Compositions and methods for reducing immune intolerance and treating autoimmune disorders - Patents.com

Abstract

Provided herein are compounds and pharma- ceutically acceptable salts thereof, lipid particles comprising such compounds or pharma- ceutically acceptable salts thereof, and compositions as described above, which can be used to reduce immune intolerance in a subject, for example to treat an autoimmune disorder, or to improve the efficacy of antigen therapy, for example in combination with protein therapy or gene therapy. The compounds have the following structural formula: JPEG2024532810000028.jpg2244 where the values ​​of the variables (e.g., X, R) are as described herein.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 233,163, filed August 13, 2021, the entire teachings of which are incorporated herein by reference. [Background technology]

[0002] Enzyme and protein replacement therapy is a successful therapeutic strategy for treating congenital disorders in which endogenous proteins are mutated, missing, or abnormal. However, clinical administration of exogenous enzymes or proteins is associated with the development of undesirable immune responses against the enzymes or proteins. Undesirable immune responses can lead to neutralization of the enzyme / protein or alteration of its pharmacokinetics. In many situations, patients have no alternative treatment options, and undesirable immune responses to treatment are a major challenge faced by recipients of enzyme and protein replacement therapy.

[0003] Similarly, gene therapy offers a promising approach to treat many congenital disorders and other diseases. The immunogenicity of the carrier and / or the genetic material carried therein is a major challenge to the clinical application of gene therapy. The presence of anti-carrier antibodies is a contraindication to some approved gene therapy treatments. Moreover, nascent anti-carrier antibodies may prevent repeated administration in subjects who receive the first dose of gene therapy.

[0004] Autoimmune disorders are a group of disorders in which the body lacks or loses tolerance to self-antigens. As a result, the body's immune system attacks healthy cells, which can have debilitating and devastating effects. Current approaches to treat autoimmune disorders rely on general immunosuppression at the humoral, cellular and / or complement levels, which renders patients immunocompromised and susceptible to opportunistic infections.

[0005] Thus, there is a need for compositions that can reduce immune intolerance to foreign antigens (e.g., enzyme replacement therapy, gene therapy) or endogenous antigens (e.g., self-antigens that cause autoimmune disorders), for example, by mitigating the immunogenicity of enzyme and protein replacement therapy and / or gene therapy, or by increasing self-tolerance to self-antigens. Summary of the Invention [Means for solving the problem]

[0006] The technology described herein relates to tolerance induction to foreign antigens (e.g., antigen-specific and / or antigen-exclusive tolerogenesis) or to self-antigens. The technology is based on engaging and regulating (e.g., activating) the T cell immunoglobulin mucin protein (TIM) family of receptors.

[0007] As used herein, compounds of the following structural formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the values ​​of the variables (e.g., R, X) are as described herein.

[0008] Also provided herein are lipid particles comprising one or more lipids, or a pharma- ceutically acceptable salt thereof, and a compound of the present disclosure.

[0009] Also provided herein are compositions (eg, pharmaceutical compositions) comprising the compounds of the present disclosure.

[0010] Also provided herein is a composition (eg, a pharmaceutical composition) comprising a plurality of lipid particles described herein.

[0011] Also provided herein is a method of tolerizing a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition described herein.

[0012] Also provided herein is a method of tolerizing a subject in need thereof to an antigen and inhibiting or reducing antigen-specific antibody titers in a subject, comprising administering to the subject an antigen and a therapeutically effective amount of a composition described herein, or administering to the subject a composition described herein comprising the antigen or an immunogenic fragment of the antigen.

[0013] Also provided herein are methods of inducing a population of regulatory T cells in a subject (e.g., in response to an antigen) and increasing the activity or level of tolerogenic T cells in a subject, comprising administering to the subject a therapeutically effective amount of a composition described herein (e.g., a composition described herein that includes an antigen or an immunogenic fragment of an antigen).

[0014] Also provided herein is a method of inducing a population of regulatory B cells in a subject (e.g., in response to an antigen), comprising administering to the subject a therapeutically effective amount of a composition described herein (e.g., a composition described herein that includes an antigen or an immunogenic fragment of an antigen).

[0015] Also provided herein are methods for treating an autoimmune disorder in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a composition described herein (e.g., a composition described herein that includes an autoantigen associated with the autoimmune disorder).

[0016] Also provided herein are methods for treating a disease, disorder, or condition in a subject in need of treatment with antigen therapy, comprising administering to the subject antigen therapy (e.g., a therapeutically effective amount of antigen therapy) and a composition described herein in an amount sufficient to immunotolerize the subject to antigen therapy, or a therapeutically effective amount of a composition described herein that includes antigen therapy.

[0017] Also provided herein are compositions (e.g., pharmaceutical compositions) for the uses described herein (e.g., treatment of an autoimmune disorder, treatment of a disease, disorder, or condition treatable with antigen therapy), wherein the composition is a composition described herein. Also provided herein are uses of the compositions described herein for the manufacture of a medicament for the uses described herein (e.g., treatment of an autoimmune disorder, treatment of a disease, disorder, or condition treatable with antigen therapy).

[0018] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0019] The above will be apparent from the following more particular description of the exemplary embodiments. [Brief description of the drawings]

[0020] [Figure 1] 1 shows a decision tree for the active ingredients described in Example 2.

[0021] [Figure 2A] Representative images of B cell clustering within a lymph node are shown, with the arrow indicating the cortex of the lymph node.

[0022] [Figure 2B] Shows DilC18(5) lymphatic uptake and B cell colocalization 60 min after oral gavage.

[0023] [Figure 2C] Shows DMPC lymphatic uptake and B cell co-localization 60 min after oral gavage.

[0024] [Figure 2D] Shows DOPC lymphatic uptake and B cell colocalization 60 min after oral gavage.

[0025] [Figure 2E]Shows DSPC lymphatic uptake and B cell co-localization 60 min after oral gavage.

[0026] [Figure 2F] Shows POPC lymphatic uptake and B cell co-localization 60 min after oral gavage.

[0027] [Figure 2G] Percentage of B cell / liposome colocalization by lipid type is shown.

[0028] [Figure 2H] Representative images of T cell clustering within lymph nodes are shown.

[0029] [Figure 2I] Shows DilC18(5) lymphatic uptake and T cell colocalization 60 min after oral gavage.

[0030] [Figure 2J] Shown is DMPC lymphatic uptake and T cell co-localization 60 min after oral gavage.

[0031] [Figure 2K] DOPC lymphatic uptake and T cell colocalization 60 min after oral gavage.

[0032] [Figure 2L] Shown is DSPC lymphatic uptake and T cell co-localization 60 min after oral gavage.

[0033] [Figure 2M] Shows POPC lymphatic uptake and T cell co-localization 60 min after oral gavage.

[0034] [Figure 2N] The percentage of T cell / liposome colocalization by lipid type is shown.

[0035] [Diagram 3]Changes in the percentage of FoxP3+ / TIM3+CD4 T cells in response to Compound 1 or Compound 2 are shown.

[0036] [Figure 4A] FIG. 1 is a schematic diagram for evaluating the pharmacodynamic (PD) effects of Compound 2 in mice.

[0037] [Figure 4B] Figure 1 shows the dose-PD relationship for Compound 2 in vivo, including dose-PD model fitting and associated confidence intervals around the mean of the model-predicted dose-PD. ED50 and ED90 values ​​are shown.

[0038] [Figure 5A] FIG. 1 is a schematic diagram of a study to determine the therapeutic use of liposomal compound 2 in an EAE multiple sclerosis (MS) model.

[0039] [Figure 5B] 1 shows time to disease onset in the EAE MS model.

[0040] [Figure 5C] 1 shows the clinical scores of treated and control groups in a preventative EAE MS model.

[0041] [Figure 5D] 1 shows the clinical scores of treated and control groups in a therapeutic EAE MS model.

[0042] [Figure 5E] FIG. 1 shows overall survival of treated and control groups in an EAE MS model.

[0043] [Figure 6] A representative transmission electron microscopy image of AAV9-CMVChry encapsulated in DMPC:GL67:compound 2 (85:5:10) liposomes is shown, in which the red arrow and circle indicate the encapsulated AAV9-CMVChry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Exemplary embodiments are described below.

[0045] definition

[0046] The compounds described herein include those described generally and further exemplified by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of the present invention, chemical elements are defined as defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Further, general principles of organic chemistry are identified in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5 th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, the relevant contents of which are incorporated herein by reference.

[0047] Unless otherwise specified herein, the nomenclature used herein generally follows the examples and rules set forth in Nomenclature of Organic Chemistry, Sections A, B, C, D, E, F, and H, Pergamon Press, Oxford, 1979, the chemical structure names and chemical structure naming rules of which are incorporated herein by reference. Optionally, the names of compounds may be generated using a chemical naming program (e.g., CHEMDRAW®, version 17.0.0.206, PerkinElmer Informatics, Inc.).

[0048] When introducing elements disclosed herein, the articles "a," "an," "the," and "said" are intended to mean that there is one or more elements. Furthermore, the one or more elements may be the same or different.

[0049] "About" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art. Usually, the acceptable error range of a particular value depends at least in part on how the value is measured or determined, e.g., on the limitations of the measurement system. For example, "about" may mean an acceptable standard deviation according to convention in the art. Alternatively, "about" may mean a range of ±20%, e.g., ±10%, ±5%, or ±1% of a given value. It should be understood that the term "about" may precede any particular value specified herein, except for the particular values ​​used in the examples.

[0050] "Alkyl" refers to a branched or straight-chain monovalent hydrocarbon radical having the specified number of carbon atoms. Thus, "(C1-C8)alkyl" refers to a radical having from 1 to 8 carbon atoms in a branched or straight-chain arrangement. In some embodiments, alkyl is any of the groups represented by (C1-C 30 ) alkyl, for example (C5-C 30 ) alkyl, (C1-C 25 )Alkyl, (C5-C 25 ) alkyl, (C 10 -C 25 ) alkyl, (C 15 -C 25 ) alkyl, (C 10 -C 20 ) alkyl, (C 15 -C 20 ) alkyl, (C1-C 15 ) alkyl, (C1-C 10)alkyl, (C1-C6)alkyl, or (C1-C5)alkyl. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, 2-methylpentyl, n-hexyl, and the like. In some embodiments, the alkyl is optionally substituted, for example, with one or more substituents described herein.

[0051] "Alkenyl" refers to a branched or straight-chain monovalent hydrocarbon radical having at least one carbon-carbon double bond and the specified number of carbon atoms. Thus, "(C2-C8)alkenyl" refers to a radical having at least one carbon-carbon double bond and from 2 to 8 carbon atoms in a branched or straight-chain arrangement. In some embodiments, alkenyl is any of the groups represented by (C1-C 30 ) alkenyl, for example (C5-C 30 ) alkenyl, (C1-C 25 ) alkenyl, (C5-C 25 ) alkenyl, (C 10 -C 25 ) alkenyl, (C 15 -C 25 ) alkenyl, (C 10 -C 20 ) alkenyl, (C 15 -C 20 ) alkenyl, (C1-C 15 ) alkenyl, (C1-C 10 )alkenyl, (C1-C6)alkenyl, or (C1-C5)alkenyl. Examples of alkenyl groups include ethenyl, 2-propenyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, allyl, 1,3-butadienyl, 1,3-dipentenyl, 1,4-dipentenyl, 1-hexenyl, 1,3-hexenyl, 1,4-hexenyl, 1,3,5-trihexenyl, 2,4-dihexenyl, and the like. In some embodiments, the alkenyl is optionally substituted, for example, with one or more substituents described herein.

[0052] "Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic, tricyclic) aromatic hydrocarbon ring system having the specified number of ring atoms and containing an aromatic ring fused to a non-aromatic ring, so long as one of the fused rings is aromatic hydrocarbon. Thus, "(C6-C 15 "Aryl" refers to a ring system having 6 to 15 ring atoms. Examples of aryl include phenyl, naphthyl, and fluorenyl. In some embodiments, aryl is optionally substituted, for example, with one or more substituents described herein.

[0053] "Heteroaryl" refers to a monocyclic or polycyclic (e.g., bicyclic, tricyclic) aromatic hydrocarbon ring system having the specified number of ring atoms, in which at least one carbon atom in the ring system is replaced with a heteroatom selected from nitrogen, sulfur, and oxygen. Thus, "(C5-C 15 "Heteroaryl" refers to a heteroaromatic ring system having 5 to 15 ring atoms consisting of carbon, nitrogen, sulfur, and oxygen. "Heteroaryl" includes a heteroaromatic ring fused to a non-aromatic ring, so long as one of the fused rings is a heteroaromatic hydrocarbon. Heteroaryl can contain 1, 2, 3, or 4 (e.g., 1, 2, or 3) heteroatoms independently selected from nitrogen, sulfur, and oxygen. Typically, heteroaryl is a heterocyclic ring system having 5 to 15 ring atoms consisting of carbon, nitrogen, sulfur, and oxygen. 20 ) Heteroaryl, for example (C5-C 15 )heteroaryl, (C5-C 12) heteroaryl, C5 heteroaryl, or C6 heteroaryl. Monocyclic heteroaryls include, but are not limited to, furan, oxazole, thiophene, triazole, triazene, thiadiazole, oxadiazole, imidazole, isothiazole, isoxazole, pyrazole, pyridazine, pyridine, pyrazine, pyrimidine, pyrrole, tetrazole, and thiazole. Bicyclic heteroaryls include, but are not limited to, indolizine, indole, isoindole, indazole, benzimidazole, benzofuran, benzothiazole, purine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, and pteridine. In some embodiments, heteroaryls are optionally substituted, for example, with one or more substituents described herein.

[0054] "Alkoxy" refers to an alkyl radical attached through an oxygen linking atom, where alkyl is as described herein. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, and the like.

[0055] "Halogen" and "halo" are used interchangeably herein and refer to fluorine, chlorine, bromine, or iodine, respectively. In some aspects, halo is fluoro, chloro, or bromo. In some aspects, halo is fluoro.

[0056] "Haloalkyl" includes mono-, poly-, and perhaloalkyl groups, where each halogen is independently selected from fluorine, chlorine, bromine, and iodine (e.g., fluorine, chlorine, and bromine), and alkyl is as described herein. In one aspect, the haloalkyl is a perhaloalkyl (e.g., perfluoroalkyl). Examples of haloalkyl include, but are not limited to, trifluoromethyl and pentafluoroethyl.

[0057] "Haloalkoxy" refers to a haloalkyl radical attached through an oxygen linking atom, where haloalkyl is as described herein. Examples of haloalkoxy include, but are not limited to, trifluoromethoxy.

[0058] The term "substituted" refers to the replacement of a hydrogen atom with a suitable substituent. Typically, a hydrogen atom bonded to a carbon atom is replaced with a suitable substituent, but a hydrogen bonded to a heteroatom, such as a nitrogen, oxygen, or sulfur atom, may also be replaced with a substituent. It will be understood that "substituted" or "substituted" includes the implicit condition that such substitution is in accordance with the allowed valence of the substituted atom. It is also preferred that the substituents and substitutions result in stable compounds that do not undergo spontaneous transformation, for example, by rearrangement, cyclization, elimination, and the like. Suitable substituents for use herein include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For example, suitable substituents may include halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkyl, alkoxy, alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, cycloalkyl, heterocyclyl, aralkyl, aryl, or heteroaryl.Those skilled in the art will understand that the substituents themselves can be substituted where appropriate.Thus, the substituents may further include, for example, acetamide.

[0059] The permissible substituents can be one or more and the same or different for appropriate organic compounds. Thus, "optionally substituted" groups can in some embodiments, independently, be halo, (C-C)alkoxy, (C-C)haloalkoxy, (C-C)alkyl, or (C-C)haloalkyl, or optionally substituted (C-C 15)Aryl or (C5-C 15 )heteroaryl. In some embodiments, an "optionally substituted" aryl or heteroaryl is substituted with 0-5 (e.g., 0-3, 0, 1, 2, 3, 4, 5) substituents independently selected from halo, (C1-C6)alkoxy, (C1-C6)haloalkoxy, (C1-C6)alkyl, or (C1-C6)haloalkyl. In some embodiments, an "optionally substituted" aryl or heteroaryl is substituted with 0-5 (e.g., 0-3, 0, 1, 2, 3, 4, 5) substituents independently selected from halo, (C1-C3)alkoxy, (C1-C3)haloalkoxy, (C1-C3)alkyl, or (C1-C3)haloalkyl. In some embodiments, the optionally substituted alkyl or alkenyl is independently halo (e.g., fluoro), (C-C)alkoxy, (C-C)haloalkoxy (e.g., (C-C)fluoroalkoxy), (C-C 15 )Aryl or (C5-C 15 ) heteroaryl.

[0060] As used herein, the term "optionally substituted" means that substitution is optional, and thus an atom or moiety designated as "optionally substituted" may be unsubstituted or substituted. In some embodiments, an optionally substituted group is unsubstituted. In some embodiments, an optionally substituted group is substituted. For example, unless otherwise indicated by the term "substituted" or "optionally substituted," a group designated herein is unsubstituted.

[0061] As used herein, the term "compounds of the disclosure" refers to any compound of the structural formulae set forth herein (e.g., compounds of structural formula I, exemplary compounds), as well as isomers such as stereoisomers (including diastereoisomers, enantiomers, and racemates) and tautomers thereof, isotopically labeled variants thereof (including those with deuterium substitutions), and inherently formed moieties thereof (e.g., polymorphs and / or solvates such as hydrates). Where moieties capable of forming salts are present, salts are likewise included, particularly pharma- ceutically acceptable salts thereof.

[0062] The compounds of the present disclosure may have asymmetric centers, chiral axes, and chiral planes (e.g., as described in E.L. Eliel and S.H. Wilen, Stereo-chemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), unless otherwise specified, and may exist as racemic mixtures, individual isomers (e.g., diastereomers, enantiomers, geometric isomers (including cis- and trans-double bond isomers), conformational isomers (including rotamers and atropisomers), tautomers) and mixtures of intermediates, along with all possible isomers and mixtures thereof.

