NZ727956B2 - Methods and compositions for treating inflammation - Google Patents

Abstract

Disclosed is a nanoparticle comprising an antigen-MHC complex, wherein the complex comprises a MHC protein complexed to an antigen derived from a microbe of the gastrointestinal tract or is a GI-associated antigen.

Description

METHODS AND ITIONS FOR TREATING INFLAMMATION CROSS-REFERENCE TO RELATED APPLICATION This application claims priority under 35 U.S.C. § 119(e) to US. Provisional Application Serial No. 61/615,743, filed March 26, 2012, the content of which is incorporated herein by reference in its entirety.

FIELD OF DISCLOSURE This disclosure is directed to compositions and methods related to immunotherapy and medicine. In particular, this disclosure is related to therapeutics for the treatment of inflammation.e.g., inflammation of the intestinal tract.

BACKGROUND Inflammatory bowel disease (IBD) is the name of a group of disorders that cause the intestines to become inflamed (red and swollen). More than 600,000 Americans have some kind of inflammatory bowel disease every year. This group of diseases is often chronic in nature and associated with symptoms such as abdominal pain, vomiting, diarrhea, rectal bleeding, severe internal cramps/muscle spasms in the region of the pelvis, and weight loss. The symptoms associated with IBD can limit the quality of life and affect those ed on a daily basis.

Treatment modalities of IBD mainly include immunosuppressives that lower the overall immunity of the t. Such treatment is risky and often puts the patient at risk for infection and disease due to compromised immunity.

There is a need in the art for target therapies that treat the disease but do not mise the overall immunity of the patient. This disclosure satisfies this need and provides related advantages as well.

In se to a need in the art, described herein are therapeutic methods and compositions that activate and amplify pre-existing endogenous mechanisms directed to suppressing c inflammation responses. In one aspect, compositions and methods are provided for treating inflammation of the gastrointestinal tract.

One aspect relates to a method for inducing an anti-inflammatory response in a cell or tissue by administering an effective amount of an antigen-MHC-nanoparticle complex; wherein the antigen is an antigen derived from a microbe that resides within or infects the gastrointestinal tract or is a GI—associated antigen. Also ed is an antigen-MHC-nanoparticle complex for use in inducing an anti-inflammatory response in a cell or tissue, wherein the n is an antigen derived from a microbe that resides within or infects the intestinal tract or is a GI- associated antigen. Also provided is the use of an antigen-MHC-nanoparticle complex in the cture of a ment useful for inducing an anti-inflammatory response in a cell or tissue, wherein the antigen is an antigen derived from a microbe that resides within or infects the gastrointestinal tract or is a GI-associated antigen.

In another aspect, a method is provided for treating inflammation in a patient in need f by administering an effective amount of an antigen-MHC-nanoparticle complex; wherein the antigen is an antigen derived from a microbe that s within or infects the intestinal tract or is a GI—associated antigen. Also provided is an antigen-MHC-nanoparticle complex for use in treating ation in a patient in need thereof, wherein the antigen is an antigen derived from a microbe that resides within or infects the gastrointestinal tract or is a GI- associated antigen. Also provided is the use of an antigen-MHC-nanoparticle complex in the manufacture of a medicament for ng ation in the gastrointestinal tract in a patient in need thereof, wherein the antigen is an antigen derived from a microbe that resides within or infects the gastrointestinal tract or is a GI—associated antigen.

In yet a r aspect, a method for accumulating anti-inflammatory T cells in a patient in need thereof is ed by administering an effective amount of an antigen-MHC- nanoparticle complex; wherein the antigen is an antigen derived from a microbe that resides within or infect the gastrointestinal tract or is a GI-associated antigen. Also ed is an antigen-MHC-nanoparticle complex for use in accumulating nflammatory T cells in a patient in need thereof, wherein the antigen is an antigen derived from a microbe that resides within or infects the gastrointestinal tract or is a GI-associated antigen. Also provided is the use of an antigen-MHC-nanoparticle complex in the manufacture of a medicament useful for accumulating anti-inflammatory T cells in a patient in need thereof, wherein the antigen is an 2013/052352 antigen derived from a microbe that resides within or infects the gastrointestinal tract or is a GI- associated antigen.

Other aspects relate to a complex comprising, consisting essentially or yet further consisting of, a nanoparticle, a MHC protein, and an antigen derived from a microbe that resides within or infects of the gastrointestinal tract or is a GI—associated antigen. Also ed are compositions comprising, consisting essentially of, or yet further consisting of, the antigen- MHC-nanoparticle as described herein and a carrier.

Also provided is a kit comprising, or alternatively consisting essentially of, or yet further consisting of, a ition as described herein and instructions to use the compositions for their intended purpose.

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 tood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Fig. 1A-1C demonstrate that BacIYL binds to H-2Kd with high affinity and the resulting pMHC complex binds to 6_214-specif1c T-cells. A, Peptide-induced stabilization of KCl molecules on RMA-SKd cells. TUM is a positive control and Gp33 is a ve (Db- binding) control. B and C, BacIYL/Kd tetramers bind specifically to 8.3-CD8+ s, albeit with lower avidity than NRP-V7/Kd tetramers.

Fig. 2A-2D show that BacIYL functions as an antagonist in isolation, but as a partial t in the ce of LPS and its donor protein is ive cross-presented by dendritic cells. A, sion of CD44 and CD69 in 8.3-CD8+ T-cells ed in the presence of BacIYL, IGRP206_214 (positive control) or TUM (negative control). B, Antagonism assay. TUM is used as a negative control. Note how increasing concentrations of BacIYL (but not TUM, a negative control that binds Kd) antagonize IGRP206_214-induced 8+ T-cell responses (IFNg secretion, top; and proliferation, bottom). C, BacIYL functions as an t in the presence of LPS. NTG, non-transgenic (CD8+ T-cells). D, DCs can process BacIYL or BACIGRp206_214-like WO 44811 epitopes from recombinant wild-type integrase or recombinant mutant Integrase (where the BacIYL epitope is mutated to encode IGRP206_214).

Fig. 3A-3D show that the BacIYL peptide induces memory CD8+ T-cell formation in vitro. A and B, Phenotype of 8+ T-cells 28 days after culture in the presence of peptide- pulsed (10 or 0.001 ug/ml) DCs. 17.6-CD8+ T-cells are very low avidity IGRP206_214-specif1c CD8+ T-cells; as expected they remain naive after 28 days in culture with BacIYL. C, Intracellular IFNy content in response to peptide nge. BacIYL-cultured 8.3-CD8+ s rapidly produce IFNy in se to IGRP206_214 stimulation. D, Secretion of IFNy by, and proliferation of memory-like 8.3-CD8+ s (induced by BacIYL) in response to peptide challenge.

Fig. 4A-4H shows that a BacIYL36_44-reactive CD8+ T-cell response affords protection from DSS-induced colitis. A and B show weight curves (A) and disease activity scores (B) of 8.3-NOD, 17.6-NOD upon DSS treatment vs. untreated mice. Figs. C and D show weight curves (C) and disease activity scores (D) of 8.3-NOD vs. Ith7’/’ 8.3-NOD mice upon DSS treatment.

Figs. E and F show the survival curves for the mice studied in A-D. Fig. G demonstrates that IGRP206_214'/' NOD, but not NOD mice are resistant to weight loss in response to colitis induced by 4% DSS. Fig. H shows that ve transfer of 36_44-crossreactive CD8+ CTL to IGRP206_214'/' NOD mice resulted in a significant reduction of disease activity scores as compared to their non-CTL-transfused counterparts.

Fig. 5A-5B shows BacIYL36_44-reactive CD8+ CTL protect 17.6-NOD mice from DSS- induced s. Fig. 5A shows weight , and Fig. 5B shows disease activity scores 17.6- NOD mice in response to DSS treatment plus 8.3-CTL transfer, to DSS treatment alone, and to no treatment at all. Note how adoptive transfer of BaclYL36_44-crossreactive CD8+ CTL to 17.6- NOD mice cantly reduced disease activity scores and weight loss in response to DSS treatment, as ed to their non-CTL—transfused counterparts.

Fig. 6 demonstrates the recruitment of Trl-like autoregulatory CD4+ T-cells to gut- associated lymphoid tissue in IGRP4_22/I-Ag7-NP-treated NOD mice. Data on two mice are shown. 2013/052352 Fig. 7 depicts a map of BacInt40_54-I-Ab-C-Jun in pMT/V5. DNA construct between Nco I (854) to Xho I (1738) sites encodes HA-BacInt40_54-I-Abeta (b)-C-Jun fusion protein (293 a.a). The fusion protein es 15 a.a HA leader sequence followed by Baclnt40_54 (TNV) peptide (15 a.a.). DNA sequence encoding peptide was linked to I-Abeta (b) (199 a.a.) through a 16 a.a GS linker. C—terminal of I-Abeta (b) was linked to C-Jun sequence (40 a.a.) thorough a 8 a.a GS linker. a.a. = amino acid.

Fig. 8 shows the protein and DNA sequences of BacInt40_54-I-Abeta (b)-C-Jun construct. The sequences of individual components in the fusion n are HA leader underline) and: sequences. GS linkers are not highlighted.

Fig. 9 depicts a map of I-Aalpha Fos-BirA-His6 in pMT/V5. DNA construct sites encoding HA leader- I—Aalpha (b)-C—Fos-BirA-His X 6 fusion protein (284 a.a) was cloned into pMT/V5 fly cell expression vector between Nco I (854) to Xbal . The fusion protein includes I-Aalpha (d) (195 a.a.), followed by C-Fos though a GS linker ( 6 a.a.), and then BirA sequence and 6 X His.

Fig. 10 shows the protein and DNA sequences of I-Aalpha (b)-C-Fos construct. The sequences of individual components in the fusion protein are HA leader (underline) followed by I—Aalpha ] b] (double underline), gigs (dotted underline), shaded) and 6 X His sequences.

GS linkers are not highlighted.

Fig. 11 depicts a map of BacInt81_95-I-Ab-C-Jun in pMT/V5. DNA construct n Nco I (854) to Xho I (1738) sites encodes HA-BacInt81_95-I-Abeta (b)-C-Jun fusion protein (293 a.a). The fusion protein includes 15 a.a HA leader sequence followed by BecInt81_95 (LGY) peptide (15 a.a.). DNA sequence ng peptide was linked to I-Abeta (b) (199 a.a.) through a 16 a.a GS linker. C—terminal of a (b) was linked to C-Jun sequence (40 a.a.) thorough a 8 a.a GS linker.

Fig. 12 shows the protein and DNA sequences of BacInt81_95-I-Abeta (b)-C-Jun uct. The sequences of individual components in the fusion protein are HA leader ine) and (shaded) sequences. GS linkers are not highlighted.

Fig. 13 depicts a map of BacInt365_379-I-Ab-C-Jun in pMT/V5. DNA uct between Nco I (854) to Xho I (1738) sites encodes HA-BacInt365_379-I-Abeta (b)-C-Jun fusion protein (293 a.a). The fusion protein includes 15 a.a HA leader sequence followed by BacInt365_379 (TQI) peptide (15 a.a.). DNA sequence encoding peptide was linked to I-Abeta (b) (199 a.a.) h a 16 a.a GS linker. C—terminal of I-Abeta (b) was linked to C-Jun sequence (40 a.a.) thorough a 8 a.a GS linker.

Fig. 14 shows the protein and DNA sequences of BacInt365_379-I-Abeta (b)-C-Jun construct. The sequences of individual components in the fusion protein are HA leader underline) and (shaded) sequences. GS linkers are not highlighted.

Fig. 15 depicts a map of BacInt57_71-I-Ab-C-Jun in pMT/V5. DNA construct between Nco I (854) to Xho I (1738) sites encodes HA-BacInt57_71-I-Abeta (b)-C-Jun fusion n (293 a.a). The fusion protein includes 15 a.a HA leader sequence ed by BacInt57_71(INH) peptide (15 a.a.). DNA sequence encoding e was linked to I-Abeta (b) (199 a.a.) through a 16 a.a GS linker. C—terminal of I-Abeta (b) was linked to C-Jun sequence (40 a.a.) thorough a 8 a.a GS linker.

Fig. 16 shows the protein and DNA sequences of BacInt57_71-I-Abeta (b)-C-Jun construct. The sequences of dual components in the fusion protein are ghted: fl d ine) and :. shaded) sequences. GS linkers are not highlighted.

Fig. 17 depicts a map of Baclntgg_102-I-Ab-C-Jun in pMT/V5. DNA construct between Nco I (854) to Xho I (173 8) sites encodes HA-BacIntgg_102-I-Abeta (b)-C-Jun fusion protein (293 a.a). The fusion protein es 15 a.a HA leader sequence followed by gg_102 (IPA) peptide (15 a.a.). DNA sequence encoding peptide was linked to I-Abeta (b) (199 a.a.) through a 16 a.a GS linker. C—terminal of I-Abeta (b) was linked to C-Jun sequence (40 a.a.) thorough a 8 a.a GS linker.

