Tolerizing immune modifying nanoparticles for treatment of narcolepsy

Abstract

The present application is directed, in general, to tolerizing immune mediated particles comprising narcolepsy with cataplexy (NT1) antigens for use in treating NT1.

Description

TOLERIZING IMMUNE MODIFYING NANOPARTICLES FOR TREATMENT OF NARCOLEPSYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of U.S. Provisional Patent Application No. 63 / 672,200 filed July 16, 2024, herein incorporated by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE DISCLOSURE

[0002] The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification as a computer readable file. The name of the file containing the Sequence Listing is “70593_SeqListing.xml", which was created on July 9, 2025, and is 742,412 bytes in size. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference,FIELD OF THE DISCLOSURE

[0003] The present application is directed to tolerizing immune modifying particles (TIMPs) comprising antigens associated with narcolepsy.BACKGROUND

[0004] Narcolepsy with cataplexy (NT 1 ) is a chronic neurological disorder characterized by excessive daytime sleepiness, cataplexy, and sleep paralysis. Hypocretins (also called orexin) are a set of neuropeptides that control an array of bodily processes, including wakefulness and REM sleep. The loss of hypocretin / orexin neurons is thought to precede narcolepsy onset.

[0005] The pathology for NT1 suggests an immunological basis due to its association with the MHC class II allele, HLA-DQB1 *06:02, polymorphisms in the TCRa locus, TNF-a receptor, TNF2 receptor and P2Y11 receptor. The increased incidence of NT1 observed after vaccination with the influenza vaccine Pandremix, a monovalent 2009 H1 N1 influenza vaccine (Vaccine. 2018 36(41 ):6202-6211 ), also suggests an immune-mediated pathology. T-cell mediated mechanisms leading to decreased hyporectin signaling have been implicated in the development of NT1 . It has been hypothesized that an autoimmune-mediated process targeting hypothalamic hypocretin / orexin neurons contributes to narcolepsy.

[0006] The prevalence of NT1 in Western countries has been estimated at 20 to 50 cases per 100,000.

[0007] In many patients with narcolepsy, sleepiness and cataplexy substantially interfere with daily life, impacting school, work, relationships, and social life. Narcolepsy is associated with higher-than-expected rates of hypertension. Patients with narcolepsy also have increased rates of obesity and diabetes. People with narcolepsy have a three- to fourfold increased risk of having a car crash, and over one-third have had an accident due to sleepiness. There is currently no cure for NT1 . Management of narcolepsy is symptomatic, and there are no diseasemodifying therapies yet available.SUMMARY OF THE DISCLOSURE

[0008] Tolerizing immune modifying particles (TIMPs), comprising one or more antigens, have been previously described for the treatment of immune-mediated disorders (e.g. autoimmune diseases and allergies) via induction of antigen-specific immune tolerance (WO201313192532 and WO2015023796 incorporated herein by reference). Encapsulation of one or more antigens within the TIMP core is an advantage as it ensures delivery of encapsulated proteins to APCs safely and effectively without inducing immune activation. In several preclinical models of autoimmune diseases and allergies, TIMPs have demonstrated efficacy at inducing T-cell tolerance. Induction of antigen-specific tolerance to NT1 autoantigens using TIMPs encapsulating NT1 -associated antigens (TIMP-NT1 ) could potentially cure narcolepsy.

[0009] The present disclosure provides compositions and methods for inducing antigenspecific tolerance to NT 1 -associated antigens using TIMP-NT 1 .

[0010] Provided herein is a TIMP-NT1 composition comprising a negatively charged particle encapsulating an antigen, wherein the antigen comprises one or more NT1 associated antigens and / or a portion thereof, or combinations of antigens or portions thereof. In some embodiments, the NT 1 associated antigen is a peptide, a protein, a homologue, a derivative, a mimotope, or a combination thereof. In various embodiments, the TIMP-NT1 particle comprises a polymer and has a negative zeta potential. In various embodiments, the polymer is a biodegradable polymer. In various embodiments, the TIMP-NT1 particle comprises a liposome, lipid nanoparticle, polymersomes, micelles, polymeric nanoparticles, acid-ionizable iron nanoadjuvant (INOP),mesoporus silica, metal-organic frameworks (MOFs), lipid nanodiscs, supramolecular nanoparticles, extracellular vesicles, or sono-driven nanoparticles.

[0011] In various embodiments, TIMP-NT1 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.

[0012] 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.

[0013] In various embodiments, TIMP-NT1 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, 89:1 1 , 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.

[0014] In various embodiments, TIMP-NT1 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-NT1 particles have a negative zeta potential of between -30 mV to -80 mV. In various embodiments, the TIMP-NT1 particles have a negative zeta potential of between -30 mV to -60 mV. In various embodiments, thenegative zeta potential is achieved by surface functionalization of the particles. In various embodiments, the surface functionalization is carboxylation.

[0015] In various embodiments, the size, or diameter, of the TIMP-NT1 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-NT 1 particles is between 400 nm to 800 nm. In various embodiments, the diameter of the negatively charged TIMP-NT1 particles is between 350 nm to 800 nm.

[0016] In various embodiments, TIMP-NT1 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 about 0.05 pm and about 10 pm, between about 0.1 pm and about 10 pm, between about 0.1 pm and about 5 pm, between about 0.1 pm and about 3 pm, , between about 0.3 pm and about 5 pm, or between 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 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 adiameter 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, 1 100 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.

[0017] In various embodiments, the TIMP-NT1 particles encapsulate one or more NT1 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-NT1 particles encapsulate one or more NT1 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.

[0018] In various embodiments, TIMP-NT1 particles encapsulate one or more NT1 associated antigens, portions, or combinations thereof. In some embodiments, the NT1 associated antigen is a peptide, a protein, a homologue, a derivative, a mimotope, or a combination thereof. In various embodiments, the TIMP-NT1 particles encapsulate one or more polynucleotides encoding NT1 associated antigens. In some embodiments, the polynucleotides comprise DNA, RNA, messenger RNA (mRNA), or circular RNA.

[0019] In various embodiments, administering TIMP-NT1 reduces immune reactivity to NT1 associated antigens.

[0020] In various embodiments, the NT1 associated antigens are selected from the group consisting of hypocretin (also known as orexin (HCRT)), HCRT cell proteins, HCRT receptor 1 (HCRTR1 ), HCRT receptor 2 (HCRTR2), muscarinic receptors, protein O-mannosyltransferase (POMT) enzyme complex (POMT1), regulatory factor X4 (RFX4), Tribbles homologue 2 (TRIB2), Lim homeobox 9 (LHX9), prostaglandin D2 receptor (DP1 ), ganglioside M3 (GM3), B cell lymphoma 6 protein, neuropeptide glutamic acid-isoleucine / a-melanocyte-stimulating hormone (NEI / aMSH), [32-adrenergic receptor (ADRB2), methyltransferase-like 22 (METTL22), 5'-nucleotidase cytosolic IA (NT5C1 A), neurexin 1 a (NRXN1 ), ubiquitin carboxyl-terminal hydrolase (MINDY-2 / FAM63B), LOC401464, protein mono-ADP-ribosyltransferase (PARP3), or nociception receptor. In various embodiments, the HRCT antigen is a peptide with amino acids 1 -13 (HCRT1.13), amino acids 56-86 (HCRT56-68), amino acids 87-99 (HCRT87-99), amino acids 86-97 (HCRT86-97), N-pyroglutamate (pyro) HCRT3448, pyro HCRT38-52, pyro HCRT33-47, pyro HCRT375i, amidated C-terminal epitope of HCRT1 (HCRT54-66-NH2), and amidated C-terminal epitope of HCRT1 (HCRT86-97-NH2). In various embodiments, the antigen is a viral epitope, protein or peptide. In various embodiments, the antigen is an influenza virus epitope, protein or peptide. In various embodiments, the virus is selected from pandemic 2009 H1 N1 (pH1 N1 ) influenza A, Puerto Rico 8 influenza A (PR8). In various embodiments, the antigen is selected from Pandemrix vaccine peptides, pandemic 2009 H1 N1 (pH1 N1 ) influenza A, hemagglutinin (HA), neuraminidase (NA), influenza RNA-dependent RNA polymerase subunit 1 (PB1 ), or nucleoprotein (NP). In various embodiments, the antigen is selected from HA273-287, HA209-223, HA321 -335, HA437-45I , HA45-59, HA217-231 , HA26I -275, NP17-31 , NPl7-31 -pH1N1 , NP437-451-pH1N1. In VaHOUS embodiments, the antigen is an influenza H1 N1 virus epitope, protein or peptide. In various embodiments, the antigen is a bacterial protein or peptide. In various embodiments, the antigen is a Streptococcus epitope, protein or peptide. In various embodiments, the antigen is Streptolysin O antigen. In various embodiments, the NT 1 associated antigens are selected from the listing in Table 1. In various embodiments, the NT1 associated antigens are selected from SEQ ID NO: 1 to SEQ ID NO: 839 (Fig. 1 ). In various embodiments, the antigens are cryptic, post-translationally modified, or hybrid HCRT peptides.

[0021] In various embodiments, TIMP-NT1 encapsulates a viral vector encoding NT1 associated antigens. In various embodiments, TIMP-NT1 encapsulates a viral vector encoding HCRT / orexin. In various embodiments, TIMP-NT1 encapsulates an AAV vector encodingHCRT / orexin. In various embodiments, the AAV is selected from the group comprising AAV-1 , AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-12, Anc80, a synthetic AAV, combinations or engineered versions thereof. In various embodiments, the antigen is one or more AAV capsid proteins. In various embodiments, the AAV capsid protein is VP-1 , VP-2, and VP-3.

[0022] In various embodiments, the TIMP-NT 1 particles encapsulate one or more NT 1 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-NT1 particles encapsulate one or more NT1 associated antigens selected from Table 1 , 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-NT1 particles encapsulate one or more NT 1 associated antigens selected from Figure 1 , 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-NT1 particles encapsulate hypocretin / orexin, have a negative zeta potential of between -100 mV and 0 mV, and wherein the particles are between 100 and 1000 nm in diameter.

