Treatment of liver disease

Abstract

Described herein are compositions and methods useful for treating liver inflammatory disorders that utilize ubiquitous, non-tissue-specific antigens associated with major histocompatibility complexes (MHC) and coupled to nanoparticle cores to induce regulatory T cells and regulatory B cells.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 62 / 852,194, filed May 23, 2019, which is incorporated herein by reference in its entirety. Summary of the Invention [Means for solving the problem]

[0002] Provided herein are compositions comprising multiple antigen-major histocompatibility complexes coupled to nanoparticle cores. The compositions are useful for treating hepatic inflammatory diseases. Many hepatic inflammatory diseases involve general inflammation of the liver. Exemplary hepatic inflammatory diseases include hepatitis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), cirrhosis, or pyogenic liver abscess. Nonspecific immune inhibitors are often available for treating these diseases, but such inhibitors are associated with significant systemic side effects. Described herein are compositions comprising ubiquitous autoantigen-MHC complexes coupled to nanoparticles (uaMHC-NPs), useful for treating hepatic inflammatory diseases or expanding the intrahepatic T regulatory cell population, which suppresses autoreactive (i.e., autoimmune or inflammatory) T cells.

[0003] First, the therapeutic agents described herein are multipurpose in that they can treat multiple liver inflammatory disease disorders that are mechanistically and pathologically unrelated with a single composition. Many such multipurpose therapeutic agents, such as corticosteroids and antibodies against common inflammatory mediators, result in systemic immunosuppression, increasing the treated patient's risk of developing secondary infections and systemic immunological complications. While multipurpose, the compositions described herein also preserve systemic immunity, leaving intact the patient's ability to fight off viral, bacterial, fungal infections, or tumors.

