Anti-ige antibodies with ph-dependent binding characteristics

Abstract

The present invention provides antibodies and antigen-binding fragments thereof that specifically bind human immunoglobulin E (IgE) with greater affinity at neutral pH than at acidic pH.

Description

ANTI-IGE ANTIBODIES WITH PH-DEPENDENT BINDING CHARACTERISTICS CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No.63 / 669,649 filed on July 10, 2024 which is herein incorporated by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (92VF-350822-WO.xml; Size: 270,394 bytes; and Date of Creation: July 9, 2025) is herein incorporated by reference in its entirety. BACKGROUND

[0003] Immunoglobulin E (IgE) is the last of the five human immunoglobulins to be discovered and is associated with several of allergic diseases and reactions including allergic rhinitis, atopic dermatitis, asthma, urticaria, food allergies, and anaphylaxis. Omalizumab and ligelizumab are two humanized anti-IgE antibodies that were developed for various therapeutic uses. Omalizumab was first approved in the U.S. and Europe for the treatment of asthma, and has since been approved for use to treat urticaria and food allergy.

[0004] Neither omalizumab nor ligelizumab exhibit pH-dependent binding properties to IgE, however, which may have advantages in certain applications. A need exists in the art for new therapeutic molecules that exhibit pH-dependent binding properties to IgE. SUMMARY

[0005] This disclosure provides for antibodies and antigen-binding fragments thereof that exhibit pH- dependent binding characteristics towards IgE. Included herein are mutant forms of omalizumab and ligelizumab. These mutants include mutants wherein one or more amino acids of the parent omalizumab or ligelizumab sequences have been changed to a histidine. Certain mutant antibodies and antibody fragments described herein which exhibit pH-dependent binding characteristics towards IgE have weaker binding to IgE at acidic pH than at a neutral pH. Certain mutant antibodies and antibody fragments described herein which exhibit pH-dependent binding characteristics towards IgE have faster dissociation rates with IgE at acidic pH than at a neutral pH.

[0006] In one aspect, provided herein is an isolated antibody or fragment thereof, which binds IgE and wherein the antibody or fragment comprises histidine at one or more amino acid positions selected from the group consisting of: 31, 35, 52, 53, 54, 64, 73, 95, 99, 100d, 100e, and 101 of a heavy chain variable region (VH) and 25, 27c, 29, 30, 49, 51, 52, 53, 54, 66, 91, 92, 93, 96, and 97 of a light chain variable region (VL), according to Kabat numbering.

[0007] In some embodiments, the antibody or fragment thereof further comprises histidine at one or more amino acid positions selected from the group consisting of: 33, 55 and 100b of a heavy chain variable region (VH) and 31, 94 and 50 of a light chain variable region (VL), according to Kabatnumbering. In some embodiments, the antibody or fragment thereof comprises histidine at one or more amino acid positions selected from the group consisting of: 31, 100e, of a heavy chain variable region (VH) and 29, 49, 51, 53, 96, and 97 of a light chain variable region (VL), according to Kabat numbering. In some embodiments, the antibody or fragment thereof binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH with a KD (acidic pH / neutral pH) ratio greater than 20. In some embodiments, the antibody or fragment thereof binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH with a KD (acidic pH / neutral pH) ratio greater than 50. In some embodiments, the antibody or fragment thereof binds IgE with a higher dissociation rate (koff) at an acidic pH than at a neutral pH. In some embodiments, the antibody or fragment thereof binds IgE with a higher dissociation rate (koff) at an acidic pH than at a neutral pH with a koff (acidic pH / neutral pH) ratio greater than 2. In some embodiments, the antibody or fragment thereof binds IgE with a higher dissociation rate (koff) at an acidic pH than at a neutral pH with a koff (acidic pH / neutral pH) ratio greater than 3.

[0008] In another aspect, provided herein is an isolated antibody or fragment thereof, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH and comprises a CDRH1 comprises the amino acid sequence of SEQ ID NO: 210, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 210; a CDRH2 comprises the amino acid sequence of SEQ ID NO: 211 or 216, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 211 or 216; a CDRH3 comprises the amino acid sequence of SEQ ID NO: 212 or 217, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 212 or 217; a CDRL1 comprises the amino acid sequence of SEQ ID NO: 213, 218, 219, 220, or 221, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 213, 218, 219, 220, or 221; a CDRL2 comprises the amino acid sequence of SEQ ID NO: 214, 222, 223, or 224, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 214, 222, 223, or 224; and a CDRL3 comprises the amino acid sequence of SEQ ID NO: 215 or 225, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 215 or 225, wherein each of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 does not comprise SEQ ID NO: 210, 211, 212, 213, 214, and 215, respectively.

[0009] In some embodiments, the antibody or fragment thereof binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH with a KD (acidic pH / neutral pH) ratio greater than 1.1. In some embodiments, the antibody or fragment thereof binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH with a KD (acidic pH / neutral pH) ratio greater than 1.5. In some embodiments, the antibody or fragment thereof comprises a heavy chain having at least one amino acid substitution with a histidine at positions selected from the group consisting of K64, D73, and W100b, according to Kabat numbering. In some embodiments, the antibody or fragment thereofcomprises a light chain having at least one amino acid substitution with a histidine at positions selected from the group consisting of D27c, G29, D30, S31, Y49, A50, A51, Y53, G66, and S91, according to Kabat numbering. In some embodiments, the at least one amino acid replaced with a histidine is selected from S31, Y49 and Y53, according to Kabat numbering. In some embodiments, the antibody or fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 1, 46 or an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO: 1 or 46, and a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 51, 63, 67, 71, 89, 90, 91, 92 or an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO: 51, 63, 67, 71, 89, 90, 91, 92. In some embodiments, the antibody or fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 1 or 46, and a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 51, 63, 67, 71, 89, 90, 91, or 92. In some embodiments, a VH / VL amino acid sequence pair comprises the amino acid sequence pair of any one of SEQ ID Nos: 1 / 89, 1 / 90, 1 / 91, 1 / 92, 46 / 63, 46 / 67, 46 / 71, 46 / 89, 46 / 90, 46 / 91, 46 / 92. In some embodiments, the antibody or fragment thereof comprising: (a) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 210, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 211, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 217, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 221, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 214, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 215; (b) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 210, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 211, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 217, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 221, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 224, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 215; (c) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 210, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 211, a CDRH3 comprising an amino acid sequence of SEQ ID NO:217, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 221, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 224, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 215; or (d) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 210, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 211, a CDRH3 comprising an amino acid sequence of SEQ ID NO:217, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 221, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 224, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 214.

[0010] In another aspect, provided herein is an isolated antibody or fragment thereof, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH and comprises a CDRH1 comprises the amino acid sequence of SEQ ID NO: 226, 232, 233, 234, or 235, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 226, 232, 233, 234, or 235; aCDRH2 comprises the amino acid sequence of SEQ ID NO: 227, 236, 237, 238, or 239, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 227, 236, 237, 238, or 239; a CDRH3 comprises the amino acid sequence of SEQ ID NO: 228.240, 241, 242, 243, 244, 245, or 282, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 228. 240, 241, 242, 243, 244, 245, or 282; a CDRL1 comprises the amino acid sequence of SEQ ID NO: 229, 246, or 247, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 229, 246, or 247; a CDRL2 comprises the amino acid sequence of SEQ ID NO: 230, 248, 249, 250, 251, or 281, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 230, 248, 249, 250, 251, or 281; and a CDRL3 comprises the amino acid sequence of SEQ ID NO: 231, 252, 253, 254, 255, 256, or 257, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 231, 252, 253, 254, 255, 256, or 257, wherein each of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 does not comprise SEQ ID NO: 226, 227, 228, 229, 230, and 231, respectively.

[0011] In some embodiments, the antibody or fragment thereof binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH with a KD (acidic pH / neutral pH) ratio greater than 20. In some embodiments, the antibody or fragment thereof comprises a heavy chain having at least one amino acid substitution with a histidine at positions selected from the group consisting of W31, E35, D52, G53, T54, F55, F95, S99, D100d, Y100e, and D101. In some embodiments, the antibody or fragment thereof comprises a light chain having at least one amino acid substitution with a histidine at positions selected from the group consisting of A25, I29, A51, S52, S54, S91, W92, S93, W94, T96, and T97. In some embodiments, the antibody or fragment thereof has at least one amino acid substitution with a histidine at positions selected from the group consisting of W31 (heavy chain), Y100e (heavy chain), I29 (light chain), A51 (light chain), T96 (light chain), and T97 (light chain). In some embodiments, the antibody or fragment thereof comprises: (a) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 248, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256; (b) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 247, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 281, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231; (c) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 282, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 248, a CDRL3 comprising an amino acid sequence of SEQ IDNO: 257; (d) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 243, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 257; (e) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 245, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 249, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231; (f) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256; or (h) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 243, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256. In some embodiments, the antibody or fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH / VL amino acid sequence pair comprises the amino acid sequence pair of any one of SEQ ID NOs: 96 / 175, 96 / 176, 96 / 180, 96 / 185, 158 / 143, 158 / 156, 158 / 179, 159 / 181, 161 / 136, 161 / 181, 162 / 143, 162 / 156, 164 / 130, 165 / 142, 165 / 157, 166 / 157, 166 / 181, 167 / 143.167 / 157, or 168 / 130.

[0012] In another aspect, provided herein is an isolated antibody or fragment thereof, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH and comprises a CDRH1 comprises the amino acid sequence of SEQ ID NO: 210, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 210; a CDRH2 comprises the amino acid sequence of SEQ ID NO: 211 or 216, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 211 or 216; a CDRH3 comprises the amino acid sequence of SEQ ID NO: 212 or 217, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 212 or 217; a CDRL1 comprises the amino acid sequence of SEQ ID NO: 213, 218, 219, 220, or 221, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 213, 218, 219, 220, or 221; a CDRL2 comprises the amino acid sequence of SEQ ID NO: 214, 222, 223, or 224, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 214, 222, 223, or 224; and a CDRL3 comprises the amino acid sequence of SEQ ID NO: 215 or 225, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 215 or 225, wherein the antibody or fragment thereof comprises histidine at one or more amino acid positions selected fromthe group consisting of: 64 and 73 of a heavy chain variable region (VH) and 27c, 29, 30, 49, 50, 51, 53, 66, and 91of a light chain variable region (VL), according to Kabat numbering.