Fig. 18 shows the protein and DNA sequences of Baclntgg_102-I-Abeta (b)-C-Jun construct. The sequences of individual components in the fusion protein are highlighted: & d underline) and shaded) sequences. GS linkers are not highlighted.

Fig. 19 shows representative TEM image of pMHC-coated gold NPs (~14 nm) concentrated at high densities (~5X1013/11’ll) and monodispersed. Mag: 50,000X.

Fig. 20 shows the effects ofpMHC (GNP) dose and pMHC valency on the agonistic properties of pMHC-coated GNPs. The Figure compares the amounts of IFNy secreted by cognate 8.3-CD8+ T-cells in response to two different pMHC-GNP samples (both ting of ~2x10” GNPs of 14 nm in diameter/ml). Au-022410 and Au-219lO carried ~250 and ~120 pMHCs/GNP, respectively. Au—Ol 18 l O-C carried ~120 control pMHCs/GNP.

Fig. 21 demonstrates the P-induced secretion of IFNy by 8.3-CD8+ T cells as a function ofpMHC y. 8.3-CD8+ T-cells (2.5x105 cells/ml) were cultured with increasing numbers ofNPs coated with three different IGRP206_214/Kd valencies.

Fig. 22 shows that the lower agonistic activity ofpMHC-NPs can be compensated by increasing the pMHC-NP density but only above a threshold ofpMHC valency. Graph compares the tic activity of three different pMHC-NP preparations (carrying three different valencies ofpMHC) over a range ofNP ies. Note that NPs carrying 8 pMHCs, unlike those carrying 11 pMHCs, cannot adequately trigger IFNy secretion even at high pMHC- NP densities, as compared to NPs carrying 54 pMHCs.

Fig. 23 shows the effects ofpMHC y threshold on the agonistic properties of pMHC-NPs as a function of total pMHC input.

Fig. 24 shows the effects ofpMHC valency on the agonistic activity -NPs produced with larger iron oxide NP cores.

Fig. 25 shows the effect of size on agonistic activity. Au15 were 14 nm GNPs coated with a relatively low pMHC valency but prepared at a high density; Au—0323-40 were 40 nm GNPs coated with high pMHC valency but at low density. Au—0224-15 had superior tic activity than the Au40 .

Fig. 26 shows the effect of protective PEGs on the function of NPs. Au- 021910 consisted of ~2x1013 GNPs of 14 nm in diameter/ml protected by 2 kD thiol-PEGs and coated with ~120 pMHCs/GNP. Au—0128 10 GNPs (also ~2x1013 14 nm GNPs/ml) were protected by 5 kD thiol-PEGs and were coated with ~175 pMHCs/GNP. Sample Au-021910 had superior tic ty.

Fig. 27 shows the Efficient expansion ofNRP-V7-reactive CD8+ T-cells by NRP- coated gold NPs. 3 X 1012 NPs (~10 nm in size) ng 25 ug of pMHC (150 pMHC/NP) were used. Pre-diabetic 10 wk-old NOD mice were d with two weekly injections ofNRP-V7/kd—coated gold NPs for 5 weeks. TUM/Kd tetramer is a negative control.

Each column of panels ponds to a different mouse.

Fig. 28 depicts the large expansion of cognate CD8+ T-cells in mice treated with pMHC-coated NPs. 3 X 1012 IGRP206_214/Kd-NPS (~10 nm in size) ng 25 ug of pMHC (150 pMHC/NP) were used. Upper panel: profile of a mouse sacrificed after 4 doses. Bottom panel: profile of two different mice after 10 injections (blood only; alive at the time of this submission).

DETAILED PTION It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be tood 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 ed claims, the singular forms (4 a) “an”, and “the” include plural referents unless the context clearly dictates ise. Thus, for example, reference to “an excipient” includes a plurality of excipients. 1. DEFINITIONS Unless defined otherwise, 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 following 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 elements, but not excluding 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 composition 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 the ability to treat inflammatory bowel disease in a subject in need of such treatment and/or inducing an anti-inflammatory se. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these tion terms are within the scope of this invention.

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 biocompatibility, rs and excipients that have had history of safe use in humans or with GRAS (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 s to the body. For a composition to be biocompatible, and be regarded 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 material in the patient is avoided. 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 material used, and other factors well recognized by the skilled artisan. A mixture of orbable, biocompatible materials can be used to form the nanoparticles used in this ion. In one embodiment, iron oxide and a biocompatible, bioabsorbable polymer can be combined. For e, iron oxide and PGLA can be combined to form a nanoparticle An antigen-MHC-nanosphere complex refers to presentation of a peptide, carbohydrate, lipid, or other antigenic segment, fragment, or epitope of an nic molecule or protein (i.e., self peptide or autoantigen) on a surface, such as a biocompatible radable nanosphere. "Antigen" as used herein refers to all, part, fragment, or segment of a molecule that can induce an immune response in a subject or an expansion of anti-pathogenic cells.

The term “about” when used before a numerical designation, e.g., ature, time, amount, and concentration, including range, indicates approximations which may vary by ( + ) or (—)lO%,5%,orl%.

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 g profile similar 2013/052352 to the ligand except the mimic has one or more functional 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 “immune cell" refers to a cell of the immune system. Cells of the immune system include, for example, adult splenocytes, T lymphocytes, B lymphocytes, and cells of bone marrow origin, such as antigen presenting cells of a mammal, that have actiVity towards the organism from which the immune cell is derived. Also included are cells of the innate immune system such as, for example, natural killer cells, mast cells, eosinophils, basophils, and phagocytic cells such as macrophages, neutorphils, and dendritic cells.

The term “anti-inflammatory T cell” refers to a T cell that promotes an anti- inflammatory response. The anti-inflammatory function of the T cell may be accomplished through production and/or ion of anti-inflammatory proteins, cytokines, chemokines, and the like. Anti-inflammatory proteins are also ed to ass anti-proliferative signals that suppress immune responses. nflammatory proteins include IL-4, IL-10, IL- 1 3, IFN—(x, TGF-B, IL-lra, G—CSF, and soluble receptors for TNF and IL-6. Also included are anti- inflammatory cells that have an inflammatory phenotype but kill antigen-presenting cells orchestrating a particular autoimmune response. In n embodiments, these cells make IFNy and TNFu, among other cytokines. In certain embodiments, the anti-inflammatory T cell is one that recognizes the gut bacterial epitope with low aVidity. In further embodiments, the anti- inflammatory T cell is a cytotoxic T cell.

The term “IL-10” or “Interleukin-10” refers to a cytokine d by the IL-10 gene.

The IL-10 sequence is represented by the k Accession No.: NM_000572.2 (mRNA) and NP_000563.1 (protein).

The term ” or “Transforming growth factor beta” refers to a protein that can have an anti-inflammatory effect. TGF-B is a ed protein that exists in at least three ms called , TGF-BZ and TGF-B3. 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- miillerian hormone, bone morphogenetic protein, decapentaplegic and Vg— l.

WO 44811 The term “gastrointestinal tract" refers to both the upper and lower gastrointestinal tract. The upper gastrointestinal tract consists of the esophagus, stomach, and duodenum. The lower gastrointestinal tract includes the small intestine and the large intestine.

The term “microbe” refers to a unicellular microscopic organism. rgansims include, for example, bacteria, fungi, archaea, and protists.

A "an effective amount" is an amount sufficient to e the intended purpose, non- limiting examples of such include: initiation 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 eutic purpose, e.g., a therapeutically 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 t disclosure.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more, H Hat least one," and "one or more than one." The term “Integrase” refers to a protein expressed in Bacteroides. The GenBank Accession No. corresponding to the sequence of Integrase is YP_00130008l.l. This sequence is represented by SEQ ID No. 2. SEQ ID No. 3 represents an encoding DNA sequence of Integrase. SEQ ID No. 1 corresponds to an epitope in the integrase n. This epitope is IYLKTNVYL (SEQ ID No. l). Bactemides strains that are known to have the IYLKTNVYL (SEQ ID No. l) epitope include, for e, oidcs sp 9___l___42l~"AA, Bactcroides sp D4, Bacteroidcs sp. 3_1_33E‘AA, Bacteroides dorei 5_l_36/D4, Bactcroides dorei DSM 17855, Bacteroides vulgatus ATCC 8482, Bacteroides sp. 4___3___47FAA, Bacteroides vulgatus PCSIO.

By "nanosphere," "NP," or “nanoparticle” herein is meant a small discrete particle that is stered singularly or pluraly to a subject, cell specimen or tissue specimen as appropriate. In certain embodiments, the nanospheres are substantially spherical in shape. The term "substantially spherical," as used herein, means that the shape of the particles does not deviate from a sphere by more than about 10%. In certain embodiments, the rticle is not a liposome or viral particle. In further ments, the nanoparticle is solid. Various known n or peptide complexes of the invention may be d to the particles. The nanospheres of this invention range in size fiom about 1 nm to about 1 um and, preferably, fiom about 10 nm to about 1 um and in some s refers to the average or median diameter of a plurality of nanospheres when a plurality of nanospheres are intended. Smaller nanosize particles can be obtained, for example, by the process of onation whereby the larger particles are allowed to settle in an aqueous solution. The upper portion 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 average 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 (mediated by antigen-specific T cells or their ion products) directed against gastrointestinal tract-microbe-specific antigens or a d epitope of antigens specific to microbes of the gastrointestinal tract. A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules, to activate antigen-specific CD4+ T helper cells and/or CD8+ cytotoxic T cells. The response may also involve activation of other ents.

The terms "inflammatory response" and "inflammation" as used herein indicate the complex ical response of vascular tissues of an dual to l stimuli, such as pathogens, d cells, or irritants, and includes ion 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. Exemplary pro- inflammatory cytokines include but are not limited to IL-1, IL-6, TNF-a, IL-l7, IL21, IL23, and TGF-B. Exemplary inflammations e acute inflammation and chronic inflammation. Acute inflammation indicates a short-term process characterized by the classic signs of inflammation (swelling, redness, pain, heat, and loss of on) due to the infiltration of the tissues by plasma and leukocytes. An acute inflammation typically occurs as long as the injurious stimulus is present and ceases once the stimulus has been removed, broken down, or walled off by scarring (fibrosis). Chronic inflammation indicates a condition characterized by concurrent active 2013/052352 inflammation, tissue ction, and attempts at repair. Chronic ation 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, macrophages, lymphocytes, and plasma cells), tissue destruction, and attempts at healing, which e angiogenesis and fibrosis. An inflammation can be inhibited in the sense of the present disclosure by affecting and in particular inhibiting anyone of the events that form the complex biological response ated with an inflammation in an individual.

The terms "epitope" and "antigenic determinant" 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 protein. 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 ring solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. s of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Glenn E. , Epitope Mapping Protocols (1996). T-cells recognize continuous epitopes of about nine amino acids for CD8 cells or about 13-15 amino acids for CD4 cells. T cells that recognize the e can be identified by in vitro assays that measure antigen- dependent proliferation, as ined by 3H-thymidine incorporation by primed T cells in response to an epitope (Burke et al., J. Inf. Dis., 170:1110-1119, 1994), by antigen-dependent killing (cytotoxic T cyte assay, Tigges et al., J. Immunol., 156(10):3901-3910, 1996) or by ne secretion. The presence of a ediated immunological se can be determined by proliferation assays (CD4+ T cells) or CTL oxic T lymphocyte) assays.

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

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

As used in this application, the term "polynucleotide" refers to a nucleic acid le that either is recombinant or has been isolated free of total genomic c acid. Included within the term ucleotide" are ucleotides (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 substantially 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 ptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide ofthe following s: 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 nucleotides, 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 having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially r protein, polypeptide, or peptide.

A polynucleotide is composed of a specific sequence 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 representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a er having a central processing unit and used for bioinformatics ations such as functional genomics and homology searching.

The term “isolated” or binant” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively 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 occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polynucleotides, polypeptides and proteins that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term “isolated or recombinant” means separated from constituents, cellular and otherwise, in which the cell, tissue, cleotide, e, ptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an ed cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype.

An isolated polynucleotide is separated from the 3’ and 5’ contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome. As is apparent to those of skill in the art, a turally 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 d, that percentage of bases (or amino acids) are the same in ing 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 described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7. l. Preferably, default parameters are used for alignment. A preferred alignment m is BLAST, using default parameters. In particular, preferred programs are BLASTN and , using the following t parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = 62; 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 ing Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.

