Compositions and methods for inducing immune tolerance

Abstract

The present disclosure is directed to compositions comprising antigen-specific IgA immune complexes and methods of using the compositions to induce immune tolerance to an allergen in a subject that has an established Th2-biased immune response at one or more mucosal sites prior to administration of the composition.

Description

[Technical field]

[0001] The present disclosure relates to compositions comprising antigen-specific IgA immune complexes and methods of using the compositions to induce immune tolerance to allergens. [Background technology]

[0002] Rates of allergic diseases such as atopic dermatitis, asthma, and food allergies have increased substantially in the past 30 years, especially in infants and young children in Western countries. The increase in prevalence of allergies is believed to be due to several variables, including birth mode, breastfeeding, and exposure to the right environment that allows for the proper building of the microbiome. Severe food allergy-related reactions, also known as food-induced anaphylaxis, are severe, life-threatening reactions that account for 30,000-120,000 emergency department visits, 2,000-3,000 hospitalizations, and approximately 150 deaths annually in the United States (Sampson et al., Pediatrics, 111:1601-1608 (2003); and Ross et al., J. Allergy Clin. Immunol., 121:166-171 (2008)). Symptoms vary in onset, occurring within seconds to hours of exposure to a food allergen, and often affect multiple organ systems, including the gastrointestinal (GI), skin, respiratory, and circulatory systems (Wang et al., Clin. Exp. Allergy, 37:651-660 (2007)). Cutaneous symptoms (e.g., urticaria and angioedema) are the most common, occurring in approximately 80% of cases. Gastrointestinal symptoms, including cramps, abdominal pain, nausea, vomiting, and diarrhea, occur in as many as 40% of cases (Sampson et al., The New England Journal of Medicine, 327:380-384 (1992)).Recent clinical data suggest an association between gastrointestinal symptoms and a more severe anaphylactic phenotype, including hypotension and hypoxia (Schrander et al., J. Pediatr. Gastroenterol. Nutr., 10:189-192 (1990); Troncone et al., Allergy, 49:142-146 (1994); Van Elburg et al., Pediatr Allergy Immunol, 4:79-85 (1993); Calvani et al., Pediatric Allergy and Immunology, 22:813-819 LID - 810.1111 / j.1399-3038.2011.01200.x[doi] (2011); and Brown, SGA, J Allergy Clin. Immunol, 114:371-376 (2004)).

[0003] IgA is the most abundant immunoglobulin and is found mainly on mucosal surfaces. Its function appears to be related to binding pathogens, agglutinating and immobilizing pathogenic microorganisms and materials in mucosal tissues, preventing colonization and invasion of underlying tissues, including the lungs and intestinal tract. Many studies have linked IgA levels to the development of modifying or tolerogenic immune responses. For example, total IgA in colostrum has been shown to provide early protection against infectious organisms and to be inversely correlated with the development of atopic dermatitis in the first two years of life (Orivuri, clin exp allergy, 2014). Breast milk also contains TGFβ1, which promotes IgA class switching in B cells and reduces inflammatory immune responses. Finally, supplementation with certain bacteria, such as Lactobacillus acidophilus, has been shown to increase levels of total and specific IgA in mucosal tissues through interactions with dendritic cells and production of retinoic acid, leading to reduced allergic responses (Mikulic et al., Cell Mol Immunol., 14(6):546-556 (2017); and Prescott et al., Clin Exp Allergy, 38(10):1606-14 (2008)). [Prior art documents] [Non-patent literature]

[0004]

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[0005] There remains a need for methods and compositions for treating allergies, such as food allergies. [Means for solving the problem]

[0006] (Brief Overview) The present disclosure provides the use of a composition comprising an immunoconjugate comprising a pharma- ceutically acceptable carrier and an allergen bound to an IgA immunoglobulin or an antigen-binding fragment thereof, for inhibiting an allergic response to a food allergen in a subject, the IgA immunoglobulin being specific for the allergen. The composition may be formulated for one or more routes of administration, including topical and systemic delivery. Exemplary formulations include formulations for pulmonary (e.g., inhalation) administration, as well as formulations for oral administration.

[0007] The present disclosure also provides a method of inhibiting an allergic response in a subject, the method comprising administering to the subject a composition comprising an immunoconjugate and a pharma- ceutically acceptable carrier, the immunoconjugate comprising an allergen bound to an IgA immunoglobulin specific for the allergen. Administration of a composition comprising an immunoconjugate of the present disclosure may be used to prophylactically treat a subject to prevent and / or reduce an allergic immune response in the subject (e.g., prevent and / or reduce a Th2-type immune response). Administration of a composition comprising an immunoconjugate of the present disclosure may also be used to therapeutically treat a subject to improve and / or reduce an allergic immune response in the subject (e.g., a Th2-type immune response at one or more mucosal sites). The present disclosure is not limited by the route or means of administration of a composition comprising an immunoconjugate. Examples of routes of administration include, but are not limited to, pulmonary administration (e.g., by inhalation), enteral administration (e.g., oral, gastric or duodenal (e.g., by feeding tube), and / or rectal administration), as well as other routes described herein. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows (A) an exemplary administration regimen of IgA immune complexes to the airway mucosa of allergic animals during systemic sensitization: (B and C) it protects the animals from mucus hypersecretion, (D) alters immune cytokine responses, and (E) increases the total number of Treg cells. [Diagram 2] Figure 2 shows that (A) an exemplary administration regimen of IgA immune complexes to the airway mucosa during sensitization protects animals from developing food allergy upon challenge: (B) it blocks clinically relevant diarrhea and hypothermia indicative of anaphylaxis, (C) it reduces systemic IgE and evidence of mast cell degranulation (mMCPt1), and (D) it reduces Th2 cytokines while increasing the inhibitory cytokine IL-10. The lack of effect with a different allergen, peanut (PE), demonstrates the allergen specificity of the response. [Diagram 3]FIG. 3 shows that (A) transfer of Th2-committed cells from a modified food allergy model and TCR transgenic DO11 Balb / c mice does not result in Treg cells, and that IgA immune complexes induce (B) significant clinical changes and (C) significant cytokine changes, and (D) the reduction in IL-4+ transferred DO11 Th2-committed cells does not overlap with the Foxp3+ Treg cells generated during the tolerizing response, indicating that the induction of Treg cells associated with the tolerizing effect of immune complexes is generated from non-Th2-committed cells. [Figure 4] FIG. 4 shows that (A) IgA immune complexes alter mast cell accumulation, (B) reduce IgE and mMCPT-1, and (C) promote a tolerogenic environment in the intestine of food-allergic mice, even when highly biased allergen-specific Th2 cells are transferred into sensitized mice. [Diagram 5] Figure 5 shows that (A) the incidence of diarrhea and hypothermia as clinical indicators of anaphylaxis was controlled only in IgA-TNP-ova immunoconjugate-treated animals. (B) Measurement of serum IgE and Mcpt indicated a decrease in the levels of both mast cell activators and products. (C) The increase in Treg cells in the mesenteric lymph nodes was highly significant in animals treated with oral IgA immunoconjugates. Data represent the mean ± SE from 5-6 mice per group. *P<0.05, **P<0.01, ***P<0.005. [Figure 6] Figure 6 shows isolated mRNA assessed by qPCR analysis for (A) TGFb or (B) IL-10. (C) In another study, BMDCs were incubated with IgA immune complexes or appropriate controls, and after overnight incubation, cells were washed and combined with naive splenic CD4+ T cells from ovalbumin TCR transgenic DO11 IL-4-GFP reporter mice and incubated for 48 hours in the presence of ovalbumin without TNP. Data represent the mean ± SE from three replicate experiments. *P<0.05, **P<0.01, ***P<0.005. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] (definition) To facilitate the understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0010] The term "allergy" as used herein refers to a chronic condition involving an abnormal or pathological immune response to a substance (i.e., an "allergen") that is normally harmless in a normal / healthy individual. Allergy is a type of immune system response called a hypersensitivity reaction. The terms "hypersensitivity" and "hypersensitivity reaction" as used herein refer to a harmful immune response that can cause tissue damage and lead to serious disease. Hypersensitivity reactions are classified into four types, and allergies are often equated with type I hypersensitivity (immediate hypersensitivity reaction mediated by IgE). "Allergen" refers to any substance (e.g., antigen) that induces an allergic reaction in a subject. Examples of allergens include, but are not limited to, airborne allergens (e.g., plant pollens such as dust mites, molds, spores, tree, weed, and grass pollen), foods (milk, egg, soy, wheat, nuts, or fish proteins), animal products (e.g., cat or dog hair), drugs (e.g., penicillin), insect venom, and latex. As used herein, the term "allergic response" refers to a pathological reaction of the immune system caused by exposure of an individual to a foreign, typically harmless substance. Thus, "inhibiting" an allergic response refers to suppressing, ameliorating, or preventing a pathological reaction to an allergen (e.g., by redirecting a Th2-biased immune response in a subject (e.g., a subject with a Th2-mediated disease) toward a Th1-type immune response (e.g., biasing toward a Th1-type immune response and / or generating a more balanced Th1 / Th2-type immune response)).

