; M428L and N434S; T307A, E380A, and N434A; M428L and N434S; D54N; D30N; E55Q; D30N and D55Q; D54N, D30N, and E55Q; E345Q and E380Q; D249N and D312N; N297G; F243L, R292P, Y300L, V305I, and P396L; S239D and I332E; K326W and E333S; S298A, E333A, and K334A; L234F, L235E, and D265A; E233P, L234V, L235A, and delta G236; S267E, H268F, and S324T; P238D, E233D, G237D, H268D, P271G, and A330R; G236A, S239D, and I332E; L235V, F243L, R292P, Y300L, and P396L; L234A, L235A, and P329G; S239D, I332E, and A330L; L234A and L235A; N325S and L328F; S267E and L328F; T366W (knob) and T366S, L368A, and Y407V (hole); H3 ION; H435N; H3 ION, H433N, and H435N; or H97N, HlOOaN, and HlOOcN; wherein residues 233, 234, 235, 236, 237, 238, 239, 243, 249, 252, 254, 256, 265, 267, 268, 271, 292, 297, 298, 300, 305, 307, 310, 312, 324, 325, 326, 328, 329, 330, 332, 333, 334, 345, 366, 368, 380, 396, 407, 428, 433, 434, and 435 are numbered according to the EU index, and residues 30, 54, 55, and 97, 100a, and 100c are numbered according to Kabat; manufacturing the antibody having reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0039] In certain aspects, provided herein is a method of screening for an antibody having a reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: S254T; T256E; S254T and T256E; S254T and M252Y; T256E and M252Y; M252Y, S254T, and T256E; N297G M428L and N434S; T307A, E380A, and N434A; M428L and N434S; D54N; D30N; E55Q; D30N and D55Q; D54N, D30N, and E55Q; E345Q and E380Q; D249N and D312N; N297G; F243L, R292P, Y300L, V305I, and P396L; S239D and I332E; K326W and E333S; S298A, E333A, and K334A; L234F, L235E, and D265A; E233P, L234V, L235A, and delta G236; S267E, H268F, and S324T; P238D, E233D, G237D, H268D, P271G, and A330R; G236A, S239D, and I332E; L235V, F243L, R292P, Y300L, and P396L; L234A, L235A, and P329G; S239D, I332E, and A330L; L234A and L235A; N325S and L328F; S267E and L328F; T366W (knob) and T366S, L368A, and Y407V (hole); H310N; H435N; H310N, H433N, and H435N; and H97N, HlOOaN, and HlOOcN; wherein residues 233, 234, 235, 236, 237, 238, 239, 243, 249, 252, 254, 256, 265, 267, 268, 271, 292, 297, 298, 300, 305, 307, 310, 312, 324, 325, 326, 328, 329, 330, 332, 333, 334, 345, 366, 368, 380, 396, 407, 428, 433, 434, and 435 are numbered according to the EU index, and residues 30, 54, 55, and 97, 100a, and 100c are numbered according to Kabat; selecting the antibody having a reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0040] In certain aspects, provided herein is a method of manufacturing an antibody having an increased half-life and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: M252Y, S254T, and T256E; M428L and N434S; and T307A, E380A, and N434A; or M428L and N434S, wherein the residues are numbered according to the EU index; and manufacturing the antibody having an increased half-life and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0041] In certain aspects, provided herein is a method of screening for an antibody having an increased half-life and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: M252Y, S254T, and T256E; M428L and N434S; and T307A, E380A, and N434A; or M428L and N434S, wherein the residues are numbered according to the EU index; and selecting the antibody having an increased half-life and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0042] In certain aspects, provided herein is a method of manufacturing an antibody having an increased Fc gamma receptor interaction and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: F243L, R292P, Y300L, V305I, and P396L; S267E and L328F; S239E and I332E; S239D, I332E, and A330L; P238D, E233D, G237D, H268D, P271G, and A330R; G236A, S239D, and I332E; N325S and L328F; or L235V, F243L, R292P, Y300L, and P396L, wherein the residues are numbered according to the EU index; and manufacturing the antibody having an increased Fc gamma receptor interaction and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0043] In certain aspects, provided herein is a method of screening for an antibody having an increased Fc gamma receptor interaction and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: F243L, R292P, Y300L, V305I, and P396L; S267E and L328F; S239E and I332E; S239D, I332E, and A330L; P238D, E233D, G237D, H268D, P271G, and A330R; G236A, S239D, and I332E; N325S and L328F; or L235V, F243L, R292P, Y300L, and P396L, wherein the residues are numbered according to the EU index; and selecting the antibody having an increased Fc gamma receptor interaction and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0044] In certain aspects, provided herein is a method of manufacturing an antibody having an increased ADCC function and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: F243L, R292P, Y300L, V305I, P396L; S239E and I332E; S239D, I332E, and A330L; or L235V, F243L, R292P, Y300L, and P396L, wherein the residues are numbered according to the EU index; and manufacturing the antibody having an increased ADCC function and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0045] In certain aspects, provided herein is a method of screening for an antibody having an increased ADCC function and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: F243L, R292P, Y300L, V305I, P396L; S239E and I332E; S239D, I332E, and A330L; or L235V, F243L, R292P, Y300L, and P396L, wherein the residues are numbered according to the EU index; and selecting the antibody having an increased ADCC function and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0046] In certain aspects, provided herein is a method of manufacturing an antibody having an increased aglycosylation and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: N297G, wherein the residues are numbered according to the EU index; and manufacturing the antibody having an increased aglycosylation and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0047] In certain aspects, provided herein is a method of screening for an antibody having an increased aglycosylation and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: N297G, wherein the residues are numbered according to the EU index; and selecting the antibody having an increased aglycosylation and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0048] In certain aspects, provided herein is a method of manufacturing an antibody having a reduced effector function and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: L234F, L235E, and D265A; L234A and L235A; L234A, L235A, and P329G; E233P, L234V, L235A, and del G236; or N297G, wherein the residues are numbered according to the EU index; andmanufacturing the antibody having a reduced effector function and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0049] In certain aspects, provided herein is a method of screening for an antibody having a reduced effector function and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: L234F, L235E, and D265A; L234A and L235A; L234A, L235A, and P329G; E233P, L234V, L235A, and del G236; or N297G, wherein the residues are numbered according to the EU index; and selecting the antibody having a reduced effector function and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0050] In some embodiments, the method further comprises measuring the viscosity of the parent antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is of an IgGl subclass.

[0051] In certain aspects, provided herein is an anti-IgE antibody comprising one or more viscosity-reducing mutations, the anti-IgE antibody comprising: a heavy chain variable domain (VH) comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO:5; CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; a light chain variable domain (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO:8; CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; and the one or more viscosity-reducing mutations results in a variant of the anti-IgE antibody having reduced glycosylation as compared to a parent antibody not having the one or more viscosity reducing mutations. In some embodiments, the one or more viscosity-reducing mutations comprises N297A, wherein residue 297 is numbered according to the EU index.

[0052] In certain aspects, provided herein is an antibody comprising one or more mutations providing a reduction in viscosity, wherein a parent of the antibody not comprising the one or more mutations has a high viscosity, the one or more mutations comprising: M252Y, S254T, and T256E; or M428L and N434S, wherein residues 252, 254, 256, 428, and 434 are numbered according to the EU index. In some embodiments, the high viscosity is at least about 20 cP (such as measured according to the description herein).

[0053] In some embodiments, the is a monoclonal antibody. In some embodiments, the antibody is of an IgGl subclass.BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIGS. 1A and IB provide a schematic representation of exemplary antibody formats and the Fc region alone (FIG. 1A) and bar graph showing the viscosity of exemplary antibody formats and the Fc region alone compared to the full-length omalizumab and trastuzumab antibodies (FIG. IB). Viscosity data were obtained by rheometry at a total protein concentration of 180 mg / mL in 20 mM histidine acetate, pH 5.5 at 25.0 °C for omalizumab and trastuzumab. Data shown are the mean viscosity values (n = 2-5) ± SD analyzed using one-way ANOVA with *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001 versus the corresponding parent antibody. cP, centipoise (unit of viscosity).

[0055] FIGS. 2A-2C show bar graphs of the viscosity of various antibody variants of omalizumab (FIG. 2A) and trastuzumab (FIGS. 2B and 2C). Data shown are mean viscosity values (n = 1-5) ± SD analyzed using one-way ANOVA with *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001 versus the corresponding parent antibody. cP, centipoise. The variant substitutions for each variant are defined in Table 7.

[0056] FIGS. 3A-3D provide diagrams of portions of the Fc region with regions and / or residues that are important to antibody viscosity. FIG. 3A shows the protein structure of the Fc region, where the boxes labeled “Y” indicate regions where mutations resulted in the largest changes in viscosity compared to omalizumab, and the boxes labeled “X” indicate regions where mutations resulted in the largest changes in viscosity compared to trastuzumab. FIG. 3B shows the CH2-CH3 elbow region where the residues M252, S254, and T256 are located that, when mutated (i.e., “YTE” variant), decreases the viscosity compared to omalizumab. FIG. 3C shows the upper CH2 residues that, when mutated, result in the largest increases in viscosity compared to trastuzumab. FIG. 3D show the CH2-CH3 elbow region where the residues M428 and N434 (i.e., “LS” or “MLNS” variant) are located that, when mutated, increases the viscosity compared to omalizumab.

[0057] FIGS. 4A-4B provide representative plots showing the viscosity (FIGS. 4A and 4C) of exemplary parent IgGl antibodies and the corresponding YTE and LS variants and the percent change of viscosity (FIGS. 4B and 4D) of the variants compared to the corresponding parent IgGl antibodies. Data shown in FIGS. 4A and 4B are the mean values (n = 1-5) ± SD. Data shown in FIGS. 4C and 4D are the mean values (n = 2-5) ± SD. cP, centipoise.

[0058] FIGS. 5A-5C provide representative plots showing the viscosity of omalizumab variants (FIG. 5 A), including using different antibody solutions (FIGS. 5B and 5C). FIG. 5Ashows the viscosity of the parent omalizumab antibody, the YTE omalizumab variant, and omalizumab variants comprising single or double point mutations selected from the three substituted YTE residues. FIG. 5B compares parent and YTE variant omalizumab viscosity across different concentrations of NaCl. FIG. 5C compares parent and YTE variant omalizumab viscosity across different concentrations of Arg-HCl. Data shown are mean viscosity values (n = 2-5) ± SD. Data were analyzed using one-way ANOVA with *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus the YTE (M252Y:S254T:T256E) variant. cP, centipoise.

[0059] FIGS. 6A and 6B provide models showing potential self-association interaction sites for a representative IgG molecule (FIG. 6A) and for omalizumab (FIG. 6B), including the parent, Fab and Fc fragments of the parent, and the YTE variant. A representative IgG molecule has been hypothesized to self-associate by 1. Fab-to-Fab interactions, 2. Fab-to-Fc interactions, and / or 3. Fc-to-Fc interactions. Omalizumab has been empirically determined to interact by 1. Fab-to-Fab interactions and / or 2. Fab-to-Fc interactions, but not measurably by Fc-to-Fc interactions.

[0060] FIGS. 7A-7D show the viscosity of the parent and YTE variant omalizumab antibodies across different temperatures (FIG. 7A) different antibody concentrations at 25.0°C (FIGS. 7B and 7D), and different rheometer shear rates at 25.0°C (FIG. 7C). Viscosity measurements were made by rheometry with 180 mg / mL antibody solutions in 20 mM histidine acetate, pH 5.5. cP, centipoise.

[0061] FIG. 8 provides a diagram showing a superposition of Fc variant crystallographic structures from the parent Fc region, the YTE variant, and the aglycosylated variant. The root mean square deviation for YTE and aglycosylated variant versus parent Fc structures were calculated using ChimeraX as 0.36A and 1.15 A, respectively. ChimeraX is described in Pettersen et al. (2021), Protein Sci 30:70-82, hereby incorporated by reference in its entirety.

[0062] FIG. 9 provides a representative bar graph of the average viscosity of omalizumab and trastuzumab YTE, NG, and YTENG Fc variants. Rheometry measurements were made at 180 mg / mL IgGl antibody solutions in 20 mM histidine acetate, pH 5.5 at 25.0°C. Data shown are the mean viscosity values (n = 1-5) ± SD analyzed using one-way ANOVA with ****p < 0.0001 versus the corresponding parent antibody. cP, centipoise.

[0063] FIG. 10 provides diagrams of omalizumab Fab selection and YTE variant Fc CH2 / CH3 elbow region containing histidine scanning residue locations and relevant negatively charged residues.

