WO2008154350A2

WO2008154350A2 - Use of tight junction agonists to suppress immune responses

Info

Publication number: WO2008154350A2
Application number: PCT/US2008/066057
Authority: WO
Inventors: Amir Tamiz; Niranjan Pandey; Blake Paterson
Original assignee: Alba Therapeutics Corporation
Priority date: 2007-06-08
Filing date: 2008-06-06
Publication date: 2008-12-18
Also published as: WO2008154350A3

Abstract

The present invention provides materials and methods to suppress an excessive or undesired immune response. In some embodiments, agonists of biological pathways responsible for opening and closing tight junctions (e.g., tight junction agonists) are used in compositions with antigens to suppress an immune response to the antigen.

Description

USE OF TIGHT JUNCTION AGONISTS TO SUPPRESS TMMUNE RESPONSES

FIELD OF THE INVENTION

[0001] The present invention provides materials and methods to suppress an excessive or undesired immune response. In some embodiments, agonists of biological pathways responsible for opening and closing tight junctions (e.g., tight junction agonists) are used in compositions with antigens to suppress an immune response to the antigen.

BACKGROUND OF THE INVENTION

[0002] The immune system serves as the body's defense mechanism against pathogens present in the environment. In some cases, exposure to certain substances causes an excessive immune response in sensitive individuals. Substances that induce the excessive immune response are referred to as allergens and the immune response is typically referred to as an allergic response. Common allergens include pollen, mold, animal dander, and cockroach and house dust mite droppings. The allergic response is specific to the allergen although a person can be allergic to multiple allergens. Typically, the severity of the response increases with repeated exposure to the allergen.

[0003] Allergic responses can cause swelling, inflammation, wheezing, excessive production of mucous, rhinitis, or "hay fever," conjunctivitis (an eye reaction), atopic dermatitis, or allergic skin reactions, and urticaria, also known as hives. In severe case, allergic reactions can be life threatening.

[0004] Allergy is currently treated with immune suppressing drugs such as corticosteroids or with an immunotherapy that results in desensitizing the subject by injecting the allergen. Despite these treatments, there remains a need in the art for materials and methods for suppressing an excessive or undesirable immune response. These and other needs are met by the present invention.

SUMMARY OF THE INVENTION

[0005] The present invention provides materials and methods for the suppression of an excessive or undesired immune response. Examples of such immune responses include, but are not limited to, allergic responses and transplant rejection responses. [0006] In one embodiment, the present invention provides compositions for suppression of an immune response. Such compositions may comprise one or more antigens and an immune-suppressing amount of one or more tight junction agonists. As used herein, a "tight junction agonist" is a compound that mediates or facilitates or augments the physiological, transient opening of tight junctions, for example, the tight junctions between adjacent epithelial cells. An example of a tight junction agonist is zonula occludens toxin (ZOT), which is produced by Vibrio cholerae. A ZOT receptor agonist is a compound which is believed to mediate tight junction opening through the same receptor utilized by ZOT. In another embodiment, a tight junction agonist may comprise zonulin. In some embodiments, a tight junction agonist may comprise a peptide. In some embodiments, a tight junction agonist may be a fragment of ZOT and/or zonulin. In some embodiments, a tight junction agonist comprising a peptide may comprise the amino acid sequence FCIGRL (SEQ ID NO:1). A tight junction agonist comprising a peptide may comprise from about 6 to about 50 amino acids, from about 6 to about 25 amino acids, or from about 6 to about 10 amino acids.

[0007] In some embodiments, a peptide tight junction agonist may comprise a sequence selected from the group consisting of Xaal Cys lie GIy Arg Leu (SEQ ID NO: 2), Phe Xaa2 He GIy Arg Leu (SEQ ID NO: 3), Phe Cys Xaa3 GIy Arg Leu (SEQ ID NO: 4), Phe Cys He Xaa4 Arg Leu (SEQ ID NO: 5), Phe Cys He GIy Xaa5 Leu (SEQ ID NO: 6), and Phe Cys He GIy Arg Xaa6 (SEQ ID NO: 7), wherein Xaal is selected from the group consisting of Ala, VaI, Leu, lie, Pro, Trp, Tyr, and Met; Xaa2 is selected from the group consisting of GIy, Ser, Thr, Tyr, Asn, and GIn; Xaa3 is selected from the group consisting of Ala, VaI, Leu, lie, Pro, Trp, and Met; Xaa4 is selected from the group consisting of GIy, Ser, Thr, Tyr, Asn, Ala, and GIn; Xaa5 is selected from the group consisting of Lys and His; Xaa6 is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, and Met.

