EP1214429A1 - Modulierung der ige-rezeptorexpression an der zelloberfläche - Google Patents

Modulierung der ige-rezeptorexpression an der zelloberfläche

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Publication number
EP1214429A1
EP1214429A1 EP00968378A EP00968378A EP1214429A1 EP 1214429 A1 EP1214429 A1 EP 1214429A1 EP 00968378 A EP00968378 A EP 00968378A EP 00968378 A EP00968378 A EP 00968378A EP 1214429 A1 EP1214429 A1 EP 1214429A1
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EP
European Patent Office
Prior art keywords
expression
cell
fcεriβ
chain variant
fcεri
Prior art date
Legal status (The legal status 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 status listed.)
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Application number
EP00968378A
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English (en)
French (fr)
Inventor
Jean-Pierre Kinet
Emmanuel Donnadieu
Marie-Helene Jouvin
William Cookson
Miriam Fleur Moffatt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oxford University Innovation Ltd
Beth Israel Deaconess Medical Center Inc
Original Assignee
Oxford University Innovation Ltd
Beth Israel Deaconess Medical Center Inc
Beth Israel Hospital Association
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Application filed by Oxford University Innovation Ltd, Beth Israel Deaconess Medical Center Inc, Beth Israel Hospital Association filed Critical Oxford University Innovation Ltd
Publication of EP1214429A1 publication Critical patent/EP1214429A1/de
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to methods and related compositions for modulating cell surface expression of the high affinity receptor for immunoglobulin E, the Fc ⁇ RI receptor.
  • the invention also relates to methods and related compositions for the treatment and/or prevention of conditions mediated by IgE such as allergic conditions.
  • Fc ⁇ RI complex is the high affinity cell surface receptor for the Fc region of antigen specific immunoglobulin E (IgE) molecules.
  • IgE immunoglobulin E
  • Fc ⁇ RI is multimeric and is a member of a family of related antigen/Fc receptors which have conserved structural features and which exhibit similar functional activities in initiating intracellular signaling cascades.
  • Fc ⁇ RI controls the activation of mast cells and basophils, and participates in IgE mediated antigen presentation.
  • Multivalent antigens bind and crosslink IgE molecules held at the cell surface by Fc ⁇ RI.
  • Receptor aggregation induces multiple signaling pathways that control diverse effector responses, including secretion of allergic mediators and the induction of cytokine gene transcription (such as IL-4, IL-6, TNF ⁇ and GM-CSF).
  • Fc ⁇ RI therefore is central to the induction and maintenance of an allergic response and physiologically may confer protection in parasitic infections.
  • Fc ⁇ RI farnesoid receptor
  • the receptor is composed of three distinct polypeptides.
  • the ⁇ chain (Fc ⁇ RI ⁇ ) binds the Fc portion of IgE with high affinity, and the ⁇ chain (Fc ⁇ RI ⁇ ) has four transmembrane domains between amino- and carboxyl-terminal cytoplasmic tails.
  • a homodimer of two disulfide linked ⁇ chains (Fc ⁇ RI ⁇ ) completes the tetrameric structure. In humans, the tetrameric structure is not obligatory, and an alternate ⁇ 2 trimer is present.
  • the ⁇ chain contains two immunoglobulin type domains, Dl and D2, that mediate binding to IgE.
  • the ⁇ and ⁇ chains contain conserved Immunoreceptor Tyrosine-based Activation Motifs (ITAM) in their cytoplasmic tails (2, 3). These motifs reportedly are phosphoacceptors, through which the receptor subunits interact with signaling proteins.
  • ITAM Immunoreceptor Tyrosine-based Activation Motifs
  • the events that control mast cell activation via Fc ⁇ RI are sequential.
  • IgE binds via its Fc fragment to the Fc ⁇ RI ⁇ chain.
  • IgE molecules are cross-linked by multivalent antigen, causing aggregation of ⁇ chains in the plane of the plasma membrane.
  • Third, information concerning productive ⁇ chain aggregation is transmitted to the ⁇ signaling subunits, via an unknown mechanism. The resulting initiation of intracellular signaling pathways controls downstream events such as allergic mediator production and cytokine gene transcription.
  • Antigenic crosslinking of the Fc ⁇ RI initiates a chain of phosphate transfer events within the receptor microenvironment.
  • the ⁇ and ⁇ chains of the Fc ⁇ RI contain ITAMs, where the tyrosine residues are phosphoacceptor sites for the action of receptor-associated protein tyrosine kinases (PTKs) (12, 13).
  • Phospho-ITAMs link receptor and signal transduction cascades.
  • the ⁇ and ⁇ ITAMs have slightly different structures and serve distinct functions.
  • There are two species of Fc ⁇ RI associated PTK the src family kinase Lyn and the p72 Syk kinase.
  • Syk " " cells have intact ⁇ and ⁇ phosphorylation but still lack downstream signaling events (16, 17, 18).
  • Lyn SH2 domain An obvious candidate for the mediation of this interaction is the Lyn SH2 domain, since ⁇ is slightly tyrosine phosphorylated under resting conditions.
  • others have reported that the 'unique' (SH4 containing) domain of Lyn interacts with Fc ⁇ RI ⁇ (19).
  • Syk is then recruited to the receptor ⁇ chain via one of the tandem SH2 domains in the kinase.
  • Lyn may transphosphorylate ITAM in other receptor complexes.
  • Syk binding to Fc ⁇ RI ⁇ leads to Lyn-dependent tyrosine phosphorylation and activation of the kinase. This step finally potentiates the productive interaction of active Syk with its many targets.
  • aggregation leads to Zyn-dependent ITAM phosphorylation.
  • Rodent Fc ⁇ RI receptor complexes have an obligatory tetrameric ⁇ 2 structure. In humans, both ⁇ 2 and ⁇ 2 complexes are observed at the cell surface. Rodent Fc ⁇ RI receptor complexes are confined to the surface of mast cells and basophils. In humans, however, it is now recognized that there is a far wider distribution of Fc ⁇ RI. On the mast cell and basophil surface there is a mixture of ⁇ 2 and ⁇ 2 complexes, while monocytes, Langerhans cells, eosinophils and dendritic cells express surface ⁇ 2 . Finally, in rodent but not human, there is crosstalk between IgE and IgG mediated cellular activation.
  • the IgE Fc region can bind to two classes of low affinity Fc ⁇ R, activatory and inhibitory isotypes. Both of these are expressed on mast cells and so there is a route by which IgE or IgG immune complexes may regulate mast cell function independently of Fc ⁇ RI.
  • Fc ⁇ RI receptor complexes are first assembled in the ER.
  • nascent ⁇ , ⁇ and ⁇ chains are thought to interact non-covalently.
  • Only the trafficking of ⁇ chains to the cell surface has been monitored extensively.
  • the ⁇ chain reportedly is core- glycosylated.
  • the core, high-mannose glycosylations of ⁇ are replaced by complex sugar, terminal glycosylations.
  • This difference can be exploited experimentally since the latter are insensitive to the action of Endoglycosidase H.
  • the reason for this two stage glycosylation may be that both during biosynthesis and at the cell surface, it is important that ⁇ chains do not aggregate in the absence of antigen.
  • the invention is based, in part, on the discovery that an Fc ⁇ RI ⁇ chain variant modulates the expression of an Fc ⁇ RI receptor (the high affinity receptor for IgE) in cells. Accordingly, the invention provides methods and compositions for modulating cell surface expression of an Fc ⁇ RI receptor in a cell (e.g., contained in a tissue or a subject) which expresses an Fc ⁇ RI receptor.
