TW202430207A - Treatment of peanut allergy with tolerizing nanoparticles - Google Patents

Abstract

The present disclosure relates, in general to methods of treating peanut allergy using nanoparticles encapsulating peanut antigens to induce antigen-specific tolerance.

Description

Treating peanut allergy with tolerizing nanoparticles

The present invention relates to a method for treating peanut allergy using immune tolerance modulating nanoparticles encapsulating purified peanut extract containing allergenic peanut protein or antigenic fragments thereof.

Peanut allergy is one of the most common food allergies, affecting approximately 1.2% of the U.S. population and 2.5% of the child population, with an increasing incidence over the past decade (Cannon HE. Am J Manag Care. 2018;24(Suppl 19):S428-s433). Peanut allergy is caused by a pathological hyperimmune response, in which exposure to peanuts can cause mild to severe symptoms such as nausea, vomiting, rash, decreased breathing, decreased blood pressure, and even death.

Normal individuals with healthy immune systems are able to remain unresponsive to antigens encountered in common foods such as peanuts; however, in individuals with peanut allergy, loss of immune tolerance to peanut antigens drives a pathological hyperimmune response. To date, eight peanut proteins (Ara h1 to Ara h8) have been identified as the major antigenic peanut proteins driving allergic hyperimmune responses (Keet et al., J Allergy Clin Immunol Pract. 2013;1(1):101-103). Additional peanut allergens have recently been described, with a total of up to 18 allergenic peanut proteins reported (Ozias-Akins et al., Allergy. 2019 May;74(5):888-898). Allergic immune responses to peanut antigenic proteins are mediated by T cell-dependent mechanisms involving upregulation of T helper type 2 (Th2) interleukin production (e.g., IL-4, IL-5, IL-9, and IL-13), and B cell class switching leading to IgE antibody production and degranulation of mast cells and phils (Sampath et al., J Clin Invest. 2019;129(4):1431-1440).

Currently, there is no cure for peanut allergy, and strict avoidance of exposure to peanut antigens and management of systemic allergic reactions are the only options available to patients. Immune tolerance therapy, which can induce T cell tolerance to peanut antigens, is considered the gold standard for the treatment of peanut allergy, but has been elusive to date. Several attempts to develop immune tolerance therapy have been conducted using oral immunotherapy (OIT), subcutaneous immunotherapy (SCIT), epicutaneous immunotherapy (EPIT), and sublingual immunotherapy (SLIT) approaches (Feuille et al., Allergy Asthma Immunol Res. 2018;10(3):189-206). The success of these treatments varies widely and only desensitization to peanut protein has been reported, which provides only protection from accidental exposure but not cure (Chinthrajah et al., Lancet. 2019;394(10207):1437-1449; Vickery et al., N Engl J Med. 2018;379(21):1991-2001; Fleischer et al., J. Am Med Assoc. 2019;321(10):946-955).

Tolerogenic immunomodulatory particles (TIMPs) comprising one or more antigens have been previously described for treating immune-mediated disorders (e.g., autoimmune diseases and allergies) by inducing antigen-specific immune tolerance (WO20131319253 and WO2015023796, which are incorporated herein by reference). TIMPs have demonstrated efficacy in inducing T cell tolerance in several preclinical models of autoimmune diseases and allergies. Peanut allergy (PA) can be improved or potentially cured by inducing antigen-specific T cell tolerance to peanut antigens using encapsulated purified peanut extract (PPE) and/or TIMPs comprising peanut extract or one or more peanut proteins or antigenic fragments thereof (including Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, Ara h13, Ara h14, Ara h15, Ara h16, Ara h17 and Ara h18).

Provided herein is a method of treating peanut allergy in an individual, comprising administering TIMP-PPE to the individual, wherein TIMP-PPE is administered at a dose of 0.001 to 12 mg/kg. Also provided herein is a method of reducing an allergic immune response to a peanut antigen in an individual suffering from PA, comprising administering TIMP-PPE to the individual, wherein TIMP-PPE is administered at a dose of about 0.001 to 12 mg/kg. In various embodiments, TIMP-PPE is administered in an amount of about 0.001 to 10 mg/kg, about 0.005 to 12 mg/kg, about 0.01 to 12 mg/kg, about 0.05 to 12 mg/kg, about 0.1 to 12 mg/kg, about 0.5 to 10 mg/kg, about 1 to 8 mg/kg, about 1.5 to 10 mg/kg, about 2 to 12 mg/kg, about 2 to 10 mg/kg, about 3 to 10 mg/kg, about 4 to 10 mg/kg, about 4 to 12 mg/kg, or about 5 to 12 mg/kg. In various embodiments, TIMP-PPE is administered at a dose of about 0.001 mg/kg, 0.0025 mg/kg, 0.005 mg/kg, 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5 mg/kg, 6 mg/kg, 8.0 mg/kg, 10 mg/kg, or 12 mg/kg. In various embodiments, TIMP-PPE is administered in an amount of about 0.1 mg, 0.25 mg, 0.5 mg, 1 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, or 800 mg.

In various embodiments, TIMP-PPE is administered at a concentration between about 0.0005 mg/mL and about 50 mg/mL. In various embodiments, TIMP-PPE is administered at a concentration of about 0.0005 mg/mL, 0.001 mg/mL, 0.005 mg/mL, 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 12.5 mg/mL, 15 mg/mL, 17.5 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 40 mg/mL, or 50 mg/mL. In various embodiments, TIMP-PPE is administered via intravenous infusion lasting about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 18, or 20 hours.

In various embodiments, TIMP-PPE is administered as a single dose or multiple doses. In various embodiments, TIMP-PPE is administered as two doses one week apart. In various embodiments, TIMP-PPE is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year.

In various embodiments, additional doses of TIMP-PPE are administered, i.e., after an initial or preliminary administration of TIMP-PPE, additional doses of TIMP-PPE are administered in a single dose or multiple doses. In various embodiments, additional doses of TIMP-PPE are administered once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year. In various embodiments, TIMP-PPE is administered in two doses one week apart, followed by additional doses of TIMP-PPE administered in a single dose once every three months.

In various embodiments, an additional dose of TIMP-PPE is administered at a dose of 0.001 mg/kg to 12 mg/kg. In various embodiments, the additional dose of TIMP-PPE is about 0.001 to 10 mg/kg, about 0.005 to 12 mg/kg, about 0.01 to 12 mg/kg, about 0.05 to 12 mg/kg, about 0.1 to 12 mg/kg, about 0.5 to 10 mg/kg, about 1 to 8 mg/kg, about 1.5 to 10 mg/kg, about 2 to 12 mg/kg, about 2 to 10 mg/kg, about 3 to 10 mg/kg, about 4 to 10 mg/kg, about 4 to 12 mg/kg, or about 5 to 12 mg/kg, or about 0.001 mg/kg, 0.0025 mg/kg, 0.005 mg/kg, 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.25, 0.5 mg/kg, 1.0 The drug was then administered at a dose of 10 mg/kg, 2.0 mg/kg, 4.0 mg/kg, 6 mg/kg, 8.0 mg/kg, 10 mg/kg or 12 mg/kg. In various embodiments, an additional dose of TIMP-PPE is administered in an amount of about 0.1 mg, 0.25 mg, 0.5 mg, 1 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, or 800 mg. In various embodiments, the additional dose of TIMP-PPE is administered at a concentration between about 0.0005 mg/mL and about 50 mg/mL. In various embodiments, an additional dose of TIMP-PPE is administered at a concentration of about 0.0005 mg/mL, 0.001 mg/mL, 0.005 mg/mL, 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 12.5 mg/mL, 15 mg/mL, 17.5 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 40 mg/mL, or 50 mg/mL.

In various embodiments, the TIMP-PPE booster dose is administered via intravenous infusion over about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 18, or 20 hours.

In various embodiments, TIMP-PPE encapsulates peanut protein and or antigenic fragments of peanut protein Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, Ara h13, Ara h14, Ara h15, Ara h16, Ara h17 or Ara h18. In various embodiments, TIMP-PPE encapsulates peanut protein and or antigenic fragments of peanut protein Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, Ara h13, Ara h14, Ara h15, Ara h16, Ara h17, Ara h18 or a combination thereof. In various embodiments, the TIMP-PPE comprises a peanut extract or one or more peanut proteins or antigenic fragments thereof selected from the group consisting of Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, Ara h13, Ara h14, Ara h15, Ara h16, Ara h17 and Ara h18.

In various embodiments, TIMP-PPE consists of poly(lactic-co-glycolic acid) (PLGA) particles encapsulating one or more peanut antigens and a suitable buffer or excipient. In various embodiments, the TIMP-PPE particles are surface functionalized. In various embodiments, the TIMP-PPE particles are surface functionalized by carboxylation. In various embodiments, the TIMP-PPE particles have a negative zeta potential. In various embodiments, the negative zeta potential of the TIMP-PPE particles is between about -100 mV and about 0 mV. In various embodiments, the zeta potential of the particle is about -100 mV to about -25 mV, about -100 mV to about -30 mV, about -80 mV to about -30 mV, about -75 mV to about -30 mV, about -70 mV to about -30 mV, about -75 to about -35 mV, about -70 to about -25 mV, about -60 mV to about -30 mV, about -60 to about -35 mV, or about -50 mV to about -30 mV. In various embodiments, the zeta potential is about -25 mV, -30 mV, -35 mV, -40 mV, -45 mV, -50 mV, -55 mV, -60 mV, -65 mV, -70 mV, -75 mV, -80 mV, -85 mV, -90 mV, -95 mV, or -100 mV.

In various embodiments, the size or diameter of the TIMP-PPE particles is between 0.05 μm and about 10 μm. In various embodiments, the size or diameter of the TIMP-PPE particles is between 0.1 μm and about 10 μm. In various embodiments, the size or diameter of the TIMP-PPE particles is between 0.1 μm and about 5 μm. In various embodiments, the size or diameter of the TIMP-PPE particles is between 0.1 μm and about 3 μm. In various embodiments, the size or diameter of the TIMP-PPE particles is between 0.3 μm and about 5 μm. In various embodiments, the size or diameter of the TIMP-PPE particles is between 0.3 μm and about 3 μm. In various embodiments, the size or diameter of the TIMP-PPE particles is between 0.3 μm and about 1 μm. In various embodiments, the size or diameter of the TIMP-PPE particles is between 0.4 μm and about 1 μm. In various embodiments, the TIMP-PPE particles have a diameter of about 100 to 10,000 nm, about 100 to 5,000 nm, about 100 to 3,000 nm, about 100 to 2,000 nm, about 300 to 5,000 nm, about 300 to 3,000 nm, about 300 to 1,000 nm, about 300 to 800 nm, about 400 to 800 nm, or about 200 to 700 nm. In various embodiments, the TIMP-PPE particles have a diameter of about 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm. In various embodiments, the diameter of the negatively charged particles is between 400 nm and 800 nm.

In various embodiments, TIMP-PPE is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally.

In various embodiments, TIMP-PPE is administered alone or in combination with one or more additional therapeutic agents. In various embodiments, the present invention provides a method of treating peanut allergy or peanut allergy-related symptoms in an individual in need thereof, comprising administering to the individual a composition comprising TIMP-PPE alone or in combination with a therapeutic agent suitable for treating peanut allergy.

In various embodiments, the therapeutic agent for treating peanut allergy induces regulatory T cells (Tregs). In various embodiments, the therapeutic agent for treating peanut allergy increases the frequency and/or number of Tregs. In various embodiments, the therapeutic agent for treating peanut allergy is IL-2 therapy that induces Tregs. In various embodiments, the IL-2 therapeutic agent is low-dose IL-2, an IL-2 mutant protein engineered to expand Tregs, an IL-2 variant engineered to expand Tregs, an IL-2 molecule engineered to be selective for a high affinity IL-2 receptor, a PEGylated IL-2, an IL-2 complex, or an IL-2/CD25 fusion protein. In various embodiments, the additional therapeutic agent is an IgE inhibitor, a philoglobulin activation inhibitor, a mast cell activation inhibitor, an antihistamine, or a small molecule or biological therapeutic. In various embodiments, the additional therapeutic agent is an IgE inhibitor, an IgE competitor for allergen binding sites, a philoglobulin activation inhibitor, a mast cell activation inhibitor, an antihistamine, an interleukin inhibitor, a microbiome therapy, a small molecule or biological therapeutic. In various embodiments, the additional therapeutic agent inhibits IgE. In various embodiments, the additional therapeutic agent inhibits IgE antibodies. In various embodiments, the additional therapeutic agent inhibits basophil activation. In various embodiments, the additional therapeutic agent inhibits mast cell activation. In various embodiments, the additional therapeutic agent is a biologic or a small molecule. In various embodiments, the additional therapeutic agent is an anti-IgE antibody, an anti-IL-4Rα antibody, an anti-IL13 antibody, an anti-IL-33 antibody, an antihistamine, a steroid, a corticosteroid, a leukotriene modifier, or a nonsteroidal anti-inflammatory drug (NSAID).

In various embodiments, the therapeutic agent for treating peanut allergy is an IgE inhibitor, an IgE competitor for allergen binding sites, an inhibitor of philoglobulin activation, an inhibitor of mast cell activation, an antihistamine, an interleukin inhibitor, a microbiota therapy, a small molecule or a biological therapeutic. In various embodiments, the therapeutic agent for treating peanut allergy inhibits IgE. In various embodiments, the therapeutic agent for treating peanut allergy inhibits IgE antibodies. In various embodiments, the therapeutic agent for treating peanut allergy inhibits philoglobulin activation. In various embodiments, the therapeutic agent for treating peanut allergy inhibits mast cell activation. In various embodiments, the therapeutic agent for treating peanut allergy is a biologic or a small molecule. In various embodiments, the therapeutic agent for treating peanut allergy is an anti-IgE antibody, an anti-IL-4Rα antibody, an anti-IL13 antibody, an anti-IL-33 antibody, an antihistamine, a steroid, a corticosteroid, a leukotriene modifier, or a nonsteroidal anti-inflammatory drug (NSAID).

In various embodiments, the additional therapeutic agent is an antihistamine. In various embodiments, the therapeutic agent suitable for treating peanut allergy is an antihistamine. In various embodiments, the antihistamine is a first generation antihistamine. In various embodiments, the antihistamine is a second generation antihistamine. In various embodiments, the antihistamine is selected from the group consisting of brompheniramine, carbinoxamine maleate, chlorpheniramine, clemastine, diphenhydramine, hydroxyzine, triprolidine, azelastine, cetirizine, desloratadine, fexofenadine, levocetrizine, doxylamine, ebastine, embramine, epinephrine, fexofenadine, loratadine, and olopatadine.

In various embodiments, the therapeutic agent administered in combination with TIMP-PPE is an anti-IgE antibody, an anti-IL-4Rα antibody, an anti-IL13 antibody, an anti-IL-33 antibody, an antihistamine, a steroid, a corticosteroid, a leukotriene modifier, a low-dose IL-2, an IL-2 mutant protein engineered to expand Tregs, an IL-2 variant engineered to expand Tregs, an IL-2 molecule engineered to be selective for a high affinity IL-2 receptor, a polyethylene glycol, or a poly(ethylene glycol)-containing molecule. Diolated IL-2, IL-2 complex, IL-2/CD25 fusion protein, probiotics, probiotics, histone deacetylase inhibitors, short-chain fatty acids (e.g. acetate, butyrate, propionate, butyrate polymers), IgE inhibitors, IgE competitors for allergen binding sites, phagocytophil activation inhibitors, mast cell activation inhibitors, interleukin inhibitors, microbiome therapy, small molecule or biological therapeutics, or nonsteroidal anti-inflammatory drugs (NSAIDs).

In various embodiments, the additional therapeutic agent is a steroid. In various embodiments, the therapeutic agent suitable for treating peanut allergy is a steroid. In various embodiments, the steroid is selected from the group consisting of beclomethasone, ciclesonide, fluticasone furoate, mometasone, budenoside, fluticasone, triamcinolone, and loteprednol.

In various embodiments, the additional therapeutic agent is a corticosteroid. In various embodiments, the therapeutic agent suitable for treating peanut allergy is a corticosteroid. In various embodiments, the corticosteroid is selected from the group consisting of cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, and hydrocortisone.

In various embodiments, the additional therapeutic agent is a nonsteroidal anti-inflammatory drug (NSAID). In various embodiments, the therapeutic agent suitable for treating peanut allergy is a nonsteroidal anti-inflammatory drug (NSAID). In various embodiments, the NSAID is a non-selective NSAID. In various embodiments, the NSAID is a COX-2 selective NSAID. In various embodiments, the NSAID is a COX-1 selective NSAID. In various embodiments, the NSAID is a prostaglandin synthase inhibitor. In various embodiments, the NSAID is selected from the group consisting of diclofenac, diclofenac potassium, diclofenac sodium, diflunisal, etodolac, flurbiprofen, fenoprofen, fenoprofen calcium, ketorolac, ketorolac sulfamethoxazole ... tromethamine, ketoprofen, tolmetin, tolmetin sodium, acetylsalicylic acid, aspirin, ibuprofen, naproxen, indomethacin, indomethacin sodium, sulindac, felbinac, piroxicam, mefenamic acid, meclofenamic acid sodium, meloxicam, nabumetone, oxaprozin, piloxicam, celecoxib, etoricoxib, lumiracoxib, rofecoxib, and valdecoxib.

In various embodiments, the additional therapeutic agent is a leukotriene modulator. In various embodiments, the therapeutic agent suitable for treating peanut allergy is a leukotriene modulator. In various embodiments, the leukotriene modulator is an anti-leukotriene. In various embodiments, the leukotriene modulator is a leukotriene receptor antagonist. In various embodiments, the leukotriene modulator is a leukotriene synthesis inhibitor. In various embodiments, the leukotriene modulator is selected from the group consisting of montelukast, zileuton, and zafirlukast.

In various embodiments, the biologic is an antibody. In various embodiments, the antibody is an anti-IgE, anti-IL-4Rα, anti-IL-13, or anti-IL-33 antibody. In various embodiments, the anti-IgE antibody is omalizumab (XOLAIR®). In various embodiments, the anti-IL-4Rα antibody is dupilumab (DUPIXENT®). In various embodiments, the anti-IL-33 antibody is etokinumab. In various embodiments, the additional therapeutic agent is administered before, during, or after administration of TIMP-PPE. In various embodiments, the additional therapeutic agent is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally.

In various embodiments, the therapeutic agent for treating peanut allergy is administered before, during, or after the administration of TIMP-PPE. In various embodiments, the therapeutic agent for treating peanut allergy is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally.

In various embodiments, the therapeutic agent is administered prior to, concurrently with, or after administration of TIMP-PPE. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-PPE. In various embodiments, the therapeutic agent is administered 1, 2, 3, or 4 weeks prior to administration of TIMP-PPE. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months prior to administration of TIMP-PPE. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years prior to administration of TIMP-PPE. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-PPE. In various embodiments, the therapeutic agent is administered 1, 2, 3, or 4 weeks after administration of TIMP-PPE. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration of TIMP-PPE. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years after administration of TIMP-PPE.

The present invention describes a method of treating peanut allergy in an individual comprising administering to the individual a combination of TIMP-PPE and an anti-IgE antibody, wherein the TIMP-PPE is administered at a dose of about 0.001 to 12 mg/kg, and wherein the anti-IgE antibody is administered at a dose of about 10 mg to about 500 mg. In various embodiments, the anti-IgE antibody is omalizumab (XOLAIR®). In various embodiments, TIMP-PPE is present at about 0.001 to 10 mg/kg, about 0.005 to 12 mg/kg, about 0.01 to 12 mg/kg, about 0.05 to 12 mg/kg, about 0.1 to 12 mg/kg, about 0.5 to 10 mg/kg, about 1 to 8 mg/kg, about 1.5 to 10 mg/kg, about 2 to 12 mg/kg, about 2 to 10 mg/kg, about 3 to 10 mg/kg, about 4 to 10 mg/kg, about 4 to 12 mg/kg, or about 5 to 12 mg/kg, or about 0.001 mg/kg, 0.0025 mg/kg, 0.005 mg/kg, 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 1.0 The drug was administered at a dosage of 1.0 mg/kg, 2.0 mg/kg, 4.0 mg/kg, 6 mg/kg, 8.0 mg/kg, 10 mg/kg or 12 mg/kg. In various embodiments, TIMP-PPE is administered in an amount of about 0.1 mg, 0.25 mg, 0.5 mg, 1 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, or 800 mg. In various embodiments, the anti-IgE antibody administered in combination with TIMP-PPE is administered in an amount of about 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg or 500 mg. In various embodiments, the dose of the anti-IgE antibody is determined based on serum IgE levels. In various embodiments, the serum IgE level is between 30-100 IU/mL, 100-200 IU/mL, 200-300 IU/mL, 300-400 IU/mL, 400-500 IU/mL, 500-600 IU/mL, 600-700 IU/mL, 700-800 IU/mL, 800-900 IU/mL, 900-1000 IU/mL, 1000-1100 IU/mL, 1100-1200 IU/mL, 1200-1300 IU/mL, 1300-1400 IU/mL, or 1400-1500 IU/mL. In various embodiments, the dose of anti-IgE antibody is determined based on the individual's weight. In various embodiments, the individual weighs between 30-40 kg, 40-50 kg, 50-60 kg, 60-70 kg, 70-80 kg, 80-90 kg, 90-125 kg, or 125-150 kg.

In various embodiments, the anti-IgE antibody is administered in a single dose or multiple doses. In various embodiments, the anti-IgE antibody is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In various embodiments, the anti-IgE antibody is administered before, at the same time, or after the administration of TIMP-PPE. In various embodiments, the anti-IgE antibody is administered one week, two weeks, three weeks, four weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months before the administration of TIMP-PPE. In various embodiments, the anti-IgE antibody is administered one week, two weeks, three weeks, or four weeks after administration of TIMP-PPE.

The present invention provides a method for treating peanut allergy comprising administering to a subject a combination of TIMP-PPE and an anti-IgE antibody, wherein the anti-IgE antibody is administered to the subject once a week for two weeks, or once a week for four weeks prior to administration of TIMP-PPE, wherein TIMP-PPE is administered in two doses, at a dose of between 0.001 mg/kg and 12 mg/kg, one week apart, and wherein the anti-IgE antibody is administered in a dose of between about 50 mg and 500 mg.

The present invention provides methods for treating peanut allergy comprising administering to a subject a combination of TIMP-PPE and an anti-IgE antibody, wherein the anti-IgE antibody is administered to the subject once a week for two weeks, or once a week for four weeks prior to administration of TIMP-PPE, wherein TIMP-PPE is administered in two doses, in doses between 0.1 mg and 800 mg, one week apart, and wherein the anti-IgE antibody is administered in a dose between about 50 mg and 500 mg.