[0063] When a disclosed compound is depicted by a structure without showing stereochemistry and the compound has one or more chiral centers, the structure should be understood to encompass one enantiomer or diastereomer of the compound separated or substantially separated from the corresponding optical isomer(s), racemic mixtures of the compound, and mixtures in which one enantiomer or diastereomer is enriched relative to its corresponding optical isomer(s). When a disclosed compound is depicted by a structure showing stereochemistry and the compound has one or more chiral centers, the stereochemistry refers to the absolute configuration of the substituents around the one or more chiral centers. "R" and "S" can also, or alternatively, be used to indicate the absolute configuration of the substituents around one or more chiral carbon atoms. D- and L- can also, or alternatively, be used to designate stereochemistry.

[0064] "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of one another, most commonly because they contain an asymmetrically substituted carbon atom that acts as a chiral center.

[0065] "Diastereomers" are stereoisomers that are not mirror-image related, most commonly because they contain two or more asymmetrically substituted carbon atoms.

[0066] As used herein, a "racemate" or "racemic mixture" refers to a mixture containing equimolar amounts of two enantiomers of a compound. Such a mixture does not exhibit optical activity (i.e., does not rotate the plane of polarized light).

[0067] Enantiomeric excess (ee) is defined as the absolute difference between the mole fractions of each enantiomer multiplied by 100% and is represented by the following formula:

number

[0068] Diastereomeric excess (de) is defined as the absolute difference between the mole fractions of each diastereomer multiplied by 100% and is represented by the following formula:

number

[0069] Unless otherwise specified, compounds of the present disclosure include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, by replacing a hydrogen with deuterium or tritium, or a carbon with 13 C or 14 Compounds produced by substituting C-enriched carbons are within the scope of the invention. In all structures provided, any hydrogen atom may be independently substituted with a deuterium ( 2 H), tritium ( 3 H) and / or fluorine ( 18 F). Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents according to the present invention.

[0070] The phrase "pharmacologically acceptable" means that the substance or composition which it modifies is, within the normal scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, or the like, commensurate with a reasonable benefit / risk ratio.

[0071] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is suitable for use in contact with mammalian tissue without excessive toxicity, irritation, allergic response, etc., within the normal scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, the relevant teachings of which are incorporated herein by reference in their entirety. Pharmaceutically acceptable salts of the compounds described herein include salts derived from suitable inorganic and organic acids and suitable inorganic and organic bases.

[0072] Examples of salts derived from suitable acids include salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art such as ion exchange. Other pharma- ceutically acceptable salts derived from suitable acids include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cinnamate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutarate, glycolate, hemisulfate, heptanoate, hexanoate, hydroiodide, hydroxybenzoate, 2-hydroxyethanesulfonate, Examples of the salts include hydroxymaleate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 2-phenoxybenzoate, phenylacetate, 3-phenylpropionate, phosphate, pivalate, propionate, pyruvate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.

[0073] Mono-, di-, or tri-acid salts can be formed, and such salts can exist in either a hydrated, solvated or substantially anhydrous form.

[0074] Salts derived from appropriate bases include salts derived from inorganic bases such as alkali metal bases, alkaline earth metal bases, and ammonium bases; salts derived from aliphatic, alicyclic, and aromatic organic amines such as methylamine, trimethylamine, picoline, and the like; and salts derived from N +Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, barium, and the like. Further pharma- ceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed where appropriate using counterions such as halides, hydroxides, carboxyls, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates.

[0075] The compounds described herein may exist as "solvates" or "hydrates". A "hydrate" is a compound that exists in a composition that contains one or more water molecules. A hydrate may contain stoichiometric amounts of water, such as monohydrates or dihydrates, or may contain random amounts of water. A "solvate" is similar to a hydrate, except that water is replaced with a solvent other than water, such as methanol, ethanol, dimethylformamide, diethyl ether, etc. Mixtures of such solvates or hydrates can also be prepared. The source of such solvates or hydrates may be from the solvent of crystallization, may be inherent in the solvent of preparation or crystallization, or may be adventitious to such solvent.

[0076] "Pharmaceutically acceptable carrier" refers to a non-toxic carrier or excipient that does not destroy the pharmacological activity of the drug with which it is formulated and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the drug. Pharmaceutically acceptable carriers that may be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, saturated vegetable fatty acids such as glycine, sorbic acid, potassium sorbate, protamine sulfate, water, partial glyceride mixtures of salts or electrolytes, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0077] As used herein, "antigen" refers to any substance that can be recognized by the immune system. "Antigen" broadly encompasses proteins such as enzymes, peptides such as polypeptides, carbohydrates such as polysaccharides, haptens, nucleic acids, and grafts. Antigens can be self-antigens, antigens produced by the body under normal conditions or as part of a disorder, or foreign antigens, non-self-antigens. Examples of self-antigens include autoantigens associated with autoimmune disorders, including any of the autoantigens described herein. Examples of foreign antigens include antigen therapy (e.g., therapeutic proteins, gene therapy, cell therapy), allergens, and alloantigens.

[0078] As used herein, "treating" refers to taking steps to deliver therapy to a subject, such as a mammal in need of treatment (e.g., by administering one or more therapeutic agents to the mammal). "Treating" includes inhibiting a disease or condition (e.g., by slowing or halting its progression or causing regression of the disease or condition), as well as alleviating symptoms caused by a disease or condition.

[0079] A "therapeutically effective amount" is an amount effective to achieve a desired therapeutic result (e.g., induction of immune tolerance, treatment, cure, suppression, or remission of a physiological response or pathology, etc.) at the dosage and for the period of time required. A complete therapeutic effect does not necessarily occur by administration of one dose, but may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the mammal, the mode of administration, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in an individual.

[0080] As used herein, a "subject" includes humans, domestic animals, such as laboratory animals (e.g., dogs, monkeys, pigs, rats, mice, etc.), household pets (e.g., cats, dogs, rabbits, etc.), and livestock (pigs, cows, sheep, goats, horses, etc.), as well as non-domestic animals. In some embodiments, the subject is a human.

[0081] compound A first embodiment is a compound of the following structural formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein: X is -N(R 1 )C(O)-, -N(R 1 )C(O)O-, -N(R 1 )C(O)N(R 2 )-, -N(R 1 )-, -N(R 1 )SO2-, -O-, -S-, -S(O)-, -S(O)2-, or -OP(O)2O-; R is optionally substituted with one or more fluoro (C1-C 30 ) alkyl or (C1-C 30 ) alkenyl, R 1 is H or (C1-C5) alkyl, R 2is H or (C1-C5) alkyl.

[0082] In a first aspect of the first embodiment, X is -N(R 1 )C(O)- or -N(R 1 )C(O)O-. The remaining variables are as described in the first embodiment.

[0083] In a second aspect of the first embodiment, X is -N(R 1 )C(O)- * or -N(R 1 )C(O)O- * and * indicates the point of attachment of X to R. The remainder of the variables are as described in the first embodiment or first aspect thereof.

[0084] In a third aspect of the first embodiment, X is -N(R 1 )C(O)- * The values ​​of the remaining variables are as described in the first embodiment, or the first or second aspect thereof.

[0085] In a fourth aspect of the first embodiment, X is -N(R 1 )C(O)O- * The values ​​of the remaining variables are as described in the first embodiment or the first to third aspects thereof.

[0086] In a fifth aspect of the first embodiment, R is optionally substituted with one or more fluoro (C5-C 30 ) alkyl or (C5-C 30 ) alkenyl. The values ​​of the remaining variables are as described in the first embodiment, or its first through fourth aspects.

[0087] In a sixth aspect of the first embodiment, R is optionally substituted with one or more fluoro (C5-C 30 ) alkyl. The values ​​of the remaining variables are as described in the first embodiment, or aspects 1 to 5 thereof.

[0088] In a seventh aspect of the first embodiment, R 1 and R 2 are each H. The values ​​of the remaining variables are as described in the first embodiment or the first to sixth aspects thereof.

[0089] In an eighth aspect of the first embodiment, X is -N(R 1 )C(O)- * , -N(R 1 )C(O)O- * , -N(R 1 )C(O)N(R 2 )- * , -N(R 1 )-, -N(R 1 )SO2- * , -O-, -S-, -S(O)-, -S(O)2-, or -OP(O)2O-, * indicates the point of attachment of X to R. The values ​​of the remaining variables are as described in the first embodiment, or the first to seventh aspects thereof.

[0090] In a ninth aspect of the first embodiment, R is optionally substituted with one or more fluoro (C 10 -C 25 ) alkyl or (C 10 -C 25 ) alkenyl. The values ​​of the remaining variables are as described in the first embodiment, or aspects 1 to 8 thereof.

[0091] In a tenth aspect of the first embodiment, R is optionally substituted with one or more fluoro (C 10 -C 20 ) alkyl or (C 10 -C 20 ) alkenyl. The values ​​of the remaining variables are as described in the first embodiment, or aspects 1 to 9 thereof.

[0092] In an eleventh aspect of the first embodiment, R is optionally substituted with one or more fluoro (C 15 -C 25 ) alkyl or (C 15 -C 25) alkenyl. The values ​​of the remaining variables are as described in the first embodiment, or aspects 1 to 10 thereof.

[0093] In a twelfth aspect of the first embodiment, R is optionally substituted with one or more fluoro (C 15 -C 20 ) alkyl or (C 15 -C 20 ) alkenyl. The values ​​of the remaining variables are as described in the first embodiment, or aspects 1 to 11 thereof.

[0094] A second embodiment is a compound of the following structural formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein: X is -N(R 1 )C(O)- * or -N(R 1 )C(O)O- * where: * indicates the point of attachment of X to R, R is optionally substituted with one or more fluoro (C5-C 30 ) alkyl or (C5-C 30 ) alkenyl, R 1 is H or (C1-C5) alkyl, With the proviso that the compound is not (S)-2-amino-3-((2E,4E)-hexa-2,4-diamido)propanoic acid, (S)-2-amino-3-hexanamidopropanoic acid, (S)-2-amino-3-heptanamidopropanoic acid, (S)-2-amino-3-octanamidopropanoic acid, or (S)-2-amino-3-palmitamidopropanoic acid, or a salt of any of the foregoing. The alternative values ​​of the variables are as described in the first embodiment, or any aspect thereof.

[0095] In a first aspect of the second embodiment, R 1is H. The values ​​of the remaining variables are as described in the first embodiment, or any aspect thereof, or the second embodiment.

[0096] Examples of compounds of structural formula I include: [ka] Or a pharma- ceutically acceptable salt of the foregoing.

[0097] Methods for preparing compounds of formula I are described in the examples herein.

[0098] Compositions and kits Usually, the compound of the present disclosure is formulated with one or more pharma- ceutically acceptable carriers for administration to a subject.The present disclosure provides such compositions, including pharmaceutical compositions.Thus, one embodiment is a composition (e.g., pharmaceutical composition) that comprises the compound of the present disclosure and a pharma- ceutically acceptable carrier.For example, the composition described herein can be used in the method described herein to deliver the compound of the present disclosure.

[0099] The compounds and compositions described herein may be in the form of a lipid particle formulation, such as a liposomal formulation. Thus, one embodiment is a lipid particle (e.g., a liposome) that comprises one or more lipids and a compound of the present disclosure.

[0100] Also included herein is at least one phospholipid (e.g., a saturated C4-C phospholipid such as dimyristoylphosphatidylcholine (DMPC)). 30 C4-C of acyl chain etc. 30Also provided are solid lipid particles (e.g., liposomes) comprising a phospholipid (containing an acyl chain) and a therapeutic agent (e.g., a compound of the present disclosure) that can be embedded in the lipid bilayer of the lipid particle. It has been found that oral administration of such solid lipid particles can be used, for example, to target the lipid particle (and thereby the therapeutic agent) to immune cells and / or lymph node(s), thereby enhancing co-localization of the lipid particle and immune cells (e.g., in the lymph node) and / or enhancing uptake of the lipid particle into the lymph node.

[0101] As used herein, "lipid particle" refers to a particle that includes at least one lipid, e.g., a phospholipid, such as a lysophospholipid. Examples of lipid particles include liposomes, micelles, and lipid nanoparticles. Lipid particles, such as liposomes, can be unilamellar or multilamellar. Lipid particles, such as liposomes, can have a fluid lipid membrane, or a gel-like or solid lipid membrane, e.g., a lipid membrane that melts at a temperature above normal human body temperature, i.e., about 37°C. In some embodiments, the lipid particle is a liposome. In some embodiments, the lipid particle is a lipid nanoparticle. In some embodiments, the lipid particle is solid. In some embodiments, the lipid particle has a melting temperature above about 37°C, e.g., above about 40°C, above about 45°C, above about 50°C, above about 55°C, or about 55°C.

[0102] Examples of phospholipids include dimyristoylphosphatidylcholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine 18:1 Δ9-Cis PC (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine 18:0 (DSPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine 16:0-18:1 (POPC), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylinositol, bisphosphatidylglycerol, phosphatidic acid, phosphatidylalcohol, and phosphatidylglycerol. Phospholipids may be saturated or unsaturated, i.e., contain one or more unsaturated units, and may contain acyl chains of various lengths. In some embodiments, phospholipids are C4-C 30 Acyl chain, e.g., C8-C 26 , C 12 -C 22 , C 10 -C 25 , C 14 -C 18 , or C 16 -C 26 Contains acyl chains. Phospholipids can be obtained from various sources, natural and synthetic. For example, PS can be obtained from pig brain PS or plant-based soybean (soybean) PS. Egg PC, PS, and synthetic PC are commercially available. In some embodiments, phospholipid is not PS or its salt (e.g., its pharmaceutically acceptable salt).

[0103] Other lipids suitable for inclusion in the lipid particles described herein include N 4 -cholesteryl-spermine, or a salt thereof, e.g., N 4 -cholesteryl-spermine HCl salt. 4 -Cholesteryl-spermine HCl salt, also known as Genzyme Lipid 67 (GL67), is cholesterol derivatized with spermine to produce the HCl salt of the cationic lipid.

[0104] Typically, the molar percentage of the therapeutic agent (e.g., a compound of the present disclosure) in a lipid particle (e.g., a liposome) containing the therapeutic agent is about 1% to about 50%, e.g., about 1% to about 35%, about 1% to about 25%, about 1% to about 15%, about 3% to about 10%, about 5% to about 50%, about 5% to about 45%, about 15% to about 40%, about 25% to about 35%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 25%, about 30%, or about 35%. In some embodiments, the molar percentage of the therapeutic agent (e.g., a compound of the present disclosure) in a lipid particle (e.g., a liposome) containing the therapeutic agent is less than 35%, e.g., less than 30%, less than 15%, or about 1% to about 10%.

[0105] Typically, the molar percentage of lipids (taken individually or collectively) in the lipid particles (e.g., liposomes) described herein is from about 50% to about 99%, e.g., from about 50% to about 75%, from about 85% to about 99%, about 70%, about 75%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or 99%. The molar percentage of each lipid in the lipid particles (e.g., liposomes) described herein can be from about 1% to about 99%, e.g., from about 1% to about 50%, from about 1% to about 35%, from about 1% to about 25%, from about 1% to about 15%, from about 3% to about 10%, from about 5% to about 50%, from about 5% to about 45%, from about 15% to about 40%, from about 25% to about 35%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 25%, about 30%, or about 35%.

[0106] The compounds of the present disclosure can be encapsulated in lipid particles such as liposomes described herein, or can be attached (covalently or non-covalently) to lipid head groups, or can be embedded in whole or in part in lipid bilayers (e.g., liposomes) covalently or non-covalently.Without wishing to be bound by any particular theory, it is believed that the compounds of the present disclosure can be embedded in the lipid bilayers of liposomes such that the amino acid residues of the compounds of the present disclosure remain exposed to the outside of the liposome, thereby mimicking natural surface presentation, e.g., PS.

[0107] In some embodiments, the one or more lipids comprise a phospholipid or a pharma- ceutically acceptable salt thereof, such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or a pharma- ceutically acceptable salt thereof. In some embodiments, the phospholipid is a saturated phospholipid, such as C4-C 30 In some embodiments, the phospholipid is an unsaturated, e.g., C4-C 30 It is the unsaturated phospholipid that contains acyl chain.In some embodiments, phospholipid is selected from DMPC, DSPC, DOPC or POPC, or its pharmaceutically acceptable salt.In some embodiments, phospholipid is DMPC or DSPC, or its pharmaceutically acceptable salt.

[0108] In some embodiments, the lipid particle (e.g., liposome) further comprises an antigen, e.g., any of the antigens described herein. Thus, in some embodiments, the lipid particle further comprises a gene therapy. In some further embodiments, the gene therapy comprises DNA and / or RNA and a viral vector. In some embodiments, the viral vector is derived from an adeno-associated virus (AAV), e.g., a recombinant AAV. In some embodiments, the AAV is AAV9. Other examples of viral vectors suitable for use with the present disclosure include viral vectors derived from retroviruses, herpes viruses, adenoviruses, lentiviruses, rabies viruses, lentiviruses, VSV, poxviruses (e.g., vaccinia viruses, smallpox viruses, canarypox), reoviruses, Semliki Forest viruses, yellow fever viruses, Sindbis viruses, togaviruses, baculoviruses, bacteriophages, alphaviruses, and flavaviruses. In some embodiments, the antigen, e.g., a gene therapy agent comprising DNA and / or RNA and a viral vector, is encapsulated within the lipid particle.

[0109] Lipid particles further comprising an antigen, and formulations comprising such lipid particles, are believed to be particularly useful for applications involving delivery of gene therapy agents (e.g., gene therapy agents comprising DNA and / or RNA) to a subject. The lipid particles are expected to facilitate co-presentation of gene therapy and compounds of the present disclosure to the immune system. Such particles can be formulated for oral and / or parenteral (e.g., subcutaneous, intramuscular, intravenous, intradermal) administration, e.g., injection.

