Tolerizing immune modifying nanoparticles for treatment of alpha gal syndrome

Abstract

The present application is directed, in general, to tolerizing immune modifying particles comprising antigens for use in treating alpha-gal syndrome, for example αGal containing proteins.

Description

[0001] TOLERIZING IMMUNE MODIFYING NANOPARTICLES FOR TREATMENT OF ALPHA GAL SYNDROME

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims the priority benefit of U.S. Provisional Patent Application No. 63 / 669,519, filed July 10, 2024, U.S. Provisional Patent Application No. 63 / 705,248, filed October 9, 2024 and U.S. Provisional Patent Application No. 63 / 705,793, filed October 10, 2024, which are incorporated by reference in their entirety.

[0004] FIELD OF THE DISCLOSURE

[0005] The present application is directed, in general, to tolerizing immune modifying particles comprising antigens associated with alpha-gal syndrome.

[0006] STATEMENT REGARDING FEDERAL FUNDING

[0007] This invention was made with government support under AI172112, CA044579, AI155678, and GM007863 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] BACKGROUND

[0009] Alpha-gal syndrome (AGS) is an atypical IgE-mediated food allergy to the oligosaccharide galactose-a-l,3-galactose (aGal) and was first described 15 years ago. Patients with AGS are mostly adult and are clinically identified due to the presentation of allergic symptoms ranging from urticaria and gastrointestinal discomfort to anaphylaxis starting between 2-6 hours after ingestion of non-primate mammalian meat, dairy, or other aGal-containing foods. In the United States, bites from the Lone Star tick, Amblyomma americanum, induce IgE sensitization to aGal. Importantly, AGS is becoming a global health problem with increasing cases reported in all continents with additional tick species implicated in aGal sensitization. AGS is therefore markedly different from traditional food allergies, which typically arise early in life and involve acute hypersensitivity reactions to protein allergens.

[0010] Mammalian meat avoidance is the primary means of AGS clinical management, which can result in significant social and economic consequences. A few reports suggest oral immunotherapy with red meat may desensitize AGS patients; however, the limited data from these reports did not explore the effects of oral immunotherapy on the modulation of innate and adaptive immune responses associated with IgE-mediated food allergy. The identification of the aGal allergen eliciting IgE responses in AGS holds promise for leading to strategies to identify and intervene in individuals at risk with allergen avoidance or other immunomodulatory approaches. Thus, there is an unmet clinical need to determine whether an aGal glycoprotein-containing immunotherapy can effectively desensitize recipients with AGS and identify effects on underlying immune mechanisms associated with carbohydrate immune tolerance, which may differ from those involved in food allergies elicited by protein allergens.

[0011] Summary of the disclosure

[0012] The present disclosure describes TIMP-AGS compositions and methods for inducing antigenspecific tolerance to AGS-associated antigens.

[0013] Provided herein is a TIMP-AGS composition comprising a negatively charged particle encapsulating an antigen, wherein the antigen comprises one or more AGS associated antigens and / or a portion thereof, or combinations of antigens or portions thereof. In some embodiments, the AGS associated antigen is a carbohydrate, a peptide, a protein, a glycoprotein, a glycolipid, a homolog, a derivative, a mimotope, or a combination thereof. In various embodiments, the TIMP-AGS particle comprises a polymer and has a negative zeta potential. In various embodiments, the polymer is a biodegradable polymer.

[0014] In various embodiments, TIMP-AGS particles comprise a polymer selected from the group consisting of polyglycolic acid (PGA), polylactic acid (PLA), polysebacic acid (PSA), poly(lactic-co-glycolic) (PLGA), poly(lactic-co-sebacic) acid (PLSA), poly(glycolic-co-sebacic) acid (PGSA), polypropylene sulfide, poly(caprolactone), chitosan, a polysaccharide, or a lipid, polystyrene, diamond, a liposome, PEG, cyclodextran, a lipid or a metal such as Iron (Fe), zinc (Zn), cadmium (Cd), gold, or silver, or combinations thereof.

[0015] In various embodiments, the polymer is a co-polymer. In various embodiments, the co-polymer has varying molar ratios of constituent polymers. In various embodiments, the molar ratio is 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.

[0016] In various embodiments, TIMP-AGS particles comprise poly (lactic-co-glycolic acid) (PLGA). In various embodiments, the particle comprises about 50:50, about 80:20 to about 100:0 polylactic acid: polyglycolic acid or from about 50:50, about 80:20 to about 100:0 polyglycolic acid: polylactic acid. In various embodiments, the particles comprise 50:50 polylactic acid: polyglycolic acid. In various embodiments, the particle comprises polylactic acid: polyglycolic acid from about 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, including all values and ranges that lie in between these values.

[0017] In various embodiments, TIMP-AGS particles have a negative zeta potential. In various embodiments, the zeta potential of the particles is from about -100 mV to about 0 mV, from about -100 mV to about -25 mV, from about -100 to about -30 mV, from about -80 mV to about -30 mV, from about -75 mV to about -30 mV, from about -70 mV to about -30 mV, from about - 75 to about -35 mV, from about -70 to about -25 mV, from about -60 mV to about -30 mV, from about -60 mV to about -35 mV, or from 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, including all values and ranges therein. In various embodiments, TIMP-AGS particles have a negative zeta potential of between -30 mV to -80 mV. In various embodiments, the TIMP-AGS particles have a negative zeta potential of between -30 mV to -60 mV. In various embodiments, the negative zeta potential is achieved by surface functionalization of the particles. In various embodiments, the surface functionalization is carboxylation.

[0018] In various embodiments, the size, or diameter, of the TIMP-AGS particles is between 0.05 pm to about 10 pm. In various embodiments, diameter of the particles is between 0.1 pm and about 10 pm. In various embodiments, diameter of the particles is between 0.1 pm and about 5 pm. In various embodiments, diameter of the particles is between 0.1 pm and about 3 pm. In various embodiments, diameter of the particles is between 0.3 pm and about 5 pm. In various embodiments, diameter of the particles is about 0.3 pm to about 3 pm. In various embodiments, diameter of the particles is between about 0.3 pm to about 1 pm. In various embodiments, diameter of the particles is between about 0.4 pm to about 1 pm. In various embodiments, the particles have a diameter of about 100 to 10000 nm, about 100 to 5000 nm, about 100 to 3000 nm, about 100 to 2000 nm, about 100 to 1500 nm, about 300 to 5000 nm, about 300 to 3000 nm, about 300 to 1000 nm, about 300 to 800 nm, about 400 to 800 nm, or about 200 to 700 nm. In various embodiments, the 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, including all values and ranges therein. In various embodiments, the diameter of the negatively charged TIMP-AGS particles is between 400 nm to 800 nm. In various embodiments, the diameter of the negatively charged TIMP-AGS particles is between 350 nm to 800 nm.

[0019] In various embodiments, TIMP-AGS particles have a homogenous size distribution. In various embodiments, the particles have a homogenous size distribution wherein at least 90% of the particles have a diameter of between 0.05 pm and about 10 pm, between 0.1 pm and about 10 pm, 0.1 pm and about 5 pm, 0.1 pm and about 3 pm, 0.3 pm and about 5 pm, 0.3 pm to about 3 pm including all values and ranges therein. In various embodiments, the particles have a homogenous size distribution wherein at least 90% of the particles have a diameter of about 100 to 10000 nm, about 100 to 5000 nm, about 100 to 3000 nm, about 100 to 2000nm, about 300 to 5000 nm, about 300 to 3000 nm, about 300 to 1000 nm, about 300 to 800 nm, about 400 to 800 nm, or about 200 to 700 nm including all values and ranges therein. In various embodiments, the 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 including all values and ranges therein. In various embodiments, the particles have a homogenous size distribution wherein at least 50% of the particles have a diameter of between about 0.05 pm and about 10 pm, about 0.1 pm and about 10 pm, about 0.1 pm and about 5 pm, about 0. 1 pm and about 3 pm, about 0.3 pm and about 5 pm, and about 0.3 pm and about 3 pm including all values and ranges therein. In various embodiments, the particles have a homogenous size distribution wherein at least 50% of the particles have a diameter of about 100 to 10000 nm, about 100 to 5000 nm, about 100 to 3000 nm, about 100 to 2000nm, about 300 to 5000 nm, about 300 to 3000 nm, about 300 to 1000 nm, about 300 to 800 nm, about 400 to 800 nm, or about 200 to 700 nm including all values and ranges therein. In various embodiments, the 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 including all values and ranges therein. In various embodiments, the particles have a homogenous size distribution wherein at least 10% of the particles have a diameter of between about 0.05 pm and about 10 pm, about 0.1 pm and about 10 pm, about 0.1 pm and about 5 pm, about 0. 1 pm and about 3 pm, about 0.3 pm and about 5 pm, and about 0.3 pm and about 3 pm including all values and ranges therein. In various embodiments, the particles have a homogenous size distribution wherein at least 10% of the particles have a diameter of about 100 to 10000 nm, about 100 to 5000 nm, about 100 to 3000 nm, about 100 to 2000nm, about 300 to 5000 nm, about 300 to 3000 nm, about 300 to 1000 nm, about 300 to 800 nm, about 400 to 800 nm, or about 200 to 700 nm including all values and ranges therein. In various embodiments, the carrier 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 including all values and ranges therein.

[0020] In various embodiments, the TIMP-AGS particles encapsulate one or more AGS associated antigens, have a negative zeta potential of between -100 mV and 0 mV, and the particles are between 100 and 1000 nm in diameter. In various embodiments, the TIMP-AGS particles encapsulate one or more AGS associated antigens, wherein the size of the particles is between 400 and 800 nm and the particles have a negative zeta potential between -30 mV and -80 mV.

[0021] In various embodiments, TIMP-AGS particles encapsulate one or more AGS associated antigens, portions, or combinations thereof. In some embodiments, the AGS associated antigen is a carbohydrate, a peptide, a protein, a glycoprotein, a glycolipid, a homolog, a derivative, a mimotope, or a combination thereof. In various embodiments, the TIMP-AGS particles encapsulate one or more polynucleotides encoding AGS associated antigens. In some embodiments, TIMP-AGS particles encapsulate one or more polynucleotides encoding AGS associated antigens and ocgal. In some embodiments, the polynucleotides comprise DNA, RNA, messenger RNA (mRNA), or circular RNA.

[0022] In various embodiments, the AGS associated antigens are selected from the group consisting of galactose, galactose-6 / / / ? / z«-l,3-galactose (ocgal), ocgal containing glycoprotein, or ocgal containing glycolipid. In some embodiments, the antigen is ocgal (Fig. 10). In some embodiments, the AGS associated antigens are selected from the group consisting of ocgal containing meat proteins. In various embodiments, the AGS associated antigens are selected from the group consisting of AGS-associated tick antigens. In various embodiments, the AGS associated ticks are selected from the group consisting Amblyomma americanum, Ixodes ricinus. Ixodes holocyclus, Ixodes australiensis, Amblyomma cajennense, Amblyomma hebraeum. Amblyomma testudinarium, Ixodes nipponensis. Amblyomma sail plum. Amblyomma variegatum, Rhipicephalus evertsi, Rhipicephalus burst Hyaloma marginatum, or Haemaphysalis longicornis.

[0023] In various embodiments, the TIMP-AGS particles encapsulate one or more AGS associated antigens, have a negative zeta potential of between -100 mV and 0 mV, and wherein the particles are between 100 and 1000 nm in diameter. In various embodiments, the TIMP-AGS particles encapsulate ocgal containing glycoprotein, have a negative zeta potential of between -100 mV and 0 mV, and wherein the particles are between 100 and 1000 nm in diameter. In various embodiments, the TIMP-AGS particles encapsulate otgal containing glycoprotein, have a negative zeta potential of between -100 mV and 0 mV, and wherein the particles are between 100 and 1000 nm in diameter.

[0024] In various embodiments, the TIMP-AGS particles encapsulate one or more AGS associated antigens, have a negative zeta potential of between -80 mV and -30 mV, and wherein the particles are between 400 nm and 800 nm in diameter. In various embodiments, the TIMP-AGS particles encapsulate agal containing glycoprotein, have a negative zeta potential of between -80 mV and -30 mV, and wherein the particles are between 400 and 800 nm in diameter.

[0025] In various embodiments, TIMP-AGS particles comprise PLGA, encapsulate one more AGS associated antigens, and have a negative zeta potential of between -80 mV and -30 mV. In various embodiments, TIMP-AGS particles comprise PLGA, encapsulate ocgal containing glycoprotein, have a negative zeta potential of between -80 mV and - 30 mV, and wherein the particles are between 400 nm and 800 nm in diameter.

[0026] The present disclosure provides methods of inducing antigen-specific immune tolerance using TIMPs encapsulating antigens as described herein comprising, administering to a subject a composition comprising negatively charged particles encapsulating an AGS associated antigen. In various embodiments, the antigen is ocgal. In various embodiments, the antigen is ocgal containing glycoprotein. In various embodiments, administration of TIMPs encapsulating one or more antigens associated with AGS to a subject in need thereof induces antigen specific tolerance to one or more antigens associated with AGS. In various embodiments, the administration alleviates one or more symptoms of AGS.

[0027] Also provided is a method of inducing tolerance in a subject in need thereof comprising administering to the subject a composition comprising a liposome as described herein.

[0028] In various embodiments, subjects are administered TIMPs encapsulating one or more AGS associated antigens.

[0029] In various embodiments, TIMP-AGS particles are administered intravenously, intramuscularly, ocularly, intraperitoneally, transdermally, nasally, orally, intra-lymphatically and / or subcutaneously.