[0008] In some embodiments, a peptide tight junction agonist may comprise a sequence selected from the group consisting of Xaal Xaa2 He GIy Arg Leu (SEQ ID NO: 8), Xaal Cys Xaa3 GIy Arg Leu (SEQ ID NO: 9), Xaal Cys He Xaa4 Arg Leu (SEQ ID NO: 10), Xaal Cys He GIy Xaa5 Leu (SEQ ID NO: 1 1), Xaal Cys He GIy Arg Xaa6 (SEQ ID NO: 12), Phe Xaa2 Xaa3 GIy Arg Leu (SEQ ID NO: 13), Phe Xaa2 He Xaa4 Arg Leu (SEQ ID NO: 14), Phe Xaa2 He GIy Xaa5 Leu (SEQ ID NO: 15), Phe Xaa2 He GIy Arg Xaa6 (SEQ ID NO: 16), Phe Cys Xaa3 Xaa4 Arg Leu (SEQ ID NO: 17), Phe Cys Xaa3 GIy Xaa5 Leu (SEQ ID NO: 18), Phe Cys Xaa3 GIy Arg Xaa6 (SEQ ID NO: 19), Phe Cys He Xaa4 Xaa5 Leu (SEQ ID NO: 20), Phe Cys He Xaa4 Arg Xaa6 (SEQ ID NO: 21), and Phe Cys He GIy Xaa5 Xaa6 (SEQ ID NO: 22), wherein Xaal is selected from the group consisting of Ala, VaI₅ Leu, He, Pro, Trp, Tyr, and Met; Xaa2 is selected from the group consisting of GIy, Ser, Thr, Tyr, Asn, and GIn; Xaa3 is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, and Met; Xaa4 is selected from the group consisting of GIy, Ser, Thr, Tyr, Asn, Ala, and GIn; Xaa5 is selected from the group consisting of Lys and His; Xaa6 is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, and Met.

[0009] In some embodiments, the antigen may be an allergen. As used herein an antigen is any substance that evokes an immune response and an allergen is any substance that induces allergy. Allergy is an excessive immune response to an antigen. Examples of suitable allergens that can be used in the compositions of the invention include, but are not limited to, pollen, dust mite allergens, mold spores, pet dander, food, insect stings, and medicines. In some embodiments, an antigen (e.g., an allergen) may be a protein or fragment thereof. In a particular embodiment, compositions of the invention may comprise a dust mite allergen, for example, Der pi . In another particular embodiment, compositions of the invention may comprise ragweed pollen.

[0010] The present invention also provides methods of treating (i.e., suppressing, reducing, ameliorating) an undesired or excessive immune response. Such methods may include administering to the subject a composition comprising the antigen and an immune-suppressing amount of a tight junction agonist. As used herein, a subject is any mammal, for example, a human. In some embodiments, a tight junction agonist may comprise a peptide. Suitable tight junction agonists for use in the methods of the invention include those listed above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011 ] Figure 1 is a bar graph showing the results of antibody titer experiments showing an increase in all types of IgG except subclass lgG2a.

[0012] Figure 2 is a bar graph showing the effects of the tight junction agonist on the relative titers of the subclasses IgGl and IgG2a. [0013] Figure 3 is a bar graph showing the effects of the tight junction agonist on the relative titers of the total IgG and on the subclasses IgGl and IgG2a as well as on IgA as a function of the amount of tight junction agonist delivered with the antigen.

[0014] Figure 4 shows the fluorescent microscopy results of a cytoskeletal rearrangement assay showing the effects of tight junction agonist and PT-gliadin on tight junction structure.

[0015] Figure 5 is a schematic showing the synthesis of AT1002.

[0016] Figure 6 is a graph of transepithelial electrical resistance (TEER) as a function of time showing the administration of tight junction agonist causes a reduction in TEER.

DETAILED DESCRIPTION OF THE INVENTION [0017] Definitions

[0018] As used herein, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.

[0019] As used herein, a "tight junction agonist" is a compound that mediates or facilitates or augments the physiological, transient opening of tight junctions. Tight junctions are structures that form a barrier between adjacent epithelial cells (Johnson and Quay, Expert Opin DrugDeliv. 2005 Mar;2(2):281-98). An example of a tight junction agonist is zonula occludens toxin (ZOT), which is produced by Vibrio cholerae. A ZOT receptor agonist is a tight junction agonist which is believed to mediate tight junction opening through the same receptor utilized by ZOT. Tight junction agonists also include zonulin.