[0064] FIGS. HA and 11B provide representative plots showing the viscosity (FIG. 11 A) of exemplary parent IgGl antibodies and the corresponding NG variant and the percent change of viscosity (FIG. 11B) of the variants compared to the corresponding parent IgGl antibodies. Data shown are the mean values (n = 2-3) ± SD. cP, centipoise.DETAILED DESCRIPTION OF THE INVENTION

[0065] Provided herein, in certain aspects, are antibody variants exhibiting lower viscosity as compared to the parent antibody. The disclosure of the present application is based, at least in part, on the inventors’ unexpected discovery that certain antibody variants described herein show unexpectedly low viscosity. The disclosure especially shows anti-IgE antibody variants (e.g., omalizumab variants) exhibiting lower viscosity as compared to the parent antibody (e.g., omalizumab). Specifically, as reported in the Examples, the inventors found that variants of omalizumab that comprised specific mutations in the Fc region had significantly lower viscosity as compared to the parental omalizumab antibody. Omalizumab provides significant therapeutic value to patients but has a relatively high viscosity. High viscosity of a therapeutic protein can complicate production and formulation, including formulation for subcutaneous administration to patients, and such findings provided herein represent a significant advancement in the field.

[0066] Accordingly, in one aspect, the present invention provides an antibody comprising one or more viscosity-reducing mutations. In a preferred embodiment, an anti-IgE antibody comprising one or more viscosity-reducing mutations is provided, the anti-IgE antibody comprising: a heavy chain variable domain (VH) comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO:5; CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; and a light chain variable domain (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO:8; CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; wherein the one or more viscosityreducing mutations comprise: S254T; T256E; or S254T and T256E; wherein the residues are numbered according to the EU index. In some embodiments, the one or more viscosity-reducing mutations further comprise M252Y. In some embodiments, the one or more viscosityreducing mutations further comprise N297G. In some embodiments, the one or more viscosityreducing mutations further comprises one or more mutations each selected from the group consisting of: D54N; D30N; E55Q; D30N and D55Q; D54N, D30N, and E55Q; E345Q and E380Q; D249N and D312N; and any combination thereof, wherein residues 249, 312, 345, and 380 are numbered according to the EU index, and residues 30, 54, and 55 are numbered according to Kabat. In some embodiments, the one or more viscosity-reducing mutations comprise any one or more mutations that are each selected from the group consisting of D30N, D54N, E55Q, D249N, M252Y, S254T, T256E, N297G, D312N, E345Q, and E380Q. In some embodiments, the anti-IgE antibody comprises: S254T; T256E; S254T and T256E; S254T, T256E, and M252Y; S254T, T256E, M252Y, andN297G; S254T, T256E, M252Y, and D54N; S254T, T256E, M252Y, and D30N; S254T, T256E, M252Y, and E55Q; S254T, T256E, M252Y, D30N and E55Q; S254T, T256E, M252Y, D30N, D54N and E55Q; S254T, T256E, M252Y, E345Q, and E380Q; or S254T, T256E, M252Y, D249N, and D312N.

[0067] In another aspect, the present invention provides an anti-IgE antibody comprising one or more viscosity-reducing mutations, the anti-IgE antibody comprising: a heavy chain variable domain (VH) comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO:5; CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; and a light chain variable domain (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO:8; CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; wherein the one or more viscosity -reducing mutations are each selected from the group consisting of: N297G; F243L, R292P, Y300L, V305I, and P396L; S239D and I332E; K326W and E333S; S298A, E333A, and K334A; L234F, L235E, and D265A; E233P, L234V, L235A, and delta G236; S267E, H268F, and S324T; P238D, E233D, G237D, H268D, P271G, and A330R; G236A, S239D, and I332E; L235V, F243L, R292P, Y300L, and P396L; L234A, L235A, and P329G; S239D, I332E, and A330L; L234A and L235A; N325S and L328F; S267E and L328F; T366W (knob) and T366S, L368A, and Y407V (hole); and any combination thereof, wherein the residues are numbered according to the EU index. In some embodiments, the one or more viscosity-reducing mutations comprise any one or more mutations each selected from the group consisting of E233D, E233P, L234A, L234F, L234V, L235A, L235E, G236A, delta G236, G237D, P238D, S239D, F243L, D265A, S267E, H268D, H268F, P271G,R292P, N297G, S298A, Y300L, V305I, S324T, N325S, K326W, L328F, P329G, A330L, A330R, I332E, E333S, E333A, K334A, T366S, T366W, L368A, P396L, and Y407V.

[0068] In another aspect, the present invention provides an anti-IgE antibody comprising one or more viscosity-reducing mutations, the anti-IgE antibody comprising: a heavy chain variable domain (VH) comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO:5; CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; and a light chain variable domain (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO:8; CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; wherein the one or more viscosity -reducing mutations are each selected from the group consisting of: H310N; H435N; H310N, H433N, and H435N; H97N, HlOOaN, and HlOOcN; and any combination thereof, wherein residues 310, 433, and 435 are numbered according to the EU index, and residues 97, 100a, and 100c are numbered according to Kabat. In some embodiments, the one or more viscosity-reducing mutations comprise any one or more mutations each selected from the group consisting of H97N, HlOOaN, HlOOcN, H310N, H433N, and H435N.

[0069] Also provided are nucleic acids, vectors, host cells, pharmaceutical compositions, and kits comprising any of the anti-IgE antibody variants (e.g., omalizumab variants) described herein. Further provided are methods of use of the anti-IgE antibody variants or pharmaceutical compositions thereof, such as for treating conditions, such as inflammatory diseases, e.g., allergic asthma, chronic rhinosinusitis with nasal polyps, chronic spontaneous / idiopathic urticaria, or food allergy.Definitions

[0070] Before describing the invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, 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.

[0071] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless indicated otherwise. For example, “a food allergy” includes one or more food allergies.

[0072] The phrase “comprising” as used herein is open-ended, indicating that such embodiments may include additional elements. In contrast, the phrase “consisting of’ is closed, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase “consisting essentially of’ is partially closed, indicating that such embodiments may further comprise elements that do not materially change the basic characteristics of such embodiments. It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0073] The term “total serum IgE” or “serum total IgE” refers to a total amount of IgE present in a serum sample.

[0074] The term “allergen-specific IgE” refers to IgE that is specific to a particular antigen, resulting from an initial exposure to allergen in a process known as allergy sensitization, and which binds the surface of mast cells and basophils and which can result in the activation of mast cells and basophils upon subsequent exposure to the same allergen.

[0075] As used herein, a “baseline” level (such as baseline level for serum total IgE, and allergen-specific IgE) in a human refers to the level before an administration of an anti-IgE antibody described herein to the human.

[0076] As used herein, the term “prevention” includes providing prophylaxis with respect to occurrence or recurrence of an IgE-mediated disorder in an individual (e.g., human subject).

[0077] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. “Pharmaceutically acceptable” excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.

[0078] As used herein, the term “treatment” refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. For example, an individual is successfully “treated” if one or more symptoms associated with the disease (e.g., asthma) are mitigated or eliminated, including, but are not limited to, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, and / or prolonging survival of individuals.

[0079] As used herein, “delaying progression of a disease” means to defer, hinder, slow, retard, stabilize, and / or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, the onset of a disease or disorder, such as asthma, may be delayed.

[0080] An “effective amount” is at least the minimum amount required to effect a measurable improvement or prevention of a particular disorder. An effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and / or prolonging survival. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

[0081] As used herein, “in conjunction with” or “in combination with” refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” or “in combination with” refers to administration of one treatment modality before, during, or after administration of the other treatment modality to the individual.

[0082] The term “viscosity” herein refers to a measure of a fluid’s resistance to deformation at a given rate. Viscosity = Stress / Shear Rate. The SI unit of dynamic viscosity is the pascal- second (Pa»s), but is also commonly expressed as centipoise (cP), which is equal to one millipascal-second (mPa»s). In some embodiments, the viscosity is measured using a cone- and-plate rheometer (e.g., TA Instruments, New Castle, Delaware, US).

[0083] A “disorder” is any condition that would benefit from treatment including, but not limited to, chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.

[0084] A “subject”, “patient” or an “individual” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.

[0085] The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

[0086] An “isolated” antibody is one which has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, an antibody is purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of, for example, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using, for example, Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0087] “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by threeconstant heavy domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain.

[0088] The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the CHI, CH2 and CH3 domains (collectively, CH) of the heavy chain and the CL domain of the light chain.

[0089] The “variable region” or “variable domain” of an antibody refers to the aminoterminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as “VH .” The variable domain of the light chain may be referred to as “VL .” These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0090] The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0091] The “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“K”) and lambda (“X”), based on the amino acid sequences of their constant domains.

[0092] The term IgG “isotype” or “subclass” as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.

[0093] Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, y, e, y, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4thed. (W.B. Saunders, Co., 2000). An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[0094] The terms “full length antibody,” “intact antibody” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region.

[0095] A “naked antibody” for the purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0096] “Antibody fragments” comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof. In some embodiments, the antibody fragment described herein is an antigen-binding fragment. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0097] Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin or IdeS treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0098] “Fv” is the minimum antibody fragment which contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of theVH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0099] The Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0100] “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see, e.g., Pluckthiin, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0101] The term “diabodies” refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

[0102] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequencewas obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0103] The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nded. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467- 12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812- 813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, NatureBiotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

[0104] The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)). Chimeric antibodies include PRIMATTZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.

[0105] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a HVR of the recipient are replaced by residues from a HVR of a non- human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and / or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1 : 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0106] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and / or has been made using any ofthe techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol., 147(l):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos.6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.

[0107] A “species-dependent antibody” is one which has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species. Normally, the species-dependent antibody “binds specifically” to a human antigen (e.g., has a binding affinity (Kd) value of no more than about 1 x 10'7M, preferably no more than about 1 * 10'8M and preferably no more than about 1 x 10'9M) but has a binding affinity for a homologue of the antigen from a second nonhuman mammalian species which is at least about 50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity for the human antigen. The species-dependent antibody can be any of the various types of antibodies as defined above, but preferably is a humanized or human antibody.

[0108] The term “hypervariable region,” “HVR,” or “HV,” when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248: 1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e.g., Hamers-Casterman et al., Nature 363:446- 448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0109] A number of HVR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd.Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software. The“contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.Loop Kabat AbM Chothia ContactLI L24-L34 L24-L34 L26-L32 L30-L36L2 L50-L56 L50-L56 L50-L52 L46-L55L3 L89-L97 L89-L97 L91-L96 L89-L96Hl H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering)Hl H31-H35 H26-H35 H26-H32 H30-H35 (Chothia Numbering)H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

[0110] HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (LI), 46-56 or SO-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (Hl), 50-65 or 49-65 (H2) and 93-102,94- 102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.[OHl] HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (LI), 46-56 or SO- 56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (Hl), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0112] “Framework” or “FR” residues are those variable domain residues other than the HVR residues as herein defined.

[0113] The term “variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

[0114] The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra, Edelman et al, (1969), Proc Natl Acad Sci USA 63:78-85). The “EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody.

[0115] As use herein, the term “binds”, “specifically binds to” or is “specific for” refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody that binds to or specifically binds to a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and / or with greater duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of < IpM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species. In another embodiment, specific binding can include, but does not require exclusive binding.

[0116] A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays as disclosed, for example, in definitions herein.

[0117] As used herein, “Percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in thespecific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.Anti-IgE Antibodies

[0118] Omalizumab (CAS Registry No. 242138-07-4) is a recombinant DNA-derived, humanized IgGl monoclonal antibody with a molecular weight of approximately 149 kDa that selectively binds to human IgE. Amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of omalizumab are set forth herein as SEQ ID NO: 3 and SEQ ID NO:4, respectively. Amino acid sequences of the full-length heavy chain and the full-length light chain of omalizumab are set forth herein as SEQ ID NO: 1 and SEQ ID NO:2, respectively. See Table 1 below. In some embodiments, the omalizumab full-length heavy chain sequence comprises a C-terminal lysine (K). In some embodiments, the omalizumab full- length heavy chain sequence does not comprise a C-terminal lysine (K), such as shown in SEQ ID NO: 1. Such variation may be attributed to, e.g., enzymatic cleavage that occurs during antibody production.

[0119] Omalizumab is designed to treat IgE-mediated disease by reducing the concentration of free IgE in blood and in tissue. Omalizumab selectively binds to human IgE at the same site of the IgE molecule that binds to the high affinity IgE Receptor (FCERI), thereby reducing IgE bound to the surface of basophils, mast cells, and dendritic cells and reducing basophil, mast cell, and dendritic cell-triggered Type 2 inflammation.

[0120] XOLAIR® (omalizumab marketed by Genentech USA, Inc., South San Francisco, CA, and Novartis Pharmaceuticals Corporation, East Hanover, NJ) is approved by the European Commission and the US FDA for treatment of indications in older children and adults, to be administered by subcutaneous (SC) injection, including commercial availability as a prefilled syringe. Specifically, omalizumab is approved for patients with moderate-to- severe persistent asthma, chronic rhinosinusitis with nasal polyps, and chronic spontaneous / idiopathic urticaria.