[0030] In various embodiments, administering TIMP-AGS to a subject in need thereof induces antigen specific tolerance to an AGS associated antigen. In various embodiments, administering TIMP- AGS to a subject reduces the inflammatory immune response to AGS associated antigens. In various embodiments, administering the TIMP-AGS to a subject ameliorates the allergic or immunogenic response to AGS associated antigens. In various embodiments, administering TIMP-AGS to a subject induces cGAS-STING pathway. In various embodiments, administering the TIMP-AGS to a subject induces type-1 IFN pathway. In various embodiments, administering TIMP-AGS to a subject in need thereof reduces Th2 cytokine IL-4, IL-5, or IL-13. In various embodiments, the allergic, immunogenic, and / or inflammatory immune response is an innate immune response, humoral immune response and / or an adaptive immune response. In various embodiments, the immune response is an antibody response. In various embodiments, the antibody response is an IgA, IgG, IgE, or IgM response. In various embodiments, the antibody response is a neutralizing antibody response, for example the formation of neutralizing antibodies against the AGS associated antigens. In various embodiments, the innate immune response is a proinflammatory APC response. In various embodiments, the adaptive immune response is an activated B-cell, CD4+, CD8+ or a proinflammatory cytokine response. In various embodiments, the immune response is a T cell, B cell, NKT cell, NK cell, monocyte, macrophage, mast cell, eosinophil, or a basophil response. In various embodiments, the immune response is reduced immune infiltrate into tissues and / or organs. In various embodiments, administering the TIMP-AGS to a subject in need thereof reduces the allergic, immunogenic, and / or inflammatory response to the one or more antigens not encapsulated within the particle. Such an immune regulatory response has been referred to in the literature as ‘infectious tolerance’ or “bystander tolerance”.

[0031] In various embodiments, administering TIMP-AGS to a subject in need thereof induces an antigen specific immune regulator response. In various embodiments, administering TIMP-AGS to a subject induces regulatory T cells, B cells, monocytes, dendritic cells, and / or macrophages. In various embodiments, administering TIMP-AGS to a subject induces antigen specific regulatory T cell (Treg), Tri, regulatory macrophages (Mreg), and / or regulatory B cells (Breg) cells.

[0032] In various embodiments, subjects are administered liposomes encapsulating one or more AGS associated antigens. In various embodiments, subjects are administered liposomes encapsulating ocgal containing glycoprotein. In various embodiments, subjects are administered liposomes encapsulating agal. In various embodiments, subjects are administered liposomes encapsulating polynucleotides encoding one or more AGS associated antigens. Administering liposomes encapsulating AGS associated antigens induces antigen specific tolerance to AGS associated antigens.

[0033] In various embodiments, the liposome is negatively charged. In various embodiments, the liposome has a negative zeta potential. In various embodiments, the negative zeta potential is between -100 mV to 0 mV. In various embodiments, the negatively charged liposomes have a zeta potential from about -100 mV to about 0 mV, from about -100 mV to about -25 mV, from about -100 to about -30 mV, from about -80 mV to about -30 mV, from about -75 mV to about - 30 mV, from about -70 mV to about -30 mV, from about -75 to about -35 mV, from about -70 to about -25 mV, from about -60 mV to about -30 mV, from about -60 mV to about -35 mV, or from 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, including all values and ranges therein. In various embodiments, the liposomes have a negative zeta potential of between -30 mV to -80 mV. In various embodiments, the liposomes have a negative zeta potential of between -30 mV to -60 mV. In various embodiments, the liposome is between 100-1000 nm, or between 300-800 nm or between 400-800 nm. In various embodiments, the liposome is about 100 nm, about 200nm, about 300nm, about 400nm, about 500 nm, about 600nm, about 700 nm, about 800 nm, about 900 nm or about 1000 nm.

[0034] In various embodiments, the liposomes are administered intravenously, intramuscularly, ocularly, intraperitoneally, transdermally, nasally, orally, intra-lymphatically and / or subcutaneously.

[0035] In various embodiments, administering the liposomes to a subject in need thereof induces antigen specific tolerance to AGS associated antigens. In various embodiments, administering the liposomes to a subject reduces the inflammatory and / or immunogenic or allergic response to AGS associated antigens. In various embodiments, administering the liposomes to a subject induces cGAS-STING pathway. In various embodiments, administering the liposomes to a subject induces type-1 IFN pathway. In various embodiments, administering liposomes to a subject in need thereof reduces Th2 cytokine IL-4, IL-5, or IL-13. In various embodiments, the immune response is an innate immune response, humoral immune response and / or an adaptive immune response. In various embodiments, the humoral immune response is an antibody response. In various embodiments, the antibody response is an IgA, IgG, IgE, or IgM response. In various embodiments, the antibody response is a neutralizing antibody response, for example the formation of neutralizing antibodies against AGS associated antigen. In various embodiments, the innate immune response is a proinflammatory APC response. In various embodiments, the adaptive immune response is an activated B-cell, CD4+, CD8+ or a proinflammatory cytokine response. In various embodiments, the immune response is a T cell, B cell, NK cell monocyte, macrophage, eosinophil, or a basophil response. In various embodiments, administering the liposomes to a subject in need thereof reduces the inflammatory and / or autoimmune response to the one or more antigens not encapsulated within the particle. Such an immune regulatory response has been referred to in the literature as ‘infectious tolerance’ or “bystander tolerance”.

[0036] In various embodiments, administering the liposomes to a subject in need thereof induces an antigen specific immune regulator response. In various embodiments, administering the liposomes to a subject induces regulatory T cells, B cells, monocytes, dendritic cells, and / or macrophages. In various embodiments, administering liposomes to a subject induces antigen specific Treg, Tri, Mreg, and / or Breg cells.

[0037] Also contemplated are compositions described herein comprising a negatively charged particle encapsulating an antigen, wherein the antigen is one or more AGS associated antigens for use in inducing tolerance in a subject.

[0038] The disclosure also provides for use of a composition described herein comprising a negatively charged particle encapsulating an antigen, wherein the antigen is one or more AGS associated antigens in the preparation of a medicament for inducing tolerance in a subject.

[0039] In various embodiments, the negatively charged particle is a TIMP or a liposome as described herein.

[0040] It is understood that each feature or embodiment, or combination, described herein is a nonlimiting, illustrative example of any of the aspects of the invention and, as such, is meant to be combinable with any other feature or embodiment, or combination, described herein. For example, where features are described with language such as “one embodiment”, “some embodiments”, “certain embodiments”, “further embodiment”, “specific exemplary embodiments”, and / or “another embodiment”, each of these types of embodiments is a nonlimiting example of a feature that is intended to be combined with any other feature, or combination of features, described herein without having to list every possible combination. Such features or combinations of features apply to any of the aspects of the invention. Where examples of values falling within ranges are disclosed, any of these examples are contemplated as possible endpoints of a range, any and all numeric values between such endpoints are contemplated, and any and all combinations of upper and lower endpoints are envisioned.

[0041] The headings herein are for the convenience of the reader and not intended to be limiting. Additional aspects, embodiments, and variations of the invention will be apparent from the Detailed Description and / or Drawings and / or claims.

[0042] BRIEF DESCRIPTION OF THE FIGURES

[0043] Figure la-ld: Prophylactic treatment with NP-o.Gal prevents IgE-mediated allergic responses to aGal. Fig. la, Schematic of prophylactic NP treatment in a mouse model of tick extract induced IgE sensitization to aGal. AGKO mice received two doses of NP-aGal-HSA or control NP-HSA, two weeks apart, prior to intradermal injections (i.d.) of whole-body protein extract prepared from lone star seed ticks supplemented with 50 pg aGal-BSA on day 0 and identically prepared booster injections on days 7 and 31. On day 35, mice received an intragastric challenge with beef extract and analyzed 1.5 hrs later. Fig. lb, Concentration of MCPT-1 in the sera of mice after challenge with beef extract measured by ELISA. Fig. 1c Fold increase in histamine released from basophil activation test (BAT) with cetuximab measured by ELISA. Fig. Id, Serum levels of total IgE, tick-specific IgE, and aGal-specific IgE from mice at day 35 measured by ELISA or Luminex. All data are expressed as mean ± SEM. Results shown are representative of two independent experiments. P = *0.05, **0.01 and ****0.0001, or ns = not significant, with unpaired, two-tailed / -test.

[0044] Figure 2a-2e: Prophylactic administration of NP-aGal reduces the frequency and activation of basophils in the mesenteric lymph nodes of mice following intragastric challenge with beef extract. Fig. 2a, b Frequencies, and activation of circulating CD45+CD49b+Fc£Rl+IgE+c-kif basophils in peripheral blood from mice at 90 minutes after oral gavage were measured by flow cytometry. Fig. 2c, d Frequencies (top), number (bottom), and activation of basophils in mesenteric lymph nodes of the same mice as in panel a,b. Fig. 2e, Frequencies (top) and numbers (bottom) of FcsRl+c-kit+mast cells in mesenteric lymph nodes. The results shown are representative of two independent experiments. All data are expressed as mean ± SEM. P = *0.05, ***0.001, and ****0.0001, with unpaired, two-tailed / -test.

[0045] Figure 3a-3c: Prophylactic administration of NP-aGal reduces IL-4, IL-5, IL-6, and IL-13, and increases IL- 10. Fig. 3a-c, Cytokine secretion in culture supernatants from splenocyte recall assays to cetuximab were measured by Luminex multiplex. Results shown are representative of two independent experiments. All data are expressed as mean ± SEM. P = *0.05, **0.01, and ***0.001, with a non-parametric Mann-Whitney test.

[0046] Fig. 4a-4d: Therapeutic administration of NP-aGal reduces hypersensitivity responses in mice following intragastric challenge with beef extract. Fig. 4a, Schematic of therapeutic NP treatment. AGKO mice received intradermal injections (i.d.) of whole-body protein extract prepared from lone star seed ticks supplemented with 50 p ocGal-BSA on day 0 and identically prepared booster injections on days 7 and 31. On days 33 and 47, mice were intravenously given NP-aGal-HSA or control NP-HAS and, two weeks later, were given a booster of tick extract. On day 68, mice received an intragastric challenge with beef extract and analyzed 1.5 hrs later. Fig. 4b Concentration of MCPT-1 in the sera of mice after challenge with beef extract measured by ELISA. Fig. 4c Fold increase in histamine released from basophil activation test (BAT) with cetuximab measured by ELISA. Fig. 4d, Serum levels of total IgE, tick-specific IgE, and ocGal- specific IgE from mice at day 35 measured by ELISA or Luminex. All data are expressed as mean ± SEM. P = *0.05 and ****0.0001, or ns = not significant, with unpaired, two-tailed / -test.

[0047] Figure 5a-5d: Therapeutic administration of NP-aGal reduces the activation of basosphils in the mesenteric lymph nodes of mice following intragastric challenge with beef extract. Fig. 5a, b, Frequencies of circulating CD45+CD49b+FcsRl+IgE+c-kit' basophils and activation by CD200R and CD41 expression in peripheral blood from mice at 90 minutes after beef gavage were measured by flow cytometry. Fig. 5c, d, Frequencies (top), numbers (bottom), and activation of basophils in mesenteric lymph nodes of the same mice as in panel a,b. e, Frequencies (top) and numbers (bottom) of FcsRl c-kif mast cells in mesenteric lymph nodes. All data are expressed as mean ± SEM. P = *0.05 and **0.01, with unpaired, two-tailed / -test.

[0048] Figure 6a-6d: Therapeutic administration of NP-aGal reduces IL-4, IL-5, IL-6, and IL-13, and increases IL- 10. Fig. 6a-c, Splenocytes harvested at day 68 from all groups of sensitized mice therapeutically administered NPs were stimulated with cetuximab, a source of aGal, and cytokine secretion in culture supernatants from splenocyte recall assays were measured by Luminex multiplex. Fig. 6d, Frequencies of CD103+CDl lc+F4 / 80‘ dendritic cells in skindraining inguinal lymph nodes of the same mice as in panels a-c were measured by flow cytometry. All data are expressed as mean ± SEM. P = *0.05, **0.01, and ***0.001, with a nonparametric Mann-Whitney test (a-c), or unpaired, two-tailed / -test (d).

[0049] Figure 7a-7b. Prophylactic administration of NP-otGal does not affect the numbers of germinal center B cells and IgE+ plasma cells. Fig. 7a, Total numbers of germinal center (GC) B cells and Fig. 7b, IgE+ plasma cells (PCs) in the skin-draining inguinal lymph nodes of mice given two doses of NPs or PBS, followed by three injections of tick extract, and analyzed at day 35. All data are expressed as mean ± SEM, with unpaired, two-tailed / -test. Figure 8. Manual gating strategy for analyzing basophils and mast cells using flow cytometry. Gates were set for single cells, live, CD45+, and then separated into FcsRl+CDl 17 (c-kit)+to identify mast cells and FcsRl+CDl 17" cells. FcsR I CD I 17" cells were further gated on CD49b to identify basophils and the expression of activation markers CD200R and CD41, with gates set based on fluorescence-minus-one (FMO) controls.

[0050] Figure 9. Basophil activation test (BAT) for release of histamine after aGal stimulation with cetuximab. Heparinized blood from mice was incubated with and without cetuximab, and basophil release of histamine was measured by ELISA. All data are expressed as mean ± SEM. P = *0.05, with paired, two-tailed / -test.

[0051] Figure 10. ocgal structure.