[0004] In one aspect, described herein is a composition for use in treating hepatic inflammatory disease. The composition includes: (a) a plurality of antigen-major histocompatibility complex (antigen-MHC) molecules, each of the plurality of antigen-MHC molecules comprising a ubiquitous autoantigen associated with the binding groove of the MHC molecule, wherein the ubiquitous autoantigen is not a liver-specific antigen; and (b) a nanoparticle core having a diameter of 1 nanometer to about 100 nanometers, wherein the antigen-MHC is coupled to the nanoparticle core or to a biocompatible layer surrounding the nanoparticle core. In certain embodiments, the MHC molecule is an MHC class II molecule. In certain embodiments, the nanoparticle core is a metal or metal oxide. In certain embodiments, the metal is iron. In certain embodiments, the metal oxide is iron oxide. In certain embodiments, the diameter is greater than 15 nanometers and less than or equal to about 30 nanometers. In certain embodiments, the diameter is about 5 nanometers to about 50 nanometers. In certain embodiments, the diameter is about 5 nanometers to about 25 nanometers. In certain embodiments, the plurality of antigen-MHC is coupled to the nanoparticle core at an antigen-MHC to nanoparticle core ratio of at least 10:1. In certain embodiments, the plurality of antigen-MHC is coupled to the nanoparticle core at an antigen-MHC to nanoparticle core ratio of about 150:1 or less. In certain embodiments, the plurality of antigen-MHC is coupled to the nanoparticle core at an antigen-MHC to nanoparticle core ratio of about 150:1 or less. In certain embodiments, the plurality of antigen-MHC is coupled to the nanoparticle core at an antigen-MHC to nanoparticle core ratio of at least 100 nm surface area. 2The antigen-MHC is coupled to the nanoparticle core at a density of about 0.4 to about 13 antigen-MHC per nanoparticle. In certain embodiments, the antigen-MHC is covalently coupled to the nanoparticle core. In certain embodiments, the antigen-MHC is coupled to the nanoparticle core by a polyethylene glycol (PEG) linker having a mass of less than about 5 kilodaltons. In certain embodiments, the nanoparticle core further comprises a biocompatible coating. In certain embodiments, the ubiquitous autoantigen comprises a polypeptide derived from a protein present at steady state in or on an intracellular compartment. In certain embodiments, the intracellular compartment is the cytosol, mitochondria, Golgi apparatus, endoplasmic reticulum, nucleus, or plasma membrane. In certain embodiments, the intracellular compartment is a mitochondrion. In certain embodiments, the ubiquitous autoantigen is Mdh1, Actg1, Vim, Ldha, Gapdh, Ywhaz, Fabp3, Atox1, Prdx1, Txndc17, Ncl, Hnrnpf, Cops9, Lsm5, Pcna, Hnrnpa2b1, Tkt, Rbbp4, Rbbp7, Nme1, Rack1, Tfrc, Gab1, Lifr, gfr, Tfrc, S100a6, Fadd, Cnrip1, Eps15l1, Nptp, Hsp e1, Bax, Hspa9, Gstp1, Ndufab1, Mdh2, Hspd1, Atp5f1a, Hspd1, Atp5f1e, Arf3, Arf4, Arf5, Dpy30, Pitpnb, Ap1b1, Arl1, Prrc1, Copz1, Sar1b, Pgrmc1, Cyp2f2, Atp2a2, Fkbp2, Cyb5a, Erp44, Canx, Hsp90b1, Vcp, and Lman1. In certain embodiments, the ubiquitous autoantigen is pyruvate dehydrogenase complex E2 component (PDC-E2) or a polypeptide derived therefrom. In certain embodiments, the ubiquitous autoantigen is cytochrome P450 2D6 (CYP2D6) or a polypeptide derived therefrom. In certain embodiments, the ubiquitous autoantigen is actin (ACTB) or a polypeptide derived therefrom. In certain embodiments, the ubiquitous autoantigen is soluble liver antigen (SLA) or a polypeptide derived therefrom.In certain embodiments, the ubiquitous autoantigen is formimidoyltransferase-cyclodeaminase (FTCD) or a polypeptide derived therefrom. In certain embodiments, the ubiquitous autoantigen is myeloperoxidase (MPO). In certain embodiments, the ubiquitous autoantigen is PDC-E2. 353-367 , PDC-E2 72-86 , PDC-E2 422-436 , PDC-E2 353-367 , PDC-E2 80-94 , PDC-E2 535-549 , PDC-E2 629-648 , PDC-E2 122-135 PDC-E2 249-263 , PDC-E2 249-263 and any combination thereof. In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of PDC-E2 422-436 , PDC-E2 80-94 , and PDC-E2 422-436 and PDC-E2 80-94 In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of CYP2D6 284-298 , CYP2D6 289-303 , CYP2D6 318-332 , CYP2D6 313-332 , CYP2D63 93-412 , CYP2D6 192-206 , CYP2D 65-19 , CYP2D6 293-307 , CYP2D6 219-233 , CYP2D6 237-251 , CYP2D6 15-29 , CYP2D6 235-249 , CYP2D6 317-331 , CYP2D6 293-307 , CYP2D6 428-442 , CYP2D6 237-251 , CYP2D6 14-28 , CYP2D6 199-213 , CYP2D6 450-464 , CYP2D6 301-315 , CYP2D6 452-466 , CYP2D6 59-73 , CYP2D6 130-144 , CYP2D6 193-212 , CYP2D6305-324 , CYP2D6 15-29 In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of ACTB 202-216 , ACTB 170-184 , ACTB 245-259 , ACTB 187-201 , ACTB 172-186 , ACTB 131-145 , ACTB 131-145 , ACTB 171-185 , ACTB 129-143 , ACTB 164-178 , ACTB 25-39 , ACTB 323-337 In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of ACTB 146-160 , ACTB 18-32 , ACTB 171-185 In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of SLA, 334-348 , SLA 196-210 , SLA 115-129 , SLA 373-386 , SLA 186-197 , SLA 342-256 , SLA 110-124 , SLA 299-313 , SLA 49-63 , SLA 260-27 4. SLA 119-133 , SLA 86-100 , SLA 26-40 , SLA 331-345 , SLA 317-331 , SLA 171-185 , SLA 417-431 , SLA 359-373 , SLA 215-229 , SLA 111-125 In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of FTCD, 439-453 , FTCD 381-395 , FTCD 297-311 , FTCD 525-539 , FTCD 218-232 , FTCD 495-509 , FTCD 262-276 , FTCD3 00-314 , FTCD259-273 , FTCD 490-504 , FTCD 389-403 , FTCD 295-309 In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of FTCD, 271-285 , FTCD 498-512 , FTCD 301-315 In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of MPO, 322-336 , MPO 714-728 , MPO 617-631 , MPO 504-518 , MPO 462-476 , MPO 617-631 , MPO 444-458 , MPO 689-703 , MPO 248-262 , MPO 511-525 , MPO 97-111 , MPO 616-630 and any combination thereof. In certain embodiments, described herein is a composition comprising the uaMHC of the uaMHC-NP and a pharmaceutically acceptable stabilizer, excipient, diluent, or combination thereof. In certain embodiments, the composition is formulated for intravenous administration. In certain embodiments, the liver inflammatory disease is selected from the group consisting of hepatitis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, and pyogenic liver abscess.