[0110] Another embodiment is a composition (e.g., a pharmaceutical composition) comprising a plurality of lipid particles (e.g., a plurality of lipid particles comprising a compound of the present disclosure). In some aspects, the composition further comprises a pharma- ceutically acceptable carrier.

[0111] The compositions described herein, and thus the compounds of the present disclosure, may be administered orally, parenterally (including subcutaneously, intramuscularly, intravenously, and intradermally), by inhalation spray, topically, rectally, nasally, bucally, intravaginally, or via an implanted reservoir. As used herein, the terms "parenteral" and "parenterally" include subcutaneous, intradermal, intravenous, intramuscular, intraocular, intravitreal, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, intrahepatic, intraperitoneal, intralesional, and intracranial injection or infusion techniques. In some embodiments, the compositions described herein can be administered intravenously and / or intraperitoneally. In some embodiments, the compositions described herein can be administered orally. In some embodiments, the compositions described herein can be administered subcutaneously. Preferably, the compositions described herein are administered orally, subcutaneously, intraperitoneally, or intravenously.

[0112] The compositions provided herein can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions, dispersions, and liquids.For tablets for oral use, commonly used carriers include lactose and cornstarch.Lubricants such as magnesium stearate are also commonly added.For oral administration in capsule form, useful diluents include lactose and dried cornstarch.When aqueous suspensions and / or emulsions are required for oral use, the active ingredient can be suspended or dissolved in an oil phase and mixed with emulsifying and / or suspending agents.If desired, certain sweeteners, flavors, or colorants can also be added.

[0113] In some embodiments, the oral formulations are formulated for immediate release or sustained / delayed release.

[0114] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharma- ceutically 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 carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) wetting agents, such as glycerol; (d) disintegrating agents, such as agar-agar, calcium carbonate, potato starch, or tapioca starch; (e) solution retarding agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium salts; (g) wetting agents, such as acetyl 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 forms may also comprise buffering agents.

[0115] Liquid dosage forms for oral administration include pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.In addition to the compounds of the present disclosure, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethyl alcohol (ethanol), isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (specifically cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, or mixtures thereof.In addition to inert diluents, oral compositions may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, coloring agents, aromatic agents, and preservatives.

[0116] Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, where the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.

[0117] Similar types of solid compositions may also be used as fillers in soft and hard-filled gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. These dosage forms may optionally contain opacifying agents, and may be of a composition that releases the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0118] The compound of the present disclosure can also be in microencapsulated form with one or more of the above-mentioned excipients.In such solid dosage form, compound can be mixed with at least one inert diluent such as sucrose, lactose or starch.According to common practice, such dosage form can also contain additional material other than inert diluent, such as tableting lubricant and other tableting aids, such as magnesium stearate and microcrystalline cellulose.

[0119] Compositions for oral administration may be designed to protect the active ingredient against degradation as it passes through the digestive tract, for example by an outer coating of the formulation on a tablet or capsule.

[0120] In another embodiment, the compounds of the present disclosure can be provided as extended (or "delayed" or "sustained") release compositions. The delayed release compositions include the compounds of the present disclosure and a delayed release component. Such compositions allow for targeted release of the compounds, for example, to the lower gastrointestinal tract, for example, the small intestine, large intestine, colon, and / or rectum. In certain embodiments, the delayed release compositions further include enteric or pH-dependent coatings, such as cellulose acetate phthalate and other phthalates (e.g., polyvinyl acetate phthalate, methacrylates (Eudragits)). Alternatively, the delayed release compositions can provide controlled release to the small intestine and / or colon by providing pH-sensitive methacrylate coatings, pH-sensitive polymer microspheres, or polymers that undergo degradation by hydrolysis. The delayed release compositions can be formulated with hydrophobic or gelling excipients or coatings. Colonic delivery can further be provided by coatings that are digested by bacterial enzymes such as amylose or pectin, by pH-dependent polymers, by hydrogel plugs that swell over time (Pulsincap), by time-dependent hydrogel coatings, and / or by acrylic acid linked to an azoaromatic bond coating.

[0121] The compositions described herein can also be administered subcutaneously, intraperitoneally, or intravenously, for example, in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oily suspension. This suspension can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween® 80) and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Acceptable vehicles and solvents that can be used include mannitol, dextrose, water, Ringer's solution, lactated Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any non-irritating fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharma- ceutically acceptable oils such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, or carboxymethylcellulose or similar dispersants commonly used in the formulation of pharma- ceutically acceptable dosage forms such as emulsions and / or suspensions. Other commonly used surfactants such as Tween® or Span® and / or other similar emulsifiers or bioavailability enhancers commonly used in the manufacture of pharma-ceutically acceptable solid, liquid or other dosage forms may also be used for formulation purposes.

[0122] The compositions described herein can also be administered in the form of suppositories for rectal administration. These can be prepared by mixing the compounds of the present disclosure with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, thereby melting in the rectum and releasing the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0123] The compositions described herein may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, such as diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

[0124] Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches can also be used.

[0125] For topical application, the composition can be formulated into a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds described herein include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water, and penetration enhancers. Alternatively, the composition can be formulated into a suitable lotion or cream containing the active compound suspended or dissolved in one or more pharma- ceutically acceptable carriers. Alternatively, the composition can be formulated in a suitable lotion or cream containing the active compound suspended or dissolved in a carrier with a suitable emulsifier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. Suitable carriers also include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water, as well as penetration enhancers.

[0126] For ophthalmic use, the composition may be formulated as a micronized suspension in isotonic, pH-adjusted, sterile saline, or preferably as a solution in isotonic, pH-adjusted, sterile saline, either in the presence or absence of a preservative, such as benzylalkonium chloride. Alternatively, for ophthalmic use, the composition may be formulated in an ointment, such as petrolatum.

[0127] The composition can also be administered by nasal aerosol or inhalation. Such compositions can be prepared according to well-known techniques in the field of pharmaceutical formulations, and can be prepared as a solution in saline, using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other conventional solubilizers or dispersants. Without being bound to any particular theory, it is believed that the local delivery of the compositions described herein, such as can be achieved by nasal aerosol or inhalation, can reduce the risk of systemic effects of the composition, such as effects on red blood cells.

[0128] Other pharma- ceutically acceptable carriers that can be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopherol polyethylene glycol 1000 succinate, surfactants such as Tween® used in pharmaceutical dosage forms, or other similar polymeric delivery matrices, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat. For example, to enhance delivery of the agents described herein, cyclodextrins, e.g., α-, β-, and γ-cyclodextrin, or chemically modified derivatives thereof, e.g., hydroxyalkyl cyclodextrins, including hydroxylpropyl-β-cyclodextrin, e.g., 2- and / or 3-hydroxypropyl-β-cyclodextrin, or other solubilized derivatives, can also be effectively used as pharma- ceutically acceptable carriers in the compositions described herein.

[0129] One embodiment is a composition comprising a compound of the present disclosure (e.g., a plurality of lipid particles comprising a compound of the present disclosure, e.g., any of the lipid particles described herein) and a cyclodextrin or a chemically modified derivative thereof. In some aspects, the cyclodextrin or a chemically modified derivative thereof comprises a hydroxyalkyl cyclodextrin, e.g., a hydroxypropyl-β-cyclodextrin. In some aspects, the compound of the present disclosure and the cyclodextrin (e.g., a hydroxyalkyl cyclodextrin, such as hydroxypropyl-β-cyclodextrin) are present in a ratio of about 1 to about 50 weight / weight (w / w) to about 1 to about 250 w / w, e.g., about 1 to about 50 w / w to about 1 to about 100 w / w, about 1 to about 80 w / w, or about 1 to about 166 w / w. In some aspects, the composition further comprises a diluent, such as water. In some aspects, the composition further comprises a sweetener and / or a flavoring agent.

[0130] In some aspects, the composition is in a liquid dosage form, and in further aspects, the composition is in a liquid dosage form for oral administration.

[0131] In some embodiments, the compositions described herein further comprise one or more additional therapeutic agents, eg, for use in combination with a compound of the present disclosure.

[0132] Some embodiments provide combinations (e.g., pharmaceutical combinations) that include a compound of the present disclosure (e.g., a composition described herein that includes a compound of the present disclosure) and one or more additional therapeutic agents (e.g., one or more compositions that include one or more additional therapeutic agents). Such combinations are particularly useful, for example, when the compound of the present disclosure and the one or more additional therapeutic agents are administered separately. In the combinations provided herein, the compound of the present disclosure and the one or more additional therapeutic agents can be administered by the same route of administration or by different routes of administration.

[0133] One embodiment is a kit comprising a compound of the present disclosure (e.g., a composition described herein comprising a compound of the present disclosure) and an antigen (e.g., any of the antigens described herein, such as an antigen therapy). In one aspect, the kit comprises a therapeutically effective amount of a compound of the present disclosure (e.g., an amount sufficient to immunotolerize the subject to the antigen to be administered; a therapeutically effective amount of the compound for treating a disease, disorder, or condition described herein). In some aspects, when the antigen is an antigen therapy, the kit comprises a therapeutically effective amount of the antigen therapy for treating the disease, disorder, or condition. In some aspects, the kit further comprises additional therapeutic agent(s) (e.g., a composition comprising additional therapeutic agent(s)). In some aspects, the kit further comprises written instructions for administering to the subject a compound of the present disclosure and / or the antigen and / or additional agent(s) for treating a disease, disorder, or condition described herein.

[0134] Suitable additional therapeutic agents include those agents described herein in connection with combination therapy.

[0135] The compositions described herein can be provided in unit dosage form. The amount of active ingredient that can be combined with a carrier to produce a unit dosage form varies, for example, depending on the subject being treated and the particular mode of administration. Usually, a unit dosage form contains about 1 to about 1,000 mg of active ingredient(s), for example, about 1 to about 500 mg, about 1 to about 250 mg, about 1 to about 150 mg, about 0.5 to about 100 mg, or about 1 to about 50 mg of active ingredient(s). In some embodiments, a unit dosage form contains about 0.01 mg to about 100 mg of active ingredient(s), for example, about 0.1 mg to about 50 mg, about 0.1 mg to about 10 mg, about 0.5 mg to about 50 mg of active ingredient(s). In some embodiments, the unit dosage form contains from about 1 mg to about 5,000 mg of the active ingredient(s), e.g., from about 10 mg to about 2,500 mg, from about 15 mg to about 1,000 mg, or from about 100 mg to about 1,000 mg of the active ingredient(s). In some embodiments, the unit dosage form contains about 15 mg, about 30 mg, about 50 mg, about 100 mg, about 125 mg, or about 150 mg of the active ingredient(s).

[0136] In some embodiments, the concentration of one or more therapeutic agents provided in the pharmaceutical composition is 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05 ... %, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w / w, w / v, or v / v, and / or 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or greater than 0.0001% w / w, w / v, or v / v.

[0137] In some embodiments, the concentration of one or more therapeutic agents provided in the pharmaceutical composition is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24%, from about 0.08% to about 26%, from about 0.09% to about 27%, from about 0.10% to about 12%, from about 0.11% to about 12%, from about 0.12% to about 12%, from about 0.13% to about 13%, from about 0.14% to about 13%, from about 0.15% to about 13%, from about 0.16% to about 13%, from about 0.17% to about 13%, from about 0.18% to about 13%, from about 0.19% to about 14%, from about 0.20% to about 25%, from about 0.21% to about 25%, from about 0.22% to about 25%, from about 0.23% to about 25%, from about 0.24% to about 25%, from about 0.25% to about 25%, from about 0.26% to about 25%, from about 0.27% to about 25%, from about 0.28% to about 25%, from about 0.29% to about 25%, from about 0.30% to about 25%, from about 0.31% to about 25%, from about 0.32% to about 25%, from about 0.33% to about 25%, The range of the active ingredient is from about 0.08% to about 23%, from about 0.09% to about 22%, from about 0.1% to about 21%, from about 0.2% to about 20%, from about 0.3% to about 19%, from about 0.4% to about 18%, from about 0.5% to about 17%, from about 0.6% to about 16%, from about 0.7% to about 15%, from about 0.8% to about 14%, from about 0.9% to about 12%, or from about 1% to about 10% w / w, w / v, or v / v. In some embodiments, the concentration of the one or more therapeutic agents provided in the pharmaceutical composition is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w / w, w / v, or v / v.

[0138] How to use It has now been found that various compounds of the present disclosure and compositions described herein can bind to TIM with greater affinity than its natural ligand, phosphatidylserine (PS), for example, and reduce the immune response.

[0139] One embodiment is a method of modulating T cell immunoglobulin and mucin domain (TIM) receptor expression or activity, comprising contacting a cell (e.g., a cell expressing a TIM receptor, such as an immune cell) with a compound of the present disclosure (e.g., a therapeutically effective amount of a compound of the present disclosure). TIM receptors are type 1 cell surface glycoproteins, and the TIM receptors expressed in humans, TIM1, TIM3, and TIM4, have been identified as phosphatidylserine receptors. TIM1 is preferentially expressed on T helper 2 cells and functions as a potent co-stimulatory molecule for T cell activation. TIM3 is preferentially expressed on T helper 1 cells, type 1 T cells, and dendritic cells, and generates an inhibitory signal that triggers apoptosis of T helper 1 cells and type 1 T cells. TIM4 is expressed on antigen presenting cells and mediates phagocytosis of apoptotic cells to promote tolerance. In some aspects, the TIM receptor is a TIM3 receptor. In some aspects, the TIM receptor is a TIM4 receptor. In some aspects, the TIM receptor is a TIM1 receptor. "TIM" is also referred to in the literature as, for example, "Tim".

[0140] Agonists of TIM receptors have now also been found to inhibit the activity of at least toll-like receptors (TLR) 3 and TLR 7, without substantially inhibiting the activity of TLR 2 and 4, which primarily recognize patterns presented by bacteria. Toll-like receptors (TLRs) form a family of pattern recognition receptors expressed on innate immune cells and constitute the first line of defense of the immune system against microorganisms. To date, ten human TLR subtypes have been identified. TLR 1, 2, 4, 5, 6, and 10 are expressed on the cell surface, whereas TLR 3, 7, 8, and 9 are localized in the endoplasmic reticulum, endosomes, and lysosomes. TLR 1, 2, and 6 recognize and bind bacterial lipoproteins and glycolipids. TLR 3, 7, 8, and 9 recognize and bind nucleic acids such as viral dsRNA (TLR 3), ssRNA (TLR 7, TLR 8), and unmethylated CpG DNA (TLR 9). TLR 4 recognizes and binds fibronectin and LPS. TLR5 recognizes and binds to bacterial flagellin. Without being bound to any particular theory, it is believed that the compounds of the present disclosure do not provide general immunosuppression, but may exert their effects in a more selective and specific manner.

[0141] Another embodiment is a method of modulating (e.g., inhibiting) the activity of TLR3, TLR7, TLR8, and / or TLR9, comprising contacting a cell (e.g., a cell expressing TLR3, TLR7, TLR8, and / or TLR9; an immune cell) with a compound of the disclosure (e.g., a therapeutically effective amount of a compound of the disclosure). In some aspects, the compound of the disclosure selectively modulates (e.g., inhibits) the activity of TLR3, TLR7, TLR8, and / or TLR9, e.g., modulates (e.g., inhibits) the activity of TLR3, TLR7, TLR8, and / or TLR9 to a greater extent than it modulates the activity of TLR1, 2, 4, 5, 6, and / or 10. For example, modulation (e.g., inhibition) of TLR3, TLR7, TLR8 and / or TLR9 activity by a compound of the disclosure can be more than 2-fold, e.g., more than 5-fold, more than 10-fold, more than 25-fold, or more than 100-fold, compared to modulation (e.g., inhibition) by the compound of activity of TLR1, 2, 4, 5, 6, and / or 10. In some embodiments, the compound does not measurably modulate (e.g., inhibit) the activity of TLR1, 2, 4, 5, 6, and / or 10.

[0142] In some embodiments of the methods described herein, the cell is an immune cell, e.g., a T cell, such as a regulatory T cell, a natural killer (NK) cell, a macrophage, a neutrophil, a myeloid-derived suppressor cell, or a dendritic cell. In some embodiments, the immune cell is a FoxP3+ and / or CD4+, e.g., a FoxP3+ and / or CD4+ T cell. In some embodiments, the immune cell is a B cell, such as a regulatory B cell. In some embodiments, the immune cell (e.g., a regulatory B cell) is CD19+, CD71+, IgM+, CD24+, CD38+, and / or CD27+.

[0143] In some embodiments of the methods described herein, the methods are performed in vitro. In other embodiments of the methods described herein, the methods are performed in vivo. Thus, in some embodiments, the cell (e.g., an immune cell) is in a subject (e.g., a subject having a disease, disorder, or condition described herein).

[0144] Another embodiment is a method of tolerizing a subject in need of tolerization (e.g., a subject having an autoimmune disorder, such as an autoimmune disorder described herein), comprising administering to the subject a therapeutically effective amount of a compound of the disclosure, e.g., in the form of a composition described herein.

[0145] Another embodiment is a method of immune tolerization (e.g., antigen therapy) of a subject in need of immune tolerization to an antigen, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure, e.g., in the form of a composition described herein. Some aspects include administering to the subject an antigen, or an immunogenic fragment thereof, and a therapeutically effective amount of a compound of the present disclosure, e.g., in the form of a composition described herein. Some aspects include administering to the subject a composition comprising a compound of the present disclosure and an antigen, or an immunogenic fragment thereof, e.g., a composition comprising a plurality of lipid particles, each lipid particle comprising a compound of the present disclosure and an antigen, or an immunogenic fragment thereof. In some aspects, the antigen, or an immunogenic fragment thereof, and the compound of the present disclosure are administered to the subject in separate formulations.

