NZ719771B2 - Methods and compositions for sustained immunotherapy - Google Patents

Abstract

This disclosure provides compositions and methods for promoting the formation, expansion and recruitment of TR1 cells and /or Breg cells in an antigen-specific manner and treating autoimmune diseases and disorders in a subject in need thereof. The compositions comprise antigen-MHC class II-nanoparticle complexes. In an embodiment the antigen-MHC class II complex is linked to the nanoparticle via a PEG linker. PEG functionalized iron oxide nanoparticles can be prepared by thermally decomposing iron acetyl acetonate in the presence of functionalized PEG molecules, with or without benzyl ether. This simplifies the production of these nanoparticles. cle complexes. In an embodiment the antigen-MHC class II complex is linked to the nanoparticle via a PEG linker. PEG functionalized iron oxide nanoparticles can be prepared by thermally decomposing iron acetyl acetonate in the presence of functionalized PEG molecules, with or without benzyl ether. This simplifies the production of these nanoparticles.

Description

METHODS AND COMPOSITIONS FOR SUSTAINED IMMUNOTHERAPY CROSS-REFERENCE TO RELATED APPLICATION This application claims priority under 35 U.S.C. § 119(e) to US. Provisional Patent Application No. 61/899,826, filed November 4, 2013, the entire content of which is hereby incorporated by reference into the present disclosure.

FIELD OF DISCLOSURE This sure is directed to compositions and methods related to immunotherapy and medicine.

BACKGROUND Throughout and within this disclosure are technical and patent publications, referenced by an identifying citation or by an Arabic number. The full bibliographic citation corresponding to the Arabic number is found in the specification, preceding the claims. The disclosures of all references cited herein are incorporated by reference into the present application to more fully describe the state of the art to which this invention ns.

Autoimmune diseases are caused by an attack of self-tissues by the immune .

An ideal therapy would be one capable of selectively blunting the autoimmune se (against all antigenic epitopes targeted in that disease) without impairing systemic immunity (immune responses to foreign antigens). Unfortunately, the lymphocyte specif1cities ed in any one autoimmune disease are many and incompletely defined, making this a challenging goal.

SUMMARY In se to this need in the art, described herein are therapeutic compositions useful in ng mune disorders. One aspect relates to a method for expanding and/or developing populations of anti-pathogenic autoreactive T cells and/or B-cells in a subject in need thereof, which method comprises, or consists essentially of, or yet further consists of, administering to that subject an antigen-MHC class II-nanoparticle (“NP”) complex (“NP- x”), wherein the antigen is an autoimmunity related antigen or tigen. In some aspects all the antigens on the particular NP are identical or they can be different. In another aspect, the antigens on the NP have different amino acid sequences but are isolated from the same antigenic protein. In a fiarther aspect, the antigens on the NP are from different antigens. In r aspect, the MHCII are the same or different.

In one aspect, this disclosure provides a NP-complex comprising, or alternatively ting essentially of, or yet further consisting of, a nanoparticle; a MHC class II protein and a e-relevant antigen that can be in the form of an antigen/MHCII complex, for use in expanding and/or developing one or more populations of B-regulatory cells and TRl cells (e.g., TRl and CD4+ cells), in a t, wherein the nanoparticle has a diameter selected from the group of: from about 1 nm to about 100 nm in diameter; from about 1 nm to about 50 nm in diameter or from about 1 nm to about 20 nm or from about 5 nm to about 20 nm in diameter and the ratio of the number of antigen-MHCII complexes to nanoparticles is from about 10:1 to about 1000: 1. In one aspect, the complex has a MHC class II density from about 0.05 pMHCII/lOO nm2 NP surface area (including coating) to about 25 pMHCII/lOO nm2 NP surface area (including coating). The antigen is an autoantigen involved in an autoimmune response or mimic thereof such as, for example, pre-diabetes, diabetes, multiple sis (“MS”) or a multiple sclerosis-related disorder, and optionally wherein when the disease is pre-diabetes or diabetes, the autoantigen is an epitope from an n expressed by atic beta cells or the autoantigen IGRP, Insulin, GAD or IA-2 protein. In another aspect, the MHC class II component comprises all or part of a HLA-DR, , or HLA- DP. The antigen-MHC class II complex is covalently or valently linked to the nanoparticle. The nanoparticle can be orbable and/or biodegradable.

In a further aspect, the nanoparticle is non-liposomal and/or has a solid core, preferably a gold or iron oxide core. When covalently linked, the antigen-MHC class II complex is covalently linked to the nanoparticle through a linker less than 5 kD in size. In one aspect, the linker comprises polyethylene glycol (PEG). The pMHC can be linked to the nanoparticle or the nanoparticle coating by any structure, including but not limited to linkers or by cross-linking. In one aspect, the MHC is linked to the nanoparticle or the coating directionally through the C-terminus. ant has discovered that the density of the n-MHC class II complexes on the nanoparticle contributes to the therapeutic benefit. Thus as disclosed herein, the antigen-MHCII nanoparticle complex can have a defined y in the range of from about 0.05 MHC molecules per 100 nm2 of surface area of the nanoparticle (the e area measured to e any coating), assuming at least 2 MHCII, or alternatively at least 8, or alternatively at least 9, or atively at least 10, or alternatively at least 11, or alternatively at least 12, MHCII complexed to the nanoparticle. In one aspect the complex has a density of MHCII from about 0.01 MHCII per 100 nm2 (0.05 100 nmz) to about 30 MHCII/100 nmz, or alternatively from 0.1 100 nm2 to about 25 MHCII/100 nmz, or atively from about 0.3 MHCII/100 nm2 to about 25 MHCII/100 nmz, or alternatively from about 0.4 MHCII/100 nm2 to about 25 MHCII/100 nmz, or alternatively from about 0.5 MHCII/100 nm2 to about 20 MHCII/100 nmz, or alternatively from 0.6 MHCII/100 nm2 to about 20 MHCII/100 nmz, or atively from about 1.0 MHCII/100 nm2 to about 20 MHCII/100 nmz, or alternatively from about 5.0 MHCII/100 nm2 to about 20 MHCII/100 nm2, or alternatively from about 10.0 MHCII/100 nm2 to about 20 MHCII/100 nmz, or alternatively from about 15 MHCII/100 nm2 to about 20 MHCII/100 nmz, or alternatively at least about 0.5, or alternatively at least about 1.0, or alternatively at least about 5.0, or alternatively at least about 10.0, or alternatively at least about 15.0 MHCII/100 nm2, the nm2 surface area of the nanoparticle to include any coating. In one aspect, when 9 or at least 9 MHCII are complexed to a nanoparticle, the density range is from about 0.3 MHCII/100 nm2 to about 20 MHCII/100 nmz.

This disclosure also provides a composition comprising a therapeutically effective amount of the NP-complex as described herein and a carrier, e.g., a pharmaceutically able carrier. In one aspect, all plexes in the composition are identical. In another aspect, the NP-complexes of the composition include diverse or different MHC- antigen complexes.

Methods to make the complexes and compositions are further provided herein. The method can comprise, or alternatively consist essentially of, or yet fiarther consist of, non- covalently coating or covalently complexing antigen-MHC complexes (e. g., MHCII complexes) onto a nanoparticle. l and stic methods are also provided. In one aspect, a method is provided for promoting the formation, expansion and recruitment of B-regulatory cells and/or TRl cells (e. g., TRl and CD4+ cells) in an antigen-specific manner in a subject in need thereof, comprising, or alternatively ting essentially of, or yet r consisting of, stering to the subject an effective amount of the NP-complex or composition as described herein.

In r aspect, a method for treating or preventing an autoimmune disease or disorder as described herein, e. g., MS, a MS-related er, diabetes or pre-diabetes, in a subject in need thereof is provided, the method comprising, or alternatively consisting essentially of, or yet further consisting of, stering to the subject an ive amount of the NP-complex or composition as bed herein, n the autoantigen is disease- relevant for the disease to be treated, e.g., for the prevention or treatment of diabetes, the antigen is a diabetes-relevant antigen. In a further aspect, the autoimmune disease is MS or a MS-related disorder and the antigen is evant.

Kits are also ed. The kits comprise, or alternatively t essentially of, or yet fiarther consist of a NP-complex as bed herein or a composition and instructions for use.

In one aspect, provided herein is a method of making nanoparticles comprising thermally decomposing or heating a nanoparticle precursor. In one embodiment, the nanoparticle is a metal or a metal oxide nanoparticle. In one embodiment, the nanoparticle is an iron oxide nanoparticle. In one embodiment, the nanoparticle is a gold nanoparticle. In one embodiment, provided herein are the nanoparticles prepared in accordance with the t technology. In one embodiment, provided herein is a method of making iron oxide nanoparticles comprising a l decomposition reaction of iron acetyl acetonate. In one embodiment, the iron oxide nanoparticle obtained is water-soluble. In one aspect, iron oxide nanoparticle is suitable for protein conjugation. In one embodiment, the method comprises a single-step thermal osition reaction.

In one aspect, the thermal decomposition occurs in the presence of fianctionalized PEG molecules. Certain non-limiting examples of fianctionalized PEG linkers are shown in Table 1.

In one , the thermal decomposition ses heating iron acetyl acetonate.

In one embodiment, the thermal decomposition comprises heating iron acetyl acetonate in the presence of fianctionalized PEG molecules. In one embodiment, the thermal decomposition comprises heating iron acetyl acetonate in the presence of benzyl ether and functionalized PEG molecules.

Without being bound by theory, in one embodiment, functionalized PEG molecules are used as reducing reagents and as surfactants. The method of making nanoparticles provided herein simplifies and improves conventional methods, which use surfactants that are difficult to be displaced, or are not displaced to tion, by PEG molecules to render the particles water-soluble. tionally, surfactants can be expensive (e.g., phospholipids) or toxic (e.g., Oleic acid or oleilamine). In another aspect, without being bound by theory, the method ofmaking rticles obviates the need to use conventional tants, thereby achieving a high degree of molecular purity and water solubility.

In one embodiment, the thermal decomposition involves iron acetyl acetonate and benzyl ether and in the absence of tional surfactants other than those employed herein.

In one embodiment, the ature for the thermal decomposition is about 80 to about 300°C, or about 80 to about 200°C, or about 80 to about 150°C, or about 100 to about 250°C, or about 100 to about 200°C, or about 150 to about 250°C, or about 150 to about 250°C. In one embodiment, the thermal decomposition occurs at about 1 to about 2 hours of time .

In one embodiment, the method of making the iron oxide nanoparticles comprises a purification step, such as by using Miltenyi Biotec LS magnet column.

In one embodiment, the nanoparticles are stable at about 4°C in phosphate buffered saline (PBS) without any detectable degradation or aggregation. In one embodiment, the nanoparticles are stable for at least 6 months.

In one aspect, provided herein is a method of making nanoparticle complexes comprising contacting pMHC with iron oxide rticles ed herein. Without being bound by theory, pMHC encodes a Cysteine at its carboxyterminal end, which can react with the maleimide group in filnctionalized PEG at about about pH 6.2 to about pH 6.5 for about 12 to about 14 hours.

In one , the method of making nanoparticle xes comprises a purification step, such as by using Miltenyi Biotec LS magnet column.

DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in ation with the detailed description of specific embodiments presented herein.

FIGS. lA-lC show schematics ofNP-complexes. is a schematic of a single-chain pMHC-class I expression construct (top) and a representative flow cytometric profile of the binding of the corresponding pMHC tetramer (fluorochrome-labeled) to cognate CD8+ T-cells. is a schematic showing the linkers and two dimensional structure ofNP-complexes. As can be seen, one NP can contain the same antigen complexed to the nanoparticle core through various chemical linkers. shows maleimide- functionalized NPs conjugated to NPs. shows the structure of a typical pMHC class II monomer (top) and a representative FACS profile of cognate CD4+ T-cells d with the ponding pMHC tetramer or left unstained.

FIGS. 3A-3B show different TlD-relevant pMHC class II-NPs reverse hyperglycemia in newly diabetic NOD mice. shows dual mouse blood glucose curves. Mice were considered ‘cured’ when stably normoglycemic for 4 wk, after which treatment was withdrawn. HEL14_22, a foreign antigen, was used as l. shows incidence of disease reversal. shows intraperitoneal e-tolerance tests (IPTGTTs) and insulin- production ty in long-term cured mice. IDDM, diabetic untreated mice; Cured, mice with normoglycemia at 50 wk of age (>30 wk after treatment withdrawal); Control, age- matched non-diabetic untreated mice (50 wk-old). shows that TlD-relevant pMHC class II-NPs expand cognate autoreactive CD4+ T-cells. Data correspond to mice treated with 2.5mi/I-Ag7-NPs. Bottom right, expansion is specific for the pMHC on the NPs, as mice treated with 2.5mi/I-Ag7-NPs did not show increased percentages of two other autoreactive CD4+ T-cell specif1cities. PLN, pancreatic lymph nodes; MN, eric lymph nodes; BM, bone marrow (a reservoir of memory T-cells). shows that TlD-relevant pMHC class II-NPs expand cognate autoreactive CD4+ T-cells. Expansion is shown for spleen but similar patterns are seen in the pancreative lymph nodes, blood and marrow. “Onset” correspond to pre-treatment values; “Cured” are mice ed normoglycemic with pMHC-NP (analyzed at >3Owk of treatment withdrawal); “IDDM” are mice that relapsed upon ent awal (~25%); “50 wk-old” corresponds to age-matched untreated abetic controls. shows that TlD-relevant pMHC class II-NPs expand cognate memory-like latory-l (“Trl or TRl”) cells. shows that the autoreactive CD4+ T-cells expanded by pMHC class II-NP are IL-lO producers. IGRP126_145/I-Ag7 tetramer+ cells from mice treated with IGRP126_145/I- Ag7-NPs or control NPs were , challenged with cognate and non-cognate peptides and the sups assayed for cytokine content with luminex technology. shows that pMHC class II-NPs reverse hyperglycemia in an IL-10 and TGFb-dependent manner. shows ability of IGRP4_22/IAg7-NPs to restore lycemia (top), expand cognate Trl cells (bottom left) and ss autoantigen presentation in the PLNs (to IGRP206_214-reactive CD8+ T-cells; bottom right) of mice treated with cytokine blocking dies ). Anti-ILlO and anti-TGFB Abs partially restore autoantigen tation and inhibit the therapeutic effect of pMHC-NPs, without impairing Trl cell expansion.

FIGS. 10A-10B show that pMHC class II-NP therapy does not compromise systemic immunity. A shows that pMHC-NP-treated NOD mice can y clear an acute viral (vaccinia virus) infection (bottom, compare day 4 versus day 14 after infection) despite systemic expansion of autoregulatory Trl CD4+ T-cells (top). B shows that pMHC-NP-treated mice (10 doses) can mount antibody ses against KLH-DNP upon immunization in CFA, as ed to untreated and unvaccinated mice. shows that pMHC class II-NP therapy reduces the severity of established EAE in C57BL/6 mice. B6 mice were immunized with pMOG35-55 in CFA and d with pertussis toxin i.v. Mice were scored for signs of EAE using ished criteria over a 15- point scale. Affected mice were treated with two weekly doses of 7.5-22.5 ug of pMOGgg- 49/IAb-coated NPs, beginning 21 days after zation.

FIGS. 12A-12C show structure and properties ofpMHC class II-NPs. A is a cartoon depicting the different chemistries that can be used to covalently coat pMHCs onto functionalized, biocompatible iron oxide NPs. FIG. IZB is a transmission electron micrograph ofpMHC-coated NPs. C shows Dynamic Light Scattering profiles of pMHC-coated vs. uncoated NPs.