[0005] In another aspect, described herein is a method for treating a liver inflammatory disease in an individual, comprising administering to the individual a composition comprising: (a) a plurality of antigen-major histocompatibility complex (antigen-MHC) molecules, each of the plurality of antigen-MHC molecules comprising a ubiquitous autoantigen associated with the binding groove of the MHC molecule, wherein the ubiquitous autoantigen is not a tissue-specific antigen; and (b) a nanoparticle core having a diameter of 1 nanometer to about 100 nanometers, wherein the antigen-MHC is coupled to the nanoparticle core or to a biocompatible layer surrounding the nanoparticle core. In certain embodiments, the MHC molecule is an MHC class II molecule. In certain embodiments, the nanoparticle core is a metal or metal oxide. In certain embodiments, the metal is iron. In certain embodiments, the metal oxide is iron oxide. In certain embodiments, the diameter is greater than 15 nanometers and less than or equal to about 30 nanometers. In certain embodiments, the diameter is between about 5 nanometers and about 50 nanometers. In certain embodiments, the diameter is between about 5 nanometers and about 25 nanometers. In certain embodiments, the antigen-MHC is coupled to the nanoparticle core at an antigen-MHC to nanoparticle core ratio of at least 10:1. In certain embodiments, the antigen-MHC is coupled to the nanoparticle core at an antigen-MHC to nanoparticle core ratio of about 150:1 or less. In certain embodiments, the antigen-MHC is coupled to the nanoparticle core at an antigen-MHC to nanoparticle core ratio of less than or equal to 100 nm of nanoparticle core surface area. 2The nanoparticle core is coupled to the nanoparticle core at a density of about 0.4 to about 13 antigen-MHC molecules per nanoparticle. In certain embodiments, the antigen-MHC is covalently coupled to the nanoparticle core. In certain embodiments, the antigen-MHC is coupled to the nanoparticle core by a polyethylene glycol (PEG) linker having a mass of less than about 5 kilodaltons. In certain embodiments, the nanoparticle core further comprises a biocompatible coating. In certain embodiments, the ubiquitous autoantigen comprises a polypeptide derived from a protein present at steady state in or on an intracellular compartment. In certain embodiments, the intracellular compartment is the cytosol, mitochondria, Golgi apparatus, endoplasmic reticulum, nucleus, or plasma membrane. In certain embodiments, the intracellular compartment is mitochondria. In certain embodiments, the ubiquitous autoantigen is pyruvate dehydrogenase complex E2 component (PDC-E2) or a polypeptide derived therefrom. In certain embodiments, the ubiquitous autoantigen is cytochrome P450 2D6 (CYP2D6) or a polypeptide derived therefrom. In certain embodiments, the ubiquitous autoantigen is actin (ACTB) or a polypeptide derived therefrom. In certain embodiments, the ubiquitous autoantigen is soluble liver antigen (SLA) or a polypeptide derived therefrom. In certain embodiments, the ubiquitous autoantigen is formimidoyltransferase-cyclodeaminase (FTCD) or a polypeptide derived therefrom. In certain embodiments, the ubiquitous autoantigen is myeloperoxidase (MPO) or a polypeptide derived therefrom. In certain embodiments, the ubiquitous autoantigen is PDC-E2 353-367 , PDC-E2 72-86 , PDC-E2 422-436 , PDC-E2 353-367 , PDC-E2 80-94 , PDC-E2 535-549 , PDC-E2 629-648 , PDC-E2 122-135 PDC-E2 249-263 , PDC-E2 249-263 and any combination thereof. In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of PDC-E2422-436 , PDC-E2 80-94 , and PDC-E2 422-436 and PDC-E2 80-94 In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of CYP2D6 284-298 , CYP2D6 289-303 , CYP2D6 318-332 , CYP2D6 313-332 , CYP2D63 93-412 , CYP2D6 192-206 , CYP2D 65-19 , CYP2D6 293-307 , CYP2D6 219-233 , CYP2D6 237-251 , CYP2D6 15-29 , CYP2D6 235-249 , CYP2D6 317-331 , CYP2D6 293-307 , CYP2D6 428-442 , CYP2D6 237-251 , CYP2D6 14-28 , CYP2D6 199-213 , CYP2D6 450-464 , CYP2D6 301-315 , CYP2D6 452-466 , CYP2D6 59-73 , CYP2D6 130-144 , CYP2D6 193-212 , CYP2D6 305-324 , CYP2D6 15-29 In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of ACTB 202-216 , ACTB 170-184 , ACTB 245-259 , ACTB 187-201 , ACTB 172-186 , ACTB 131-145 , ACTB 131-145 , ACTB 171-185 , ACTB 129-143 , ACTB 164-178 , ACTB 25-39 , ACTB 323-337 In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of ACTB 146-160 , ACTB 18-32 , ACTB 171-185In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of SLA, 334-348 , SLA 196-210 , SLA 115-129 , SLA 373-386 , SLA 186-197 , SLA 342-256 , SLA 110-124 , SLA 299-313 , SLA 49-63 , SLA 260-27 4. SLA 119-133 , SLA 86-100 , SLA 26-40 , SLA 331-345 , SLA 317-331 , SLA 171-185 , SLA 417-431 , SLA 359-373 , SLA 215-229 , SLA 111-125 In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of FTCD, 439-453 , FTCD 381-395 , FTCD 297-311 , FTCD 525-539 , FTCD 218-232 , FTCD 495-509 , FTCD 262-276 , FTCD3 00-314 , FTCD 259-273 , FTCD 490-504 , FTCD 389-403 , and FTCD 295-309 In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of FTCD 271-285 , FTCD 498-512 , and FTCD 301-315 and any combination thereof. In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of MPO 322-336 , MPO 714-728 , MPO 617-631 , MPO 504-518 , MPO 462-476 , MPO 617-631 , MPO 444-458 , MPO 689-703 , MPO 248-262 , MPO 511-525 , MPO 97-111 , MPO616-630 and any combination thereof. In certain embodiments, the composition further comprises a pharmaceutically acceptable stabilizer, excipient, diluent, or any combination thereof. In certain embodiments, the composition is formulated for intravenous administration. In certain embodiments, the liver inflammatory disease is selected from the group consisting of hepatitis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, and pyogenic liver abscess.