The present invention describes a method of treating peanut allergy in an individual comprising administering to the individual a combination of TIMP-PPE and an anti-IL-4Rα antibody, wherein TIMP-PPE is administered at a dose of about 0.001 mg/kg to 12 mg/kg, and wherein the anti-IL-4Rα antibody is administered at a dose of about 10 mg to about 500 mg. In various embodiments, the anti-IL-4Rα antibody is dupilumab (DUPIXENT®). In various embodiments, TIMP-PPE is present at about 0.001 to 10 mg/kg, about 0.005 to 12 mg/kg, about 0.01 to 12 mg/kg, about 0.05 to 12 mg/kg, about 0.1 to 12 mg/kg, 0.5 to 10 mg/kg, about 1 to 8 mg/kg, about 1.5 to 10 mg/kg, about 2 to 12 mg/kg, about 2 to 10 mg/kg, about 3 to 10 mg/kg, about 4 to 10 mg/kg, about 4 to 12 mg/kg, or about 5 to 12 mg/kg, or about 0.001 mg/kg, 0.0025 mg/kg, 0.005 mg/kg, 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 The drug was administered at a dose of 10 mg/kg, 4.0 mg/kg, 6 mg/kg, 8.0 mg/kg, 10 mg/kg or 12 mg/kg. In various embodiments, TIMP-PPE is administered in an amount of about 0.1 mg, 0.25 mg, 0.5 mg, 1 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, or 800 mg. In various embodiments, the anti-IL-4Rα antibody is administered at a dose of about 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, or 600 mg. In various embodiments, the dose of the anti-IL-4Rα antibody is determined based on serum IgE levels. In various embodiments, the serum IgE level is between 30-100 IU/mL, 100-200 IU/mL, 200-300 IU/mL, 300-400 IU/mL, 400-500 IU/mL, 500-600 IU/mL, 600-700 IU/mL, 700-800 IU/mL, 800-900 IU/mL, 900-1000 IU/mL, 1000-1100 IU/mL, 1100-1200 IU/mL, 1200-1300 IU/mL, 1300-1400 IU/mL, or 1400-1500 IU/mL. In various embodiments, the anti-IL-4Rα antibody dose is determined based on the individual's weight. In various embodiments, the individual weighs between 30-40 kg, 40-50 kg, 50-60 kg, 60-70 kg, 70-80 kg, 80-90 kg, 90-125 kg or 125-150 kg. In various embodiments, the anti-IL-4Rα antibody is administered in a single dose or multiple doses. In various embodiments, the anti-IL-4Rα antibody is administered once a week, once every two weeks, once every three weeks or once every four weeks. In various embodiments, the anti-IL-4Rα antibody is administered before, simultaneously with or after the administration of TIMP-PPE. In various embodiments, the anti-IL-4Rα antibody is administered one week, two weeks, three weeks or four weeks before the administration of TIMP-PPE. In various embodiments, the anti-IL-4Rα antibody is administered one week, two weeks, three weeks, or four weeks after administration of TIMP-PPE. In various embodiments, the anti-IL-4Rα antibody is administered twice at an initial dose of between 400 mg and 600 mg, followed by subsequent doses at a maintenance dose of between 200 mg and 300 mg.

In various embodiments, the antihistamine administered in combination with TIMP-PPE is administered in an amount of about 0.05 mg to 2000 mg. In various embodiments, the antihistamine is administered in an amount of about 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 1000 mg, 1500 mg, or 2000 mg. In various embodiments, the antihistamine is administered in a single dose or multiple doses. In various embodiments, the antihistamine is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In various embodiments, the antihistamine is administered twice, three times, four times, five times, or six times a day. In various embodiments, the antihistamine is administered before, simultaneously with, or after the administration of TIMP-PPE. In various embodiments, the antihistamine is administered one week, two weeks, three weeks, four weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months before the administration of TIMP-PPE. In various embodiments, the antihistamine is administered one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months after the administration of TIMP-PPE. In various embodiments, the antihistamine is administered 5, 10, 15, 30, 45, or 60 minutes before the administration of TIMP-PPE. In various embodiments, the antihistamine is administered 5, 10, 15, 30, 45, or 60 minutes after the administration of TIMP-PPE. In various embodiments, the antihistamine is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours before the administration of TIMP-PPE. In various embodiments, the antihistamine is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours after the administration of TIMP-PPE. In various embodiments, the antihistamine is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-PPE. In various embodiments, the antihistamine is administered 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-PPE.

In various embodiments, the steroid administered in combination with TIMP-PPE is administered in an amount of about 0.05 mg to 2000 mg. In various embodiments, the steroid is administered in an amount of about 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 1000 mg, 1500 mg, or 2000 mg. In various embodiments, the steroid is administered in a single dose or multiple doses. In various embodiments, the steroid is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In various embodiments, the steroid is administered twice, three times, four times, five times, or six times a day. In various embodiments, the steroid is administered before, simultaneously with, or after the administration of TIMP-PPE. In various embodiments, the steroid is administered one week, two weeks, three weeks, four weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months before the administration of TIMP-PPE. In various embodiments, the steroid is administered one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months after TIMP-PPE is administered. In various embodiments, the steroid is administered 5, 10, 15, 30, 45, or 60 minutes prior to TIMP-PPE administration. In various embodiments, the steroid is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours prior to TIMP-PPE administration. In various embodiments, the steroid is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-PPE.

In various embodiments, the corticosteroid administered in combination with TIMP-PPE is administered in an amount of about 0.05 mg to 2000 mg. In various embodiments, the corticosteroid is administered in an amount of about 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 1000 mg, 1500 mg, or 2000 mg. In various embodiments, the corticosteroid is administered in a single dose or multiple doses. In various embodiments, the corticosteroid is administered once a day, once a week, once every two weeks, once every three weeks, or once every four weeks. In various embodiments, the corticosteroid is administered twice, three times, four times, five times, or six times a day. In various embodiments, the corticosteroid is administered before, simultaneously with, or after the administration of TIMP-PPE. In various embodiments, the corticosteroid is administered one week, two weeks, three weeks, four weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months before the administration of TIMP-PPE. In various embodiments, the corticosteroid is administered one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months after the administration of TIMP-PPE. In various embodiments, the corticosteroid is administered 5, 10, 15, 30, 45, or 60 minutes prior to the administration of TIMP-PPE. In various embodiments, the corticosteroid is administered 5, 10, 15, 30, 45, or 60 minutes after the administration of TIMP-PPE. In various embodiments, the corticosteroid is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours prior to administration of TIMP-PPE. In various embodiments, the corticosteroid is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours after administration of TIMP-PPE. In various embodiments, the corticosteroid is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-PPE. In various embodiments, the corticosteroid is administered 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-PPE.

In various embodiments, the NSAID administered in combination with TIMP-PPE is administered in an amount of about 0.05 mg to 2000 mg. In various embodiments, the NSAID is administered in an amount of about 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 1000 mg, 1500 mg, or 2000 mg. In various embodiments, the NSAID is administered in a single dose or multiple doses. In various embodiments, the NSAID is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In various embodiments, the NSAID is administered twice, three times, four times, five times, or six times a day. In various embodiments, the NSAID is administered before, simultaneously with, or after the administration of TIMP-PPE. In various embodiments, the NSAID is administered one week, two weeks, three weeks, four weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months before the administration of TIMP-PPE. In various embodiments, the NSAID is administered one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months after the administration of TIMP-PPE. In various embodiments, the NSAID is administered 5, 10, 15, 30, 45, or 60 minutes before the administration of TIMP-PPE. In various embodiments, the NSAID is administered 5, 10, 15, 30, 45, or 60 minutes after the administration of TIMP-PPE. In various embodiments, the NSAID is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours prior to administration of TIMP-PPE. In various embodiments, the NSAID is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours after administration of TIMP-PPE. In various embodiments, the NSAID is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-PPE. In various embodiments, the NSAID is administered 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-PPE.

In various embodiments, the leukotriene modulator administered in combination with TIMP-PPE is administered in an amount of about 0.05 mg to 2000 mg. In various embodiments, the leukotriene modulator is administered in an amount of about 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 1000 mg, 1500 mg, or 2000 mg. In various embodiments, the leukotriene modulator is administered in a single dose or multiple doses. In various embodiments, the leukotriene modulator is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In various embodiments, the leukotriene modulator is administered two, three, four, five, or six times a day. In various embodiments, the leukotriene modulator is administered prior to, concurrently with, or after the administration of TIMP-PPE. In various embodiments, the leukotriene modulator is administered one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months prior to the administration of TIMP-PPE. In various embodiments, the leukotriene modulator is administered one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months after the administration of TIMP-PPE. In various embodiments, the leukotriene modulator is administered 5, 10, 15, 30, 45, or 60 minutes before the administration of TIMP-PPE. In various embodiments, the leukotriene modulator is administered 5, 10, 15, 30, 45, or 60 minutes after the administration of TIMP-PPE. In various embodiments, the leukotriene modulator is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours before or after administration of TIMP-PPE. In various embodiments, the leukotriene modulator is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours before or after administration of TIMP-PPE. In various embodiments, the leukotriene modulator is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-PPE. In various embodiments, the leukotriene modulator is administered 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-PPE.

It is contemplated that when TIMP-PPE is administered with an additional therapeutic agent described herein, TIMP-PPE is administered in a single dose or in multiple doses. In various embodiments, TIMP-PPE is administered in two doses one week apart.

It is contemplated that when TIMP-PPE is administered in combination with a therapeutic agent described herein for treating peanut allergy, TIMP-PPE is administered in a single dose or in multiple doses. In various embodiments, TIMP-PPE is administered in two doses one week apart. In various embodiments, TIMP-PPE is administered in two doses one week apart, followed by a single dose of TIMP-PPE administered once every three months as an additional dose.

In various embodiments, administration of TIMP-PPE alone or in combination with one or more additional therapeutic agents to a subject in need thereof relieves one or more peanut allergy symptoms. In various embodiments, the peanut allergy symptoms are selected from the group consisting of skin reactions, hives, skin redness, skin swelling, itching, throat tightening, difficulty breathing, shortness of breath, and systemic anaphylaxis.

In various embodiments, administration of TIMP-PPE alone or in combination with one or more therapeutic agents useful for treating peanut allergy to a subject in need thereof relieves one or more peanut allergy symptoms. In various embodiments, peanut allergy symptoms are selected from the group consisting of: skin reactions; hives; skin redness; skin swelling; itching; throat tightening; difficulty breathing; shortness of breath; digestive problems such as diarrhea, angina, nausea, or vomiting; decreased blood pressure; and systemic allergic reactions.

In various embodiments, administration of TIMP-PPE alone or in combination with one or more additional therapeutic agents to an individual in need thereof shortens the duration and reduces the severity of an allergic immune response to peanut protein. In various embodiments, administration of TIMP-PPE alone or in combination with one or more additional therapeutic agents to an individual in need thereof shortens the duration and reduces the severity of an allergic immune response following exposure to peanut protein. In various embodiments, the allergic immune response is a Th2 T cell response, B cell activation, basophil activation, eosinophil activation, mast cell activation, and/or IgE induction.

In various embodiments, the efficacy of TIMP-PPE in relieving one or more peanut allergy symptoms and/or shortening the duration and reducing the severity of an allergic immune response to peanut protein is determined from analysis of one or more biological samples from an individual. In various embodiments, the biological sample is selected from the group consisting of whole blood, peripheral blood, peripheral blood mononuclear cells (PBMC), serum, plasma, urine, cerebrospinal fluid (CSF), feces, tissue biopsy, and/or bone marrow biopsy. In various embodiments, the efficacy of TIMP-PPE in relieving one or more peanut allergy symptoms and/or shortening the duration and reducing the severity of an allergic immune response to peanut protein is determined by a double-blind, placebo-controlled food challenge (DBPCFC). In various embodiments, the efficacy of TIMP-PPE in alleviating one or more peanut allergy symptoms and/or shortening the duration and severity of an allergic immune response to peanut protein is determined by skin prick testing (SPT).

In various embodiments, administration of TIMP-PPE alone or in combination with an additional therapeutic agent to a subject reduces the proportion of Th2a+ T cells present in the total T cell population in peripheral blood.

In various embodiments, administration of TIMP-PPE alone or in combination with a therapeutic agent to a subject reduces the proportion of Th2a+ T cells present in the total T cell population in peripheral blood.

In various embodiments, administration of TIMP-PPE alone or in combination with an additional therapeutic agent to a subject reduces the ratio of activated peanut protein-specific T cells to inactivated peanut protein-specific T cells in peripheral blood.

In various embodiments, administration of TIMP-PPE alone or in combination with a therapeutic agent to a subject reduces the proportion of activated peanut protein-specific T cells in peripheral blood.

In various embodiments, administering TIMP-PPE alone or in combination with an additional therapeutic agent to a subject increases the level of peanut protein-specific Treg cells in the blood. In various embodiments, administering TIMP-PPE alone or in combination with a therapeutic agent to a subject increases the level of peanut protein-specific Treg cells in the blood.

In various embodiments, administration of TIMP-PPE alone or in combination with an additional therapeutic agent to a subject reduces basophil activation. In various embodiments, administration of TIMP-PPE alone or in combination with a therapeutic agent to a subject reduces basophil activation.

In various embodiments, administering TIMP-PPE alone or in combination with an additional therapeutic agent to a subject reduces the level of peanut protein-specific IgE in the blood. In various embodiments, administering TIMP-PPE alone or in combination with a therapeutic agent to a subject reduces the level of peanut protein-specific IgE in the blood.

In various embodiments, administering TIMP-PPE alone or in combination with an additional therapeutic agent to an individual reduces the ratio of peanut protein-specific IgE to IgG levels in the blood. In various embodiments, administering TIMP-PPE alone or in combination with a therapeutic agent to an individual reduces the ratio of peanut protein-specific IgE to IgG levels in the blood.

In various embodiments, administering TIMP-PPE alone or in combination with an additional therapeutic agent to an individual reduces the level of Th2 interleukin content in the blood. In various embodiments, administering TIMP-PPE alone or in combination with a therapeutic agent to an individual reduces the level of Th2 interleukin content in the blood. In various embodiments, the Th2 interleukin is selected from the group consisting of IL-4, IL-5, IL-9, and IL-13.

In various embodiments, TIMP-PPE alone or in combination with an additional therapeutic agent is administered to an individual to increase tolerance to peanut protein. In various embodiments, TIMP-PPE alone or in combination with a therapeutic agent is administered to an individual to increase tolerance to peanut protein.

Also contemplated are compositions comprising a TIMP-PPE described herein for use in treating peanut allergy. In various embodiments, the invention provides the use of a composition comprising a TIMP-PPE described herein for the preparation of a medicament for treating peanut allergy.

It should be understood that each feature or embodiment or combination described herein is a non-limiting illustrative example of any one of the aspects of the invention, and is therefore intended to be combined with any other feature or embodiment or combination described herein. For example, where features are described using language such as "one embodiment," "some embodiments," "certain embodiments," "further embodiments," "particular exemplary embodiments," and/or "another embodiment," each of these types of embodiments is a non-limiting example of a feature that is intended to be combined with any other feature or combination of features described herein without necessarily listing every possible combination. These features or combinations of features apply to any one of the aspects of the invention. Where examples of values falling within a range are disclosed, any of these examples are considered as possible endpoints of the range, any and all values between these endpoints are considered, and any and all combinations of upper and lower endpoints are contemplated.

The titles herein are for the convenience of the reader and are not intended to be limiting. Additional aspects, embodiments and variations of the invention will be apparent from the implementation and/or drawings and/or claims.

Cross-references to related applications

This application claims priority to U.S. Provisional Patent Application No. 63/380,173 filed on October 19, 2022 and U.S. Provisional Patent Application No. 63/486,812 filed on February 24, 2023, which are incorporated herein by reference in their entirety.

The contents of the electronically submitted text file are incorporated herein by reference in their entirety: a computer-readable copy of the sequence listing (file name: 58512_SeqListing.XML, creation date: October 18, 2023, file size 27,434 bytes).

The present invention provides methods for monitoring the induction and maintenance of immune tolerance in an individual following immunotherapy .

Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below.

As used in this specification and the accompanying claims, the indefinite articles "a" and "an" and the definite article "the" include plural as well as singular referents, unless the context clearly dictates otherwise.

The term "about" or "approximately" means the acceptable error of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. When the term "about" or "approximately" precedes the first value in a series of two or more numerical values, it is understood that the term "about" or "approximately" applies to each of the numerical values in the series.

As used herein, "particle" refers to any non-tissue derived material composition, which may be a sphere or sphere-like entity, a bead or a liposome. Depending on the context, the term "particle", the term "immune modified particle", the term "carrier particle" and the term "bead" may be used interchangeably. In addition, the term "particle" may be used to cover both beads and spheres.

As used herein, "negatively charged particles" refer to particles that have been modified to have a net surface charge of less than zero.

"Carboxylated particles" or "carboxylated beads" or "carboxylated spheres" include any particle that has been modified to contain carboxyl groups on its surface. In some embodiments, the addition of carboxyl groups enhances uptake of the particle from circulation by phagocytes/monocytes, for example, via interaction with scavenger receptors such as MARCO. Carboxylation of the particle can be achieved using any compound that adds carboxyl groups.

As used herein, "TIMP-PPE" refers to negatively charged immune tolerance immunomodulatory particles (TIMPs), which include peanut extract or one or more peanut proteins or antigenic fragments thereof, including Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, Ara h13, Ara h14, Ara h15, Ara h16, Ara h17 and Ara h18.

As used herein, the term "Th cell" or "helper T cell" refers to a CD4 ⁺ cell. CD4 ⁺ T cells assist other white blood cells in the immune process, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. T cells become activated when they present peptide antigens via MHC class II molecules expressed on the surface of antigen presenting cells (APCs).

As used herein, the term "Th1 cells" refers to a subset of Th cells that produce pro-inflammatory mediators. Th1 cells secrete interleukins to promote immune responses and play a role in host defense against pathogens, in part by mediating the recruitment of neutrophils and macrophages to infected tissues. Th1 cells secrete interleukins including IFN-γ, IL-2, IL10, and TNF α/β to coordinate defense against intracellular pathogens (such as viruses and some bacteria).

As used herein, the term "Th2 cells" refers to a subset of Th cells that mediate activation and maintenance of antibody-mediated immune responses to extracellular parasites, bacteria, allergens and toxins. Th2 cells mediate these functions by producing various interleukins, such as IL-4, IL-5, IL-6, IL-9, IL-13 and IL-17E (IL-25), which are responsible for producing antibodies, activating eosinophils and inhibiting certain macrophage functions, thereby providing a protective response that is independent of phagocytes.

"Polypeptide" and "protein" refer to polymers composed of amino acid residues linked by peptide bonds or peptide bond equivalent isomers, naturally occurring related structural variants and non-naturally occurring synthetic analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The terms "polypeptide" and "protein" are not limited to the minimum length of the product. The term "protein" generally refers to larger polypeptides. The term "peptide" generally refers to shorter polypeptides. Therefore, peptides, oligopeptides, dimers, polymers and the like are included in the definition. Full-length proteins and fragments thereof are covered by the definition. The terms "polypeptide" and "protein" also include post-expression modifications of the polypeptide or protein, such as glycosylation, acetylation, phosphorylation and the like. In addition, for the purposes of the present invention, "polypeptides" may also include "modifications" of the native sequence, such as deletions, additions, substitutions (which may be conservative in nature or may include substitutions with any of the 20 amino acids commonly present in human proteins, or any other naturally or non-naturally occurring or atypical amino acids) and chemical modifications (e.g., addition of a peptide mimetic or substitution with a peptide mimetic). Such modifications may be intentional, such as by site-directed mutagenesis or by chemical modification of amino acids to remove or attach chemical moieties, or may be accidental, such as by mutations generated by the host cell producing the protein or by errors caused by PCR amplification prior to transfection of the host cell.

As used herein, "antigenic portion" or "antigen" refers to any portion, such as a peptide, that is recognized by the host's immune system. Examples of antigenic portions include, but are not limited to, self-antigens, allergens, enzymes and/or bacterial or viral proteins, peptides, drugs or components.

"Pharmaceutically acceptable carrier" refers to any of standard pharmaceutical carriers, buffers, and the like, such as phosphate-buffered saline solutions, 5% dextrose in water, and emulsions (e.g., oil/water or water/oil emulsions). Non-limiting examples of excipients include adjuvants, binders, fillers, diluents, disintegrants, emulsifiers, wetting agents, lubricants, glidants, sweeteners, flavorings, and coloring agents. Suitable pharmaceutical carriers, excipients, and diluents are described in Remington's Pharmaceutical Sciences, 19th edition (Mack Publishing Co., Easton, 1995). The preferred pharmaceutical carrier depends on the intended mode of administration of the active agent. Typical modes of administration include enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous or intraperitoneal injection; or topical, transdermal or transmucosal administration) or by inhalation.

"Pharmaceutically acceptable" or "pharmacologically acceptable" means a substance that is not biologically or otherwise undesirable, that is, a substance that can be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any component of a composition in which it is contained or with any component present on or in the subject's body.

As used herein, the term "individual" encompasses both mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class mammalia: humans, non-human primates (such as chimpanzees and other apes and monkey species); farm animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. The term does not indicate a specific age or sex.

The term "antigenic determinant" refers to the part of any molecule that can be recognized and bound by a selective binding agent at one or more antigen binding regions. Antigenic determinants are usually composed of chemically active surface groups of molecules (e.g., amino acids or carbohydrate side chains) and have specific three-dimensional structural characteristics and charge-mass characteristics. As used herein, antigenic determinants can be contiguous or non-contiguous. In addition, antigenic determinants can be mimetics (mimetic antigenic determinants) because they contain a three-dimensional structure consistent with an antigenic determinant used to generate antibodies, but do not contain or only contain some of the amino acid residues found in the target that are used to stimulate the antibody immune response. As used herein, a mimetic epitope is not considered to be an antigen that is different from the epitope bound by the selective binding agent; the selective binding agent recognizes the same three-dimensional structure of the epitope and the mimetic epitope.

The term "therapeutically effective amount" as used herein refers to an amount of the antigen-specific composition of the present invention that is effective in improving or alleviating the symptoms or signs of the disease to be treated.

The terms "treat," "treated," "treating," and "treatment" as used herein with respect to the methods herein refer to the temporary or permanent, partial or complete elimination, reduction, inhibition, or improvement of one or more clinical symptoms, manifestations, or course of an event, disease, or condition. Such treatment may not necessarily be absolutely useful.

As used herein, the term "symptom" refers to any physical or observable manifestation of a disorder, whether or not it is generally characteristic of the disorder. The term "symptom" may mean all such manifestations or any subset thereof.

As used herein, "add-on dose" refers to the administration of a TIMP following an initial or preliminary administration. Administration or re-administration of a supplemental dose enhances the tolerance immune response induced by the initial administration. The supplemental dose may be greater than, equal to, or less than the initial dose of TIMP particles administered to the individual. Monitor individuals treated with immune tolerance therapy to confirm maintenance of immune tolerance. The decision to administer a supplemental dose is based on the observation of a change, weakening, or loss of immune tolerance. Methods for monitoring the immune tolerance state of individuals treated with immune tolerance therapy have been previously described (see International Patent Publication WO 2022/221622, which is incorporated herein by reference). Additional doses of TIMP-PPE may be administered with or without the same, different, or combination therapies described herein, which can be administered with the initial TIMP-PPE dose.

The size and charge of the particles are critical to tolerance induction. Although the particles will vary in size and charge based on the antigen encapsulated therein, in general, the particles described herein are effective in inducing tolerance when they are between about 100 nanometers and about 1500 nanometers and have a negative charge between 0 and about -100 mV. In various embodiments, the particles have a diameter of 400 to 800 nanometers and have a charge between about -25 mV and -70 mV. In various embodiments, the particles have a diameter of 400-800 nanometers and have a charge between about -30 mV and -60 mV. In various embodiments, the particles have a diameter of 400-800 nanometers and have a charge between about -30 mV and -80 mV. The average particle size and charge of the particles may change slightly during the freeze-drying process, therefore, the average as-synthesized and average after freeze-drying are described. As used herein, the terms "size as-synthesized" and "charge as-synthesized" refer to the size and charge of the particles before freeze-drying. The terms "size after freeze-drying" and "charge after freeze-drying" refer to the size and charge of the particles after freeze-drying.