[0121] In some embodiments, the anti-IgE antibody described herein comprises CDR-H1 (GYSITSGY, set forth as SEQ ID NO:5), CDR-H2 (TYDGS, set forth as SEQ ID NO:6), CDR- H3 (GSHYFGHWHFAV, set forth as SEQ ID N0:7), CDR-L1 (RASQSVDYDGDSYMN, set forth as SEQ ID NO: 8), CDR-L2 (AASYLES, set forth as SEQ ID NO: 9) and CDR-L3 (QQSHEDPYT, set forth as SEQ ID NO: 10). In some embodiments, the anti-IgE antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO:3, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO:4. In some embodiments, the anti-IgE antibody is a monoclonal antibody. In some embodiments, the anti-IgE antibody is a humanized or chimeric antibody. In some embodiments, the anti-IgE antibody is a full-length IgGl antibody.

[0122] In some embodiments, there is provided an anti-IgE antibody comprising variants described herein that is compared to a parent anti-IgE antibody, wherein the parent anti-IgE antibody is omalizumab or a monoclonal antibody comprising: CDR-H1 (GYSITSGY, set forth as SEQ ID NO: 5), CDR-H2 (TYDGS, set forth as SEQ ID NO: 6), CDR-H3 (GSHYFGHWHFAV, set forth as SEQ ID NO:7), CDR-L1 (RASQSVDYDGDSYMN, set forth as SEQ ID NO: 8), CDR-L2 (AASYLES, set forth as SEQ ID NOV) and CDR-L3 (QQSHEDPYT, set forth as SEQ ID NO: 10); and a wildtype Fc region. In some embodiments, the parent anti-IgE antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO:3, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO:4. In some embodiments, the parent anti-IgE antibody comprises a full-length heavy chain of SEQ ID NO: 1, and a full-length light chain of SEQ ID NO:2. In some embodiments, the full-length heavy chain sequence further comprises a C- terminal lysine (K). In some embodiments, the parent anti-IgE antibody is omalizumab. In some embodiments, the parent anti-IgE antibody is a monoclonal antibody. In some embodiments, the parent anti-IgE antibody is a humanized or chimeric antibody. In some embodiments, the parent anti-IgE antibody is a full-length IgGl antibody. In other embodiments, the parent anti-IgE antibody is a biosimilar of omalizumab.Table 1. Exemplary anti-IgE antibody sequences.Low Viscosity Variants

[0123] Provided herein are antibodies comprising variants (e.g., Fc region variants) that show significantly reduced antibody viscosity. In preferred embodiments, the antibody variants are anti-IgE antibodies.

[0124] Self-association of antibodies can contribute to viscosity of an antibody. Selfassociation of antibodies and other proteins has been analyzed as proxy for measurement ofviscosity. Assessing self-association of antibody solutions can be measured with a variety of instrumentation, either directly or indirectly, including dynamic light scattering (DLS), selfinteraction nanoparticle spectroscopy, analytical ultracentrifugation, and fluorescence correlation spectroscopy. The present disclosure assesses variants (e.g., Fc region variants) for their impact on antibody viscosity. E.g., those listed in Table 2 below.

[0125] In some embodiments, there is provided an antibody, or especially an anti-IgE antibody, comprising one or more viscosity-reducing mutations, wherein the one or more viscosity-reducing mutations are each selected from the group consisting of D30N, D54N, E55Q, D249N, M252Y, S254T, T256E, N297G, D312N, E345Q, E380Q, E233D, E233P, L234A, L234F, L234V, L235A, L235E, G236A, delta G236, G237D, P238D, S239D, F243L, D265A, S267E, H268D, H268F, P271G, R292P, S298A, Y300L, V305I, S324T, N325S, K326W, L328F, P329G, A330L, A330R, I332E, E333S, E333A, K334A, T366S, T366W, L368A, P396L, Y407V, H97N, HlOOaN, HlOOcN, H310N, H433N, and H435N, wherein residues are according to the EU index, except that residues 30, 54, 55, 97, 100a, and 100c are numbered according to Kabat. In some embodiments, the one or more viscosity -reducing mutations comprise any one or more mutations each selected from the group consisting of: S254T; T256E; S254T and T256E; M252Y and S254T; M252Y and T256E; M252Y, S254T, and T256E; N297G; D54N; D30N; E55Q; D30N and D55Q; D54N, D30N, and E55Q; E345Q and E380Q; D249N and D312N; F243L, R292P, Y300L, V305I, and P396L; S239D and I332E; K326W and E333S; S298A, E333A, and K334A; L234F, L235E, and D265A; E233P, L234V, L235A, and delta G236; S267E, H268F, and S324T; P238D, E233D, G237D, H268D, P271G, and A330R; G236A, S239D, and I332E; L235V, F243L, R292P, Y300L, and P396L; L234A, L235A, and P329G; S239D, I332E, and A330L; L234A and L235A; N325S and L328F; S267E and L328F; T366W (knob) and T366S, L368A, and Y407V (hole); H310N; H435N; H310N, H433N, and H435N; and H97N, HlOOaN, and HlOOcN, wherein residues are according to the EU index, except that residues 30, 54, 55, 97, 100a, and 100c are numbered according to Kabat. In some embodiments, the mutations are relative to a reference antibody, such as omalizumab. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized or chimeric antibody. In some embodiments, the antibody is a full-length IgGl antibody.

[0126] In some embodiments, there is provided an antibody, or especially an anti-IgE antibody comprising one or more viscosity-reducing mutations, wherein the one or more viscosity-reducing mutations comprise S254T and / or T256E, wherein the residues arenumbered according to the EU index. In some embodiments, the one or more viscosityreducing mutations further comprise M252Y. In some embodiments, the one or more viscosityreducing mutations is M252Y and S254T. In some embodiments, the one or more viscosityreducing mutations is M252Y and T256E. In some embodiments, the one or more viscosityreducing mutations is M252Y, S254T, and T256E. In some embodiments, the one or more viscosity-reducing mutations further comprise N297G. In some embodiments, the one or more viscosity-reducing mutations further comprises one or more mutations each selected from the group consisting of: D54N; D30N; E55Q; D30N and D55Q; D54N, D30N, and E55Q; E345Q and E380Q; D249N and D312N; and any combination thereof, wherein residues 249, 312, 345, and 380 are numbered according to the EU index, and residues 30, 54, and 55 are numbered according to Kabat. In some embodiments, the one or more viscosity-reducing mutations comprise any one or more mutations each selected from the group consisting of D30N, D54N, E55Q, D249N, M252Y, S254T, T256E, N297G, D312N, E345Q, and E380Q. In some embodiments, the mutations are relative to a reference antibody, such as omalizumab. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized or chimeric antibody. In some embodiments, the antibody is a full-length IgGl antibody.

[0127] In some embodiments, there is provided an antibody, or especially an anti-IgE antibody comprising one or more viscosity-reducing mutations, wherein the one or more viscosity-reducing mutations comprise any one or more mutations each selected from the group consisting of: N297G; F243L, R292P, Y300L, V305I, and P396L; S239D and I332E; K326W and E333S; S298A, E333A, and K334A; L234F, L235E, and D265A; E233P, L234V, L235A, and delta G236; S267E, H268F, and S324T; P238D, E233D, G237D, H268D, P271G, and A330R; G236A, S239D, and I332E; L235V, F243L, R292P, Y300L, and P396L; L234A, L235A, and P329G; S239D, I332E, and A330L; L234A and L235A; N325S and L328F; S267E and L328F; T366W (knob) and T366S, L368A, and Y407V (hole); and any combination thereof, wherein the residues are numbered according to the EU index. In some embodiments, the one or more viscosity-reducing mutations comprise any one or more mutations each selected from the group consisting of E233D, E233P, L234A, L234F, L234V, L235A, L235E, G236A, delta G236, G237D, P238D, S239D, F243L, D265A, S267E, H268D, H268F, P271G, R292P, N297G, S298A, Y300L, V305I, S324T, N325S, K326W, L328F, P329G, A330L, A330R, I332E, E333S, E333A, K334A, T366S, T366W, L368A, P396L, and Y407V. In some embodiments, the mutations are relative to a reference antibody, such as omalizumab. In someembodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized or chimeric antibody. In some embodiments, the antibody is a full-length IgGl antibody.

[0128] In some embodiments, there is provided an antibody, or especially an anti-IgE antibody comprising one or more viscosity-reducing mutations, wherein the one or more viscosity-reducing mutations comprise any one or more mutations each selected from the group consisting of: H310N; H435N; H310N, H433N, and H435N; H97N, HlOOaN, and HlOOcN; and any combination thereof, wherein residues 310, 433, and 435 are numbered according to the EU index, and residues 97, 100a, and 100c are numbered according to Kabat. In some embodiments, the one or more viscosity-reducing mutations comprise any one or more mutations each selected from the group consisting of H97N, HlOOaN, HlOOcN, H310N, H433N, and H435N. In some embodiments, the mutations are relative to a reference antibody, such as omalizumab. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized or chimeric antibody. In some embodiments, the antibody is a full-length IgGl antibody.

[0129] In some embodiments, there is provided an anti-IgE antibody comprising one or more viscosity-reducing mutations, the anti-IgE antibody comprising a heavy chain variable domain (VH) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:5, a CDR- H2 comprising the amino acid sequence of SEQ ID NO:6, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; a light chain variable domain (VL) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; and wherein the one or more viscosity-reducing mutations comprise S254T and / or T256E, wherein the residues are numbered according to the EU index. In some embodiments, the one or more viscosity-reducing mutations further comprise M252Y. In some embodiments, the one or more viscosity-reducing mutations is M252Y and S254T. In some embodiments, the one or more viscosity-reducing mutations is M252Y and T256E. In some embodiments, the one or more viscosity -reducing mutations is M252Y, S254T, and T256E. In some embodiments, the one or more viscosity-reducing mutations further comprise N297G. In some embodiments, the one or more viscosity-reducing mutations further comprises one or more mutations each selected from the group consisting of: D54N; D30N; E55Q; D30N and D55Q; D54N, D30N, and E55Q; E345Q and E380Q; D249N and D312N; and any combination thereof, wherein residues 249, 312, 345, and 380 are numbered according to the EU index, andresidues 30, 54, and 55 are numbered according to Kabat. In some embodiments, the one or more viscosity -reducing mutations comprise any one or more mutations each selected from the group consisting of D30N, D54N, E55Q, D249N, M252Y, S254T, T256E, N297G, D312N, E345Q, and E380Q. In some embodiments, the anti-IgE antibody comprises a VH comprising at least about 90% (e.g., at least about any of 95%, 96%, 97%, 98%, 99%, or more) sequence homology to the amino acid sequence of SEQ ID NO:3, and a VL comprising at least about 90% (e.g., at least about any of 95%, 96%, 97%, 98%, 99%, or more) sequence homology to the amino acid sequence of SEQ ID NO:4. In some embodiments, the anti-IgE antibody comprises: a full-length heavy chain amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least about 95% (e.g., at least about any of 96%, 97%, 98%, 99%, or more) sequence identity therewith; and / or a full-length light chain amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least about 95% (e.g., at least about any of 96%, 97%, 98%, 99%, or more) sequence identity therewith. In some embodiments, the anti-IgE antibody comprises a full-length heavy chain selected from the group consisting of SEQ ID NOs: 11-13, and a full-length light chain of SEQ ID NO:2. In some embodiments, the full- length heavy chain sequence further comprises a C-terminal lysine (K). In some embodiments, the anti-IgE antibody is a monoclonal antibody. In some embodiments, the anti-IgE antibody is a humanized or chimeric antibody. In some embodiments, the anti-IgE antibody is a full- length IgGl antibody.

[0130] In some embodiments, there is provided an anti-IgE antibody comprising one or more viscosity-reducing mutations, the anti-IgE antibody comprising a heavy chain variable domain (VH) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:5, a CDR- H2 comprising the amino acid sequence of SEQ ID NO:6, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; a light chain variable domain (VL) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; and wherein the one or more viscosity-reducing mutations comprise any one or more mutations each selected from the group consisting of: N297G; F243L, R292P, Y300L, V305I, and P396L; S239D and I332E; K326W and E333S; S298A, E333A, and K334A; L234F, L235E, and D265A; E233P, L234V, L235A, and delta G236; S267E, H268F, and S324T; P238D, E233D, G237D, H268D, P271G, and A330R; G236A, S239D, and I332E; L235V, F243L, R292P, Y300L, and P396L; L234A, L235A, and P329G; S239D, I332E, and A330L; L234A and L235A; N325S and L328F; S267E and L328F; T366W (knob) and T366S, L368A,and Y407V (hole); and any combination thereof, wherein the residues are numbered according to the EU index. In some embodiments, the one or more viscosity -reducing mutations comprise any one or more mutations each selected from the group consisting of E233D, E233P, L234A, L234F, L234V, L235A, L235E, G236A, delta G236, G237D, P238D, S239D, F243L, D265A, S267E, H268D, H268F, P271G, R292P, N297G, S298A, Y300L, V305I, S324T, N325S, K326W, L328F, P329G, A330L, A330R, I332E, E333S, E333A, K334A, T366S, T366W, L368A, P396L, and Y407V. In some embodiments, the anti-IgE antibody comprises a VH comprising at least about 90% (e.g., at least about any of 95%, 96%, 97%, 98%, 99%, or more) sequence homology to the amino acid sequence of SEQ ID NO:3, and a VL comprising at least about 90% (e.g., at least about any of 95%, 96%, 97%, 98%, 99%, or more) sequence homology to the amino acid sequence of SEQ ID NO:4. In some embodiments, the anti-IgE antibody comprises: a full-length heavy chain amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least about 95% (e.g., at least about any of 96%, 97%, 98%, 99%, or more) sequence identity therewith; and / or a full-length light chain amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least about 95% (e.g., at least about any of 96%, 97%, 98%, 99%, or more) sequence identity therewith. In some embodiments, the anti-IgE antibody comprises a full-length heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 14-15, and a full-length light chain amino acid sequence of SEQ ID NO:2. In some embodiments, the full-length heavy chain sequence further comprises a C-terminal lysine (K). In some embodiments, the anti-IgE antibody is a monoclonal antibody. In some embodiments, the anti-IgE antibody is a humanized or chimeric antibody. In some embodiments, the anti-IgE antibody is a full-length IgGl antibody.