[0052] DETAILED DESCRIPTION

[0053] There is a need for therapeutics to address the immune imbalance in AGS leading to improved disease symptoms and improved outcomes without the risk of toxic side-effects. Embodiments of the present application employ compositions comprising TIMPs to address this need. TIMPs are surface functionalized negatively charged particles made of biodegradable material encapsulating antigenic proteins or peptide epitopes associated with inflammatory conditions such as autoimmune disease and allergies. TIMPs are designed for targeted delivery of encapsulated proteins / peptides to antigen presenting cells (APCs) of the mononuclear phagocyte system resulting in APC mediated T cell reprogramming via non-inflammatory pathways.

[0054] TIMPs encapsulating one or more antigens implicated in or associated with AGS (TIMP-AGS) find use to treat AGS by reprogramming the immune system and inducing antigen-specific tolerance to AGS associated antigens. There is a current need for immune tolerizing therapies which can induce tolerance to autoimmune AGS associated antigens for long term therapeutic benefit without exposing patients to risk of adverse events.

[0055] IgE antibodies against the mammalian oligosaccharide allergen galactose-a-l,3-galactose (aGal) can result in a severe allergic disease known as alpha-gal syndrome (AGS). This syndrome, acquired by tick bites that cause aGal sensitization, leads to allergic reactions after ingestion of non-primate mammalian meat and mammalian-derived products that contain aGal. Allergen- specific immunotherapies for this tickborne allergic syndrome are understudied, as are the immune mechanisms of allergic desensitization that induce clinical tolerance to aGal. As described herein, administration of aGal glycoprotein-containing nanoparticles (TIMP-AGS) to mice prior to tick protein-induced aGal IgE sensitization blunts the production of Th2 cytokines IL-4, IL-5, and IL-13 in an aGal-dependent manner. Furthermore, these effects correlate with suppressed production of aGal-specific IgE and hypersensitivity reactions as measured by reduced basophil activation and histamine release and the systemic release of mast cell protease- 1 (MCPT-1). Therapeutic administration of two doses of TIMP-AGS nanoparticles to mice sensitized to aGal have efficacy by reducing the production of Th2 cytokines, aGal-specific IgE production, and MCPT-1 release without reducing basophil activation or histamine release. These data identify nanoparticles carrying encapsulated aGal glycoprotein as a diseasemodifying treatment strategy for inducing aGal-specific immune tolerance and reveals diverse mechanisms by which aGal nanoparticles modify immune responses for established aGal- specific IgE-mediated allergic reactions, although an understanding of the mechanisms of action is not required to practice the invention.

[0056] The present disclosure provides compositions of negatively charged particles encapsulating one or more AGS associated antigens. Also included are methods of inducing antigen specific tolerance using the negatively charged particles described herein.

[0057] Definitions

[0058] Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below.

[0059] As used in the specification and the appended 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.

[0060] The term “about” or “approximately” means an acceptable error for 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. Whenever the term “about” or “approximately” precedes the first numerical value in a series of two or more numerical values, it is understood that the term “about” or “approximately” applies to each one of the numerical values in that series.

[0061] “Particle” as used herein refers to any non-tissue derived composition of matter suitable for biological use, it may be a sphere or sphere-like entity, bead, or liposome. The term “particle”, the term “tolerizing immune modifying particle”, the term “carrier particle”, and the term “bead” may be used interchangeably depending on the context. Additionally, the term “particle” may be used to encompass beads and spheres.

[0062] “Negatively charged particle” as used herein refers to particles which have been modified to possess a net surface charge that is less than zero.

[0063] “Surface-functionalized” as used herein refers to particles which have one or more functional groups on its surface. In some embodiments, the surface functionalization occurs by the introduction of one or more functional groups to the surface of a particle. In various embodiments, surface functionalization may be achieved by carboxylation (i.e., addition of one or more carboxyl groups to the particle surface) or addition of other chemical groups (e.g., other chemical groups that impart a negative surface charge).

[0064] “Carboxylated particles” or “carboxylated beads” or “carboxylated spheres” includes any particle that has been modified to contain a carboxyl group on its surface. In some embodiments the addition of the carboxyl group enhances phagocyte / monocyte uptake of the particles from circulation, for instance through the interaction with scavenger receptors such as MARCO. Carboxylation of the particles can be achieved using any compound which adds carboxyl groups, including, but not limited to, poly (ethylene-maleic anhydride) (PEMA).

[0065] As used herein, the term “Th cell” or “helper T cell” refers to CD4+cells. CD4+T cells assist other white blood cells with immunologic processes, 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 are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs).

[0066] As used herein, the term “Thl cell” refers to a subset of Th cells which produce pro- inflammatory mediators. Thl cells secrete cytokines to facilitate immune response and play a role in host defense against pathogens in part by mediating the recruitment of neutrophils and macrophages to infected tissues. Thl cells secrete cytokines including IFN-gamma, IL-2, IL-10, and TNF alpha / beta to coordinate defense against intracellular pathogens such as viruses and some bacteria.

[0067] As used herein, the term “Th2 cell” refers to a subset of Th cells that mediate the activation and maintenance of the antibody-mediated immune response against extracellular parasites, bacteria, allergens, and toxins. Th2 cells mediate these functions by producing various cytokines such as IL-4, IL-5, IL-6, IL-9, IL-13, and IL-17E (IL-25) that are responsible for antibody production, eosinophil activation, and inhibition of several macrophage functions, thus providing phagocyteindependent protective responses.

[0068] As used herein, the term ‘Thl7 cell’ refers to a subset of Th cells that produce pro-inflammatory responses against extracellular bacteria and fungi. Thl7 cells mediate these functions by producing cytokines such as IFN-gamma, IL-17A, IL-17F, IL-21, IL-22, TNF- alpha, and GM- CSF that are responsible for neutrophil, myeloid cell and B-cell recruitment.

[0069] “Polypeptide" and “protein” refer to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof, linked via peptide bonds or peptide bond isosteres. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The terms “polypeptide” and “protein” are not limited to a minimum length of the product. The term "protein" typically refers to large polypeptides. The term "peptide" typically refers to short polypeptides. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full- length proteins and fragments thereof are encompassed by the definition. The terms “polypeptide” and “protein” also include post-expression modifications of the polypeptide or protein, for example, glycosylation. Furthermore, for purposes of the present disclosure, a “polypeptide” can include “modifications,” such as deletions, additions, substitutions (which may be conservative in nature or may include substitutions with any of the 20 amino acids that are commonly present in human proteins, or any other naturally or non-naturally-occurring or atypical amino acids), and chemical modifications (e.g., addition of or substitution with peptidomimetics), to the native sequence. These modifications may be deliberate, as through site-directed mutagenesis, or through chemical modification of amino acids to remove or attach chemical moieties, or may be accidental, such as through mutations arising via hosts cells that produce the proteins or through errors due to PCR amplification prior to host cell transfection.

[0070] The term “glycoprotein” refers to proteins or peptides containing glycans attached to amino acid side chains. The term “glycolipids” refers to lipids containing glycan attached to lipid residues. Glycans are oligosaccharide chains; which are saccharide polymers, that can attach to amino acids (glycoproteins). Typically, these bonds are formed through a process called glycosylation. There are different forms of glycosylation that attach specific glycans to proteins and lipids. For example, N-glycosylation (attachment of glycans to nitrogen on the amine side chain of asparagine) and O-glycosylation (attachment of glycans to oxygen on serine and / or threonine).

[0071] “Antigenic moiety” or “antigen” as used herein refers to any moiety, for example a peptide, that is recognized by the host’s immune system. Examples of antigenic moieties include, but are not limited to, autoantigens, allergens, enzymes, and / or bacterial, viral or tick proteins, peptides, drugs or components.

[0072] “AGS associated antigen” as used herein refers to an antigen, or a portion or fragment thereof, that results in an immune reaction against the agal carbohydrate moiety, agal containing protein, agal containing lipid, or portion or fragment thereof that has been determined to be related to onset of, a symptom of or progression of AGS. agal structure and related information can be found in Figure 10 and Table 1. An AGS associated antigen can include the whole protein or other protein or agal glycolipid lipid, the agal carbohydrate or a moiety associated with the protein that may elicit an immune response in a subject. An AGS associated antigen can include a post-translational modification that results in an immune reaction against the protein or portion or fragment thereof.

[0073] “Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, buffers, and the like, such as a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions (e.g., an oil / water or water / oil emulsion). Non-limiting examples of excipients include adjuvants, binders, fillers, diluents, disintegrants, emulsifying agents, wetting agents, lubricants, glidants, sweetening agents, flavoring agents, and coloring agents. Suitable pharmaceutical carriers, excipients and diluents are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995). Preferred pharmaceutical carriers depend upon the intended mode of administration of the active agent. Typical modes of administration include enteral (e. ., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous or intraperitoneal injection; or topical, transdermal, or transmucosal administration) or via inhalation.

[0074] By “pharmaceutically acceptable” or “pharmacologically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual without causing any undesirable biological effects or without interacting in a deleterious manner with any of the components of the composition in which it is contained or with any components present on or in the body of the individual.

[0075] As used herein, the term “subject” encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, nonhuman primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of nonmammals include, but are not limited to, birds, fish, and the like. The term does not denote a particular age or gender.

[0076] The term “epitope” refers to that portion of any molecule capable of being recognized by and bound by a selective binding agent at one or more of the antigen binding regions. Epitopes usually consist of chemically or immunologically active surface groupings of molecules, such as, amino acids or carbohydrate side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes as used herein may be contiguous or non-contiguous.

[0077] The term mimotope as used herein relates to a peptide that mimics the structure of an epitope. Mimotopes are epitope-mimicking structures suitable for vaccination. They may be linear or circular peptides, or anti -idiotypic antibodies. By virtue of molecular mimicry these mimotopes can induce an immune response not only directed against the immunogen, but also towards the natural epitope expressed or overexpressed on a cell.

[0078] Moreover, epitopes may be mimetic (mimotopes) in that they comprise a three-dimensional structure that is identical to the epitope used to generate the antibody, yet comprise none or only some of the amino acid residues found in the target that were used to stimulate the antibody immune response. As used herein, a mimotope is not considered a different antigen from the epitope bound by the selective binding agent; the selective binding agent recognizes the same three-dimensional structure of the epitope and mimotope.

[0079] The term “therapeutically effective amount” is used herein to indicate the amount of antigenspecific composition of the disclosure that is effective to ameliorate or lessen one or more symptoms or signs of disease to be treated.

[0080] The terms “treat”, “treated”, “treating” and “treatment”, as used with respect to methods herein refer to eliminating, reducing, suppressing or ameliorating, either temporarily or permanently, either partially or completely, a clinical symptom, manifestation or progression of an event, disease or condition. Such treating need not be absolute to be useful.

[0081] Particles

[0082] The size and charge of the TIMPs may be selected to optimize tolerance induction. While the particles will differ in size and charge based on the antigen encapsulated within them, in general, particles described herein are effective at inducing tolerance when they are between about 100 nanometers and about 1500 nanometers and have a charge of between 0 to about -100 mV. In various embodiments, the particles are 400-800 nanometers in diameter and have a charge of between about -25mV and -70mV. In various embodiments, the particles are 400-800 nanometers in diameter and have a charge of between about -30mV and -80mV. In various embodiments, the particles are 400-800 nanometers in diameter and have a charge of between about -30mV and -60mV. The average particle size and charge of the particles can be slightly altered in the lyophilization process, therefore, both post-synthesis averages and postlyophilization averages are described. As used herein, the term “post-synthesis size” and “post synthesis charge” refer to the size and charge of the particle prior to lyophilization. The term “post lyophilization size” and “post lyophilization charge” refer to the size and charge of the particle after lyophilization.

[0083] In some embodiments, the particle is non-metallic. In these embodiments the particle may be formed from a polymer. In a preferred embodiment, the particle is biodegradable in an individual. In this embodiment, the particles can be provided in an individual across multiple doses without there being an accumulation of particles in the individual. 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. Interactions between the particle surface and the interstitium may be a factor that plays a role in lymphatic uptake. The particle surface may be coated with a material to prevent or decrease non-specific interactions. Steric stabilization by coating particles with hydrophilic layers such as poly(ethylene glycol) (PEG) and its copolymers such as PLURONICS® (including copolymers of polyethylene glycol)-bl-poly(propylene glycol)-bl- poly(ethylene glycol)) may reduce the non-specific interactions with proteins of the interstitium as demonstrated by improved lymphatic uptake following subcutaneous injections. All of these facts suggest relevance of the physical properties of the particles in terms of lymphatic uptake. Biodegradable polymers may be used to make all or some of the polymers and / or particles and / or layers. Biodegradable polymers may undergo degradation, for example, by a result of functional groups reacting with the water in the solution. The term "degradation" as used herein refers to becoming soluble, either by reduction of molecular weight or by conversion of hydrophobic groups to hydrophilic groups. Polymers with ester groups are generally subject to spontaneous hydrolysis, e.g., polylactides and polyglycolides.

[0084] Particles disclosed herein may also contain additional components. For example, carriers may have imaging agents incorporated or conjugated to the carrier. An example of a carrier nanosphere having an imaging agent that is currently commercially available is the Kodak X- sight nanospheres. Inorganic quantum-confined luminescent nanocrystals, known as quantum dots (QDs), have emerged as ideal donors in FRET applications: their high quantum yield and tunable size-dependent Stokes Shifts permit different sizes to emit from blue to infrared when excited at a single ultraviolet wavelength. (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 known as dendrimers, may be used in biological labeling, 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, unstable reagents. (Etienne, et al., Appl. Phys. Lett. 87, 181913, 2005).