In some embodiments, the particles are non-metallic. In such embodiments, the particles may be formed from a polymer. In a preferred embodiment, the particles are biodegradable in a subject. In this embodiment, the particles may be provided in a subject across multiple doses without particle accumulation in the subject. Examples of suitable particles include polystyrene particles, PLGA particles, PLURONICS stabilized polypropylene sulfide particles, and diamond particles.

Preferably, the particle surface is composed of a material that minimizes non-specific or unwanted biological interactions. The interaction between the particle surface and the stroma can be a factor that plays a role in lymphatic absorption. The particle surface can be coated with a material that prevents or reduces non-specific interactions. Improved lymphatic absorption after subcutaneous injection shows that spatial stabilization of particles coated with hydrophilic layers such as poly(ethylene glycol) (PEG) and copolymers thereof such as PLURONICS® (including copolymers of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) can reduce non-specific interactions with proteins of the stroma. All of these facts indicate the relevance of the physical properties of the particles in terms of lymphatic absorption. Biodegradable polymers can be used to prepare all or some of the polymers and/or particles and/or layers. Biodegradable polymers can be degraded, for example, by functional groups that react with water in solution. The term "degradation" as used herein refers to becoming soluble by reducing molecular weight or by converting hydrophobic groups to hydrophilic groups. Polymers with ester groups generally undergo spontaneous hydrolysis, such as polylactide and polyglycolide.

The particles disclosed herein may also contain additional components. For example, the carrier may have an imaging agent incorporated into or bound to the carrier. An example of a carrier nanosphere with a currently commercially available imaging agent is the Kodak X-sight nanosphere. Inorganic quantum confined luminescent nanocrystals called quantum dots (QDs) have emerged as ideal donors in FRET applications: their high quantum yield when attacked at a single UV wavelength and tunable size-dependent Stokes shift allow different sizes to emit from blue to infrared. (Bruchez et al., Science, 1998, 281, 2013; Niemeyer, C. M Angew. Chem. Int. Ed. 2003, 42, 5796; Waggoner, A. Methods Enzymol. 1995, 246, 362; Brus, L. E. J. Chem. Phys. 1993, 79, 5566). Quantum dots, such as hybrid organic/inorganic quantum dots based on a class of polymers called dendrimers, can be used in biomarkers, imaging, and optical biosensing systems. (Lemon et al., J. Am. Chem. Soc. 2000, 122, 12886). Unlike the traditional synthesis of inorganic quantum dots, the synthesis of these hybrid quantum dot nanoparticles does not require high temperatures or highly toxic and unstable reagents (Etienne et al., Appl. Phys. Lett. 87, 181913, 2005).

The particles can be formed from a wide range of materials. The particles are preferably composed of materials suitable for biological use. For example, the particles can be composed of glass, silica, polyesters of hydroxy carboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxy carboxylic acids and dicarboxylic acids. More generally, the carrier particles can be composed of polyesters of linear or branched chain, substituted or unsubstituted, saturated or unsaturated, linear or cross-linked alkyl, haloalkyl, sulfanyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl or alkoxy hydroxy acids; or linear or branched chain, substituted or unsubstituted, saturated or unsaturated, linear or cross-linked alkyl, haloalkyl, sulfanyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl or alkoxy dicarboxylic acids. In addition, the carrier particles can be quantum dots or can be composed of quantum dots, such as quantum dot polystyrene particles (Joumaa et al. (2006) Langmuir 22: 1810-6). Carrier particles comprising mixtures of ester and anhydride bonds (e.g., copolymers of glycolic acid and sebacic acid) can also be used. For example, the carrier particles can include materials including polyglycolic acid polymers (PGA), polylactic acid polymers (PLA), polysebacic acid polymers (PSA), poly(lactic-co-glycolic) acid copolymers (PLGA or PLG; the terms are interchangeable), poly(lactic-co-sebacic) acid copolymers (PLSA), poly(ethanol-co-sebacic) acid copolymers (PGSA), polypropylene sulfide polymers, poly(caprolactone), polyglucosamine, and the like. Other biocompatible biodegradable polymers suitable for use in the present invention include polymers or copolymers of caprolactones, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates, and degradable urethanes, and copolymers of these with linear or branched, substituted or unsubstituted alkyl, halogen, sulfanyl, aminoalkyl, alkenyl, or aromatic hydroxycarboxylic acids or dicarboxylic acids. In addition, biologically important amino acids with reactive side chain groups (such as lysine, arginine, aspartic acid, glutamine, serine, threonine, tyrosine, and cysteine or their mirror image isomers) may be included in copolymers with any of the foregoing to provide reactive groups for binding to antigenic peptides and proteins or binding moieties. Biodegradable materials suitable for use in the present invention include diamond, PLA, PGA, polypropylene sulfide and PLGA polymers. Biocompatible but non-biodegradable materials can also be used in the carrier particles of the present invention. For example, acrylates, ethyl-vinyl acetate, acyl-substituted cellulose acetate, non-degradable urethanes, styrene, vinyl chloride, vinyl fluoride, vinyl imidazole, chlorosulfonated olefins, ethylene oxide, vinyl alcohol, TEFLON® (DuPont, Wilmington, Del.) and resistant non-biodegradable polymers can be used.

In certain embodiments, the particles are copolymers having a molar ratio of about 80:20 to about 100:0. Suitable copolymer ratios for the immunomodified particles of the invention may be 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0. In certain embodiments, the particles are PLURONICS stabilized polypropylene sulfide particles, polyglycolic acid particles (PGA), polylactic acid particles (PLA), or poly(lactic-co-glycolic acid) particles. In certain embodiments, the particles have a copolymer ratio of polylactic acid/polyglycolic acid 80:20, polylactic acid/polyglycolic acid 90:10, or polylactic acid/polyglycolic acid 50:50. In various embodiments, the particles are poly(lactic-co-glycolic acid) particles and have a copolymer ratio of about 50:50 polylactic acid:polyglycolic acid.

It is contemplated that the particles may further comprise a surfactant. The surfactant may be anionic, cationic or nonionic. Surfactants in the poloxamer and polaxamine families are commonly used in particle synthesis. Surfactants that may be used include, but are not limited to, PEG, Tween-80, gelatin, polydextrose, pluronic L-63, PVA, PAA, methylcellulose, lecithin, DMAB and PEMA. In addition, biodegradable and biocompatible surfactants include, but are not limited to, vitamin E TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate), polyamino acids (e.g., polymers of lysine, arginine, aspartic acid, glutamine, serine, threonine, tyrosine, and cysteine or their mirror image isomers), and sulfate polymers. In certain embodiments, two surfactants are used. For example, if the particles are produced by a double emulsion method, the two surfactants may include a hydrophobic surfactant for the first emulsion and a hydrophobic surfactant for the second emulsion.

In certain embodiments, polypeptide antigens are encapsulated in particles by a single emulsion method. In further embodiments, polypeptide antigens are more hydrophobic. Sometimes, double emulsion methods cause the formation of larger particles, which can cause leakage of hydrophilic active components and low interception efficiency. Coagulation and Ostwald ripening are two mechanisms that can make double emulsion droplets unstable, and diffusion through the organic phase of the hydrophilic active component is the main mechanism that causes low levels of encapsulated active components. In some embodiments, reducing the size of nanoparticles can be beneficial. One strategy to achieve this goal is to apply the second strongest shear rate. The leakage effect can be reduced by using high polymer concentrations and high polymer molecular weights, accompanied by an increase in the viscosity of the internal water phase and an increase in the molecular weight of the surfactant. In certain embodiments, the antigen-encapsulated particles are made by nanoprecipitation, coprecipitation, inert gas condensation, sputtering, microemulsion, sol-gel, layer-by-layer technique, or ion gelation. Several methods for making nanoparticles have been described in the literature and are incorporated herein by reference (Sánchez, Mejía, and Orozco 2020; Zielińska et al. 2020). Antigen

Antigen refers to the hidden part of a molecule that can be recognized by host immune cells, such as polypeptide or peptide sequences, 3D structures of polypeptides or peptides, polysaccharides or polynucleotides. Antigenic specificity refers to the ability of individual host cells to recognize an antigen and produce an immune response to the antigen alone, or to produce an immune response to molecules that are very similar to the antigen (such as antigenic determinants or mimic antigenic determinants).

"Anergy," "tolerance," or "antigen-specific tolerance" refers to the insensitivity of T cells to stimulation mediated by T cell receptors. Such insensitivity is generally antigen-specific and persists after exposure to the antigenic peptide has ceased. For example, T cell anergy is characterized by insufficient production of cytokines (e.g., IL-2). T cell anergy occurs when a T cell is exposed to an antigen and receives a first signal (a signal mediated by a T cell receptor or CD-3) in the absence of a second signal (a co-stimulatory signal). Under these conditions, re-exposure of the cell to the same antigen (even in the presence of co-stimulatory molecules) results in the inability to produce cytokines and, therefore, to proliferate. Therefore, the inability to produce interleukins will block proliferation. However, if cultured with interleukins (e.g., IL-2), anergic T cells can still proliferate.

It is contemplated that the immune tolerance therapies described herein are antigen-specific. For example, the TIMPs administered as immune tolerance therapies encapsulate one or more antigens associated with the immune tolerance therapies and the associated diseases or conditions being treated. It is contemplated that the TIMPs used in the immune tolerance therapies comprise one or more peanut antigens. The one or more peanut antigens are derived from peanut protein extracts or may be peptides derived from known peanut proteins.

More than 15 peanut allergens have been officially recognized by the WHO/IUIS Subcommittee on Allergen Nomenclature (www.allergen.org), namely Ara h1 to Ara h18, including Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, Ara h13, Ara h14, Ara h15, Ara h16, Ara h17 and Ara h18. Peanut allergens are classified into different groups based on their structure (e.g., trimers, monomers, cupin, albumin, prolamin, actin, oleosin, defense peptides, vincillin, and non-specific lipid transfer protein (nsLTP)) based on Ara h1, h2, h3, h5, h6, and h8, and each of these groups has a different degree of allergenic potency (Ozias-Akins et al., Allergy 74:888-898, 2019). Known peanut allergens include those derived from peanut Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, and Ara h18. See, e.g., UNIPROT database No. E5G076, which shows the Ara h 1 polypeptide sequence (SEQ ID NO: 1); the Ara h2 polypeptide, UNIPROT database No. A0A445BYI5 (SEQ ID NO: 2); the Ara h3 polypeptide, UNIPROT database No. E5G077 (SEQ ID NO: 3) (see, e.g., Ara h3 heteroallergens 1 and 2 (formerly Ara h4), UNIPROT database Nos. O82580 (SEQ ID NO: 4) and Q9SQH7 (SEQ ID NO: 5) respectively); the Ara h5 polypeptide, UNIPROT database No. L7QH52 (SEQ ID NO: 6); the Ara h6 polypeptide, UNIPROT database No. A5Z1R0 (SEQ ID NO: 7); the Ara h7 polypeptide, UNIPROT database number B4XID4 (SEQ ID NO: 8); Ara h8 polypeptide sequence, UNIPROT database number Q6VT83 (SEQ ID NO: 9); Ara h9, heteroallergens 1 and 2, UNIPROT database numbers B6CEX8 and B6CG41, respectively (SEQ ID NOs: 10 and 11); Ara h10, heteroallergens 1 and 2, UNIPROT database numbers Q647G5 and Q647G4, respectively (SEQ ID NOs: 12 and 13); Ara h11, heteroallergens 1 and 2, UNIPROT database numbers Q45W87 and Q45W86, respectively (SEQ ID NOs: 14 and 15); Ara h12, UNIPROT database number B3EWP3 (SEQ ID NO: 16); Ara h13, heteroallergens 1 and 2, UNIPROT database numbers B3EWP4 and C0HJZ1, respectively (SEQ ID NOs: 17 and 18); Ara h14, heteroallergens 1, 2 and 3, UNIPROT database numbers Q9AXI1, Q9AXI0 and Q6J1J8, respectively (SEQ ID NOs: 19-21); Ara h15, UNIPROT database number Q647G3 (SEQ ID NO: 22); Ara h16, UNIPROT database number A0A509ZX51 (SEQ ID NO: 23); Ara h17, UNIPROT A database number 0A510A9S3 (SEQ ID NO: 24); and Ara h18, UNIPROT database number A0A444XS96 (SEQ ID NO: 25).

In various embodiments, the allergenic peanut protein comprises one or more of Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, Ara h13, Ara h14, Ara h15, Ara h16, Ara h17, and Ara h18. In various embodiments, the peptide derived from peanut protein comprises an allergenic antigenic determinant from one or more of Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, Ara h13, Ara h14, Ara h15, Ara h16, Ara h17, and Ara h18 proteins.

In certain embodiments, one, two, three or more antigens or antigenic peptides are used in the TIMPs. In certain embodiments, one or more peanut antigens are encapsulated in the TIMPs by covalently linking to the inner surface of the particle (see, e.g., U.S. Patent Publication No. US20190282707, which is incorporated herein by reference). In certain embodiments, it is contemplated that sequences of two or more peanut proteins, e.g., from Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, and/or Ara h8, are linked in a fusion protein and encapsulated within the TIMPs described herein. In certain embodiments, it is contemplated that sequences from two or more peanut proteins, such as Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, Ara h13, Ara h14, Ara h15, Ara h16, Ara h17, and Ara h18, are linked in a fusion protein and encapsulated within a TIMP described herein. Methods for preparing TIMPs with linked antigenic determinants are described in U.S. Patent Publication No. US20190365656, which is incorporated herein by reference. Methods of Use

Peanut allergy is the most common food allergy in the United States, with up to 0.6% of adults and 0.8% of children diagnosed with peanut allergy. In addition, due to the high prevalence of peanuts in the population and the widespread use of peanut-derived products as fillers in many packaged foods, peanut allergy is the most common cause of fatal food-related systemic anaphylaxis. Therefore, peanut allergy poses a serious health hazard in the United States.

The four major peanut allergens are Ara h 1, Ara h 2, Ara h 3 and Ara h 6. Ara h 1 and Ara h 2 are identified in more than 95% of patients with peanut allergy.

Avoidance of peanuts by modifying the diet has long been the most effective treatment for peanut allergy. However, severe cases of peanut allergy can also be triggered by food handling in close proximity to peanuts and/or inhalation of nearby peanut products. When complete avoidance of peanut products and areas contaminated with peanut products is not achievable, severe allergic reactions may occur requiring the patient to receive medications such as adrenaline, antihistamines, and/or oral steroids.

Currently approved therapies and therapies in clinical trials for the treatment of peanut allergy (such as PALFORZIA™) are designed only to protect individuals from harm from accidental peanut exposure by desensitizing them. The treatment regimen for PALFORZIA™ also involves a fairly extensive daily dose escalation and maintenance schedule and can challenge individual compliance and still require the individual to maintain a peanut-free diet.

Therapies that render T cells tolerant to peanut proteins could potentially cure peanut allergy, eliminating the burden associated with strictly avoiding peanut products.

Provided herein is a method of treating peanut allergy in an individual, comprising administering TIMP-PPE to the individual, wherein TIMP-PPE is administered in an amount determined based on the individual's weight. It is also contemplated that TIMP-PPE may be administered in a fixed dose regardless of the individual's weight. Provided herein is a method of treating peanut allergy in an individual, comprising administering TIMP-PPE to the individual, wherein TIMP-PPE is administered in an amount of 0.1 to 12 mg/kg. Provided herein is a method of treating peanut allergy in an individual, comprising administering TIMP-PPE to the individual, wherein TIMP-PPE is administered in an amount of 0.001 to 12 mg/kg. In various embodiments, a method of treating peanut allergy in a subject is contemplated, comprising administering TIMP-PPE to the subject, wherein the TIMP-PPE is administered at a dose of 0.001 to 12 mg/kg based on the subject's body weight or at a fixed dose of between 0.1 mg and 800 mg. Also provided herein is a method of reducing an allergic immune response to a peanut antigen in a subject suffering from PA, comprising administering TIMP-PPE to the subject, wherein the TIMP-PPE is administered at a dose of 0.001 to 12 mg/kg. Further encompassed herein are methods of reducing an allergic immune response to peanut antigens in an individual suffering from PA comprising administering to the individual TIMP-PPE, wherein the TIMP-PPE is administered at a dose of 0.001 to 12 mg/kg based on the individual's body weight or at a fixed dose of between 0.1 mg and 800 mg.

It is also contemplated that TIMP-PPE is administered in an amount of about 0.001 to 10 mg/kg, about 0.005 to 12 mg/kg, about 0.01 to 12 mg/kg, about 0.05 to 12 mg/kg, about 0.1 to 12 mg/kg, about 0.5 to 10 mg/kg, about 1 to 8 mg/kg, about 1.5 to 10 mg/kg, about 2 to 12 mg/kg, about 2 to about 10 mg/kg, about 3 to 10 mg/kg, about 4 to 10 mg/kg, about 4 to 12 mg/kg, or about 5 to 12 mg/kg. Optionally, TIMP-PPE is administered at a dose of about 0.001 mg/kg, 0.0025 mg/kg, 0.005 mg/kg, 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 4.0 mg/kg, 6 mg/kg, 8.0 mg/kg, 10 mg/kg, or 12 mg/kg. Alternatively, TIMP-PPE is administered in an amount of about 0.1 mg, 0.25 mg, 0.5 mg, 1 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, or 800 mg. In another embodiment, TIMP-PPE is administered at a concentration of between about 0.0005 mg/mL and about 50 mg/mL, optionally about 0.0005 mg/mL, 0.001 mg/mL, 0.005 mg/mL, 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 12.5 mg/mL, 15 mg/mL, 17.5 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 40 mg/mL or 50 mg/mL.

It is contemplated that TIMP-PPE is administered in a single dose or in multiple doses. In various embodiments, TIMP-PPE is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year. In certain embodiments, TIMP-PPE is administered in two doses one week apart.

In various embodiments, TIMP-PPE is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally. It is contemplated that if TIMP-PPE is administered intravenously, it may be administered by intravenous infusion for about 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 18, or 20 hours, or by intravenous infusion for about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 18, or 20 hours.

Also provided herein is a method of extending the duration or enhancing the efficacy of tolerance to peanut allergen induced by TIMP-PPE treatment, comprising administering an additional dose of TIMP-PPE. In various embodiments, after the initial or preliminary administration of TIMP-PPE, an additional dose of TIMP-PPE is administered in a single dose or multiple doses. In various embodiments, the additional dose of TIMP-PPE is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year. In various embodiments, the additional dose of TIMP-PPE is administered every three months in a single dose.

It is further contemplated that TIMP-PPE is administered alone or in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents include, but are not limited to, IgE inhibitors, basophil activation inhibitors, mast cell activation inhibitors, antihistamines, nonsteroidal anti-inflammatory drugs (NSAIDs), or small molecule or biological therapeutics.

In various embodiments, TIMP-PPE is administered alone or in combination with one or more therapeutic agents useful for treating peanut allergy. Exemplary therapeutic agents include, but are not limited to, IgE inhibitors, basophil activation inhibitors, mast cell activation inhibitors, antihistamines, nonsteroidal anti-inflammatory drugs (NSAIDs), probiotics, probiotics, histone deacetylase inhibitors, short chain fatty acids (e.g., acetate, butyrate, propionate, butyrate polymers), IgE competitors for allergen binding sites, interleukin inhibitors, microbiome therapy, steroids, corticosteroids, leukotriene modifiers, or small molecules or biological therapeutics. Exemplary additional therapeutic agents or therapeutic agents suitable for treating peanut allergy also include therapeutic agents that increase the number, frequency, or activity of Tregs.

In various embodiments, the biologic is an antibody. In various embodiments, the antibody is an anti-IgE, anti-IL-4Rα, anti-IL-13, or anti-IL-33 antibody. In various embodiments, the anti-IgE antibody is omalizumab (XOLAIR®). Exemplary anti-IL IL-4Rα antibodies include dupilumab (DUPIXENT®), and anti-IL-33 antibodies include etronizumab.

In various embodiments, the antihistamine is a first generation antihistamine. In various embodiments, the antihistamine is a second generation antihistamine. In various embodiments, the antihistamine is selected from the group consisting of brompheniramine, carbinoxamine maleate, chlorpheniramine, clemastine, diphenhydramine, hydroxyzine, triprolidine, azelastine, cetirizine, desloratadine, fexofenadine, levocetirizine, lolatadine and olopatadine. In various embodiments, the additional therapeutic agent is a steroid. In various embodiments, the steroid is selected from the group consisting of beclomethasone, ciclesonide, fluticasone furoate, mometasone, butenide, fluticasone, triamcinolone and loteprednol. In various embodiments, the additional therapeutic agent is a corticosteroid. In various embodiments, the corticosteroid is selected from the group consisting of corticosterone, prasone, prasunol, methylprasunol, dexamethasone, betamethasone, and hydrocorticosterone.

In various embodiments, the additional therapeutic agent is a nonsteroidal anti-inflammatory drug (NSAID). In various embodiments, the therapeutic agent suitable for treating peanut allergy is a nonsteroidal anti-inflammatory drug (NSAID). In various embodiments, the NSAID is a non-selective NSAID. In various embodiments, the NSAID is a selective NSAID. In various embodiments, the NSAID is a COX-2 selective NSAID. In various embodiments, the NSAID is a COX-1 selective NSAID. In various embodiments, the NSAID is a prostaglandin synthase inhibitor. In various embodiments, the NSAID is selected from the group consisting of diclofenac, diclofenac potassium, diclofenac sodium, diflunisal, etodolac, flurbiprofen, fenoprofen, fenoprofen calcium, ketorolac, ketorolac sulphonamide, ketoprofen, tolmetin, tolmetin sodium, aspirin, ibuprofen, naproxen, indomethacin, indomethacin sodium, sulindac, felbinac, piloxetine, mefenamic acid, meclofenamic acid sodium, meloxicam, nabumetone, oxaprozin, piloxetine, celecoxib, etodolac, etoricoxib, lumirox, rofecoxib and valdecoxib.

In various embodiments, the additional therapeutic agent is a leukotriene modulator. In various embodiments, the therapeutic agent suitable for treating peanut allergy is a leukotriene modulator. In various embodiments, the leukotriene modulator is an anti-leukotriene. In various embodiments, the leukotriene modulator is a leukotriene receptor antagonist. In various embodiments, the leukotriene modulator is a leukotriene synthesis inhibitor. In various embodiments, the leukotriene modulator is selected from the group consisting of montelukast, zileuton, and zalukast.

In various embodiments, the additional therapeutic agent is administered before, during, or after the administration of TIMP-PPE. In various embodiments, a therapeutic agent useful for treating peanut allergy is administered before, during, or after the administration of TIMP-PPE.

In various embodiments, the additional therapeutic agent is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, by inhalation, or orally. In various embodiments, the therapeutic agent suitable for treating peanut allergy is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, by inhalation, or orally.

