WO2014009707A1 - Variant ige fc regions - Google Patents

Variant ige fc regions Download PDF

Info

Publication number
WO2014009707A1
WO2014009707A1 PCT/GB2013/051804 GB2013051804W WO2014009707A1 WO 2014009707 A1 WO2014009707 A1 WO 2014009707A1 GB 2013051804 W GB2013051804 W GB 2013051804W WO 2014009707 A1 WO2014009707 A1 WO 2014009707A1
Authority
WO
WIPO (PCT)
Prior art keywords
ige
region
variant
antibody
binding
Prior art date
Application number
PCT/GB2013/051804
Other languages
French (fr)
Inventor
Andrew John BEAVIL
James Michael MCDONNELL
Original Assignee
King's College London
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by King's College London filed Critical King's College London
Publication of WO2014009707A1 publication Critical patent/WO2014009707A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification

Definitions

  • the present invention relates to antibodies, and in particular to immunoglobulin E (IgE) antibodies.
  • the invention is primarily concerned with variant Fc regions of IgE antibodies and their use in the creation of novel engineered or modified IgE antibodies.
  • the invention extends to methods for modifying antibodies and to pharmaceutical compositions comprising such antibodies.
  • the invention also covers uses of the modified antibodies in a wide range of therapeutic applications.
  • Immunoglobulin E (IgE) antibodies play a key role in the mechanisms of allergic disease, not only recognizing allergens through their Fab regions, but also interacting via their Fc regions with two very different cell surface receptors.
  • the IgE-binding oc-chain of FcsRI consists of two extracellular Ig-like domains (sFcsRIoc).
  • FcsRII also known as CD23
  • B cells consists of three C- type lectin "head” domains connected to the membrane by a trimeric oc-helical coiled- coil "stalk".
  • Membrane CD23 (1T1CD23) is cleaved from the cell surface by
  • CD23 expressed on B cells also has the potential to contribute to the clinically serious phenomenon of the spreading of allergic reactivity to unrelated allergens, through its ability to internalize IgE-allergen complexes irrespective of the allergen, in contrast to mlgE-mediated allergen-specific presentation through the B cell receptor.
  • CD23 expressed on gastrointestinal epithelial cells also contributes to IgE-allergen transport across the gut epithelial barrier to trigger food allergenic reactions and similarly on respiratory tract epithelial cells to contribute to airway allergic inflammation. Understanding the IgE-CD23 interaction thus has implications for many aspects of allergic disease.
  • the inventors have now accurately defined the CD23 binding site on the Fc region of IgE, and have clearly demonstrated that the binding site is located in the Ce3 domain of IgE. Based on these results, the inventors have created a series of variant IgE Fc regions, which may be used in the creation of novel and clinically safer IgE-based antibody therapies.
  • a variant IgE Fc region comprising at least one amino acid modification in the Ce3 domain relative to a wild- type IgE Fc region, wherein the at least one amino acid in the wild-type IgE Fc region which is modified is part of a CD23 binding site, and wherein the variant IgE Fc region exhibits a reduced binding affinity to CD23 compared to the wild-type IgE Fc region.
  • the variant IgE Fc region of the first aspect exhibits reduced, and preferably ablated, binding to CD23 (FceRII), but is capable of binding to FceRI.
  • the variant IgE Fc region may still bind to antigens and trigger mast cells through its interaction with FceRI, but, because it cannot bind (or only poorly binds) to CD23, it avoids the problematic side effects that are commonly associated with CD23 binding, including epitope spreading. Therefore, the Fc region of the invention has considerable utility in IgE antibody engineering.
  • the inventors have surprisingly shown that the CD23 and FceRI binding sites occur on opposite ends of the Ce3 domain of IgE, and that allosteric inhibition prohibits simultaneous binding of these two receptors, thereby preventing engagement and cross-linking of IgE bound to mast cells by soluble CD23.
  • immunoglobulin E is a class of antibodies found only in mammals. IgE is a dimeric antibody with four Ig-like domains (Cei- Ce4). The ability to mediate cytotoxic and phagocytic effector functions are potent mechanisms by which antibodies destroy targeted cells.
  • the Fc region links the recognition domain of antibodies to these effector functions through an interaction with Fc receptors and ligands. Manipulation of these effector functions by alteration of the Fc region has important implications in the treatment of numerous medical conditions, e.g. cancer, autoimmune disease and infectious diseases.
  • the Fc region comprises the constant region of an antibody excluding the first constant region immunoglobulin domain.
  • Wild type Fc region refers to the amino acid or nucleotide sequences that are found in nature, including allelic variations.
  • Variant Fc region refers to an Fc sequence that differs from the wild type sequence by virtue of at least one amino acid modification.
  • An Fc variant may only encompass an Fc region, or it may exist in the context of an antibody, Fc fusion, isolated Fc, Fc fragment, or other polypeptide that is substantially encoded by Fc.
  • Fc variant may refer to the Fc polypeptide itself, compositions comprising the Fc variant polypeptide or the amino acid sequence.
  • the DNA sequence encoding wild-type human IgE-Fc is provided herein as SEQ ID NO: 1
  • polypeptide sequence of wild-type human IgE-Fc is provided herein as SEQ ID NO: 1
  • the variant IgE Fc region of the invention is derived from the wild- type human IgE-Fc comprising an amino acid sequence substantially as set out in SEQ ID No: 2, or a functional variant or fragment thereof.
  • the variant IgE Fc region is derived from the wild-type human IgE-Fc which is encoded by a nucleic acid sequence substantially as set out in SEQ ID No:i, or a functional variant or fragment thereof.
  • the Ce3 domain of IgE is defined as amino acid residues 329-441 of SEQ ID No: 2.
  • the at least one amino acid which is modified may be any one of amino acid residues 329-441 of SEQ ID No:2.
  • the inventors are the first to demonstrate that the A-B helix of Cs3 does not form part of the CD23 binding site, and so it cannot be directly involved in CD23 binding. Therefore, preferably the at least one amino acid modification in the variant IgE Fc region is not in the A-B helix of the Ce3 domain of IgE. It will be appreciated that the A-B helix of the Ce3 domain of IgE is defined as amino acid residues 344-353 of SEQ ID No:2.
  • the variant IgE Fc region comprises at least one amino acid modification in the E-F helix of the Ce3 domain of IgE, in the C-D loop of the Ce3 domain of IgE and/or in the C- terminal region of the Ce3 domain of IgE.
  • the E-F helix of the Ce3 domain of IgE is defined as amino acid residues 404-415 of SEQ ID No: 2
  • the C-D loop of the Ce3 domain of IgE is defined as amino acid residues 376-380 of SEQ ID No: 2
  • the C-terminal region of the Ce3 domain of IgE is defined as amino acid residues 433-441 of SEQ ID No: 2. It will be appreciated that the numbering of amino acid residues given herein is equivalent to the wild-type sequence of human Ig Fc region as shown in SEQ ID No:2.
  • the variant IgE Fc region comprises at least one amino acid modification at a position corresponding to wild-type IgE Fc region selected from the group consisting of: (i) amino acids 404-415 from the E-F helix of IgE; (ii) amino acids 376-380 from the C-D loop of IgE; and (iii) residue 433-441 from the C- terminal region of IgE.
  • the variant IgE region may comprise at least one amino acid modification at a position corresponding to wild-type IgE Fc region selected from the group consisting of: 376; 377; 378; 379; 380; 404; 405; 406; 407; 408; 409; 410; 411; 412; 413; 414; 415; 433; 434; 435; 436; 437; 438; 439; 440; and 441 of SEQ ID No:2.
  • the variant IgE region may comprise at least one amino acid modification at a position corresponding to wild-type IgE Fc region selected from the group consisting of: 376; 378; 381; 408; 409; 411; 412; 435; 439; and 440.
  • the variant IgE region comprises at least one amino acid modification at a position equivalent to 409, 412, 376, 380 and/or 435 of the wild-type human IgE Fc region.
  • the at least one modification may comprise an amino acid deletion or an insertion. However, preferably the at least one modification comprises an amino acid substitution. Preferably, the at least one modification may comprise a non- conservative amino acid change. For example, the charge (i.e. pH) of amino acid may be changed, or the residue may be converted from being hydrophilic so that it is hydrophobic, or vice versa. For example, a K residue may be substituted for a D, or an E may be replaced with a R. Alternatively, a small amino acid may be replaced with a large amino acid (e.g., Y for A).
  • a large amino acid may be replaced with a small or constraining amino acid, for example alanine, glycine, proline or serine (e.g., F may be replaced with S).
  • substitution with alanine may be preferred, and the skilled person would readily appreciate the codons which encode alanine, and therefore how to modify the wild- type DNA sequence of SEQ ID No: i to produce variant Fc regions of the invention.
  • the variant Fc region may comprise at least one amino acid modification selected from: D409A; E412A; R376A; K380A and K435A.
  • the variant human IgE Fc region may comprise more than one modification in the Ce3 domain of the Fc region equivalent to the human IgE Fc region of a wild-type antibody.
  • the variant human IgE Fc region may comprise at least two, three, four or five modifications in the Ce3 domain, as described herein.
  • the variant Fc region may comprise a double or treble mutant, and so on.
  • the variant Fc region may comprise a modification at two, three, four, five or more of the amino acids at positions equivalent to 376; 377; 378; 379; 380; 404; 405; 406; 407; 408; 409; 410; 411; 412; 413; 414; 415; 433; 434; 435; 436; 437; 438; 439; 440; and 441 of wild-type human IgE-Fc.
  • the variant Fc region may comprise a modification at each of the amino acids at positions equivalent to 409, 412, 376, 380 and/ or 435 of wild- type human IgE-Fc.
  • polypeptide sequence of the variant IgE Fc region (denoted as D409A) is provided herein as SEQ ID No:3, as follows:
  • polypeptide sequence of the variant IgE Fc region is identical to the polypeptide sequence of the variant IgE Fc region
  • E412A is provided herein as SEQ ID No:4, as follows:
  • polypeptide sequence of the variant IgE Fc region is provided herein as SEQ ID No:5, as follows:
  • polypeptide sequence of the variant IgE Fc region (K380A) is provided herein as SEQ ID No:6, as follows:
  • polypeptide sequence of the variant IgE Fc region (K435A) is provided herein as SEQ ID No ⁇ , as follows:
  • TTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRP 3 43 SPFDLF IRKSPT I TCLWDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTL 403 PVGTRDWIEGETYQCRVTHPHLPRALMRSTTATSGPRAAPEVYAFATPEWPGSRDKRTLA 463 CLI QNFMPED I SVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEF I 523 CRAVHEAASP S QTVQRAVS VNP
  • a preferred variant IgE Fc region of the invention comprises an amino acid sequence substantially as set out in any one of SEQ ID No: 3-7, or a functional variant or fragment thereof.
  • the IgE Fc variants disclosed herein exhibit reduced binding affinity to the CD23 receptor compared to that of the wild-type or parent Fc region.
  • reduced affinity compared to the wild-type Fc region can mean that the Fc variant binds to CD23 with a lower equilibrium constant of association (K A or Ka) or higher equilibrium constant of dissociation (K D or Kd) than the wild-type Fc region when the amounts of variant and wild type region in the binding assay that is used are essentially the same.
  • the Fc variant with reduced binding affinity for CD23 may display from about a 5-fold to about 10,000-fold reduction in CD23 binding affinity compared to the wild type Fc region, where CD23 binding affinity is determined, for example, by the binding methods disclosed herein.
  • Reduced affinity can also be defined relative to an absolute level of affinity.
  • the preferred variants exhibit surprisingly high K D values.
  • the equilibrium constant of dissociation (K D or Kd) of the variant IgE Fc region for CD23 maybe at least 5 ⁇ , ⁇ , 25 ⁇ or more.
  • the variant IgE Fc region of the first aspect is capable of binding to FceRI.
  • the binding affinity of the variant Fc region for FceRI is at least the same as that of the wild-type Fc region.
  • the variant IgE Fc region is capable of binding to FceRI with a K A value of at least io? M 1 , io? M 1 , 10 8 M 1 , ⁇ M 1 , or at least 10 10 M 1 .
  • a nucleic acid encoding the variant IgE Fc region according to the first aspect is provided herein as SEQ ID No:8, as follows:
  • the DNA sequence encoding the variant IgE Fc region (E412A) is provided herein as SEQ ID No :g, as follows:
  • DNA sequence encoding the variant IgE Fc region (R376A) is provided herein as SEQ ID No:io, as follows:
  • SEQ ID No: 10 the DNA sequence encoding the variant IgE Fc region (K380A) is provided herein as SEQ ID No:ii, as follows:
  • DNA sequence encoding the variant IgE Fc region (K435A) is provided herein as SEQ ID No: 12, as follows:
  • the nucleic acid according to the second aspect may comprise a nucleotide sequence substantially as set out in any one of SEQ ID No: 8-12, or a functional variant or fragment thereof.
  • the codon which is shown in bold and underlined represents a modification which would encode alanine.
  • alanine may be encoded by GCT, GCC, GCA and GCG and so any of these codons would be suitable as a modification to create the same amino acid mutation.
  • codons for glycine GGT, GGC, GGA and GGG
  • proline CCC, CCA, CCG
  • serine TCT, TCC, TCA, TCG, AGT and AGC
  • the nucleic acid may be an isolated or purified nucleic acid sequence.
  • the nucleic acid sequence may be a DNA sequence.
  • Also described herein are methods for producing the variant IgE Fc region of the first aspect using the nucleic acids of the second aspect.
  • the nucleic acid sequence may be incorporated into a genetic construct for cloning purposes.
  • a genetic construct comprising the nucleic acid of the second aspect.
  • Genetic constructs of the invention maybe in the form of an expression cassette, which maybe suitable for expression of the encoded polypeptide (i.e. the variant IgE Fc region) in a host cell.
  • the genetic construct may be introduced in to a host cell without it being incorporated in a vector.
  • the genetic construct which maybe a nucleic acid molecule, maybe incorporated within a liposome or a virus particle.
  • a purified nucleic acid molecule e.g. histone-free DNA, or naked DNA
  • the genetic construct may be introduced directly in to cells of a host subject (e.g. a bacterial or eukaryotic cell) by transfection, infection,
  • genetic constructs of the invention may be introduced directly into a host cell using a particle gun.
  • the genetic construct may be harboured within a recombinant vector, for expression in a suitable host cell.
  • a recombinant vector comprising the genetic construct according to the third aspect.
  • the recombinant vector may be a plasmid, cosmid or phage.
  • Such recombinant vectors are useful for transforming host cells with the genetic construct of the third aspect, and for replicating the expression cassette therein.
  • the skilled technician will appreciate that genetic constructs of the invention may be combined with many types of backbone vector for expression purposes.
  • Recombinant vectors may include a variety of other functional elements including a suitable promoter to initiate gene expression. For instance, the recombinant vector may be designed such that it autonomously replicates in the cytosol of the host cell.
  • the recombinant vector may be designed such that it integrates into the genome of a host cell.
  • DNA sequences which favour targeted integration are envisaged.
  • the recombinant vector may also comprise DNA coding for a gene that may be used as a selectable marker in the cloning process, i.e. to enable selection of cells that have been transfected or transformed, and to enable the selection of cells harbouring vectors incorporating heterologous DNA.
  • the selectable marker gene may be in a different vector to be used simultaneously with vector containing the gene of interest.
  • the vector may also comprise DNA involved with regulating expression of the coding sequence, or for targeting the expressed polypeptide to a certain part of the host cell.
  • a host cell comprising the genetic construct according to the third aspect, or the recombinant vector according to the fourth aspect.
  • the host cell may be a bacterial cell.
  • the host cell maybe an animal cell.
  • the host cell may be a mammalian cell, for example a mouse or rat cell. It is preferred that the host cell is not a human cell.
  • the host cell may be transformed with genetic constructs or vectors according to the invention, using known techniques. Suitable means for introducing the genetic construct into the host cell will depend on the type of cell.
  • a method of preparing a variant IgE Fc region comprising- (i) culturing at least one cell according to the fifth aspect under conditions suitable for the expression of a variant IgE Fc region; and (ii) isolating the variant IgE Fc region.
  • the inventors believe that knowledge of the sequences of IgE Fc region which form the binding site for the CD23 receptor can be harnessed in a method for reducing the binding affinity of any existing IgE antibody or in the production of novel IgE antibodies, in order to reduce or avoid the risks of patients suffering from any CD23- mediated side effects that may be associated with an IgE antibody therapy.
  • a method of producing a variant IgE Fc region which exhibits a reduced binding affinity for CD23 comprising modifying at least one amino acid in the Ce3 domain of a wild-type IgE Fc region, wherein the at least one amino acid in the wild-type IgE Fc region which is modified is part of the CD23 binding site, such that the variant IgE Fc region exhibits a reduced binding affinity to CD23 compared to the wild-type IgE Fc region.
  • the variant IgE Fc region produced by the method is incapable of binding to CD23 (FceRII), but is capable of binding to FceRI.
  • the method does not comprise modifying an amino acid in the A-B helix of the Ce3 domain of IgE.
  • the method comprises modifying at least one amino acid in the E-F helix, in the C-D loop and/or in the C-terminal region of the Ce3 domain of IgE.
  • the method comprises modifying at least one amino acid at a position corresponding to wild-type IgE Fc region selected from the group consisting of: (i) amino acids 404-415 from the E-F helix of IgE; (ii) amino acids 376- 380 from the C-D loop of IgE; and (iii) residue 433-441 from the C-terminal region of IgE.
  • the method may comprise modifying at least one amino acid at a position corresponding to wild-type IgE Fc region selected from the group consisting of: 376; 377; 378; 379; 380; 404; 405; 406; 407; 408; 409; 410; 411; 412; 413; 414; 415; 433; 434; 435; 436; 437; 438; 439; 440; and 441 of SEQ ID No:2.
  • the method comprises modifying at least one amino acid at a position equivalent to 409, 412, 376, 380 and/or 435 of the wild-type human IgE Fc region.
  • the method may comprise modifying at least two, three, four, five or more amino acids in the Ce3 domain relative to a wild-type IgE Fc region.
  • the wild-type IgE Fc region which is modified in the method may be part of a therapeutically active IgE antibody.
  • IgE antibody which may be modified include IgE antibodies directed against tumour antigens, as described by Karagiannis et al. (Karagiannis, 2011, Cancer Immunol Immunotherapy; PMID 22139135).
  • the method may be used to modify the IgE antibody such that it cannot bind to CD23, thereby avoiding the risk of CD23-mediated side effects.
  • the method may comprise initially creating the variant IgE Fc region and then fusing it to a functional fragment of an IgE antibody to produce an Fc fusion, for example a chimera.
  • the functional fragment may comprise variable regions exhibiting immunospecificity for a target epitope.
  • the functional fragment maybe selected from a group consisting of VH, VL, Fd, Fv, Fab, Fab', scFv and F(ab') 2 .
  • the variant IgE Fc region of the first aspect or the method of the seventh aspect can be used to form antibody or a functional fragment thereof.
  • an antibody or a functional fragment thereof comprising the variant IgE Fc region of the first aspect.
  • the antibody or functional fragment thereof is incapable of binding to CD23 (FceRII), or exhibits a reduced binding affinity thereto, but is capable of binding to FceRI.
  • the invention extends both to whole antibodies (i.e. immunoglobulins) with immunospecificity for a certain target epitope, as well as to functional fragments thereof. Such fragments retain at least one antigen binding region of a corresponding full-length antibody as well as the variant IgE Fc region of the invention.
  • the antibody or functional fragment thereof may comprise a monoclonal or polyclonal antibody or functional fragment thereof.
  • the antibody or functional fragment may be monovalent, divalent or polyvalent.
  • Monovalent antibodies are dimers (HL) comprising a heavy (H) chain associated by a disulfide bridge with a light chain (L).
  • Divalent antibodies are tetramer (H2L2) comprising two dimers associated by at least one disulfide bridge.
  • Polyvalent antibodies may also be produced, for example by linking multiple dimers.
  • the basic structure of an antibody molecule consists of two identical light chains and two identical heavy chains which associate non-covalently and can be linked by disulphide bonds. Each heavy and light chain contains an amino-terminal variable region of about 110 amino acids, and constant sequences in the remainder of the chain. The variable region includes several hypervariable regions, or
  • CDRs complementarity determining regions
  • the constant region consists of one of five heavy chain sequences ( ⁇ , ⁇ , ⁇ , a, or ⁇ ) and one of two light chain sequences ( ⁇ or ⁇ ).
  • the heavy chain constant region sequences determine the isotype of the antibody and the effector functions of the molecule.
  • the antibody or functional fragment thereof is an IgE.
  • the antibody or fragment thereof may be a human antibody.
  • the term "human antibody” can mean an antibody, such as a monoclonal antibody, which comprises substantially the same heavy and light chain CDR amino acid sequences as found in a particular human antibody exhibiting immunospecificity for certain epitope.
  • human monoclonal antibody can include a monoclonal antibody with substantially human CDR amino acid sequences produced, for example by recombinant methods such as production by a phage library, by lymphocytes or by hybridoma cells.
  • humanised antibody can mean an antibody from a non-human species (e.g. mouse) whose protein sequences have been modified to increase their similarity to antibodies produced naturally in humans.
  • the antibody may be a recombinant antibody.
  • the term "recombinant human antibody” can include a human antibody produced using recombinant DNA technology.
  • the term "antigen binding region” can mean a region of the antibody having specific binding affinity for its target antigen.
  • the binding region may be a hypervariable CDR or a functional portion thereof.
  • the term "functional portion” of a CDR can mean a sequence within the CDR which shows specific affinity for the target antigen.
  • the functional portion of a CDR may comprise a ligand which specifically binds to its epitope.
  • CDR can mean a hypervariable region in the heavy and light variable chains. There maybe one, two, three or more CDRs in each of the heavy and light chains of the antibody. Normally, there are at least three CDRs on each chain which, when configured together, form the antigen-binding site, i.e.
  • CDR the three-dimensional combining site with which the antigen binds or specifically reacts. It has however been postulated that there may be four CDRs in the heavy chains of some antibodies.
  • the definition of CDR also includes overlapping or subsets of amino acid residues when compared against each other. The exact residue numbers which encompass a particular CDR or a functional portion thereof, 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.
  • the term "functional fragment" of an antibody can mean a portion of the antibody which retains a functional activity.
  • a functional activity can be, for example antigen binding activity or specificity.
  • a functional activity can also be, for example, an effector function provided by an antibody constant region.
  • the term "functional fragment” is also intended to include, for example, fragments produced by protease digestion or reduction of a human monoclonal antibody and by recombinant DNA methods known to those skilled in the art.
  • Human monoclonal antibody functional fragments include, for example individual heavy or light chains and fragments thereof, such as VL, VH and Fd; monovalent fragments, such as Fv, Fab, and Fab' ; bivalent fragments such as F(ab single chain Fv (scFv); and Fc fragments.
  • VL fragment can mean a fragment of the light chain of a human monoclonal antibody which includes all or part of the light chain variable region, including the CDRs.
  • a VL fragment can further include light chain constant region sequences.
  • VH fragment can means a fragment of the heavy chain of a human monoclonal antibody which includes all or part of the heavy chain variable region, including the CDRs.
  • Fd fragment can mean the light chain variable and constant regions coupled to the heavy chain variable and constant regions, i.e. VL CL and VH CH-i.
  • Fv fragment can mean a monovalent antigen-binding fragment of a human monoclonal antibody, including all or part of the variable regions of the heavy and light chains, and absent of the constant regions of the heavy and light chains.
  • the variable regions of the heavy and light chains include, for example, the CDRs.
  • an Fv fragment includes all or part of the amino terminal variable region of about no amino acids of both the heavy and light chains.
  • Fab fragment means a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than an Fv fragment.
  • a Fab fragment includes the variable regions, and all or part of the first constant domain of the heavy and light chains.
  • a Fab fragment additionally includes, for example, amino acid residues from about no to about 220 of the heavy and light chains.
  • Fab' fragment can means a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than a Fab fragment.
  • a Fab' fragment includes all of the light chain, all of the variable region of the heavy chain, and all or part of the first and second constant domains of the heavy chain.
  • a Fab' fragment can additionally include some or all of amino acid residues 220 to 330 of the heavy chain.
  • F(ab') 2 fragment can mean a bivalent antigen-binding fragment of a human monoclonal antibody.
  • An F(ab') 2 fragment includes, for example, all or part of the variable regions of two heavy chains-and two light chains, and can further include all or part of the first constant domains of two heavy chains and two light chains.
  • single chain Fv can mean a fusion of the variable regions of the heavy (VH) and light chains (VL) connected with a short linker peptide.
  • VH variable regions of the heavy
  • VL light chains
  • scFv single chain Fv
  • the functional fragment may include fragments wherein at least one of the binding region sequences has substantially the same amino acid sequence as the binding region sequences of the antibody.
  • the functional fragment may comprise any of the fragments selected from a group consisting of VH, VL, Fd, Fv, Fab, Fab', scFv, F (ab') 2 and Fc fragment.
  • the invention comprises a functional fragment of an antibody (e.g. Fv, Fab, and Fab', bivalent fragments such as F(ab') 2 , single chain Fv (scFv), fused to the Fc region of the first aspect.
  • the functional fragment may comprise any one of the antigen binding region sequences of the VL, any one of the antigen binding region sequences of the VH, or a combination of VL and VH antigen binding regions of a human antibody.
  • the appropriate number and combination of VH and VL antigen binding region sequences may be determined by those skilled in the art depending on the desired affinity and specificity and the intended use of the functional fragment.
  • Functional fragments of antibodies may be readily produced and isolated using methods well known to those skilled in the art. Such methods include, for example, proteolytic methods, recombinant methods and chemical synthesis. Proteolytic methods for the isolation of functional fragments comprise using human antibodies as a starting material.
  • Enzymes suitable for proteolysis of human immunoglobulins may include, for example, papain, and pepsin.
  • the appropriate enzyme may be readily chosen by one skilled in the art, depending on, for example, whether monovalent or bivalent fragments are required.
  • papain cleavage results in two monovalent Fab' fragments that bind antigen and an Fc fragment.
  • Pepsin cleavage results in a bivalent F (ab') fragment.
  • An F (ab') 2 fragment of the invention maybe further reduced using, for example, DTT or 2-mercaptoethanol to produce two monovalent Fab' fragments.
  • Functional fragments produced by proteolysis may be purified by affinity and column chromatographic procedures. For example, undigested antibodies and Fc fragments may be removed by binding to protein A. Additionally, functional fragments may be purified by virtue of their charge and size, using, for example, ion exchange and gel filtration chromatography. Such methods are well known to those skilled in the art.
  • the human antibody or functional fragment thereof may be produced by any means.
  • Such regions may include, for example, all or part of the variable region of the heavy and light chains.
  • regions can particularly include the antigen binding regions of the heavy and light chains, preferably the antigen binding sites, most preferably, the CDRs.
  • the polynucleotide encoding the human antibody or functional fragment of the invention may be produced using methods known to those skilled in the art.
  • the polynucleotide encoding the antibody or a functional fragment thereof may be directly synthesized by methods of oligonucleotide synthesis known in the art.
  • smaller fragments may be synthesized and joined to form a larger functional fragment using recombinant methods known in the art.
  • immunospecificity can mean the binding region is capable of immunoreacting with a certain target epitope, by specifically binding therewith.
  • immunoreact can mean the binding region is capable of eliciting an immune response upon binding with an epitope.
  • epitope can mean any region of an antigen with ability to elicit, and combine with, a binding region of the antibody or fragment thereof.
  • IgE antibodies play a central role in the initiation and regulation of allergic disorders
  • clinical applications of the variant IgE Fc region of the first aspect, and of the antibodies (i.e. modified antibodies) of the eighth aspect include their use in treating a wide range of medical conditions (including cancer, autoimmune disease or infectious disease), while simultaneously avoiding the deleterious allergic side effects that are often observed in patients who are treated with IgE antibody therapies. In addition, they may also have utility in diagnosis.
  • a method for treating, preventing or ameliorating a CD23-mediated side effect in a subject comprising administering, to a subject in need of such a treatment, a therapeutically effective amount of a variant IgE Fc region according to the first aspect, or an antibody or a functional fragment thereof according to the eighth aspect.
  • the variant IgE Fc region, or the antibody or a functional fragment thereof is used for preventing a CD23-mediated side effect.
  • the CD23-mediated side effect, which is prevented or avoided, may be caused by IgE antibody treatment or therapy, of which there are many types.
  • the CD23-mediated side effect which is prevented is one which may otherwise be caused during the treatment of cancer, autoimmune disease or infectious disease with a therapeutic IgE antibody.
  • the antibody or fragment thereof maybe a therapeutic antibody.
  • a CD23-mediated side effect which may be prevented may include CD23-dependent epitope spreading, which will be known to the skilled person.
  • Epitope spreading is the process by which an antibody response to one epitope on an antigen leads to the production of antibodies specific for other epitopes on the same antigen, or for epitopes on entirely unrelated antigens. This results from the internalisation of whole antigen and subsequent display of a range of peptides derived from that antigen, leading to the generation of T cells with different epitope specificities. Simultaneous processing of two unrelated antigens by an antigen-presenting cell can lead to the production of antibodies directed against both antigens (Gould and Sutton, 2008, Nature Reviews Immunology, 8, 205-217).
  • agents may be used in a monotherapy (e.g. the use of an antibody or fragment thereof alone), for treating, ameliorating or preventing a CD23-mediated side effect.
  • agents according to the invention maybe used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing CD23-mediated side effects.
  • the agents according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used.
  • the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment.
  • the vehicle of medicaments according to the invention should be one which is well -tolerated by the subject to whom it is given, and preferably enables delivery of the agents across the blood-brain barrier.
  • Medicaments comprising agents of the invention maybe used in a number of ways.