[0020] Tight junction agonists

[0021 ] Compositions of the invention typically comprise one or more tight junction agonists. A tight junction agonist as used herein is a compound that mediates the physiological, transient opening of tight junctions. In some embodiments, a tight junction agonist may operate by binding to the ZOT receptor, i.e., may be a ZOT receptor agonist. [0022] In some embodiments, a tight junction agonist may comprise a peptide comprising the amino acid sequence FCIGRL and/or functional derivatives of this sequence. Functional derivatives of peptide FCIGRL include, for example, Xaai Cys Ue GIy Arg Leu (SEQ ID NO: 2), Phe Xaa₂ He GIy Arg Leu (SEQ ID NO: 3), Phe Cys Xaa₃ GIy Arg Leu (SEQ ID NO: 4), Phe Cys He Xaa₄ Arg Leu (SEQ ID NO: 5), Phe Cys He GIy Xaa₅ Leu (SEQ ID NO: 6), and Phe Cys lie GIy Arg Xaae (SEQ ID NO: 7). Xaaj is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, Tyr, and Met; Xaa₂ is selected from the group consisting of GIy, Ser, Thr, Tyr, Asn, and GIn; Xaa₃ is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, and Met; Xaa₄ is selected from the group consisting of GIy, Ser, Thr, Tyr, Asn, Ala, and GIn; Xaas is selected from me group consisting of Lys and His; Xaa6 is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, and Met. In some embodiments, a tight junction agonist may consist of a peptide having the sequence FCIGRL and/or functional derivatives of this sequence as described herein.

[0023] Further, functional derivatives of peptide FCIGRL include: Xaai Xaa₂ He GIy

Arg Leu (SEQ ID NO: 8), Xaa, Cys Xaa₃ GIy Arg Leu (SEQ ID NO: 9), Xaa, Cys He Xaa, Arg Leu (SEQ ID NO: 10), Xaa] Cys He GIy Xaa₅ Leu (SEQ ID NO: 11), Xaa, Cys lie GIy Arg XaO₆ (SEQ ID NO: 12), Phe Xaa₂ Xaa₃ GIy Arg Leu (SEQ ID NO: 13), Phe Xaa₂ Ue XaEi₄ Arg Leu (SEQ ID NO: 14), Phe Xaa₂ lie GIy Xaa_s Leu (SEQ ID NO: 15), Phe Xaa₂ He GIy Arg Xaa* (SEQ ID NO: 16), Phe Cys Xaa₃ Xaa, Arg Leu(SEQ ID NO: 17), Phe Cys Xaa₃ GIy Xaa_s Leu (SEQ ID NO: 18), Phe Cys Xaa₃ GIy Arg Xaa* (SEQ ID NO: 19), Phe Cys He Xaa₄ Xaa₅ Leu (SEQ ID NO: 20), Phe Cys He Xasu Arg XZa₆ (SEQ ID NO: 21), and Phe Cys He GlyXaa₅ Xaa<; (SEQ ID NO: 22). Xaai is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, Tyr, and Met; Xaai is selected from the group consisting of GIy, Ser, Thr, Tyr, Asn, and GIn; Xaa₃ is selected from the group consisting of Ala, VaI, Leu, lie, Pro, Trp, and Met; Xaa₄ is selected from the group consisting of GIy, Ser, Thr, Tyr, Asn, Ala, and GIn; Xaas is selected from the group consisting of Lys and His; Xaa<s is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, and Met.

[0024] When the tight junction agonist is a peptide, any length of peptide may be used. For example, an agonist may be about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14 or about 15 amino acids in length. In some embodiments, a peptide tight junction agonist may be from about 3 to about 12, from about 4 to about 12, from about 5 to about 12, from about 6 to about 12, from about 7 to about 12, from about 8 to about 12, from about 9 to about 12, from about 10 to about 12, from about 3 to about 10, from about 4 to about 10, from about 5 to about 10, from about 6 to about 10, from about 7 to about 10, from about 8 to about 10, from about 9 to about 10 amino acids in length. In some embodiments, a peptide tight junction agonist may be 9 amino acids or less in length. In some embodiments of the invention, peptides tight junction agonists do not encompass full length ZOT or zonulin.

[0025] Peptide agonists can be chemically synthesized and purified using well-known techniques, such as described in High Performance Liquid Chromatography of Peptides and Proteins: Separation Analysis and Conformation, Eds. Mant et al., C.R.C. Press (1991), and a peptide synthesizer, such as Symphony (Protein Technologies, Inc.); or by using recombinant DNA techniques, i.e., where the nucleotide sequence encoding the peptide is inserted in an appropriate expression vector, e.g., an E. coli or yeast expression vector, expressed in the respective host cell, and purified from the cells using well-known techniques.

[0026] Formulations

[0027] Compositions of the invention may formulated for any type of delivery, for example, parenteral, mucosal or intestinal delivery.

[0028] Compositions of the invention may formulated for pulmonary delivery (e.g., may be pulmonary dosage forms). Typically such compositions may be provided as pharmaceutical aerosols, e.g., solution aerosols. Those of skill in the art are aware of many different methods and devices for the formation of pharmaceutical aerosols, for example, those disclosed by Sciarra and Sciarra, Aerosols, in Remington: The Science and Practice of Pharmacy, 20th Ed., Chapter 50, Gennaro et a Eds., Lippincott, Williams and Wilkins Publishing Co., (2000).