The present invention provides a method of treating peanut allergy in an individual, comprising administering to the individual a combination of TIMP-PPE and an anti-IgE antibody, wherein TIMP-PPE is administered at a dose of about 0.001 mg/kg to 12 mg/kg, and wherein the anti-IgE antibody is administered at a dose of about 10 mg to about 500 mg. In various embodiments, the anti-IgE antibody is omalizumab (XOLAIR®), quilizumab, or ligelizumab.

In various embodiments, the anti-IgE antibody administered in combination with TIMP-PPE is administered in an amount of about 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, or 500 mg. The dosage of anti-IgE antibody is determined based on serum IgE levels, which may be between about 30-100 IU/mL, 100-200 IU/mL, 200-300 IU/mL, 300-400 IU/mL, 400-500 IU/mL, 500-600 IU/mL, 600-700 IU/mL, 700-800 IU/mL, 800-900 IU/mL, 900-1000 IU/mL, 1000-1100 IU/mL, 1100-1200 IU/mL, 1200-1300 IU/mL, 1300-1400 IU/mL, or between 1400-1500 IU/mL. Alternatively, the dosage of anti-IgE antibody is determined based on the individual's weight. In various embodiments, the individual weighs between 30-40 kg, 40-50 kg, 50-60 kg, 60-70 kg, 70-80 kg, 80-90 kg, 90-125 kg, or 125-150 kg.

In various embodiments, the anti-IgE antibody is administered in a single dose or multiple doses. In various embodiments, the anti-IgE antibody is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In various embodiments, the anti-IgE antibody is administered before, simultaneously with, or subsequently/after the administration of TIMP-PPE. In various embodiments, the anti-IgE antibody is administered one week, two weeks, three weeks, or four weeks before the administration of TIMP-PPE. In various embodiments, the anti-IgE antibody is administered one week, two weeks, three weeks, or four weeks after the administration of TIMP-PPE.

Administration of two therapeutic agents simultaneously or concurrently does not require that the agents be administered at the same time or by the same route, as long as there is overlap in the time periods during which the agents exert their therapeutic effects. Contemporaneous or sequential administration is encompassed, such as administration on different days or weeks. Upon further consideration, the therapeutic agents are administered in separate formulations and concurrently or simultaneously, where concurrently refers to administration of the agents within 30 minutes of each other. Prior administration refers to administration of the therapeutic agent within a range of one week prior to treatment with TIMP-PPE, up to 30 minutes prior to administration of TIMP-PPE. Subsequent administration is intended to describe administration from 30 minutes after TIMP-PPE treatment to one week after TIMP-PPE administration.

The present invention provides a method for treating peanut allergy comprising administering to a subject a combination of TIMP-PPE and an anti-IgE antibody, wherein the anti-IgE antibody is administered to the subject once a week for two weeks, or once a week for four weeks prior to administration of TIMP-PPE, wherein TIMP-PPE is administered in two doses, at a dose of between 0.001 mg/kg and 12 mg/kg, one week apart, and wherein the anti-IgE antibody is administered in a dose of between about 50 mg and 500 mg.

Also provided is a method of treating peanut allergy in a subject comprising administering to the subject a combination of TIMP-PPE and an anti-IL-4Rα antibody, wherein TIMP-PPE is administered at a dose of about 0.001 mg/kg to 12 mg/kg, and wherein the anti-IL-4Rα antibody is administered at a dose of about 10 mg to about 500 mg. In various embodiments, the anti-IL-4Rα antibody is dupilumab (DUPIXENT®).

In various embodiments, the anti-IL-4Rα antibody is administered at a dose of about 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, or 600 mg. In various embodiments, the dosage of the anti-IL-4Rα antibody is determined based on serum IgE levels, which may be between about 30-100 IU/mL, 100-200 IU/mL, 200-300 IU/mL, 300-400 IU/mL, 400-500 IU/mL, 500-600 IU/mL, 600-700 IU/mL, 700-800 IU/mL, 800-900 IU/mL, 900-1000 IU/mL, 1000-1100 IU/mL, 1100-1200 IU/mL, 1200-1300 IU/mL, 1300-1400 IU/mL, or between 1400-1500 IU/mL. Alternatively, the anti-IL-4Rα antibody dosage is determined based on the subject's weight. In various embodiments, the subject's weight is between 30-40 kg, 40-50 kg, 50-60 kg, 60-70 kg, 70-80 kg, 80-90 kg, 90-125 kg, or 125-150 kg.

In various embodiments, the anti-IL-4Rα antibody is administered in a single dose or multiple doses. In various embodiments, the anti-IL-4Rα antibody is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In various embodiments, the anti-IL-4Rα antibody is administered before, simultaneously with, or after the administration of TIMP-PPE. In various embodiments, the anti-IL-4Rα antibody is administered one week, two weeks, three weeks, or four weeks before the administration of TIMP-PPE. In various embodiments, the anti-IL-4Rα antibody is administered one week, two weeks, three weeks, or four weeks after the administration of TIMP-PPE. In various embodiments, the anti-IL-4Rα antibody is administered in two doses at an initial dose of between 400 mg and 600 mg, followed by subsequent doses at a maintenance dose of between 200 mg and 300 mg.

In various embodiments, the antihistamine administered in combination with TIMP-PPE is administered in an amount of about 0.05 mg to 2000 mg. In various embodiments, the antihistamine is administered in an amount of about 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 1000 mg, 1500 mg, or 2000 mg. In various embodiments, the antihistamine is administered in a single dose or multiple doses. In various embodiments, the antihistamine is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In various embodiments, the antihistamine is administered twice, three times, four times, five times, or six times a day. In various embodiments, the antihistamine is administered before, simultaneously with, or after the administration of TIMP-PPE. In various embodiments, the antihistamine is administered one week, two weeks, three weeks, four weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months before the administration of TIMP-PPE. In various embodiments, the antihistamine is administered one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months after the administration of TIMP-PPE. In various embodiments, the antihistamine is administered 5, 10, 15, 30, 45, or 60 minutes before the administration of TIMP-PPE. In various embodiments, the antihistamine is administered 5, 10, 15, 30, 45, or 60 minutes after the administration of TIMP-PPE. In various embodiments, the antihistamine is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours prior to administration of TIMP-PPE. In various embodiments, the antihistamine is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after administration of TIMP-PPE. In various embodiments, the antihistamine is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-PPE. In various embodiments, the antihistamine is administered 1, 2, 3, 4, 5, 6 or 7 days after administration of TIMP-PPE. In various embodiments, the antihistamine is a first generation antihistamine or a second generation antihistamine. In various embodiments, the antihistamine is selected from the group consisting of brompheniramine, carbinoxamine maleate, chlorpheniramine, clemastine, diphenhydramine, hydroxyzine, triprolidine, azelastine, cetirizine, desloratadine, fexofenadine, levocetirizine, dualamin, ebastine, embramine, adrenaline, fexofenadine, rolatadine and olopatadine.

In various embodiments, the steroid administered in combination with TIMP-PPE is administered in an amount of about 0.05 mg to 2000 mg. In various embodiments, the steroid is administered in an amount of about 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 1000 mg, 1500 mg, or 2000 mg. In various embodiments, the steroid is administered in a single dose or multiple doses. In various embodiments, the steroid is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In various embodiments, the steroid is administered twice, three times, four times, five times, or six times a day. In various embodiments, the steroid is administered before, simultaneously with, or after the administration of TIMP-PPE. In various embodiments, the steroid is administered one week, two weeks, three weeks, four weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months before the administration of TIMP-PPE. In various embodiments, the steroid is administered one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months after the administration of TIMP-PPE. In various embodiments, the steroid is administered 5, 10, 15, 30, 45, or 60 minutes before the administration of TIMP-PPE. In various embodiments, the steroid is administered 5, 10, 15, 30, 45, or 60 minutes after the administration of TIMP-PPE. In various embodiments, the steroid is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours prior to administration of TIMP-PPE. In various embodiments, the steroid is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after administration of TIMP-PPE. In various embodiments, the steroid is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-PPE. In various embodiments, the steroid is administered 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-PPE. In various embodiments, the steroid is selected from the group consisting of beclomethasone, ciclesonide, fluticasone furoate, mometasone, butenide, fluticasone, triamcinolone, and loteprednol.

In various embodiments, the additional therapeutic agent is a corticosteroid. In various embodiments, the therapeutic agent suitable for treating peanut allergy is a corticosteroid. In various embodiments, the corticosteroid administered in combination with TIMP-PPE is administered in an amount of about 0.05 mg to 2000 mg. In various embodiments, the corticosteroid is administered in an amount of about 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 1000 mg, 1500 mg, or 2000 mg. In various embodiments, the corticosteroid is administered in a single dose or multiple doses. In various embodiments, the corticosteroid is administered once a day, once a week, once every two weeks, once every three weeks, or once every four weeks. In various embodiments, the corticosteroid is administered two, three, four, five, or six times per day. In various embodiments, the corticosteroid is administered prior to, concurrently with, or after the administration of TIMP-PPE. In various embodiments, the corticosteroid is administered one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months prior to the administration of TIMP-PPE. In various embodiments, the corticosteroid is administered one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months after the administration of TIMP-PPE. In various embodiments, the corticosteroid is administered 5, 10, 15, 30, 45, or 60 minutes prior to the administration of TIMP-PPE. In various embodiments, the corticosteroid is administered 5, 10, 15, 30, 45, or 60 minutes after the administration of TIMP-PPE. In various embodiments, the corticosteroid is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours prior to administration of TIMP-PPE. In various embodiments, the corticosteroid is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after administration of TIMP-PPE. In various embodiments, the corticosteroid is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-PPE. In various embodiments, the corticosteroid is administered 1, 2, 3, 4, 5, 6, or 7 days after the administration of TIMP-PPE. In various embodiments, the corticosteroid is selected from the group consisting of corticosterone, prasone, prasone, methylprasone, dexamethasone, betamethasone, and hydrocorticosterone.

In various embodiments, the NSAID administered in combination with TIMP-PPE is administered in an amount of about 0.05 mg to 2000 mg. In various embodiments, the NSAID is administered in an amount of about 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 1000 mg, 1500 mg, or 2000 mg. In various embodiments, the NSAID is administered in a single dose or multiple doses. In various embodiments, the NSAID is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In various embodiments, the NSAID is administered twice, three times, four times, five times, or six times a day. In various embodiments, the NSAID is administered before, simultaneously with, or after the administration of TIMP-PPE. In various embodiments, the NSAID is administered one week, two weeks, three weeks, four weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months before the administration of TIMP-PPE. In various embodiments, the NSAID is administered one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months after the administration of TIMP-PPE. In various embodiments, the NSAID is administered 5, 10, 15, 30, 45, or 60 minutes before the administration of TIMP-PPE. In various embodiments, the NSAID is administered 5, 10, 15, 30, 45, or 60 minutes after the administration of TIMP-PPE. In various embodiments, the NSAID is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours prior to administration of TIMP-PPE. In various embodiments, the NSAID is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours after administration of TIMP-PPE. In various embodiments, the NSAID is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-PPE. In various embodiments, the NSAID is administered 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-PPE. In various embodiments, the NSAID is a non-selective NSAID, a COX-2 selective NSAID, or a COX-1 selective NSAID. In various embodiments, the NSAID is a prostaglandin synthase inhibitor. In various embodiments, the NSAID is selected from the group consisting of diclofenac, diclofenac potassium, diclofenac sodium, diflunisal, etodolac, flurbiprofen, fenoprofen, fenoprofen calcium, ketorolac, ketorolac sulphonamide, ketoprofen, tolmetin, tolmetin sodium, acesalic acid, aspirin, ibuprofen, naproxen, indomethacin, indomethacin sodium, sulindac, felbinac, piloxetine, mefenamic acid, meclofenamic acid sodium, meloxicam, nabumetone, oxaprozin, piloxetine, celecoxib, etodolac, etoricoxib, lumirox, rofecoxib, valdecoxib.

In various embodiments, the leukotriene modulator administered in combination with TIMP-PPE is administered in an amount of about 0.05 mg to 2000 mg. In various embodiments, the leukotriene modulator is administered in an amount of about 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 1000 mg, 1500 mg, or 2000 mg. In various embodiments, the leukotriene modulator is administered in a single dose or multiple doses. In various embodiments, the leukotriene modulator is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In various embodiments, the leukotriene modulator is administered two, three, four, five, or six times a day. In various embodiments, the leukotriene modulator is administered prior to, concurrently with, or after the administration of TIMP-PPE. In various embodiments, the leukotriene modulator is administered one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months prior to the administration of TIMP-PPE. In various embodiments, the leukotriene modulator is administered one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months after the administration of TIMP-PPE. In various embodiments, the leukotriene modulator is administered 5, 10, 15, 30, 45, or 60 minutes before the administration of TIMP-PPE. In various embodiments, the leukotriene modulator is administered 5, 10, 15, 30, 45, or 60 minutes after the administration of TIMP-PPE. In various embodiments, the leukotriene modulator is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours prior to administration of TIMP-PPE. In various embodiments, the leukotriene modulator is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours after administration of TIMP-PPE. In various embodiments, the leukotriene modulator is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-PPE. In various embodiments, the leukotriene modulator is administered 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-PPE. In various embodiments, the leukotriene modulator is an anti-leukotriene, a leukotriene receptor antagonist, or a leukotriene synthesis inhibitor. In various embodiments, the leukotriene modulator is selected from the group consisting of montelukast, zileuton, and zalukast.

It is contemplated that administration of TIMP-PPE alone or in combination with one or more additional treatments to individuals in need may relieve one or more symptoms of peanut allergy. Symptoms of peanut allergy include skin reactions, hives, skin redness, skin swelling, itching, throat tightening, difficulty breathing, shortness of breath, and systemic anaphylaxis. Additional symptoms include digestive problems such as diarrhea, stomach cramps, nausea or vomiting, and a drop in blood pressure.

It is contemplated that administration of TIMP-PPE alone or in combination with one or more therapeutic agents useful for treating peanut allergy to an individual in need thereof may alleviate one or more symptoms of peanut allergy. Symptoms of peanut allergy include skin reactions; hives; skin redness; skin swelling; itching; throat tightening; difficulty breathing; shortness of breath; digestive problems such as diarrhea, stomach cramps, nausea or vomiting; decreased blood pressure; and systemic allergic reactions.

It is also contemplated that administration of TIMP-PPE alone or in combination with one or more additional therapeutic agents to an individual in need thereof may shorten the duration and reduce the severity of an allergic immune response to or following exposure to peanut protein. Allergic immune responses encompassed herein include Th2 T cell responses, B cell activation, basophil activation, eosinophil activation, mast cell activation, and/or IgE induction. Other immune responses include T cell-dependent mechanisms involving upregulation of helper T cell type 2 (Th2) cytokine production (e.g., IL-4, IL-5, IL-9, and IL-13), B cell class switching leading to IgE antibody production, and/or IgE:IgG ratios.

It is also contemplated that administration of TIMP-PPE alone or in combination with one or more therapeutic agents useful for treating peanut allergy to an individual in need thereof may shorten the duration and reduce the severity of an allergic immune response to or following exposure to peanut protein. Allergic immune responses encompassed herein include T cell-dependent mechanisms involving upregulation of helper T cell type 2 (Th2) cytokine production (e.g., IL-4, IL-5, IL-9, and IL-13), B cell activation, B cell class switching leading to IgE antibody production, basophil activation, eosinophil activation, mast cell activation, and/or IgE:IgG ratio. Screening Methods

It is contemplated to monitor the induction and maintenance of immune tolerance in individuals who suffer from, are being treated for, or are about to undergo treatment for peanut allergy, wherein the antigen-specific immune tolerance therapy consists of the peanut allergen-encapsulated TIMPs described herein.

Methods for screening cell types, interleukins or other tolerance measures in individuals undergoing the immune tolerance therapy described herein are known in the art. Methods for assessing tolerance use such techniques, such as flow cytometry, mass cytometry (CyTOF), ELISA, ELISPOT, in vivo/in vitro cell stimulation assays (including but not limited to cell proliferation assays, basophil activation test (BAT), macrophage stimulation assays), such as measuring autoantibodies by ImmunoCap analysis or measuring Ig serotypes.

In various embodiments, the immune tolerance state of an individual is determined based on analysis of one or more biological samples from the individual. The biological samples include whole blood, peripheral blood, peripheral blood mononuclear cells (PBMC), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and/or bone marrow biopsy. In various embodiments, the analysis of the biological sample includes analyzing the amount and/or presence of cell surface proteins, extracellular proteins, intracellular proteins, nucleic acids, metabolites, and/or combinations thereof.

Cells analyzed from biological samples include immune cells, non-immune cells and/or combinations thereof. Immune cells include innate immune cells, acquired immune cells and/or combinations thereof. Innate immune cells analyzed from biological samples include antigen presenting cells (APCs). Exemplary innate immune cells analyzed from biological samples include monocytes, macrophages, neutrophils, granulocytes, dendritic cells, mast cells, eosinophils, basophils and/or combinations thereof. Adaptive immune cells analyzed from biological samples include effector immune cells, such as CD4+ T cells, CD8+ T cells, B cells, NK cells, NK-T cells and/or combinations thereof. In various embodiments, the T cells are Th1 cells, Th2a cells, Treg cells and Tr1 cells.

In certain embodiments, the cells analyzed from the biological sample are epithelial cells, stromal cells, endothelial cells, fibroblasts, coat cells, adipocytes, mesenchymal stem cells, hematopoietic stem cells, hematopoietic progenitor cells, liver sinus endothelial cells (LSEC) and/or Kupffer cells.

One aspect of the immune tolerance state and immune profile of an individual is determined by analyzing one or more proteins from one or more biological samples from the individual. In various embodiments, the protein is an interleukin and/or an interleukin. In various embodiments, the protein is a cell signaling protein. In various embodiments, the interleukins and chemokines are selected from the group consisting of IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12p70, IL-13, IL-14, IL-15, IL-16, IL-17, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-27b, IL-2 8. IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2 0. CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2 (MCP-1), CXCL3 (MIP-1α, CXCL4 (MIP-1β, CXCL5 (RANTES), CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, GM-CSF, IFN-α, IFN-β, IFN-γ, TNF-α, TGF-β1, TGF-β2, TGF-β3, soluble CD14 and/or combinations thereof

In various embodiments, the protein is a protease. In various embodiments, the protease is an aspartic acid protease, a cysteine protease, a metalloprotease, a serine protease and/or a threonine protease. In various embodiments, the protease is selected from the group consisting of ADAM1, ADAM2, ADAM7, ADAM8, ADAM9, ADAM10, ADAM11, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAM20, ADAM21, ADAM22, ADAM23, ADAM28, ADAM29, ADAM30, ADAM33, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28. In various embodiments, the protein associated with apoptosis is selected from the group consisting of: P53, apoptosis proteinase 1, apoptosis proteinase 2, apoptosis proteinase 3, apoptosis proteinase 4, apoptosis proteinase 5, apoptosis proteinase 6, apoptosis proteinase 7, apoptosis proteinase 8, apoptosis proteinase 9, apoptosis proteinase 10, apoptosis proteinase 11, apoptosis proteinase 12, apoptosis proteinase 13, apoptosis proteinase 14, BCL-2, BCL-XL, MCL-1, CED-9, A1, BFL1, BAX, BAK, DIVA, BCL-XS, BIK, BIM, BAD, BID and EGL-1. Several methods for analyzing proteins in biological samples have been described in the literature, including enzyme-linked immunosorbent assay (ELISA), Western blot and mass spectrometry. In various embodiments, the protein is one or more immunoglobulins (Ig). In various embodiments, Ig is selected from the group consisting of: IgA, IgD, IgE, IgM and/or variants thereof. In various embodiments, the immunoglobulin is antigen-specific. Several methods for detecting immunoglobulins from biological samples have been described in the literature, including ELISA and ImmunoCap.

A list of human metabolites that can be analyzed from biological samples can be found in the literature, including (Psychogios et al., 2011), (Wishart et al., HMDB: the Human Metabolome Database. Nucleic Acids Res. 2007 Jan;35(Database J):D521-6, 2007) and the Human Metabalome Database (HMDB) and are incorporated herein by reference.

One aspect of the immune tolerance state and immune profile of an individual is determined by analyzing one or more cell surface proteins from a biological sample. In various embodiments, the cell surface proteins include CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RA, CD47, CD45RA, CD4 5RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66b, CD69, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103, CD107, CD112, CD120a, CD120b, CD123, CD125, CD12 7. CD134, CD135, CD140a, CD141, CD154, CD155, CD160, CD161, CD163, CD172a, XCR1, CD203c, CD204, CD206, CD207 CD226, CD244, CD267, CD268, CD269, CD355, CD358, CRTH2, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL 3. KIR3DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS, NKp44, NKp46, TCR, BCR, integrin, FcβεRI, MHC-I, MHC-II, IL -1R, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-3Rα, CSF2RB, IL-4R, IL-5Rα, CSF2RB, IL-6Rα, gp130, IL-7Rα, IL-9R, IL-10R, IL-12Rβ1, IL-12Rβ2, IL-13Rα1, IL-13Rα2, IL-15Rα, IL-21R, IL -23R, IL-27Rα, IL-31Rα, OSMR, CSF-1R, cell surface IL-15, IL-10Rα, IL-10Rβ, IL-20Rα, IL-20Rβ, IL-22Rα1, IL-22Rα2, IL-22Rβ, IL-28RA, PD-1, PD-1H , BTLA, CTLA-4, PD-L1, PD-L2, 2B4, B7-1, B7-2, B7-H1, B7-H4, B7-DC, DR3, LIGHT, LAIR, LTα1β2, LTβR, TIM-1, TIM-3, TIM-4, TIGIT, LAG-3, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR , GITRL, TL1A, HVEM, 41-BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF3, TRAF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, AITR, TRAMP, CCR1, CCR2 , CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CLECL9a, DC-SIGN, IGSF4A, SIGLEC, EGFR, PDGFR, VEGFR, FAP, α-SMA, FAS, FAS-L, FC , ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM-1, MICA, MICB, UL16, ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1 α, β, γ, δ and ε, H60a, H60b, H60c, GPR15, ST2 and/or combinations thereof. Integrins include α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8 and/or combinations thereof. TCRs include α, β, γ, δ, ε, ζ chains and/or combinations thereof. Several methods for analyzing cell surface protein expression, including flow cytometry and mass cytometry (CyTOF), have been described in the literature.

One aspect of an individual's immune tolerance state and immune profile is determined by analyzing one or more metabolites from a biological sample. In various embodiments, the metabolite is an inflammatory metabolite. In various embodiments, the metabolite is an anti-inflammatory metabolite. In various embodiments, examples of inflammatory metabolites include acids, lipids, sugars, amino acids, lactate, trimethylamine N-oxide, O-acetylcreatine, L-carnitine, choline, succinate, glutamine, fatty acids, cholesterol, 3-hydroxybutyrate, 3'-sialyllactose, arachidonic acid, prostaglandins (G2 and H2), PGD2, PGE2, PGF2a, PGI2, TXA2, leukotrienes (A4, B4, C4, D4, E4), kynurenine, 3-hydroxykynurenine, lipoxin A4, and lipoxin B4. In various embodiments, examples of anti-inflammatory metabolites include 2-amino-3-carboxymuconate 6-semialdehyde, picolinic acid, oxadiaminobenzoic acid, 3-hydroxyoxadiaminobenzoic acid, glutaryl coenzyme A, NAD+, quinolinic acid, arginine, butyrate, and adenosine. Lists of human metabolites that can be analyzed from biological samples can be found in the literature, including (Psychogios et al., 2011 PLoS One 6(2):e16957), (Wishart et al., HMDB: the Human Metabolome Database. Nucleic Acids Res. 2007 Jan; 35(Database Journal):D521-6, 2007) and the Human Metabalome Database (HMDB), each of which is incorporated herein by reference.