[0013] In some embodiments, the antibody or fragment thereof further comprises histidine at one or more amino acid positions selected from the group consisting of: 100b of a heavy chain variable region (VH) and 31 of a light chain variable region (VL), according to Kabat numbering. In some embodiments, the antibody or fragment thereof binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH and comprises a CDRH1 comprises the amino acid sequence of SEQ ID NO: 226, 232, 233, 234, or 235, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 226, 232, 233, 234, or 235; a CDRH2 comprises the amino acid sequence of SEQ ID NO: 227, 236, 237, 238, or 239, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 227, 236, 237, 238, or 239; a CDRH3 comprises the amino acid sequence of SEQ ID NO: 228.240, 241, 242, 243, 244, 245, or 282, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 228.240, 241, 242, 243, 244, 245, or 282; a CDRL1 comprises the amino acid sequence of SEQ ID NO: 229, 246, or 247, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 229, 246, or 247; a CDRL2 comprises the amino acid sequence of SEQ ID NO: 230, 248, 249, 250, 251, or 281, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 230, 248, 249, 250, 251, or 281; and a CDRL3 comprises the amino acid sequence of SEQ ID NO: 231, 252, 253, 254, 255, 256, or 257, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 231, 252, 253, 254, 255, 256, or 257, wherein the antibody or fragment thereof comprises histidine at one or more amino acid positions selected from the group consisting of: 31, 35, 52, 53, 54, 55, 95, 99, 100d, 100e, 101 of a heavy chain variable region (VH) and 25, 29, 51, 52, 54, 91, 92, 93, 94, 96, and 97 of a light chain variable region (VL), according to Kabat numbering. In some embodiments, the antibody or fragment thereof further comprises histidine at one or more amino acid positions selected from the group consisting of: 33, 58, 100b, 100c of a heavy chain variable region (VH) and 50 of a light chain variable region (VL), according to Kabat numbering. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG.1 illustrates a scheme to identify pH-dependent IgE-binding antibodies.

[0015] FIG.2 shows graphs regarding IgE binding of omalizumab mutants at pH 7.4 and pH 6.0.

[0016] FIG.3 shows graphs regarding IgE binding of ligelizumab mutants at pH 7.4 and pH 6.0.

[0017] FIG.4 shows graphs regarding IgE binding of omalizumab mutants at pH 7.4 and pH 6.0.

[0018] FIG.5 shows graphs regarding IgE binding of ligelizumab mutants at pH 7.4 and pH 6.0.

[0019] FIG.6 shows graphs regarding IgE binding of ligelizumab mutants at pH 7.4 and pH 6.0.DETAILED DESCRIPTION Definitions

[0020] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “an antibody,” is understood to represent one or more antibodies. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0021] As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non- naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

[0022] The term “isolated” as used herein with respect to cells, nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule. The term “isolated” as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to cells or polypeptides which are isolated from other cellular proteins or tissues. Isolated polypeptides is meant to encompass both purified and recombinant polypeptides.

[0023] As used herein, the term “recombinant” as it pertains to polypeptides or polynucleotides intends a form of the polypeptide or polynucleotide that does not exist naturally, a non-limiting example of which can be created by combining polynucleotides or polypeptides that would not normally occur together.

[0024] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, though preferably less than 25% identity, with one of the sequences of the present disclosure.

[0025] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 %) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Biologically equivalent polynucleotides are those having the above-noted specified percent homology and encoding a polypeptide having the same or similar biological activity.

[0026] As used herein, an “antibody” or “antigen-binding polypeptide” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. Thus, the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen. Examples of such include, but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein.

[0027] The terms “antibody fragment” or “antigen-binding fragment”, as used herein, is a portion of an antibody such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term “antibody fragment” includes aptamers, spiegelmers, and diabodies. The term “antibody fragment” also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0028] A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins. In some aspects, the regions are connected with a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus ofthe VHwith the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019.

[0029] The term antibody encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, µ, α, δ, ε) with some subclasses among them (e.g., γ l- γ4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.

[0030] Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VK or VH domain, fragments produced by a Fab expression library, and anti- idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to LIGHT antibodies disclosed herein). Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

[0031] Light chains are classified as either kappa or lambda (Κ, λ). Each heavy chain class may be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

[0032] Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VK) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains of the light chain (CK) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen- binding site or amino- terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CK domains actually comprise the carboxy- terminus of the heavy and light chain, respectively.

[0033] As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VK domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VK chains (i.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In some instances, e.g., certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, a complete immunoglobulin molecule may consist of heavy chains only, with no light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).

[0034] In naturally occurring antibodies, the six “complementarity determining regions” or “CDRs” present in each antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domains, referred to as “framework” regions, show less inter-molecular variability. The framework regions largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β -sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined (see “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987)).

[0035] In the case where there are two or more definitions of a term which is used and / or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term “complementarity determining region” (“CDR”) to describe the non-contiguous antigen combining sites found withinthe variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. MoI. Biol.196:901-917 (1987), which are incorporated herein by reference in their entireties. The CDR definitions according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth in the table below as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0036] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0037] In addition to table above, the Kabat number system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the thirty third amino acid residue after the end of CDR-H2; includes 3-25 amino acids; and ends at the sequence W- G-X-G, where X is any amino acid. CDR-L1 begins at approximately residue 24 (i.e., following a cysteine residue); includes approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at approximately the sixteenth residue after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins at approximately the thirty third residue after the end of CDR-L2 (i.e., following a cysteine residue); includes approximately 7-11 residues and ends at the sequence F or W-G-X-G, where X is any amino acid. Throughout this application, amino acid numbers are according to Kabat numbering system, unless specified otherwise.

[0038] Antibodies disclosed herein may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be condricthoid in origin (e.g., from sharks).

[0039] As used herein, the term “heavy chain constant region” includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of: a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the disclosure may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the disclosure comprises a polypeptide chain comprising a CH3 domain. Further, an antibody for use in the disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As set forth above, it will be understood by one of ordinary skill in the art that the heavy chain constant region may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.

[0040] The heavy chain constant region of an antibody disclosed herein may be derived from different immunoglobulin molecules. For example, a heavy chain constant region of a polypeptide may comprise a CH1 domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain constant region can comprise a hinge region derived, in part, from an IgGlmolecule and, in part, from an IgG3molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGlmolecule and, in part, from an IgG4molecule.

[0041] As used herein, the term “light chain constant region” includes amino acid sequences derived from antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or constant lambda domain.

[0042] A “light chain-heavy chain pair” refers to the collection of a light chain and heavy chain that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0043] As previously indicated, the subunit structures and three dimensional configuration of the constant regions of the various immunoglobulin classes are well known. As used herein, the term “VH domain” includes the amino terminal variable domain of an immunoglobulin heavy chain and the term “CH1 domain” includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.

[0044] As used herein the term “CH2 domain” includes the portion of a heavy chain molecule that extends, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983). The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 residues.

[0045] As used herein, the term “hinge region” includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

[0046] By “specifically binds” or “has specificity to,” it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody “A” may be deemed to have a higher specificity for a given epitope than antibody “B,” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.”

[0047] As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0048] By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.

[0049] As used herein, phrases such as “to a patient in need of treatment” or “a subject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and / or for treatment.

[0050] The present disclosure provides antibodies (including antigen-binding fragments thereof) that exhibit pH-dependent binding characteristics. As used herein, the expression "pH-dependent binding" means that the antibody exhibits reduced binding to IgE at acidic pH as compared to neutral pH.

[0051] As used herein, the expression "acidic pH" means a pH of 6.0 or less, including pH values of about 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, and less. In some embodiments, “acidic pH” means a pH of about 6.0. In some embodiments, “lysosomal pH” can be “acidic pH.”

[0052] As used herein, the expression "neutral pH" means a pH of about 7.0 to about 7.4. In some embodiments, “neutral pH” means a pH of about 7.0, 7.1, 7.2, 7.3, and 7.4. In some embodiments, “neutral pH” means a pH of about 7.4. In some embodiments, “endosomal pH” can be “neutral pH.”

[0053] The "affinity" of an antibody for an antigen (e.g., IgE for purposes of the present disclosure) is expressed in terms of the KD of the antibody. The KD of an antibody refers to the equilibrium constant for the dissociation equilibrium of the antibody-antigen interaction, where KDis equal to kon / koff. The dissociation constant, KD, and affinity are inversely related; the greater the KDvalue for an antibody binding to an antigen, the weaker the binding affinity of that antibody to that antigen. Accordingly, as used herein, the expression "higher affinity at neutral pH than at acidic pH" (or the equivalent expression "pH-dependent binding") means that the KD for the antibody binding to IgE at acidic pH is greater than the KD for the antibody binding to IgE at neutral pH.

[0054] The binding properties of an antibody for a particular antigen may also be expressed in terms of the koffof the antibody. The koffof an antibody refers to the first-order rate constant for the dissociation of the antibody-antigen complex. Assuming a constant kon, a higher koffcorresponds to a higher KDand a lower affinity. Anti-IgE Antibodies

[0055] Immunoglobulin E (IgE) is the last of the five human immunoglobulins to be discovered and is associated with several of allergic diseases and reactions including allergic rhinitis, atopic dermatitis, asthma, urticaria, food allergies, and anaphylaxis. Omalizumab and ligelizumab are two humanized anti-IgE antibodies that were developed for various therapeutic uses. Omalizumab was first approved in the U.S. and Europe for the treatment of asthma, and has since been approved for use to treat urticaria and food allergy. However, neither omalizumab nor ligelizumab exhibit pH- dependent binding properties.

[0056] On the other hand, antibodies or fragments thereof described herein show superior pH- dependent IgE binding properties. One application of such antibodies is using in LYTACs (lysosome- targeting chimeras) for targeted protein degradation, where a high acidic:neutral KD or koff may bedesired. Mutant antibodies described herein may also have favorable physical properties as measured by, for example, transition melting temperature, aggregation temperature, size exclusion chromatography, hydrophilic interaction chromatography, cross-interaction chromatography, and poly-specificity.

[0057] The present invention provides anti-IgE antibodies with pH-dependent binding characteristics, wherein such antibodies possess one or more amino acid differences as compared to a parental anti- IgE antibody. As used herein, a “parental” anti-IgE antibody is an anti- IgE antibody which does not exhibit pH-dependent binding characteristics or which exhibits only intermediate pH-dependent binding characteristics (e.g., wherein the binding affinity of the parental antibody to IgE at neutral pH is no more than 3 times greater than the binding affinity of the antibody to IgE at acidic pH; or wherein the parental antibody binds IgE with a t1 / 2 at acidic pH that is no more than 3 times shorter than the t1 / 2 for the antibody binding to IgE at neutral pH). In some cases, a “parental” anti- IgE antibody may be an anti-IgE antibody that exhibits enhanced binding to IgE at acidic pH as compared to neutral pH. In some embodiments, a “parental” anti-IgE antibody is an antibody which is obtained by standard antibody production / isolation methods (e.g., mouse immunization, phage display, etc.) without any amino acid modifications artificially introduced in the complementarity determining regions (CDRs). In some embodiments, a parental anti-IgE antibody is omalizumab or ligelizumab.