[0011] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids comprising at least two or more contiguous amino acids, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0012] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. Preferably, the effect is therapeutic, i.e., the effect partially or completely alleviates or cures the injury, disease, or condition, and / or adverse symptoms resulting from the injury, disease, or condition. Similarly, a "therapeutic agent" is a substance, molecule, or compound that, when administered to a subject in need thereof, can alleviate or cure the injury, disease, condition, and / or adverse symptoms. To this end, the methods described herein desirably include administering a "therapeutically effective amount" of an immune complex comprising IgA. A "therapeutically effective amount" refers to an amount effective at the dosage and duration of administration necessary to achieve a desired therapeutic result (e.g., suppression of an allergic response). A therapeutically effective amount may vary according to factors such as the severity of the disease or condition, age, sex, and weight of the individual, and the ability of the therapeutic agent to elicit a desired response in the individual.

[0013] As used herein, the terms "immunogen" and "antigen" refer to an active agent (e.g., an allergen or a microorganism (e.g., a bacterium, virus, or fungus)) and / or portions or components thereof that are capable of eliciting an immune response in a subject.

[0014] As used herein, the term "immunoglobulin" or "antibody" refers to a protein found in the blood or other body fluids of vertebrates, which is used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. Typically, an immunoglobulin or antibody is a protein that contains at least one complementarity determining region (CDR). The CDRs form the "hypervariable region" of the antibody, which is responsible for antigen binding. An immunoglobulin typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (V H ) region and three C-terminal constant (C H 1. C H 2. C H 3) region, and each light chain contains one N-terminal variable (V L ) region and one C-terminal constant (C L ) region. The light chain of an antibody can be classified into one of two different types, κ (kappa) and λ (lambda), based on the amino acid sequence of its constant domain. In a typical antibody, each light chain is linked to a heavy chain by a disulfide bond, and the two heavy chains are linked to each other by disulfide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.

[0015] As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of B lymphocytes directed against a single epitope on an antigen. Monoclonal antibodies are typically produced using hybridoma technology as first reported by Kohler and Milstein, Eur. J. Immunol., 5:511-519 (1976). Monoclonal antibodies may also be produced using recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), isolated from phage display antibody libraries (see, e.g., Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J. Mol. Biol., 222:581-597 (1991)), or produced from transgenic mice carrying a fully human immunoglobulin system (see, e.g., Lonberg, Nat. Biotechnol., 23(9):1117-25 (2005); and Lonberg, Handb. Exp. Pharmacol., 181:69-97 (2008)). In contrast, "polyclonal" antibodies are antibodies secreted by different B cell lineages within an animal. Polyclonal antibodies are a population of immunoglobulin molecules that recognize multiple epitopes on the same antigen.

[0016] The terms "fragment of an antibody," "antibody fragment," and "antigen-binding fragment" of an antibody are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see generally Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005)). Desirably, an antibody fragment contains, for example, one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or a combination thereof. Examples of antibody fragments include: (i) V L , V H , C L , and C H1 (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bond at the hinge region; and (iii) a V fragment of a single arm of an antibody.L and V H (iv) Fab' fragments resulting from cleavage of the disulfide bonds of the F(ab')2 fragment using mild reducing conditions; (v) disulfide-stabilized Fv fragments (dsFv fragments); and (vi) antibody single variable region domains (V H Or V L ) polypeptides.

[0017] As used herein, when an antibody or other entity (e.g., an antigen-binding domain) "specifically recognizes," "specifically binds," or is "specific" for an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules and binds to the antigen or epitope with substantially higher affinity than other entities that do not display the antigen or epitope. In this regard, "substantially higher affinity" means an affinity that is high enough to allow detection of the antigen or epitope as distinguished from the entity using a desired assay or measurement device. Typically, it is at least 10 7 M -1 (For example, >10 7 M -1 , >10 8 M -1 , >10 9 M -1 , >10 10 M -1 , >10 11 M -1 , >10 12 M -1 , >10 13 M -1 etc.) binding constant (K a ) means a binding affinity having a specific epitope. In certain such embodiments, the antibody can bind to different antigens, so long as the different antigens contain that particular epitope. In certain instances, for example, homologous proteins from different species may contain the same epitope.

[0018] As used herein, the term "host" or "subject" refers to an individual to be treated (e.g., administered) by the compositions and methods of the present invention. Subjects include, but are not limited to, mammals (e.g., murine, simian, equine, bovine, porcine, canine, feline, etc.), and most preferably, humans. In the context of the present invention, the term "subject" generally refers to an individual suspected of or diagnosed as suffering from an allergy (e.g., food allergy).

