WO2024107889A1 - Compositions and methods for treating primary biliary cholangitis - Google Patents

Abstract

Provided herein are methods and compositions related to compositions comprising synthetic nanocarriers for treating primary biliary cholangitis (PBC).

Description

COMPOSITIONS AND METHODS FOR TREATING PRIMARY BILIARY CHOLANGITIS

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63/425,660, filed November 15, 2022, the contents of which are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

Provided herein are methods and compositions related to synthetic nanocarriers comprising an immunosuppressant for treating primary biliary cholangitis in a subject.

SUMMARY OF THE INVENTION

In one aspect, provided herein are methods for treating primary biliary cholangitis (PBC) in a subject comprising administering a composition comprising synthetic nanocarriers comprising an immunosuppressant, to the subject wherein the subject has primary biliary cholangitis. In one embodiment, the synthetic nanocarriers may further comprise a PBC- associated antigen, such as a PBC-associated autoantigen, such as PDC-E2, the method further comprises administering a composition comprising synthetic nanocarriers comprising a PBC-associated antigen, such as a PBC-associated autoantigen, such as PDC-E2.

In one embodiment of any one of the methods or compositions provided herein, the PBC-associated antigen is a whole protein or protein fragment. Preferably, in some embodiments, the protein fragment comprises one or more T cell epitopes of the PBC- associated antigen.

In one embodiment of any one of the methods or compositions provided herein, the synthetic nanocarriers comprising the immunosuppressant and/or the synthetic nanocarriers comprising the PBC-associated antigen are the same population of synthetic nanocarriers.

In one embodiment of any one of the methods or compositions provided herein, the synthetic nanocarriers comprising the immunosuppressant and/or the synthetic nanocarriers comprising the PBC-associated antigen are different populations of synthetic nanocarriers.

In one embodiment of any one of the methods or compositions provided herein, a number of T cell epitopes are administered to the subject, and each of the T cell epitopes can be comprised in separate populations of synthetic nanocarriers, respectively, or can be comprised in the same population of synthetic nanocarriers. In some embodiments, these T cell epitopes can be provided as a protein or protein fragment.

In one embodiment of any one of the methods or compositions provided herein, the T cell epitopes can be provided as a concatenated polypeptide and comprised in a population of synthetic nanocarriers that can be the same or different from the population of synthetic nanocarriers that comprise the immunosuppressant, wherein the concatenated polypeptide comprises a protease cleavage site, such as one cleavable by an endogenous protease, such as cathepsin, between at least two of the T cell epitopes. In some embodiments, there is such a protease cleavage site between each set of two T cell epitopes within the concatenated polypeptide.

In one embodiment of any one of the methods or compositions provided herein, when the synthetic nanocarriers comprising the immunosuppressant and the synthetic nanocarriers comprising the PBC-associated antigen are different populations of synthetic nanocarriers, the different populations of synthetic nanocarriers are admixed prior to administration to the subject. Thus, in one embodiment of any one of the methods provided herein, the method further comprises a step of admixing the different populations of synthetic nanocarriers prior to administration to the subject.

In some embodiments of any one of the methods provided, the method comprises reducing an immune response. In some embodiments of any one of the methods provided, the administration of the synthetic nanocarriers increases a tolerogenic phenotype. In some embodiments of any one of the methods provided, the method further comprises identifying and/or providing the subject having PBC.

In one embodiment of any one of the methods provided, the synthetic nanocarriers are administered concomitantly. In one embodiment of any one of the methods provided, the synthetic nanocarriers are administered simultaneously.

In one embodiment of any one of the methods provided, the method further comprises providing the subject having PBC.

In one embodiment of any one of the methods provided herein, the method further comprises identifying the subject as being in need of a method provided herein.

In one embodiment of any one of the methods or compositions provided herein, the synthetic nanocarriers are in effective amounts for modulating any one of the immune responses provided herein and/or for treating PBC. In one embodiment of any one of the methods or compositions provided, the immunosuppressant is an mTOR inhibitor. In one embodiment of any one of the methods or compositions provided, the mTOR inhibitor is rapamycin or a rapalog.

In one embodiment of any one of the methods or compositions provided, the immunosuppressant is encapsulated in the synthetic nanocarriers.

In one embodiment of any one of the methods or compositions provided, the PBC- associated antigen is encapsulated in the synthetic nanocarriers.

In one embodiment of any one of the methods or compositions provided, the synthetic nanocarriers comprise lipid nanoparticles, polymeric nanoparticles, metallic nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles or peptide or protein particles. In one embodiment of any one of the methods or compositions provided, the polymeric nanoparticles comprise a polyester, polyester attached to a polyether, polyamino acid, polycarbonate, polyacetal, polyketal, polysaccharide, polyethyloxazoline or polyethyleneimine. In one embodiment of any one of the methods or compositions provided, the polymeric nanoparticles comprise a polyester or a polyester attached to a polyether. In one embodiment of any one of the methods or compositions provided, the polyester comprises a poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid) or polycaprolactone. In one embodiment of any one of the methods or compositions provided, the polymeric nanoparticles comprise a polyester and a polyester attached to a polyether. In one embodiment of any one of the methods or compositions provided, the polyether comprises polyethylene glycol or polypropylene glycol.

In one embodiment of any one of the methods or compositions provided, the mean of a particle size distribution obtained using dynamic light scattering of a population of the synthetic nanocarriers is a diameter greater than 1 lOnm, greater than 150nm, greater than 200nm, or greater than 250nm. In one embodiment of any one of the methods or compositions provided, the mean of a particle size distribution obtained using dynamic light scattering of a population of the synthetic nanocarriers is less than 5pm, less than 4pm, less than 3pm, less than 2pm, less than 1pm, less than 750nm, less than 500nm, less than 450nm, less than 400nm, less than 350nm, or less than 300nm.

In one embodiment of any one of the methods or compositions provided, the load of immunosuppressant comprised in the synthetic nanocarriers, on average across the synthetic nanocarriers, is between 0.1% and 50% (weight/weight), between 4% and 40%, between 5% and 30%, or between 8% and 25%. In one embodiment of any one of the methods or compositions provided, an aspect ratio of a population of the synthetic nanocarriers is greater than or equal to 1: 1, 1: 1.2, 1: 1.5, 1:2, 1:3, 1:5, 1:7 or 1: 10.

In another aspect, a composition comprising any one or more of the populations of synthetic nanocarriers provided herein is provided.

In another aspect, a composition as described in any one of the methods provided or any one of the Examples is provided. In one embodiment, the composition is any one of the compositions of synthetic nanocarriers for administration or combinations of compositions of synthetic nanocarriers for administration according to any one of the methods provided.

In another aspect, any one of the compositions is for use in any one of the methods provided.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows the induction of the tolerogenic liver sinusoidal endothelial cells (PD- Ll⁺, CD80^low, CD86^low) following ImmTOR treatment.

Fig. 2 shows the induction of the hepatic regulatory T cells (CD4⁺, CD25¹¹¹, PD-1⁺) following ImmTOR treatment.

Fig. 3 shows the alanine transaminase (ALT) reduction in an inflamed liver following ImmTOR treatment.

Fig. 4 shows disease scores from paraffin-embedded liver sections of NOD.c3c4 mice stained with hematoxylin and eosin. Bile duct degeneration (left graft), biliary hypertrophy (center graft), and liver inflammation (right graph) were graded on a 5-point severity scale.

Fig. 5 shows representative histology sections from female (left column) and male (right column) mice are shown at 5x magnification. The mice were untreated (top row) or treated with ImmTOR (bottom row).

Figs. 6A-6C characterize the efficacy of ImmTOR in a concanavalin A-induced model of autoimmune hepatitis. Fig. 6A shows the percentage of activated T cells (top left graph), the total number of activated T cells (top right graph), the activated cytotoxic T cell (CTL) fraction (bottom left graph) and the total number of activated CTLs (bottom right graph). Fig. 6B shows serum levels of interferon-gamma (left graph), IL-6 (center graph), and CXCL1 (right graph). Fig. 6C shows FGF21 serum levels. Error bars indicate mean +/- SD. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified materials or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting of the use of alternative terminology to describe the present invention.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety for all purposes.

As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a polymer" includes a mixture of two or more such molecules or a mixture of differing molecular weights of a single polymer species, reference to "a synthetic nanocarrier" includes a mixture of two or more such synthetic nanocarriers or a plurality of such synthetic nanocarriers, and the like.

As used herein, the term “comprise” or variations thereof such as “comprises” or “comprising” are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, elements, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein, the term “comprising” is inclusive and does not exclude additional, unrecited integers or method/process steps.

In embodiments of any one of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of’ or “consisting of’. The phrase “consisting essentially of’ is used herein to require the specified integer(s) or steps as well as those which do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, elements, characteristics, properties, method/process steps or limitations) alone.

A. INTRODUCTION

Synthetic nanocarriers comprising an immunosuppressant administered, optionally, in combination with synthetic nanocarriers comprising a PBC-associated antigen can have beneficial effects for subjects with PBC. Without being bound by theory, it is believed that these effects can be achieved, at least in part, due to a decreased immune reaction to antigens or immune systems associated with PBC. For example, tolerogenic immune effects can be induced in the subject having PBC with the administration of the synthetic nanocarriers.