[0131] In some embodiments, there is provided an anti-IgE antibody comprising one or more viscosity-reducing mutations, the anti-IgE antibody comprising a heavy chain variable domain (VH) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:5, a CDR- H2 comprising the amino acid sequence of SEQ ID NO:6, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; a light chain variable domain (VL) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; and wherein the one or more viscosity-reducing mutations comprise any one or more mutations each selected from the group consisting of: H310N; H435N; H310N, H433N, and H435N; H97N, HlOOaN, and HlOOcN; and any combination thereof, wherein residues 310, 433, and 435 are numbered according to the EU index, and residues 97, 100a, and 100c arenumbered according to Kabat. In some embodiments, the one or more viscosity -reducing mutations comprise any one or more mutations each selected from the group consisting of H97N, HlOOaN, HlOOcN, H310N, H433N, and H435N. In some embodiments, the anti-IgE antibody comprises a VH comprising at least about 90% (e.g., at least about any of 95%, 96%, 97%, 98%, 99%, or more) sequence homology to the amino acid sequence of SEQ ID NO:3, and a VL comprising at least about 90% (e.g., at least about any of 95%, 96%, 97%, 98%, 99%, or more) sequence homology to the amino acid sequence of SEQ ID NO:4. In some embodiments, the anti-IgE antibody comprises: a full-length heavy chain amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least about 95% (e.g., at least about any of 95%, 96%, 97%, 98%, 99%, or more) sequence identity therewith; and / or a full-length light chain amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least about 95% (e.g., at least about any of 96%, 97%, 98%, 99%, or more) sequence identity therewith. In some embodiments, the full-length heavy chain sequence further comprises a C-terminal lysine (K). In some embodiments, the anti-IgE antibody is a monoclonal antibody. In some embodiments, the anti-IgE antibody is a humanized or chimeric antibody. In some embodiments, the anti-IgE antibody is a full-length IgGl antibody.Table 2. Exemplary omalizumab low-viscosity variants.

[0132] In some embodiments, the anti-IgE antibody binds human IgE with an affinity of (KA) >106M’1, such as >107M’1, preferably >108M’1, more preferably >109M'1. For example, the KA value of the anti-IgE antibody in binding to human IgE is between about 108M'1and aboutIO10M'1. In some embodiments, the anti-IgE antibody binds human IgE with a KA of from about 107M'1to about 1011M'1(e.g., from about 107M'1to about 108M’1, from about 107M’1to about 109M’1, from about 108M'1to about 109M’1, from about 107M'1to about IO10M’1, from about 108M'1to about IO10M’1, from about 109M'1to about IO10M’1, from about 107M’1to about 1011M’1, from about 108M'1to about 1011M’1, from about 109M'1to about 1011M’ or from about IO10M'1to about 1011M'1). In some embodiments, the anti-IgE antibody binds human IgE with an affinity of at least about KA ~ 109M'1. See, e.g., Pennington et al. (2016), Nat Comm. 7: 11610.

[0133] In some embodiments, the anti-IgE antibody has a viscosity of about 180 cP or less at 180 mg / mL in 20 mM histidine acetate, pH 5.5. For example, in some embodiments, the anti-IgE antibody has a viscosity of less than about any of 170 cP, 160 cP, 150 cP, 140 cP, 130 cP, 120 cP, 110 cP, 100 cP, 90 cP, 80 cP, 75 cP, 70 cP, 60 cP, 50 cP, 40 cP, 30 cP, 25 cP, 20 cP, 10 cP, 9 cP, 8 cP, 7 cP, 6 cP, 5 cP, 4 cP, 3 cP, 2 cP, 1 cP, or less at 180 mg / mL in 20 mM histidine acetate, pH 5.5. in some embodiments, the anti-IgE antibody has a viscosity from about any of 1 cP to 200 cP, 80 cP to 180 cP, 100 cP to 200 cP, 50 cP to 150 cP, 75 cP to 175 cP, or 25 cP to 125 cP at 180 mg / mL in 20 mM histidine acetate, pH 5.5. In some embodiments, the anti-IgE antibody has a viscosity of about 100 cP or less at 180 mg / mL in 20 mM histidine acetate, pH 5.5.

[0134] In some embodiments, the anti-IgE antibody has a viscosity of about 180 cP or less at 150 mg / mL in 20 mM histidine acetate, pH 5.5. For example, in some embodiments, the anti-IgE antibody has a viscosity of less than about any of 170 cP, 160 cP, 150 cP, 140 cP, 130 cP, 120 cP, 110 cP, 100 cP, 90 cP, 80 cP, 75 cP, 70 cP, 60 cP, 50 cP, 40 cP, 30 cP, 25 cP, 20 cP, 10 cP, 9 cP, 8 cP, 7 cP, 6 cP, 5 cP, 4 cP, 3 cP, 2 cP, 1 cP, or less at 150 mg / mL in 20 mM histidine acetate, pH 5.5. in some embodiments, the anti-IgE antibody has a viscosity from about any of 1 cP to 200 cP, 80 cP to 180 cP, 100 cP to 200 cP, 50 cP to 150 cP, 75 cP to 175 cP, 25 cP to 125 cP, or 5 cP to 50 cP at 150 mg / mL in 20 mM histidine acetate, pH 5.5. In some embodiments, the anti-IgE antibody has a viscosity of about 100 cP or less at 150 mg / mL in 20 mM histidine acetate, pH 5.5.

[0135] In some embodiments, the viscosity of the anti-IgE antibody is measured using a cone-and-plate rheometer at a temperature of about 0°C to about 100°C. For example, the temperature can be from about 10°C to about 30°C, from about 15°C to about 35°C, from about 20°C to about 40°C, from about 25°C to about 45°C, from about 30°C to about 50°C, from about 35°C to about 55°C, from about 40°C to about 60°C, from about 45°C to about 65°C, from about50°C to about 70°C, from about 55°C to about 75°C, from about 60°C to about 80°C, from about 60°C to about 85°C, from about 70°C to about 90°C, from about 75°C to about 95°C, from about 80°C to about 100°C, or higher. In some embodiments, the viscosity of the anti-IgE antibody is measured using a cone-and-plate rheometer at a temperature of about 20°C. In some embodiments, the viscosity of the anti-IgE antibody is measured using a cone-and-plate rheometer at a temperature of about 25°C. In some embodiments, the viscosity of the anti-IgE antibody is measured using a cone-and-plate rheometer at a temperature of about 30°C. In some embodiments, the viscosity of the anti-IgE antibody is measured using a cone-and-plate rheometer at a temperature of about 35°C. In some embodiments, the viscosity of the anti-IgE antibody is measured using a cone-and-plate rheometer at a temperature of about 40°C.Vectors, Host Cells and Recombinant Methods

[0136] The invention also provides isolated nucleic acid encoding a variant antibody as disclosed herein, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibody described herein.

[0137] Antibodies may be produced using recombinant methods and compositions, e.g., as described in US 4,816,567. For these methods one or more isolated nucleic acid(s) encoding an antibody are provided.

[0138] In case of a native antibody or native antibody fragment, two nucleic acids are required, one for the light chain or a fragment thereof and one for the heavy chain or a fragment thereof. Such nucleic acid(s) encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chain(s) of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.

[0139] In one aspect, isolated nucleic acids encoding an antibody as used in the methods as reported herein are provided.

[0140] In one aspect, a method of making an antibody is provided, wherein the method comprises culturing a host cell comprising nucleic acid(s) encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0141] For recombinant production of an antibody, nucleic acids encoding the antibody, e.g., as described above, are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids may be readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) or produced by recombinant methods or obtained by chemical synthesis.

[0142] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0143] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gemgross, T.U., Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0144] Suitable host cells for the expression of a (glycosylated) antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0145] Plant cell cultures can also be utilized as hosts. See, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0146] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham, F.L. et al., J. Gen Virol. 36 (1977) 59- 74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as described, e.g., in Mather, J.P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Otheruseful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NSO and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255- 268.

[0147] In one aspect, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NSO, Sp20 cell).

[0148] Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Suitable conditions for expression of polynucleotides may include, without limitation, suitable medium, suitable density of host cells in the culture medium, presence of necessary nutrients, presence of supplemental factors, suitable temperatures and humidity, and absence of microorganism contaminants. A person with ordinary skill in the art can select the suitable conditions as appropriate for the purpose of the expression. In some embodiments, the method of making further comprises purifying any of the obtained antibodies described herein.Pharmaceutical Compositions

[0149] Also provided herein are pharmaceutical compositions and formulations, e.g., for the treatment of a condition (c.g, allergic asthma, chronic rhinosinusitis with nasal polyps, chronic spontaneous / idiopathic urticaria, or food allergy) as described herein. In some embodiments, the pharmaceutical compositions and formulations further comprise a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions and formulations comprise a low-viscosity anti-IgE antibody as described herein.

[0150] After preparation of the antibody, such as an omalizumab variant (e.g., techniques for producing antibodies which can be formulated as disclosed herein are elaborated herein and are known in the art), the pharmaceutical formulation comprising said antibody is prepared. The therapeutically effective amount of antibody present in the formulation is determined by, e.g., taking into account the desired dose volumes and mode(s) of administration. In some embodiments, the anti-IgE antibody is formulated at concentrations from about 25mg / mL to about 400 mg / mL, for example, from about 25 mg / mL to about 400 mg / mL, or from about 50mg / mL to about 350 mg / mL, or from about 35 mg / mL to about 300 mg / mL, or from about 40 mg / mL to about 250 mg / mL, or from about 45 mg / mL to about 200 mg / mL, or from about 25 mg / mL to about 150 mg / mL, or from about 30 mg / mL to about 140 mg / mL, or from about 35 mg / mL to about 130 mg / mL, or from about 40 mg / mL to about 120 mg / mL, or from about 50 mg / mL to about 130 mg / mL, or from about 50 mg / mL to about 125 mg / mL, or from about 50 mg / mL to about 120 mg / mL, or from about 50 mg / mL to about 110 mg / mL, or from about 50 mg / mL to about 100 mg / mL, or from about 50 mg / mL to about 90 mg / mL, or from about 50 mg / mL to about 80 mg / mL, or from about 54 mg / mL to about 66 mg / mL. In certain embodiments, the anti-IgE antibody to be formulated has not been subjected to prior lyophilization, and the formulation of interest herein is an aqueous formulation.

[0151] Pharmaceutical compositions and formulations as described herein can be prepared by mixing the active ingredients (such as an antibody or a polypeptide) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington ’s Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-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 polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral -active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos.2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

[0152] Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and W02006 / 044908, the latter formulations including a histidine-acetate buffer. E.g., Strickley & Lambert (2021), J Pharm Sci 110:2590-2608 e2556, hereby incorporated by reference in its entirety, for further description of appropriate antibody formulations.

[0153] The composition and formulation herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0154] Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington ’s Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980).

[0155] Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g, films, or microcapsules. The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g, by filtration through sterile filtration membranes.