[0023] In various embodiments, the TIMP-NT 1 particles encapsulate one or more NT 1 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-NT1 particles encapsulate one or more NT1 associated antigens selected from Table 1 , have a negative zeta potential of between -80 mV and -30 mV, and wherein the particles are between 400 and 800 nm in diameter. In various embodiments, the TIMP-NT1 particles encapsulate one or more NT 1 associated antigens selected from Figure 1 , have a negative zeta potential of between -80 mV and -30 mV, and wherein the particles are between 400 and 800 nm in diameter. In various embodiments, the TIMP-NT1 particles encapsulate hypocretin / orexin, have a negative zeta potential of between -80 mV and -30 mV, and wherein the particles are between 400 and 800 nm in diameter.

[0024] In various embodiments, TIMP-NT1 particles comprise PLGA, encapsulate one or more NT 1 associated antigens, and have a negative zeta potential between -80 mV and -30 mV. In various embodiments, TIMP-NT1 particles comprise PLGA, encapsulate one or more NT 1 associated antigens, have a negative zeta potential between -80 mV and -30 mV, and wherein the particles are between 400 nm and 800 nm in diameter.

[0025] In various embodiments, TIMP-NT1 particles comprise PLGA, encapsulate hypocretin / orexin, and have a negative zeta potential of between -80 mV and -30 mV. In various embodiments, TIMP-NT1 particles comprise PLGA, encapsulate one or more NT1 associated antigens selected from Table 1 , have a negative zeta potential between -80 mV and -30 mV, and wherein the particles are between 400 nm and 800 nm in diameter. In various embodiments, TIMP-NT1 particles comprise PLGA, encapsulate one or more NT1 associated antigens selected from Figure 1 , have a negative zeta potential 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 NT1 associated antigen. In various embodiments, the antigen is hypocretin / orexin. In various embodiments, administration of TIMPs encapsulating one or more antigens associated with NT1 to a subject in need thereof induces antigen specific tolerance to one or more antigens associated with NT1 . In various embodiments, the administration alleviates one or more symptoms of NTI.

[0027] In various embodiments, subjects are administered TIMPs encapsulating one or more NT 1 associated antigens.

[0028] In various embodiments, TIMP-NT1 particles are administered intravenously, intramuscularly, ocularly, intraperitoneally, transdermally, nasally, orally, intra-lymphatically and / or subcutaneously.

[0029] In various embodiments, administering TIMP-NT1 in a subject in need thereof induces antigen specific tolerance to an NT1 associated antigen. In various embodiments, administering TIMP-NT1 in a subject reduces the inflammatory immune response to NT1 associated antigens, e.g., relative to before the administration. In various embodiments, administering the TIMPs in a subject ameliorates the autoimmune response to NT1 associated antigens, e.g., relative to before the administration. In various embodiments, the autoimmune and / or inflammatory immune response is an innate immune response, a 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 the NT1 associated antigens. In various embodiments, the innate immune response is a proinflammatory APC response. Invarious embodiments, the adaptive immune response is an activated CD4+, CD8+ or a proinflammatory cytokine response. In various embodiments, the immune response includes a T cell, B cell, NK cell, monocyte, macrophage, eosinophil, or a basophil response. In various embodiments, the immune response is reduced immune infiltrate into tissues and / or organs. In some embodiments, administering TIMP-NT 1 decreases the number and / or frequency of antigen-specific CD4+, or CD8+ T-cells compared to before the administration. In some embodiments, administering TIMP-NT1 decreases CD4+, or CD8+ T-cell infiltration compared to before the administration. In some embodiments, administering TIMP-NT1 decreases number and / or frequency of activated antigen-specific CD4+, or CD8+ T-cells compared to before the administration. In various embodiments, administering the TIMP-NT1 to a subject in need thereof reduces the autoimmune and / or inflammatory response to 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”. In various embodiments, administering the TIMP-NT1 in a subject induces cGAS-STING pathway. In various embodiments, administering the TIMP-NT1 in a subject induces type-1 IFN pathway.

[0030] In various embodiments, administering TIMP-NT1 to a subject in need thereof induces an antigen specific immune regulator response. In various embodiments, administering TIMP- NT1 to a subject induces regulatory T cells, B cells, monocytes, and / or macrophages. In various embodiments, administering TIMP-NT1 to a subject induces antigen specific regulatory T cell (Treg), Tr1 , regulatory macrophages (Mreg), and / or regulatory B cells (Breg) cells.

[0031] Also provided is a method of inducing tolerance in a subject in need thereof comprising administering to the subject a composition comprising a liposome, or lipid nanoparticle as described herein.

[0032] In various embodiments, subjects are administered liposomes encapsulating one or more NT-1 associated antigens. In various embodiments, subjects are administered liposomes encapsulating polynucleotides encoding one or more NT-1 associated antigens. Administering liposomes encapsulating NT1 associated antigens induces antigen specific tolerance to NT1 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.

[0034] 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.

[0035] In various embodiments, the liposomes are administered intravenously, intramuscularly, ocularly, intraperitoneally, transdermally, nasally, orally, intra-lymphatically and / or subcutaneously.

[0036] In various embodiments, administering the liposomes in a subject in need thereof induces antigen specific tolerance to NT1 associated antigens. In various embodiments, administering the liposomes in a subject reduces the inflammatory and / or autoimmune response to NT1 associated antigens. In various embodiments, administering the liposomes in a subject reduces the inflammatory and / or autoimmune response to one or more encapsulated antigens. In various embodiments, administering the liposomes in 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’. In various embodiments, administering the liposomes in a subject induces cGAS-STING pathway. In various embodiments, administering the liposomes in a subject induces type-1 IFN pathway. 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 NT1 associated antigen. In various embodiments, the innate immune response is a proinflammatory APC response. In various embodiments, the adaptive immune response is an activated CD4+, CD8+ or a proinflammatory cytokineresponse. In various embodiments, the immune response is a T cell, B cell, NK cell, monocyte, macrophage, eosinophil, or a basophil response.

[0037] In various embodiments, administering the liposomes in a subject in need thereof induces an antigen specific immune regulator response. In various embodiments, administering the liposomes in a subject induces regulatory T cells, B cells, monocytes, and / or macrophages. In various embodiments, administering liposomes in a subject induces antigen specific Treg, Tr1 , Mreg, and / or Breg cells.

[0038] In various embodiments, subjects are administered lipid nanoparticles encapsulating one or more NT-1 associated antigens. In various embodiments, subjects are administered lipid nanoparticles (LNP) encapsulating polynucleotides encoding one or more NT-1 associated antigens. Administering lipid nanoparticles encapsulating NT1 associated antigens induces antigen specific tolerance to NT 1 associated antigens.

[0039] In some embodiments, the lipids comprising the LNP consist of an anionic or cationic ionizable lipid, helper lipid, cholesterol, and PEG-lipids. In some embodiments, the lipids are selected from the group consisting of 1 ,2-di-0-octadecenyl-3-trimethylammonium-propane (DOTMA), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB), 2,3- dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1 -propanaminium trifluoroacetate (DOSPA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane (DLin-KC2-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31 -tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA; MC3), (2S)-2,5-bis(3-aminopropylamino)-N-[2- (dioctadecylamino)acetyl]pentanamide (DOGS; Transfectam), N1-[2-((1 S)-1 -[(3- aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]- benzamide (MVL5), 3|3-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), N4-cholesteryl-spermine (GL67), Di((Z)-non-2-en-1 -yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319), Heptadecan-9-yl 8-((2- hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5), Heptadecan-9-yl 8-((2- hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (Lipid H (SM-102)), ((4- hydroxybutyl)azanediyl)bis(hexane-6,1 -diyl)bis(2-hexyldecanoate) (ALC-0315), 1 ,1 '-((2-(4-(2- ((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl) piperazin-1 - yl)ethyl)azanediyl) bis(dodecan-2-ol) (C12-200), Tetrakis (8-methylnonyl) 3, 3', 3", 3"'- (((methylazanediyl)bis(propane-3,1 diyl))bis(azanetriyl)) tetrapropionate (306Oi10), 3,6-bis(4- (bis(2-hydroxydodecyl)amino)butyl) piperazine-2, 5-dione (cKK-E12), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1 -diyl))bis(azanetriyl))tetrakis(ethane-2,1 -diyl)(9Z,97,9"Z,9'"Z,12Z,127,12'7,12'"Z)- tetrakis(octadeca-9,12-dienoate) (OF-Deg-Lin), ethylphosphatidylcholine (ePC), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, -sitosterol, 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000 (PEG2000-DMG), 1 ,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DSG), DODAP (1 ,2-dioleoyl-3-dimethylammonium-propane), DODMA (1 ,2-dioleoyl- 3-dimethylammonium-propane), 1 ,2-dioleoyl-sn-glycero-3-phosphate (18PA), 1 ,2-dioleoyl-sn- glycero-3-phospho-l-serine (DOPS), Phosphatidic Acid (PA) derivatives, cardiolipin, 1 ,2- Dioleoyl-sn-glycero-3-phosphate (DOPA), PEG2000-DSPE (1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), PEG2000-DOPE (1 ,2-dioleoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). In some embodiments, lipids comprising the LNP are apoptosis inducing and selected from the group consisting of phosphatidylserine, lysophosphatidylcholine, ceramide, oxidized phospholipids, prostaglandins. In various embodiments, the LNP encapsulates or is complexed to RNA, mRNA, circular RNA, or DNA encoding one or more antigens. In some embodiments, the carrier particles comprise a lipid nanoparticle (LNP) encapsulating or complexed to (i) RNA encoding one or more antigens. In some embodiments, the lipid nanoparticles further comprise one or more immunomodulatory agents. In some embodiments, the immunomodulatory agent is selected from the group consisting of immunosuppressive agents, cytokines or chemokines, immunostimulants, or immune cell function modulators. In some embodiments, the immunomodulatory agent is selected from the group consisting of mTOR inhibitors, glucocorticoids, vitamins and vitamin derivatives, calcineurin inhibitors, nuclear factor kappa B inhibitors, kynurenine, aryl hydrocarbon receptor agonists, glycan and glycan binding proteins, apoptotic signaling pathway agonists, TLR agonists, inhibitory receptor activating agents, T-cell modulators, immunological synapse modulators, or Janus kinase inhibitors. In various embodiments, the immunomodulatory agents are selected from the group consisting of dexamethasone, rapamycin, cyclosporine A, FK506, ITE, andrographolide, tofacitinib, rosiglitazone, IL-10, PD-1 , PD-L1 / L2, IL-2, TGF-[3, TGF-[3-signaling agonist, GM-CSF, IFN-J3, IL- 35, IL-27, IL-4, hepatocyte growth factor, tumor necrosis factor- related apoptosis-inducing ligand (TRAIL) agonist, vitamin D, retinoic acid. In some embodiments, the immunomodulators are encoded by mRNA.