[0006] The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features and advantages thereof will be obtained by reference to the following detailed description that sets forth illustrative examples in which the principles of the features described herein are utilized, and the accompanying drawings in which: [Brief explanation of the drawings]

[0007] [Figures 1A-1G]This figure illustrates the expansion of primary biliary cirrhosis (PBC)-associated regulatory T cells by nanoparticles (PBC-associated peptide-major histocompatibility complex-nanoparticles (pMHC-NPs)) coupled to MHC class II associated with peptides derived from the pyruvate dehydrogenase complex E2 component (PDC-E2). Figure 1A shows the percentage of tetramer+ CD4+ T cells in the blood of NOD mice versus NOD.c3c4 mice as a function of age. Figure 1B shows the percentage of tetramer+ CD4+ T cells in the peripheral blood of pMHC-NP-treated NOD.c3c4 mice compared with untreated NOD or NOD.c3c4 mice or NOD.c3c4 mice treated with control Cys-NP. Figure 1C shows the percentage of tetramer+ CD4+ T cells in various organs in mice from panel B at the end of pMHC-NP therapy. Figure 1D shows the percentage of tetramer+ CD4+ T cells in NOD.c3c4 mice treated with type 1 diabetes-associated pMHC-NPs. Figure 1E shows the percentage of tetramer+ CD4+ T cells in various lymphoid organs and the liver of NOD.c3c4 mice treated with NPs coated with one of two different PBC-associated pMHCs. Figure 1F shows the expression of TR1-like cell surface markers by tetramer+ CD4+ T cells expanded in NOD.c3c4 mice by pMHC-NP therapy. Figure 1G shows the cytokine secretion profiles of tetramer+ CD4+ TR1-like cells versus tetramer-negative CD4+ T cells stimulated with peptide-pulsed DCs and sorted ex vivo. [Figures 2A-2C] We illustrate the expansion of PBC-associated regulatory T cells by PBC-associated pMHC-NPs. (Figures 2A-B) Upregulation of TR1-like markers on tetramer+ CD4+ T cells expanded in vivo in response to PDC166 / IAg7-NP or PDC82 / IAg7-NP therapy. Figure 2A shows representative FACS profiles. Figure 2B shows mean fluorescence intensity values. Figure 2C shows mean fluorescence intensity values ​​of the TR1 cell surface marker on tetramer+ CD4+ T cells generated in 38-44 week-old NOD.c3c4 mice in response to PDC166 / IAg7-NP therapy. [Figures 3A-3F]Figure 3 illustrates the clinical, phenotypic, immunological, and pathological characteristics of liver disease in NOD.c3c4 mice. Figure 3A shows age-related changes in serum TB and ALT levels. Figure 3B shows the microscopic scoring system (left) and the progression of microscopic disease scores with age (right). Figure 3C shows representative CBD images and the progression of CBD diameter and score with age. Figure 3D shows representative liver images (top) and the progression of liver scores and liver weight with age (bottom). Figure 3E shows that NOD.c3c4 mice spontaneously develop anti-PDC-E2-specific autoantibodies (left) and ANA (right). Figure 3F shows representative images of liver inflammation mediated by CD4+ and CD8+ T cells. [Figures 4A-4I] We demonstrate disease reversal in a PBC mouse model using PBC-associated pMHC-NPs. Figure 4A shows changes in serum total bile acid (TBA) and alanine aminotransferase (ALT) levels in NOD.c3c4 mice treated with PDC166 / IAg7-NP, PDC82 / IAg7-NP, or control (Cys-NP). Figure 4B shows representative histological images (top) and average histological scores (bottom) of the livers of NOD.c3c4 mice treated with PDC166 / IAg7-NP, PDC82 / IAg7-NP, or Cys-NP. Figure 4C shows representative macroscopic images of the common bile duct (top) and average common bile duct scores and diameters (bottom). Figure 4D shows representative macroscopic images of the liver (top) and average liver scores and liver weights (bottom). Figure 4E shows representative whole-body images of NOD.c3c4 mice treated with PDC166 / IAg7-NPs or control Cys-NPs. Figure 4F shows the changes in anti-mitochondrial (PDC-E2) antibody and antinuclear autoantibody (ANA) titers after treatment (top two left and right panels, respectively), as well as