  • the invention is based, in part, on the discovery that expression of a variant of one of the constituent Fc ⁇ RI receptor chains (e.g., an Fc ⁇ RI ⁇ chain variant, an Fc ⁇ RI ⁇ chain variant, or an Fc ⁇ RI ⁇ chain variant) in a cell that expresses a wild type Fc ⁇ RI receptor, results in the abrogation or decrease of Fc ⁇ RI receptor cell surface expression.
  • a variant of one of the constituent Fc ⁇ RI receptor chains e.g., an Fc ⁇ RI ⁇ chain variant, an Fc ⁇ RI ⁇ chain variant, or an Fc ⁇ RI ⁇ chain variant
  • expression of a variant of one of the constituent Fc ⁇ RI receptor chains e.g., an Fc ⁇ RI ⁇ chain variant, an Fc ⁇ RI ⁇ chain variant, or an Fc ⁇ RI ⁇ chain variant
  • expression of a variant of one of the constituent Fc ⁇ RI receptor chains e.g., an Fc ⁇ RI ⁇ chain variant, an Fc ⁇ RI ⁇ chain variant, or an Fc ⁇ RI ⁇ chain variant
  • a method for inhibiting expression of an Fc ⁇ RI receptor in a cell involves contacting a cell expressing (i.e., a cell expressing or capable of expressing), an Fc ⁇ RI receptor with an Fc ⁇ RI ⁇ chain variant in an effective amount to inhibit expression of the Fc ⁇ RI receptor in the cell.
  • the Fc ⁇ RI ⁇ chain variant is an isolated nucleic acid molecule that inhibits expression of an Fc ⁇ RI receptor in the cell.
  • the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:3.
  • the Fc ⁇ RI ⁇ chain variant is an isolated peptide molecule that inhibits expression of an Fc ⁇ RI receptor in the cell.
  • the isolated peptide molecule comprises the nucleotide sequence of SEQ ID NO:4. The contacting can occur in vitro or in vivo.
  • a method for inhibiting expression of an Fc ⁇ RI receptor in a subject to treat a condition mediated by IgE involves administering to a subject in need of such treatment an Fc ⁇ RI ⁇ chain variant in an effective amount to inhibit Fc ⁇ RI receptor expression in a cell of the subject.
  • the condition mediated by IgE is an allergic condition.
  • the allergic condition is atopy, rhinitis, conjunctivitis, anaphylaxis, urticaria, or angioedema.
  • conditions mediated by IgE are IgE-dependent late phase reactions.
  • the Fc ⁇ RI ⁇ chain variant is an isolated nucleic acid molecule that inhibits expression of an Fc ⁇ RI receptor in the cell.
  • the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:3.
  • the Fc ⁇ RI ⁇ chain variant is an isolated peptide molecule that inhibits expression of an Fc ⁇ RI receptor in the cell.
  • the isolated peptide molecule comprises the amino acid sequence of SEQ ID NO:4. Any of the foregoing embodiments can further comprise co-administering to the subject an anti-allergic (anti- atopic) agent other than an Fc ⁇ RI ⁇ chain variant.
  • a method of screening for Fc ⁇ RI receptor expression modulating agents involves (a) contacting a putative Fc ⁇ RI receptor expression modulating agent with cell expressing (i.e., a cell expressing or capable of expressing) an Fc ⁇ RI receptor, (b) measuring Fc ⁇ RI receptor expression by the cell, and (c) determining whether Fc ⁇ RI receptor expression by the cell is altered compared to Fc ⁇ RI receptor expression by a control cell, wherein the control cell is contacted with an Fc ⁇ RI ⁇ chain variant.
  • the Fc ⁇ RI ⁇ chain variant is an endogenous nucleic acid molecule of the cell.
  • the Fc ⁇ RI ⁇ chain variant is a heterologous nucleic acid molecule of the cell.
  • the Fc ⁇ RI ⁇ chain variant comprises the nucleotide sequence of SEQ ID NO:3.
  • measuring Fc ⁇ RI receptor expression by the cell comprises using an anti-Fc ⁇ RI chain-specific antibody.
  • a method of screening for Fc ⁇ RI ⁇ chain variant expression modulating agents involves (a) contacting a putative Fc ⁇ RI ⁇ chain variant expression modulating agent with a test cell expressing (i.e., a cell expressing or capable of expressing) an Fc ⁇ RI ⁇ chain variant, (b) measuring Fc ⁇ RI ⁇ chain variant expression by the cell, and (c) determining whether Fc ⁇ RI ⁇ chain variant expression by the cell is altered, compared to a control cell expressing an Fc ⁇ RI ⁇ chain variant in the absence of a putative Fc ⁇ RI ⁇ chain variant expression modulating agent.
  • control cell expresses an Fc ⁇ RI ⁇ chain variant identical to the Fc ⁇ RI ⁇ chain variant expressed by the test cell.
  • measuring Fc ⁇ RI ⁇ chain variant expression by the cell comprises using reverse transcription-polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcription-polymerase chain reaction
  • a method for inhibiting expression of an Fc ⁇ RI ⁇ chain in a cell involves contacting a cell expressing (i.e., a cell expressing or capable of expressing), an Fc ⁇ RI ⁇ chain with an Fc ⁇ RI ⁇ chain variant in an effective amount to inhibit expression of the Fc ⁇ RI ⁇ chain in the cell.
  • the Fc ⁇ RI ⁇ chain variant is an isolated nucleic acid molecule that inhibits expression of an Fc ⁇ RI ⁇ chain.
  • the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:3. The contacting can occur in vitro or in vivo.
  • a method for determining whether a subject has a condition mediated by IgE or a predisposition thereto involves determining Fc ⁇ RI ⁇ chain variant expression in a subject suspected of having a condition mediated by IgE or a predisposition thereto, and comparing the Fc ⁇ RI ⁇ chain variant expression to a control. Lower levels of Fc ⁇ RI ⁇ chain variant expression in the subject as compared to the control are indicative for the presence of, or a predisposition to, a condition mediated by IgE in the subject.
  • Fc ⁇ RI ⁇ chain variant expression is mRNA expression.
  • Fc ⁇ RI ⁇ chain variant expression is peptide expression.
  • Figure 1 Diagrammatic sketches of: A. The domain structure of WT (wild type) and ⁇ T (variant) Fc ⁇ RI ⁇ chains; and B. Predicted topologies of WT and ⁇ T Fc ⁇ RI ⁇ chains.
  • Figure 2. Graph depicting levels of surface Fc ⁇ RI ⁇ polypeptide chains in U937 cells stably transfected with ⁇ T ⁇ , ⁇ , and ⁇ Fc ⁇ RI ⁇ isoforms. Each point represents a different clone.
  • FIG. 1 Graph depicting Fc ⁇ RI receptor cell surface expression in U937 ⁇ (WT) Fc ⁇ RI stable transfectants, transiently retransfected with either control or ⁇ T Fc ⁇ RI ⁇ cDNA.
  • SEQ ID NO:l is the wild type (WT) human Fc ⁇ RI ⁇ chain cDNA sequence.
  • SEQ ID NO:2 is the wild type (WT) human Fc ⁇ RI ⁇ chain polypeptide sequence encoded by the cD A sequence set forth in SEQ ID NO: 1.
  • SEQ ID NO:3 is the variant ( ⁇ T or Fc ⁇ RI ⁇ T) Fc ⁇ RI ⁇ chain cDNA sequence.