[0058] Mutant antibodies described herein show superior pH-dependent IgE binding properties. For example, an antibody according to one embodiment, comprising VH comprising an amino acid sequence of SEQ ID NO.159; and VL comprising an amino acid sequence of SEQ ID NO.181, was shown to have a KD ratio (acidic pH 6.0: neutral pH 7.4) of about 45.5 as measured by biolayer interferometry (BLI), and a koff ratio (acidic pH 6.0: neutral pH 7.4) of about 3.2 as measured by BLI. Similarly, an antibody according to one embodiment, comprising VH comprising an amino acid sequence of SEQ ID NO.166; and VL comprising an amino acid sequence of SEQ ID NO.181, was shown to have KDratio (acidic pH 6.0: neutral pH 7.4) of about 52.9 as measured by BLI, and a koff ratio (acidic pH 6.0: neutral pH 7.4) of about 4.2 as measured by BLI.

[0059] In accordance with one embodiment of the present disclosure, therefore, provided is an isolated antibody or fragment thereof which has specificity to a human IgE protein and binds to one or more amino acid residues selected from the human IgE protein. The human IgE protein, as known in the art, includes 428 amino acid residues, as shown in SEQ ID NO: 209 in the table below. In the present disclosure, “immunoglobulin E”, “human immunoglobulin E”, “IgE”, or human “IgE” may refer to this human IgE protein having an amino acid sequence of SEQ ID NO: 209.Table A. Amino Acid Sequence of hIgE Sequence:

[0060] In accordance with one embodiment of the present disclosure, provided is an isolated antibody or fragment thereof that bind human IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH. For example, the isolated antibody or fragment thereof. In certain embodiments, such isolated antibody or fragment thereof includes one or more amino acid substitutions with histidine from a regular anti-IgE antibodies or fragments thereof (“parental antibody”; e.g., omalizumab, ligelizumab, etc.). For example, the isolated antibody or fragment thereof may have histidine at least one of the following positions: 31, 33, 52, 53, 54, 55, 64, 73, 95, 99, 100b, 100d, 100e, and 101 of a heavy chain variable region (VH) and 25, 27c, 29, 30, 31, 33, 34, 35, 50, 51, 52, 53, 54, 66, 91, 92, 93, 94, 96, and 97 of a light chain variable region (VL), according to Kabat numbering. In some embodiments, the isolated antibody or fragment thereof may have histidine at least two, three, or four of the following positions: 31, 33, 52, 53, 54, 55, 64, 73, 95, 99, 100b, 100d, 100e, and 101 of a heavy chain variable region (VH) and 25, 27c, 29, 30, 31, 33, 34, 35, 50, 51, 52, 53, 54, 66, 91, 92, 93, 94, 96, and 97 of a light chain variable region (VL), according to Kabat numbering.

[0061] In some embodiments, the isolated antibody or fragment thereof may comprise histidine at one or more amino acid positions selected from the group consisting of: 31, 35, 52, 53, 54, 64, 73, 95, 99, 100d, 100e, and 101 of a heavy chain variable region (VH) and 25, 27c, 29, 30, 49, 51, 52, 53, 54, 66, 91, 92, 93, 96, and 97 of a light chain variable region (VL), according to Kabat numbering. In some embodiments, the isolated antibody or fragment thereof may comprise histidine at two, three, four or more amino acid positions selected from the group consisting of: 31, 35, 52, 53, 54, 64, 73, 95, 99, 100d, 100e, and 101 of a heavy chain variable region (VH) and 25, 27c, 29, 30, 49, 51, 52, 53, 54, 66, 91, 92, 93, 96, and 97 of a light chain variable region (VL), according to Kabat numbering. In some embodiments, the isolated antibody or fragment thereof may further comprise histidine at one or more amino acid positions selected from the group consisting of: 33, 55 and 100b of a heavy chain variable region (VH) and 31, 94 and 50 of a light chain variable region (VL), according to Kabat numbering.

[0062] In some embodiments, the isolated antibody or fragment thereof may comprise histidine at one or more amino acid positions selected from the group consisting of: 31, 100e, of a heavy chain variable region (VH) and 29, 49, 51, 53, 96, and 97 of a light chain variable region (VL), according to Kabat numbering.

[0063] In some embodiments, the isolated antibody or fragment thereof may have histidine at least one of the following positions: 31, 33, 100b of a heavy chain variable region (VH) and 29, 31, 49, 50, 51, 53, 96, and 97 of a light chain variable region (VL), according to Kabat numbering. In some embodiments, the isolated antibody or fragment thereof may have histidine at least two, three, four, five, six, or more of the following positions: 31, 33, 100b of a heavy chain variable region (VH) and 29, 31, 49, 50, 51, 53, 96, and 97 of a light chain variable region (VL), according to Kabat numbering.

[0064] In some embodiments, the isolated antibody or fragment thereof may have histidine at one or more amino acid positions selected from the group consisting of: 31 and 100e of a heavy chain variable region (VH) and 29, 49, 51, 53, 96, and 97 of a light chain variable region (VL), according to Kabat numbering. The antibody or fragment thereof may further comprising histidine at one or more amino acid positions selected from the group consisting of: 33 and 100b of a heavy chain variable region (VH) and 31 and 50 of a light chain variable region (VL), according to Kabat numbering.

[0065] In some embodiments, VHdoes not comprise amino acid sequence of SEQ ID NO: 263, 264, 265, 266, 273, 274, 275, or 276 or VLdoes not comprise amino acid sequence of SEQ ID NO: 267, 268, 269, 270, 271, 272, 277, 278, 279, or 280.

[0066] For example, the isolated antibody or fragment thereof binds to IgE exhibits a KDratio (acidic pH (i.e., pH 6.0): neutral pH (i.e., pH 7.4)) of about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or greater. In other words, the isolated antibody or fragment thereof binds to IgE at a KD which is about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 times or more of KDat a neutral pH (i.e., pH 7.4). In some embodiments, the isolated antibody or fragment thereof binds IgE at a higher KD(lower affinity) at an acidic pH (i.e., pH 6.0) than at a neutral pH (i.e., pH 7.4) with a KD(acidic pH / neutral pH) ratio greater than about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, or 100.

[0067] In accordance with one embodiment of the present disclosure, the isolated antibody or fragment thereof of claim 1, wherein the antibody binds IgE with a higher dissociation rate (koff) at an acidic pH than at a neutral pH. For example, the isolated antibody or fragment thereof binds to IgE with a koffratio (acidic pH (i.e., pH 6.0): neutral pH (i.e., pH 7.4)) of about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or greater. In other words, the isolated antibody or fragment thereof binds to IgE at a koff which is about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 times or more of koff at a neutral pH (i.e., pH 7.4). In some embodiments, the isolated antibody or fragment thereof binds IgE at a higher koff (higherdissociation) at an acidic pH (i.e., pH 6.0) than at a neutral pH (i.e., pH 7.4) with a koff(acidic pH / neutral pH) ratio greater than about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

[0068] In certain embodiments, such isolated antibody or fragment thereof includes one or more amino acid substitutions with histidine from a parental anti-IgE antibodies or a fragment thereof. Mutant of Parental Antibody 1

[0069] In accordance with one embodiment of the present disclosure, a reference anti-IgE antibody (“Parental Antibody 1”) comprises a heavy chain variable region (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable region (VL) comprising a CDRL1, a CDRL2 and a CDRL3 as described in tables below. In some embodiments, the Parent Antibody 1 is omalizumab, or a variant or fragment thereof. Table B. CDR Sequences of Parental Antibody 1:Table C. VH / VL Sequences of Parental Antibody 1:

[0070] In accordance with one embodiment of the present disclosure, provided is a mutant of Parental Antibody 1or fragment thereof, which bind human IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH. The mutant antibody or fragment thereof may have one or more amino acid substitutions to histidine. In some embodiments, the mutant antibody or fragment thereof may have substitution to histidine in positions selected from the group consisting of: 31, 35, 52, 53, 54, 55, 65, 74, 95, 99, 100b, 100d, 100e, 101 of a heavy chain variable region (VH) and 25, 29, 30, 33, 34, 35, 51, 52, 53, 54, 55, 57, 70, 91, 92, 93, 94, 95, 96, and 97 of a light chain variable region (VL), according to Kabat numbering. In some embodiments, the mutant antibody or fragment thereof may have substitution to histidine in position 65 or 100b of a heavy chain variable region (VH) and 35, 53, 57 of a light chain variable region (VL), according to Kabat numbering.

[0071] For example, an isolated antibody or fragment thereof comprises a heavy chain variable region (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable region (VL)comprising a CDRL1, a CDRL2 and a CDRL3, wherein: the CDRH1 comprises the amino acid sequence of SEQ ID NO: 210, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 210; the CDRH2 comprises the amino acid sequence of SEQ ID NO: 211 or 216, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 211 or 216; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 212 or 217, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 212 or 217; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 213, 218, 219, 220, or 221, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 213, 218, 219, 220, or 221; the CDRL2 comprises the amino acid sequence of SEQ ID NO: 214, 222, 223, or 224, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 214, 222, 223, or 224; and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 215 or 225, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 215 or 225. Table D. CDR Sequences of an Example pH-dependent anti-IgE antibody

[0072] In some embodiment, an isolated antibody or fragment thereof comprises a heavy chain variable region (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable region (VL) comprising a CDRL1, a CDRL2 and a CDRL3, wherein: the CDRH1 comprises the amino acid sequence of SEQ ID NO: 210; the CDRH2 comprises the amino acid sequence of SEQ ID NO: 211 or 216; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 212 or 217; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 213, 218, 219, 220, or 221; the CDRL2 comprises the amino acid sequence of SEQ ID NO: 214, 222, 223, or 224; and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 215 or 225. In some embodiment, an isolated antibody or fragment thereof comprises a heavy chain variable region (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable region (VL) comprising a CDRL1, a CDRL2 and a CDRL3, wherein: the CDRH1 comprises the amino acid sequence of SEQ ID NO: 210; the CDRH2 comprisesthe amino acid sequence of SEQ ID NO: 211; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 212 or 217; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 213 or 221; the CDRL2 comprises the amino acid sequence of SEQ ID NO: 214 or 224; and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 215. In some embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 210; the CDRH2 comprises the amino acid sequence of SEQ ID NO: 211; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 212; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 221; the CDRL2 comprises the amino acid sequence of SEQ ID NO: 214; and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 215. In some embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 210; the CDRH2 comprises the amino acid sequence of SEQ ID NO: 211; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 212; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 221; the CDRL2 comprises the amino acid sequence of SEQ ID NO: 224; and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 215. In some embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 210; the CDRH2 comprises the amino acid sequence of SEQ ID NO: 211; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 212; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 210; the CDRL2 comprises the amino acid sequence of SEQ ID NO: 224; and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 215. In some embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 210; the CDRH2 comprises the amino acid sequence of SEQ ID NO: 211; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 212; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 210; the CDRL2 comprises the amino acid sequence of SEQ ID NO: 214; and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 215.