[0146] As used herein, "immune tolerization" refers to, for example, reducing and / or eliminating an immune response to an antigen. The immune response may be evidenced, for example, by immunological hyperactivity, inflammatory cytokine release, and / or activation of immune cells, such as macrophages, neutrophils, eosinophils, T cells, and B cells. As used herein, "immune tolerization" refers, for example, to reducing immunological hyperactivity, inhibiting inflammatory cytokine release, and / or inhibiting activation and / or neutralizing immune cells, such as macrophages, neutrophils, eosinophils, T cells, and B cells. In a clinical setting, immune tolerization may be evidenced, for example, by a reduction in the severity of an autoimmune disease and / or an improvement in the activity of an antigen therapy administered.

[0147] Thus, the process of immune tolerization can be viewed along a continuum from immunological hyperactivity to immunological hypoactivity to, for example, immunological unresponsiveness to an antigen. By "immune tolerization" we contemplate the gradual progression along this continuum towards immunological unresponsiveness, as well as the induction of immunological hypoactivity or immunological unresponsiveness. In other words, immune tolerization includes reducing the level of immune intolerance and inducing immune tolerance. In certain preferred embodiments described herein, the method induces immune tolerance.

[0148] In some embodiments, a subject exhibiting immune intolerance or a subject with immune intolerance has a measurable immune response, e.g., to an antigen, e.g., measurable antibody production in response to the antigen. In some embodiments, a subject exhibiting immune tolerance or a subject with immune intolerance does not have a measurable immune response, e.g., to an antigen, e.g., measurable antibody production in response to the antigen. ELISAs and / or activity assays, including those described herein, are known in the art and can be used to measure antibody production indicative of immune intolerance.

[0149] In some autoimmune diseases, antibodies are not always present. Immune intolerance in such cases may be evidenced by clinical symptoms of the autoimmune disease, and / or the presence of autoreactive T cells or B cells, and / or an increase in other inflammatory immune cells, such as neutrophils, eosinophils, etc. In some embodiments, subjects who exhibit immune intolerance or subjects who are immune intolerant (e.g., subjects who have an autoimmune disease, such as an autoimmune disease described herein) have a measurable cytokine response. For example, subjects suffering from rheumatoid arthritis may have a measurable TNF-α response. In some embodiments, subjects who exhibit immune tolerance or subjects who are immune tolerant (e.g., subjects who have an autoimmune disease, such as an autoimmune disease described herein) do not have a measurable cytokine response.

[0150] Tolerization can be achieved in a general or antigen-specific manner, e.g., resulting in general or antigen-specific tolerance (e.g., general or specific, acquired or adaptive tolerance), respectively. Indicators of general tolerization include, for example: (a) immunological hyperactivity and / or lack and / or reduction in anti-inflammatory cytokine release, (b) neutralization of immune cells such as macrophages, neutrophils, eosinophils, T cells, and B cells, (c) increased numbers of regulatory T cells and / or increased activity or levels of tolerogenic T cells (e.g., FoxP3+ / CD4+ T cells, CD4+ / CD25 ... hi / Foxp3+ / CTLA4+ / Tim3+ / NRP1+ / ICOS-T cells;CD4+ / CD25 hi / Foxp3+ / CTLA4+ / Tim3+ T cells; and / or CD4+ / CD25 hi Indicators of antigen-specific immune tolerization include, for example: (a) an increase in the number of antigen-specific regulatory T cells (e.g., CD4+ / FoxP3+ T cells; CD4+ / CD25+ / FoxP3+ / CTLA4+ / NRP1+ / ICOS- T cells); and / or (d) an increase in the number of regulatory B cells (e.g., CD19+ / CD71+ / IgM+ / CD24+ / CD38+ / CD27+ B cells; and / or CD19+ / CD71+ / IgM+ B cells). hi / Foxp3+ / CTLA4+ / Tim3+ / NRP1+ / ICOS-T cells;CD4+ / CD25 hi / Foxp3+ / CTLA4+ / Tim3+ T cells; and / or CD4+ / CD25 hi / Foxp3+ / CTLA4+ / NRP1+ / ICOS- T cells), (b) a decrease in antigen-specific antibody titers and / or B cell numbers, including antigen-specific memory B cells, (c) a decrease in IL-6 and / or IL-17, (d) an increase in TGF-β, IL-10, IL-35, CD40, CD80, and / or CD86, (e) a decrease in responsiveness after rechallenge with antigen, and / or (f) an increase in the number of antigen-specific regulatory B cells (e.g., CD19+ / CD71+ / IgM+ / CD24+ / CD38+ / CD27+ B cells, and / or CD19+ / CD71+ / IgM+ B cells). Techniques for assessing these indicators are known in the art and described herein. For example, some of the aforementioned indicators can be assessed using culture conditions.

[0151] In autoimmune diseases, treatment with the compounds of the present disclosure results in the proliferation of natural regulatory T cells. Such treatment does not prevent innate immune responses such as those triggered by innate immune cells that respond to danger signals from pathogens, but results in systemic adaptive immune tolerance. Thus, immune tolerization can be achieved herein without systemic innate immune suppression, so that, for example, a subject can still mount an innate immune response to an antigen (e.g., a pathogen). In some embodiments, immune tolerization is systemic adaptive immune tolerization. In some embodiments, immune tolerization is antigen-specific, for example, resulting in a reduction in immune intolerance to a particular antigen(s), or immune tolerance to a particular antigen(s).

[0152] In some aspects, the tolerization is systemic, eg, reducing systemic immune intolerance or resulting in systemic immune tolerance.

[0153] It is understood that antigen-specific immune tolerization can be achieved by administering to a subject a particular antigen and a therapeutically effective amount of a compound of the disclosure or a composition described herein, in accordance with the methods described herein, and additionally, or alternatively, by administering to a subject an immunogenic fragment of the particular antigen and a therapeutically effective amount of a compound of the disclosure or a composition described herein.

[0154] As used herein, an "immunogenic fragment" of an antigen refers to a fragment of the antigen that induces an immune response against the antigen. The immunogenic fragment of an antigen may induce an immune response in a subject to the same extent as the immune response induced by the antigen itself, but does not necessarily induce an immune response to the same extent as the antigen itself, as long as the fragment has an immune tolerizing effect when administered according to the methods described herein.

[0155] Another embodiment is a method for inhibiting or reducing antigen-specific antibody titers in a subject, comprising administering to the subject an antigen, or an immunogenic fragment thereof, and a therapeutically effective amount of a compound of the present disclosure, for example in the form of a composition described herein. Some aspects include administering to the subject a composition described herein that comprises a compound of the present disclosure and an antigen, or an immunogenic fragment thereof, for example a composition that comprises a plurality of lipid particles, each lipid particle comprising a compound of the present disclosure and an antigen, or an immunogenic fragment thereof. In some aspects, the antigen, or an immunogenic fragment thereof, and the compound of the present disclosure are administered to the subject in separate formulations.

[0156] In some aspects of the methods described herein, the antigen is an allergen, such as a food allergen or a latex allergen. Examples of food allergens include peanut allergens, such as Ara hI or Ara hII, walnut allergens, such as Jug rI, Brazil nut allergens, such as albumin, shrimp allergens, such as Pen aI, egg allergens, such as ovomucoid, milk allergens, such as bovine β-lactoglobin, wheat gluten antigens, such as gliadin, and fish allergens, such as parvalbumin. An example of a latex allergen is Hey b7. Other allergens include antigen E, or protein antigens from grasses, such as Amb aI (ragweed pollen), Lol p1 (grass), house dust mite allergens, such as Der pI and Der PII (house dust mite), Fel dI (house cat), Bet v1 (birch), and protein antigens from tree pollens, such as Cry j1 and Cry j2 (cedar). The source of the allergen listed in brackets following each allergen indicates the source with which the indicated allergen is normally associated.

[0157] Another embodiment is a method for inducing a population of regulatory T cells in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure, for example in the form of a composition described herein. Some aspects further comprise administering to the subject an antigen, or an immunogenic fragment thereof, in response to which a population of regulatory T cells is induced. Some aspects comprise administering to the subject a composition as described herein, comprising a compound of the present disclosure and an antigen, or an immunogenic fragment thereof, for example a composition comprising a plurality of lipid particles, each lipid particle comprising a compound of the present disclosure and an antigen, or an immunogenic fragment thereof. In some aspects, the antigen, or an immunogenic fragment thereof, and the compound of the present disclosure are administered to the subject in separate formulations.

[0158] Without wishing to be bound by any particular theory, it is believed that the compounds of the present disclosure primarily stimulate natural regulatory T cells (nT regsThe compounds of the present disclosure are also believed to induce a population of regulatory T cells by expanding a population of regulatory T cells that are, for example, FoxP3+ / NRP1+. regs , e.g., FoxP3+ T cells, FoxP3+ / TIM3+ T cells). Thus, in some embodiments, the method of inducing a regulatory T cell population is, for example, a method of expanding a population of natural regulatory T cells (e.g., regulatory T cells that are FoxP3+ / NRP1+) without substantially inducing inducible regulatory T cells. As described herein, for example, neuropilin-1 (Nrp1) expression can be used to distinguish natural regulatory T cells from inducible regulatory T cells. Thus, in some embodiments, the method of inducing a regulatory T cell population is, for example, a method of inducing a population of regulatory T cells (e.g., FoxP3+ / NRP1+ T cells) that express Nrp1 by expanding a population of natural regulatory T cells. Without wishing to be bound by any particular theory, for example, the ability to expand a population of natural regulatory T cells (e.g., regulatory T cells that are FoxP3+ / NRP1+) without substantially inducing inducible regulatory T cells is believed to be useful in treating autoimmune diseases without affecting general immune suppression.

[0159] In some embodiments, the regulatory T cells are FoxP3+, for example, FoxP3+ / TIM3+, FoxP3+ / NRP1+.Regulatory T cells can be determined to be positive (+) or negative (-) for any of the above markers, for example, by flow cytometry analysis.

[0160] Another embodiment is a method of increasing the activity or level of tolerogenic T cells in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the disclosure, e.g., in the form of a composition described herein.

[0161] Another embodiment is a method for inducing a population of regulatory B cells in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure, for example in the form of a composition described herein. Some aspects further comprise administering to the subject an antigen, or an immunogenic fragment thereof, in response to which a population of regulatory B cells is induced. Some aspects comprise administering to the subject a composition as described herein comprising a compound of the present disclosure and an antigen, or an immunogenic fragment thereof, for example a composition comprising a plurality of lipid particles, each lipid particle comprising a compound of the present disclosure and an antigen, or an immunogenic fragment thereof. In some aspects, the antigen, or an immunogenic fragment thereof, and the compound of the present disclosure are administered to the subject in separate formulations.

[0162] Compounds of the present disclosure have been found to increase the expression of certain regulatory markers on B cells, such as CD19, CD71, and IgM, thereby inducing a population of CD19+ / CD71+ / IgM+ B cells. In some embodiments, regulatory B cells are CD19+, CD71+, IgM+, CD24+, CD38+, and / or CD27+, e.g., CD19+ / CD71+ / IgM+. Whether regulatory B cells are positive (+) or negative (-) for any of the aforementioned markers can be determined, for example, by flow cytometry analysis.

[0163] Another embodiment is a method of treating an autoimmune disorder in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure, for example in the form of a composition described herein. It is understood that in an autoimmune disorder, it may be desirable to induce general adaptive immune tolerization (e.g., immune tolerance), for example by inducing a population of regulatory T cells, or specific immune tolerization (e.g., immune tolerance), for example by tolerizing the subject to an autoantigen or immunogenic fragment thereof associated with the autoimmune disorder. Thus, in some aspects of the method of treating an autoimmune disorder, the method further comprises administering (e.g., co-administering) to the subject an autoantigen or immunogenic fragment thereof associated with the autoimmune disorder. Some aspects comprise administering to the subject a composition described herein comprising a compound of the present disclosure and an autoantigen, or an immunogenic fragment thereof, for example a composition comprising a plurality of lipid particles, each lipid particle comprising a compound of the present disclosure and an autoantigen, or an immunogenic fragment thereof. In some aspects, the autoantigen, or an immunogenic fragment thereof, and the compound of the present disclosure are administered to the subject in separate formulations.

[0164] Specific examples of autoimmune disorders treatable according to the methods described herein include achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal neuropathy (AMAN), Baro's disease, Behcet's disease, and benign mucosal keratoderma. Pemphigoid, Bullous Pemphigoid, Castleman's Disease (CD), Celiac Disease, Chagas Disease, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Recurrent Multifocal Osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or Eosinophilic Granulomatosis (EGPA), Cicatricial Pemphigoid, Cogan's Syndrome, Cold Agglutinin Disease, Congenital Heart Block, Coxsackie Myocarditis, CREST Syndrome, Crohn's Disease, Dermatitis Herpetiformis, Dermatomyositis, Devic's Disease (Neuromyelitis Optica), Discoid Lupus, Dressler's Syndrome, Endometriosis, Eosinophilic Esophagitis (EoE), Eosinophilic Fasciitis, Erythema Nodosum, Essential Mixed Combined cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch purpura (HSP), herpes gestationis or pemphigoid of pregnancy (PG), hidradenitis suppurativa (HS) (acne contralateral), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis , Juvenile Onset Diabetes Mellitus (Type 1 Diabetes), Juvenile Myositis (JM), Kawasaki Disease, Lambert-Eaton Syndrome, Leukocytoclastic Vasculitis, Lichen Planus, Lichen Sclerosus, Lignin Conjunctivitis, Linear Immunoglobulin A Disease (LAD), Lupus, Chronic Lyme Disease, Meniere's Disease, Microscopic Polyangiitis (MPA), Mixed Connective Tissue Disease (MCTD), Mooren's Ulcer, Mucha-Habermann Disease, Multifocal Motor Neuropathy (MMN) or MMNCB, Multiple Sclerosis, Myasthenia Gravis, Myositis, Narcolepsy, Neonatal Lupus, Neuromyelitis Optica, Neutropenia, Ocular Cicatricial Pemphigoid, Optic Neuritis, Relapsing Rheumatism (PR), PANDAS,Paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome types I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, recurrent multiple myelopathy, These include: chronic chondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-body syndrome (SPS), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), thyroid eye disease (TED), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes mellitus, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada syndrome.

[0165] In some embodiments, the autoimmune disorder is a neurological autoimmune disorder. Examples of neurological autoimmune disorders include multiple sclerosis, neuromyelitis optica, myasthenia gravis, anti-myelin oligodendrocyte glycoprotein antibody disease (MOG), MOG antibody-associated disorder (MOGAD, e.g., MOG-associated pediatric demyelinating disease), autoimmune encephalitis, acute disseminated encephalomyelitis (ADEM), chronic meningitis, central nervous system vasculitis, Guillain-Barre syndrome, Hashimoto's thyroiditis, steroid-responsive encephalopathy with autoimmune thyroiditis (SREAT), neurosarcoidosis, optic neuritis, and transverse myelitis.

[0166] In some embodiments, the autoimmune disorder is rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (IBD), multiple sclerosis, type 1 diabetes, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, or vasculitis. In some embodiments, the autoimmune disorder is systemic lupus erythematosus. In some embodiments, the autoimmune disorder is IBD.

[0167] In some embodiments, the autoimmune disorder is multiple sclerosis, neuromyelitis optica, myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), rheumatoid arthritis, or myasthenia gravis. In some embodiments, the autoimmune disorder is multiple sclerosis. In some embodiments, the autoimmune disorder is neuromyelitis optica. In some embodiments, the autoimmune disorder is MOGAD. In some embodiments, the autoimmune disorder is rheumatoid arthritis. In some embodiments, the autoimmune disorder is myasthenia gravis.

[0168] Clinical management of multiple sclerosis usually follows one of two paradigms: the escalation paradigm or the induction / maintenance paradigm. In the escalation paradigm, a more effective and efficacious disease-modifying treatment (DMT) is administered after treatment with a less effective and efficacious DMT has failed. Standard treatment in the escalation paradigm usually includes treatment with glatiramer acetate, interferon beta, and / or teriflunimide, escalating to fingolimod and / or dimethyl fumarate upon treatment failure, further escalating to natalizumab and / or anti-B cell upon treatment failure, and still further escalating to alemtuzumab and / or mitoxantrone upon treatment failure.

[0169] The induction / maintenance treatment paradigm for the clinical management of multiple sclerosis involves an induction phase followed by a maintenance phase in which patients are treated with a high-potency DMT to induce disease control, followed by a switch to a safer, lower-potency DMT as maintenance therapy.

[0170] Disease-modifying therapies (DMTs) used to treat multiple sclerosis include interferon beta-1a (e.g., AVONEX®, REBIF®), interferon beta-1b (e.g., BETASERON®, EXTAVIA®), glatiramer acetate (e.g., COPAXONE®, GLATOPA®), ofatumumab (e.g., KESIMPTA®), pegylated interferon beta-1a (e.g., PLEGRIDY®), teriflunomide (e.g., AUBAGIO®), monomethyl fumarate (e.g., BAFIERTAM™), dimethyl fumarate (e.g., TECFIDERA®), fingolimod (e.g., GILENYA®), cladribine (e.g., MAVENCLAD®), siponimod (e.g., MAYZENT®), ponesimob (e.g., PONVORY®), diroximel fumarate (e.g., VUMERITY®), ozanimob (e.g., ZEPOSIA®), alemtuzumab (e.g., LEMTRADA®), mitoxantrone (e.g., NOVANTRONE®), ocrelizumab (e.g., OCREVUS®), and natalizumab (e.g., TYSABRI®). Examples of high efficacy DMTs include, but are not limited to, natalizumab, alemtuzumab, anti-B cells, and mitoxantrone. Examples of lower potency DMTs include, but are not limited to, glatiramer acetate, interferon beta, teriflunimide, DMF, and fingolimov.

[0171] Natalizumab is a recombinant humanized IgG4κ monoclonal antibody produced in mouse myeloma cells. Natalizumab binds to the α4 subunit of α4β1 and α4β7 integrins expressed on the surface of all leukocytes except neutrophils, and inhibits α4-mediated adhesion of leukocytes to their counter-receptor(s). Natalizumab injection is indicated as monotherapy for the treatment of relapsing forms of multiple sclerosis, including clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease, in adults. Glatiramer acetate injection is indicated for the treatment of relapsing forms of multiple sclerosis, including clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease, in adults. Dimethyl fumarate for oral use is indicated for the treatment of relapsing forms of multiple sclerosis.