Thus, provided herein are methods, and related compositions, for treating a subject with PBC, for example, by administering synthetic nanocarriers comprising an immunosuppressant and, optionally, synthetic nanocarriers comprising a PBC-associated antigen.

The invention will now be described in more detail below.

B. DEFINITIONS

"Administering" or "administration" or “administer” means giving a material to a subject in a manner such that there is a pharmacological result in the subject. This may be direct or indirect administration, such as by inducing or directing another subject, including another clinician or the subject itself, to perform the administration.

“Amount effective” in the context of a composition or dose for administration to a subject refers to an amount of the composition or dose that produces one or more desired responses in the subject, e.g., treating PBC as is described herein. Therefore, in some embodiments, an amount effective is any amount of a composition or dose provided herein that produces one or more of the desired therapeutic effects as provided herein. This amount can be for in vitro or in vivo purposes. For in vivo purposes, the amount can be one that a clinician would believe may have a clinical benefit for a subject in need thereof. Any one of the compositions or doses, including label doses, as provided herein can be in an amount effective.

Amounts effective can involve reducing the level of an undesired response, although in some embodiments, it involves preventing an undesired response altogether. Amounts effective can also involve delaying the occurrence of an undesired response. An amount that is effective can also be an amount that produces a desired therapeutic endpoint or a desired therapeutic result. In other embodiments, the amounts effective can involve enhancing the level of a desired response, such as a therapeutic endpoint or result. Amounts effective, preferably, result in a therapeutic result or endpoint with respect to a condition related to PBC in any one of the subjects provided herein. The achievement of any of the foregoing can be monitored by routine methods.

Amounts effective will depend, of course, on the particular subject being treated; the severity of a condition, disease or disorder; the individual patient parameters including age, physical condition, size and weight; the duration of the treatment; the nature of concurrent therapy (if any); the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reason.

“Assessing a therapeutic response” refers to any measurement or determination of the level, presence or absence, reduction in, increase in, etc. of a therapeutic response in vitro or in vivo. Such measurements or determinations may be performed on one or more samples obtained from a subject. Such assessing can be performed with any of the methods provided herein or otherwise known in the art. The assessing may be assessing any one or more of the biomarkers associated with immune responses or PBC or otherwise known in the art.

“Attach” or “Attached” or “Couple” or “Coupled” (and the like) means to chemically associate one entity (for example a moiety) with another. In some embodiments, the attaching is covalent, meaning that the attachment occurs in the context of the presence of a covalent bond between the two entities. In non-covalent embodiments, the non-covalent attaching is mediated by non-covalent interactions including but not limited to charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and/or combinations thereof. In embodiments, encapsulation is a form of attaching.

“Average” refers to the mean unless indicated otherwise.

“Concomitantly” means administering two or more materials/agents to a subject in a manner that is correlated in time, preferably sufficiently correlated in time such that a first composition (e.g., synthetic nanocarriers comprising an immunosuppressant) has an effect on a second composition, such as increasing the efficacy of the second composition, preferably the two or more materials/agents are administered in combination. In embodiments, concomitant administration may encompass administration of two or more compositions within a specified period of time. In some embodiments, the two or more compositions are administered within 1 month, within 1 week, within 1 day, or within 1 hour. In some embodiments, concomitant administration encompasses simultaneous administration of two or more compositions.

"Dosage form" means a pharmacologically and/or immunologically active material in a medium, carrier, vehicle, or device suitable for administration to a subject. Any one of the compositions or doses provided herein may be in a dosage form.

“Dose” refers to a specific quantity of a pharmacologically and/or immunologically active material for administration to a subject for a given time. Unless otherwise specified, the doses recited for compositions comprising synthetic nanocarriers comprising an immunosuppressant or antigen refer to the weight of the immunosuppressant or antigen (z.e., without the weight of the synthetic nanocarrier material). When referring to a dose for administration, in an embodiment of any one of the methods, compositions or kits provided herein, any one of the doses provided herein is the dose as it appears on a label/label dose.

“Encapsulate” means to enclose at least a portion of a substance within a synthetic nanocarrier. In some embodiments, a substance is enclosed completely within a synthetic nanocarrier. In other embodiments, most or all of a substance that is encapsulated is not exposed to the local environment external to the synthetic nanocarrier. In other embodiments, no more than 50%, 40%, 30%, 20%, 10% or 5% (weight/weight) is exposed to the local environment. Encapsulation is distinct from absorption, which places most or all of a substance on a surface of a synthetic nanocarrier, and leaves the substance exposed to the local environment external to the synthetic nanocarrier. In embodiments of any one of the methods or compositions provided herein, the immunosuppressants are encapsulated within the synthetic nanocarriers.

“Identifying a subject” is any action or set of actions that allows a clinician to recognize a subject as one who may benefit from the methods or compositions provided herein or some other indicator as provided. Preferably, the identified subject is one who is in need of therapeutic treatment for PBC. Such subjects include any subject that has PBC. In some embodiments, the subject is suspected of having or determined to have a likelihood of having PBC based on symptoms (and/or lack thereof), patterns of behavior (e.g., that would put a subject at risk), and/or based on one or more tests (e.g., biomarker assays). In some embodiments of any one of the methods provided herein, the subject is one that will benefit or is in need of a reduced or weakened immune response in view of having PBC.

In one embodiment of any one of the methods provided herein, the method further comprises identifying a subject in need of a composition or method as provided herein. The action or set of actions may be either directly oneself or indirectly, such as, but not limited to, an unrelated third party that takes an action through reliance on one’s words or deeds.

“Immunosuppressant” means a compound that can cause a tolerogenic effect through its effects on APCs. A tolerogenic effect generally refers to the modulation by the APC or other immune cells that reduces, inhibits or prevents an undesired immune response to an antigen in a durable fashion. In one embodiment of any one of the methods or compositions provided, the immunosuppressant is one that causes an APC to promote a regulatory phenotype in one or more immune effector cells. For example, the regulatory phenotype may be characterized by the inhibition of the production, induction, stimulation or recruitment of antigen- specific CD4+ T cells or B cells, the inhibition of the production of antigen- specific antibodies, the production, induction, stimulation or recruitment of Treg cells (e.g., CD4+CD25highFoxP3+ Treg cells), etc. This may be the result of the conversion of CD4+ T cells or B cells to a regulatory phenotype. This may also be the result of induction of FoxP3 in other immune cells, such as CD8+ T cells, macrophages and iNKT cells. In one embodiment of any one of the methods or compositions provided, the immunosuppressant is one that affects the response of the APC after it processes an antigen. In another embodiment of any one of the methods or compositions provided, the immunosuppressant is not one that interferes with the processing of the antigen. In a further embodiment of any one of the methods or compositions provided, the immunosuppressant is not an apoptotic- signaling molecule. In another embodiment of any one of the methods or compositions provided, the immunosuppressant is not a phospholipid.

Immunosuppressants include, but are not limited to mTOR inhibitors, such as rapamycin or a rapamycin analog (i.e., rapalog); TGF-|3 signaling agents; TGF-|3 receptor agonists; histone deacetylase inhibitors, such as Trichostatin A; corticosteroids; inhibitors of mitochondrial function, such as rotenone; P38 inhibitors; NF-K|3 inhibitors, such as 6Bio, Dexamethasone, TCPA-1, IKK VII; adenosine receptor agonists; prostaglandin E2 agonists (PGE2), such as Misoprostol; phosphodiesterase inhibitors, such as phosphodiesterase 4 inhibitor (PDE4), such as Rolipram; proteasome inhibitors; kinase inhibitors; etc. “Rapalog”, as used herein, refers to a molecule that is structurally related to (an analog) of rapamycin (sirolimus). Examples of rapalogs include, without limitation, temsirolimus (CCI-779), everolimus (RAD001), ridaforolimus (AP-23573), and zotarolimus (ABT-578). Additional examples of rapalogs may be found, for example, in WO Publication WO 1998/002441 and U.S. Patent No. 8,455,510, the rapalogs of which are incorporated herein by reference in their entirety. Further immunosuppressants are known to those of skill in the art, and the invention is not limited in this respect.

In embodiments, when coupled to the synthetic nanocarriers, the immunosuppressant is an element that is in addition to the material that makes up the structure of the synthetic nanocarrier. For example, in one such embodiment, where the synthetic nanocarrier is made up of one or more polymers, the immunosuppressant is a compound that is in addition and coupled to the one or more polymers. As another example, in one such embodiment, where the synthetic nanocarrier is made up of one or more lipids, the immunosuppressant is again in addition and coupled to the one or more lipids.