  • compositions comprising agents and medicaments of the invention may be administered by inhalation (e.g. intranasally).
  • Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin.
  • Agents and medicaments according to the invention may also be incorporated within a slow- or delayed-release device.
  • Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months.
  • the device may be located at least adjacent the treatment site.
  • Such devices may be particularly advantageous when long-term treatment with agents used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).
  • agents and medicaments according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment. Injections maybe intravenous (bolus or infusion) or
  • the amount of the antibodies, fragments, peptides and nucleic acids (i.e. agent) that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the
  • Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the disease. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.
  • a daily dose of between o.oo ⁇ g/kg of body weight and lomg/kg of body weight of agent according to the invention may be used for treating, ameliorating, or preventing a CD23-mediated side effect. More typically, the daily dose of agent is between o.o ⁇ g/kg of body weight and lmg/kg of body weight, more preferably between body weight, and most preferably between approximately body weight.
  • the agent may be administered before, during or after onset of the side effect. Daily doses may be given as a single administration (e.g. a single daily injection).
  • the agent may require administration twice or more times during a day.
  • agents may be administered as two (or more depending upon the severity of the condition being treated) daily doses of between 0.07 ⁇ g and 700 mg (i.e. assuming a body weight of 70 kg).
  • a patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter.
  • a slow release device may be used to provide optimal doses of agents according to the invention to a patient without the need to administer repeated doses.
  • Known procedures such as those conventionally employed by the pharmaceutical industry (e.g.
  • a pharmaceutical composition comprising a variant IgE Fc region according to the first aspect, or an antibody or fragment thereof according to the eighth aspect; and optionally a pharmaceutically acceptable vehicle.
  • the composition may be CD23-mediated side effect treatment composition.
  • CD23-mediated side effect treatment composition can mean a pharmaceutical formulation used in the therapeutic amelioration, prevention or treatment of a CD23- mediated side effect.
  • the invention also provides in a thirteenth aspect, a process for making the composition according to the tenth aspect, the process comprising combining a therapeutically effective amount of a variant IgE Fc region according to the first aspect, or an antibody or a functional fragment thereof as defined in the eighth aspect, with a pharmaceutically acceptable vehicle.
  • a "subject" may be a vertebrate, mammal, or domestic animal.
  • medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications. Most preferably, the subject is a human being.
  • a “therapeutically effective amount” of the antibody or fragment thereof is any amount which, when administered to a subject, is the amount of agent that is needed to treat CD23-mediated side effect, or to produce the desired effect.
  • the therapeutically effective amount of antibody or fragment thereof used may be from about o.ooi ng to about ⁇ mg, and typically from about o.oi ng to about loo ng. It is preferred that the amount of antibody or fragment is an amount from about o.i ng to about 10 ng, and most preferably from about 0.5 ng to about 5 ng.
  • a "pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.
  • the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet.
  • a solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents.
  • the vehicle may also be an encapsulating material.
  • the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention.
  • the active agent may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain up to 99% of the active agents.
  • Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
  • the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.
  • the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution.
  • Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
  • the active agent according to the invention maybe dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
  • the liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators.
  • liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil).
  • the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate.
  • Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration.
  • the liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.
  • Liquid pharmaceutical compositions which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection.
  • the agent may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
  • compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 8o (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
  • solutes or suspending agents for example, enough saline or glucose to make the solution isotonic
  • bile salts for example, enough saline or glucose to make the solution isotonic
  • bile salts for example, enough saline or glucose to make the solution isotonic
  • bile salts for example, enough saline or glucose to make the solution isotonic
  • acacia gelatin
  • sorbitan monoleate sorbitan monoleate
  • polysorbate 8o oleate esters of sorbito
  • administration include sterile solutions, emulsions, and suspensions.
  • nucleic acid or peptide or variant, derivative or analogue thereof which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof.
  • substantially the amino acid/nucleotide/peptide sequence can be a sequence that has at least 40% sequence identity with the amino acid/ nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID No:2 (i.e. wild type human IgE Fc region) or the nucleotide identified as SEQ ID No:i (i.e.
  • amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 50%, more preferably greater than 65%, 70%, 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged.
  • the amino acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90%, 92%, 95%, 97%, 98%, and most preferably at least 99% identity with any of the sequences referred to herein.
  • the skilled technician will appreciate how to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences.
  • the percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g.
  • percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.
  • acid/polynucleotide/polypeptide sequences may then be calculated from such an alignment as (N/T)*ioo, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs.
  • a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to the sequences shown in SEQ ID No's: 1, 8-12 or their complements under stringent conditions.
  • stringent conditions we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/ sodium citrate (SSC) at approximately 45°C followed by at least one wash in o.2x SSC/ 0.1% SDS at approximately 20-65°C.
  • a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ ID No: 2, 3-7.
  • nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.
  • Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change.
  • Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
  • small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine.
  • Large non- polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.
  • the polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
  • the positively charged (basic) amino acids include lysine, arginine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids
  • Figure l shows NMR mapping of the CD23 and IgE interaction surfaces.
  • a small number of residues from Ce3 show chemical shift perturbation upon addition of derCD23. Increasing amounts of unlabelled derCD23 were added to a 200 ⁇ sample of ⁇ N-labelled Ce3; five spectra of the titration are overlaid (red, zero derCD23;
  • the IgE-CD23 interaction site is formed by complementary charged surfaces
  • Figure 2 shows competition binding experiments between derCD23 and sFceRIa for IgE-Fc.
  • A-D Surface plasmon resonance and
  • E-F TR-FRET-based competitive binding experiments were used to investigate the mechanisms of receptor interactions.
  • the binding of derCD23 was tested against (A) IgE-Fc immobilized on a sensor surface and (B) IgE-Fc captured on an FceRIa-immobilized surface; the start of the derCD23 injection is indicated with an arrow.
  • Figure 3 shows that soluble CD23 does not cross-link IgE bound to FceRI on mast cells.
  • the ability of soluble CD23 to engage IgE on B cells and mast cells was tested.
  • A After preincubation of IgE, the addition of anti-IgE antibody results in activation of the FceRIa + LAD-2 mast cell line, as measured by release of ⁇ -hexosaminidase.
  • Neither monomeric derCD23 nor trimeric triCD23 is able to cross-link IgE and activate mast cells in this assay.
  • triCD23 effectively cross-links mlgE on the surface of IgE + human tonsillar B cells, resulting in activation of these cells and increased secretion of IgE.
  • B The effects of ionic strength on the binding of IgE-Fc to derCD23. Binding affinities are measured over a range of ionic strengths and plotted as log (K A ) versus log (ionic strength). The steep slope (-0.94) of the curve confirms a strong electrostatic contribution to binding;
  • Figure 5 shows the structure of the derCD23-Fcs3-4 complex.
  • the two molecules of derCD23 (light and dark blue Coc traces with surfaces) bind one to each heavy chain between the Cs3 (dark red and green) and Cs4 domains (light red and green).
  • the carbohydrate is shown in all-atom representation (red and yellow, without surfaces) and can be seen behind the (red) Cs3 domain.
  • the adjacent N- and C- termini of each derCD23 molecule, the former being the connection to the "stalk", the latter to the "tail” region, can be seen at the extreme left and right of the figure.
  • the complex shown here comprises chains A (red), B (green), G (light blue) and H (dark blue));
  • Figure 6 shows salt bridges and hydrogen bonds at the derCD23-Fcs3-4 interface.
  • the H-bonds associated with the four salt bridges are shown in red, additional H- bonds present in all six independent interactions are shown in green, and a further H-bond present in 5/6 molecules is shown in yellow;
  • Figure 7 shows composite images of the derCD23 and sFcsRIoc complexes with Fcs3"4 to show the mutual incompatibility of their binding modes, a) derCD23-Fcs3- 4 and sFcsRIa-Fcs3-4 [PDB 1F6A] complexes superposed on their (Cs4) 2 domain pairs.
  • the receptors are shown as surfaces (derCD23 light & dark blue; sFcsRIoc red) and the Fcs3-4 structures are shown as Coc traces (in corresponding colours).
  • the closed (derCD23-binding) and open (sFcsRIoc-binding) conformations of the Cs3 domains may be seen, b) Steric clashes between the sFcsRIa structure (red Coc trace) and both chains of the derCD23-Fcs3-4 complex (blue) are indicated (orange surfaces), c) Steric clashes of both derCD23 molecules (blue Coc traces) with the sFcsRIoc-Fcs3-4 complex (red) are indicated (green surfaces);
  • Figure 9 shows electron density in the interface region. Electron density 2 0 - F c map contoured at ⁇ for key side-chains involved in salt bridges at the
  • derCD23/Fcs3-4 interface The side-chains shown in blue are from derCD23, those in purple from Fcs3-4;
  • FIG. 10a The six independent Fcs3-4 heavy chains superposed on their Cs4 domains, showing the variation in the relative positions of the Cs3 domains, b) The six independent derCD23 molecules superposed, showing virtually no overall structural variation;
  • Figure 11 shows a comparison of Fcs3-4 heavy chains taken from various Fcs3-4 and IgE-Fc structures superposed by their Cs4 domains upon the set of six independent heavy chains of the derCD23-Fcs3-4 complex (shown lightly coloured), a) Free IgE- Fc (light and dark red) [PDB 2WQR; 28], b) sFcsRIoc-IgE-Fc complex (light and dark cyan) [PDB 2Y7Q; 21], c) sFcsRIoc-Fcs3-4 complex (light and dark purple) [PDB 1F6A; 20], d) Single chain of free Fcs3-4 (green), the most closed structure observed to date [PDB 3HA0; 27]; and
  • Figure 12 shows the superposition of apo- and calcium-bound derCD23 structures.
  • the apo-CD23 (dark blue) and calcium-bound CD23 (dark red) structures [PDB 2H2R, 2H2T; 40] superposed upon the six derCD23 molecules from the Fcs3-4 complex (lightly coloured), showing differences in loops 1 and 4.
  • the Ca 2+ ion is shown (red sphere); note the disordered loop 4 between residues Ser252 and Asp258 in the calcium-bound structure.
  • MAb 7.12 was produced from a B cell hybridoma (Kanowith-Klein et al., 1988, Clin Immunol Immunopathol 48, 214-224), and was a kind gift of Dr. Rebecca Beavil (King's College London, UK).
  • NMR spectroscopy was performed on protein samples in a buffer containing 25mM Tris, 125 mM NaCl, 41 ⁇ CaCl 2 , pH 6.8, at protein concentrations between 120 and 900 ⁇ . Data were collected at 25°C on Bruker spectrometers equipped with CryoProbes operating at 500 and 700 MHz. For chemical shift perturbation experiments, unlabelled derCD23 ligands were concentrated to 2mM and then added in small aliquots to samples of 200 ⁇ ⁇ N-labelled Ce3 until saturation was seen. The NMR chemical shifts of the urea denatured and native state Ce3 domain are available from the BioMagResBank database under accession numbers 18482 and 18483.
  • Thermodynamic measurements were performed at 288K, 293K, 298K and 303K.
  • approximately 90RU of IgE-Fc was captured on an ⁇ -fusion protein surface during a l-min injection of a ⁇ IgE-Fc sample; after a 3-min stabilization period, ⁇ - ⁇ derCD23 was injected for l-min followed by a 15-min dissociation phase. All measurements were done independently at least twice, using standard double reference subtraction methods for data analysis
  • FRET assay Inhibition assays were performed by competing ⁇ terbium-chelate labelled derCD23 and 0-20 ⁇ Alexa 647-labelled IgE-Fc with a range of
  • LAD-2 The human mast cell line LAD-2 (NIH) was cultured in Stem Pro-34 SFM medium (Gibco) supplemented with L-glutamine (2 mM), penicillin (10 U/ml), and streptomycin (1 g/ml; all from Life Technologies). LAD-2 cells were suspended in culture medium at 1.6x1 ⁇ 6 cells/ml and 50 ⁇ 1 aliquots were placed in v-bottomed 96-well plates (Greiner Bio-One). Cells were primed by addition of 2.5nM IgE (NIBSC) or a buffer-only control for one hour, before addition of cross-linking reagents.
  • NIBSC 2.5nM IgE
  • Polyclonal rabbit anti-human IgE (Dako) was added at 2onM and soluble CD23 constructs at 0.1, 1 and ⁇ , and incubated for 1 hour at 37°C. Supernatants were harvested and tested for ⁇ -hexosaminidase release, measured fluorometrically as described previously (Hammond, 2006, In Cell Biology, J.E. Celis, editor. Elsevier. Amsterdam). Controls included cells treated with wash buffer plus 1% Triton-X for total release, with buffer-only to measure background release, typically about 10% of total release, with 2.5nM IgE-only, and with ⁇ CD23-only. The level of degranulation measured for Triton-X treated cells was defined as 100% release and all samples were defined relative to that.
  • B cell activation assays Human tonsillar B cells were cultured in 24-well plates (Nunc) at 5 x 10 5 cells/ml in RPMI with penicillin (lOOlU/ml), streptomycin glutamine (2mM) (Invitrogen), 10% FCS (Hyclone, Perbio Biosciences Ltd), insulin ⁇ g/ml) and transferrin (35 g/ml) (Sigma-Aldrich). Cells were activated with IL-4 (200lU/ml) (R&D Systems), anti-CD40 antibody ( ⁇ g/ml) (G28.5; ATCC), and either ⁇ derCD23 or ⁇ triCD23. Supernatants were harvested on day 12 for IgE measurements.
  • IgE ELISA assays were performed as described previously (Cooper et al., 2012, J Immunol 188, 3199-3207), using Maxisorp plates (Nunc) coated with polyclonal mouse anti-human IgE (1:7000) (Dako), in pH 9.8 carbonate buffer (0.2M Na 2 C0 3 , 0.2M NaHC0 3 ). Unbound sites were blocked with 2% milk powder in PBS + 0.05% Tween20 (Sigma-Aldrich).
  • Binding was detected by mouse anti-human IgE-HRP (1:1000) (Dako) in 1% milk powder in PBS-T for 2 hours at 37°C. Standard curves were derived using human serum IgE (NIBSC), with a minimal detectable concentration of about 2ng/ml.
  • the inventors identified the IgE binding site on CD23 using NMR chemical shift perturbation studies (Hibbert et al., 2005, J Exp Med 202, 751-760). In this work, the inventors have now performed the reciprocal NMR binding experiment, mapping the interaction site of CD23 onto the Ce3 domain from IgE. Using an approach described by Schulman et al. (Schulman et al., 1997, Nat Struct Biol 4, 630-634), they then assigned the backbone resonances of the molten globule Ce3 domain by first performing resonance assignments of Ce3 denatured in 6M urea and then, through gradual titration of buffer conditions, tracking those resonances to the native state Ce3 domain.
  • the identified residues from three discontinuous sequences (amino acids 405-407, 409-411 and 413 from the E-F helix of the Ce3 domain of IgE, amino acids 377-380 from the C-D loop, and residue 436 from the C-terminal region) form a contiguous surface representing the binding site on Ce3 for CD23 ( Figure lB).
  • This region is at the end of the Ce3 domain, near to the interface with Ce4, in contrast to the interaction site for FceRI, which is at the other end of Ce3 near the interface with Ce2 (Garman et al., 2000, Nature 406, 259-266; Holdom et al., 2011, Nat Struct Mol Biol 18, 571-576) (Fig.
  • thermodynamic characteristics as was also observed for the FcaRI-IgA interaction.
  • the two binding affinities imply an asymmetry of the two CD23 binding sites, which may possibly be allosterically induced.
  • CD23 mutations D227A, E257A, R224A and R188A all show a change in binding free energy (AAG) of about +6 kJ mol 1 (Table 1). Uncharged residues also contribute to the binding energy; a prominently exposed tyrosine residue (Y189) in the center of CD23's IgE binding site makes a substantial contribution to binding energy.
  • the CD23 binding surface on IgE is also predominantly electrostatic, with residues D409, E412, R376 and K380 showing the largest effects on CD23 binding energetics.
  • the inventors performed a set of competitive binding assays to confirm this experimentally. Firstly, using an SPR assay, they showed that derCD23 can bind to IgE-Fc immobilized to an SPR chip but cannot bind to IgE-Fc captured by immobilized FceRIa (Fig. 2A,B); a positive control, a Fab fragment of the anti-IgE antibody 7.12, directed against the Ce2 domain, did bind to FceRIa-captured IgE-Fc (data not shown). Secondly, they showed that IgE-Fc can bind to immobilized derCD23, but an IgE-Fc-sFceRIa complex cannot bind to derCD23 (Fig. 2C,D).
  • a competitive inhibitor affects the apparent binding affinity, with inhibitor I reducing the apparent affinity by a ratio of (i+[I]/Ki); whereas an allosteric inhibitor affects the apparent B ma x of the interaction without changing the apparent K D (Fersht, 1999, Structure and Mechanism in Protein Science. New York, W.H. Freeman.).
  • FceRIa as the inhibitor of the IgE-Fc- derCD23 interaction results in a decrease of apparent B ma x without affecting the apparent K D of the interaction (Fig. 2E,F).
  • Soluble trimeric CD23 has been shown to bind to and cross-link membrane IgE on B cells, resulting in B cell activation. However, it is essential that trimeric CD23 not cross-link IgE bound to FceRI on the surface of mast cells. If this were to occur then high levels of CD23 would result in mast cell activation in the absence of allergens.
  • the data from binding experiments (Fig. 2B) predict that soluble CD23 cannot directly cross-link IgE bound to FceRI on mast cells. The inventors tested this prediction in a mast cell degranulation assay using the FceRI + LAD-2 human mast cell line.
  • cells are first primed by adding IgE, followed by addition of potential cross-linking reagents and measurement of release of the mast cell granule- associated enzyme ⁇ -hexosaminidase.
  • An anti-IgE antibody results in FceRI- mediated activation of the mast cell and robust ⁇ -hexosaminidase release, but the addition of either the monomeric derCD23 or a trimeric CD23 construct (triCD23) fails to induce mast cell degranulation (Fig. 3A).
  • trimeric CD23 effectively cross-links IgE on B cells, resulting in activation of these cells and increased production of soluble IgE (Fig. 3B).
  • Immunoglobulins have evolved two separate sites for binding to receptors.
  • IgE One site is near the hinge region in IgG and at the Ce2-C83 interface in IgE, while the other is at the interface of the C-terminal domain and the penultimate domain: the Ce3-C84 interface in IgE.
  • a mechanism of communication has evolved within the IgE molecule between these two distant sites to prevent simultaneous engagement of CD23 and FceRI. This may be a unique property of IgE. Since IgE and CD23 both exist in membrane bound and soluble forms, and soluble FceRIa has also recently been shown to exist at functionally relevant concentrations, there is considerable potential for receptor cross-regulation. Mutually exclusive receptor binding assures independent functions for IgE-FceRI and IgE-CD23 interactions. IgE is a clinically important drug target.
  • An anti-IgE antibody (omalizumab) is an effective therapy, currently used in the treatment of moderate to severe asthma that is not controlled by corticosteroids.
  • Omalizumab binds to the Ce3 domain of IgE and competitively inhibits FceRI binding, although its in vivo activity relies on more than just inhibition of this interaction. Results presented here demonstrate that IgE is amenable to allosteric inhibition, an approach that may have significant advantages over competitive inhibition, and lay the foundation for the development of allosteric modulators of IgE-receptor interactions. Summary
  • IgE the antibody that mediates allergic responses, acts as part of a self-regulating protein network. Its unique effector functions are controlled through interactions of its Fc region with two cellular receptors, FceRI on mast cells and basophils and CD23 on B cells. IgE cross-linked by allergen triggers mast cell activation via FceRI, while IgE-CD23 interactions control IgE expression levels.
  • the inventors have determined the CD23 binding site on IgE, using a combination of NMR chemical shift mapping and site-directed mutagenesis. They have shown that the CD23 and FceRI interaction sites are at opposite ends of the Ce3 domain of IgE, but that receptor binding is mutually inhibitory, mediated by an allosteric mechanism.
  • Human IgE-Fc (N265Q, N371Q) was expressed in NSo cells and purified by affinity chromatography with sFc8RIa-IgG 4 -Fc fusion protein as previously described (Shi, 1997, Biochemistry, 36, 2112-2122).
  • the genes for recombinant human Fc£3 ⁇ 4 (Cys328-Lys547, with N-terminal ADP) and sFcsRIoc-Cys-His (Vali-Lysi76, with C- terminal cysteine and His6 tag) were synthesized by DNA2.0 and cloned as
  • Human Fcs3-4 was purified by cation exchange chromatography on a SPHP matrix (GE Healthcare) in 50 mM NaOAc buffer pH 6.0, followed by gel filtration on a Superdex S200 matrix (GE Healthcare) in PBS pH 7.4.
  • Human sFcsRIoc-Cys-His was purified on a Ni-NTA column (Qiagen) followed by gel filtration on a Superdex S200 matrix (GE Healthcare) in PBS pH 7.4 and stored under nitrogen to prevent reactivity of the free cysteine residue.
  • Fce3-4 was concentrated to 20 mg/ml, and derCD23 to 18 mg/ml, in 25 mM Tris-HCl pH 7.5, 20mM NaCl, 0.05% sodium azide ('crystallization buffer').
  • the complex was formed with 0.4 mM derCD23 (6.2 mg/ml), 0.2 mM Fce3-4 (10 mg/ml) and 4 mM CaCl 2 , diluted with equal volume of 3% PEG 8,000, 0.1 M Tris-HCl pH 7.5 as the precipitant.
  • Inhibition assays were performed by competing 5 % of 1 ⁇ terbium labeled derCD23 and 5 % of 5 ⁇ Alexa Fluor 647 labeled IgE-Fc or Fce3-4 with a dilution series of unlabeled sFceRIa-Cys-His. Assays were conducted in 384 well hi-base, white plates (Greiner BioOne) using Lanthascreen buffer (Invitrogen) as a diluent. The plate was left to incubate for 1 hr at room temperature with shaking and read by the Artemis plate reader (Berthold Technologies).
  • TR-FRET ratios were then calculated for each well as the emission of acceptor at 665 nm divided by the emission of donor at 620 nm multiplied by 10 4 . Data were analyzed using GraphPad Prism 5. Similarly, 2 nM terbium labeled sFceRIa and 10 nM Alexa Fluor 647 labeled IgE-Fc or Fce3-4 were competed with a dilution series of unlabeled derCD23. Assays were conducted as described above with the exception of an overnight incubation.
  • Crystallization Crystals with a different morphology were also found after 6 days with 12% PEG 4,000, 0.1 M Tris-HCl pH 8.75, 0.2 M sodium acetate trihydrate as precipitant after streak seeding. Diffraction data were collected to 3.6A and the crystal form was found to be the same as that reported in Table 2.
  • Crystallogr D, 62, 439-450 were generated using the TLSMD web server (Painter, 2006, JAppl Crystallogr, 39, 109-111). Carbohydrate atoms and a single water molecule were subsequently incorporated into the structure. No electron density was observed for Fcs3-4 residues 363 - 364 of chain B; 367 - 371 and 421 -428 of chain C; 367 - 370, 419 - 422, 429 - 430, 479 - 480 and 517 - 519 of chain E; and derCD23 residues 256 - 257 of chains G, H, I, J and K. Therefore, these residues were not built into the model. Refinement statistics are shown in Table 2.
  • IgE-Fc and Fce3-4 were labeled with acceptor fluorophore by reacting 4 mg/ml protein in 100 mM sodium bicarbonate, 50 mM NaCl, pH 9.3, with a 5-fold molar excess of Alexa Fluor 647 succinimidyl ester (Invitrogen). After 1 hr incubation at room temperature with agitation, excess unreacted fluorophore was removed by dialyzing into HBS (10 mM Hepes, 150 mM NaCl, 4mM CaCl 2 , pH 7.4).
  • derCD23 and the sFceRIa-Cys-His mutant were labeled with donor fluorophore.
  • derCD23 at 3.5 mg/ml in 100 mM Hepes, 125 mM NaCl, pH 8.3 was reacted with a 5-fold molar excess of terbium chelate isothiocyanate
  • Outliers o * Values in parentheses are for the outer resolution shell.
  • the crystal form contained three independent copies of the complex in the asymmetric unit and the six heavy chains (labeled A to F) bound to six derCD23 heads (labeled G to L) showed essentially identical modes of interaction, exemplified by the structure depicted (Fig. 5; chains A, B, G & H).
  • the angle between the Cs3 and Cs4 domains varies only slightly between the six heavy chains (Fig. 10a) and there are virtually no differences between the six derCD23 head domains (Fig. 10b). All three complexes thus display approximate two-fold symmetry, although one (consisting of chains C, D, I & J) includes the two "extremes" in terms of the angle between the Cs3 and Cs4 domains, but these differ by only 7 degrees.
  • electron density was present for five N-linked sugar units at Asn394 ([N- acetylglucosamine] 2 [Mannose] 3 ; Fig. 5), and an additional two mannose residues were visible on chain C.
  • the buried surface area for each interaction ranges from 860 to 890A 2 for all except one (chains F & L) at 920A 2 ; in this latter case the derCD23 loop at residues Ser256 and GIU257 is not as disordered as it is in the other five
  • the Cs3 domain dominates the interface with 63% of the contact area, a further 25% involves the linker region (residues 437 to
  • IgE-Fc adopts an asymmetrically bent structure with the (Cs2) 2 domain pair folded back against one of the Cs3 domains, but although the two Cs3- Cs4 pairs have different inter-domain angles, both are more open than that seen in the derCD23-Fcs3-4 complex (Fig. 11a).
  • the (Cs2) 2 domain pair was modeled onto the derCD23-Fcs3-4 complex. This was achieved by superimposing the Cs3 domain of free IgE-Fc that contacts the (Cs2) 2 domain pair, onto the Cs3 domain (chain A) of the derCD23-Fcs3-4 complex.
  • the closed conformation for the Cs3 domains in the derCD23 complex is clearly incompatible with FcsRI binding. This may be seen by superimposing the derCD23- Fcs3"4 and the sFcsRIoc-Fcs3-4 (Garman, 2000, Nature, 406:259-266) complexes on their (Cs4) 2 domain pair, and noting the very different orientations of the Cs3 domains (Fig. 7a).
  • IgE and CD23 The interaction between IgE and CD23 is critically involved in the allergic response at several stages, including allergen presentation, the regulation of IgE synthesis and transport of IgE and immune complexes across epithelial barriers in the gut and airways.
  • 1T1CD23 At the cell surface, 1T1CD23 is trimeric, and SCD23 fragments shed from the membrane that contain sufficient stalk region are also trimeric, although the structure of the trimer has only been modeled based either upon the structures of other C-type lectins or guided by NMR chemical shift data.
  • two derCD23 "heads" bind to IgE, one to each heavy- chain at a location between the Cs3 and Cs4 domains, and remote from the FcsRI binding site.
  • the interaction is predominantly hydrophilic and dominated by salt bridges between positively charged CD23 residues and negatively charged IgE residues, despite the overall net positive charge (+9) of the Cs3 domain.
  • the site on CD23 in agreement with that identified by NMR chemical shift mapping by titration of 15 N-labeled derCD23 with monomeric Cs3 (titration with IgE-Fc led to the formation of high molecular weight oligomers), is diametrically opposed to the connection to the oc-helical coiled-coil stalk region (Fig. 5).
  • This topology is such that an IgE molecule could not engage two heads from the same (modeled) CD23 trimer (as depicted in earlier cartoons), but could readily cross-link two mCD23 molecules.
  • IgE-Fc and IgE additionally contain the Cs2 domains, and it is important to consider their effect upon the crystallographic results presented here for Fcs3-4.
  • IgE-Fc the (Cs2) 2 domain pair packs asymmetrically against one of the Cs3 domains in the bent IgE-Fc structure, and moves together with that Cs3 domain when it opens up to accommodate sFcsRIoc binding.
  • Modeling the (Cs2) 2 domains onto the derCD23- Fcs3"4 complex shows that although there is no steric conflict that would prevent binding of either derCD23 molecule, the Cs2 domains lie immediately adjacent to one of the derCD23 molecules and suggest that an interaction (stabilizing or
  • CD23 belongs to the C-type (calcium-dependent) lectin superfamily and the presence of Ca 2+ is known to enhance the affinity for IgE approximately 7-fold, although it is not essential for binding.
  • a crystal structure of the head domain with a single bound Ca 2+ ion has been solved, together with the Ca 2+ -free form, and an NMR structure also reports Ca 2+ binding, but at an alternative site.
  • no Ca 2+ ions were observed in any of the six derCD23 molecules in the Fcs3-4 complex, despite the presence of 2 mM Ca 2+ in the crystallization medium.
  • the existence of a disordered loop in derCD23 at the edge of the interface is intriguing.
  • the crystal structure of the lectin head domain showed Ca 2+ bound at a site involving residues GIU249 and Thr25i of loop 4.
  • residues 253-257 of loop 4 were also disordered, paradoxically becoming ordered in the absence of Ca 2+ due to a rearrangement of the side-chain of Arg253, which occupied the Ca 2+ site.
  • the conformation of loop 4 seen in the complex differs from either the apo- or Ca 2+ -bound structures; together with the adjacent loop 1, they represent the only main-chain conformational changes in derCD23 upon Fcs3"4 binding.
  • FcsRI cannot be cross-linked either by soluble IgE-CD23 complexes, or IgE bound to 1T1CD23 (on B cells or APCs) in the absence of allergen.
  • the mutual exclusion of FcsRI and CD23 binding is thus an important aspect of IgE biology, allowing it to function independently through its two receptors.
  • the inventors have hypothesized that through such interactions, the former leading to up-regulation of IgE synthesis and the latter to down-regulation, CD23 contributes to the mechanism of IgE homeostasis, and this notion has received experimental support from studies with monomelic and oligomeric SCD23 species.
  • the structure of the complex is also consistent with the co-crosslinking of mCD2i and mlgE by trimeric SCD23 (proposed to enhance IgE up-regulation), since CD21 binds to the "tail" sequence that is, although only partially present in derCD23, located adjacent to the connection to the stalk [Fig. 5;].
  • the precise spatial orientation of mCD2i and mlgE by trimeric SCD23 proposed to enhance IgE up-regulation
  • immunoglobulin E immunoglobulin E
  • FcsRI immunoglobulin E
  • B cells The role of immunoglobulin E (IgE) in allergic disease mechanisms is performed principally through its interactions with two receptors, FcsRI on mast cells and basophils, and CD23 (FcsRII) on B cells.
  • the former mediates allergic hypersensitivity, the latter regulates IgE levels, and both receptors, also expressed on antigen presenting cells, contribute to allergen uptake and presentation to the immune system.
  • the inventors have solved the crystal structure of the soluble lectin-like "head" domain of CD23 (derCD23) bound to a sub- fragment of IgE-Fc consisting of the dimer of Cs3 and Cs4 domains (Fcs3-4).
  • CD23 head binds to each heavy chain at the interface between the two domains, explaining the known 2:1 stoichiometry and suggesting mechanisms for cross-linking membrane-bound trimeric CD23 by IgE, or membrane IgE by soluble trimeric forms of CD23, both of which may contribute to the regulation of IgE synthesis by B cells.
  • the two symmetrically located binding sites are distant from the single FcsRI binding site, which lies at the opposite ends of the Cs3 domains. Structural comparisons with both free IgE-Fc and its FcsRI complex reveal not only that the conformational changes in IgE-Fc required for CD23 binding are incompatible with FcsRI binding, but also that the converse is true.
  • the two binding sites are allosterically linked.
  • the inventors demonstrate experimentally the reciprocal inhibition of CD23 and FcsRI binding in solution, and suggest that the mutual exclusion of receptor binding allows IgE to function independently through its two receptors.