[0019] As used herein, the term "immune response" refers to a response by the immune system of a subject. For example, immune responses include, but are not limited to, a detectable change (e.g., an increase) in activation of Toll-like receptors (TLRs), expression and / or secretion of lymphokines (e.g., cytokines (e.g., Th1 or Th2 type cytokines) or chemokines), activation of macrophages, activation of dendritic cells, activation of T cells (e.g., CD4+ or CD8+ T cells), activation of NK cells, and / or activation of B cells (e.g., production and / or secretion of antibodies). Further examples of immune responses include the binding of an immunogen (e.g., an antigen) to an MHC molecule to induce a cytotoxic T lymphocyte ("CTL") response, a B cell response (e.g., antibody production) and / or a T helper lymphocyte response, and / or a delayed type hypersensitivity (DTH) response to the antigen from which the immunogenic polypeptide is derived, expansion (e.g., proliferation of cell populations) of cells of the immune system (e.g., T cells, B cells (e.g., at any stage of development (e.g., plasma cells)), and increased processing and presentation of antigens by antigen-presenting cells. An immune response can be against an immunogen that the subject's immune system recognizes as foreign (e.g., a non-self antigen from a microorganism (e.g., a pathogen), or a self antigen that is recognized as foreign). Thus, as used herein, an "immune response" should be understood to refer to all types of immune responses, including, but not limited to, innate immune responses (e.g., activation of the Toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells (e.g., antigen-specific T cells) and non-specific cells of the immune system), and humoral immune responses (e.g., responses mediated by B cells (e.g., via the production and secretion of antibodies into plasma, lymph, and / or tissue fluids)). The term "immune response" is meant to encompass all aspects of the ability of a subject's immune system to respond to antigens and / or immunogens (e.g., both initial responses to immunogens (e.g., pathogens) and adaptive (e.g., memory) responses that are the result of an adaptive immune response).

[0020] As used herein, the term "mucosal immunity" refers to immune responses that arise at surfaces that come into contact with the environment, such as the epidermis, gums, nose, intestine, uterus and prostate gland. In health, the mucosal immune system provides protection against pathogens while remaining tolerant to harmless resident microorganisms and benign environmental agents.

[0021] As used herein, the term "immune tolerance" or "tolerance" refers to a state of immune system unresponsiveness to substances or tissues that have the potential to induce an immune response. "Central tolerance" is the primary way that the immune system distinguishes self from non-self, and is established by eliminating autoreactive lymphocyte clones before they develop into fully immunocompetent cells. Central tolerance occurs during lymphocyte development in the thymus and bone marrow for T and B lymphocytes, respectively. "Peripheral tolerance" is important to prevent immune system hyperreaction to various environmental factors (allergens, gut microbes, etc.), and occurs after T and B cells mature and enter peripheral tissues and lymph nodes.

[0022] As described herein, any suitable sample type may be obtained from a subject suspected of having an allergy. A subject (e.g., a human) is "suspected of having an allergy" if he or she is predisposed to experiencing an allergy or is exhibiting allergic symptoms. Predisposition may be genetic (e.g., a certain genetic tendency to experience an allergy) or due to other factors (e.g., environmental conditions, exposure to immunogenic compounds present in certain foods, etc.). Thus, the present invention is not limited to a particular risk, nor is the present invention limited to a particular allergy (e.g., any human may experience any allergy).

[0023] (Detailed Description) The present disclosure is based, at least in part, on the discovery that IgA complexed with a cognate antigen provides immune tolerance in animals with a pre-existing allergic response. Although an understanding of the mechanism is not required to practice the present disclosure, and the present disclosure is not limited to a particular mechanism, in some embodiments, a composition comprising IgA complexed with an antigen provides a tolerizing signal and / or generation of regulatory T cells ("Treg" or "Treg cells") in a subject (e.g., a subject with a pre-existing allergic response). In some embodiments, the immune tolerance provided by the compositions and methods of the present disclosure (e.g., via induction of Treg cells) prevents progression to chronic severe allergic responses at mucosal surfaces (e.g., lung and gut) and blocks anaphylaxis. In other embodiments, the immune tolerance provided by the compositions and methods of the present disclosure (e.g., via induction of Treg cells) occurs systemically within a subject.

[0024] In some embodiments, the disclosure provides a method of inhibiting an allergic response in a subject, the method comprising administering to the subject a composition comprising an immunoconjugate and a pharma- ceutically acceptable carrier, wherein the immunoconjugate comprises an allergen bound to an IgA immunoglobulin specific for the allergen, and wherein the subject has an established Th2-polarized immune response at one or more mucosal sites prior to administering the composition.

[0025] As used herein, the term "immune complex (IC)" refers to an antibody bound to a soluble antigen. Immune complexes may also be referred to as "antigen-antibody complexes" or "antigen-binding antibodies." IgA is the most abundant immunoglobulin produced and is found primarily at mucosal surfaces. Its function appears to be related to binding pathogens, agglutinating and immobilizing pathogenic microorganisms and substances within mucosal tissues to prevent colonization and invasion of underlying tissues, including the lungs and intestinal tract. In addition to IgA, mucosal surfaces obtain protection from pathogens through a tight epithelial barrier and secretion of mucus and other substances to prevent colonization. Patients with partial or total deficiencies in the production of secretory IgA have been shown to experience increased allergies to environmental antigens (including foods), as well as an increased prevalence of autoimmune responses. These and other data suggest that IgA is involved in blocking sensitization of immune system responses and inducing tolerance at mucosal surfaces. In particular, the total amount of IgA in colostrum and breast milk can provide early protection against infectious organisms and has also been shown to be inversely correlated with the development of atopic dermatitis in the first two years of life (Orivuri, clin exp allergy, 2014). Breast milk also contains TGFβ1, which promotes IgA class switching in B cells. Studies have also found a dynamic correlation between Treg cell development, TGFβ production, IgA levels, and tolerance induction.

[0026] The immune complex of the composition may comprise an antigen-binding fragment of an IgA antibody, such as a whole IgA antibody or any of the antibody fragments described herein. The IgA antibody or antigen-binding fragment thereof specifically binds to one or more antigens. In some embodiments, the IgA immunoglobulin is specific for a single allergen, as demonstrated in the experiments described herein indicating that the tolerizing response induced by the IgA immune complex is allergen specific. The allergen may be any suitable allergen disclosed herein or known in the art. In some embodiments, the allergen is an airborne allergen, such as dust mites, molds, spores, plant pollens, such as tree, weed, and grass pollens. In other embodiments, the allergen is a food allergen. It is understood that food allergy is an atopic disease that is mechanistically distinct from non-atopic diseases such as celiac disease. Food allergies may be broadly classified into those mediated by IgE, those mediated by both IgE-dependent and IgE-independent pathways (mixed type), and those that are not IgE-mediated. The immune mechanisms underlying food allergies are further described, for example, in Wong et al., Nat Rev Immunol., 16(12):751-765 (2016). The immune complexes can include any one or combination of IgA immunoglobulins and food allergens. For example, the food allergens can be peanut allergens, tree nut allergens, dairy allergens, wheat allergens, sesame allergens, soy allergens, egg allergens, shellfish allergens, meat allergens, and / or corn allergens.