  • SEQ ID NO:4 is the variant ( ⁇ T or Fc ⁇ RI ⁇ T) Fc ⁇ RI ⁇ chain polypeptide sequence encoded by the cDNA sequence set forth in SEQ ID NO: 3.
  • SEQ ID NO:5 is a sense PCR primer from exon 2 of Fc ⁇ RI ⁇ .
  • SEQ ID NO:6 is an antisense PCR primer from exon 7 of Fc ⁇ RI ⁇ .
  • SEQ ID NO:7 is the wild type (WT) human Fc ⁇ RI ⁇ chain cDNA sequence.
  • SEQ ID NO: 8 is the wild type (WT) human Fc ⁇ RI ⁇ chain cDNA sequence.
  • SEQ ID NO: 9 is the wild type (WT) mouse Fc ⁇ RI ⁇ chain cDNA sequence.
  • the invention is based, in part, on the discovery that a variant form of the ⁇ chain (Fc ⁇ RI ⁇ ) of the Fc ⁇ RI receptor, Fc ⁇ RI ⁇ T (SEQ ID NO:4), inhibits Fc ⁇ RI receptor expression in a cell expressing (i.e., a cell expressing or capable of expressing), an Fc ⁇ RI receptor. More specifically, we have discovered that the Fc ⁇ RI ⁇ chain variant prevents maturation (i.e., post- translational modifications) of Fc ⁇ RI receptor chain Fc ⁇ RI ⁇ , leading to inhibition of Fc ⁇ RI receptor expression in a cell expressing (i.e., a cell expressing or capable of expressing), an Fc ⁇ RI receptor.
  • the invention provides methods and compositions for modulating cell surface expression of an Fc ⁇ RI receptor in a cell (e.g., contained in a tissue or a subject) which expresses an Fc ⁇ RI receptor.
  • a cell e.g., contained in a tissue or a subject
  • the phrase "cell expressing” is meant to embrace cells already expressing a particular polypeptide, as well as cells capable of such expression.
  • the invention is based, in part, on the discovery that expression of a variant of one of the constituent Fc ⁇ RI receptor chains (e.g., an Fc ⁇ RI ⁇ chain variant, an Fc ⁇ RI ⁇ chain variant, or an Fc ⁇ RI ⁇ chain variant ) in a cell that expresses a wild type Fc ⁇ RI receptor, results in the abrogation or decrease of Fc ⁇ RI receptor cell surface expression.
  • a variant of one of the constituent Fc ⁇ RI receptor chains e.g., an Fc ⁇ RI ⁇ chain variant, an Fc ⁇ RI ⁇ chain variant, or an Fc ⁇ RI ⁇ chain variant
  • expression of a variant of one of the constituent Fc ⁇ RI receptor chains e.g., an Fc ⁇ RI ⁇ chain variant, an Fc ⁇ RI ⁇ chain variant, or an Fc ⁇ RI ⁇ chain variant
  • results in the abrogation or decrease of Fc ⁇ RI receptor cell surface expression results in the abrogation or decrease of Fc ⁇ RI receptor cell surface expression.
  • activity usually associated with such a receptor and mediation of IgE signals
  • inhibit expression refers to inhibiting (i.e., reducing to a detectable extent) replication, transcription, and/or translation of one or more Fc ⁇ RI receptor constituent polypeptide genes [i.e., the gene encoding the ⁇ chain (Fc ⁇ RI ⁇ ), the ⁇ chain (Fc ⁇ RI ⁇ ), or the ⁇ chain (Fc ⁇ RI ⁇ )], since inhibition of any of these processes results in the inhibition of expression of an Fc ⁇ RI receptor constituent polypeptide encoded by its respective gene.
  • the term also refers to inhibition of post-translational modifications on the Fc ⁇ RI receptor constituent polypeptide, since inhibition of such modifications will also prevent proper expression (i.e., expression as in a wild type cell) of the encoded polypeptide.
  • the inhibition of gene expression can be directly determined by detecting a decrease in the level of mRNA for the gene, or the level of protein expression of the gene, using any suitable means known to the art, such as nucleic acid hybridization or antibody detection methods, respectively. Inhibition of gene expression can also be determined indirectly by detecting a change in Fc ⁇ RI receptor activity as a whole (e.g., histamine and/or cytokine release by the cell upon triggering with IgE, etc.), or activity of each of the constituent Fc ⁇ RI receptor chains (e.g., phosphorylation, glycosylation, etc.).
  • Fc ⁇ RI ⁇ chain variant refers to a wild type Fc ⁇ RI ⁇ chain nucleic acid or polypeptide which contains one or more modifications (as described below) in its primary sequence, giving rise to a peptide with functional properties that differ from those of the wild type. In certain instances, for example, the variant will have "dominant negative" peptide properties. In other instances, the variant will have a direct effect on the expression of the Fc ⁇ RI ⁇ and Fc ⁇ RI ⁇ receptor chains (e.g., at the nucleotide and/or amino acid level - transcriptionally, translationally, post-translationaly), resulting in inhibition of Fc ⁇ RI receptor expression.
  • a "dominant negative" peptide is an inactive variant of a protein, which, by interacting with the cellular machinery, displaces an active protein (e.g., wild type Fc ⁇ RI ⁇ chain) from its interaction with the cellular machinery or competes with the active protein, thereby reducing the effect of the active protein.
  • an active protein e.g., wild type Fc ⁇ RI ⁇ chain
  • a dominant negative receptor which binds a ligand but does not transmit a signal in response to binding of the ligand can reduce the biological effect of expression of the ligand.
  • a dominant negative catalytically-inactive kinase which interacts normally with target proteins but does not phosphorylate the target proteins can reduce phosphorylation of the target proteins in response to a cellular signal.
  • a dominant negative transcription factor which binds to a promoter site in the control region of a gene but does not increase gene transcription can reduce the effect of a normal transcription factor by occupying promoter binding sites without increasing transcription.
  • the end result of the expression of a dominant negative polypeptide in a cell is a reduction in function of active proteins.
  • an Fc ⁇ RI ⁇ chain variant refers to a nucleic acid with a nucleotide sequence as set forth in SEQ ID NO:3, a polypeptide with an amino acid sequence as set forth in SEQ ID NO:4, and structurally related nucleic acids and polypeptides, respectively, which share a common function with these nucleic acids and polypeptides, namely, the ability to function in a dominant negative fashion and inhibit expression of the Fc ⁇ RI receptor in screening assays such as those described herein.
  • structurally related refers to nucleic acids and polypeptides that are homologous and or allelic to the Fc ⁇ RI ⁇ chain variant.
  • homologs and alleles typically will share at least 40% nucleotide identity and/or at least 50% amino acid identity to SEQ ID NO:3 and SEQ ID NO:4, respectively, in some instances will share at least 50% nucleotide identity and/or at least 65% amino acid identity and in still other instances will share at least 60% nucleotide identity and/or at least 75% amino acid identity.
  • the homology can be calculated using various, publicly available software tools developed by NCBI (Bethesda, Maryland).
  • Exemplary tools include the heuristic algorithm of Altschul SF, et al., (JMol Biol, 1990, 215:403-410), also known as BLAST. Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well as Kyte-Doolittle hydropathic analysis can be obtained using public (EMBL, Heidelberg, Germany) and commercial (e.g., the MacVector sequence analysis software from Oxford Molecular Group/enetics Computer Group, Madison, WI). Watson-Crick complements of the foregoing nucleic acids also are embraced by the invention.
