WO2000040713A1 - Procede permettant de prevenir des reactions immunes et d'hypersensibilite - Google Patents

Procede permettant de prevenir des reactions immunes et d'hypersensibilite Download PDF

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Publication number
WO2000040713A1
WO2000040713A1 PCT/CA2000/000015 CA0000015W WO0040713A1 WO 2000040713 A1 WO2000040713 A1 WO 2000040713A1 CA 0000015 W CA0000015 W CA 0000015W WO 0040713 A1 WO0040713 A1 WO 0040713A1
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Prior art keywords
antigen
ige
hrp
receptor
sensitized
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PCT/CA2000/000015
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English (en)
Inventor
Mary H. Perdue
Ping-Chang Yang
M. Cecilia Berin
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Mcmaster University
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Priority to AU19601/00A priority Critical patent/AU1960100A/en
Publication of WO2000040713A1 publication Critical patent/WO2000040713A1/fr

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    • 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
    • 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
    • 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 present invention is generally concerned with modulating the immune system, and in particular inhibiting allergic reactions, and more particularly methods and compositions for preventing immune hypersensitivity responses such as in food allergy or respiratory allergies.
  • the immune system is a complex, multifactorial defense system that protects the body from a wide range of infectious diseases including viruses, bacteria, parasites and fungi. Although critical for our survival, in certain instances, such as autoimmune disease, transplant rejection, allergies and inflammation, the immune system can be the cause of illness. Allergic diseases are common in Western countries, affecting more than 20 percent of the U.S. population with a cost of more than $10 billion each year (Kluger, H. (1998)), and are increasing in prevalence in most countries of the developed world (Woolcock and Peat, 1997). In such conditions it is desirable to suppress or tolerize the immune response. Extrinsic antigens are responsible for initiating pathophysiology in most cases of allergy.
  • the epithelium that theoretically acts as a barrier restricting passage of macromolecules (Robbins and Rennard (1997); Madara (1989)), clearly allows uptake of immunologically intact antigens to the site of tissue effector cells that are subsequently activated.
  • transepithelial transport of proteins normally takes approximately 20 min (Bomsel et al. (1989)); however, allergic symptoms may begin very rapidly after encounter with antigen (Bentley et al. (1997)).
  • the intestinal epithelium With respect to food allergy, one of the roles of the intestinal epithelium is to act as a barrier to limit the uptake of antigens and noxious materials from the gut lumen.
  • the intestinal epithelium (individual cells referred to as enterocytes) also plays an important role in sampling luminal contents and delivering antigens to the mucosal immune system. Small quantities of macromolecules are transported intact across the intestinal epithelium by endocytic uptake at the apical membrane and transcytosis through the cell (Sanderson and Walker (1993)). It is well understood that antigen delivered to the lamina intestinal does not normally generate an immune reaction (Strobel and Mowat (1998)). However, in allergic individuals, antigen that reaches mucosal effector cells results in a local hypersensitivity reaction involving stimulated ion and water secretion and enhanced motility (Crowe and Perdue (1992)).
  • IgE has been shown to be present in human intestinal secretions of individuals with food allergies (Belut et al. (1980)). In parasitized rats, the concentration of IgE was shown to be greater in intestinal secretions than in serum or mesenteric lymph (Negrao-Correa et al. (1996)), suggesting the possibility of an epithelial receptor for IgE.
  • Intestinal hypersensitivity reactions are mediated by antigen cross-linking of IgE antibodies bound to the high affinity receptor (FceRI) on mast cells (Metzger (1992)).
  • FceRI high affinity receptor
  • FceRIL low-affinity IgE receptor
  • B cells B cells, eosinophils, macrophages and dendritic cells
  • This receptor has also been shown by immunohistochemistry to be located on human intestinal epithelial cells, with maximal expression in patients with food allergies or inflammatory bowel disease (Kaiserlian et al. (1993)).
  • CD23/IgE can enhance the efficiency of antigen sampling and presentation in B cells by trapping antigen present at low concentrations (Bonnefoy et al. (1995)). Therefore, IgE is thought to provide an "antigen-focusing" function in such cells. Understanding the mechanism responsible for the rapid appearance of the physiological changes (within minutes after oral antigen challenge) in a hypersensitivity reaction would provide opportunities to prevent such reactions.
  • the present inventors have demonstrated that rapid local hypersensitivity reactions in the gut occur following transepithelial transport of specific antigen (the one responsible for the reaction) via IgE bound to epithelial cells by means of CD23 receptors.
  • the inventors have demonstrated this system in rats passively and actively sensitized to a model protein antigen, horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • Sensitization stimulates the expression of CD23 receptors on the apical membrane of enterocytes and their expression is reduced following antigen challenge due to internalization.
  • High power electron microscopy demonstrated co-localization of antigen and CD23 in endocytic vesicles inside epithelial cells.
  • CD23 A functional role for CD23 in antigen sampling has been shown by inhibition of enhanced epithelial antigen uptake by a specific antibody against CD23.
  • the present inventors have demonstrated the same type of rapid specific antigen transport across epithelial cells of the airway of specifically sensitized rats.
  • the present invention provides a method of modulating antigen uptake by CD23 receptors.
  • the invention provides methods of modulating an immune response, preferably inhibiting or preventing the response, more preferably inhibiting or preventing a hypersensitivity reaction.
  • the present invention in one embodiment provides a method of modulating an immune reaction arising through interaction of a CD 23 receptor and an IgE-antigen complex comprising administering an effective amount of an agent which interferes with the interaction, preferably the modulation is inhibition of the interaction.
  • the agent is an antibody to the CD 23 receptor.
  • the agent inhibits the expression of the CD23 receptor; is an antisense oligonucleotide that is complimentary to a nucleic acid sequence from the CD 23 receptor gene; is any non-antibody reagent which interacts with the CD23 receptor and inhibits IgE-antigen complex binding, preferably a non protein antagonist of IgE-antigen complex binding to CD23.
  • the agent interacts with the IgE-antigen complex. In respect of all such agents, the prefered modulation is to inhibit the interaction of CD 23 receptor and an IgE-antigen complex.
  • the method of the invention is in respect of a CD 23 receptor is localized in epithelial cells of the airway or gut, preferably the immune reaction is a hypersensitivity reaction, more preferably the hypersensitivity reaction is an allergy.
  • the epithelial cells are from the gut, a prefered allergy is a food allergy.
  • a preferred allergy is an airborne.
  • the immune reaction occurs in an animal, preferably a human.
  • the present invention has important implications for preventing or controlling allergic reactions by providing a method of interfering with antigen uptake at the level of the epithelium in all mucosal surfaces thereby reducing or preventing inappropriate immune responses including a hypersensitivity reaction.
  • a method of treating a hypersensitivity reaction in an animal comprising administering an effective amount of a substance which interferes with the internalization of CD23-IgE-antigen complexes into cells of the animal having CD23-IgE-antigen complexes.
  • the CD 23 receptor is localized in epithelial cells of the airway or gut of the animal, and the hypersensitivity reaction is an allergy.
  • the allergy is in the gut it is a food allergy, and when in the airway it is asthma.
  • a method of treating a hypersensitivity reaction in an animal comprising administering an effective amount of a substance which interferes with the transport of CD23-IgE-antigen complexes across cells of the animal having CD23-IgE-antigen complexes.
  • the CD 23 receptor is localized in epithelial cells of the airway or gut of the animal, and the hypersensitivity reaction is an allergy.
  • the allergy is in the gut it is a food allergy, and when in the airway it is asthma.
  • the present invention provides methods for assaying for a substance which modulates the interaction of CD23 receptor and IgE-antigen complex.
  • substances identified which modulate uptake of antigen by CD23 may be formulated into pharmaceutical compositions for administration to individuals suffering from disorders of the immune system.
  • the present invention also provides in another aspect compositions for use in modulating an immune reaction arising through interaction of a CD23 receptor and an IgE-antigen complex in an animal in need of modulation thereof.
  • Figures 1A, IB, and 1C show high power electron photomicrographs of antigen (HRP) uptake into enterocytes comparing epithelial cells from ( Figure 1A) rats actively sensitized to HRP; (Figure IB) rats actively sensitized to OVA; (Figure 1C) and naive controls.
  • Figure ID shows a graph illustrating a significant increase in the area of HRP endosomes in sensitized rats.
  • Figures 2A and 2B show high power electron photomicrographs of antigen (HRP) uptake into enterocytes in rats passively sensitized by injection with: (Figure 2A) high IgE containing serum; and (Figure 2B) non-immune serum.
  • Figure 2C shows a graph illustrating that IgE is required for the enhanced uptake of HRP.