[0172] In some embodiments, the multiple sclerosis has not been previously treated. In alternative embodiments, the multiple sclerosis has been previously treated with standard therapies, such as, for example, natalizumab (TYSABRI®), glatiramer acetate and / or dimethyl fumarate, or DMT.

[0173] In some embodiments, the multiple sclerosis is primary progressive multiple sclerosis (PPMS). In some embodiments, the multiple sclerosis is relapsing remitting multiple sclerosis (RRMS). In some embodiments, the multiple sclerosis is clinically isolated syndrome (CIS). In some embodiments, the multiple sclerosis is secondary progressive multiple sclerosis (SPMS).

[0174] In some embodiments (e.g., when the autoimmune disease is multiple sclerosis), the method includes administering to the subject a therapeutically effective amount of an induction therapy comprising a compound of the present disclosure, e.g., in the form of a pharmaceutical composition. In some embodiments (e.g., when the autoimmune disease is multiple sclerosis), the method includes administering to the subject a therapeutically effective amount of a maintenance therapy comprising a compound of the present disclosure, e.g., in the form of a pharmaceutical composition. In some embodiments (e.g., when the autoimmune disease is multiple sclerosis), the method includes administering to the subject a therapeutically effective amount of an induction therapy comprising a compound of the present disclosure, e.g., in the form of a pharmaceutical composition, and (e.g., followed by) a therapeutically effective amount of a maintenance therapy comprising a compound of the present disclosure, e.g., in the form of a pharmaceutical composition.

[0175] In some embodiments (e.g., where the autoimmune disease is multiple sclerosis), the compound of the disclosure is administered in combination with a DMT, such as natalizumab and / or glatiramer acetate and / or dimethyl fumarate, and in some further embodiments, the method further comprises administering to the subject a DMT, such as natalizumab and / or glatiramer acetate and / or dimethyl fumarate.

[0176] In some embodiments, the autoimmune disorder has not been previously treated, hi alternative embodiments, the autoimmune disorder has been previously treated with a standard of care therapy, such as natalizumab (TYSABRI®) or glatiramer acetate for multiple sclerosis.

[0177] Examples of autoantigens associated with autoimmune disorders include the thyroid stimulating hormone receptor of the thyroid gland (Graves' disease), thyroid antigens such as thyroid peroxidase (Hashimoto's thyroiditis), beta cell antigens such as glutamic acid decarboxylase and insulin (type I diabetes), cytochrome P450 antigens (Addison's disease), myelin proteins such as myelin basic protein (multiple sclerosis), uveal antigens (uveitis), H +These include gastric parietal cell antigens such as ATPase and intrinsic factor (pernicious anemia), transglutaminase (gluten enteropathy), cardiomyocyte proteins such as myosin (myocarditis, rheumatic heart disease), platelet antigens such as GP IIb / IIIa (idiopathic thrombocytopenic purpura), erythrocyte membrane proteins (autoimmune hemolytic anemia), neutrophil membrane proteins (autoimmune neutropenia), basement membrane antigens such as type IV collagen α3 chain (Goodpasture's disease), intrahepatic bile duct / mitochondrial antigens such as 2-oxoacid dehydrogenase complex (primary biliary cirrhosis), hepatocyte antigens such as cytochrome P450 and 206 (autoimmune hepatitis), acetylcholine receptors (myasthenia gravis), and desmoglein (pemphigus and other bullous diseases). The disorder listed in parentheses next to each autoantigen indicates the autoimmune disorder with which the indicated autoantigen is usually associated.

[0178] The compounds of the present disclosure and compositions described herein are expected to be useful adjunctive therapies for antigen therapy, e.g., gene therapy, for example, by inhibiting undesired immune responses to the antigen therapy and / or by allowing administration and / or repeated administration of the antigen therapy. Another embodiment is a method of treating a disease, disorder, or condition in a subject in need of treatment with antigen therapy, comprising administering a compound of the present disclosure to the subject, e.g., in the form of a composition described herein. In some aspects, the compound of the present disclosure is administered in an amount sufficient to immunotolerize the subject to the antigen therapy. In some aspects, the method further comprises administering (e.g., co-administering) an antigen therapy (e.g., a therapeutically effective amount of the antigen therapy) to the subject, e.g., simultaneously or sequentially with the compound of the present disclosure. Some aspects include administering to the subject a composition described herein comprising a compound of the present disclosure and an antigen therapy, e.g., a composition comprising a plurality of lipid particles, each lipid particle comprising a compound of the present disclosure and an antigen therapy. In some aspects, the antigen therapy and the compound of the present disclosure are administered to the subject in separate formulations.

[0179] In some aspects, the antigen therapy is an antibody therapy (e.g., monoclonal antibody therapy), including chimeric antibody therapy, humanized antibody therapy, and fully human antibody therapy. Specific examples of antibody therapy include anti-tumor necrosis factor (anti-TNF) therapy, such as adalimumab (Humira®; for rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, plaque psoriasis, hidradenitis suppurativa, uveitis) and infliximab (Remicade®; for Crohn's disease, pediatric Crohn's disease, ulcerative colitis, pediatric ulcerative colitis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis), golimumab (Humira®; for rheumatoid arthritis, juvenile idiopathic arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis), golimumab (Humira®; for rheumatoid ... mab (Simponi®, for rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, polyarticular juvenile idiopathic arthritis), etanercept (Enbrel®, for rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, plaque psoriasis), and certolizumab pegol (Cimzia®, for Crohn's disease, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, non-radiographic axial spondyloarthritis, plaque psoriasis).

[0180] In some embodiments, the antigen therapy is a protein replacement therapy, e.g., enzyme replacement therapy. Examples of protein replacement therapy include replacement therapy for clotting disorders, such as Factor VIII and Factor IX for hemophilia A and B, enzyme replacement therapy for lysosomal storage diseases, such as alglucosidase alfa (Myozyme® and Lumizyme®) for Pompe disease, alpha-L-iduronidase for Hurler syndrome, and adenosine deaminase for adult adenosine deaminase deficiency.

[0181] In some embodiments, the antigen therapy is a gene therapy. Gene therapy typically works by one of three mechanisms: (1) by providing the subject with a healthy copy of the disease-causing gene (e.g., as voretigene neparvovec-rzyl (Luxturna®) does), (2) by inactivating the disease-causing gene (e.g., ASO and siRNA), or (3) by introducing a gene into the body to help treat the disease. Gene therapy includes DNA (e.g., antisense oligonucleotides (ASO)) and / or RNA (e.g., siRNA), which can be delivered to the subject in vivo or ex vivo via a variety of products. In vivo gene delivery products include plasmid DNA, viral vectors (AAV, such as AAV9), and non-viral vectors, such as bacterial vectors or lipid nanoparticles. Other examples of non-viral vectors suitable for in vivo gene delivery include exosomes, polymer particles, inorganic particles, and lipid-polymer hybrid particles. Ex vivo gene delivery products include subject-derived cellular gene therapy products. Gene therapy also includes gene editing technologies, such as CRISPR. Gene editing technologies such as CRISPR can be conveniently delivered to a subject via any of the in vivo gene delivery products described herein.Specific examples of gene therapy include the voretigene neparvovec-rzyl (Luxturna®, for retinal dystrophy) and the onasemnogene abeparvovec-xioi (Zolgensma®, for pediatric spinal muscular atrophy).

[0182] In some embodiments, gene therapy includes DNA and / or RNA and viral vectors. In some embodiments, the viral vector is derived from an adeno-associated virus (AAV), such as a recombinant AAV. In some embodiments, the AAV is AAV9. Other examples of viral vectors suitable for use with the present disclosure include viral vectors derived from retroviruses, herpes viruses, adenoviruses, lentiviruses, rabies viruses, lentiviruses, VSV, poxviruses (e.g., vaccinia viruses, smallpox viruses, canarypox), reoviruses, Semliki Forest viruses, yellow fever viruses, Sindbis viruses, togaviruses, baculoviruses, bacteriophages, alphaviruses, and flavaviruses.

[0183] In some embodiments, the antigen therapy is a cell therapy. One example of a cell therapy is axicabtagene ciloleucel (Yescarta®, for relapsed or refractory large B-cell lymphoma). Another example of a cell therapy is CAR-T cells.

[0184] Alloantigens are antigens that are present in some but not all individuals of a species and are recognized as foreign by those who do not possess them, and are often the basis of transplant rejection.Accordingly, another embodiment is a method for treating graft-versus-host disease in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a composition described herein.

[0185] Examples of alloantigens include, but are not limited to, major histocompatibility complex (MHC) class I and class II antigens, minor histocompatibility antigens, endothelial glycoproteins such as blood group antigens, and carbohydrate determinants.

[0186] Another embodiment is a method of promoting wound healing in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure, e.g., in the form of a composition described herein.

[0187] In some embodiments of any method described herein, the method further comprises administering an antigen or its immunogenic fragment to the subject. In some further embodiments, the antigen or its immunogenic fragment is co-administered with the compound of the present disclosure. For example, it may be sometimes desirable to induce antigen-specific immune tolerance (e.g., when the compound of the present disclosure is administered to tolerize the subject to antigen therapy). When antigen-specific immune tolerance is desired, the antigen or its immunogenic fragment is preferably co-administered with the compound of the present disclosure, for example in the form of a composition as described herein.

[0188] As used herein, "co-administer," "co-administration," and the like refer to the simultaneous or near simultaneous but sequential administration of two or more agents (e.g., a compound of the disclosure and an antigen) via the same route of administration at the same or near the same site on a subject's body.

[0189] When co-administration is simultaneous (e.g., concurrent), the first agent (e.g., a compound of the present disclosure) and the second agent (e.g., an additional therapeutic agent, an antigen, or an immunogenic fragment thereof) can be present in separate formulations or in the same formulation. Alternatively, the first and second agents can be administered sequentially as separate compositions. When co-administration is sequential, administration of the subsequent composition(s) is performed within 24 hours, preferably within 12 hours, for example, within 10 hours, 5 hours, 4 hours, 3 hours, 2 hours, 60 minutes, 30 minutes, 15 minutes, 10 minutes, or 5 minutes, of administration of the first composition. Usually, when co-administration is sequential, administration of the next composition(s) follows immediately after completion of administration of the first composition, taking into account the actions that the clinician or subject administering the composition may need to take to prepare for administration of the next composition(s).

[0190] When co-administration is oral, administration site is mouth, and whether co-administered two or more drugs are administered in a single formulation or in separate formulations, they are orally administered at the same site.However, when co-administration is by injection of two or more compositions, administration site is more generally approximately the same.In such a situation, the anatomical sites of administration are generally less than 2 inches apart from each other, for example, less than about 0.5 inches, less than about 1 inch, or less than about 1.5 inches apart from each other.

[0191] In some aspects, the antigen or immunogenic fragment thereof and the compound of the present disclosure are orally co-administered. In a further aspect, the administration of the antigen or immunogenic fragment thereof precedes the administration of the compound of the present disclosure. In an alternative further aspect, the administration of the compound of the present disclosure precedes the administration of the antigen or immunogenic fragment thereof. In yet another further aspect, the administration of the compound of the present disclosure and the antigen or immunogenic fragment thereof is simultaneous.

[0192] Co-administration can be by any route of administration described herein. In some aspects, the compounds of the present disclosure and the antigen or immunogenic fragment thereof are co-administered orally. In some aspects, the compounds of the present disclosure and the antigen or immunogenic fragment thereof are co-administered subcutaneously.

[0193] Without wishing to be bound by any particular theory, it is believed that it may be desirable for the subject's immune system to encounter the antigen and the compound of the present disclosure together, or for the antigen and the compound of the present disclosure to be "co-presented" to the subject's immune system. When the compound of the present disclosure is co-administered with the antigen, and the antigen is a protein, such as in protein replacement therapy, co-administration, for example by injecting the antigen and the compound of the present disclosure in separate formulations, is expected to provide effective co-presentation of the compound of the present disclosure and the antigen to the subject's immune system. In such applications, the compound of the present disclosure may, but need not, be incorporated into lipid particles. In a preferred embodiment of such applications, the co-administration is subcutaneous administration, for example by injection. In applications involving the delivery of gene therapy (e.g., gene therapy including DNA and / or RNA), it may be desirable to formulate the gene therapy and the compound of the present disclosure into lipid particles that include the gene therapy and the compound of the present disclosure to facilitate effective co-presentation of the gene therapy and the compound of the present disclosure to the subject's immune system. In preferred embodiments, such particles are formulated for oral and / or parenteral (eg, subcutaneous, intramuscular, intravenous, intradermal) administration, eg, by injection.

[0194] Also, without wishing to be bound by any particular theory, it is believed that certain compounds of the present disclosure identified herein are embedded in liposomes.For example, the use of such compounds according to the methods disclosed herein can be advantageous in aspects in which the effective co-presentation of the compounds of the present disclosure and antigens to the immune system of a subject is promoted by incorporating the compounds of the present disclosure and antigens into lipid particles that contain the compounds of the present disclosure and antigens.

[0195] The compounds of the present disclosure may also be administered in combination with one or more non-antigenic therapies to treat a disease, disorder, or condition. When administered "in combination" with such a non-antigenic therapy, the compounds of the present disclosure may be administered before, after, or simultaneously with the other therapy(s) (e.g., additional therapeutic agent(s)). When administered simultaneously (e.g., concurrently), the compounds of the present disclosure and the other therapy may be in separate formulations or in the same formulation. Alternatively, the compounds of the present disclosure and the other therapy may be administered as separate compositions, at about the same time or sequentially at different times. When the compounds of the present disclosure and the other therapy (e.g., therapeutic agent) are administered as separate formulations or compositions, the compounds of the present disclosure and the other therapy may be administered by the same route of administration or by different routes of administration. A skilled clinician can determine the appropriate timing for administering each therapy used in combination (e.g., sufficient timing to allow for overlap of the pharmaceutical effects of the therapies). Typically, the combination therapy provides the beneficial effects of the drug combination in treating the disease, condition, or disorder described herein.

[0196] In some aspects, the methods described herein further include administering to the subject an additional non-antigenic therapy(s) (e.g., a therapeutically effective amount), e.g., in combination with a compound disclosed herein or a composition described herein. In some aspects, the compound or composition disclosed herein is administered prior to the additional therapy(s). In some aspects, the compound or composition disclosed herein is administered after the additional therapy(s). In some aspects, the compound or composition disclosed herein is administered simultaneously with the additional therapy(s).

[0197] The therapeutically effective amount of the agent to be administered can be determined by a clinician of ordinary skill in the art using the guidance provided herein and other methods known in the art. For example, a suitable dose may be about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.01 mg / kg to about 1 mg / kg body weight per treatment. Determining the dosage for a particular agent, subject, and disease is well within the capabilities of a person of ordinary skill in the art. Preferably, the dosage will not cause adverse side effects or will cause minimal side effects.

[0198] The compounds of the present disclosure, compositions described herein, antigens, or other therapeutic agents can be administered via a variety of routes of administration, including, for example, oral, feeding, topical, transdermal, rectal, parenteral (e.g., intra-arterial, intravenous, intramuscular, subcutaneous injection, intradermal injection), intravenous injection, and inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal instillation), depending on the compound, antigen, and / or therapeutic agent, respectively, and the particular disease to be treated. Administration can be local or systemic. The preferred mode of administration can vary depending on the particular compound or agent.

[0199] In some embodiments, administration (e.g., of a compound of the present disclosure or a composition and / or antigen described herein) is oral. In some embodiments, administration (e.g., of a compound of the present disclosure or a composition and / or antigen described herein) is intravenous. In some embodiments, administration (e.g., of a compound of the present disclosure or a composition and / or antigen described herein) is subcutaneous.

[0200] The disclosed compounds or compositions described herein can be administered prophylactically according to the methods disclosed herein, as well as when the disclosed compounds or compositions described herein are co-administered with antigen therapy to a subject with no known immune intolerance to antigen therapy. The disclosed compounds or compositions described herein can also or alternatively be administered therapeutically according to the methods disclosed herein, as when a subject has demonstrated immune intolerance to an antigen (e.g., allergic reaction, graft rejection). Thus, in some embodiments, the subject does not have a known immune intolerance to the antigen, for example, because the subject is naive to the antigen. In some embodiments, the subject does not have a known immune intolerance to the antigen after the antigen is administered and / or after exposure to the antigen. In some embodiments, the subject is immune intolerant to the antigen, for example, develops immune intolerance after administration and / or exposure to the antigen, or is inherently immune intolerant to the antigen.

[0201] The compounds of the present disclosure or compositions described herein can be administered (e.g., co-administered) according to the methods disclosed herein at the time of the first exposure to the antigen, as when the compounds of the present disclosure or compositions described herein are administered together with the first dose of the antigen therapy. Additionally or alternatively, the compounds of the present disclosure or compositions described herein can be administered (e.g., co-administered) according to the methods disclosed herein at the time of the second or further exposure to the antigen, as when the compounds of the present disclosure or compositions described herein are administered together with the second or further additional dose (e.g., repeated dose) of the antigen therapy.

[0202] The methods described herein aim to reduce immune intolerance to an antigen over the long term of a subject's life, e.g., for the period of time necessary to treat a disease, disorder, or condition with an antigen therapy described herein. Thus, in some aspects of the methods described herein, the method further comprises administering an antigen or an immunogenic fragment thereof (e.g., an antigen therapy, such as a therapeutically effective amount of an antigen therapy) to the subject in the absence of a compound of the present disclosure or a composition described herein.

[0203] However, the subject's immune intolerance may increase over time following the methods described herein, e.g., following subsequent exposure(s) to the antigen. In such cases, the methods described herein can be repeated, e.g., in the same manner that a "booster" vaccine is repeated, to re-tolerize the subject to the antigen.