[0085] Particles can be formed from a wide range of materials. The particle is preferably composed of a material suitable for biological use. For example, particles may be composed of glass, silica, polyesters of hydroxy carboxylic acids, poly anhydrides of dicarboxylic acids, or copolymers of hydroxy carboxylic acids and dicarboxylic acids. More generally, the carrier particles may be composed of polyesters of straight chain or branched, substituted or unsubstituted, saturated or unsaturated, linear or cross-linked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy hydroxy acids, or polyanhydrides of straight chain or branched, substituted or unsubstituted, saturated or unsaturated, linear or cross-linked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy dicarboxylic acids. Additionally, carrier particles can be quantum dots, or composed of quantum dots, such as quantum dot polystyrene particles (Joumaa et al. (2006) Langmuir 22: 1810-6). Carrier particles including mixtures of ester and anhydride bonds (e.g., copolymers of glycolic and sebacic acid) may also be employed. For example, carrier particles may comprise 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(glycolic-co-sebacic) acid copolymers (PGSA), polypropylene sulfide polymers, poly(caprolactone), chitosan, dextran acetate etc. Other biocompatible, biodegradable polymers useful in the present disclosure include polymers or copolymers of caprolactones, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates and degradable urethanes, as well as copolymers of these with straight chain or branched, substituted or unsubstituted, alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy- or di-carboxylic acids. Yet other biocompatible, biodegradable polymers include poly(P-amino esters), hyaluronic acid, citric acid, sialic acid, 3’-sialyllactose, polysaccharide A, glucans, salicyclic acids, bile acids (e.g. ursodeoxycholic acid, cholic acid, lithocholic acid, deoxycholic acid, cheno-deoxycholic acid, urso-deoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, taurodeoxycholic acid, lithocholic acid, taurolitholic acid, taurochenodeoxy cholic acid, tauroursodeoxycholic acid, glycolithocholic acid, glycochenodeoxy cholic acid, and taurine conjugates of 3-alpha-7-alpha-12-alpha-22-xi- tetrahydroxy-5-beta-cholestan-26-oic acid (tetrahydroxystero-cholanic acid) and 3-alpha-12 alpha-22 xi-trihydroxy-5-beta-cholestan-26-oic acid). Monomeric bile acids can be esterified to produce polymeric bile acids. In some embodiments, particle surface is coated with polymeric bile acids. In addition, the biologically important amino acids with reactive side chain groups, such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine and cysteine, or their enantiomers, may be included in copolymers with any of the aforementioned materials to provide reactive groups for conjugating to antigen peptides and proteins or conjugating moieties. Biodegradable materials suitable for the present disclosure include diamond, PLA, PGA, polypropylene sulfide, and PLGA polymers. Biocompatible but non-biodegradable materials may also be used in the carrier particles of the disclosure. For example, non-biodegradable polymers of silicones, P-cyclodextrin-containing polycation, hexadimethrine bromide (HDMBr), and 1,4-diaminebutane core-PAMAM-G3, acrylates, ethylene-vinyl acetates, acyl substituted cellulose acetates, non-degradable urethanes, styrenes, vinyl chlorides, vinyl fluorides, vinyl imidazoles, chlorosulphonated olefins, ethylene oxide, vinyl alcohols, TEFLON (DuPont, Wilmington, Del.), and nylons may be employed.

[0086] In certain embodiments, the particle is a co-polymer having a molar ratio from about 80:20 to about 100:0. Suitable co-polymer ratio of present immune modified particles 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 various embodiments, the particle is a PLURONICS stabilized polypropylene sulfide particle, a polyglycolic acid particle (PGA), a polylactic acid particle (PLA), or a poly(lactic-co-glycolic acid) particle. In various embodiments, the particle is a carboxylated PLGA particle. In various embodiments, the particle has 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 particle is a poly(lactic-co-glycolic acid) particle and has a copolymer ratio of about 50:50 polylactic acid:polyglycolic acid. In various embodiments, the particle comprises about 50:50, about 80:20 to about 100:0 polylactic acid: polyglycolic acid or from about 50:50, about 80:20 to about 100:0 polyglycolic acid: polylactic acid. In various embodiments, the particle comprises 50:50 polylactic acid: polyglycolic acid. In various embodiments, the particle comprises polylactic acid: polyglycolic acid from about 99: 1 to about 1 :99, e.g., about 99: 1, about 95:5, about 90: 10, about 85: 15, about 80:20, about 75:25, about 70:30, about 65:35, about 60:40, about 55:45, about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, about 10:90, about 5:95, and about 1 :99, including all values and ranges that lie in between these values. It is contemplated that the particle may further comprise a surfactant and / or stabilizer. The surfactant can be anionic, cationic, or nonionic. Surfactants in the poloxamer and poloxamines family are commonly used in particle synthesis. Surfactants that may be used, include, but are not limited to PEG, Tween-80, gelatin, dextran, pluronic L-63, polyvinyl alcohol (PVA), polyacrylic acid (PAA), methylcellulose, lecithin, didodecyldimethylammonium bromide (DMAB) and poly(ethylene-alt-maleic acid) (PEMA). Additionally, biodegradable and biocompatible surfactants including, but not limited to, vitamin E TPGS (D-a-tocopheryl polyethylene glycol 1000 succinate), poly amino acids (e.g., polymers of lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine and cysteine, or their enantiomers), sodium cholate, and sulfate polymers. In some embodiments, two surfactants are used. For example, if the particle is produced by a double emulsion method, the two surfactants can include a hydrophobic surfactant for the first emulsion, and a hydrophobic surfactant for the second emulsion.

[0087] In various embodiments, the polypeptide antigens are encapsulated in the particles by a singleemulsion process. In a further embodiment, the polypeptide antigens are more hydrophobic. Sometimes, the double emulsion process leads to the formation of large particles which may result in the leakage of the hydrophilic active component and low entrapment efficiencies. The coalescence and Ostwald ripening are two mechanisms that may destabilize the double-emulsion droplet, and the diffusion through the organic phase of the hydrophilic active component is the main mechanism responsible of low levels of entrapped active component. In some embodiments, it may be beneficial to reduce the nanoparticle size. One strategy to accomplish this is to apply a second strong shear rate. The leakage effect can be reduced by using a high polymer concentration and a high polymer molecular mass, accompanied by an increase in the viscosity of the inner water phase and in increase in the surfactant molecular mass. In certain embodiments, the particles encapsulating antigens are manufactured by nanoprecipitation, coprecipitation, inert gas condensation, sputtering, microemulsion, sol-gel method, layer-by-layer technique or ionic gelation method. Several methods for manufacturing nanoparticles have been described in the literature and are incorporated herein by reference1,2.

[0088] In some embodiments, the particle is a liposome. Liposomes may be prepared from a variety of lipid materials including, but not limited to, lipids of phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidic acid, dicetyl phosphate, monosialoganglioside, polyethylene glycol, stearyl armine, ovolecithin, modified lipids and cholesterol, as well as mixtures of these in varying stoichiometries. Liposomes, as used herein, may also be formed from non-lipid amphipathic molecules, such as block copolymers of poly(oxyethylene-b-isoprene-b-oxye-thylene) and the like. In preferred embodiments, the liposomes are prepared from lipids or incorporate lipids that will form negatively charged liposomes, such as those produced from phosphatidyl serine, dicetyl phosphate, and dimyristoyl phosphatidic acid. In various embodiments, the negatively charged liposomes have a zeta potential from about -100 mV to about 0 mV, from about -100 mV to about -25 mV, from about -100 to about -30 mV, from about -80 mV to about -30 mV, from about -75 mV to about -30 mV, from about -70 mV to about -30 mV, from about -75 to about - 35 mV, from about -70 to about -25 mV, from about -60 mV to about -30 mV, from about -60 mV to about -35 mV, or from 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, including all values and ranges therein. In various embodiments, the liposomes have a negative zeta potential of between -30 mV to -80 mV. In various embodiments, the liposomes have a negative zeta potential of between -30 mV to -60 mV.

[0089] In various embodiments, the liposomes encapsulate one or more AGS associated antigens. In various embodiments, the liposomes encapsulate one or more AGS associated antigens, portions, or combinations thereof. In some embodiments, the AGS associated antigen is a carbohydrate, a peptide, a protein, a glycoprotein, a glycolipid, a homolog, a derivative, a mimotope, or a combination thereof. In various embodiments, the AGS associated antigens are selected from the group consisting of galactose, galactose-a / ?Ar / -l, 3 -galactose (agal), agal containing glycoprotein, or agal containing glycolipid. In some embodiments, the antigen is agal. In various embodiments, the AGS associated antigens are selected from the group consisting of AGS associated tick antigens. In various embodiments, the AGS associated ticks are selected from the group consisting of Amblyomma americanum, Ixodes ricinus, Ixodes holocyclus, Ixodes australiensis, Amblyomma cajennense, Amblyomma hebraeum, Amblyomma testudinarium, Ixodes nipponensis, Amblyomma sculptum, Amblyomma variegatum, Rhipicephalus evertsi, Rhipicephalus burst, Hyaloma marginatum, or Haemaphysalis longicornis. In various embodiments, the TIMP-AGS particles encapsulate one or more polynucleotides encoding AGS associated antigens. In some embodiments, TIMP-AGS particles encapsulate one or more polynucleotides encoding AGS associated antigens and agal. In some embodiments, the polynucleotides comprise DNA, RNA, messenger RNA (mRNA), or circular RNA.

[0090] In various embodiments, the liposomes encapsulate one or more AGS associated antigens, have a negative zeta potential of between -100 mV and 0 mV, and wherein the size of liposomes is between 100 and 1000 nm in diameter. In various embodiments, the liposomes encapsulate one or more AGS associated antigens, portions thereof or combinations thereof, wherein the size of the liposomes is between 400 and 800 nm and the liposomes have a negative zeta potential between -30 mV and -80 mV.

[0091] In various embodiments, the liposomes encapsulate one or more AGS associated antigens, wherein the size of the liposomes is between 100 and 1000 nm, and the liposomes have a negative zeta potential between -100 mV and 0 mV. In various embodiments, the liposomes are used to induce tolerance in a subject with AGS. In various embodiments, the liposome administration is intravenous.

[0092] Antigens

[0093] An antigen refers to a discreet portion of a molecule, such as a polypeptide or peptide sequence, a 3-D structural formation of a polypeptide or peptide, a polysaccharide, polynucleotide or a post-translational or post-expression modification that can be recognized by a host immune cells. Antigen-specific refers to the ability of a subject’s host cells to recognize and generate an immune response against an antigen alone, or to molecules that closely resemble the antigen, as with an epitope or mimotope.

[0094] "Anergy," "tolerance," or "antigen-specific tolerance" refers to insensitivity of T cells to T cell receptor-mediated stimulation. Such insensitivity is generally antigen- specific and persists after exposure to the antigenic peptide has ceased. For example, anergy in T cells is characterized by lack of cytokine production, e.g., IL-2. T-cell anergy occurs when T cells are exposed to antigen and receive a first signal (a T cell receptor or CD-3 mediated signal) in the absence of a second signal (a costimulatory signal). Under these conditions, re-exposure of the cells to the same antigen (even if re-exposure occurs in the presence of a costimulatory molecule) results in failure to produce cytokines and subsequently failure to proliferate. Thus, a failure to produce cytokines prevents proliferation. Anergic T cells can, however, proliferate if cultured with cytokines (e.g., IL-2).

[0095] It is contemplated that the tolerizing therapy described herein is antigen-specific or carbohydrate- antigen specific. For example, TIMPs administered as tolerizing therapy encapsulate one or more antigens associated with said tolerizing therapy and associated disease or condition being treated. It is contemplated that the TIMPs used in tolerizing therapy comprise one or more AGS associated antigens, portions thereof, or combinations thereof. It is also contemplated that the AGS associated antigen is a carbohydrate, a peptide, a protein, a glycoprotein, a glycolipid, a homolog, a derivative, a mimotope, or a combination thereof.

[0096] Table 1

[0097] The term "homolog" as used herein with reference to the epitopes used in the context of the disclosure, refers to molecules having at least 50%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% amino acid sequence identity with the naturally occurring epitope, thereby maintaining the ability of the epitope to bind an antibody or cell surface receptor of a B and / or T cell. Particular homologs of an epitope correspond to the natural epitope modified in, for example, three, two, or one amino acid.

[0098] The term "polynucleotide (or nucleic acid) encoding AGS associated antigen" as used herein refers to a nucleotide sequence, which, when expressed in an appropriate environment, results in the generation of the relevant amino acid sequence or a derivative or homolog thereof. Such polynucleotides or nucleic acids include the normal sequences encoding the peptide, as well as derivatives and fragments of these nucleic acids. The term "sequence identity" of two sequences as used herein relates to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the sequences, when the two sequences are aligned. In particular, the sequence identity is from 50% to 100%, from 60% to 70%, from 70% to 80%, from 81% to 85%, from 86% to 90%, from 91% to 95%, from 96% to 100%, or 100%.

[0099] The term "derivative" as used herein with reference to the antigens described herein refers to molecules which contain at least the peptide active portion and, in addition thereto comprises a complementary portion which can have different purposes such as stabilizing the peptides or altering the pharmacokinetic or pharmacodynamic properties of the peptide e.g. a thioredox motif, or conjugation of a STING agonist.

