WO2026015652A2 - Anticorps anti-ige à caractéristiques de liaison ph-dépendantes - Google Patents

Anticorps anti-ige à caractéristiques de liaison ph-dépendantes

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Publication number
WO2026015652A2
WO2026015652A2 PCT/US2025/036999 US2025036999W WO2026015652A2 WO 2026015652 A2 WO2026015652 A2 WO 2026015652A2 US 2025036999 W US2025036999 W US 2025036999W WO 2026015652 A2 WO2026015652 A2 WO 2026015652A2
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amino acid
seq
acid sequence
antibody
fragment
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WO2026015652A3 (fr
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Jeffrey IWIG
Isabel JOHNSON
Christos KOUGENTAKIS
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Lycia Therapeutics Inc
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Lycia Therapeutics Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4283—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
    • C07K16/4291—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig against IgE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • Immunoglobulin E 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.
  • Neither omalizumab nor ligelizumab exhibit pH-dependent binding properties to IgE, however, which may have advantages in certain applications.
  • 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.
  • 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • 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 Kabat numbering.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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 D27c, G29, D30, S31, Y49, A50, A51, Y53, G66, and S91, according to Kabat numbering.
  • the at least one amino acid replaced with a histidine is selected from S31, Y49 and Y53, according to Kabat numbering.
  • 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • 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.
  • 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
  • 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.
  • 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
  • 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 23
  • 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.
  • 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.
  • 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.
  • 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).
  • 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
  • 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.
  • VH heavy chain variable region
  • 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
  • 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
  • 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.
  • 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
  • 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.
  • FIG.1 illustrates a scheme to identify pH-dependent IgE-binding antibodies.
  • FIG.2 shows graphs regarding IgE binding of omalizumab mutants at pH 7.4 and pH 6.0.
  • FIG.3 shows graphs regarding IgE binding of ligelizumab mutants at pH 7.4 and pH 6.0.
  • FIG.4 shows graphs regarding IgE binding of omalizumab mutants at pH 7.4 and pH 6.0.
  • FIG.5 shows graphs regarding IgE binding of ligelizumab mutants at pH 7.4 and pH 6.0.
  • 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.
  • 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).
  • polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
  • polypeptides include 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, and the term “polypeptide” may be used instead of, or interchangeably with any of these terms.
  • 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.
  • isolated refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule.
  • 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.
  • 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.
  • 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.
  • 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.
  • Homology 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 compared sequence 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.
  • 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.
  • Biologically equivalent polynucleotides are those having the above-noted specified percent homology and encoding a polypeptide having the same or similar biological activity.
  • 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.
  • 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.
  • 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.
  • antibody fragment includes aptamers, spiegelmers, and diabodies.
  • 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.
  • 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 of the V H with the C-terminus of the V L , 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.
  • heavy chains are classified as gamma, mu, alpha, delta, or epsilon ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) with some subclasses among them (e.g., ⁇ l- ⁇ 4). 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 e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc. are well characterized and are known to confer functional specialization.
  • IgG immunoglobulin molecule
  • 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.
  • 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.
  • Light chains are classified as either kappa or lambda ( ⁇ , ⁇ ). Each heavy chain class may be bound with either a kappa or lambda light chain.
  • 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.
  • 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.
  • the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens.
  • 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).
  • 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.
  • CDR complementarity determining region
  • 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.
  • amino acid numbers are according to Kabat numbering system, unless specified otherwise.
  • Antibodies disclosed herein may be from any animal origin including birds and mammals.
  • the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.
  • the variable region may be condricthoid in origin (e.g., from sharks).
  • an antibody 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.
  • 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.
  • 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.
  • 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.
  • the present disclosure provides antibodies (including antigen-binding fragments thereof) that exhibit pH-dependent binding characteristics.
  • pH-dependent binding means that the antibody exhibits reduced binding to IgE at acidic pH as compared to neutral pH.
  • 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.
  • the KD of an antibody refers to the equilibrium constant for the dissociation equilibrium of the antibody-antigen interaction, where K D is equal to k on /k off .
  • the dissociation constant, K D , and affinity are inversely related; the greater the K D value for an antibody binding to an antigen, the weaker the binding affinity of that antibody to that antigen.
  • the expression "higher affinity at neutral pH than at acidic pH” 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.
  • the binding properties of an antibody for a particular antigen may also be expressed in terms of the k off of the antibody.
  • 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.
  • 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).
  • 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.
  • KD ratio acidic pH 6.0: neutral pH 7.4
  • BLI biolayer interferometry
  • 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 K D ratio (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.
  • K D ratio acidic pH 6.0: neutral pH 7.4
  • a koff ratio acidic pH 6.0: neutral pH 7.4
  • 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.).
  • 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • 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 (V H ) 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 (V L ), according to Kabat numbering.
  • V H heavy chain variable region
  • V L light chain variable region