[0073] In some embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 210; the CDRH2 comprises the amino acid sequence of SEQ ID NO: 211; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 217; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 221; the CDRL2 comprises the amino acid sequence of SEQ ID NO: 214; and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 215. In some embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 210; the CDRH2 comprises the amino acid sequence of SEQ ID NO: 211; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 217; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 221; the CDRL2 comprises the amino acid sequence of SEQ ID NO: 224; and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 215. In some embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 210; the CDRH2 comprises the amino acid sequence of SEQ ID NO: 211; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 217; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 210; the CDRL2 comprises the amino acid sequence of SEQ ID NO: 224; and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 215. In some embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 210; the CDRH2 comprises the amino acid sequence of SEQ ID NO:211; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 217; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 210; the CDRL2 comprises the amino acid sequence of SEQ ID NO: 214; and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 215.

[0074] In some embodiments, the isolated antibody or fragment thereof may comprise: (a) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 210, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 211, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 217, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 221, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 214, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 215; (b) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 210, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 211, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 217, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 221, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 224, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 215; (c) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 210, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 211, a CDRH3 comprising an amino acid sequence of SEQ ID NO:217, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 221, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 224, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 215; or (d) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 210, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 211, a CDRH3 comprising an amino acid sequence of SEQ ID NO:217, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 221, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 224, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 214.

[0075] In some embodiments, an isolated antibody or fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO:1, or a variant of any of the foregoing amino acid sequences in which one or more amino acids is substituted with a histidine residue, and a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 51, or a variant of any of the foregoing amino acid sequences in which one or more amino acids is substituted with a histidine residue.Table E. VH / VL Sequences of an Example pH-dependent anti-IgE antibody

[0076] In some embodiments, the heavy chain variable region (VH) and / or the light chain variable region (VL) includes one or more amino acid substitutions to histidine in a non-CDR framework region. In one aspect, the present disclosure provides anti-IgE antibodies or fragments thereof which exhibit pH-dependent binding characteristics, and which comprise a heavy chain variable region (VH) having amino acid sequence of SEQ ID NO: 1 or 46, and a light chain variable region (VL) having amino acid sequence of SEQ ID NO: 51, 63, 67, 71, 89-92. In some embodiments, a VH / VLamino acid sequence pair comprises the amino acid sequence pair of any one of SEQ ID Nos: 1 / 89, 1 / 90, 1 / 91, 1 / 92, 46 / 63, 46 / 67, 46 / 71, 46 / 89, 46 / 90, 46 / 91, 46 / 92.

[0077] In one aspect, the present disclosure provides anti-IgE antibodies or fragments thereof which exhibit pH-dependent binding characteristics, and which comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 1 or a variant of SEQ ID NO: 1 having at least 90% sequence identity with SEQ ID NO: 1, and VL comprises SEQ ID NO: 51 or a variant of SEQ ID NO: 51 having at least 90% sequence identity with SEQ ID NO: 51. Thevariant of SEQ ID NO: 1 for VHmay comprise one or more amino acid substitutions selected from the group consisting of K64H, D73H, and W100bH. The variant of SEQ ID NO: 1 for VHmay comprise one or more amino acid substitutions of W100bH. The variant of SEQ ID NO: 51 for VL may comprise one or more amino acid substitutions selected from the group consisting of D27cH, G29H, D30H, S31H, Y49H, A50H, A51H, Y53H, G66H, and S91H. The variant of SEQ ID NO: 51 for VL may comprise one or more amino acid substitutions selected from the group consisting of D27cH, G29H, S31H, Y49H, and Y53H. The variant of SEQ ID NO: 51 for VL may comprise one or more amino acid substitutions selected from the group consisting of S31H, Y49H, and Y53H.

[0078] In some embodiments, an isolated antibody or fragment thereof which binds IgE at a higher KD(lower affinity) at an acidic pH than at a neutral pH, may comprise a CDRH1 comprising the amino acid sequence of SEQ ID NO: 210, or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 210; a CDRH2 comprising the amino acid sequence of SEQ ID NO: 211 or 216, or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 211 or 216; a CDRH3 comprising the amino acid sequence of SEQ ID NO: 212 or 217, or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 212 or 217; a CDRL1 comprising the amino acid sequence of SEQ ID NO: 213, 218, 219, 220, or 221, or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 213, 218, 219, 220, or 221; a CDRL2 comprising the amino acid sequence of SEQ ID NO: 214, 222, 223, or 224, or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 214, 222, 223, or 224; and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 215 or 225, or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 215 or 225, wherein the antibody or fragment thereof comprises histidine at one or more amino acid positions selected from the group consisting of: 64 and 73 of a heavy chain variable region (VH) and 27c, 29, 30, 49, 50, 51, 53, 66, and 91of a light chain variable region (VL), according to Kabat numbering.

[0079] In some embodiments, the antibody or fragment thereof may further comprise histidine at one or more amino acid positions selected from the group consisting of: 100b of a heavy chain variable region (VH) and 31 of a light chain variable region (VL), according to Kabat numbering. Mutant of Parental Antibody 2

[0080] In accordance with one embodiment of the present disclosure, a reference anti-IgE antibody (“Parental Antibody 2”) comprises a heavy chain variable region (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable region (VL) comprising a CDRL1, a CDRL2 and a CDRL3 as described in tables below. In some embodiments, the Parent Antibody 2 is ligelizumab, or a variant or fragment thereof.Table F. CDR Sequences of Parental Antibody 2:Table G. VH / VL Sequences of Parental Antibody 2:

[0081] In accordance with one embodiment of the present disclosure, provided is a mutant of Parental Antibody 2 or fragment thereof, which bind human IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH. The mutant antibody or fragment thereof may have one or more amino acid substitutions to histidine. In some embodiments, the mutant antibody or fragment thereof may have one or more substitutions to histidine.

[0082] For example, an isolated antibody or fragment thereof comprises a heavy chain variable region (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable region (VL) comprising a CDRL1, a CDRL2 and a CDRL3, wherein: the CDRH1 comprises the amino acid sequence of SEQ ID NO: 226, 232, 233, 234, or 235, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 226, 232, 233, 234, or 235; the CDRH2 comprises the amino acid sequence of SEQ ID NO: 227, 236, 237, 238, or 239, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 227, 236, 237, 238, or 239; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 228, 240, 241, 242, 243, 244, 245, or 282, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 228, 240, 241, 242, 243, 244, 245, or 282; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 229, 246, or 247, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 229, 246, or 247; the CDRL2 comprises the amino acid sequence of SEQ ID NO: 230, 248, 249, 250, 251, or 281, an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 230, 248, 249, 250, 251, or 281; and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 231, 252, 253, 254, 255, 256, or 257, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 231, 252, 253, 254, 255, 256, or 257.

[0083] In some embodiments, the antibody or fragment thereof may comprise: (a) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acidsequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 248, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256; (b) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 247, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 281, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231; (c) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 282, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 248, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 257; (d) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 243, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 257; (e) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 245, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 249, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231; (f) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 249, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231; (g) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256; or (h) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 243, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256.

[0084] In some embodiments, the antibody or fragment thereof comprises a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising anamino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 248, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256. In some embodiments, the antibody or fragment thereof comprises a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 247, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 281, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231. In some embodiments, the antibody or fragment thereof comprises a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 282, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 248, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 257. In some embodiments, the antibody or fragment thereof comprises a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 243, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 257. In some embodiments, the antibody or fragment thereof comprises a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 245, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 249, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231. In some embodiments, the antibody or fragment thereof comprises a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 249, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231. In some embodiments, the antibody or fragment thereof comprises a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256. In some embodiments, the antibody or fragment thereof comprises a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 243, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256.In some embodiments, the antibody or fragment thereof comprises a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, aCDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 248, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256. In some embodiments, the antibody or fragment thereof comprises a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 247, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 281, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231. In some embodiments, the antibody or fragment thereof comprises a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 282, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 248, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 257. In some embodiments, the antibody or fragment thereof comprises a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 243, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 257. In some embodiments, the antibody or fragment thereof comprises a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 245, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 249, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231. In some embodiments, the antibody or fragment thereof comprises a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 249, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231. In some embodiments, the antibody or fragment thereof comprises a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256. In some embodiments, the antibody or fragment thereof comprises a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 243, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256.Table H. CDR Sequences of an Example pH-dependent anti-IgE antibody

[0085] In some embodiments, an isolated antibody or fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 96, 158, 161, 162, 164, 165, 166, 167, 168, or a variant of any of the foregoing amino acid sequences in which one or more amino acids is substituted with a histidine residue, and a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NOs: 130, 142, 143, 156, 157, 175, 176, 179, 180, 181, 185, or a variant of any of the foregoing amino acid sequences in which one or more amino acids is substituted with a histidine residue. Table I. VH / VL Sequences of an Example pH-dependent anti-IgE antibody

[0086] In one aspect, the present disclosure provides anti-IgE antibodies or fragments thereof which exhibit pH-dependent binding characteristics, and which comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH / VL amino acid sequence pair comprises the amino acid sequence pair of any one of SEQ ID Nos: 96 / 175, 96 / 176, 96 / 180, 96 / 185, 158 / 143, 158 / 156, 158 / 179, 159 / 181, 161 / 136, 161 / 181, 162 / 143, 162 / 156, 164 / 130, 165 / 142, 165 / 157, 166 / 157, 166 / 181, 167 / 143.167 / 157, or 168 / 130. In some embodiments, VH / VL amino acid sequence pair comprises: 96 / 180, 96 / 185, 161 / 181, 165 / 157, or 167 / 143.