[0100] In certain embodiments, one, two, three, or a higher number of antigens or antigenic peptides are used in the TIMPs. In certain embodiments, the one or more antigens are encapsulated in the TIMP by covalent linkage to the interior surface of the particle (See e.g., US Patent Publication US20190282707, herein incorporated by reference). In certain embodiments, it is contemplated that sequences of two or more antigens are linked in a fusion protein and encapsulated within a TIMP described herein. Methods for making TIMPs with linked epitopes are described in US Patent Publication US20190365656, herein incorporated by reference.

[0101] Methods of Use

[0102] Provided herein is a method of inducing tolerance in a subject in need thereof comprising administering to the subject a composition comprising negatively charged particles encapsulating an antigen, wherein the antigen is one or more AGS associated antigens, portions, homologs, derivatives, mimotopes, or combinations thereof.

[0103] In various embodiments, the subject is a subject who has been diagnosed with AGS, a subject who is receiving therapy for AGS, a subject who is sensitized to a-gal, a subject who is at risk of developing atherosclerosis associated with a-gal IgE, or a subject who is at risk of developing AGS. AGS is diagnosed through a detailed patient history, physical examination, and a blood test. The main diagnostic test for AGS is a blood test for IgE antibodies specific to agal. Skin tests for reactions to allergens like pork or beef may also be used to help diagnose AGS. A subject who is sensitized to a-gal serum is positive for IgE to a-gal, but not diagnosed with AGS. Sensitized individuals make IgE to a-gal but do not yet show allergic reactions after eating mammalian meat. These individuals are at a risk of developing AGS. Subjects bitten by a tick are also at a risk of developing IgE to a-gal and at a risk of developing AGS.

[0104] AGS is also known as mammalian meat allergy, alpha-gal allergy, red meat allergy, tick bite meat allergy, or red meat allergy.

[0105] If TIMP-AGS particles comprising a single antigen are administered in combination with another carrier particle comprising a different antigen, or second agent, the particles and or second agent can be administered concurrently or sequentially. Concomitant or concurrent administration of two therapeutic agents does not require that the agents be administered at the same time or by the same route, as long as there is an overlap in the time period during which the agents are exerting their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks. It is further contemplated that the therapeutics are administered in a separate formulation and administered concurrently or concomitantly, with concurrently referring to agents given within 30 minutes of each other. Prior administration refers to administration of a therapeutic within the range of one week prior to treatment with a carrier particle, up to 30 minutes before administration of a carrier particle. Subsequent administration is meant to describe administration from 30 minutes after treatment up to one week after administration.

[0106] In various embodiments, TIMP-AGS is administered at a dose of about 0.1 mg, 0.25 mg, 0.5 mg, Img, 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-AGS is administered at a dose from about 0.001 to about 10 mg / kg, from about 0.005 to about 12 mg / kg, from about 0.01 to about 12 mg / kg, from about 0.05 to about 12 mg / kg, from about 0.1 to about 12 mg / kg, about 0.5 to 10 mg / kg, from about 1 to 8 mg / kg, from about 1.5 to 10 mg / kg, from about 2 to 12 mg / kg, from about 2 to 10 mg / kg, from about 3 to 10 mg / kg, from about 4 to 10 mg / kg, from about 4 to 12 mg / kg, or from about 5 to 12 mg / kg. Optionally, TIMP-AGS is administered in a dose of about 0.001 mg / kg, about 0.0025 mg / kg, about 0.005 mg / kg, about 0.01 mg / kg, about 0.025 mg / kg, about 0.05 mg / kg, 0.1 mg / kg, 0.25, 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. In another embodiment, particles are administered at a concentration of between about 0.0005 mg / mL and about 50 mg / mL between about 0.05 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.

[0107] TIMP-AGS particles can be given in any dose effective to dampen the allergic or immunogenic response in a subject in need thereof or to treat AGS in a subject in need thereof. In certain embodiments, about 102to about 1020particles are provided to the subject in need thereof. In a further embodiment between about 103to about 1015particles are provided. In yet a further embodiment between about 106to about 1012particles are provided. In still a further embodiment between about 108to about 1010particles are provided. In a further embodiment the dose is 0.1% solids / ml. Therefore, for 0.5 pm particles, a preferred dose is approximately 4*109particles, for 0.05 pm particles, a preferred dose is approximately 4* 1012beads, for 3 pm particles, a preferred dose is 2* 107particles. However, any dose that is effective in treating AGS is encompassed by the current invention.

[0108] In various embodiments, the disclosure provides a method of treating AGS (mammalian meat allergy / alpha-gal allergy / red meat allergy / tick bite meat allergy / red meat allergy) or AGS related symptoms, in a subject in need thereof comprising administering to the subject a composition comprising TIMP-AGS alone or in combination with a therapeutic useful to treat AGS. In various embodiments, the therapeutic useful to treat AGS induces regulatory T-cells (Tregs). In various embodiments, the therapeutic useful to treat AGS increases the frequency and / or the number Tregs. In various embodiments, the therapeutic useful to treat AGS is IL-2 therapy to induce Tregs. In various embodiments, the IL-2 therapy is low dose IL-2, IL-2 muteins engineered to expand Tregs, IL-2 variants engineered to expand Tregs, IL-2 molecules engineered to be selective for the high-affinity IL-2 receptor, PEGylated IL-2, IL-2 complexes, or IL-2 / CD25 fusion proteins. In various embodiments, the therapeutic useful to treat AGS is a prebiotic, a probiotic, histone deacetylase inhibitor, short chain fatty acids (e.g. acetate, butyrate, propionate, butyrate polymer), an inhibitor of IgE, competitor of IgE for allergen binding sites, an inhibitor of basophil activation, an inhibitor of mast cell activation, an antihistamine, a cytokine inhibitor, microbiome therapy, or a small molecule or biological therapeutic. In various embodiments, the therapeutic inhibits IgE antibodies. In various embodiments, the therapeutic inhibits basophil activation. In various embodiments, the therapeutic inhibits mast cell activation. In various embodiments, the therapeutic is a biologic or a small molecule. In various embodiments, the additional therapeutic is an anti-IgE antibody, an anti-IL-4Ra antibody, an anti-IL13 antibody, an anti-IL-33 antibody, an antihistamine, a steroid, a corticosteroid, a leukotriene modifier, or a nonsteroid anti-inflammatory drug (NSAID).

[0109] In various embodiments, the therapeutic 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 antihistamines are 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.

[0110] In various embodiments, the therapeutic 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.

[0111] In various embodiments, the therapeutic is a corticosteroid. In various embodiments, the corticosteroid is selected from the group consisting of cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, and hydrocortisone.

[0112] In various embodiments, the therapeutic is a nonsteroid 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 diclofenac, diclofenac potassium, diclofenac sodium, diflunisal, etodolac, flurbiprofen, fenoprofen, fenoprofen calcium, ketorolac, ketorolac tromethamine, ketoprofen, tolmetin,tolmetin sodium, acetylsalicylic acid, aspirin, ibuprofen, naproxen, indomethacin, indomethacin sodium, sulindac, felbinac, piroxicam, mefenamic acid, meclofenamate sodium, meloxicam, nabumetone, oxaprozin, piroxicam, celecoxib, etodolac, etoricoxib, lumiracoxib, rofecoxib, and valdecoxib. In various embodiments, the therapeutic is a leukotriene modifier. In various embodiments the leukotriene modifier is an antileukotriene. In various embodiments the leukotriene modifier is a leukotriene receptor antagonist. In various embodiments the leukotriene modifier is a leukotriene synthesis inhibitor. In various embodiments the leukotriene modifier is selected from the group consisting of montelukast, zileuton, and zafirlukast.

[0113] In various embodiments, the biologic is an antibody. In various embodiments, the antibody is an anti-IgE, anti-lL-4Ra, anti-IL-13, or an anti-LL-33 antibody. In various embodiments, the anti- IgE antibody is omalizumab (XOLALR®). In various embodiments, the anti-IL-4Ra antibody is dupilumab (DUPIXENT®). In various embodiments, the anti-IL-33 antibody is etokinumab. In various embodiments, the therapeutic is administered prior to, during, or after the administration of TIMP-AGS.

[0114] In various embodiments, the one or more therapeutic is administered prior to, concurrently with or subsequent to administration of TIMP-AGS particles described herein. In various embodiments, the therapeutic is administered 0.5, 1, 2, 4, 5, 8, 10, 12, 16 or 24 hours prior to administration of TIMP-AGS including all ranges and values that lie between those ranges. In various embodiments, the therapeutic is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-AGS. In various embodiments, the therapeutic is administered 1, 2, 3, or 4 weeks prior to administration of TIMP-AGS. In various embodiments, the therapeutic is administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months prior to administration of TIMP-AGS. In various embodiments, the therapeutic is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years prior to administration of TIMP-AGS. In various embodiments, the therapeutic is administered concurrently with TIMP-AGS. In various embodiments, the therapeutic is administered 0.5 1, 2, 4, 5, 8, 10, 12, 16 or 24 hours subsequent to administration of TIMP-AGS including all ranges and values that lie between those ranges. In various embodiments, the therapeutic is administered 1, 2, 3, 4, 5, 6, or 7 days subsequent to administration of the TIMP- AGS. In various embodiments, the therapeutic is administered 1, 2, 3, or 4 weeks subsequent to administration of TIMP-AGS. In various embodiments, the therapeutic is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months subsequent to administration of the TIMP-AGS. In various embodiments, the therapeutic is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years subsequent to administration of TIMP-AGS described herein. Screening Methods

[0115] It is contemplated that induction of, and maintenance of immunological tolerance is monitored in a subject suffering from AGS who is treated, or about to undergo treatment, with carrier particles as described herein.

[0116] Methods of screening for cell types, cytokines or other measures of tolerance from a subject undergoing tolerizing therapy as described herein are known in the art. Methods of assessing tolerance are done using such techniques as flow cytometry, Mass Cytometry (CyTOF), ELISA, ELISPOT, in vitro! ex vivo cell stimulation assays (including, but not limited to, cell proliferation assays, basophil activation test (BAT), macrophage stimulation assays), measuring autoantibodies or measuring Ig serotype, e.g., by ImmunoCap assay.

[0117] A list of human metabolites that can be assayed from a biological sample can be found in the literature including in (Psychogios et al., 2011), (Wishart et al., HMDB: the Human Metabolome Database. Nucleic Acids Res. 2007 Jan; 35(Database issue):D521-6, 2007), and the Human Metabalome Database (HMDB) and are incorporated herein by reference.

[0118] One aspect of a subject’s immune tolerance status, and immune signature, is determined by analyzing one or more cell-surface proteins from a biological sample(s). In various embodiments, the cell-surface proteins include CDlc, CD2, CD3, CD4, CD5, CD8, CD9, CD 10, CDl lb, CDl lc, 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, CD45RO, 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, CD127, 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, KIR3DL3, KIR3DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS, NKp44, NKp46, TCR, BCR, Integrins, FcPsRI, MHC-I, MHC-II, IL-1R, IL- 2Ra, IL-2RP, IL-2Ry, IL-3Ra, CSF2RB, IL-4R, IL-5Ra, CSF2RB, IL-6Ra, gp!30, IL-7Ra, IL- 9R, IL-10R, IL-12RP1, IL-12RP2, IL-13Ral, IL-13Ra2, IL-15Ra, IL-21R, IL-23R, IL-27Ra, IL-31Ra, OSMR, CSF-1R, cell-surface IL-15, IL-lORa, IL-1OR0, IL-20Ra, IL-20RP, IL-22Ral, IL-22Ra2, IL-22RP, 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, LTaip2, LTPR, 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,a-SMA, FAS, FAS-L, FC, ICAM- 1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM-1, MICA, MICB, ULI 6, ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULTI, RAE1 a,P,y,6, and s, H60a, H60b, H60c, GPR15, ST2, and / or combinations thereof. Integrins include al, a2, allb, a3, a4, a5, a6, a7, a8, a9, alO, al l, aD, aE, aL, aM, aV, aX, pi, P2, p3 , P4, P5, P6, P7, P8 and / or combinations thereof. TCR include a, P, y, 8, s, , chains and / or combinations thereof. Several methods have been described in the literature for assaying of cell-surface protein expression, including Flow Cytometry and Mass Cytometry (CyTOF).

[0119] In various embodiments, treatment with TEMP -AGS decreases the expression of inflammatory cell surface proteins by 5%-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%, inclusive of all values and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30- 70%, 35-65%, 40-60%, 45-55%, or 50% or by about 2-100-fold (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-fold inclusive of all values and ranges between these values) relative to the subject’s baseline measurement and / or relative to a healthy subject. In various embodiments, treatment with TIMP-AGS increases the expression of anti-inflammatory cell surface proteins by 5%-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%, inclusive of all values and ranges between these values), 10-95%, 15-90%, 20- 85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or by about 2-100-fold (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-fold inclusive of all values and ranges between these values) relative to the subject’s baseline measurement and / or relative to a healthy subject.