FIGS. 13A-13C show expansion and differentiation of cognate B-cells into Breg cells in pMHC class II-NP-treated mice. In A, l:l mixtures of PKH26-labeled/pulsed with 2.5mi peptide B-cells (bottom) (or dendritic cells, top) plus CFSE-labeled/GPI peptide- pulsed B-cells (bottom) (or tic cells, top) were injected into 2.5mi/IAg7-NP-treated NOD mice. Seven days later, the hosts were analyzed for presence of both subsets of B-cells (bottom) or dendritic cells (top). Left panels show representative results and Right histograms show a summary of the results obtained over several experiments. The data indicate that 2.5mi-peptide-pulsed B-cells (but not DCs) expand in 2.5mi/IAg7-NP-treated NOD mice. In B (left panel), Applicant compared the B-cell content in the pancreatic (PLN) and mesenteric (MLN) lymph nodes ofNOD mice treated with 2.5mi/IAg7-NPs versus NPs coated with l (diabetes-irrelevant) pMHC-NPs. Data show increased recruitment of B-cells in the . In B (right panel), Applicant compared the recruitment of B-cells to the PLNs as a function of Trl cell recruitment. Data were obtained using l different pMHC-NP ations. Data show a statistically-significant correlation between recruitment ofpMHC- NP-expanded TRl cells and B-cell recruitment to the PLNs. In B, Applicant ered B-cells, pulsed with 2.5mi or control peptides, from ILlO-eGFP knock-in NOD mice into several different donor mouse types (top labels). After 7 days, spleens were analyzed for conversion of the transfused B-cells into ILlO-producing (eGFP+) B-cells expressing high levels of CDld and CD5 (B-regulatory cells). Data show robust expansion and conversion of cognate (2.5mi-loaded) B-cells into B-reg cells only in 2.5mi/IAg7-NP- treated hosts. shows synthesis of surface onalized iron oxide nanoparticle by thermal decomposition of iron acetylacetonate and bioconjugation.

DETAILED DESCRIPTION It is to be understood that this invention is not limited to particular embodiments bed, 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 only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, the ar forms “ 3, (C , an”, and “the” include plural referents unless the t y dictates otherwise.

Thus, for example, reference to “an excipient” es a plurality of excipients.

I. DEFINITIONS Unless defined ise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein the ing terms have the following meanings.

As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited ts, but not ing others.

“Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a ition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention, such as itions for treating or preventing multiple sclerosis. sting of” shall mean excluding more than trace elements of other ingredients and ntial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

An “auto-reactive T cell” is a T cell that recognizes an “auto-antigen”, which is a molecule produced and contained by the same individual that contains the T cell.

A genic T cell” is a T cell that is harmful to a subject containing the T cell.

Whereas, a non-pathogenic T cell is not substantially harmful to a subject, and an anti- pathogenic T cells reduces, ameliorates, inhibits, or negates the harm of a pathogenic T cell.

As used herein the terms regulatory B-cells or B-regulatory cells gs”) intend those cells that are responsible for the anti-inflammatory effect, that is characterized by the expression of CDld, CD5 and the secretion of IL-10. B-regs are also identified by expression of Tim-l and can be induced through Tim-l on to promote tolerance. The ability of being B-regs was shown to be driven by many stimulatory factors such as toll-like receptors, CD40-ligand and others. However, filll terization of B-regs is ongoing. B- regs also express high levels of CD25, CD86, and TGF-B. This subset of B cells is able to suppress Thl proliferation, thus contributing to the maintenance of self-tolerance. The potentiation of B-reg function should become the aim ofmany immunomodulatory drugs, contributing to a better control of autoimmune diseases. See for example: ncbi.nlm.nih.gov/pubmed/23707422, last accessed on October 31, 2013. 2014/003014 T Regulatory 1 cells (Trl) are a subset of CD4+ T cells that have tory properties and are able to suppress antigen-specific immune responses in vitro and in vivo.

These T-regulatory 1 (Trl) cells are defined by their unique profile of cytokine production and make high levels of IL-10 and TGF-beta, but no IL-4 or IL-2. The IL-10 and TGF-beta produced by these cells mediate the inhibition of primary naive T cells in vitro. There is also eVidence that Tr1 cells exist in vivo, and the presence of high IL-lO-producing CD4(+) T cells in ts with severe combined deficiency who have received allogeneic stem- cell transplants have been documented. Tr1 cells are involved in the regulation of peripheral tolerance and they could potentially be used as a cellular therapy to modulate immune responses in vivo. See for example: ncbi.nlm.nih.gov/pubmed/10887343 last accessed on October 31, 2013, Type-1 T regulatory (Trl) cells are defined by their ability to produce high levels of IL-10 and TGF-beta. Tr1 cells specific for a variety of antigens arise in vivo, but may also differentiate from naive CD4+ T cells in the presence of IL-10 in vitro. Tr1 cells have a low proliferative capacity, which can be overcome by IL-15. Tr1 cells suppress naive and memory T helper type 1 or 2 responses via production of IL-10 and TGF-beta. Further characterization of Tr1 cells at the molecular level will define their mechanisms of action and clarify their relationship with other subsets of Tr cells. The use of Tr1 cells to identify novel s for the development ofnew therapeutic agents, and as a cellular y to modulate peripheral tolerance, can be foreseen. See for example, ncbi.nlm.nih.gov/pubmed/l1722624, last accessed on October 31, 2013.

The terms “inhibiting,” “reducing,” or “prevention,” or any variation of these terms, when used in the claims and/or the specification includes any able se or complete inhibition to achieve a desired result.

Throughout this application, the term “about” is used to indicate that a value es the standard deviation of error for the device or method being employed to determine the value. The term “about” when used before a cal designation, e.g., temperature, time, amount, and concentration, including range, indicates approximations whichmayvaryby(+)or(—)10%,5%,or1%.

By "biocompatible", it is meant that the components of the delivery system will not cause tissue injury or injury to the human biological system. To impart patibility, polymers and excipients that have had history of safe use in humans or with GRAS 2014/003014 (Generally Accepted As Safe) status, will be used preferentially. By biocompatibility, it is meant that the ingredients and excipients used in the composition will ultimately be "bioabsorbed" or cleared by the body with no e effects to the body. For a composition to be biocompatible, and be ed as non-toxic, it must not cause toxicity to cells.

Similarly, the term “bioabsorbable” refers to nanoparticles made from materials which undergo bioabsorption in viva over a period of time such that long term accumulation of the al in the patient is d. In a preferred embodiment, the biocompatible nanoparticle is bioabsorbed over a period of less than 2 years, preferably less than 1 year and even more preferably less than 6 months. The rate of bioabsorption is related to the size of the particle, the al used, and other factors well recognized by the skilled artisan. A mixture of bioabsorbable, biocompatible materials can be used to form the nanoparticles used in this invention. In one embodiment, iron oxide and a biocompatible, bioabsorbable polymer can be combined. For example, iron oxide and PGLA can be combined to form a nanoparticle.

An antigen-MHC-nanoparticle complex (“NP-complex”) refers to tation of a peptide, carbohydrate, lipid, or other antigenic segment, fragment, or epitope of an antigenic molecule or protein (i.e., self peptide or autoantigen) on a surface, such as a biocompatible biodegradable nanosphere. en" as used herein refers to all, part, fragment, or segment of a molecule that can induce an immune response in a t or an ion of anti- pathogenic cells.

A "mimic" is an analog of a given ligand or peptide, wherein the analog is substantially similar to the ligand. "Substantially similar" means that the analog has a binding profile similar to the ligand except the mimic has one or more fianctional groups or modifications that collectively accounts for less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5% of the molecular weight of the ligand.

The term "anti-pathogenic autoreactive T cell" refers to a T cell with anti- pathogenic properties (i.e., T cells that ract an autoimmune disease such as MS, a MS- related disease or disorder, or abetes). These T cells can include anti-inflammatory T cells, effector T cells, memory T cells, low-avidity T cells, T helper cells, autoregulatory T cells, cytotoxic T cells, natural killer T cells, TRl cells, CD4+ T cells, CD8+ T cells and the like.

The term “anti-inflammatory T cell” refers to a T cell that es an anti- inflammatory response. The anti-inflammatory fianction of the T cell may be accomplished through production and/or secretion of anti-inflammatory proteins, cytokines, chemokines, and the like. Anti-inflammatory proteins are also intended to encompass anti-proliferative signals that suppress immune ses. Anti-inflammatory proteins include IL-4, IL-lO, IL- 13, IL-21, IL-23, IL-27, IFN—u, TGF-B, IL-lra, G-CSF, and soluble receptors for TNF and IL-6. Accordingly, aspects of the sure relate to methods for treating, in a patient, an autoimmune disorder, such as MS, a MS-related disorder, diabetes or abetes, the method comprising, consisting essentially of or yet further consisting of administering to that patient an antigen-MHCII-nanoparticle complex, n the antigen is a disease-relevant antigen.

The term “IL-10” or “Interleukin-10” refers to a ne encoded by the IL-lO gene. The IL-lO sequence is represented by the GenBank Accession No.: NM_000572.2 (mRNA) and NP_000563.1 (protein).

The term “TGF-B” or “Transforming growth factor beta” refers to a protein that can have an anti-inflammatory effect. TGF-B is a secreted protein that exists in at least three isoforms called TGF-Bl, TGF-B2 and . It was also the original name for TGF-Bl, which was the founding member of this family. The TGF-B family is part of a superfamily of proteins known as the transforming growth factor beta superfamily, which includes inhibins, activin, anti-mullerian e, bone morphogenetic protein, decapentaplegic and Vg-l.

A "an effective amount" is an amount sufficient to e the intended purpose, non-limiting examples of such include: tion of the immune se, modulation of the immune response, suppression of an inflammatory response and modulation of T cell activity or T cell populations. In one aspect, the effective amount is one that functions to achieve a stated therapeutic purpose, e.g., a eutically effective amount. As described herein in detail, the effective amount, or dosage, depends on the purpose and the composition, component and can be determined according to the present disclosure.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the cation may mean "one," but it is also consistent with the meaning of "one or more, at least one," and "one or more than one." By phere," "NP," or “nanoparticle” herein is meant a small discrete particle that is administered singularly or pluraly to a subject, cell specimen or tissue specimen as appropriate. In certain embodiments, the rticles are substantially spherical in shape.

In certain embodiments, the nanoparticle is not a me or viral particle. In further embodiments, the nanoparticle is solid or has a solid core. The term "substantially spherical," as used herein, means that the shape of the particles does not e from a sphere by more than about 10%. s known antigen or peptide complexes of the invention may be applied to the particles. The nanoparticles of this invention range in size from about 1 nm to about 1 um and, preferably, from about 1 nm to about 100 nm or alternatively from about 1 nm to about 50 nm, or alternatively from about 5 to 50 nm or alternatively from about 5 nm to 100 nm, and in some aspects refers to the average or median diameter of a plurality of nanoparticles when a plurality of nanoparticles are intended. r nanosize les can be obtained, for example, by the process of fractionation whereby the larger particles are allowed to settle in an aqueous solution. The upper n of the solution is then recovered by methods known to those of skill in the art. This upper portion is enriched in smaller size particles. The process can be repeated until a desired e size is generated.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein the phrase "immune response" or its equivalent "immunological response" refers to the development of a cell-mediated response ted by antigen- specific T cells or their ion products). A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules, to treat or prevent a viral infection, expand antigen-specific Breg cells, TCl, CD4+ T helper cells and/or CD8+ cytotoxic T cells and/or disease generated, autoregulatory T cell and B cell y” cells. The response may also involve activation of other components.

The terms matory response" and "inflammation" as used herein indicate the complex biological response of vascular tissues of an individual to harmful stimuli, such as pathogens, damaged cells, or irritants, and includes secretion of cytokines and more particularly of pro-inflammatory cytokines, i.e. cytokines which are produced predominantly by activated immune cells and are involved in the amplification of inflammatory reactions. ary pro-inflammatory cytokines include but are not limited to IL-1, IL-6, IL-lO, TNF- 2014/003014 a, IL-l7, IL21, IL23, IL27 and TGF-B. Exemplary inflammations include acute inflammation and c inflammation. Acute ation indicates a short-term process characterized by the classic signs of inflammation (swelling, redness, pain, heat, and loss of filnction) due to the infiltration of the tissues by plasma and leukocytes. An acute inflammation typically occurs as long as the ous stimulus is present and ceases once the stimulus has been removed, broken down, or walled off by scarring (fibrosis). Chronic inflammation indicates a ion characterized by concurrent active inflammation, tissue destruction, and attempts at repair. Chronic inflammation is not characterized by the classic signs of acute inflammation listed above. Instead, chronically inflamed tissue is characterized by the infiltration of mononuclear immune cells (monocytes, hages, lymphocytes, and plasma cells), tissue destruction, and attempts at healing, which include angiogenesis and fibrosis. An inflammation can be inhibited in the sense of the present disclosure by affecting and in particular inhibiting any one of the events that form the complex biological response associated with an inflammation in an dual.

An autoimmune disorder may include, but is not limited to, diabetes melitus, pre- diabetes, transplantation rejection, multiple sclerosis,a le-sclerosis related disorder, premature ovarian failure, scleroderm, Sjogren's disease, lupus, vilelego, alopecia (baldness), polyglandular failure, Grave's disease, yroidism, polymyosititis, pemphigus, s disease, colititis, autoimmune hepatitis, hypopituitarism, myocardititis, Addison's disease, autoimmune skin diseases, uveitis, pernicious anemia, hypoparathyroidism, and/or rheumatoid arthritis. In n s, a peptide component of an antigen/MHCII/particle complex is derived or designed from an autoantigen or an autoantigen epitope, or a mimic thereof, involved in the mune response to be probed, modulated, or blunted by the treatment. In particular aspects, the autoantigen is a peptide, carbohydrate, or lipid. In certain aspects, an autoantigen is a fragment, epitope, or peptide of a protein, carbohydrate, or lipid expressed by n cells of a subject, such as pancreatic beta cells, and e, but is not limited to a fragment of IGRP, Insulin, GAD or IA-2 protein. Various such proteins or epitopes have been identified for a variety of autoimmune conditions. The autoantigen may be a peptide, carbohydrate, lipid or the like derived from a second endocrine or neurocrine component, such as peri-islet Schwann cell or the like.

As used , the term “disease-relevant” n s an antigen or fragment thereof selected to treat a selected disease. For e, a diabetes-relevant antigen is an antigen or fragment thereof that will treat diabetes. A MS-relevant antigen is selected to treat MS. A diabetes-relevant antigen would not be selected to treat MS. Similarly, an autoimmunity related antigen is an antigen that is relevant to an autoimmune e and would not be selected for the treatment of a disorder or disease other than autoimmunity, e.g., As used herein, the term “diabetes” intends a variable disorder of carbohydrate metabolism caused by a combination of hereditary and environmental factors and is usually characterized by inadequate secretion or utilization of insulin, by excessive urine production, by excessive amounts of sugar in the blood and urine, and by thirst, hunger, and loss of weight. Diabetes is characterized by Type 1 diabetes and Type 2 diabetes. The se ic (“NOD”) mouse is an accepted animal model for the study and treatment of diabetes.