representative images (bottom) of Hep2 cells stained with serum from pMHC-NP-treated versus Cys-NP-treated NOD.c3c4 mice. Figure 4G shows the percentage of tetramer+ cells in mice treated starting at 24 weeks of age (from 38 to 44 weeks). Figures 4H and 4I show the microscopic scores (Figure 4H) and macroscopic scores (Figure 4I) of the mice examined in Figure 4G. [Figures 5A-5E]Figure 5 illustrates the change in circulating frequency of tetramer+ CD4+ T cells in response to periodic retreatment with PBC-associated nanoparticles. Figure 5A shows two different mice, and Figure 5B shows the average values ​​corresponding to the cohort of mice treated with PDC166 / IAg7-NPs or left untreated. Figure 5C shows the percentage of tetramer+ CD4+ T cells, and Figure 5D shows the mean fluorescence intensity staining of the TR1 marker for tetramer+ CD4+ T cells from the mice tested in Figure 5A. Figure 5E shows the mean macroscopic CBD score and liver score for the mice tested in Figures 5A-5D. [Figures 6A-6F] Figure 6 illustrates the effect of treatment with PBC-associated pMHC-NPs or standard of care for PBC (UDCA) on macroscopic disease scores and serum ALT levels (Figure 6A) and microscopic disease scores (Figure 6B) when treatment is initiated early in the disease process. Figure 6C shows the percentage of tetramer+ CD4+ T cells in the mice tested in Figures 6A-6B. Figures 6D and 6E show the effect of treatment with PBC-associated pMHC-NPs or standard of care for PBC (UDCA) on macroscopic disease scores (Figure 6D) and microscopic disease scores (Figure 6E) when treatment is initiated at an advanced stage of disease. Figure 6F shows the percentage of tetramer+ CD4+ T cells in the mice tested in Figures 6D-6E. [Figure 7A-7M]We demonstrate that PBC-associated pMHC-NPs expand regulatory B cells. Figure 7A shows the percentage of tetramer+ CD4+ T cells in mice treated with pMHC-NPs, rat IgG (control), or blocking rat mAbs against mouse IL-10 or TGF-beta. Figures 7B and 7C show the macroscopic (Figure 7B) and microscopic (Figure 7C) scores of the mice tested in Figure 7A. Figure 7D shows the percentage of tetramer+ CD4+ T cells in the blood and lymphoid organs of NOD.c3c4.scid hosts reconstituted with whole splenocytes from untreated NOD.c3c4 donors and then infused with splenic CD4+ T cells from PDC166-181 / IAg7-NP-treated NOD.c3c4 mice. The latter were either left untreated or treated with PDC166-181 / IAg7-NP after CD4+ T cell transfer. Figure 7E shows representative FACS staining histograms (top) and mean fluorescence intensity values ​​(bottom) of the TR1 marker on tetramer+ CD4+ vs. tetramer- CD4+ T cells from hosts treated with PDC166-181 / IAg7-NP. Figure 7F shows the macroscopic scores and measurements for the mice tested in A. Figure 7G shows the cytokine profiles of LPS-challenged CD11b+ cells isolated from liver-draining (PLN) or non-draining (MLN) lymph nodes of pMHC-NP-treated vs. Cys-NP-treated NOD.c3c4 mice. Figure 7H shows the cytokine profiles of hepatic Kupffer cells from pMHC-NP-treated vs. Cys-NP-treated NOD.c3c4 mice. Figure 7I shows the absolute numbers of B cells in liver-draining (PCLN) or non-draining (ILN) lymph nodes or livers of pMHC-NP-treated vs. Cys-NP-treated NOD.c3c4 mice. Figure 7J shows the correlation between the absolute number of B cells and the absolute number of tetramer+ CD4+ T cells in pMHC-NP-treated mice. (Figure 7K shows the IL-10 secretion levels of LPS-challenged B cells isolated from liver-draining and non-draining lymph nodes or livers of pMHC-NP-treated vs. Cys-NP-treated NOD.c3c4 mice.) Figure 7L shows representative FACS plots demonstrating the conversion of B cells to IL-10-producing Breg cells only in experiments conducted in pMHC-NP-treated hosts.Figure 7M shows the percentage of conventional B cell-derived Breg cells in pMHC-NP or Cys-NP-treated hosts in the liver and peripheral lymphoid organs harboring (spleen and PCLN) or lacking (MLN) pMHC-NP-induced TR1-like CD4+ T cells. [Figures 8A-8C] Figure 8 illustrates the expansion of T regulatory cells by PBC-associated pMHC-NPs in humanized mice. Figure 