“Load”, when coupled to a synthetic nanocarrier, is the amount of the immunosuppressant and/or antigen coupled to the synthetic nanocarrier based on the total dry recipe weight of materials in an entire synthetic nanocarrier (weight/weight). Generally, such a load is calculated as an average across a population of synthetic nanocarriers. In one embodiment of any one of the methods or compositions provided, the load of immunosuppressant on average across the synthetic nanocarriers is between 0.1% and 50%. In another of any one of the methods or compositions provided, the load of immunosuppressant on average across the synthetic nanocarriers is between 4%, 5%, 65, 7%, 8% or 9% and 40% or between 4%, 5%, 65, 7%, 8% or 9% and 30%. In another of any one of the methods or compositions provided, the load of immunosuppressant on average across the synthetic nanocarriers is between 10% and 40% or between 10% and 30%. In another embodiment of any one of the methods or compositions provided, the load of immunosuppressant is between 0.1% and 20%. In a further embodiment of any one of the methods or compositions provided, the load of immunosuppressant and/or antigen is between 0.1% and 10%. In still a further embodiment of any one of the methods or compositions provided, the load of immunosuppressant and/or antigen is between 1% and 10%. In still a further embodiment of any one of the methods or compositions provided, the load of immunosuppressant is between 7% and 20%. In yet another embodiment of any one of the methods or compositions provided, the load or immunosuppressant is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19% at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29% or at least 30% on average across the population of synthetic nanocarriers. In yet a further embodiment of any one of the methods or compositions provided, the load of immunosuppressant is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% on average across the population of synthetic nanocarriers. In some embodiments of any one of the above embodiments, the load of immunosuppressant is no more than 35%, 30% or 25% on average across a population of synthetic nanocarriers. In any one of the methods, compositions or kits provided herein, the load of the immunosuppressant, such as rapamycin, and/or antigen may be any one of the loads provided herein. In embodiments of any one of the methods or compositions provided, the load is calculated as known in the art.

In some embodiments, the immunosuppressant or antigen load of the nanocarrier in suspension is calculated by dividing the immunosuppressant or antigen content of the nanocarrier as determined by HPLC analysis of the test article by the nanocarrier mass. The total polymer content is measured either by gravimetric yield of the dry nanocarrier mass or by the determination of the nanocarrier solution total organic content following pharmacopeia methods and corrected for PVA content.

“Maximum dimension of a synthetic nanocarrier” means the largest dimension of a nanocarrier measured along any axis of the synthetic nanocarrier. “Minimum dimension of a synthetic nanocarrier” means the smallest dimension of a synthetic nanocarrier measured along any axis of the synthetic nanocarrier. For example, for a spheroidal synthetic nanocarrier, the maximum and minimum dimension of a synthetic nanocarrier would be substantially identical, and would be the size of its diameter. Similarly, for a cuboidal synthetic nanocarrier, the minimum dimension of a synthetic nanocarrier would be the smallest of its height, width or length, while the maximum dimension of a synthetic nanocarrier would be the largest of its height, width or length. In an embodiment, a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or greater than 100 nm. In an embodiment, a maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or less than 5 pm. Preferably, a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is greater than 110 nm, more preferably greater than 120 nm, more preferably greater than 130 nm, and more preferably still greater than 150 nm. Aspects ratios of the maximum and minimum dimensions of inventive synthetic nanocarriers may vary depending on the embodiment. For instance, aspect ratios of the maximum to minimum dimensions of the synthetic nanocarriers may vary from 1: 1 to 1,000,000: 1, preferably from 1: 1 to 100,000: 1, more preferably from 1: 1 to 10,000: 1, more preferably from 1: 1 to 1000: 1, still more preferably from 1: 1 to 100: 1, and yet more preferably from 1: 1 to 10: 1.

Preferably, a maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample is equal to or less than 3 pm, more preferably equal to or less than 2 pm, more preferably equal to or less than 1 pm, more preferably equal to or less than 800 nm, more preferably equal to or less than 600 nm, and more preferably still equal to or less than 500 nm. In preferred embodiments, a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or greater than lOOnm, more preferably equal to or greater than 120 nm, more preferably equal to or greater than 130 nm, more preferably equal to or greater than 140 nm, and more preferably still equal to or greater than 150 nm.

Measurement of synthetic nanocarrier dimensions (e.g., diameter) may be obtained by suspending the synthetic nanocarriers in a liquid (usually aqueous) media and using dynamic light scattering (DLS) (e.g. using a Brookhaven ZetaPALS instrument). For example, a suspension of synthetic nanocarriers can be diluted from an aqueous buffer into purified water to achieve a final synthetic nanocarrier suspension concentration of approximately 0.01 to 0.1 mg/mL. The diluted suspension may be prepared directly inside, or transferred to, a suitable cuvette for DLS analysis. The cuvette may then be placed in the DLS, allowed to equilibrate to the controlled temperature, and then scanned for sufficient time to acquire a stable and reproducible distribution based on appropriate inputs for viscosity of the medium and refractive indices of the sample. The effective diameter, or mean of the distribution, can then reported. “Dimension” or “size” or “diameter” of synthetic nanocarriers means the mean of a particle size distribution obtained using dynamic light scattering in some embodiments.

“PBC-associated antigen” means an antigen that is associated with having PBC or is associated with risk of having PBC, such as associated with the pathogenesis or etiology of PBC, and can preferably be presented for recognition by cells of the immune system, such as presented by antigen presenting cells, including but not limited to dendritic cells, B cells or macrophages. The PBC-associated antigen can be presented for recognition by cells, such as recognition by T cells. Such antigens can be recognized by and trigger an immune response in a T cell via presentation of the antigen or portion thereof bound to a Class I or Class II major histocompatibility complex molecule (MHC), or bound to a CD1 complex. In one embodiment, the antigen may be an autoantigen. An “autoantigen” or “self antigen” is generally an antigen of one’s own cells or cell products. In one embodiment, the PBC- associated autoantigen is PDC-E2.

The PBC-associated antigen can be comprised in synthetic nanocarriers as provided herein as a whole protein or protein fragment. Preferably, in some embodiments, the protein fragment comprises one or more T cell epitopes of the PBC-associated antigen. In some embodiments, a number of T cell epitopes of a PBC-associated antigen are administered to the subject within one or more populations of synthetic nanocarriers, and each of the T cell epitopes can be comprised in separate populations of synthetic nanocarriers, respectively, or can be comprised in the same population of synthetic nanocarriers. In some embodiments, these T cell epitopes can be provided within a protein or protein fragment. In some embodiments, the T cell epitopes can be provided as a concatenated polypeptide and comprised in a population of synthetic nanocarriers, wherein the concatenated polypeptide comprises a protease cleavage site. The protease cleavage site can be cleavable by an endogenous protease, such as cathepsin. In some embodiments, the protease cleavage site is between at least two of the T cell epitopes. In some embodiments, there is such a protease cleavage site between each set of two T cell epitopes within the concatenated polypeptide. Such polypeptides can be produced recombinantly or synthetically, and methods for doing so are known in the art. For example, when produced recombinantly, bacterial, mammalian or insect cells can be used.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” means a pharmacologically inactive material used together with a pharmacologically active material to formulate the compositions. Pharmaceutically acceptable excipients comprise a variety of materials known in the art, including but not limited to saccharides (such as glucose, lactose, and the like), preservatives such as antimicrobial agents, reconstitution aids, colorants, saline (such as phosphate buffered saline), and buffers. Any one of the compositions provided herein may include a pharmaceutically acceptable excipient or carrier.

“Promoting tolerogenic immune effect,” or the like means modulating, such as decreasing or increasing, the levels of immune responses such that tolerance is promoted. The immune response can be relative to a control such as the immune response without administration of the synthetic nanocarriers as provided herein. In some embodiments, the immune response is decreased, e.g., is decreased at least 20-40% relative to a control. Preferably the decrease is at least two-fold.

“Protocol” refers to any dosing regimen of one or more substances to a subject. A dosing regimen may include the amount, frequency, rate, duration and/or mode of administration. In some embodiments, such a protocol may be used to administer one or more compositions of the invention to one or more test subjects. Therapeutic responses in these test subjects can then be assessed to determine whether or not the protocol was effective in generating a desired response, such as treatment of PBC. Whether or not a protocol had a desired effect can be determined using any of the methods provided herein or otherwise known in the art. For example, a population of cells may be obtained from a subject to which a composition provided herein has been administered according to a specific protocol in order to determine whether or not specific enzymes, biomarkers, etc. were generated, activated, etc. Useful methods for detecting the presence and/or number of biomarkers include, but are not limited to, flow cytometric methods (e.g., FACS) and immunohistochemistry methods. Antibodies and other binding agents for specific staining of certain biomarkers, are commercially available kits. Such kits typically include staining reagents for multiple antigens that allow for FACS -based detection, separation and/or quantitation of a desired cell population from a heterogeneous population of cells. Any one of the methods provided herein can include a step of determining a protocol and/or the administering is done based on a protocol determined to have any one of the beneficial results or desired beneficial result as provided herein.

“Providing a subject” is any action or set of actions that causes a clinician to come in contact with a subject and administer a composition provided herein thereto or to perform a method provided herein thereupon. Preferably, the subject is one who is in need of treatment of PBC. The action or set of actions may be taken either directly oneself or indirectly. In one embodiment of any one of the methods provided herein, the method further comprises providing a subject.