In certain embodiments, the tolerance state of an individual is determined by analyzing nucleic acids from a biological sample. In various embodiments, the nucleic acid is DNA and/or RNA, including but not limited to single-stranded DNA, double-stranded DNA, mRNA, rRNA, tRNA, siRNA, miRNA, long non-coding RNA (long ncRNA, lncRNA) and non-coding RNA (ncRNA), mitochondrial RNA. In various embodiments, the immune tolerance state of an individual is determined by analyzing gene expression from a biological sample. In various embodiments, the immune tolerance state is determined by analyzing the expression of genes associated with immune function, antibodies, xenobiotic reactions, metabolism, apoptosis, cell death, necrosis, ferroptosis, autophagy, cell migration, endocytosis, phagocytosis, endocytosis, tight junction regulation, cell adhesion, differentiation and/or combinations thereof. In various embodiments, the immune tolerance state is determined by analyzing the expression of genes associated with immunosuppression. In various embodiments, the immune tolerance state is determined by analyzing the expression of genes associated with immune activation. In various embodiments, the immune tolerance state is determined by analyzing the expression of genes associated with immunomodulatory function. In various embodiments, nucleic acid analysis is used to generate immune tolerance characteristics. Several methods have been described in the literature for high-throughput gene expression analysis, including RNA sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq), exome sequencing, and microarray-based analysis.

The biological sample is analyzed after stimulation with one or more stimuli (such as antigens, allergens, and one or more activators) in vivo and/or in vitro, as appropriate. It is contemplated that the T cells, B cells, and immunoglobulins used for analysis are antigen-specific. Exemplary T cells include effector memory T cells, antigen-specific T cells, activated antigen-specific T cells, Th1 cells, pathogenic Th2a+ cells, Th17 cells, follicular helper T cells (TFH), TH0 cells, or other antigen-specific T cells. B cells include effector B cells, memory B cells, plasma cells, and regulatory B (Breg) cells. In certain embodiments, T cells or B cells are identified based on the expression of proteins described in Table 1. Table 1 Cell type Performance Markers Effector memory (EM) T cells CD45RA ^LO CD4 ⁺ Disease-specific T cells CD45RA ^LO CD4 ⁺ CD154 ⁺ CD137 ⁺ Activated disease-specific T cells CD38 ⁺ Pathogenic Th2a+ cells CD45RA ^LO CD4 ⁺ CD154 ⁺ CD137 ⁺ CXCR5 ^- CRTH2 ^+/- CD27 ^- CD200R ⁺ CD161 ⁺ Th1 cells CXCR5 ^- CRTH2 ^- CXCR3 ⁺ CCR6 ^- Th17 cells CXCR5 ^- CRTH2 ^- CXCR3 ^- CCR6 ⁺ TFH cells CD45RA ^LO CD4 ⁺ CXCR5 ⁺ Treg CD45RA ^LO CD4 ⁺ CD154 ^- CD127 ^- CD25 ⁺ TIGIT ⁺ CD137 ⁺ GARP ^+/- OX40 ^+/- Th0 cells CXCR5 ^- CRTH2 ^- CXCR3 ^- CCR6 ^- CCR4 ^- Activated antigen-specific CD8 T cells CD8 ⁺ CD137 ⁺ CD69 ⁺ CD355 ⁺ Intestinal homing T cells CD4 ⁺ α4β7 ⁺ and CD8 ⁺ αEβ7 ⁺ Brain homing T cells CD45RO ⁺ CCR7 ⁺ CD27 ⁺ and/or CD45RO ⁺ CCR7 ⁺ CCR5 ⁺ CXCR3 ⁺ CD27 ⁺ Exhausted T cells CD4 ⁺ TIGIT ⁺ KLRG1 ⁺ PD-1 ⁺ Plasmablast B cells (effector B cells) CD3 ^- CD19 ⁺ CD137 ⁺ CD45RA ⁺ CD27 ⁺ 38 ⁺

In various embodiments, the immune tolerance state of an individual is determined by obtaining one or more samples, such as whole blood, from the individual on the day of the first administration of TIMP-PPE before dosing (Day 1), 14 days after the second dose, and then every 90 days after the second dose (e.g., Day 90, Day 180, Day 270, and Day 360 after the second dose). The whole blood can then be processed to isolate peripheral blood mononuclear cells (PBMCs), basophils, neutrophils, plasma, and serum for downstream analysis. Cells isolated from one or more samples collected from the individual are analyzed and analyzed using the methods described below.

In various embodiments, the immune tolerance state of an individual is determined by obtaining one or more samples, such as whole blood, from the individual on the day of the first administration of TIMP-PPE before dosing (Day 1), 14 days after the second dose (Day 15), 60 days, and then, as appropriate, every 90 days after the second dose (e.g., Day 90, Day 180, Day 270, and Day 360 after the second dose). The whole blood can then be processed to isolate peripheral blood mononuclear cells (PBMCs), basophils, neutrophils, plasma, and serum for downstream analysis. Cells isolated from one or more samples collected from an individual are analyzed and analyzed using the methods described below.

In various embodiments, the immune tolerance state of the individual determined before the administration of TIMP-PPE is used as a baseline. In various embodiments, the baseline of the individual is determined based on the analysis of one or more biological samples 1, 2, 3, 4, 5, 6, or 7 days before the administration of TIMP-PPE. In various embodiments, the baseline of the individual is determined based on the analysis of one or more biological samples 1, 2, 3, or 4 weeks before the administration of TIMP-PPE. In various embodiments, the baseline of the individual is determined based on the analysis of one or more biological samples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months before the administration of TIMP-PPE.

In various embodiments, the immune tolerance state of the individual is determined after administration of TIMP-PPE. In various embodiments, the immune tolerance state of the individual is determined based on analysis of one or more biological samples 1, 2, 3, 4, 5, 6 or 7 days after administration of TIMP-PPE. In various embodiments, immune tolerance is determined based on analysis of one or more biological samples 1, 2, 3 or 4 weeks after administration of TIMP-PPE. In various embodiments, immune tolerance is determined based on analysis of one or more biological samples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after administration of TIMP-PPE. In various embodiments, the immune tolerance state of the individual determined after administration of TIMP-PPE is compared to baseline. In various embodiments, the immune tolerance status of an individual determined following administration of TIMP-PPE is compared to healthy individuals or individuals administered a placebo.

In various embodiments, the immune tolerance status of an individual is determined following administration of TIMP-PPE, whether an initial or initial dose of TIMP-PPE or an additional dose of TIMP-PPE.

After consideration, the following analysis follows the immune status and tolerance induction of individuals receiving TIMP-PPE immune tolerance therapy.

The ratio of peanut-specific Th2a+ cells stimulated in vitro with purified antigenic peanut protein (Th2a+ cells/total peanut-specific T cells) can be measured, for example, by flow cytometry. Th2a+ cells are defined as CRTH2+/CD161+/CD154+/CD27-. Total peanut-responsive cells are defined as CRTH2-/CD161+/CD154+/CD27-. In various embodiments, administration of TIMP-PPE to an individual maintains or reduces peanut-specific Th2a+ cells by about 1%-100% relative to placebo administration and/or one or more baseline measurements obtained from the individual during treatment. 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values and ranges between such values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or about 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times, including all values and ranges between such values).

The ratio of activated peanut-specific T cells after ex vivo stimulation with peanut protein is measured by flow cytometry (activated peanut-specific T cells/total peanut-specific T cells). Activated peanut-specific T cells are defined as CD154+/CD38+. Non-activated peanut-specific T cells are defined as CD154+. In various embodiments, administration of TIMP-PPE to an individual maintains or reduces activated peanut-specific T cells by about 1%-100% relative to placebo administration and/or one or more baseline measurements obtained from the individual during treatment. 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values and ranges between such values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or about 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times, including all values and ranges between such values).

The frequency of regulatory T cell populations (CD4+/CD25+/FoxP3+/Helios+/IL-10+) or (CD4+ ^CD45RAlowCD4 +CD137+CD25+ ^CD127low ) is determined by flow cytometry. Multicolor flow analysis is performed to provide the ratio of peanut-specific regulatory T cells (peanut-specific regulatory T cells/peanut-specific CD4+ effector memory cells). In various embodiments, administration of TIMP-PPE to an individual increases Treg cells by about 1%-100% relative to placebo administration and/or one or more baseline measurements obtained from the individual during treatment. 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values and ranges between such values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or an increase of about 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times, including all values and ranges between such values).

The ratio of IL-5 to IFN-γ in the PBMC culture supernatant is then measured, for example, by Luminex 200 detection.

The following indicators of immune tolerance status can be determined based on analysis of phagocytic globules isolated from one or more blood samples collected from an individual and stimulated ex vivo with purified antigenic peanut protein: the ratio of activated CD203+/CD63+ phagocytic globules after ex vivo stimulation with purified antigenic peanut protein, using the phagocytic globulin activation assay (BAT) (Santos and Lack 2016 Clin Transl Allergy. 6: 10); and the effective concentration for 50% maximal phagocytic globulin activation (EC50) after ex vivo stimulation with purified antigenic peanut protein, measured using the phagocytic globulin activation assay, wherein the activated phagocytic globules are CD203+/CD63+/-. Assays were performed to provide the effective concentration for 50% maximal phagocytic activation (EC50).

The following indicators of immune tolerance state can be determined based on analysis of serum separated from one or more blood samples obtained from the individual: the ratio of peanut-specific IgE to IgG, as measured by the ImmunoCap assay. In various embodiments, administration of TIMP-PPE to the individual reduces the peanut-specific IgE:IgG ratio by about 1%-100% relative to placebo administration and/or one or more baseline measurements obtained from the individual during treatment. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all values and ranges between such values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or about 2-100 times lower (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times lower, including all values and ranges between such values).

Changes in peanut-specific IgE are measured by ImmunoCap analysis. In various embodiments, administration of TIMP-PPE to an individual maintains or reduces the level of peanut-specific IgE by about 1%-100% relative to placebo administration and/or one or more baseline measurements obtained from the individual during treatment. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all values and ranges between such values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or about 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times, including all values and ranges between such values).

The following indicators of immune tolerance status can be determined based on analysis of phagocytic globules isolated from one or more blood samples collected from an individual and stimulated ex vivo with purified antigenic peanut protein: the ratio of activated CD203+/CD63+ phagocytic globules after ex vivo stimulation with purified antigenic peanut protein, using the phagocytic globulin activation assay (BAT) (Santos and Lack 2016 Clin Transl Allergy. 6: 10); and the effective concentration for 50% maximal phagocytic globulin activation (EC50) after ex vivo stimulation with purified antigenic peanut protein, measured using the phagocytic globulin activation assay, wherein the activated phagocytic globules are CD203+/CD63+/-. The analysis is performed to provide an effective concentration for 50% maximum phagocytic globulin activation (EC50). In various embodiments, administration of TIMP-PPE reduces phagocytic globulin activation. In various embodiments, administration of TIMP-PPE to an individual increases the EC50 for maximum phagocytic globulin activation by about 1%-100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values and ranges between such values), 10-95%, 15-90%, 20-85%, 25- 75%, 30-70%, 35-65%, 40-60%, 45-55% or 50%, or about 2-10,000 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1000 or 10,000 times, including all values and ranges between these values).

In certain embodiments, the efficacy of TIMP-PPE in alleviating one or more symptoms of peanut allergy, improving tolerance to peanut protein, and/or shortening the duration and reducing the severity of allergic immune responses to peanut protein is determined by a double-blind, placebo-controlled food challenge (DBPCFC), administration of a cumulative tolerance-altering dose of peanut protein during DBPCFC, and/or skin prick testing (SPT). The procedures for conducting DBPCFC and SPT have been previously described (Sampson et al., J Allergy Clin Immunol. 2012;130(6):1260-1274; Heinzerling et al. The skin prick test - European standards. Clin Transl Allergy. 2013;3(1):3).

The efficacy of TIMP-PPE in alleviating one or more symptoms of peanut allergy and/or shortening the duration and severity of an allergic immune response to peanut protein is determined based on analysis of one or more biological samples from an individual. The biological samples include whole blood, peripheral blood, peripheral blood mononuclear cells (PBMC), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and/or bone marrow biopsy. In various embodiments, the analysis of the biological sample includes analysis of the amount and/or presence of cell surface proteins, extracellular proteins, intracellular proteins, nucleic acids, metabolites, and/or combinations thereof.

The immune tolerance profile of an individual is generated using one or more of the following parameters, which are analyzed based on one or more biological samples obtained from the individual and stimulated in vivo and/or ex vivo: a.  The proportion of effector T cells in the total T cell population, b.  The proportion of Treg cells in the total T cell population, c.  The proportion of effector B cells in the total B cell population, d.  The level of specific IgG, IgA, IgM and/or IgE, e.  The level of inflammatory cytokines and interleukins, f.   The level of anti-inflammatory cytokines and interleukins, g.  The level of inflammatory metabolites, and h.  The level of anti-inflammatory metabolites.

If 1, 2, 3, 4, 5, 6, 7 or 8 of the parameters listed in (a)-(h) above indicate maintenance of immune tolerance, then the immune tolerance signature indicates maintenance of immune tolerance. In various embodiments, if at least 2/8 of the parameters listed in (a)-(h) indicate maintenance of immune tolerance, then the immune tolerance signature indicates maintenance of immune tolerance. In various embodiments, if 1, 2, 3, 4, 5, 6, 7 or 8 of the parameters listed in (a)-(h) above indicate maintenance of immune tolerance, then it is determined that the individual does not need treatment with a TIMP. In various embodiments, if at least 3/8 of the parameters listed in (a)-(h) above indicate maintenance of immune tolerance, then it is determined that the individual does not need treatment with a TIMP.

An immune tolerance profile of an individual generated using one or more of the parameters described herein indicates weakening and/or absence of immune tolerance before or after treatment with TIMP-PPE if the following conditions are met:

a. the proportion of effector T cells in the total T cell population is between 5% and 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100%, including all values and ranges between such values), and/or

b. The proportion of Treg cells in the total T cell population is between 1-3%, and/or

c. the proportion of effector B cells in the total B cell population is between 5% and 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values and ranges between such values), and/or

d. An increase of approximately 5%-100% in IgG, IgA, IgM and/or IgE levels relative to one or more baseline values in healthy individuals and/or obtained from individuals during treatment about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values and ranges between such values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or about 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times, including all values and ranges between such values), and/or

e.   Inflammatory interleukins/chemokines increased by approximately 5%-100% relative to one or more baseline values measured in healthy individuals and/or obtained from individuals during treatment about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values and ranges between such values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or about 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times, including all values and ranges between such values), and/or

f.   The level of anti-inflammatory interleukins/chemokines is reduced by about 5%-100% compared to one or more baseline values obtained from healthy individuals and/or individuals during treatment about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values and ranges between such values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or about 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times, including all values and ranges between such values), and/or

g. Levels of inflammatory metabolites increased by approximately 5%-100% relative to one or more baseline values measured in healthy individuals and/or during treatment about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values and ranges between such values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or about 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times, including all values and ranges between such values), and/or

h. A decrease of approximately 5%-100% in the level of anti-inflammatory metabolites relative to one or more baseline values obtained from healthy individuals and/or during treatment 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values and ranges between such values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or about 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times, including all values and ranges between such values).

Assume that at the pre-dose time point on day 1, the proportion of Th2a+ cells in a peanut allergic individual is expected to be >15%. Treatment with TIMP-PPE is expected to reduce the proportion of Th2a+ cells to <15% 14 days after the second dose, indicating induced immune tolerance. An increase in the proportion of Th2a+ cells to >15% at any subsequent time point (e.g., day 90, day 180, day 270, and day 360 post-dose) would indicate weakened immune tolerance and the need for re-administration of TIMP-PPE to restore immune tolerance. In summary, the results from the above analyses can be used to determine the characteristics of immune tolerance, regardless of whether the individual has maintained immune tolerance. If such analyses indicate weakening and/or loss of immune tolerance, TIMP-PPE can be re-administered to the individual to restore immune tolerance. Pharmaceutical preparations

Depending on the route of administration, the pharmaceutical composition of the present invention containing the TIMP-PPE described herein as an active ingredient may contain a pharmaceutically acceptable carrier or additive. Examples of such carriers or additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymers, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble polydextrose, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin, agar, diglycerol, glycerol, propylene glycol, polyethylene glycol, vaseline, wax, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, pharmaceutically acceptable surfactants and the like. Depending on the dosage form of the present invention, the additives used are selected from (but not limited to) the above or a combination thereof as needed.

The formulation of the pharmaceutical composition will vary depending on the chosen route of administration (e.g., solution, emulsion). Suitable compositions comprising the administered therapeutic agent may be prepared in the form of a physiologically acceptable vehicle or carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcohol/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or non-volatile oils. Intravenous vehicles may include various additives, preservatives, or fluids, nutrients, or electrolyte supplements.

There are various aqueous carriers, such as sterile phosphate-buffered saline, bacteriostatic water, water, buffered water, 0.4% saline, 0.3% glycine, and the like, and may include other proteins (such as albumin, lipoprotein, globulin, etc.) for enhanced stability, which have undergone mild chemical modification, or the like.

Therapeutic formulations of the inhibitor are prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing the inhibitor having the desired purity with a physiologically acceptable carrier, excipient or stabilizer, as appropriate (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexahydroxyammonium chloride; benzathonium chloride, benzathonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins , such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counter ions, such as sodium ions; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

Formulations for intravenous administration must be sterile. This is easily accomplished by filtration through sterile filter membranes.

Aqueous suspensions may contain the active compound in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic. acacia); the dispersant or wetting agent may be a naturally occurring phospholipid, such as lecithin, or a condensation product of an alkylene oxide with a fatty acid, such as polyoxyethylene stearate, or a condensation product of an alkylene oxide with a long chain aliphatic alcohol, such as heptadecyl-enoxyhexadecyl alcohol, or a condensation product of an alkylene oxide with a partial ester derived from a fatty acid and a hexitol, such as polyoxyethylene sorbitol monooleate, or a condensation product of an alkylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride, such as polyethylene dehydrated sorbitan monooleate. The aqueous suspension may also contain one or more preservatives, such as ethyl p-hydroxybenzoate or n-propyl p-hydroxybenzoate.

The TIMP-PPE described herein can be stored freeze-dried and reconstituted in a suitable carrier prior to use.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the modified particles are mixed with at least one inert, pharmaceutically acceptable excipient or carrier (such as sodium citrate or dicalcium phosphate) and/or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; c) moisturizers. The invention relates to a pharmaceutical composition comprising: a) a disintegrant such as glycerol; b) a disintegrant such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) a solution retardant such as wax; f) an absorption enhancer such as quaternary ammonium compounds; g) a wetting agent such as cetyl alcohol and glyceryl monostearate; h) an adsorbent such as kaolin and bentonite clay, and i) a lubricant such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and a mixture thereof. In the case of capsules, tablets and pills, the dosage form may also include a buffering agent.

As another aspect, the invention includes a kit comprising one or more compounds or compositions packaged in a manner useful for practicing the methods of the invention. In one embodiment, such a kit includes a compound or composition described herein (e.g., a composition comprising a TIMP alone or in combination with a second agent) packaged in a container (e.g., a sealed bottle or can), and a label affixed to the container or included in the package describing the use of the compound or composition in practicing the method. Preferably, the compound or composition is packaged in unit dose form. The kit may further include a device suitable for administering the composition according to a specific route of administration or suitable for practicing a screening assay. Preferably, the kit contains a label describing the use of the inhibitor composition.

Additional aspects and details of the present invention will be apparent from the following examples, which are intended to be illustrative and not limiting. Example 1 - Phase I/II Trial of TIMP-PPE in Peanut Allergy

This example describes a Phase 1b/2a, randomized, double-blind, placebo-controlled, two-part study to evaluate the safety, tolerability, pharmacodynamics, and efficacy of TIMP-PPE (CNP-201) in individuals aged 16-55 years with peanut allergy.

CNP-201 consists of PLGA nanoparticles encapsulating purified peanut extract. The average diameter of CNP-201 particles is 400-800 nm and the negative zeta potential is between -32 mV and -50 mV. CNP-201 particles are supplied as a lyophilized formulation. Prior to administration, CNP-201 particles are reconstituted in sterile water for injection and diluted in sterile saline (0.9% sodium chloride).

The study consists of Part A and Part B.

Part A: Part A is a randomized, double-blind, placebo-controlled study of the tolerability and safety of CNP-201 at escalating doses. Three groups will be selected in Part A to receive CNP-201 or placebo at multiple escalating doses. This will be followed by Part B, which is a randomized, double-blind, placebo-controlled repeated-dose study using the safe and tolerable CNP-201 dose determined in Part A.

After the initial screening assessment, subjects who met all inclusion criteria and did not meet exclusion criteria underwent a skin prick test (SPT) followed by a baseline double-blind, placebo-controlled food challenge (DBPCFC) (peanut and placebo (oatmeal) challenge, administered on two separate days) to confirm peanut allergy. The SPT and DBPCFC were performed by study physicians or staff trained to manage clinical emergencies present in the field, with immediate access to emergency medications and equipment, and within the confines of an adjacent hospital emergency department (for rapid provision of emergency care if needed). Subjects who continued to meet all inclusion criteria and did not meet exclusion criteria were eligible for study enrollment after completing two days of DBPCFC.

After DBPCFC, all subjects who continued to meet all I/E criteria received subcutaneous injections of omalizumab (XOLAIR®). The dose of omalizumab (XOLAIR®) followed the product labeling as specified in the protocol and was determined based on the subject's body weight and serum IgE at the initial screening. Subjects were given medication every 2 weeks or every 4 weeks according to the product labeling.

Subjects who continued to meet the inclusion/exclusion criteria were randomized to receive CNP-201 or placebo (0.9% sodium chloride USP) by intravenous (IV) infusion in a 2:1 ratio (Part A) or a 1:1 ratio (Part B) on Day 1. Subjects were given CNP-201 or placebo on Days 1 and 8.

Peanut-allergic individuals eligible for inclusion in this study were defined based on the following inclusion criteria: 1. Males and non-pregnant females aged 16 to 55 years (inclusive). 2. Individuals with a body mass index (BMI) ≥18 and ≤32 and a weight >30 kg and ≤150 kg at screening. 3. Individuals with serum IgE ≥30 IU/mL and ≤1500 IU/mL at screening. 4. Individuals diagnosed with peanut allergy by a physician or with a documented history of peanut allergy. 5. Individuals with a documented history of non-severe systemic allergic reaction to peanut (grade ≤3) (including mild wheezing or dyspnea but no hypoxia). 6.  Individuals with peanut-specific IgE >5 kU/L as measured by ImmunoCAP at screening. 7.  Individuals who reported no peanut dietary intake and no suspected peanut exposure (including any peanut food challenge) for at least 14 days before screening and agreed to continue limiting peanut exposure during the study period except for the study DBPCFC. 8.  Individuals with a change in water prick diameter >3 mm on a positive skin prick test (SPT) for peanut compared to a negative control (50% glycerol) at screening. 9.  Individuals who are willing and able to provide written informed consent approved by the Institutional Review Board (IRB). 10. Subjects with positive peanut DBPCFC (baseline DBPCFC) at a peanut protein priming dose of ≥10 mg and ≤300 mg at screening. 11. Subjects with ≥15% peanut-specific Th2a+ T cells (peanut-specific Th2a+ cells/total peanut-specific T cells) after ex vivo stimulation of PBMC at screening.