[0027] In some embodiments, the composition is desirable to include a carrier such as a pharma- ceutically acceptable carrier. As used herein, the term "pharma-ceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, including, but not limited to, phosphate buffered saline, water, and various types of wetting agents (e.g., sodium lauryl sulfate), any solvent, dispersion medium, coating, sodium lauryl sulfate, isotonicity and absorption retarding agents, disintegrants (e.g., potato starch or sodium starch glycolate), polyethylene glycol, and the like. The composition may also include stabilizers and preservatives. Examples of carriers, stabilizers, and adjuvants are described and known in the art (see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Edition, Mack Publ. Co., Easton, PA (1975)).

[0028] The choice of carrier is determined in part by the particular immunoconjugate used and the method of administration. For example, the pharmaceutical composition may contain a preservative, such as, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. Optionally, a mixture of two or more preservatives may be used. In addition, a buffer may be used in the composition. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. Optionally, a mixture of two or more buffers may be used. Methods for preparing administrable (e.g., parenterally administrable) compositions are known to those skilled in the art and are described in detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st Edition (May 1, 2005).

[0029] In some embodiments, the composition may employ time-release, delayed release, and sustained release delivery systems so that delivery of the composition occurs before and within sufficient time to cause sensitization of the site to be treated.Many types of release delivery systems are available and known to those skilled in the art.Such systems may avoid repeated administration of the composition, thereby increasing the convenience of the subject and the physician, and may be particularly suitable for certain embodiments.

[0030] The compositions desirably comprise an "effective amount" of the immunoconjugate, i.e., a dose or concentration of the immunoconjugate that induces a desired immune response (e.g., tolerance) in a recipient (e.g., a human). For example, the composition may comprise a therapeutically effective amount of the immunoconjugate, as described above. Alternatively, the pharmacological and / or physiological effect may be prophylactic, i.e., the effect completely or partially prevents a disease or condition or its symptoms. In this regard, the compositions of the present disclosure comprise a "prophylactically effective amount" of the immunoconjugate. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of administration necessary, to achieve a desired prophylactic result (e.g., prevention of allergies or allergic reactions). For example, a composition comprising the immunoconjugate may be administered to a subject (e.g., an infant) prior to the onset of allergies.

[0031] An effective amount may be administered in one or more administrations (e.g., via the same or different routes) or applications, but is not intended to be limited to a particular formulation or route of administration. A composition comprising an effective amount of an IgA-allergen immune complex may be administered to a mammal (e.g., a human) using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. The composition is preferably suitable for parenteral administration. As used herein, the term "parenteral administration" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to a mammal using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. In some embodiments, the composition is formulated for nasal administration.

[0032] The present disclosure provides compositions and methods for treating (e.g., prophylactically and / or therapeutically) a subject having an allergy or predisposed to experiencing an allergy, comprising providing to the subject an immunoconjugate of the present disclosure (e.g., thereby reducing and / or ameliorating a Th2-type allergic immune response and / or promoting mucosal tolerance to an IgA-targeted allergen in the subject). In some embodiments, the composition comprising the immunoconjugate is delivered enterally. In other embodiments, the composition comprising the immunoconjugate is delivered by pulmonary administration (e.g., by inhalation). In still further embodiments, the composition comprising the immunoconjugate is administered by two or more routes as described herein.

[0033] In some embodiments, direct delivery and / or encapsulation of IgA immune complexes via oral administration is used to promote mucosal tolerance (e.g., locally in the gut and / or at remote mucosal sites such as in the respiratory tract). Delivery of IgA immune complexes can be independent of other compositions. In some embodiments, encapsulation (e.g., by biodegradable microparticles) may be used (e.g., to mitigate degradation of IgA immune complexes during passage through the low pH of the stomach). The present disclosure is not limited to a particular encapsulation technique. In some embodiments, encapsulation of immune complexes utilizes polymeric poly(ethylene glycol)-block-polycaprolactone (PEG-b-PCL)-based nanoparticles or reagents such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG).

[0034] According to the methods of the present disclosure, a composition comprising an immunoconjugate is desirably administered to a subject in which a Th2 biased immune response has been established (e.g., at one or more mucosal sites) prior to administration of the composition. It is understood that there are two major subsets of T lymphocytes, distinguished by the presence of cell surface molecules known as CD4 and CD8. T lymphocytes expressing CD4 are also known as helper T cells, which are the most abundant cytokine producing cells. CD4+ T cells can be further subdivided into Th1 and Th2 cells, the cytokines they produce being known as Th1 and Th2 type cytokines. Th1 type cytokines (e.g., IFN-γ and / or tumor necrosis factor (TNF)) tend to generate inflammatory responses responsible for killing intracellular parasites and perpetuating autoimmune responses. Excessive pro-inflammatory responses can lead to uncontrolled tissue damage, which can be countered by Th2 type cytokines. Th2-type cytokines include interleukin 4 (IL-4), 5 (IL-5), and 13 (IL-13), which are associated with promoting IgE and eosinophil responses in atopy, as well as interleukin 10, which indicates a more anti-inflammatory response. Thus, in some embodiments, administration of a composition comprising an IgA immune complex increases expression of IL-10, TGF-β1, IFN-γ, IL-17, and / or CCL2 in a subject. In excess, a Th2 response counteracts Th1-mediated microbicidal effects. Humans ideally generate a balanced Th1 and Th2 response. Th2 immune responses are characterized by the production of IgE antibodies and high levels of Th2 cytokines, and are associated with inadequate defense against some pathogens, as well as cancer, colitis, asthma, and allergies. Th2 cells predominate in most allergy and asthma patients and differentiate from undifferentiated precursor T cells under the influence of IL-4. Th2 cells control allergic inflammation through the release of the Th2 cytokines IL-4, IL-5, IL-9, and IL-13.

[0035] In some embodiments, administration of a composition comprising an immunoconjugate as described herein results in modulation of an existing Th2 immune response in a subject. For example, the disclosed method provides the ability to redirect a Th2-biased immune response in a subject (e.g., in a subject with a Th2-mediated disease) toward a Th1-type immune response (e.g., bias toward a Th1-type immune response and / or generate a more balanced Th1 / Th2-type immune response) through exposure of the subject to an IgA immune conjugate. In some embodiments, administration of a composition comprising an immunoconjugate to a subject increases the expression of Th1-type cytokines (e.g., IFN-γ and / or tumor necrosis factor (TNF)) in the subject. Thus, the disclosed method can be used to vaccinate an individual against allergies (e.g., peanut or other food allergies, respiratory allergies, etc.). In one embodiment, the present invention provides a more effective benefit (e.g., a more significant reduction in signs, symptoms, or causes of allergy, or a longer-lasting reduction in signs, symptoms, or conditions of allergy) than that achieved with conventional allergy injections.