  • 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 (V H ) and 25, 27c, 29, 30, 49, 51, 52, 53, 54, 66, 91, 92, 93, 96, and 97 of a light chain variable region (V L ), according to Kabat numbering.
  • 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.
  • 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.
  • 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 (V H ) and 29, 31, 49, 50, 51, 53, 96, and 97 of a light chain variable region (VL), according to Kabat numbering.
  • 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.
  • 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 (V H ) and 31 and 50 of a light chain variable region (V L ), according to Kabat numbering.
  • V H does not comprise amino acid sequence of SEQ ID NO: 263, 264, 265, 266, 273, 274, 275, or 276 or V L does not comprise amino acid sequence of SEQ ID NO: 267, 268, 269, 270, 271, 272, 277, 278, 279, or 280.
  • the isolated antibody or fragment thereof binds IgE at a higher K D (lower affinity) at an acidic pH (i.e., pH 6.0) than at a neutral pH (i.e., pH 7.4) with a K D (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.
  • K D acidic pH/neutral pH
  • the isolated antibody or fragment thereof of claim 1 wherein the antibody binds IgE with a higher dissociation rate (k off ) at an acidic pH than at a neutral pH.
  • the isolated antibody or fragment thereof binds to IgE with a k off ratio (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.
  • a k off ratio acidic pH (i.e., pH 6.0): neutral pH (i.e., pH 7.4)
  • 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).
  • a neutral pH i.e., pH 7.4
  • the isolated antibody or fragment thereof binds IgE at a higher koff (higher dissociation) at an acidic pH (i.e., pH 6.0) than at a neutral pH (i.e., pH 7.4) with a k off (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.
  • 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.
  • 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • the Parent Antibody 1 is omalizumab, or a variant or fragment thereof.
  • 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 (V L ) 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.
  • VH heavy chain variable region
  • V L light chain variable region
  • 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 (V L ) 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; 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.
  • VH heavy chain variable region
  • V L light chain variable region
  • 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.
  • an isolated antibody or fragment thereof which binds IgE at a higher K D (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,
  • 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 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
  • 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: 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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: 229, a CDRL2 comprising an amino acid sequence of SEQ ID NO: 248, a CDRL3 comprising an amino amino acid sequence of SEQ ID
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 (V L ) 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.
  • VH heavy chain variable region
  • V L light chain variable region
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the antibodies may contain one or more non-classical amino acids.
  • the antibodies may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
  • 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.
  • 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.
  • 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
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • the antibodies can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • 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.
  • 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.
  • Methods of making antibodies are well known in the art and described herein.
  • 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.
  • 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 (V H ) and 25, 27c, 29, 30, 49, 51, 52, 53, 54, 66, 91, 92, 93, 96, and 97 of a light chain variable region (V L ), according to Kabat numbering.
  • V H heavy chain variable region
  • V L light chain variable region
  • 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.
  • 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.
  • Embodiment 5 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.
  • 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.
  • 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 (k off ) at an acidic pH than at a neutral pH with a k off (acidic pH/neutral pH) ratio greater than 2.
  • Embodiment 9 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 k off (acidic pH/neutral pH) ratio greater than 3.
  • koff dissociation rate at an acidic pH than at a neutral pH with a k off (acidic pH/neutral pH) ratio greater than 3.
  • Embodiment 13 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.
  • 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.
  • Embodiment 14 Embodiment 14.
  • 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • 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 K D (acidic pH/neutral pH) ratio greater than 20.
  • 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.
  • Embodiment 22 Embodiment 22.
  • Embodiment 23 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.
  • 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).
  • 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
  • Embodiment 25 The antibody or fragment thereof of Embodiment 18, comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the V H / V L 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.
  • Embodiment 26 Embodiment 26.
  • 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 (V H ) and 31 of a light chain variable region (V L ), according to Kabat numbering.
  • Embodiment 28 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 (V H ) and 31 of a light chain variable region (V L ), according to Kabat numbering.
  • Table 4 Physical properties of ligelizumab mutants EXAMPLE 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).
  • 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.
  • IgE was flowed over at increasing concentrations in a multi-cycle kinetic assay with 120 s association and 600 s dissociation (FIG.2).
  • 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 were regenerated 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.
  • 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.
  • Table 9 IgE binding by single histidine mutants of ligelizumab as measured by SPR at pH 7.4
  • Table 10 IgE binding by single histidine mutants of ligelizumab as measured by SPR at pH 6.0
  • Example 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.
  • Binding to recombinant human IgE 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 ligelizumab binding data was fit to a one-to-one binding model (FIG.4) with Biacore T200 Evaluation Software 3.2.1.
  • 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.
  • Table 17 IgE binding by combination histidine mutants of ligelizumab as measured by SPR at pH 6.0.
  • 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).
  • 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.
  • 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 7.4
  • Table 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.)

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Abstract

La présente invention concerne des anticorps et des fragments de liaison à l'antigène de ceux-ci qui se lient spécifiquement à l'immunoglobuline E (IgE) humaine avec une plus grande affinité à un pH neutre qu'à un pH acide.
PCT/US2025/036999 2024-07-10 2025-07-09 Anticorps anti-ige à caractéristiques de liaison ph-dépendantes Pending WO2026015652A2 (fr)

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GB201707484D0 (en) * 2017-05-10 2017-06-21 Argenx Bvba Method of preparing ph-dependent antibodies
CN120271714A (zh) * 2022-12-07 2025-07-08 南开大学 一种针对IgE的抗体或其抗原结合片段以及Fc变体

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US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6458592B1 (en) 1995-03-29 2002-10-01 Abgenix, Inc. Production of antibodies using cre-mediated site-specific recombination
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