Type 1 Diabetes (TlD) in mice is associated with autoreactive CD8+ T-cells. Nonobese diabetic (NOD) mice develop a form of TlD, closely resembling human TlD, that results from selective destruction of pancreatic B cells by T-cells recognizing a growing list of autoantigens. Although initiation of T1D clearly requires the contribution of CD4+ cells, there is compelling ce that TlD is CD8+ T-cell-dependent. It has been discovered that a significant fraction of islet-associated CD8+ cells in NOD mice use CDR3-invariant VOLl7- J(142+ TCRs, referred to as ‘8.3-TCR—like’. These cells, which recognize the mimotope NRP-A7 (defined using combinatorial peptide ies) in the context of the MHC molecule Kd, are already a significant component of the earliest NOD islet CD8+ infiltrates, are ogenic, and target a peptide from islet-specific glucosephosphatase catalytic subunit- related protein , a n of unknown on. The CD8+ cells that recognize this peptide (IGRP206_214, similar to NRP-A7) are unusually frequent in the circulation (>l/200 CD8+ cells). Notably, progression of insulitis to diabetes in NOD mice is invariably accompanied by cyclic expansion of the ating IGRP206_214-reactive CD8+ pool, and by avid maturation of its islet-associated counterpart. More ly, it has been shown that islet-associated CD8+ cells in NOD mice recognize multiple IGRP epitopes, ting that IGRP is a dominant autoantigen for CD8+ cells, at least in murine TlD. NOD islet- associated CD8+ cells, particularly those found early on in the disease process also recognize an insulin epitope (Ins B15_23).

Association studies have suggested that certain HLA class I s (i.e., HLA- ) afford susceptibility to human TlD. Pathology studies have shown that the insulitis lesions of newly diagnosed patients consist mostly of (HLA class I-restricted) CD8+ s, which are also the predominant cell population in patients d by transplantation with pancreas isografts (from identical twins) or allografts (from related donors).

Insulin is a key target of the antibody and CD4+ response in both human and murine TlD. The human insulin B chain epitope hInsB10_1g is ted by HLA-A*0201 to autoreactive CD8+ cells both in islet transplant recipients and in the course of spontaneous disease. In addition, four onal peptides have been identified from mouse pre-proinsulin l or 2 that are recognized by islet-associated CD8+ T-cells from HLA-A*0201-transgenic mice in the context of HLA-A*0201.

As used herein, the term “pre-diabetes” intends an asymptomatic period ing a diabetic condition characterized by subclinical beta cell damage wherein the patient exhibits normal plasma glucose levels. It also is characterized by the presence of islet cell autoantibodies (ICAs) and, when close to the onset of clinical symptoms, it may be anied by rance to glucose.

As used herein, the term “multiple sclerosis” or “MS” intends the autoimmune disorder in which the body's immune system eats away at the protective sheath that covers nerves. This interferes with the communication between the brain and the rest of the body.

Ultimately, this may result in deterioration of the nerves themselves, a process that is not reversible.

As used herein, the term “multiple sis-related disorder” intends a disorder that co-presents with a susceptibility to MS or with MS. Non-limiting examples of such include neuromyelitis optica (NMO), uveitis, athis pain sis, sclerosis, arteriosclerosis, sclerosis disseminata systemic sclerosis, spino-optical MS, primary ssive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis, and ataxic sclerosis, The terms "epitope" and "antigenic determinan " are used interchangeably to refer to a site on an antigen to which B and/or T cells respond or recognize. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a n. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, -l6- and more usually, at least 5 or 8-20 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional r magnetic resonance. See, e. g., Glenn E. , Epitope Mapping Protocols (1996). T-cells recognize uous epitopes of about nine amino acids for CD8 cells or about 13-15 amino acids for CD4 cells. T cells that recognize the epitope can be identified by in Vitro assays that measure antigen-dependent proliferation, as determined by midine incorporation by primed T cells in response to an epitope (Burke et al., J. Inf.

Dis., 170:1 1 10-1 1 19, 1994), by antigen-dependent killing (cytotoxic T cyte assay, Tigges et al., J. Immunol., 156(10):3901-3910, 1996) or by cytokine secretion. The presence of a cell-mediated immunological response can be ined by proliferation assays (CD4+ T cells) or CTL (cytotoxic T lymphocyte) assays.

Optionally, an antigen or preferably an epitope of an antigen, can be chemically conjugated to, or sed as, a fusion protein with other ns, such as MHC and MHC related proteins.

As used herein, the terms “patient” and ct” are used synonymously and refer to a mammal. In some ments the patient is a human. In other embodiments the patient is a mammal commonly used in a laboratory such as a mouse, rat, simian, canine, feline, bovine, equine, or ovine.

As used in this ation, the term "polynucleotide" refers to a nucleic acid molecule that either is recombinant or has been isolated free of total genomic nucleic acid.

Included within the term "polynucleotide" are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, Viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, ed ntially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be RNA, DNA, analogs thereof, or a combination thereof A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide of the following lengths: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1095, 1100, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000, or more tides, nucleosides, or base pairs. It also is contemplated that a particular polypeptide from a given species may be encoded by nucleic acids containing natural variations that have slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein, polypeptide, or peptide.

A polynucleotide is composed of a specific ce of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical entation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.

The term “isolated” or “recombinant” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, tively that are present in the natural source of the macromolecule as well as polypeptides. The term “isolated or recombinant nucleic acid” is meant to include nucleic acid fragments which are not naturally ing as fragments and would not be found in the l state. The term “isolated” is also used herein to refer to polynucleotides, polypeptides and proteins that are ed from other cellular proteins and is meant to ass both purified and recombinant ptides. In other embodiments, the term “isolated or recombinant” means separated from constituents, cellular and otherwise, in which the cell, , polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype. An isolated cleotide is separated from the 3 ’ and 5 ’ contiguous tides with which it is normally associated in its native or natural environment, e.g., on the some. As is apparent to those of skill in the art, a non- naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart.

A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those bed in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement , section 7.7.18, Table 7.7. l. Preferably, default parameters are used for alignment. A preferred ent program is BLAST, using default parameters. In particular, red programs are BLASTN and BLASTP, using the following default ters: Genetic code = rd; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + te + PIR.

Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.

It is to be inferred without explicit recitation and unless otherwise intended, that when the present invention relates to an antigen, ptide, protein, polynucleotide or antibody, an equivalent or a biologically equivalent of such is intended within the scope of this invention. As used herein, the term "biological equivalent thereof ’ is intended to be synonymous with "equivalent thereof’ when referring to a nce antigen, protein, antibody, fragment, polypeptide or nucleic acid, and intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof In one aspect, an equivalent polynucleotide is one that hybridizes under stringent conditions to the cleotide or ment of the polynucleotide as bed herein for use in the described methods. In another aspect, an equivalent antibody or antigen binding polypeptide intends one that binds with at least 70 % , or alternatively at least 75 % or alternatively at least 80 % or alternatively at least 85 %, or , , alternatively at least 90 %, or alternatively at least 95 % affinity or higher affinity to a reference antibody or antigen binding nt. In another aspect, the equivalent thereof competes with the binding of the antibody or antigen binding fragment to its antigen under a competitive ELISA assay. In another aspect, an equivalent intends at least about 80 % gy or identity and alternatively, at least about 85 %, or alternatively at least about 90 %, or alternatively at least about 95 %, or alternatively 98 % percent homology or identity and exhibits ntially equivalent biological activity to the reference protein, polypeptide or nucleic acid.

"Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the tide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two s forming a duplex structure, three or more strands forming a multi-stranded complex, a single selfhybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the tion of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about 10x SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC. Examples of high ency conditions include: tion temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about 0. lx SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, 0.lx SSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with l, 2, or more washing steps, and wash tion times are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.

“Homology” or ity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid les. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of ng or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% ty, or alternatively less than 25% identity, with one of the ces of the present invention.

"Homology" or ity" or "similarity" can also refer to two nucleic acid molecules that hybridize under ent conditions.

As used herein, the terms "treating," "treatment" and the like are used herein to WO 63616 mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect attributable to the disorder. In one aspect, treatment indicates a ion in the signs of the disease using an established scale.

IGRP, which is encoded by a gene ed on chromosome 2q28-32 that ps a TlD susceptibility locus, IDDM7 (2q3 1), has also been recently fied as a ell autoantigen of potential relevance in human TlD. Two HLA-A*O20l-binding epitopes of human IGRP (hIGRP228_236 and hIGRP265_273) are recognized by islet-associated CD8+ cells from murine MHC class I-deficient NOD mice expressing an HLA-A*0201 transgene. Non- limited examples of IGRP antigens binding to the murine MHC class II molecule (IAg7) include for example, IGRP206_214, which comprises the antigenic peptide VYLKTNVFL and IGRP“; which comprises the antigenic peptide LHRSGVLIIHHLQEDYRTY or an equivalent thereof, and IGRP128_145 ,which comprises the antigenic peptide TAALSYTISRMEESSVTL or an equivalent thereof.

“To prevent” intends to prevent a disorder or effect in vitro or in vivo in a system or subject that is predisposed to the disorder or effect.

A “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant. In certain embodiments, the composition does not contain an adjuvant.

A “pharmaceutical composition” is intended to include the ation of an active agent with a r, inert or active, making the ition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

The term "fianctionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode ically equivalent amino acids (see below Table).

Codon Table WO 63616 As used herein, a "protein" or "polypeptide" or "peptide" refers to a molecule comprising at least five amino acid residues.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, r, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. 11. DESCRIPTIVE EMBODIMENTS There is currently no therapeutic platform that enables te suppression of polyclonal autoimmune responses without compromising systemic immunity. Applicant’s disclosure described herein enables the design of autoimmune disease-specific nes that turn active disease-specific CD4+ T-cells and B-cells into e, mono-specific tory CD4+ T-cells and B-cells that coordinately suppress all other autoreactive T and B-cell responses of the host, regardless of their fine nic specificity, and yet with exquisite disease-specificity and without impairing ic immunity.

The autoantigenic complexity of Type 1 Diabetes (TlD).

TlD is caused by a chronic autoimmune se that progressively erodes the pancreatic Beta-cell mass. B-cell destruction in both humans and NOD mice is ed by T- cells recognizing many autoantigens (Tsai, S. et al. (2008) Adv. Immunol. 100:79-124; Lieberman, S. et al. (2003) Tissue Antigens -377). Although the precise sequence of events remains ill defined, current evidence suggests that TlD requires CD4+ and CD8+ cells; that active T cells entiate into killers by engaging B-cell antigens on local APCs; and that these T-cells target a wide repertoire of autoantigens (Tsai, S. et al. (2008) Adv. Immunol. 100:79-124; Santamaria, P. (2010) Immunity 32:437-445).

It has been shown that soluble peptides can induce peptide-specific T-cell tolerance in viva, but cannot blunt poly-specific autoimmune responses (Han et al. (2005) Nature Medicine ll(6):645-652). Unexpectedly, it was found that, unlike therapy with soluble peptide, therapy with NPs coated with a single levant pMHC class I (originally used as a negative contol) blunted the progression of TlD in pre-diabetic NOD mice and restored normoglycemia in diabetic animals (Tsai, S. et al. (2010) Immunity 32:568-5 80). Subsequent work led to the unexpected discovery that pMHC-NP y ons by expanding, in an epitope-specific manner, autoantigen-experienced autoreactive CD8+ cells that suppressed the recruitment of other autoantigenic T cell cities by inhibiting and killing autoantigen-loaded APCs. More recently, Applicant has found that this therapeutic platform can be harnessed for the in vivo ion of active T-regulatory CD4+ cells.

Specifically, Applicant discovered that NPs coated with individual TlD-relevant pMHC class II expand disease-specific TRl CD4+ T-cells, expressing the TRl markers CD49b and LAG3 (Gagliani, N. et al. (2013) Nature Medicine 19:739-746) and producing the cytokines ILlO and TGF-B (see below).

Collectively, these observations t a new paradigm in the progression of autoimmunity, stating that chronic stimulation of naive autoreactive CD8+ or CD4+ T cells by nous epitopes triggers their differentiation into memory-like autoreactive regulatory T cells; and that these memory autoreactive regulatory cells suppress the activation of both cognate and non-cognate high-avidity autoreactive T cell specificities by suppressing and/or killing autoantigen-loaded APCs (Tsai, S. et al. (2010) Immunity 32:568-5 80).

Importantly, and without being bound by theory, any single epitope (pMHC) city involved in an autoimmune disease (among many) can be used, when coated as a ligand onto NPs, to blunt complex autoimmune responses. It is Applicant’s belief that these NP preparations cannot activate na'1've T-cells, hence induce or T-cell responses, because they lack key co-stimulatory molecules, such as CD80 and CD86. In fact, cognate naive and effector autoreactive cells are deleted by this approach. Therefore, the therapeutic approach that enabled its discovery provide a platform for a new class of therapeutics in autoimmunity, potentially capable of resolving polyclonal autoimmune responses in a e- and organspecif1c manner without compromising systemic immunity.

III. METHODS Medical and diagnostic methods are also provided. In one aspect, a method is provided for promoting the ion, expansion and recruitment of B-regulatory cells and/or TRl cells (e. g., TRl and CD4+ cells) in an antigen-specific manner in a subject in need thereof, comprising, or alternatively ting essentially of, or yet further consisting of, stering to the subject an effective amount of the NP-complex or composition as described herein.

In another aspect, a method for treating or preventing an autoimmune disease or disorder as described herein, e. g., MS, a MS-related disorder, diabetes or pre-diabetes, in a subject in need thereof is provided, the method comprising, or alternatively consisting essentially of, or yet further consisting of, administering to the subject an effective amount of the NP-complex or composition as bed herein, n the autoantigen is disease- relevant for the e to be treated, e.g., for the tion or treatment of diabetes, the antigen is a diabetes-relevant antigen. In a further aspect, the autoimmune disease is multiple-sclerosis or a multiple-sclerosis d disorder and the antigen is MS-relevant.

Peptide antigens for the treatment or prevention of pre-diabetes or es, include, but are not limited to hInsB10_1g (HLVEALYLV), hIGRP228_236 LWSV), hIGRP265_273 (VLFGLGFAI), IGRP206_214 (VYLKTNVFL), NRP-A7 (KYNKANAFL), NRP-I4 (KYNIANVFL), NRP-V7 NVFL), YAI/Db (FQDENYLYL) and/or INS 315,23 (LYLVCGERG), GAD65114_123, VMNILLQYVV; 36_545, RMMEYGTTMV; GFAP143_151, NLAQTDLATV; GFAP214_222, QLARQQVHV; IA-2172_180, SLSPLQAEL; IA- 2482490, SLAAGVKLL; IA-2805_813, VIVMLTPLV; ppIAPP5_13, KLQVFLIVL; ppIAPP9_17, FLIVLSVAL; IGRP152_160, MLI; IGRP211_219, NLFLFLFAV; IGRP215_223, FLFAVGFYL; IGRP222_230, YLLLRVLNI; IGRP228_236, LNIDLLWSV; IGRP265_273, VLFGLGFAI; IGRP293_301, TSL; Pro-insulinL2_10, ALWMRLLPL; Pro-insulinL3_11, LWMRLLPLL; sulinL6_14, RLLPLLALL; Pro-insulinBs_14, HLCGSHLVEA; Pro- insulinBlo_1g, YLV; sulinBl4_22, ALYLVCGER; Pro-insulin315_24, ERGF; Pro-insulinBl7_25, LVCGERGFF; Pro-insulinBlg_27, VCGERGFFYT; Pro- insulin1320_27, GERGFFYT; sulin1321_29, ERGFFYTPK; Pro-insulin1325_01, FYTPKTRRE; Pro-insulinBz7_cs, TPKTRREAEDL; Pro-insulinczo_28, SLQPLALEG; Pro-insulin025_33, ALEGSLQKR; Pro-insulin029_A5, SLQKRGIVEQ; Pro-insulinA1_10, GIVEQCCTSI; Pro- insulinA2_10, IVEQCCTSI; Pro-insulinA12_20, SLYQLENYC or equivalents and/or combinations f. Additional examples include ProIns 76-90, SLQPLALEGSLQKRG, ProIns 79-89, PLALEGSLQKR, ProIns 90-109, GIVEQCCTSICSLYQLENYC, ProIns 94- 105, QCCTSICSLYQL, GAD 247-266, NMYAMMIARFKMFPEVKEKG, GAD 255-265, RFKMFPEVKEK, GAD 555-567, NFFRMVISNPAAT, IGRP 13-25, QHLQKDYRAYYTF, IGRP 8-27, GVLIIQHLQKDYRAYYTFLN, ProIns B24-C36, FFYTPMSRREVED and equivalents of each thereof.