[0204] The compounds of the present disclosure, or other therapeutic agents described herein, can be administered via a variety of routes of administration, including, for example, oral, feeding, topical, transdermal, rectal, parenteral (e.g., intra-arterial, intravenous, intramuscular, subcutaneous injection, intradermal injection), intravenous infusion, and inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal instillation) routes of administration, depending on the compound and the particular disease to be treated. Administration can be local or systemic. In some embodiments, administration (e.g., of a compound of the present disclosure) is oral. In some embodiments, administration (e.g., of a compound of the present disclosure) is intravenous. Preferred modes of administration can vary depending on the particular compound or agent. Typically, a compound of the present disclosure or other therapeutic agent is administered about 1 to about 6 times per day (e.g., 1, 2, 3, 4, 5, or 6 times), also or alternatively, as an infusion (e.g., continuous infusion). In some aspects, administration (e.g., of a compound of the present disclosure) is QD or BID (e.g., QD). In some embodiments, administration (eg, of a compound of the present disclosure) is daily.

[0205] It is shown herein that orally administered liposomes as described herein can reach lymph nodes and co-localize with immune cells, including B cells and T cells, in lymph nodes. Accordingly, also provided herein is a method of delivering a therapeutic agent (e.g., a compound of the present disclosure) to a lymph node of a subject, comprising orally administering to the subject a therapeutically effective amount of a composition containing a plurality of lipid particles (e.g., solid lipid particles), wherein each lipid particle comprises at least one phospholipid (e.g., C4-C in dimyristoylphosphatidylcholine (DMPC)). 30 Acyl chains, e.g., saturated C4-C 30The lipid particle includes a lipid bilayer that contains a phospholipid containing an acyl chain, and a therapeutic agent (e.g., a compound of the present disclosure) that can be embedded in the lipid bilayer of the lipid particle.

[0206] The compounds of the present disclosure or other therapeutic agents can be administered at a dose ranging from about 0.001 mg / kg body weight to about 100 mg / kg body weight, or at a dose ranging from about 1 mg / dose to about 5,000 mg / dose every 4 to 120 hours, or according to the requirements of the particular agent. For example, a suitable dose can be about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.01 mg / kg to about 1 mg / kg body weight per treatment. In some embodiments, a suitable dose (e.g., daily dose) is about 0.1 mg / kg to about 10 mg / kg body weight per treatment, e.g., about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to 2.5 mg / kg, or about 0.2 mg / kg to about 2.4 mg / kg body weight. A suitable dose may be about 0.001 mg / dose to about 100 mg / dose, about 0.01 mg / dose to about 100 mg / dose, about 0.1 mg / dose to about 50 mg / dose, about 0.1 mg / dose to about 10 mg / dose, about 0.5 mg / dose to about 50 mg / dose, about 1 mg / dose to about 10,000 mg / dose, about 1 mg / dose to about 7,500 mg / dose, about 1 mg / dose to about 5,000 mg / dose, about 10 mg / dose to about 2,500 mg / dose, or about 100 mg / dose to about 1,000 mg / dose. In some embodiments, a suitable dose (e.g., daily dose) is from about 10 mg / dose to about 1,000 mg / dose, e.g., from about 15 mg / dose to about 1,000 mg / dose, from about 10 mg / dose to about 500 mg / dose, from about 10 mg / dose to about 250 mg / dose, or from about 15 mg / dose to about 150 mg / dose.

[0207] Doses lower or higher than those listed above may be necessary.The specific dosage and treatment regimen for any particular patient depends on a variety of factors, including, for example, the activity of the specific drug used, age, body weight, general health, sex, diet, administration time, excretion rate, drug combination, severity and course of disease, condition or symptom, predisposition of the subject to disease, condition or symptom, and the judgment of the treating physician.Determining the dosage for a particular drug, subject, and disease, disorder, or condition is within the capabilities of those skilled in the art. EXAMPLES

[0208] Example 1. Synthesis of Compound 1 and Compound 2 The synthesis of Compound 1 and Compound 2 used the following Boc-Dap-OH (Boc-Dap-OH) as a common starting material: [ka]

[0209] Compound 1: (S)-2-amino-3-(((hexadecyloxy)carbonyl)amino)propanoic acid [ka]

[0210] Boc-Dap-OH (0.500 g) was dissolved in 10% sodium carbonate solution (7 mL) at 0° C., and a solution of cetyl chloroformate (0.866 g) in dioxane (7 mL) was added dropwise at 0° C. The reaction mixture was stirred at 0° C. for 1 h and then at room temperature (RT) for 1.5 h. The reaction was then quenched with 60 mL of water. The heterogeneous mixture was extracted with ether. The solid was suspended between the aqueous and ether layers. The aqueous layer was separated, the solid was filtered and washed with excess ether. The solid was suspended in water, acidified to pH 1 and immediately extracted with EtOAc (2×35 mL). The separated organic layer was washed with water (3×20 mL) and brine (15 mL) and dried over Na2SO4. The solvent was removed under reduced pressure to give (S)-2-((tert-butoxycarbonyl)amino)-3-(((hexadecyloxy)carbonyl)amino)propanoic acid.

[0211] To generate (S)-2-amino-3-(((hexadecyloxy)carbonyl)amino)propanoic acid HCl salt, a solution of (S)-2-((tert-butoxycarbonyl)amino)-3-(((hexadecyloxy)carbonyl)amino)propanoic acid (0.423 g) in 4N HCl / dioxane (7 mL) was stirred at 0° C. for 2.5 h. The solvent was then removed under reduced pressure to give (S)-2-((tert-butoxycarbonyl)amino)-3-(((hexadecyloxy)carbonyl)amino)propanoic acid. Traces of solvent were further removed at 70° C. under vacuum for 5 h.

[0212] Compound 2: (S)-2-amino-3-stearamidopropanoic acid [ka]

[0213] Boc-Dap-OH (0.500 g) was dissolved in 10% sodium carbonate solution (7 mL) at 0° C. and a solution of stearoyl chloride (0.86 g) in dioxane (7 mL) was added dropwise at 0° C. The reaction mixture, thickened with a white precipitate, was stirred at 0° C. for 10 min and then at room temperature for 2 h. The reaction was quenched with 60 mL of water. Ether was added to emulsify the solution. More ether was added. The emulsion was washed with ether (3×25 mL). The emulsion was acidified to pH 1, causing the ether layer to separate and the aqueous layer to become cloudy. The ether layer was separated and dried over Na2SO4. The solvent was removed under reduced pressure to give (S)-2-((tert-butoxycarbonyl)amino)-3-(stearamido)propanoic acid.

[0214] To prepare (S)-2-amino-3-stearamidopropanoic acid HCl salt, a solution of (S)-2-((t-butoxycarbonyl)amino)-3-(stearamido)propanoic acid (0.502 g) in 4N HCl / dioxane (9 mL) was stirred at 0° C. for 2.5 h. Ether was added. The precipitated compound was filtered and washed with excess ether. Traces of solvent were removed under high vacuum overnight.

[0215] Example 2. Liposome synthesis The day before liposome preparation, the synthetic route was determined based on the decision tree shown in Figure 1. For options A and B, the active ingredient was dissolved in an appropriate solvent (e.g., chloroform (Sigma-Aldrich, #650498-1L, St. Louis, MO)) and stored at -80°C in amber glass vials. For option C, stock solutions were not made until the day of liposome preparation.

[0216] On the day of liposome preparation, the amount of base lipid and active ingredient was calculated. In Option A, if the test substance was dissolved in chloroform, the volume of test substance required to obtain the desired molar concentration of the test substance was calculated. The amount of base lipid required to make up the remaining molar ratio was also calculated. For example, if the molar ratio of test substance:base lipid is 30:70, the amount of base lipid is the amount of test substance multiplied by 70 and divided by 30. The appropriate amount of test substance and base lipid were aliquoted into 5 ml or 15 ml round bottom flasks and 1-2 ml of chloroform was added.

[0217] In Option B, when the test substance and base lipid were dissolved in immiscible solvents or when the two solvents required different temperature and vacuum settings (e.g., DMSO and chloroform), the amount of test substance and the amount of base carrier lipid required to obtain the desired molar concentration were calculated as in Option A. Evaporation was started in the solvent requiring higher temperature and vacuum before adding the substance to the solvent requiring lower temperature and vacuum. Temperature and vacuum were adjusted accordingly.

[0218] For option C, if the test article was water soluble, the liposomes were composed of 100% base lipid. The amount of base lipid was determined based on the amount of test article to be loaded into the fully synthesized liposomes. For example, if the molar ratio of test article:base lipid was 30:70, the amount of test article was multiplied by 100 and divided by 30 to determine the amount of base lipid. The appropriate amount of base lipid was aliquoted into a 5 ml or 15 ml round bottom flask and 1-2 ml of chloroform was added.

[0219] The round-bottom flask was attached to a rotary evaporator (R100 rotary evaporator with V100 and I100 vacuum pumps and interfaces, BUCHI Labortechnik AG, Flawil, Switzerland) and the flask was partially immersed in the water bath by lowering the adjustable arm. The vacuum pump was turned on. To rotate the round-bottom flask, the rotating shaft was turned on and the speed was set to "3". The vacuum was reduced as necessary to avoid bubbling / boiling of the solvent in the flask. Once a uniform dry film was formed, the rotating shaft was turned off. In option B, a second solvent containing the second substance was added and the above steps were repeated.

[0220] For rehydration, an appropriate aqueous buffer (e.g., PBS (Thermo Fisher Scientific (Gibco), #10010049, Waltham, MA)) was added (1 ml for option A or B, 0.5 ml for option C, pH was adjusted as necessary). The round-bottom flask was vortexed until the solution was milky / turbid. The flask was replaced on the rotary evaporator and the arm was lowered so that the bottom of the flask was immersed in the water bath. The rotary shaft was turned on and set to "3", but the pump was not turned on. After 15 minutes, the lipid film was fully hydrated, liposomes were formed, and ready for sizing.

[0221] A manual extruder (Avanti Corporation, Alexandria, VA, AVANTI manual extruder; 610023-1EA, PC membrane 0.8 μm: 610009-1EA) was assembled for extrusion and sizing. The entire volume (up to 1 ml) was drawn into one of two syringes provided by the manual extruder. The liposome preparation was extruded 20 times. The final extruded liposome formulation was transferred and the volume was made up as needed to obtain the final desired molar concentration of the test article.

[0222] Example 3. Effect of acyl chain length and saturation on lymphatic uptake and immune cell colocalization of orally administered Compound 2 containing liposomes The oral route is a patient-friendly route of administration. It also offers a unique opportunity to target the immune system via the mesenteric lymph nodes, which may prove useful in administering immunomodulatory therapies. Compound 2, a novel amphiphilic molecule designed to span the lipid bilayer of liposomal membranes, is believed to be a TIM agonist. Preliminary screening with compound 2 showed that it is pharmacologically active and can indeed induce tolerogenic immune responses in vitro and in vivo. Compound 2 inhibits FoxP3 in vivo. + / CD4 + It can increase T cells (Example 5), and in vitro FoxP3 + / TIM3 + CD4 T cells can be increased (Example 4).

[0223] The goal was to optimize a liposomal formulation that could target both cortical follicles for B cell binding and paracortical follicles for T cell binding. Natural phospholipids with a phosphatidylcholine (PC) head group were selected as the base lipid, due in part to the neutral charge of PC. The effect of different acyl chain lengths and saturation levels on lymphatic uptake and immune cell colocalization after oral administration of liposomes was evaluated in vivo.

[0224] Table 1 shows the materials and reagents used in this experiment. [Table 1]

[0225] To optimize the lipid:DilC18(5)-DS fluorescent dye ratio in vitro, DMPC liposomes (AVANTI Polar Lipids Inc., Birmingham, AL) were prepared as described in Example 2. Lipid films were hydrated with PBS containing various concentrations of DilC18(5)-DS fluorescent dye (100 nM to 2 μM). Fluorescence intensity was measured at Ex / Em 650 nm / 670 nm in a 96-well plate to identify the minimum concentration of DilC18(5) required to obtain reliable fluorescence measurements.

[0226] Test substances were prepared as follows: Compound 2 was dissolved in DMSO. Four different formulations (one for each lipid in Table 1) were prepared with a molar ratio of Compound 2:lipid of 30:70 as described in Example 2. 100 nM DilC 18 (5) The lipid film was hydrated using PBS containing the dye. The fluorescence intensity of each preparation was measured at Ex / Em 650 nm / 670 nm in a 96-well plate.

[0227] Animals (n=3 per group) were administered 200 μl of 160 μM of each fluorescent liposome by oral gavage. All animals received the same dose of lipid regardless of fluorescence intensity. The control group received 100 nM DilC 18 (5) was administered by oral gavage with 200 μl of PBS. To quantify lymphatic uptake and immune cell colocalization of each liposomal formulation, animals were sacrificed 60 min after oral gavage. Mesenteric lymph nodes were extracted and stored in optimal cutting temperature (OCT) compound for sectioning, labeling, and imaging.

[0228] For staining of T and B cells, mesenteric lymph nodes were excised 60 min after oral gavage and snap frozen in OCT compound. Ten micrometer sections were cut at -20°C and frozen sections were fixed in 2% paraformaldehyde (PFA) for 15 min at room temperature. Sections were washed in 1×PBS and subsequently incubated overnight at 4°C with fluorochrome-conjugated (FITC) primary antibodies for either CD45R or TCR-β at a dilution of 1:100 in 1×PBS. The next day, slides were washed three times in 1×PBS and mounted with DAPI-containing mounting medium.

[0229] For fluorescence imaging, stained sections of mesenteric lymph nodes were visualized under an epifluorescence microscope (Nikon E-800; Nikon Inc., Melville, NY) using channel Cy5 (Ex / Em 650 nm / 670 nm) for DilC-18(5)-DS fluorescent dye (red), channel FITC (Ex / Em 494 nm / 518 nm) for CD45R or TCR-β (green), and channel DAPI (Ex / Em 358 nm / 461 nm) for nuclei (blue).

[0230] Visual inspection of immunofluorescence provided very good qualitative detection of the location of liposomes within lymph node sections, as well as the level of colocalization with B and T cells. However, visual inspection did not explain the differences in fluorescence intensity of each liposome, as seen in Table 2. Furthermore, visual inspection did not allow objective differentiation between liposomal formulations. For objective evaluation, a colocalization analysis was performed to determine the percentage of colocalization.

[0231] Colocalization of liposomes with B and T cells was measured with ImageJ. Images of each channel (FITC, Cy5, and DAPI) were merged. The area percentage of colocalization was calculated by thresholding the merged image in the yellow spectrum, which represents the area where green (immune cells) and red (liposomes) were colocalized. Quantified results from image analysis were normalized by subtracting the background obtained from the control group (free DilC- 18(5)-DS dye alone). Correction for the different fluorescence intensities of each liposome was performed by normalizing the results of image thresholding to the fluorescence intensity (FI) of each liposome as previously described to obtain a corrected average FI for each section.

[0232] Because tissue sections were not homogenous, it was expected that immunofluorescence staining would have a high variability from one section to another. To determine the effectiveness of liposome / immune cell interactions, the mean FI of the colocalized areas was normalized to the mean FI of immune cells using the following formula:

number

[0233] Colocalization analysis was performed on all sections of all groups except the free dye control group, as free dye / immune cell colocalization was not observed in those sections.

[0234] DilC in PBS at concentrations ranging from 100 nM to 2 μM 18 Fluorescence intensity measurements of (5) confirmed that the fluorescence of the dye in aqueous media was poor compared to its fluorescence once entrapped in liposomes (Table 2). The relative fluorescence intensity (RFI) of the free dye remained low in the concentration range of 100 nM to 1 μM. At 2 μM, DiC 18 The fluorescence of (5) increased in PBS, but not in liposomal DilC. 18 (5). 18 The RFI of (5) was increased by >2000% in the range of 100–500 nM compared to the RFI of the dye in PBS. This was even greater in the liposomal formulation containing 1 μM dye. The RFI was decreased in the 2 μM liposomal formulation, which could be the result of self-quenching of the dye or insufficient uptake into the liposomes at such high concentrations (Table 2). The results of this screen showed that the RFI of 100 nM DilC 18(5) is sufficient to detect liposomal fluorescence, and furthermore, the poor RFI of the dye in PBS at that concentration suggests that a purification step to remove free dye is not necessary. [Table 2]

[0235] Fluorescence intensity measurements of each liposome formulation revealed differences between them: liposomes made with unsaturated DOPC and POPC had higher RFIs (73.2 and 61.8, respectively) than those made with saturated DMPC and DSPC (35.8 and 20.4, respectively) (Table 3). [Table 3]

[0236] Sectioning and immunofluorescence staining was successful for all samples except one from the free dye control group. Staining revealed that this sample was from mesenteric fat, not mesenteric lymph node. All other sections were successfully stained for image analysis.

[0237] Immunofluorescence staining was performed to assess colocalization of B cells with liposomes. FITC-conjugated anti-CD45R was observed in all samples, indicating the presence of B cells. Both mature (light) and immature (dark) B cells were observed in all sections from all treatment groups. In some sections, B cells were clustered around a dark center, possibly in the lymph node cortex (arrow in Figure 2A), representing an area of ​​the lymph node space of B cells (e.g., paracortex or medulla).

[0238] Lymph node uptake and B cell colocalization were evaluated 60 min after oral administration of 200 μl PBS containing 100 nM DilC18(5). DAPI staining shows the general structure of lymph node sections. CD45R-FITC shows B cell localization. The Cy5 channel detecting DilC18(5) fluorescence showed a signal in one sample, mainly concentrated at the periphery of the section (see Fig. 2B, M2, DilC18(5) panel). However, no colocalization was observed (Fig. 2B, M2, CD45R-FITC / DilC18(5) panel). DilC18(5) was not detected in lymph nodes from the other samples (Fig. 2B, M3).