[0036] In some embodiments, administration of a composition comprising an IgA immunoconjugate as described herein primes, enables, and / or enhances the induction of both a humoral immune response (e.g., the generation of specific antibodies) and a cellular immune response (e.g., cytotoxic T lymphocytes) (e.g., thereby ameliorating the signs, symptoms, or conditions of an allergic disease). Ideally, administration of an IgA immunoconjugate composition induces the development of regulatory T cells (Tregs) in a subject. As used herein, the term "regulatory T cells (Tregs)" refers to a specialized subpopulation of T cells that act to suppress immune responses, thereby maintaining homeostasis and self-tolerance. Tregs can inhibit T cell proliferation and cytokine production, and play an important role in preventing autoimmunity. Tregs can inhibit immune responses through inhibition by inhibitory cytokines, inhibition by cytolysis, inhibition by impairing metabolism, and inhibition by modulating dendritic cell (DC) maturation or function. With regard to inhibitory cytokines, interleukin-10 (IL-10), transforming growth factor-β (TGFβ), and IL-35 are important mediators of Treg cell function. Both mouse and human Treg cells have been shown to mediate cytolysis via granzyme A and / or granzyme B and perforin in vitro and in vivo.

[0037] Tregs are characterized by the expression of the master transcription factor forkhead box P3 (Foxp3). Although Foxp3 expression has been widely used as a marker of the Treg lineage, recent data indicate that Treg fate is determined by multifactorial signaling pathways including cytokines, nuclear factors, and epigenetic modifications. Based on the expression levels of FOXP3 and CD45RA, several subpopulations of human Treg cells have been identified. Such subpopulations include naive / resting (CD45RA + FoxP3 low ), effector type (CD45RA - FoxP3 high ), and cytokine-producing (CD45RA - FoxP3 low) Naive Tregs have a completely demethylated FoxP3 locus and arise from the thymus. Effector Tregs are an active population in vivo, while cytokine-producing Tregs contain cells that can suppress immune responses while producing proinflammatory cytokines such as IL-17 and IFN-γ. Tregs are further described, for example, in Kondelkova et al., ACTA MEDICA (Hradec Kralove) 2010;53(2):73-77; Vignali et al., Nat Rev Immunol. 2008 July;8(7):523-532.doi:10.1038 / nri2343; and Romano et al., Front.Immunol., 2019 January 31;doi.org / 10.3389 / fimmu.2019.00043.

[0038] Furthermore, in some embodiments, a composition comprising an IgA immune complex (e.g., when administered to a subject) induces both a systemic and a mucosal immune response (e.g., generates systemic and / or mucosal immunity (e.g., thereby reducing or preventing the signs, symptoms, or conditions of an allergic disease)). Thus, in some embodiments, administration of a composition of the present disclosure provides protection against exposure to one or more allergens and / or allergens (e.g., food allergens). Ideally, administration of a composition comprising an IgA immune complex results in reduced hypersensitivity to the allergen in a subject subsequently exposed to the allergen.

[0039] The disclosed method may be performed in combination with other therapeutic methods to achieve the desired biological effect in the patient. Ideally, the disclosed method may include or be performed in combination with one or more therapeutic agents or regimens that improve allergy symptoms and signs and / or Th2-biased immune responses at one or more mucosal sites. For example, the disclosed method may be performed in combination (e.g., simultaneously, by the same route, and / or with a similar ascending dose scheme) with one or more immunotherapeutic agents or treatment regimens for desensitization of an individual to potential food allergens. Desensitization immunotherapy is generally delivered sublingually, orally, or through the skin. Sublingual immunotherapy, or SLIT, involves administering a liquid extract of an allergen under the tongue and holding it there for several minutes. Daily allergen dosages start in the submilligram range and gradually increase over several days or weeks. The first double-blind, placebo-controlled trial of SLIT for food allergy was published in 2005 (Enrique et al., J. Allergy Clin. Immunol., 116:1073-1079 (2005)), and a large multicenter, randomized, placebo-controlled, double-blind, crossover study in 2013 evaluated SLIT for peanut allergy (Fleischer et al., J. Allergy Clin. Immunol., 131:119-127 (2013)). In oral immunotherapy, or OIT, low doses (in the milligram range) of allergen are taken daily and gradually increased over a period of several months (e.g., every 2 weeks). OIT uses a higher dose of allergen than other forms of immunotherapy, so patients are often desensitized to sufficient amounts of allergen to avoid life-threatening reactions from accidental exposure, but can also be desensitized to the ability to ingest gram quantities of allergenic foods. Epidermal immunotherapy, or EPIT, uses an adhesive that contains microgram quantities of allergen to deliver antigens to the skin surface. This route of administration appears to cause fewer and less severe side effects than OIT, and some subjects may prefer to wear a skin patch rather than ingesting the same food allergen orally every day.

[0040] In other embodiments, the methods of the present disclosure may be performed in combination with monoclonal antibody therapy. Several monoclonal antibodies have been developed to block processes related to allergic immune responses. For example, the monoclonal antibody omalizumab (XOLAIR®) binds to the Fc region of IgE antibodies, blocking IgE binding to FcεRI, thus preventing Fc receptor-mediated activation and degranulation of mast cells and basophils (Pennington et al., Nat Commun., 7:11610 (2016)). Omalizumab was originally approved for the treatment of allergic asthma, but has been tested in a series of small studies in combination with OIT as a treatment for food allergies (Nadeau et al., Clin. Immunol., 127:1622-1624 (2011); Schneider et al., J. Allergy Clin. Immunol., 132:1368-1374 (2013); Wood et al., J. Allergy Clin. Immunol., 137:1103-1110 (2016); and Begin et al., Allergy Asthma Clin. Immunol., 10:7 (2014)). Monoclonal antibodies that target upstream mediators of food allergy may also be used in the methods described herein. For example, monoclonal antibodies that bind IL-5, such as mepolizumab (NUCALA®) and reslizumab (CINQAIR®), have been evaluated for the treatment of eosinophilic esophagitis (EoE), which can be induced by cow's milk allergens (Assa'ad et al., Gastroenterology, 141:1593-1604 (2011); and Spergel et al., J. Allergy Clin. Immunol., 129:456-463 (2012)). Therapeutic monoclonal antibodies can be administered in combination with the immunotherapies described above. Current and future potential therapeutic agents for food allergies are described in detail, for example, in Yu et al., Nat Rev Immunol., 16(12):751-765 (2016), any one or more of which can be used in combination with the compositions and methods disclosed herein.

[0041] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLES

[0042] The following materials and methods were used in the experiments described in the Examples.

[0043] mouse BALB / c mice, 6-8 weeks of age, were obtained from The Jackson Laboratory (Bar Harbor, ME). DO11.10-Lky-IL-4GFP mice were a kind gift from Dr. Simon Hogan at the University of Michigan Medical School (Ann Arbor, MI). Mice were maintained in a clean barrier facility and handled under protocols approved by the Institutional Animal Care and Use Committee of the University of Michigan Animal Facility.