[0029] The compositions of the invention may be formulated for enteric delivery, for example, may comprise one or more coatings, for example, delayed release coating containing one or more enteric agents. A delayed release coating is typically substantially stable in gastric fluid and substantially unstable (e.g., dissolves rapidly or is physically unstable) in intestinal fluid, thus providing for substantial release of the tight junction antagonist from the composition in the duodenum or the jejunum. [0030] The terms "stable in gastric fluid" or "stable in acidic environments" refers to a composition that releases 30% or less by weight of the total tight junction antagonist in the composition in gastric fluid with a pH of 5 or less, or simulated gastric fluid with a pH of 5 or less, in approximately sixty minutes.

[0031 ] Nasal dosage compositions for nasal delivery are well-known in the art. Such nasal dosage compositions generally comprise water-soluble polymers that have been used extensively to prepare pharmaceutical dosage forms (Martin et al, In: Physical Chemical Principles of Pharmaceutical Sciences, 3rd Ed., pages 592-638 (1983)) that can serve as carriers for peptides for nasal administration (Davis, In: Delivery Systems for Peptide Drugs, 125:1-21 (1986)). The nasal absorption of peptides embedded in polymer matrices has been shown to enhance through retardation of nasal mucociliary clearance (Ilium et al, Int. J. Pharm., 46:261-265 (1988)).

[0032] Typically, compositions comprising a tight junction agonist (e.g., peptide agonist) comprise an immune suppressive amount of the agonist. The immune suppressive amount of agonist (e.g., peptide agonist) employed may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0033] Compositions of the invention may comprise one or tight junction agonist at a level of from about 0.000001 wt% to about 50 wt%, from about 0.000001 wt% to about 45 wt%, from about 0.000001 wt% to about 40 wt%, from about 0.000001 wt% to about 35 wt%, from about 0.000001 wt% to about 30 wt%, from about 0.000001 wt% to about 25 wt%, from about 0.000001 wt% to about 20 wt%, from about 0.000001 wt% to about 15 wt%, from about 0.000001 wt% to about 10 wt%, from about 0.000001 wt% to about 5 wt%, from about 0.000001 wt% to about 2.5 wt%, from about 0.000001 wt% to about 1 wt%, from about 0.000001 wt% to about 0.1 wt%, from about 0.000001 wt% to about 0.01 wt%, from about 0.000001 wt% to about 0.001 wt%, from about 0.000001 wt% to about 0.0001 wt%, from about 0.000001 wt% to about 0.00005 wt%, from about 0.0001 wt% to about 50 wt%, from about 0.0001 wt% to about 45 wt%, from about 0.0001 wt% to about 40 wt%, from about 0.0001 wt% to about 35 wt%, from about 0.0001 wt% to about 30 wt%, from about 0.0001 wt% to about 25 wt%, from about 0.0001 wt% to about 20 wt%, from about 0.0001 wt% to about 15 wt%, from about 0.0001 wt% to about 10 wt%, from about 0.0001 wt% to about 5 wt%, from about 0.0001 wt% to about 2.5 wt%, from about 0.0001 wt% to about 1 wt%, from about 0.0001 wt% to about 0.1 wt% from about 0.0001 wt°/o to about 0.01 wt%, from about 0.0001 wt% to about 0.001 wt%, from about 0.0001 wt% to about 0.0005 wt%, from about 0.1 wt% to about 50 wt%, from about 0.1 wt% to about 45 wt%, from about 0.1 wt% to about 40 wt%, from about 0.1 wt% to about 35 wt%, from about 0.1 wt% to about 30 wt%, from about 0.1 wt% to about 25 wt% from about 0.1 wt% to about 20 wt%, from about 0.1 wt% to about 15 wt%, from about 0.1 wt% to about 10 wt%, from about 0.1 wt% to about 5 wt%, from about 0.1 wt% to about 2.5 wt%, from about 0.1 wt% to about 1 wt%, from about 0.1 wt% to about 0.5 wt%, from about 0.1 wt% to about 0.2 wt%, from about 1 wt% to about 50 wt%, from about 1 wt% to about 45 wt%, from about 1 wt% to about 40 wt%, from about 1 wt% to about 35 wt%, from about 1 wt% to about 30 wt%, from about 1 wt% to about 25 wt%, from about 1 wt% to about 20 wt%, from about 1 wt% to about 15 wt%, from about 1 wt% to about 10 wt%, from about 1 wt% to about 5 wt%, from about 1 wt% to about 2.5 wt%, from about 5 wt% to about 50 wt%, from about 5 wt% to about 45 wt%, from about 5 wt% to about 40 wt%, from about 5 wt% to about 35 wt%, from about 5 wt% to about 30 wt%, from about 5 wt% to about 25 wt%, from about 5 wt% to about 20 wt%, from about 5 wt% to about 15 wt%, from about 5 wt% to about 10 wt%, from about 5 wt% to about 9 wt%, from about 5 wt% to about 8 wt%, from about 5 wt% to about 7 wt%, or from about 5 wt% to about 6 wt% of the total weight of the composition. Compositions of the invention may comprise one or more tight junction agonists at a level of about 0.00001 wt%, about 0.00005 wt%, about 0.0001 wt%, about 0.0005 wt%, about 0.001 wt%, about 0.005 wt%, about 0.01 wt%, about 0.05 wt%, about 0.1 wt%, about 0.5 wt%, about 1 wt%, about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, or about 50 wt% based on the total weight of the composition.