Whent the method is directed to the treatment of MS or MS-related disorders, the complex includes antigens related to multiple sclerosis. Such antigens include, for example, those disclosed in US. Patent Publication No. 2012/0077686, and ns derived from myelin basic protein, myelin associated glycoprotein, myelin oligodendrocyte protein, proteolipid n, oligodendrocyte myelin oligoprotein, myelin associated oligodendrocyte basic protein, endrocyte specific protein, heat shock proteins, oligodendrocyte specific ns NOGO A, glycoprotein Po, peripheral myelin protein 22, and 2'3'-cyclic nucleotide 3'-phosphodiesterase. In certain embodiments, the antigen is derived from Myelin Oligodendrocyte Glycoprotein (MOG). Non-limited examples include, for example, MAG287_295, SLLLELEEV; MAG509_517, LMWAKIGPV; MAG556_564, VLFSSDFRI; MBP110_ 118, SLSRFSWGA; MOG114_122, KVEDPFYWV; MOG166_175, RTFDPHFLRV; MOG172_180, FLRVPCWKI; MOG179_188, KITLFVIVPV; MOG188_196, VLGPLVALI; MOG181_189, PVL; MOG205_214, RLAGQFLEEL; PLP80_88, FLYGALLLA or equivalents or ations thereof.

Additional non-limiting examples of antigens that can be used in this invention comprise polypeptides comprising, or atively consisting essentially of, or yet r ting of the polypeptides of the group: MOG35_55, MEVGWYRSPFSRVVE-H..YRNG K; MOG36_55, EVGWYRSPFSRVVI-HXRNGK; MAG287_295, SLLLELEEV; MAG509_517, LMWAKIGPV; MAG556_564, VLFSSDFRI; MBPImng, SLSRFSWGA; _122, KVEDPFYWV; MOG166_175, RTFDPHFLRV; MOG172_180, WKI; MOG179_188, KITLFVIVPV; _196, VLGPLVALI; MOG181_189, TLFVIVPVL; MOG205_214, RLAGQFLEEL; PLP80_88, FLYGALLLA, or an equivalent of each thereof, or combinations thereof.

Methods to determine and monitor the therapy are known in the art and briefly described herein. When red in vitro, administration is by contacting the composition with the tissue or cell by any appropriate method, e.g., by administration to cell or tissue culture medium and is useful as a screen to determine if the y is appropriate for an individual or to screen for alternative therapies to be used as a substitute or in combination with the disclosed compositions. When stered in vivo, stration is by systemic or local administration. In vivo, the methods can be ced on a non-human animal to screen alternative therapies to be used as a substitute or in combination with the disclosed compositions prior to human administration. In a human or non-human , they are also useful to treat the disease or disorder.

The above methods require stration of an effective amount of a NP-complex.

The MHC of the antigen-MHC-nanoparticle complex can be MHC I, MHC II, or assical MHC but preferably MHCII. MHC proteins are described herein. In one embodiment, the MHC of the antigen-MHC-nanoparticle complex is a MHC class I. In another embodiment, the MHC is a MHC class II. In other embodiments, the MHC component of the antigen-MHC-nanoparticle complex is MHC class II or a non-classical MHC molecule as described herein. In one aspect, the antigen comprises, or alternatively consists essentially of, or yet further consists of the polypeptide GWYRSPFSRVVH or an equivalent of GWYRSPFSRVVH.

The size of the nanoparticle can range from about 1 nm to about 1 um. In certain embodiments, the nanoparticle is less than about 1 um in diameter. In other embodiments, the nanoparticle is less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm in diameter. In further embodiments, the nanoparticle is from about 1 nm to about 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 75 nm, or 100 nm in diameter. In specific embodiments, the nanoparticle is from about 1 nm to about 100 nm, about 1 nm to about 50 nm, about 1 nm to about 20 nm, or about 5 nm to about 20 nm.

The size of the complex can range from about 5 nm to about 1 um. In certain embodiments, the complex is less than about 1 um or alternatively less than 100 nm in diameter. In other embodiments, the complex is less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm in diameter. In further embodiments, the complex is from about 5 nm or 10 nm to about 50 nm, or about 5 nm to about 75 nm, or about 5 nm to about 50 nm, or about 5 nm to about 60 nm, or from about 10 nm to about 60 nm, or in one aspect about 55 nm.

Applicant has discovered that the density of the antigen-MHC complexes on the nanoparticle contributes to the therapeutic benefit. Thus, as disclosed herein the antigen- MHC nanoparticle x can have a defined density in the range of from about 0.05 MHC molecules per 100 nm2 of surface area of the nanoparticle including the complex, assuming at least 2 MHC, or alternatively at least 8, or alternatively at least 9, or atively at least 10, or alternatively at least 11, or alternatively at least 12, MHC complexed to the nanoparticle.

In one aspect the complex has a y of MHC from about 0.01 MHC per 100 nm2 (0.05 MHC/100 nmz) to about 30 MHC/100 nm2, or alternatively from 0.1 MHC/100 nm2 to about 0 nm2, or alternatively from about 0.3 MHC/100 nm2 to about 25 MHC/100 nmz, or alternatively from about 0.4 MHC/100 nm2 to about 25 MHC/100 nmz, or alternatively from about 0.5 MHC/100 nm2 to about 20 MHC/100 nm2, or alternatively from 0.6 MHC/100 nm2 to about 20 MHC/100 nmz, or atively from about 1.0 MHC/100 nm2 to about 20 MHC/100 nmz, or alternatively from about 5.0 MHC/100 nm2 to about 20 MHC/100 nmz, or alternatively from about 10.0 MHC/100 nm2 to about 20 MHC/100 nmz, or alternatively from about 15 MHC/100 nm2 to about 20 MHC/100 nm2, or alternatively at least about 0.5, or atively at least about 1.0, or atively at least about 5.0, or alternatively at least about 10.0, or alternatively at least about 15.0 MHC/100 nm2 .

In one aspect, When 9 or at least 9 MHC are complexed to a nanoparticle, the y range is from about 0.3 MHC/100nm2 to about 20 MHC/100nm2.

In one of its method aspects, there is provided a method for accumulating B- tory cells and/or anti-inflammatory T cells in a t in need thereof. In a further embodiment, the T cell is a CD4+ or CD8+ T cell. In a related embodiment, the T cell secretes IL-10 or TGFB. The method comprises, consists essentially of, or yet further consists of administering to a patient in need thereof an effective amount of the antigen-MHC nanoparticle x as described .

In one embodiment, the compositions and methods described herein are for treating an autoimmune disorder such as MS, MS-associated disorder, diabetes or pre-diabetes. The method comprises, consists essentially of, or yet further consists of administering to a t in need thereof an effective amount of the antigen-MHCII nanoparticle complex as described herein.

Details regarding modes of administration in vitro and in vivo are described within.

This disclosure also provides use of the NP-complexes for the ation of ments for the treatment and/or prevention of diseases and disorders as described herein.

IV. ANTIGEN-MHC-NANOPARTICLE COMPLEXES A. Polypeptides and Polynucleotides Further aspects relate to an isolated or purified polypeptide antigens, comprising, or consisting essentially of, or yet further consisting of, the amino acid sequences as described herein, or a polypeptide having at least about 80% sequence identity, or alternatively at least 85 %, or alternatively at least 90%, or alternatively at least 95 %, or alternatively at least 98 % sequence identity to the amino acid sequences of the ns, or polypeptides encoded by polynucleotides having at about 80% sequence identity, or alternatively at least 85 %, or alternatively at least 90%, or alternatively at least 95 %, or alternatively at least 98 % sequence identity to the polynucleotide ng the amino acid sequences of the antigen, or its complement, or a polypeptide encoded by a polynucleotide that hybridizes under conditions of moderate to high stringency to a polynucleotide encoding the amino acid ce of the antigens, or its complement. Also provided are ed and purified polynucleotides encoding the antigen polypeptides disclosed herein, or amino acids having at least about 80% sequence identity thereto, or atively at least 85 %, or atively at least 90%, or alternatively at least 95 %, or alternatively at least 98 % sequence identity to the disclosed sequences, or an equivalent, or a polynucleotide that hybridizes under stringent conditions to the polynucleotide, its equivalent or its complement and ed or purified polypeptides encoded by these polynucleotides. The polypeptides and polynucleotides can be ed with non-naturally occurring nces with which they are not associated with in , e. g., carriers, pharmaceutically acceptable rs, vectors and MHC molecules, nanoparticles as known in the art and as described herein. ns, including segments, fragments and other molecules derived from an antigenic species, including but not limited to peptides, carbohydrates, lipids or other molecules presented by classical and non-classical MHC molecules of the invention are typically complexed or operatively coupled to a MHC molecule or derivative f.

Antigen recognition by T lymphocytes is major ompatibility complex (MHC)- restricted. A given T lymphocyte will recognize an antigen only when it is bound to a particular MHC molecule. In general, T lymphocytes are stimulated only in the presence of self -MHC molecules, and antigen is recognized as fragments of the antigen bound to self MHC les. MHC restriction def1nes T lymphocyte city in terms of the antigen recognized and in terms of the MHC molecule that binds its antigenic fragment(s). In particular aspects n ns will be paired with certain MHC molecules or polypeptides derived therefrom.

The term "operatively coupled" or "coated" as used , refers to a situation where individual polypeptide (e. g., MHC) and antigenic (e.g., peptide) components are combined to form the active complex prior to binding at the target site, for example, an immune cell. This includes the situation where the individual polypeptide complex components are synthesized or recombinantly expressed and subsequently isolated and combined to form a complex, in vitro, prior to administration to a subject; the situation where a chimeric or fusion polypeptide (i.e., each discrete protein component of the complex is ned in a single polypeptide chain) is synthesized or recombinantly expressed as an intact x. Typically, polypeptide complexes are added to the nanoparticles to yield nanoparticles with adsorbed or coupled polypeptide complexes having a ratio of number of molecules:number of nanoparticle ratios from about, at least about or at most about about 0.1, 0.5, 1, 3, 5, 7, 10, 15, 20, 25, 30, 35, 40, 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500 or more to:1, more typically 0.1 :1, 1:1 to 50:1 or 300: 1. The polypeptide content of the nanoparticles can be determined using standard techniques.

B. MHC Molecules ellular and extracellular antigens present quite different challenges to the immune system, both in terms of recognition and of appropriate response. Presentation of 2014/003014 antigens to T cells is mediated by two distinct classes of molecules MHC class I (MHC-I) and MHC class II (MHC-II) (also fied as “pMHC” ), which utilize distinct antigen processing pathways. Peptides derived from intracellular antigens are presented to CD8+ T cells by MHC class I molecules, which are expressed on virtually all cells, while extracellular antigen-derived peptides are presented to CD4+ T cells by MHC-II molecules. However, there are certain exceptions to this dichotomy. Several studies have shown that peptides generated from endocytosed particulate or soluble ns are presented on MHC-I molecules in macrophages as well as in dendritic cells. In certain embodiments of the invention, a particular antigen is identified and presented in the antigen-MHC-nanoparticle complex in the t of an appropriate MHC class I or II polypeptide. In certain aspects, the genetic makeup of a t may be assessed to determine which MHC polypeptide is to be used for a particular patient and a particular set of peptides. In certain embodiments, the MHC class 1 component comprises all or part of a HLA-A, HLA-B, HLA-C, HLA-E, HLA- F, HLA-G or CD-l molecule. In embodiments wherein the MHC component is a MHC class II component, the MHC class II component can se all or a part of a HLA-DR, HLA- DQ, or .

Non-classical MHC molecules are also plated for use in MHC complexes of the invention. Non-classical MHC molecules are non-polymorphic, conserved among species, and possess narrow, deep, hydrophobic ligand binding pockets. These binding pockets are capable of presenting glycolipids and phospholipids to Natural Killer T (NKT) cells or certain subsets of CD8+ T-cells such as Qal or restricted CD8+ T-cells.

NKT cells represent a unique lymphocyte population that co-express NK cell markers and a semi-invariant T cell receptor (TCR). They are implicated in the regulation of immune responses associated with a broad range of diseases.

C. Antigenic Components Certain aspects of the invention include methods and compositions concerning antigenic itions including segments, fragments, or epitopes of polypeptides, peptides, nucleic acids, ydrates, lipids and other molecules that provoke or induce an antigenic response, lly referred to as antigens. In particular, antigenic segments or fragments of antigenic determinants, which lead to the ction of a cell via an autoimmune response, can be fied and used in making an antigen-MHC-nanoparticle complex described herein. Embodiments of the invention include compositions and methods for the modulation of an immune response in a cell or tissue of the body.

Antigenic polypeptides and peptides of the invention may be modified by various amino acid deletions, insertions, and/or substitutions. In particular embodiments, modified ptides and/or peptides are capable of modulating an immune response in a subject. In some embodiments, a wild-type version of a protein or peptide are employed, r, in many embodiments of the invention, a modified protein or polypeptide is employed to generate an antigen-MHC-nanoparticle complex. An antigen-MHC-nanoparticle complex can be used to generate an nflammatory immune response, to modify the T cell population of the immune system (i.e., re-educate the immune system), and/or foster the recruitment and lation of anti-inflammatory T cells to a particular tissue. The terms described above may be used interchangeably herein. A "modified protein" or "modified polypeptide" or "modified e" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some embodiments, a modified n or polypeptide or peptide has at least one modified activity or function (recognizing that proteins or polypeptides or peptides may have le ties or filnctions). It is cally contemplated that a modified protein or polypeptide or peptide may be altered with respect to one ty or function yet retains a wild-type activity or function in other respects, such as immunogenicity or ability to interact with other cells of the immune system when in the context of an MHC-nanoparticle Non-limiting examples, of peptide antigens e, but are not limited to hInsB10_1g (HLVEALYLV), hIGRP22g_236 (LNIDLLWSV), hIGRP265_273 GFAI), IGRP206_214 (VYLKTNVFL), NRP-A7 (KYNKANAFL), NRP-I4 (KYNIANVFL), NRP-V7 (KYNKANVFL), YAI/Db (FQDENYLYL) and/or INS B1543 (LYLVCGERG), as well as peptides and proteins disclosed in US. Patent Application Publication No. 2005/0202032 and lents and/or combinations thereof.

In certain aspects, a peptide antigen for treatment of T1D is GAD65114_123, VMNILLQYVV; GAD65536_545, RMMEYGTTMV; GFAP143_151, NLAQTDLATV; GFAP214_ 222, QLARQQVHV; 2_1go, SLSPLQAEL; IA-24g2_490, SLAAGVKLL; IA-2805_813, VIVMLTPLV; 5_13, KLQVFLIVL; ppIAPP9_17, FLIVLSVAL; IGRP152_160, FLWSVFMLI; IGRP211_219, FAV; IGRP215_223, FLFAVGFYL; IGRP222_230, YLLLRVLNI; IGRP228_236, LNIDLLWSV; IGRP265_273, VLFGLGFAI; IGRP293_301, RLLCALTSL; Pro-insulinL2_10, ALWMRLLPL; Pro-insulinL3_11, LWMRLLPLL; Pro- insulinL6_14, RLLPLLALL; Pro-insulin135_14, HLCGSHLVEA; Pro-insulin1310_1g, HLVEALYLV; Pro-insulinBl4_22, ALYLVCGER; Pro-insulin315_24, ERGF; Pro- insulinBl7_25, LVCGERGFF; Pro-insulinBlg_27, VCGERGFFYT; Pro-insulinBzo_27, GERGFFYT; Pro-insulin1321_29, ERGFFYTPK; sulin1325_01, FYTPKTRRE; Pro- insulint7_Cs, EAEDL; Pro-insulinczo_28, SLQPLALEG; Pro-insulin025_33, ALEGSLQKR; Pro-insulin029_A5, SLQKRGIVEQ; Pro-insulinA1_10, GIVEQCCTSI; Pro- insulinA2_10, IVEQCCTSI; Pro-insulinA12_20, SLYQLENYC or equivalents and/or combinations thereof.