[0040] In various embodiments, the LNP is negatively charged. In various embodiments, the LNP 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 LNPs have a zetapotential 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 LNPs have a negative zeta potential of between -30 mV to -80 mV. In various embodiments, the LNPs have a negative zeta potential of between -30 mV to -60 mV.

[0041] In various embodiments, the LNP is between 100-1000 nm, or between 300-800 nm or between 400-800 nm. In various embodiments, the LNP 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.

[0042] Also contemplated are compositions described herein comprising a negatively charged particle encapsulating an antigen, wherein the antigen is one or more NT1 associated antigens for use in inducing tolerance in a subject.

[0043] 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 NT1 associated antigens in the preparation of a medicament for inducing tolerance in a subject.

[0044] In various embodiments, the negatively charged particle is a TIMP or a liposome as described herein.

[0045] It is understood that each feature or embodiment, or combination, described herein is a non-limiting, 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 contemplatedas 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.

[0046] 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.BRIEF DESCRIPTION OF THE FIGURES

[0047] Figure 1. Amino acid sequences of NT 1 associated antigens.DETAILED DESCRIPTION

[0048] There is a need for therapeutics to address the immune imbalance in NT 1 leading to improved disease symptoms and improved outcomes without the risk of toxic side-effects. 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.

[0049] In pre-clinical models of autoimmune diseases and allergies, TIMPs have demonstrated therapeutic efficacy at inducing T-cell tolerance to antigenic / allergenic proteins and peptides resulting in improved disease symptoms. TIMPs encapsulating one or more antigens implicated in or associated with narcolepsy with cataplexy (TIMP-NT1 ) can potentially treat NT1 by reprogramming the immune system and inducing antigen-specific T cell tolerance to NT1 associated antigens. There is a current need for immune tolerizing therapies which can induce tolerance to autoimmune NT1 associated antigens for long term therapeutic benefit without exposing patients to risk of adverse events.

[0050] The present disclosure provides compositions of negatively charged particles encapsulating one or more NT1 associated antigens. Also included are methods of inducing antigen specific tolerance using the negatively charged particles described herein.Definitions

[0051] Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below.

[0052] 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.

[0053] 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.

[0054] “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.

[0055] “Negatively charged particle” as used herein refers to particles which have been modified to possess a net surface charge that is less than zero.

[0056] “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 a 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).

[0057] “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 asMARCO. Carboxylation of the particles can be achieved using any compound which adds carboxyl groups, including, but not limited to, poly (ethylene-maleic anhydride) (PEMA).

[0058] As used herein the term “D90” refers to particle size distribution in which 90% of the total volume of material in the sample is contained or found. For example, if the D90 is 1000 nm, this indicates that 90% of the sample has a size of 1000 nm or smaller. Similarly, D50 or D10 refers to a particle size distribution in which 50% or 10%, respectively, of the total volume of material in the sample is contained or found. D [4,3] refers to volume or mass moment mean or De Brouckere Mean Diameter. In some embodiments, the D90 / D50 / D10 are number (count or frequency) based measurements or volume-based measurements which can be carried out by Laser Diffraction (LD), SPOS, or SPOS with nanoparticle tracking analysis (SPOS-NTA). Particle size is measured using techniques known in the art.

[0059] 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).

[0060] As used herein, the term “Th1 cell” refers to a subset of Th cells which produce pro- inflammatory mediators. Th1 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. Th1 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.

[0061] 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 phagocyte-independent protective responses.

[0062] As used herein, the term ‘Th 17 cell’ refers to a subset of Th cells that produce pro- inflammatory responses against extracellular bacteria and fungi. Th17 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.

[0063] “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, acetylation, phosphorylation and the like. 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.

[0064] “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 or viral proteins, peptides, drugs or components.

[0065] “NT1 associated antigen” as used herein refers to an antigen, or a portion or fragment thereof, that results in an immune reaction against the protein or portion or fragment thereof that has been determined to be related to onset, a symptom of or progression of NT1 . An NT 1 associated antigen can include the whole protein or other protein or moiety associated with the protein that may elicit an immune response in a subject. An NT1 associated antigen can include a post-translational modification that results in an immune reaction against the protein or portion or fragment thereof.

[0066] “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-limitingexamples 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.g., oral) or parenteral e.g., subcutaneous, intramuscular, intravenous or intraperitoneal injection; or topical, transdermal, or transmucosal administration) or via inhalation.

[0067] 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.

[0068] 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, non-human 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 non-mammals include, but are not limited to, birds, fish, and the like. The term does not denote a particular age or gender.

[0069] 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 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 noncontiguous.

[0070] 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.

[0071] 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.

[0072] The term “therapeutically effective amount” is used herein to indicate the amount of antigen-specific composition of the disclosure that is effective to ameliorate or lessen one or more symptoms or signs of disease to be treated.

[0073] 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.Particles

[0074] Tolerizing Immune Modifying Particles (TIMPs), comprising one or more antigens, have been previously described for the induction of antigen specific tolerance for treating inflammatory conditions (e.g., autoimmune diseases and allergies) (WO201313192532 and WO2015023796 incorporated herein by reference). In several preclinical models of autoimmune diseases and allergies, TIMPs have demonstrated efficacy at inducing antigen specific tolerance and inhibition of pathologic inflammatory immune responses.

[0075] The size and charge of the particles may be selected to optimize tolerance induction and are important for 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 -25 mV and -70 mV. 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 -30 mV and -80 mV. The average particle size and charge of the particles can be slightly altered in the lyophilization process, therefore, both post-synthesis averages and post-lyophilization averages are described. As used herein, the term “post-synthesis size” and “post synthesis charge” refer to the size andcharge 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.

[0076] 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.

[0077] 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 polyethylene glycol) (PEG) and its copolymers such as PLURONICS® (including copolymers of poly(ethylene 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.

[0078] 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 beused 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).

[0079] 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, polyanhydrides 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 invention 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. 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 invention include diamond, PLA, PGA, polypropylene sulfide, and PLGA polymers. Biocompatible but non-biodegradable materials may also be used in the carrier particles of the invention. For example, non-biodegradable polymers of 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.

[0080] In certain embodiments, the particle is a co-polymer having a molar ratio from about 50:50 or 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.

[0081] 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 caninclude a hydrophobic surfactant for the first emulsion, and a hydrophobic surfactant for the second emulsion.

[0082] In various embodiments, the polypeptide antigens are encapsulated in the particles by a single-emulsion 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, co-precipitation, 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.

[0083] 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 zetapotential 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.

[0084] In various embodiments, the liposomes encapsulate one or more NT1 associated antigens. In various embodiments, the liposomes encapsulate one or more NT1 associated antigens, portions, or combinations thereof. In some embodiments, the NT1 associated antigen is a peptide, a protein, a homologue, a derivative, a mimotope, or a combination thereof. In various embodiments, the TIMP-NT1 particles encapsulate one or more polynucleotides encoding NT1 associated antigens. In some embodiments, the polynucleotides comprise DNA, RNA, messenger RNA (mRNA), or circular RNA. In various embodiments, the antigen is selected from the group consisting of hypocretin / orexin (HCRT), HCRT cell proteins, HCRT receptor 1 (HCRTR1 ), HCRT receptor 2 (HCRTR2), muscarinic receptors, protein O- mannosyltransferase (POMT) enzyme complex (POMT1 ), regulatory factor X4 (RFX4), Tribbles homologue 2 (TRIB2), Lim homeobox 9 (LHX9), prostaglandin D2 receptor (DP1 ), ganglioside M3 (GM3), B cell lymphoma 6 protein, neuropeptide glutamic acid-isoleucine / a-melanocyte- stimulating hormone (NEI / aMSH), 2-adrenergic receptor (ADRB2), methyltransferase-like 22 (METTL22), 5'-nucleotidase cytosolic IA (NT5C1 A), neurexin 1 a (NRXN1 ), ubiquitin carboxyl- terminal hydrolase (MINDY-2 / FAM63B), LOC401464, protein mono-ADP-ribosyltransferase (PARP3), or nociception receptor. In various embodiments, the HRCT antigen is a peptide with amino acids 1 -13 (HCRT1.13), amino acids 56-86 (HCRTse es), amino acids 87-99 (HCRT87-99), amino acids 86-97 (HCRT86-97), N-pyroglutamate (pyro) HCRT34-48, pyro HCRT38-52, pyro HCRT33- 47, pyro HCRT37-51, amidated C-terminal epitope of HCRT1 (HCRT54-66-NH2), and amidated C- terminal epitope of HCRT1 (HCRT86-97-NH2). In various embodiments, the antigen is a viral epitope, protein or peptide. In various embodiments, the antigen is an influenza virus epitope, protein or peptide. In various embodiments, the virus is selected from pandemic 2009 H1 N1 (pH1 N1 ) influenza A, Puerto Rico 8 influenza A (PR8). In various embodiments, the antigen is selected from Pandemrix vaccine peptides, pandemic 2009 H1 N1 (pH1 N1 ) influenza A, hemagglutinin (HA), neuraminidase (NA), influenza RNA-dependent RNA polymerase subunit 1 (PB1 ), or nucleoprotein (NP). In various embodiments, the antigen is selected from HA273-287, HA209 -223, HA321 -335, HA437 -451 , HA45 -59, HA217-231 , HA261 -275, N P 17-31 , NP 17-31 -pH1 N1 , NP437 -451-pH1 N1. I n various embodiments, the antigen is an influenza H1 N1 virus epitope, protein or peptide. In various embodiments, the antigen is a bacterial protein or peptide. In various embodiments, theantigen is a Streptococcus epitope, protein or peptide. In various embodiments, the antigen is Streptolysin O antigen. In various embodiments, the NT 1 associated antigens are selected from the listing in Table 1. In various embodiments, the NT1 associated antigens are selected from Fig. 1.