  • Figure 3A is a low power photomicrograph illustrating immunohistochemical staining of a jejunal section from a control rat illustrating the apical membrane of enterocytes lining the intestine.
  • Figure 3B is a low power photomicrograph illustrating immunohistochemical staining of a jejunal section from a sensitized rat illustrating CD23 on the apical membrane of enterocytes lining the intestine.
  • Figure 4A is an extremely high power electron photomicrograph illustrating specific labeling with antibody-linked colloidal gold particles demonstrating CD23 along the microvilli of enterocytes in sensitized rats.
  • Figure 4B is an extremely high power electron photomicrograph illustrating an endosome within an enterocyte from a sensitized rat. The endosome is positive for both HRP and anti-CD23.
  • Figure 4C shows a graph illustrating that expression of CD23 is upregulated by sensitization and subsequently reduced by antigen challenge.
  • Figure 5 shows a graph of the hypersensitivity reaction (short-circuit current (Isc) and conductance (G) response) in sensitized tissues + treatment with increasing concentrations of B3B4 (antibody against the CD23 receptor). There is clear inhibition of the reaction by blocking the CD23 receptor.
  • Figure 6 is a drawing illustrating a histogram of area of HRP-containing endosomes in enterocytes of IL-4 (+/+) mice, either naive control mice or mice actively sensitized to HRP (HRP-sens) or OVA (OVA-sens).
  • Figure 7 is a drawing illustrating a histogram of area of HRP-containing endosomes in enterocytes of IL-4 (+/+) mice, either naive control mice or mice actively sensitized (AS) to HRP or passively sensitized (PS) with untreated serum (IgE (+)) or IgE depleted serum (IgE (-)).
  • Figure 8 is a drawing illustrating a histogram of area of HRP-containing endosomes in enterocytes of IL-4 (+/+) or IL-4 (-/-) mice, either naive control mice or mice actively sensitized (AS) to HRP.
  • Figure 9 is a drawing illustrating a histogram of area of HRP-containing endosomes in enterocytes of IL-4 (-/-) mice, either naive control mice or mice passively sensitized (PS) with whole (untreated) serum or serum depleted of IgE (IgE (-) serum) or neutralized with anti-IL-4 (IL-4 (-) serum).
  • PS passively sensitized
  • Figure 10 is a figure illustrating immunohistochemistry showing CD23 immunoreactivity on intestinal epithelial cells.
  • Figure 11 is a drawing illustrating a histogram of effect of anti-CD23
  • Figure 12 is a drawing illustrating a histogram of area of HRP-containing endosomes in enterocytes of CD23 (+/+) or CD23 (-/-) mice, either naive control mice or mice actively sensitized (AS) to HRP.
  • Figure 13 provides an illustration of representative tracings of short-circuit current in tracheas from: (i) naive control rats; (ii) OVA sensitized rats; and (iii) HRP sensitized rats.
  • Figure 14 panels A, B and C provide electron photomicrographs of mast cells in rat trachea lamina basement.
  • Figure 15 shows representative photomicrographs prepared from tracheal tissues fixed at 2 min (before hypersensitivity reaction) or 90 min (after hypersensitivity reaction) after addition of HRP to the luminal side of the trachea.
  • Figure 16 illustrates the effect of sensitization on uptake of HRP into tracheal epithelial cells in HRP sensitized versus control rats.
  • Figure 17 illustrates distribution of HRP endosomes within ciliated and non-ciliated epithelial cells.
  • Figure 18 shows a scanning electron photomicrograph from (A) naive controls, (B) OVA sensitized rats and (C) HRP sensitized rats.
  • Figure 19 illustrates the effect of sensitization and antigen challenge on mucosal to serosal flux of HRP.
  • Figure 20 provides photomicrographs of tracheal epithelial cells from an HRP sensitized rat at 2 min (A) or 90 min (B) after HRP challenge. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides a method of modulating an immune response, in an animal, to a specific antigen.
  • the method comprises administering an effective amount of an agent capable of interfering with the interaction between an IgE-antigen complex (where the antigen provokes the immune response), and CD23 receptors, to an animal in need thereof.
  • the expressions “interfere with” and “interfering with” and like expressions mean inhibiting, reducing, diminishing or blocking the interaction between IgE and CD23 receptors.
  • the term “interaction” as used herein means the binding, association, or complexing of a CD23 receptor with IgE-antigen, regardless of the degree of association between molecules, however the association is formed.
  • CD23 “CD23 receptor”, and “CD23/Fc ⁇ RII” all have the same meaning and refer to the receptor with low affinity for IgE. Therapeutic Uses
  • the present invention provides a method of modulating an immune reaction arising through interaction of a CD23 receptor and an IgE-antigen complex comprising administering an effective amount of an agent which interferes with the interaction to an animal in need thereof.
  • effective amount means an amount effective, at dosages and for periods of time necessary to achieve the desired results.
  • animal as used herein means any member of the animal kingdom.
  • the animal is a human and the CD23 receptors are located on epithelial cells.
  • the epithelial cells are located in the gut or airway passages, however, the present invention is applicable to any epithelium expressing CD23 receptors, including in all mucosal surfaces, whereby CD23 receptors bind IgE-antigen complex and rapidly transport the complex across the cell thereby providing antigen to result in an inappropriate immune response, including, without unduly limiting the foregoing, a hypersensitivity reaction.
  • the modulation of the immune reaction is inhibition and a preferred immune response is allergy.
  • the method of the present invention may also be used to treat or prevent an allergic reaction.
  • the immune system mounts an attack against a generally harmless, innocuous antigen or allergen.
  • Allergies that may be prevented or treated using the methods of the invention include, but are not limited to, hay fever, asthma, atopic eczema as well as allergies to poison oak and ivy, house dust mites, bee pollen, nuts, shellfish, penicillin and numerous others.
  • the present invention provides a method of preventing or treating an allergy comprising administering an effective amount of an agent which interferes with the interaction of CD23 receptors and IgE-antigen complex to an animal having or suspected of having an allergy.
  • the present invention provides a method of preventing a food allergy response in an animal to a specific antigen preferably nuts, eggs, fish, shellfish, meats, milk, soya, fruits, vegetables, cereals, tea, coffee, chocolate, synthetic chemicals, and food additives (it would be understood by those skilled in the art that almost any food containing protein can be included in this list, although some are more common) comprising administering to the gut, preferably prior to eating a food substance known or thought to trigger an allergic reaction, an effective amount of an agent capable of interfering with the interaction between antigen specific IgE complexed with the antigen and CD23 receptors of enterocytes of an animal expressing a hypersensitivity reaction to the antigen.
  • the animal is a human and the agent is an antibody.
  • the present invention provides a method of preventing a respiratory allergy response to a specific antigen or group of antigens, such as in hayfever or extrinsic asthma, in an animal comprising administering to the airway an effective amount of an agent capable of interfering with the interaction between antigen specific IgE complexed with the antigen and CD23 receptors of the epithelial cells of the airways of the animal in need thereof.
  • a specific antigen or group of antigens such as in hayfever or extrinsic asthma
  • an agent capable of interfering with the interaction between antigen specific IgE complexed with the antigen and CD23 receptors of the epithelial cells of the airways of the animal in need thereof.
  • the animal is a human and the agent is an antibody.
  • the agents are administered in a suitable pharmaceutical formulation.
  • the present invention has important implications for preventing or controlling allergic reactions by providing a method of interfering with antigen uptake at the level of the epithelium in all mucosal surfaces thereby reducing or preventing inappropriate immune responses including a hypersensitivity reaction.
  • a substance or substances which reduce or block the expression of CD23 receptors may also be identified by comparing the pattern and level of expression of the CD23 receptor, and /or the level of allergic response, in tissues and cells in the presence, and in the absence of the substance or substances.
  • a related aspect of the present invention and included within the scope of the present invention are substances which can suppress or inhibit the action of substances which increase expression of the CD23 receptor.
  • One substance which is shown herein to increase expression of the CD23 receptor is IL-4, accordingly, the present invention includes those substances capable of inhibiting the activity of IL-4 in respect of the CD23 receptor.
  • Part of this aspect includes substances which are regulators of promoter activity responsible for expression of CD23 and the pathways responsible for suppression or activation of CD23 are within the scope of the present invention
  • Substances which affect a CD23 promoter's activity may be identified by comparing the pattern and level of expression of a CD23 gene, in cells in the presence, and in the absence of the substance. Accordingly a method for assaying for the presence of an antagonist of CD23 promoter activity is provided comprising providing a cell containing a reporter gene under the control of the promoter with a substance which is a suspected antagonist under conditions which permit interaction and assaying for the decrease of reporter gene product. Accordingly there is provided a method for assaying for a substance which is an antagonist of CD23 promoter activity comprising:
  • the selected substance decreases product by blocking upregulation of the promoter.