“Repeat dose” or “repeat dosing” or the like means at least one additional dose or dosing that is administered to a subject subsequent to an earlier dose or dosing of the same material or materials. Repeat dosing is considered to be efficacious if it results in a beneficial effect for the subject. Preferably, efficacious repeat dosing results in a decreased immune response and/or the promotion of a tolerogenic phenotype. In any one of the methods provided herein, the repeat dosing can occur monthly. “Subject” means animals, including warm blooded mammals such as humans and primates; avians; domestic household or farm animals such as cats, dogs, sheep, goats, cattle, horses and pigs; laboratory animals such as mice, rats and guinea pigs; fish; reptiles; zoo and wild animals; and the like. In any one of the methods, compositions and kits provided herein, the subject is human. Any one of the methods provided herein can include a step of repeat dosing.

“Synthetic nanocarrier(s)” means a discrete object that is not found in nature, and that possesses at least one dimension that is less than or equal to 5 microns in size. Synthetic nanocarriers may be a variety of different shapes, including but not limited to spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and the like. Synthetic nanocarriers comprise one or more surfaces.

A synthetic nanocarrier can be, but is not limited to, one or a plurality of lipid-based nanoparticles (also referred to herein as lipid nanoparticles, i.e., nanoparticles where the majority of the material that makes up their structure are lipids), polymeric nanoparticles, metallic nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, viruslike particles (i.e., particles that are primarily made up of viral structural proteins but that are not infectious or have low infectivity), peptide or protein-based particles (also referred to herein as protein particles, i.e., particles where the majority of the material that makes up their structure are peptides or proteins) (such as albumin nanoparticles) and/or nanoparticles that are developed using a combination of nanomaterials such as lipid-polymer nanoparticles. Synthetic nanocarriers may be a variety of different shapes, including but not limited to spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and the like. Examples of synthetic nanocarriers include (1) the biodegradable nanoparticles disclosed in US Patent 5,543,158 to Gref et al., (2) the polymeric nanoparticles of Published US Patent Application 20060002852 to Saltzman et al., (3) the lithographically constructed nanoparticles of Published US Patent Application 20090028910 to DeSimone et al., (4) the disclosure of WO 2009/051837 to von Andrian et al., (5) the nanoparticles disclosed in Published US Patent Application 2008/0145441 to Penades et al., (6) the nanoprecipitated nanoparticles disclosed in P. Paolicelli et al., “Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles” Nanomedicine. 5(6): 843-853 (2010), and (7) those of Look et al., Nanogel-based delivery of mycophenolic acid ameliorates systemic lupus erythematosus in mice” J. Clinical Investigation 123(4): 1741-1749(2013).

Synthetic nanocarriers may have a minimum dimension of equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not comprise a surface with hydroxyl groups that activate complement or alternatively comprise a surface that consists essentially of moieties that are not hydroxyl groups that activate complement in some embodiments. In an embodiment, synthetic nanocarriers that have a minimum dimension of equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not comprise a surface that substantially activates complement or alternatively comprise a surface that consists essentially of moieties that do not substantially activate complement. In a more preferred embodiment, synthetic nanocarriers according to the invention that have a minimum dimension of equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not comprise a surface that activates complement or alternatively comprise a surface that consists essentially of moieties that do not activate complement. In embodiments, synthetic nanocarriers exclude virus-like particles. In embodiments, synthetic nanocarriers may possess an aspect ratio greater than or equal to 1: 1, 1: 1.2, 1: 1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1: 10.

“Treating” refers to the administration of one or more compositions with the expectation that the subject may have a resulting benefit due to the administration. Treating may be direct or indirect, such as by inducing or directing another subject, including another clinician or the subject itself, to treat the subject.

C. METHODS AND RELATED COMPOSITIONS

Provided herein are methods and related compositions useful for treating PBC.

Synthetic Nanocarriers

A wide variety of synthetic nanocarriers can be used according to the invention. In some embodiments, synthetic nanocarriers are spheres or spheroids. In some embodiments, synthetic nanocarriers are flat or plate- shaped. In some embodiments, synthetic nanocarriers are cubes or cubic. In some embodiments, synthetic nanocarriers are ovals or ellipses. In some embodiments, synthetic nanocarriers are cylinders, cones, or pyramids.

In some embodiments, it is desirable to use a population of synthetic nanocarriers that is relatively uniform in terms of size or shape so that each synthetic nanocarrier has similar properties. For example, at least 80%, at least 90%, or at least 95% of the synthetic nanocarriers of any one of the compositions or methods provided, based on the total number of synthetic nanocarriers, may have a minimum dimension or maximum dimension that falls within 5%, 10%, or 20% of the average diameter or average dimension of the synthetic nanocarriers. Synthetic nanocarriers can be solid or hollow and can comprise one or more layers. In some embodiments, each layer has a unique composition and unique properties relative to the other layer(s). To give but one example, synthetic nanocarriers may have a core/shell structure, wherein the core is one layer (e.g. a polymeric core) and the shell is a second layer (e.g. a lipid bilayer or monolayer). Synthetic nanocarriers may comprise a plurality of different layers.

In some embodiments, synthetic nanocarriers may optionally comprise one or more lipids. In some embodiments, a synthetic nanocarrier may comprise a liposome. In some embodiments, a synthetic nanocarrier may comprise a lipid bilayer. In some embodiments, a synthetic nanocarrier may comprise a lipid monolayer. In some embodiments, a synthetic nanocarrier may comprise a micelle. In some embodiments, a synthetic nanocarrier may comprise a core comprising a polymeric matrix surrounded by a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In some embodiments, a synthetic nanocarrier may comprise a non- polymeric core (e.g., metal particle, quantum dot, ceramic particle, bone particle, viral particle, proteins, nucleic acids, carbohydrates, etc.) surrounded by a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.).

In other embodiments, synthetic nanocarriers may comprise metal particles, quantum dots, ceramic particles, etc. In some embodiments, a non-polymeric synthetic nanocarrier is an aggregate of non-polymeric components, such as an aggregate of metal atoms (e.g., gold atoms).

In some embodiments, synthetic nanocarriers may optionally comprise one or more amphiphilic entities. In some embodiments, an amphiphilic entity can promote the production of synthetic nanocarriers with increased stability, improved uniformity, or increased viscosity. In some embodiments, amphiphilic entities can be associated with the interior surface of a lipid membrane (e.g., lipid bilayer, lipid monolayer, etc.). Many amphiphilic entities known in the art are suitable for use in making synthetic nanocarriers in accordance with the present invention. Such amphiphilic entities include, but are not limited to, phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine (DOPE); dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine; cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate; diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid, such as palmitic acid or oleic acid; fatty acids; fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides; sorbitan trioleate (Span®85) glycocholate; sorbitan monolaurate (Span®20); polysorbate 20 (Tween®20); polysorbate 60 (Tween®60); polysorbate 65 (Tween®65); polysorbate 80 (Tween®80); polysorbate 85 (Tween®85); polyoxyethylene monostearate; surfactin; a poloxomer; a sorbitan fatty acid ester such as sorbitan trioleate; lecithin; lysolecithin; phosphatidylserine; phosphatidy linositol;sphingomyelin; phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid; cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerol ricinoleate; hexadecyl sterate; isopropyl myristate; tyloxapol; poly(ethylene glycol)5000- phosphatidylethanolamine; poly(ethylene glycol)400-monostearate; phospholipids; synthetic and/or natural detergents having high surfactant properties; deoxy cholates; cyclodextrins; chaotropic salts; ion pairing agents; and combinations thereof. An amphiphilic entity component may be a mixture of different amphiphilic entities. Those skilled in the art will recognize that this is an exemplary, not comprehensive, list of substances with surfactant activity. Any amphiphilic entity may be used in the production of synthetic nanocarriers to be used in accordance with the present invention.

In some embodiments, synthetic nanocarriers may optionally comprise one or more carbohydrates. Carbohydrates may be natural or synthetic. A carbohydrate may be a derivatized natural carbohydrate. In certain embodiments, a carbohydrate comprises monosaccharide or disaccharide, including but not limited to glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose, arabinose, glucoronic acid, galactoronic acid, mannuronic acid, glucosamine, galatosamine, and neuramic acid. In certain embodiments, a carbohydrate is a polysaccharide, including but not limited to pullulan, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen, hydroxyethylstarch, carageenan, glycon, amylose, chitosan, N,O-carboxylmethylchitosan, algin and alginic acid, starch, chitin, inulin, konjac, glucommannan, pustulan, heparin, hyaluronic acid, curdlan, and xanthan. In embodiments, the synthetic nanocarriers do not comprise (or specifically exclude) carbohydrates, such as a polysaccharide. In certain embodiments, the carbohydrate may comprise a carbohydrate derivative such as a sugar alcohol, including but not limited to mannitol, sorbitol, xylitol, erythritol, maltitol, and lactitol.

In some embodiments, synthetic nanocarriers can comprise one or more polymers. In some embodiments, the synthetic nanocarriers comprise one or more polymers that is a non- methoxy-terminated, pluronic polymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight) of the polymers that make up the synthetic nanocarriers are non-methoxy-terminated, pluronic polymers. In some embodiments, all of the polymers that make up the synthetic nanocarriers are non-methoxy-terminated, pluronic polymers. In some embodiments, the synthetic nanocarriers comprise one or more polymers that is a non-methoxy-terminated polymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight) of the polymers that make up the synthetic nanocarriers are non-methoxy-terminated polymers. In some embodiments, all of the polymers that make up the synthetic nanocarriers are non-methoxy-terminated polymers. In some embodiments, the synthetic nanocarriers comprise one or more polymers that do not comprise pluronic polymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight) of the polymers that make up the synthetic nanocarriers do not comprise pluronic polymer. In some embodiments, all of the polymers that make up the synthetic nanocarriers do not comprise pluronic polymer. In some embodiments, such a polymer can be surrounded by a coating layer (e.g., liposome, lipid monolayer, micelle, etc.). In some embodiments, elements of the synthetic nanocarriers can be attached to the polymer.