[0034] Compositions of the invention may comprise one or more antigens (e.g., allergens) at a concentration sufficient to cause the desired biological response. Compositions of the invention may comprise one or more antigens at from about 0.1 wt% to about 50 wt%, from about 0.1 wt% to about 45 wt%, from about 0.1 wt% to about 40 wt%, from about 0.1 wt% to about 35 wt%, from about 0.1 wt% to about 30 wt%, from about 0.1 wt% to about 25 wt%, from about 0.1 wt% to about 20 wt%, from about 0.1 wt% to about 15 wt%, from about 0.1 wt% to about 10 wt%, from about 0.1 wt% to about 5 wt%, from about 0.1 wt% to about 2.5 wt%, from about 0.1 wt% to about 1 wt%, from about 0.1 wt% to about 0.5 wt%, from about 0.1 wt% to about 0.2 wt%, from about 1 wt% to about 50 wt%, from about 1 wt% to about 45 wt%, from about 1 wt% to about 40 wt%, from about 1 wt% to about 35 wt%, from about 1 wt% to about 30 wt%, from about 1 wt% to about 25 wt%, from about 1 wt% to about 20 wt%, from about 1 wt% to about 15 wt%, from about 1 wt% to about 10 wt%, from about 1 wt% to about 5 wt%, from about 1 wt% to about 2.5 wt%, from about 5 wt% to about 50 wt%, from about 5 wt% to about 45 wt%, from about 5 wt% to about 40 wt%, from about 5 wt% to about 35 wt%, from about 5 wt% to about 30 wt%, from about 5 wt% to about 25 wt%, from about 5 wt% to about 20 wt%, from about 5 wt% to about 15 wt%, from about 5 wt% to about 10 wt%, from about 5 wt% to about 9 wt%, from about 5 wt% to about 8 wt%, from about 5 wt% to about 7 wt%, or from about 5 wt% to about 6 wt% of the total weight of the composition. Compositions of the invention may comprise one or more antigens at a level of about 0.1 wt%, about 1 wt%, about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, or about 50 wt% based on the total weight of the composition.

[0035] Compositions of the invention may comprise one or pharmaceutically acceptable excipients at a level of from about 0.1 wt% to about 50 wt%, from about 0.1 wt% to about 45 wt%, from about 0.1 wt% to about 40 wt%, from about 0.1 wt% to about 35 wt%, from about 0.1 wt% to about 30 wt%, from about 0.1 wt% to about 25 wt%, from about 0.1 wt% to about 20 wt%, from about 0.1 wt% to about 15 wt%, from about 0.1 wt% to about 10 wt%, from about 0.1 wt% to about 5 wt%, from about 0.1 wt% to about 2.5 wt%, from about 0.1 wt% to about 1 wt%, from about 0.1 wt% to about 0.5 wt%, from about 0.1 wt% to about 0.2 wt%, from about 1 wt% to about 50 wt%, from about 1 wt% to about 45 wt%, from about 1 wt% to about 40 wt%, from about 1 wt% to about 35 wt%, from about 1 wt% to about 30 wt%, from about 1 wt% to about 25 wt%, from about 1 wt% to about 20 wt%, from about 1 wt% to about 15 wt%, from about 1 wt% to about 10 wt%, from about 1 wt% to about 5 wt%, from about 1 wt% to about 2.5 wt%, from about 5 wt% to about 50 wt%, from about 5 wt% to about 45 wt%, from about 5 wt% to about 40 wt%, from about 5 wt% to about 35 wt%, from about 5 wt% to about 30 wt%, from about 5 wt% to about 25 wt%, from about 5 wt% to about 20 wt%, from about 5 wt% to about 15 wt%, from about 5 wt% to about 10 wt%, from about 5 wt% to about 9 wt%, from about 5 wt% to about 8 wt%, from about 5 wt% to about 7 wt%, or from about 5 wt% to about 6 wt% of the total weight of the composition. Compositions of the invention may comprise one or more pharmaceutically acceptable excipients at a level of about 0.1 wt%, about 1 wt%, about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, or about 50 wt% based on the total weight of the composition.