  • the selected substance decreases product by downregulation of the promoter.
  • antibodies to CD23 receptors or antisense oligonucleotides complimentary to a nucleic acid encoding the CD23 receptor may be prepared as discussed below.
  • the present invention provides methods for identifying substances which interfere with the internalization and transport of
  • CD23-IgE-antigen complexes and which may be used in the treatment of conditions involving CD23 mediated transport of IgE antibody bound to specific antigen.
  • agents which are capable of interfering with the interaction of CD23 receptors and IgE-antigen complex are contemplated.
  • Agents that inhibit the activity of the CD23 receptor include antibodies to CD23 receptor.
  • Agents that inhibit the expression of the CD23 gene include antisense oligonucleotides to a CD23 nucleic acid sequence. Described in further detail below are examples of such agents including methods of their preparation.
  • Antibodies that bind a CD23 receptor can be prepared using techniques known in the art such as those described by Kohler and Milstein, Nature 256, 495 (1975) and in U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993 which are incorporated herein by reference. (See also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are also incorporated herein by reference).
  • antibodies are understood to include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab') 2 ) and recombinantly produced binding partners.
  • Antibodies are understood to be reactive against the receptor encoded by the nucleic acid molecule of the CD23 receptor of the animal to be treated if they bind to the receptor with an affinity of greater than or equal to 10" 6 M.
  • antibodies may be developed which not only bind to the protein, but which bind to a regulator of the protein, and which also block the biological activity of the protein.
  • Polyclonal antibodies may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, various fowl, rabbits, mice, or rats. Briefly, a CD23 receptor, or portions thereof, may be used to immunize an animal. An animal may be immunized through intraperitoneal, intramuscular, intraocular, or subcutaneous injections, in conjunction with an adjuvant such as Freund's complete or incomplete adjuvant. Following several booster immunizations, samples of serum are collected and tested for reactivity to the protein. Particularly preferred polyclonal antisera will give a signal on one of these assays that is at least three times greater than background. Once the titer of the animal has reached a plateau in terms of its reactivity to the receptor protein, larger quantities of antisera may be readily obtained either by weekly bleedings, or by exsanguinating the animal.
  • Monoclonal antibodies may also be readily generated using conventional techniques as described herein.
  • hybridoma cell lines are prepared by a process involving the fusion under appropriate conditions of an immortalizing cell line and spleen cells from an animal appropriately immunized to produce the desired antibody.
  • Immortalizing cell lines may be murine in origin however, cell lines of other mammalian species may be employed including those of rat, bovine, canine, human origin, and the like.
  • the immortalizing cell lines are most often of tumor origin, particularly myeloma cells but may also include normal cells transformed with, for example, Epstein Barr Virus. Any immortalizing cell may be used to prepare the hybridomas of the present invention.
  • Antibody producing cells may be employed as fusion partners such as spleen cells or peripheral blood lymphocytes.
  • the animal from which the cells are to be derived may be immunized at intervals with peptides derived from CD23 receptors.
  • the immortalizing cells and lymphoid cells may be fused to form hybridomas according to standard and well-known techniques employing polyethylene glycol as a fusing agent. Alternatively, fusion may be accomplished by electrofusion.
  • Hybridomas are screened for appropriate monoclonal antibody secretion by assaying the supernatant or protein purified from the ascites for reactivity using the method described herein.
  • the hybridomas are screened for antibodies which have the desired properties e.g. interfere with the binding site of the CD23 receptor and inhibit binding with IgE-antigen. The binding site of the receptor may vary depending upon the IgE-antigen complex under consideration.
  • the monoclonal antibodies produced by the hybridoma cell lines of the invention are also part of the present invention. It is understood that immunoglobulins may exist in acidic, basic, or neutral form depending on their amino acid composition and environment, and they may be found in association with other molecules such as saccharides or lipids.
  • the monoclonal antibodies produced by hybridoma cell lines of the invention may be directed against one or more epitopes of the CD23 receptor. Any characteristic epitope associated with a CD23 receptor may provide the requisite antigenic determinant. It is contemplated that monoclonal antibodies produced by the hybridoma cell lines fall within the scope of the present invention so long as they remain capable of selectively reacting with peptides from the CD23 receptor.
  • antibody as used herein is intended to include fragments thereof which also specifically react with a CD23 receptor, or peptide thereof, having the activity of the CD23 receptor.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments.
  • Chimeric antibody derivatives i.e., antibody molecules that combine a non-human animal variable region and a human constant region are also contemplated within the scope of the invention.
  • Chimeric antibody molecules can include, for example, the antigen binding domain from an antibody of a mouse, rat, or other species, with human constant regions.
  • Conventional methods may be used to make chimeric antibodies containing the immunoglobulin variable region which recognizes the gene product of CKIP-1 antigens of the invention (See, for example, Morrison et al., Proc. Natl Acad. Sci. U.S.A. 81,6851 (1985); Takeda et al., Nature 314, 452 (1985), Cabilly et al., U.S. Patent No.
  • Monoclonal or chimeric antibodies specifically reactive with a CD23 receptor as described herein can be further humanized by producing human constant region chimeras, in which parts of the variable regions, particularly the conserved framework regions of the antigen-binding domain, are of human origin and only the hypervariable regions are of non-human origin.
  • Such immunoglobulin molecules may be made by techniques known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279 (1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)), and PCT Publication WO92/06193 or EP 0239400).
  • Humanized antibodies can also be commercially produced (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.)
  • Specific antibodies, or antibody fragments, reactive against CD23 receptors may also be generated by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria with peptides of CD23 receptors.
  • complete Fab fragments, VH regions and FV regions can be expressed in bacteria using phage expression libraries (See for example Ward et al., Nature 341, 544-546: (1989); Huse et al., Science 246, 1275-1281 (1989); and McCafferty et al. Nature 348, 552-554 (1990)).
  • phage expression libraries See for example Ward et al., Nature 341, 544-546: (1989); Huse et al., Science 246, 1275-1281 (1989); and McCafferty et al. Nature 348, 552-554 (1990)).
  • SCID-hu mouse for example the model developed by Genpharm, can be used to produce antibodies, or fragments thereof.
  • Antisense oligonucleotides that are complimentary to a nucleic acid sequence from a CD23 receptor gene can also be used in the methods of the present invention to inhibit CD-23 receptors. Accordingly, the present invention provides a method of preventing or reducing immune reactions arising by virtue of interaction between CD 23 receptors and IgE-antigen complexes comprising administering an effective amount of an antisense oligonucleotide that is complimentary to a nucleic acid sequence from a CD 23 receptor gene to an animal in need thereof.
  • the term "antisense oligonucleotide” as used herein means a nucleotide sequence that is complimentary to its target.
  • oligonucleotide refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages.
  • the term also includes modified or substituted oligomers comprising non-naturally occurring monomers or portions thereof, which function similarly. Such modified or substituted oligonucleotides may be preferred over naturally occurring forms because of properties such as enhanced cellular uptake, or increased stability in the presence of nucleases.
  • the term also includes chimeric oligonucleotides which contain two or more chemically distinct regions. For example, chimeric oligonucleotides may contain at least one region of modified nucleotides that confer beneficial properties (e.g. increased nuclease resistance, increased uptake into cells), or two or more oligonucleotides of the invention may be joined to form a chimeric oligonucleotide.
  • the antisense oligonucleotides of the present invention may be ribonucleic or deoxyribonucleic acids and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil.
  • the oligonucleotides may also contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines,
  • antisense oligonucleotides of the invention may contain modified phosphorous, oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
  • the antisense oligonucleotides may contain phosph oroth i oa tes, p h osph otri esters, m eth yl p h osph onates, a nd phosphorodithioates.
  • phosphorothioate bonds link all the nucleotides.
  • the antisense oligonucleotides of the invention may also comprise nucleotide analogs that may be better suited as therapeutic or experimental reagents.
  • An example of an oligonucleotide analogue is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides (P.E. Nielsen, et al Science 1991, 254, 1497). PNA analogues have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro.
  • PNA peptide nucleic acid
  • oligonucleotides may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones.
  • the nucleotides may have morpholino backbone structures (U.S. Pat. Nol 5,034, 506).
  • Oligonucleotides may also contain groups such as reporter groups, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an antisense oligonucleotide.
  • Antisense oligonucleotides may also have sugar mimetics.
  • the antisense nucleic acid molecules may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • the antisense nucleic acid molecules of the invention or a fragment thereof may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with mRNA or the native gene e.g. phosphorothioate derivatives and acridine substituted nucleotides.
  • the antisense sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.