Immunosuppressants or antigens can be coupled to the synthetic nanocarriers by any of a number of methods. Generally, the attaching can be a result of bonding between the immunosuppressants or antigens and the synthetic nanocarriers. This bonding can result in the immunosuppressants or antigens being attached to the surface of the synthetic nanocarriers and/or contained (encapsulated) within the synthetic nanocarriers. In some embodiments of any one of the methods or compositions provided, however, the immunosuppressants or antigens are encapsulated by the synthetic nanocarriers as a result of the structure of the synthetic nanocarriers rather than bonding to the synthetic nanocarriers. In preferable embodiments of any one of the methods or compositions provided, the synthetic nanocarrier comprises a polymer as provided herein, and the immunosuppressants or antigens are coupled to the polymer.

When coupling occurs as a result of bonding between the immunosuppressants or antigens and synthetic nanocarriers, the coupling may occur via a coupling moiety. A coupling moiety can be any moiety through which an immunosuppressant or antigen is bonded to a synthetic nanocarrier. Such moieties include covalent bonds, such as an amide bond or ester bond, as well as separate molecules that bond (covalently or non-covalently) the immunosuppressant or antigen to the synthetic nanocarrier. Such molecules include linkers or polymers or a unit thereof. For example, the coupling moiety can comprise a charged polymer to which an immunosuppressant or antigen electrostatically binds. As another example, the coupling moiety can comprise a polymer or unit thereof to which it is covalently bonded.

In preferred embodiments of any one of the methods or compositions provided, the synthetic nanocarriers comprise a polymer as provided herein. These synthetic nanocarriers can be completely polymeric or they can be a mix of polymers and other materials.

In some embodiments of any one of the methods or compositions provided, the polymers of a synthetic nanocarrier associate to form a polymeric matrix. In some of these embodiments of any one of the methods or compositions provided, a component, can be covalently associated with one or more polymers of the polymeric matrix. In some embodiments of any one of the methods or compositions provided, covalent association is mediated by a linker. In some embodiments of any one of the methods or compositions provided, a component can be non-covalently associated with one or more polymers of the polymeric matrix. For example, in some embodiments of any one of the methods or compositions provided, a component can be encapsulated within, surrounded by, and/or dispersed throughout a polymeric matrix. Alternatively or additionally, a component can be associated with one or more polymers of a polymeric matrix by hydrophobic interactions, charge interactions, van der Waals forces, etc. A wide variety of polymers and methods for forming polymeric matrices therefrom are known conventionally.

Polymers may be natural or unnatural (synthetic) polymers. Polymers may be homopolymers or copolymers comprising two or more monomers. In terms of sequence, copolymers may be random, block, or comprise a combination of random and block sequences. Typically, polymers in accordance with the present invention are organic polymers.

In some embodiments, the polymer comprises a polyester, polycarbonate, polyamide, or poly ether, or unit thereof. In other embodiments, the polymer comprises poly (ethylene glycol) (PEG), polypropylene glycol, poly(lactic acid), poly(glycolic acid), poly(lactic-co- glycolic acid), or a polycaprolactone, or unit thereof. In some embodiments, it is preferred that the polymer is biodegradable. Therefore, in these embodiments, it is preferred that if the polymer comprises a polyether, such as poly(ethylene glycol) or polypropylene glycol or unit thereof, the polymer comprises a block-co-polymer of a polyether and a biodegradable polymer such that the polymer is biodegradable. In other embodiments, the polymer does not solely comprise a polyether or unit thereof, such as poly(ethylene glycol) or polypropylene glycol or unit thereof.

Other examples of polymers suitable for use in the present invention include, but are not limited to polyethylenes, polycarbonates (e.g. poly(l,3-dioxan-2one)), polyanhydrides (e.g. poly(sebacic anhydride)), polypropylfumerates, polyamides (e.g. polycaprolactam), polyacetals, polyethers, polyesters (e.g., polylactide, polyglycolide, polylactide-co-glycolide, polycaprolactone, polyhydroxyacid (e.g. poly(P-hydroxyalkanoate))), poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, poly acrylates, polymethacrylates, polyureas, polystyrenes, and polyamines, polylysine, polylysine-PEG copolymers, and poly (ethyleneimine), poly(ethylene imine)-PEG copolymers.

In some embodiments, polymers in accordance with the present invention include polymers which have been approved for use in humans by the U.S. Food and Drug Administration (FDA) under 21 C.F.R. § 177.2600, including but not limited to polyesters (e.g., polylactic acid, poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone, poly(l,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; poly acrylates; and polycyanoacrylates.

In some embodiments, polymers can be hydrophilic. For example, polymers may comprise anionic groups (e.g., phosphate group, sulphate group, carboxylate group); cationic groups (e.g., quaternary amine group); or polar groups (e.g., hydroxyl group, thiol group, amine group). In some embodiments, a synthetic nanocarrier comprising a hydrophilic polymeric matrix generates a hydrophilic environment within the synthetic nanocarrier. In some embodiments, polymers can be hydrophobic. In some embodiments, a synthetic nanocarrier comprising a hydrophobic polymeric matrix generates a hydrophobic environment within the synthetic nanocarrier. Selection of the hydrophilicity or hydrophobicity of the polymer may have an impact on the nature of materials that are incorporated within the synthetic nanocarrier.

In some embodiments, polymers may be modified with one or more moieties and/or functional groups. A variety of moieties or functional groups can be used in accordance with the present invention. In some embodiments, polymers may be modified with polyethylene glycol (PEG), with a carbohydrate, and/or with acyclic poly acetals derived from polysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Certain embodiments may be made using the general teachings of US Patent No. 5543158 to Gref et al., or WO publication W02009/051837 by Von Andrian et al. In some embodiments, polymers may be modified with a lipid or fatty acid group. In some embodiments, a fatty acid group may be one or more of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, or lignoceric acid. In some embodiments, a fatty acid group may be one or more of palmitoleic, oleic, vaccenic, linoleic, alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic, eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, polymers may be polyesters, including copolymers comprising lactic acid and glycolic acid units, such as poly (lactic acid-co-glycolic acid) and poly (lactide - co-glycolide), collectively referred to herein as “PLGA”; and homopolymers comprising glycolic acid units, referred to herein as “PGA,” and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L- lactide, collectively referred to herein as “PLA.” In some embodiments, exemplary polyesters include, for example, poly hydroxy acids; PEG copolymers and copolymers of lactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEG copolymers, and derivatives thereof. In some embodiments, polyesters include, for example, poly (caprolactone), poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine), poly(serine ester), poly(4-hydroxy-L-proline ester), poly[a-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible and biodegradable co-polymer of lactic acid and glycolic acid, and various forms of PLGA are characterized by the ratio of lactic acid:glycolic acid. Lactic acid can be L-lactic acid, D- lactic acid, or D, L-lactic acid. The degradation rate of PLGA can be adjusted by altering the lactic acid:glycolic acid ratio. In some embodiments, PLGA to be used in accordance with the present invention is characterized by a lactic acid:glycolic acid ratio of approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, or approximately 15:85.

In some embodiments, polymers may be one or more acrylic polymers. In certain embodiments, acrylic polymers include, for example, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymer, poly (methyl methacrylate), poly (methacrylic acid anhydride), methyl methacrylate, polymethacrylate, poly (methyl methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate copolymer, glycidyl methacrylate copolymers, polycyanoacrylates, and combinations comprising one or more of the foregoing polymers. The acrylic polymer may comprise fully-polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general, cationic polymers are able to condense and/or protect negatively charged strands of nucleic acids. Amine-containing polymers such as poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, Bioconjugate Chem., 6:7), poly(ethylene imine) (PEI; Boussif et al., 1995, Proc. Natl. Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers (Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993, Bioconjugate Chem., 4:372) are positively-charged at physiological pH, form ion pairs with nucleic acids. In embodiments, the synthetic nanocarriers may not comprise (or may exclude) cationic polymers.

In some embodiments, polymers can be degradable polyesters bearing cationic side chains (Putnam et al., 1999, Macromolecules, 32:3658; Barrera et al., 1993, J. Am. Chem. Soc., 115: 11010; Kwon et al., 1989, Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633; and Zhou et al., 1990, Macromolecules, 23:3399). Examples of these polyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J. Am. Chem. Soc., 115: 11010), poly(serine ester) (Zhou et al., 1990, Macromolecules, 23:3399), poly(4- hydroxy-L-proline ester) (Putnam et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc., 121:5633).