[0156] In some embodiments, the pharmaceutical formulation of an antibody described herein does not include a viscosity reducing agent. In some embodiments, the pharmaceutical formulation of an antibody, e.g., an anti-IgE antibody, described herein comprises a high concentration of the anti-IgE antibody (e.g., >100 mg / mL, such as about any of 105 mg / mL, 110 mg / mL, 115 mg / mL, 120 mg / mL, 125 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 175 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 350 mg / mL, 400 mg / mL, or more). In some embodiments, the viscosity of the pharmaceutical formulation is about 180 cP or less (e.g., less than about any of 170 cP, 160 cP, 150 cP, 140 cP, 130 cP, 120 cP, 110 cP, 100 cP, 90 cP, 80 cP, 75 cP, 70 cP, 60 cP, 50 cP, 40 cP, 30 cP, 25 cP, 20 cP, 10 cP, 9 cP, 8 cP, 7 cP, 6 cP, 5 cP, 4 cP, 3 cP, 2 cP, 1 cP, or less) at 150 mg / mL in 20 mM histidine acetate, pH 5.5. in some embodiments, the viscosity of the pharmaceutical formulation is from about any of 1 cP to 200 cP, 80 cP to 180 cP, 100 cP to 200 cP, 50 cP to 150 cP, 75 cP to 175 cP, 25 cP to 125 cP, or 5 cP to 50 cP at 150 mg / mL in 20 mM histidine acetate, pH 5.5. In some embodiments,the viscosity of the pharmaceutical formulation is about 100 cP or less at 150 mg / mL in 20 mM histidine acetate, pH 5.5.Kits and Articles of Manufacture

[0157] The present application further provides articles of manufacture comprising the compositions (such as pharmaceutical compositions) described herein in suitable packaging. Suitable packaging for compositions (such as pharmaceutical compositions) described herein are known in the art, and include, for example, vials (such as sealed vials), vessels, ampules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may further be sterilized and / or sealed.

[0158] The present disclosure also provides kits for use in the methods described above. In some embodiments, the kits, comprise: i) a pharmaceutical composition comprising an antibody described herein, such as an anti-IgE antibody (such as an omalizumab variant comprising one or more viscosity-reducing mutations); and ii) instructions for administration of the pharmaceutical composition by subcutaneous injection to a human subject suffering from a condition as described herein (e.g., allergic asthma, chronic spontaneous / idiopathic urticaria, chronic rhinosinusitis with nasal polyps, or food allergy); optionally wherein the pharmaceutical composition is administered at, e.g., an anti-IgE antibody (e.g., omalizumab comprising one or more viscosity-reducing mutations) dose and a dosing interval determined from measurement of body weight and baseline total serum IgE level of the subject. In some embodiments, the pharmaceutical composition is an aqueous solution further comprising L- arginine hydrochloride, L-histidine, and L-histidine hydrochloride monohydrate. In some embodiments, the pharmaceutical composition further comprises sucrose. In specific embodiments, the pharmaceutical composition contains a low-viscosity omalizumab variant at a concentration of about 150 g / L in 0.02 M histidine, 0.2 M arginine-HCl, and 0.04 % polysorbate 20, pH 6. In some embodiments, the pharmaceutical composition is present in a pre-filled syringe. In some embodiments, the pharmaceutical composition is a lyophilized powder present in a vial, and the kit further comprises instructions for reconstituting the lyophilized powder in water for injection.

[0159] As a matter of convenience, the antibody of the present invention can be provided in a diagnostic kit, i.e., packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay. Where the antibody of the present invention is labeled with an enzyme, the kit will include substrates and cofactors required by theenzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore). In addition, other additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay. Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.Methods of Treating a Condition

[0160] Also provided herein are methods of treating an individual (e.g., a subject, such as a human subject or human patient) having a condition associated with IgE hypersensitivity comprising administering to the individual an effective amount of an antibody described herein, such as the anti-IgE antibody described herein, or the pharmaceutical composition thereof. In some embodiments, the method comprises treating an individual (e.g., a subject, such as a human subject or human patient) having a condition, wherein the condition is selected from the group consisting of an asthma, chronic rhinosinusitis with nasal polyps, chronic spontaneous / idiopathic urticaria, and food allergy, the method comprising administering to the individual an effective amount of the anti-IgE antibody described herein or the pharmaceutical composition thereof. In some embodiments, the asthma is moderate to severe. In some embodiments, the anti-IgE antibody comprises one or more viscosity-reducing mutations. In some embodiments, the anti-IgE antibody is an omalizumab variant that displays low viscosity. In some embodiments, the anti-IgE antibody described herein further can be co-administered with a corticosteroid and / or an immunusuppressor and / or a long-acting beta agonist (LABA).

[0161] IgE is a member of the immunoglobulin family that mediates allergic responses, such as asthma, food allergies, type 1 hypersensitivity, and sinus inflammation. IgE is secreted by and expressed on the surface of B-cells. IgE binds to (e.g.) B-cells, monocytes, eosinophils, and platelets through its Fc region to the low affinity IgE receptor, FcsRII. Upon exposure of a subject to an allergen, B-cells bearing a surface-bound IgE antibody specific for the antigen are activated and develop into IgE-secreting plasma cells. The resulting allergen-specific IgE then circulates through the bloodstream and binds to the surface of mast cells in tissues and basophils in the blood via the high affinity receptor, FcsRI. As a result, the mast cells and basophils become sensitized for the allergen such that subsequent exposure(s) causes a cross linking of the basophil and mast cell FcsRI, which results in a release of histamine, leukotrienesand platelet activating factors, eosinophil and neutrophil chemotactic factors, and pro- inflammatory cytokines such as IL-3, IL-4, IL-5, and GM-CSF. These factors are responsible for clinical hypersensitivity and anaphylaxis. See, e.g., US7157085B2, hereby incorporated by reference in its entirety.

[0162] In some embodiments, the condition associated with IgE hypersensitivity can include, but is not limited to, type 1 diabetes, glomerulonephritis, allergic encephalomyelitis, multiple sclerosis, inflammatory bowel diseases, autoimmune gastritis, myasthenia gravis, autoimmune thyroiditis, acquired aplastic anemia, autoimmune encephalitis, Parkinson’s disease, FoxP3- deficiency, IPEX syndrome, immuno-dysregulation (e.g., Treg dysfunction), polyendocrinopathy, enteropathy, anti -tumor immunity, transplant rejection, asthma (e.g. allergic asthma, moderate to severe persistent allergic asthma), chronic spontaneous urticaria, rhinitis (i.e., chronic idiopathic urticaria; e.g., Seasonal allergic rhinitis, such as Cedar pollinosis), atopic dermatitis, bullous pemphigoid, chronic inducible urticarias (CINDUs), nasal polyposis, IgE-driven food allergy to one or more allergens (e.g., milk, eggs, fish, Crustacean shellfish, tree nuts, peanuts, wheat, and soybeans), idiopathic angioedema, systemic anaphylaxis, mastocytosis, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IFF), eosinophilic esophagitis, etc. (see, e.g., WO2021048678 Al, WO2021250533A1, WO2019157358A1, US17 / 642,319, US16 / 987,958, and US7157085B2, hereby each incorporated by reference in its entirety).

[0163] In some embodiments, the method comprises treating or preventing a disease or disorder involving Treg cell dysfunction in an individual in need thereof, comprising administering to the individual an effective amount of the anti-IgE antibody described herein or the pharmaceutical composition thereof. In some embodiments, the method comprises treating or preventing a mast cell-mediated inflammatory disease in an individual in need thereof, comprising administering to the individual an effective amount of the anti-IgE antibody described herein or the pharmaceutical composition thereof. In some embodiments, the mast cell-mediated inflammatory disease is selected from the group consisting of asthma, atopic dermatitis, urticaria (e.g., CSU or CIU), systemic anaphylaxis, mastocytosis, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IFF), and eosinophilic esophagitis.

[0164] In some embodiments, the method comprises treating an individual (e.g., a subject, such as a human subject or human patient) asthma, comprising administering to the individual an effective amount of the anti-IgE antibody described herein or the pharmaceuticalcomposition thereof. In some embodiments, the asthma is persistent chronic severe asthma with acute events of worsening symptoms (exacerbations or flares) that can be life threatening. In some embodiments, the asthma is atopic (also known as allergic) asthma, non-allergic asthma (e.g., often triggered by infection with a respiratory virus such as influenza, parainfluenza, rhinovirus, human metapneumovirus, or respiratory syncytial virus), or inhaled irritant (e.g., air pollutants, smog, diesel particles, volatile chemicals, gases indoors or outdoors, or cold, dry air). In some embodiments, the asthma is i. intermittent or exercise-induced; ii. Due to acute or chronic primary or second-hand exposure to smoke (e.g., cigarettes, cigars, or pipes) or by inhaling or vaping (e.g., tobacco, marijuana, or other such substances); or iii. Triggered by recent ingestion of aspirin or related NSAIDS. In some embodiments, the asthma is mild, or corticosteroid-naive asthma, newly diagnosed and untreated asthma, or otherwise not previously requiring the chronic use of inhaled topical or systemic steroids to control symptoms (e.g., cough, wheeze, shortness of breath / breathlessness, or chest pain). In some embodiments, the asthma is chronic corticosteroid resistant asthma, corticosteroid refractory asthma, or asthma uncontrolled by corticosteroids or other medications. In some embodiments, the asthma is moderate to severe asthma. In certain embodiments, the asthma is TH2-high asthma. In some embodiments, the asthma is eosinophilic asthma, e.g., wherein the individual has been determined to be Eosinophilic Inflammation Positive (EiP) (see, e.g., W02015 / 061441, hereby incorporated by reference in its entirety).

[0165] In some embodiments, the method comprises treating an individual (e.g, a subject, such as a human subject or human patient) having chronic rhinosinusitis with nasal polyps, comprising administering to the individual an effective amount of the anti-IgE antibody described herein or the pharmaceutical composition thereof. In some embodiments, patients suffer from any one or more of nasal congestion, loss of smell, and / or rhinorrhea. In some embodiments, the method further comprises co-treatment with a corticosteroid and / or an immunosuppressor and / or surgical removal of the nasal polyps. In some embodiments, the individual is non-responsive to nasal corticosteroids.

[0166] In some embodiments, the method comprises treating an individual (e.g, a subject, such as a human subject or human patient) having chronic spontaneous urticaria (i.e., chronic idiopathic urticaria), comprising administering to the individual an effective amount of the anti- IgE antibody described herein or the pharmaceutical composition thereof. In some embodiments, the individual suffers urticaria at least 3 times per week and lasting for 6 weeks or longer (e.g., at least about any of 7 weeks, 8 weeks, 9 weeks, ten weeks, 2 months, 3 months,4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, or longer). In some embodiments, the method further comprises co-treatment with an Hl antihistamine, H2 antihistamine, leukotriene modifier, and / or glucocorticoid. In some embodiments, the individual is non-responsive to Hl antihistamines. In some embodiments, the individual is non-responsive to Hl antihistamines in combination with H2 antihistamines, leukotriene modifiers, and / or glucocorticoids.

[0167] In some embodiments, the method comprises treating an individual (e.g., a subject, such as a human subject or human patient) having a food allergy, comprising administering to the individual an effective amount of the anti-IgE antibody described herein or the pharmaceutical composition thereof. In some embodiments, the method comprises preventing an allergic reaction to consumption of a food allergen in a human subject with a food allergy. In some embodiments, the individual suffers from one or more food allergies, for example but not limited to any one or more of peanut, milk, egg, wheat, cashew, hazelnut, and walnut. In some embodiments, the allergic reaction is a mild-to-moderate allergic reaction triggered by consumption of as little as about 100 mg to about 300 mg of the food allergen by the subject. In some embodiments, the allergic reaction is moderate-to-severe. In some embodiments, the method reduces the likelihood the individual will require rescue treatment after consumption of the food allergen.

[0168] In some embodiments, the individual in need thereof has had an inadequate response to conventional therapy, including corticosteroids, or is intolerant to or has medical contraindications for such therapies. In some embodiments, the individual in need thereof does not respond to treatment with corticosteroids. In some embodiments, the individual in need thereof displays inadequate response to corticosteroids; is in need of corticosteroid-sparing; or is someone for whom corticosteroid treatment is inappropriate. In some embodiments, the individual in need thereof cannot tolerate corticosteroids.

[0169] In some embodiments, the individual is a human. In some embodiments, the individual is a human subject or patient. In some embodiments, the human subject or patient is an adolescent or an adult. In some embodiments, the human subject or patient is at least about 12 years of age, for example at least about any of 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, or more years of age. In some embodiments, the human subject or patient is pediatric. In some embodiments, the human subject or patient is between about 2 years to about 6 years of age, or about 6 years to about 12years of age, for example about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years of age.

[0170] Provided with the description of method of treatment are alternate formats thereof. For example, in some embodiments, provided is an antibody variant described herein, such as an anti-IgE variant, e.g., an omalizumab variant, for use in a method of treating a condition in an individual in need thereof. In some embodiments, provided herein is an antibody variant described herein, such as an anti-IgE variant, e.g., an omalizumab variant, for use in the manufacture of a medicament for treating a condition in an individual in need thereof.EXEMPLARY EMBODIMENTS

[0171] Embodiment 1. An antibody comprising one or more viscosity -reducing mutations, the one or more viscosity -reducing mutations comprising: S254T; T256E; or S254T and T256E, wherein the residues are numbered according to the EU index.

[0172] Embodiment 2. The antibody of embodiment 1, wherein the one or more viscosity reducing mutations further comprise M252Y.

[0173] Embodiment 3. The antibody of embodiment 1 or 2, wherein the one or more viscosity-reducing mutations is M252Y and S254T.

[0174] Embodiment 4. The antibody of embodiment 1 or 2, wherein the one or more viscosity-reducing mutations is M252Y and T256E.