Additional miting examples of antigens include MS and MS-relevant or related antigens that can be used in this invention comprise polypeptides comprising, or alternatively consisting essentially of, or yet fiarther consisting of the polypeptides of the group: MOG35_55, MEVGWYRSE’FSRVVHLYEKN’GK; MOG36_55, EV’SWWRSPFSRVVE-ELYRNGK; MAG287_295, EEV; _517, LMWAKIGPV; MAG556_564, VLFSSDFRI; MBP1110_118, SLSRFSWGA; MOG114_122, KVEDPFYWV; MOG166_175, RTFDPHFLRV; MOG172_180, FLRVPCWKI; MOG179_1gg, KITLFVIVPV; MOG188_196, VLGPLVALI; MOG181_189, TLFVIVPVL; MOG205_214, LEEL; PLP80_ gg, FLYGALLLA, or an equivalent of each thereof, or ations thereof.

In still filther aspects peptide antigens for the treatment of MS and MS-related disorders include without limitation: MOG35_55, MEV’GWY RSPFSRVVH LYRNGK; MOG36_ 55, ‘WYRSPFSRVVHLYRNGK; MAG287_295, SLLLELEEV; MAG509_517, LMWAKIGPV; MAG556_564, VLFSSDFRI; MBP110_118, SLSRFSWGA; MOG114_122, KVEDPFYWV; MOG166_175, RTFDPHFLRV; MOG172_180, FLRVPCWKI; MOG179_1gg, KITLFVIVPV; _196, VLGPLVALI; MOG181_189, TLFVIVPVL; MOG205_214, RLAGQFLEEL; 88, LLA MAG287_295, SLLLELEEV; MAG509_517, LMWAKIGPV; MAG556_564, VLFSSDFRI, and equivalents and/or combinations f Antigens for the treatment ofMS and ated disorders include, those disclosed in US. Patent Application Publication No. 2012/0077686, and antigens derived from myelin basic protein, myelin associated glycoprotein, myelin endrocyte protein, proteolipid protein, oligodendrocyte myelin rotein, myelin associated oligodendrocyte basic protein, oligodendrocyte specific protein, heat shock ns, oligodendrocyte specific proteins NOGO A, glycoprotein Po, peripheral myelin n 22, and 2'3'-cyclic nucleotide 3'-phosphodiesterase. In certain embodiments, the antigen is derived from Myelin Oligodendrocyte rotein (MOG).

In certain embodiments, the size of a protein or polypeptide (wild-type or modified), including any x of a protein or peptide of st and in ular a MHC- e filSlOIl, may comprise, but is not limited to 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino molecules or greater, including any range or value derivable therein, or derivative thereof. In certain aspects, 5, 6, 7, 8, 9, 10 or more contiguous amino acids, including derivatives thereof, and fragments of an antigen, such as those amino acid sequences disclosed and referenced herein, can be used as antigens. It is contemplated that polypeptides may be d by truncation, rendering them shorter than their corresponding wild-type form, but also they might be altered by fusing or conjugating a heterologous protein sequence with a ular function (e.g., for presentation as a protein complex, for enhanced immunogenicity, etc.).

Proteinaceous compositions may be made by any technique known to those of skill in the art, including (i) the expression of proteins, polypeptides, or peptides through standard molecular biological techniques, (ii) the isolation of proteinaceous compounds from l sources, or (iii) the chemical synthesis of proteinaceous materials. The nucleotide as well as the protein, polypeptide, and peptide sequences for various genes have been previously sed, and may be found in the recognized computerized databases. One such database is the National Center for Biotechnology Information's GenBank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov/). The all or part of the coding regions for these genes may be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.

Amino acid sequence variants of autoantigenic epitopes and other polypeptides of these itions can be substitutional, insertional, or deletion variants. A modification in a pfideoftheinvenfionrnayaflmctl,2,3,4,5,6,7,8,9,lO,ll,l2,l3,l4,lS,l6,l7, 18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,4l,42, 43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,6l,62,63,64,65,66,67, 68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,9l,92, 93,94,95,96,97,98,99,lOO,lOO,lOl,lOZ,103,104,105,106,107,108,109,110,11L 112,113,ll4,llS,ll6,ll7,118,ll9,120,121,122,123,124,125,126,127,128,129, 130,l3l,l32,l33,l34,l35,l36,l37,138,139,140,141,l42,l43,l44,l45,l46,l47, 148,149,150,151,152,153,154,155,156,157,158,159,l60,l6l,l62,l63,l64,l65, 166,167,l68,l69,l70,l7l,l72,l73,174,175,176,177,178,179,180,181,182,183, 184,185,186,187,188,189,l90,l9l,192,193,194,195,l96,l97,l98,l99,200,201, 202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219, 220,221,222,223,224,225,226,227,228,229,230,23l,232,233,234,235,236,237, 9,240,241,242,235,236,237,238,239,240,24l,242,243,244,245,246,247, 248,249,250,251,252,253,254,255,256,257,258,259,260,26l,262,263,264,265, 7,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283, 284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,301, 302,303,304,305,306,307,308,309,310,311,312,3l3,3l4,315,316,317,318,3l9, 320,321,322,323,324,325,326,327,328,329,330,33l,332,333,334,335,336,337, 9,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355, 356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373, 374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,39L 392,393,394,395,396,397,398,399,400,401,402,403,404,405,406,407,408,409, 410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427, 428,429,430,431,432,433,434,435,436,437,438,439,440,44l,442,443,444,445, 446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463, 464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481, 482,483,484,485,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500 or more non-contiguous or contiguous amino acids of a peptide or polypeptide, as compared to wild-type.

Deletion variants typically lack one or more residues of the native or Wild-type amino acid ce. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein. Insertional mutants typically 2014/003014 involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of one or more residues. Terminal additions, called fusion proteins, may also be generated.

Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the ptide, with or without the loss of other functions or properties.

Substitutions may be conservative, that is, one amino acid is ed with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; ne to serine; glutamine to asparagine; glutamate to aspartate; e to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to ine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative such that a fianction or actiVity of a polypeptide or peptide is affected, such as aVidity or affinity for a cellular receptor(s).

Non-conservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.

Proteins of the invention may be inant, or sized in Vitro.

Alternatively, a recombinant protein may be isolated from ia or other host cell.

It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N— or C-terminal amino acids, or 5' or 3' nucleic acid ces, respectively, and yet still be ially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological n actiVity (e.g., genicity). The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences g either of the 5' or 3' portions of the coding region.

It is plated that in compositions of the invention, there is between about 0.001 mg and about 10 mg of total protein per ml. Thus, the concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 50, 100 ug/ml or mg/ml or more (or any range derivable n). Of this, about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 1,12,13,14,15,16,17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% may be antigen-MHC-nanoparticle x.

In addition, US. Patent No. 4,554,101 (Hopp), which is incorporated herein by reference, teaches the identification and preparation of epitopes from primary amino acid sequences on the basis of hydrophilicity. Through the methods disclosed in Hopp, one of skill in the art would be able to identify potential epitopes from within an amino acid sequence and confirm their immunogenicity. us ific publications have also been devoted to the prediction of secondary structure and to the identification of epitopes, from es of amino acid sequences (Chou & Fasman, Adv. Enzymol., 47:45-148, 1978; Chous and Fasman, Annu, Rev. Biochem., 47:251-276, 1978, Chou and Fasman, Biochemistry, 13(2):211-222, 1974; Chau and Fasman, Biochemistry, 13(2):222-245, 1974, Chou and Fasman, Biophys. J of these may be used, if desired, ., 26(3):385-399, 1979). Any to supplement the teachings of Hopp in US. Patent No. 4,554,101.

For any given autoimmune e the antigen MHC compex can be identified and pre-selected using known methods in the art. Algorithms exist - d from a set of aligned peptides known to bind to a given MHC molecule, which can be used as a predictor of both peptide-MHC binding and T-cell es. See, e.g., Reche and Reinherz (2007) Methods Mol. Biol. 409: 185-200.

Molecules other than peptides can be used as antigens or antigenic fragments in complex with MHC molecules, such molecules include, but are not limited to carbohydrates, lipids, small molecules, and the like. Carbohydrates are major components of the outer surface of a variety of cells. Certain carbohydrates are teristic of different stages of differentiation and very often these carbohydrates are recognized by specific antibodies.

Expression of distinct carbohydrates can be restricted to specific cell types.

D. Substrates/Nanoparticles In certain aspect, antigen/MHC complexes are ively coupled to a substrate which can be bound covalently or non-covalently to the substrate. A substrate can be in the form of a nanoparticle that optionally comprises a biocompatible and/or bioabsorbable material. Accordingly, in one embodiment, the nanoparticle is biocompatible and/or bioabsorbable. In r aspect, the nanoparticle has a solid core and/or is not a liposome.

A substrate can also be in the form of a nanoparticle such as those described usly in US. Patent Publication No. 2009/0155292. rticles can have a structure of variable dimension and known variously as a nanosphere, a nanoparticle or a biocompatible biodegradable nanosphere or a patible biodegradable rticle. Such particulate formulations containing an antigen/MHC complex can be formed by covalent or non- covalent coupling of the complex to the nanoparticle.

The nanoparticles typically consist of a substantially spherical core and optionally one or more . The core may vary in size and composition. In addition to the core, the nanoparticle may have one or more layers to provide functionalities appropriate for the applications of interest. The thicknesses of layers, if present, may vary depending on the needs of the c applications. For example, layers may impart useful optical properties.

Layers may also impart al or biological fianctionalities, referred to herein as chemically active or biologically active layers, and for these functionalities the layer or layers may typically range in thickness from about 0.001 micrometers (l nanometer) to about 10 micrometers or more ding on the d nanoparticle diameter), these layers typically being applied on the outer surface of the nanoparticle.

The compositions of the core and layers may vary. Suitable materials for the particles or the core include, but are not limited to polymers, ceramics, glasses, minerals, and the like. Examples include, but are not limited to, standard and specialty glasses, silica, polystyrene, polyester, polycarbonate, acrylic polymers, polyacrylamide, polyacrylonitrile, polyamide, fluoropolymers, silicone, celluloses, silicon, metals (e.g., iron, gold, ), minerals (e. g., ruby), nanoparticles (e.g., gold nanoparticles, colloidal particles, metal oxides, metal sulfides, metal des, and magnetic materials such as iron oxide), and composites thereof. The core could be of homogeneous composition, or a composite of two or more s of material depending on the properties desired. In certain aspects, metal nanoparticles will be used. These metal les or rticles can be formed from Au, Pt, Pd, Cu, Ag, Co, Fe, Ni, Mn, Sm, Nd, Pr, Gd, Ti, Zr, Si, and In, precursors, their binary alloys, their ternary alloys and their intermetallic compounds. See US. Patent No. 6,712,997. In certain embodiments, the compositions of the core and layers may vary provided that the rticles are biocompatible and bioabsorbable. The core could be of homogeneous composition, or a composite of two or more classes of material depending on the properties d. In certain aspects, metal nanospheres will be used. These metal rticles can be formed from Fe, Ca, Ga and the like. In certain embodiments, the nanoparticle comprises a core sing metal or metal oxide such as gold or iron oxide.

As previously stated, the nanoparticle may, in addition to the core, include one or more layers. The nanoparticle may include a layer consisting of a biodegradable sugar or other polymer. Examples of biodegradable layers include but are not limited to dextran; poly(ethylene glycol); poly(ethylene oxide); mannitol; poly(esters) based on polylactide (PLA), ycolide (PGA), polycaprolactone (PCL); poly(hydroxalkanoate)s of the PHB- PHV class; and other modified poly(saccharides) such as starch, cellulose and chitosan.

Additionally, the rticle may include a layer with suitable surfaces for attaching chemical onalities for chemical binding or coupling sites.

Layers can be produced on the nanoparticles in a y of ways known to those skilled in the art. Examples e sol-gel chemistry techniques such as described in Iler, Chemistry of Silica, John Wiley & Sons, 1979; Brinker and Scherer, Sol-gel Science, Academic Press, (1990). Additional approaches to producing layers on nanoparticles include surface chemistry and encapsulation techniques such as described in Partch and Brown, J.

Adhesion, 67:259-276, 1998; Pekarek et al., Nature, 367:258, (1994); Hanprasopwattana, Langmuir, 12:3173-3179, (1996); Davies, Advanced Materials, 10: 270, (1998); and references therein. Vapor deposition techniques may also be used; see for example Golman and Shinohara, Trends Chem. Engin., 6:1-6, (2000); and US. Pat. No. 6,387,498. Still other approaches e layer-by-layer self-assembly techniques such as described in Sukhorukov et al., Polymers Adv. Tech., 9(10-11):759-767, (1998); Caruso et al., Macromolecules, 2317-2328, (1998); Caruso et al., J.Amer. Chem. Soc., 121(25):6039-6046, (1999); US. Pat. No. 6,103,379 and nces cited therein.

Nanoparticles may be formed by contacting an aqueous phase containing the antigen/MHC/co-stimulatory le complex and a polymer and a nonaqueous phase followed by evaporation of the nonaqueous phase to cause the coalescence of particles from the aqueous phase as taught in US. Patent No. 330 or 542. Preferred polymers for such preparations are natural or synthetic copolymers or polymers selected from the group consisting of gelatin agar, starch, arabinogalactan, albumin, collagen, polyglycolic acid, polylactic acid, glycolide-L(-) lactide poly(episilon-caprolactone, poly(epsilon-caprolactone- tic acid), poly(epsilon-caprolactone-CO-glycolic acid), poly(B-hydroxy butyric acid), poly(ethylene oxide), polyethylene, poly(alkylcyanoacrylate), poly(hydroxyethyl rylate), polyamides, poly(amino acids), poly(2-hydroxyethyl DL-aspartamide), poly(ester urea), poly(L-phenylalanine/ethylene glycol/l,6-diisocyanatohexane) and poly(methyl rylate). Particularly preferred polymers are polyesters, such as polyglycolic acid, polylactic acid, glycolide-L(-) lactide pisilon-caprolactone), poly(epsilon-caprolactone-CO-lactic acid), and poly(epsilon-caprolactone-CO-glycolic acid).

Solvents useful for dissolving the polymer include: water, hexafluoroisopropanol, methylenechloride, tetrahydrofuran, , benzene, or hexafluoroacetone sesquihydrate.

The size of the nanoparticle can range from about 1 nm to about 1 um. In certain embodiments, the rticle is less than about 1 um in diameter. In other embodiments, the rticle is less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm in diameter. In further embodiments, the nanoparticle is from about 1 nm to about 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 75 nm, or 100 nm in diameter. In specific embodiments, the nanoparticle is from about 1 nm to about 100 nm, about 1 nm to about 50 nm, about 1 nm to about 20 nm, or about 5 nm to about 20 nm.

The size of the complex can range from about 5 nm to about 1 um. In certain embodiments, the complex is less than about 1 um or alternatively less than 100 nm in diameter. In other embodiments, the complex is less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm in diameter. In further embodiments, the complex is from about 10 nm to about 50 nm, or about 20 nm to about 75 nm, or about 25 nm to about 60 nm, or from about 30 nm to about 60 nm, or in one aspect about 55 nm.