  • Modifications which create an Fc ⁇ RI ⁇ chain variant are typically made to the nucleic acid which encodes the Fc ⁇ RI ⁇ chain polypeptide.
  • Other similar methods for creating and testing variants of a protein according to the invention will be apparent to one of ordinary skill in the art.
  • functionally equivalent variants of Fc ⁇ RI ⁇ T chain polypeptides i.e., variants of Fc ⁇ RI ⁇ polypeptides which retain the above-identified function of the Fc ⁇ RI ⁇ T polypeptide, are contemplated by the invention.
  • Fc ⁇ RI ⁇ chain variant polypeptides can be screen for a dominant negative variant of a protein, and using standard mutagenesis techniques to create one or more dominant negative Fc ⁇ RI ⁇ chain variant polypeptides.
  • Fc ⁇ RI ⁇ chain variant nucleic acids and polypeptides one of ordinary skill in the art can modify the Fc ⁇ RI ⁇ nucleotide (SEQ ID NO:l) and/or peptide sequence (SEQ ID NO:2) by site-specific mutagenesis, scanning mutagenesis, partial gene deletion or truncation, and the like. See, e.g., U.S. Patent No.
  • the invention provides a method for inhibiting expression of an Fc ⁇ RI receptor in a cell of a subject to treat a condition mediated by IgE.
  • the method involves administering to a subject in need of such treatment an Fc ⁇ RI ⁇ chain variant in an effective amount to inhibit Fc ⁇ RI receptor expression in a cell of the subject.
  • a "subject” as used herein, is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent.
  • a "cell” of a subject, as used herein, refers to a cell that expresses, or is capable of expressing, an Fc ⁇ RI receptor, and it includes cells of hematopoietic origin, and more specifically mast cells and/or basophils.
  • a “condition mediated by IgE” as used herein, refers to Type I hypersensitivity responses (also known as immediate hypersensitivity reactions) and IgE-dependent "late phase reactions,” characterized by an infiltration of inflammatory cells that appears days after exposure to an allergen (e.g., as in the inflammatory reaction present in the airways of asthmatic patients).
  • the immune system of humans and animals normally functions to protect its host from infectious organisms or from cancerous transformation by host cells. In many instances however, the immune system manifests a response that itself results in considerable damage to otherwise healthy cells and organs. Such over-reactivity of immune responsiveness is responsible for many serious conditions or diseases including allergies and autoimmune diseases.
  • immunologists In order to classify the processes by which the immune system produces cellular damage, immunologists have divided immune responses into four broad classes (Type I, II, III and IV) (Roitt, I. M., et al., Immunology, C. V. Mosby, N.Y., 1985, p. 19.1).
  • Type I hypersensitivity responses are also called immediate hypersensitivity reactions and refer to those conditions which produce the symptoms classically associated with “allergies” or the “allergic syndrome” including atopy [e.g., allergic rhinitis (hay fever), allergic asthma, allergic conjunctivitis], urticaria, angioedema, and allergic reactions to inseGt stings or foods (anaphylaxis). These allergic conditions are characterized by a rapid clinical manifestation of allergic symptoms within minutes after exposure to an antigen (allergen) to which the subject has been previously sensitized.
  • IgE immunoglobulin E
  • IgE directed toward an antigen must bind to a receptor (Fc ⁇ RI receptor) on mast cells and basophils which specifically bind to the Fc region of IgE.
  • Fc ⁇ RI receptor a receptor on mast cells and basophils which specifically bind to the Fc region of IgE.
  • Mast cells and basophils that have anti-allergen-IgE bound to them are considered to be sensitized or "armed" for subsequent exposure to allergen.
  • the cells are automatically stimulated or "triggered” to release histamine and other vasoactive chemicals (e.g., lipid mediators such as leukotrienes B 4 and C 4 , prostaglandin D 2, platelet-activating factor, cytokines such as IL-4, IL-5, IL-6, TNF- ⁇ , etc.) which produce the familiar "allergic symptoms" characteristic of allergic conditions.
  • vasoactive chemicals e.g., lipid mediators such as leukotrienes B 4 and C 4 , prostaglandin D 2, platelet-activating factor, cytokines such as IL-4, IL-5, IL-6, TNF- ⁇ , etc.
  • the hypersensitivity states characterized by types II, III and IV hypersensitivity are very distinct from type I hypersensitivity.
  • allergic inflammation in type I hypersensitivity begins within minutes after allergen exposure.
  • other hypersensitivity states exhibit inflammation only after hours to days following reexposure to the sensitizing agent.
  • the sensitizing agent allergen
  • Type I hypersensitivity the sensitizing agent
  • Type II, III and IV hypersensitivity may have immune responses directed towards antigens located on cells and molecules that are normal constituents of the body.
  • autoimmune diseases Such immune responses toward normal constituents of the body are termed "autoimmune diseases" and constitute a medically class of diseases distinct from conditions mediated by IgE (e.g., allergies).
  • IgE e.g., allergies
  • a further distinction is the degree to which cell killing occurs.
  • the IgE mediated triggering reaction which causes the release of vasoactive allergic mediators does not result in the death of the releasing mast cell or basophil. Instead, the "trigger" reaction is the result of an active secretory process that may recur after a length of time.
  • the effect of the vasoactive allergic mediators on surrounding cells is regulatory, not cytotoxic.
  • Allergic mediators serve to increase the permeability of small blood vessels and activate a variety of vasoregulatory and immunoregulatory processes that do not normally result in cell death.
  • Types II, III and IV hypersensitivity by contrast, have as a principal function cell killing reactions which normally lead to the destruction of infectious agents or cancer cells.
  • the invention involves a method of screening for Fc ⁇ RI receptor expression modulating agents.
  • the method involves (a) contacting a putative Fc ⁇ RI receptor expression modulating agent with a cell expressing (i.e., a cell expressing or capable of expressing) an Fc ⁇ RI receptor, (b) measuring Fc ⁇ RI receptor expression by the cell, and (c) determining whether Fc ⁇ RI receptor expression by the cell is altered compared to Fc ⁇ RI receptor expression by a control cell, wherein the control cell is contacted with an Fc ⁇ RI ⁇ chain variant. Additional controls may also include measuring Fc ⁇ RI receptor expression by a cell in the absence of a putative Fc ⁇ RI receptor expression modulating agent.
  • Fc ⁇ RI receptor expression by the cell is accomplished using a number of different methods, most of which are well known by a person of ordinary skill in the art. For example, a direct way would be to measure mRNA expression for one of the constituent Fc ⁇ RI receptor chains using Northern blotting, RT-PCR, etc.
  • the human nucleic acid sequences encoding each of the three constituent Fc ⁇ RI receptor chains are known in the art and are publicly available through NCBI's GenBank databases (Accession nos.: X06948 for Fc ⁇ RI ⁇ -SEQ ID NO:7; M89796 for Fc ⁇ RI ⁇ -SEQ ID NO:l; and L03533 for Fc ⁇ RI ⁇ -SEQ ID NO: 8).
  • Another direct way to measure Fc ⁇ RI receptor expression by the cell is to use antibodies specific for one of the constituent Fc ⁇ RI receptor chains and a number of irnmunocyto- and immunohisto- chemical protocols.
  • Antibodies specific for each of the constituent Fc ⁇ RI receptor chains are commercially available and can be obtained, for example, through Upstate Biotechnology, Lake Placid, NY [rabbit polyclonal anti-human Fc ⁇ RI ⁇ peptide (997) cat. 06-725; mouse monoclonal anti -human Fc ⁇ RI ⁇ chain (clone 3G6) cat. 05-491; rabbit polyclonal anti-human Fc ⁇ RI ⁇ peptide cat. 06-726; rabbit polyclonal anti- human Fc ⁇ RI ⁇ peptide (934) cat.