[0239] Lymph node uptake and B cell colocalization were assessed 60 min after oral administration of 200 μl of 160 μM Compound 2 / DMPC DilC18(5)-DS labeled liposomes. DAPI staining shows the general structure of lymph node sections. CD45R-FITC shows B cell localization. Cy5 channel detecting DilC18(5) fluorescence showed DMPC liposome penetration into lymph nodes. Merging of FITC and Cy5 channels showed colocalization of B cells and DMPC liposomes (see arrow in Figure 2C, CD45R-FITC / DilC18(5) panel). DMPC colocalization occurred mainly in the bright fluorescent areas, suggesting colocalization with mature B cells.

[0240] 160 μM Compound 2 / DOPC DiC 18 Lymph node uptake and B cell colocalization were evaluated 60 min after oral administration of 200 μl of (5)-DS-labeled liposomes. DAPI staining was used to show the general structure of lymph node sections. CD45R-FITC was used to show B cell localization. Dilc staining was used to show DMPC liposome penetration into lymph nodes. 18(5) The Cy5 channel was used to detect fluorescence. Merging of the FITC and Cy5 channels showed colocalization of B cells with DOPC liposomes. Unlike DMPC, DOPC seemed to penetrate deeper into the lymph nodes. Colocalization was observed in both bright and dark fluorescent areas, suggesting colocalization in both mature and immature B cells (arrow in Figure 2D, CD45R-FITC / DilC 18 (See panel (5)).

[0241] 160 μM Compound 2 / DSPC DiC 18 (5) Lymph node uptake and B cell colocalization were evaluated 60 min after oral administration of 200 μl of DS-labeled liposomes. DAPI staining shows the general structure of lymph node sections. CD45R-FITC shows B cell localization. 18 (5) The Cy5 channel, which detects fluorescence, showed DSPC liposome penetration into lymph nodes. Merging of FITC and Cy5 showed colocalization of B cells with DSPC liposomes. Similar to DOPC, DSPC appeared to penetrate deep into lymph nodes. Colocalization could be seen in both bright and dim fluorescent areas, suggesting colocalization in both mature and immature B cells (arrow in Figure 2E, CD45R-FITC / DilC 18 (See panel (5)).

[0242] 160 μM Compound 2 / POPC DiC 18 (5) Lymph node uptake and B cell colocalization were evaluated 60 min after oral administration of 200 μl of DS-labeled liposomes. DAPI staining was used to show the general structure of lymph node sections. CD45R-FITC was used to show B cell localization. 18(5) The Cy5 channel, which detects fluorescence, showed POPC liposome penetration into lymph nodes. Merging of the FITC and Cy5 channels showed colocalization of POPC liposomes with B cells. Similar to DOPC and DSPC, POPC appeared to penetrate deep into lymph nodes. Colocalization was observed in both bright and dim fluorescent areas, suggesting colocalization in both mature and immature B cells (Figure 2F, CD45R-FITC / DilC 18 (5)See arrow in panel).

[0243] B cell colocalization was analyzed. The averaged mean fluorescence intensity (FI) of B cells in the analyzed sections was similar for all groups: 17.8 (5.9) (mean (SD)) for DMPC, 16.3 (8.7) for DOPC, 18.3 (7.8) for DSPC, and 25.4 (8.8) for POPC. For all formulations, both the uncorrected and corrected mean FI areas showed high variability. However, the percentage of localization showed less variability since it was corrected for the number of B cells in each section (Table 4). Liposomes formulated with saturated lipids (DSPC or DMPC) had better B cell colocalization than liposomes formulated with unsaturated lipids (DOPC or POPC). The percentage of colocalization (mean (SD)) was 20.2% (3.9%) for DSPC and 10.3% (0.8%) for DMPC, while the percentage of colocalization was 6.8% (2.5%) for DOPC and 5.0% (1.6%) for POPC. Statistical analysis showed that DSPC liposomes were statistically better than the other three formulations (Figure 2G). [Table 4]

[0244] FITC-conjugated anti-TCRβ was observed in all samples, indicating the presence of T cells. In most sections, T cells were found to be distributed uniformly throughout. T cells were expected to be localized in the paracortex of the lymph node. However, localized structures were observed in some but not all sections (Figure 2H).

[0245] 100nM DilC 18 Lymph node uptake and T cell colocalization were evaluated 60 min after oral administration of 200 μl of PBS containing (5). DAPI staining shows the general structure of lymph node sections. TCRβ-FITC shows T cell localization. 18 (5) The Cy5 channel, which detects fluorescence, showed no signal, indicating no dye uptake into the lymph nodes (Figure 2I).

[0246] 160 μM Compound 2 / DMPC DiC 18 (5) Lymph node uptake and T cell colocalization were evaluated 60 min after oral administration of 200 μl of DS-labeled liposomes. DAPI staining was used to show the general structure of lymph node sections. TCRβ-FITC was used to show T cell localization. 18 (5) The Cy5 channel, which detects fluorescence, showed DMPC liposome penetration into the lymph node. Red fluorescence can be seen in clusters surrounding the dark spots. Merging of the FITC and Cy5 channels showed colocalization of T cells with DMPC liposomes (Figure 2J, TCRβ-FITC / DilC 18 (5)See arrow in panel).

[0247] 160 μM Compound 2 / DOPC DiC 18 (5) Lymph node uptake and T cell colocalization were evaluated 60 min after oral administration of 200 μl of DS-labeled liposomes. DAPI staining was used to show the general structure of lymph node sections. TCRβ-FITC was used to show T cell localization. 18(5) The Cy5 channel, which detects fluorescence, showed DOPC liposome penetration into the lymph node. Similar to the DMPC-treated group, red fluorescence was observed within clusters surrounding dark spots. Merging of the FITC and Cy5 channels showed colocalization of T cells with DOPC liposomes (Figure 2K, TCRβ-FITC / DilC 18 (5)See arrow in panel).

[0248] 160 μM Compound 2 / DSPC DiC 18 (5) Lymph node uptake and T cell colocalization were evaluated 60 min after oral administration of 200 μl of DS-labeled liposomes. DAPI staining was used to show the general structure of lymph node sections. TCRβ-FITC was used to show T cell localization. 18 (5) The Cy5 channel, which detects fluorescence, showed DSPC liposome penetration into the lymph node. Similar to the DOPC and DMPC treatment groups, red fluorescence was detected in circular clusters. However, the red fluorescence was more generally distributed throughout the lymph node section. Merging of the FITC and Cy5 channels showed colocalization of T cells with DSPC liposomes (Figure 2L, TCRβ-FITC / DilC 18 (5)See arrow in panel).

[0249] 160 μM Compound 2 / POPC DiC 18 (5) Lymph node uptake and T cell colocalization were evaluated 60 min after oral administration of 200 μl of DS-labeled liposomes. DAPI staining was used to show the general structure of lymph node sections. TCRβ-FITC was used to show T cell localization. 18 (5) The Cy5 channel, which detects fluorescence, showed POPC liposome penetration into the lymph node. Similar to the DSPC-treated group, red fluorescence was observed in circular clusters and more generally distributed throughout the lymph node section. Merging of the FITC and Cy5 channels showed colocalization of T cells with DSPC liposomes (Figure 2M, TCRβ-FITC / DilC 18 (5)See arrow in panel).

[0250] T cell colocalization was analyzed. The averaged mean fluorescence intensity (FI) of T cells in the analyzed sections was similar between all groups, 13.7 (7.7) for DMPC, 11.8 (3.8) for DOPC, 8.1 (3.0) for DSPC, and 11.9 (3.3) for POPC (mean (SD)). Both uncorrected and corrected mean FI areas showed a greater variability than that observed in the sections used to evaluate B cell colocalization regardless of formulation. However, the percentage of localization showed substantially smaller variability for all formulations except DMPC (Table 5). Statistical analysis of the different groups showed no differences between the groups. This is mainly driven by the variability of the DMPC group (global ANOVA reported in Figure 2N). However, pairwise comparisons showed that DSPC was statistically better than DOPC and POPC (Figure 2N). [Table 5]

[0251] Acyl chain length and saturation level affect the physical properties of liposomes. Lipids with high unsaturation or shorter chains tend to have lower melting temperatures and can produce fluid lipid membranes. In contrast, saturated lipids and lipids with longer acyl chains tend to have higher melting temperatures and can produce solid liposomes with gel-like lipid membranes.

[0252] After oral administration, liposomes are expected to fully reach the absorptive site in the intestine, be absorbed, and excrete into the afferent lymph nodes where they undergo immune sampling. The data support the idea that orally administered liposomes can reach the mesenteric lymph nodes. Furthermore, exposure to immune cells in the lymph nodes was improved by varying the acyl chain length and saturation level.

[0253] DMPC and DSPC, both of which have saturated acyl chains, both outperformed DOPC and POPC. Furthermore, DSPC, with its 18-carbon acyl chain, outperformed DMPC, which has a 14-carbon acyl chain. DSPC has the highest melting temperature (T m = 55.6°C). Liposomes synthesized with DSPC are expected to be solid at body temperature. Even with the incorporation of compound 2, DSPC liposomes are expected to have the highest melting temperature of all other liposomes tested in this study.

[0254] The negative side of having solid liposomes is the difficulty in loading the payload. 18 Despite being rehydrated in the same buffer containing (5), both DMPC and DSPC liposomes were visible as having lower fluorescence compared to DOPC and POPC, which is not believed to affect the loading of compound 2. Compound 2 has a 16-carbon tail that mimics a lysophospholipid and is designed to function as a lipid, incorporating into the liposomal membrane prior to the hydration step.

[0255] These data indicate that it is possible to target the lymphatic system with orally administered liposomes. The data further suggest that choosing the appropriate acyl chain on the lipid carrier may be useful for targeting specific structures, such as immune cells within lymph nodes.

[0256] DSPC liposomes had better B cell colocalization than DMPC, DOPC, and POPC. With respect to T cell colocalization, DSPC performed better than DOPC and POPC, but was not statistically different from DMPC.

[0257] Example 4. In vitro dose-response analysis of Compound 1 and Compound 2 in mouse T cells Compound 1 and Compound 2 were each formulated with dimyristoylphosphatidylcholine (DMPC) at a drug to lipid molar ratio of 30:70. Liposomes were synthesized in PBS according to Example 2.

[0258] Spleen cells from naive C57BL / 6 mice were stained with CFSE, prepared, cultured, and administered. 2 × 10 cells were 5 Cells were seeded at 1000 cells / well. Each compound was dosed at 48 μM to 1.5 μM using a log2 dilution scheme. FoxP3 + / TIM3 + Cells were incubated for 72 hours before phenotypic analysis of CD4 T cells was performed. Flow cytometric analysis was performed to determine FoxP3 expression as a function of compound and dose. + / TIM3 + Changes in the proportion of CD4 T cells were assessed. Concentration responses were fitted to a 4- or 5-parameter log-logistic model (using the 'drc' package in 'R'). Model fitting revealed that EC 50 and E.C. 90 The value was obtained.

[0259] At the end of the incubation period, the cells were observed under a microscope. Cells treated with 48 μM Compound 1 did not appear healthy and were excluded from further analysis. All other cells appeared healthy and suitable for flow cytometry analysis.

[0260] FoxP3 + / TIM3 + For double positive CD4 T cells, the mean values ​​increased from 3.65% (SD=0.2) and 3.21% (SD=1.47) for low doses of Compound 1 and Compound 2, respectively, to 8.26% (SD=1.2) and 7.36% (SD=1.9) for high doses of Compound 1 and Compound 2, respectively. Visual inspection of the model fit suggests that a four-parameter dose-response model is adequate to capture the data from Compound 2. However, due to the lack of a plateau for Compound 1, the model did not show a significant improvement in E max and E.C. 50could not provide a reliable estimate of E max was fixed at 7.9%, the average response for Compound 1 and Compound 2 at 10 μM, and 10 and 30 μM, respectively. Figure 3A shows the results of fitting the data to a four-parameter dose-response model. Table 6 shows the EC values ​​obtained from the model fitting. 50 and E.C. 90 This is a summary of the values ​​of . [Table 6]

[0261] Both Compound 1 and Compound 2 bind to FoxP3 + / TIM3 + Treatment with 10 μM compound 1 resulted in a dose-dependent increase in CD4 T cells. Treatment with 10 μM compound 1 resulted in a greater increase in FoxP3 + / TIM3 + Compound 2 at 30 μM increased FoxP3 T cells by 126% compared to lower doses of Compound 2. + / TIM3 + CD4 T cells increased by 129%.

[0262] Compound 2 is expected to be more stable than compound 1 because the ester bond present in compound 1 is replaced in compound 2. Furthermore, compound 2 has been shown to have lower in vitro toxicity compared to compound 1 at a dose of 30 μM. Although both compound 1 and compound 2 are attractive candidates for further development, compound 2 may have some advantages from the standpoint of stability and formulation.

[0263] Example 5. FoxP3 uptake by Compound 2 in mice + CD4 + Increase in T cells Without wishing to be bound by theory, it is believed that binding of compound 2 to the TIM family of receptors induces a tolerogenic immune response, e.g., tolerogenic FoxP3 + / CD4 +It is hypothesized to increase T cells (T-reg). In this example, the pharmacodynamic (PD) effect of a single oral dose of compound 2 in mice was evaluated after 5 days of administration.

[0264] All animal studies were conducted in accordance with Tufts University / Tufts Medical Center & Human Nutrition Research Center on Aging under IACUC number B2020-91. Animals (The Jackson Laboratory, Bar Harbor, ME, B57BL6) were housed 4 per cage and provided with food and water ad libitum.

[0265] Compound 2 was dissolved in DMSO and formulated with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in a molar ratio of Compound 2:DMPC of 10:90 as described in Example 2. Animals were dosed according to Table 7. Dose was selected based on preliminary data suggesting 42 μM as the maximally effective dose in vitro. [Table 7]

[0266] To evaluate the PD effect of a single dose of Compound 2 in mice, animals were administered a single dose by oral gavage according to group assignment (Table 7). Five days after oral dosing, animals were sacrificed and spleens were removed for T cell phenotyping. The study included ex vivo analysis followed by dose-response analysis (Figure 4A). Spleens harvested for splenocyte analysis were prepared into single cell suspensions for cell phenotyping by flow cytometry (eBioscience, Inc., San Diego, CA). Cells were stained and gated for CD4. FoxP3. + / CD4 + The proportion of T cells was assessed using flow cytometry. Dose-response curves were fitted to a 4- or 5-parameter log-logistic model (using the "drc" package in "R"). Model fitting revealed that EC50 and E.C. 90 The value was obtained.

[0267] FoxP3 + / CD4 + The percentage of T cells increased in a dose-dependent manner, from a mean of 3.87% (SD=0.37) in the low dose group to 11.53% (SD=1.47) in the high dose group. The data were fitted to a four-parameter dose-response model. Table 8 shows the model-estimated pharmacodynamic parameters and associated standard errors. Based on this analysis, the ED 50 is estimated at 43.1 μM, ED 90 is estimated at 218 μM (Figure 4B). [Table 8]

[0268] The data from this study support the hypothesis that oral administration of compound 2 can induce a tolerogenic immune response in a dose-dependent manner. FoxP3 was upregulated in the high-dose group compared to the low-dose group. + / CD4 + A 219% increase in T cells (T-reg) was observed. Over 100% increase (e.g., FoxP3 + / CD4 + T cell doubling) usually results in tolerance to the target antigen. 50 Repeated administration at 100 mg / kg / day appears to be sufficient to induce tolerance.

[0269] Example 6. MOG 35-55 Preventive and therapeutic effects of Compound 2 in an induced mouse EAE model Multiple sclerosis is a chronic, often disabling disease of the human central nervous system (CNS). When tolerance to myelin is lost, the immune system attacks the myelin, resulting in the clinical symptoms of the disease. This is mediated by pathogenic autoreactive T cells that recognize autoantigenic peptides complexed with major histocompatibility complex (MHC) molecules. There is no known cure for MS, but blocking the ability of autoreactive T cells to invade the CNS has proven to be an effective treatment option to ameliorate MS symptoms. Anti-α4 mAbs such as Tysabri have been approved for the management of MS. Other treatment options rely on systemic immunosuppressants such as steroids.

[0270] Addressing the presence of autoreactive T cells and inducing their transition to more tolerogenic T cells may provide a curative therapy for MS. Compound 2 has been proposed as a T cell immunoglobulin mucin protein family receptor (TIM) agonist that can induce tolerogenic T cells. TIMs play an important role in adaptive and innate immune responses and are associated with the control of autoimmunity and cancer. Several ligands, including the phospholipid phosphatidylserine (PS), are known to bind to TIMs.

[0271] The affinity of PS for different members of the TIM family varies substantially, with TIM3 having a lower affinity for PS than TIM4, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 109, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,

[0272] The tolerogenic potential of PS has been exploited by tumors. For example, PS in the human ovarian tumor microenvironment can induce T cell signaling arrest. Moreover, PS-mediated T cell arrest was blocked by anti-PS antibodies. Taken together, published data on TIM and its role in immune tolerance suggest that TIM is a potential target for the treatment of autoimmune disorders.

[0273] This study evaluates the effect of Compound 2 (a TIM agonist) on disease onset, disease progression, and overall survival in mice with experimental autoimmune (allergic) encephalomyelitis (EAE), which is considered the best model of multiple sclerosis (MS).

[0274] All animal studies will be conducted in accordance with Tufts University / Tufts Medical Center & Human Nutrition Research Center on Aging under IACUC number B2020-91. Animals will be housed in designated facilities at Tufts University-Tufts Medical Center & Human Nutrition Research Center on Aging. Animals will be housed four per cage with free access to food and water.