  • the antisense oligonucleotides may be introduced into tissues or cells using techniques in the art including vectors (retroviral vectors, adenoviral vectors and DNA virus vectors) or physical techniques such as microinjection.
  • the antisense oligonucleotides may be directly administered in vivo or may be used to transf ect cells in vitro which are then administered in vivo.
  • the antisense oligonucleotide may be delivered to macrophages and /or endothelial cells in a liposome formulation. Screening for Other Agents
  • the invention provides a method for assaying for a substance which modulates the interaction of
  • CD23 receptor and IgE-antigen complex comprising:
  • the CD23 receptor is preferably localized in epithelial cells of the airway or gut of an animal, and may be in the animal, but, for the purposes of identifying substances as discussed herein, are preferably in vitro on epithelial cells, or, immobilized on for example agarose or other appropriate medium as disussed below.
  • Substances which affect CD23 receptor activity can be identified based on their ability to bind to the CD23 receptor. Therefore, the invention also provides methods for identifying substances which are capable of binding to the CD23 receptor. In particular, the methods may be used to identify substances which are capable of binding to, and blocking the activity of, i.e., inactivating the CD23 receptor.
  • Substances which can bind with a CD23 receptor may be identified by reacting the CD23 receptor with a substance which potentially binds to the CD23 receptor, and assaying for complexes, for free substance, or for non-complexed CD23 receptor.
  • a yeast two hybrid assay system may be used to identify proteins which interact with the CD23 receptor (Fields, S. and Song, O., 1989, Nature, 340:245-247).
  • Conditions which permit the formation of substance and CD23 receptor complexes may be selected having regard to factors such as the nature and amounts of the substance and the protein.
  • the substance-protein complex, free substance or non-complexed proteins may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof.
  • antibody against the CD23 receptor or the substance, or labelled CD23 receptor, or a labelled substance may be utilized.
  • the antibodies, proteins, or substances may be labelled with a detectable substance as described above.
  • the invention further provides a method for assaying for a substance that antagonizes CD23 receptor intracellular trafficking comprising administering to a non-human animal or to a tissue of an animal, a substance suspected of antagonizing
  • the present invention provides a method for assaying for a substance which modulates the internalization of CD23-IgE-antigen complex into cells of an animal having CD23-IgE-antigen complexes comprising:
  • the invention also provides a method for assaying for a substance which modulates the transcellular transport of CD23-IgE-antigen complex in cells of an animal having CD23-IgE-antigen complexes comprising:
  • transcellular transport includes endosomal transcellular transport. Agents affecting such transport include those which act on microtubules and/or microfilaments. Further it is readily apparent that any substance selected per se according to any of the methods outlined herein is within the scope of the present invention.
  • the CD23 receptor, or the substance used in the methods of the invention may be insolubilized.
  • the CD23 receptor or substance may be bound to a suitable carrier.
  • suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc.
  • the carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc.
  • the insolubilized protein or substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.
  • the reagents suitable for applying the methods of the invention to identify substances that affect the CD23 receptor-IgE-antigen complex interaction may be packaged into convenient kits providing the necessary materials packaged into suitable containers.
  • the kits may also include suitable supports useful in performing the methods of the invention.
  • the present invention provides a composition for use in modulating an immune reaction arising through interaction of a CD 23 receptor and an IgE-antigen complex in an animal in need of modulation thereof comprising an agent which modulates the reaction.
  • the agent is: (a) an antibody specific for a CD 23 receptor; (b) antisense nucleic acid molecules complimentary to the CD 23 receptor gene; (c) a protein or non-protein reagent which interacts with the CD 23 receptor; (d) a substance which interacts with the IgE-antigen complex; or (e) a substance which modulates IL-4 activity.
  • the modulation of the immune reaction is inhibition of the interaction between a CD 23 receptor and an IgE-antigen complex.
  • the agent includes any non-antibody reagent which binds with or interacts with a CD23 receptor or IgE-antigen complex which inhibits interaction of a CD 23 receptor and an IgE-antigen complex (eg, a non protein antagonist).
  • any composition contemplated herein may comprise more than one agent of the invention.
  • All of the above described substances may be formulated into pharmaceutical compositions for adminstration to subjects in a biologically compatible form suitable for administration in vivo.
  • biologically compatible form suitable for administration in vivo is meant a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects.
  • the substances may be administered to living organisms including humans, and animals.
  • animal includes all members of the animal kingdom.
  • a therapeutically active amount of the pharmaceutical compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • a therapeutically active amount of a substance may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage procedures may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • the active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, intranasal, transdermal application, or rectal administration. Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.
  • the active substance is a nucleic acid encoding an antisense oligonucleotide it may be delivered using techniques known in the art.
  • Recombinant molecules comprising an antisense sequence or oligonucleotide may be directly introduced into cells or tissues in vivo using delivery vehicles such as retroviral vectors, adeno viral vectors and DNA virus vectors. They may also be introduced using physical techniques such as microinjection and electroporation or chemical methods such as co-precipitation and incorporation of DNA into liposomes.
  • compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
  • Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
  • the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • a composition according to the invention is preferably administered in situations where the immune reaction is a hypersensitivity reaction, preferably an allergy.
  • the allergy is in the gut, it is a food allergy; where the allergy is localized in epithelial cells of the airway, it is an allergy of the airway.
  • Serum for passive sensitization was generated in high responder Brown-Norway rats (250-300 g, Harlan Sprague-Dawley Inc., Indianapolis, Indiana) sensitized to HRP as above, with a boost injection (10 ⁇ g HRP) at day 28. Serum was collected at day 32, pooled and stored at -70°C. This serum had an anti-HRP titer of 1:1024 by passive cutaneous anaphylaxis. Sprague-Dawley rats were passively sensitized by i.p. injection of 1.5 ml of this serum that was either untreated, heat treated at 56°C for 2 h to destroy heat-labile IgE antibodies, or depleted of IgE by immunoprecipitation.
  • IgE antibodies were removed from rat serum by immunoprecipitation with monoclonal mouse anti-rat IgE (MARE-1, Serotec, Raleigh, NC) coupled to Sepharose 4B beads. Serum was incubated overnight at 4°C overnight, and centrifuged to remove the IgE/bead complexes. IgE titres were tested before and after immunoprecipitation by passive cutaneous anaphylaxis (Ovary (1964)). Rats injected with serum from naive rats served as controls. Experiments were conducted 3 days after passive/sham sensitization. All animal experiments were conducted with approval from the McMaster University Animal Care Committee. Ussing Chamber Studies
  • Rats were anesthetized and a 10-15 cm segment of jejunum was removed beginning 5-10 cm distal to the ligament of Treitz.
  • the external muscle layer was stripped off and mucosal sheets were mounted in Using chambers (surface area 0.6 cm 2 ).
  • the tissue was bathed in 10 ml of oxygenated Krebs buffer (in mM: 115.0 NaCl, 8.0 KCI, 1.25 CaCl 2 , 1.2 MgCl 2 , 2.0 KH 2 P0 4 , and 25.0 NaHC0 3 , pH 7.35 + 0.02, 37°C).
  • the buffer in the serosal compartment contained 10 mM glucose and was osmotically balanced by 10 mM mannitol in the luminal compartment of the chamber.
  • Tissues were short-circuited at zero volts with an automated voltage clamp (WPI Instruments, Narco Scientific, Mississauga, ON, Canada), and the short-circuit current (Isc, mA/cm 2 ) was continuously monitored as an indication of net ion transport. At intervals, the Isc was turned off and the spontaneous potential difference was recorded. Tissue conductance (in mS/cm 2 ) was calculated according to Ohm's Law. Tissues were allowed to equilibrate for 20 min before addition of 5 x 10" 5 M HRP to the apical compartment. Two min after HRP addition, tissues were removed and fixed for electron microscopy.
  • Tissues were fixed in 2% glutaraldehyde in 0J M sodium cacodylate buffer (pH 7.4), washed, incubated in 3,3'-diaminobenzadine tetrahydrochlorine (Sigma) and H 2 0 2 , and subsequently processed for transmission electron miscroscopy. Ultrathin sections of mid-villus epithelium (cut in the longitudinal plane) were stained with uranyl acetate and lead citrate. Photomicrographs of epithelial cells were taken at a magnification of 8,000.
  • the high affinity receptor for IgE (Fc ⁇ RI) is restricted to mast cells and basophils due to the necessity of the beta chain for receptor stability and transport to the cell surface, while in humans, expression requires only the alpha and gamma chains and distribution is more extensive (Kinet 1999).
  • Fc ⁇ RI the high affinity receptor for IgE
  • the low affinity receptor (Fc ⁇ RII/CD23) has been identified on epithelial cells in the intestine (Kaiserlian et al. 1993) and airways (Campbell et al. 1994). Therefore, we determined the effect of sensitization of rats on epithelial expression of CD23.