It is to be inferred without explicit recitation and unless otherwise ed, that when the t invention relates to a polypeptide, 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 gical equivalent thereof” is ed to be synonymous with "equivalent thereof” when referring to a reference protein, antibody, fragment, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically d 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 izes under stringent conditions to the polynucleotide or complement of the polynucleotide as described herein for use in the described methods. In another aspect, an equivalent dy or antigen binding polypeptide intends one that binds with at least 70 % or atively at least 75 % or alternatively at least 80 % or alternatively at least 85 %, or , , alternatively at least 90 %, or atively at least 95 % affinity or higher y to a reference dy or antigen binding fragment. In r 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 lent 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 substantially equivalent ical 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 nucleotide 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 strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PC on, or the tic 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 ons 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 stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about 0.1x SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, 0.1x SSC, or deionized water. In general, ization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation 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 molecules. 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 n sequences is a function of the number of matching or homologous positions shared by the ces. An “unrelated” or “non- homologous” sequence shares less than 40% identity, or atively less than 25% identity, with one of the sequences of the present invention.

"Homology" or "identity" or "similarity" can also refer to two nucleic acid les that hybridize under stringent conditions.

As used herein, the terms "treating," "treatment" and the like are used herein to mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or lly preventing a disorder or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect utable to the disorder. In one aspect, treatment indicates a reduction in inflammation in a patient.

Methods to measure such include t limitation vasodilation, production of inflammation markers, and leukocyte infiltration cessation. Markers for inflammation include, for example, IL-6, IL-8, IL-18, TNF-alpha, and CRP. Any appropriate method to measure and monitor such markers are known in the art.

To prevent s 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 A aceutical composition” is intended to include the combination of an active WO 44811 agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

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

Codon Table Amino Acids Codons Alanine GCA GCC GCG GCU Cysteine 0 UGC UGU Aspartic acid GAC GAU Glutamic acid Glu GAA GAG Phenylalanine Phe UUC UUU Glycine GGA GGC GGG GGU Histidine His CAC CAU Isoleucine Ile AUA AUC AUU Lysine r AAA AAG Leucine r UUA UUG CUA CUC CUG CUU Methionine AUG gine AAC AAU Proline CCA CCC CCG CCU Glutamine CAA CAG Arginine AGA AGG CGA CGC CGG CGU Serine m AGC AGU UCA UCC UCG UCU Threonine a ACA ACC ACG ACI Valine GUA GUC GUG GUU Tryptophan a UGG Tyrosine iiliifilllliillfillfiifi a iiiiiiiiiiiiliiiiiii UAC UAU As used herein, a in" 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 fiom the ing detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of ration 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.

Descriptive Embodiments It was previously unknown that antigenic peptides from the symbiotic ia of the intestinal tract were specifically recognized by endogenous host s upon being processed by professional n-presenting cells (APCs, such as dendritic cells or DCs), and that this antigen-driven interaction between a e T-cell and the APC can inhibit IBD.

Without being bound by theory, Applicants believe that proteins from the bacteria that reside in or infect the gastrointestinal tract are processed by the proteasome or in the endosome and the resulting peptides shuttled to the endoplasmic reticulum for binding to endogenous MHC class I or class II molecules, which are then transported to the APC's plasma membrane, which then activates e T-cells.

Applicants e that this is the first disclosure that antigens of gastrointestinal- associated bacteria are processed and presented to e endogenous T-cells with the capacity to suppress inflammatory bowel disease, and therefore, Applicants believe that these antigens could be used as a target to foster the recruitment and accumulation of autoregulatory (anti- inflammatory) s to, for example, the gut in inflammatory bowel disease. Antigen-MHC- nanoparticle complexes have previously been shown to expand eutic populations of T cells in other diseases (see for e.g. US Patent Pub. No.: 2009/0155292), but it was unknown that this technology could suppress inflammation, in for example, the gastrointestinal tract or treat atory bowel es. Compositions and methods described herein are useful for the suppression of inflammation and for the treatment of diseases ated therewith.

II. METHODS The methods as described herein comprise, or alternatively consist essentially of, or yet further consist of the stration of an effective amount of an antigen-MHC-nanoparticle complex to a cell, tissue or subject for the purpose of one or more of: (l) inducing an anti- inflammatory response in a cell or tissue; (2) treating or reducing inflammation in a patient in need thereof; (3) accumulating autoregulatory, anti-inflammatory T cells in a patient in need thereof and/or (4) transfering cytotoxic T-lymphocytes targeting gut bacterial epitopes in a patient in need thereof. In one embodiment, the cytotxic T-lymphocytes recognize the gut bacterial epitope with low avidity.

In one embodiment, ation of the gastrointestinal tract is reduced or treated.

Methods to determine and monitor the therapy are known in the art and briefly described herein.

When delivered in vitro, administration is by contacting the composition with the tissue or cell by any appropriate method, e.g., by stration to cell or tissue culture medium and is useful as a screen to determine if the therapy is appropriate for an individual or to screen for alternative therapies to be used as a substitute or in combination with the sed itions. When administered in vivo, stration is by systemic or local administration. In vivo, the methods can be practiced 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 e or disorder.

In certain embodiments, the patient to be treated by the methods of this disclosure suffers from a gastrointestinal disease having as a symptom or condition thereof inflammation of the GI tissue. Non-limiting examples of gastrointestinal diseases include inflammatory bowel disease, colitis, Crohn’s disease, allergic reactions in the gastrointestinal tract, food allergies, eosinophilic diseases in the gastrointestinal system, irritable bowel syndrome, celiac disease and gastric haemorrhagia. In one embodiment, the disease is ed from the group of: atory bowel disease, colitis, Crohn’s disease, allergic inflammation of the gastrointestinal tract, and celiac disease. In a related embodiment, the disease is inflammatory bowel disease.

Methods described herein are useful for inducing an anti-inflammatory response in a cell or tissue. In one embodiment, the cell is a cell or tissue of the gastrointestinal tract. The upper intestinal tract consists of the esophagus, stomach, and duodenum. The exact ation between "upper" and " can vary. Upon gross dissection, the duodenum may appear to be a unified organ, but it is often divided into two parts based upon function, arterial supply, or logy. The lower gastrointestinal tract includes the small intestine and the large intestine. The small intestine has three parts: the duodenum, jejunum, and ileum. In the duodenum, the digestive enzymes from the as and the gallbladder (bile) mix together.

Digestive enzymes break down proteins and bile and emulsify fats into micelles. The duodenum contains Brunner's glands which produce bicarbonate, and pancreatic juice which contains bicarbonate to neutralize hydrochloric acid of the stomach. The jejunum is the midsection of the Intestine, connecting the duodenum to the ileum. It contains the plicae ares, and villi to se the surface area of that part of the GI Tract. The ileum has villi, where all soluble molecules are absorbed into the blood (capillaries and lacteals). The large intestine has three parts: the cecum, colon, and rectum. The orm appendix is attached to the cecum. The colon includes the ascending colon, transverse colon, descending colon and sigmoid flexure.

The main function of the colon is to absorb water, but it also contains bacteria that produce beneficial ns.

In another embodiment, the anti-inflammatory se is induced in an immune cell or tissue containing such. Immune cells include, for example, adult splenocytes, T lymphocytes, B lymphocytes, and cells of bone marrow origin, such as defective antigen presenting cells of a mammal, that have ty s the organism from which the immune cell is derived.

The MHC of the antigen-MHC-nanoparticle complex can be MHC I, MHC II, or nonclassical MHC. MHC proteins are described herein. In one ment, the MHC of the n-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 bed herein.

In one of its method aspects, there is provided a method for accumulating anti- inflammatory (gut microbe-specif1c or gastrointestinal-microbe c) T cells in a patient in need thereof. In one embodiment, the T cells are accumulated in the gastrointestinal tract of the patient. In another embodiment, the T cell is a tional CD8+ T-cell recognizing any gastrointestinal tract microbial antigen. In a further embodiment, the T cell is a memory-like autoregulatory CD8+ T cell. In yet a further embodiment, the T cell is a CD4+ T cell. In a related embodiment, the T cell secretes IL-10 or TGFB.

Details regarding modes of administration in vitro and in vivo are described within. 111. ANTIGEN-MHC-NANOPARTICLE COMPLEXES Certain aspects relate to processes for producing gut antigen-specific anti-IBD medicaments that cally target gut inflammation without compromising systemic immunity. Example 2 describes the production of antigen-MHC-nanoparticle complexes.

Antigen-MHC—nanoparticle complexes useful in this ion comprise an n derived fiom a microbe of the gastrointestinal tract. It is contemplated that administering nanoparticles coated with gut-specific antigen-MHC complexes to a patient will result in a an expansion of circulating gut antigen-specific T cells that are fiom about 0.5% to about 90% of total circulating T cells, or from about 1% to about 80%, or from about 5% to about 80%, or from about 10% to about 80%, or from about 10% to about 50%, or from about 50% to about 90%, or from about 20% to about 50%, or from about 30% to about 60%, or from about 35% to about 65%, or from about 40% to about 70%, or from about 45% to about 75%, or from about 50% to about 80%, or from about % to about 55%, or from about 0.5% to about 1%, or from about 1% to about 2.5%, or from about 2.5% to about 5%, or from about 0.1% to about 5%, or from about 1% to about 5%, or from about 0.1% to about 10%, A. Polypeptides and Polynucleotides Further aspects relate to an isolated or purified polypeptide sing, or consisting essentially of, or yet further consisting of, the amino acid ce of SEQ ID No. 1 or a ptide having at least about 80% sequence identity, or alternatively at least 85 %, or alternatively at least 90%, or atively at least 95 %, or alternatively at least 98 % sequence identity to SEQ ID No. 1. Also provided are isolated and d polynucleotides encoding the polypeptide corresponding to SEQ ID No. 1, at least about 80% sequence identify to SEQ ID No. 1, or alternatively at least 85 %, or atively at least 90%, or alternatively at least 95 %, or alternatively at least 98 % sequence identity to SEQ ID No. 1 or an equivalent, or a polynucleotide that hybridizes under stringent conditions to the polynucleotide, its equivalent or its complement and isolated or purified polypeptides encoded by these polynucleotides.

Other s relate to an isolated or d polypeptide comprising, or consisting essentially of, or yet further consisting of, the amino acid sequence of SEQ ID Nos. 4, 5, 6, 7, or 8 or a polypeptide having at least about 80% sequence identity at least about 80% sequence identify to SEQ ID No. 4-8, or alternatively at least 85 %, or alternatively at least 90%, or alternatively at least 95 %, or alternatively at least 98 % sequence identity to SEQ ID Nos. 48 Also provided are isolated and purified polynucleotides encoding the polypeptide corresponding to SEQ ID Nos. 4-8, or an equivalent, or a polynucleotide that hybridizes under stringent conditions to the polynucleotide, its equivalent or its complement and isolated or purified polypeptides encoded by these cleotides or one having at least about 80% sequence identify to polynucleotides encoding SEQ ID No. 4-8, or alternatively at least 85 %, or alternatively at least 90%, or alternatively at least 95 %, or alternatively at least 98 % sequence identity to polynucleotides encoding SEQ ID Nos. 4-8.

CE LISTINGS SEQ ID No. l: BacIYL e: I‘r’LKTNVYL SEQ ID No 2: Integrase protein (Baden ides vufgatus) L?KIRYQ-VFNRQKKL KQGTA-VQVEAYPNQRKIY.K"NVY.KPflCWSRflGAQVINHPQSNEL A LY EYI.Y.QGI?.GYWKRGIPAT-S--KDAVKKKSAVNVSFSTFAKSAIDNSDKKQSTKDNLHSTAAVL DF KD.TYTbLRDbflQYLRflKGNAVNTIAKHMRQ.R"LVN'AI.‘J QGYMIADAYPFRKYKIKQEKGRH fit.TPDfl.KK.flTVflVflxKSMRIVJDAFLFCCYTGLRYSDFCQ.TPENFIRVNGKRWLYFKSVKTGVEIR .P-{L.F?SRA.GI.DRYPDIGSLVSLPCNSEVNKQLRKLTGLCGIKKRITYHVSRHTCATLLVHQGVAI TTVQK..GHTSVKTTQIYSSV.SSTIVRDLK VQRKRKKVKMFPDKGLRTSDFIDNRJ.