[0036] Compositions of the invention may be formulated to deliver an immune suppressive amount of a tight junction agonist in combination with an antigen. An immune suppressive amount is typically greater than 1 mg tight junction agonist/kg body weight of the subject. An immune suppressive amount may be from about 5 mg/kg to about 100 mg/kg, from about 5 mg/kg to about 50 mg/kg, from about 5 mg/kg to about 25 mg/kg, or from about 5 mg/kg to about 10 mg/kg.

[0037] Methods of use

[0038] The pharmaceutical compositions of the invention can be used for treating, ameliorating, and/or preventing an excessive or undesired immune response to an antigen. Any allergy may be treated using the compositions of the invention by selection of an appropriate antigen.

[0039] The following examples arc provided for illustrative purposes only, and are in no way intended to limit the scope of the present invention.

EXAMPLE l

[0040] The experiments described in the attached figures were performed with tight junction agonist peptide FCIGRL also known as ATI 002.

[0041 ] Adult female mice were immunized intranasally with ovalbumin (Ova) in the presence or absence of varying doses of AT-1002. The test regiment is detailed in the following table:

[0042] In this study, female Balb/c mice were immunized intranasally with

Ovalbumin and AT- 1002 simultaneously, four times in 21 day intervals and complete antibody responses to Ovalbumin were measured in the serum and in vaginal washes using quantitative Ova-specific ELISA. Effects of AT- 1002 stimulation on IgG subclasses (IgGl , and IgG2a) and IgA were studied. Mice immunized with Ova in the presence of AT- 1002 exhibit significantly higher antibody titers than those induced by immunization with antigen alone when antigen is administered with a low dose of tight junction agonist.

[0043] Female Balb/c mice (6-7 weeks of age) were obtained from the Jackson

Laboratory (Bar Harbor, ME) and acclimated in a semi-rigid isolator with exclusive usage for this study. Total of four immunizations were performed in 21 day intervals. Complete antibody responses to ovalbumin were measured in the serum and in vaginal washes 3 weeks post-immunization using quantitative ovalbumin-specific ELISA assays. Antibody responses measured included ovalbumin-specific total IgG, IgGl, IgG2a, and IgA. Responses from sample pools from each group as well as from individual mice were measured.

[0044] Figures 1-3 show the results of the assays. Figure 1 shows an increase in all types of IgG except subclass IgG2a was seen when ovalbumin was delivered with tight junction agonist at low dose. Figure 2 shows that subclass IgGl was increased when ovalbumin was administered with tight junction agonist while subclass IgG2a was decreased. Figure 3 shows the effects of the tight junction agonist on the relative titers of the total IgG and on the subclasses IgGl and IgG2a as well as on IgA as a function of the amount of tight junction agonist delivered with the antigen. Mice immunized with ovalbumin in the presence of low doses of AT-1002 exhibit significantly higher antibody titers than those induced by immunization with antigen alone. AT-1002 exhibits Th2 biased adjuvant effect at low dose. Unexpectedly, at higher dosesAT1002 exhibits immunosuppressive effects. Higher doses of AT 1002 caused a reduction in antibody titer when compared to the administration of ovalbumin alone. Thus, high doses of tight junction agonist, for example, greater than about 1 mg/kg cause the suppression of the immune response to the antigen ovalbumin.

EXAMPLE 2 [0045] Assay of cytoskeletal rearrangement induced by PT-Gliadin and AT 1002

[0046] Gliadin treated with the peptidases pepsin and trypsin (termed PT-gliadin or

PTG) induces a cytoskeletal arrangement in CaCo2 cells grown in monolayers. The rearrangement can be visualized using a Nikon-TE2000 epifluorescence microscope and a 4Ox objective and Alexa Fluor 555 conjugated phalloidin (Invitrogen, Carlsbad, CA), which binds specifically to F-actin. Exposure times were identical for control and agonist/antagonist treated samples. The figures were generated using Adobe Photoshop CS2 v 9.0.2. Figure 4 shows the rearrangement induced by tight junction agonist ATI 002.

EXAMPLE 3 [0047] Measurement Of Trans Epithelial Electric Resistance (TEER)

[0048] CaCo2 cells form monolayers that exhibit tight junctions between adjacent cells. Treatment of CaCo2 monolayers with peptide FCIGRL decreased TEER 16- fold in CaCo2 monolayers compared to vehicle alone. The TEER assay may be performed as follows:

[0049] Prepare Hank's Balanced Salt solution by weighing obtaining 1 L bottle of

HBSS removing 10ml of HBSS and replacing it with 10ml HEPES buffer pH 7.0. Adjust pH to 7.4 ±0.1 using concentrated NaOH (10N).

[0050] Remove Caco-2 cells from incubator, grown on 12-well, 3.0 μM, polycarbonate Transwell® filters (Corning) and record passage#, date cells seeded and age in days.