[0120] In various embodiments, administering TIMP-AGS to a subject in need thereof, alone or in combination with one or more additional therapeutics, reduces the duration and severity of an allergic or immunogenic immune response to ocgal. In various embodiments, administering TIMP-AGS to a subject in need thereof, alone or in combination with one or more additional therapeutics, reduces the duration and severity of an allergic or immunogenic immune response following exposure to ocgal. In various embodiments, the allergic or immunogenic 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, administering TIMP-AGS to a subject in need thereof reduced Th2 cytokine IL-4, IL-5, or IL-13. In various embodiments, administering TIMP-AGS to a subject in need thereof reduces total IgE production, tick-specific IgE production or aGal-specific IgE production. In various embodiments, administration of TIMP-AGS decreases frequencies and activation of basophils and mast cells. In various embodiments, administering TIMP-AGS reduces total, tick-specific or aGal-specific IgE / IgG ratios. Basophil activation can be measured by Basophil Activation Test (BAT), or histamine levels and mast cell reactivity by MCPT-1. In various embodiments, the reduction is by about 1%-100% (e.g. about 1%, about 2%, 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%, inclusive of all values and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35- 65%, 40-60%, 45-55%, or 50% or by about 2-100-fold (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-fold inclusive of all values and ranges between these values) relative to placebo administration and / or one or more baseline measurements taken from the subject during treatment.

[0121] In various embodiments, administering TIMP-AGS increases total IgG production, tick-specific IgG production or aGal-specific IgG. In various embodiments, administering TIMP-AGS increases IL-10 production. In various embodiments, the increase is by about 1%-100% (e.g. about 1%, about 2%, 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%, inclusive of all values and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or by about 2-100-fold (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-fold inclusive of all values and ranges between these values) relative to placebo administration and / or one or more baseline measurements taken from the subject during treatment.

[0122] In various embodiments, administering TIMP-AGS to a subject in need thereof, alone or in combination with one or more therapeutics ameliorates AGS hypersensitivity following ocgal containing meat consumption (e.g., red meat like cows, pigs, lambs, rabbits, buffalos, bison, and kangaroos), xenotransplation with bovine or porcine organs, consumption of ocgal containing pharmaceuticals, medicines, gelatin capsules. In various embodiments, administering TIMP- AGS to a subject in need thereof, alone or in combination with one or more therapeutics useful to AGS, relieves one or more symptoms of AGS following ocgal containing meat consumption (e.g., red meat like beef, pork), xenotransplation with bovine or porcine organs, consumption of ocgal containing pharmaceuticals, medicines, gelatin, collagen. In various embodiments, the symptoms of AGS are selected from the group consisting of skin reactions, hives, skin redness, skin swelling, itching, tightening of the throat, difficulty breathing, shortness of breath, digestive problems such as diarrhea, stomach cramps, nausea, vomiting, drop in blood pressure, and anaphylaxis.

[0123] In certain embodiments, the subject’s tolerance status is determined by analyzing nucleic acids from the biological sample(s). In various embodiments, the nucleic acids are DNA and / or RNA, including, but not limited to, single stranded DNA, double stranded DNA, mRNA, rRNA, tRNA, siRNA, microRNA (miRNA), long non-coding RNAs (long ncRNAs, IncRNA), and non-coding RNA (ncRNA), mitochondrial RNA. In various embodiments, the subject’s immune tolerance status is determined by assaying gene expression from the biological sample(s). In various embodiments, the immune tolerance status is determined by assaying gene expression associated with immune function, an antibody, foreign body response, metabolism, apoptosis, cell death, necrosis, ferroptosis, autophagy, cell migration, endocytosis, phagocytosis, pinocytosis, tight- junction regulation, cell adhesion, differentiation, and / or combinations thereof. In various embodiments, the immune tolerance status is determined by assaying gene expression associated with immune suppression. In various embodiments, the immune tolerance status is determined by assaying gene expression associated with immune activation. In various embodiments, the immune tolerance status is determined by assaying gene expression associated with immune regulatory functions. In various embodiments, nucleic acid analysis is used to generate an immune tolerance signature. Several methodologies 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 analyses.

[0124] The biological sample is optionally assayed after in vivo and / or ex vivo stimulation with one or more stimuli such as an antigen, an allergen, and one or more activating agents. It is contemplated that the T cells, B cells, and immunoglobulins used in the assay are antigen specific. Exemplary T cells include effector memory T cells, antigen specific T cells, activated antigen specific T cells, Thl cells, pathogenic Th2a+ cells, Thl7 cells, T follicular helper (TFH) cells, THO cells, or other antigen-specific T cells. B cells include effector B cells, memory B cells, plasma cells, and regulatory B (Breg) cells.

[0125] In various embodiments, the immune tolerance status of the subject is determined by obtaining one or more samples, e.g., whole blood, from the subject pre-dose on the day of the first TIMP administration (Day 1), and at a date(s) after administration. Whole blood can then be processed to isolate peripheral blood mononuclear cells (PBMCs), basophils, neutrophils, plasma, and serum for downstream analyses. Assay of cells isolated from one or more samples collected from the subject and analyzed.

[0126] Pharmaceutical Formulations

[0127] Pharmaceutical compositions of the present disclosure containing the TIMP described herein and an antigen may contain pharmaceutically acceptable carriers or additives depending on the route of administration. Examples of such carriers or additives include water, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carbox-yvinyl polymer, carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, Vaseline®, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptable surfactant and the like. Additives used are chosen from, but not limited to, the above or combinations thereof, as appropriate, depending on the dosage form of the present disclosure.

[0128] Formulation of the pharmaceutical composition will vary according to the route of administration selected (e.g., solution, emulsion). An appropriate composition comprising the therapeutic to be administered can be prepared in a physiologically acceptable vehicle or carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s or fixed oils. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers.

[0129] A variety of aqueous carriers, e.g., sterile phosphate buffered saline solutions, bacteriostatic water, water, buffered water, 0.4% saline, 0.3% glycine, and the like, and may include other proteins for enhanced stability, such as albumin, lipoprotein, globulin, etc., subjected to mild chemical modifications or the like.

[0130] Therapeutic formulations of the inhibitors are prepared for storage by mixing the inhibitor having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl para-bens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; 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 dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn- protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0131] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0132] 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 acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate.

[0133] The TIMP comprising antigen as described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use.

[0134] 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 di calcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0135] Kits

[0136] The disclosure also provides kits which comprise one or more compounds or compositions packaged in a manner which facilitates their use to practice methods of the disclosure. 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 another agent), packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the compound or composition in practicing the method.

[0137] Preferably, the compound or composition is packaged in a unit dosage form. The kit may further include a device suitable for administering the composition according to a specific route of administration or for practicing a screening assay. Preferably, the kit contains a label that describes use of the particle compositions.

[0138] Additional aspects and details of the disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting.

[0139] EXAMPLES

[0140] Materials and methods

[0141] Mice. The alpha- 1,3 -galactosyl transferase' ' (AGKO) mice have been described and were bred on a C57BL / 6 background. Studies used 2-month-old, age-matched mice of both sexes with a mean weight of 18-22 gm and were randomly allocated to each experimental or control group. No mice were excluded from analysis. All mice were bred and maintained in the specificpathogen free animal facilities at the University of Virginia with the approval of the Institutional Animal Care and Use Committee protocol #3506 and were used in compliance with the Association for Assessment and Accreditation of Laboratory Animals Care policies.

[0142] Generation and Characterization of aGal and Control Nanoparticles (NP). A 12.5 w / w% solution of aGal-human serum albumin (aGal-P-l,4-GlcNAc-HSA with 3 atom spacer; Dextra UK) in human serum albumin (Sigma) was prepared and dissolved at 200 mg / mL in phosphate buffered saline (PBS) for generation of NP-aGal. Alternatively, a 200 mg / mL HSA protein (Sigma) solution in PBS was used as a starting point to produce control HSA NPs. 150 pL of either the aGal glycoprotein or HSA solution was then added to 2 mL of 20% w / v 50:50 poly(lactide-co-glycolide)-COOH (i.v.=0.18; Evonik) dissolved in dichloromethane. Following sonication, 10 mL of 2% w / v poly(ethylene-alt-maleic anhydride) (PEMA; MW 400 kDa; Polysciences, Inc.) was added and the solution was sonicated again. This solution was subsequently added to a 0.5% w / v PEMA solution and continuously stirred overnight to allow for dichloromethane evaporation. The resulting solid NP-aGal or NP-HSA were washed with 0.1 M sodium bicarbonate-sodium carbonate buffer, pH 9.6 (Polysciences, Inc.) and then lyophilized in cryoprotectant consisting of 3% w / v aqueous D-mannitol and 4% w / v aqueous sucrose. Dynamic light scattering was performed on each batch of NPs using a Zetasizer Nano ZSP to ensure a diameter of between 400 and 700 nm, a surface zeta potential of <-35 mV, and a polydispersity index of <0.3.

[0143] Nanoparticle Treatment and IgE Sensitization For prophylactic NP studies, AGKO mice were treated with 2.5 mg of i.v. aGal-HSA NPs, 2.5 mg of i.v. HSA NPs, or an equivalent volume (100 pL) of PBS at days -28 and -14 (Fig. la). Each mouse was subsequently given three intradermal (i.d.) injections of 50 pg whole body protein extract prepared from Amblyomma americanum seed ticks (Oklahoma Tick Rearing Facility) and 50 pg aGal-BSA on days 0, 7, and 31 to induce IgE sensitization to aGal as previously described. On day 35, the mice were administered 250 pg beef extract via i.g. challenge and serum and tissues were subsequently harvested after 90 minutes for further analysis. For therapeutic NP studies, AGKO mice were given three intradermal (i.d.) injections of 50 pg whole body protein extract prepared from Amblyomma americanum seed ticks and 50 pg aGal-BSA on days 0, 7, and 31 (Fig. 4a). On days 33 and 47, mice were treated with 2.5 mg of i.v. NP-aGal, 2.5 mg of i.v. NP-HSA, or PBS. On day 63, the mice were given an additional i.d. injection of 50 pg whole body protein extract and after five days were administered 500 pg beef extract via i.g. challenge. Serum and tissues were subsequently harvested after 90 minutes for further analysis.

[0144] Splenocyte Recall Assays Spleens were harvested 90 minutes after i.g. beef extract challenge and manually disrupted to generate single-cell suspensions. Red blood cells were depleted using ACK lysing buffer. Lymphocytes were resuspended in culture medium (DMEM, 5% FBS, 2 mM L-glutamine, 1% NEAA, 1 mM sodium pyruvate, lO mM MOPS, 50 pM 2-mercaptoethanol, 100 IU penicillin, and 100 pg / mL streptomycin) and plated at 800,000 cells per well in tissue culture- treated 96-well flat-bottom plates. Cells were stimulated with 10 pg / mL cetuximab anti- epidermal growth factor receptor (EGFR) antibody (Lilly) or left unstimulated in cell culture medium for use as controls. After 72 hours, secretion of the cytokines TFNy, IL-4, IL-5, IL-6, IL- 10, IL-13, and TNFa was measured in cell culture supernatants using a Luminex Multiplex detection system (EMD Millipore). For each sample, data were determined as follows: [Stimulated] - [Unstimulated] = Total (pg / mL) for each cytokine (mean of duplicate determinations) to show antigen-specific cytokine production.

[0145] Serum ELISA for Tick and aGal-Specific IgE and MCPT-1 Quantification. Total serum IgE and tick-specific IgE were determined by ELISA. For total serum titers, Costar high binding plates (Corning) were coated with 0.83 pg / ml unlabeled anti-mouse IgE in PBS (Southern Biotechnologies). For tick-specific ELISAs, high binding plates were coated with tick extract at a concentration of 10 pg / ml in PBS. Serum was diluted at 1 : 100 for IgE and serially titrated in 3- fold increments. HRP -labeled anti-mouse IgE (Southern Biotechnologies) served as the detection antibody, and the assay was developed using tetramethylbenzidine (BD Pharmingen) with 2N H2SO4 used as the stop solution. Total IgE antibody titers were quantified through a standard curve obtained using unlabeled IgE (Southern Biotech), whereas tick-specific IgE OD values were calculated as arbitrary units using a standard curve with pooled sera (starting at 1 : 50 and followed by 3-fold dilutions) from previous experiments. Alpha-gal IgE was detected by Luminex using a modified version of the Milliplex MAP mouse IgE single plex magnetic bead kit (Millipore Sigma, MGAMMAG-300E), where the kappa PE was replaced by alpha-gal conjugated with biotin followed by streptavidin PE. Briefly, mouse sera (diluted 1 : 100 in assay buffer) was incubated with anti-mouse IgE beads for Ih at RT. After washing, beads were incubated with 25 pg / mL biotinylated alpha-gal (Dextra Laboratories, NGB1334) for 18 hr at 4°C, washed again, and incubated with 25 pL streptavidin-PE (Millipore Sigma, L-SAPE12) for 30 min at RT, washed, and resuspended in 150 pL PBS. Samples were run through a Luminex XMAP INTELLIFLEX in the Flow Cytometry Core at the University of Virginia. Data are expressed as PE mean fluorescence intensity (MFI) of the samples minus the PE MFI of the blank. All samples were assessed in duplicate. Mouse MCPT-1 serum levels were measured by ELISA according to the manufacturer’s protocol (BioLegend). All ELISA assays were analyzed at 450 nm using a BioTek plate reader. Flow Cytometry. Mouse peripheral blood, inguinal and mesenteric lymph nodes were collected 90 minutes after oral gavage with beef extract and analyzed for frequencies of CD45+CD49b+Fc£Rl+c-kit" basophils, CD45+FcsRl+c-kit+mast cells, or CD45+CD103+CDl lc+F4 / 80‘ dendritic cells as previously described(14). Cells were stained with CD45-APC (Clone 104; eBioscience), CD49b-PerCP-Cy5.5 (Clone DX5; BioLegend), FcsRlalpha-FITC (Clone MAR-1; BioLegend), c-kit-PE-Cy7 (Clone 2B8; eBioscience), CD103-BV711 (Clone M290; BD Biosciences), CD1 Ic-PE (Clone HL3; BD Biosciences), and F4 / 80-PerCP-Cy5.5 (Clone T45-2342; BD Biosciences). In stains as indicated, cells were also stained with CD200R-PE (Clone OX- 108; BioLegend) and CD41 -Brilliant Violet 421 (Clone WMReg30; BD Biosciences). Cell viability was determined using LIVE / DEAD Aqua (Invitrogen) and doublets were excluded based on forward scatter and pulse width. Samples were fixed in 1% paraformaldehyde, washed, and acquired on an Attune Nxt cytometer and analyzed using FlowJo software version 10.8.2 (Tree Star). Gates were determined using fluorescence minus one staining controls.