SEQ ID No. 3: Integrase DNA sequence (Bactemides Wiggins) ATGCTAGAGAAGA"ACGATACAGGTTGGTCT"TAACCGCCAAAAGAAAC"GAA"AAGCAAGGCACGGCCC"TGTACA GGTTGAAGCC"A""TGAACCAAAGGAAAA"C"ACC"GAAGACCAA"GT""ACC"CAAACCGGAG"GCTGGAGCCGTG AGGGGGCACAAG"CA""AACCACCCCCAA"C"AACGAACTCAACGCAA"GCTC"ATGAA”ACATCCTGTA”CTGCAA GGCATAGAG""GGGG"A"TGGAAGCGCGGAA"ACC"GCCACACTC"CAC"ACTGAAGGA"GCTG"CAAGAAGAAAAG TGCCG"GAA"GTCAGC""C"CCAC"TTCGCCAAATCAGCCATTGACAA""CGGACAAGAAGCAG"CCACCAAGGACA AC"CGACAC"GGCGGTCC"GAA"GAC"TCCGTTCCGGA""GGACTTCAAGGATCT”ACCTATACATTCCTT CG"GA"TT"GAGCAA"A"""AAGGGAAAAGGGCAATGCGGTCAA"ACGA"AGCCAAGCACA"GAGACAGCTCCGTAC CAATGAGGCAA"CAACCAGGGA"A"A"GCACGCGGACGC"TA"CCGT"CAGAAAG"ACAAAATCAAACAGG AGAAAGGCAGACATGAG"""CT"ACCCCGGACGAGCTGAAGAAGC"GGAAACGGTCGAAG"GGAAGAGAAG"CCATG CGCCA"GTGCTCGA"GCC""CC"GTTC"GC"G""ATACCGGAT"GCGC"ATTC"GACT"C"GCCAGCTCACACCTGA GAAT'1'1CAT”AGAG”AAACGGCAAACGGTGGC"G"AC'1TCAAA"CCG"CAAGACAGGGGTGGAAATCCG"C"GCCGr1 r1ACA"C"GC"GTT"GAAAGCAGGGCA""GGGCA""CT"GACCG"TATCCGGA"ATAGG"AGTCT"GTA"CCCTACCC C"CGGAAG"GAATAAGCAGC'1'1CGAAAGC"GACCGGA"'1GTG"GGTA"CAAAAAACGGA"AACC"ACCATGr1 GAGCCG"CA"ACC"GTGCCACCC"GC"GG"TCA"CAGGGAGT"GCGA""ACAACAGTCCAGAAGCTGC"CGGACATA CT"CCG"AAAGACCACACAGA"""A""CGGAGG"AC""TCCAGCACCA"TG"GCG"GACTTGAAAAATG"TCAAAGG AAAAGGAAAAAAG”AAAGATG”"”CC”GA”AAAGGC””GAGAACATC”GAT”TTA”AGACAACCGGTAG SEQ ID No. 4: BacInt40_54 peptide sequence: WWLKPECWSREGA SEQ ID No. 5: BacInt81_95 peptide sequence: LGYWKRGIPATLSLL SEQ ID No. 6: BacInt365_379 peptide sequence: TQIYSEVLSSTIVRD SEQ ID No. 7: BacInt57_71 peptide sequence: INHPQSNELNAMLYE SEQ ID No. 8: BacIntgg_102 peptide ce: IPATLSLLKDAVKKK Antigens, including segments, fragments and other molecules derived from an antigenic species, ing but not limited to peptides, carbohydrates, lipids or other les presented by cal and non-classical MHC molecules of the invention are typically complexed or operatively coupled to a MHC molecule or derivative thereof. Antigen recognition by T cytes is major histocompatibility 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 selfMHC molecules. MHC restriction defines T lymphocyte specificity in terms of the antigen recognized and in terms of the MHC molecule that binds its antigenic nt(s). In particular aspects n antigens will be paired with certain MHC molecules or polypeptides derived there from.

The term "operatively coupled" or d" as used herein, refers to a situation where individual polypeptide (e.g., MHC) and antigenic (e.g., peptide) ents 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 sed 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 contained in a single polypeptide chain) is synthesized or inantly expressed as an intact complex. lly, polypeptide complexes are added to the nanoparticles to yield nanoparticles with adsorbed or d polypeptide complexes having a ratio of number of molecules:number of nanoparticle ratios from about, at least about or at most 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. In a specific embodiment, the ratio of the number of antigen-MHC molecules to the number of nanoparticles is from about 10:1 to about 1000: 1. The polypeptide content of the nanoparticles can be determined using standard techniques.

The peptides and proteins described herein can also be used in conventional methods to treat inflammation of the gastrointestinal tract. Accordingly, certain aspects relate to methods for inducing an anti-inflammatory response in a cell or tissue, comprising contacting the cell or tissue with an effective amount of an antigen, wherein the antigen is an antigen derived from a e that s within or infects a cell or tissue of the gastrointestinal tract (GI) or is a GI- associated n. Another aspect relates to a method for treating inflammation in a patient in need thereof comprising administering an effective amount of an antigen to the patient, wherein the antigen is d from a microbe that resides within or s a cell or tissue of the gastrointestinal tract or is a GI-associated antigen. A r aspect relates to a method for accumulating anti-inflammatory T cells in the GI tract of a patient in need thereof comprising administering an effective amount of an antigen to the patient, wherein the antigen is an antigen derived from a microbe that resides within or infects a cell or tissue of the gastrointestinal tract or is a ociated antigen. The antigen may be, for example, an antigen that corresponds to a peptide having at least 80% identity to the peptide sequence of the group: SEQ ID Nos. 1, 2, 4, 5, 6, 7, or 8. In certain ments, the antigen is complexed with MHC molecules prior to stration. In other embodiments, the antigen is administered with an adjuvant. Examples of suitable adjuvants include, but are not limited to Freund’s Complete and Incomplete, mineral salts and polynucleotides. Other non-limiting examples of suitable adjuvants include monophosphoryl lipid A (MPL), mutant derivatives of the heat labile enterotoxin of E. coli, mutant derivatives of cholera toxin, CPG oligonucleotides, and adjuvants derived from squalene B. MHC Molecules Intracellular and extracellular antigens present quite different challenges to the immune system, both in terms of ition and of appropriate response. Presentation of antigens to T cells is mediated by two distinct classes of molecules MHC class I (MHC-I) and MHC class II (MHC-II), which utilize distinct antigen sing ys. 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 les. 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 ion, a particular antigen is identified and presented in the antigen-MHC-nanoparticle complex in the context of an appropriate MHC class I or II polypeptide. In certain aspects, the genetic makeup of a subject may be assessed to determine which MHC polypeptide is to be used for a particular patient and a particular set of peptides.

Non-classical MHC molecules are also contemplated 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 ting glycolipids and olipids to Natural Killer T (NKT) cells or certain subsets of CD8+ T-cells such as Qal or HLA-E-restricted CD8+ T-cells. NKT cells ent a unique cyte population that ress NK cell markers and a nvariant 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 compositions including segments, fragments, or epitopes of polypeptides, peptides, nucleic acids, carbohydrates, lipids and other molecules that provoke or induce an antigenic response, generally referred to as antigens. In ular, antigenic segments or fragments of antigenic determinants, which lead to the destruction of a cell via an autoimmune response, can be identified 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.

Polypeptides and peptides of the invention may be modified by various amino acid deletions, insertions, and/or substitutions. In ular embodiments, modif1ed polypeptides and/or peptides are capable of ting an immune response in a subject. In some ments, a ype version of a protein or peptide are employed, however, in many embodiments of the invention, a modified protein or polypeptide is employed to generate an antigen-MHC-nanoparticle complex. An antigen-MHC-nanoparticle x can be used to generate an anti-inflammatory immune response, to modify the T cell tion of the immune system (i.e., re-educate the immune system), and/or foster the recruitment and accumulation of anti-inflammatory T cells to a particular tissue, such as, for example, a tissue of the gastrointestinal tract. The terms described above may be used interchangeably herein. A ed protein" or "modified polypeptide" or "modified e" refers to a protein or ptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some embodiments, a modified protein or polypeptide or peptide has at least one modified activity or function (recognizing that proteins or polypeptides or peptides may have multiple activities or functions). It is specifically contemplated that a modified protein or polypeptide or peptide may be altered with respect to one activity or function yet s 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 complex.

Antigens of the invention include antigens derived from proteins of a microbe common to the gastrointestinal tract. Microbes common to the gastrointestinal tract include, for example, obacter spp, Acidaminococcusfermentans, Acinetobacter cac0aceticus, Actinomyces spp, Actinomyces viscosus, Actinomyces naeslundii, Aeromonas spp, atibacter actinomycetemcomitans, Alistipes putredinis, Anaerotruncus colihominis, biospirillum spp, Alcaligenesfaecalis, Arachnia propionica, Bacillus spp, Bacteroides spp, Bacteroides caccae, Bacteriodes capillosus, Bacteroides dorei, Bacteroides hii, Bacteroides alis, Bacteroidesfinegoldii, Bacteroidesfragilis, Bacteroides intermedius, Bacteroides intestinalis, Bacteroides melaninogenicus, oides , Bacteroides pectinophilus, Bacteroides pneumosintes, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides mis, Bacteroides vulgatus, oides xylanisolvens, Bacterionema matruchotii, Blautia hansenii, Corynebacterium matruchotii, Bifidobacterium spp, Buchnera aphidicola, Bulyrivibrio crossotus, Bulyriviberiofibrosolvens, Campylobacter spp, Campylobacter coli, Campylobacter um, Campylobacter upsaliensis, Candida albicans, Capnocytophaga spp, idium spp, Citrobacterfreundii, Clostridium asparagiforme, Clostridium dijficile, Clostridium Zeptum, Clostridium nexile, Clostridium scindens, Clostridium sordellii, sella aerofaciens, C0pr0c0ccus comes, C0pr0c0ccus eutactus, Corynebacterium spp, Doreaformicigenerans, Dorea longicatena, Eikenella corrodens, Enterobacter cloacae, Enterococcus spp, Enterococcus faecalis, Enterococcusfaecium, Escherichia coli, Eubacterium spp, Eubacterium hallii, Eubacterium rectale, Eubacterium siraeum, Eubacterium ventriosum, Faecalibacterium prausnitzii, Flavobacterium spp, Fusobacterium spp, Fusobacterium tum, Gordonia Bacterium spp, Haemophilius parainfluenzae, Haemophilus paraphrophilus, Holdemania filiformis, Lactobacillus spp, Leptotrichia buccalis, ella morganii, Mycobacteria spp, Mycoplasma spp, Micrococcus spp, Mycoplasma spp, Mycobacterium chelonae, Neisseria spp, Neisseria sicca, Parabacteroides distasonis, Parabacteroidesjohnsonii, Parabacteroides merdae, Peptococcus spp, Peptostreptococcus spp, Plesiomonas loides, Porphyromonas gingivalis, Propionibacterium spp, Propionibacterium acnes, Providencia spp, Pseudomonas nosa, Roseburia intestinalis, Ruminococcus bromii, Ruminococcus gnavus, Ruminococcus torques, Ruminococcus lactaris, Ruminococcus obeum, Rothia dentocariosa, coccus spp, Sarcina spp, Staphylococcus aureus, lococcus epidermidis, Streptococcus anginosus, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus sobrinus, Streptococcus thermophilus, Streptococcus viridans, Subdoligranulum variabile, Torulopsis glabrata, Treponema denticola, Treponema refringens, Veillonella spp, Vibrio spp, Vibrio sputorum, lla ogenes, and ia enterocolitica. Qin et al., (2010) Nature, Vol. 464:4 describes prevalent bacteria in the gastrointestinal tract. In certain embodiments, the antigen is derived from a bacteria belonging to the genera of the group: Bacteroides, Clostridium, Dorea, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, and Bifidobacterium. In a related ment, the n is derived from oides. In a further embodiment, the antigen is derived from a protein of Bacteroides. In yet another embodiment, the antigen is derived from the n Integrase. In a further ment, the antigen corresponds to a peptide having at least 80% identity, or at least about 80% sequence identify to SEQ ID No. l, or atively at least 85 %, or alternatively at least 90%, or alternatively at least 95 %, or alternatively at least 98 % sequence identity to the peptide sequence of SEQ ID No. 1. In other embodiments, the antigen corresponds to a peptide having at least 80% identity to the e sequence of SEQ ID Nos. 4-8. Other useful antigens include those that induce T cells that can cross-react with an antigen of a gut e. For example, IGRP206_214 epitope (expressed by pancreatic beta cells) and NRP-V7 or NRP-A7 (mimics of IGRP206_214) can be used to induce 8.3-like CD8+ T-cells that can cross-react with the BacIYL sequence.

Antigens of the invention also include GI—associated antigens such as known inflammatory bowel disease-related antigens (e.g. ovalbumin), dieteray antigens such as yeast mannan, gliadin and known celiac disease related antigens such as gliadin from gluten. 2013/052352 In certain embodiments, the size of a protein or ptide (wild-type or modified), including any complex of a protein or peptide of interest and in particular a MHC-peptide fusion, may comprise, but is not limited to 5, 6, 7, 8, 9,10,11, 12, 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 n, or derivative thereof.

In certain aspects, 5, 6, 7, 8, 9, 10 or more uous amino acids, including tives 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 mutated by tion, rendering them shorter than their ponding wild-type form, but also they might be altered by fusing or ating a heterologous protein sequence with a particular function (e. g., for presentation as a protein complex, for enhanced immunogenicity, etc.).