[0085] In various embodiments, the liposomes encapsulate one or more NT1 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 NT 1 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.

[0086] In various embodiments, the liposomes encapsulate antigens selected from Table 1 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 NT1 . In various embodiments, the liposome administration is intravenous.

[0087] In some embodiments, the particle is a administered lipid nanoparticle (LNP). In some embodiments, the LNPs encapsulate one or more NT-1 associated antigens.

[0088] In some embodiments, the lipids comprising the LNP consist of an anionic or cationic ionizable lipid, helper lipid, cholesterol, and PEG-lipids. In some embodiments, the lipids are selected from the group consisting of 1 ,2-di-0-octadecenyl-3-trimethylammonium-propane (DOTMA), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB), 2,3- dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1 -propanaminium trifluoroacetate (DOSPA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane (DLin-KC2-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31 -tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA; MC3), (2S)-2,5-bis(3-aminopropylamino)-N-[2- (dioctadecylamino)acetyl]pentanamide (DOGS; Transfectam), N1-[2-((1 S)-1 -[(3- aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]- benzamide (MVL5), 3p-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), N4-cholesteryl-spermine (GL67), Di((Z)-non-2-en-1 -yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319), Heptadecan-9-yl 8-((2- hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5), Heptadecan-9-yl 8-((2- hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (Lipid H (SM-102)), ((4-hydroxybutyl)azanediyl)bis(hexane-6, 1 -diyl)bis(2-hexyldecanoate) (ALC-0315), 1 ,1 '-((2-(4-(2- ((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl) piperazin-1 - yl)ethyl)azanediyl) bis(dodecan-2-ol) (C12-200), Tetrakis (8-methylnonyl) 3, 3', 3", 3'"- (((methylazanediyl)bis(propane-3,1 diyl))bis(azanetriyl)) tetrapropionate (3060i10), 3,6-bis(4- (bis(2-hydroxydodecyl)amino)butyl) piperazine-2, 5-dione (cKK-E12), (((3,6-dioxopiperazine-2,5- diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl)(9Z,9'Z,9"Z,9'"Z,12Z,12'Z,12"Z,12"'Z)- tetrakis(octadeca-9,12-dienoate) (OF-Deg-Lin), ethylphosphatidylcholine (ePC), 1 ,2-distearoyl-sn-glycero-3-phosphocholine(DSPC), cholesterol, -sitosterol, 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000 (PEG2000-DMG), 1 ,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DSG), DODAP (1 ,2-dioleoyl-3-dimethylammonium-propane), DODMA (1 ,2-dioleoyl- 3-dimethylammonium-propane), 1 ,2-dioleoyl-sn-glycero-3-phosphate (18PA), 1 ,2-dioleoyl-sn- glycero-3-phospho-l-serine (DOPS), Phosphatidic Acid (PA) derivatives, cardiolipin, 1 ,2- Dioleoyl-sn-glycero-3-phosphate (DOPA), PEG2000-DSPE (1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), PEG2000-DOPE (1 ,2-dioleoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). In some embodiments, lipids comprising the LNP are apoptosis inducing and selected from the group consisting of phosphatidylserine, lysophosphatidylcholine, ceramide, oxidized phospholipids, prostaglandins. In various embodiments, the LNP encapsulates or is complexed to RNA, mRNA, circular RNA, or DNA encoding one or more antigens. In some embodiments, the carrier particles comprise a lipid nanoparticle (LNP) encapsulating or complexed to (i) RNA encoding one or more antigens. In some embodiments, the lipid nanoparticles further comprise one or more immunomodulatory agents. In some embodiments, the immunomodulatory agent is selected from the group consisting of immunosuppressive agents, cytokines or chemokines, immunostimulants, or immune cell function modulators. In some embodiments, the immunomodulatory agent is selected from the group consisting of mTOR inhibitors, steroids, corticosteroids, glucocorticoids, vitamins and vitamin derivatives, calcineurin inhibitors, nuclear factor kappa B inhibitors, kynurenine, aryl hydrocarbon receptor agonists, glycan and glycan binding proteins, apoptotic signaling pathway agonists, TLR agonists, inhibitory receptor activating agents, T-cell modulators, immunological synapse modulators, or Janus kinase inhibitors. In various embodiments, the immunomodulatory agents are selected from the group consisting of dexamethasone, rapamycin, cyclosporine A, FK506, ITE, andrographolide, tofacitinib, rosiglitazone, IL-10, PD-1 , PD-L1 / L2, IL-2, TGF-p, TGF-J3-signaling agonist, GM- CSF, IFN-p, IL-35, IL-27, IL-4, hepatocyte growth factor, tumor necrosis factor- relatedapoptosis-inducing ligand (TRAIL) agonist, vitamin D, retinoic acid. In some embodiments, the immunomodulators are encoded by mRNA.Antigens

[0089] 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. Post-translational modifications include phosphorylation, glycosylation (e.g. O-linked glycosylation, N-linked glycosylation, C-linked glycosylation), ubiquitination, nitrosylation, methylation, acetylation, lipidation, disulfide bonds, oxidation, or, succinylation, malonylation, sumoylation, glutathionylation, hydroxylation, palmitoylation, pyrrolidone carboxymic acid, glutarylation, gamma-carboxyglutamic acid, crotonylation, citrullination, myristoilation, sulfation, formylation or amidation.

[0090] "Anergy," ''tolerance,'1or "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).

[0091] It is contemplated that the tolerizing therapy described herein is 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 NT 1 associated antigens, portions thereof, or combinations thereof. It is also contemplated that the NT 1 associated antigen is a peptide, a protein, a homologue, a derivative, a mimotope, or a combination thereof.

[0092] Exemplary NT1 associated antigen include those listen in Table 1 or Figure 1 e.g. hypocretin / orexin (HCRT), HCRT cell proteins, HCRT receptor 1 (HCRTR1 ), HCRT receptor 2 (HCRTR2), muscarinic receptors, protein O-mannosyltransferase (POMT) enzyme complex (POMT1 ), regulatory factor X4 (RFX4), Tribbles homologue 2 (TRIB2), Lim homeobox 9 (LHX9), prostaglandin D2 receptor (DP1 ), ganglioside M3 (GM3), B cell lymphoma 6 protein, neuropeptide glutamic acid-isoleucine / a-melanocyte-stimulating hormone (NEI / aMSH), 02- adrenergic receptor (ADRB2), methyltransferase-like 22 (METTL22), 5'-nucleotidase cytosolic IA (NT5C1 A), neurexin 1 a (NRXN1 ), ubiquitin carboxyl-terminal hydrolase (MINDY-2 / FAM63B), LOC401464, protein mono-ADP-ribosyltransferase (PARP3), nociception receptor, HCRT1-13, HCRT56-68, HCRT87-99, HCRT86 -97, N-pyroglutamate (pyro) HCRT34-48, pyro HCRT38-52, pyro HCRT33-47, pyro HCRT37-5i, arnidated C-terminal epitope of HCRT1 (HCRT54-66-NH2), amidated C- terminal epitope of HCRT1 (HCRT86-97-NH2), an influenza virus epitope, hemagglutinin (HA), neuraminidase (NA), influenza RNA-dependent RNA polymerase subunit 1 (PB1 ), nucleoprotein (NP), HA273-287, HA209 -223, HA321 -335, HA437 -451 , HA45 -59, HA217-231 , HA261 -275, NP 17-31 , NP 17-31 -pH1 N1 , NP437-45I-PHINI , or Streptolysin O.

[0093] The preprohypocretin gene encodes two mature, homologous, carboxy-terminal- amidated, neuroactive peptides known as HCRT1 and HCRT2 (referred to collectively here as HCRT; also known as orexin-A and orexin-B).

[0094] Table 1

[0095] The term "homologue" 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 homologues of an epitope correspond to the natural epitope modified in, for example, three, two, or in one amino acid.

[0096] The term "polynucleotide (or nucleic acid) encoding NT 1 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 homologue thereof. Such polynucleotides or nucleic acids include the normal sequences encoding the peptide, as well as derivatives and fragments of these nucleic acids.

[0097] 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%.

[0098] The term "derivative" as used herein with reference to the antigens of the disclosure 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, introduction of D-amino acids.

[0099] 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.Methods of Use

[0100] 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 NT1 associated antigens, portions, homologues, derivatives, mimotopes, or combinations thereof.

[0101] In various embodiments, the subject is a subject who has been diagnosed with NT1 , a subject who is receiving therapy for NT1 , or a subject who is at a risk of developing with NT1 , or a subject who is at risk of developing co-morbidities, conditions, or diseases associated with NT 1 . The diagnosis NT 1 is based upon the presence of cataplexy and / or a cerebrospinal fluid(CSF) hypocretin / orexin level < 110 pg / mL. Subjects with intermediate levels of hypocretin / orexin (>110pg / mL - 200 pg / mL) combined with excessive daytime sleepiness can be considered to be at a risk of developing NT 1 .

[0102] Also provided herein are methods for treating additional autoimmune, neurodegenerative, psychiatric, or hypothalamic conditions in which one or more NT1- associated antigens encapsulated within the TIMP-NT1 particles are implicated. Such conditions may include, for example, narcolepsy type 2, idiopathic hypersomnia, Kleine-Levin syndrome, obstructive sleep apnea, primary insomnia, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, multiple system atrophy, depression, anxiety disorders, schizophrenia, autism spectrum disorder, hypothalamic obesity, craniopharyngioma-associated hypothalamic dysfunction, Prader-Willi syndrome, diabetes insipidus, and hypopituitarism, each of which has been associated with orexin dysregulation, loss of orexin-producing neurons, or broader hypothalamic dysfunction. The method comprises inducing immune tolerance in a subject in need thereof by administering a composition comprising negatively charged particles encapsulating one or more NT1 -associated antigens, or fragments, homologues, derivatives, mimotopes, or combinations thereof.