Subjects who met all inclusion criteria and did not meet exclusion criteria after completing two screening visits, including two baseline DBPCFCs (peanut and placebo challenges, administered on separate days), were enrolled in one of three dose-escalation groups. Subjects were randomized in a 2:1 ratio to receive CNP-201 or placebo (0.9% sodium chloride injection) as a 200 mL intravenous infusion on Days 1 and 8.

The dosages of the three groups were as follows: Group 1: 250 mg, Group 2: 450 mg, Group 3: 650 mg. The dosing interval between subjects in each dosage group was at least 48 hours.

After the initial screening assessment, individuals who meet all inclusion criteria and do not meet exclusion criteria will undergo a skin prick test (SPT) followed by a baseline double-blind, placebo-controlled food challenge (DBPCFC) (peanut and placebo (oatmeal) challenge, administered on two separate days) to confirm peanut allergy. If a reaction occurs and is treated, the peanut and placebo challenges will be performed at least 48 hours apart. A timeline of the study procedures is depicted in Figure 1C.

After completing the second set of DBPCFC and the 2-hour observation period, subjects who continued to meet all inclusion criteria and did not meet the exclusion criteria received the first subcutaneous injection of omalizumab. Omalizumab (XOLAIR®) dosing followed the product label and was determined based on the subject's body weight and serum IgE at screening as described in Table 2. Subjects were dosed every 2 weeks (Day -29, Day -15, and Day -1) or every 4 weeks (Day -29 and Day -1) according to the product label. Table 2 : Omalizumab Dosing Determination Screening serum IgE (IU/mL) Dosing frequency Weight(kg) ＞30-40 ＞40-50 ＞50-60 ＞60-70 ＞70-80 ＞80-90 ＞90-125 ＞125-150 Dosage(mg) ＞30-100 Every 4 weeks 75 150 150 150 150 150 300 300 ＞ 100-200 150 300 300 300 300 300 450 600 ＞ 200-300 225 300 300 450 450 450 600 375 ＞ 300-400 300 450 450 450 600 600 450 525 ＞ 400-500 450 450 600 600 375 375 525 600 ＞ 500-600 450 600 600 375 450 450 600 ＞ 600-700 450 600 375 450 450 525 ＞ 700-800 Every 2 weeks 300 375 450 450 525 600 ＞ 800-900 300 375 450 525 600 ＞900-1000 375 450 525 600 ＞1000-1100 375 450 600 ＞1100-1200 450 525 600 ＞1200-1300 450 525 ＞1300-1500 525 600

Any subject who experienced a severe hypersensitivity reaction to omalizumab was provided appropriate treatment and discontinued from the study and then replaced with another subject at the same dose.

Subjects returned to the clinic on Day 1 for final eligibility assessment and collection of laboratory samples. Subjects who continued to meet all inclusion and no exclusion criteria were randomized to dose groups at that time. Subjects were given CNP-201 or placebo on Day 1 and on Day 8. CNP-201 or placebo was administered by intravenous infusion over approximately 3-4 hours using a progressive infusion rate. Subjects underwent 4 hours of medical observation in the clinic after infusion for acute adverse events (AEs), including infusion reactions (IR). If allergic reactions occurred, antihistamine/epinephrine was available for immediate treatment.

Subjects returned to the clinic 2 days after each infusion (Day 3 and Day 10) for safety laboratory data collection, drug review, and AE assessment, and followed daily telephone interviews between infusions (Days 33-36) to assess and record any AEs and drug changes. During the post-dose period, subjects returned to the clinic on Day 15 for safety laboratory data collection, PD measurements, and assessment of AEs and drug changes.

After all subjects in a dose cohort complete the Day 15 clinic visit (7 days after the second dose), if cohort expansion is necessary (a minimum of three additional subjects randomized 2:1 to receive CNP-201 or placebo (0.9% sodium chloride USP)), or if any other clinical recommendations should be made, a Data Monitoring Committee (DMC) will be convened to review all available safety data and determine whether it is acceptable to proceed to the next escalating dose cohort.

Subjects will return to the clinic on Day 60 for collection of immunological safety laboratory data, PD measurements, and a second SPT, followed by a DBPCFC (completed on Day 61). Subjects will return to the clinic on Day 90 for an end-of-study visit for collection of safety laboratory data, PD measurements, and final assessment of AEs and drug product changes. After all subjects complete the Day 15 visit, recommendations will be made regarding continuation of Part B with CNP-201 at the dose identified as safe and tolerable in Part A.

Part B : Subjects in Part B were randomized in a 1:1 ratio to receive CNP-201 or placebo (0.9% sodium chloride USP) at a dose identified as safe and tolerable in Part A. After the initial screening assessment, subjects who met all inclusion criteria and did not meet exclusion criteria underwent a skin prick test (SPT) followed by a baseline double-blind, placebo-controlled food challenge (DBPCFC) (peanut and placebo (oatmeal) challenge, administered on separate days) to confirm peanut allergy. If a reaction occurred and was treated, the peanut and placebo challenges were performed at least 48 hours apart. After completion of the second DBPCFC and a 2-hour observation period, subjects who continued to meet all inclusion criteria and did not meet exclusion criteria received the first subcutaneous injection of omalizumab.

Dosage of omalizumab (XOLAIR®) follows the product labeling as specified in the protocol and is determined based on the subject's body weight and serum IgE at screening and is described in Table 2. Subjects were dosed every 2 weeks or every 4 weeks according to the product labeling. Any subject who experienced a severe hypersensitivity reaction to omalizumab was provided appropriate treatment and discontinued from the study. These subjects were replaced in Part B.

Subjects returned to the clinic on Day 1 for final eligibility assessment and collection of laboratory samples. Subjects who continued to meet all inclusion criteria and no exclusion criteria were randomized to receive CNP-201 or placebo.

Subjects will receive CNP-201 or placebo on Days 1 and 8. The study product will be administered by IV infusion over approximately 3-4 hours using a progressive infusion rate. Subjects will undergo 4 hours of medical observation in the clinic following infusion for acute AEs. If an allergic reaction occurs, antihistamine/epinephrine may be used immediately for treatment. Subjects will follow daily telephone interviews between infusions (Days 2-7 and Days 9-14) to assess and record any AEs and medication changes.

During the post-dose period, subjects returned to the clinic on Day 15 for collection of safety laboratory data, PD measurements, and assessment of AEs and drug changes. Subjects also returned to the clinic on Day 60 for collection of immunological safety laboratory data, PD measurements, and a second SPT, followed by a DBPCFC (completed on Day 61). Subjects returned to the clinic on Day 90 for an end-of-study visit for collection of safety laboratory data, PD measurements, and final assessment of AEs and drug changes.

The safety and tolerability data generated were monitored in Part B. The Medical Monitor was informed of any serious adverse event (SAE) and any adverse event > Grade 2 (CTCAE v.5.0) within 24 hours of becoming aware of the event. The Medical Monitor may subsequently convene a dedicated DMC meeting to evaluate the safety and tolerability data to determine whether continued dosing is still acceptable and to make recommendations, including but not limited to continuing dosing and stopping or suspending dosing for the individual. The DMC may recommend stopping or suspending the study at any time during the study if, in its opinion, continued dosing would pose a safety risk to the individual.

In Parts A and B of the study, subjects received CNP-201 via intravenous infusion over approximately 3-4 hours at the following graduated infusion rates: 20 mL/h for the first 15 minutes, 40 mL/h for the next 15 minutes, and 80 mL/h for the remainder of the infusion.

Study duration: 2 doses, 7 days apart (Part A and Part B). Total duration of study for individual subjects is approximately 134 days; 14 days of screening, 30 days of omalizumab dosing, 60 days of IP dosing, and 30 days of follow-up.

Primary endpoints (Part A and Part B) included: frequency of adverse events (AEs) and serious adverse events (SAEs), MedDRA 23.0 (CTCAE v.5.0); laboratory safety assessments (hematology, serum chemistry, coagulation panel, urinalysis); physical examination, including vital signs (blood pressure, heart rate, temperature); 12-lead electrocardiogram (ECG); serum interleukins (TNF-α, IL-2, IL-6, IL-8, IL-1β, MCP-1, MIP-1β, MIP-1α, IFN-γ, IL-12p70); peanut-specific Th2a ⁺ after ex vivo stimulation of PBMCs at baseline (before dosing on Day 1) and between placebo and CNP-201 on Day 15 Changes in the ratio of T cells (peanut-specific Th2a ⁺ cells/total peanut-specific T cells); and changes in the ratio of activated peanut-specific T cells to total peanut-specific T cells after ex vivo stimulation of PBMCs between placebo and CNP-201 at baseline (before dosing on Day 1) and on Day 15.

Secondary endpoints (Part A and Part B) included: Changes in the ratio of IL-5 to IFN-γ after ex vivo stimulation of PBMCs between placebo and CNP-201 at baseline (before dosing on Day 1) and on Day 15.

Exploratory endpoints (Part A and Part B) included: changes in the ratio of peanut-specific regulatory T cells (peanut-specific regulatory T cells/peanut-specific CD4+ effector memory cells) after ex vivo stimulation of PBMCs between placebo and CNP-201 at baseline (before dosing on Day 1) and on Day 15; effective concentration for 50% maximal phagocytosis as measured by the phagocytosis activation test (CD203c+/CD63+/- phagocytosis activation) (EC ₅₀ 50% maximal phagocytosis activation) between placebo and CNP-201 at baseline (before DBPCFC on Day -30) and on Day 60. ); change in the ratio of peanut-specific IgE to IgG measured by ImmunoCap analysis between placebo and CNP-201 at baseline (days [-30]-[-29]) and on days 60-61; and change in the cumulative tolerated dose (CTD) of peanut protein (mg) administered during DBPCFC between placebo and CNP-201 at baseline (days [-30]-[-29]) and on days 60-61 . Cell markers used to assess exploratory indices are described in Table 3 below: Table 3. Cell markers used to assess exploratory indices Cell population Mark Effect memory cells CD45RA ^low , CD4+ Peanut-specific T cells EM [CD45RA ^low , CD4+], CD154+, CD137+ Peanut-specific Th2a+ cells EM [CD45RA ^low , CD4+], CD154+, CD137+, CXCR5-, CRTh2+/-, CD27-, CD200R+, CD161+ Activated peanut-specific T cells EM [CD45RA ^low , CD4+], CD154+, CD137+, CD38+ Peanut-specific CD4+ effector memory cells EM [CD45RA ^low , CD4+], CD137+ Peanut-specific regulatory T cells EM CD4+ [CD45RA ^low , CD4+, CD137+], CD25 ⁺ , CD127 ^low Alkaliphilic globules CD45+, CD3-, SSCA ^low , CRTh2+, HLADR-, CD123+ Activated alkaliphilic globulin CD45+, CD3-, SSCA ^low , CRTh2+, HLADR-, CD123+, CD203c+/CD63+/- Example 1A - Phase I/II Trial of TIMP-PPE in Peanut Allergy

Provides an alternative for conducting a Phase 1b/2a, randomized, double-blind, placebo-controlled, two-part study to evaluate the safety, tolerability, efficacy, and efficacy of TIMP-PPE (CNP-201) in individuals aged 16-35 years with peanut allergy.

CNP-201 consists of PLGA nanoparticles encapsulating purified peanut extract with an average diameter of 400-800 nm and a negative zeta potential between -30 mV and -60 mV. CNP-201 particles are supplied as a lyophilized formulation. Prior to administration, CNP-201 particles are reconstituted in sterile water for injection and diluted in sterile saline (0.9% sodium chloride).

The study consists of Part A and Part B. Part A: Part A is a randomized, double-blind, placebo-controlled study of the tolerability and safety of CNP-201 at increasing doses. Three groups will be selected in Part A to receive CNP-201 or placebo at multiple increasing doses. This is followed by Part B, which is a randomized, double-blind, placebo-controlled repeated-dose study using CNP-201 at the safe and tolerable dose determined in Part A.

All subjects meeting inclusion criteria (except IgG and IgE results that may not be available at Question 2) and without exclusion criteria after the initial screening assessment underwent skin prick testing (SPT) followed by a baseline double-blind, placebo-controlled food challenge (DBPCFC) (peanut and placebo (oatmeal) challenge, administered on two separate days) to confirm peanut sensitization. SPT and DBPCFC were performed by study physicians or staff trained to manage clinical emergencies present in the field, with immediate access to emergency medications and equipment, and within the confines of an adjacent hospital emergency department (for rapid provision of emergency care if needed). After completing the two-day DBPCFC, subjects who continued to meet all inclusion criteria and did not meet exclusion criteria were eligible for inclusion in the study.

After DBPCFC, all subjects who continued to meet all I/E criteria received subcutaneous injections of omalizumab (XOLAIR®). The dose of omalizumab (XOLAIR®) followed the product labeling as specified in the protocol and was determined based on the subject's body weight and serum IgE at the initial screening. Subjects were given medication every 2 weeks or every 4 weeks based on the product labeling.

Subjects who continued to meet all inclusion/exclusion criteria were randomized on Day 1 to receive CNP-201 or placebo (0.9% sodium chloride USP) by intravenous (IV) infusion in a 2:1 ratio (Part A) or a 1:1 ratio (Part B). Subjects were administered CNP-201 or placebo on Days 1 and 8. Subjects remained in the clinic on Days 1 and 8 from admission (before administration of CNP-201 or placebo) to the last procedure performed 4 hours after dosing on the same day, unless infusion reactions, systemic allergic reactions, or other adverse events required extended monitoring. Subjects were discharged if safety parameters were acceptable to the investigator.

Peanut allergic individuals eligible for inclusion in this study were defined based on the following inclusion criteria: 1. Males and non-pregnant females aged 16 to 35 years (inclusive). 2. Individuals with a body mass index (BMI) ≥18 and ≤32 and a weight >30 kg and ≤150 kg at screening. Individuals outside this range may be included at the discretion of the investigator. 3. Individuals with serum IgE ≥30 IU/mL and ≤1500 IU/mL at screening. Individuals outside this range may be included at the discretion of the investigator. 4. Individuals diagnosed by a physician as having peanut allergy or with a history of peanut allergy. 5. Subjects with a documented history of non-severe systemic allergic reaction (grade ≤3) to peanut (including mild wheezing or dyspnea but no hypoxia). 6. Subjects with peanut-specific IgE >2 kU/L as measured by ImmunoCAP at screening, and/or a change in blisters diameter >3 mm on a positive skin prick test (SPT) to peanut compared to a negative control (50% glycerol) at screening. 7. Subjects who self-reported no peanut dietary intake for at least 14 days prior to screening and no suspected peanut exposure (including any peanut food challenge), and who agree to continue limiting peanut exposure during the study period, except for the study DBPCFC. 8. Female subjects and male subjects and their female spouses/partners who are willing to implement highly effective contraceptive methods starting at screening and continuing throughout the study until Day 90, which may include but are not limited to abstinence, having sex only with people of the same sex, monogamous relationships with vasectomized partners, vasectomy, hysterectomy, bilateral tubal ligation, approved hormonal methods, intrauterine devices (IUDs), or the use of spermicides and barrier methods (e.g., condoms, diaphragms) in combination (EOS/ET). 9. Female subjects who agree not to breastfeed starting at the initial screening and throughout the study until Day 90 (EOS/ET). 10. Female individuals who agree not to donate eggs from the time of initial screening and throughout the study until day 90 (EOS/ET). 11. Individuals who are willing and able to provide written informed consent approved by the Institutional Review Board (IRB). 12. Individuals who are willing to perform and comply with all study procedures, including attending study visits and completing two DBPCFCs as planned. 13. Male individuals who agree not to donate sperm from the time of screening and throughout the study until day 90 (EOS/ET).

Subjects must have a positive peanut DBPCFC at screening and a priming dose of peanut protein ≥10 mg and ≤300 mg to be included in the statistical analysis of exploratory outcomes. Subjects who tolerate >444 mg peanut (cumulative tolerated dose) will be followed up separately for safety and assessed.

Subjects who met all inclusion criteria and did not meet exclusion criteria after completing two screening visits, including two baseline DBPCFCs (peanut and placebo challenges, administered on separate days), were enrolled in one of three dose-escalation groups. Subjects were randomized in a 2:1 ratio to receive CNP-201 or placebo (0.9% sodium chloride injection) as a 200 mL intravenous infusion on Days 1 and 8. The doses for the 3 groups were as follows: Group 1: 250 mg, Group 2: 450 mg, Group 3: 650 mg. Dosing was separated for subjects within a dose group by at least 48 hours.

After the initial screening assessment, individuals who meet all inclusion criteria and do not meet exclusion criteria will undergo a skin prick test (SPT) followed by a baseline double-blind, placebo-controlled food challenge (DBPCFC) (peanut and placebo (oatmeal) challenge, administered on two separate days) to confirm peanut allergy. If a reaction occurs and is treated, the peanut and placebo challenges will be performed at least 48 hours apart.

After the second challenge of DBPCFC and the 2-hour observation period, subjects who continued to meet all inclusion criteria and did not meet exclusion criteria would proceed with the first subcutaneous injection of XOLAIR®.

The XOLAIR® dose and dosing frequency (every 2 weeks or every 4 weeks) were determined based on the individual's serum IgE at screening as described in Table 2 and body weight measured at XOLAIR® dose 1.

Subjects returned to the clinic on Day 1 for final eligibility assessment and collection of laboratory samples. Subjects who continued to meet all inclusion and no exclusion criteria were randomized to dose groups at that time. Subjects were administered CNP-201 or placebo on Day 1 and on Day 8. CNP-201 or placebo was administered by intravenous infusion over approximately 3-4 hours using a progressive infusion rate. Subjects underwent 4 hours of medical observation in the clinic after infusion for acute adverse events (AEs), including infusion reactions (IRs). If an allergic reaction occurs, antihistamine/epinephrine will be available for immediate treatment.

If two identical Grade 3 adverse events (AEs) or one Grade ≥ 4 (CTCAE v.5.0 or CoFAR v.1 for allergy-related AEs) considered possibly related to CNP-201 occur, dosing will be withheld and the DMC will be convened to review all available safety data obtained to date. Grade 4 or 5 AEs considered serious AEs (SAEs) will be reported to the institution. After reviewing all available safety data, the DMC will make recommendations, which may include but are not limited to discontinuing dosing, reducing dosing, continuing dosing, expanding the current arm, adding an arm to Part A, or identifying dose-limiting toxicities. The DMC may also be convened specifically to address safety concerns that arise during dosing of any individual in Part A based on ongoing routine monitoring of safety data by the trial sponsor’s medical monitors. The DMC will evaluate available safety data, including but not limited to AEs, physical examinations, vital signs, 12-lead ECGs, and available laboratory results.

Subjects returned to the clinic 2 days after each infusion (Day 3 and Day 10) for safety laboratory data collection, drug review, and AE assessment, and followed daily telephone interviews after each infusion (Days 4-7 and Days 11-14) to assess and record any AEs and drug changes. During the post-dose period, subjects returned to the clinic 7 days after administration of Dose 2 of CNP-201 or placebo for safety laboratory data collection, PD measurements, and AE and drug change assessments.

After all subjects in a dose cohort have completed the Day 15 clinic visit (7 days after the second dose), if cohort expansion is necessary (a minimum of three additional subjects randomized 2:1 to receive CNP-201 or placebo), or if any other clinical recommendation should be made (e.g., expansion to Part B), the DMC is convened to review all available safety data and determine whether it is acceptable to proceed to the next escalating dose cohort.

Subjects returned to the clinic on Day 60 for collection of immunological safety laboratory data, PD measurements, and a second SPT, followed by a post-dose DBPCFC, and returned to the clinic on Day 90 for an end-of-study visit for collection of safety laboratory data, PD measurements, and final assessment of AEs and drug product changes. Emerging safety and tolerability data were monitored continuously. The Medical Monitor was informed of any serious adverse event (SAE) and any >Grade 2 adverse event (CTCAE v.5.0 or CoFAR v.1) that may be related to the test product within 24 hours of becoming aware of the event. The Medical Monitor may subsequently convene a dedicated DMC meeting to evaluate the safety and tolerability data to determine if continued dosing is still acceptable and to make recommendations, including but not limited to continuing dosing and stopping or suspending dosing for the individual. The DMC may recommend stopping or suspending the study at any time during the study if, in its opinion, continued dosing would pose an unacceptable safety risk to the individual. Recommendations regarding continuation of Part B with the dose of CNP-201 identified as safe and tolerable in Part A will be made after all subjects complete the Day 15 visit.

Part B: Subjects in Part B were randomized in a 1:1 ratio to receive either CNP-201 or placebo at a dose identified as safe and tolerable in Part A. Subjects in Parts A and B were assessed identically, with the only difference between the two parts being dose escalation in Part A.

After an initial screening assessment, individuals who met all inclusion criteria and did not meet exclusion criteria underwent a skin prick test (SPT) followed by a baseline double-blind, placebo-controlled food challenge (DBPCFC) (peanut and placebo (oatmeal) challenge, administered on separate days) to confirm peanut allergy. If a reaction occurred and was treated, the peanut and placebo challenges were performed at least 48 hours apart.

After the second challenge of DBPCFC and the 2-hour observation period were completed, subjects who continued to meet all inclusion criteria and did not meet exclusion criteria continued with the first subcutaneous injection of XOLAIR®. The dose and frequency of XOLAIR® administration (every 2 weeks or every 4 weeks) were determined based on the subject's serum IgE at screening and body weight measured at XOLAIR® dose 1 as described in Table 2.

Subjects returned to the clinic on Day 1 for final eligibility assessment and collection of laboratory samples. Subjects who continued to meet all inclusion criteria and no exclusion criteria were randomized to receive CNP-201 or placebo.

Subjects were administered CNP-201 or placebo on Day 1 and on Day 8. CNP-201 or placebo was administered by intravenous infusion over approximately 3-4 hours using a gradual infusion rate. Subjects underwent 4-hour medical observation in the clinic following infusion for acute adverse events (AEs), including infusion reactions (IRs). If allergic reactions occurred, antihistamine/epinephrine was available for immediate treatment.

Subjects returned to the clinic 2 days after each infusion (Day 3 and Day 10) for safety laboratory data collection, drug review, and AE assessment, and followed daily telephone interviews after each infusion (Days 4-7 and Days 11-14) to assess and record any AEs and drug changes. During the post-dose period, subjects returned to the clinic 7 days after administration of Dose 2 of CNP-201 or placebo for safety laboratory data collection, PD measurements, and AE and drug change assessments.

Subjects returned to the clinic on Day 60 for collection of immunological safety laboratory data, PD measurements, and a second SPT, followed by a post-dose DBPCFC, and returned to the clinic on Day 90 for an end-of-study visit for collection of safety laboratory data, PD measurements, and final assessment of AEs and drug product changes. Emerging safety and tolerability data were monitored continuously. The Medical Monitor was informed of any serious adverse event (SAE) and any >Grade 2 adverse event (CTCAE v.5.0 or CoFAR v.1) that may be related to the test product within 24 hours of becoming aware of the event. The Medical Monitor may then convene a dedicated DMC meeting to evaluate the safety and tolerability data to determine whether continued dosing is still acceptable and make recommendations, including but not limited to continuing dosing and stopping or suspending dosing for the individual. If, in the opinion of the DMC, continued dosing would pose an unacceptable safety risk to the individual, it may recommend stopping or suspending the study at any time during the study.