[0175] Embodiment 5. The antibody of embodiment 1 or 2, wherein the one or more viscosity-reducing mutations is M252Y, S254T, and T256E.

[0176] Embodiment 6. The antibody of embodiment 5, wherein the one or more viscosityreducing mutations further comprise N297G.

[0177] Embodiment 7. The antibody of any one of embodiments 1-5, wherein the one or more viscosity -reducing mutations further comprises one or more mutations each selected from the group consisting of: D54N; D30N; E55Q; D30N and D55Q; D54N, D30N, and E55Q; E345Q and E380Q; D249N and D312N; and any combination thereof, wherein residues 249, 312, 345, and 380 are numbered according to the EU index, and residues 30, 54, and 55 are numbered according to Kabat.

[0178] Embodiment 8. The antibody of any one of embodiments 1-7, wherein the antibody is an anti-IgE antibody comprising: a heavy chain variable domain (VH) comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO:5; CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; a light chain variable domain (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO:8; CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0179] Embodiment 9. An antibody comprising one or more viscosity -reducing mutations, wherein the one or more viscosity-reducing mutations are each selected from the group consisting of: N297G; F243L, R292P, Y300L, V305I, and P396L; S239D and I332E; K326W and E333S; S298A, E333A, and K334A; L234F, L235E, and D265A; E233P, L234V, L235A, and del.G236; S267E, H268F, and S324T; P238D, E233D, G237D, H268D, P271G, and A330R; G236A, S239D, and I332E; L235V, F243L, R292P, Y300L, and P396L; L234A, L235A, and P329G; S239D, I332E, and A330L; L234A and L235A; N325S and L328F; S267E and L328F; T366W (knob) and T366S, L368A, and Y407V (hole); and any combination thereof, wherein the residues are numbered according to the EU index.

[0180] Embodiment 10. The antibody of embodiment 9, wherein the antibody is an anti-IgE antibody comprising: a heavy chain variable domain (VH) comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO:5; CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; a light chain variable domain (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO:8; CDR-L2 comprising the amino acid sequence of SEQ ID NOV; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0181] Embodiment 11. An antibody comprising one or more viscosity-reducing mutations, wherein the one or more viscosity-reducing mutations are each selected from the group consisting of: H310N; H435N; H310N, H433N, and H435N; H97N, HlOOaN, and HlOOcN; and any combination thereof, wherein residues 310, 433, and 435 are numbered according to the EU index, and residues 97, 100a, and 100c are numbered according to Kabat.

[0182] Embodiment 12. The antibody of embodiment 11, wherein the antibody is an anti- IgE antibody comprising: a heavy chain variable domain (VH) comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO:5; CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and CDR-H3 comprising the amino acid sequence of SEQ IDN0:7; a light chain variable domain (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO:8; CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0183] Embodiment 13. The antibody of any one of embodiments 1-12, wherein the antibody is a monoclonal antibody.

[0184] Embodiment 14. The antibody of any one of embodiments 1-13, wherein the antibody is a humanized or chimeric antibody.

[0185] Embodiment 15. The antibody of any one of embodiments 1-14, wherein the antibody is an anti-IgE antibody comprising a heavy chain variable region (VH) of SEQ ID NO:3, or an amino acid sequence having at least about 95% sequence identity therewith, and a light chain variable region (VL) of SEQ ID NON, or an amino acid sequence having at least about 95% sequence identity therewith.

[0186] Embodiment 16. The antibody of any one of embodiments 1-15, wherein the antibody is an anti-IgE antibody comprising a full-length heavy chain of SEQ ID NO: 1, or an amino acid sequence having at least about 95% sequence identity therewith.

[0187] Embodiment 17. The antibody of embodiment 16, wherein the full-length heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-15.

[0188] Embodiment 18. The antibody of any one of embodiments 1-17, comprising a full- length light chain of SEQ ID NO:2, or an amino acid sequence having at least about 95% sequence identity therewith.

[0189] Embodiment 19. The antibody of any one of embodiments 1-18, wherein the antibody is a full-length IgGl antibody.

[0190] Embodiment 20. The antibody of any one of embodiments 1-19, wherein the antibody binds human IgE with an affinity of at least about KA ~ 109M'1.

[0191] Embodiment 21. The antibody of any one of embodiments 1-20, wherein the antibody has a viscosity of about 180 cP or less at 180 mg / mL in 20 mM histidine acetate, pH 5.5.

[0192] Embodiment 22. The antibody of any one of embodiments 1-21, wherein the antibody has a viscosity of about 100 cP or less at 180 mg / mL in 20 mM histidine acetate, pH 5.5.

[0193] Embodiment 23. An isolated nucleic acid encoding the antibody of any one of embodiments 1-22.

[0194] Embodiment 24. A vector comprising the isolated nucleic acid of embodiment 23.

[0195] Embodiment 25. A host cell comprising the nucleic acid of embodiment 23 or the vector of embodiment 24.

[0196] Embodiment 26. A method of producing an antibody comprising culturing the host cell of embodiment 25 under conditions suitable for the expression of the antibody.

[0197] Embodiment 27. The method of embodiment 26, further comprising recovering the antibody produced by the host cell.

[0198] Embodiment 28. A pharmaceutical composition comprising the antibody of any one of embodiments 1-22 and a pharmaceutically acceptable carrier.

[0199] Embodiment 29. The pharmaceutical composition of embodiment 28, wherein the pharmaceutical composition is formulated for subcutaneous administration.

[0200] Embodiment 30. A syringe comprising the pharmaceutical composition of embodiment 28 or 29.

[0201] Embodiment 31. A method of treating an individual having a condition, wherein the condition is selected from the group consisting of an asthma, chronic rhinosinusitis with nasal polyps, chronic spontaneous or idiopathic urticaria, and food allergy, the method comprising administering to the individual an effective amount of the antibody of any one of embodiments 1-22 or the pharmaceutical composition of embodiment 28 or 29.

[0202] Embodiment 32. The method of embodiment 31, wherein the asthma is moderate-to- severe asthma.

[0203] Embodiment 33. The method of embodiment 31 or 32, wherein the individual is human.

[0204] Embodiment 34. A method of manufacturing an antibody having a reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: S254T; T256E; S254T and T256E; S254T and M252Y; T256E and M252Y; M252Y, S254T, and T256E; N297G; M428L and N434S; T307A, E380A, and N434A; M428L and N434S; D54N; D30N; E55Q; D30N and D55Q; D54N, D30N, and E55Q; E345Q and E380Q; D249N and D312N; N297G; F243L, R292P, Y300L, V305I, and P396L; S239D and I332E; K326W and E333S; S298A, E333A, and K334A; L234F, L235E, and D265A; E233P, L234V, L235A, and delta G236; S267E, H268F, and S324T; P238D, E233D, G237D, H268D, P271G, and A330R; G236A, S239D, and I332E; L235V, F243L, R292P,Y300L, and P396L; L234A, L235A, and P329G; S239D, I332E, and A330L; L234A and L235A; N325S and L328F; S267E and L328F; T366W (knob) and T366S, L368A, and Y407V (hole); H310N; H435N; H310N, H433N, and H435N; or H97N, HlOOaN, and HlOOcN; wherein residues 233, 234, 235, 236, 237, 238, 239, 243, 249, 252, 254, 256, 265, 267, 268, 271, 292, 297, 298, 300, 305, 307, 310, 312, 324, 325, 326, 328, 329, 330, 332, 333, 334, 345, 366, 368, 380, 396, 407, 428, 433, 434, and 435 are numbered according to the EU index, and residues 30, 54, 55, and 97, 100a, and 100c are numbered according to Kabat; manufacturing the antibody having reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0205] Embodiment 35: A method of screening for an antibody having a reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: S254T; T256E; S254T and T256E; S254T and M252Y; T256E and M252Y; M252Y, S254T, and T256E; N297G M428L and N434S; T307A, E380A, and N434A; M428L and N434S; D54N; D30N; E55Q; D30N and D55Q; D54N, D30N, and E55Q; E345Q and E380Q; D249N and D312N; N297G; F243L, R292P, Y300L, V305I, and P396L; S239D and I332E; K326W and E333S; S298A, E333A, and K334A; L234F, L235E, and D265A; E233P, L234V, L235A, and delta G236; S267E, H268F, and S324T; P238D, E233D, G237D, H268D, P271G, and A330R; G236A, S239D, and I332E; L235V, F243L, R292P, Y300L, and P396L; L234A, L235A, and P329G; S239D, I332E, and A330L; L234A and L235A; N325S and L328F; S267E and L328F; T366W (knob) and T366S, L368A, and Y407V (hole); H310N; H435N; H310N, H433N, and H435N; and H97N, HlOOaN, and HlOOcN; wherein residues 233, 234, 235, 236, 237, 238, 239, 243, 249, 252, 254, 256, 265, 267, 268, 271, 292, 297, 298, 300, 305, 307, 310, 312, 324, 325, 326, 328, 329, 330, 332, 333, 334, 345, 366, 368, 380, 396, 407, 428, 433, 434, and 435 are numbered according to the EU index, and residues 30, 54, 55, and 97, 100a, and 100c are numbered according to Kabat; selecting the antibody having a reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0206] Embodiment 36. A method of manufacturing an antibody having an increased halflife and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: M252Y, S254T, and T256E; M428L and N434S; and T307A, E380A, and N434A; or M428L and N434S, wherein the residues are numbered according to the EU index; and manufacturing the antibody having an increased half-life and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0207] Embodiment 37. A method of screening for an antibody having an increased half-life and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: M252Y, S254T, and T256E; M428L and N434S; and T307A, E380A, and N434A; or M428L and N434S, wherein the residues are numbered according to the EU index; and selecting the antibody having an increased half-life and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0208] Embodiment 38. A method of manufacturing an antibody having an increased Fc gamma receptor interaction and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: F243L, R292P, Y300L, V305I, and P396L; S267E and L328F; S239E and I332E; S239D, I332E, and A330L; P238D, E233D, G237D, H268D, P271G, and A330R; G236A, S239D, and I332E; N325S and L328F; or L235V, F243L, R292P, Y300L, and P396L, wherein the residues are numbered according to the EU index; and manufacturing the antibody having an increased Fc gamma receptor interaction and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0209] Embodiment 39. A method of screening for an antibody having an increased Fc gamma receptor interaction and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: F243L, R292P, Y300L, V305I, and P396L; S267E and L328F; S239E and I332E; S239D, I332E, and A330L; P238D, E233D, G237D, H268D, P271G, and A330R; G236A, S239D, and I332E; N325S and L328F; or L235V, F243L, R292P, Y300L, and P396L, wherein the residues are numbered according to the EU index; and selecting the antibody having an increased Fc gamma receptor interaction and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0210] Embodiment 40. A method of manufacturing an antibody having an increased ADCC function and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: F243L, R292P, Y300L, V305I, P396L; S239E and I332E; S239D, I332E, and A330L; or L235V, F243L, R292P, Y300L, and P396L, wherein the residues are numbered according to the EU index; and manufacturing the antibodyhaving an increased ADCC function and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0211] Embodiment 41. A method of screening for an antibody having an increased ADCC function and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: F243L, R292P, Y300L, V305I, P396L; S239E and I332E; S239D, I332E, and A330L; or L235V, F243L, R292P, Y300L, and P396L, wherein the residues are numbered according to the EU index; and selecting the antibody having an increased ADCC function and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0212] Embodiment 42. A method of manufacturing an antibody having an increased aglycosylation and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: N297G, wherein the residues are numbered according to the EU index; and manufacturing the antibody having an increased aglycosylation and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0213] Embodiment 43. A method of screening for an antibody having an increased aglycosylation and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: N297G, wherein the residues are numbered according to the EU index; and selecting the antibody having an increased aglycosylation and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0214] Embodiment 44. A method of manufacturing an antibody having a reduced effector function and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: L234F, L235E, and D265A; L234A and L235A; L234A, L235A, and P329G; E233P, L234V, L235A, and del G236; or N297G, wherein the residues are numbered according to the EU index; and manufacturing the antibody having a reduced effector function and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0215] Embodiment 45. A method of screening for an antibody having a reduced effector function and reduced viscosity, the method comprising: measuring the viscosity of an antibody comprising one or more of the following mutations: L234F, L235E, and D265A; L234A and L235A; L234A, L235A, and P329G; E233P, L234V, L235A, and del G236; or N297G,wherein the residues are numbered according to the EU index; and selecting the antibody having a reduced effector function and reduced viscosity as compared to a parent antibody not having the one or more mutations.

[0216] Embodiment 46. The method of any one of embodiments 34-45, further comprising measuring the viscosity of the parent antibody.

[0217] Embodiment 47. The method of any one of embodiments 34-46, wherein the antibody is a monoclonal antibody.

[0218] Embodiment 48. The method of any one of embodiments 34-47, wherein the antibody is of an IgGl subclass.