Proteinaceous compositions may be made by any que known to those of skill in the art, including (i) the expression of proteins, polypeptides, or peptides h standard molecular biological techniques, (ii) the isolation of proteinaceous compounds from natural 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 disclosed, 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 compositions can be substitutional, insertional, or deletion variants. A ation in a polypeptide ofthe invention may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 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, 71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,9l,92,93,94,95,96, 97,98,99,100,100,101,102,103,104,105,106,107,108,109,110,lll,ll2,113,114,115, 116,117,118,ll9,120,121,122,123,124,125,126,127,128,129,130,l3l,132,133,134, 135,136,137,l38,l39,l40,l4l,l42,l43,l44,l45,l46,147,148,149,150,151,152,153, 154,155,156,157,158,159,l60,l6l,l62,l63,l64,l65,166,l67,l68,l69,l70,l7l,l72, l73,l74,l75,l76,l77,l78,l79,l80,l8l,182,183,184,185,l86,l87,l88,l89,l90,l9l, 192,193,194,195,196,197,198,l99,200,201,202,203,204,205,206,207,208,209,210, 211,212,213,214,215,216,2l7,218,219,220,22l,222,223,224,225,226,227,228,229, 1,232,233,234,235,236,237,238,239,240,24l,242,235,236,237,238,239,240, 241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259, 260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278, 279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297, 298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,3l6, 317,318,319,320,321,322,323,324,325,326,327,328,329,330,33l,332,333,334,335, 336,337,338,339,340,34l,342,343,344,345,346,347,348,349,350,351,352,353,354, 355,356,357,358,359,360,36l,362,363,364,365,366,367,368,369,370,37l,372,373, 374,375,376,377,378,379,380,38l,382,383,384,385,386,387,388,389,390,391,392, 393,394,395,396,397,398,399,400,401,402,403,404,405,406,407,408,409,410,41L 412,413,414,415,416,417,418,4l9,420,42l,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, 450,451,452,453,454,455,456,457,458,459,460,46l,462,463,464,465,466,467,468, 469,470,471,472,473,474,475,476,477,478,479,480,48l,482,483,484,485,486,487, 9,490,49l,492,493,494,495,496,497,498,499,500cu1norenon:configuousor contiguous amino acids of a peptide or polypeptide, as compared to wild-type.

Deletion variants lly lack one or more residues of the native or wild-type amino ammmmmehmwwmmflmwwnmdwmdmammMNfiwmgmmmmmmmbwnm d. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein. Insertional mutants typically involve the addition of material at a non-terminal point in the ptide. This may e 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 polypeptide, with or without the loss of other ons or ties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge.

Conservative substitutions are well known in the art and e, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to e or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; 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 on or ty of a polypeptide or peptide is ed, 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 synthesized in vitro. Alternatively, a recombinant protein may be isolated from bacteria or other host cell.

It also will be understood that amino acid and nucleic acid sequences may include additional es, such as onal N— or C—terminal amino acids, or 5' or 3' nucleic acid sequences, respectively, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the ia set forth above, including the maintenance of ical protein activity (e.g., immunogenicity). The addition of al sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.

It is contemplated that in itions 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 therein). Of this, about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 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 complex.

The present invention contemplates the administration of an antigen-MHC- nanoparticle complex to effect a treatment against a disease or condition associated with inflammation of the gastrointestinal tract.

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. Numerous scientific publications have also been devoted to the prediction of secondary structure and to the fication of epitopes, fiom analyses of amino acid sequences (Chou & , Adv. Enzymol., 47:45-148, 1978; Chous and Fasman, Annu, Rev. Biochem., 47:251-276, 1978, Chou and Fasman, mistry, 13(2):211-222, 1974; Chau and Fasman, Biochemistry, 13(2):222-245, 1974, Chou and Fasman, Biophys. J ., 26(3):385-399, 1979). Any of these may be used, if d, to supplement the teachings of Hopp in US. Pat.

No. 101.

Molecules other than es 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 y of cells. Certain carbohydrates are characteristic of different stages of differentiation and very often these carbohydrates are ized by specific antibodies. Expression of distinct carbohydrates can be restricted to specific cell types.

D. Substrates/Nanoparticles In certain , antigen/MHC complexes are operatively d to a substrate. A substrate can be in the form of a nanoparticle that optionally comprises a biocompatible, bioabsorbable material. Accordingly, in one embodiment, the nanoparticle is patible and/or bioabsorbable. A substrate can also be in the form of a nanoparticle such as those described previously in US Patent Pub. No.: 2009/0155292 which is herein incorporated by reference in its entirety. Nanoparticles can have a structure of variable dimension and known variously as a nanosphere, a nanoparticle or a biocompatible biodegradable nanosphere or a biocompatible biodegradable nanoparticle. Such particulate ations ning an antigen/MHC complex can be formed by covalent or non-covalent coupling of the x to the nanoparticle.

The nanoparticles typically consist of a substantially spherical core and optionally one or more layers. 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 chemical or biological onalities, referred to herein as chemically active or biologically active layers, and for these onalities 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 desired 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 rs, 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, olymers, silicone, celluloses, silicon, metals (e.g., iron, gold, silver), minerals (e.g., ruby), nanoparticles (e.g., gold nanoparticles, dal les, metal , metal sulf1des, 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 particles or nanoparticles 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 6,712,997, which is orated herein by reference in its entirety. In certain embodiments, the compositions of the core and layers may vary provided that the nanoparticles are biocompatible and bioabsorbable. The core could be of homogeneous composition, or a ite of two or more classes of material depending on the properties desired. In certain s, metal nanospheres will be used. These metal nanoparticles can be formed from Fe, Ca, Ga and the like.

As previously stated, the nanoparticle may, in addition to the core, include one or more . The nanoparticle may e a layer consisting of a biodegradable sugar or other polymer. Examples of biodegradable layers include but are not limited to dextran; thylene glycol); poly(ethylene oxide); mannitol; poly(esters) based on polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL); poly(hydroxalkanoate)s of the PHB-PHV class; and other modified poly(saccharides) such as starch, cellulose and chitosan. Additionally, the nanoparticle may include a layer with suitable surfaces for attaching chemical onalities for al binding or coupling sites.

Layers can be produced on the nanoparticles in a y of ways known to those skilled in the art. Examples include sol-gel chemistry techniques such as described in Iler, Chemistry of , John Wiley & Sons, 1979; Brinker and Scherer, Sol-gel Science, Academic Press, (1990). Additional approaches to producing layers on rticles e 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); sopwattana, Langmuir, 12:3173-3179, (1996); Davies, Advanced Materials, 10: 1264-1270, ; 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 include 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, 32(7):2317-2328, (1998); Caruso et al., J.Amer. Chem. Soc., 121(25):6039-6046, (1999); US. Pat. No. 6,103,379 and references cited therein.

Nanoparticles may be formed by contacting an aqueous phase containing the antigen/MHC/co-stimulatory molecule complex and a polymer and a nonaqueous phase followed by evaporation of the eous phase to cause the coalescence of particles from the aqueous phase as taught in US. Pat. No. 4,589,330 or 4,818,542. red 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 pisilon-caprolactone, poly(epsilon-caprolactone-CO-lactic acid), poly(epsilon-caprolactone-CO-glycolic acid), poly(B-hydroxy butyric acid), poly(ethylene oxide), polyethylene, poly(alkylcyanoacrylate), poly(hydroxyethyl methacrylate), polyamides, poly(amino acids), poly(2-hydroxyethyl DL—aspartamide), poly(ester urea), poly(L- phenylalanine/ethylene glycol/l,6-diisocyanatohexane) and poly(methyl methacrylate).

Particularly preferred polymers are polyesters, such as polyglycolic acid, polylactic acid, glycolide-L(-) lactide poly(episilon-caprolactone, psilon-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 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 further ments, the nanoparticle is from about 1 nm to about 15 nm or to about 30 nm, 50 nm, 75 nm, or 100 nm. In r embodiments, the nanoparticle is from about 5 nm to about 50 nm. In a related embodiment, the nanoparticle is from about 5 to about 15 nm in diameter.

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

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

The term "linking molecule" means a substance capable of linking with the substrate or nanoparticle and also capable of linking to an n-MHC complex.

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

The functional groups or the linking molecules bearing them may be selected from amino groups, carbonic acid groups, thiols, thioethers, disulfides, guanidino, hydroxyl groups, amine , l dioles, aldehydes, alpha-haloacetyl groups, mercury les, ester groups, acid halide, acid ter, 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 yl compounds, arylhalogenides or their derivatives.

Non-limiting examples for other linking molecules with higher molecular weights are c acid molecules, polymers, copolymers, polymerizable ng 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 ty molecules containing themselves nucleic acid les, though with a complementary sequence with respect to the linking molecule.

A specific example of a covalent linker includes poly(ethylene) glycol (PEG). The PEG linker may be a thiol-PEG-NHZ linker.

In certain embodiments, the linker as bed 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 fiom about 1 to about or 1.5 kD. , 4.5, 4, 3.5, 3, 2.5, 2, As examples for polymerizable coupling agents, diacetylene, styrene butadiene, vinylacetate, acrylate, acrylamide, vinyl compounds, styrene, silicone oxide, boron oxide, phosphorous oxide, borates, e, polypyrrole and phosphates can be cited.

The surface of the substrate or nanoparticle can be chemically modified, for ce by the binding of phosphonic acid derivatives having functional reactive groups. One example of these phosphonic acid or phosphonic acid ester derivates is imino-bis(methylenphosphono) carbonic acid which can be synthesized according to the "Mannich-Moedritzer” reaction. This binding on can be performed with substrate or nanosphere as directly obtained from the preparation process or after a pre-treatment (for instance with trimethylsilyl bromide). In the first case the onic 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 bromide, on the other hand, is believed to dealkylate alkyl group-containing phosphorous-based complexing agents, thereby creating new binding sites for the phosphonic acid (ester) derivative. The phosphonic acid (ester) derivative, or linking molecules bound thereto, may display the same functional groups as given above. A further example of the surface treatment of the substrate or nanosphere involves g in a diole such as ethylene glycol. It should be noted that this treatment may be redundant if the sis already proceeded in a diole. Under these circumstances the synthesis product directly obtained is likely to show the necessary onal . This ent is r applicable to ate or rticle that were produced in N— or P-containing complexing agents. If such substrate or le 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 diole 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 functional 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 e may also be coated by homo- or copolymers. Examples for polymerizable ng agents are. N—(3-aminopropyl) mercaptobenzamidine, 3-(trimethoxysilyl)propylhydrazide and 3- trimethoxysilyl)propylmaleimide. Other non-limiting es of rizable coupling agents are mentioned herein. These ng agents can be used singly or in combination depending on the type of copolymer to be ted as a coating.

Another 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 orides are capable of reacting 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 bi-functional linker, such as idopropionic acid hydrazide.

For valent linking techniques, chain-type molecules having a polarity or charge opposite to that of the substrate or nanosphere surface are particularly suitable. Examples for linking molecules which can be non-covalently linked to hell nanospheres involve anionic, cationic or zwitter—ionic surfactants, acid or basic proteins, polyamines, polyamides, polysulfone or polycarboxylic acid. The hydrophobic interaction between substrate or nanosphere and amphiphilic reagent having a functional reactive group can generate the necessary link. In particular, chain-type molecules with amphiphilic character, such as phospholipids or derivatised ccharides, which can be crosslinked with each other, are useful. The absorption of these les on the e can be achieved by coincubation. The binding between y molecule and substrate or nanoparticle can also be based on non-covalent, self-organising bonds.

One example thereof involves simple detection probes with biotin as linking molecule and avidin- 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 biological molecule (e.g., MHC molecule or derivative f) can be coupled to the linking molecule, covalently or non-covalently, in line with standard procedures of organic chemistry such as oxidation, halogenation, alkylation, acylation, on, substitution or amidation. These methods for coupling the covalently or non-covalently bound linking molecule can be applied prior to the ng of the linking molecule to the substrate or WO 44811 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 abilities to elicit or te an immune response. 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 tic synthesizers are commercially available and can be used in accordance with known protocols. See, for e, Stewart and Young, Solid Phase Peptide Synthesis, 2nd. Ed., Pierce Chemical Co.l, (1984); Tam et al., J. Am. Chem. Soc., 105:6442, (1983); eld, Science, 232(4748):341-347, (1986); and Barany and Merrifield, The Peptides, Gross and Meinhofer (Eds.), Academic Press, NY, 1- 284, (1979), each incorporated herein by nce. Alternatively, inant DNA technology may be employed wherein a nucleotide 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 ion includes the use of gene transfer to cells, including microorganisms, 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 ions. A nucleic acid encoding virtually any ptide 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 discussed herein. Examples of mammalian host cell lines include, but are not d to Vero and HeLa cells, other B- and T-cell lines, such as CEM, 721.221, H9, Jurkat, Raji, as well as cell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cells. In addition, a host cell strain may be chosen that tes the expression of the inserted sequences, or that modifies and ses the gene product in the manner desired. Such modifications (e.g., glycosylation) and processing (e.g., ge) of protein products may be important for the function of the protein. Different host cells have characteristic and c mechanisms for the ranslational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and sing of the foreign protein expressed.