In Parts A and B of the study, subjects received CNP-201 via intravenous infusion over approximately 3-4 hours at the following graduated infusion rates: 20 mL/h for the first 15 minutes, 40 mL/h for the next 15 minutes, and 80 mL/h for the remainder of the infusion.

In Parts A and B, subjects randomized to the placebo group received 0.9% sodium chloride for injection (normal saline [NS]). On Days 1 and 8, placebo was administered as a 200 mL intravenous infusion at the following progressive infusion rate: 20 mL/h for the first 15 minutes, 40 mL/h for the next 15 minutes, and 80 mL/h for the remainder of the infusion.

Study Duration: 2 doses, 7 days apart (Part A and Part B). Total duration of study for individual subjects was approximately 134 days; 14 days of screening, approximately 30 days of XOLAIR® dosing, 60 days of test product dosing, and approximately 30 days of post-dosing follow-up.

The main endpoints (Part A and Part B) included: frequency of adverse events (AEs) and serious adverse events (SAEs), MedDRA 23.0 (CTCAE v.5.0 or CoFAR v.1 for allergy-related AEs); laboratory safety assessment (blood, serum chemistry, coagulation panel, and urinalysis); physical examination, including vital signs (blood pressure, heart rate, and temperature); 12-lead electrocardiogram (ECG); serum interleukins (TNF-α, IL-2, IL-6, IL-8, IL-1β, MCP-1, MIP-1β, MIP-1α, IFN-γ, and IL-12p70).

Exploratory endpoints (Part A and Part B) included: the proportion of peanut-specific Th2a+ T cells after ex vivo stimulation of PBMCs between placebo and CNP-201 at baseline (before dosing on Day 1) and on Day 15 (peanut-specific Th2a+ =Changes in 50% maximal basophil activation (EC50) measured by the basophil activation test (CD203c+/CD63+/- basophil activation) between placebo and CNP-201 at baseline (screening, visit 2, before DBPCFC) and at visit 11; Changes in peanut-specific IgE measured by ImmunoCap assay between placebo and CNP-201 at baseline (screening, visit 2, before DBPCFC) and at visit 11; Changes in peanut-specific IgE measured by ImmunoCap assay between placebo and CNP-201 at baseline (screening, visit 2, before DBPCFC) and at visit 11; Changes in peanut-specific IgE measured by ImmunoCap assay between placebo and CNP-201 at baseline (screening, visit 2, before DBPCFC) and at visit 11 Change in the ratio of peanut-specific IgE to IgG measured by ImmunoCap assay between placebo and CNP-201 at baseline (screening, DBPCFC, visits 2 and 3) and after dosing (post-dose DBPCFC, visits 11 and 12) in the cumulative tolerated dose (CTD) of peanut protein (mg) administered during DBPCFC between placebo and CNP-201; Change in the ratio of activated peanut-specific T cells (activated peanut-specific T cells/total peanut-specific T cells) after ex vivo stimulation of PBMCs between placebo and CNP-201 at baseline (pre-dose on day 1) and on day 15. Example 2. Phase I/II trial of TIMP-PPE in peanut allergy without IgE inhibitors

This example describes a Phase 1b/2a randomized, double-blind, placebo-controlled study to evaluate the safety, tolerability, and efficacy of TIMP-PPE (CNP-201) in individuals aged 16-55 years with peanut allergy without administration of an IgE inhibitor.

CNP-201 consists of PLGA nanoparticles encapsulating purified peanut extract with an average diameter of 400-800 nm and a negative zeta potential between -30 mV and -60 mV. CNP-201 particles are supplied as a lyophilized formulation. Prior to administration, CNP-201 particles are reconstituted in sterile water for injection and diluted in sterile saline (0.9% sodium chloride).

This study is a randomized, double-blind, placebo-controlled study of the safety and tolerability of increasing doses of CNP-201. Three groups will be selected to receive CNP-201 or placebo in multiple increasing doses.

Subjects who met all inclusion criteria and did not meet exclusion criteria were enrolled in the study. Subjects who continued to meet the inclusion/exclusion criteria were randomized in a 2:1 ratio on Day 1 to receive CNP-201 or placebo (0.9% sodium chloride USP) by intravenous (IV) infusion. Subjects were given CNP-201 or placebo on Days 1 and 8.

Subjects may receive pre-dose antihistamines (e.g., 10 mg IV cetirizine) and corticosteroids (e.g., 125 mg IV methylprednisolone) 30 minutes prior to each infusion of CNP-201 or placebo. Subjects will maintain their current SoC during the study, excluding a 12-hour washout period (for beta-agonists, theophylline, and cromolyn) and a 7-day washout period for antihistamines prior to skin prick testing (SPT). Subjects will restart their SoC regimen after the SPT and remain on SoC during the dosing/duration of the study.

Peanut allergic subjects eligible for inclusion in this study were defined based on the following inclusion criteria: 1. Males and non-pregnant females aged 16 to 55 years (inclusive). 2. Subjects diagnosed by a physician as having peanut allergy or with a documented history of peanut allergy. 3. Subjects weighing ≥31.25 kg at screening, subjects outside this range may be included at the discretion of the investigator. 4. Subjects with a documented history of non-severe systemic allergic reaction to peanuts (grade ≤3) (including mild wheezing or dyspnea but not hypoxia). 5. Individuals with peanut-specific IgE >5 kU/L as measured by ImmunoCAP at screening. 6. Individuals who reported no peanut dietary intake and no suspected peanut exposure (including any peanut food challenge) for at least 14 days before screening and agreed to continue limiting peanut exposure during the study. 7. Individuals with a change in water prick diameter >3 mm in a positive skin prick test (SPT) for peanut compared to a negative control (50% glycerol) at screening. 8. Female subjects and male subjects and their female spouses/partners who are willing to implement highly effective contraceptive methods starting at screening and continuing throughout the study until Day 38, which may include but are not limited to abstinence, having sex only with people of the same sex, monogamous relationships with vasectomized partners, vasectomy, hysterectomy, bilateral tubal ligation, approved hormonal methods, intrauterine devices (IUDs), or the use of spermicides and barrier methods (such as condoms, diaphragms) in combination (EOS/ET). 9. Female subjects who agree not to breastfeed starting at the initial screening and throughout the study until Day 38 (EOS/ET). 10. Female individuals who agree not to donate eggs from the time of initial screening and throughout the study until day 38 (EOS/ET). 11. Individuals who are willing and able to provide written informed consent approved by the Institutional Review Board (IRB). 12. Individuals who are willing to perform and comply with all study procedures. 13. Male individuals who agree not to donate sperm from the time of screening and throughout the study until day 38 (EOS/ET).

Subjects who met all inclusion criteria and did not meet exclusion criteria after completing the screening interview were enrolled in one of three dose escalation groups. Subjects were randomized in a 2:1 ratio to receive CNP-201 or placebo (0.9% sodium chloride injection) as a 200 mL intravenous infusion on Days 1 and 8. The doses in the three groups were as follows: Group 1: 250 mg, Group 2: 450 mg, and Group 3: 650 mg. Dosing was separated for subjects within the dose groups by at least 48 hours.

Subjects who meet all inclusion criteria and do not meet exclusion criteria at screening are enrolled in the study. Subjects will return to the clinic on Day 1 for final eligibility assessment and collection of laboratory samples. Subjects who continue to meet all inclusion and no exclusion criteria are randomized to dose groups at that time. Subjects may receive a pre-dose antihistamine (e.g., 10 mg IV cetirizine) and a corticosteroid (e.g., 125 mg IV methylprednisolone) 30 minutes prior to each infusion of CNP-201 or placebo on Days 1 and 8. CNP-201 or placebo is administered by intravenous infusion using a gradual infusion rate over approximately 3-4 hours. Subjects experienced medical observation for 4 hours following infusion in the clinic for acute adverse events (AEs), including infusion reactions (IRs). If allergic reactions occurred, antihistamines/epinephrines, including intramuscular (IM) and intravenous (IV) epinephrine, were used for immediate treatment.

Subjects followed daily telephone interviews after each infusion (Days 2-7 and Days 9-14) to assess and record any AEs and drug changes. During the post-dose period, subjects returned to the clinic 7 days after administration of Dose 2 of CNP-201 or placebo (Day 15) for collection of safety laboratory data, PD measurements, and assessment of AEs and drug changes.

After all subjects in a dose cohort have completed the Day 15 clinic visit (7 days after the second dose), the DMC is convened to review all available safety data and determine whether it is acceptable to proceed to the next escalating dose cohort.

Subjects returned to the clinic on Day 38 for an end-of-study visit for the collection of safety laboratory data, PD measurements, and final assessment of AEs and drug changes, as well as a second SPT.

Monitor emerging safety and tolerability data on an ongoing basis. Inform the Medical Monitor of any serious adverse event (SAE) and any Grade ≥ 2 adverse event (CTCAE v.5.0 or CoFAR v.3.0) that may be related to the test product within 24 hours of becoming aware of the event. The Medical Monitor may convene a dedicated DMC meeting to evaluate the safety and tolerability data to determine whether continued dosing is still acceptable and make recommendations, including but not limited to continuing dosing and stopping or suspending dosing for the individual. The DMC may recommend stopping or suspending the study at any time during the study if, in its opinion, continued dosing would pose an unacceptable safety risk to the individual.

Subjects received CNP-201 via intravenous infusion over approximately 3-4 hours at the following progressive infusion rates: 20 mL/h for the first 15 minutes, 40 mL/h for the next 15 minutes, and 80 mL/h for the remainder of the infusion.

Study Duration: 2 doses, 7 days apart. Total duration of study for individual subjects is approximately 45 days; screening 7 days; test product dosing 8 days; and post-dosing evaluation 30 days.

The main indicators included: the frequency of adverse events (AEs) and serious adverse events (SAEs), MedDRA 23.0 (CTCAE v.5.0 or CoFAR v.3.0 for allergy-related AEs); laboratory safety assessment (blood, serum chemistry, coagulation panel, and urinalysis); physical examination, including vital signs (blood pressure, heart rate, and temperature); 12-lead electrocardiogram (ECG); serum interleukins (TNF-α, IL-2, IL-6, IL-8, IL-1β, MCP-1, MIP-1β, MIP-1α, IFN-γ, and IL-12p70).

Exploratory endpoints included: change in the ratio of peanut-specific IgE to IgG measured by the ImmunoCap assay between placebo and CNP-201 at baseline and at day 38; change in peanut-specific IgE measured by the ImmunoCap assay between placebo and CNP-201 at baseline and at day 38.

Preferred embodiments of the invention are described herein. Variations of the preferred embodiments may become apparent to those of ordinary skill in the art after reading the foregoing description. The inventors expect those of ordinary skill in the art to employ such variations as appropriate, and the inventors intend to practice the invention in other ways than those specifically described herein. Therefore, the invention includes all modifications and equivalents of the subject matter set forth in the claims appended hereto as permitted by applicable law. In addition, unless otherwise indicated herein or otherwise clearly contradicted by the context, the invention encompasses any combination of the above elements in all possible variations thereof. Example 3-Phase I/II Trial of Low-Dose TIMP-PPE in Peanut Allergy

This example describes a Phase 1b/2a randomized, double-blind, placebo-controlled study to evaluate the safety, tolerability, and efficacy of TIMP-PPE (CNP-201) in individuals aged 16-55 years with peanut allergy.

CNP-201 consists of PLGA nanoparticles encapsulating purified peanut extract with an average diameter of 400-800 nm and a negative zeta potential between -30 mV and -60 mV. CNP-201 particles are supplied as a lyophilized formulation. Prior to administration, CNP-201 particles are reconstituted in sterile water for injection and diluted in sterile saline (0.9% sodium chloride).

This study is a randomized, double-blind, placebo-controlled study of the safety and tolerability of increasing doses of CNP-201. Four groups will be selected to receive CNP-201 or placebo in multiple increasing doses.

Subjects who met all inclusion criteria and did not meet exclusion criteria were enrolled in the study. Subjects who continued to meet the inclusion/exclusion criteria were randomized in a 2:1 ratio on Day 1 to receive CNP-201 or placebo (0.9% sodium chloride USP) by intravenous (IV) infusion. Subjects were given CNP-201 or placebo on Days 1 and 8.

Subjects may receive a pre-administration NSAID (e.g., 325 mg oral acetylsalicylic acid) daily 2 days prior and 60 minutes prior to each infusion of CNP-201 or placebo. Subjects may receive a pre-administration and/or post-administration leukotriene modifier (e.g., 10 mg oral montelukast) 12 hours prior, 45 minutes prior, and 12 hours after each infusion of CNP-201 or placebo. Subjects may receive a pre-administration antihistamine (e.g., 50 mg IV diphenhydramine) and a corticosteroid (e.g., 125 mg IV methylprednisolone) 30 minutes prior to each infusion of CNP-201 or placebo.

Subjects will maintain their current SoC during the study, excluding the 12-hour washout period (for beta-agonists, theophylline, and cromolyn) and the 7-day washout period for antihistamines prior to skin prick testing (SPT). Subjects will restart their SoC regimen after SPT and remain on SoC during the dosing/duration of the study.

Peanut allergic subjects eligible for inclusion in this study were defined based on the following inclusion criteria: 1. Males and non-pregnant females aged 16 to 55 years (inclusive). 2. Subjects diagnosed by a physician as having peanut allergy or with a documented history of peanut allergy. 3. Subjects weighing ≥31.25 kg at screening, subjects outside this range may be included at the discretion of the investigator. 4. Subjects with a documented history of non-severe systemic allergic reaction to peanut (grade â§3) (including mild wheezing or dyspnea but not hypoxia). 5. Unless OIT was previously performed for their peanut allergy, subjects with peanut-specific IgE ≥5 kU/L as measured by ImmunoCAP at screening, subjects who had previously undergone OIT for peanut allergy and peanut-specific IgE ≥5 kU/L as measured by ImmunoCap at screening may be included at the investigator’s discretion; or subjects with a positive SPT for peanut at screening with a change in blisters ≥3 mm in diameter compared to a negative control (50% glycerol), subjects who had previously undergone OIT for peanut allergy and did not have a positive skin prick test (SPT) for peanut at screening with a change in blisters ≥3 mm in diameter may be included at the investigator’s discretion. 6. Individuals who self-reported no peanut diet for at least 14 days prior to screening and no suspected peanut exposure (including any peanut food challenge), and agreed to continue limiting peanut exposure during the study. 7. Individuals with a change in blisters diameter of >3 mm in a positive skin prick test (SPT) for peanut compared to a negative control (50% glycerol) at screening. 8. Female subjects and male subjects and their female spouses/partners who are willing to implement highly effective contraceptive methods starting at screening and continuing throughout the study until Day 38, which may include but are not limited to abstinence, having sex only with people of the same sex, monogamous relationships with vasectomized partners, vasectomy, hysterectomy, bilateral tubal ligation, approved hormonal methods, intrauterine devices (IUDs), or the use of spermicides and barrier methods (such as condoms, diaphragms) in combination (EOS/ET). 9. Female subjects who agree not to breastfeed starting at the initial screening and throughout the study until Day 38 (EOS/ET). 10. Female individuals who agree not to donate eggs from the time of initial screening and throughout the study until day 38 (EOS/ET). 11. Individuals who are willing and able to provide written informed consent approved by the Institutional Review Board (IRB). 12. Individuals who are willing to perform and comply with all study procedures. 13. Male individuals who agree not to donate sperm from the time of screening and throughout the study until day 38 (EOS/ET).

Subjects who met all inclusion criteria and did not meet exclusion criteria after completing the screening interview were enrolled in one of several dosing groups. Subjects were randomized in a 2:1 ratio to receive CNP-201 or placebo (0.9% sodium chloride injection) as a 200 mL intravenous infusion on Days 1 and 8. The dose in the first group was 25 mg. Additional doses in the remaining 3 groups were reduced to 0.1 mg, 0.25 mg, 0.5 mg, 1 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, or could be increased to 650 mg. Dosing of subjects within a dose group was separated by at least 48 hours.

Subjects who meet all inclusion criteria and do not meet exclusion criteria at screening are enrolled in the study. Subjects will return to the clinic on Day 1 for final eligibility assessment and collection of laboratory samples. Subjects who continue to meet all inclusion criteria and do not meet exclusion criteria are randomized to dose groups at that time. Subjects may receive a pre-dose NSAID (e.g., 325 mg oral acetylsalicylate) daily 2 days prior to and 60 minutes prior to each infusion of CNP-201 or placebo on Days 1 and 8. Subjects may receive a pre-dose and/or post-dose leukotriene modifier (e.g., 10 mg oral montelukast) 12 hours prior to, 45 minutes prior to, and 12 hours after each infusion of CNP-201 and placebo on Days 1 and 8. Subjects may receive pre-administration antihistamines (such as 50 mg IV diphenhydramine) and corticosteroids (such as 125 mg IV methylprednisolone) 30 minutes prior to each infusion of CNP-201 or placebo on Days 1 and 8.

CNP-201 or placebo was administered by intravenous infusion using a gradual infusion rate over approximately 3-4 hours. Subjects underwent medical observation for 4 hours following infusion for acute adverse events (AEs), including infusion reactions (IRs), in the clinic. If allergic reactions occurred, antihistamines/epinephrines, including intramuscular (IM) and intravenous (IV) epinephrine, were used immediately for treatment.

Subjects followed daily telephone interviews after each infusion (Days 2-7 and Days 9-14) to assess and record any AEs and drug changes. During the post-dose period, subjects returned to the clinic 7 days after administration of Dose 2 of CNP-201 or placebo (Day 15) for collection of safety laboratory data, PD measurements, and assessment of AEs and drug changes.

After all subjects in a dose cohort have completed the Day 15 clinic visit (7 days after the second dose), the DMC is convened to review all available safety data and determine whether it is acceptable to proceed to the next escalating dose cohort.

Subjects will return to the clinic on Day 38 for an end-of-study visit for safety laboratory data collection and PD measurements, assessment of AEs and drug changes, and a second SPT. On Day 60, subjects will return to the clinic for safety laboratory data, PD measurements, and DBPCFC. On Day 90, 120 subjects will return to the clinic for safety laboratory data collection and PD measurements, as appropriate. Subjects will return on Day 180 for a second DBPCFC consisting of a peanut and placebo (oatmeal) challenge, safety laboratory data, PD measurements, and final assessment of AEs and drug changes, as appropriate.

Monitor emerging safety and tolerability data on an ongoing basis. Inform the Medical Monitor of any serious adverse event (SAE) and any Grade ≥ 2 adverse event (CTCAE v.5.0 or CoFAR v.3.0) that may be related to the test product within 24 hours of becoming aware of the event. The Medical Monitor may convene a dedicated DMC meeting to evaluate the safety and tolerability data to determine whether continued dosing is still acceptable and make recommendations, including but not limited to continuing dosing and stopping or suspending dosing for the individual. The DMC may recommend stopping or suspending the study at any time during the study if, in its opinion, continued dosing would pose an unacceptable safety risk to the individual.

Subjects received CNP-201 via intravenous infusion over approximately 3-4 hours at the following progressive infusion rate: 1 mL/h for the first 10 minutes, 2 mL/h for the next 10 minutes, 5 mL/h for the next 10 minutes, 10 mL/h for the next 10 minutes, 20 mL/h for the next 15 minutes, 40 mL/h for the next 15 minutes, and 80 mL/h for the remainder of the infusion.

Study Duration: 2 doses, 7 days apart. Total duration of study for individual subjects is approximately 67 days; screening 7 days; test product dosing 8 days; and post-dose evaluation 52 days. Additional 120 days of post-dose evaluation as appropriate.

The primary objectives include: safety and tolerability of CNP-201.

Exploratory objectives include: changes in responses to DBPCFC in patients treated with CNP-201 or placebo, changes in the ratio of peanut-specific IgE to IgG in patients treated with CNP-201 or placebo, changes in peanut-specific IgE in patients treated with CNP-201 or placebo, changes in the proportion of peanut-specific Th2a+ T cells in patients treated with CNP-201 or placebo, changes in responses to the basophil activation test in patients treated with CNP-201 or placebo, changes in the proportion of peanut-specific regulatory T cells in patients treated with CNP-201 or placebo.

The main indicators included: the frequency of adverse events (AEs) and serious adverse events (SAEs), MedDRA 23.0 (CTCAE v.5.0 or CoFAR v.3.0 for allergy-related AEs); laboratory safety assessment (blood, serum chemistry, coagulation panel, and urinalysis); physical examination, including vital signs (blood pressure, heart rate, and temperature); 12-lead electrocardiogram (ECG); serum interleukins (TNF-α, IL-2, IL-6, IL-8, IL-1β, MCP-1, MIP-1β, MIP-1α, IFN-γ, and IL-12p70).

Exploratory endpoints included: DBPCFC between placebo and CNP-201 at Day 60 and Day 180 (with a cumulative 4043 The percentage difference of individuals in the DBPCFC group was 1.37 ± 1.13, 1.34 ± 1.12, 1.37 ± 1.16, 1.38 ± 1.16, 1.39 ± 1.17, 1.37 ± 1.16, 1.38 ± 1.16, 1.39 ± 1.17, 1.37 ± 1.16, 1.39 ± 1.16, 1.39 ± 1.16, 1.39 ± 1.16, 1.39 ± 1.16, 1.39 ± 1.16, 1.39 ± 1.16, 1.39 ± 1.16, 1.39 ± 1.16, 1.39 ± 1.16, 1.39 ± 1.16, 1.39 ± 1.16, 1.39 ± 1.16, 1.39 ± 1.16, 1.39 ± 1.16, 1.39 ± 1.16, 1.39 ± 1.16, Changes in the ratio of T cells (peanut-specific Th2a+ cells/total peanut-specific T cells), changes in the effective concentration of 50% maximal phagocytic activation (EC50) measured by the phagocytic activation test (CD203c+/CD63+/- phagocytic activation) between placebo and CNP-201 at baseline and on days 60 and 180, changes in the ratio of peanut-specific regulatory T cells after ex vivo stimulation of PBMCs (peanut-specific regulatory T cells/peanut-specific CD4+ effector memory cells) between placebo and CNP-201 at baseline and on day 15. Example 4 - Interim results from a phase I / II trial of low-dose TIMP - PPE in peanut allergy

This example describes the results of a Phase 1b/2a randomized, double-blind, placebo-controlled study showing that administration of low doses of CNP-201 is effective in treating peanut allergy.

Subjects received a single intravenous dose of either 1 mg or 25 mg CNP-201 on day 1. Figure 2 summarizes the results of peanut-allergic subjects who received low doses of CNP-201 particles in peanut allergy. Three-quarters of patients who received low doses of CNP-201 had increased BAT thresholds, with 2 patients having persistence extended to day 60. Peanut-allergic patients who received up to a 25 mg dose of CNP-201 exhibited a reduction in the ratio of peanut-specific IgE to IgG on days 15 and 38. Peanut-allergic patients who received a single 25 mg dose of CNP-201 exhibited induction of antigen-specific Tregs and a reduction in pathogenic peanut-specific T cell subsets (Th2a, TFH, B cytoplasmic blasts).