[0044] reagent Purified IgA from MOPC315 cells (ECACC 85022106), which recognizes the DNP-TNP substituted protein, was purchased from MP Biochemicals (USA). 2,4,6-trinitrophenyl hapten conjugated to ovalbumin protein (TNP-Ova) was purchased from Biosearch Technologies (USA).

[0045] OVA-induced intestinal anaphylaxis Mice aged 6–8 weeks were sensitized twice by intraperitoneal injection to OVA (50 μg OVA per mg alum) in sterile saline on days 0 and 14. During the second sensitization, mice were treated intratracheally (it) with IgA+TNP-Ova; IgA alone; TNP-Ova alone; and saline, respectively. Two weeks after treatment, mice were subjected to repeated oral gavage (og) challenges with OVA (50 mg OVA in 250 μl saline). Before each Og challenge, mice were fasted for 4–5 h. Rectal temperatures were measured before challenge and every 15 min thereafter until 60 min. Diarrhea was assessed by visual observation of the mice until 60 min after the og challenge, and mice that showed large amounts of liquid stool were scored as diarrhea positive. Mice that showed symptoms of anaphylaxis (hypothermia with a body temperature drop of >1.5°C and diarrhea) after the fourth challenge were considered allergic. Mice that showed liquid stools were scored as positive for diarrhea.

[0046] Adoption T lymphocytes were harvested from the spleens of female DO11.10-Lky-IL-4GFP mice. Spleens were placed in chilled RPMI 1640 (Bio-Whittaker, Walkersville, MD) and cells were isolated through a 40 μm nylon mesh filter. After red blood cell lysis, splenocytes were resuspended in RPMI 1640 and diluted to 5 × 10 6 10 cells were plated and incubated with Ova peptide (100ug / mL) in in vitro Th2 biased conditions (IL-4 (20ng / mL); anti-IFN-γ (10ug / mL); IL-2 (10U / mL)) for 72-96 hours. CD4+KJ1.26+IL4-GFP+ cells were sorted using a cell sorter (BD Melody) and transferred to mice sensitized with Ova (50µg OVA per mg alum, intraperitoneally) 7 days prior to adoptive transfer at 1x10 cells per mouse. 6Cells were adoptively transferred. 24 hours after adoptive transfer, mice were treated intratracheally (it) with IgA+TNP-Ova; IgA alone; TNP-Ova alone; and saline, respectively (day 8). On day 14, mice were subjected to repeated oral gavage (og) challenges with OVA (50 mg OVA in 250 μl saline) and rectal temperature and diarrhea were recorded as described above. Flow cytometry analysis was performed on mesenteric lymph nodes to identify IL-4-GFP cells.

[0047] Lymph node restimulation Lung-draining lymph nodes (LDLN), mesenteric lymph nodes, and Peyer's patches were enzymatically digested in RPMI 1640 containing 10% FCS for 45 min at 37°C using 1 mg / ml collagenase A (Roche) and 20 U / ml DNase I (Sigma-Aldrich). Tissues were further dispersed with an 18-gauge needle (1 ml syringe). Red blood cells were lysed and samples were filtered through a 100 μm nylon mesh. Cells (5 × 10 5 ) were seeded into 96-well plates and restimulated with Ova for 48 h. Levels of IL-4, IL-5, IL-13, IL-17A, and IFN-γ in the supernatants were measured using Bio-Plex cytokine assays (Bio-Rad Laboratories).

[0048] quantitative PCR Lung tissues were homogenized in TRIzol reagent and RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). cDNA was synthesized using murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA) and incubated at 37°C for 1 h, followed by 95°C for 10 min to stop the reaction. Real-time quantitative PCR (qPCR) was multiplexed using Taqman primers with FAM-conjugated probes to measure transcription of Il10, foxp3, Il4, Il5, Il13, Il17a, Ifng, and Ccl2. Fold changes were quantified using the 2-ΔΔ cycle threshold (CT) method normalized to 18s RNA or untreated animals. To measure mRNA levels of Muc5ac and Gob5, custom primers were designed as described (Miller et al., 2004). All reactions were performed on an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA).

[0049] Serum IgE and mMCPT-1 assays Serum samples of blood collected after cardiac puncture were analyzed using ELISA kits for OVA-specific IgE (MD Bioproducts, Oakdale, MN, USA) and mMCPT-1 (Invitrogen, Carlsbad, CA, USA). Steady-state analysis was performed for total IgE (Bioscience, San Diego, CA, USA) and mMCPT-1 according to the manufacturer's instructions.

[0050] Flow cytometry Lungs were removed and single cells were isolated by enzymatic digestion with 2.5 mg / ml LIBERASE™ (Roche) and 20 U / ml DNaseI (Sigma, St. Louis, MO) in RPMI 1640 for 45 min at 37°C or with 1 mg / mL collagenase (Roche) and 20 U / ml DNaseI (Sigma, St. Louis, MO) in RPMI 1640 + 10% FCS for 60 min at 37°C. Tissues were further dispersed through an 18-gauge needle (5 ml syringe) to lyse red blood cells, and samples were filtered twice through a 100 μm nylon mesh. Cells were resuspended in PBS. Live cells were identified using a LIVE / DEAD Fixable Yellow Dead Cell Stain kit (Thermo Fisher Scientific, Waltham, MA), then washed and resuspended in PBS with 1% FCS. Fc receptors were blocked with purified anti-CD16 / 32 (clone 93; BioLegend, San Diego, Calif.). Surface markers were identified using antibodies (clones) against the following antigens (all from BioLegend): anti-CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD25 (3C7), CD69 (H1.2F3), CD19 (1D3 / CD19), F4 / 80 (BM8), CD11c (N418), MHC II (M5 / 114.15.2), CD11b (M1 / 70), CD45 (30-F11), CD127 (A7R34), CD90 (30-H12), ST2 (DIH4), Gr-1 (RB6-8C5), B220 (RA3-6B2), and Ter119 (Ter-119). Lineage markers for staining of innate lymphoid cells were anti-CD3, CD11b, B220, Gr-1, and TER119. ILC2: CD45+ / Lin- / CD90+ / ST2+. Data were collected using a NovoCyte flow cytometer (ACEA Bioscience, Inc., San Diego, CA). Data analysis was performed using FlowJo software (Tree Star, OR, USA).