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

G. Nucleic Acids The present invention may e recombinant polynucleotides encoding the proteins, polypeptides, peptides of the invention, such as, for e, SEQ ID No. l, 2, or 3. The nucleic acid sequences for ary antigens and MHC molecules for presenting the antigens, are included and can be used to prepare an antigen-MHC complex.

In particular embodiments, the invention concerns isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode an autoantigen and/or a MHC molecule. The term "recombinan " may be used in ction with a polypeptide or the name of a specific ptide, and this lly refers to a polypeptide produced from a nucleic acid molecule that has been lated 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 restriction enzyme sites, multiple cloning sites, other coding segments, 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 limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, ranslational modification, or for therapeutic benefits such as targeting or efficacy. A tag or other logous polypeptide may be added to the modified polypeptide-encoding sequence, wherein "heterologous" refers to a polypeptide that is not the same as the d polypeptide.

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

A. Pharmaceutical Compositions Compositions of the invention may be conventionally administered parenterally, by injection, for example, intravenously, subcutaneously, or intramuscularly. Additional formulations which are le for other modes of administration include oral formulations.

Oral formulations include such normally employed excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, es, ned release formulations or powders and contain about % to about 95% of active ient, preferably about 25% to about 70%. The preparation of an s composition that contains a antigen-MHC-nanoparticle complex that s the subject's immune ion will be known to those of skill in the art in light of the present disclosure. In certain embodiments, a composition may be inhaled (e.g., US. Patent No. 6,651,655, which is specifically incorporated by reference in its ty). 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 ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of ten to several hundred nanograms or micrograms antigen-MHC-nanoparticle complex per administration. Suitable regimes for l stration and boosters are also variable, but are typified by an initial administration followed by subsequent administrations.

In many instances, it will be desirable to have le administrations of a e- 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 0.5-5 years, usually two years, may be desirable to maintain the condition of the immune system. The course of the administrations may be followed by assays for atory immune responses and/or gulatory anti- inflammatory T cell activity.

In some embodiments, pharmaceutical compositions are administered to a subject.

Different aspects of the present invention involve stering an effective amount of a antigen-MHC-nanoparticle complex composition to a subject. Additionally, such compositions can be administered in combination with modifiers of the immune system. Such compositions will generally be dissolved or dispersed in a pharmaceutically able carrier or s medium.

The s "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular es 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 r" includes any and all ts, dispersion media, coatings, antibacterial and antifungal agents, isotonic 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 injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous ation of sterile inj ectable solutions or dispersions.

In all cases the form must be sterile and must be fluid to the extent that it may be easily injected.

It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

The compositions may be formulated into a l or salt form. Pharmaceutically acceptable salts, include the acid addition 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 inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric ides, and such organic bases as pylamine, 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 poly(ethylene glycol), and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as in, by the nance of the ed 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, osal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, ed by ization. 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 s ized active ingredients into a sterile e which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable 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 ion is meant to include any method of sterilization that does not significantly se the therapeutic properties of the antigen-MHC-nanoparticle complexes. s of sterilization that involve intense heat and pressure, such as autoclaving, may mise the tertiary structure of the complex, thus significantly decreasing the therapeutic properties of the antigen-MHC-nanoparticle complexes.

WO 44811 An ive amount of therapeutic composition is determined based on the intended goal. The term "unit dose" or "dosage" refers to ally discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses sed above in association 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 composition also depend on the judgment of the practitioner and are peculiar 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 a antigen-MHC-nanoparticle complex, may also be used in ation with the administration of traditional therapies. These include, but are not limited to, anti- inflammatory drugs such as alazine, corticosteroids such as prednisone, and immune system suppressors such as oprine and mercaptopurine. An antibiotic, such as metronidazole, may also be helpful for killing germs in the intestines.

To help treat symptoms, a doctor may recommend anti-diarrheals, ves, pain relievers or other over-the-counter (OTC) drugs. Steroids are generally used for people who have more severe form of Crohn’s disease. In more aggressive disease, steroids may be used with immunosuppressants or with a newer medicine called infliximab.

When combination therapy is ed, various combinations may be employed, for example n-MHC-nanoparticle complex stration 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 A/A/B/B B A/B/B/A/ B/B/A/A B/A/B/A B/A/A/B 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/subject will follow general ols for the administration of such compounds, 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 01‘ Ex Vivo Administration As used herein, the term in vitro administration refers to manipulations performed on cells removed from or outside of a subject, including, but not d 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 vivo administration includes all manipulations performed within a subject, including administrations.

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

V. EXAMPLES The following examples are given for the purpose of illustrating s embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will iate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those s, ends and advantages inherent herein. The t examples, along with the methods described herein are presently representative of embodiments and are exemplary, and are not ed 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 Bacteroides Integrase as an antigenic target of memory-like gulatory T-cells It was investigated whether a novel e eroides Integrase (BacIYL: SEQ ID No. 1) could bind to the NOD mouse major histocompatibility complex class 1 molecule H-2Kd over a range of concentrations, as ed to TUM (a positive control), IGRP206_214, and LCMV-encoded Gp33 (a Db-binding negative l). As shown in Figure 1A, the BacIYL sequence (SEQ ID No. l) bound Kd molecules on the surface of Transporter-Associated with Antigen-Processing (TAP)-deficient RMA—SKd cells as efficiently as 6_214 and TUM.

To ascertain if the BacIYL/Kd peptide-MHC (pMHC) complex could be recognized by IGRP206_214-reactive CD8+ T-cells, naive splenic CD8+ T-cells from 8.3-TCR-transgenic NOD mice (8.3-NOD) were stained with fiuorochrome-conjugated TUM/Kd ive control), NRP- V7/Kd (positive control) and BacIYL/Kd pMHC tetramers. As shown in Fig. 1B, 8.3-CD8+ T- cells bound Bac-IYL/Kd tetramers efficiently, albeit with lower mean fluorescence intensity (mfi) as IGRP206_214/Kd tetramers, suggesting that the 8.3-TCR binds this pMHC complex with low affinity. This was confirmed by carrying out Scatchard plot analyses of tetramer binding at equilibrium. As shown in Fig. 1C, Bac-IYL/Kd ers bound 8.3-CD8+ T-cells with ~2-fold lower avidity.

To investigate if the L sequence had agonistic ty on naive 8.3-CD8+ T- cells, naive 8.3-CD8+ T-cells were cultured with TUM (negative control), IGRP206_214 (positive control) and Bac-IYL for 24h. Unlike 6_214, which elicited upregulation of both CD44 and CD69, Bac-IYL was only able to induce CD69 upregulation (Fig. 2A). This indicated that Bac- IYL had partial agonistic activity, consistent with the low-binding avidity of the corresponding tetramers seen in Fig. 1C. Since differentiated 8.3-cytotoxic T-lymphocytes (8.3-CTL) do not kill BacIYL-pulsed targets or Integrase-encoding cDNA-transfected HEK293-Kd cells these data show that BacIYL can bind to and ‘tickle’ the 8.3-TCR without driving most T-cell activation programs downstream of the TCR.

Because certain idity TCR-binding ligands have antagonistic properties (in addition to partial agonistic activity at higher ligand densities), it was igated whether Bac- IYL might be able to antagonize IGRP206_214-induced 8.3-CD8+ T-cell responses. As shown in Fig. 2B, L but not TUM (a Kd-binding peptide that is not recognized by the 8.3-TCR) was able to antagonize IGRP206_214-induced 8.3-CD8+ T-cell ses (IFNy secretion and proliferation) over a range of concentrations (above 1 uM). Thus, when presented to 8.3-CD8+ T-cells in isolation, Bac-IYL binds to 8.3-like TCRs with low avidity, antagonizes agonist- induced responses at relatively low ligand densities, and induces partial agonistic responses at high ligand densities.

Without being bound by theory, it was then believed that in vivo, Bac-IYL, encoded in prevalent gut bacterial strains, would not be presented in isolation, but rather in the context of bacterial toll-like receptor ligands, such as LPS. This, in turn, might abrogate the antagonistic properties of Bac-IYL and afford it agonistic activity. In agreement with this hypothesis, naive 8.3-CD8+ T-cells mounted efficient IFNy and proliferative responses to Bac-IYL in the presence of LPS (Fig. 2C).

Antigenic peptides encoded in bacteria must be processed from the donor full-length protein by professional antigen-presenting cells (APCs, such as tic cells —DCs—). In the case of the Bac-IYL peptide, its donor protein, the Bacteroides Integrase, would have to be processed by the proteasome and the resulting peptides shuttled to the ER for binding to endogenous MHC (Kd) molecules, which would then be transported to the APC's plasma membrane for re to T-cells. To investigate if DCs could s oides Integrase protein and generate Bac-IYL/Kd complexes capable of eliciting 8+ T-cell activation, recombinant GST-fused Integrase preparations encoding the wild-type Bac-IYL sequence or a mutated L epitope identical to 6_214 were produced and purified. DCs were then then fed the recombinant proteins (in the presence of LPS) and 8+ s, to measure 8.3-CD8+ T-cell activation. As shown in Fig. 2D, both types of recombinant Integrase preparations induced 8.3-CD8+ T-cell tion, particularly the one encoding IGRP206_214, as expected. Thus, DCs can process Bacteroides Integrase and generate epitopes capable of activating cognate T-cells.

Because low-avidity active T-cells tend to differentiate into memory-like anergic (non-proliferating, but ne-secreting) autoregulatory (autoimmune disease-suppressing) T- cells in response to chronic autoantigenic ation, it was contemplated that Bac-IYL might be able to induce memory-like 8.3-CD8+ T-cells in vitro. As shown in Fig. 3A, 8.3-CD8+ T- cells (but not low-avidity 6_214-reactive l7.6-CD8+ T-cells) cultured in the presence of Bac-IYL peptide for 28 days sed the late T-cell activation marker CD44 and low levels of the naive T-cell marker CD62L. In addition, these cells expressed the early activation marker CD69 and CD122, a memory T-cell marker (Fig. 3B). Functionally, these cells behaved like memory T-cells. Thus, they rapidly produced IFNg in response to t (IGRP206_214)-pulsed DCs (Figs. 3C and D). However, unlike conventional memory-like CD8+ T-cells, and like gulatory CD8+ T-cells, they displayed proliferative unresponsiveness (anergy) as compared to naive 8.3-CD8+ T-cells (Fig. 3D). Accordingly, these Bac-IYL-activated CD8+ T- cells have all the hallmarks of the autoregulatory CD8+ T-cells that arise spontaneously, in vivo, in se to chronic autoantigenic stimulation.

It has been nted that TCRa—/— mice can develop spontaneous IBD (see, for example, Mombaerts, P., et al. (1993) Cell 75:274-282.) or DSS-induced IBD (see, for example, Mahler, M., et al. (1998) Am JPhysiol 274:G544-551.) and the NOD strain is also tible to DSS-induced IBD (see, for e, Mahler, M., et al. (1998) Am JPhysiol 274:G544-551.).

Several s such as genetic, environmental, composition of the gut ial flora, the structure of the intestinal epithelial layer as well as elements of the innate and adaptive immune systems are all known to contribute to the initiation, progression and regulation of IBD, albeit h poorly understood mechanisms. IBD is defined as inflammation underneath the mucosal and epithelia layers of the gut wall (see, for example, Nell, S., et al. Nat Rev Microbiol 8:564- 577; Maloy, K. J., et al. Nature 474:298-306; Khor, B., et al. Nature 474:307-317; and Kaser, A., et al. (2010) Annu Rev Immunol 28:573-621.). To investigate the biological significance of BaclYL36_44 recognition by cognate CD8+ T-cells in the context of IBD, Applicants compared the susceptibility of 8.3- vs. 17.6-TCR-transgenic NOD.IGRP206_214’/’ mice (carrying IGRP206_ 214-specific CD8+ T-cells capable of recognizing or not recognizing BaclYL36_44, respectively).

Mice were exposed to 2% DSS in the drinking water for 1 wk, to compromise gut epithelial integrity and expose the gut microbiota to the gut-associated lymphoid tissue (GALT) without inducing overt disease (bleeding or weight loss). After an additional week on 0% DSS, these mice were exposed to three cycles of 3.5% DSS (wk l)/0% DSS (wk 2 and 3). As shown in Figs. 4A, 4B and 4E, 8.3-NOD mice exhibited significant resistance to colitis and no mortality as compared to 17.6-NOD mice, suggesting that in viva activation of 8+ cells by the Bac- IYL36_44 epitope rendered the hosts resistant to colitis. Furthermore, 8.3-NOD mice lacking integrin B7 were highly tible to s (Figs. 4C, 4D and 4F). These results support the idea that 8+ T-cells’ anti-colitogenic effect requires recruitment to the GALT.