  • expression of more than one chain is measured at the same time.
  • expression of the Fc ⁇ RI ⁇ chain is measured at the same time as Fc ⁇ RI ⁇ chain expression.
  • Another indirect way to measure Fc ⁇ RI receptor expression by the cell is to measure Fc ⁇ RI receptor activity.
  • Fc ⁇ RI receptor activity can be measured by the downstream effects of IgE binding to the receptor. Such downstream effects include secretion of chemical compounds by the cell, supra, in response to antigen-IgE binding to the receptor.
  • the Fc ⁇ RI ⁇ chain variant is an endogenous nucleic acid molecule of the cell. By “endogenous” it is meant that it is naturally present in the cell genome.
  • the Fc ⁇ RI ⁇ chain variant is a heterologous nucleic acid molecule of the cell.
  • heterologous or foreign nucleic acid molecule (DNA and RNA) are used interchangeably and refer to DNA or RNA that does not occur naturally as part of the genome in which it is present or which is found in a location or locations in the genome that differ from that in which it occurs in nature.
  • it is DNA or RNA that is not endogenous to the cell and has been artificially introduced into the cell.
  • heterologous DNA examples include, but are not limited to, DNA that encodes an Fc ⁇ RI ⁇ chain variant polypeptide according to the invention, and DNA that encodes RNA or proteins that mediate or alter expression of endogenous DNA (for example, that of wild type Fc ⁇ RI ⁇ chain) by affecting transcription, translation, or other regulatable biochemical processes.
  • the cell that expresses the heterologous DNA such as DNA encoding an Fc ⁇ RI ⁇ chain variant polypeptide, may contain DNA encoding the same or different Fc ⁇ RI ⁇ chain variant polypeptide.
  • the screening methods of the invention involve assaying for compounds which interfere with Fc ⁇ RI receptor expression activity, and can be detected by any of the above-identified methods (e.g., downstream effects after the Fc ⁇ RI receptor binds its natural ligand, IgE). Such methods are adaptable to automated, high throughput screening of compounds.
  • the target therapeutic indications for agents detected by the screening methods are limited only in that the target cellular function be subject to modulation by alteration of the formation of a complex comprising an Fc ⁇ RI receptor, and its natural ligand, IgE.
  • Target indications can include cellular processes mediated by the Fc ⁇ RI receptor following its binding to IgE (e.g., histamine or cytokine release, etc.).
  • a screening assay mixture includes a binding partner for the receptor, e.g., a naturally occurring ligand (i.e., IgE) that is capable of binding to the Fc ⁇ RI receptor or, alternatively, is comprised of an analog which mimics the Fc ⁇ RI receptor binding properties of the naturally occurring ligand for purposes of the assay.
  • the screening assay mixture also comprises a candidate agent (e.g., an agent that modulates expression of the Fc ⁇ RI receptor, and most preferably inhibits expression of the Fc ⁇ RI receptor).
  • a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a different response to the various concentrations.
  • one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection.
  • An essential control according to the invention is the comparison of the effects of the inhibitory agent on Fc ⁇ RI receptor expression with the Fc ⁇ RI receptor expression in a cell expressing (i.e., a cell expressing or capable of expressing) an Fc ⁇ RI ⁇ chain variant of the invention.
  • Candidate agents encompass numerous chemical classes, although typically they are organic compounds.
  • the candidate agents are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500, preferably less than about 1000 and, more preferably, less than about 500.
  • Candidate agents comprise functional chemical groups necessary for structural interactions with polypeptides and/or nucleic acids, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups.
  • the candidate agents can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups.
  • Candidate agents also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like.
  • the agent is a nucleic acid
  • the agent typically is a DNA or RNA molecule, although modified nucleic acids as defined herein are also contemplated.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily be modified through conventional chemical, physical, and biochemical means. Further, known pharmacological agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs of the agents.
  • reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce non-specific or background interactions of the reaction components.
  • Other reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used.
  • the mixture of the foregoing assay materials is incubated under conditions whereby, but for the presence of the candidate putative inhibitor agent, the Fc ⁇ RI receptor specifically binds its natural binding target, a portion thereof or analog thereof.
  • incubation temperature typically are between 4°C and 40°C.
  • Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 0.1 and 10 hours.
  • the presence or absence of specific binding between the Fc ⁇ RI receptor and one or more binding targets is detected by any convenient method available to the user, supra.
  • the therapeutics of the invention embrace isolated nucleic acids and polypeptides.
  • isolated as used herein in reference to a nucleic acid molecule, means a nucleic acid sequence: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) synthesized by, for example, chemical synthesis; (iii) recombinantly produced by cloning; or (iv) purified, as by cleavage and gel separation.
  • PCR polymerase chain reaction
  • isolated means a polypeptide encoded by an isolated nucleic acid sequence, as well as polypeptides synthesized by, for example, chemical synthetic methods, and polypeptides separated from biological materials, and then purified using conventional protein analytical procedures.
  • the therapeutics of the invention are in effective amounts.
  • the "effective amount” will depend upon the mode of administration, the particular condition being treated and the desired outcome. It will also depend upon, the stage of the condition, the age and physical condition of the subject, the nature of concurrent therapy, if any, and like factors well known to the medical practitioner. For therapeutic applications, it is that amount sufficient to achieve a medically desirable result. In some cases, for example, this is a decrease in a subject's immune hypersensitivity to allergens as evidenced by a decrease in circulating histamines, cytokines, etc. (i.e., inhibition of allergen effects).
  • doses of active compounds of the present invention would be from about 0.01 mg/kg per day to 1000 mg/kg per day. It is expected that doses ranging from 50-500 mg/kg will be suitable.
  • a variety of administration routes are available. The methods of the invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical, nasal, intradermal, or parenteral routes.
  • parenteral includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. They could, however, be preferred in emergency situations.
  • Oral administration will be preferred for prophylactic treatment because of the convenience to the patient as well as the dosing schedule.
  • a desirable route of administration is by pulmonary aerosol.
  • Techniques for preparing aerosol delivery systems containing peptides are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the peptides, for example, the paratope binding capacity of a peptide (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences. 18th edition, 1990, pp 1694-1712; incorporated by reference).
  • Those of skill in the art can readily determine the various parameters and conditions for producing antibody or peptide aerosols without resort to undue experimentation.
  • Preferred methods for administering Fc ⁇ RI ⁇ variants or agents that induce Fc ⁇ RI ⁇ variant expression of the invention also include spliceosome-mediated RNA trans-splicing as described by Puttaraju, M., et al (53) and using agents commercially available from Intronn LLC (Durham, NC), and ribozymes using methods well known in the art and reviewed by Woolf, TM (58).
  • compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active agent.
  • Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Lower doses will result from other forms of administration, such as intravenous administration.
  • the Fc ⁇ RI ⁇ chain variant polypeptides or functionally equivalent fragments thereof may be combined, optionally, with a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration into a human.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions.
  • Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
  • the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
  • pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • Fc ⁇ RI ⁇ chain variant polypeptides, or functionally equivalent fragments thereof, preferably are produced recombinantly, although such polypeptides may be isolated from biological extracts.