[0051] Isolation of lamina propria mononuclear cells To remove Peyer's patches and mesenteric vessels, ileal loop tissues were opened longitudinally and the LP-enriched fraction was enzymatically dissociated at 37 °C under mechanical agitation as described by Luissint et al. (2019). Briefly, mucus was removed by washing the tissue for 20 min in PBS supplemented with 2% FBS and 5 mM DTT (Fisher BioReagents). IEC lining was removed by three successive washes for 10 min in chelating buffer (PBS containing 2% FBS and 5 mM EDTA). Tissues were minced and digested for 30 min in HBSS+ supplemented with 10 mM HEPES, LIBERASE™ (37.5 U / mL; Roche Applied Science, Indianapolis, IN), and DNase I (300 Kuntz units / mL). The cell suspension was filtered, washed in PBS solution supplemented with 10% FBS and 2 mM EDTA, and cells were counted and stained for flow cytometry analysis.

[0052] Histological examination Serial 6 μm sections were obtained from paraffin-embedded, 10% formalin-fixed left lung and small intestine stained with H&E. To select representative slides, two lung / small intestine sections per mouse and five sections per animal were analyzed. PAS staining was performed to identify mucus in the airways. Chloroacetate esterase staining was performed to identify mast cells in the small intestine.

[0053] statistical analysis Data were analyzed by Prism 7 (GraphPad Software). Data presented are mean ± SEM. Comparisons of two groups were performed by unpaired two-tailed Student's t-test. Comparisons of more than two groups were analyzed by one-way ANOVA, followed by individual comparisons by two-tailed Student's t-test. A p-value <0.05 was considered significant.

[0054] [Example 1] This example demonstrates that administration of IgA immune complexes to the airways inhibits the allergic asthmatic response in mice.

[0055] The role of IgA in regulating immune responses has not been addressed in previous studies. Most of the support for an important role for IgA in tolerogenic and inhibitory responses is circumstantial, either related to deficiency or correlated with IgA levels. We investigated whether antigen-specific IgA, with or without antigen, could alter ongoing allergic immune responses. To this end, we used a model of allergic airway responses with two alum-ovalbumin systemic sensitizations to elicit a strong Th2 response. At the time of the second intraperitoneal alum-ovalbumin sensitization, animals received an intratracheal supplementation of TNP-specific IgA with or without TNP-ovalbumin. The latter immune complex allowed IgA to induce a specific response similar to that occurring at mucosal surfaces during antigen exposure. Other controls were TNP-ova only and saline (vehicle). Two weeks after the second alum-ovalbumin challenge, animals were given seven ovalbumin challenges to the airways over a two-week period. 24 hours after the final ovalbumin airway challenge, animals were examined for evidence of allergic airway disease (Figure 1A). Examination of histopathology stained with PAS suggested that animals treated with IgA-TNP-ova immune complexes had reduced mucus staining of the airways compared to all other groups (Figure 1B), which was confirmed by examining expression of muc5b, the major mucin expressed in the lung by goblet cells (Figure 1C). Restimulation of lung-draining lymph node cells demonstrated that the IgA / TNP-ova treated group had a significant reduction in the Th2 cytokines IL-4, IL-5, IL-13, and an increase in IL-10 (Figure 1D). Furthermore, flow cytometric analysis of lymph node cells demonstrated a significant increase in CD4+CD25+Foxp3+ T cells only in the IgA / TNP-ova treatment group, a nearly 10-fold increase compared to allergic animals treated with saline controls (Figure 1E). Thus, mucosal administration of IgA / TNP-ova in animals with a systemic allergic response to ova protected the animals from local allergen challenge, with a corresponding increase in Treg cells.

[0056] The reduction in established immune responses in IgA-immunocomplex-sensitized mice to the mucosal airway challenge described above pointed to an association with the development of Treg cell responses. To understand whether this response was local or whether IgA immune complexes applied to the airway had a systemic effect, a study was designed to investigate intestinal food allergy using a similar strategy. Similar to the airway model, a systemic sensitization model was used to follow the application of similar IgA immune complexes to the airway, in which alum-ovalbumin was administered IP to induce an allergic response (Figure 2A). Furthermore, to confirm allergen-specific responses, mice were immunized with alum-precipitated peanut allergen (Greer) (Figure 2A). Just before the second alum-ova sensitization, animals were administered immune complexes or appropriate controls to the airway (IT). Intragastric ovalbumin food challenge was initiated 2 weeks later, followed by four oral gavage doses as indicated. To determine whether the alteration of ovalbumin responses by IgA immune complexes was allergen specific, peanut-alum sensitized mice were given PE-saline or PE-IgA+TNF-Ova.

[0057] The primary outcome of the response was the onset of diarrhea within 30 min after oral ovalbumin challenge. Examination of diarrhea in the different groups demonstrated that TNP-ova given to the airways alone did not alter the ongoing allergic response, whereas IgA-TNP-ova immune complexes mitigated the onset of the diarrhea response (Figure 2B). In contrast, peanut-alum sensitized mice treated with IgA-TNP-ova immune complexes did not show a reduction in diarrhea compared to peanut control mice (Figure 2B). Another sign of anaphylaxis, reduction in body temperature, was significantly reduced when animals were challenged but not in animals treated with IgA immune complexes. To further examine the onset of the allergic response, serum IgE was examined along with mast cell-derived MCPt 60 min after oral challenge (Figure 2C). Both IgE and MCPt were significantly reduced only in animals exposed to IgA immune complexes in the airways of animals challenged with ovalbumin. We also examined the overall immune response by restimulating gut-draining lymph node cells with ovalbumin. The resulting supernatants demonstrated a significant decrease in IL-4 and IL-13 and an increase in IL-10 in animals treated with IgA immune complexes (Figure 2E). In contrast, none of the immune parameters, including IgE, MCPT1, or cytokines, were altered in peanut-sensitized and challenged mice with IgA-TNP-ova immune complexes (Figure 2C-E). Together, these data demonstrate that the significant changes induced by exposure to airway IgA / TNP-ova immune complexes during the systemic allergic response protect animals from severe Th2-induced oral antigen responses and are allergen-specific.

[0058] To better understand how IgA immune complexes alter allergic responses in sensitized mice, a T cell transfer model was utilized with Th2-biased DO11 ova-specific TCR transgenic T cells. The model was constructed by isolating GFP IL-4 reporter DO11 naive splenocytes. Spleen cells from naive DO11 mice were biased in vitro with rIL-4, anti-IFN, rIL-2, and TCR activation (see Materials and Methods) and sorted for GFP expression, which indicated the production of IL-4. Because the model uses a subset of already biased Th cells, the immunization protocol was modified to treat animals by IP injection of sorted IL-4-producing obligate T cells into Balb / c mice on day 7 after a single round of alum-ova sensitization, followed 24 hours later by administration of IgA / TNP-ova immune complexes to the airways (Figure 3A). Gavage challenge was initiated on day 14, and animals were examined for diarrhea at the fourth challenge. The IgA / TNP-ova composition almost completely protected mice from severe diarrhea and hypothermia, both signs of anaphylaxis (Figure 3B). When draining lymph nodes were restimulated ex vivo, a significant reduction in IL-4, IL-5, and IL-13 and a significant increase in IL-10 were observed (Figure 3C). When flow cytometry was performed to identify GFP+IL-4+ T cells, a significant reduction and a corresponding increase in Foxp3+ Treg cells were found in the IgA / TNP-ova treated group (Figure 3D). Importantly, none of the Foxp3+ T cells were IL-4+, indicating that they were derived from naive host T cells and not Th2-committed cells.