For the BAT test, blood was drawn before and after low-dose CNP-201 treatment. PBMCs isolated from the blood were stimulated with increasing concentrations of peanut allergen. Degranulation of basophils was measured by flow cytometry by CD203 and CD63 positive markers expressed on basophils. The activation threshold, i.e. the cutoff for a positive reaction, was 50% CD203 ⁺ CD63 ⁺ expression. In Figure 3, basophil activation was detected before treatment with 25 mg CNP-201. Peanut-allergic individuals receiving a single 25 mg intravenous dose of CNP-201 demonstrated an increase in the EC50 (peanut allergen concentration) required to increase the expression of activation markers on the surface of phagophores following allergen stimulation. Following treatment with CNP-201, phagophore degranulation was below the activation threshold at days 15, 38, and 60. Increases in BAT indicate anergy to peanut allergen.

Peanut-specific IgE and IgG levels were measured from the patients' sera. Peanut-allergic patients who received up to 25 mg intravenous doses of CNP-201 showed an increase in peanut-specific IgG, while peanut-specific IgE levels did not change (Figure 4). A decrease in the peanut-specific IgE/IgG ratio was detected on days 15 and 38 after dosing compared to placebo (Figure 4).

PBMCs obtained from the blood of peanut allergic patients who received a single dose of 25 mg CNP-201 were stimulated with whole peanut extract and CD40 ligand. Subpopulations of peanut-specific activated T cells were further characterized by the expression of lineage-specific markers. Figure 5 shows that peanut allergy-associated immune cell subsets are reduced up to 100 days after administration of a single 25 mg dose of CNP-201. Peanut allergic patients who received a single 25 mg intravenous dose of CNP-201 exhibited a reduction in the allergenic subsets helper T cells 2 (Th2A), filtration helper T cells (TFH), terminally differentiated effector memory cells (TEMRA), and B-cell plasma blasts. Figure 6 shows that there were fewer pathogenic activated peanut-specific CD4 ⁺ T cells after administration of 25 mg CNP-201. In patients treated with low doses of CNP-201, peanut-specific activated T cell subsets CD4 ⁺ CD25 ⁺ , CD4 ⁺ CD69 ⁺ , and CD4 ⁺ PD-1 ⁺ were reduced compared to placebo. Peanut-allergic individuals who received a single 25 mg intravenous dose of CNP-201 demonstrated an increase in peanut-specific Tregs (CD4 ⁺ CD137 ⁺ CD25 ^{+ CD127LO} ) compared to placebo (Figure 7). Example 5 : Phase I / II Trial of 1 mg Dose of TIMP-PPE in Peanut Allergy

This example describes a Phase 1b/2a randomized, double-blind, placebo-controlled study to evaluate the safety, tolerability, and efficacy of TIMP-PPE (CNP-201) in individuals aged 16-55 years with peanut allergy.

CNP-201 consists of PLGA nanoparticles encapsulating purified peanut extract with an average diameter of 400-800 nm and a negative zeta potential between -30 mV and -80 mV. CNP-201 particles are supplied as a lyophilized formulation. Prior to administration, CNP-201 particles are reconstituted in sterile water for injection and diluted in sterile saline (0.9% sodium chloride).

The study is a randomized, double-blind, placebo-controlled study of the safety, efficacy and tolerability of multiple ascending doses of CNP-201 (escalation phase). Its purpose is to identify a safe and tolerable dose for further evaluation in a larger number of individuals (expansion phase). The study will enroll 4 groups to receive CNP-201 or placebo in multiple ascending doses.

Subjects will maintain their current SoC during the study, excluding the 12-hour washout period (for beta-agonists, theophylline, and cromolyn) and the 7-day washout period for antihistamines prior to skin prick testing (SPT). Subjects will restart their SoC regimen after SPT and remain on SoC during the dosing/duration of the study.

Peanut allergic subjects eligible for inclusion in this study were defined based on the following inclusion criteria: 1. Males and non-pregnant females aged 16 to 55 years (inclusive). 2. Subjects diagnosed by a physician as having peanut allergy or with a documented history of peanut allergy. 3. Subjects weighing ≥31.25 kg at screening, subjects outside this range may be included at the discretion of the investigator. 4. Subjects with a documented history of non-severe systemic allergic reaction to peanuts (grade ≤3) (including mild wheezing or dyspnea but not hypoxia). 5. Subjects with peanut-specific IgE ≥5 kU/L as measured by ImmunoCAP at screening, unless they had previously undergone OIT for peanut allergy, and subjects who had previously undergone OIT for peanut allergy and had peanut-specific IgE ≥5 kU/L as measured by ImmunoCap at screening may be included at the investigator’s discretion. 6. Subjects with a change in blister diameter of ≥5 mm on a positive SPT for peanut compared to a negative control (50% glycerol) at screening, and subjects who had previously undergone OIT for peanut allergy and did not have a change in blister diameter of ≥5 mm on a positive skin prick test (SPT) for peanut at screening may be included at the investigator’s discretion. 7. Individuals who self-reported no peanut diet and no suspected peanut exposure (including any peanut food challenge) for at least 14 days before screening, and agreed to continue to limit peanut exposure during the study. 8. Female individuals and male individuals and their female spouses/partners who are willing to implement highly effective contraceptive methods starting at screening and continuing throughout the study until Day 38, which may include but are not limited to abstinence, sexual intercourse only with people of the same sex, monogamous relationships with vasectomized partners, vasectomy, hysterectomy, bilateral tubal ligation, approved hormonal methods, intrauterine devices (IUDs), or the use of spermicides and barrier methods (e.g., condoms, diaphragms) in combination (EOS/ET). 9. Female subjects who agree not to breastfeed starting from the initial screening and continuing until day 60. 10. Female subjects who agree not to donate eggs starting from the initial screening and continuing until day 60. 11. Subjects who are willing and able to provide written informed consent approved by the Institutional Review Board (IRB). 12. Subjects who are willing to perform and comply with all study procedures. 13. Male subjects who agree not to donate sperm starting from the screening and continuing until day 60.

Subjects who meet all inclusion criteria and do not meet exclusion criteria after completing the screening interview are enrolled in one of several dosing groups. During the escalation phase, subjects will receive CNP-201 as an intravenous infusion on Days 1 and 8. The dose for the first group is 1 mg. Additional doses for the remaining 3 groups are decreased to 0.1 mg, 0.25 mg, 0.5 mg, 1 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, or can be increased to 650 mg.

Individuals who meet all inclusion criteria and do not meet exclusion criteria at screening are enrolled in the study. CNP-201 or placebo will be administered by intravenous infusion using a graduated infusion rate. CNP-201 will be administered using the following graduated infusion rate: 1 mL/h for the first 10 minutes, 2 mL/h for the next 10 minutes, 5 mL/h for the next 10 minutes, 10 mL/h for the next 10 minutes, and 20 mL/h for the remainder of the infusion time.

Subjects remained in the clinic from admission (before TP administration) to the last procedure performed 4 hours after dosing on the same day on Days 1 and 8, unless infusion reactions, systemic allergic reactions, or other adverse events necessitated extended monitoring. Subjects were discharged if safety parameters were acceptable to the investigator.

Subjects followed daily telephone interviews following each infusion (Days 2-7 and Days 9-14) to assess and record any AEs and drug changes.

Seven days after the second dose of CNP-201 (Day 15), subjects were required to return to the clinic for the collection of safety laboratory data, assessment of AEs and drug changes, and collection of PD assessments. Subjects were followed up for safety and tolerability during the post-dose period.

On Day 60 (end of study), subjects returned to the clinic for collection of immunological safety laboratory data, PD measurements, and underwent a double-blind, placebo-controlled food challenge (DBPCFC) consisting of peanut and placebo (oatmeal) challenges. The DBPCFC was performed by study physicians or staff trained to manage clinical emergencies present in the field, with immediate access to emergency medications and equipment, and within the confines of an adjacent hospital emergency room (to provide rapid provision of emergency care if needed).

Subjects then returned to the clinic on Day 60 for an end-of-study visit for collection of safety laboratory data, PD measurements, and final assessment of AEs and drug changes.

The total duration of the study for individual subjects was approximately 240 days; screening up to 180 days, 8 days of dosing with CNP-201 or placebo, and 52 days of post-dosing assessments (Figure 8).

After all subjects in a dose cohort have completed the Day 15 clinic visit (7 days after the second dose), the DMC is convened to review all available safety data. At this time, the DMC determines whether it is acceptable to proceed to the next dose cohort and makes a recommendation regarding the dose for that cohort.

If a safe and effective dose is identified during the escalation phase, the study will proceed to the expansion phase. Subjects in the expansion phase will be randomized in a 1:1 ratio to receive either CNP-201 or placebo at the dose identified as safe and tolerable during the escalation phase. Subjects will undergo the same assessments during the escalation and expansion phases.

The primary objectives include: safety and tolerability of CNP-201.

Exploratory objectives include: changes in responses to DBPCFC in patients treated with CNP-201 or placebo, changes in the ratio of peanut-specific IgE to IgG in patients treated with CNP-201 or placebo, changes in peanut-specific IgE in patients treated with CNP-201 or placebo, changes in the proportion of peanut-specific Th2a+ T cells in patients treated with CNP-201 or placebo, changes in responses to the basophil activation test in patients treated with CNP-201 or placebo, changes in the proportion of peanut-specific regulatory T cells in patients treated with CNP-201 or placebo.

The main indicators included: the frequency of adverse events (AEs) and serious adverse events (SAEs), MedDRA 23.0 (CTCAE v.5.0 or CoFAR v.3.0 for allergy-related AEs); laboratory safety assessment (blood, serum chemistry, coagulation panel, and urinalysis); physical examination, including vital signs (blood pressure, heart rate, and temperature); 12-lead electrocardiogram (ECG); serum interleukins (TNF-α, IL-2, IL-6, IL-8, IL-1β, MCP-1, MIP-1β, MIP-1α, IFN-γ, and IL-12p70).

Exploratory endpoints included: DBPCFC between placebo and CNP-201 at day 60 (with a cumulative 4043 The percentage difference of individuals in the DBPCFC group (mg), the change in the cumulative tolerated dose (CTD) of peanut protein (mg) administered during DBPCFC between placebo and CNP-201 at day 60, the change in the ratio of peanut-specific IgE to IgG measured by ImmunoCap assay between placebo and CNP-201 at baseline and at day 60, the change in peanut-specific IgE measured by ImmunoCap assay between placebo and CNP-201 at baseline and at day 60, the change in peanut-specific Th2a+ after ex vivo stimulation of PBMCs between placebo and CNP-201 at baseline and at day 15 Changes in the ratio of T cells (peanut-specific Th2a+ cells/total peanut-specific T cells), changes in the effective concentration of 50% maximal phagocytic activation (EC50) measured by the phagocytic activation test (CD203c+/CD63+/- phagocytic activation) between placebo and CNP-201 at baseline and on days 15 and 60, changes in the ratio of peanut-specific regulatory T cells after ex vivo stimulation of PBMCs (peanut-specific regulatory T cells/peanut-specific CD4+ effector memory cells) between placebo and CNP-201 at baseline and on day 15.

Preferred embodiments of the invention are described herein. After reading the foregoing description, variations of the preferred embodiments may become apparent to those skilled in the art. The inventors expect those skilled in the art to adopt such variations as appropriate, and the inventors intend to implement the invention in other ways than those specifically described herein. Therefore, the invention includes all modifications and equivalents of the subject matter set forth in the claims attached hereto as permitted by applicable law. In addition, unless otherwise indicated herein or otherwise clearly contradictory to the context, the invention covers any combination of the above elements in all possible variations thereof.

FIG1A. Time course of events for subjects participating in Part A of the human study of CNP-201 particles. FIG1B. Time course of events for subjects participating in Part B of the human study of CNP-201 particles. FIG1C. Time course of events for subjects participating in the low starting dose study of CNP-201 particles.

Figure 2. Summary of results for patients receiving low-dose CNP-201 particles in peanut allergy: Patients received a single dose of approximately 1 mg or 25 mg of CNP-201 intravenously. Patients receiving low-dose CNP-201 had increased BAT thresholds, decreased peanut-specific IgE to IgG ratios, decreased induction of antigen-specific Tregs, and/or decreased pathogenic peanut-specific T cell subsets (Th2a, TFH, B cytoplasmic blasts).

Figure 3. Alkalophil sensitivity test using crude peanut extract: Peanut-allergic patients who received a single 25 mg intravenous dose of CNP-201 demonstrated an increase in the EC50 (peanut allergen concentration) required to increase the expression of activation markers on the surface of alkalophils following stimulation with peanut allergen.

FIG4. Change in IgE to IgG Phenotype at Doses up to 25 mg CNP-201: Peanut-allergic patients receiving up to 25 mg intravenous doses of CNP-201 demonstrated an increase in peanut-specific IgG and a decrease in the peanut-specific IgE/IgG ratio at days 15 and 38 post-dose compared to placebo.

Figure 5. Reduced peanut allergy-associated immune cell subsets. Peanut allergy patients who received a single 25 mg intravenous dose of CNP-201 showed a reduction in the allergenic subsets T helper 2A (Th2A), filtrating T helper (TFH), terminally differentiated effector memory cells (TEMRA), and B cell plasma blasts.

Figure 6. Fewer pathogenically activated peanut-specific CD4+ T cells were observed after a single 25 mg dose of CNP-201. In patients treated with low doses of CNP-201, peanut-specific activated T cell subsets CD4+CD25+, CD4+CD69+, and CD4+PD-1+ were reduced compared to placebo.

FIG. 7. Induction of Antigen-Specific Regulatory T Cells (Tregs): Peanut-allergic patients receiving a single 25 mg intravenous dose of CNP-201 exhibited an increase in peanut-specific Tregs compared to placebo.

Figure 8. Time course of events for subjects enrolled in the study with a 1 mg starting dose of CNP-201 particles.

TW202430207A_112139979_SEQL.xml

Claims (65)

A method of treating peanut allergy in an individual comprises administering to the individual a tolerogenic immunomodulatory particle encapsulating peanut protein (TIMP-PPE), alone or in combination with one or more therapeutic agents, wherein the TIMP-PPE is administered at a dose of between about 0.001 mg/kg and 12 mg/kg. The method of claim 1, wherein the TIMP-PPE particles have an average diameter between 100 nm and 1500 nm. The method of claim 1 or 2, wherein the TIMP-PPE particles have a negative zeta potential. The method of any of the preceding claims, wherein the particles have a negative zeta potential between -30 mV and -100 mV. The method of any of the preceding claims, wherein TIMP-PPE is administered at a concentration between about 0.0005 mg/mL and about 50 mg/mL, optionally about 0.0005 mg/mL, 0.001 mg/mL, 0.005 mg/mL, 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 12.5 mg/mL, 15 mg/mL, 17.5 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 40 mg/mL, or 50 mg/mL. The method of any of the preceding claims, wherein TIMP-PPE is administered at a dose of about 0.001 mg/kg, 0.0025 mg/kg, 0.005 mg/kg, 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 4.0 mg/kg, 6 mg/kg, 8.0 mg/kg, 10 mg/kg or 12 mg/kg. The method of any of the preceding claims, wherein TIMP-PPE is administered in an amount between about 0.1 mg and 800 mg. The method of any of the preceding claims, wherein TIMP-PPE is administered in an amount of about 0.1 mg, 0.25 mg, 0.5 mg, 1 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, or 800 mg. The method of any of the preceding claims, wherein TIMP-PPE is administered in a single dose or in multiple doses. The method of any of the preceding claims, wherein TIMP-PPE is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year. The method of any preceding claim, wherein TIMP-PPE is administered in two doses one week apart. The method of any of the preceding claims, wherein TIMP-PPE is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally. The method of any of the preceding claims, wherein TIMP-PPE is administered at a concentration between 0.0005 mg/mL and 50 mg/mL. The method of any of the preceding claims, wherein TIMP-PPE is administered in two doses one week apart, followed by additional doses of TIMP-PPE administered once every three months as a single dose. The method of any one of claims 1 to 14, wherein the therapeutic agent administered in combination with TIMP-PPE is an anti-IgE antibody, an anti-IL-4Rα antibody, an anti-IL13 antibody, an anti-IL-33 antibody, an antihistamine, a steroid, a corticosteroid, a leukotriene modifier, a low-dose IL-2, an IL-2 mutant protein engineered to expand Tregs, an IL-2 variant engineered to expand Tregs, an IL-2 engineered to be selective for a high affinity IL-2 receptor, or 2 molecules, PEGylated IL-2, IL-2 complex, IL-2/CD25 fusion protein, probiotics, probiotics, histone deacetylase inhibitors, short-chain fatty acids (e.g., acetate, butyrate, propionate, butyrate polymers), IgE inhibitors, IgE competitors for allergen binding sites, philoglobulin activation inhibitors, mast cell activation inhibitors, interleukin inhibitors, microbiome therapy, small molecule or biological therapeutics, or nonsteroidal anti-inflammatory drugs (NSAIDs). The method of claim 15, wherein the anti-IgE antibody is omalizumab. The method of claim 15 or 16, wherein the anti-IgE antibody is administered subcutaneously. The method of any one of claims 15 to 17, wherein the anti-IgE antibody is administered in a single dose or multiple doses. The method of any one of claims 15 to 18, wherein the anti-IgE antibody is administered before, simultaneously with, or after administration of TIMP-PPE. The method of any one of claims 15 to 19, wherein the anti-IgE antibody is administered three doses two weeks apart, or two doses four weeks apart, prior to administration of TIMP-PPE. The method of any one of claims 15 to 20, wherein the anti-IgE antibody is administered in an amount between about 10 mg and 500 mg. The method of claim 21, wherein the anti-IgE antibody is administered in an amount of about 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg or 500 mg. The method of claim 21 or 22, wherein the dose of the anti-IgE antibody is determined based on the IgE level in the individual's blood. The method of claim 21 or 22, wherein the dose of the anti-IgE antibody is determined based on the individual's weight. The method of any of the preceding claims, wherein administering TIMP-PPE alone or in combination with an additional therapeutic agent to the individual reduces one or more symptoms of peanut allergy. The method of claim 19, wherein the one or more peanut allergy symptoms are selected from the group consisting of: skin reactions; hives; skin redness; skin swelling; itching; throat tightening; difficulty breathing; shortness of breath; digestive problems such as diarrhea, angina, nausea or vomiting; decreased blood pressure; and systemic allergic reactions. The method of any of the preceding claims, wherein TIMP-PPE alone or in combination with a therapeutic agent is administered to the subject to reduce the duration and severity of an allergic immune response to peanut protein. The method of claim 27, wherein the allergic immune response is a Th2 cell response, B cell activation, basophil activation, eosinophil activation, mast cell activation and/or IgE induction. The method of claim 28, wherein one or more biological samples obtained from the individual are analyzed for the Th2 cell response, B cell activation, basophil activation, eosinophil activation, mast cell activation and/or IgE induction. The method of claim 29, wherein the biological sample is selected from the group consisting of whole blood, peripheral blood, peripheral blood mononuclear cells (PBMC), serum, plasma, urine, cerebrospinal fluid (CSF), feces, tissue biopsy and/or bone marrow biopsy. The method of claim 27, wherein TIMP-PPE alone or in combination with a therapeutic agent is administered to the individual to reduce the proportion of Th2a+ T cells present in the total T cell population in peripheral blood. The method of claim 27, wherein TIMP-PPE alone or in combination with an additional therapeutic agent is administered to the individual to reduce the proportion of activated peanut protein-specific T cells in peripheral blood. The method of claim 27, wherein administration of TIMP-PPE alone or in combination with a therapeutic agent to the individual increases the level of peanut protein-specific Treg cells in the blood. The method of claim 27, wherein administering TIMP-PPE alone or in combination with a therapeutic agent to the subject reduces basophil activation. The method of claim 34, wherein basophil activation is analyzed by stimulating basophils in vitro with peanut protein in a basophil activation test (BAT). The method of claim 27, wherein TIMP-PPE alone or in combination with a therapeutic agent is administered to the individual to reduce the level of peanut protein-specific IgE in the blood. The method of claim 27, wherein TIMP-PPE alone or in combination with a therapeutic agent is administered to the individual to reduce the ratio of peanut protein-specific IgE to IgG levels in the blood. The method of any one of claims 1 to 37, wherein TIMP-PPE alone or in combination with a therapeutic agent is administered to the individual to reduce the level of Th2 interleukin content in the blood. The method of claim 38, wherein the Th2 cytokines are selected from the group consisting of IL-4, IL-5, IL-9 and IL-13. The method of any of the preceding claims, wherein TIMP-PPE alone or in combination with a therapeutic agent is administered to the individual to increase tolerance to peanut protein. The method of claim 40, wherein tolerance to peanut protein is determined by a double-blind placebo-controlled food challenge. The method of claim 40, wherein tolerance to peanut protein is determined by skin prick testing (SPT). The method of any of the preceding claims, wherein the TIMP-PPE comprises peanut extract or one or more peanut proteins or antigenic fragments thereof selected from the group consisting of Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, Ara h13, Ara h14, Ara h15, Ara h16, Ara h17 and Ara h18. The method of any of the preceding claims, wherein the TIMP-PPE comprises one or more proteins or antigenic fragments of Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7 or Ara h8, or a combination thereof. The method of any of the preceding claims, wherein a combination of an antihistamine, a corticosteroid, an NSAID and/or a leukotriene modulator and TIMP-PPE is administered to the individual. The method of claim 45, wherein the antihistamine is diphenhydramine. The method of claim 45 or 46, wherein the antihistamine is administered in an amount of about 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 1000 mg, 1500 mg or 2000 mg. The method of any one of claims 45 to 47, wherein the antihistamine is administered intravenously. The method of any one of claims 45 to 48, wherein the antihistamine is administered in a single dose or in multiple doses. The method of any one of claims 45 to 49, wherein the antihistamine is administered 30 minutes prior to administration of TIMP-PPE. The method of claim 45, wherein the corticosteroid is methylprednisone. The method of any one of claims 45 to 51, wherein the corticosteroid is administered in an amount of about 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 1000 mg, 1500 mg or 2000 mg. The method of any one of claims 45 to 52, wherein the corticosteroid is administered intravenously. The method of any one of claims 45 to 53, wherein the corticosteroid is administered in a single dose or in multiple doses. The method of any one of claims 45 to 54, wherein the corticosteroid is administered 30 minutes prior to administering TIMP-PPE. The method of claim 45, wherein the NSAID is acetylsalicylic acid. The method of any of claims 45 and 56, wherein the NSAID is administered in an amount of about 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 1000 mg, 1500 mg or 2000 mg. The method of any one of claims 45 or 56 to 57, wherein the NSAID is administered orally. The method of any one of claims 45 or 56 to 58, wherein the NSAID is administered in a single dose or in multiple doses. The method of any one of claims 45 or 56 to 59, wherein the NSAID is administered daily for two days and is administered 60 minutes prior to administration of TIMP-PPE. The method of claim 45, wherein the leukotriene modulator is montelukast. The method of any of claims 45 or 61, wherein the leukotriene modulator is administered in an amount of about 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 1000 mg, 1500 mg or 2000 mg. The method of any one of claims 45 or 61-62, wherein the leukotriene modulator is administered orally. The method of any one of claims 45 or 61 to 63, wherein the leukotriene modulator is administered in a single dose or in multiple doses. The method of any one of claims 45 or 61 to 64, wherein the leukotriene modulator is administered 12 hours before, 45 minutes before, and/or 12 hours after administration of TIMP-PPE.