  • Fc ⁇ RI ⁇ chain variant polypeptides include chimeric proteins comprising a fusion of a Fc ⁇ RI ⁇ chain variant peptide with another polypeptide, e.g., a polypeptide capable of providing or enhancing protein-protein binding, sequence specific nucleic acid binding (such as GAL4), enhancing stability of Fc ⁇ RI ⁇ chain variant polypeptide under assay conditions, or providing a detectable moiety, such as green fluorescent protein.
  • a polypeptide fused to a Fc ⁇ RI ⁇ chain variant polypeptide or fragment may also provide means of readily detecting the fusion protein, e.g., by immunological recognition or by fluorescent labeling.
  • Various techniques may be employed for introducing Fc ⁇ RI ⁇ chain variant encoding nucleic acids of the invention into cells, depending on whether the nucleic acids are introduced in vitro or in vivo in a host.
  • Such techniques include transfection of nucleic acid- CaPO 4 precipitates, transfection of nucleic acids associated with DEAE, transfection with a retro virus including the nucleic acid of interest, liposome mediated transfection, and the like.
  • a vehicle used for delivering a nucleic acid of the invention into a cell e.g., a retrovirus, or other virus; a liposome
  • a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or incorporated within the nucleic acid delivery vehicle.
  • proteins which bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake.
  • proteins include capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like.
  • Polymeric delivery systems also have been used successfully to deliver nucleic acids into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acids.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the anti-inflammatory agent, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109.
  • Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • hydrogel release systems such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • sylastic systems such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • peptide based systems such as fatty acids
  • wax coatings such as those described in U.S. Patent Nos.
  • Long-term sustained release means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days.
  • Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
  • the agents of the invention are "co-administered," which means administered substantially simultaneously, with another anti-allergic agent other than an Fc ⁇ RI ⁇ chain variant molecule.
  • substantially simultaneously it is meant that an Fc ⁇ RI ⁇ chain variant related molecule of the invention is administered to a subject, as an admixture in a single composition, or sequentially, close enough in time with the administration of the other anti-allergic agent, whereby the two compounds may exert an additive or even synergistic effect, e.g. decreasing or completely eliminating a Type I hypersensitivity response.
  • the invention also embraces determining whether a subject has a condition mediated by IgE or a predisposition thereto.
  • the method involves determining whether an Fc ⁇ RI ⁇ chain variant according to the invention is present in a subject, and or whether this variant is expressed at different levels in the subject when compared to a control.
  • the method involves determining whether the Fc ⁇ RI ⁇ chain variant is present and or expressed at lower levels (amounts) in the subject in comparison to a control.
  • control refers to Fc ⁇ RI ⁇ chain variant expression in a control subject.
  • a "control subject” is an apparently healthy subject with no symptoms indicative of a condition mediated by IgE, or predisposition to a condition mediated by IgE (e.g., no family history of a condition mediated by IgE ).
  • the Fc ⁇ RI ⁇ chain variant is a polypeptide designated herein as Fc ⁇ RI ⁇ T having an amino acid sequence as set forth in SEQ ID NO:4.
  • Variant polypeptide Fc ⁇ RI ⁇ T arises from an Fc ⁇ RI ⁇ chain mRNA (having a cDNA as depicted in SEQ ID NO: 3), in which intron 5 (402 nucleotides in length) of the genomic Fc ⁇ RI ⁇ chain gene, is not spliced out and forms part of the mRNA.
  • the resulting translated variant polypeptide, Fc ⁇ RI ⁇ T is shorter than the wild type Fc ⁇ RI ⁇ polypeptide because of an in frame early termination signal in the the fifth intron and, as a result, is missing the C- terminus of the wild type protein (see Figure 1).
  • a suitable tissue sample is obtained from the subject suspected of having a condition mediated by IgE or a predisposition thereto.
  • Lymph or blood are the preferred tissues where samples can be obtained, but other tissues that contain cells of hematopoietic origin (such as mast cells and basophils) can also be used according to the invention.
  • a preferred method for determining the presence and/or levels of Fc ⁇ RI ⁇ chain variant expression according to this aspect of the invention is quantitative RT-PCR (54-57).
  • primers that span intron 5 e.g., from exons 2 and 7 to perform the detection and/or quantitation of the variant trancript are selected.
  • examples of such primers include those whose sequences are set forth in SEQ ID NOs 5 and 6.
  • the assay is performed in parallel with or in reference to a control assay in which samples obtained from control subjects, preferably of like tissue, are used to establish a control amount of the variant.
  • the wild type transcript optionally serves as an internal control for the reaction and/or quantitation.
  • a lower level (amount) of the Fc ⁇ RI ⁇ chain variant transcript relative to an Fc ⁇ RI ⁇ chain variant transcript of a control subject is indicative of the subject having a condition mediated by IgE or a predisposition thereto.
  • NIH3T3 ⁇ transfectants were maintained in DMEM (Biofluids, Rockville, MD) with 10% calf serum (Biofluids, Rockville, MD) and 300 ⁇ g/ml of hygromycin (Calbiochem, La olla, CA) and 500 ⁇ g/ml of neomycin (Gibco/BRL, Baltimore, MD). All the other NIH- 3T3 transfected cell lines were maintained in the same medium without neomycin. U937 transfected cell lines were maintained in RPMI-1640 (Biofluids) with 20% fetal bovine serum (Biofluids) and 1 mg/ml of neomycin.
  • BMMC Mouse Bone Marrow-derived Mast Cells
  • Human Cord Blood Mast Cells were generated as previously described (44).
  • mononucleated cells were isolated from human umbilical cord blood by centrifugation over Histopaque-1077 (Sigma), and then were cultured in Iscove's modified Dulbecco's medium supplemented with 10 % FBS, 100 ng/ml recombinant human stem cell factor (Biosource), 10 ng/ml recombinant human IL-6 (Endogen) and 1 mM prostaglandin E2 (Cayman Chemical, Ann Arbor, MI).
  • the human ⁇ subunit and mutated forms of rat ⁇ cDNA ( ⁇ YM, tyrosines at position 218, 224, and 228 with the ⁇ lTAM were changed to phenylalanines) of Fc ⁇ RI were subcloned into pCDL-SR ⁇ 296.
  • the rat ⁇ and ⁇ subunits of Fc ⁇ RI were subcloned into pSHSX, a pCDL-SR ⁇ 296 derivative containing a hygromycin resistance cassette.
  • rat ⁇ and ⁇ subunit, rat ⁇ subunit cDNAs and the mutated form of rat ⁇ cDNA were subcloned into pBJlneo, a neomycin derivative of pCDL-SR ⁇ 296 (15).
  • pBJlneo a neomycin derivative of pCDL-SR ⁇ 296
  • the trunctated forms of rat ⁇ ( ⁇ NT, the first 57 amino acids of NH 2 -tennmal cytoplasmic domain were deleted; ⁇ CT, the last 38 amino acids of COOH-terminal cytoplasmic domain were deleted) have been described (40).
  • ⁇ NT and ⁇ CT were subcloned into pCDL-SR ⁇ 296.
  • the WT ⁇ cDNA subcloned in the eukaryotic expression vector pBJlneo was used as a template for site-directed mutagenesis with the Quick-change kit (Stratagene) using two different oligonucleotides containing the sequences from the 181L-183L and 237G variants. Confirmation of the mutations was obtained by DNA sequencing.
  • the spice variant of human Fc ⁇ RI ⁇ see RT PCR paragraph below.
  • the ⁇ WT and splice variant cDNAs were FLAG-tagged at the N terminus using the FLAG epitope (Kodak, New Haven, CO).
  • the flagged cDNAs were subcloned in pBJlneo.