[0059] To further explain the pathogenesis of the disease, we performed tests to determine the number of mast cells accumulated in the small intestine, as this may correlate with the severity of the anaphylactic disease response. Animals treated with IgA / TNP-ova immune complexes showed a significant reduction in mast cells in the small intestine (Figure 4A). Serum IgE and mMCPt-1 levels were also significantly reduced only in animals treated with IgA / TNP-ova immune complexes (Figure 4B), further demonstrating a reduction in Th2 and mast cell activation. Furthermore, when the expression levels of IL-10, TGF-β, and foxp3 were examined in intestinal tissue, a significant upregulation of these genes was observed (Figure 4C). Thus, the ability to control Th2 immune responses corresponds to the development of increased mast cell numbers in the small intestine of allergic mice and can be modulated by mucosal treatment with allergen-specific IgA immune complexes.

[0060] One potential mechanism of how IgA immune complexes promote altered responses is through differential activation of antigen-presenting cells (APCs), particularly dendritic cells (DCs). To examine this, bone marrow-derived DCs were cultured in GM-CSF for 6–7 days and exposed to IgA immune complexes or appropriate controls. After overnight incubation, DCs were assessed for expression of innate immune system cytokines, IL-10 and TGFβ, by quantitative PCR analysis (Figure 6). The data demonstrate that IgA immune complexes induced a significant increase in mRNA expression of TGFβ (Figure 6A) and IL-10 (Figure 6B), while IgA alone increased them to a much lesser extent. To better understand whether immune complex activation alters the ability of DCs to trigger antigen-specific primary immune responses, we used DO11 ovalbumin peptide transgenic TCR naive splenic CD4 T cells. After overnight incubation with IgA immune complexes or appropriate controls, DCs were washed and re-seeded with DO11 naive CD4 T cells at a ratio of 1:10 with fresh ovalbumin for processing and presentation to T cells. The data in Figure 6C indicate that in response to ovalbumin, substantial production of all cytokines occurred compared to the DC only control (no ovalbumin administration), while DCs pre-incubated with IgA immune complexes had significant decreases in IL-4, IL-13, and IL-17, and increases in IL-10 and IFNγ in the supernatant after 48 hours of incubation. Together, these data indicate that IgA immune complexes induce regulatory cytokines in DCs that correspond to changes in T cell responses.

[0061] [Example 2] This example describes oral administration of IgA immune complexes.

[0062] To promote a similar modulatory effect in anaphylactic disease, IgA immune complexes with TNP-ovalbumin were given by oral gavage to systemically sensitized mice. Systemically sensitized animals were given IgA:TNP-ovalbumin immune complexes or appropriate control treatments by oral gavage on day 14 prior to the second alum-ovalbumin IP sensitization. As shown in Figure 2 above, an oral gavage challenge was given with ovalbumin on protocol day 28, 14 days after the second alum-ovalbumin sensitization. After the final challenge, animals were observed for diarrhea and temperature changes, which showed that only animals receiving IgA IC by oral gavage were protected from anaphylactic disease, as assessed by the incidence of diarrhea and temperature changes (Figure 5). Furthermore, IgE and MCPT1 were also significantly reduced only in IgA-TNP-ova IC-treated animals. Lymph nodes of animals treated with IgA IC showed an approximately five-fold increase in Treg cells, whereas IgA alone showed a more modest increase in Treg cells but was insufficient to alter the severe anaphylactic ova-induced disease. Thus, IgA IC against ovalbumin can be delivered at both airway and intestinal mucosal surfaces to induce regulatory responses that protect against severe allergen-induced sequelae.

[0063] All references cited in this specification, including publications, patent applications, and patents, are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0064] The use of the terms "a" and "an" as well as "the" and "at least one" and similar references in the context of describing the present invention (particularly in the context of the claims below) should be construed to cover both the singular and the plural, unless otherwise stated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item selected from the listed items (A or B), or any combination of two or more of the listed items (A and B), unless otherwise stated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open ended terms (i.e., meaning "including but not limited to"), unless otherwise stated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each individual value within the range, unless otherwise stated herein, and each individual value is incorporated into the specification as if it were individually stated herein. All methods described herein can be performed in any suitable order, unless otherwise stated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better clarify the invention, and does not impose limitations on the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0065] Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that such variations will be adopted by those skilled in the art as appropriate, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, combinations of the above-described elements in all possible variations thereof are encompassed by the present invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. 1. A method for inhibiting an allergic reaction in a subject, the method comprising administering to the subject a composition comprising an immunoconjugate and a pharmaceutically acceptable carrier, wherein the immunoconjugate comprises an allergen bound to an IgA immunoglobulin specific for the allergen, and the subject has an established Th2-biased immune response at one or more mucosal sites prior to administering the composition.

2. 10. The method of claim 1, wherein the allergen is a food allergen, an airborne allergen, an animal product, a drug, an insect venom, or latex.

3. 3. The method of claim 2, wherein the allergen is a food allergen selected from peanut allergens, tree nut allergens, dairy allergens, wheat allergens, soy allergens, egg allergens, shellfish allergens, meat allergens, sesame allergens, and corn allergens.

4. The method of claim 1 , wherein the subject is a human.

5. 10. The method of claim 1, wherein administering the composition increases expression of IL-10, TGF-β1, IFN-γ, IL-17, and / or CCL2 in the subject.

6. 10. The method of claim 1, wherein administering the composition induces the development of regulatory T cells (Tregs) in the subject.

7. The method of claim 1, wherein administering the composition to a subject increases expression of Th1-type cytokines in the subject.

8. The method of claim 1 , wherein the composition is administered to the subject intranasally.

9. 10. The method of claim 1, wherein hypersensitivity to the allergen is reduced in a subject subsequently exposed to the allergen.

10. Use of a composition comprising an immunoconjugate comprising a pharmaceutically acceptable carrier and an allergen bound to an IgA immunoglobulin or an antigen-binding fragment thereof, for inhibiting an allergic reaction to a food allergen in a subject, wherein the IgA immunoglobulin is specific for the allergen.

11. 11. The use according to claim 10, wherein the allergen is a food allergen, an airborne allergen, an animal product, a drug, an insect venom, or latex.

12. 12. The use according to claim 11, wherein the allergen is a food allergen selected from peanut allergens, tree nut allergens, dairy allergens, wheat allergens, soy allergens, egg allergens, shellfish allergens, meat allergens, and corn allergens.

13. 11. The use according to claim 10, wherein the composition is formulated for enteral administration.

14. The use of claim 10, wherein the composition is formulated for oral administration.

15. 11. The use of claim 10, wherein the composition is formulated for mucosal administration.

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