[0146] Basophil Activation Test Heparinized blood from individual mice was obtained and the resultant cells were cultured in basophil culture medium (5% AB serum RPMI supplemented with penicillin / streptomycin and 1 mM L-glutamine) in 96-well v-shaped plates for 1.5 hour at 37°C with or without 10 pg / ml cetuximab as previously described. The cells were then spun down, and the culture supernatant was collected and frozen at -20°C. Histamine levels were subsequently measured by a competitive ELISA kit (Enzo Life Science) in the supernatant according to manufacturer’s instructions. Fold increase was calculated as cetuximab divided by non-stimulated sample for each mouse. All plates were read at 450 nm using a BioTek plate reader.

[0147] Statistical Analysis. Statistics were determined using Prism software vlO (GraphPad Software, Boston, MA). To assess differences between groups, the unpaired, two-tailed / -test or the nonparametric Mann-Whitney test was used. Error bars shown in each figure indicate mean ± SEM. Significance was defined as p values < 0.05 and are stated in the figure legends.

[0148] EXAMPLE 1

[0149] Prophylactic treatment with aGal glycoprotein-containing nanoparticles reduces sensitization to the aGal oligosaccharide. PLG NPs encapsulating protein allergens imitate the size and charge of apoptotic cellular debris and directly associate with antigen presenting cells (APCs), leading to a reduction in the secretion of Th2 cytokines. Experiments were performed to determine whether NPs containing aGal glycoprotein would induce a level of tolerance sufficient to prevent sensitization to aGal when NPs were administered prophylactically to an established mouse model of cutaneous sensitization to aGal. This mouse model uses the proallergic adjuvant effects of lone star tick protein extracts and the glycoprotein aGal-bovine serum albumin (BSA) to reliably induce aGal-specific IgE production in mice deficient in aGal (AGKO). Nanoparticles containing the glycoconjugate aGal-human serum albumin (NP-aGal), and as controls, with human serum albumin alone (NP-HSA), were generated with a diameter of -500 nm and a zeta potential of — 40 mV. To test the effects of prophylactic treatment with NPs on immune cells responding to cutaneous aGal exposure, two doses of allergen-encapsulating NPs or an equivalent volume of phosphate buffered saline (PBS) were intravenously delivered two weeks apart to AGKO mice (Fig. la). AGKO mice were subsequently given a series of intradermal injections with lone star tick protein extract and aGal-BSA prior to intragastric challenge with aGal-containing beef extract as previously described. Unsensitized controls consisted of naive mice. At day 35, mice that were sensitized after PBS i.v. injection displayed significant reactions to intragastric beef extract challenge as measured by increased serum MCPT-1 levels (Fig. lb) and histamine levels released by circulating blood basophils after in vitro stimulation with cetuximab, a monoclonal antibody containing aGal moieties in the Fab portion of its heavy chain (Fig. 1c). As expected, neither MCPT-1 in serum nor histamine released by cetuximab-stimulated basophils were detected from naive mice, demonstrating that sensitization to aGal through the skin contributes to a hypersensitivity response following meat consumption. Mice that were sensitized after treatment with NPs that contained aGal (NP-aGal) showed significantly reduced levels of histamine and MCPT-1 compared to levels from mice treated with PBS following intragastric beef extract challenge, supporting reduced sensitization to aGal in mice that were prophylactically treated with NP-aGal. Mice that were intradermally exposed to lone star tick protein extract and aGal-BSA after treatment with NPs that contained the irrelevant HSA protein (NP-HSA) showed increased levels of histamine released by basophils similar to the levels observed from mice treated with PBS. Increased levels of MCPT-1 were found in sera from mice treated with NP-HSA following intragastric beef extract challenge but were significantly less compared to mice treated with PBS. Sera from AGKO mice administered NPs or PBS and then immunized with tick protein extract and aGal-BSA was examined for levels of total IgE and tick antigen- and aGal-specific IgE by ELISA. First, it was confirmed that total IgE, tick-, and aGal-specific IgE levels were induced in sera from sensitized mice treated with PBS compared to naive controls (Fig. Id). These IgE antibodies were also induced in sensitized mice treated with control NP-HSA. Total IgE and tick-specific IgE levels were induced in sensitized mice treated with NP-aGal, similar to PBS- and NP -EISA-treated mice, with tick-specific IgE levels significantly reduced relative to PBS treatment. aGal-specific IgE levels were significantly reduced in NP-aGal -treated mice with 36% of mice expressing aGal-specific IgE compared to the PBS- and NP-HSA-treated groups that had 82% and 80% of mice expressing aGal-specific IgE, respectively (Fig. Id, right panel). The total numbers of germinal center B cells (B220+GL-7+CD95+) and IgE+plasma cells (B220- CD138+) within the skin draining inguinal lymph nodes were increased in all groups of sensitized mice compared to naive controls, with a trend to fewer numbers of both cell types measured in NP-aGal-treated animals though not statistically significant (Fig. 7). These results suggest that the mechanism by which MCPT-1 and histamine release levels were reduced in mice prophylactically treated with NP-aGal and orally challenged with beef extract was in part through reduced aGal-specific IgE production.

[0150] EXAMPLE 2

[0151] Prophylactic treatment with aGal glycoprotein-containing nanoparticles reduces basophil frequencies and activation in mesenteric lymph nodes. In murine models of food allergy, basophils play a significant role in the sensitization phase. To determine the impact of prophylactic administration of NPs on basophils, AGKO mice were sensitized with tick extract plus aGal-BSA after treatment with NP-aGal, NP-HSA, or PBS and assessed for the frequency and activation of basophils by flow cytometry. Circulating and mesenteric lymph node basophils that drain the gastrointestinal tract were a focus of analysis because of the oral challenge. Analysis of peripheral blood basophils (CD45+CD49b+Fc£Rl+IgE+c-kit‘) from all groups of sensitized mice showed that the percentages of basophils significantly increase after intragastric challenge with beef extract compared to naive controls (Fig. 2a; Fig. 8). Sensitized mice also exhibited increased frequencies of blood basophils that expressed the basophil activation markers CD200R and CD41, following oral challenge with beef extract regardless of prophylactic treatment (Fig. 2b). The groups of sensitized mice that were prophylactically treated with NP- HSA and PBS also showed that both the percentages and the numbers of basophils significantly increase in the mesenteric lymph nodes compared to naive controls (Fig. 2c). Moreover, the percentages and the total numbers of basophils in the mesenteric lymph nodes that expressed CD41 and CD200R were similarly increased in these groups of mice (Fig. 2d). In contrast, sensitized mice that were prophylactically treated with NP-aGal showed significantly reduced frequencies and activation of basophils in the mesenteric lymph nodes compared to sensitized mice that were administered NP-HSA and PBS (Fig. 2c, d). Mast cells are found throughout the gastrointestinal tract and are increased in food-allergic subjects. As expected, increased percentages and numbers of FcsR l c-kit mast cells in the mesenteric lymph nodes were found in sensitized mice prophylactically treated with NP-HSA and PBS compared to naive controls (Fig. 2e). Mice treated with NP-aGal showed reduced percentages of mast cells and fewer numbers in mesenteric lymph nodes but did not reach significance. Taken together, these results demonstrate that tick-induced sensitization to aGal promotes increased intestinal basophils and mast cells which are reduced by prophylactic NP-aGal treatment. Decreased activation of mesenteric lymph node basophils (Fig. 2d) and serum levels of MCPT-1 (Fig. lb) in sensitized mice treated with NP-aGal suggests that NP-aGal treatment also reduces degranulation of basophils and mast cells in response to intragastric challenge with aGal-containing beef extract.

[0152] EXAMPLE 3

[0153] Prophylactic treatment with aGal glycoprotein-containing nanoparticles reduces Th2 cytokine production. PLG NPs were given intravenously to deliver protein cargo to antigen presenting cells in the spleen reduce Th2 cell activation. The effects of prophylactic administration of NPs on allergic cytokine production by splenic cells following stimulation with aGal were investigated. Splenocytes from mice that were sensitized after treatment with NP- aGal, NP-HSA, or PBS were obtained and stimulated with cetuximab to induce aGal-dependent cytokine production and assessed 3 days later for the presence of Thl, Th2, and regulatory T cell cytokines in the cell culture medium. Splenocytes from naive mice served as negative controls. Analysis of the response to cetuximab in recall assays of splenic cells from NP-aGal -treated mice but not mice treated with NP-HSA or PBS showed a significant reduction in secretion of the Th2 cytokines IL-4, IL-5, IL-6, and IL-13 (Fig. 3a). No effects were detected in secretion of the Thl cytokines IFN-y and TNF-a regardless of the NP treatment group compared to PBS controls (Fig. 3b). Antigen-specific T cell suppression through the production of IL-10 contributes to the development of oral tolerance. Increased IL- 10 secretion from recall assays of splenocytes of NP-aGal -treated mice but not mice treated with NP-HSA or PBS was found, suggesting that aGal-dependent induction of IL-10 secretion contributes to reduced sensitization to aGal (Fig. 3c). Taken together, these results show that prophylactic NP-aGal treatment blocks the secretion of Th2 cytokines associated with food allergy.

[0154] EXAMPLE 4

[0155] Therapeutic treatment with aGal glycoprotein-containing nanoparticles reduces allergic responses to aGal. The ability of therapeutically administered NPs containing aGal to reduce allergic reactivity in AGKO mice with established sensitization to aGal was determined. Mice were sensitized intradermally with tick extract plus aGal -B SA and received two intravenous doses of NPs or PBS, followed two weeks later by a booster with tick extract, and four days after that with an intragastric challenge with beef (Fig. 4a). At day 68, mice that were sensitized and treated with PBS displayed significant reactions to intragastric beef extract challenge as measured by increased serum MCPT-1 levels (Fig. 4b) and histamine levels released by circulating blood basophils after in vitro stimulation with cetuximab (Fig. 4c). Neither MCPT-1 in serum nor histamine released by cetuximab-stimulated basophils were detected from naive mice. Mice that were sensitized and treated with NP-aGal showed significantly reduced levels of MCPT-1 and lower levels (though not statistically significant) of histamine compared to levels from mice treated with PBS or control NP-HSA following intragastric beef extract challenge, supporting reduced hypersensitivity to aGal in mice that were therapeutically treated with NP- aGal. In contrast, basophils activated with cetuximab showed no increase in histamine release over unstimulated basophils from the peripheral blood of mice treated with NP-HSA (Fig. 4c), which resulted from greater basal levels of histamine released from unstimulated basophils (Fig. 9). The levels of total IgE and tick-specific IgE were induced in sera from all groups of sensitized mice regardless of therapeutic treatment compared to naive controls (Fig. 4d). aGal- specific IgE levels were significantly reduced in mice treated with NP-aGal or NP-HSA compared to the PBS-treated and naive groups.

[0156] An increase in peripheral blood basophils expressing the activation markers CD200R and CD41 was observed in all groups of sensitized mice regardless of therapeutic treatment after intragastric challenge with beef extract compared to naive controls (Fig. 5a, b). Analysis of basophil frequencies in mesenteric lymph nodes from sensitized mice showed no differences between treatment groups (Fig. 5c). However, sensitized mice that were therapeutically treated with NP-aGal demonstrated reduced percentages and numbers of basophils expressing CD41 and CD200R compared to mice treated with NP-HSA and PBS (Fig. 5d). No differences in the percentages and numbers of mast cells in the mesenteric lymph nodes were found in mice among the treatment groups. These results indicate that while therapeutic administration of NP-aGal using this treatment regimen and experimental timeline does not affect basophil or mast cell frequencies upon oral challenge, basophil activation was significantly reduced in mesenteric lymph nodes.

[0157] Ex vivo analysis of the response of spleen cells from control and NP treated mice demonstrated reduced aGal-specific IL-4, IL-5, and IL-13 production following therapeutic administration of NP-aGal (Fig. 6a). Cetuximab stimulation of splenocytes from NP-aGal-treated mice did not increase IFNy and TNFa production (Fig. 6b), but significantly increased IL- 10 production (Fig. 6c) compared to controls and NP-HSA treated mice. CD103+dendritic cells (DC) at sites of allergen drainage play a critical role in the generation of natural oral tolerance, and prior studies have suggested that they also mediate desensitization in food allergic oral immunotherapy subjects. Thus, sensitized mice treated with NPs or PBS, followed two weeks later by an intradermal booster with tick extract, and four days after that were evaluated for the frequencies of CD103+DCs in the skin-draining inguinal lymph nodes. The percentage of

[0158] CD103+CDl lc+F4 / 80' DCs and numbers (though not statistically significant) were increased in mice treated with NPs compared to PBS controls (Fig. 6d), suggesting that therapeutic administration of NPs may induce DCs away from a Th2 skewing phenotype. Reduced levels of IL-4, IL-5, and IL-13, known to promote IgE class switching, and increased levels of IL-10 produced by splenocytes from NP-aGal treated mice following cetuximab stimulation, suggests that the effects of NP-aGal were sufficient to suppress allergic cytokine production.