[0087] In one aspect, the present disclosure provides anti-IgE antibodies or fragments thereof which exhibit pH-dependent binding characteristics, and which comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VHcomprises SEQ ID NO: 96 or a variant of SEQ ID NO: 96 having at least 90% sequence identity with SEQ ID NO: 96, and VL comprises SEQ ID NO: 130 or a variant of SEQ ID NO: 130 having at least 90% sequence identity with SEQ ID NO: 130. The variant of SEQ ID NO: 96 for VH may comprise one or more amino acid substitutions selected from the group consisting of W31H, E35H, D52H, G53H, T54H, F55H, F95H, S99H, D100dH, Y100eH, and D101H. The variant of SEQ ID NO: 130 for VL may comprise one or more amino acid substitutions selected from the group consisting of A25H, I29H, A51H, S52H, S54H, S91H, W92H, S93H, W94H, T96H, and T97H. In some embodiments, either VHor VLcomprise at least one amino acid substitutions selected from the group consisting of W31H (VH), Y100eH (VH), I29H (VL), A51H (VL), T96H (VL), and T97H (VL).

[0088] In some embodiments, an isolated antibody or fragment thereof, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH, may comprise a CDRH1 comprising the amino acid sequence of SEQ ID NO: 226, 232, 233, 234, or 235, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 226, 232, 233, 234, or 235; a CDRH2 comprising the amino acid sequence of SEQ ID NO: 227, 236, 237, 238, or 239, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 227, 236, 237, 238, or 239; a CDRH3 comprising the amino acid sequence of SEQ ID NO: 228, 240, 241, 242, 243, 244, 245, or 282, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 228, 240, 241, 242, 243, 244, 245, or 282; a CDRL1 comprising the amino acid sequence of SEQ ID NO: 229, 246, or 247, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 229, 246, or 247; a CDRL2 comprising the amino acid sequence of SEQ ID NO: 230, 248, 249, 250, 251, or 281, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 230, 248, 249, 250, 251, or 281; and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 231, 252, 253, 254, 255, 256, or 257, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 231, 252, 253, 254, 255, 256, or 257, wherein the antibody or fragment thereof comprises histidine at one or more amino acid positions selected from the group consisting of: 31, 35, 52, 53, 54, 55, 95, 99, 100d, and 100e, 101 of a heavy chain variable region (VH) and 25, 29,51, 52, 54, 53, 91, 92, 93, 94, 96, and 97 of a light chain variable region (VL), according to Kabat numbering.

[0089] In some embodiments, an isolated antibody or fragment thereof, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH, may comprise: (a) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 248, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256; (b) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 247, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 281, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231; (c) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 282, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 248, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 257; (d) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 243, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 257; or (e) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 245, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 249, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231; (f) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 249, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231; (g) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256; (h) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 243, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256.

[0090] In some embodiments, the antibody or fragment thereof further comprises histidine at one or more amino acid positions selected from the group consisting of: 33, 58, 100b, 100c of a heavy chain variable region (VH) and 31 of a light chain variable region (VL), according to Kabat numbering.

[0091] It will also be understood by one of ordinary skill in the art that antibodies as disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.

[0092] In certain embodiments, the antibody comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, an antibody of the disclosure may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[0093] Antibodies, variants, or derivatives thereof of the disclosure include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to the epitope. For example, but not by way of limitation, the antibodies can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the antibodies may contain one or more non-classical amino acids.

[0094] In some embodiments, the antibodies may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.

[0095] The antibodies may be conjugated or fused to a therapeutic agent, which may include detectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in the art.

[0096] The antibodies can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

[0097] The antibodies can also be detectably labeled using fluorescence emitting metals such as152Eu, or others of the lanthanide series. These metals can be attached to the antibody using suchmetal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to an antibody are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2ndEd.), Robinson et al., (eds.), Marcel Dekker, Inc., pp.623- 53 (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies ‘84: Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp.303-16 (1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. (52:119-58 (1982)). Polynucleotides Encoding the Antibodies and Methods of Preparing the Antibodies

[0098] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies, variants or derivatives thereof of the disclosure. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of the antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of the antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules.

[0099] Methods of making antibodies are well known in the art and described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in U.S. patents: 6,150,584; 6,458,592; 6,420,140 which are incorporated by reference in their entireties. Other Embodiments

[0100] Embodiment 1. An isolated antibody or fragment thereof, which binds IgE and wherein the antibody or fragment comprises histidine at one or more amino acid positions selected from the group consisting of: 31, 35, 52, 53, 54, 64, 73, 95, 99, 100d, 100e, and 101 of a heavy chain variable region (VH) and 25, 27c, 29, 30, 49, 51, 52, 53, 54, 66, 91, 92, 93, 96, and 97 of a light chain variable region (VL), according to Kabat numbering.

[0101] Embodiment 2. The antibody or fragment thereof of Embodiment 1, further comprising histidine at one or more amino acid positions selected from the group consisting of: 33, 55 and 100b of a heavy chain variable region (VH) and 31, 94 and 50 of a light chain variable region (VL), according to Kabat numbering.

[0102] Embodiment 3. The antibody or fragment thereof of Embodiment 1, comprising histidine at one or more amino acid positions selected from the group consisting of: 31, 100e, of a heavy chain variable region (VH) and 29, 49, 51, 53, 96, and 97 of a light chain variable region (VL), according to Kabat numbering.

[0103] Embodiment 4. The isolated antibody or fragment thereof of any one of Embodiments 1-3, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH with a KD (acidic pH / neutral pH) ratio greater than 20.

[0104] Embodiment 5. The isolated antibody or fragment thereof of any one of Embodiments 1-4, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH with a KD (acidic pH / neutral pH) ratio greater than 50.

[0105] Embodiment 6. The isolated antibody or fragment thereof of any one of Embodiments 1-5, wherein the antibody binds IgE with a higher dissociation rate (koff) at an acidic pH than at a neutral pH.

[0106] Embodiment 7. The isolated antibody or fragment thereof of any one of Embodiments 1-6, wherein the antibody binds IgE with a higher dissociation rate (koff) at an acidic pH than at a neutral pH with a koff(acidic pH / neutral pH) ratio greater than 2.

[0107] Embodiment 8. The isolated antibody or fragment thereof of any one of Embodiments 1-7, wherein the antibody binds IgE with a higher dissociation rate (koff) at an acidic pH than at a neutral pH with a koff(acidic pH / neutral pH) ratio greater than 3.

[0108] Embodiment 9. An isolated antibody or fragment thereof, which binds IgE at a higher KD(lower affinity) at an acidic pH than at a neutral pH and comprises a CDRH1 comprises the amino acid sequence of SEQ ID NO: 210, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 210; a CDRH2 comprises the amino acid sequence of SEQ ID NO: 211 or 216, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 211 or 216; a CDRH3 comprises the amino acid sequence of SEQ ID NO: 212 or 217, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 212 or 217; a CDRL1 comprises the amino acid sequence of SEQ ID NO: 213, 218, 219, 220, or 221, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 213, 218, 219, 220, or 221; aCDRL2 comprises the amino acid sequence of SEQ ID NO: 214, 222, 223, or 224, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 214, 222, 223, or 224; and a CDRL3 comprises the amino acid sequence of SEQ ID NO: 215 or 225, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 215 or 225, wherein each of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 does not comprise SEQ ID NO: 210, 211, 212, 213, 214, and 215, respectively.

[0109] Embodiment 10. The antibody or fragment thereof of Embodiment 9, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH with a KD (acidic pH / neutral pH) ratio greater than 1.1.

[0110] Embodiment 11. The antibody or fragment thereof of Embodiment 9 or 10, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH with a KD (acidic pH / neutral pH) ratio greater than 1.5.

[0111] Embodiment 12. The antibody or fragment thereof of any one of Embodiments 9-11, comprising a heavy chain having at least one amino acid substitution with a histidine at positions selected from the group consisting of K64, D73, and W100b, according to Kabat numbering.

[0112] Embodiment 13. The antibody or fragment thereof of any one of Embodiments 9-12, comprising a light chain having at least one amino acid substitution with a histidine at positions selected from the group consisting of D27c, G29, D30, S31, Y49, A50, A51, Y53, G66, and S91, according to Kabat numbering.

[0113] Embodiment 14. The antibody or fragment thereof of any one of Embodiments 9-13, wherein the at least one amino acid replaced with a histidine is selected from S31, Y49 and Y53, according to Kabat numbering.

[0114] Embodiment 15. The antibody or fragment thereof of any one of Embodiments 9-14, comprising a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 1, 46 or an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO: 1 or 46, and a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 51, 63, 67, 71, 89, 90, 91, 92 or an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO: 51, 63, 67, 71, 89, 90, 91, 92.

[0115] Embodiment 16. The antibody or fragment thereof of any one of Embodiments 9-15, comprising a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 1 or 46, and a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 51, 63, 67, 71, 89, 90, 91, or 92.

[0116] Embodiment 17. The antibody or fragment thereof of any one of Embodiments 9-16, wherein a VH / VLamino acid sequence pair comprises the amino acid sequence pair of any one of SEQ ID Nos: 1 / 89, 1 / 90, 1 / 91, 1 / 92, 46 / 63, 46 / 67, 46 / 71, 46 / 89, 46 / 90, 46 / 91, 46 / 92.

[0117] Embodiment 18. The antibody or fragment thereof of Embodiment 17, comprising: (a) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 210, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 211, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 217, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 221 a CDRH2 comprising an amino acid sequence of SEQ ID NO: 214, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 215; (b) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 210, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 211, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 217, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 221 a CDRH2 comprising an amino acid sequence of SEQ ID NO: 224, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 215; a CDRH1 comprising an amino acid sequence of SEQ ID NO: 210, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 211, a CDRH3 comprising an amino acid sequence of SEQ ID NO:217, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 221, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 224, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 215; or (d) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 210, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 211, a CDRH3 comprising an amino acid sequence of SEQ ID NO:217, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 221, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 224, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 214.

[0118] Embodiment 19. An isolated antibody or fragment thereof, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH and comprises a CDRH1 comprises the amino acid sequence of SEQ ID NO: 226, 232, 233, 234, or 235, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 226, 232, 233, 234, or 235; a CDRH2 comprises the amino acid sequence of SEQ ID NO: 227, 236, 237, 238, or 239, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 227, 236, 237, 238, or 239; a CDRH3 comprises the amino acid sequence of SEQ ID NO: 228.240, 241, 242, 243, 244, 245, or 282, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 228.240, 241, 242,243, 244, 245, or 282; a CDRL1 comprises the amino acid sequence of SEQ ID NO: 229, 246, or 247, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 229, 246, or 247; a CDRL2 comprises the amino acid sequence of SEQ ID NO: 230, 248, 249, 250, 251, or 281, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 230, 248, 249, 250, 251, or 281; and a CDRL3 comprises the amino acid sequence of SEQ ID NO: 231, 252, 253, 254, 255, 256, or 257, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 231, 252, 253, 254, 255, 256, or 257, wherein each of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 does not comprise SEQ ID NO: 226, 227, 228, 229, 230, and 231, respectively.