Abstract

The invention relates to antibodies, and in particular to IgE antibodies. The invention is primarily concerned with variant Fc regions of IgE antibodies and their use in the creation of novel engineered or modified IgE antibodies. The invention extends to methods for modifying antibodies and to pharmaceutical compositions comprising such antibodies. The invention also covers uses of the modified antibodies in a wide range of therapeutic applications.

Description

VARIANT IGE FC REGIONS
The present invention relates to antibodies, and in particular to immunoglobulin E (IgE) antibodies. The invention is primarily concerned with variant Fc regions of IgE antibodies and their use in the creation of novel engineered or modified IgE antibodies. The invention extends to methods for modifying antibodies and to pharmaceutical compositions comprising such antibodies. The invention also covers uses of the modified antibodies in a wide range of therapeutic applications.
Immunoglobulin E (IgE) antibodies play a key role in the mechanisms of allergic disease, not only recognizing allergens through their Fab regions, but also interacting via their Fc regions with two very different cell surface receptors. FcsRI, the receptor found on mast cells and basophils, binds IgE with high affinity (KA = io10-iou M 1) and is responsible for allergic sensitization and the immediate (Type I)
hypersensitivity reaction in which minute amounts of allergen cross-link receptor- bound IgE and trigger cell degranulation. The IgE-binding oc-chain of FcsRI consists of two extracellular Ig-like domains (sFcsRIoc).
In contrast, FcsRII (also known as CD23), expressed on B cells, consists of three C- type lectin "head" domains connected to the membrane by a trimeric oc-helical coiled- coil "stalk". A single head domain binds to IgE-Fc with lower affinity (KA = io5-io6 M 1) than FcsRI, although avidity of the trimer can substantially enhance this interaction. Membrane CD23 (1T1CD23) is cleaved from the cell surface by
endogenous proteases such as ADAMio to yield soluble trimeric and monomeric forms (SCD23), which have been implicated in both positive and negative feedback mechanisms for the regulation of IgE synthesis by B cells that have switched to IgE production. Both FcsRI and CD23 are also expressed on a range of antigen- presenting cells (APC) where they play similar roles in trapping IgE-allergen complexes and promoting the allergic response, but the functional interplay (i.e. cooperation or competition) between these two receptors in the context of APCs is not well understood.
CD23 expressed on B cells also has the potential to contribute to the clinically serious phenomenon of the spreading of allergic reactivity to unrelated allergens, through its ability to internalize IgE-allergen complexes irrespective of the allergen, in contrast to mlgE-mediated allergen-specific presentation through the B cell receptor. CD23 expressed on gastrointestinal epithelial cells also contributes to IgE-allergen transport across the gut epithelial barrier to trigger food allergenic reactions and similarly on respiratory tract epithelial cells to contribute to airway allergic inflammation. Understanding the IgE-CD23 interaction thus has implications for many aspects of allergic disease.
As described in the Examples, using x-ray crystallography, NMR spectroscopy and site-directed mutagenesis, the inventors have now accurately defined the CD23 binding site on the Fc region of IgE, and have clearly demonstrated that the binding site is located in the Ce3 domain of IgE. Based on these results, the inventors have created a series of variant IgE Fc regions, which may be used in the creation of novel and clinically safer IgE-based antibody therapies.
Therefore, in a first aspect of the invention, there is provided a variant IgE Fc region comprising at least one amino acid modification in the Ce3 domain relative to a wild- type IgE Fc region, wherein the at least one amino acid in the wild-type IgE Fc region which is modified is part of a CD23 binding site, and wherein the variant IgE Fc region exhibits a reduced binding affinity to CD23 compared to the wild-type IgE Fc region. Advantageously, and preferably, the inventors have found that the variant IgE Fc region of the first aspect exhibits reduced, and preferably ablated, binding to CD23 (FceRII), but is capable of binding to FceRI. Thus, the variant IgE Fc region may still bind to antigens and trigger mast cells through its interaction with FceRI, but, because it cannot bind (or only poorly binds) to CD23, it avoids the problematic side effects that are commonly associated with CD23 binding, including epitope spreading. Therefore, the Fc region of the invention has considerable utility in IgE antibody engineering. The inventors have surprisingly shown that the CD23 and FceRI binding sites occur on opposite ends of the Ce3 domain of IgE, and that allosteric inhibition prohibits simultaneous binding of these two receptors, thereby preventing engagement and cross-linking of IgE bound to mast cells by soluble CD23.
It will be appreciated that immunoglobulin E (IgE) is a class of antibodies found only in mammals. IgE is a dimeric antibody with four Ig-like domains (Cei- Ce4). The ability to mediate cytotoxic and phagocytic effector functions are potent mechanisms by which antibodies destroy targeted cells. The Fc region links the recognition domain of antibodies to these effector functions through an interaction with Fc receptors and ligands. Manipulation of these effector functions by alteration of the Fc region has important implications in the treatment of numerous medical conditions, e.g. cancer, autoimmune disease and infectious diseases. The Fc region comprises the constant region of an antibody excluding the first constant region immunoglobulin domain. Wild type Fc region refers to the amino acid or nucleotide sequences that are found in nature, including allelic variations. Variant Fc region refers to an Fc sequence that differs from the wild type sequence by virtue of at least one amino acid modification. An Fc variant may only encompass an Fc region, or it may exist in the context of an antibody, Fc fusion, isolated Fc, Fc fragment, or other polypeptide that is substantially encoded by Fc. Fc variant may refer to the Fc polypeptide itself, compositions comprising the Fc variant polypeptide or the amino acid sequence.
The DNA sequence encoding wild-type human IgE-Fc is provided herein as SEQ ID
No:i, as follows:
TGCTCCAGGGACTTCACCCCGCCCACCGTGAAGATCTTACAGTCGTCCTGCGACGGCGGC GGGCACTTCCCCCCGACCATCCAGCTCCTGTGCCTCGTCTCTGGGTACACCCCAGGGACT ATCAACATCACCTGGCTGGAGGACGGGCAGGTCATGGACGTGGACTTGTCCACCGCCTCT ACCACGCAGGAGGGTGAGCTGGCCTCCACACAAAGCGAGCTCACCCTCAGCCAGAAGCAC TGGCTGTCAGACCGCACCTACACCTGCCAGGTCACCTATCAAGGTCACACCTTTGAGGAC AGCACCAAGAAGTGTGCAGATTCCAACCCGAGAGGGGTGAGCGCCTACCTAAGCCGGCCC AGCCCGTTCGACCTGTTCATCCGCAAGTCGCCCACGATCACCTGTCTGGTGGTGGACCTG GCACCCAGCAAGGGGACCGTGAACCTGACCTGGTCCCGGGCCAGTGGGAAGCCTGTGAAC CACTCCACCAGAAAGGAGGAGAAGCAGCGCAATGGCACGTTAACCGTCACGTCCACCCTG CCGGTGGGCACCCGAGACTGGATCGAGGGGGAGACCTACCAGTGCAGGGTGACCCACCCC CACCTGCCCAGGGCCCTCATGCGGTCCACGACCAAGACCAGCGGCCCGCGTGCTGCCCCG GAAGTCTATGCGTTTGCGACGCCGGAGTGGCCGGGGAGCCGGGACAAGCGCACCCTCGCC TGCCTGATCCAGAACTTCATGCCTGAGGACATCTCGGTGCAGTGGCTGCACAACGAGGTG CAGCTCCCGGACGCCCGGCACAGCACGACGCAGCCCCGCAAGACCAAGGGCTCCGGCTTC TTCGTCTTCAGCCGCCTGGAGGTGACCAGGGCCGAATGGGAGCAGAAAGATGAGTTCATC TGCCGTGCAGTCCATGAGGCAGCGAGCCCCTCACAGACCGTCCAGCGAGCGGTGTCTGTA AATCCCG
The polypeptide sequence of wild-type human IgE-Fc is provided herein as SEQ ID
No:2, as follows:
CSRDFTPPTVKI LQS SCDGGGHFPPT I QLLCLVSGYTPGT INI TWLEDGQVMDVDLSTAS 2 83
TTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRP 3 43
SPFDLF IRKSPT I TCLWDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTL 403
PVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLA 463
CL I QNFMPED I SVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEF I 523 CRAVHEAASP S QTVQRAVS VNP
[SEQ ID No: 2]
Thus, preferably the variant IgE Fc region of the invention is derived from the wild- type human IgE-Fc comprising an amino acid sequence substantially as set out in SEQ ID No: 2, or a functional variant or fragment thereof. Preferably, the variant IgE Fc region is derived from the wild-type human IgE-Fc which is encoded by a nucleic acid sequence substantially as set out in SEQ ID No:i, or a functional variant or fragment thereof.
The Ce3 domain of IgE is defined as amino acid residues 329-441 of SEQ ID No: 2. Thus, the at least one amino acid which is modified may be any one of amino acid residues 329-441 of SEQ ID No:2.
As described in the Examples, and as illustrated in Figure lB, the inventors are the first to demonstrate that the A-B helix of Cs3 does not form part of the CD23 binding site, and so it cannot be directly involved in CD23 binding. Therefore, preferably the at least one amino acid modification in the variant IgE Fc region is not in the A-B helix of the Ce3 domain of IgE. It will be appreciated that the A-B helix of the Ce3 domain of IgE is defined as amino acid residues 344-353 of SEQ ID No:2. The inventors have observed that a number of amino acid residues from three discontinuous sequences in the Fc region of IgE form a contiguous surface, which together represent the binding site on the Ce3 domain of IgE for the CD23 receptor. These three spaced apart sequences are located in the E-F helix, the C-D loop and the C-terminal region of the Ce3 domain of IgE. Accordingly, it is preferred that the variant IgE Fc region comprises at least one amino acid modification in the E-F helix of the Ce3 domain of IgE, in the C-D loop of the Ce3 domain of IgE and/or in the C- terminal region of the Ce3 domain of IgE.
It will be appreciated that the E-F helix of the Ce3 domain of IgE is defined as amino acid residues 404-415 of SEQ ID No: 2, the C-D loop of the Ce3 domain of IgE is defined as amino acid residues 376-380 of SEQ ID No: 2, and that the C-terminal region of the Ce3 domain of IgE is defined as amino acid residues 433-441 of SEQ ID No: 2. It will be appreciated that the numbering of amino acid residues given herein is equivalent to the wild-type sequence of human Ig Fc region as shown in SEQ ID No:2.
Therefore, preferably the variant IgE Fc region comprises at least one amino acid modification at a position corresponding to wild-type IgE Fc region selected from the group consisting of: (i) amino acids 404-415 from the E-F helix of IgE; (ii) amino acids 376-380 from the C-D loop of IgE; and (iii) residue 433-441 from the C- terminal region of IgE. Thus, the variant IgE region may comprise at least one amino acid modification at a position corresponding to wild-type IgE Fc region selected from the group consisting of: 376; 377; 378; 379; 380; 404; 405; 406; 407; 408; 409; 410; 411; 412; 413; 414; 415; 433; 434; 435; 436; 437; 438; 439; 440; and 441 of SEQ ID No:2.
The inventors have analysed the bound structure of the IgE/CD23 complex and used considerable inventive endeavour to determine the solvent-exposed IgE residues that make contact with CD23. Accordingly, the variant IgE region may comprise at least one amino acid modification at a position corresponding to wild-type IgE Fc region selected from the group consisting of: 376; 378; 381; 408; 409; 411; 412; 435; 439; and 440.
The inventors observed that mutations of amino acid residues at positions 409, 412, 376, 380 and 435 displayed the largest effects on CD23 binding energetics. Therefore, preferably the variant IgE region comprises at least one amino acid modification at a position equivalent to 409, 412, 376, 380 and/or 435 of the wild-type human IgE Fc region.
The at least one modification may comprise an amino acid deletion or an insertion. However, preferably the at least one modification comprises an amino acid substitution. Preferably, the at least one modification may comprise a non- conservative amino acid change. For example, the charge (i.e. pH) of amino acid may be changed, or the residue may be converted from being hydrophilic so that it is hydrophobic, or vice versa. For example, a K residue may be substituted for a D, or an E may be replaced with a R. Alternatively, a small amino acid may be replaced with a large amino acid (e.g., Y for A).
Alternatively, a large amino acid may be replaced with a small or constraining amino acid, for example alanine, glycine, proline or serine (e.g., F may be replaced with S). Substitution with alanine may be preferred, and the skilled person would readily appreciate the codons which encode alanine, and therefore how to modify the wild- type DNA sequence of SEQ ID No: i to produce variant Fc regions of the invention. For example, in one embodiment the variant Fc region may comprise at least one amino acid modification selected from: D409A; E412A; R376A; K380A and K435A.
The variant human IgE Fc region may comprise more than one modification in the Ce3 domain of the Fc region equivalent to the human IgE Fc region of a wild-type antibody. For example, the variant human IgE Fc region may comprise at least two, three, four or five modifications in the Ce3 domain, as described herein. Thus, the variant Fc region may comprise a double or treble mutant, and so on. The variant Fc region may comprise a modification at two, three, four, five or more of the amino acids at positions equivalent to 376; 377; 378; 379; 380; 404; 405; 406; 407; 408; 409; 410; 411; 412; 413; 414; 415; 433; 434; 435; 436; 437; 438; 439; 440; and 441 of wild-type human IgE-Fc. The variant Fc region may comprise a modification at each of the amino acids at positions equivalent to 409, 412, 376, 380 and/ or 435 of wild- type human IgE-Fc.
In one embodiment, the polypeptide sequence of the variant IgE Fc region (denoted as D409A) is provided herein as SEQ ID No:3, as follows:
CSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTAS 283
TTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRP 343
SPFDLFIRKSPTITCLWDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTL 403
PVGTRAWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLA 463
CLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFI 523 CRAVHEAASPSQTVQRAVSVNP
[SEQ ID No:3]
In another embodiment, the polypeptide sequence of the variant IgE Fc region
(E412A) is provided herein as SEQ ID No:4, as follows:
CSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTAS 283
TTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRP 343
SPFDLFIRKSPTITCLWDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTL 403
PVGTRDWIAGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLA 463
CLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFI 523 CRAVHEAASPSQTVQRAVSVNP
[SEQ ID No:4]
In another embodiment, the polypeptide sequence of the variant IgE Fc region (R376A) is provided herein as SEQ ID No:5, as follows:
CSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTAS 283
TTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRP 343
SPFDLFIRKSPTITCLWDLAPSKGTVNLTWSAASGKPVNHSTRKEEKQRNGTLTVTSTL 403
PVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLA 463
CLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFI 523 CRAVHEAASPSQTVQRAVSVNP
[SEQ ID No:5]
In another embodiment, the polypeptide sequence of the variant IgE Fc region (K380A) is provided herein as SEQ ID No:6, as follows:
CSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTAS 283
TTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRP 343
SPFDLFIRKSPTITCLWDLAPSKGTVNLTWSRASGAPVNHSTRKEEKQRNGTLTVTSTL 403
PVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLA 463
CLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFI 523 CRAVHEAASPSQTVQRAVSVNP [SEQ ID No:6]
In another embodiment, the polypeptide sequence of the variant IgE Fc region (K435A) is provided herein as SEQ ID No η, as follows:
CSRDFTPPTVKI LQS SCDGGGHFPPT I QLLCLVSGYTPGT INI TWLEDGQVMDVDLSTAS 2 83
TTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRP 3 43 SPFDLF IRKSPT I TCLWDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTL 403 PVGTRDWIEGETYQCRVTHPHLPRALMRSTTATSGPRAAPEVYAFATPEWPGSRDKRTLA 463 CLI QNFMPED I SVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEF I 523 CRAVHEAASP S QTVQRAVS VNP
[SEQ ID No:7]
Thus, a preferred variant IgE Fc region of the invention comprises an amino acid sequence substantially as set out in any one of SEQ ID No: 3-7, or a functional variant or fragment thereof.
The IgE Fc variants disclosed herein exhibit reduced binding affinity to the CD23 receptor compared to that of the wild-type or parent Fc region. The term "reduced affinity" compared to the wild-type Fc region can mean that the Fc variant binds to CD23 with a lower equilibrium constant of association (KA or Ka) or higher equilibrium constant of dissociation (KD or Kd) than the wild-type Fc region when the amounts of variant and wild type region in the binding assay that is used are essentially the same. For example, the Fc variant with reduced binding affinity for CD23 may display from about a 5-fold to about 10,000-fold reduction in CD23 binding affinity compared to the wild type Fc region, where CD23 binding affinity is determined, for example, by the binding methods disclosed herein.
Reduced affinity can also be defined relative to an absolute level of affinity. As shown in Table 1, the preferred variants exhibit surprisingly high KD values. For example, the equilibrium constant of dissociation (KD or Kd) of the variant IgE Fc region for CD23 maybe at least 5μΜ, ιθμΜ, 25μΜ or more.
Preferably, the variant IgE Fc region of the first aspect is capable of binding to FceRI. Preferably, the binding affinity of the variant Fc region for FceRI is at least the same as that of the wild-type Fc region. For example, the variant IgE Fc region is capable of binding to FceRI with a KA value of at least io? M 1, io? M 1, 108 M 1, ιο^ M 1, or at least 1010 M 1. In a second aspect, there is provided a nucleic acid encoding the variant IgE Fc region according to the first aspect. In one embodiment, the DNA sequence encoding the variant IgE Fc region (D409A) is provided herein as SEQ ID No:8, as follows:
TGCTCCAGGGACTTCACCCCGCCCACCGTGAAGATCTTACAGTCGTCCTGCGACGGCGGC GGGCACTTCCCCCCGACCATCCAGCTCCTGTGCCTCGTCTCTGGGTACACCCCAGGGACT ATCAACATCACCTGGCTGGAGGACGGGCAGGTCATGGACGTGGACTTGTCCACCGCCTCT ACCACGCAGGAGGGTGAGCTGGCCTCCACACAAAGCGAGCTCACCCTCAGCCAGAAGCAC TGGCTGTCAGACCGCACCTACACCTGCCAGGTCACCTATCAAGGTCACACCTTTGAGGAC AGCACCAAGAAGTGTGCAGATTCCAACCCGAGAGGGGTGAGCGCCTACCTAAGCCGGCCC AGCCCGTTCGACCTGTTCATCCGCAAGTCGCCCACGATCACCTGTCTGGTGGTGGACCTG GCACCCAGCAAGGGGACCGTGAACCTGACCTGGTCCCGGGCCAGTGGGAAGCCTGTGAAC CACTCCACCAGAAAGGAGGAGAAGCAGCGCAATGGCACGTTAACCGTCACGTCCACCCTG CCGGTGGGCACCCGAGC TGGATCGAGGGGGAGACCTACCAGTGCAGGGTGACCCACCCC CACCTGCCCAGGGCCCTCATGCGGTCCACGACCAAGACCAGCGGCCCGCGTGCTGCCCCG GAAGTCTATGCGTTTGCGACGCCGGAGTGGCCGGGGAGCCGGGACAAGCGCACCCTCGCC TGCCTGATCCAGAACTTCATGCCTGAGGACATCTCGGTGCAGTGGCTGCACAACGAGGTG CAGCTCCCGGACGCCCGGCACAGCACGACGCAGCCCCGCAAGACCAAGGGCTCCGGCTTC TTCGTCTTCAGCCGCCTGGAGGTGACCAGGGCCGAATGGGAGCAGAAAGATGAGTTCATC TGCCGTGCAGTCCATGAGGCAGCGAGCCCCTCACAGACCGTCCAGCGAGCGGTGTCTGTA AATCCCG
[SEQ ID No:8]
In another embodiment, the DNA sequence encoding the variant IgE Fc region (E412A) is provided herein as SEQ ID No :g, as follows:
TGCTCCAGGGACTTCACCCCGCCCACCGTGAAGATCTTACAGTCGTCCTGCGACGGCGGC GGGCACTTCCCCCCGACCATCCAGCTCCTGTGCCTCGTCTCTGGGTACACCCCAGGGACT ATCAACATCACCTGGCTGGAGGACGGGCAGGTCATGGACGTGGACTTGTCCACCGCCTCT ACCACGCAGGAGGGTGAGCTGGCCTCCACACAAAGCGAGCTCACCCTCAGCCAGAAGCAC TGGCTGTCAGACCGCACCTACACCTGCCAGGTCACCTATCAAGGTCACACCTTTGAGGAC AGCACCAAGAAGTGTGCAGATTCCAACCCGAGAGGGGTGAGCGCCTACCTAAGCCGGCCC AGCCCGTTCGACCTGTTCATCCGCAAGTCGCCCACGATCACCTGTCTGGTGGTGGACCTG GCACCCAGCAAGGGGACCGTGAACCTGACCTGGTCCCGGGCCAGTGGGAAGCCTGTGAAC CACTCCACCAGAAAGGAGGAGAAGCAGCGCAATGGCACGTTAACCGTCACGTCCACCCTG CCGGTGGGCACCCGAGACTGGATCSCSGGGGAGACCTACCAGTGCAGGGTGACCCACCCC CACCTGCCCAGGGCCCTCATGCGGTCCACGACCAAGACCAGCGGCCCGCGTGCTGCCCCG GAAGTCTATGCGTTTGCGACGCCGGAGTGGCCGGGGAGCCGGGACAAGCGCACCCTCGCC TGCCTGATCCAGAACTTCATGCCTGAGGACATCTCGGTGCAGTGGCTGCACAACGAGGTG CAGCTCCCGGACGCCCGGCACAGCACGACGCAGCCCCGCAAGACCAAGGGCTCCGGCTTC TTCGTCTTCAGCCGCCTGGAGGTGACCAGGGCCGAATGGGAGCAGAAAGATGAGTTCATC TGCCGTGCAGTCCATGAGGCAGCGAGCCCCTCACAGACCGTCCAGCGAGCGGTGTCTGTA AATCCCG
[SEQ ID No:9]
In another embodiment, the DNA sequence encoding the variant IgE Fc region (R376A) is provided herein as SEQ ID No:io, as follows:
TGCTCCAGGGACTTCACCCCGCCCACCGTGAAGATCTTACAGTCGTCCTGCGACGGCGGC GGGCACTTCCCCCCGACCATCCAGCTCCTGTGCCTCGTCTCTGGGTACACCCCAGGGACT ATCAACATCACCTGGCTGGAGGACGGGCAGGTCATGGACGTGGACTTGTCCACCGCCTCT ACCACGCAGGAGGGTGAGCTGGCCTCCACACAAAGCGAGCTCACCCTCAGCCAGAAGCAC TGGCTGTCAGACCGCACCTACACCTGCCAGGTCACCTATCAAGGTCACACCTTTGAGGAC AGCACCAAGAAGTGTGCAGATTCCAACCCGAGAGGGGTGAGCGCCTACCTAAGCCGGCCC AGCCCGTTCGACCTGTTCATCCGCAAGTCGCCCACGATCACCTGTCTGGTGGTGGACCTG GCACCCAGCAAGGGGACCGTGAACCTGACCTGGTCCGCGGCCAGTGGGAAGCCTGTGAAC CACTCCACCAGAAAGGAGGAGAAGCAGCGCAATGGCACGTTAACCGTCACGTCCACCCTG CCGGTGGGCACCCGAGACTGGATCGAGGGGGAGACCTACCAGTGCAGGGTGACCCACCCC CACCTGCCCAGGGCCCTCATGCGGTCCACGACCAAGACCAGCGGCCCGCGTGCTGCCCCG GAAGTCTATGCGTTTGCGACGCCGGAGTGGCCGGGGAGCCGGGACAAGCGCACCCTCGCC TGCCTGATCCAGAACTTCATGCCTGAGGACATCTCGGTGCAGTGGCTGCACAACGAGGTG CAGCTCCCGGACGCCCGGCACAGCACGACGCAGCCCCGCAAGACCAAGGGCTCCGGCTTC TTCGTCTTCAGCCGCCTGGAGGTGACCAGGGCCGAATGGGAGCAGAAAGATGAGTTCATC TGCCGTGCAGTCCATGAGGCAGCGAGCCCCTCACAGACCGTCCAGCGAGCGGTGTCTGTA AATCCCG
[SEQ ID No: 10] In another embodiment, the DNA sequence encoding the variant IgE Fc region (K380A) is provided herein as SEQ ID No:ii, as follows:
TGCTCCAGGGACTTCACCCCGCCCACCGTGAAGATCTTACAGTCGTCCTGCGACGGCGGC GGGCACTTCCCCCCGACCATCCAGCTCCTGTGCCTCGTCTCTGGGTACACCCCAGGGACT ATCAACATCACCTGGCTGGAGGACGGGCAGGTCATGGACGTGGACTTGTCCACCGCCTCT ACCACGCAGGAGGGTGAGCTGGCCTCCACACAAAGCGAGCTCACCCTCAGCCAGAAGCAC TGGCTGTCAGACCGCACCTACACCTGCCAGGTCACCTATCAAGGTCACACCTTTGAGGAC AGCACCAAGAAGTGTGCAGATTCCAACCCGAGAGGGGTGAGCGCCTACCTAAGCCGGCCC AGCCCGTTCGACCTGTTCATCCGCAAGTCGCCCACGATCACCTGTCTGGTGGTGGACCTG GCACCCAGCAAGGGGACCGTGAACCTGACCTGGTCCCGGGCCAGTGGGSs GCCTGTGAAC
CACTCCACCAGAAAGGAGGAGAAGCAGCGCAATGGCACGTTAACCGTCACGTCCACCCTG CCGGTGGGCACCCGAGACTGGATCGAGGGGGAGACCTACCAGTGCAGGGTGACCCACCCC CACCTGCCCAGGGCCCTCATGCGGTCCACGACCAAGACCAGCGGCCCGCGTGCTGCCCCG GAAGTCTATGCGTTTGCGACGCCGGAGTGGCCGGGGAGCCGGGACAAGCGCACCCTCGCC TGCCTGATCCAGAACTTCATGCCTGAGGACATCTCGGTGCAGTGGCTGCACAACGAGGTG CAGCTCCCGGACGCCCGGCACAGCACGACGCAGCCCCGCAAGACCAAGGGCTCCGGCTTC TTCGTCTTCAGCCGCCTGGAGGTGACCAGGGCCGAATGGGAGCAGAAAGATGAGTTCATC TGCCGTGCAGTCCATGAGGCAGCGAGCCCCTCACAGACCGTCCAGCGAGCGGTGTCTGTA AATCCCG