[0219] Embodiment 49. An anti-IgE antibody comprising one or more viscosity-reducing mutations, the anti-IgE antibody comprising: a heavy chain variable domain (VH) comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO:5; CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; a light chain variable domain (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO:8; CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; and the one or more viscosity-reducing mutations results in a variant of the anti-IgE antibody having reduced glycosylation as compared to a parent antibody not having the one or more viscosity reducing mutations.

[0220] Embodiment 50. The anti-IgE antibody of embodiment 36, wherein the one or more viscosity-reducing mutations comprises N297A, wherein residue 297 is numbered according to the EU index.

[0221] Embodiment 51. An antibody comprising one or more mutations providing a reduction in viscosity, wherein a parent of the antibody not comprising the one or more mutations has a high viscosity, the one or more mutations comprising: M252Y, S254T, and T256E; or M428L and N434S, wherein residues 252, 254, 256, 428, and 434 are numbered according to the EU index.

[0222] Embodiment 52. The antibody of embodiment 51, wherein the high viscosity is at least about 20 cP (such as measured according to the description herein).

[0223] Embodiment 53. The antibody of any one of embodiments 49-52, wherein the antibody is a monoclonal antibody.

[0224] Embodiment 54. The antibody of any one of embodiments 49-53, wherein the antibody is of an IgGl subclass.SEQUENCES

[0225] Omalizumab, full-length heavy chain; SEQ ID NO: 1EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASI TYDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFG HWHFAVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0226] Omalizumab, full-length light chain; SEQ ID NO:2DIQLTQSPSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQKPGKAPKLLI YAASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC

[0227] Omalizumab, VH; SEQ ID NO: 3EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITYDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFG HWHFAVWGQGTLVTVSS

[0228] Omalizumab, VL; SEQ ID NO:4DIQLTQSPSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGT KVEIK

[0229] Omalizumab, CDR-H1; SEQ ID NO:5GYSITSGY

[0230] Omalizumab, CDR-H2; SEQ ID NO: 6TYDGS

[0231] Omalizumab, CDR-H3; SEQ ID NO:7GSHYFGHWHFAV

[0232] Omalizumab, CDR-L 1 ; SEQ ID NO : 8RASQSVDYDGDSYMN

[0233] Omalizumab, CDR-L2; SEQ ID NO: 9AASYLES

[0234] Omalizumab, CDR-L3 ; SEQ ID NO : 10QQSHEDPYT

[0235] Omalizumab variant, Full-length heavy chain, YTE variant, Low-viscosity Fc mutations bolded and underlined; SEQ ID NO: 11EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASI TYDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFG HWHFAVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEV TCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0236] Omalizumab variant, Full-length heavy chain, NG variant, Low-viscosity Fc mutations bolded and underlined; SEQ ID NO: 12EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASI TYDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFG HWHFAVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0237] Omalizumab variant, Full-length heavy chain, YTENG variant, Low-viscosity Fc mutations bolded and underlined; SEQ ID NO: 13EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASI TYDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFG HWHFAVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEV TCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0238] Omalizumab variant, Full-length heavy chain, LPLIL variant, Low-viscosity Fc mutations bolded and underlined; SEQ ID NO: 14EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASI TYDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFG HWHFAVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLLPPKPKDTLMISRTPEV TCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PPEEQYNSTLRVVSILTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0239] Omalizumab variant, Full-length heavy chain, LPLIL-NG variant, Low-viscosity Fc mutations bolded and underlined; SEQ ID NO: 15EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASI TYDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFG HWHFAVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLLPPKPKDTLMISRTPEV TCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PPEEQYGSTLRVVSILTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGEXAMPLES

[0240] 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 experimentsperformed. 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.Example 1: Generation of parent and mutagenized IgGl antibodies.

[0241] IgGl antibodies: Antibodies such as omalizumab and trastuzumab, etc., and variants thereof (e.g., Fc variants as described in the present application) were generated using gene synthesis to produce DNA fragments encoding for the parent and the modified heavy chain (HC). Gene fragments then were inserted into a mammalian expression vector and transient transfection was carried out in high titer Chinese hamster ovary (HT CHO) cells followed by two-step purification by protein A chromatography and size exclusion chromatography (SEC).

[0242] Ffab'h generation: Intact IgGl antibodies were IdeS digested and purified with CHI- XL Affinity Matrix. Samples were buffer-exchanged against 20 mM histidine acetate, pH 5.5 using Slide- A-Lyzer 10 kDa MWCO Dialysis Cassettes (Thermo Fisher Scientific). Dialyzed antibody samples were concentrated to greater than 180 mg / mL using Amicon Ultra- 15 10 kDa MWCO centrifugal filter units (MilliporeSigma) by centrifugation at 4,000 g. Concentration determinations were made by gravimetric analysis in duplicate with 2-3 pL of sample diluted approximately 500-fold prior to taking the A280 measurement on an 8453 UV / Vis Spectrometer (Agilent). The A280 was then multiplied by the dilution factor and the product was divided by the antibody extinction coefficient, individually calculated based upon corresponding primary sequence, to yield the sample concentration. Extinction coefficients and pl for corresponding IgGl antibodies were determined with internal software and recapitulated with the ExPASy ProtParam tool. Positive displacement pipettes were used for dispensing high concentration antibody samples to enable accurate volume transfers.

[0243] Parent IgGl antibodies, F(ab')2 fragments, Fc regions (e.g., Matsumiya et al. (2007), J Mol Biol 368:767-79, and mixtures of F(ab')2 fragments and Fc regions then were assessed for their self-association by testing for their viscosity as described below in Example 2 (FIG.1A and FIG. 6B)Example 2: Contributions of variable domains, Fc region, and IgGl format to the viscosity of omalizumab.

[0244] Alternative possible molecular mechanisms underlying the high IgGl viscosity were considered, focusing on omalizumab, and the domain location of interaction sites. A priori, IgG self-association sites may include Fab-Fab, Fab-Fc and Fc-Fc interactions (FIG. 6A). Omalizumab has a high viscosity compared to trastuzumab. Trastuzumab is a structurally similar IgGl antibody that differs from omalizumab only in the antigen-binding variable domain. Given the structural similarities and differences of omalizumab and trastuzumab, the viscosity of F(ab')2 fragments, Fc regions, and a mixture of F(ab')2 fragments and Fc regions (e.g., see FIG. 1A) was tested and compared to the viscosity of the parent antibody in order to identify what region(s) impact antibody self-association and thus the antibody viscosity.

[0245] To determine viscosity, samples were analyzed on a Discovery HR 30 cone-and-plate rheometer (TA Instruments) using 20 mm diameter stainless steel 1° cone angle Peltier plate geometry. Concentrated antibody samples were prepared by dilution in 20 mM histidine acetate, pH 5.5, prior to loading 40 pL onto the rheometer plate. The cone was lowered, and samples were measured at a range of temperatures or temperature-controlled 25.0°C with fixed shear-rate of 1,000 s'1. Each reported value was representative of the mean of 12 measurements collected over 1 minute. The rheometer was calibrated with 8 cP (s6) and 30 cP (s20) viscosity standards (Cannon Instrument Company) to ±5% at the start of each day.

[0246] The viscosity of omalizumab and trastuzumab was measured as above and determined to be 176 cP and 8 cP, respectively. The omalizumab-corresponding F(ab')2 fragment showed an 89% reduction in viscosity from the parental omalizumab IgGl antibody, while the Fc region displayed very low viscosity (3 cP), as shown in FIG. IB. The viscosity of an equimolar mixture of omalizumab F(ab')2 at a concentration of 117 mg / mL and Fc region at a concentration of 63 mg / mL failed to restore the high viscosity of the parental omalizumab antibody. These results suggest that there is some dependence on the intactness of this IgGl and that the Fab-Fc association may be important contributors to the high viscosity of omalizumab (e.g., see FIG. 6B).

[0247] In contrast to omalizumab, the viscosity of trastuzumab IgGl and its corresponding F(ab')2 fragment was comparable, consistent with no major role for the Fc regions in viscosity, including in the viscosity of trastuzumab (FIG. IB). The viscosity of an equimolar mixture of the corresponding F(ab')2 and Fc fragments of trastuzumab was also measured and found toresult in a moderate decrease in viscosity, indicating some dependence on intact IgGl format for trastuzumab viscosity.Example 3: Parental omalizumab histidine scanning and YTE variant with modified near-histidine negatively charged residues impact on viscosity.

[0248] The viscosity of omalizumab variants that comprised mutations within the omalizumab Fab and / or the Fc region was assessed (FIG. 10). In one experiment, the parental omalizumab antibody was scanned for histidines in the variable domains and the Fc region, and substituted for asparagine. In a second experiment, omalizumab variants were generated by removing potential charge repulsion from the variable domains and / or the Fc region of the omalizumab YTE variant antibody. Results from both experiments are presented in Table 6 below. The YTE Fc mutations are described in, e.g., Oganesyan et al. (2009), Mol Immunol 46: 1750-1755.Table 6. Viscosity results of omalizumab variants generated by histidine scanning or potential charge repulsion removal. Charge repulsion removal was always performed in combination with YTE mutations. Rheometry measurements were made at 180 mg / mL IgGl antibody solutions in 20 mM histidine acetate, pH 5.5 at 25.0°C.

[0249] These results demonstrate that the fragmentation of omalizumab into F(ab')2 and Fc components greatly reduces viscosity (FIG. IB). This may reflect lowering of the number of potential self-association sites per molecule from four for omalizumab IgGl down to two for the corresponding F(ab')2 plus Fc mixture (FIG. 6B). In lolo, these observations suggest that Fab-Fab and Fab-Fc but not Fc-Fc interactions make significant contributions to the high viscosity of omalizumab.Example 4: Effects of Fc variants on omalizumab viscosity.

[0250] Generation of antibodies and antibody variants and measurement of viscosities were performed as described in Examples 1-3. Common Fc variants that were tested are provided in Table 7 below.Table 7. Common Fc region variants, including their respective effects upon effector functions, plasma half-life and heavy chain heterodimerization. Residues are numbered using the EU numbering scheme.D, increased binding or function; U, decreased binding or function.Listed exemplary antibody therapeutics are approved or currently undergoing regulatory review that contain the listed mutations (*)alone or (**)in combination with additional Fc mutations, (e.g., The Antibody Society, Inc. Antibody therapeutics approved or in regulatory review in the EU or US. [accessed 2023 Oct 21], https: / / www.antibodysociety.org / resources / approved-antibodies / .)

[0251] The effects of commonly used Fc variants were tested with regards to the viscosity of omalizumab. Many Fc variants resulted in only small perturbations in viscosity, whereas large increases or decreases in viscosity were seen in some cases (see FIGS. 2 A and 2B). The Fc region variant YTE is known for extending plasma half-life extension. The Fc region variant NG is known for causing aglycosylation (e.g., Borrok et al. (2012), ACS Chem Biol 7: 1596- 1602). The Fc region variant LPLIL is known for increasing effector function. These variants were tested and found to decrease the viscosity compared to omalizumab by 91%, 55%, and 38%, respectively (FIG. 2A). In contrast, an alternative plasma half-life extending variant, LS,actually increased the viscosity compared to omalizumab by 45%. Other Fc variants tested had viscosity comparable to that of omalizumab.

[0252] The effects of the same Fc variants on the viscosity of trastuzumab were also tested. In contrast to the effects seen with variants of omalizumab, none of the Fc variants significantly reduced the already low viscosity of trastuzumab (FIG. 2B). However, three Fc variants increased the viscosity of trastuzumab: EFT (102%), V12 (94%), and SELF (59%). Replicate analyses were conducted on trastuzumab and variants thereof and the results are provided in FIG. 2C illustrating that three Fc variants increased the viscosity of trastuzumab: EFT (102%), V12 (94%), and SELF (47%). These data demonstrate that unwanted self-association can sometimes result from Fc modifications that are commonly used in clinical-stage antibodies.

[0253] Further, the combination of the YTE and the NG variants on the viscosity of omalizumab and trastuzumab were tested, as shown in FIG. 9. Combining these Fc variants (i.e., YTENG) resulted in an intermediate viscosity (25 cP), suggesting these Fc variants reduce omalizumab viscosity through distinct mechanisms.Example 5: Spatial Localization of Fc variant substitutions with significant viscosity effects.

[0254] Next, possible mechanisms by which the YTE variant might reduce the viscosity compared to the parental omalizumab antibody were considered. The spatial characteristics of low-viscosity antibody variants identified in Example 2-3 above were analyzed. The large collection of different substitutions analyzed in multiple antibodies presented a unique ability to identify topological areas linked to self-association in the Fc region.

[0255] As a whole, the spatial orientation of viscosity effecting substitutions introduced to omalizumab was significantly different from that of trastuzumab, as shown in FIG. 3A. Regions associated with viscosity-effecting substitutions introduced to omalizumab are shown in boxes labeled Y, while regions associated with viscosity-effecting substitutions in trastuzumab are shown in boxes labeled X (FIG. 3A). All variants identified that substantially increase the viscosity of trastuzumab involve replacement of residues that are located in the upper CH2 domain, as shown in FIG. 3C.