[0119] Embodiment 20. The antibody or fragment thereof of Embodiment 19, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH with a KD(acidic pH / neutral pH) ratio greater than 20.

[0120] Embodiment 21. The antibody or fragment thereof of Embodiment 19 or 20, comprising a heavy chain having at least one amino acid substitution with a histidine at positions selected from the group consisting of W31, E35, D52, G53, T54, F55, F95, S99, D100d, Y100e, and D101.

[0121] Embodiment 22. The antibody or fragment thereof of any one of Embodiments 19-21, comprising a light chain having at least one amino acid substitution with a histidine at positions selected from the group consisting of A25, I29, A51, S52, S54, S91, W92, S93, W94, T96, and T97.

[0122] Embodiment 23. The antibody or fragment thereof of any one of Embodiments 19-22, having at least one amino acid substitution with a histidine at positions selected from the group consisting of W31 (heavy chain), Y100e (heavy chain), I29 (light chain), A51 (light chain), T96 (light chain), and T97 (light chain).

[0123] Embodiment 24. The antibody or fragment thereof of any one of Embodiments 19-23, comprising: (a) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 248, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256; (b) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228,a CDRL1 comprising an amino acid sequence of SEQ ID NO: 247, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 281, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231; (c) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 282, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 248, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 257; (d) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 243, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 257; (e) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 245, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 249, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231; (f) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256; or (h) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 243, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256.

[0124] Embodiment 25. The antibody or fragment thereof of Embodiment 18, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH / VLamino acid sequence pair comprises the amino acid sequence pair of any one of SEQ ID NOs: 96 / 175, 96 / 176,96 / 180, 96 / 185, 158 / 143, 158 / 156, 158 / 179, 159 / 181, 161 / 136, 161 / 181, 162 / 143, 162 / 156, 164 / 130, 165 / 142, 165 / 157, 166 / 157, 166 / 181, 167 / 143.167 / 157, or 168 / 130.

[0125] Embodiment 26. An isolated antibody or fragment thereof, which binds IgE at a higher KD(lower affinity) at an acidic pH than at a neutral pH and comprises a CDRH1 comprises the amino acid sequence of SEQ ID NO: 210, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 210; a CDRH2 comprises the amino acid sequence of SEQ ID NO: 211 or 216, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 211 or 216; a CDRH3 comprises the amino acid sequence of SEQ ID NO: 212 or 217, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 212 or 217; a CDRL1 comprises the amino acid sequence of SEQ ID NO: 213, 218, 219, 220, or 221, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 213, 218, 219, 220, or 221; a CDRL2 comprises the amino acid sequence of SEQ ID NO: 214, 222, 223, or 224, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 214, 222, 223, or 224; and a CDRL3 comprises the amino acid sequence of SEQ ID NO: 215 or 225, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 215 or 225, wherein the antibody or fragment thereof comprises histidine at one or more amino acid positions selected from the group consisting of: 64 and 73 of a heavy chain variable region (VH) and 27c, 29, 30, 49, 50, 51, 53, 66, and 91of a light chain variable region (VL), according to Kabat numbering.

[0126] Embodiment 27. The antibody or fragment thereof of Embodiment 26, further comprising histidine at one or more amino acid positions selected from the group consisting of: 100b of a heavy chain variable region (VH) and 31 of a light chain variable region (VL), according to Kabat numbering.

[0127] Embodiment 28. An isolated antibody or fragment thereof, which binds IgE at a higher KD(lower affinity) at an acidic pH than at a neutral pH and comprises a CDRH1 comprises the amino acid sequence of SEQ ID NO: 226, 232, 233, 234, or 235, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 226, 232, 233, 234, or 235; a CDRH2 comprises the amino acid sequence of SEQ ID NO: 227, 236, 237, 238, or 239, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 227, 236, 237, 238, or 239; a CDRH3 comprises the amino acid sequence of SEQ ID NO: 228.240, 241, 242, 243, 244, 245, or 282, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 228.240, 241, 242, 243, 244, 245, or 282; a CDRL1 comprises the amino acid sequence of SEQ ID NO: 229, 246, or 247, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 229, 246, or 247; a CDRL2 comprises the amino acid sequence of SEQ ID NO: 230, 248, 249, 250, 251, or 281, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 230, 248, 249, 250, 251, or 281; and a CDRL3 comprises the amino acid sequence of SEQ ID NO: 231, 252, 253, 254, 255, 256, or 257, or an amino acid sequence having one or two amino acid substitution fromSEQ ID NO: 231, 252, 253, 254, 255, 256, or 257, wherein the antibody or fragment thereof comprises histidine at one or more amino acid positions selected from the group consisting of: 31, 35, 52, 53, 54, 55, 95, 99, 100d, 100e, 101 of a heavy chain variable region (VH) and 25, 29, 51, 52, 54, 91, 92, 93, 94, 96, and 97 of a light chain variable region (VL), according to Kabat numbering.

[0128] Embodiment 29. The antibody or fragment thereof of Embodiment 28, further comprising histidine at one or more amino acid positions selected from the group consisting of: 33, 58, 100b, 100c of a heavy chain variable region (VH) and 50 of a light chain variable region (VL), according to Kabat numbering. EXAMPLES EXAMPLE 1: General Method of Histidine Mutagenesis

[0129] FIG.1 shows the mutagenesis workflow for omalizumab and ligelizumab mutants. Single amino acid histidine substitutions were introduced into the variable region of omalizumab and ligelizumab. In total, each of 86 residues of omalizumab (residues of SEQ ID NOs: 258, 211, 259, 260, 213, 261, 262, and 216) and 63 residues (residues of SEQ ID Nos: 226-231) of ligelizumab were substituted with histidine (Tables 1-2). Single histidine residue substitutions demonstrating pH- dependent binding to IgE by BLI and SPR (methods described below) were integrated to make every possible combination. For omalizumab, 4 single mutations resulted in mutants having pH-dependent IgE binding. Using those 4 mutation sites, 16 combination mutants with 1 to 4 histidine residue mutations were generated. For ligelizumab, 9 single histidine mutation sites demonstrated pH- dependent IgE binding. Using those 9 mutation sites, 520 combination mutants with between 1 and 9 histidine mutation sites were generated. Histidine combination mutants with no expression were removed from the screen. The remaining combination mutants were screened for pH-dependent IgE binding using the BLI and SPR methods described below. Table 1: Omalizumab amino acid sequences that were substituted (underlining indicates CDR regions)Table 2: Ligelizumab amino acid sequences that were substituted (underlining indicates CDR regions).EXAMPLE 2: Expression and Purification

[0130] All anti-IgE mutants were cloned into a pTT5 vector (Thermo Fisher) with a hIgK single peptide and human IgG1 Fc backbone. They were transiently expressed in HEK293 according to the manufacturer’s protocol (Thermo Fisher). HPLC Protein A affinity chromatography was used to measure expression titers (Thermo Scientific mAbPac Protein A). Mutants were purified on FPLC using Protein A affinity chromatography (mAb select Sure) according to the manufacturers protocol (Cytiva). In certain circumstances, additional purification steps, size exclusion chromatography (Superdex 200, Cytiva), were completed to remove high molecular weight species before binding properties were measured. Purity was measured with HPLC size exclusion chromatography (Sepax ZENIX-C SEC-300) with 1X Phosphate buffered saline as the mobile phase. Table 3: Physical properties of omalizumab mutantsTable 4: Physical properties of ligelizumab mutantsEXAMPLE 3. Evaluation of IgE binding by single histidine mutants of omalizumab and ligelizumab by biolayer interferometry (BLI) and surface plasmon resonance (SPR)

[0131] Referring to FIG.1, step 1, initial binding assessments of 86 omalizumab and 63 ligelizumab single mutants were performed using a Gator Prime BLI instrument. Binding to IgE was measured in 20 mM citric acid / phosphate buffer (pH 7.4 or 6.0), 150 mM NaCl, and 0.05% Tween-20 (BLI buffer). Mutant antibodies were diluted to 0.5 or 1.0 µg / ml, for omalizumab and ligelizumab mutants, respectively, and immobilized using Gator anti-human IgG Fc Gen II (HFCII) probes. For initial screening of single variants, recombinant human IgE Lambda (BioRad) was diluted to 10 nM in BLI buffer. Probes were equilibrated in BLI buffer with shaking for 10 min before experiments. Five cycles of regeneration (5 s, Gator Regeneration Buffer, No Salt, #120063) and neutralization (5 s, BLI buffer) were performed before assay. Probes were baselined in BLI buffer for 120 s, loaded with mutant antibody for 180-400 s (until response of 0.2-0.5 nm was reached), followed by an additional baselining step for 120 s. The antibody-loaded probes were then allowed to associate with IgE for 180 s, followed by 300-500 s of disassociation in BLI buffer (300-350 s for omalizumab, 350-500 s for ligelizumab). Multiple runs (10-12) of the wild type (WT) parent antibody (omalizumab and ligelizumab) were used to generate cutoffs for pH sensitivity. Single variants with KD pH 6.0 / pH 7.4 ratios greater than 2 standard deviations from mean ratios of the WT, and which showed less than 10- fold loss of affinity (KD) versus the parental antibody were purified by SEC and analyzed by SPR.

[0132] Single histidine variants meeting these criteria were then screened for IgE binding at pH 7.4 and pH 6.0 by SPR on Biacore T200 instrument. For omalizumab mutant Y53H-LC, a polyclonal anti-human IgG antibody (Invitrogen A18827) was bound to flow cells of a Series S SA chip using streptavidin biotin chemistry. For all other single-site omalizumab and ligelizumab mutants, a monoclonal anti-human IgG antibody (Thermo Fisher A55735) was conjugated to flow cells of a Series S CM5 chips using amine coupling. Recombinant human IgE binding was performed in 20 mM citric acid phosphate at either pH 7.4 or 6.0, 150 mM NaCl, 0.05% Tween-20. Omalizumab or ligelizumab mutants were immobilized on the anti-IgG chip, paired with reference flow cells where no IgG was immobilized. For omalizumab mutants P-062 (S31H-LC), P-066 (Y49H-LC), and P-070 (Y53H-LC), IgE was flowed over at increasing concentrations in a multi-cycle kinetic assay with 120 s association and 600 s dissociation (FIG.2). For all other omalizumab and ligelizumab single-site histidine mutants, IgE was flowed over at 5 increasing concentrations in a single-cycle kinetic assay, with 120 second association and 600 s final dissociation (FIG.3). All flow cells of the chip wereregenerated between variants with 10 mM pH 2.0 glycine buffer for subsequent immobilization and binding cycles. Omalizumab and ligelizumab with parental CDR regions were used as controls. Omalizumab binding data was fit to a bivalent analyte binding model and ligelizumab binding data was fit to a one-to-one binding model with Biacore T200 Evaluation Software 3.2.1.