[0103] If TIMP-NT1 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.

[0104] In various embodiments, TIMP-NT1 is administered at a dose of about 0.1 mg, 0.25 mg, 0.5 mg, 1 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg,750 mg, 775 mg, or 800 mg. In various embodiments, TIMP-NT 1 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-NT1 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.

[0105] TIMP-NT1 particles can be given in any dose effective to dampen the autoimmune response in a subject in need thereof or to treat NT1 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 beads, a preferred dose is approximately 4x109beads, for 0.05 pm beads, a preferred dose is approximately 4x1012beads, for 3 pm beads, a preferred dose is 2x 107beads, however, any dose that is effective in treating NT 1 is encompassed by the current invention.

[0106] In some embodiments, TIMP-NT1 particles are administered in combination with one or more therapeutics. In various embodiments, the therapeutic is selected from the group comprising methylphenidate, amphetamines, oxybates, sodium oxybate, or mixed oxybate salts, modafinil, armodafinil, pitolisant, venlafaxine, fluoxetine, solriamfetol, duloxetine, benzodiazepine receptor agonist, orexin receptor agonist, antidepressants. In various embodiments, the therapeutic induces regulatory T-cells (e.g. IL-2 cytokine or PD-1 agonist). In various embodiments, the therapeutic decreases effector T-cells (e.g. CD3 antagonist). In various embodiments, the therapeutic decreases neuronal senescence, epigenetic silencing, or revives neuronal function compared to before administration of TIMPs.

[0107] In various embodiments, the one or more therapeutic is administered prior to, concurrently with or subsequent to administration of TIMP-NT1 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-NT1 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-NT 1 . In various embodiments, the therapeutic is administered 1 , 2, 3, or 4 weeks prior to administration of TIMP-NT 1 . 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-NT 1 . 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-NT 1 . In various embodiments, the therapeutic is administered concurrently with TIMP-NT 1 . 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-NT 1 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-NT 1 . In various embodiments, the therapeutic is administered 1 , 2, 3, or 4 weeks subsequent to administration of TIMP-NT 1 . 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-NT 1 . 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-NT1 described herein.

[0108] In various embodiments, administering TIMP-NT1 in a subject in need thereof induces antigen specific tolerance to an NT1 associated antigen. In various embodiments, administering TIMP-NT1 in a subject reduces the inflammatory immune response to NT1 associated antigens. In various embodiments, administering the TIMPs in a subject ameliorates the autoimmune response to NT1 associated antigens. Inducing tolerance or removing autoinflammatory mediators by administering TIMP-NT1 will restore hypocretin expression by not only stopping destruction of neurons but also reviving silenced or senescent neurons. In various embodiments, administration of TIMP-NT1 induces immunologic anergy. In various embodiments, the administration results in apoptosis of macrophages or monocytes in the subject. In some embodiments, administering TIMP-NT 1 decreases number and / or frequency of antigen-specific CD4+, or CD8+ T-cells compared to before the administration. In some embodiments, administering TIMP-NT1 decreases CD4+, or CD8+ T-cell infiltration. In some embodiments, administering TIMP-NT1 decreases number and / or frequency of activated antigen-specific CD4+, or CD8+ T-cells. In some embodiments, administering TIMP-NT1 decreases number and / or frequency of activated antigen-specific B-cells. In someembodiments, administering TIMP-NT1 induces regulatory T-cells (Tregs), or Trl cells. In various embodiments, administration of TIMP-NT1 induces neuronal regeneration, increases or stabilizes neuronal mass / number, decreases neuronal destruction, increases Treg:Teff ratio, reduces epitope spread, induces bystander tolerance, induces infectious tolerance, induces linked suppression, induces T-cell exhaustion, induces T-cell angergy, induces T-cell deviation, induces T-cell senescence, induces T-cell apoptosis, or induces T-cell deletion. In various embodiments, administration of TIMP-NT1 increases expression of regulatory receptors / ligands (e.g. PD-1 , PD-L1 , CTLA-4, IL-10, TGF-(3. In various embodiments, administration of TIMP-NT 1 increases a anti-inflammatory cytokine response (e.g. IL-10, TGF-£, IL-33).

[0109] In various embodiments, administering TIMP-NT1 in a subject induces cGAS-STING pathway. In various embodiments, administering the TIMP-NT1 in a subject induces type-1 IFN pathway. In various embodiments, the autoimmune and / or inflammatory 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 the NT1 associated antigens. In various embodiments, the immune response is a T cell, B cell, NK cell, monocyte, macrophage, eosinophil, or a basophil response. In various embodiments, the innate immune response is a proinflammatory APC response. In various embodiments, the adaptive immune response is an activated CD4+, CD8+ or a proinflammatory cytokine response. In various embodiments, the immune response is reduced immune infiltrate into tissues and / or organs. In various embodiments, administering the TIMP-NT1 to a subject in need thereof reduces the autoimmune and / or inflammatory response to 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”.

[0110] Administration of TIMP-NT1 to a subject in need thereof stabilizes or reduces narcoleptic symptoms. Administration of TIMP-NT1 to a subject in need thereof stabilizes or reduces disturbances in rapid eye movement (REM) sleep with the presence of hallucinations upon falling asleep (hypnagogic) or waking up (hypnopompic). Administration of TIMP-NT1 to a subject in need thereof stabilizes or reduces sleep attacks and / or sleep paralysis. Administration of TIMP-NT1 to a subject in need thereof stabilizes or reduces excessive daytime sleepiness symptoms. Administration of TIMP-NT1 to a subject in need thereof stabilizes or reducescataplexy symptoms. Administration of TIMP-NT1 decreases destruction of hypocretinproducing neurons. Administration of TIMP-NT1 increases hypocretin expression and / or decreases hypocretin deficiency. Administration of TIMP-NT1 preserve and / or restore function of hypothalamic neurons. Administration of TIMP-NT1 reverses epigenetic modifications. Administration of TIMP-NT 1 to a subject in need thereof stabilizes or reduces the number antigen-specific CD4+ and / or CD8+ T-cells. Administration of TIMP-NT1 to a subject in need thereof stabilizes or reduces the number activated antigen-specific CD4+ and / or CD8+ T-cells. Administration of TIMP-NT1 to a subject in need thereof increases the number of antigenspecific regulatory T-cells. Administration of TIMP-NT 1 to a subject in need thereof decreases pro-inflammatory cytokines and / or increases anti-inflammatory cytokines. Administration of TIMP-NT 1 to a subject in need thereof stabilizes or reduces the number of proinflammatory APCs. Administration of TIMP-NT1 to a subject in need thereof stabilizes or increases hypocretin / orexin levels in the CSF. In another aspect, the invention provides compositions and methods for diagnosing or characterizing narcolepsy, stratifying patient populations, differentiating narcolepsy sub-types, distinguishing narcolepsy from other hypersomnia disorders. Narcolepsy is increasingly recognized as an autoimmune disorder; however, definitive evidence remains limited. The present disclosure not only offers a therapeutic strategy but may also serve as functional proof-of-concept that NT 1 is mediated by autoimmune mechanisms. Successful induction of immune tolerance using TIMP-NT1 would strengthen the autoimmune hypothesis underlying narcolepsy. The present composition targets narcolepsyspecific antigens, including post-translationally modified hypocretin peptides and viral mimotopes.Screening Methods

[0111] It is contemplated that induction of and maintenance of immunological tolerance is monitored in a subject suffering from NT1 treated, or about to undergo treatment, with particles as described herein.

[0112] 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 v / o 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.

[0113] In various embodiments, the immune tolerance status of a subject is determined from the assay of one or more biological samples from the subject. Biological samples include whole-blood, peripheral blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, cerebrospinal fluid (CSF), stool, a tissue biopsy, and / or a bone-marrow biopsy. In various embodiments, the assay of the biological sample(s) includes analyzing levels of, and or presence or absence of, cell-surface proteins, extracellular proteins, intracellular proteins, nucleic acids, metabolites, enzymes, and / or combinations thereof relevant in the disease or disorder.

[0114] Cells assayed from the biological sample include immune cells, non-immune cells, and / or combinations thereof. Immune cells include innate immune cells, adaptive immune cells, and / or combinations thereof. Innate immune cells assayed from the biological sample(s) are antigen-presenting cells (APCs). Exemplary innate immune cells assayed from the biological sample include monocytes, macrophages, neutrophils, granulocytes, dendritic cells, mast cells, eosinophils, basophils, and / or combinations thereof. Adaptive immune cells assayed from the biological sample(s) include effector immune cells, such as CD4+ T cells, CD8+ T cells, B cells, NK cells, NK-T cells, and / or combinations thereof. In various embodiments, the T cells are Th1 cells, Th2a cells, Treg cells, and Tr1 cells.

[0115] In certain embodiments, the cells assayed from the biological sample(s) are epithelial cells, stromal cells, endothelial cells, fibroblasts, pericytes, adipocytes, mesenchymal stem cells, hematopoietic stem cells, hematopoietic progenitor cells, hepatocytes, liver sinusoidal endothelial cells (LSECs), and / or Kupffer cells.