The properties of these and other polymers and methods for preparing them are well known in the art (see, for example, U.S. Patents 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al., 2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc., 123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J. Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181). More generally, a variety of methods for synthesizing certain suitable polymers are described in Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles of Polymerization by Odian, John Wiley & Sons, Fourth Edition, 2004; Contemporary Polymer Chemistry by Allcock et al., Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S. Patents 6,506,577, 6,632,922, 6,686,446, and 6,818,732. In some embodiments, polymers can be linear or branched polymers. In some embodiments, polymers can be dendrimers. In some embodiments, polymers can be substantially cross-linked to one another. In some embodiments, polymers can be substantially free of cross-links. In some embodiments, polymers can be used in accordance with the present invention without undergoing a cross-linking step. It is further to be understood that the synthetic nanocarriers may comprise block copolymers, graft copolymers, blends, mixtures, and/or adducts of any of the foregoing and other polymers. Those skilled in the art will recognize that the polymers listed herein represent an exemplary, not comprehensive, list of polymers that can be of use in accordance with the present invention.

In some embodiments, synthetic nanocarriers do not comprise a polymeric component. In some embodiments, synthetic nanocarriers may comprise metal particles, quantum dots, ceramic particles, etc. In some embodiments, a non-polymeric synthetic nanocarrier is an aggregate of non-polymeric components, such as an aggregate of metal atoms (e.g., gold atoms).

Immunosuppressants

Any immunosuppressant as provided herein can be, in some embodiments of any one of the methods or compositions provided, coupled to synthetic nanocarriers. Immunosuppressants include, but are not limited to, statins; mTOR inhibitors, such as rapamycin or a rapamycin analog (rapalog); TGF-|3 signaling agents; TGF-|3 receptor agonists; histone deacetylase (HD AC) inhibitors; corticosteroids; inhibitors of mitochondrial function, such as rotenone; P38 inhibitors; NF-K|3 inhibitors; adenosine receptor agonists; prostaglandin E2 agonists; phosphodiesterase inhibitors, such as phosphodiesterase 4 inhibitor; proteasome inhibitors; kinase inhibitors; G-protein coupled receptor agonists; G- protein coupled receptor antagonists; glucocorticoids; retinoids; cytokine inhibitors; cytokine receptor inhibitors; cytokine receptor activators; peroxisome proliferator-activated receptor antagonists; peroxisome proliferator-activated receptor agonists; histone deacetylase inhibitors; calcineurin inhibitors; phosphatase inhibitors and oxidized ATPs. Immunosuppressants also include IDO, vitamin D3, cyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol, azathiopurine, 6-mercaptopurine, aspirin, niflumic acid, estriol, tripolide, interleukins (e.g., IL-1, IL- 10), cyclosporine A, siRNAs targeting cytokines or cytokine receptors and the like. Examples of statins include atorvastatin (LIPITOR®, TORVAST®), cerivastatin, fluvastatin (LESCOL®, LESCOL® XL), lovastatin (MEVACOR®, ALTOCOR®, ALTOPREV®), mevastatin (COMPACTIN®), pitavastatin (LIVALO®, PIAVA®), rosuvastatin (PRAVACHOL®, SELEKTINE®, LIPOSTAT®), rosuvastatin (CRESTOR®), and simvastatin (ZOCOR®, LIPEX®).

Examples of mTOR inhibitors include rapamycin and analogs thereof (e.g., CCL-779, RAD001, AP23573, C20-methallylrapamycin (C20-Marap), C16-(S)- butylsulfonamidorapamycin (C16-BSrap), C16-(S)-3-methylindolerapamycin (C16-iRap) (Bayle et al. Chemistry & Biology 2006, 13:99-107)), AZD8055, BEZ235 (NVP-BEZ235), chrysophanic acid (chrysophanol), deforolimus (MK-8669), everolimus (RAD0001), KU- 0063794, PL103, PP242, temsirolimus, and WYE-354 (available from Selleck, Houston, TX, USA).

“Rapalog”, as used herein, refers to a molecule that is structurally related to (an analog) of rapamycin (sirolimus). Examples of rapalogs include, without limitation, temsirolimus (CCI-779), everolimus (RAD001), ridaforolimus (AP-23573), and zotarolimus (ABT-578). Additional examples of rapalogs may be found, for example, in WO Publication WO 1998/002441 and U.S. Patent No. 8,455,510, the rapalogs of which are incorporated herein by reference in their entirety.

When coupled to a synthetic nanocarrier, the amount of the immunosuppressant coupled to the synthetic nanocarrier based on the total dry recipe weight of materials in an entire synthetic nanocarrier (weight/weight), is as described elsewhere herein. Preferably, in some embodiments of any one of the methods or compositions or kits provided herein, the load of the immunosuppressant, such as rapamycin or rapalog, is between 4%, 5%, 65, 7%, 8%, 9% or 10% and 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40% by weight.

In regard to synthetic nanocarriers coupled to immunosuppressants, methods for coupling components to synthetic nanocarriers may be useful. Elements of the synthetic nanocarriers may be coupled to the overall synthetic nanocarrier, e.g., by one or more covalent bonds, or may be attached by means of one or more linkers. Additional methods of functionalizing synthetic nanocarriers may be adapted from Published US Patent Application 2006/0002852 to Saltzman et al., Published US Patent Application 2009/0028910 to DeSimone et al., or Published International Patent Application WO/2008/127532 Al to Murthy et al. In some embodiments, the coupling can be a covalent linker. In embodiments, immunosuppressants according to the invention can be covalently coupled to the external surface via a 1,2, 3 -triazole linker formed by the 1,3-dipolar cycloaddition reaction of azido groups with immunosuppressant containing an alkyne group or by the 1,3-dipolar cycloaddition reaction of alkynes with immunosuppressants containing an azido group. Such cycloaddition reactions are preferably performed in the presence of a Cu(I) catalyst along with a suitable Cu(I)-ligand and a reducing agent to reduce Cu(II) compound to catalytic active Cu(I) compound. This Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) can also be referred as the click reaction.

Additionally, covalent coupling may comprise a covalent linker that comprises an amide linker, a disulfide linker, a thioether linker, a hydrazone linker, a hydrazide linker, an imine or oxime linker, an urea or thiourea linker, an amidine linker, an amine linker, and a sulfonamide linker.

Alternatively or additionally, synthetic nanocarriers can be coupled to components directly or indirectly via non-covalent interactions. In non-covalent embodiments, the non- covalent attaching is mediated by non-covalent interactions including but not limited to charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and/or combinations thereof. Such couplings may be arranged to be on an external surface or an internal surface of a synthetic nanocarrier. In embodiments of any one of the methods or compositions provided, encapsulation and/or absorption is a form of coupling.

For detailed descriptions of available conjugation methods, see Hermanson G T “Bioconjugate Techniques”, 2nd Edition Published by Academic Press, Inc., 2008. In addition to covalent attachment the component can be coupled by adsorption to a pre-formed synthetic nanocarrier or it can be coupled by encapsulation during the formation of the synthetic nanocarrier.

D. METHODS OF MAKING AND USING THE METHODS AND RELATED COMPOSITIONS

Synthetic nanocarriers may be prepared using a wide variety of methods known in the art. For example, synthetic nanocarriers can be formed by methods such as nanoprecipitation, flow focusing using fluidic channels, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, milling, microemulsion procedures, microfabrication, nanofabrication, sacrificial layers, simple and complex coacervation, and other methods well known to those of ordinary skill in the art. Alternatively or additionally, aqueous and organic solvent syntheses for monodisperse semiconductor, conductive, magnetic, organic, and other nanomaterials have been described (Pellegrino et al., 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; and Trindade et al., 2001, Chem. Mat., 13:3843). Additional methods have been described in the literature (see, e.g., Doubrow, Ed., “Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5: 13; Mathiowitz et al., 1987, Reactive Polymers, 6:275; and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755; US Patents 5578325 and 6007845; P. Paolicelli et al., “Surface- modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853 (2010)).

Materials may be encapsulated into synthetic nanocarriers as desirable using a variety of methods including but not limited to C. Astete et al., “Synthesis and characterization of PLGA nanoparticles” J. Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K. Avgoustakis “Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide) Nanoparticles: Preparation, Properties and Possible Applications in Drug Delivery” Current Drug Delivery 1:321-333 (2004); C. Reis et al., “Nanoencapsulation I. Methods for preparation of drug- loaded polymeric nanoparticles” Nanomedicine 2:8- 21 (2006); P. Paolicelli et al., “Surface- modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853 (2010). Other methods suitable for encapsulating materials into synthetic nanocarriers may be used, including without limitation methods disclosed in United States Patent 6,632,671 to Unger issued October 14, 2003.

In certain embodiments, synthetic nanocarriers are prepared by a nanoprecipitation process or spray drying. Conditions used in preparing synthetic nanocarriers may be altered to yield particles of a desired size or property (e.g., hydrophobicity, hydrophilicity, external morphology, “stickiness,” shape, etc.). The method of preparing the synthetic nanocarriers and the conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) used may depend on the materials to be attached to the synthetic nanocarriers and/or the composition of the polymer matrix.

If synthetic nanocarriers prepared by any of the above methods have a size range outside of the desired range, synthetic nanocarriers can be sized, for example, using a sieve. Compositions provided herein may comprise inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonate, acetate, or citrate) and pH adjustment agents (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citrate or acetate, amino acids and their salts) antioxidants (e.g., ascorbic acid, alpha- tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene9-10 nonyl phenol, sodium desoxy cholate), solution and/or cryo/lyo stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic adjustment agents (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity-adjustment agents (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethylene glycol, ethanol).