[0051 ] Aspirate cell culture medium from both the apical (AP) and basolateral (BL) compartments, replacing with 0.5 ml and 1.5 ml of HBSS, respectively. Incubate cells at 37°C for 30 minutes. [0052] Add the tight junction agonist at the indicated concentrations to the apical solution and using the MilliCell-ERS instrument (Millipore), measure and record the transepithelial electrical resistance (TEER) across each filter and record.

[0053] The results of the assays are provided in Figure 6.

[0054] While the invention has been described in detail, and with reference to specific embodiments thereof, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof and such changes and modifications may be practiced within the scope of the appended claims. All patents and publications herein are incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in their entirety.

Claims

What is claimed is:

1. A composition for suppressing an immune response to an antigen, comprising: the aantigcn; and an immune-suppressing amount of one or more tight junction agonists.

2. A composition according to claim 1, wherein at least one agonist comprises a peptide.

3. A composition according to claim 2, wherein the peptide comprises the sequence FCIGRL

4. A composition according to claim 2, wherein the peptide comprises a sequence selected from the group consisting of Xaai Cys lie GIy Arg Leu (SEQ ID NO: 2), Phe Xaa₂ He GIy Arg Leu (SEQ ID NO: 3), Phe Cys Xaa₃ GIy Arg Leu ^"(SEQ ID NO: 4), Phe Cys He Xaa₄ Arg Leu (SEQ ID NO: 5), Phe Cys lie GIy Xaa₅ Leu (SEQ ID NO: 6), and Phe Cys He GIy Arg Xaa$ (SEQ ID NO: 7), wherein Xaai is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, Tyr, and Met; Xaa₂ is selected from the group consisting of GIy, Ser, Thr, Tyr, Asn, and GIn; Xaa₃ is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, and Met; Xaa^ is selected from the group consisting of GIy, Ser, Thr, Tyr, Asn, Ala, and GIn; Xaa₅ is selected from the group consisting of Lys and His; Xaa^ is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, and Met.

5. A composition according to claim 2, wherein the peptide comprises a sequence selected from the group consisting of Xaai Xaa₂ He GIy Arg Leu (SEQ ID NO: 8), Xaai Cys Xaa₃ GIy Arg Leu (SEQ ID NO: 9), Xaa, Cys He XaJ_-4 Arg Leu (SEQ ID NO: 10), Xaai Cys He GIy Xaa₅ Leu (SEQ ID NO: 11), Xaa, Cys He GIy Arg Xaae (SEQ ID NO: 12), Phe Xaa₂ Xaa₃ GIy Arg Leu (SEQ ID NO: 13), Phe Xaa₂ He XaB₄ Arg Leu (SEQ ID NO: 14), Phe Xaa₂ He GIy Xaa₅ Leu (SEQ ID NO: 15), Phe Xaa₂ He GIy Arg Xaae (SEQ ID NO: 16), Phe Cys Xaa₃ Xaa4 Arg Leu (SEQ ID NO: 17), Phe Cys Xaa₃ GIy Xaa₅ Leu (SEQ ID NO: 18), Phe Cys Xaa₃ GIy Arg Xaa_δ (SEQ ID NO: 19), Phe Cys He Xaa₄ Xaa₅ Leu (SEQ ID NO: 20), Phe Cys He Xaa4 Arg Xaa* (SEQ ID NO: 21 ), and Phe Cys He GIy Xaa₅ Xaae (SEQ ID NO: 22), wherein Xaai is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, Tyr, and Met; Xaa₂ is selected from the group consisting of GIy, Ser, Thr, Tyr, Asn, and GIn; Xaa₃ is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, and Met; Xaaα is selected from the group consisting of GIy, Ser, Thr, Tyr, Asn, Ala, and Gb; Xaa₅ is selected from the group consisting of Lys and His; Xaa<; is selected from the group consisting of Ala, VaI, Leu, Ue, Pro, Trp, and Met.

6. A composition according to claim 2, wherein the peptide comprises from about 6 to about 10 amino acids.

7. A composition according to claim 1, wherein the antigen is an allergen.

8. A composition according to claim 7, wherein the allergen is selected from a group consisting of pollen, dust mite allergens, mold spores, pet dander, food, insect stings, and medicines.

9. A composition according to claim 7, wherein the allergen is a protein.

10. A composition according to claim 7, wherein the allergen is a dust mite allergen.

1 1. A composition according to claim 7, wherein the allergen is dust mite allergen Der pi .

12. A composition according to claim 7, wherein the allergen is ragweed pollen.

13. A composition according to claim 7, wherein the composition is in aqueous solution.

14. A composition according to claim 1 , wherein the composition is in a saline solution.

15. A composition according to claim 1 , wherein the composition further comprises one or more pharmaceutically acceptable excipients.