The above data predicted that NOD.IGRP206_214’/’ mice, which export sed numbers of vidity 6_214-reactive (BacIYL36_44 cross-reactive) CD8+ cells to the periphery, should display a relative resistance to DSS-induced colitis vs. wild-type NOD mice, in which a significant fraction of these higher-avidity CD8+ T-cells are deleted. Indeed, as shown in Fig. 4G, NOD.IGRP206_214’/’ mice, unlike NOD mice, were resistant to weight loss resulting from 4% DSS. To directly investigate a role for a cytotoxic CD8+ T-cell response against 36_44-loaded APCs in colitis resistance, 4% DSS was fed to NOD.IGRP206_214’/’ hosts along with i.v. ions of in vitro-differentiated 8.3-CTL (cytotoxic T lymphocytes) . As shown in Fig. 4H, 8.3-CTL—transfused hosts had lower disease activity scores than non- transfused mice.

To further substantiate these results, Applicants ascertained the ability of 8.3-CTL to protect OD mice, which are highly susceptible to DSS-induced colitis, from disease. As shown in Fig. 5A, 8.3-CTL—transferred l7.6-NOD mice (one CTL er per week) did not significantly lose weight over a 35-day -up, as compared to non-CTL-transferred 17.6- NOD mice. rmore, 8.3-CTL transfer significantly reduced the disease activity scores in these animals (Fig. 5B). Together, these data support the idea that a CTL response against a gut bacterial epitope s resistance to colitis. Accordingly, approaches capable of eliciting in the in vivo activation and expansion of gut microbiota-specif1c CTLs should have therapeutic significance in IBD.

The data described herein sively demonstrates that the Bacteroides Integrase is a bona-fide antigenic target of anti-IBD T-cells in the gut-associated lymphoid .

Accordingly, this antigen could be used as a target to foster the recruitment and accumulation of autoregulatory (anti-inflammatory) T-cells to the gut in inflammatory bowel disease. In one embodiment, systemic treatment of subjects with nanoparticles coated with peptide-MHC class I complexes induces antigen-specif1c CD8+ T cells (8.3-like, both conventional and memory-like autoregulatory). In another embodiment, systemic treatment of subjects with nanoparticles coated with peptide-MHC class II complexes induces antigen-specific T-regulatory-l (IL- lO/TGFb-producing) CD4+ T-cells. In fact, Trl-like CD4+ T-cells expanded by nanoparticles 2013/052352 coated with the NOD mouse class II MHC molecule I—Ag7 presenting an IGRP-derived autoantigenic epitope accumulate in gut-associated lymphoid tissue, including Peyer's Patches and intra-epithelial lymphocyte aggregates. Fig. 6 shows data from two mice cured from diabetes by treatment with IGRP4_22/I-Ag7-coated nanoparticles - these mice were analyzed at 50 wk of age; GPI/I-Ag7 tetramer is a negative control tetramer).

Accordingly, nanoparticles coated with MHC class 1 and/or II molecules presenting epitopes from Bacteroides Integrase elicit the expansion of Integrase-specific CD8+ or ke CD4+ T-cells, most of which will accumulate in the gut, helping restore immune tasis in individuals affected with IBD. Thus, the compositions of this disclosure provide this method of treatment as well.

Example 2 Process for making antigen-MHC-nanoparticle complexes.

Inorganic nanoparticles (iron oxide =IONP; NPs) of a desired size. IONPs are produced via l decomposition. IONPs sized as such are biocompatible and can be PEGylated for protein conjugation. To coat pMHC and/or other proteins onto IONPs, surfactant- coated NPs are reacted with functionalized PEG linkers of the riate length. The linkers are purified by HPLC and characterized by 1H-NMR, MALDI/GPC and GPC, to confirm chemical identity, purity, molecular weight and polydispersity. Similar linkers and approaches can be used to coat GNPs, except that the linkers will have a thiol (SH) group at their NP- binding end.

Example 3 Size, Density, and Exposure of pMHC-coated Nanoparitcles. 1. Synthesis and characterization of gold-based pMHC-coated NP.

Gold nanoparticles (GNPs) of specific sizes were synthesized. The size, density, surface charge and spersity of the GNP preparations are measured using spectrophotometry, ission electron copy (TEM) and dynamic light scattering. The GNP samples are then concentrated and conjugated with mono-specific pMHC xes using different approaches as described below. Applicants have developed methods to quantitate the pMHC valency per GNP and to trate the pMHC-coated GNP preparations of different sizes at high densities (~1014/ml) without compromising monodispersion (Fig. 19).

II. terization of the pMHC binding capacity of GNPs. pMHC complexes were coated onto GNPs of various sizes using two ent approaches: (i) random binding ofpMHC to the GNP surface via electrostatic interactions; and (ii) directional binding through a thiol-PEG—NH2 linker (in this case, an additional thiol-PEG linker as GNP stabilizer was used to t aggregation). It was believed that the first approach would enable very high ligand densities (ofpMHC per GNP) while compromising the directionality ofpMHC binding (i.e. only a fraction of the molecules might become available for recognition by cognate T-lymphocytes). The second approach aimed to generate pMHC-coated GNPs carrying lower densities ofpMHC but bound directionally, via their ini. Both approaches were tested on GNPs of various diameters, ranging from 14 to 40 nm. It was confirmed that, for both approaches, the inding capacity of GNPs is a function of size, and more specifically surface area (higher number ofpMHCs on bigger NPs). Surprisingly, it was found that PEG ed-binding not only ensures the directionality of binding but also es the binding capacity of individual GNPs (contrary to initial expectations). Table 1 below summarizes the data.

Table 1. pMHC binding capacity of GNPs Diameter (nm) Surface area: pMHCs/GNP pMHCs/GNP x 102 nm2 absor n tion linker ———— ———3,750 ———— 2,850 5,250 III. Agonistic ty versus pMHC content.

The effects of pMHC valency, GNP size, GNP density and g strategy on the functional (agonistic) activity of pMHC-coated GNPs in vitro were tested. The ability of various 6_214-Kd-GNP preparations to activate cognate (IGRP206_214-speciflc) naive CD8+ T cells (herein referred to as '8.3-CD8+ T-cells') derived from T-cell receptor (TCR) transgenic NOD mice (or 8.3-NOD mice) were compared. The first set of experiments aimed to compare the effects of 6_214-Kd (pMHC) valency over a range of GNP densities in the culture. GNPs conjugated with a control ognate) pMHC complex (Tum-Kd) were used as negative controls. As expected, IGRP206_214-Kd-coated (but not -coated) GNPs activated these T cells (as measured by IFNy production), and they did so in a GNP dose- (hence pMHC dose)- dependent manner. Fig. 20 shows an experiment using ~14 nm GNPs coated with different numbers ofpMHC molecules/GNP using the linker method. Fig. 20 compares the amounts of IFNy secreted by cognate 8.3-CD8+ T-cells in response to two different pMHC-GNP samples (both consisting of ~2x1013 GNPs of 14 nm in diameter/ml). Au-0224lO and Au-2l9lO carried ~250 and ~l20 pMHCs/GNP, respectively. Au-Ol 1810-C carried ~l20 control pMHCs/GNP.

GNPs coated with ~2-fold higher numbers ofpMHC complexes/GNP had superior tic activity. Thus, the tic activity of pMHC-coated GNPs is a function of total pMHC (GNP) content. These results were counter-intuitive as the state of the art would suggest that, in the absence of ulatory molecules on the NPs, sing the numbers ofpMHCs on individual NPs would also increase avidity and should promote deletion (cell death), rather than proliferation and cytokine secretion from cognate T-cells. This would be true for both low avidity and high avidity s. For example, previous work by the Applicants (Han et al., Nature ne, 2005) and others indicated that peptides recognized with high avidity or es recognized with low avidity but given a high concentrations have an increased ability to delete cognate T cells in vivo. Therefore, in the context of therapeutic delivery of enous antigen-MHC-coated nanoparticles or soluble peptides, cognate T-cells should undergo deletion in a peptide affinity and dose-dependent . This expectation was not met by the data shown in Fig. 20.

IV. A valency threshold in the agonistic ty of peptide-MHC-nanoparticle complexes To further investigate the role of peptide-MHC (pMHC) valency on the tic properties of pMHC-conjugated nanoparticles (pMHC-NPs), the ability of 8nm diameter iron- oxide (Fe304) NPs covalently coupled with increasing numbers of IGRP206_214/Kd pMHC monomers, to r the secretion of IFN-gamma (IFNy) by cognate (IGRP206_214/Kd-specif1c) CD8+ T cells (herein referred to as 8.3-CD8+ T-cells) in vitro was compared. As shown in Table 2, 8.3-CD8+ T cells produced negligible amounts of IFNy when cultured in the presence ofNPs coated with 8 pMHC monomers per NP, but produced substantially higher amounts of IFNy in response to NPs coated with higher pMHC valencies, even as low as 11 pMHC monomers/NP, in a dose-response manner.

Table 2 Secretion of IFNy by 8.3-CD8+ T cells in response to NPs conjugated with increasing pMHC valencies (at 5X1011 NPs/mL) —————110512 02912 —————012011 —————031511 —————051211 ————"100711 —————011411 This positive effect of pMHC valency on the agonistic activity of pMHC-NPs was maintained over a range ofpMHC-NP densities (Fig. 21). ably, however, Whereas 25x1011 NPs (per ml) carrying 11 pMHCs/NP had similar agonistic activity as 5x1011 NPs (per ml) carrying 54 NP, increasing the number ofNPs carrying 8 pMHCs/NP to values as high as 40x1011 NPs/ml had minimal s (Fig. 22). Taken er, these results indicate that there is a threshold of pMHC valency, lying between 9 and 11 pMHCs/NP, below which relatively small increases in the number ofNPs (i.e. 5-fold) cannot overcome the low agonistic activity of pMHC-NPs coated at low valencies (it is noted that that the use of >50x1011 NPs in these in vitro experiments is not informative due to cellular toxicity caused by high NP densities).

This pMHC valency threshold effect is r illustrated in Fig. 23, where the IFNy secretion data are ized to the tration of total pMHC delivered by the coated NPs in the cultures. NPs carrying 11 pMHCs/NP triggered significantly higher IFNy responses over a 2013/052352 range of pMHC concentrations than those triggered by NPs ng 8 pMHCs/NP.

Furthermore, differences in the agonistic ties of these two NP preparations increased substantially with total pMHC content. That is, differences in the agonistic properties of 2.4 ug/ml of pMHC delivered by the NPs as octamers versus monodecamers were much higher than differences in the agonistic properties of the same formulations at 10-fold lower concentrations of total pMHC.

Fig. 24 shows that these profound effects of pMHC valency on the agonistic properties ofpMHC-NPs can also be seen when using larger NPs (which can accept much higher pMHC valencies than the 8 nm NPs studied in Figs. 21-23) used at lower NP densities (to normalize the total iron oxide content in the cultures). Whereas l8nm diameter NPs carrying <10 pMHCs/NP had virtually no biological activity up to 4x1011 , the agonistic ty of l8nm diameter NPs carrying higher pMHC valencies increased linearly with NP density. Comparison of Figs. 23 and 24 r shows that 2x1011 l8nm NPs delivering 61 pMHCs/NP have similar tic activity than 2x1011 8nm NPs delivering a similar number (54) of pMHCs/NP, indicating that the effects ofpMHC valency are not significantly affected by NP volume.

Taken together, these data trate that pMHC-coated NPs acquire powerful agonistic activity above a certain pMHC valency threshold (lying between 9 and 11 pMHCs/NP).

Increases in either pMHC valency or NP density can enhance the agonistic properties ofpMHC- NPs carrying “threshold” or “supra-threshold” pMHC-valencies but not the agonistic properties of NPs carrying -threshold” pMHC valencies.

V. Agonistic activity versus NP size and density.

Further analysis indicated that total pMHC content is not the only factor affecting the agonistic activity of pMHC-NPs in vitro and that NP size also plays an important independent role. This was investigated by comparing the agonistic activity of two pMHC-GNP s of different size (14 and 40 nm in diameter, respectively) and different pMHC ies but under conditions of similar total pMHC content. In the experiment shown in Fig. 25, 14 nm GNPs carrying ~200 pMHC molecules/GNP, and 40 nm GNPs carrying ~5,000 pMHCs/GNP were used. The GNP densities of these two samples was ed (to 3x1013 and 1012 GNPs/mL, respectively) to adjust the total pMHC content in each sample to ~450 ug/ml. Notably, 8.3- CD8+ T cells responded significantly better to the 14 nm pMHC/GNP compound than to the 40 nm one over a range of total pMHC contents, despite the fact that the latter were decorated with significantly more pMHC complexes than the former. This suggested that GNP density (more GNPs/cognate T-cell) is key. In other words, 4x40 nm NPs carrying 1000 pMHCs/GNP (4000 pMHCs) would be less desirable than 40x10 nm NPs carrying 100 pMHCs/GNP (4000 pMHCs).