Compositions according to the invention can comprise pharmaceutically acceptable excipients, such as preservatives, buffers, saline, or phosphate buffered saline. The compositions may be made using conventional pharmaceutical manufacturing and compounding techniques to arrive at useful dosage forms. In an embodiment of any one of the methods or compositions provided, compositions are suspended in sterile saline solution for injection together with a preservative. Techniques suitable for use in practicing the present invention may be found in Handbook of Industrial Mixing: Science and Practice, Edited by Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004 John Wiley & Sons, Inc.; and Pharmaceutics: The Science of Dosage Form Design, 2nd Ed. Edited by M. E. Auten, 2001, Churchill Livingstone. In an embodiment of any one of the methods or compositions provided, compositions are suspended in sterile saline solution for injection with a preservative.

It is to be understood that the compositions of the invention can be made in any suitable manner, and the invention is in no way limited to compositions that can be produced using the methods described herein. Selection of an appropriate method of manufacture may require attention to the properties of the particular moieties being associated.

In some embodiments of any one of the methods or compositions provided, compositions are manufactured under sterile conditions or are terminally sterilized. This can ensure that resulting compositions are sterile and non-infectious, thus improving safety when compared to non-sterile compositions. This provides a valuable safety measure, especially when subjects receiving the compositions have immune defects, are suffering from infection, and/or are susceptible to infection. Administration

Administration according to the present invention may be by a variety of routes, including but not limited to subcutaneous, intravenous, and intraperitoneal routes. For example, the mode of administration for the composition of any one of the treatment methods provided may be by intravenous administration, such as an intravenous infusion that, for example, may take place over about 1 hour. The compositions referred to herein may be manufactured and prepared for administration using conventional methods.

The compositions of the invention can be administered in effective amounts, such as the effective amounts described herein. In some embodiments of any one of the methods or compositions provided, repeated multiple cycles of administration of synthetic nanocarriers is undertaken. Doses of dosage forms may contain varying amounts according to the invention. The amounts present in the dosage forms can be varied according to the nature of the synthetic nanocarrier and/or immunosuppressant and/or antigen, the therapeutic benefit to be accomplished, and other such parameters. In embodiments, dose ranging studies can be conducted to establish optimal therapeutic amounts of the component(s) to be present in dosage forms. In embodiments, the component(s) are present in dosage forms in an amount effective to generate a therapeutic response to PBC. Dosage forms may be administered at a variety of frequencies.

Aspects of the invention relate to determining a protocol for the methods of administration as provided herein. A protocol can be determined by varying at least the frequency, dosage amount of the synthetic nanocarriers and subsequently assessing a desired or undesired response, such as an immune response. The protocol can comprise at least the frequency of the administration and doses of the synthetic nanocarriers. Any one of the methods provided herein can include a step of determining a protocol or the administering steps are performed according to a protocol that was determined to achieve any one or more of the desired results as provided herein.

Dosing

The compositions provided herein may be administered according to a dosing schedule.

EXAMPLES

Example 1: Synthesis of Synthetic Nanocarriers Comprising an Immunosuppressant (Prophetic) Synthetic nanocarriers (e.g., PLA and/or PLA-PEG nanocarriers) comprising an immunosuppressant, such as rapamycin, can be produced using any method known to those of ordinary skill in the art. Preferably, in some embodiments of any one of the methods or compositions provided herein the synthetic nanocarriers comprising an immunosuppressant are produced by any one of the methods of US Publication No. US 2016/0128986 Al and US Publication No. US 2016/0128987 Al, the described methods of such production and the resulting synthetic nanocarriers being incorporated herein by reference in their entirety. In any one of the methods or compositions provided herein, the synthetic nanocarriers comprising an immunosuppressant are such incorporated synthetic nanocarriers.

Example 2: Co-administer Synthetic Nanocarriers Encapsulating Rapamycin (e.g., ImmTOR) with PDC-E2 (Prophetic)

PBC is an autoimmune disorder where the body mistakenly attacks tissue in the liver. This can lead to inflammation, damage and scarring of the small bile ducts. PBC occurs with an incidence of 1:2500 in the U.S. and is more common in females. Patients with PBC can benefit from a targeted, liver-directed approach to treating the root cause of the disorder. An autoantigen, PDC-E2, is implicated in PBC. PBC is a T cell-mediated disease, driven by an antigen. As shown in FIGs. 1-3, ImmTOR biodistributes to the liver and induces a tolerogenic environment (FIGs. 1-2) and ImmTOR shows hepatoprotective properties in liver injury models (FIG. 3).

Example 3: ImmTOR Treatment in a PBC Mouse Model

Materials

Rapamycin containing nanoparticles (ImmTOR) were manufactured as described earlier (1,2). ImmTOR doses were based on rapamycin content ranging from 50 to 300 pg per mouse. Rapamycin (sirolimus) was manufactured by Concord Biotech (Ahmedabad, India). Antigen-containing nanoparticles (NP) were prepared using a water/oil/water (W/O/W) double-emulsion solvent evaporation method. Briefly, PUGA (50:50) and pegylated polylactic acid (PLA-PEG) were dissolved in dichloromethane to form the oil phase. An aqueous solution of antigen (chicken ovalbumin or OVA protein, hybrid insulin peptide HIP6.9) or PDC-ED-ILD (amino acids 213-314), was then added to the oil phase and emulsified by sonication (Branson Digital Sonifier 250A). Following emulsification of the antigen solution into the oil phase, a double emulsion was created by adding an aqueous solution of polyvinyl alcohol and sonicating a second time. The double emulsion was added to a beaker containing PBS and stirred at RT for 4 h to allow the dichloromethane to evaporate. The resulting NPs were washed twice by centrifuging at 75,600 x g for 50 min at 4 °C followed by resuspension of the pellet in PBS. Concentration of extracted antigens was measured by HPLC. Dynamic Light Scattering (DLS) analysis of particle size and PDI was performed using a Malvern Zetasizer Nano-ZS ZEN 3600. All the nanoparticles loaded with antigens exhibited a particle size distribution ranging between 140-155 nm with a low polydispersity index (<0.15). Recombinant PDC-E2-ILD was manufactured by Genscript (Piscataway, NJ) using its proprietary E. coli expression system.

PBC Mouse Model

NOD.c3c4 mice were enrolled in the study at 8 weeks and treated for the first time at 10 weeks of age. After termination at 24 weeks, livers were fixed, embedded in paraffin, sectioned, stained with hematoxylin and eosin and then slide images were taken. The resulting slides were evaluated by a certified veterinary pathologist, and microscopic findings were scored as follows: Grade 0 (normal): finding not present, Grade 1 (minimal): a focal, subtle, or trivial change, Grade 2 (mild): an easily identifiable change of limited severity and/or distribution, Grade 3 (moderate): an obvious change with normal tissue remaining, Grade 4 (marked): an extensive change that obliterates much of the normal tissue, Grade 5 (severe): a maximal change.

Results

The activity of ImmTOR in NOD.C3C4 mice, which spontaneously develop an autoimmune disease of the liver which closely resembles primary biliary cholangitis (PBC) was assessed (6, 7). The primary T cell epitope has been mapped to a peptide in the inner lipoyl domain of the E2 component of the pyruvate dehydrogenase complexes (PDC-E2-ILD) (6). Mice were treated starting at 10 weeks of age, prior to the onset of disease, with three monthly doses of ImmTOR (at weeks 10, 14, and 18). Samples were taken and the mice were analyzed at week 24. Fig. 4 shows the results, demonstrating that ImmTOR treatment produces a reduction in bile duct epithelium degeneration, biliary hyperplasia, and liver inflammation relative to the untreated control group. Co-administration of NP-PDC-E2-ILD provided additional benefit. Liver histology showed striking biliary pathology, with marked peri-biliary mononuclear cell infiltrates, biliary hypercellularity and ductular ectasia in both female (Fig. 5, panels C and D) and male mice (Fig. 5, panels G and H). Treatment with ImmTOR (panels D and H), showed progressive improvement of all histologic features relative to the untreated group (panels C and G). In Fig. 5, the asterisk (*) indicates inflammation and the arrows point to multifocal duct ectasia.

Example 4: ImmTOR Treatment in an Autoimmune Hepatitis Mouse Model

Rapamycin containing nanoparticles (ImmTOR) and antigen-containing nanoparticles were prepared as described in Example 3.

Autoimmune Hepatitis Mouse Model

Concanavalin A (Con A) induced liver toxicity model was employed essentially as earlier described (5). Briefly, mice were injected (i.v., r.o.) Con A at 12 mg/kg and then terminally bled at 6 or 12 hours post-challenge with cytokine levels in serum determined by MSD as described above and liver tissues collected simultaneously for single-cell suspension analysis by flow cytometry as described above or for hematoxylin-eosin staining followed by microscopic evaluation.

Results

The activity of the ImmTOR was evaluated in a model of autoimmune hepatitis induced by systemic administration of the concanavalin A, a lectin that causes polyclonal lymphocyte activation and hepatic infiltration of activated immune cells. Previous studies have shown that Treg depletion with anti-IL-2Ra antibodies exacerbated disease while adoptive transfer of Treg ameliorated disease (8).