  • the antibody was isolated from culture supernatant by ammonium sulfate precipitation followed by affinity purification in a protein G-Sepharose column (Pharmacia Biotech, Uppsala, Sweden). In preliminary experiments, we confirmed that this antibody similarly identified CD23 expression on rat as well as mouse splenic B cells.
  • the B3B4 antibody was used for immunohistochemical detection of CD23 on intestinal epithelial cells. Light Microscopy
  • Tissues were fixed in 2% paraformaldehyde mixed with 0.75% glutaraldehyde for 3 h at room temperature. Dehydration was carried out with a series of graded ethanols. The specimens were saturated in 50% (3 h), 75% (3 h) and 100% (overnight) LR White at 4%C and embedded with LR White and polymerized in a freezer under UV radiation at -10°C for 24 h. Ultrathin sections were cut (98 nm) and placed on formvar coated nickel grids. The grids were treated with 1% BSA mixed with 5% rabbit serum for 30 min, incubated with primary antibody for 1 h at room temperature, then incubated with gold-conjugated rabbit anti-rat IgG antibody for 1 h.
  • the grids were washed 6 times in PBS after each incubation step. Sections were post-fixed by exposing the grids to osmium tetroxide vapour for 5 min. They were then stained with uranyl acetate and lead citrus. Sections were observed with the electron microscope, and photomicrographs were prepared. Immunogold labels were counted on the microvillus membrane of epithelial cells and expressed per 100 ⁇ m. Controls for electron microscopy included sections where the B3B4 primary antibody was omitted or replaced with an irrelevant isotype-matched antibody. Effect of Blocking CD23 on Antigen Uptake
  • B3B4 was injected i.p. into sensitized rats (1 mg on each of 3 days prior to the experiment).
  • B3B4 antibody (10-40 ⁇ g/ml) or isotype control antibody was added to the mucosal buffer bathing tissues from rats actively sensitized to HRP.
  • tissues were removed 2 minutes after challenge and processed to identify HRP in tissues. The area of HRP-containing endosomes in enterocytes was measured in 12 windows/rat group. Effect of Blocking CD23 on the Hypersensitivity Reaction
  • Immunohistochemical staining of rat jejunal sections with anti-CD23 mAb was performed on intestinal sections from actively sensitized and naive rats. In sections from sensitized rats, immunoreactivity was most prominent in the brush borders of enterocytes, with lighter staining in the apical portion of these cells ( Figure 3 A, and B). Almost all epithelial cells on the villi were labeled, including enterocytes and goblet cells ( Figure 3A). Some lamina intestinal cells were also positive for CD23 immunoreactivity. In contrast, in sections from control rats, immunoreactivity was localized to only a few cells in the epithelial layer and some in the laminalitis (Figure 3B).
  • Antigen challenge causes a reduction in surface expression of CD23
  • Antigen challenge significantly (p ⁇ 0.001) reduced expression of CD23 on the surface of the enterocyte microvilli, but CD23 expression was still significantly greater than in controls (P ⁇ 0.001) ( Figure 4C).
  • Anti-CD23 pre-treatment of tissues reduced the total area of HRP endosomes inside enterocytes in a concentration-dependent manner (Table 1). At the highest concentration of antibody used (40 ⁇ g/ml), the value for the area of HRP-containing endosomes in sensitized rats was similar to the value in control unsensitized rats ( Figure ID). This data provides functional evidence for epithelial antigen sampling through a CD23-mediated mechanism.
  • Anti-CD23 Antibodies Inhibit the Intestinal Hypersensitivity Reaction
  • Anti-CD23 antibody added in excess to the luminal surface of epithelial cells before antigen challenge also inhibited functional changes characteristic of the intestinal hypersensitivity reaction (Figure 5).
  • the Isc response to HRP challenge of tissues from sensitized rats was reduced, and the magnitude of the inhibition depended on the concentration of antibody used. In fact, at the highest concentration (40 ⁇ g/ml), there was no secretory response at all to luminal antigen.
  • Figure 5 also shows that the rise in conductance was similarly inhibited by the antibody.
  • mast cells bear IgE receptors and reside in close proximity to epithelial cells in intestinal tissues, it was possible that mast cells were responsible for the identification of specific antigen. Therefore, similar studies in mast cell-deficient Ws/Ws rats were conducted (Berin et al. 1998). It was confirmed that these animals had no mast cells in the intestinal tract, but produced normal levels of IgE antibodies. Jejunal tissues from sensitized Ws/Ws rats also demonstrated enhanced epithelial uptake of HRP, but did not display any hypersensitivity reaction based on the complete lack of Isc or conductance response following luminal antigen challenge.
  • phase I early phase
  • phase IT phase I
  • antigen is transported via the transcellular route in endosomes which rapidly traverse epithelial cells; this phase in inducted by sensitization and is specific for the sensitizing antigen, but is mast cell-independent.
  • phase II begins after mast cell activation and involves recruitment of the paracellular route which amplifies the barrier defect, resulting in non-specific uptake of antigens and other luminal molecules.
  • the experiments outlined in the example establish the mechanism responsible for the first phase of specific antigen uptake across the intestinal epithelium.
  • the first phase may be the most crucial in delivering antigen into the body, we determined the effect of inhibiting phase I antigen transport on the hypersensitivity reaction.
  • IgE is present in human intestinal secretions of individuals with food allergies (Belut et al. 1980). Impressively, in parasitized rats, the concentration of IgE (but not antibodies of other isotypes) was shown to be greater in intestinal fluid than in serum or mesenteric lymph (Negrao-Correa et al. 1996), suggesting a receptor-mediated mechanism for transepithelial transport of IgE into the lumen. In this study, enhanced epithelial uptake from the lumen of a model antigen, HRP,was documented in rats sensitized to the specific antigen compared with those sensitized to an irrelevant protein, OVA, or naive rats. This finding also implicates an immunoglobulin recognition system at the level of the epithelium.
  • Intestinal hypersensitivity reactions are mediated by antigen cross-linking of IgE antibodies bound to its high affinity receptor (Fc ⁇ RI) on mast cells.
  • Fc ⁇ RI high affinity receptor
  • This receptor has not been demonstrated on epithelial cells.
  • the low-affinity IgE receptor (Fc ⁇ RII, also known as CD23) has been identified on the apical membrane of enterocytes in biopsies from humans, with increased expression in individuals with food allergy and inflammatory bowel disease (Kaiserlian et al. 1993). CD23 has also been demonstrated on airway epithelial cells in asthmatics (Campbell et al. 1994). Therefore, we determined the effect of sensitization of rats on epithelial expression of CD23.
  • the B3B4 anti-CD23 antibody used in the expression studies has been well-characterized as identifying CD23 on mouse B cells and blocking binding of IgE to its receptor (Rao et al. 1999). There is a high degree of homology between mouse and rat (and even human) CD23 (Conrad et al. 1994). It was confirmed that the B3B4 antibody similarly identifies CD23 on splenic B cells from rats and mice. Immunohistochemistry of light microscopic sections showed minimal CD23 expression on epithelial cells in control animals, although there was some positive staining in the lamina limbal. In sensitized rats, CD23 immunoreactivity on enterocytes was dramatically enhanced; the expression appeared to be mainly on and immediately below the apical membrane.
  • IL-4 (-/-) mice were bred in the Central Animal Facility at McMaster University (original breeders from Harvard School of Medicine); CD23 (-/-) mice (C57/BL6 background) were bred at McMaster University (original breeders from University of Virginia).
  • IL-4 (+/+) (BALB/c) and CD23 (+/+) (C57/BL6) controls were purchased from Harlan Breeding Laboratories. Mice were used at -10 weeks of age. All animal experiments were conducted with the approval from the McMaster University Animal Care Committee. Mice were actively sensitized to horseradish peroxidase (HRP) (type
  • mice were passively sensitized by ip injection of 0.5 ml of high IgE-containing serum (titer 1:1024) obtained from actively sensitized and boosted Brown Norway rats, a high responder strain.
  • the serum was either untreated, heated (56° for 2 h) to destroy heat labile IgE or depleted of IgE by passing the serum through a column containing sepharose 4B beads conjugated to anti-IgE (MARE-1, anti-rat IgE heavy chain, Serotec).
  • MARE-1 anti-rat IgE heavy chain, Serotec
  • IL-4 was neutralized by incubating serum with anti-IL-4 antibody (rabbit anti-rat, Biosource) for 1 hour at 22°. Naive mice injected with serum from non-sensitized rats served as controls. Experiments were conducted 2-3 days post passive sensitization. Ussing Chambers
  • the tissue was pulsed with 1 mV for a duration of 1 s.