E. Coupling n-MHC Complex with the Nanoparticle In order to couple the substrate or nanospheres to the antigen-MHC complexes the following techniques can be applied.

The binding can be generated by chemically modifying the substrate or nanoparticle which typically involves the generation of ional groups" on the surface, said fianctional groups being capable ofbinding to an antigen-MHC complex, and/or linking the optionally chemically modified surface of the substrate or nanoparticle with covalently or non- ntly bonded so-called "linking molecules," followed by reacting the antigen-MHC complex with the nanoparticles obtained.

The term "linking le" means a substance capable of linking with the substrate or nanoparticle and also capable of g to an antigen-MHC complex. In certain embodiments, the antigen-MHC complexes are coupled to the rticle by a linker. Non- limiting examples of suitable linkers include dopamine (DPA)-polyethylene glycol (PEG) linkers such as DPA-PEG-NHS ester, DPA-PEG-orthopyridyl-disulfide (OPSS) and/or DPA- PEG-Azide. Other linkers e peptide s, ethylene glycol, biotin, and strepdavidin.

The term "fianctional groups" as used herein before is not restricted to ve chemical groups forming covalent bonds, but also includes chemical groups leading to an ionic interaction or hydrogen bonds with the antigen-MHC complex. er, it should be noted that a strict distinction between "fianctional groups" generated at the surface and linking molecules bearing "functional groups" is not possible, since sometimes the modification of the surface requires the reaction of r linking molecules such as ethylene glycol with the nanosphere surface.

The functional groups or the linking molecules bearing them may be ed from amino groups, carbonic acid groups, thiols, hers, disulfides, guanidino, hydroxyl groups, amine , l diols, aldehydes, alpha-haloacetyl groups, mercury organyles, ester , acid halide, acid thioester, acid anhydride, isocyanates, isothiocyanates, sulfonic acid halides, imidoesters, diazoacetates, diazonium salts, l,2-diketones, phosphonic acids, phosphoric acid esters, sulfonic acids, azolides, imidazoles, indoles, N—maleimides, alpha- beta-unsaturated carbonyl compounds, arylhalogenides or their derivatives.

Non-limiting examples for other g molecules with higher lar weights are nucleic acid molecules, polymers, copolymers, polymerizable coupling agents, silica, proteins, and chain-like molecules having a surface with the opposed polarity with respect to the substrate or nanoparticle. Nucleic acids can provide a link to affinity molecules containing themselves nucleic acid molecules, though with a complementary sequence with t to the g molecule.

A specific example of a covalent linker includes poly(ethylene) glycol (PEG) such as functionalized PEGs. As used herein, “functionalized PEGs” refer to PEG moieties including terminal functional group, non-limiting examples of which include amino, mercapto, thioether, carboxyl, and the likes. Non-limiting examples of functionalized PEG linkers on various nanoparticle cores are provided in Tables 1 and 2 attached hereto, e.g., the PEG linker thiol-PEG-NHZ linker.

In certain embodiments, the linker as described herein has a defined size. In some embodiments, the linker is less that about 10 kD, less than about 5 kD, less than about 4.5 kD, less than about 4 kD, less than about 3.5 kD, less than about 3 kD, less than about 2.5 kD, less than about 2 kD, or less than about 1 kD. In further embodiments, the linker is from about 0.5 kD to about 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1 kD. In yet further embodiments, the linker is from about 1 to about, 4.5, 4, 3.5, 3, 2.5, 2, or 15 kD.

As examples for polymerizable coupling agents, diacetylene, styrene ene, vinylacetate, te, acrylamide, vinyl compounds, styrene, silicone oxide, boron oxide, phosphorous oxide, borates, pyrrole, polypyrrole and phosphates can be cited.

The surface of the substrate or nanoparticle can be chemically modified, for instance by the binding of phosphonic acid derivatives having functional reactive groups.

One example of these phosphonic acid or phosphonic acid ester tes is imino- bis(methylenphosphono) ic acid which can be synthesized according to the "Mannich- Moedritzer" on. This binding reaction can be performed with substrate or here as directly obtained from the ation process or after a pre-treatment (for instance with trimethylsilyl bromide). In the first case the phosphonic acid (ester) derivative may for instance displace components of the reaction medium which are still bound to the surface.

This displacement can be enhanced at higher temperatures. Trimethylsilyl e, on the other hand, is believed to dealkylate alkyl group-containing orous-based complexing agents, thereby creating new binding sites for the phosphonic acid (ester) derivative. The phosphonic acid (ester) derivative, or g molecules bound thereto, may y the same functional groups as given above. A r e of the surface treatment of the substrate or nanosphere involves heating in a diole such as ethylene glycol. It should be noted that this treatment may be redundant if the synthesis already proceeded in a diol. Under these circumstances the synthesis product directly obtained is likely to show the ary functional . This treatment is however applicable to substrate or nanoparticle that were produced in N— or P-containing complexing agents. If such substrate or particle are subjected to an after-treatment with ethylene glycol, ingredients of the reaction medium (e.g. complexing agent) still binding to the surface can be replaced by the diol and/or can be dealkylated.

It is also possible to replace N-containing complexing agents still bound to the particle surface by primary amine derivatives having a second fianctional group. The surface of the substrate or nanoparticle can also be coated with silica. Silica allows a relatively simple chemical conjugation of organic molecules since silica easily reacts with organic linkers, such as triethoxysilane or chlorosilane. The nanoparticle surface may also be coated by homo- or copolymers. Examples for polymerizable coupling agents are N—(3- ropyl)mercaptobenzamidine, 3-(trimethoxysilyl)propylhydrazide and 3- trimethoxysilyl)propylmaleimide. Other non-limiting es of polymerizable ng agents are mentioned herein. These coupling agents can be used singly or in combination depending on the type of copolymer to be generated as a coating. r surface modification technique that can be used with substrates or nanoparticles containing oxidic transition metal nds is conversion of the oxidic transition metal compounds by chlorine gas or organic chlorination agents to the corresponding oxychlorides. These oxychlorides are e of ng with nucleophiles, such as hydroxy or amino groups as often found in biomolecules. This technique allows generating a direct conjugation with proteins, for instance-via the amino group of lysine side chains. The conjugation with proteins after surface modification with oxychlorides can also be effected by using a ctional linker, such as maleimidopropionic acid hydrazide.

For non-covalent g techniques, type molecules having a polarity or charge opposite to that of the substrate or nanosphere surface are particularly le.

Examples for linking molecules which can be non-covalently linked to core/shell nanospheres involve anionic, cationic or zwitter—ionic surfactants, acidic or basic proteins, polyamines, ides, polysulfone or polycarboxylic acid. The hydrophobic interaction between substrate or nanosphere and amphiphilic reagent having a functional reactive group can te the necessary link. In particular, chain-type les with amphiphilic character, such as phospholipids or derivatized polysaccharides, which can be crosslinked with each other, are useful. The absorption of these molecules on the surface can be achieved by coincubation. The binding between ty molecule and substrate or nanoparticle can also be based on non-covalent, self-organising bonds. One example thereof involves simple 2014/003014 detection probes with biotin as linking molecule and - or strepdavidin-coupled molecules.

Protocols for coupling reactions of functional groups to biological molecules can be found in the literature, for instance in "Bioconjugate Techniques" (Greg T. Hermanson, Academic Press 1996). The ical molecule (e.g., MHC molecule or derivative thereof) can be d to the g molecule, covalently or non-covalently, in line with standard procedures of organic chemistry such as oxidation, halogenation, alkylation, acylation, addition, tution or amidation. These methods for coupling the covalently or non- covalently bound linking molecule can be d prior to the coupling of the linking molecule to the substrate or nanosphere or thereafter. Further, it is possible, by means of incubation, to effect a direct binding of molecules to correspondingly pre-treated substrate or nanoparticle (for instance by trimethylsilyl bromide), which display a modified surface due to this pre-treatment (for instance a higher charge or polar surface).

F. Protein Production The present invention describes polypeptides, peptides, and proteins for use in various embodiments of the present invention. For example, specific peptides and their complexes are assayed for their ies to elicit or modulate an immune se. In specific embodiments, all or part of the peptides or proteins of the invention can also be synthesized in solution or on a solid support in accordance with conventional techniques.

Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2Ild Ed., Pierce Chemical Co.l, (1984); Tam et al., J. Am. Chem. Soc., 105:6442, (1983); ield, Science, 232(4748):34l-347, (1986); and Barany and Merrifield, The Peptides, Gross and Meinhofer (Eds.), Academic Press, NY, 1-284, (1979), each orated herein by reference. Alternatively, recombinant DNA technology may be employed wherein a tide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.

One embodiment of the invention includes the use of gene transfer to cells, including rganisms, for the production of ns. The gene for the protein of interest may be transferred into appropriate host cells followed by culture of cells under the appropriate conditions. A nucleic acid ng virtually any polypeptide may be employed.

The generation of recombinant expression vectors, and the elements included therein, are known to one skilled in the art and are briefly sed herein. Examples of mammalian host cell lines include, but are not limited to ero and HeLa cells, other B- and T-cell lines, such as CEM, 721.221, H9, Jurkat, Raji, as well as cell lines of e hamster ovary (CHO), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cells. In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or that modifies and processes the gene product in the manner desired. Such modifications (e. g., glycosylation) and processing (e.g., cleavage) of n products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.

A number of selection systems may be used including, but not limited to HSV thymidine kinase, hypoxanthine-guanine oribosyltransferase, and adenine oribosyltransferase genes, in tk-, hgprt— or aprt-cells, respectively. Also, anti- metabolite resistance can be used as the basis of selection: for dhfr, which confers ance to trimethoprim and methotrexate; gpt, which confers ance to mycophenolic acid; neo, which confers resistance to the lycoside G418; and hygro, which confers resistance to hygromycin.

G. Nucleic Acids The present invention may include recombinant polynucleotides encoding the proteins, polypeptides, es of the invention, such as those encoding antigenic peptides.

In particular ments, the invention concerns isolated nucleic acid segments and recombinant vectors orating nucleic acid sequences that encode an autoantigen and/or a MHC molecule. The term "recombinant" may be used in conjunction with a polypeptide or the name of a ic polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

The nucleic acid segments used in the present invention, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional ction enzyme sites, multiple cloning sites, other coding ts, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being d by the ease of preparation and use in the ed recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional logous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. A tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

V. PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION Provided herein are pharmaceutical compositions useful for the treatment of disease.

A. ceutical Compositions The antigen-MHC nanoparticle complexes can be administred alone or in combination with a r, such as a pharmaceutically acceptable carrier in a composition.

Compositions of the invention may be conventionally administered parenterally, by injection, for example, intravenously, aneously, or intramuscularly. Additional formulations which are suitable for other modes of administration include oral formulations. Oral formulations include such normally employed excipients such as, for example, pharmaceutical grades of mannitol, lactose, , magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ient, preferably about 25% to about 70%. The ation of an aqueous ition that contains an antigen-MHC-nanoparticle complex that modifies the subj ect's immune condition will be known to those of skill in the art in light of the t disclosure. In certain embodiments, a composition may be inhaled (e.g., US.

Patent No. 6,651,655, which is specifically orated by nce in its entirety). In one embodiment, the antigen-MHC-nanoparticle complex is administered systemically.

Typically, compositions of the invention are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immune modifying. The quantity to be administered depends on the subject to be treated.

Precise amounts of active ient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of ten to l hundred nanograms or micrograms antigen-MHC-nanoparticle complex per administration. Suitable s for initial administration and boosters are also variable, but are typified by an initial administration followed by subsequent strations.

In many ces, it will be desirable to have multiple administrations of a peptide- MHC-nanoparticle complex, about, at most about or at least about 3, 4, 5, 6, 7, 8, 9, 10 or more. The administrations will normally range from 2 day to twelve week intervals, more usually from one to two week intervals. Periodic boosters at intervals of 025-5 years, y two years, may be desirable to maintain the condition of the immune system. The course of the administrations may be followed by assays for inflammatory immune responses and/or autoregulatory T cell activity.

In some embodiments, pharmaceutical compositions are stered to a t.

Different aspects of the present invention involve administering an effective amount of a antigen-MHC-nanoparticle complex ition to a subject. Additionally, such compositions can be administered in combination with modifiers of the immune system.

Such itions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, or human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifilngal agents, ic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated.

The pharmaceutical forms suitable for able use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile inj ectable ons or dispersions. In all cases the form must be e and must be fluid to the extent that it may be easily injected. It also should be stable under the ions of manufacture and storage and must be preserved against the inating action of microorganisms, such as bacteria and fiangi.

The compositions may be formulated into a neutral or salt form. ceutically acceptable salts, include the acid on salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.

Salts formed with the free carboxyl groups can also be derived from nic bases such as, for example, sodium, ium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid thylene glycol), and the like, suitable mixtures f, and vegetable oils. The proper y can be ined, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the inj ectable compositions can be t about by the use in the itions of agents ng absorption, for example, aluminum monostearate and gelatin. e injectable solutions are prepared by incorporating the active compounds in the ed amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by sterilization. Sterilization of the solution will be done in such a way as to not diminish the therapeutic properties of the antigen-MHC- nanoparticle complex. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ients from those enumerated above. In the case of sterile powders for the preparation of sterile inj ectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterilized solution thereof. One such method of sterilization of the solution is sterile filtration, however, this invention is meant to e any method of sterilization that does not significantly decrease the therapeutic properties of the antigen-MHC-nanoparticle complexes. Methods of ization that involve intense heat and re, such as aVing, may compromise the tertiary structure of the complex, thus significantly sing the therapeutic properties of the antigen-MHC-nanoparticle xes.

An ive amount of therapeutic ition is determined based on the intended goal. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in ation with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and/or protection desired. Precise amounts of the ition also depend on the nt of the practitioner and are ar to each indiVidual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.

B. Combination Therapy The compositions and related methods of the present invention, particularly administration of an antigen-MHC-nanoparticle x, may also be used in combination with the administration of traditional therapies. These e, but are not limited to, Avonex (interferon beta-la), Betaseron (interferon beta-lb), Copaxone ramer acetate), Novantrone (mitoxantrone), Rebif (interferon beta-la), Tysabri (natalizumab), Gilenya (fingolimod), Glatiramer, steroids, Cytoxan, Imuran, Baclofen, deep brain stimulation, Ampyra (dalfampridine), acupuncture, and physical therapy.

When ation therapy is employed, various combinations may be employed, for example antigen-MHC-nanoparticle complex administration is "A" and the additional agent is "B": A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A/ B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A Administration of the peptide-MHC complex compositions of the present ion to a patient/subj ect will follow general protocols for the administration of such nds, taking into account the toxicity, if any. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, such as hydration, may be applied in combination with the described therapy.

C. In Vitro 0r Ex Vivo Administration As used herein, the term in vitro stration refers to manipulations med on cells d from or outside of a subject, including, but not limited to cells in culture.

The term ex vivo administration refers to cells which have been manipulated in vitro, and are subsequently administered to a subject. The term in viva administration includes all manipulations performed within a subject, including administrations.

In certain aspects of the t invention, the compositions may be administered either in vitro, ex vivo, or in vivo. In certain in vitro embodiments, autologous T cells are ted with compositions of this invention. The cells or tissue can then be used for in vitro analysis, or alternatively for ex vivo administration.

VI. EXAMPLES The ing examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any n.

One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present es, along with the methods described herein are tly representative of embodiments and are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1. Preparation and analysis of pMHC nanoparticles. pMHC production Two ent methods were used to express recombinant pMHC class I complexes.