Female C57BL/6 mice (n=5 per cohort) were either left untreated or pre-treated with ImmTOR (200 pg). Mice were challenged 4 days later with 12 mg/kg of concanavalin A (Con A, groups dosed with Con A are indicated by the bracket to the right of the treatment groups) or left unchallenged (no Con A). At 12 hours after Con A challenge, serum was drawn for cytokine analysis and livers were harvested and hepatic T cells were assessed by flow cytometry. Activated (CD69⁺) and highly activated (CD69^hlgh) T cells (CD3⁺) and CTL (CD3⁺CD8⁺) are shown either as fractions of total or as absolute cell numbers. A representative experiment of 4 studies that resulted in similar outcomes is shown and error bars indicate mean +/- SD (Fig. 6A). Prophylactic treatment with ImmTOR monotherapy inhibited infiltration of activated effector T cells (Fig. 6A). Serum levels at 12 hours after Con A challenge were also measured (summary of 2 independent experiments, n=8- 10/group), and ImmTOR also reduced production of serum interferon-y (IFN-y) and, to a lesser extent, of CXCL1 chemokine (Fig. 6B). ImmTOR administration also induced increased production of FGF21, a hepatoprotective stress-response growth factor relative to the untreated control group (Fig. 6C).

References (Examples 3-4)

1. T. K. Kishimoto et al., Improving the efficacy and safety of biologic drugs with tolerogenic nanoparticles. Nat Nano technol 11, 890-899 (2016).

2. R. A. Maldonado et al., Polymeric synthetic nanoparticles for the induction of antigen- specific immunological tolerance. Proc Natl Acad Sci U S A 112, E156-165 (2015).

3. J. Quinn et al., Expression and lipoylation in Escherichia coli of the inner lipoyl domain of the E2 component of the human pyruvate dehydrogenase complex. Biochem J 289 ( Pt 1), 81-85 (1993).

4. L. Khoryati et al., An IL-2 mutein engineered to promote expansion of regulatory T cells arrests ongoing autoimmunity in mice. Sci Immunol 5, (2020)

5. P. O. Ilyinskii, C. J. Roy, J. LePrevost, G. L. Rizzo, T. K. Kishimoto, Enhancement of the Tolerogenic Phenotype in the Liver by ImmTOR Nanoparticles. Front Immunol 12, 637469 (2021).

6. J. Irie et al., NOD.c3c4 congenic mice develop autoimmune biliary disease that serologically and pathogenetically models human primary biliary cirrhosis. J Exp Med 203, 1209-1219 (2006).

7. S. Koarada et al., Genetic control of autoimmunity: protection from diabetes, but spontaneous autoimmune biliary disease in a nonobese diabetic congenic strain. J Immunol 173, 2315-2323 (2004).

8. H. X. Wei et al., CD4+ CD25+ Foxp3+ regulatory T cells protect against T cell- mediated fulminant hepatitis in a TGF-beta-dependent manner in mice. J Immunol 181, 7221- 7229 (2008).

Claims

CLAIMS What is claimed is:

1. A method of treating primary biliary cholangitis (PBC) in a subject comprising: administering a composition comprising synthetic nanocarriers comprising an immunosuppressant to the subject; and optionally, administering a composition comprising synthetic nanocarriers comprising a PBC-associated antigen, wherein the subject has PBC.

2. The method of claim 1, wherein the PBC-associated antigen is PDC-E2.

3. The method of claim 1 or 2, wherein the population of synthetic nanocarriers comprising the immunosuppressant and the optional population of synthetic nanocarriers comprising the PBC-associated antigen are the same.

4. The method of claim 1 or 2, wherein the population of synthetic nanocarriers comprising the immunosuppressant and the optional population of synthetic nanocarriers comprising the PBC-associated antigen are different.

5. The method of claim 4, wherein the population of synthetic nanocarriers comprising the immunosuppressant and the optional population of synthetic nanocarriers comprising the PBC-associated antigen are administered concomitantly.

6. The method of any one of the preceding claims, wherein more than one T cell epitope of a PBC-associated antigen is administered to the subject.

7. The method of claim 6, wherein the more than one T cell epitope are on synthetic nanocarriers different from the synthetic nanocarriers that comprise the immunosuppressant.

8. The method of claim 7, wherein each of the T cell epitopes are comprised in different populations of synthetic nanocarriers (different from the population of synthetic nanocarriers comprising the immunosuppressant and different from the population of synthetic nanocarriers that comprise another T cell epitope).

9. The method of any one of the preceding claims, wherein when there are different populations of synthetic nanocarriers, the different populations are admixed prior to administration.

10. The method of claim 6, wherein the more than one T cell epitopes are on the same synthetic nanocarriers that comprise the immunosuppressant.

11. The method of claim 6, wherein the more than one T cell epitopes are on the same population of synthetic nanocarriers but different from the synthetic nanocarriers that comprise the immunosuppressant.

12. The method of claim 10 or 11, wherein the more than one T cell epitopes are comprised in a concatenated polypeptide.

13. The method of claim 12, wherein the concatenated polypeptide comprises a protease cleavage site between two of the more than one T cell epitopes.

14. The method of claim 12 or 13, wherein the concatenated polypeptide comprises a protease cleavage site between each pair of the more than one T cell epitopes.

15. The method of claim 13 or 14, wherein the protease cleavage site is a cathepsin cleavage site.

16. The method of any one of the preceding claims, wherein the administration of the synthetic nanocarriers increases a tolerogenic phenotype.

17. The method of any one of the preceding claims, wherein the method further comprises identifying and/or providing the subject having or suspected of having PBC.

18. The method of any one of the preceding claims, wherein the immunosuppressant is an mTOR inhibitor.

19. The method of claim 18, wherein the mTOR inhibitor is rapamycin or a rapalog.

20. The method of any one of the preceding claims, wherein the immunosuppressant and/or PBC-associate autoantigen(s) is/are encapsulated in the synthetic nanocarriers.

21. The method of any one of the preceding claims, wherein the synthetic nanocarriers comprise lipid nanoparticles, polymeric nanoparticles, metallic nanoparticles, surfactantbased emulsions, dendrimers, buckyballs, nanowires, virus-like particles or peptide or protein particles.

22. The method of claim 21, wherein the synthetic nanocarriers comprise polymeric nanoparticles.

23. The method of claim 22, wherein the polymeric nanoparticles comprise a polyester, polyester attached to a polyether, polyamino acid, polycarbonate, polyacetal, polyketal, polysaccharide, polyethyloxazoline or polyethyleneimine.

24. The method of claim 22, wherein the polymeric nanoparticles comprise a polyester or a polyester attached to a polyether.

25. The method of claim 23 or 24, wherein the polyester comprises a poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid) or polycaprolactone.

26. The method of any one of claims 23-25, wherein the polymeric nanoparticles comprise a polyester and a polyester attached to a polyether.

27. The method of any one of claims 23-26, wherein the polyether comprises polyethylene glycol or polypropylene glycol.

28. The method of any one of the preceding claims, wherein the mean of a particle size distribution obtained using dynamic light scattering of a population of the synthetic nanocarriers is a diameter greater than 1 lOnm.

29. The method of claim 28, wherein the diameter is greater than 150nm.

30. The method of claim 29, wherein the diameter is greater than 200nm.

31. The method of claim 30, wherein the diameter is greater than 250nm.

32. The method of any one of claims 28-31, wherein the diameter is less than 5|am.

33. The method of claim 32, wherein the diameter is less than 4|am.

34. The method of claim 33, wherein the diameter is less than 3|am.

35. The method of claim 34, wherein the diameter is less than 2|am.

36. The method of claim 35, wherein the diameter is less than 1pm.

37. The method of claim 36, wherein the diameter is less than 750nm.

38. The method of claim 37, wherein the diameter is less than 500nm.

39. The method of claim 38, wherein the diameter is less than 450nm.

40. The method of claim 39, wherein the diameter is less than 400nm.

41. The method of claim 40, wherein the diameter is less than 350nm.

42. The method of claim 41, wherein the diameter is less than 300nm.

43. The method of any one of the preceding claims, wherein the load of immunosuppressant comprised in the synthetic nanocarriers, on average across the synthetic nanocarriers, is between 0.1% and 50% (weight/weight).

44. The method of claim 43, wherein the load is between 4% and 40%.

45. The method of claim 44, wherein the load is between 5% and 30%.

46. The method of claim 45, wherein the load is between 8% and 25%.

47. The method of any one of the preceding claims, wherein the load of PBC-associated antigen comprised in the synthetic nanocarriers, on average across the synthetic nanocarriers, is between 1% and 10% (weight/weight).

48. The method of any one of the preceding claims, wherein an aspect ratio of a population of the synthetic nanocarriers is greater than or equal to 1: 1, 1: 1.2, 1: 1.5, 1:2, 1:3, 1:5, 1:7 or 1: 10.

49. A composition comprising any one of or combinations of the populations of synthetic nanocarriers as described in any one of the preceding claims.

50. A kit comprising any one of or combinations of the populations of synthetic nanocarriers as described in any one of the preceding claims.