  • the monoclonal anti-phosphotyrosine antibody (4G10) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). PE-streptavidin was from PharMingen (San Diego, CA).
  • the anti-human syk antibody 996 was raised against a synthetic peptide representing amino acids 150-159 of human syk sequence (15).
  • Monoclonal anti-rat Fc ⁇ RI ⁇ C tail antibody (NB) was from Dr. D Holowka, and chimeric anti-Nip human IgE were from Dr. Z. Eshhar.
  • the monoclonal FLAG antibody covalently attached to agarose beads was from Sigma (Saint Louis, MO). Recombinant Vaccinia Virus Construction
  • ⁇ 2 cells The generation of ⁇ 2 cells has been previously described (14). Construction of other NIH-3T3 sublines expressing FceRI variants was similar. In brief, cells were co- electroporated (270V, 960 ⁇ F) with appropriate constructs. Resistant clones were selected, and screened for surface Fc ⁇ RI expression by flow cytometry with FITC-IgE. U937 cells were co-transfected by electroporation (260V, 960 ⁇ F) with human ⁇ , ⁇ and ⁇ constructs or with ⁇ and ⁇ constructs only.
  • Transient transfection KU812 cells (5 x 10 ) were co-transfected by electroporation (300V, 960 ⁇ F) with 10 ⁇ g of ⁇ T expression vector and l ⁇ g of green fluorescent protein (GFP; pGreen Lantern, Gibco). Human CD81 subcloned in PBJlneo was used as a control vector (Fleming 1997).
  • FceRI expression was analysed on GFP positive gated cells.
  • Infection with Recombinant Vaccinia Viruses Adherent NIH 3T3 cells in one 150cm 2 culture flask were infected with the appropriate virus at 5 pfu/cell in 5 ml DMEM with 2.5% calf serum for 30 min at 4°C with gentle rocking, then for 30 min at 37°C. Control virus bearing the pSC65 vaccinia recombinant vector was added to single recombinant virus infection in order to allow valid comparisons with recombinant virus coinfections.
  • Cell Activation with Antigen and Lysis Cell Activation with Antigen and Lysis
  • Infected NIH-3T3 sublines (3-5 x 10 6 cells/sample) or U937 sublines (3 x 10 7 cells/sample) were harvested in 1 ml medium. Cells were stimulated with 200 ng/ml DNP24- 40-HSA (Sigma, St Louis, MO) in 1 ml for 4 min at room temperature with rocking.
  • DNP24- 40-HSA Sigma, St Louis, MO
  • Immunoprecipitations were performed as previously described (14), except for the cells lysed in 10 mM CHAPS. In this case immunoprecipitates were washed with lysis buffer containing 2 mM CHAPS. In vitro kinase assays were performed as described (15), and analyzed by SDS-PAGE and autoradiography. Where indicated, immunoprecipitates were treated with endo- ⁇ -N-acetylglucosamidase (Endo H) (New England Biolabs) as previously described (47). After immunoprecipitation with appropriate antibodies, samples were resolved on SDS polyacrylamide gel, transferred to PVDF membrane, and blotted with the antibodies indicated.
  • Endo H endo- ⁇ -N-acetylglucosamidase
  • Immunoreactive proteins were visualized by using alkaline phosphatase- coupled second-step reagents and enhanced chemifluorescence (ECF, Amersham). Fluorescence was quantified using a Storm scanner (Molecular Dynamics). Where appropriate, cells were treated with proteasome inhibitors (ALLN 250 ⁇ M) (Calbiochem) or vehicle for 2 hours at 37°C before lysis. Measurement of Calcium Moblization
  • U937 transfectant cells were washed and incubated with 2 ⁇ M Fura-2 acetoxymethyl ester (Moleular Probes, Portland, OR) and 0.08% Pluronic F-127 (Molecular Probes) in calcium buffer (135 mM NaCl, 5 mM KC1, 1 mM CaCl 2 , 1 mM MgCl 2 , 5.6 mM glucose, 10 mM HEPES, pH 7.4, 0.1% BSA and 2.5 mM Probenecid [Sigma, St. Louis, MO]) for 1 hr with gentle rocking at room temperature.
  • 2 ⁇ M Fura-2 acetoxymethyl ester Moleular Probes, Portland, OR
  • Pluronic F-127 Molecular Probes
  • calcium buffer (135 mM NaCl, 5 mM KC1, 1 mM CaCl 2 , 1 mM MgCl 2 , 5.6 mM glucose, 10 mM HEPES, pH 7.4, 0.1% BSA and 2.5
  • FcR ⁇ Gene was isolated from a 129 ⁇ -DASH library (49) (a gift from Dr. P. Love, NIH, Bethesda, MD).
  • exons IV- VI corresponding to bp 344-577 of the mouse FcR ⁇ cDNA sequence -SEQ ID NO:9) (GenBank Ace. No. AB033617, and 50) with a Neo cassette.
  • a genomic 4 kb fragment encompassing exons I-IV bp 38-343 of the mouse FcR ⁇ cDNA sequence
  • a genomic 1.4 kb fragment encompassing exons VI -VII were amplified by polymerase chain reaction using appropriate primers and cloned respectively into the Xhol and Xbal of pJNS2 vector (42).
  • the construct was linearized by Notl and electroporated into D3 or El 4 TG2a ES cells.
  • ES cells were grown and selected as previously described (48). Genomic DNA was extracted from clonal ES cells, digested with Dral and hybridized by Southern blot to a 440 bp fragment located 16 bp downstream of exon VII. The hybridizing bands were 3.4 kb for the WT and 2.8kb for the disrupted allele. Two out of 109 G418 resistant-ganciclovir sensitive clones were found positive for homologous recombination. Animals Chimera, heterozygous (+/-) and homozygous (-/-) animals for the disrupted FcR ⁇ allele were generated as previously described (48); FcR ⁇ -/- animals were on a Balb/c F2 background.
  • FceRI ⁇ -/-animals have been previously described (42), and were on a Balb/c F6 background.
  • FcR ⁇ -/- animals were compared to age-matched Fc ⁇ RI ⁇ -/- for all relevant experiments.
  • FcR ⁇ -/- were also crossed with the previously described transgenic mice for the human Fc ⁇ RI ⁇ gene (hFc ⁇ RI ⁇ Tg) (43).
  • Progeny of the second generation thus contained hFc ⁇ RI ⁇ Tg/FcR ⁇ -/- or hFc ⁇ RI ⁇ Tg/FcR ⁇ +/- animals. Littermates were used in all appropriate experiments.
  • Cellular degranulation was measured by the release of ⁇ -hexoseaminidase as described before with minor modifications (51). Cells were incubated with 50 ng/ml/10 6 cells humanlgE over night or with various doses of anti- Fc ⁇ RI ⁇ (15-1) for 2 hours, washed twice and stimulated with NIP-BSA or with 10 ⁇ g/ml goat anti-mouse IgG F(ab') 2 for 20 min.
  • RNA was isolated with RNAzol B (Tel-Test, Inc., Friendswood, TX) following manufacturer instructions. Aliquots of 1 ⁇ g of total RNA were converted to cDNA by using poly(dT)18 primers and transcribed with 100 units of Moloney murine leukemia virus reverse transcriptase (Clontech, Palo Altp, CA) at 42°C for 60 min. For PCR, aliquots of DNA w equivalent to 0.1 ⁇ g total RNA were used in each reaction (50 ⁇ l) containing 50 pmol of each primer, 200 ⁇ M of each deoxynucleoside trisphosphate, and 1.25 units of Taq polymerase (Fisher Scientific, Pittsburg, PA).