[0159] Using an established mouse model of tick-induced IgE sensitization to the mammalian oligosaccharide aGal, intravenous administration of glycoprotein allergen-encapsulating NPs was investigated for their ability to attenuate allergic responses after intragastric challenge with aGal-containing beef extract. Glycoprotein-encapsulating NPs may be used to prophylactically prevent tick-induced sensitization to the carbohydrate aGal while simultaneously providing insight into the immune pathways involved in desensitization to aGal by NPs. No prior studies have investigated the pro-tolerogenic immunological changes occurring following carbohydrate antigen-specific reprogramming by NPs. NP-aGal prophylactic treatment alters a range of immune pathways involving T cells, basophils, and mast cells. When delivered prior to sensitization, NPs containing aGal-HSA effectively reduced serum MCPT-1 levels and aGal- specific basophil histamine levels, which corresponded to reduced frequencies and activation of basophils in mesenteric lymph nodes. Further, mast cell frequency trended downwards in the mesenteric lymph nodes. Alpha-gal-specific stimulation of splenocytes from mice prophylactically treated with NP-aGal significantly reduced IL-4, IL-5, and IL-13 production and increased the production of the regulatory cytokine IL- 10 without skewing towards a Thl phenotype. These findings suggest that NP-aGal treatment can reprogram cytokine production in the spleen, a central tolerogenic organ, to prevent elevated Th2 responses and subsequently leads to reduced aGal-specific IgE production. The effects of NP-aGal on Th2 cytokine responses is consistent with a previous report showing that IgE sensitization to aGal is dependent on CD41T cell help. The role of CD4+T cells has not yet been definitively determined in humans with AGS nor in another mouse model of AGS involving subcutaneous sensitization of AGKO mice with tick salivary gland extract.

[0160] Analysis of sensitized mice therapeutically treated with NPs revealed that NP-aGal significantly reduced serum MCPT-1 levels as well as histamine released by basophils though the latter was not statistically significant. Moreover, activation but not frequencies of basophils in mesenteric lymph nodes was reduced in mice treated with NP-aGal. No changes in the frequencies of mast cells in mesenteric lymph nodes of mice were detected. The modest effects of NP-aGal on basophil and mast cell frequencies when given therapeutically compared to prophylactically suggests that therapeutic administration of NPs may not affect expansion of these cell types in the mesenteric lymph nodes when pre-existing IgE antibodies are bound to FcsR. Therapeutic delivery of NP-aGal to sensitized mice suppressed IL-4, IL-5, and IL-13 production and increased production of the immunosuppressive cytokine IL- 10 from splenocytes stimulated with aGal. Allergen immunotherapy has been shown to induce IL- 10 following grass pollen subcutaneous immunotherapy and sublingual immunotherapy (42) and HDM subcutaneous immunotherapy (43, 44). Generation of T regulatory cells that produce IL-10 in vivo after tolerance induction with oral antigens has been reported (45, 46). Thus, increased production of IL-10 from splenocytes stimulated with aGal may suggest that therapeutic administration of NP- aGal to sensitized mice induces a T regulatory cell subset that plays a role in oral tolerance. Using a mouse model of egg allergy, protein allergen-encapsulating NPs can reprogram pathogenic allergen-specific Th2 cells toward a T regulatory phenotype in the small intestine lamina propria. A trending increase in the frequency of CD103+DCs was found in the skindraining lymph nodes of mice treated with NP-aGaL CD103+DCs isolated from the mesenteric lymph nodes of mice and humans induce differentiation of naive T cells into Tregs.

[0161] Current treatment modalities for patients with AGS are limited to the prevention of new tick bites and avoidance of mammalian meats and mammalian-derived food products and drugs. However, allergen avoidance leaves patients susceptible to accidental allergen exposures, as well as economic and social consequences related to avoidance-imposed lifestyle changes. Alternatively, recent reports have shown that patients with AGS who underwent oral immunotherapy with red meat became tolerant to red meat. Oral immunotherapy for any allergen currently relies on continuous daily dosing, which despite careful surveillance of young and old patients alike results in high exposure to treatment-related adverse effects.

[0162] However, the effects of NPs containing encapsulating a carbohydrate allergen have never been tested. As demonstrated herein, in AGKO mice aGal NPs administered prophylactically suppress Th2 cytokines IL-4, IL-5, and IL- 13, correlating with reduced aGal-specific IgE production and hypersensitivity responses, as measured by decreased frequencies and activation of basophils and mast cell reactivity. Therapeutic delivery of aGal NPs to sensitized mice also suppresses Th2 cytokines, aGal-specific IgE production, and mast cell reactivity but does not affect frequencies of basophils and mast cells. These results demonstrate the ability of aGal NPs to suppress a carbohydrate allergen-specific IgE response when given prophylactically and therapeutically

[0163] In these studies, prophylactic treatment with only two doses of NP-aGal is sufficient to prevent sensitization to carbohydrate antigen-specific responses and reduce allergic burden following subsequent exposure to beef extract. Moreover, therapeutic administration of two doses of aGal- containing nanoparticles to mice sensitized to aGal had partial efficacy by reducing the production of Th2 cytokines, aGal-specific IgE production, and MCPT-1 release but did not reduce basophil activation or histamine release. Thus, NP-aGal can be successfully exploited to improve allergen-specific immunotherapy outcomes. In conclusion, these studies demonstrate the first therapeutic strategy using NPs to treat AGS, an understudied tick-borne food allergy to mammalian meat. Prophylactic treatment with aGal glycoprotein-containing NPs reduces splenocyte Th2 cytokine production following stimulation with a different aGal -containing glycoprotein. aGal-glycoprotein NPs subsequently reduce total, tick-, and aGal-specific IgE levels in the blood while simultaneously reducing the reactivity of circulating basophils and mast cells. Additionally, while the current studies demonstrate that these NPs hold prophylactic efficacy in preventing AGS formation, therapeutic treatment with aGal glycoprotein-containing NPs also reduces Th2 cytokines, aGal-specific IgE levels, and mast cell activity. This is the first demonstration of immunological tolerance induction to an oligosaccharide. The therapeutic potential of NP-aGal to reduce AGS in recipients with preexisting disease is highlighted.

[0164] All publications, patents, and patent applications discussed and cited herein are hereby incorporated by reference in their entireties. It is understood that the disclosed invention is not limited to the particular methodology, protocols and materials described as these can vary. It is also understood that the terminology used herein is for the purposes of describing particular embodiments only and is not intended to limit the scope of the appended claims.

[0165] Those skilled in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

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Claims

1. WE CLAIM:

1. A composition comprising tolerizing immune modifying particles encapsulating Alpha- Gal-Syndrome-associated antigens (TIMP-AGS) particles encapsulating one or more AGS associated antigens, portions thereof, or combinations thereof.

2. The composition of claim 1, wherein the particles comprise a biodegradable polymer.

3. The composition of claims 1-2, wherein the biodegradable polymer is polyglycolic acid (PGA), poly (lactide-co-glycolide) (PLG), polylactic acid (PLA), a co-polymer of PLG and PLA (PLGA), poly caprolactone (PCL), polystyrene, polysebacic acid (PSA), dextran acetate, poly(lactic-co-sebacic) acid (PLSA), polypropylene sulfide, cyclodextran, poly ethylene glycol (PEG), chitosan, a polysaccharide, one or more lipids, a liposome, diamond, iron, zinc, cadmium, gold or silver.

4. The composition of any one of claims 1-3, wherein the particles have a negative zeta potential.

5. The composition of claim 4, wherein the particles have a zeta potential between -100 mV and 0 mV.

6. The composition of claim 4, wherein the particles have a zeta potential between -100 mV and -30 mV.

7. The composition of claims 4-6, wherein the particles have a zeta potential between -30 and -80 mV.

8. The composition of any one of claims 1-7, wherein the particle size is between 100 nm and 1000 nm.

9. The composition of claim 8, wherein the particle size is between 400-800 nm10. The composition of any one of claims 1-9, wherein the antigen comprises one or more carbohydrate, peptide, protein, glycoprotein, glycolipid, homolog, derivative, mimotope, or combination.

11. The composition of claim 10, wherein the antigen is selected from the group consisting of galactose, gal actose-a / / ? / ?«-l ,3 -galactose (agal), agal containing glycoprotein, or agal containing glycolipid.

12. The composition of claim 1 1, wherein the antigen is gal actose-r / / / ; / 7< -l , 3 -galactose (ocgal).

13. The composition of claim 10, wherein the antigen is an AGS-associated tick antigen.

14. The composition of claim 10, wherein the AGS associated ticks are selected from the group consisting of Amblyomma americanum, Ixodes ricinus, Ixodes holocyclus, Ixodes australiensis, Amblyomma cajemense, Amblyomma hebraeum, Amblyomma testudmarium, Ixodes nipponensis, Amblyomma sculptum, Amblyomma variegatum, Rhipicephalus evertsi, Rhipicephalus burst Hyaloma marginatum, or Haemaphysalls longicornis .

15. A method of inducing tolerance in a subject in need thereof comprising administering to the subject a composition comprising tolerizing immune modifying particles encapsulating one or more Alpha Gal Syndrome associated antigens (TIMP-AGS).

16. The method of claim 15, wherein the particle comprises polyglycolic acid (PGA), poly (lactide-co-glycolide) (PLG), polylactic acid (PLA), a co-polymer of PLG and PLA (PLGA), polycaprolactone (PCL), polystyrene, polysebacic acid (PSA), poly(lactic-co- sebacic) acid (PLSA), polypropylene sulfide, cyclodextran, poly ethylene glycol (PEG), chitosan, a polysaccharide, one or more lipids, a liposome, diamond, iron, zinc, cadmium, gold or silver.

17. The method of any one of claims 15-16, wherein the particles have a negative zeta potential.

18. The method of any one of claims 15-17, wherein the particles have a zeta potential between 0 mV and -100 mV.

19. The method of any one of claims 15-18, wherein the particles have a zeta potential between -30 mV and -100 mV.

20. The method of any one of claims 15-19, wherein the size of the particle is between 100 nm and 1000 nm.

21. The method of any one of claims 15-20, wherein the size of the particle is between 400 nm and 800 nm.

22. The method of any one of claims 15-21, wherein the antigen comprises one or more carbohydrate, peptide, protein, glycoprotein, glycolipid, homolog, derivative, mimotope, or combination.

23. The method of claim 22, wherein the antigen is selected from the group consisting of galactose, gal actose-c / / / ? / ?«-l ,3 -galactose (agal), agal containing glycoprotein, or agal containing glycolipid.

24. The method of claim 22, wherein the antigen is galactose-tz / / ? / ?a-l,3-galactose (ocgal).

25. The method of claim 22, wherein the antigen is an AGS associated tick- antigens.

26. The method of claim 22, wherein the AGS associated ticks are selected from the group consisting of Amblyomma americanum, Ixodes ricinus, Ixodes holocyclus, Ixodes australiensis, Amblyomma cajennense, Amblyomma hebraeum, Amblyomma testudinarium, Ixodes nlpponensis. Amblyomma sculptum, Amblyomma variegatum, Rhipicephalus evertsi, Rhipicephalus burst Hyaloma marginatum, or Haemaphysalis longicornis .

27. The method of claim 15, wherein the subject has an allergy or hypersensitivity.

28. The method of claim 27, wherein the allergy is AGS.

29. The method of any one of claims 15-28, wherein administration of the said composition reduces an AGS associated immune response.

30. The method of claim 29, wherein the immune response is an inflammatory, allergic and / or immunogenic response.

31. The method of claim 30, wherein the immune response is a humoral immune response.

32. The method of claim 30, wherein the immune response is an adaptive immune response.

33. The method of claim 30, wherein the immune response is an innate immune response.

34. The method of claim 30, wherein the immune response is an antibody response.

35. The method of claim any one of claims 15-34, wherein the composition is administered intravenously, intramuscularly, ocularly, intraperitoneally, transdermally, nasally, orally, intra-lymphatically and / or subcutaneously.

36. The method of any one of claims 15-35, wherein administering TIMP-AGS alone or in combination with one or more additional therapeutics, reduces the duration and severity of an allergic or immunogenic immune response to ocgal.

37. The method of claim 36, wherein the allergic immune response is a Th2 T-cell response, B-cell activation, basophil activation, eosinophil activation, mast cell activation, and / or IgE induction.

38. The method of claim 37, wherein administering TIMP-AGS reduces IL-4, IL-5, or IL-13 levels.

39. The method of claim 37, wherein administering TIMP-AGS reduces total IgE production, tick-specific IgE production, aGal-specific IgE production or IgE: IgG ratios.

40. The method of claim 36, wherein administering TIMP-AGS, increases total IgG production, tick-specific IgG production or aGal-specific IgG.

41. The method of claim 36, wherein administering TIMP-AGS increases IL-10 production.

42. The method of any one of claims 36-41, wherein administering TIMP-AGS ameliorates one or more symptoms of AGS.

43. The method of claim 42, wherein the symptoms are skin reactions, hives, skin redness, skin swelling, itching, tightening of the throat, difficulty breathing, shortness of breath, digestive problems such as diarrhea, stomach cramps, nausea, or vomiting, drop in blood pressure, and anaphylaxis.

44. The method of any one of claims 36-43, wherein administering TIMP-AGS ameliorates AGS hypersensitivity.

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