8A shows representative tetramer staining in NSG hosts transplanted with PBMCs from a DRB4*0101+ PBC patient and treated with three different PBC-associated pMHC-NPs. Figure 8B shows the mean percentage and absolute number of tetramer+ CD4+ cells in reactive vs. non-reactive pMHC-NP-treated mice or untreated littermates. Figure 8C shows the mean fluorescence intensity values ​​(top) and two-dimensional FACS plots (bottom) of the TR1 markers CD49b and LAG3 in the mice tested in Figures 8A-B. [Figures 9A-9E] These results demonstrate that PBC-associated pMHC-NPs can treat hepatic autoimmune models distinct from PBC, specifically primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH). Figure 9A shows the percentage of tetramer+ CD4+ T cells in NOD.Abcb4- / - mice responding to pMHC-NP therapy. Figure 9B shows the mean primary sclerosing cholangitis score (top) and representative H&E- or Picrosirius-stained liver sections (bottom), as well as TBA and ALT (right column), corresponding to the mice tested in Figure 10A. Figure 9C shows the percentage of tetramer+ CD4+ T cells in NOD mice infected with hFTCD-encoding adenovirus (resulting in AIH) and treated with three different pMHC-NP types. Figure 9D shows the autoimmune hepatitis histopathological score (top) and representative H&E- and Picrosirius-stained liver sections (bottom) in the mice tested in Figure 9C. Figure 9E shows serum ALT levels in the mice in Figure 9C [n = 11, 5, 9, and 10 mice (from left to right), 2-3 experiments]. [Figures 10A-10B]We demonstrate that PBC- and AIH-associated pMHC-NPs expand T regulatory cells in a PSC mouse model. Figure 10A shows representative FACS staining histograms, and Figure 10B shows the mean fluorescence intensity values ​​of the TR1 marker on tetramer+ CD4+ T cells versus tetramer- CD4+ T cells in NOD.Abcb4- / - mice (which spontaneously develop PSC) treated with PBC- or AIH-associated pMHC-NPs. [Figures 11A-11B] We illustrate that PBC-associated pMHC-NPs expand T regulatory cells in an AIH mouse model. Figure 11A shows representative FACS staining histograms, and Figure 11B shows the mean fluorescence intensity values ​​of the TR1 marker on tetramer+ CD4+ cells versus tetramer- CD4+ T cells in NOD mice infected with adenovirus encoding hFTCD (which spontaneously develop AIH) treated with PBC-associated or AIH-associated pMHC-NPs. [Figures 12A-12C] This demonstrates the ability of ubiquitous autoantigen-based pMHC-NPs to alleviate liver inflammatory disease in an organ-specific, rather than disease-specific, manner. Figure 12A shows the percentage of tetramer+ CD4+ T cells in NOD.c3c4 mice treated with AIH-associated pMHC-NP types. Figure 12B shows the mean macroscopic liver scores in NOD.c3c4 mice treated with PBC-associated, AIH-associated, or T1D-associated pMHC-NP types. Figure 12C shows the percentage of tetramer+ CD4+ T cells in NOD mice treated with PBC-associated or AIH-associated pMHC-NP types. [Figures 13A-13C]Figure 13 illustrates the therapeutic effect of PBC-associated pMHCII-NPs in (NODxB6.Ifng-ARE-Del- / -)F1 mice. Figure 13A shows the percentage of tetramer+ CD4+ T cells in mice (pooled males and females) treated with Cys-NPs or PDC166-181 / IAg7-NPs. Figure 13B shows serum TBA and ALT levels in the mice tested in A. Figure 13C shows the microscopic score (left) and representative H&E-stained liver sections (upper right) and picrosirius red-stained liver sections (lower right) of the female mice tested in A. Data in Figures 13A-13C correspond to n = 7 and 12 mice / treatment type, respectively, from 2-3 experiments. [Figures 14A-14B] 14A illustrates non-limiting embodiments of pMHC-NPs of the present disclosure. Figure 14A shows the alpha chain of an MHC class II dimer fused to the CH2 and CH3 domains of an immunoglobulin molecule containing an engineered knob (SEQ ID NO: 100). Figure 14B shows the beta chain of an MHC class II with an N-terminal ubiquitous polypeptide (E2122-135) fused to the CH2 and CH3 domains of an immunoglobulin molecule containing an engineered hole (SEQ ID NO: 101). DETAILED DESCRIPTION OF THE INVENTION