  • RNAzol B Tel-Test, Inc., Friendswood, TX
  • ⁇ WT and its splice variant were amplified using a sense primer located in exon 2 (5'-GTGCCTGCATTTGAAGTCTTG-3', SEQ ID NO:5) and antisense primer located in exon 7 (5'-TGGATCCTTGGCTGTGAATC-3 ⁇ SEQ ID NO:6).
  • PCR reactions were performed under the following conditions : lmin at 94°C, followed by 22 to30 cycles of 45 sec at 94°C, 45 sec at 60°C, 1 min at 72°C. PCR products were visualised on 1.2 % agarose gels stained with ethidium bromide. Cloning of PCR Products
  • PCR products were isolated after agarose gel separation, purified with QIAquick PCR
  • RNA from untransfected or transfected U937 cells was prepared with RNAzol B (Tel-Test, Inc., Friendswood, TX). Poly A RNA from CBMC was purified using the
  • RNA probes were generated by in vitro transcription using the Maxiscript kit (Ambion, Austin, TX). The ⁇ probe was designed to overlap the end of exon 5 and the beginning of intron 5. Probes were also designed in the ⁇ and ⁇ cDNAs and used as controls. RPA was performed using the RPAIII kit (Ambion, Austin, TX). Ten ⁇ g total RNA from U937 cells and the mRNA
  • RNAse A 3o obtained from 30 x 10 6 CBMC were incubated with 10 5 cpm freshly prepared probe, and probe was then digested in the presence of 0.375 units RNAse A and 15 units RNAse Tl or buffer. Protected probe fragments were separated by denaturing polyacrylamide gel electrophoresis. Gels were exposed to fluorescent screens that were read on a Storm 840 scanner (Molecular Dynamics, CA), and analyzed using the ImageQuant software (Molecular Dynamics, CA). Pulse-Chase Analysis
  • the Fc ⁇ RI ⁇ molecule is a unique subunit among human antigen receptors. It has a topology where both tails are cytoplasmic, and contains an atypical ITAM motif with an extra tyrosine and decreased spacing relative to the consensus.
  • NIH3T3 stably transfected with human Fc ⁇ RI ⁇ cDNAs are infected with vaccinia viruses separately encoding the Lyn and Syk tyrosine kinases.
  • Surface ⁇ levels were assessed by flow cytometry and then antigen stimulation of equivalent numbers of Fc ⁇ RI between clones was performed.
  • ⁇ 2 receptors are stimulated, there is substantially enhanced Syk tyrosine phosphorylation, Syk kinase activity, and Fc ⁇ RI ⁇ tyrosine phosphorylation relative to that achieved by stimulation of ⁇ receptors.
  • These data were subsequently reproduced precisely in a hematopoietic cell line system, the U937.
  • the latter cell line also enabled us to perform a functional assay to assess whether the biochemical amplification observed translated to a significant increase in a functional response to antigen stimulation.
  • Stable cell lines expressing Fc ⁇ RI isoforms are important tools in our analyses of Fc ⁇ RI biology.
  • NIH 3T3 and U937 hematopoietic cell backgrounds
  • ⁇ 2 and ⁇ receptors There is, in all cases, a statistically significant difference between surface expression of ⁇ 2 and ⁇ receptors.
  • the presence of ⁇ correlates with increased expression of receptor at the cell surface in both NIH3T3 and U937 cells.
  • these results were subsequently reproduced in transient expression systems.
  • Fc ⁇ RI surface levels We tested the idea that ⁇ could inducibly alter Fc ⁇ RI expression levels.
  • Fc ⁇ RI ⁇ was transiently transfected into KU812 which had been stably transfected with ⁇ receptors.
  • KU812 are a transformed mast cell line with no endogenous expression of Fc ⁇ RI at their surface.
  • introduction of Fc ⁇ RI ⁇ induces expression of Fc ⁇ RI complexes at the cell surface.
  • ⁇ could be promoting the trafficking of Fc ⁇ RI subunits from their sites of synthesis in the ER to the plasma membrane.
  • ⁇ may affect the stability of surface and nascent complexes, decreasing their turnover and causing a cumulative increase in surface receptor numbers. We then addressed whether either or both of these mechanisms were likely to operate.
  • Fc ⁇ RI complexes undergo the following maturation process: 1) In the ER, subunits associate non-covalently and ⁇ is core-glycosylated, and thus sensitive to the action of Endoglycosidase H (Endo H). 2) Trafficking from ER to Golgi follows; in the Golgi, terminal glycosylation occurs which places complex sugars (Endo H resistant) on the ⁇ chain. We can use SDS-PAGE to look at trafficking of nascent ⁇ 2 / ⁇ complexes, differentiating between stages on the basis of molecular weight and EndoH sensitivity.
  • Terminally glycosylated ⁇ runs as a diffuse species at around 66 kDa. In contrast with the 48 kDa species, mature, complex-sugar glycosylated, ⁇ is resistant to Endo H. Clear differences in ⁇ content are observed between ⁇ and ⁇ 2 cell lines. In the presence of ⁇ chains there is less of the Endo H sensitive (immature) ⁇ species and an increased amount of mature ⁇ chains. Densitometry was performed to quantitate these differences and a ratio of mature to immature ⁇ chains was calculated. Thus, the presence of ⁇ affects the intracellular trafficking of ⁇ chains, apparently accelerating their progress towards a mature form.
  • Example 3 A novel splice variant of ⁇ , ⁇ T, is an intrinsic downregulator of Fc ⁇ RI surface expression
  • ⁇ T The splice variant of ⁇ would produce a truncated protein, ⁇ T, lacking the C-terminal transmembrane and cytoplasmic domains. Since the new 16 aa sequence has some hydrophobic character, it is possible that there is some interaction with the membrane to form an imperfect transmembrane domain.
  • ⁇ T may be topologically distinct from wild-type ( ⁇ WT), with the C- and N-termini on opposite sides of the membrane.
  • RNAse protection assays RPA
  • ⁇ T was a transcript that could be detected in normal cell and was not a PCR artifact.
  • RPA can detect as little as 5 fg RNA.
  • U937 cells untransfected, or transfected with either ⁇ 2 or ⁇ T ⁇ 2 ), or primary human cord blood derived mast cells.
  • Fc ⁇ RI ⁇ or ⁇ T were immunoprecipitated from lysates using their FLAG epitope tags and samples were resolved by SDS-PAGE. After autoradiography, we observed that wild-type ⁇ was stable over the 30 min time course. In contrast, we observed that ⁇ T had an extremely short half-life. Densitometry indicated that ⁇ T had a half-life of approximately 10 min under the conditions of this experiment. We observed herein that ⁇ T is an expressed variant protein. It is reasonable to assume, therefore, that ⁇ T may form Fc ⁇ RI complexes. However, due to its variant structure and rapid turnover, we might expect that Fc ⁇ RI formed of ⁇ T ⁇ would behave differently than their wild-type counterparts.
  • Fc ⁇ RI ⁇ can be identified based on their Endo H sensitivity, and apparent molecular weight after separation by polyacrylamide gel electrophoresis (PAGE).
  • PAGE polyacrylamide gel electrophoresis
  • CD20 a regulator of cell-cycle progression of B lymphocytes. Immunol Today 15, no. 9:450-4.
  • Allergy-associated FcRbeta is a molecular amplifier of IgE- and IgG- mediated in vivo responses. Immunity 8, no. 4:517-29.

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