  • the change in Isc caused by the pulse was used to determine the conductance (G, in mS/cm 2 ) according to Ohm's law.
  • Tissues were allowed to equilibrate for 15 min until the Isc stabilized.
  • HRP (5 X 10 "5 M) was added to the mucosal buffer at 30 min. Antigen uptake by enterocytes
  • Tissues were removed from the Ussing chamber at various times after HRP challenge and processed for electron microscopy to determine the route and amount of HRP uptake in the epithelium.
  • Tissues were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h at room temperature, left in 0.1 M sodium cacodylate buffer overnight at 4°C and then washed three times for 5 min in 0.05 M Tris buffer (pH 7.6).
  • Tissues were incubated for 30 min at 20° in 3,3'- diaminobensidine tetrahydrochlorine (Sigma) (5 mg in 10 ml of 0.05 M Tris buffer and 0.01% H 2 0 2 .
  • mice were killed by cervical dislocation and a laparotomy was performed.
  • the spleen was removed and meshed through a metal screen to isolate single cells.
  • Erythrocytes were lyzed with ammonium chloride potassium buffer and the remaining cells were washed and incubated in 24 well plates with with PHA (10 mg/ml) or ConA (10 mg/ml) for 48 hrs.
  • the supernatant was collected and tested for IL-4 by ELISA (Biosource). The limit detection level was 5 pg/ml.
  • Jejunal tissues from actively sensitized BALB/c mice were mounted in Ussing Chambers where basal parameters were determined. Baseline conductance values of tissues from naive and sensitized mice were not statistically different (Table 2), indicating that intestinal permeability to ions in the two groups was comparable. Jejunal tissues were then challenged with HRP antigen added to the mucosal (luminal) buffer. A rise in Isc, indicating the secretory response characteristic of the hypersensitivity reaction, began after 2 min. Therefore, to examine antigen uptake in enterocytes before the hypersensitivity reaction, tissues were removed at 2 min and immediately fixed for electron microscopy. A dramatic increase in HRP uptake was found in jejunum from sensitized mice compared with controls ( Figure 6).
  • Enhanced antigen uptake in enterocytes of sensitized mice is dependent on IgE.
  • the rate of antigen transport across epithelial cells was also enhanced in both actively and passively sensitized mice.
  • the percent of enterocytes containing HRP positive endosomes in all cell regions was significantly greater in actively sensitized mice vs controls (apical: 71.3 ⁇ 4.6 % vs 18 ⁇ 1.4 %*, mid: 25.5 ⁇ 2.4 % vs 2.3 ⁇ 0.5 %*, basal: 17.5 ⁇ 1.3 % vs 0 ⁇ 0 %*, lamina intestinal: 6.3 ⁇ 1.8 % vs 0 ⁇ 0 %*; p ⁇ 0.05) (Table 3).
  • mice were similarly significantly higher for passively sensitized mice (apical: 58.5 ⁇ 4.5 %; mid: 32.0 ⁇ 0.7; basal: 29.5 ⁇ 5.0 %; lamina basement membrane: 21.5 + 2.6 %).
  • This result indicates that in sensitized mice, antigen was more rapidly transport across intestinal epithelial cells.
  • mice that were injected with IgE-depleted serum showed a slower rate of antigen transport, with the majority of HRP endosomes being confined to the apical region (Table 3), a situation comparable to that in naive control mice.
  • IL-4 is required for enhanced transepithelial antigen transport in sensitized mice.
  • IL-4 is a cytokine produced in excess in allergic conditions.
  • HRP uptake into enterocyte endosomes was compared in IL-4 (+/+) and IL-4 (-/-) mice actively sensitized to HRP.
  • the absence of IL-4 production in IL-4 (-/-) mice was confirmed by ELISA for IL-4 in culture media from spleen cells (undetectable for both naive and sensitized IL-4 (-/-) mice vs 39.3 ⁇ 1.5 pg/ml for sensitized IL-4 (+/+) mice).
  • mice The area of endosomes in both sensitized and non-sensitized IL-4 (-/-) mice was not different from values in naive control IL-4 (+/+) mice, indicating that IL-4 was required for the enhanced antigen uptake in enterocytes of actively sensitized mice.
  • actively sensitized IL-4 (-/-) mice did not demonstrate an enhanced rate of transport compared with naive mice (Table 3).
  • the absence of increased epithelial antigen uptake may have been due simply to lack of IgE in these mice. In fact, no IgE at all was measured in serum from these mice. Therefore, experiments were conducted in passively sensitized IL-4 (-/-) mice.
  • CD23/Fc ⁇ RII the low affinity IgE receptor
  • Fc ⁇ RI the high affinity receptor
  • IL-4 was shown to upregulate the expression of CD23 on B cells and monocytes. Therefore, we postulated that CD23 on mouse enterocytes is involved in enhanced antigen uptake. Expression of CD23 was examined by immunohistochemistry.
  • Immunohistochemistry showed a very low-level expression of CD23 on the apical membrane of enterocytes in non-sensitized naive mice (Figure 10B).
  • the expression of CD23 was upregulated by sensitization, shown by increased staining on apical membrane as well as in intracytoplasmic regions of enterocytes in actively sensitized mice ( Figure 10A).
  • No staining of CD23 was seen on the apical membrane of epithelial cells in actively sensitized IL-4 (-/-) mice (not shown).
  • Enhanced transepithelial antigen uptake in sensitized mice is inhibited by anti-CD23 antibody.
  • mice that lack CD23 were used to investigate the role of CD23 in enhanced antigen uptake.
  • Baseline conductance values were similar for jejunal tissues from naive and actively sensitized CD23 (+/+) mice and CD23 (-/-) mice (Table 4).
  • the titer of IgE in serum measured by passive cutaneous anaphylaxis was 1:256 (median value) for actively sensitized CD23 (-/-) versus 1:128 (median value) for actively sensitized CD23 (+/+) mice.
  • Ws/Ws mast cell deficient rats that were obtained by breeding male and female Ws/+ heterozygous rats (breeding colony at McMaster University, original rats provided by Dr. Y. Kitamura, Osaka University Medical School, Japan).
  • Ws/Ws rats have a 12-base deletion in the tyrosine kinase domain of the c-kit gene (Tsujimura et al. (1991)) that results in a lack of melanocytes, erythrocytes, and mast cells (Niwa et al. (1991)). All animal experiments were conducted with approval from the McMaster University Animal Care Committee.
  • Rats were sensitized to HRP (type II, Sigma Chemical Co, St. Louis, MO) by sc injection of 1 mg protein in 1 ml alum, plus i.p. injection of 1 ml Bordetella pertussis vaccine (Connaught Laboratories, Mississauga, ON, Canada).
  • HRP type II, Sigma Chemical Co, St. Louis, MO
  • sc injection of 1 mg protein in 1 ml alum plus i.p. injection of 1 ml Bordetella pertussis vaccine (Connaught Laboratories, Mississauga, ON, Canada).
  • OVA ovalbumin
  • Naive control rats were injected with saline. Fourteen days after sensitization, the rats were anesthetized with urethane and the trachea was removed and immediately immersed in warmed oxygenated Krebs buffer.
  • the buffer in the serosal compartment contained 10 mM glucose and was osmotically balanced by 10 mM mannitol in the luminal compartment of the chamber.
  • Tissues were short-circuited at zero volts with an automated voltage clamp (WPI Instruments, Narco Scientific, Mississauga, ON, Canada), and the short-circuit current (Isc, ⁇ A/cm 2 ) was continuously monitored as an indication of net ion transport.
  • the circuit was opened at 10 min intervals to obtain the PD, and tissue conductance (G, mS/cm 2 ) was calculated according to Ohm's law. Tissues were allowed to equilibrate for 20 min before baseline values were recorded. Tissues with abnormal baseline values of Isc or conductance were considered damaged and were excluded. Flux of HRP
  • HRP was added to the luminal buffer at a final concentration of 5 x 10 "5 M.
  • the buffers were sampled at 30 min intervals for 90 min.
  • the HRP concentration in the samples was determined by assaying enzyme activity using a modified
  • tracheal tissues were obtained from all groups of rats for electron microscopy (EM) at 2 and 90 min after HRP addition to the luminal buffer. Tissues were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h at room temperature, washed and left overnight (at 4°C) in the same buffer, and washed 3 times for 5 min each in 0.05 M Tris buffer (pH 7.6).
  • Tissues were incubated for 30 min in 5 mg of 3,3'-diaminobenzadine tetrahydrochlorine (Sigma) in 10 ml 0.05 M Tris buffer and 0.01% H 2 0 2 (pH 7.6, 22°C), and were subsequently processed for routine transmission EM. Tissues were cut to obtain longitudinal sections of epithelium. Ultrathin sections were placed on copper grids, stained with uranyl acetate and lead citrate, and observed with a transmission electron microscope. EM photomicrographs of tracheal epithelial cells were taken at magnification of 8,000.