16. A composition according to claim 7, wherein the tight junction agonist is a peptide comprising the sequence FCIGRL and the composition is in aqueous solution and the composition comprises one or more allergens selected from the group consisting of pollen, dust mite allergens, mold spores, pet dander, food, insect stings, and medicines.

17. A method of suppressing an immune response to an antigen in a subject, comprising: administering to the subject a composition comprising the antigen and an immune- suppressing amount of a tight junction agonist.

18. A method according to claim 17, wherein the subject is a mammal.

19. A method according to claim 17, wherein the subject is a human.

20. A method according to claim 17, wherein at least one agonist comprises a peptide.

21. A method according to claim 20, wherein the peptide comprises the sequence FCIGRL.

22. A method according to claim 20, wherein the peptide comprises a sequence selected from the group consisting of Xaai Cys lie GIy Arg Leu (SEQ ID NO: 2), Phe Xaa₃ lie GIy Arg Leu (SEQ ID NO: 3), Phe Cys Xaa₃ GIy Arg Leu (SEQ ID NO: 4), Phe Cys He XaO₄ Arg Leu (SEQ ID NO: 5), Phe Cys He GIy Xaa_s Leu (SEQ ID NO: 6), and Phe Cys He GIy Arg Xaa^ (SEQ ID NO: 7), wherein Xaai is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, Tyr, and Met; Xaa₂ is selected from the group consisting of GIy, Ser, Thr, Tyr, Asn, and GIn; Xaa3 is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, and Met; Xaa₄is selected from the group consisting of GIy, Ser, Thr, Tyr, Asn, Ala, and GIn; Xaas is selected from the group consisting of Lys and His; Xaa^is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, and Met.

23. A method according to claim 20, wherein the peptide comprises a sequence selected from the group consisting of Xaai Xaa₂ He GIy Arg Leu (SEQ ID NO: 8), Xaai Cys Xaa₃ GIy Arg Leu (SEQ ID NO: 9), Xaa_t Cys He XaJi₄ Arg Leu (SEQ ID NO: 10), Xaa, Cys He GIy Xaa₅ Leu (SEQ ID NO: 11), Xaa, Cys He GIy Arg Xaa^ (SEQ ID NO: 12), Phe Xaa₂ Xaa₃ GIy Arg Leu (SEQ ID NO: 13), Phe Xaa₂ He Xaa,, Arg Leu (SEQ ID NO: 14), Phe Xaa₂ He GIy Xaa₅ Leu (SEQ ID NO: 15), Phe Xaa₂ He GIy Arg Xaa* (SEQ ID NO: 16), Phe Cys Xaa₃ Xaa4 Arg Leu (SEQ ID NO: 17), Phe Cys Xaa₃ GIy Xaa₅ Leu (SEQ ID NO: 18), Phe Cys Xaa₃ GIy Arg Xaaδ (SEQ ID NO: 19), Phe Cys He XaJi₄ Xaa₅ Leu (SEQ ID NO: 20), Phe Cys He Xaa4 Arg Xaae (SEQ ID NO: 21), and Phe Cys He GlyXaa₅ Xaas (SEQ ID NO: 22), wherein Xaai is selected from the group consisting of Ala, VaI, Leu, He, Pro, Trp, Tyr, and Met; Xaa₂ is selected from the group consisting of GIy, Ser, Thr, Tyr, Asn, and GIn; Xaa₃ is selected from the group consisting of Ala, VaI₅ Leu, Ue, Pro, Trp, and Met; Xaa₄ is selected from the group consisting of GIy, Ser, Thr, Tyr, Asn, Ala, and GIn; Xaa₅ is selected from the group consisting of Lys and His; Xaa<sis selected from the group consisting of Ala, VaI, Leu, lie, Pro, Trp, and Met.

24. A method according to claim 20, wherein the peptide comprises from about 6 to about 10 amino acids.

25. A method according to claim 17, wherein the antigen is an allergen.

26. A method according to claim 25, wherein the allergen is selected from a group consisting of pollen, dust mite allergens, mold spores, pet dander, food, insect stings, and medicines.

27. A method according to claim 25, wherein the allergen is a protein.

28. A method according to claim 25, wherein the allergen is a dust mite allergen.

29. A method according to claim 25, wherein the allergen is dust mite allergen Der p 1.

30. A method according to claim 25, wherein the allergen is ragweed pollen.

31. A method according to claim 17, wherein the composition is in aqueous solution.

32. A method according to claim 17, wherein the composition is in a saline solution.

33. A method according to claim 17, wherein the composition further comprises one or more pharmaceutically acceptable excipients.

34. A method according to claim 25, wherein the tight junction agonist is a peptide comprising the sequence FClGRL and the composition is in aqueous solution and the composition comprises one or more allergens selected from the group consisting of pollen, dust mite allergens, mold spores, pet dander, food, insect stings, and medicines.