[0116] One aspect of a subject’s immune tolerance status, and immune signature, is determined by analyzing one or more proteins from one or more biological sample(s) from the subject. In various embodiments, the proteins are cytokines and / or chemokines. In various embodiments the proteins are cell signaling proteins. In various embodiments, the cytokines and chemokines are selected from the group consisting of IL-1 a, IL-1 p, IL-2, IL-3, IL-4, IL-5, IL- 6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-12p70, IL-13, IL-14, IL-15, IL-16, IL-17, IL-17, IL-18, IL- 20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-27b, IL-28, IL-29, IL-30, IL-31 , IL-32, IL-33, IL-35, IL-36, CCL1 , CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11 , CCL12, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21 , CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1 , CXCL2 (MCP-1), CXCL3 (MIP-1 a, CXCL4 (MIP-1 p, CXCL5 (RANTES), CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11 , CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, GM-CSF, IFN-a, IFN-p, IFN-y, TNF-a, TGF-pi ,TGF-P2, TGF-|33, soluble CD14, and / or combinations thereof. In various embodiments, the protein is a protease. In various embodiments, the protease is an aspartic protease, a cysteine protease, a metalloprotease, a serine protease, and / or a threonine protease. In various embodiments, the protease is selected from the group consisting of ADAM1 , ADAM2, ADAM7, ADAM8, ADAM9, ADAM10, ADAM11 , ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAAM20, ADAM21 , ADAM22, ADAM23, ADAM28, ADAM29, ADAM30, ADAM33, MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21 , MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28. In various embodiments, proteins associated with apoptosis are selected from the group consisting of P53, Caspase 1 , Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 11 , Caspase 12, Caspase 13, Caspase 14, BCL-2, BCL-XL, MCL-1 , CED-9, A1 , BFL1 , BAX, BAK, DIVA, BCL-XS, BIK, BIM, BAD, BID, and EGL-1 . Several methods for assaying proteins from a biological sample have been described in the literature including enzyme-linked immunosorbent assay (ELISA), western blots, and mass spectrometry. In various embodiments the protein is one or more immunoglobulins (Ig). In various embodiments, the Ig are selected from the group consisting of IgA, IgD, IgE, IgM, and / or variants thereof. In various embodiments the immunoglobulins are antigen specific. Several methods for the detection of immunoglobulins from a biological sample have been described in the literature including ELISA and ImmunoCap.

[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., 201 1), (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 CD1 c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11 b, CD1 1c, 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, CD41 b, 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, FcpeRI, MHC-I, MHC-II, IL-1 R, IL-2Ra, IL-2Rp, IL-2Ry, IL-3Ra, CSF2RB, IL-4R, IL-5Ra, CSF2RB, IL-6Ra, gp130, IL-7Ra, IL-9R, IL-10R, IL-12R 1 , IL-12R|32, IL-13Ra1 , IL-13Ra2, IL-15Ra, IL-21 R, IL-23R, IL-27Ra, IL-31 Ra, OSMR, CSF-1 R, cell-surface IL-15, IL-10Ra, IL-1 ORp, IL-20Ra, IL-2ORJ3, IL-22Ra1 , IL-22Ra2, IL-22Rp, IL-28RA, PD-1 , PD- 1 H, BTLA, CTLA-4, PD-L1 , PD-L2, 2B4, B7-1 , B7-2, B7-H1 , B7-H4, B7-DC, DR3, LIGHT, LAIR, LTai p2, LT[3R, TIM-1 , TIM-3, TIM-4, TIGIT, LAG-3, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41 -BB, 41 BB-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, CCR1 1 ,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, MICE, UL16, ULBP1 , ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULTI , RAE1 a,p,y,6, and £, H60a, H60b, H60c, GPR15, ST2, and / or combinations thereof. Integrins include a1 , a2, allb, a3, a4, a5, a6, a7, a8, a9, a10, a11 , aD, aE, aL, aM, aV, aX, [31 , [32, [33, [34, [35, [36, [37, [38 and / or combinations thereof. TCR include a, [3, y, 5, E, 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 TIMP-NT1 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-NT 1 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] The immune tolerance signature of a subject is generated using one or more of the following parameters assayed from one or more biological samples obtained from the subject and stimulated in vivo and / or ex vivo\ . proportion of effector T cells in the total T cell population,B. proportion of Treg cells in the total T cell population,C. proportion of effector B cells in the total B cell population,D. levels of specific IgG, and / or IgM,E. levels of inflammatory cytokines and chemokines,F. levels of anti-inflammatory cytokines and chemokines,G. levels of liver enzymes,H. levels of inflammatory metabolites, and l. levels of anti-inflammatory metabolites.

[0121] The immune tolerance signature is indicative of maintenance of immune tolerance if 1 , 2, 3, 4, 5, 6, 7, 8, or 9 parameters listed in (a)-(i) above indicate maintenance of immune tolerance. In various embodiments, the immune tolerance signature is indicative of maintenance of immune tolerance if at least 2 / 9 parameters listed in (a)-(i) indicate maintenance of immune tolerance. In various embodiments, the subject is determined to not require treatment with TIMPs if 1 , 2, 3, 4, 5, 6, 7, 8, or 9 parameters listed in (a)-(i) above indicate maintenance of immune tolerance. In various embodiments, the subject is determined to not require treatment with TIMPs if at least 3 / 9 parameters listed in (a)-(i) above indicate maintenance of immune tolerance.

[0122] The immune tolerance signature of a subject generated using one or more parameters described herein indicates weakening and / or absence of immune tolerance prior to or after treatment with TIMP-T 1 D, if:

[0123] a. the proportion of effector T cells in the total T cell population is between 0.01%- 100% (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, 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), relative to the subject’s baseline measurement and / or relative to a placebo control and / or

[0124] b. the proportion of Treg cells in the total T cell population is between 0.01 -100% (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, 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) relative to the subject’s baseline measurement and / or relative to a placebo control, and / or

[0125] c. the proportion of effector B cells in the total B cell population is between 0.01%- 100% (e.g about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1 %, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, 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), relative to the subject’s baseline measurement and / or relative to a placebo control and / or

[0126] d. the levels of IgG and / or IgM are increased by about 0.01 %-100% (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, 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 placebo control, and / or

[0127] e. levels of inflammatory cytokines / chemokines are increased by about 0.01%- 100% (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, 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 allvalues and ranges between these values) relative to the subject’s baseline measurement and / or relative to a placebo control, and / or

[0128] f. levels of anti-inflammatory cytokines and chemokines are decreased by about 0.01%-100% (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, 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 a relative to the subject’s baseline measurement and / or relative to a placebo control, and / or

[0129] g. levels of liver enzymes are increased by about 0.01 %-100% (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, 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 placebo control, and / or

[0130] h. levels of inflammatory metabolites are increased by about 0.01 %-100% (e.g., about 0.01 %, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, 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 placebo control, and / or

[0131] i. levels of anti-inflammatory metabolites are decreased by about 0.01 %-100% (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, 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 placebo control.

[0132] 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.

[0133] 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, activatedantigen specific T cells, Th1 cells, pathogenic Th2a+ cells, Th17 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.

[0134] 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.Pharmaceutical Formulations

[0135] 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.

[0136] 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.

[0137] 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 mayinclude other proteins for enhanced stability, such as albumin, lipoprotein, globulin, etc., subjected to mild chemical modifications or the like.

[0138] 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).

[0139] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0140] 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.

[0141] The TIMP comprising antigen as described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use.

[0142] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the modified particles are mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as 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.Kits

[0143] 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. 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.

[0144] Additional aspects and details of the disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting.EXAMPLESExample 1 - Efficacy of TIMPs at improving sleep attacks and catapletic episodes in an adoptive transfer model of narcolepsy

[0145] The efficacy of TIMP-NT1 (CNP-NT1 ) at inducing antigen specific tolerance and improving sleep attacks and cataplexy will be examined in a preventative and therapeutic mouse model.

[0146] CNP-NT1 consists of PLGA particles encapsulating recombinant orexin. CNP-NT1 particles have an average diameter of 400-800 nm and a zeta potential between -30 and -80 mV. Two other particle formulations, (i) PLGA particles, 400-800 nm, -30 to -80 mV encapsulating viral hemagglutinin (CNP-HA) and (ii) PLGA particles, 400-800 nm, -30 to -80 mV encapsulating both orexin and viral hemagglutinin (CNP-orexHA) were also tested.

[0147] F1 -Orex-HA mice, expressing HA specifically in hypothalamic orexin-i- neurons will be used in this study. F1 -Orex-HA mice are divided into five groups and injected intravenously with CNP-NT1 (2.5 mg / mouse), CNP-HA (2.5 mg / mouse), CNP-orexHA (2.5 mg / mouse), Unloaded CNPs (Control, 2.5 mg / mouse), and PBS respectively on Day 0, Day +7, and Day +14. On day 0, 3x107HA-specific TCR-transgenic CD8+cytotoxic T-lymphocytes (CTLs) and / or HA-specific CD4+Th1 cells are adoptively transferred intravenously to F1 -Orex-HA mice.

[0148] CNP-NT 1 , CNP-HA, CNP-orexHA treated mice will show reduced neuronal loss and manifestations mimicking human narcolepsy compared to mice administered unloaded CNPs and PBS.

[0149] Results of this study will demonstrate that TIMPs encapsulating NT 1 associated antigens are effective at inducing antigen specific tolerance leading to improved narcoleptic symptoms and neuronal loss.Example 2 - Intravenous administration of TIMPs in a process mimicking narcolepsy elicited by vaccination

[0150] The F1-Orex-HA mouse model will be used to assess the efficacy of CNPs to induce tolerance to influenza vaccine antigens in a prophylactic manner. Five groups of Orex-HA mice will be dosed with naive HA-specific TCR-transgenic CD4+ and / or CD8+ T cells on day -8. Each group will be treated with CNP-NT 1 (2.5 mg / mouse), CNP-HA (2.5 mg / mouse), CNP-orexHA (2.5 mg / mouse), unloaded CNPs (2.5 mg / mouse), or PBC on day -7 and day 0. On day 0, mice are vaccinated with Pandremix influenza vaccine. Mice treated with unloaded CNPs or PBS will result in long-lasting T cell infiltration of the hypothalamus associated with progressive loss of HCRT / orexin-expressing neurons which will not observed in mice treated with CNP-NT 1 , CNP- HA or CNP-orexHA.

[0151] The F1-Orex-HA mouse model will be used to assess the efficacy of CNPs to induce tolerance to influenza vaccine antigens in a therapeutic setting. Five groups of Orex-HA mice will be dosed with naive HA-specific TCR-transgenic CD4+ and / or CD8+ T cells on day -1 . On day 0, mice are vaccinated with Pandremix influenza vaccine. Mice develop narcoleptic disease that peaks between day 7 and day 21 . Each group will be treated with CNP-NT 1 (2.5 mg / mouse), CNP-HA (2.5 mg / mouse), CNP-orexHA (2.5 mg / mouse), unloaded CNPs (2.5 mg / mouse), or PBC on day 21 , day 28 and day 35. Mice treated with unloaded CNPs or PBS will result in long-lasting T cell infiltration of the hypothalamus associated with progressive loss of HCRT-expressing neurons which will not observed in mice treated with CNP-NT 1 , CNP-HA or CNP-orexHA.