[SEQ ID No:ii]
In another embodiment, the DNA sequence encoding the variant IgE Fc region (K435A) is provided herein as SEQ ID No: 12, as follows:
TGCTCCAGGGACTTCACCCCGCCCACCGTGAAGATCTTACAGTCGTCCTGCGACGGCGGC GGGCACTTCCCCCCGACCATCCAGCTCCTGTGCCTCGTCTCTGGGTACACCCCAGGGACT ATCAACATCACCTGGCTGGAGGACGGGCAGGTCATGGACGTGGACTTGTCCACCGCCTCT ACCACGCAGGAGGGTGAGCTGGCCTCCACACAAAGCGAGCTCACCCTCAGCCAGAAGCAC TGGCTGTCAGACCGCACCTACACCTGCCAGGTCACCTATCAAGGTCACACCTTTGAGGAC AGCACCAAGAAGTGTGCAGATTCCAACCCGAGAGGGGTGAGCGCCTACCTAAGCCGGCCC AGCCCGTTCGACCTGTTCATCCGCAAGTCGCCCACGATCACCTGTCTGGTGGTGGACCTG GCACCCAGCAAGGGGACCGTGAACCTGACCTGGTCCCGGGCCAGTGGGAAGCCTGTGAAC CACTCCACCAGAAAGGAGGAGAAGCAGCGCAATGGCACGTTAACCGTCACGTCCACCCTG CCGGTGGGCACCCGAGACTGGATCGAGGGGGAGACCTACCAGTGCAGGGTGACCCACCCC CACCTGCCCAGGGCCCTCATGCGGTCCACGACCSCSACCAGCGGCCCGCGTGCTGCCCCG GAAGTCTATGCGTTTGCGACGCCGGAGTGGCCGGGGAGCCGGGACAAGCGCACCCTCGCC TGCCTGATCCAGAACTTCATGCCTGAGGACATCTCGGTGCAGTGGCTGCACAACGAGGTG CAGCTCCCGGACGCCCGGCACAGCACGACGCAGCCCCGCAAGACCAAGGGCTCCGGCTTC TTCGTCTTCAGCCGCCTGGAGGTGACCAGGGCCGAATGGGAGCAGAAAGATGAGTTCATC TGCCGTGCAGTCCATGAGGCAGCGAGCCCCTCACAGACCGTCCAGCGAGCGGTGTCTGTA AATCCCG
[SEQ ID No:l2]
Thus, the nucleic acid according to the second aspect may comprise a nucleotide sequence substantially as set out in any one of SEQ ID No: 8-12, or a functional variant or fragment thereof. For each of the nucleic acid sequences represented by SEQ ID No: 8-12, the codon which is shown in bold and underlined represents a modification which would encode alanine. However, it will be appreciated that alanine may be encoded by GCT, GCC, GCA and GCG and so any of these codons would be suitable as a modification to create the same amino acid mutation.
Similarly, the codons for glycine (GGT, GGC, GGA and GGG), proline (CCT, CCC, CCA, CCG) and serine (TCT, TCC, TCA, TCG, AGT and AGC) will be known, and could be used to modify wild type sequence.
The nucleic acid may be an isolated or purified nucleic acid sequence. The nucleic acid sequence may be a DNA sequence. Also described herein are methods for producing the variant IgE Fc region of the first aspect using the nucleic acids of the second aspect. Thus, the nucleic acid sequence may be incorporated into a genetic construct for cloning purposes. In a third aspect, there is provided a genetic construct comprising the nucleic acid of the second aspect.
Genetic constructs of the invention maybe in the form of an expression cassette, which maybe suitable for expression of the encoded polypeptide (i.e. the variant IgE Fc region) in a host cell. The genetic construct may be introduced in to a host cell without it being incorporated in a vector. For instance, the genetic construct, which maybe a nucleic acid molecule, maybe incorporated within a liposome or a virus particle. Alternatively, a purified nucleic acid molecule (e.g. histone-free DNA, or naked DNA) maybe inserted directly into a host cell by suitable means, e.g. direct endocytotic uptake. The genetic construct may be introduced directly in to cells of a host subject (e.g. a bacterial or eukaryotic cell) by transfection, infection,
electroporation, microinjection, cell fusion, protoplast fusion or ballistic
bombardment. Alternatively, genetic constructs of the invention may be introduced directly into a host cell using a particle gun. Alternatively, the genetic construct may be harboured within a recombinant vector, for expression in a suitable host cell.
Therefore, in a fourth aspect, there is provided a recombinant vector comprising the genetic construct according to the third aspect. The recombinant vector may be a plasmid, cosmid or phage. Such recombinant vectors are useful for transforming host cells with the genetic construct of the third aspect, and for replicating the expression cassette therein. The skilled technician will appreciate that genetic constructs of the invention may be combined with many types of backbone vector for expression purposes. Recombinant vectors may include a variety of other functional elements including a suitable promoter to initiate gene expression. For instance, the recombinant vector may be designed such that it autonomously replicates in the cytosol of the host cell. In this case, elements which induce or regulate DNA replication may be required in the recombinant vector. Alternatively, the recombinant vector may be designed such that it integrates into the genome of a host cell. In this case, DNA sequences which favour targeted integration (e.g. by homologous recombination) are envisaged. The recombinant vector may also comprise DNA coding for a gene that may be used as a selectable marker in the cloning process, i.e. to enable selection of cells that have been transfected or transformed, and to enable the selection of cells harbouring vectors incorporating heterologous DNA. Alternatively, the selectable marker gene may be in a different vector to be used simultaneously with vector containing the gene of interest. The vector may also comprise DNA involved with regulating expression of the coding sequence, or for targeting the expressed polypeptide to a certain part of the host cell. In a fifth aspect, there is provided a host cell comprising the genetic construct according to the third aspect, or the recombinant vector according to the fourth aspect.
The host cell may be a bacterial cell. The host cell maybe an animal cell. The host cell may be a mammalian cell, for example a mouse or rat cell. It is preferred that the host cell is not a human cell. The host cell may be transformed with genetic constructs or vectors according to the invention, using known techniques. Suitable means for introducing the genetic construct into the host cell will depend on the type of cell.
Thus, according to a sixth aspect, there is provided a method of preparing a variant IgE Fc region, the method comprising- (i) culturing at least one cell according to the fifth aspect under conditions suitable for the expression of a variant IgE Fc region; and (ii) isolating the variant IgE Fc region.
The inventors believe that knowledge of the sequences of IgE Fc region which form the binding site for the CD23 receptor can be harnessed in a method for reducing the binding affinity of any existing IgE antibody or in the production of novel IgE antibodies, in order to reduce or avoid the risks of patients suffering from any CD23- mediated side effects that may be associated with an IgE antibody therapy.
Hence, in a seventh aspect, there is provided a method of producing a variant IgE Fc region which exhibits a reduced binding affinity for CD23, the method comprising modifying at least one amino acid in the Ce3 domain of a wild-type IgE Fc region, wherein the at least one amino acid in the wild-type IgE Fc region which is modified is part of the CD23 binding site, such that the variant IgE Fc region exhibits a reduced binding affinity to CD23 compared to the wild-type IgE Fc region.
Preferably, the variant IgE Fc region produced by the method is incapable of binding to CD23 (FceRII), but is capable of binding to FceRI.
Preferably, the method does not comprise modifying an amino acid in the A-B helix of the Ce3 domain of IgE. Preferably, the method comprises modifying at least one amino acid in the E-F helix, in the C-D loop and/or in the C-terminal region of the Ce3 domain of IgE. Preferably, the method comprises modifying at least one amino acid at a position corresponding to wild-type IgE Fc region selected from the group consisting of: (i) amino acids 404-415 from the E-F helix of IgE; (ii) amino acids 376- 380 from the C-D loop of IgE; and (iii) residue 433-441 from the C-terminal region of IgE. The method may comprise modifying at least one amino acid at a position corresponding to wild-type IgE Fc region selected from the group consisting of: 376; 377; 378; 379; 380; 404; 405; 406; 407; 408; 409; 410; 411; 412; 413; 414; 415; 433; 434; 435; 436; 437; 438; 439; 440; and 441 of SEQ ID No:2. Preferably, the method comprises modifying at least one amino acid at a position equivalent to 409, 412, 376, 380 and/or 435 of the wild-type human IgE Fc region. The method may comprise modifying at least two, three, four, five or more amino acids in the Ce3 domain relative to a wild-type IgE Fc region.
The wild-type IgE Fc region which is modified in the method may be part of a therapeutically active IgE antibody. Examples of such an IgE antibody which may be modified include IgE antibodies directed against tumour antigens, as described by Karagiannis et al. (Karagiannis, 2011, Cancer Immunol Immunotherapy; PMID 22139135). Thus, the method may be used to modify the IgE antibody such that it cannot bind to CD23, thereby avoiding the risk of CD23-mediated side effects. Alternatively, the method may comprise initially creating the variant IgE Fc region and then fusing it to a functional fragment of an IgE antibody to produce an Fc fusion, for example a chimera. The functional fragment may comprise variable regions exhibiting immunospecificity for a target epitope. The functional fragment maybe selected from a group consisting of VH, VL, Fd, Fv, Fab, Fab', scFv and F(ab')2. Based on the foregoing, it will be appreciated that the variant IgE Fc region of the first aspect or the method of the seventh aspect can be used to form antibody or a functional fragment thereof. Thus, in an eighth aspect of the invention, there is provided an antibody or a functional fragment thereof comprising the variant IgE Fc region of the first aspect.
Preferably, the antibody or functional fragment thereof is incapable of binding to CD23 (FceRII), or exhibits a reduced binding affinity thereto, but is capable of binding to FceRI.
The invention extends both to whole antibodies (i.e. immunoglobulins) with immunospecificity for a certain target epitope, as well as to functional fragments thereof. Such fragments retain at least one antigen binding region of a corresponding full-length antibody as well as the variant IgE Fc region of the invention. The antibody or functional fragment thereof may comprise a monoclonal or polyclonal antibody or functional fragment thereof.
The antibody or functional fragment may be monovalent, divalent or polyvalent. Monovalent antibodies are dimers (HL) comprising a heavy (H) chain associated by a disulfide bridge with a light chain (L). Divalent antibodies are tetramer (H2L2) comprising two dimers associated by at least one disulfide bridge. Polyvalent antibodies may also be produced, for example by linking multiple dimers. The basic structure of an antibody molecule consists of two identical light chains and two identical heavy chains which associate non-covalently and can be linked by disulphide bonds. Each heavy and light chain contains an amino-terminal variable region of about 110 amino acids, and constant sequences in the remainder of the chain. The variable region includes several hypervariable regions, or
complementarity determining regions (CDRs), that form the antigen-binding site of the antibody molecule and determine its specificity for the antigen in question. On either side of the CDRs of the heavy and light chains is a framework region, a relatively conserved sequence of amino acids that anchors and orients the CDRs.
The constant region consists of one of five heavy chain sequences (μ, γ, ζ, a, or ε) and one of two light chain sequences (κ or λ). The heavy chain constant region sequences determine the isotype of the antibody and the effector functions of the molecule. Preferably, the antibody or functional fragment thereof is an IgE.
The antibody or fragment thereof may be a human antibody. As used herein, the term "human antibody" can mean an antibody, such as a monoclonal antibody, which comprises substantially the same heavy and light chain CDR amino acid sequences as found in a particular human antibody exhibiting immunospecificity for certain epitope. An amino acid sequence, which is substantially the same as a heavy or light chain CDR, exhibits a considerable amount of sequence identity when compared to a reference sequence. Such identity is definitively known or recognizable as
representing the amino acid sequence of the particular human antibody.
Substantially the same heavy and light chain CDR amino acid sequence can have, for example, minor modifications or conservative substitutions of amino acids. Such a human antibody maintains its function of selectively binding to an epitope. The term "human monoclonal antibody" can include a monoclonal antibody with substantially human CDR amino acid sequences produced, for example by recombinant methods such as production by a phage library, by lymphocytes or by hybridoma cells. The term "humanised antibody" can mean an antibody from a non-human species (e.g. mouse) whose protein sequences have been modified to increase their similarity to antibodies produced naturally in humans.
The antibody may be a recombinant antibody. The term "recombinant human antibody" can include a human antibody produced using recombinant DNA technology.
The term "antigen binding region" can mean a region of the antibody having specific binding affinity for its target antigen. The binding region may be a hypervariable CDR or a functional portion thereof. The term "functional portion" of a CDR can mean a sequence within the CDR which shows specific affinity for the target antigen. The functional portion of a CDR may comprise a ligand which specifically binds to its epitope. The term "CDR" can mean a hypervariable region in the heavy and light variable chains. There maybe one, two, three or more CDRs in each of the heavy and light chains of the antibody. Normally, there are at least three CDRs on each chain which, when configured together, form the antigen-binding site, i.e. the three-dimensional combining site with which the antigen binds or specifically reacts. It has however been postulated that there may be four CDRs in the heavy chains of some antibodies. The definition of CDR also includes overlapping or subsets of amino acid residues when compared against each other. The exact residue numbers which encompass a particular CDR or a functional portion thereof, 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.
The term "functional fragment" of an antibody can mean a portion of the antibody which retains a functional activity. A functional activity can be, for example antigen binding activity or specificity. A functional activity can also be, for example, an effector function provided by an antibody constant region. The term "functional fragment" is also intended to include, for example, fragments produced by protease digestion or reduction of a human monoclonal antibody and by recombinant DNA methods known to those skilled in the art. Human monoclonal antibody functional fragments include, for example individual heavy or light chains and fragments thereof, such as VL, VH and Fd; monovalent fragments, such as Fv, Fab, and Fab' ; bivalent fragments such as F(ab single chain Fv (scFv); and Fc fragments.
The term "VL fragment" can mean a fragment of the light chain of a human monoclonal antibody which includes all or part of the light chain variable region, including the CDRs. A VL fragment can further include light chain constant region sequences.
The term "VH fragment" can means a fragment of the heavy chain of a human monoclonal antibody which includes all or part of the heavy chain variable region, including the CDRs.
The term "Fd fragment" can mean the light chain variable and constant regions coupled to the heavy chain variable and constant regions, i.e. VL CL and VH CH-i.
The term "Fv fragment" can mean a monovalent antigen-binding fragment of a human monoclonal antibody, including all or part of the variable regions of the heavy and light chains, and absent of the constant regions of the heavy and light chains. The variable regions of the heavy and light chains include, for example, the CDRs. For example, an Fv fragment includes all or part of the amino terminal variable region of about no amino acids of both the heavy and light chains.
The term "Fab fragment" means a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than an Fv fragment. For example, a Fab fragment includes the variable regions, and all or part of the first constant domain of the heavy and light chains. Thus, a Fab fragment additionally includes, for example, amino acid residues from about no to about 220 of the heavy and light chains. The term "Fab' fragment" can means a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than a Fab fragment. For example, a Fab' fragment includes all of the light chain, all of the variable region of the heavy chain, and all or part of the first and second constant domains of the heavy chain. For example, a Fab' fragment can additionally include some or all of amino acid residues 220 to 330 of the heavy chain.
The term "F(ab')2 fragment" can mean a bivalent antigen-binding fragment of a human monoclonal antibody. An F(ab')2 fragment includes, for example, all or part of the variable regions of two heavy chains-and two light chains, and can further include all or part of the first constant domains of two heavy chains and two light chains.
The term "single chain Fv (scFv)" can mean a fusion of the variable regions of the heavy (VH) and light chains (VL) connected with a short linker peptide. One skilled in the art knows that the exact boundaries of a fragment of a human monoclonal antibody are not important, so long as the fragment maintains a functional activity. Using well-known recombinant methods, one skilled in the art can engineer a polynucleotide sequence to express a functional fragment with any endpoints desired for a particular application. A functional fragment of the antibody may comprise fragments with substantially the same heavy and light chain variable regions as the human antibody.
The functional fragment may include fragments wherein at least one of the binding region sequences has substantially the same amino acid sequence as the binding region sequences of the antibody. The functional fragment may comprise any of the fragments selected from a group consisting of VH, VL, Fd, Fv, Fab, Fab', scFv, F (ab')2 and Fc fragment. Thus, in some embodiment, the invention comprises a functional fragment of an antibody (e.g. Fv, Fab, and Fab', bivalent fragments such as F(ab')2, single chain Fv (scFv), fused to the Fc region of the first aspect.
The functional fragment may comprise any one of the antigen binding region sequences of the VL, any one of the antigen binding region sequences of the VH, or a combination of VL and VH antigen binding regions of a human antibody. The appropriate number and combination of VH and VL antigen binding region sequences may be determined by those skilled in the art depending on the desired affinity and specificity and the intended use of the functional fragment. Functional fragments of antibodies may be readily produced and isolated using methods well known to those skilled in the art. Such methods include, for example, proteolytic methods, recombinant methods and chemical synthesis. Proteolytic methods for the isolation of functional fragments comprise using human antibodies as a starting material. Enzymes suitable for proteolysis of human immunoglobulins may include, for example, papain, and pepsin. The appropriate enzyme may be readily chosen by one skilled in the art, depending on, for example, whether monovalent or bivalent fragments are required. For example, papain cleavage results in two monovalent Fab' fragments that bind antigen and an Fc fragment. Pepsin cleavage, for example, results in a bivalent F (ab') fragment. An F (ab')2 fragment of the invention maybe further reduced using, for example, DTT or 2-mercaptoethanol to produce two monovalent Fab' fragments.
Functional fragments produced by proteolysis may be purified by affinity and column chromatographic procedures. For example, undigested antibodies and Fc fragments may be removed by binding to protein A. Additionally, functional fragments may be purified by virtue of their charge and size, using, for example, ion exchange and gel filtration chromatography. Such methods are well known to those skilled in the art.
The human antibody or functional fragment thereof may be produced by
recombinant methodology. Preferably, one initially isolates a polynucleotide encoding desired regions of the antibody heavy and light chains. Such regions may include, for example, all or part of the variable region of the heavy and light chains. Preferably, such regions can particularly include the antigen binding regions of the heavy and light chains, preferably the antigen binding sites, most preferably, the CDRs.
The polynucleotide encoding the human antibody or functional fragment of the invention may be produced using methods known to those skilled in the art. The polynucleotide encoding the antibody or a functional fragment thereof may be directly synthesized by methods of oligonucleotide synthesis known in the art.
Alternatively, smaller fragments may be synthesized and joined to form a larger functional fragment using recombinant methods known in the art.
As used herein, the term "immunospecificity" can mean the binding region is capable of immunoreacting with a certain target epitope, by specifically binding therewith. The term "immunoreact" can mean the binding region is capable of eliciting an immune response upon binding with an epitope.
The term "epitope" can mean any region of an antigen with ability to elicit, and combine with, a binding region of the antibody or fragment thereof.
Since IgE antibodies play a central role in the initiation and regulation of allergic disorders, clinical applications of the variant IgE Fc region of the first aspect, and of the antibodies (i.e. modified antibodies) of the eighth aspect, include their use in treating a wide range of medical conditions (including cancer, autoimmune disease or infectious disease), while simultaneously avoiding the deleterious allergic side effects that are often observed in patients who are treated with IgE antibody therapies. In addition, they may also have utility in diagnosis.