[0008] Table 1 illustrates linkers useful for coupling ubiquitous autoantigen-MHC to nanoparticles.

[0009] Tables 2, 3, and 4 illustrate the percentage and absolute numbers of tetramer+ CD4+ T cells in NSG mice engrafted with PBMCs from DRB4*0101+ PBC patients upon treatment with three different human PBC-associated pMHC-NP types.

[0010] Various embodiments are described below. It should be noted that the specific embodiments are not intended as exhaustive or as limitations on the broader aspects discussed herein. An aspect described in the context of a particular embodiment is not necessarily limited to that embodiment and may be practiced in any other embodiment.

[0011] The use of the terms "a," "an," and "the" and similar reference words in the context of describing elements (particularly in the context of the claims below) should be construed to encompass both the singular and the plural unless otherwise specified herein or otherwise clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each individual value falling within the range, unless otherwise specified herein, and each individual value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise specified herein or otherwise clearly contradicted by context. The use of any examples or exemplary language provided herein (e.g., "such as") is intended merely to facilitate a better understanding of the embodiments and does not impose limitations on the claims, unless otherwise expressly stated. No language herein should be construed as implying that any non-claimed element is required.

[0012] As used herein, "antigen" refers to all, a part, a fragment, or a segment of a molecule capable of inducing an immune response or the expansion of immune cells, preferably T cells or B cells, in a subject. Antigens can be polypeptides, lipids, carbohydrates, or nucleic acids.

[0013] As used herein, "individual" is synonymous with "subject" or "patient." An individual may be diagnosed with a disease. An individual may be suspected of having a particular disease based on exhibiting at least one symptom of the disease, having a family history of the disease, having a genotype associated with defining risk for the disease, or having one or more phenotypic measurements or "laboratory tests" at or near a level that may identify an individual at risk for the disease. An individual may be a mammal, such as a horse, cat, dog, pig, cow, goat, sheep, etc. An individual may, in certain cases, be a human individual.

[0014] As used herein, "about" will vary to some extent depending on the context in which it is used, as would be understood by one of ordinary skill in the art. If there are uses of the term that are not clear to persons of ordinary skill in the art, "about" will mean up to ±10% of the particular value of the term, taking into account the context in which it is used.

[0015] As used herein, "polypeptide" means a plurality of amino acids joined by peptide bonds having more than about 8 amino acid residues. The amino acids of a polypeptide can be naturally occurring or non-naturally occurring amino acid residues.

[0016] Percent (%) sequence identity to a reference polypeptide sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of known ways using publicly available computer software such as BLAST, BLAST-2, ALIGN, and Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes of this specification, % amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is the copyrighted work of Genentech, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559 (registered under U.S. Copyright Registration No. TXU510087). The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, Calif.) or can be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0017] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as 100 times the fraction X / Y, as follows: X is the number of amino acid residues scored as a perfect match by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0018] The uaMHC or uaMHC-NP of the present disclosure described herein can be encoded by a nucleic acid. A nucleic acid is a type of polynucleotide comprising two or more nucleotide bases. In certain embodiments, the nucleic acid is a component of a vector that can be used to transfer a polypeptide-encoding polynucleotide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting other nucleic acids to which it is linked. One type of vector is a genome-integrating vector or "integrating vector," which can become integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid capable of extrachromosomal replication. As used herein, a vector capable of directing the expression of an operably linked gene is referred to as an "expression vector." Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In expression vectors, regulatory elements, such as promoters, enhancers, and polyadenylation signals used to control transcription, can be derived from mammalian, microbial, viral, or insect genes. A selection gene that facilitates the replication ability in a host, usually conferred by an origin of replication, and the recognition of transformants may additionally be incorporated. Vectors derived from viruses such as lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses may be used. Plasmid vectors may be linearized to integrate into a chromosomal location. Vectors may contain sequences that direct site-specific integration into a defined location or set of restriction sites in the genome (e.g., AttP-AttB recombination). Additionally, vectors may contain sequences derived from transposable elements.

[0019] Either the nucleic acid encoding the uaMHC or the vector containing said nucleic acid can be transferred into a cell line suitable for producing the uaMHC. In certain embodiments, the nucleic acid or vector is stably integrated into the genome of the cell line. Suitable cell lines may be, for example, Vero cells (ATCC CRL81), CHO-K1 (ATCC CRL61) cells, HeLa cells, or L cells. Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; PER.C6™ cells (Crucell); and NSO cells.

[0020] In one aspect, described herein is a composition for use in treating liver inflammatory disease, comprising: (a) a plurality of antigen-major histocompatibility complex (antigen-MHC) molecules, each of the plurality of antigen-MHC molecules comprising a ubiquitous autoantigen associated with the binding groove of the MHC molecule, wherein the ubiquitous autoantigen is not a liver-specific antigen; and (b) a nanoparticle core having a diameter of 1 nanometer to about 100 nanometers, wherein the antigen-MHC is coupled to the nanoparticle core or to a biocompatible layer surrounding the nanoparticle core.

[0021] In another aspect, described herein is a method of treating hepatic inflammatory disease in an individual, comprising administering to the individual a composition comprising: (a) a plurality of antigen-major histocompatibility complex (antigen-MHC) molecules, wherein each of the plurality of antigen-MHC molecules comprises a ubiquitous autoantigen associated with the binding groove of the MHC molecule, wherein the ubiquitous autoantigen is not a tissue-specific antigen; and (b) a nanoparticle core having a diameter of about 1 nanometer to about 100 nanometers, wherein the antigen-MHC is coupled to the nanoparticle core or to a biocompatible layer surrounding the nanoparticle core.