[0275] Materials and reagents are shown in Table 9. [Table 9]

[0276] Compound 2 was formulated with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in a molar ratio of Compound 2:DSPC of 3.75:96.25. Two test articles were formulated and tested: Compound 2 / MOG 35-55 It is called MOG 35-55 The first formulation was made using Compound 2:DSPC containing MOG 33-55 Another formulation was made without DSPC and is designated Compound 2. DSPC was selected based on superior mesenteric lymph node targeting and excellent immune cell colocalization after oral administration. The ratio of Compound 2 to DSPC was selected based on in vitro dose response studies. Liposomes were prepared in PBS as described in Example 2 and extruded through a 100 micron filter. Animals were dosed with 100 μl of 42 μM Compound 2 or 100 μl of 42 μM / 1 μg Compound 2 / MOG per mouse. 35-55The dose was selected based on an in vitro dose-response analysis.

[0277] Experimental autoimmune (allergic) encephalomyelitis (EAE) is considered the best preclinical model of multiple sclerosis (MS). EAE is characterized by an immune response against CNS tissue and can be induced by immunizing animals against CNS proteins.

[0278] In the active EAE model, MOG emulsified in complete Freund's adjuvant (CFA) was administered 35-55 Mice are immunized with the peptides by subcutaneous injection (0.1 mL of emulsion / mouse) at the base of the tail under anesthesia. On the day of injection (day 0) and 2 days later, mice are injected intraperitoneally with pertussis toxin (PT) in PBS at 600 ng / mouse / dose (0.1 mL).

[0279] Symptoms typically develop in mice 9-14 days after immunization (day 0). Daily observation and scoring of mice begins on day 9 and continues until the end of the study. Table 10 details expected clinical signs and the scoring criteria used in this study. [Table 10-1] [Table 10-2]

[0280] This study was designed to evaluate the preventive and therapeutic effects of compound 2 in an EAE mouse model, as well as to evaluate the effect of co-formulation of compound 2 with an antigen. In the setting of autoimmune disease, it is hypothesized that antigen co-formulation / co-presentation is not necessary, since the antigen is endogenously expressed and autoreactive T cells are abundant due to the disease. Figure 5A shows an overview of the study.

[0281] Mice were treated with MOG 35-55Treatment in the prophylactic group began 4 days after immunization with either 100 μl of 42 μM compound 2 or 1 μg of MOG. 35-55 Compound 2 at 42 μM coformulated with 35-55 ) was orally administered once a day for the duration of the study.

[0282] To evaluate the therapeutic effect of Compound 2, treatment was initiated after the first clinical symptoms were observed. Mice were assigned to treatment or control groups if they had a clinical score of 1 according to Table 11. Treatment groups were orally administered 100 μL of 42 μM Compound 2 once daily for the remainder of the study. Mice in all groups were monitored and scored daily for clinical symptoms. Mice that reached a score of 5 according to Table 11 or evident death in their cage were marked as events in the survival analysis.

[0283] At the end of the study, spleens were removed from a representative sample of mice from each group for splenocyte analysis. Single cell suspensions were prepared for cell phenotyping by flow cytometry. Cells were stained for CD4 + Gates were set for T cells. FoxP3 + / CD4 + The percentage of T cells was assessed by flow cytometry.

[0284] Compound 2 and Compound 2 / MOG in EAE models 35-55 The onset of disease was evaluated to evaluate the preventive effect of Compound 2 and Compound 2 / MOG. The onset of disease was significantly higher in the control group than in the control group. 35-55 Disease onset in the control group started on day 9 post-immunization, whereas it started on day 12 in both treatment groups. All animals in the treatment groups showed symptoms by day 13, whereas it was day 11 in the control group. Survival analysis showed that this was a statistically significant delay. Compound 2 and Compound 2 / MOG 35-55 There was no difference between the two (Figure 5B).

[0285] After disease onset, clinical scores progressed as expected in all groups. However, Compound 2 and Compound 2 / MOG 35-55 Animals treated with Compound 2 / MOG showed less severe symptoms compared to the control group. Peak symptoms in the control group were observed on day 17 with a mean score of 4.77 (SD=0.67) compared to Compound 2 and Compound 2 / MOG. 35-55 In the treated group, the mean mean values ​​were 2.6 (SD = 0.23) and 2.9 (SD = 0.41), respectively. No recovery period was observed in the control group, as most of the animals in the control group died of the disease (Figure 5C). However, Compound 2 and Compound 2 / MOG 35-55 In both treatment groups, symptoms began to subside, and by the end of the study, the mean scores were 35-55 and 2.1 (SD = 0.23) and 2.4 (SD = 0.23), respectively (Figure 5C).

[0286] After disease onset, animals treated with compound 2 showed a delay in disease progression by one day and milder symptoms thereafter throughout the remainder of the study compared to untreated animals (Figure 5D). Peak symptoms in the control group were observed on day 7 after disease onset with a mean score of 4.77 (SD=0.67) compared to 2.8 (SD=0.43) in the treated group on day 6 after symptom onset. Symptoms in the treated group declined after reaching a peak of a mean of 2.33 (SD=0.29) by day 10 after symptom onset. No remission was observed in the control group due to high mortality in the untreated group.

[0287] Any mouse score of 5 or spontaneous death was defined as an event for survival analysis. Mortality in the untreated group was high in this study, with the first mouse dying 3 days after disease onset. Overall, 8 of 9 animals died spontaneously or had to be euthanized according to Table 11, compared with 0 of 9 in the compound 2 treatment group (Figure 5E). This was an unusually aggressive model, as the usual mortality rate in EAE mouse models is reported to be less than 30%.

[0288] Flow cytometric analysis of splenocytes isolated from control mice that survived to the end and from randomly selected mice from each treatment group revealed increased expression of Foxp3 in all treated mice compared to controls. + / CD4 + In the prevention group, Foxp3 + / CD4 + The percentage of T cells was determined by Compound 2 and Compound 2 / MOG 35-55 The Foxp3 expression levels in mice treated with Compound 2 after symptom onset were 10.4% and 7.02%, respectively, compared to 3.20% in control mice, representing an increase of 225% and 119%, respectively, compared to controls. + / CD4 + The percentage of T cells was 26.1%, which was a 715% increase over surviving mice in the untreated group.

[0289] In this study, an aggressive form of the EAE model was induced in mice. This is evident from the high clinical scores and mortality in the untreated group, with 8 out of 9 animals reaching a score of 5. In contrast, the average clinical scores in control / untreated animals from EAE literature samples range from 3 to 4. Despite the aggressive form of EAE, oral Compound 2 and Compound 2 / MOG 35-55 Prophylactic treatment with Compound 2 delayed disease onset by at least 3 days, which is comparable to the observations made using anti-α4 antibody treatment in the EAE model reported by Kent et al. 35-55 There was no difference between the two, suggesting that antigen co-administration / co-presentation is not required in the autoimmune model.

[0290] Animals treated with Compound 2 after the onset of symptoms had better survival rates and lower clinical scores than the untreated group. By the end of the study, animals treated with Compound 2 had a mean clinical score that was 54% lower than the mean clinical score in the untreated group. In comparison, Kent et al. reported a 66% reduction in mean clinical score at the peak of response (day 15 of the study) in anti-α4 treated mice compared to the mean clinical score in untreated mice.

[0291] Ex vivo analysis of splenocytes isolated from representative animals from each group supported the proposed mechanism of action of compound 2, with an increase in tolerogenic T cells observed in all treatment groups compared to untreated animals.

[0292] Example 7. Liposome formulation of Compound 2 with DMPC and GL67 To test the dissolution of compound 2 in DMPC and GL67 (N4-cholesteryl-spermine HCl salt), compound 2 (3 mg) was incubated in DMPC (42.47 mg) and GL67 (2.669 mg) in 3 ml of chloroform solution at room temperature (RT) in a molar ratio of compound 2:DMPC:GL67 of 10:85:5. Specifically, 3.0 mg of compound 2 was weighed and mixed with 3 ml of chloroform in a volumetric flask. The flask was sonicated for 5 minutes, then briefly vortexed, and incubated overnight at room temperature. After overnight incubation, the flask was sonicated for 5 minutes, then briefly vortexed. 42.47 mg of DMPC was added to the flask. A clear solution with no cloudiness was observed. 2.669 mg of GL67 was added to the flask, and the flask was sonicated for 1-3 minutes. A clear solution was observed after sonication.

[0293] The clear solution was transferred to a 5 ml round bottom flask, which was then loaded onto a rotary evaporator. The rotary evaporator was initially run at room temperature with a vacuum pressure of 800 mbar and a speed of 5, then the temperature of the water bath was increased to 37° C. The vacuum pressure was then reduced to 600 mbar for 15 minutes, and then further reduced to 400 mbar for 30 minutes. After approximately 1 hour on the rotary evaporator, the organic solvent was completely evaporated and a thin film was formed on the round bottom flask.

[0294] Next, a hydration buffer was prepared for rehydration of the thin film. Specifically, 880 mg of hydroxypropyl beta-cyclodextrin (HPBCD) was weighed and dissolved in saline (approximately 11 ml) in a 50 ml conical tube. Then, AAV9-CMV Chry (3×10 6 , Vigene Biosciences, Charles River company) was added to the hydration buffer.

[0295] The membrane was then rehydrated using hydration buffer. This was done by adding the buffer to a round-bottom flask and attaching the round-bottom flask to a rotary evaporator under vacuum. Gentle rotation of the rotary evaporator caused the thin film to rehydrate and form liposomes. As the thin film rehydrated into liposomes, the AAV9 particles were trapped in the aqueous center of the newly formed liposomes.

[0296] The resulting liposomes were rehydrated and then imaged using a transmission electron microscope (TEM). Figure 6 shows AAV9-CMV encapsulated in DMPC:GL67:Compound 2 (85:5:10) liposomes. Chry Representative TEM images of AAV9-CMV encapsulated in 100-well plates are shown. Chry Shows.

[0297] Example 8. FoxP3 upregulation by Compound 2 in mice + CD4 + Increase in T cells The PD efficacy of a single oral dose of DMPC:GL67:Compound 2 (85:5:10) liposomes encapsulating AAV9-GFP was evaluated in mice after 5 days of administration. Chry Liposomes were synthesized according to the procedure described in Example 7, except that was replaced with AAV9-GFP (Vigene Biosciences, a Charles River company). Experiments were performed as described in Example 5, using the treatment groups described in Table 11. [Table 11]

[0298] In the naïve group, FoxP3 + / CD4 + The T cell percentages were 4.65, 3.91, and 5.01. 6 In the group treated with the DMPC:GL67:Compound 2 (85:5:10) liposomal formulation encapsulating AAV9-GFP, FoxP3 + / CD4 + The percentages of T cells were 7.94, 9.37, and 8.15. Thus, 10 6 Oral administration of a single dose of 100 μg / mouse of AAV9-GFP resulted in T reg Expression was enhanced. References 1. Wolf, Y., Anderson, AC & Kuchroo, VK TIM3 comes of age as an inhibitory receptor. Nat Rev Immunol 20,173-185(2020). https: / / doi.org / 10.1038 / s41577-019-0224-6 2. Freeman GJ,Casasnovas JM,Umetsu DT,DeKruyff RH. TIM genes:a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity. Immunol Rev.2010;235(1):172-189. doi:10.1111 / j.0105-2896.2010.00903.x. 3. Albacker LA,Karisola P,Chang YJ,et al. TIM-4,a receptor for phosphatidylserine,controls adaptive immunity by regulating the removal of antigen-specific T cells. J Immunol.2010;185(11):6839-6849. doi:10.4049 / jimmunol.1001360 4. Santiago C,Ballesteros A,Martinez-Munoz L,et al. Structures of T cell immunoglobulin mucin protein 4 show a metal-Ion-dependent ligand binding site where phosphatidylserine binds. Immunity.2007;27(6):941-951. doi:10.1016 / j.immuni.2007.11.008 5. Sabatos-Peyton,C.A.et al. Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy. Oncoimmunology 7,e1385690(2018). 6. Kelleher RJ Jr,Balu-Iyer S,Loyall J,et al. Extracellular Vesicles Present in Human Ovarian Tumor Microenvironments Induce a Phosphatidylserine-Dependent Arrest in the T-cell Signaling Cascade. Cancer Immunol Res.2015;3(11):1269-1278. doi:10.1158 / 2326-6066.CIR-15-0086. 7. Ramadan A,Lucca LE,Carrie N,Desbois S,Axisa PP,Hayder M,Bauer J,Liblau RS,Mars LT. In situ expansion of T cells that recognize distinct self-antigens sustains autoimmunity in the CNS. Brain.2016 May;139(Pt 5):1433-46. doi:10.1093 / brain / aww032. Epub 2016 Mar 21. PMID:27000832. 8. Kent,S.J.,Karlik,S.J.,Cannon,C.,Hines,D.K.,Yednock,T.A.,Fritz,L.C.,& Horner,H.C.(1995). A monoclonal antibody to α4 integrin suppresses and reverses active experimental allergic encephalomyelitis. Journal of neuroimmunology,58(1),1-10. 9. Serre L, Girard M, Ramadan A, Menut P, ​​Rouquie N, Lucca LE, Mahiddine K, Leobon B, Mars LT, Guerder S. Thymic-Specific Serine Protease Limits Central Tolerance and Exacerbates Experimental Autoimmune Encephalomyelitis. J Immunol.2017 Dec 1;199(11):3748-3756. doi:10.4049 / jimmunol.1700667. Epub 2017 Oct 23. PMID:29061767.

[0299] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0300] Although exemplary embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

1. A compound of the following structural formula: 【Chemistry 8】 or a pharmaceutically acceptable salt thereof, wherein: X is -N(R １ ) C(O)- ＊ or -N(R １ )C(O)O- ＊ wherein: ＊ indicates the point of attachment of X to R; R is optionally substituted with one or more fluoro (C ５ -C ３０ ) alkyl or (C ５ -C ３０ ) alkenyl, R １ is H or (C １ -C ５ ) alkyl, The compound or a pharmaceutically acceptable salt thereof, provided that the compound is not (S)-2-amino-3-((2E,4E)-hexa-2,4-diamido)propanoic acid, (S)-2-amino-3-hexanamidopropanoic acid, (S)-2-amino-3-heptanamidopropanoic acid, (S)-2-amino-3-octanamidopropanoic acid, or (S)-2-amino-3-palmitamidopropanoic acid, or a salt of any of the foregoing.

2. wherein R is optionally substituted with one or more fluoro (C ５ -C ３０ ) alkyl, or 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R is (C 15 -C 20 ) alkyl or (C 15 -C 20 ) alkenyl optionally substituted with one or more fluoro.

3. In the formula, R １ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein:

4. The compound of claim 1 having the following structural formula: 【Chemistry 9】 or a pharmaceutically acceptable salt thereof, or 【Chemistry 10】 or a pharmaceutically acceptable salt thereof.

5. one or more lipids, or pharmaceutically acceptable salts thereof, and (i) a compound of claim 1, or a pharmaceutically acceptable salt thereof, or (ii) a compound of the following structural formula: 【Chemistry 11】 or a pharmaceutically acceptable salt thereof, wherein: X is -N(R １ )C(O)-,-N(R １ )C(O)O-,-N(R １ )C(O)N(R ２ ) -, -N(R １ ) -, -N(R １ ) SO ２ -, -O-, -S-, -S(O)-, or -S(O) ２ - and R is optionally substituted with one or more fluoro (C １ -C ３０ ) alkyl or (C １ -C ３０ ) alkenyl, R １ is H or (C １ -C ５ ) alkyl, R ２ is H or (C １ -C ５ ) alkyl.

6. the one or more lipids comprise a phospholipid, or a pharmaceutically acceptable salt thereof; The lipid particle of claim 5 , wherein the phospholipid is a saturated phospholipid, or a pharmaceutically acceptable salt thereof.

7. 7. The lipid particle of claim 6, wherein the phospholipid is dimyristoylphosphatidylcholine (DMPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine 18:0 (DSPC).

8. The lipid particle of claim 5 , further comprising an antigen.

9. A composition comprising a plurality of lipid particles according to claim 8.

10. A composition comprising a plurality of lipid particles according to claim 5.

11. A combination comprising a plurality of lipid particles described in claim 5 and an antigen.

12. 1. A combination or composition for immune tolerization of a subject in need thereof to an antigen, comprising: (i) the combination comprises the antigen, or an immunogenic fragment thereof, and the composition of claim 10; or (ii) the composition is the composition of claim 9; A combination or composition wherein the antigen in the composition is the antigen to which the subject is to be tolerized or an immunogenic fragment of the antigen to which the subject is to be tolerized.

13. 12. A composition according to claim 9 or 10 or a combination according to claim 11 for inducing a regulatory T cell population, increasing the activity or level of tolerogenic T cells or inducing a regulatory B cell population in a subject.

14. 1. A combination or composition for inhibiting or reducing antigen-specific antibody titers in a subject, comprising: (i) the combination comprises the antigen or immunogenic fragment thereof and the composition of claim 10; or (ii) the composition is the composition of claim 9; A combination or composition wherein the antigen in the composition is the antigen against which the antibody titer is to be inhibited or reduced, or an immunogenic fragment of the antigen against which the antibody titer is to be inhibited or reduced.

15. The lipid particle of claim 8, the composition of claim 9, or the combination of claim 11, wherein the antigen is an antigen therapy.

16. 16. The lipid particle, composition, or combination of claim 15, wherein the antigen is a therapeutic protein, enzyme replacement therapy, or gene therapy.

17. A combination or composition for the treatment of a disease, disorder, or condition by antigen therapy in a subject in need thereof, comprising: (i) the combination comprises the antigen therapy and a composition of claim 10 in an amount sufficient to immunotolerize the subject to the antigen therapy; or (ii) the composition is the composition of claim 9; The combination or composition, wherein said antigen in said composition is said antigen therapy.

18. 18. The combination or composition of claim 17, wherein the antigen therapy is enzyme replacement therapy or gene therapy.

19. A composition or combination for treating an autoimmune disorder in a subject in need thereof, comprising: (i) the composition is a composition according to claim 10, or (ii) the composition or combination is a composition according to claim 9 or a combination according to claim 11; A composition or combination wherein the antigen in the composition or combination is an autoantigen associated with the autoimmune disorder, or an immunogenic fragment thereof.

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