[0152] Results of this study will demonstrate that TIMPs encapsulating NT 1 associated antigens are effective at inducing antigen specific tolerance to mimotopes leading to improved narcoleptic symptoms and neuronal loss.Example 3 - Intravenous administration of TIMPs in an AAV-HCRT / orexin model to restore orexin signaling

[0153] Gene therapy is used to restore HCRT / orexin signaling in an Atx mouse model of narcolepsy. In these Atx mice, expression of a toxic protein (ataxin-3) selectively kills orexin neurons by 8-12 weeks of age. Atx mice between weeks 13-19 are divided into 2 groups, administered intravenously AAV8 encoding the transgene for orexin on day 0 and then administered 2 doses of either CNP-NT 1 or PBS also administered i.v.

[0154] Gene therapy is also used to restore HCRT / orexin signaling in the F1 -Orex-HA mouse model. F1 -Orex-HA mice are divided into five groups and on day 0, 3x107HA-specific TCR- transgenic CD8+cytotoxic T-lymphocytes (CTLs) and / or HA-specific CD4+Th1 cells are adoptively transferred intravenously. On day 0, Day +7 and Day +15, mice are injected intravenously with CNP-NT1 (2.5 mg / mouse), CNP-HA (2.5 mg / mouse), CNP-orexHA (2.5 mg / mouse), Unloaded CNPs (Control, 2.5 mg / mouse), or PBS. On day 30, mice are administered AAV8 encoding the transgene HCRT / orexin by microinjection into the mediobasal hypothalamus.

[0155] At the end of the study, mice dosed with CNP-NT 1 , CNP-HA, and CNP-orexHA followed by AAV8-orexin will show increased levels of HCRT / orexin compared to unloaded CNPs or PBS treated mice. Mice will also show long bouts of wakefulness and have a normal diurnal pattern of arousal without cataplexy.

[0156] Results of this study will show that inducing tolerance to HCRT / orexin can aid in restoring orexin signaling by AAV gene therapy even in cases where HCRT / orexin-producing neurons were previously eliminated by the autoimmune response.Example 4 - Intravenous administration of TIMPs in a process mimicking narcolepsy elicited by vaccination

[0157] PLGA particles (50:50 lactic:glycolic acid) were prepared via double emulsion to encapsulate hemagglutinin protein. The resulting particles had an average diameter of 402 nm, zeta potential of - 49 mV.

[0158] Activated CD8+T cells specific for influenza hemagglutinin (HA512-520, amino acid sequence IYSTVASSL) were adoptively transferred into F1 -Orex-HA mice, a transgenic model expressing HA in the hypothalamus, on Day 0. On Day +1 , mice (n=4-5) received one of the following treatments via intravenous injection: Vehicle control, Unloaded control PLGA nanoparticles (2.5 mg), or HA-loaded PLGA nanoparticles (2.5 mg).

[0159] On Day 8, central nervous system (CNS) tissues are harvested to assess various immune response, including: CD8+T cell infiltration, cytokine production (e.g., IFN-y) and microglial activation and MHO class II expression

[0160] It is hypothesized that treatment with HA-Loaded Nanoparticle will have reduced frequency of IFN-y+CD8+T cells infiltrating the CNS, and reduced expression of MHC class II on microglia, indicating dampened neuroinflammation.

[0161] It is expected that administration of the particles results in decreased CD4+ and CD8+ T-cells in the CNS. Teffreduction, Treginduction, and cytokine suppression are also measured.Example 4 - Assessment of immune tolerance in mouse models

[0162] C57BL / 6 or orexin knockout mice are primed subcutaneously with 100 pg of orexin emulsified in CFA. On Day 0, mice are randomized into five treatment groups. Mice are treated intravenously with TIMP-NT1 (PLGA nanoparticles, 400-800 nm diameter, -80 mV to -30 mV encapsulating orexin protein) at 0.25 mg, 0.5 mg, 1.25 mg, 2.5 mg doses, unloaded particle controls at 2.5 mg, saline, on Days 0, 7, and 14. On Day 21 post priming, the baseline pinna thickness of both ears of each mouse is measured. On the same day, mice are intradermally challenged with 10 pg (10 pL injection volume) OVA protein in the left ear and 10 pg (10 pL injection volume) orexin protein in the right ear. The pinna thickness of both ears of each mouse is evaluated 24 hours post challenge, and the delta pinna thickness is calculated. A significant reduction in mean ear swelling observed in the orexin-challenged ears of mice treated withTIMP-NT1 , compared to saline control or unloaded particles, indicates successful induction of antigen specific immune tolerance to orexin.

[0163] 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.

[0164] 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.REFERENCES

[0165] 1 . Sanchez A, Mejia SP, Orozco J. Recent Advances in Polymeric Nanoparticle- Encapsulated Drugs against Intracellular Infections. Molecules. 2020;25(16).

[0166] 2. Zielihska A, Carreiro F, Oliveira AM, et al. Polymeric Nanoparticles: Production, Characterization, Toxicology and Ecotoxicology. Molecules. 2020;25(16).

[0167] 3. Bernard-Valnet R, Yshii L, Queriault C, et al. CD8 T cell-mediated killing of orexinergic neurons induces a narcolepsy-like phenotype in mice. Proc Natl Acad Sci U S A. 2016;113(39):10956-10961.

[0168] 4. Kantor S, Mochizuki T, Lops SN, et al. Orexin gene therapy restores the timing and maintenance of wakefulness in narcoleptic mice. Sleep. 2013;36(8):1129-1138. Published 2013 Aug 1. doi:10.5665 / sleep.2870

Claims

WE CLAIM:1 . A composition comprising tolerizing immune modifying particles (TIMP) particles encapsulating one or more narcolepsy with cataplexy (NT1 ) associated antigens (TIMP- NT1), portions thereof, or combinations thereof.

2. The composition of claim 1 , wherein the particle comprises a liposome, lipid nanoparticle, polymersomes, micelles, polymeric nanoparticles, acid-ionizable iron nanoadjuvant (INOP), mesoporus silica, metal-organic frameworks (MOFs), lipid nanodiscs, supramolecular nanoparticles, extracellular vesicles, or sono-driven nanoparticles.

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

4. The composition of any one of claims 1 -3, wherein the biodegradable polymer is polyglycolic acid (PGA), poly (lactide-co-glycolide) (PLG), polylactic acid (PLA), a copolymer of PLG and PLA (PLGA), polycaprolactone (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.

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

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

7. The composition of claim 5, wherein the particles have a zeta potential between -100 mV and -30 mV.

8. The composition of claim any one of claims 1 -7, wherein the particles have a zeta potential between -30 and -80 mV.

9. The composition of any one of claims 1 -8, wherein the particle size is between 0.1 pm and 10 pm.

10. The composition of any one of claims 1 -9, wherein the particle size is between 100 nm and 1000 nm.1 1. The composition of claim 10, wherein the particle size is between 400-800 nm.

12. The composition of any one of claims 1 -9, wherein at least 90% of the particles have a diameter between about 0.1 pm and 5 pm, about 0.1 pm and 3 pm, about 0.3 pm and 5 pm, or about 0.3 pm to 3 pm.

13. The composition of any one of claims 1 -9, wherein at least 50% of the particles have a diameter between about 0.1 pm and 5 pm, about 0.1 pm and 3 pm, about 0.3 pm and 5 pm, or about 0.3 pm to 3 pm.

14. The composition of any one of claims 1 -9, wherein at least 10% of the particles have a diameter between about 0.1 pm and 5 pm, about 0.1 pm and 3 pm, about 0.3 pm and 5 pm, or about 0.3 pm to 3 pm.

15. The composition of any one of claims 1 -14, wherein the antigen comprises one or more proteins, peptides, homologues, derivatives, mimotopes, one or more antigenic epitopes or combinations thereof.

16. The composition of claim 15, wherein the antigen is selected from the group consisting of the antigens set out in Table 1 .

17. The composition of claim 15, wherein the antigen is selected from the antigens set out in Figure 1.

18. The composition claim 15, wherein the antigen is selected from the group consisting of hypocretin / orexin (HCRT), HCRT cell proteins, HCRT receptor 1 (HCRTR1 ), HCRT receptor 2 (HCRTR2), muscarinic receptors, protein O-mannosyltransferase (POMT) enzyme complex (POMT1 ), regulatory factor X4 (RFX4), Tribbles homologue 2 (TRIB2), Lim homeobox 9 (LHX9), prostaglandin D2 receptor (DP1 ), ganglioside M3 (GM3), B cell lymphoma 6 protein, neuropeptide glutamic acid-isoleucine / a-melanocyte-stimulating hormone (NEI / aMSH), [32-adrenergic receptor (ADRB2), methyltransferase-like 22 (METTL22), 5'-nucleotidase cytosolic IA (NT5C1A), neurexin 1 a (NRXN1 ), ubiquitin carboxyl-terminal hydrolase (MINDY-2 / FAM63B), LOC401464, protein mono-ADP- ribosyltransferase (PARP3), nociception receptor, HCRT1.13, HCRT56-68, HCRT87-99, HCRT86-97, N-pyroglutamate (pyro) HCRT34-48, pyro HCRT38-52, pyro HCRT33-47, pyro HCRT37-51, HCRT54-66-NH2, HCRT86-97-NH2, influenza virus epitope, hemagglutinin (HA), neuraminidase (NA), influenza RNA-dependent RNA polymerase subunit 1 (PB1 ), nucleoprotein (NP), and Streptolysin O antigen.