[0256] With respect to omalizumab, viscosity reducing variants NG and LPLIL have solvent accessible surface residues in the upper CH2 domain as well as glycan-facing residues in the CH2 and CH3 domains. The most significant viscosity reducing variant, YTE, is found withinthe CH2 / CH3 elbow region, as shown in FIG. 3B. This location overlaps with the single Fc variant with elevated viscosity, LS, as shown in FIG. 3D. The striking contrast in viscosity between the YTE Fc variant (17 cP) and LS Fc variant (240 cP) suggests a single selfassociation interface unique to omalizumab in the CH2 / CH3 elbow region.

[0257] The X-ray crystallographic structure of the YTE Fc region (PDB structure 3FJT) is highly similar to the wild-type Fc region (PDB structure 3 AVE) (e.g., see FIG. 8). Nevertheless, the YTE mutations have been previously reported to reduce the thermal melting temperature (Tm) of the IgGl CH2 domain by ~7°C, consistent with some structural destabilization of the CH2 domain. Hydrogen / deuterium exchange experiments with matched parent and YTE variant antibodies revealed greater flexibility of the 244-254 segment of the CH2 domain in the YTE variant. The other Fc variant identified that greatly reduces the viscosity compared to parental omalizumab, NG (55%), also lowers the thermal stability through destabilization of the CH2 domain. However, the results described herein suggest that these Fc variants reduce viscosity through distinct mechanisms, which is confirmed by the YTENG combination variant that showed intermediate viscosity.Example 6: Effect of YTE and LS variants on additional high viscosity antibodies.

[0258] The large decrease and increase in viscosity imparted by the introduction of the YTE and LS substitutions, respectively, to omalizumab (FIG. 2A) provided motivation to evaluate these Fc variants in the context of multiple different antibodies. Five additional IgGl antibodies were selected due to their high viscosity (i.e., between 23-156 cP): infliximab (anti-TNFa), anti-GCGR antibody, cetuximab (anti-EGFR), anti-IL-6 antibody, and vonlerolizumab (anti- 0X40). Antibody variants with YTE and LS Fc regions were generated for each antibody as described in Example 1 above, and the viscosity of each variant was tested and compared to the viscosity of the parent antibody, as described in Example 2 above.

[0259] The effect of the YTE and LS substitutions on the viscosity of these additional antibodies was highly dependent on the variable domain context, as shown in FIG. 4A. Among this set of antibodies, any viscosity reductions were much less pronounced than for the YTE variant of omalizumab. Indeed, the largest viscosity reductions observed were for the YTE variant of infliximab (41%) and the LS variant of anti -IL-6 antibody (41%), as shown in FIG. 4B. The incorporation of the YTE substitutions into vonlerolizumab had the opposite effect, increasing the viscosity by 91%, which further demonstrates the antibody-specific nature ofthese Fc variant effects on self-association. For each of the antibodies tested, either YTE or the LS half-life extension mutations gave rise to a small to moderate reduction (17-41%) in the viscosity compared to the corresponding parent IgGl antibody, as shown in FIG. 4B. Replicate analyses were conducted (FIGS. 4C and 4D) and an increased viscosity was again observed for YTE substitutions into, e.g., vonlerolizumab.Example 7: High viscosity of omalizumab mitigated by individual YTE substitutions.

[0260] The large reduction in viscosity imparted by the introduction of the YTE triple mutation to omalizumab was further investigated by evaluating all possible YTE component single (M252Y, S254T and T256E) and double (M252Y:S254T, M252Y:T256E, and S254T:T256E) mutations. Antibody variants were generated according to Example 1 above, and the viscosity of antibody variants was measured as described in Example 2 above.

[0261] Strikingly, the large decrease in viscosity resulting from the YTE triple mutation (91%) could be nearly recapitulated with individual S254T (84%) or T256E substitutions (83%), as shown in FIG. 5A. In contrast, the single substitution M252Y had the opposite effect, increasing the viscosity by 186%. Surprisingly, the viscosity reducing effects of either the S254T or T256E single mutations were not significantly attenuated by combining them with the M252Y mutation.Example 8: Electrostatic and hydrophobic interactions may contribute to the high viscosity of omalizumab.

[0262] The type of noncovalent interactions contributing to the high viscosity of omalizumab were studied by the addition of either of the excipients, NaCl or arginine-hydrochloride (Arg- HC1).

[0263] Omalizumab and the corresponding YTE variant were prepared as described in Example 1 above. Rheometry measurements were obtained at 180 mg / mL IgGl antibody solutions in 20 mM histidine acetate, pH 5.5 at 25.0°C in the absence or presence of different concentrations of the excipients NaCl or Arg-HCl. FIG. 5A provides the results in the absence of either excipient, whereas FIGS. 5B and 5C provide the viscosity of each variant based on the concentration curve of NaCl and Arg-HCl, respectively.

[0264] The viscosity of omalizumab variants was reduced by 42-47% compared to the parental omalizumab antibody, in the presence of NaCl (FIG. 5B), suggesting shielding of electrostatic interactions. In contrast, the viscosity of the YTE variant of omalizumab was slightly increased compared to that of omalizumab by addition of NaCl (FIG. 5B). The addition of Arg-HCl reduced viscosity of the omalizumab parental antibody in solution (51-69%) and also its YTE variant (34-43%), as shown in FIG. 5C, suggesting aromatic groups are a contributing factor to high viscosity of omalizumab. Additional analysis of the omalizumab YTE variant compared to omalizumab parental antibody revealed decreased temperature effects, reduced shear thinning at high shear rates and a significant delay in concentrationdependent pseudo-exponential growth in viscosity, is shown in FIGS. 7A-7D.Example 9: Effect of NG variants on additional antibodies.

[0265] Changes in viscosity were assessed for the introduction of the NG substitutions to fourteen IgGl antibodies (an NG substitution for omalizumab was assessed in FIG. 2A). The antibodies tested are briakinumab, an anti-IL-6 antibody, ifabotuzumab, vonlerolizumab (an anti-OX40), olinvacimab, vedolizumab, trastuzumab, infliximab (an anti-TNFa antibody), icrucumab, tezepelumab, ixekizumab, an anti-PCSK9 antibody, cetuximab (an anti-EGFR antibody), and an anti-GCGR antibody. Antibody variants with NG Fc regions were generated for each antibody as described in Example 1 above, and the viscosity of each variant was tested and compared to the viscosity of the parent antibody, as described in Example 2 above. The results from the viscosity test are illustrated in FIGS. 11A and 11B. As shown in FIGS. 11A and 11B, the reduction in viscosity observed for omalizumab extended to several additional antibodies including briakinumab, an anti-IL-6 antibody, and ifabotuzumab.

Claims

CLAIMSWhat is claimed is:

1. An anti-IgE antibody comprising one or more viscosity-reducing mutations, the anti- IgE antibody comprising: a heavy chain variable domain (VH) comprising:CDR-H1 comprising the amino acid sequence of SEQ ID NO:5;CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; andCDR-H3 comprising the amino acid sequence of SEQ ID NO:7; a light chain variable domain (VL) comprising:CDR-L1 comprising the amino acid sequence of SEQ ID NO:8;CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; andCDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; and the one or more viscosity-reducing mutations comprising:S254T;T256E; orS254T and T256E, wherein the residues are numbered according to the EU index.

2. The anti-IgE antibody of claim 1, wherein the one or more viscosity-reducing mutations further comprise M252Y.

3. The anti-IgE antibody of claim 1 or 2, wherein the one or more viscosity-reducing mutations is M252Y and S254T.

4. The anti-IgE antibody of claim 1 or 2, wherein the one or more viscosity-reducing mutations is M252Y and T256E.

5. The anti-IgE antibody of claim 1 or 2, wherein the one or more viscosity-reducing mutations is M252Y, S254T, and T256E.

6. The anti-IgE antibody of claim 5, wherein the one or more viscosity-reducing mutations further comprise N297G.

7. The anti-IgE antibody of any one of claims 1-5, wherein the one or more viscosityreducing mutations further comprise one or more mutations each selected from the group consisting of:D54N;D30N;E55Q;D30N and D55Q;D54N, D30N, and E55Q;E345Q and E380Q;D249N and D312N; and any combination thereof, wherein residues 249, 312, 345, and 380 are numbered according to the EU index, and residues 30, 54, and 55 are numbered according to Kabat.

8. An anti-IgE antibody comprising one or more viscosity-reducing mutations, the anti- IgE antibody comprising: a heavy chain variable domain (VH) comprising:CDR-H1 comprising the amino acid sequence of SEQ ID NO:5;CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; a light chain variable domain (VL) comprising:CDR-L1 comprising the amino acid sequence of SEQ ID NO:8;CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; and the one or more viscosity-reducing mutations are each selected from the group consisting of:N297G;F243L, R292P, Y300L, V305I, and P396L;S239D and I332E;K326W and E333S;S298A, E333A, and K334A;L234F, L235E, and D265A;E233P, L234V, L235A, and delta G236;S267E, H268F, and S324T;P238D, E233D, G237D, H268D, P271G, and A33 OR;G236A, S239D, and I332E;L235V, F243L, R292P, Y300L, and P396L;L234A, L235A, and P329G;S239D, I332E, and A330L;L234A and L235A;N325S and L328F;S267E and L328F;T366W (knob) and T366S, L368A, and Y407V (hole); and any combination thereof, wherein the residues are numbered according to the EU index.

9. An anti-IgE antibody comprising one or more viscosity-reducing mutations, the anti- IgE antibody comprising: a heavy chain variable domain (VH) comprising:CDR-H1 comprising the amino acid sequence of SEQ ID NO:5;CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; andCDR-H3 comprising the amino acid sequence of SEQ ID NO:7; a light chain variable domain (VL) comprising:CDR-L1 comprising the amino acid sequence of SEQ ID NO:8;CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; andCDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; and the one or more viscosity-reducing mutations are each selected from the group consisting of:H310N;H435N;H310N, H433N, and H435N;H97N, HlOOaN, and HlOOcN; and any combination thereof, wherein residues 310, 433, and 435 are numbered according to the EU index, and residues 97, 100a, and 100c are numbered according to Kabat.

10. The anti-IgE antibody of any one of claims 1-9, wherein the anti-IgE antibody is a monoclonal antibody.

11. The anti-IgE antibody of any one of claims 1-10, wherein the anti-IgE antibody is a humanized or chimeric antibody.

12. The anti-IgE antibody of any one of claims 1-11, comprising a heavy chain variable region (VH) of SEQ ID NO:3, or an amino acid sequence having at least about 95% sequence identity therewith, and a light chain variable region (VL) of SEQ ID NO:4, or an amino acid sequence having at least about 95% sequence identity therewith.

13. The anti-IgE antibody of any one of claims 1-12, comprising a full-length heavy chain of SEQ ID NO: 1, or an amino acid sequence having at least about 95% sequence identity therewith.

14. The anti-IgE antibody of claim 13, wherein the full-length heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-15.

15. The anti-IgE antibody of any one of claims 1-14, comprising a full-length light chain of SEQ ID NO:2, or an amino acid sequence having at least about 95% sequence identity therewith.

16. The anti-IgE antibody of any one of claims 1-15, wherein the anti-IgE antibody is a full-length IgGl antibody.

17. The anti-IgE antibody of any one of claims 1-16, wherein the anti-IgE antibody binds human IgE with an affinity of at least about KA ~ 109M’1.

18. The anti-IgE antibody of any one of claims 1-17, wherein the anti-IgE antibody has a viscosity of about 180 cP or less at 180 mg / mL in 20 mM histidine acetate, pH 5.5.

19. The anti-IgE antibody of any one of claims 1-18, wherein the anti-IgE antibody has a viscosity of about 100 cP or less at 180 mg / mL in 20 mM histidine acetate, pH 5.5.

20. An isolated nucleic acid encoding the anti-IgE antibody of any one of claims 1-19.

21. A vector comprising the isolated nucleic acid of claim 20.

22. A host cell comprising the nucleic acid of claim 20 or the vector of claim 21.

23. A method of producing an anti-IgE antibody comprising culturing the host cell of claim 22 under conditions suitable for the expression of the anti-IgE antibody.

24. The method of claim 23, further comprising recovering the anti-IgE antibody produced by the host cell.

25. A pharmaceutical composition comprising the anti-IgE antibody of any one of claims 1-19 and a pharmaceutically acceptable carrier.

26. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition is formulated for subcutaneous administration.

27. A syringe comprising the pharmaceutical composition of claim 25 or 26.

28. A method of treating an individual having a condition, wherein the condition is selected from the group consisting of an asthma, chronic rhinosinusitis with nasal polyps, chronic spontaneous or idiopathic urticaria, and food allergy, the method comprising administering to the individual an effective amount of the anti- IgE antibody of any one of claims 1-19 or the pharmaceutical composition of claim 25 or 26.

29. The method of claim 28, wherein the asthma is moderate-to-severe asthma.

30. The method of claim 28 or claim 29, wherein the individual is human.

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