  • the area of HRP-containing endosomes was measured within ciliated and non-ciliated epithelial cells using a computerized image processing system and expressed as area ( ⁇ m 2 )/ window (206 ⁇ m 2 ). Twenty electron photomicrographs (one window per photomicrograph with each window convering 1-2 cells) were analyzed for each rat, 60 for each experimental group all analyzed by the same observer who was unaware of the treatment group. In addition, relatively low magnification transmission EM (20 views per rat) was used to determine the relative number of mast cells in the tracheal epithelium (expressed per 1000 epithelial cells) and high magnification was used to evaluate their state of activation.
  • Scanning EM was used to determine the relative number of ciliated versus non-ciliated cells in the epithelium after sensitization.
  • Specimens from the distal trachea (just distal to the segment used for transport studies) were fixed in 2% gluteraldehyde for 2 h at room temperature. After rinsing with cacodylate buffer, the specimens were post-fixed in 1% osmium tetroxide for 1 h. This was followed by dehydration in a graded series of concentrations of ethanol and freeze dried in liquid carbon dioxide. The specimens were then coated with gold and examined under a scanning electron microscope. Ciliated cells in the surface epithelium were counted in windows measuring 7000 ⁇ m 2 at a magnification of 2,000. At least 20 views were evaluated per rat. Statistics
  • mast cells/ 1000 epithelial cells were counted in tracheal sections from all 3 rat groups.
  • tracheas from naive control rats there were 11.0 ⁇ 1.5 (mean ⁇ Standard Erro (SE)) mast cells versus a significantly greater (p ⁇ 0.001) number, 21.0 + 0.6 in the OVA-sensitized rats and 25.3 ⁇ 2.6 in the HRP- sensitized rats.
  • Mast cells in tracheas from naive controls and OVA sensitized rats were mostly normal in appearance, containing electron dense granules at 90 min after HRP challenge ( Figures 7A and B).
  • mast cells demonstrated changes typical of activation: (a) decreased granule density (82%), and (b) spaces between the granule core and its membrane (76%).
  • a typical activated mast cell from this rat group is shown in Figure 7C.
  • the area of HRP containing endosomes was measured in epithelial cells of the 3 rat groups.
  • epithelial cells of HRP sensitized rats the area of HRP endosomes was significantly increased (p ⁇ 0.01) compared with results in control groups ( Figure 9A).
  • the value for HRP sensitized rats was ⁇ 4 fold that for naive control rats; the result in OVA sensitized rats was not significantly different from that in naive control rats.
  • the area of HRP containing endosomes in OVA sensitized rats was also significantly greater (p ⁇ 0.05) than in naive controls, but still well below the value in HRP sensitized rats.
  • Representative EM photomicrographs of tissues fixed 90 minutes after 90 minutes after HRP challenge are shown in Figures 8C and D.
  • the area of HRP endosomes was no different at 90 minutes (1.29 + 0.04 ⁇ m 2 / window) than at 2 minutes.
  • Distribution of HRP endosomes within different epithelial cell types Non-ciliated and ciliated cells in the surface layer were examined to determine their relative importance in transcytosis of HRP.
  • tissue conductance a measure of the integrity of the tight junctions, began to rise post-challenge in HRP sensitized rats, but not in OVA or saline rats and was significantly elevated (p ⁇ 0.01) at 90 min (33.5 + 1.2 mS/cm 2 vs. 22.2 + 1.0 and 20.8 ⁇ 0.6, respectively).
  • HRP was located in the paracellular spaces subsequent to the hypersensitivity reaction
  • Figure 13A shows an example of HRP in the process of being endocytosed at the apical membrane of a tracheal epithelial cell.
  • DISCUSSION OF RESULTS IN EXAMPLE 3 Extrinsic antigens are responsible for initiating pathophysiology in most cases of airway allergy. Allergic reactions have been well studied, both in humans and in animal models. The accepted mechanism involves a sequence of events beginning with inhalation of the antigen that must pass through the epithelial barrier to reach and activate effector mast cells, located in the lamina limba or epithelium below the level of the tight junctions. These cells then release a host of bioactive mediators responsible for functional changes that include secretion of fluid and mucus and contraction of smooth muscle.
  • HRP as an antigen since it can be measured quantitatively by enzymatic assay, thus allowing us to accurate determination of flux of the protein across tracheal tissue.
  • reaction product can be observed by EM, it was possible to visualize its pathway across the epithelium.
  • HRP When added to the luminal side of sensitized rat trachea in Ussing chambers, HRP produced a similar pattern of increase in Isc (magnitude and timing) as documented for other antigens (Sestini et al. (1990)), indicating that HRP is a suitable model antigen for these studies.
  • the increase in Isc in HRP-challenged tracheas from HRP sensitized rats began shortly after 2 min. Therefore, tracheal tissues were fixed for EM at 2 min after luminal challenge to observe the initial pathway used for antigen uptake.
  • endosomes containing HRP were present mainly in the apical region of epithelial cells of tracheas from naive control rats and rats sensitized to OVA.
  • HRP endosomes were seen throughout epithelial cells in tracheas from HRP sensitized rats.
  • additional studies were carried out in mast cell-deficient Ws/Ws rats.
  • HRP was not present within the tight junctions or paracellular regions in any of the groups 2 minutes after HRP challenge, indicating that HRP transport to the lamina intestinal occurred via a very rapid transcellular route.
  • the finding that HRP uptake was increased in HRP sensitized, but not OVA sensitized rats, at 2 minutes suggests that there may be recognition of antigen at the apical surface of the epithelium, potentially through an immunoglobulin mediated uptake system.
  • Receptors for immunoglobulin have been demonstrated on epithelial cells, including plgR and FcRn for IgA and IgG, respectively (Daniele 1990; Israel et al. 1997).
  • immunization has been shown to affect protein transport in rat airways (Folkesson et al. 1998).
  • HRP was also apparent in the paracellular spaces and tight junctions in epithelium of HRP sensitized and challenged rats; this observation was never recorded in naive control or OVA sensitized rats, even though very thorough analysis was carried out. This finding coincided in time with significantly increased conductance from baseline of tracheal tissues in HRP sensitized and challenged rats.
  • the morphological and electrophysiological data support the conclusion that opening of the tight junctions occurred after the hypersensitivity reaction to allow paracellular antigen transport.
  • Overall HRP transport across tracheal epithelium determined by flux measurements over 90 min was greater during all 3 flux periods in HRP sensitized rats compared with naive controls and OVA sensitized rats.
  • HRP flux across tracheal epithelium from Ws/Ws rats sensitized to HRP was of the same magnitude as control rats, indicating that this phase of antigen transport was mast cell-dependent. Recruitment of the paracellular pathway after intestinal hypersensitivity is also dependent on the presence of mast cells (Berin et al. (1998)). Unlike controls, where the flux stablized with time, the magnitude of the rate of HRP transport continued to increase in HRP sensitized and challenged rats. The enhanced transepithelial antigen transport observed over the 90 min flux period was likely due to transport via both paracellular and transcellular pathways.
  • mast cell activation was confirmed in several ways: mast cell granules were decreased in density and many granules demonstrated clear zones around a central core. However, no changes in mast cells occurred after exposure to HRP in tracheas that were obtained from naive control rats or rats sensitized to OVA. Mast cells contain a number of mediators that have been shown to have effects on epithelial barrier function. Mast cells in rat trachea are predominately of the "mucosal" type, and contain the specific protease, rat mast cell protease II (RMCP II) that acts on collagen as a substrate.
  • RMCP II rat mast cell protease II
  • mast cell degranulation also induces vagal sensory neuron excitation (Kiernan (1990)).
  • Mast cells and nerves act as a functional unit to regulate intestinal epithelial ion secretion (Perdue et al. (1991)). Cholinergic stimulation of intestinal epithelium causes an increase in tight junction permeability, such that large protein tracers can leak through the paracellular pathway (Phillips et al. (1991)).
  • mast cells to regulate the integrity of the epithelial tight junction directly or indirectly through nerve activation.
  • Mast cells are also capable of releasing a number of cytokines after stimulation with IgE, including IFN- ⁇ (Burd et al.
  • TNF-a (Gordon and Galli (1990)
  • IL-4 (Madara and Stafford (1989)).
  • These cytokines have been demonstrated to decrease the resistance of cultured monolayers of polarized epithelial cells (Madara and Stafford (1989); Rodriguez et al. (1995); Colgan et al. (1994)).