[0133] Omalizumab variants with less than one quarter of the normalized binding response as wildtype at pH 7.4 were removed from subsequent analysis. Four single histidine sites [Y49H-LC, S31H-LC, Y53H-LC, W100bH-HC] for omalizumab were selected for combination mutagenesis based on the SPR results. Table 5: IgE binding by single histidine mutants of omalizumab as measured by BLI.Table 6: IgE binding by single histidine mutants of omalizumab as measured by SPR at pH 7.4.Table 7: IgE binding by single histidine mutants of omalizumab as measured by SPR at pH 6.0.

[0134] Nine single sites for ligelizumab were selected for combination mutagenesis based on pH- sensitive IgE binding and / or accelerated dissociation kinetics relative to wildtype at pH 6.0. Specifically, five variants had a 3.5-fold higher binding constant (KD) at pH 6.0 than pH 7.4 [A51H- LC, I29-LC, T96-LC, Y100eH-HC, Y50H-LC], two variants had dissociation rates at least twice as fast as wildtype at pH 6.0 [W33H-HC, W31H-HC], and two variants satisfied both criteria [T97H-LC, N100bH-HC]. Seven ligelizumab variants with half or less IgE binding than wildtype at pH 7.4 were excluded from hit selection, as defined by the ratio of maximum binding signal to ligelizumab immobilization density.Table 8: IgE binding by single histidine mutants of ligelizumab as measured by BLI.Table 9: IgE binding by single histidine mutants of ligelizumab as measured by SPR at pH 7.4Table 10: IgE binding by single histidine mutants of ligelizumab as measured by SPR at pH 6.0Example 4. Evaluation of IgE binding by combination histidine variants of omalizumab and ligelizumab BLI and SPR

[0135] Eleven omalizumab and 520 ligelizumab combination mutants were expressed and purified using Protein A chromatography, as described above. Mutants with sufficient expression were tested for IgE binding by BLI , using the same protocol as above, except that IgE was diluted to 10 nM (pH 7.4) or 50 nM (pH 6.0) (Fig.1, Step 2) (Table 10). To evaluate binding, both KD and binding response normalized to IgG immobilization (Rmax / load, a measure of binding signal) were evaluated. Ninety-five ligelizumab mutants with Rmax / load values greater than 0.7 at pH 7.4 (to screen for binders that still bound at pH 7.4), and pH-dependent ratios Rmax / load ratio (pH 6.0 / pH 7.4) less than 0.7 or pH dependent KD ratios greater than 5.0 were selected for further characterization to identifyvariants with fast off rates at pH 6.0 (Fig.1, Step 3). In these BLI experiments, the IgG molecule was immobilized and association to IgE (10 nM, 180 s) was performed at pH 7.4, but disassociation of IgE from IgG occurred in pH 6.0 buffer (300 s) (Table 11). Table 11: pH-depending binding behavior of selected omalizumab mutants by BLITable 12: pH-depending binding behavior of selected ligelizumab mutants by BLITable 13: pH-depending binding behavior of selected ligelizumab mutants by BLI

[0136] The 36 mutants with the lowest disassociation ratios (taken as the response at end of pH 6.0 disassociation period divided by the response at the end of the pH 7.4 association period) were selected for further characterization. Variants with Rmax / load under 0.85 at pH 7.4 were generally excluded, although 5 of the 36 had lower values but were brought forward during optimization of the methods.

[0137] BLI dose responses were conducted for the 36 ligelizumab combination mutants using 20 nM (pH 7.4) or 200 nM (pH 6.0) starting IgE concentrations, followed by 7 Log2 dilutions (Fig.1, step 4). An IgE-free blank was included to account for baseline shifts in dose response curves. Global fitting of the data was performed without constraining Rmax for the individual curves. The KD ratios of the BLI dose response curves were used in conjunction with those generated by SPR to select a final list of pH-sensitive IgE binders.

[0138] Four omalizumab combination mutants and thirty-six Protein A purified ligelizumab combination mutants were subsequently evaluated for IgE binding at pH 7.4 and pH 6.0 by SPR. And additional sixteen ligelizumab mutants were also evaluated for comparison. SPR was performed on Biacore T200 instrument. A monoclonal anti-human IgG antibody (Thermo Fisher A55735) was conjugated to all 4 flow cells of a Series S CM5 chips using amine coupling. Binding to recombinant human IgE (BioRad HCA171G) was performed in 20 mM citric acid phosphate at either pH 7.4 or 6.0, 150 mM NaCl, 0.05% Tween-20. Mutants were immobilized on the anti-IgG chip, paired with reference flow cells where no IgG was immobilized. IgE was flowed over at 5 increasing 1concentrations in a single-cycle kinetic assay, with 120 s association and 600 s final dissociation. All flow cells of the chip were regenerated between variants with 10 mM pH 2.0 glycine buffer for subsequent immobilization and binding cycles. Wild type omalizumab and ligelizumab were used as controls. Omalizumab binding data was fit to a bivalent analyte binding model and ligelizumabbinding data was fit to a one-to-one binding model (FIG.4) with Biacore T200 Evaluation Software 3.2.1.

[0139] Ligelizumab mutants were ranked for pH-sensitive IgE binding as measured by SPR with the ratio of binding at pH 7.4 to pH 6.0. Binding at either pH was quantified by the ratio of maximum binding signal to ligelizumab immobilization density. Seven combination variants with inferior IgE binding than wildtype at pH 7.4 were excluded from hits selection. Inferior IgE binding was defined as the ratio of maximum binding signal to ligelizumab immobilization density less than 0.65.

[0140] Some ligelizumab mutants showing weak binding at pH 6.0 were not amenable to a one-to- one curve fit. Hence, data of some mutants were omitted from Tables 14 and 15 but are shown in Table 16 where a bivalent analyte binding model was used. Table 14: IgE binding by combination histidine mutants of omalizumab as measured by SPR at pH 7.4Table 15: IgE binding by combination histidine mutants of omalizumab as measured by SPR at pH 6.0.Table 16: IgE binding by combination histidine mutants of ligelizumab as measured by SPR at pH 7.4.Table 17: IgE binding by combination histidine mutants of ligelizumab as measured by SPR at pH 6.0.

[0141] Select mutants were expressed and purified by Protein A affinity chromatography and size exclusion chromatography, then profiled by SPR with single-cycle and / or multicycle kinetics at pH 7.4 and pH 6.0 using similar experimental setups as described above (Fig.1, step 5). For single-cycle experiments, a monoclonal anti-human IgG antibody (Thermo Fisher A55735) was conjugated to all 4 flow cells of a Series S CM5 chips using amine coupling. Recombinant human IgE (BioRad HCA171G) binding was performed in 20 mM citric acid phosphate at either pH 7.4 or 6.0, 150 mM NaCl, 0.01% Tween-20. Ligelizumab mutants were immobilized on the anti-IgG chip, paired with reference flow cells where no IgG was immobilized. IgE was flowed over at 5 increasing concentrations in a single-cycle kinetic assay, with 120 s association and 600 s final dissociation. All flow cells of the chip were regenerated between variants with 10 mM pH 2.0 glycine buffer for subsequent immobilization and binding cycles. For multi-cycle kinetics experiments, mutants were immobilized on the anti-IgG chip, paired with reference flow cells where no IgG was immobilized. IgE was flowed over at 7 increasing concentrations in a multi-cycle kinetic assay with 120 s association and 600 s dissociation (FIG.6). All flow cells of the chip were regenerated between variants with 10 mM pH 2.0 glycine buffer between each concentration of IgE. Wild type ligelizumab was used as a control. Binding data was fit to both one-to-one and bivalent analyte binding models with Biacore T200 Evaluation Software 3.2.1 (FIGs.5 and 6). Table 18: Additional pH-Dependent Binding Properties of Certain Ligelizumab Mutants by BLI.Table 19: Additional pH-Dependent Binding Properties of Certain Ligelizumab Mutants by SPR at pH 7.4Table 20: Additional pH-Dependent Binding Properties of Certain Ligelizumab Mutants by SPR at pH 6.0. (N.D. indicates not determined due to poor curve fitting.)SEQUENCE LISTING

Claims

CLAIMS 1. An isolated antibody or fragment thereof, which binds IgE and wherein the antibody or fragment comprises histidine at one or more amino acid positions selected from the group consisting of: 31, 35, 52, 53, 54, 64, 73, 95, 99, 100d, 100e, and 101 of a heavy chain variable region (VH) and 25, 27c, 29, 30, 49, 51, 52, 53, 54, 66, 91, 92, 93, 96, and 97 of a light chain variable region (VL), according to Kabat numbering.

2. The antibody or fragment thereof of claim 1, further comprising histidine at one or more amino acid positions selected from the group consisting of: 33, 55 and 100b of a heavy chain variable region (VH) and 31, 94 and 50 of a light chain variable region (VL), according to Kabat numbering.

3. The antibody or fragment thereof of claim 1, comprising histidine at one or more amino acid positions selected from the group consisting of: 31, 100e, of a heavy chain variable region (VH) and 29, 49, 51, 53, 96, and 97 of a light chain variable region (VL), according to Kabat numbering.

4. The isolated antibody or fragment thereof of any one of claims 1-3, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH with a KD(acidic pH / neutral pH) ratio greater than 20.

5. The isolated antibody or fragment thereof of any one of claim 1-4, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH with a KD(acidic pH / neutral pH) ratio greater than 50.

6. The isolated antibody or fragment thereof of any one of claims 1-5, wherein the antibody binds IgE with a higher dissociation rate (koff) at an acidic pH than at a neutral pH.

7. The isolated antibody or fragment thereof of any one of claims 1-6, wherein the antibody binds IgE with a higher dissociation rate (koff) at an acidic pH than at a neutral pH with a koff (acidic pH / neutral pH) ratio greater than 2.