Accordingly, in a ninth aspect of the invention, there is provided a variant IgE Fc region according to the first aspect, or an antibody or a functional fragment thereof according to the eighth aspect, for use in therapy or diagnosis.
In a tenth aspect, there is provided provided a variant IgE Fc region according to the first aspect, or an antibody or a functional fragment thereof according to the eighth aspect, for use in treating, preventing or ameliorating a CD23-mediated side effect.
In an eleventh aspect, there is provided a method for treating, preventing or ameliorating a CD23-mediated side effect in a subject, the method comprising administering, to a subject in need of such a treatment, a therapeutically effective amount of a variant IgE Fc region according to the first aspect, or an antibody or a functional fragment thereof according to the eighth aspect. Preferably, the variant IgE Fc region, or the antibody or a functional fragment thereof is used for preventing a CD23-mediated side effect. The CD23-mediated side effect, which is prevented or avoided, may be caused by IgE antibody treatment or therapy, of which there are many types. The CD23-mediated side effect which is prevented is one which may otherwise be caused during the treatment of cancer, autoimmune disease or infectious disease with a therapeutic IgE antibody. Thus, the antibody or fragment thereof maybe a therapeutic antibody.
A CD23-mediated side effect which may be prevented may include CD23-dependent epitope spreading, which will be known to the skilled person. Epitope spreading is the process by which an antibody response to one epitope on an antigen leads to the production of antibodies specific for other epitopes on the same antigen, or for epitopes on entirely unrelated antigens. This results from the internalisation of whole antigen and subsequent display of a range of peptides derived from that antigen, leading to the generation of T cells with different epitope specificities. Simultaneous processing of two unrelated antigens by an antigen-presenting cell can lead to the production of antibodies directed against both antigens (Gould and Sutton, 2008, Nature Reviews Immunology, 8, 205-217). It will be appreciated that antibodies, fragments, peptides and nucleic acids according to the invention (collectively referred to herein as "agents") maybe used in a monotherapy (e.g. the use of an antibody or fragment thereof alone), for treating, ameliorating or preventing a CD23-mediated side effect. Alternatively, agents according to the invention maybe used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing CD23-mediated side effects.
The agents according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well -tolerated by the subject to whom it is given, and preferably enables delivery of the agents across the blood-brain barrier. Medicaments comprising agents of the invention maybe used in a number of ways. For instance, oral administration may be required, in which case the agents may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Compositions comprising agents and medicaments of the invention may be administered by inhalation (e.g. intranasally). Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin.
Agents and medicaments according to the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site. Such devices may be particularly advantageous when long-term treatment with agents used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).
In a preferred embodiment, agents and medicaments according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment. Injections maybe intravenous (bolus or infusion) or
subcutaneous (bolus or infusion), or intradermal (bolus or infusion).
It will be appreciated that the amount of the antibodies, fragments, peptides and nucleic acids (i.e. agent) that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the
physiochemical properties of the agent, and whether it is being used as a
monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half-life of the agent within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the disease. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.
Generally, a daily dose of between o.oo^g/kg of body weight and lomg/kg of body weight of agent according to the invention may be used for treating, ameliorating, or preventing a CD23-mediated side effect. More typically, the daily dose of agent is between o.o^g/kg of body weight and lmg/kg of body weight, more preferably between
Figure imgf000022_0001
body weight, and most preferably between approximately
Figure imgf000022_0002
body weight. The agent may be administered before, during or after onset of the side effect. Daily doses may be given as a single administration (e.g. a single daily injection).
Alternatively, the agent may require administration twice or more times during a day. As an example, agents may be administered as two (or more depending upon the severity of the condition being treated) daily doses of between 0.07 μg and 700 mg (i.e. assuming a body weight of 70 kg). A patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter. Alternatively, a slow release device may be used to provide optimal doses of agents according to the invention to a patient without the need to administer repeated doses. Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the agents according to the invention and precise therapeutic regimes (such as daily doses of the agents and the frequency of administration). In a twelfth aspect of the invention, there is provided a pharmaceutical composition comprising a variant IgE Fc region according to the first aspect, or an antibody or fragment thereof according to the eighth aspect; and optionally a pharmaceutically acceptable vehicle. The composition may be CD23-mediated side effect treatment composition. The term "CD23-mediated side effect treatment composition" can mean a pharmaceutical formulation used in the therapeutic amelioration, prevention or treatment of a CD23- mediated side effect. The invention also provides in a thirteenth aspect, a process for making the composition according to the tenth aspect, the process comprising combining a therapeutically effective amount of a variant IgE Fc region according to the first aspect, or an antibody or a functional fragment thereof as defined in the eighth aspect, with a pharmaceutically acceptable vehicle. A "subject" may be a vertebrate, mammal, or domestic animal. Hence, medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications. Most preferably, the subject is a human being.
A "therapeutically effective amount" of the antibody or fragment thereof is any amount which, when administered to a subject, is the amount of agent that is needed to treat CD23-mediated side effect, or to produce the desired effect. For example, the therapeutically effective amount of antibody or fragment thereof used may be from about o.ooi ng to about ι mg, and typically from about o.oi ng to about loo ng. It is preferred that the amount of antibody or fragment is an amount from about o.i ng to about 10 ng, and most preferably from about 0.5 ng to about 5 ng.
A "pharmaceutically acceptable vehicle" as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions. In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, the active agent may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active agents. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.
However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active agent according to the invention maybe dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.
Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection. The agent may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium. The agents and compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 8o (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The agents used according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral
administration include sterile solutions, emulsions, and suspensions.
It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof. The terms "substantially the amino acid/nucleotide/peptide sequence", "functional variant" and "functional fragment", can be a sequence that has at least 40% sequence identity with the amino acid/ nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID No:2 (i.e. wild type human IgE Fc region) or the nucleotide identified as SEQ ID No:i (i.e. encoding wild type human IgE Fc region), or 40% identity with the polypeptides identified as SEQ ID No:3"7 (i.e. IgE Fc variant regions) or the nucleotides identified as SEQ ID No:8- 12 (i.e. encoding IgE Fc variant regions), and so on.
Amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 50%, more preferably greater than 65%, 70%, 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90%, 92%, 95%, 97%, 98%, and most preferably at least 99% identity with any of the sequences referred to herein. The skilled technician will appreciate how to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences. In order to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g.
functional form and constants.
Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.
Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al, 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al, 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.
Preferably, calculation of percentage identities between two amino
acid/polynucleotide/polypeptide sequences may then be calculated from such an alignment as (N/T)*ioo, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs. Hence, a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:- Sequence Identity = (N/T)*ioo.
Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to the sequences shown in SEQ ID No's: 1, 8-12 or their complements under stringent conditions. By stringent conditions, we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/ sodium citrate (SSC) at approximately 45°C followed by at least one wash in o.2x SSC/ 0.1% SDS at approximately 20-65°C. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ ID No: 2, 3-7.
Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non- polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids
All of the features described herein (including any accompanying claims, abstract and drawings), and/ or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/ or steps are mutually exclusive.
For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying diagrammatic drawings, in which: -
Figure l shows NMR mapping of the CD23 and IgE interaction surfaces. (A) A small number of residues from Ce3 show chemical shift perturbation upon addition of derCD23. Increasing amounts of unlabelled derCD23 were added to a 200μΜ sample of ^N-labelled Ce3; five spectra of the titration are overlaid (red, zero derCD23;
magenta, 50 μΜ; blue, 100 μΜ; cyan, 150 μΜ; green, 200 μΜ). Insets show magnified views of the indicated regions. (B) The NMR-derived derCD23 interaction site on Ce3 was mapped onto the structure of IgE-Fc (1F6A; Garman et al., 2000, Nature 406:259-266) and shown as surface representation. For comparison, the residues of IgE that interact with FceRI are indicated in green. (C) The IgE interaction surface on CD23 was defined previously (Hibbert et al., 2005, J Exp Med 202:751-760) and shown here as a surface representation. The interacting surfaces of IgE and CD23 are coloured according to electrostatic potential and coded such that regions with a potential <-4 kBT are red, whereas those >4 kBT are blue (kB,
Boltzmann constant; T, absolute temperature). The IgE-CD23 interaction site is formed by complementary charged surfaces;
Figure 2 shows competition binding experiments between derCD23 and sFceRIa for IgE-Fc. (A-D) Surface plasmon resonance and (E-F) TR-FRET-based competitive binding experiments were used to investigate the mechanisms of receptor interactions. The binding of derCD23 was tested against (A) IgE-Fc immobilized on a sensor surface and (B) IgE-Fc captured on an FceRIa-immobilized surface; the start of the derCD23 injection is indicated with an arrow. DerCD23 binding to
immobilized IgE-Fc with a KD of 2.3 μΜ; no measureable binding was observed for derCD23 to IgE-Fc complexed to FceRIa. (C) The binding of IgE-Fc to immobilized derCD23 was compared with (D) the binding of a complex of IgE-Fc/ sFceRIa to the same surface; the start of the injection of the complex is indicated with an arrow. IgE-Fc binds to derCD23 with a KD of 2.4 μΜ, but the IgE-Fc/sFceRIa complex does not bind to derCD23. All SPR binding experiments were performed using identical two-fold serial dilutions of ligands, from 40 μΜ to 78nM. (E) Binding between terbium-labelled derCD23 and Alexa 647-labelled IgE-Fc was measured in a solution TR-FRET assay in the presence of increasing concentrations of the αγ-fusion protein (the FceRIa extracellular region fused to an IgG4 Fc) (Shi et al., 1997, Biochemistry, 36, 2112-2122); zero (black), o.snM (red), 2.5nM (blue) and 5nM (green). (F) These data are also shown as a double reciprocal plot to illustrate that addition of the inhibitor results in a decrease in the apparent binding maximum (the value at X=o), rather than a change in the apparent binding affinity (the value at Y=o); this is characteristic of allosteric inhibitors;
Figure 3 shows that soluble CD23 does not cross-link IgE bound to FceRI on mast cells. The ability of soluble CD23 to engage IgE on B cells and mast cells was tested. (A) After preincubation of IgE, the addition of anti-IgE antibody results in activation of the FceRIa+ LAD-2 mast cell line, as measured by release of β-hexosaminidase. Neither monomeric derCD23 nor trimeric triCD23 is able to cross-link IgE and activate mast cells in this assay. (B) In contrast, triCD23 effectively cross-links mlgE on the surface of IgE+ human tonsillar B cells, resulting in activation of these cells and increased secretion of IgE. B cell cultures were activated with IL-4 and anti- CD40, and soluble CD23 was added at 1 μΜ; supernatants were harvested 12 days after activation, tested for IgE levels and compared with levels for cells treated with IL-4/anti-CD40 alone (*=p<0.05; **=p<o.oi); Figure 4 shows the temperature dependence and ionic strength dependence of the interaction between IgE-Fc and derCD23. (A) The temperature dependence of the IgE-Fc and derCD23 interaction is demonstrated by plotting the natural log of the equilibrium constant as a function of the inverse of absolute temperature (the van't Hoff plot). A linear fit of the data is shown (red line), and 95% confidence levels indicated (dashed lines). The slope of this plot estimates the enthalpic changes associated with binding. At a temperature of 298K, the thermodynamic parameters of binding are as follows: AG= -32.2 (± 0.5) kJ mol 1, ΔΗ= -5.0 (± 0.3) mol 1, -TAS= - 27.2 kJ mol 1; these values are consistent with a strong electrostatic component to binding energetics. (B) The effects of ionic strength on the binding of IgE-Fc to derCD23. Binding affinities are measured over a range of ionic strengths and plotted as log (KA) versus log (ionic strength). The steep slope (-0.94) of the curve confirms a strong electrostatic contribution to binding;
Figure 5 shows the structure of the derCD23-Fcs3-4 complex. The two molecules of derCD23 (light and dark blue Coc traces with surfaces) bind one to each heavy chain between the Cs3 (dark red and green) and Cs4 domains (light red and green). The carbohydrate is shown in all-atom representation (red and yellow, without surfaces) and can be seen behind the (red) Cs3 domain. The adjacent N- and C- termini of each derCD23 molecule, the former being the connection to the "stalk", the latter to the "tail" region, can be seen at the extreme left and right of the figure. (The complex shown here comprises chains A (red), B (green), G (light blue) and H (dark blue));
Figure 6 shows salt bridges and hydrogen bonds at the derCD23-Fcs3-4 interface. The H-bonds associated with the four salt bridges are shown in red, additional H- bonds present in all six independent interactions are shown in green, and a further H-bond present in 5/6 molecules is shown in yellow;
Figure 7 shows composite images of the derCD23 and sFcsRIoc complexes with Fcs3"4 to show the mutual incompatibility of their binding modes, a) derCD23-Fcs3- 4 and sFcsRIa-Fcs3-4 [PDB 1F6A] complexes superposed on their (Cs4)2 domain pairs. The receptors are shown as surfaces (derCD23 light & dark blue; sFcsRIoc red) and the Fcs3-4 structures are shown as Coc traces (in corresponding colours). The closed (derCD23-binding) and open (sFcsRIoc-binding) conformations of the Cs3 domains may be seen, b) Steric clashes between the sFcsRIa structure (red Coc trace) and both chains of the derCD23-Fcs3-4 complex (blue) are indicated (orange surfaces), c) Steric clashes of both derCD23 molecules (blue Coc traces) with the sFcsRIoc-Fcs3-4 complex (red) are indicated (green surfaces);
Figure 8 shows that sFcsRIa displaces derCD23 and vice versa in a FRET inhibition assay, a) Competition of derCD23 binding to IgE-Fc (blue) and Fcs3-4 (red) by unlabeled sFcsRIa. b) Competition of sFcsRIa binding to IgE-Fc (blue) and Fcs3-4 (red) by unlabeled derCD23. (All data: n=3; error bars ± SEM);
Figure 9 shows electron density in the interface region. Electron density 2 0 - Fc map contoured at ΐσ for key side-chains involved in salt bridges at the
derCD23/Fcs3-4 interface. The side-chains shown in blue are from derCD23, those in purple from Fcs3-4;
Figure 10a) The six independent Fcs3-4 heavy chains superposed on their Cs4 domains, showing the variation in the relative positions of the Cs3 domains, b) The six independent derCD23 molecules superposed, showing virtually no overall structural variation;
Figure 11 shows a comparison of Fcs3-4 heavy chains taken from various Fcs3-4 and IgE-Fc structures superposed by their Cs4 domains upon the set of six independent heavy chains of the derCD23-Fcs3-4 complex (shown lightly coloured), a) Free IgE- Fc (light and dark red) [PDB 2WQR; 28], b) sFcsRIoc-IgE-Fc complex (light and dark cyan) [PDB 2Y7Q; 21], c) sFcsRIoc-Fcs3-4 complex (light and dark purple) [PDB 1F6A; 20], d) Single chain of free Fcs3-4 (green), the most closed structure observed to date [PDB 3HA0; 27]; and
Figure 12 shows the superposition of apo- and calcium-bound derCD23 structures. The apo-CD23 (dark blue) and calcium-bound CD23 (dark red) structures [PDB 2H2R, 2H2T; 40] superposed upon the six derCD23 molecules from the Fcs3-4 complex (lightly coloured), showing differences in loops 1 and 4. The Ca2+ ion is shown (red sphere); note the disordered loop 4 between residues Ser252 and Asp258 in the calcium-bound structure.
Examples
Example 1
Materials and Methods
Protein expression and purification: Recombinant human IgE-Fc (comprising domains Ce2-C84)(Wan et al., 2002, Nat Immunol 3:681-686), the αγ-fusion protein (the FceRIa extracellular region fused to an IgG4 Fc) (Shi et al., 1997, Biochemistry 36, 2112-2122), soluble FceRIa (Price et al., 2005, J Biol Chem 280, 2324-2330), derCD23 (Hibbert et al., 2005, J Exp Med 202, 751-760) and the Ce3 domain (Price et al., 2005, J Biol Chem 280, 2324-2330) were each produced and purified as described previously. Primers for site-directed mutagenesis were obtained from Sigma-Genosys (Sigma Lifescience), and mutagenesis was performed using the
QuikChange II Kit (Stratagene) according to the manufacturer's instructions. Mutant constructs were expressed and purified using the same methods as the wildtype proteins. MAb 7.12 was produced from a B cell hybridoma (Kanowith-Klein et al., 1988, Clin Immunol Immunopathol 48, 214-224), and was a kind gift of Dr. Rebecca Beavil (King's College London, UK).
NMR spectroscopy: NMR spectroscopy was performed on protein samples in a buffer containing 25mM Tris, 125 mM NaCl, 41ΏΜ CaCl2, pH 6.8, at protein concentrations between 120 and 900 μΜ. Data were collected at 25°C on Bruker spectrometers equipped with CryoProbes operating at 500 and 700 MHz. For chemical shift perturbation experiments, unlabelled derCD23 ligands were concentrated to 2mM and then added in small aliquots to samples of 200μΜ ^N-labelled Ce3 until saturation was seen. The NMR chemical shifts of the urea denatured and native state Ce3 domain are available from the BioMagResBank database under accession numbers 18482 and 18483.
Surface plasmon resonance: All experiments were performed on a Biacore T100 instrument (GE Healthcare), essentially as described previously (Hibbert et al., 2005, J Exp Med 202, 751-760; Holdom et al., 2011, Nat Struct Mol Biol 18, 571-576).
Specific binding surfaces were prepared using standard amine coupling methods for derCD23 and the αγ-fusion protein, whereas IgE-Fc was biotinylated (one biotin per molecule) and captured on a streptavidin surface. Coupling densities were kept low (<ioo RU) to minimize potential avidity effects. Ligands in HBS (lomM HEPES, pH 7.4, 15ΟΠ1Μ NaCl, 4mM CaCl2, 0.005% surfactant p2o) were injected at 25 μΐ/min with a l-min association phase followed by a 15-min dissociation phase.
Thermodynamic measurements were performed at 288K, 293K, 298K and 303K. For the sandwich binding experiments, approximately 90RU of IgE-Fc was captured on an αγ-fusion protein surface during a l-min injection of a ιοηΜ IgE-Fc sample; after a 3-min stabilization period, ο-ιοομΜ derCD23 was injected for l-min followed by a 15-min dissociation phase. All measurements were done independently at least twice, using standard double reference subtraction methods for data analysis
(Myszka, 1999, JMol Recognit 12, 279-284). Curve fitting and other analyses were performed using MicroCal Origin 8.0 software (OriginLab Corporation).
FRET assay: Inhibition assays were performed by competing ΐμΜ terbium-chelate labelled derCD23 and 0-20μΜ Alexa 647-labelled IgE-Fc with a range of
concentrations of unlabelled αγ-fusion protein. Protein mixtures were prepared in Lanthascreen buffer (Invitrogen) in triplicate, in 384-well plates (Greiner Bio-One), and equilibrated overnight at room temperature. FRET measurements were made on an Artemis plate reader (Berthold Technologies). TR-FRET ratios were calculated for each well as the emission of acceptor at 66snm divided by the emission of donor at 62onm and then multiplied by 10,000. Apparent KD and Bmax values were derived from non-linear curve fitting of inhibition titrations, and data are presented as double reciprocal plots to highlight the differences in apparent Bmax values upon addition of the inhibitor.