19. The composition of claim 15, wherein the antigen is SEQ ID NO. 827.

20. A method of inducing tolerance in a subject in need thereof comprising administering to the subject a composition comprising tolerizing immune modifying particles (TIMP-NT1) particles encapsulating one or more narcolepsy with cataplexy (NT1 ) associated antigens, portions thereof, or combinations thereof.21 . A method of inducing tolerance in a subject in need thereof comprising administering to the subject a composition comprising negatively charged particles (TIMP) encapsulating an antigen, wherein the antigen is one or more narcolepsy with cataplexy (NT1 ) associated antigens.

22. The method of claim 21 , wherein TIMP-NT1 comprises a liposome, lipid nanoparticle, polymersomes, micelles, polymeric nanoparticles, acid-ionizable iron nanoadjuvant (INOP), mesoporus silica, metal-organic frameworks (MOFs), lipid nanodiscs, supramolecular nanoparticles, extracellular vesicles, or sono-driven nanoparticles.

23. The method of claim 21 , 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.

24. The method of any one of claims 21 -23, wherein the particles have a negative zeta potential.

25. The method of claim 24, wherein the particles have a zeta potential between 0 mV and - 100 mV.

26. The method of claim 24, wherein the particles have a zeta potential between -30 mV and -100 mV.

27. The method of any one of claims 20-26, wherein the size of the particle is between 100 nm and 1000 nm.

28. The method of any one of claims 20-26, wherein the size of the particle is between 400 nm and 800 nm.

29. The method of any one of claims 20-28, wherein the antigen comprises one or more peptide, protein, homologue, derivative, mimotope, or combinations thereof.

30. The method of claim 22, wherein the antigen wherein the antigen is selected from the molecules set out in Table 1 .31 . The method of claim 22, wherein the antigen wherein the antigen is selected from the antigens set out in Figure 1 .

32. The method of claim 22, wherein the antigen is selected from the group consisting of hypocretin / orexin (HCRT), HCRT cell proteins, HCRT receptor 1 (HCRTR1 ), HCRT receptor 2 (HCRTR2), muscarinic receptors, protein O-mannosyltransferase (POMT) enzyme complex (POMT1 ), regulatory factor X4 (RFX4), Tribbles homologue 2 (TRIB2), Lim homeobox 9 (LHX9), prostaglandin D2 receptor (DP1 ), ganglioside M3 (GM3), B cell lymphoma 6 protein, neuropeptide glutamic acid-isoleucine / a-melanocyte-stimulating hormone (NEI / aMSH), [32-adrenergic receptor (ADRB2), methyltransferase-like 22 (METTL22), 5'-nucleotidase cytosolic IA (NT5C1A), neurexin 1 a (NRXN1 ), ubiquitin carboxyl-terminal hydrolase (MINDY-2 / FAM63B), LOC401464, protein mono-ADP- ribosyltransferase (PARP3), nociception receptor, HCRT1.13, HCRT56-68, HCRT87-99, HCRT86-97, N-pyroglutamate (pyro) HCRT34-48, pyro HCRT38-52, pyro HCRT33.47, pyro HCRT37-5i, HCRT54-66-NH2, HCRTs6 -97-NH2, influenza virus epitope, hemagglutinin (HA), neuraminidase (NA), influenza RNA-dependent RNA polymerase subunit 1 (PB1 ), nucleoprotein (NP), or Streptolysin O antigen.

33. The method of claim 29, wherein the antigen is SEQ ID NO. 827.

34. The method of any one of claims 20-33, wherein the subject has an autoimmune disease.

35. The method of claim 34, wherein the autoimmune disease is narcolepsy with cataplexy (NT1 ).

36. The method of any one of claims 20-35, wherein administration of the composition reduces a NT1 associated immune response.

37. The method of claim 36, wherein the immune response is an inflammatory and / or autoimmune response.

38. The method of claim 37, wherein the immune response is a humoral immune response.

39. The method of claim 37, wherein the immune response is an adaptive immune response.

40. The method of claim 37, wherein the immune response is an innate immune response.41 . The method of claim 38, wherein the immune response is an antibody response.

42. The method of claims 20-21 , wherein the composition is administered intravenously, intramuscularly, ocularly, intraperitoneally, transdermally, nasally, orally, intra- lymphatically and / or subcutaneously.

43. A composition comprising Tolerizing immune modifying particles (TIMP-NT1) particles encapsulating a viral vector encoding narcolepsy with cataplexy (NT1 ) associated antigens.

44. The composition of claim 43, wherein TIMP-NT1 encapsulates a viral vector encoding HCRT / orexin.

45. The composition of claim 43 or 44, wherein the viral vector is a AAV vector.

46. The composition of any one of claims 43-45, wherein the particles comprise a biodegradable polymer.

47. The composition of any one of claims 43-46, wherein the biodegradable polymer is polyglycolic acid (PGA), poly (lactide-co-glycolide) (PLG), polylactic acid (PLA), a copolymer of PLG and PLA (PLGA), polycaprolactone (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.

48. The composition of any one of claims 43-47, wherein the particles have a negative zeta potential.

49. The composition of claim 48, wherein the particles have a zeta potential between -100 mV and 0 mV.

50. The composition of claim 48, wherein the particles have a zeta potential between -100 mV and -30 mV.51 . The composition of any one of claims 43-50, wherein the particles have a zeta potential between -30 and -80 mV.

52. The composition of any one of claims 43-51 , wherein the particle size is between 100 nm and 1000 nm.

53. The composition of any one of claims 43-52, wherein the particle size is between 400-800 nm54. The composition of any one of claims 43-53, wherein the antigen comprises one or more proteins, peptides, homologues, derivatives, mimotopes, one or more antigenic epitopes or combinations.

55. The composition of claim 54, wherein the antigen is selected from the group comprising Table 1 .

56. The composition of claim 55, wherein the antigen is selected from the antigens set out in Figure 1 .

57. The composition of any one of claims 54-56, wherein the antigen is selected from hypocretin / orexin (HCRT), HCRT cell proteins, HCRT receptor 1 (HCRTR1 ), HCRT receptor 2 (HCRTR2), muscarinic receptors, protein O-mannosyltransferase (POMT) enzyme complex (POMT1 ), regulatory factor X4 (RFX4), Tribbles homologue 2 (TRIB2), Lim homeobox 9 (LHX9), prostaglandin D2 receptor (DP1 ), ganglioside M3 (GM3), B cell lymphoma 6 protein, neuropeptide glutamic acid-isoleucine / a-melanocyte-stimulating hormone (NEI / aMSH), [32-adrenergic receptor (ADRB2), methyltransferase-like 22 (METTL22), 5'-nucleotidase cytosolic IA (NT5C1A), neurexin 1 a (NRXN1 ), ubiquitin carboxyl-terminal hydrolase (MINDY-2 / FAM63B), LOC401464, protein mono-ADP- ribosyltransferase (PARP3), nociception receptor, HCRT1.13, HCRT56-68, HCRT87-99, HCRT86-97, N-pyroglutamate (pyro) HCRT34-48pyro HCRT38-52, pyro HCRT33-47, pyro HCRT37-51, HCRT54-66-NH2, HCRT86 -97-NH2, influenza virus epitope, hemagglutinin (HA), neuraminidase (NA), influenza RNA-dependent RNA polymerase subunit 1 (PB1 ), nucleoprotein (NP), or Streptolysin O antigen.

58. The composition of claim 57, wherein the antigen is SEQ ID NO. 827.

59. A method of treating disorders or additional autoimmune, neurodegenerative, psychiatric, or hypothalamic conditions in which one or more narcolepsy with cataplexy (NTI )-associated antigens encapsulated within the tolerizing immune modifying particles (TIMP-NT1 ) particles are implicated by administering a composition comprising TIMP- NT1.

60. The method of claim 59, wherein the disorder is selected from the group consisting of narcolepsy type 2, idiopathic hypersomnia, Kleine-Levin syndrome, obstructive sleep apnea, primary insomnia, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, multiple system atrophy, depression, anxiety disorders, schizophrenia, autism spectrum disorder, hypothalamic obesity, craniopharyngioma-associated hypothalamicdysfunction, Prader-Willi syndrome, diabetes insipidus, and hypopituitarism, autoimmune encephalitis associated with anti-Ma2 autoantibodies either of paraneoplastic origin or triggered by checkpoint inhibitors, anti-glial acid fibrillary protein astrocytopathy, neuromyelitis optica spectrum disorder (NMOSD), anti-Hu and anti- / V-methyl-D- aspartate receptor (NMDAR) autoimmune encephalitis.61 . A composition comprising lipid nanoparticles encapsulating one or more narcolepsy with cataplexy (NTI )-associated antigens, wherein the lipid nanoparticles have a diameter between 100 nm and 1000 nm and a zeta potential between -100 mV and 0 mV.

62. The composition of claim 61 , wherein the lipid nanoparticles are formulated with one or more lipids selected from the group consisting of DOTMA, DOPE, DOTAP, DDAB, DLin- MC3-DMA, SM-102, ALC-0315, DSPC, cholesterol, and PEG2000-DMG.

63. The composition of claim 61 or 62, wherein the lipid nanoparticles encapsulate mRNA encoding one or more NT1 -associated antigens.

64. The composition of any one of claims 61-63, wherein the lipid nanoparticles further comprise one or more immunomodulatory agents.

65. The composition of claim 64, wherein the immunomodulatory agent is selected from the group consisting of dexamethasone, rapamycin, cyclosporine A, FK506, ITE, andrographolide, tofacitinib, rosiglitazone, IL-10, PD-1 , PD-L1 / L2, IL-2, TGF-£, TGF- - signaling agonist, GM-CSF, IFN-p, IL-35, IL-27, IL-4, hepatocyte growth factor, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) agonist, vitamin D, retinoic acid.

66. A method of inducing antigen-specific immune tolerance comprising administering to a subject a composition comprising lipid nanoparticles encapsulating one or more narcolepsy with cataplexy (NTI )-associated antigens.

67. The method of claim 66, wherein the lipid nanoparticles encapsulate mRNA encoding the NT1 associated antigen.

68. A composition comprising liposomes encapsulating one or more narcolepsy with cataplexy (NTI )-associated antigens, wherein the liposomes have a diameter between 100 nm and 1000 nm and a zeta potential between -100 mV and 0 mV.

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