Thus, when taken together these data suggest that optimal NP preparations are those comprised of small GNPs used at high pMHC densities. Increasing pMHC y on these small NPs further increase their surprising and unexpected agonistic properties.

VI. tic activity versus pMHC exposure.

As noted above, the pMHC-coated GNP samples are produced by co-coating GNPs with a 3.4 kD thiol-PEG-NH2 linker (as acceptor ofpMHC itermini) with a thiol-PEG linker that functions as GNP stabilizer. To investigate if the length of the stabilizing PEG linker influences its GNP anti-aggregation properties, the ability of the thiol-PEG-NH2 linker to bind pMHC molecules and/or the agonistic properties of oated GNPs, pMHC-coated GNPs ed using stabilizing linkers of different sizes (2 kD and 5 kD, r and longer than the cceptor linker, respectively) were compared. It was found that both linkers had similar anti-aggregation properties, and that the 5 kD linker did not inhibit g ofpMHC to the shorter 3.4 kD thiol—PEG-NH2 linker. Notably, however, pMHC-GNPs that were ted by the shorter (2 kD) thiol-PEG had superior agonistic activity in vitro than those co-coated with the longer (5 kD) thiol-PEG (Fig. 26). This suggests that long protective thiol-PEG linkers shield pMHC molecules bound to the acceptor linker from exposure to e T cells.

VII. Small NPs covalently coupled to high densities of pMHC afford maximum autoregulatory T-cell ion effects in vivo.

Nanoparticles having an average diameter of about 10 nm and coupled to either NRP- V7/Kd (also ed to as IGRP206_214- Kd) or TUM/Kd (control) were made in accordance with the methods described herein, and tested for their ability to induce expansion of cognate autoregulatory CD8+ T cells in viva. Fig. 27 shows the results of an experiment in which antigen-MHC-GNPs were injected intravenously into 10 week-old wild-type NOD mice mice biweekly for 5 consecutive weeks. Changes in the size of the cognate T-cell population in the circulation and different lymphoid tissues in response to therapy were assessed by staining cell suspensions with fluorescently—labeled antigen-MHC tetramers (both cognate as well as irrelevant control tetramers). Administration of 10-100 fewer GNPs than what was has previously been shown in the art (See, for example, Tsai et al., Immunity, 2010 in which nanoparticles coated with 1-8 pMHCs were tested) but coated with 150 n-MHCs per GNP resulted in substantially higher expansions (Fig. 27). They expanded CD8+ T-cells in vivo to levels several fold higher (up to 44% of all circulating CD8+ T-cells) than those we typically obtain with nanoparticles coated with a pMHC at a valency of about 8 (1-2% cells in blood; See, for example, Tsai et al., Immunity, 2010, Figure 1C). The above data indicate that small rticles coated with high antigen-MHC valencies afford maximum T-cell expansion effects. These results were unexpected. Accordingly, it is not the overall avidity of the pMHC- NP-T-cell interaction that is responsible for therapeutic effect, but rather the avidity of the sor population that gives rise to the T-cells that expand in response to pMHC-NP therapy.

This retation is consistent with the data bed herein and implies that valency of pMHCs on NPs should increase the therapeutic efficacy -NPs.

Example 4 Large ion of cognate CD8+ T-cells by pMHC-GNPs coated at higher pMHC valencies. It was next determined whether pMHC-NPs have the potential to induce massive expansions of cognate T-cells in vivo. This was done by treating mice with several injections of 3x1012 10-14 nm NPs carrying 25 ug of total pMHC (~150 IGRP206_214/Kd molecules per NP). As shown in Fig. 28, mice treated with 10 doses (twice a week for 10 week) displayed massive expansions of cognate IGRP206_214 (NRP-V7)-reactive CD8+ T-cells in peripheral blood as compared to their untreated counterparts (from <0.4 to >17 or 47% CD8+ T- cells) (lower panels). Such expansion was already seen in a mouse that was sacrificed after 4 doses ofpMHC-NPs (upper panels). The pMHC-NP-expanded cells ically bound cognate but not non-cognate pM Example 5 Preparation of pMHC conjugated Gold NanoParticles pMHC conjugated Gold NanoParticle Preparation (pMHC-GNPs, 12 and 30 nm).

Preparation of GNPs. GNPs were prepared by heating D.D. water (200 mL) in a ball flask in a silicon oil bath till g. A solution of 1% HAuCL4 (4 mL) was then added into boiling water.

The solution was stirred for 10 min before adding of 1% Na Citrate solution. For 12 nm GNPs, 12 mL Na Citrate solution was added. For 30 nm GNPs, 12 mL Na Citrate solution was added. A wine color appears immediately after adding Na Citrate solution. To complete the reaction, GNP on was stirred for 30 minutes more. This is a ation of the method bed in Levy, R. et a1. (“Rational and combinatorial design of peptide capping ligands for gold nanoparticles.” J Am Chem Soc 126, 10076-84 (2004)) which is herein incorporated by reference. e modification ofGNPs. GNPs were pegylated by addition of 25 mM thiol- PEG-NH2 (M.W. 3,400) and 50 mM thiol—PEG (M. W. 2,000, PEG/GNP ratio 10,000: 1) into GNP solution. The solution was stirred for 5 hours at room temperature. Pegylated GNPs were then washed with 3 X 30 mL sterilized D. D. water to remove excess PEGs, and resuspended in 40 mL of 100 mM MES (C6H13NO4S.xH20) buffer, pH 5.5. pMHC ation. pMHCs (IGRP206_214/Kd, 4 mg) was added into solution of pegylated GNPs, drop-by-drop with mild stirring at room temperature. The mixture is stirred for one hour before the addition of 20 mg 1-Ethyl(3-dimethylaminopropyl) carbodiimide (EDC).

The mixture is stirred for additional 4 hrs. pMHC-GNPs conjugates are then washed with 40 mL Phosphate Buffered Saline (PBS, PH 4) for three times, and resuspended in 8 mL PBS.

Example 6 Preparation of pMHC conjugated Gold NanoParticles Preparation of pMHC conjugated GNPs (pMHC-GNPS, 2-10 nm). Prepare GNPs (2-5 nm). GNPs of 2-5 nm were prepared by dissolving 250 mg (for 2 nm GNPs) or 50 mg (for 4 nm GNPs) lamine in 10 mL of DDAB solution (100 mM Didodecyldimethylammonium e (DDAB) in Toluene). Secondly, 100 mg Tetrabutylammonium borohydride (TBAB) was ved in 4 mL ofDDAB solution. Solutions of Dodecylamine and TBAB were then mixed in a 50 mL three-neck flask, stirring under nitrogen. 34 mg AuC13 was resolved in 4.5 mL DDAB solution, and injected y into a mixture of TBAB and Dodecylamine solution. on becomes deep red immediately, indicating the formation of GNPs. The mixture was continuously stirred for 30 min, and 15 mLs of ethanol were added into the mixture. The mixture was then spun at 4,100 x g for 12 min to precipitate GNPs.

Prepare GNPs (6-10 nm). To e GNPs of 6-10nm Decanoic acid (172 mg) was first dissolved in 10 mL Toluene, and then mixed with various amounts of TBAB solution (4 and 1 mL for 6 and 10 nm GNPs, respectively) in a 50 mL three-neck flask, when stirring under nitrogen. AuC13 (34 mg dissolved in in 4.5 mL DDAB stock solution) was then quickly injected into the mixture of TBAB and Decanoic acid solution. The solution became deep red immediately. The mixture was continuously stirred for 30 min, and 15 mL ethanol was added into the mixture. The mixture is then spun at 4,100 x g for 12 min to precipitate GNPs.

Surface modification ofGNPs. GNPs were resuspended in 20 mL of 0.1 M mercaptopropanoic acid (MPA) in methanol, pH 10 and stirred for one hour at room ature. mL ethyl acetate was then added. The mixture was then spun at 4,100 x g for 15 min. The precipitated GNPs were then washed with 30 mL sterilized D.D. water for three times, and resuspended in 20 mL 100 mM MES (C6H13NO4S.xH20) buffer, pH 5.5. To this e, solutions of 0.5 M Polyoxyethylene bis(amine) (at 10,000:1 PEG/GNP ratio) and 0.1M l-Ethyl- 3-(3-dimethylaminopropyl) carbodiimide (EDC) (final EDC concentration 2 mM) were added.

The mixture was then stirred for 4 hours. The ted GNPs were washed with 3 X 30 mL ized D.D. water to remove excess PEG and EDC. pMHC conjugation. Pegylated GNPs were resuspended in 20 mL 100 mM MES (C6H13NO4S.xH20) buffer, pH 5.5. pMHCs (5 mg/mL, total 10 - 30 mg) were then added to resuspended GNPs (500:1 NP ratio), drop-by-drop, and stirred for 1 hour at room temperature before adding 0.1M l-Ethyl(3-dimethylaminopropyl) carbodiimide (EDC) (final EDC tration 2 mM). The mixture was stirred for 4 more hours. pMHC-GNPs conjugates were washed three with 40 mL Phosphate Buffered Saline (PBS, PH 7.2-7.4), and then resuspended in 10-20 mL PBS.

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

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

In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled 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.

Throughout this disclosure, various publications, patents and published 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 entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including def1nitions, will control.

Claims (24)

What is claimed is:

1. A nanoparticle comprising an antigen-MHC x, wherein the complex comprises a MHC protein complexed to an antigen derived from a microbe of the gastrointestinal tract wherein the ratio of antigen-MHC complex per nanoparticle is from about 10:1 to about 1000:1.

2. The nanoparticle of claim 1, wherein the nanoparticle has a diameter selected from the group of diameters of: from about 1 nm to about 100 nm; from about 5 nm to about 50 nm; or from about 5 to about 15 nm.

3. The nanoparticle of any one of the previous claims, n the n-MHC complex is covalently linked or non-covalently linked to the nanoparticle.

4. The nanoparticle of any one of the previous claims, wherein the antigen-MHC x is covalently linked to the nanoparticle h a linker less than 5 kD in size.

5. The nanoparticle of any one of the us claims, wherein the n is derived from a microbe of the group: Bacteroides, Clostridium, Fusobacterium, Eubacterium, coccus, Peptococcus, Peptostreptococcus, or Bifidobacterium.

6. The rticle of any one of the previous claims, wherein the antigen is derived from Bacteroides or from a protein of Bacteroides.

7. The nanoparticle of any one of the previous claims, wherein the antigen is derived from Integrase.

8. The nanoparticle of any one of the previous claims, wherein the antigen comprises a peptide having at least 80% identity to the peptide sequence of the group: SEQ ID Nos. 1, 4, 5, 6, 7, or 8.

9. The nanoparticle of any one of the previous claims, wherein the nanoparticle is biocompatible or bioabsorbable.

10. The nanoparticle of any one of the previous claims, wherein the MHC comprises a MHC class I or a MHC class II.

11. The nanoparticle of any one of the previous claims, wherein the nanoparticle is nonliposomal.

12. A composition comprising the nanoparticle of any one of claims 1-11 and a carrier.

13. A method for preparing or obtaining the nanoparticle of any one of claims 1-11 comprising complexing the antigen-MHC complex to the nanoparticle.

14. Use of the rticle of any one of claims 1-11 for the manufacture of a medicament for treating inflammation of the gastrointestinal tract by inducing an anti-inflammatory response in a cell or tissue in the gastrointestinal tract.

15. Use of the nanoparticle of any one of claims 1-11 for the cture of a medicament for treating inflammation in a patient in need thereof.

16. Use of the nanoparticle of any one of claims 1-11 for the cture of a medicament for treating inflammation of the gastrointestinal tract by accumulating anti-inflammatory T cells and/or anti-inflammatory cells in the GI tract of a patient in need thereof.

17. The use of claim 15 or claim 16, wherein the patient s from a gastrointestinal e of the group: inflammatory bowel disease, colitis, Crohn’s disease, allergic inflammation of the intestinal tract, or celiac disease.

18. The use of claim 14, wherein the cell or tissue is a cell or tissue of the GI tract.

19. The use of any one of claims 16-17, wherein the T cell is a CD4+ T cell or a CD8+ T cell.

20. The use of any one of claims 16-17 or 19, wherein the T cell secretes IL-10 or TGFβ.

21. The use of claim 14 or 18, wherein the anti-inflammatory response is induced in the gastrointestinal tract.

22. The use of claim 15, wherein inflammation of the gastrointestinal tract is treated.

23. Use of the rticle of any one of claims 1-12 for the manufacture of a ment for treating inflammation of the gastrointestinal tract by transferring cytotoxic T-lymphocytes targeting gut bacterial epitopes in a patient in need thereof.

24. The use of claim 23, wherein the cytotoxic T-lymphocytes recognize the gut bacterial epitope with low avidity.