Mast cell degranulation assay: The human mast cell line LAD-2 (NIH) was cultured in Stem Pro-34 SFM medium (Gibco) supplemented with L-glutamine (2 mM), penicillin (10 U/ml), and streptomycin (1 g/ml; all from Life Technologies). LAD-2 cells were suspended in culture medium at 1.6x1ο6 cells/ml and 50μ1 aliquots were placed in v-bottomed 96-well plates (Greiner Bio-One). Cells were primed by addition of 2.5nM IgE (NIBSC) or a buffer-only control for one hour, before addition of cross-linking reagents. Polyclonal rabbit anti-human IgE (Dako) was added at 2onM and soluble CD23 constructs at 0.1, 1 and ιθμΜ, and incubated for 1 hour at 37°C. Supernatants were harvested and tested for β-hexosaminidase release, measured fluorometrically as described previously (Hammond, 2006, In Cell Biology, J.E. Celis, editor. Elsevier. Amsterdam). Controls included cells treated with wash buffer plus 1% Triton-X for total release, with buffer-only to measure background release, typically about 10% of total release, with 2.5nM IgE-only, and with ιθμΜ CD23-only. The level of degranulation measured for Triton-X treated cells was defined as 100% release and all samples were defined relative to that.
B cell activation assays: Human tonsillar B cells were cultured in 24-well plates (Nunc) at 5 x 105 cells/ml in RPMI with penicillin (lOOlU/ml), streptomycin
Figure imgf000033_0001
glutamine (2mM) (Invitrogen), 10% FCS (Hyclone, Perbio Biosciences Ltd), insulin ^g/ml) and transferrin (35 g/ml) (Sigma-Aldrich). Cells were activated with IL-4 (200lU/ml) (R&D Systems), anti-CD40 antibody (^g/ml) (G28.5; ATCC), and either ΐμΜ derCD23 or ΐμΜ triCD23. Supernatants were harvested on day 12 for IgE measurements. IgE ELISA assays were performed as described previously (Cooper et al., 2012, J Immunol 188, 3199-3207), using Maxisorp plates (Nunc) coated with polyclonal mouse anti-human IgE (1:7000) (Dako), in pH 9.8 carbonate buffer (0.2M Na2C03, 0.2M NaHC03). Unbound sites were blocked with 2% milk powder in PBS + 0.05% Tween20 (Sigma-Aldrich).
Binding was detected by mouse anti-human IgE-HRP (1:1000) (Dako) in 1% milk powder in PBS-T for 2 hours at 37°C. Standard curves were derived using human serum IgE (NIBSC), with a minimal detectable concentration of about 2ng/ml.
Results and discussion
In an earlier study, the inventors identified the IgE binding site on CD23 using NMR chemical shift perturbation studies (Hibbert et al., 2005, J Exp Med 202, 751-760). In this work, the inventors have now performed the reciprocal NMR binding experiment, mapping the interaction site of CD23 onto the Ce3 domain from IgE. Using an approach described by Schulman et al. (Schulman et al., 1997, Nat Struct Biol 4, 630-634), they then assigned the backbone resonances of the molten globule Ce3 domain by first performing resonance assignments of Ce3 denatured in 6M urea and then, through gradual titration of buffer conditions, tracking those resonances to the native state Ce3 domain. Next, the inventors titrated unlabelled monomeric CD23 protein (derCD23) against an ^N-labelled Ce3 sample and used the assigned NMR spectra to identify residues that were affected by addition of ligand. A small number of Ce3 residues showed peak shifting and line broadening during the derCD23 titration, consistent with an interaction characterized by fast/intermediate exchange kinetics (see Fig. lA). When mapped onto the surface of the protein, the identified residues from three discontinuous sequences (amino acids 405-407, 409-411 and 413 from the E-F helix of the Ce3 domain of IgE, amino acids 377-380 from the C-D loop, and residue 436 from the C-terminal region) form a contiguous surface representing the binding site on Ce3 for CD23 (Figure lB). This region is at the end of the Ce3 domain, near to the interface with Ce4, in contrast to the interaction site for FceRI, which is at the other end of Ce3 near the interface with Ce2 (Garman et al., 2000, Nature 406, 259-266; Holdom et al., 2011, Nat Struct Mol Biol 18, 571-576) (Fig. lB). The identification of this CD23 interaction site on IgE provides a structural explanation for the experimentally observed 2:1 (CD23:IgE) stoichiometry, as the dimeric IgE-Fc can bind to two separate CD23 lectin head domains. The two CD23 interactions were shown to have slightly different binding affinities and
thermodynamic characteristics, as was also observed for the FcaRI-IgA interaction. The two binding affinities imply an asymmetry of the two CD23 binding sites, which may possibly be allosterically induced. CD23's capacity for inducing a
conformational change in IgE is discussed further below.
In support of the NMR-based interaction site mapping, the inventors then analysed the binding characteristics of the IgE-CD23 interaction and performed extensive site- directed mutagenesis of residues at the binding interface. Surface plasmon resonance (SPR) based measurements of binding thermodynamics indicate an interaction with a strongly favorable entropic contribution (at 298K, AG = -32.2 (±0.5) kJ mol 1, ΔΗ =-5.0 (±0.3) kJ mol 1, -TAS= -27.2 kJ mol 1) (Figure 4A). Binding experiments performed over a range of different ionic strengths gave a measure of the contributions of electrostatic forces to the overall binding energies; a plot of log(KA) versus log(ionic strength) shows an unusually steep slope of -0.94 (Fig. 4B) (in comparison, the same analysis of the IgE-Fc-sFceRIa interaction gives a slope of -0.13). Both of these measurements are consistent with the IgE-CD23 binding event being strongly electrostatic in character.
Following the NMR mapping of interaction epitopes for both proteins, the inventors used site-directed mutagenesis to validate the interaction site in the context of the full IgE-Fc construct and to define the energetic contributions of individual residues. Ten mutants from derCD23 and eleven mutants from IgE-Fc (domains Ce2-4) were produced, purified and characterized; their binding affinities were measured using an SPR assay (Hibbert et al., 2005). Table 1 summarizes the results of the site-directed mutagenesis studies.
Table 1 - Effects of mutations on the IgE-CD2 interaction
IgE-Fc KD AAG derCD23 KD AG
mutation (μΜ) (kJ mol 1) mutation (μΜ) (kJ mol 1)
wildtype 2-3 - wildtype 2-3 -
D409A 26.3 +6.0 D227A 30.9 +6.4
E412A 24.2 +5-8 E257A 26.7 +6.1
R376A 19.7 +5-3 R224A 26.2 +6.0
K380A 13-3 +4-3 R188A 25-0 +5-9
K435A 5-0 +1.9 Y189A 15-6 +4.7
K352A 3-8 +1.2 K276A 10.9 +3-9
R351A 2.6 +0.3 L226A 6.6 +2.6
D347A 2-5 +0.2 D236A 5-8 +2.3
P439A 2-5 +0.2 D192A 4-3 +1.6
Q535A 2-5 +0.2 E265A 2-5 +0.2
Q538A 2-4 +0.1
Mutations on both proteins that affect binding are entirely consistent with the NMR- defined interaction sites. Charged residues have the largest energetic effect on binding. CD23 mutations D227A, E257A, R224A and R188A all show a change in binding free energy (AAG) of about +6 kJ mol 1 (Table 1). Uncharged residues also contribute to the binding energy; a prominently exposed tyrosine residue (Y189) in the center of CD23's IgE binding site makes a substantial contribution to binding energy. The CD23 binding surface on IgE is also predominantly electrostatic, with residues D409, E412, R376 and K380 showing the largest effects on CD23 binding energetics.
An earlier study had implicated IgE residue K352 from the A-B helix as part of the binding interface. However, the inventors' NMR perturbation experiments indicate that the A-B helix is near the site of interaction, but in the assay reported herein that directly measures binding affinity, a K352A mutation had only a very small effect on CD23 binding (see Table 1). The high-resolution crystal structure of the IgE-CD23 complex indicates that K352 is not part of the binding site. The inventors postulate that it is likely that the effects of the non-conservative K352E mutation previously observed resulted from local conformational changes rather than a direct effect from K352 on CD23 binding. Because the NMR data indicated a site on Ce3 very near to the Ce4 interface (Fig. lB) and because binding sites from several other
immunoglobulin-receptor interactions involve sites analogous to the Οε3-Οε4 interface, the inventors also made a pair of mutations in the F-G loop of the Ce4 domain, close to the CD23 binding site in Ce3. However, neither Q535A nor Q538A appear to affect CD23 binding, leading the inventors to believe that CD23's binding surface on IgE is largely restricted to residues from Ce3. Earlier studies indicated that soluble CD23 can compete with FceRI binding, and this was attributed to steric competition for an overlapping binding site within the Ce3 domain. However, the data described herein show that the CD23 and FceRI binding sites are spatially distinct and suggest that the mechanism of mutual inhibition must be allosteric in nature. The inventors performed a set of competitive binding assays to confirm this experimentally. Firstly, using an SPR assay, they showed that derCD23 can bind to IgE-Fc immobilized to an SPR chip but cannot bind to IgE-Fc captured by immobilized FceRIa (Fig. 2A,B); a positive control, a Fab fragment of the anti-IgE antibody 7.12, directed against the Ce2 domain, did bind to FceRIa-captured IgE-Fc (data not shown). Secondly, they showed that IgE-Fc can bind to immobilized derCD23, but an IgE-Fc-sFceRIa complex cannot bind to derCD23 (Fig. 2C,D). These data indicate that CD23 and FceRI interactions with IgE are mutually inhibitory. Finally, they also tested the ability of the receptors to compete for binding to IgE in a solution TR-FRET experiment (Selvin, 2002, Annu Rev Biophys Biomol Struct 31, 275-302). This assay can be performed under equilibrium binding conditions, allowing a different set of mechanistic properties to be tested than in the kinetic SPR experiments. Under equilibrium conditions, different inhibition patterns are observed for competitive and allosteric inhibitors. A competitive inhibitor affects the apparent binding affinity, with inhibitor I reducing the apparent affinity by a ratio of (i+[I]/Ki); whereas an allosteric inhibitor affects the apparent Bmax of the interaction without changing the apparent KD (Fersht, 1999, Structure and Mechanism in Protein Science. New York, W.H. Freeman.). Using FceRIa as the inhibitor of the IgE-Fc- derCD23 interaction results in a decrease of apparent Bmax without affecting the apparent KD of the interaction (Fig. 2E,F). These experiments confirm mutual inhibition by the two IgE receptors and offer experimental evidence that an allosteric mechanism is involved. Given the location of the CD23 binding site, the most obvious mechanism for allostery is a conformational change around the interface between the Ce3 and Ce4 domains. Crystal structures of IgE-Fc and IgE-Fc-FceRIa complexes indicate that the Ce3 domains can exist in "open" and "closed" states, with only an open state being capable of binding FceRI. A detailed study of the open and closed states concluded that it is the motions around the Ce3 A-B helix, sitting at the Ce3-C84 interface, that control the orientation of the two Ce3 domains.
Soluble trimeric CD23 has been shown to bind to and cross-link membrane IgE on B cells, resulting in B cell activation. However, it is essential that trimeric CD23 not cross-link IgE bound to FceRI on the surface of mast cells. If this were to occur then high levels of CD23 would result in mast cell activation in the absence of allergens. The data from binding experiments (Fig. 2B) predict that soluble CD23 cannot directly cross-link IgE bound to FceRI on mast cells. The inventors tested this prediction in a mast cell degranulation assay using the FceRI+ LAD-2 human mast cell line. In this assay, cells are first primed by adding IgE, followed by addition of potential cross-linking reagents and measurement of release of the mast cell granule- associated enzyme β-hexosaminidase. An anti-IgE antibody results in FceRI- mediated activation of the mast cell and robust β-hexosaminidase release, but the addition of either the monomeric derCD23 or a trimeric CD23 construct (triCD23) fails to induce mast cell degranulation (Fig. 3A). In contrast, trimeric CD23 effectively cross-links IgE on B cells, resulting in activation of these cells and increased production of soluble IgE (Fig. 3B). Immunoglobulins have evolved two separate sites for binding to receptors. One site is near the hinge region in IgG and at the Ce2-C83 interface in IgE, while the other is at the interface of the C-terminal domain and the penultimate domain: the Ce3-C84 interface in IgE. A mechanism of communication has evolved within the IgE molecule between these two distant sites to prevent simultaneous engagement of CD23 and FceRI. This may be a unique property of IgE. Since IgE and CD23 both exist in membrane bound and soluble forms, and soluble FceRIa has also recently been shown to exist at functionally relevant concentrations, there is considerable potential for receptor cross-regulation. Mutually exclusive receptor binding assures independent functions for IgE-FceRI and IgE-CD23 interactions. IgE is a clinically important drug target. An anti-IgE antibody (omalizumab) is an effective therapy, currently used in the treatment of moderate to severe asthma that is not controlled by corticosteroids. Omalizumab binds to the Ce3 domain of IgE and competitively inhibits FceRI binding, although its in vivo activity relies on more than just inhibition of this interaction. Results presented here demonstrate that IgE is amenable to allosteric inhibition, an approach that may have significant advantages over competitive inhibition, and lay the foundation for the development of allosteric modulators of IgE-receptor interactions. Summary
IgE, the antibody that mediates allergic responses, acts as part of a self-regulating protein network. Its unique effector functions are controlled through interactions of its Fc region with two cellular receptors, FceRI on mast cells and basophils and CD23 on B cells. IgE cross-linked by allergen triggers mast cell activation via FceRI, while IgE-CD23 interactions control IgE expression levels. The inventors have determined the CD23 binding site on IgE, using a combination of NMR chemical shift mapping and site-directed mutagenesis. They have shown that the CD23 and FceRI interaction sites are at opposite ends of the Ce3 domain of IgE, but that receptor binding is mutually inhibitory, mediated by an allosteric mechanism. This prevents CD23-mediated cross-linking of IgE bound to FceRI on mast cells and resulting antigen-independent anaphylaxis. The mutually inhibitory nature of receptor binding provides a degree of autonomy for the individual activities mediated by IgE- FceRI and IgE-CD23 interactions. It will be appreciated that by inhibiting binding between the Fc region on IgE and CD23 (which has been clearly demonstrated herein), one can be confident about inhibiting the downstream functional activity and avoiding CD23-mediated side effects, which are often experienced when using therapeutic IgE antibodies. Example 2
Materials and Methods
Protein expression and purification: Recombinant human derCD23 (Seri56 - GIU298) was expressed, refolded and dialyzed into 25 mM Tris-HCl pH 7.5
('purification buffer') as previously described (Hibbert, 2005, J. Exp. Med., 202, 751- 760). It was purified on a heparin-sepharose column (GE Healthcare) pre- equilibrated with purification buffer, eluting with 25 mM Tris-HCl pH 7.5, 200 mM NaCl. Fractions were pooled, concentrated to 1 ml and loaded onto a HiLoad 16/60 Superdex G75 column (GE Healthcare), pre-equilibrated and subsequently washed with purification buffer. Folding was assessed by iD-Ή NMR at 500 MHz (large dispersion and strong signals of methyl groups between 1.0 and -1.0 ppm). Human IgE-Fc (N265Q, N371Q) was expressed in NSo cells and purified by affinity chromatography with sFc8RIa-IgG4-Fc fusion protein as previously described (Shi, 1997, Biochemistry, 36, 2112-2122). The genes for recombinant human Fc£3~4 (Cys328-Lys547, with N-terminal ADP) and sFcsRIoc-Cys-His (Vali-Lysi76, with C- terminal cysteine and His6 tag) were synthesized by DNA2.0 and cloned as
Hindlll/EcoRI fragments into proprietary mammalian expression vectors. The DNAs were transiently transfected into HEK293 cells using 293-fectin (Life
Technologies) according to the manufacturers' instructions and the supernatants were harvested 6 days post transfection. Human Fcs3-4 was purified by cation exchange chromatography on a SPHP matrix (GE Healthcare) in 50 mM NaOAc buffer pH 6.0, followed by gel filtration on a Superdex S200 matrix (GE Healthcare) in PBS pH 7.4. Human sFcsRIoc-Cys-His was purified on a Ni-NTA column (Qiagen) followed by gel filtration on a Superdex S200 matrix (GE Healthcare) in PBS pH 7.4 and stored under nitrogen to prevent reactivity of the free cysteine residue.
Crystallization and data collection: Fce3-4 was concentrated to 20 mg/ml, and derCD23 to 18 mg/ml, in 25 mM Tris-HCl pH 7.5, 20mM NaCl, 0.05% sodium azide ('crystallization buffer'). The complex was formed with 0.4 mM derCD23 (6.2 mg/ml), 0.2 mM Fce3-4 (10 mg/ml) and 4 mM CaCl2, diluted with equal volume of 3% PEG 8,000, 0.1 M Tris-HCl pH 7.5 as the precipitant. Crystals grew to -400 μπι within 8 days at 295 K, were flash-cooled to 100K (using 14% PEG 8,000, 0.1 M Tris- HCl pH 7.5, 30% PEG 200) and data collected at beamlines I02 and I04, Diamond Light Source (Harwell, U.K.). FRET assay: Labeling of the proteins is described in SI Materials and Methods.
Inhibition assays were performed by competing 5 % of 1 μΜ terbium labeled derCD23 and 5 % of 5 μΜ Alexa Fluor 647 labeled IgE-Fc or Fce3-4 with a dilution series of unlabeled sFceRIa-Cys-His. Assays were conducted in 384 well hi-base, white plates (Greiner BioOne) using Lanthascreen buffer (Invitrogen) as a diluent. The plate was left to incubate for 1 hr at room temperature with shaking and read by the Artemis plate reader (Berthold Technologies). TR-FRET ratios were then calculated for each well as the emission of acceptor at 665 nm divided by the emission of donor at 620 nm multiplied by 104. Data were analyzed using GraphPad Prism 5. Similarly, 2 nM terbium labeled sFceRIa and 10 nM Alexa Fluor 647 labeled IgE-Fc or Fce3-4 were competed with a dilution series of unlabeled derCD23. Assays were conducted as described above with the exception of an overnight incubation. Positive controls for the two experiments were provided by displacement of labeled sFcsRIa by unlabeled sFcsRIa, and labeled derCD23 by unlabeled derCD23; these defined the start- and end-point values for each titration, which were treated as zero and 100% inhibition for calculation of the "% inhibition" values for displacement of one receptor by the other (Figs. 8a & b).
Crystallization: Crystals with a different morphology were also found after 6 days with 12% PEG 4,000, 0.1 M Tris-HCl pH 8.75, 0.2 M sodium acetate trihydrate as precipitant after streak seeding. Diffraction data were collected to 3.6A and the crystal form was found to be the same as that reported in Table 2.
Crystallographic data collection and structure determination: Indexing, integration and merging of data to 3.1A resolution was carried out with the HKL2000 suite of programs (Otwinowski, 1997, Methods Enzymol., 276, 307-326). Nine previously determined Fce3-4 dimers (Wurzburg & Jardetzky, 2009, J. Mol. Biol, 393, 176-190) were used as search models in molecular replacement. However, due to the high degree of conformational flexibility of the two Fce3-4 chains (Wurzburg & Jardetzky, 2009, J. Mol. Biol, 393, 176-190), fourteen additional dimers were manually generated from crystallographically independent chains and were also used as search models. Solutions were only found using the "closed" conformations of the Fce3-4 dimer. For example, using a dimer generated from chain A of PDB 3H9Z, two copies of the Fc83"4 dimer were identified in the asymmetric unit using PHASER (MacCoy, 2007, J. Appl. Crystallogr., 40, 658-674). Subsequently, four derCD23 (PDB 2H2R) molecules were located. A third derCD23-Fcs3-4 complex could only be found by searching with one of the previously located complexes.
Due to the high degree of NCS, reflections were selected for the Rfree set in thin resolution shells (Fabiola, 2006, Acta Crystallogr D, 62, 227-238) using SFTOOLS (Winn, 2011, Acta Crystallogr D, 67, 235-242). Iterative cycles of refinement using PHENIX (Adams, 2011, Methods, 55, 94-106), REFMAC5 (Murshudov, 2011, Acta Crystallogr D, 67, 355-367) and BUSTER-TNT (Bricogne, BUSTER-TNT, Global Phasing Ltd.) alternated with manual model building with COOT (Emsley, 2010, Acta Crystallogr D„ 66, 486-501). To minimize bias, the model was built into 2 0 - Fc composite omit, 2 0 - Fc and F0 - Fc electron density maps. Initially during refinement, tight NCS restraints were used. These were gradually relaxed, and finally Local Structure Similarity Restraints (LSSR or local NCS') (Murshudov, 2011, Acta Crystallogr D, 67, 355-367) were applied. TLS groups (Painter, 2008, Acta
Crystallogr D, 62, 439-450) were generated using the TLSMD web server (Painter, 2006, JAppl Crystallogr, 39, 109-111). Carbohydrate atoms and a single water molecule were subsequently incorporated into the structure. No electron density was observed for Fcs3-4 residues 363 - 364 of chain B; 367 - 371 and 421 -428 of chain C; 367 - 370, 419 - 422, 429 - 430, 479 - 480 and 517 - 519 of chain E; and derCD23 residues 256 - 257 of chains G, H, I, J and K. Therefore, these residues were not built into the model. Refinement statistics are shown in Table 2. Labeling of proteins for FRET assay: IgE-Fc and Fce3-4 were labeled with acceptor fluorophore by reacting 4 mg/ml protein in 100 mM sodium bicarbonate, 50 mM NaCl, pH 9.3, with a 5-fold molar excess of Alexa Fluor 647 succinimidyl ester (Invitrogen). After 1 hr incubation at room temperature with agitation, excess unreacted fluorophore was removed by dialyzing into HBS (10 mM Hepes, 150 mM NaCl, 4mM CaCl2, pH 7.4). derCD23 and the sFceRIa-Cys-His mutant were labeled with donor fluorophore. derCD23 at 3.5 mg/ml in 100 mM Hepes, 125 mM NaCl, pH 8.3 was reacted with a 5-fold molar excess of terbium chelate isothiocyanate