8. The isolated antibody or fragment thereof of any one of claims 1-7, wherein the antibody binds IgE with a higher dissociation rate (koff) at an acidic pH than at a neutral pH with a koff (acidic pH / neutral pH) ratio greater than 3.

9. An isolated antibody or fragment thereof, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH and comprises a CDRH1 comprises the amino acid sequence of SEQ ID NO: 210, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 210; a CDRH2 comprises the amino acid sequence of SEQ ID NO: 211 or 216, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 211 or 216; a CDRH3 comprises the amino acid sequence of SEQ ID NO: 212 or 217, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 212 or 217; a CDRL1 comprises the amino acid sequence of SEQ ID NO: 213, 218, 219, 220, or 221, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 213, 218, 219, 220, or 221; a CDRL2 comprises the amino acid sequence of SEQ ID NO: 214, 222, 223, or 224, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 214, 222, 223, or 224; and a CDRL3 comprises the amino acid sequence of SEQ ID NO: 215 or 225, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 215 or 225, wherein each of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 does not comprise SEQ ID NO: 210, 211, 212, 213, 214, and 215, respectively.

10. The antibody or fragment thereof of claim 9, which binds IgE at a higher KD(lower affinity) at an acidic pH than at a neutral pH with a KD(acidic pH / neutral pH) ratio greater than 1.

1.

11. The antibody or fragment thereof of claim 9 or 10, which binds IgE at a higher KD(lower affinity) at an acidic pH than at a neutral pH with a KD(acidic pH / neutral pH) ratio greater than 1.

5.

12. The antibody or fragment thereof of any one of claims 9-11, comprising a heavy chain having at least one amino acid substitution with a histidine at positions selected from the group consisting of K64, D73, and W100b, according to Kabat numbering.

13. The antibody or fragment thereof of any one of claims 9-12, comprising a light chain having at least one amino acid substitution with a histidine at positions selected from the group consisting of D27c, G29, D30, S31, Y49, A50, A51, Y53, G66, and S91, according to Kabat numbering.

14. The antibody or fragment thereof of any one of claims 9-13, wherein the at least one amino acid replaced with a histidine is selected from S31, Y49 and Y53, according to Kabat numbering.

15. The antibody or fragment thereof of any one of claims 9-14, comprising a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 1, 46 or an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO: 1 or 46, and a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 51, 63, 67, 71, 89, 90, 91, 92 or an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO: 51, 63, 67, 71, 89, 90, 91, 92.

16. The antibody or fragment thereof of any one of claims 9-15, comprising a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 1 or 46, and a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 51, 63, 67, 71, 89, 90, 91, or 92.

17. The antibody or fragment thereof of any one of claims 9-16, wherein a VH / VLamino acid sequence pair comprises the amino acid sequence pair of any one of SEQ ID Nos: 1 / 89, 1 / 90, 1 / 91, 1 / 92, 46 / 63, 46 / 67, 46 / 71, 46 / 89, 46 / 90, 46 / 91, 46 / 92.

18. The antibody or fragment thereof of claim 17, comprising: (a) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 210, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 211, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 217, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 221 a CDRH2 comprising an amino acid sequence of SEQ ID NO: 214, and a CDRH3 comprising an amino acid sequence of SEQ ID NO: 215; (b) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 210, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 211, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 217, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 221, and a CDRH2 comprising an amino acid sequence of SEQ ID NO: 224,a CDRH3 comprising an amino acid sequence of SEQ ID NO: 215; (c) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 210, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 211, a CDRH3 comprising an amino acid sequence of SEQ ID NO:217, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 221, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 224, and a CDRH3 comprising an amino acid sequence of SEQ ID NO: 215; or (d) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 210, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 211, a CDRH3 comprising an amino acid sequence of SEQ ID NO:217, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 221, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 224, and a CDRH3 comprising an amino acid sequence of SEQ ID NO:

214.

19. An isolated antibody or fragment thereof, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH and comprises a CDRH1 comprises the amino acid sequence of SEQ ID NO: 226, 232, 233, 234, or 235, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 226, 232, 233, 234, or 235; a CDRH2 comprises the amino acid sequence of SEQ ID NO: 227, 236, 237, 238, or 239, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 227, 236, 237, 238, or 239; a CDRH3 comprises the amino acid sequence of SEQ ID NO: 228.240, 241, 242, 243, 244, 245, or 282, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 228.240, 241, 242, 243, 244, 245, or 282; a CDRL1 comprises the amino acid sequence of SEQ ID NO: 229, 246, or 247, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 229, 246, or 247; a CDRL2 comprises the amino acid sequence of SEQ ID NO: 230, 248, 249, 250, 251, or 281, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 230, 248, 249, 250, 251, or 281; and a CDRL3 comprises the amino acid sequence of SEQ ID NO: 231, 252, 253, 254, 255, 256, or 257, or an amino acid sequence having one or two amino acid substitution from SEQ IDNO: 231, 252, 253, 254, 255, 256, or 257, wherein each of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 does not comprise SEQ ID NO: 226, 227, 228, 229, 230, and 231, respectively.

20. The antibody or fragment thereof of claim 19, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH with a KD (acidic pH / neutral pH) ratio greater than 20.

21. The antibody or fragment thereof of claim 19 or 20, comprising a heavy chain having at least one amino acid substitution with a histidine at positions selected from the group consisting of W31, E35, D52, G53, T54, F55, F95, S99, D100d, Y100e, and D101.

22. The antibody or fragment thereof of any one of claims 19-21, comprising a light chain having at least one amino acid substitution with a histidine at positions selected from the group consisting of A25, I29, A51, S52, S54, S91, W92, S93, W94, T96, and T97.

23. The antibody or fragment thereof of any one of claims 19-22, having at least one amino acid substitution with a histidine at positions selected from the group consisting of W31 (heavy chain), Y100e (heavy chain), I29 (light chain), A51 (light chain), T96 (light chain), and T97 (light chain).

24. The antibody or fragment thereof of any one of claims 19-23, comprising: (a) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 248, and a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256; (b) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 247, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 281, and a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231; (c) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233,a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 282, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 248, and a CDRL3 comprising an amino acid sequence of SEQ ID NO: 257; (d) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 243, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, and a CDRL3 comprising an amino acid sequence of SEQ ID NO: 257; (e) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 245, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 249, and a CDRL3 comprising an amino acid sequence of SEQ ID NO: 231; (f) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 235, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 228, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, and a CDRL3 comprising an amino acid sequence of SEQ ID NO: 256; or (h) a CDRH1 comprising an amino acid sequence of SEQ ID NO: 233, a CDRH2 comprising an amino acid sequence of SEQ ID NO: 227, a CDRH3 comprising an amino acid sequence of SEQ ID NO: 243, a CDRL1 comprising an amino acid sequence of SEQ ID NO: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 230, anda CDRL3 comprising an amino acid sequence of SEQ ID NO:

256.

25. The antibody or fragment thereof of claim 19, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH / VL amino acid sequence pair comprises the amino acid sequence pair of any one of SEQ ID NOs: 96 / 175, 96 / 176, 96 / 180, 96 / 185, 158 / 143, 158 / 156, 158 / 179, 159 / 181, 161 / 136, 161 / 181, 162 / 143, 162 / 156, 164 / 130, 165 / 142, 165 / 157, 166 / 157, 166 / 181, 167 / 143.167 / 157, or 168 / 130.

26. An isolated antibody or fragment thereof, which binds IgE at a higher KD (lower affinity) at an acidic pH than at a neutral pH and comprises a CDRH1 comprises the amino acid sequence of SEQ ID NO: 210, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 210; a CDRH2 comprises the amino acid sequence of SEQ ID NO: 211 or 216, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 211 or 216; a CDRH3 comprises the amino acid sequence of SEQ ID NO: 212 or 217, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 212 or 217; a CDRL1 comprises the amino acid sequence of SEQ ID NO: 213, 218, 219, 220, or 221, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 213, 218, 219, 220, or 221; a CDRL2 comprises the amino acid sequence of SEQ ID NO: 214, 222, 223, or 224, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 214, 222, 223, or 224; and a CDRL3 comprises the amino acid sequence of SEQ ID NO: 215 or 225, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 215 or 225, wherein the antibody or fragment thereof comprises histidine at one or more amino acid positions selected from the group consisting of: 64 and 73 of a heavy chain variable region (VH) and 27c, 29, 30, 49, 50, 51, 53, 66, and 91of a light chain variable region (VL), according to Kabat numbering.

27. The antibody or fragment thereof of claim 26, further comprising histidine at one or more amino acid positions selected from the group consisting of: 100b of a heavy chain variable region (VH) and 31 of a light chain variable region (VL), according to Kabat numbering.

28. An isolated antibody or fragment thereof, which binds IgE at a higher KD(lower affinity) at an acidic pH than at a neutral pH and comprises a CDRH1 comprises the amino acid sequence of SEQ ID NO: 226, 232, 233, 234, or 235, or an amino acid sequence having one or twoamino acid substitution from SEQ ID NO: 226, 232, 233, 234, or 235; a CDRH2 comprises the amino acid sequence of SEQ ID NO: 227, 236, 237, 238, or 239, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 227, 236, 237, 238, or 239; a CDRH3 comprises the amino acid sequence of SEQ ID NO: 228.240, 241, 242, 243, 244, 245, or 282, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 228.240, 241, 242, 243, 244, 245, or 282; a CDRL1 comprises the amino acid sequence of SEQ ID NO: 229, 246, or 247, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 229, 246, or 247; a CDRL2 comprises the amino acid sequence of SEQ ID NO: 230, 248, 249, 250, 251, or 281, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 230, 248, 249, 250, 251, or 281; and a CDRL3 comprises the amino acid sequence of SEQ ID NO: 231, 252, 253, 254, 255, 256, or 257, or an amino acid sequence having one or two amino acid substitution from SEQ ID NO: 231, 252, 253, 254, 255, 256, or 257, wherein the antibody or fragment thereof comprises histidine at one or more amino acid positions selected from the group consisting of: 31, 35, 52, 53, 54, 55, 95, 99, 100d, 100e, 101 of a heavy chain variable region (VH) and 25, 29, 51, 52, 54, 91, 92, 93, 94, 96, and 97 of a light chain variable region (VL), according to Kabat numbering.

29. The antibody or fragment thereof of claim 28, further comprising histidine at one or more amino acid positions selected from the group consisting of: 33, 58, 100b, 100c of a heavy chain variable region (VH) and 50 of a light chain variable region (VL), according to Kabat numbering.

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