The first involved re-folding MHC class I heavy and light chains expressed in bacteria in the presence of peptide, followed by purification via gel filtration and anion exchange chromatography, as described (Garboczi, D.N. et al. (1992) Proc Natl. Acad Sci USA 89:3429-3433; Altman, JD. et al. (1996) Science 274:94-96). The second involved expressing MHC class I xes at high yields in lentiviral-transduced freestyle CHO cells as single chain constructs in which the peptide-coding sequence, the MHC class I light and heavy chains are sequentially tethered with flexible GS linkers (Yu, Y.Y. et al. (2002) J Immunol 168:3145-3149) followed by a carboxyterminal linker encoding a BirA site, a 6xHis tag ending with a free Cys. The secreted proteins were d from culture supematants using nickel columns and anion exchange chromatography and used directly for NP coating or biotinylated to produce pMHC tetramers using hrome-conjugated streptavidin.

Tetramers generated using entative -chain pMHC complexes encoding the IGRP206_214 autoantigenic peptide or its mimic NRP-V7 ntly bind to cognate monoclonal autoreactive CD8+ T-cells but not to their polyclonal counterparts (not , as determined by flow cytometry.

Recombinant pMHC class II monomers were initially purified from Drosophila SC2 cells transfected with constructs encoding I-AB and I-Ad chains carrying c-Jun or c-Fos leucine zippers, respectively, and a BirA and 6xHis tags as previously described (Stratmann, T. et al. (2000) J Immunol 165:3214-3225, Stratmann, T. et al. (2003) J. Clin. Invest. 14-3225). As the yields of this approach were generally low and time-consuming, Applicant developed an expression system in freestyle CHO cells transduced with lentiviruses encoding a monocistronic message in which the peptide-IAB and IA0L chains of the complex are ted by the me skipping P2A sequence (Holst, J. et al. (2006) Nat Protoc 1:406-417). As with the single chain pMHC class I constructs described above, a linker encoding a BirA site, a 6xHis tag and a free Cys was added to the carboxyterminal end of the construct. The self-assembled pMHC class II xes were purified from the cell culture supernatants by nickel chromatography followed by anion exchange and used for coating onto NPs or processed for biotinylation and tetramer formation as described above. pMHC class II tetramers generated using a representative pMHC class II complex encoding the 2.5mi autoantigenic peptide are specifically and efficiently bound by cognate monoclonal autoreactive CD4+ T-cells, as determined by flow cytometry. pMHC tetramer staining PE-conjugated TUM- and BDC2.5mi/IAg7 tetramers were prepared using biotinylated pMHC monomers as described (Stratmann, T. et al. (2000) J l 165:3214-3225; Stratmann, T. et al. (2003) J. Clin. Invest. 112:3214-3225; Amrani, A. et al. (2000) Nature 406:739-742). Peripheral blood mononuclear cells, splenocytes and lymph node CD8+ or CD4+ T-cells were stained with er (5 ug/mL) in FACS buffer (0.1% sodium azide and 1% PBS in PBS) for 1 h at 4°C, washed, and incubated with FITC-conjugated D80L or anti-CD4 (5 ug/mL) and PerCP-conjugated anti-B220 (2 ug/mL; as a 'dumb' gate) for 30 min at 4°C. Cells were washed, fixed in 1% PFA/PBS and analyzed by FACS.

NP sis Gold nanoparticles (GNPs) were sized using chemical reduction of gold chloride with sodium e as described (Perrault, S.D. et al. (2009) Nano Lett 9: 1909- 1915). , 2 mL of 1% of HAuCl4 (Sigma Aldrich) was added to 100 mL H20 under vigorous ng and the solution heated in an oil bath. Six (for 14 nm GNPs) or two mL (for 40 nm GNPs) of 1% Na Citrate were added to the boiling HAuCl4 solution, which was stirred for an additional 10 min and then cooled down to room temperature. GNPs were stabilized by the addition of 1 uMol of thiol-PEG linkers (Nanocs, MA) functionalized with —COOH or — NHz groups as acceptors ofpMHC (Tables 1 and 2). Pegylated GNPs were washed with water to remove fiee thiol-PEG, concentrated and stored in water for further analysis. NP density was via spectrophotometry and calculated according to Beer’s law.

The SFP series iron oxide NPs (SFP IONPs) were produced by thermal decomposition of iron acetate in organic solvents in the presence of surfactants, then rendered solvent in aqueous buffers by pegylation (Xie, J. et al. (2007) Adv Mater 19:3163; Xie, J. et al. (2006) Pure Appl. Chem. 78: 1003-1014; Xu, C. et al. (2007) Polymer International 56:821-826). Briefly, 2 mMol Fe(acac)3 (Sigma Aldrich, le, ON) were ved in a mixture of 10 mL benzyl ether and oleylamine and heated to 100°C for 1 hr followed by 300°C for 2 hr with reflux under the protection of a en blanket. Synthesized NPs were precipitated by on of ethanol and resuspended in hexane. For pegylation of the IONPs, 100 mg of different 3.5 kDa DPA-PEG linkers (81-85 in Table 1; Jenkem Tech USA) were dissolved in a e of CHC13 and HCON(CH3)2 (DMF). The NP solution (20 mg Fe) was then added to the DPA-PEG solution and stirred for 4 hr at room temperature. ted SFP NPs were precipitated ght by addition of hexane and then resuspended in water. Trace amounts of aggregates were removed by high-speed centrifiagation 0 xg, 30 min), and the sperse SFP NPs were stored in water for fiarther characterization and pMHC conjugation. The concentration of iron in IONP products was determined by spectrophotometry at A410 in 2N HCL. Based on the molecular structure and diameter of SFP NPs (Fe304; 8:1 nm diameter) (Xie, J. et al. (2007) Adv Mater 19:3163; Xie, J. et al. (2006) Pure Appl. Chem. 78: 1003-1014), Applicant estimates that SFP solutions containing 1 mg of iron contain 5x1014 NPs.

Applicant subsequently developed a new IONP design that allowed the formation, also by thermal decomposition but in a single step, of pegylated IONPs in the complete absence of surfactants (PF series IONPs). In this novel design, PEG molecules were used both as reducing reagents and as surfactants. In a typical reaction, 3 g PEG (2 kDa) were melted slowly in a 50 mL round bottom boiling flask at 100°C and then mixed with 7 mL of benzyl ether and 2 mMol c)3. The reaction was vigorously stirred for one hr and heated to 260°C with reflux for an additional two hr. The reaction mixture was cooled down to room temperature, transferred to a centrifilgation tube and mixed with 30 mL water. Insoluble materials were removed by centrifilgation at 2,000xg for 30 min. The free PEG molecules were removed by ultrafiltration through Amicon-15 filters (MWCO 100 kDa, Millipore, Billerica, MA). ant was able to generate IONPs with most, albeit not all of the PEG molecules tested (Table 1, P1-P5). The size of the IONPs varied depending on the onal groups of the PEG linkers used in the thermal decomposition reactions (Tables 1 and 2). The NPs could be readily purified using magnetic (MACS) columns (Miltenyi Biotec, Auburn, CA) or an IMag cell tion system (BD BioSciences, Mississauga, ON). The purified IONPs were stored in water or in various buffers (pH 5-10) at room temperature or at 4°C without any able aggregation. NP density was ated as described above for SFP NPs. pMHC conjugation of NPs pMHC conjugation to NPs produced with PEG linkers carrying distal -NH2 or — COOH groups was achieved via the formation of amide bonds in the presence of l-Ethyl ethylamin0pr0pyl]carbodiimide hydrochloride (EDC). NPs (GNP-C, SFP-C and PF-C, Table 2) with —COOH groups were first dissolved in 20 mM MES buffer, pH 5.5. N- hydroxysulfosuccinimide sodium salt (sulpha-NHS, Thermo scientific, Waltham, MA, final concentration 10 mM) and EDC (Thermo scientific, Waltham, MA, final concentration 1 mM) were added to the NP solution. After 20 min of ng at room temperature, the NP solution was added drop-wise to the solution containing pMHC monomers dissolved in 20 mM borate buffer (pH 8.2). The mixture was stirred for additional 4 hr. To conjugate pMHCs to NHz-functionalized NPs (GNP-N, SFP-N and PF-N, Table 2), pMHC complexes were first dissolved in 20 mM MES buffer, pH 5.5, containing 100 mM NaCl. Sulpha-NHS (10 mM) and EDC (5 mM) were then added to the pMHC solution. The activated pMHC molecules were then added to the NP solution in 20 mM borate buffer (pH 8.2), and stirred for 4 hr at room temperature.

To conjugate pMHC to maleimide-functionalized NPs (SFP-M and PF-M, Table 2 and ), pMHC molecules were first ted with Tributylphospine (TBP, 1 mM) for 4 hr at room temperature. pMHCs engineered to encode a free carboxyterminal Cys residue were then mixed with NPs in 40 mM phosphate buffer, pH 6.0, containing 2 mM EDTA, 150 mM NaCl, and incubated overnight at room temperature. pMHCs were covalently bound with NPs via the formation of a carbon-sulfide bond between ide groups and the Cys e.

Click chemistry was used to conjugate pMHC or avidin to NPs onalized with azide groups (SFP-Z, Table 2). For this reaction, pMHC or avidin molecules were first incubated with dibenzocyclooctyl (DBCO, Click try Tools, Scottdale, AZ) t for 2 hr at room temperature. Free DBCO molecules were removed by dialysis overnight. pMHC- 0r -DBCO conjugates were then incubated with SFP-Z for 2 hr, resulting in formation of le bonds between pMHCs or avidin molecules and NPs.

Unconjugated pMHC complexes in the different pMHC-NP conjugating reactions were removed by extensive dialysis against PBS, pH 7.4, at 4°C though 300 kDa molecular weight cut off membranes (Spectrum labs). Alternatively, pMHC-conjugated IONPs were purified by magnetic separation. The conjugated NPs were concentrated by ultrafiltration through Amicon 15 units (100 kDa MWCO) and stored in PBS.

Electron microscopy, dynamic light scattering, DLS and small angle o beam ction The core size and dispersity of unconjugated and pMHC-conjugated NPs were first assessed via transmission electron microscopy (TEM, Hitachi H7650). Dynamic light scattering (DLS) was used to determine the pMHC-NPs’ hydrodynamic size, zeta potential and monodisperity using a ZetaSizer ment (Malvem, UK). The chemical nature of the iron oxide core of the PF series ofNPs was evaluated using small angle electro beam diffraction (SEBD). r Transformation Infrared spectroscopy The surface chemical properties of the PF-series IONP designs were evaluated using Fourier Transformation Infrared spectroscopy (FTIR). The FTIR spectra of control PEG and PEG anchored on the PF-NP surface were obtained using a Nicolet FTIR ophotometer on an ATR (attenuated total reflection) mode. Each of the spectra was ed as the average of 256 scans at 4 cm"1 spectral resolution. The stretching vibration signatures of the PEG backbone C-O-C groups and their distal pMHC-acceptor filnctional groups were identified.

Agarose gel ophoresis To quickly evaluate changes on the NP charge as a function of pegylation or pMHC coating, NPs were subjected to electrophoresis on 0.8% agarose gels. Pegylated NPs migrated to ve or ve poles depending on the overall surface charge. Coomassie blue staining was done to confirm co-migration of the pMHCs with the NPs.

Native and denaturing polyacrylamide gel electrophoresis pMHC conjugated NPs were subjected to native-PAGE (10%) and SDS-PAGE (12%) analyses to confirm absence of free (unconjugated pMHC) in the pMHC-NP preparations and to confirm presence of intact trimolecular pMHC complexes on the NP’s surface. pMHC valency measurements To evaluate the number ofpMHC monomers conjugated onto individual NPs (pMHC valency), we measured the pMHC concentration of the pMHC-NP preps using different approaches, including Bradford assay (Thermo ific), amino acid analysis (HPLC-based fication of 17 different amino acids in yzed pMHC-NP preparations) (University of Toronto), dot-ELISA and signature peptide analysis by mass spectrometry) and the values converted to ratios of pMHC molecular number to NP number.

Briefly, in the “dot-ELISA” approach, pMHC-conjugated and unconjugated NPS and pMHC monomer solutions (as standards) were serially diluted in PBS and then absorbed to a PVDF ne in a ell filter plate (PALL Corporation). The plate was d to partially dry at room temperature and then ted with pMHC specific primary antibodies (i.e., ZM and anti-Kd antibodies for pMHC class I-coated NPs, clones 2M2 and SFl-l .l, BioLegend, San Diego, CA), followed by HRP- or AP-conjugated secondary antibodies.

Upon development of the enzymatic color reactions, the contents of the wells were transferred to wells in a conventional ELISA plate and their absorbances measured at 450 nm using a plate reader. For the signature e mass ometry approach, pMHC-specific trypsin es (signature peptides TWTAADTAALITR for Kd complexes and AQNSELASTANMLR for I-Ag7 complexes) were identified via mass spectrometry. The corresponding synthetic peptides were labeled with stable isotopes (AQUA peptide synthesis, Sigma Aldrich). The isotope-labeled peptides were then serially diluted to defined concentrations and mixed with pMHC-conjugated NPs for trypsin digestion. The mixtures were subjected to mass spectroscopy (Agilent QTOF6520) to quantify the ratios of isotope- labeled versus unlabeled signature peptides, as a read-out ofpMHC concentration. Since the values generated by these different methods were similar, the Bradford assay (using unconjugated NPs as blanks) became the method of choice for ease and simplicity.

Agonistic activity of pMHC-NPs in vitro FACS-sorted splenic CD8+ cells from TCR-TG mice (2.5 x105 cells/mL) were incubated with serially diluted pMHC conjugated or control NPs for 24-48 h at 37°C. The supernatants were d for IFNy by ELISA. The cultured cells were pulsed with l mCi of [3H]-thymidine and harvested after 24 h to e [3H] incorporation. pMHC-NP therapy Cohorts of 10 wk-old female NOD mice were injected iv. with pMHC-coated NPs in PBS twice a week for 5 wk (10 doses in total). Increases in the size of tetramer+ CD8+ or CD4+ T-cell pools in blood, spleen, lymph nodes and/or marrow, as well as their phenotypic properties, were assessed by flow cytometry as described (Tsai, S. et al. (2010) Immunity 32:568-5 80) (and Clemente-Casares et al., ted). In other experiments, mice displaying blood glucose levels >11 mM for 2 days were d i.v. twice a wk with pMHC-NP and monitored for hyperglycemia until stably lycemic (for 4 wk). Animals were also assessed daily for glycosuria and given human insulin isophane (l IU per day) so. if 3+.

Statistical analyses Data were compared by two-tailed Student’s t, Mann-Whitney U, Chi-Square, or two-way ANOVA tests. Statistical cance was assumed at P < 0.05.

Mice NOD/Lt mice were from the Jackson Lab (Bar Harbor, ME). 17.40L/8.3 (8.3-NOD), 17.6u/8.3u NOD) and BDC2NOD mice have been described (Katz, J.D. et al. (1993) Cell 74:1089-1100; Verdaguer, J. et al. (1997) J Exp Med 186:1663-1676; Han, B. et al. (2005) J Clin Invest 115:1879-1887). e 2. Production of TlD-relevant pMHC class II.

Several different TlD-relevant and irrelevant (i.e., negative control) peptide/I-Ag7 complexes were produced in eukaryotic (S2 or CHO cells). Studies using ers generated from these monomer preps confirm that these rs are ed into the supernatant as ly folded pMHC complexes. provides an example.