(Invitrogen) for a 3 hr incubation at room temperature with agitation. Excess fluorophore was removed by dialyzing into HBS. sFceRIa-Cys-His at 5 mg/ml in PBS (20 mM phosphate buffer saline, pH 7.4) was reacted with a 10-fold molar excess of terbium chelate maleimide (Invitrogen) overnight at 4°C via thiol coupling.
Results
Overall topology of the complex
The structure of the complex was determined at 3.1A resolution and reveals one derCD23 molecule bound to each heavy chain at the interface between Cs3 and Cs4, making contact with residues from both domains (Fig. 5 and Table 2; Fig. 9 shows the electron density map at the interface). Table 2 - Crystallographic data collection and refinement statistics
Data processing statistics
Beamline I04, Diamond Light Source Wavelength (A) 0.9763
Space group 2A2!
Unit cell parameters (A) a = 62.89, b = 110.75, c = 376 Resolution range (A)* 50.0 - 3.10 (3.21 - 3.10) Observations 162,066
Unique reflections 48,290
Average redundancy 3.6 (4.0)
Completeness (%) 92.8 (94-7)
Wilson B factor (A2) 117
I/oiX) 14-5 (1-68)
Emerge 0.086 (0.588)
Refinement statistics
Resolution range (A) 29.9 - 3.1
Total No. of reflections 48,257
No. of working reflections 45,820
No. of test reflections 2,437
R factor* 0.2649
^working 0.2640
Rir e 0.2831
No. of atoms
Protein 16,107
Others§ 389
R.m.s. bond-length deviation (A) 0.041
R.m.s. bond-angle deviation (°) 1.29
Mean B factor (A2) 111
Main chain 107
Side Chain 115
Others§ 131
R.m.s. backbone B factor deviation11 2.6
Ramachandran statistics (%)e
Favored 92.9
Allowed 100
Outliers o * Values in parentheses are for the outer resolution shell.
† Emerge =∑|/o s" <I> \/∑<I>
*R factor =∑m\ \F0(hkV)\ - \Fc(hkl)\ |/∑m\F0(hkl)\
§ Carbohydrate and one water molecule.
^R.m.s. deviation between B factors for bonded main-chain atoms.
e As defined by MolProbity (Chen, 2010, Acta Crystallogr D 66: 12-21). .
The crystal form contained three independent copies of the complex in the asymmetric unit and the six heavy chains (labeled A to F) bound to six derCD23 heads (labeled G to L) showed essentially identical modes of interaction, exemplified by the structure depicted (Fig. 5; chains A, B, G & H). The angle between the Cs3 and Cs4 domains varies only slightly between the six heavy chains (Fig. 10a) and there are virtually no differences between the six derCD23 head domains (Fig. 10b). All three complexes thus display approximate two-fold symmetry, although one (consisting of chains C, D, I & J) includes the two "extremes" in terms of the angle between the Cs3 and Cs4 domains, but these differ by only 7 degrees. On all six heavy chains, electron density was present for five N-linked sugar units at Asn394 ([N- acetylglucosamine]2[Mannose]3; Fig. 5), and an additional two mannose residues were visible on chain C.
Flexibility in the Cs3-Cs4 inter-domain angle has been well-documented through studies of unliganded Fcs3-4 structures in different crystal forms, as well as the unliganded IgE-Fc structure and the sFcsRIoc complexes with both Fcs3-4 and IgE- Fc. Amongst all of these structures the inter-domain angle varies over a range of approximately 25 degrees between the most "open" (in the sFcsRIoc complexes) and the most "closed" (chain D in the derCD23 complex reported here) (Fig. na-d).
Previously, the most closed conformation had been seen in one of the unliganded Fcs3"4 structures (chain D of PDB 3HA0), and the conformations seen in the derCD23 complex all range within 3 or 4 degrees of this closed conformation (Fig. lid).
Nature of the IgE/CD23 interface
The 2:1 stoichiometry agrees with earlier measurements in solution by analytical ultracentrifugation, recorded for both Fcs3-4 and IgE-Fc, but direct contact with Cs4 was not anticipated by previous mutagenesis or peptide inhibition studies. Three salt bridges between Fcs3-4 and derCD23 (Asp409-Argi88; Glu4i2-Argi88; GIU412- Arg224) and a potential fourth (Glu4i4-Hisi86 if protonated), together with four additional hydrogen bonds (Arg376-Tyri89; Asp409-Tyri89; Arg440-Ser254, both side-chain and main-chain) dominate the interaction (Fig. 6 and Fig. 9). In the four salt bridges, all negative charges reside on IgE, all positive charges on CD23. Arg440, while H-bonding to Ser254 of derCD23, retains its intra-chain salt bridge interaction with GIU529 of Cs4 that is seen in other, unliganded Fcs3-4 structures. These salt bridging and hydrogen bonding residues of IgE are not conserved in the other human antibody classes (Fig. 12), consistent with the specificity of CD23 for IgE.
Remarkably few additional residues make well-defined van der Waals contacts in all six interfaces (Ile4ii, Gly4i3, Pro439, GIU529, Gln535 in Fcs3-4; Trpi84, Vali85, Asp227 in derCD23) and the surface complementarity is poor (calculated Sc values for the six interfaces range from 0.63 to 0.71); indeed, there is a substantial cavity at the interface between GIU412 (in Fcs3-4), Arg224 and the Cys259-Cys273 disulphide bridge (in derCD23). The buried surface area for each interaction ranges from 860 to 890A2 for all except one (chains F & L) at 920A2; in this latter case the derCD23 loop at residues Ser256 and GIU257 is not as disordered as it is in the other five
independent views of the interaction. The Cs3 domain dominates the interface with 63% of the contact area, a further 25% involves the linker region (residues 437 to
440) and Cs4 contributes 12%. All of the salt-bridge and hydrogen bond interactions involve the Cs3 domain with the exception of Arg440 in the linker region, while Cs4 contributes only van der Waals interactions. Earlier studies had implicated the AB loop of Cs3, and residue Lys352 in particular, in CD23 binding, but this loop is not directly involved at all and Lys352 makes no contact with CD23; any effects of mutagenesis in this loop must therefore be indirect. It is the EF loop/helix of the Cs3 domain that is centrally placed at the interface (Fig. 6). Surprisingly, no Ca2+ ions were found bound to derCD23 in the complex; the role of Ca2+ in the CD23-IgE interaction is discussed below.
Conformational change upon CD2 binding to IgE-Fc
In the free state, IgE-Fc adopts an asymmetrically bent structure with the (Cs2)2 domain pair folded back against one of the Cs3 domains, but although the two Cs3- Cs4 pairs have different inter-domain angles, both are more open than that seen in the derCD23-Fcs3-4 complex (Fig. 11a). To assess whether the presence of the Cs2 domains might be expected to affect derCD23 binding, the (Cs2)2 domain pair was modeled onto the derCD23-Fcs3-4 complex. This was achieved by superimposing the Cs3 domain of free IgE-Fc that contacts the (Cs2)2 domain pair, onto the Cs3 domain (chain A) of the derCD23-Fcs3-4 complex. One of the derCD23 molecules (chain G) lay very close to the Cs2 domains, with a minor rearrangement of the N-terminal residues of Cs2 required to prevent a steric clash. Contact between side chains of this derCD23 molecule and Cs2 in a complex with IgE-Fc (or indeed IgE) certainly cannot be ruled out; the other bound derCD23 molecule (chain H) however, lay far from the Cs2 domains. Modeling of the Cs2 domains onto the Fcs3-4 complex also revealed that the AB loop of Cs2 (chain A) would have to adopt a slightly different
conformation to avoid clashing with Cs3 (chain B) in the very closed conformation that this Cs3 domain adopts in the complex.
Mutual exclusion of FcsRI and CD23 binding to IgE-Fc
The closed conformation for the Cs3 domains in the derCD23 complex is clearly incompatible with FcsRI binding. This may be seen by superimposing the derCD23- Fcs3"4 and the sFcsRIoc-Fcs3-4 (Garman, 2000, Nature, 406:259-266) complexes on their (Cs4)2 domain pair, and noting the very different orientations of the Cs3 domains (Fig. 7a). The steric clashing of sFcsRIa with the Cs3 domains in their CD23-bound conformation, and of both derCD23 molecules with the Cs3 and Cs4 domains in their sFcsRIoc-bound conformations, is clear from the superposition of the complexes upon one or other of the Cs3 domains (Figs. 7b & c). Thus IgE cannot bind both FcsRI and CD23 simultaneously: binding of sFcsRIa causes
conformational changes that are incompatible with binding of either derCD23 molecule, and the binding of either derCD23 molecule ensures that the two sub-sites required for high-affinity binding of FcsRI are not both accessible.
This surprising conclusion was verified experimentally in solution using acceptor fluorophore-labeled IgE-Fc or Fcs3-4, and competitively displacing either donor fluorophore-labeled derCD23 by unlabeled sFcsRIa, or labeled sFcsRIa by unlabeled derCD23 (Fig. 8). It can be seen that sFcsRIa readily and completely displaces derCD23 from the complex with either IgE-Fc or Fcs3-4. Similarly, derCD23 can displace sFcsRIa almost completely from either IgE-Fc or Fcs3-4, but at very much higher concentrations as expected from the considerably lower binding affinity of derCD23. Positive controls for these FRET measurements were provided by displacement of labeled sFcsRIoc by unlabeled sFcsRIoc, and labeled derCD23 by unlabeled derCD23. Discussion
The interaction between IgE and CD23 is critically involved in the allergic response at several stages, including allergen presentation, the regulation of IgE synthesis and transport of IgE and immune complexes across epithelial barriers in the gut and airways. At the cell surface, 1T1CD23 is trimeric, and SCD23 fragments shed from the membrane that contain sufficient stalk region are also trimeric, although the structure of the trimer has only been modeled based either upon the structures of other C-type lectins or guided by NMR chemical shift data. In the crystal structure of the complex reported here, two derCD23 "heads" bind to IgE, one to each heavy- chain at a location between the Cs3 and Cs4 domains, and remote from the FcsRI binding site. The interaction is predominantly hydrophilic and dominated by salt bridges between positively charged CD23 residues and negatively charged IgE residues, despite the overall net positive charge (+9) of the Cs3 domain. The site on CD23, in agreement with that identified by NMR chemical shift mapping by titration of 15N-labeled derCD23 with monomeric Cs3 (titration with IgE-Fc led to the formation of high molecular weight oligomers), is diametrically opposed to the connection to the oc-helical coiled-coil stalk region (Fig. 5). This topology is such that an IgE molecule could not engage two heads from the same (modeled) CD23 trimer (as depicted in earlier cartoons), but could readily cross-link two mCD23 molecules. (The distance of 136A between the Ca atoms of the N-terminal Phei58 residues of the two derCD23 molecules, which are immediately adjacent to the top of the predicted oc-helical coiled-coil stalk region, is such that it would require unraveling of about 30 residues, or almost one third of the stalk, to allow two heads from the same CD23 trimer to bind to the same IgE-Fc). Furthermore, the exposed location of the IgE- binding site on derCD23 strongly suggests that trimeric SCD23 could cross-link IgE molecules either in solution or as mlgE on the cell surface, but a definitive statement must await determination of the structure of the CD23 trimer.
The binding of derCD23 in the cleft between the Cs3 and Cs4 domains fixes the angle between these two domains. Comparisons between Fcs3-4 and IgE-Fc structures (the latter including Cs2 domains), both free and bound to sFcsRIoc, show that while the (Cs4)2 domain pair display no conformational variation and provide a fixed point of reference, the Cs3 domains can adopt a range of "open" and "closed"
conformations. The most open of these have been found in the complexes of Fcs3-4 and IgE-Fc with sFcsRIoc (Fig na-d) and occur in both ε-chains since this receptor engages with both Cs3 domains (Fig. 7a). In contrast, CD23 binding causes the Cs3 domains to adopt the most closed conformation that has yet been observed in any Fcs3"4 or IgE-Fc structure. This opening and closing of the Cs3 domains in fact results from a flexing within the Cs3 domain, an effect first noted in earlier structural comparisons. The AB loop/helix of Cs3 interacts closely with Cs4 (Fig. 6), and a change in the relative orientation of the EF loop/helix, which is contacted by derCD23 (Fig. 6), can clearly be seen when the FcsRI and CD23 complexes are compared (Fig. 7a). This intra-domain flexibility may be a result of the unique packing/stability profile of Cs3 compared with all other CH domains, and in fact the isolated Cs3 domain displays molten globule-like characteristics. The consequence of these extreme open (FcsRI-bound) and closed (CD23-bound) conformations for the Cs3 domains of IgE is that the binding of the two receptors are incompatible with each other (Figs. 7a-c). Clearly this is not, as previously thought, due to overlapping binding sites, but to a surprising allosteric linkage between the two distant sites. The idea that the open and closed conformations might interact differently with FcsRI and CD23 has been previously considered following a surprising discovery that CD23 bound more strongly to IgE at 4°C than 37°C, while the opposite was true for FcsRI. It was hypothesised that a change in the relative proportion of these open and closed conformational states might account for this temperature effect, and it will certainly be revealing to explore further the
temperature dependence of the kinetics and thermodynamics of these receptor interactions as well as the conformational dynamics of the IgE molecule, particularly with respect to the Cs3 domains.
IgE-Fc and IgE additionally contain the Cs2 domains, and it is important to consider their effect upon the crystallographic results presented here for Fcs3-4. In IgE-Fc the (Cs2)2 domain pair packs asymmetrically against one of the Cs3 domains in the bent IgE-Fc structure, and moves together with that Cs3 domain when it opens up to accommodate sFcsRIoc binding. Modeling the (Cs2)2 domains onto the derCD23- Fcs3"4 complex shows that although there is no steric conflict that would prevent binding of either derCD23 molecule, the Cs2 domains lie immediately adjacent to one of the derCD23 molecules and suggest that an interaction (stabilizing or
destabilizing) might occur. This is consistent with the observed 2:1 stoichiometry for derCD23 binding to both Fcs3-4 and IgE-Fc. Although there is no evidence of any significant difference between the binding affinity of derCD23 for Fcs3-4, IgE-Fc or IgE, or between the binding of the two derCD23 molecules to IgE-Fc, a slightly faster on-rate for derCD23 binding to Fcs3-4 compared with both IgE-Fc and IgE has been reported. The biphasicity or dual affinity of CD23 binding to IgE that has been reported is a function of its oligomeric state and ability to engage IgE through more than one head, although as pointed out above, two heads of one CD23 almost certainly cannot engage the same IgE molecule and the avidity effect must result from trimeric CD23 binding to more than one IgE molecule.
The structural comparison of the derCD23 and sFcsRIa complexes (Figs. 7b & c) also shows that sFcsRIa binding prevents binding of both derCD23 molecules, and similarly that binding of either derCD23 molecule will prevent sFcsRIa binding. The experimental data presented here (Fig. 8) confirm this observation. sFcsRIa readily displaces derCD23 and, as expected, at approximately equimolar (5μΜ)
concentrations of sFcsRIa and IgE-Fc (or Fcs3-4) the displacement of derCD23 is virtually complete since this concentration of sFcsRIa is very much greater than the KD ~ 1 nM for sFcsRIa binding. (The difference between the two curves in Fig.8a may reflect the slightly lower affinity reported for sFcsRIa binding to Fcs3-4 compared with IgE-Fc). derCD23 can similarly cause almost complete displacement of sFcsRIa, although much higher concentrations are required, greater than ιοομΜ, in order to overcome its lower affinity (KD ~ io~5 - io~6 M).
CD23 belongs to the C-type (calcium-dependent) lectin superfamily and the presence of Ca2+ is known to enhance the affinity for IgE approximately 7-fold, although it is not essential for binding. A crystal structure of the head domain with a single bound Ca2+ ion has been solved, together with the Ca2+-free form, and an NMR structure also reports Ca2+ binding, but at an alternative site. However, no Ca2+ ions were observed in any of the six derCD23 molecules in the Fcs3-4 complex, despite the presence of 2 mM Ca2+ in the crystallization medium. In this regard, the existence of a disordered loop in derCD23 at the edge of the interface (residues 256-257 of loop 4, following earlier terminology) is intriguing. The crystal structure of the lectin head domain showed Ca2+ bound at a site involving residues GIU249 and Thr25i of loop 4. In that structure, residues 253-257 of loop 4 were also disordered, paradoxically becoming ordered in the absence of Ca2+ due to a rearrangement of the side-chain of Arg253, which occupied the Ca2+ site. The conformation of loop 4 seen in the complex differs from either the apo- or Ca2+-bound structures; together with the adjacent loop 1, they represent the only main-chain conformational changes in derCD23 upon Fcs3"4 binding. The role of Ca2+ in IgE binding to CD23 thus remains unresolved at present, but it may involve reorganization of residues of the flexible loop 4 in the context of the complex, providing additional protein-protein interactions and thus enhancing affinity. There are precedents for the stabilizing effects of Ca2+ upon this and other loops in C-type lectin domains, such as mannose-binding protein in which carbohydrate binding is affected, and others where the binding of a protein partner is affected.
What is the functional significance of IgE's inability to engage both FcsRI and CD23 simultaneously? Although the affinity of derCD23 for IgE is considerably lower than that of FcsRI, avidity effects for trimeric CD23 can considerably enhance its ability to compete with FcsRI binding. Indeed, a recombinant SCD23 species trimerized via a leucine-zipper motif, termed ZZCD23, more effectively inhibited IgE binding to mast cells expressing FcsRI. The mutual exclusion of FcsRI and CD23 binding to IgE is essential to prevent mast cell and basophil activation by trimeric SCD23, which could otherwise cross-link FcsRI-bound IgE on these cells. Similarly, it ensures that FcsRI cannot be cross-linked either by soluble IgE-CD23 complexes, or IgE bound to 1T1CD23 (on B cells or APCs) in the absence of allergen. The mutual exclusion of FcsRI and CD23 binding is thus an important aspect of IgE biology, allowing it to function independently through its two receptors.
Furthermore, the location of the CD23 binding sites at points where the two heavy chains are most widely separated (in contrast to the FcsRI binding site where the chains approach each other most closely) and the connections to the stalk region (Fig. 5), maximize the propensity for cross-linking of mlgE on B cells committed to IgE synthesis by trimeric SCD23, and also mCD23 on B cells by IgE or IgE-allergen complexes. The inventors have hypothesized that through such interactions, the former leading to up-regulation of IgE synthesis and the latter to down-regulation, CD23 contributes to the mechanism of IgE homeostasis, and this notion has received experimental support from studies with monomelic and oligomeric SCD23 species. The structure of the complex is also consistent with the co-crosslinking of mCD2i and mlgE by trimeric SCD23 (proposed to enhance IgE up-regulation), since CD21 binds to the "tail" sequence that is, although only partially present in derCD23, located adjacent to the connection to the stalk [Fig. 5;]. However, the precise spatial
arrangement of the IgE and CD21 binding sites on each derCD23 head and the
relative positions of the heads in the CD23 trimer (as yet only modeled), together with the fact that two heads from one trimer almost certainly cannot engage a single IgE molecule, undoubtedly places topological constraints upon the formation of
signaling complexes of these three molecules at the B cell surface. The crystal structure of the derCD23-Fcs3-4 complex reported here provides the first structural basis for designing inhibitors with the potential to interfere with IgE
regulation and other CD23-mediated processes. Of wider significance to IgE and allergic disease, the structure also reveals the mechanism for an allosteric connection between the distantly located binding sites for IgE's two principal receptors, and demonstrates that allosteric inhibition is a viable strategy to target either of these receptor interactions for therapeutic purposes.
Summary
The role of immunoglobulin E (IgE) in allergic disease mechanisms is performed principally through its interactions with two receptors, FcsRI on mast cells and basophils, and CD23 (FcsRII) on B cells. The former mediates allergic hypersensitivity, the latter regulates IgE levels, and both receptors, also expressed on antigen presenting cells, contribute to allergen uptake and presentation to the immune system. The inventors have solved the crystal structure of the soluble lectin-like "head" domain of CD23 (derCD23) bound to a sub- fragment of IgE-Fc consisting of the dimer of Cs3 and Cs4 domains (Fcs3-4). One CD23 head binds to each heavy chain at the interface between the two domains, explaining the known 2:1 stoichiometry and suggesting mechanisms for cross-linking membrane-bound trimeric CD23 by IgE, or membrane IgE by soluble trimeric forms of CD23, both of which may contribute to the regulation of IgE synthesis by B cells. The two symmetrically located binding sites are distant from the single FcsRI binding site, which lies at the opposite ends of the Cs3 domains. Structural comparisons with both free IgE-Fc and its FcsRI complex reveal not only that the conformational changes in IgE-Fc required for CD23 binding are incompatible with FcsRI binding, but also that the converse is true. The two binding sites are allosterically linked. The inventors demonstrate experimentally the reciprocal inhibition of CD23 and FcsRI binding in solution, and suggest that the mutual exclusion of receptor binding allows IgE to function independently through its two receptors.