  • transepithelial antigen transport across airway epithelium in specifically sensitized rats occurred in two phases. Initially, antigen was taken up through a transcellular pathway. Sensitization increased both the amount of specific antigen taken up and the rate at which it appeared in the lamina propria. Subsequent to activation of mast cells and the hypersensitivity reaction, a large increase in protein flux was observed coincident with increased conductance and morphological evidence of paracellular protein transport. While not wishing to be bound by any one theory, these results suggest that in an allergic individual, small amounts of antigen are initially preferentially transported across the airway epithelium, subsequently activating subepithelial mast cells, resulting in a non-specific barrier defect that amplifies the hypersensitivity reaction.
  • Anti-CD23 ( ⁇ g/ml HRP endosome area ( ⁇ m2)
  • IgE" Serum 17.5 ⁇ 1.0 3.0 ⁇ 1.5 0.0 ⁇ 0.0 0.0 ⁇ 0.0
  • Gastrointestinal food hypersensitivity basic mechanisms of pathophysiology. Gastroenterology 103:1075-1095.
  • Kiernan JA. 1990 Degranulation of mast cells in the trachea and bronchi of the rat following stimulation of the vagus nerve. Int Arch Allergy Appl Immunol 91:398. Kinet, J.P. 1999. The high-affinity IgE receptor (Fc ⁇ RI): from physiology to pathology. Ann. Rev. Immunol. 17:931-972.
  • Pertussis toxin stimulates hypersensitivity and enhances nerve-mediated antigen uptake in rat intestine. Am. J. Physiol. 267 :G745-753.
  • Tumour necrosis factor- ⁇ induces morphological and functional alterations of intestinal HT29 cl.l9a cell monolayers. Cytokine 7:441.
  • Figures 1A-1C are representative electron photomicrographs showing uptake of horseradish peroxidase (HRP, electron dense product), into endosomes in intestinal enterocytes 2 min after luminal HRP challenge: (A) HRP sensitized rat, (B) ovalbumin (OVA) sensitized rat, (C) naive control rat (4,000 x magnification).
  • Figure 1(D) is a graph comparing the area of HRP endosomes in enterocytes of rats in the different groups; the area is significantly greater in HRP sensitized rats compared with control rats or rats sensitized to OVA.
  • Figures 2A-2B are electron photomicrographs showing uptake of HRP into endosomes in intestinal enterocytes 2 min after luminal HRP challenge. Rats were injected with (A) serum obtained from HRP-sensitized rats, containing high anti-HRP IgE titers (immune) or (B) IgE-depleted serum (4,000 x magnification).
  • Figure 2C is a graph comparing the area of endosomes in enterocytes of control rats with rats injected with immune serum, either untreated, heat-treated or immuno-precipitated to remove IgE antibodies. IgE was required for the enhanced uptake of HRP antigen.
  • Figures 3A-3B are light micrographs showing jejunal tissues obtained from A) naive control rat or B) actively sensitized rat. Sections were cryofixed and stained with anti-CD23 antibody. Strong immunoreactivity for CD23 is demonstrated on epithelial cells in the section from the sensitized rat but not in the section from the control rat (40 x magnification). These photomicrographs are representative of those prepared from 6 rats/group.
  • Figures 4A-4B are electron photomicrographs showing location of CD23 by immunocytochemistry using anti-CD23 antibody-linked colloidal gold particles.
  • A Section of intestine from an HRP sensitized rat prior to HRP challenge demonstrates gold labeling (CD23 receptors) on the enterocyte microvilli.
  • B Section of intestine from an HRP sensitized rat after HRP challenge shows that CD23 receptors are co-localized within an HRP-containing endosome.
  • A 30,000 x magnification
  • B 80,000 x magnification
  • Figure 4C is a graph showing the changes in numbers of gold labels on enterocyte microvilli in control rats and after sensitization and antigen challenge with HRP.
  • Figure 5 is a graph showing the intestinal hypersensitivity reaction as indicated by an increase in short-circuit current (Isc, in ⁇ A/cm 2 ) and conductance (G, in mS/cm 2 ) after HRP challenge of intestine from HRP sensitized rats.
  • Tissues were untreated (0) or treated by addition of 100, 200, or 400 ⁇ g of anti-CD23 antibody (B3B4) prior to HRP challenge. *p ⁇ 0.05 compared to baseline values.
  • Figure 8 is a drawing illustrating a histogram of area of HRP-containing endosomes in enterocytes of IL-4 (+/+) or IL-4 (-/-) mice, either naive control mice or mice actively sensitized (AS) to HRP.
  • Figure 10 is a figure illustrating immunohistochemistry showing CD23 immunoreactivity on intestinal epithelial cells.
  • A Mouse sensitized to HRP. Staining is predominant on the apical membrane of enterocytes in the mid villus region with some staining of the cytoplasm just below the apical membrane. There are also some positive cells in the lamina propria.
  • B Naive control mouse. No staining is evident on epithelial cells although some immunoreactivity is present in the lamina limbal.
  • FIG 11 is a drawing illustrating a histogram of effect of anti-CD23 (B3B4) on area of HRP-containing endosomes in enterocytes of actively sensitized IL-4 (+/+) mice.
  • Tissues from HRP sensitized mice were pre-treated with B3B4 for 30 min prior to HRP challenge (addition to the mucosal buffer).
  • Figure 13 shows representative tracings of short-circuit current (Isc) in tracheas from a naive control rat, OVA sensitized rat, and HRP sensitized rat.
  • the tissues were challenged with 50 ⁇ mol/L HRP on the luminal side of the tissue.
  • tissues were removed from the Ussing chamber and fixed for electron microscopy 2 min after HRP addition.
  • the tracings are representative of three similar results for each rat group.
  • Figure 16 shows the effect of sensitization on uptake of HRP into tracheal epithelial cells.
  • Figure 17 is a graph showing the distribution of HRP endosomes within ciliated and non-ciliated epithelial cells at (A) 2 min or (B) 90 min. EM photomicrographs of tracheal epithelial cells were taken at magnification of 8,000. The area of HRP-containing endosomes was measured within ciliated and non-ciliated epithelial cells using a computerized image processing system and expressed as HRP area ( ⁇ m 2 )/ window (206 ⁇ m 2 ). *Significance difference (p ⁇ 0.05) between ciliated and non-ciliated cells in the same A&B group; #significance at p ⁇ 0.05 compared with naive control rats.
  • Figure 19 shows the effect of sensitization and antigen challenge on mucosal to serosal flux of HRP.
  • Flux of HRP was measured across trachea obtained from rats injected with saline, rats sensitized to OVA, or rats sensitized to HRP.
  • HRP-sensitized mast cell-deficient Ws/Ws rats were also examined.
  • Figure 20 shows photomicrographs of tracheal epithelial cells from an HRP sensitized rat 2 min (A) or 90 min (B) after addition of HRP to the luminal buffer.
  • A The arrow indicates HRP in the process of being endocytosed by a tracheal epithelial cell.

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Abstract

La présente invention porte sur des procédés de modulation du système immun, et notamment de réactions allergiques, en bloquant ou faisant obstacle à l'interaction entre des récepteurs CD23 et des complexes d'IgE-antigènes. Ces procédés consistent à prévenir des réactions immunes d'hypersensibilité telles que des allergies alimentaires ou respiratoires.
PCT/CA2000/000015 1999-01-06 2000-01-06 Procede permettant de prevenir des reactions immunes et d'hypersensibilite WO2000040713A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2005083385A2 (fr) * 2004-02-26 2005-09-09 Alk-Abelló A/S Procede pour evaluer le potentiel therapeutique d'un vaccin a administration mucosale
US20130039934A1 (en) * 2009-12-01 2013-02-14 Trustees Of Boston University Treatment of ige-mediated disease
US10907151B2 (en) * 2010-11-24 2021-02-02 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Compositions and methods for treating or preventing lupus

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005083385A2 (fr) * 2004-02-26 2005-09-09 Alk-Abelló A/S Procede pour evaluer le potentiel therapeutique d'un vaccin a administration mucosale
WO2005083385A3 (fr) * 2004-02-26 2005-11-17 Alk Abello As Procede pour evaluer le potentiel therapeutique d'un vaccin a administration mucosale
US20130039934A1 (en) * 2009-12-01 2013-02-14 Trustees Of Boston University Treatment of ige-mediated disease
US8945575B2 (en) * 2009-12-01 2015-02-03 Trustees Of Boston University Treatment of IgE-mediated disease
US9617325B2 (en) 2009-12-01 2017-04-11 Boston Medical Center Corporation Treatment of IgE-mediated disease
US10907151B2 (en) * 2010-11-24 2021-02-02 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Compositions and methods for treating or preventing lupus

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