Reversal of hyperglycemia in NOD mice by treatment with TlD-relevant pMHC class II-NPs Diabetic NOD mice were treated twice a wk with 7.5 ug ofpMHC class II-coated- NPs. Mice were considered cured when lycemic for 4 wk, at which point treatment was withdrawn. As shown in Fig. 3, whereas 2.5mi/I-Ag7-, IGRP128_145/I-Ag7-, and IGRP4_22/I- Ag7-NPs reversed hyperglycemia in 90-100% of mice (n=29 mice), treatment with HEL14_22/IAg7-NPs (a foreign pMHC) had no effect. Intraperitoneal glucose tolerance tests (IPGTTs) in cured mice >30 wk after treatment withdrawal yielded curves that were very similar to those in age-matched non-diabetic untreated controls and cantly different than those ed in untreated acutely diabetic NOD mice (. Thus, NPs coated with levant pMHC class II restore glucose homeostasis in diabetic mice.

TlD-relevant pMHC class II-NPs expand cognate memory TR1 gulatory CD4+ T cells Studies of blood, spleens, pancreatic lymph nodes (PLNs), mesenteric lymph nodes (MLNs) and bone marrow of 50 wk-old diabetic mice that had been rendered normoglycemic by treatment with 2.5mi/I-Ag7-NPs ed significantly increased tages of 2.5mi/I- Ag7 tetramer+ CD4+ cells, as compared to mice studied at diabetes onset or age-matched non- ic untreated s (. CD4+ T-cell expansion was antigen-specific (.

The tempo, magnitude and distribution of expansion were similar for the three TlD-relevant pMHC class II-NPs tested (. Phenotypic analyses of the NP-expanded tetramer+ cells vs. tetramer— cells in all these cohorts revealed a memory-like TR1 phenotype ( top) with co-expression of the ecific markers described recently (Gagliani, N. et al. (2013) Nature Medicine 19:739-746) ( bottom): CD6210W/CD44high/ ICOS+/CD25’/FoxP3’ /surface TGFBV CD49b+/LAG3+. That these cells were not FoxP3+ was confirmed in NOD mice expressing FoxP3 promoter-eGFP, in which all pMHC-NP-expanded cells were eGFP- negative (not shown).

Consistent with these phenotypic data, tetramer+ CD4+ cells sorted from pMHCNP-treated mice responded to DCs pulsed with cognate peptide by almost exclusively secreting IL-10 and, to a lower extent, IFNy (and not shown). Importantly, purified CD4+ but not CD8+ T cells from pMHC-NP-treated donors ted T1D in NOD.scz'd mice transferred with diabetogenic splenocytes and hosts treated with pMHC class II-NPs were 100% protected for >100 days (not shown).

These pMHC class II-NP-expanded tetramer+ cells, unlike their er— counterparts, inhibited the proliferation of non-cognate T-cells to peptide-pulsed DCs (presenting the peptides targeted by both the responder and tetramer+ TR1 cells). on of an anti-IL10 or anti-TGFB mAbs to the cultures partially inhibited the suppression, versus es receiving anti-IFNy or rat-IgG (not shown). Most importantly, studies of diabetic mice treated with IGRP4_22 or 2.5mi/I-Ag7-NPs and blocking anti-IL-lO, anti-TGFB or anti- IFNy mAbs or rat-IgG ( indicate that restoration of normoglycemia by pMHC class II-NPs requires IL-10 and TGFB but not IFNy. However, studies in spontaneously diabetic I 07/7 and NOD.Ifng’/’ mice suggest that expression of both IL-lO and IFNy are necessary for development of the TRl cells that expand in response to pMHC class II-NPs; in these mice, P therapy expanded Th2-like cells (NOD.Ifng’/’) or IFNy+/IL-4+/IL10’ cells (NOD.IZI OT/Tmice). Studies in ic IGRP’/’ NOD mice (unable to prime IGRP- reactive T cells) showed that these mice did not respond to IGRP4_22/I-Ag7-NPs (there was no T cell expansion or restoration of normoglycemia) because these mice lacked IGRP4_22- primed cells. In contrast, all the diabetic IGRP’P NOD mice treated with 2.5mi/I-Ag7-NPs cured (not shown). Thus, pMHC class II-NPs, like pMHC class I-NPs, operate by expanding disease-primed regulatory , but cannot prime these ses de novo because they lack co-stimulatory signals.

Lastly, studies with vaccinia virus (rVV) showed that pMHC class II-NP-treated NOD mice can readily clear an acute viral infection (A). In agreement with this, d mice can mount antibody responses t a model antigen in adjuvant (B).

Example 3. Monospecific pMHC class II-NPs decrease the severity of EAE.

Applicant then tested the therapeutic potential of a pMHC class II-based nanomedicine in Experimental Autoimmune Encephalomye-litis (EAE). This model was utilized in the most stringent test possible: to igate ifpMHC-NPs can reverse ished EAE as opposed to prevent or blunt its development. This is not a trivial issue. A recent review of interventions in EAE shows that <l% of over 400 studies initiated treatment 21 days after EAE induction , J. et al. (2006) Nat Protoc 1:406-417); the reported data were obtained in mice in which treatment was initiated 21 days after EAE induction and improved disease scores in a dose-dependent manner ().

Example 4. Synthesis and quality control of pMHC class II-coated NPs.

Applicant developed an optimized iron oxide NP design that does not employ surfactants for synthesis and yields highly stable, monodispersed ations that can be loaded with optimal pMHC loads. Although several different pMHC-coating chemistries can be used (A), Applicant regularly uses NPs functionalized with maleimide-conjugated PEGs, which accept high valencies ofpMHCs engineered to encode a free Cys at their carboxyterminal end (up to more than 60 pMHCs/NP). These pMHC class II-NPs are processed h l quality control checks to define pMHC valencies per NP (dot- ELISA, amino acid analysis), NP density, NP charge and NP size (metal core, as defined by WO 63616 TEM; and hydrodynamic diameter, as defined via dynamic light scattering (DLS)). FIG. IZB shows a representative TEM image and C shows DLS profiles ofpMHC-uncoated vs. coated NPs. A typical dosing regimen involves the administration of 1-50 ug of total pMHC ated) per dose (about 2 uL of the preparation diluted in 100 uL of PBS).

Example 5. Treatment with pMHC class II-coated NPs.

The above data are consistent with data Applicant previously obtained in mice treated with pMHC class I-NPs: pMHC class II-NPs expand cognate memory regulatory T cells (in this case TRl) that ss the presentation of other autoantigenic peptides by local autoantigen-loaded APCs (Amrani, A. et al. (2000) Nature 406:739-742).

Human TRl CD4+ T-cell clones have been reported to kill certain subsets of professional APCs, such as dendritic cells (DCs) (Amrani, A. et al. (2000) Nature 406:739- 742). Applicant therefore investigated whether the antigen-specific TRl cells that expand in response top MHC class II-NP therapy suppressed autoimmunity by killing tigen- loaded APCs. This was done by tranfusing 1:1 mixtures of DCs pulsed with 2.5mi or GPI peptides and labeled with PKH26 (2.5mi-pulsed DCs) or CFSE (GPI-pulsed DCs), into NOD mice that had received 10 doses of 2.5mi/1Ag7-NPs during the ing 5 weeks, or NOD mice that had not received any treatment. The hosts were sacrificed 7 days later to compare the ratios of PKH26+ vs CFSE+ cells in the two different hosts. As shown in A (top panels), no differences were observed, suggesting that the TRl CD4+ T-cells that expanded in response to P therapy do not kill n-expressing DCs.

To investigate whether this was a peculiarity of the type ofAPC used (a DC) or a general feature of other APC types, the above ments were ed but using splenic B- cells as opposed to DCs. Unexpectedly, it was found that the numbers of 2.5mi-pulsed B- cells expanded (rather than decreased) in hosts that had been treated with 2.5mi/IAg7-NPs- coated NPs (A, bottom panels). This was cted because, based on the state-of- the-art, it was expected just the opposite outcome (a selective and specific decrease of 2.5mi- pulsed B-cells as compared to their GPI-pulsed rparts).

Applicant then ascertained whether such a B-cell-expanding effect ofpMHC class II-NP treatment could be nted by comparing the absolute numbers and percentages of B-cells in the pancreas-draining (PLN) and non-draining (MLN) lymph nodes of mice treated with 2.5mi/IAg7-NPs versus untreated controls. As shown in B, pMHC class II-NP- treated NOD mice had a marked increase in the percentage of B-cells in the PLN but not MLN. No such differences were seen in the PLN vs MLN of untreated NOD mice, indicating that these effects were a consequence ofpMHC-NP y. Notably, there was a statistically significant correlation between the frequency of 2.5mi-specif1c TRl CD4+ T-cells in the PLNs of individual mice and the frequency of PLN-associated B-cells, suggesting that such an increased recruitment of B-cells to the PLNs of the pMHC-NP-treated NOD mice was driven by the 2.5mi-specif1c TRl CD4+ T-cells that expanded in se to MHC-NP therapy.

Collectively, these data raised the possibility that the B-cells that expanded in response to MHC-NP therapy might be B-regulatory cells, that is B-cells that acquire the capacity to produce IL-lO in response to cognate interactions with the P-expanded TRl CD4+ T-cells. This case scenario posits that 2.5mi-specif1c TRl CD4+ T-cells would induce the differentiation and expansion of undifferentiated chromogranin A-specif1c B-cells (chromogranin A is the natural antigenic source of the 2.5mi epitope) that have captured chromogranin A and therefore present the corresponding 2..’5mi/IAg7 pMHC complexes on their surface, to IL-lO-producing Breg cells.

To test this hypothesis, Applicant transfused 2.5mi- or GPI-pulsed B-cells (labeled with PKH26) from a strain ofNOD mice in which one of its two ILlO loci carries a ed insertion of an IRES-eGFP cassette n the stop codon and polyadenylation signal of exon 5 (ll), into 2.5mi/1Ag7-NP-treated or untreated NOD hosts.

Seven days after transfer, the flow cytometric phenotype of the donor PKH26+ B- cells in the hosts (C, top panel) was determined. As shown in C e and bottom panels), a significant fraction of the donor B-cells expressed ILlO-encoded eGFP and were both CD5+ and gh. These are three key markers of Breg cells (Xie, J. et al. (2007) Adv Mater 19:3163; Xie, J. et al. (2006) Pure Appl. Chem. 78: 014). This was only seen with s pulsed with 2.5mi, but not with B-cells pulsed with a negative control e (GPI), and it only occurred in pMHC-NP-treated mice. Importantly, this effect was mediated, at least in part, by the IL-lO pMHC-NP-expanded TRl CD4+ s, because no such response was observed in IL-lO-deflcient NOD hosts.

Taken together, these data demonstrate that pMHC class II-NP therapy induces the differentiation and expansion of antigen-specific B-cells into B-regulatory cells.

The description of data suggesting the existence of B lymphocytes with regulatory properties can be found in literature dating back to 1974. Similarly to the TRl CD4+ T-cells that expand in response to pMHC class II-NP therapy, Breg cells express immunosuppressive cytokines, including IL-10 and TGFb, as well as other molecules that can inhibit pathogenic autoreactive T- and B-cells in an antigen-dependent and highly specific manner, via cognate, pMHC class II-driven cell-to-cell interactions (Xu, C. et al. (2007) Polymer International 56:821-826). Although ent stimuli have been shown to be able to induce Breg formation in vitro, and to a much lesser extent in vivo, to the best of Applicant’s knowledge there is currently no therapeutic approach capable of ng and expanding antigen-specific Breg cells in vivo. By eliciting highly disease-specific TR1 CD4+ T-cells, Applicant trates that pMHC class II-based nanomedicines also elicit disease-specific Breg cells.

Since Breg cells can also promote the differentiation of effector into TR1 CD4+ T-cells, pMHC class II-based nanomedicines unleash a profound and sustained immunosuppressive response that is highly n-specific and therefore capable of selectively suppressing autoimmune responses without compromising systemic immunity.

Example 6. Synthesis of surface functionalized iron oxide nanoparticle by thermal decomposition of iron acetonate, and bioconjugation thereof PEG is melted. Benzyl ether and iron acetyle acetonate is added. After 1 hr of heating at 105°C, the ature is increased to 2600C and refluxed. After about 2 hr, iron rticles form and the color of the solution turns black. The reaction is cooled down to room temperature and some water added to extract nanoparticles from the reaction vessel.

The nanoparticles are purified by Miltenyi Biotec LS magnet column. The making of iron oxide nanoparticle protein ates include adding n and the iron nanoparticle at a buffered pH of 6.2-6.5 (0.15M NaCl and 2mM EDTA), stirring at room temperature for 12- 14 hours, and purifying protein conjugated particle by Miltenyi Biotec LS magnet column.

It should be tood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the ions embodied therein herein disclosed may be ed to by those skilled in the art, and that such modifications, improvements and ions are considered to be within the scope of this ion. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as tions on the scope of the invention.

The ion has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the ion. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically d herein.

In addition, where features or aspects of the invention are described in terms of Markush groups, those d in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of s of the Markush group.

The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only atives and “and/or.” As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or ining” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Throughout this disclosure, various publications, patents and hed patent specifications are referenced by an identifying citation. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their ty, to the same extent as if each were incorporated by reference dually. In case of conflict, the present specif1cation, including def1nitions, will control.

Table 1. onalized PEG linkers Nana-n afiéfie- -. \ a makmgtcmfi ‘ " Mai—FES— wuse Slap-\m‘nniv PES- nwéiufi-fise ' " \cpaxitie Table 2. Nanoparticle designs and pMHC-binding capacity. iflfi Syméaesas mag-E: {ma-aisugaiésm RFEEEi§3§a§fi=fi§ figags’egatian we Rammm agifiw '1‘) m 3%E} (-1!

Claims (15)

What is claimed 1. is:

1. A method for making PEG functionalized iron oxide nanoparticles comprising thermally decomposing iron acetyl acetonate in the presence of functionalized PEG molecules or the ce of functionalized PEG molecules and benzyl ether, and wherein the temperature of the thermal decomposition is n 80 to 300° C, and wherein the functionalized PEG is a maleimide functionalized PEG.

2. The method of claim 1, wherein the iron oxide nanoparticle is soluble.

3. The method of claim 1, wherein the thermal decomposition comprises a single-step on.

4. The method of claim 3, wherein the thermal decomposition is carried out in the presence of benzyl ether.

5. The method of claim 1, n the temperature for the thermal osition is 80 to about 200°C.

6. The method of claim 1, wherein the thermal decomposition is carried out for about 1 to about 2 hours.

7. The method of claim 1, wherein the nanoparticles are stable at about 4°C in PBS without any detectable degradation or aggregation.

8. The method of claim 7, wherein the rticles are stable for at least 6 months.

9. The method of any one of claims 1-8, wherein the method further comprises ing the nanoparticles with a magnetic column.

10. A method for making major histocompatibility complex peptide (pMHC) nanoparticle complexes comprising: a) thermally decomposing iron acetyl acetonate in the ce of functionalized PEG molecules or the presence of functionalized PEG molecules and benzyl ether, wherein the temperature for the thermal decomposition is between 80 to 300 ºC, thereby providing PEG functionalized iron oxide nanoparticles; and b) contacting the PEG functionalized iron oxide nanoparticles with pMHC thereby providing pMHC nanoparticle complexes, and wherein the functionalized PEG is a maleimide functionalized PEG.

11. The method of claim 10, wherein the method further comprises purifying the nanoparticles with a magnetic .

12. The method of claim 1, wherein the temperature for the thermal decomposition is 80 to about 150°C.

13. The method of claim 1, n the temperature for the thermal decomposition is about 100 to about 250°C.

14. The method of claim 1, wherein the temperature for the thermal decomposition is about 100 to about 200°C.

15. The method of claim 1, wherein the temperature for the thermal decomposition is about 150 to about 250°C W0