Claims

Claims
1. A variant IgE Fc region comprising at least one amino acid modification in the Ce3 domain relative to a wild-type IgE Fc region, wherein the at least one amino acid in the wild-type IgE Fc region which is modified is part of a CD23 binding site, and wherein the variant IgE Fc region exhibits a reduced binding affinity to CD23 compared to the wild-type IgE Fc region.
2. A variant IgE Fc region according to claim 1, wherein the variant IgE Fc region exhibits ablated binding to CD23 (FceRII), but is capable of binding to FceRI.
3. A variant IgE Fc region according to either claim 1 or 2, wherein the variant IgE Fc region is derived from the wild-type human IgE Fc comprising an amino acid sequence substantially as set out in SEQ ID No: 2, or a functional variant or fragment thereof.
4. A variant IgE Fc region according to any preceding claim, wherein the variant IgE Fc region is derived from the wild-type human IgE Fc region which is encoded by a nucleic acid sequence substantially as set out in SEQ ID No:i, or a functional variant or fragment thereof.
5. A variant IgE Fc region according to any preceding claim, wherein the at least one amino acid which is modified is any one of amino acid residues 329-441 of SEQ ID No:2.
6. A variant IgE Fc region according to any preceding claim, wherein the at least one amino acid modification in the variant IgE Fc region is not in the A-B helix of the Ce3 domain of IgE.
7. A variant IgE Fc region according to any preceding claim, wherein the variant IgE Fc region comprises at least one amino acid modification in the E-F helix of the
Ce3 domain of IgE, in the C-D loop of the Ce3 domain of IgE and/or in the C-terminal region of the Ce3 domain of IgE.
8. A variant IgE Fc region according to any preceding claim, wherein the variant IgE Fc region comprises at least one amino acid modification at a position
corresponding to wild-type IgE Fc region selected from the group consisting of: (i) amino acids 404-415 from the E-F helix of IgE; (ii) amino acids 376-380 from the C- D loop of IgE; and (iii) residue 433-441 from the C-terminal region of IgE.
9. A variant IgE Fc region according to any preceding claim, wherein the variant IgE region comprises at least one amino acid modification at a position
corresponding to wild-type IgE Fc region selected from the group consisting of: 376; 377; 378; 379; 380; 404; 405; 406; 407; 408; 409; 410; 411; 412; 413; 414; 415; 433; 434; 435; 436; 437; 438; 439; 440; and 441 of SEQ ID No:2, and preferably at position 376; 378; 381; 408; 409; 411; 412; 435; 439; and/or 440 of SEQ ID No:2.
10. A variant IgE Fc region according to any preceding claim, wherein the variant IgE region comprises at least one amino acid modification at a position equivalent to 409, 412, 376, 380 and/or 435 of the wild-type human IgE Fc region.
11. A variant IgE Fc region according to any preceding claim, wherein the at least one modification comprises an amino acid substitution.
12. A variant IgE Fc region according to any preceding claim, wherein the at least one modification comprises a non-conservative substitution.
13. A variant IgE Fc region according to any preceding claim, wherein the variant Fc region comprises at least one amino acid modification selected from: D409A; E412A; R376A; K380A and K435A.
14. A variant IgE Fc region according to any preceding claim, wherein the variant human IgE Fc region comprises more than one modification in the Ce3 domain of the Fc region equivalent to the human IgE Fc region of a wild-type antibody.
15. A variant IgE Fc region according to any preceding claim, wherein the variant human IgE Fc region comprises at least two, three, four or five modifications at any of the amino acids at positions equivalent to 376; 377; 378; 379; 380; 404; 405; 406; 407; 408; 409; 410; 411; 412; 413; 414; 415; 433; 434; 435; 436; 437; 438; 439; 440; and 441 of wild-type human IgE-Fc.
16. A variant IgE Fc region according to any preceding claim, wherein the variant Fc region comprises a modification at each of the amino acids at positions equivalent to 409, 412, 376, 380 and/or 435 of wild-type human IgE-Fc.
17. A variant IgE Fc region according to any preceding claim, wherein the variant IgE Fc region comprises an amino acid sequence substantially as set out in any one of SEQ ID No: 3-7, or a functional variant or fragment thereof.
18. A variant IgE Fc region according to any preceding claim, wherein the equilibrium constant of dissociation (KD or Kd) of the variant IgE Fc region for CD23 is at least 5μΜ, ιθμΜ, 25μΜ or greater.
19. A variant IgE Fc region according to any preceding claim, wherein the variant IgE Fc region is capable of binding to FceRI with a KA value of at least io? M 1.
20. A nucleic acid encoding the variant IgE Fc region according to any one of claims 1-19.
21. A nucleic acid according to claim 20, wherein the nucleic acid comprises a nucleotide sequence substantially as set out in any one of SEQ ID No: 8-12, or a functional variant or fragment thereof.
22. A genetic construct comprising the nucleic acid according to either claim 20 or claim 21.
23. A recombinant vector comprising the genetic construct according to claim 22.
24. A host cell comprising the genetic construct according to either claim 20 or claim 21, or the recombinant vector according to claim 23.
25. A method of preparing a variant IgE Fc region, the method comprising- (i) culturing at least one cell according to claim 24 under conditions suitable for the expression of a variant IgE Fc region; and (ii) isolating the variant IgE Fc region.
26. A method of producing a variant IgE Fc region which exhibits a reduced binding affinity for CD23, the method comprising modifying at least one amino acid in the Ce3 domain of a wild-type IgE Fc region, wherein the at least one amino acid in the wild-type IgE Fc region which is modified is part of the CD23 binding site, and wherein the variant IgE Fc region exhibits a reduced binding affinity to CD23 compared to the wild-type IgE Fc region.
27. A method according to claim 26, wherein the variant IgE Fc region produced by the method is as defined in any one of claims 1-19.
28. A method according to either claim 25 or claim 26, wherein the wild-type IgE Fc region which is modified is part of a therapeutically active IgE antibody.
29. A method according to either claim 25 or claim 26, wherein the method comprises initially creating the variant IgE Fc region and then fusing it to a functional fragment of an IgE antibody to produce an Fc fusion, for example a chimera.
30. An antibody or a functional fragment thereof comprising the variant IgE Fc region of any one of claims 1-19.
31. An antibody or a functional fragment thereof according to claim 30, wherein the antibody or functional fragment thereof is incapable of binding to CD23 (FceRII), or exhibits a reduced binding affinity thereto, but is capable of binding to FceRI.
32. An antibody or a functional fragment thereof according to either claim 30 or claim 31, wherein the antibody or functional fragment thereof is monoclonal or polyclonal.
33. An antibody or a functional fragment thereof according to any one of claims 30-32, wherein the functional fragment comprises any of the fragments selected from a group consisting of VH, VL, Fd, Fv, Fab, Fab', scFv, F (ab and Fc fragment.
34. A variant IgE Fc region according to any one of claims 1-19, or an antibody or a functional fragment thereof according to any one of claims 30-33, for use in therapy or diagnosis.
35. A variant IgE Fc region according to any one of claims 1-19, or an antibody or a functional fragment thereof according to any one of claims 30-33, for use in treating, preventing or ameliorating a CD23-mediated side effect.
36. A variant IgE Fc region, or an antibody or a functional fragment thereof according to claim 35, wherein the CD23-mediated side effect is prevented in IgE antibody treatment or therapy.
37. A variant IgE Fc region, or an antibody or a functional fragment thereof according to either claim 35 or 36, wherein the CD23-mediated side effect is prevented during the treatment of cancer, autoimmune disease or infectious disease.
38. A variant IgE Fc region, or an antibody or a functional fragment thereof according to any one of claims 35-37, wherein the CD23-mediated side effect is
CD23-dependent epitope spreading.
39. A pharmaceutical composition comprising a variant IgE Fc region according to any one of claims 1-19, or an antibody or a functional fragment thereof according to any one of claims 30-33; and optionally a pharmaceutically acceptable vehicle.
40. A process for making the composition according to claim 39, the process comprising combining a therapeutically effective amount of a variant IgE Fc region according to any one of claims 1-19, or an antibody or a functional fragment thereof according to any one of claims 30-33, with a pharmaceutically acceptable vehicle.
PCT/GB2013/051804 2012-07-09 2013-07-09 Variant ige fc regions WO2014009707A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1212185.1 2012-07-09
GB1212185.1A GB2503883A (en) 2012-07-09 2012-07-09 Variant IgE with reduced affinity to CD23

Publications (1)

Publication Number Publication Date
WO2014009707A1 true WO2014009707A1 (en) 2014-01-16

Family

ID=46766371

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2013/051804 WO2014009707A1 (en) 2012-07-09 2013-07-09 Variant ige fc regions

Country Status (2)

Country Link
GB (1) GB2503883A (en)
WO (1) WO2014009707A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10947295B2 (en) 2017-08-22 2021-03-16 Sanabio, Llc Heterodimers of soluble interferon receptors and uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993004173A1 (en) * 1991-08-14 1993-03-04 Genentech, Inc. Immunoglobulin variants for specific fc epsilon receptors

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004013158A2 (en) * 2002-08-01 2004-02-12 Northwestern University MUTANTS OF IgE PROTEINS AND USES THEREOF

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993004173A1 (en) * 1991-08-14 1993-03-04 Genentech, Inc. Immunoglobulin variants for specific fc epsilon receptors

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10947295B2 (en) 2017-08-22 2021-03-16 Sanabio, Llc Heterodimers of soluble interferon receptors and uses thereof

Also Published As

Publication number Publication date
GB2503883A (en) 2014-01-15
GB201212185D0 (en) 2012-08-22

Similar Documents

Publication Publication Date Title
AU2021218004B2 (en) Constructs having a SIRP-alpha domain or variant thereof
US20210388329A1 (en) Sirp-alpha variant constructs and uses thereof
KR20200119833A (en) Antibodies that bind to GPRC5D
TWI821197B (en) Engineered transferrin receptor binding polypeptides
KR20220028035A (en) Antibodies that bind to GPRC5D
AU2021215151B2 (en) SIRP-alpha variant constructs and uses thereof
JP2022543551A (en) Antibody that binds to GPRC5D
KR20230025673A (en) Antibodies that bind to CD3 and FolR1
US20240002484A1 (en) ANTIBODIES TO HUMAN COMPLEMENT FACTOR C2b AND METHODS OF USE
CA3084105A1 (en) Antibodies binding to hla-a2/wt1
WO2014009707A1 (en) Variant ige fc regions
KR20220092859A (en) Anti-CXCR2 antibodies and uses thereof
US11739142B2 (en) Bispecific anti-CCL2 antibodies
US20240018240A1 (en) Antibodies binding to cd3 and plap
KR20240021859A (en) Bispecific anti-CCL2 antibody
NZ721147B2 (en) Bispecific antigen binding molecules

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13739247

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13739247

Country of ref document: EP

Kind code of ref document: A1