[0022] Ubiquitous autoantigens Described herein are nanoparticle compositions and methods useful for treating liver inflammatory disorders. The nanoparticle compositions comprise multiple MHC-associated antigens coupled to nanoparticles. The nanoparticle compositions and methods utilize broadly expressed ubiquitous autoantigens to induce the development of regulatory T and B lymphocytes.

[0023] In certain aspects, the antigen associated with the MHC molecule is a ubiquitous autoantigen or a polypeptide derived from a ubiquitous autoantigen. Ubiquitous autoantigens are differentiated from tissue-specific antigens in that they are antigens generally expressed by at least a plurality of different, unrelated cell types. In certain embodiments, the ubiquitous autoantigen is generally expressed by ontogenetically distinct tissues. In certain embodiments, the ubiquitous autoantigen is expressed by at least two cell types derived from tissues originating from the list consisting of ectoderm, mesoderm, and endoderm. In certain embodiments, the ubiquitous autoantigen is generally expressed by functionally distinct tissues. In certain embodiments, the ubiquitous autoantigen is expressed in at least two tissues selected from the list consisting of neural tissue, endocrine tissue, connective tissue, hematopoietic cells, liver tissue, cardiac tissue, skin tissue, lung tissue, vascular tissue, intestinal tissue, and stomach tissue. In certain embodiments, the ubiquitous autoantigen is expressed in both neural tissue and liver tissue. In certain embodiments, the ubiquitous autoantigen is expressed in both neural and pancreatic tissues. In certain embodiments, the ubiquitous autoantigen is a polypeptide derived from a protein involved in a cellular process common to at least one, two, three, four, five, six, seven, eight, nine, ten, or more cell types. A ubiquitous autoantigen may be a sequence common to two or more closely related proteins recognized as paralogs or homologs within the same family, yet exhibiting differential expression across various tissues. In certain embodiments, the two or more closely related proteins may, for example, perform the same or similar function in two different, unrelated tissues. In certain embodiments, the ubiquitous autoantigen is a polypeptide derived from a protein involved in a cellular process, wherein the cellular process is a metabolic process selected from glycolysis, oxidative phosphorylation, glycogen production, nucleotide biosynthesis, beta-oxidation, and omega-oxidation.In certain embodiments, the ubiquitous autoantigen is selected from the list consisting of pyruvate dehydrogenase complex E2 component (PDC-E2), cytochrome P450 2D6 (CYP2D6), formimidoyltransferase-cyclodeaminase (FTCD, also known as formiminotransferase cyclodeaminase), soluble liver antigen (SLA), actin (ACTB), and myeloperoxidase (MPO).

[0024] Ubiquitous autoantigens are often encoded by housekeeping genes that are utilized by various cell types. For example, actin is a cytoskeletal protein that contributes to cell structure, cell mobility, cell division, and vesicle mobility, and is ubiquitously expressed. Therefore, many ubiquitous autoantigens are intracellular and reside in specific subcellular compartments at steady state. Antigens reside at steady state in the subcellular location where they can be found in their highest abundance (e.g., as determined by microscopy or cell fractionation). For example, despite the fact that actin can be found extracellularly in association with exosomes, the vast majority of actin is found in the cytosol of cells. Similarly, many antigens can transit various organelles but reside primarily in a single organelle. For example, many endoplasmic reticulum (ER)-resident proteins will transiently transit the cis-Golgi but then immediately return to the ER for steady-state residence.

[0025] In certain embodiments, the ubiquitous autoantigen used in the nanoparticle compositions described herein is a polypeptide derived from the pyruvate dehydrogenase complex E2 component (PDC-E2). In certain embodiments, the PDC-E2-derived polypeptide is PDC-E2 for DRB3*0202. 353-367 , PDC-E2 72-86 , and PDC-E2 422-436 , PDC-E2 for DRB5*0101 353-367 , PDC-E2 80-94 , and PDC-E2 535-549 , PDC-E2 for DRB4*0101 629-648 , PDC-E2 122-135, and PDC-E2 249-263 , and PDC-E2 for DRB1*0801 249-263 In certain embodiments, the polypeptide derived from PDC-E2 is any one or more of: 422-436 and PDC-E2 80-94 In certain embodiments, the polypeptide derived from PDC-E2 is any one or more of SEQ ID NOs: 1-12.

[0026] In certain embodiments, the ubiquitous autoantigen used in the nanoparticle compositions described herein is a polypeptide derived from cytochrome P450 2D6 (CYP2D6). In certain embodiments, the polypeptide derived from CYP2D6 is CYP2D6 284-298 , CYP2D6 289-303 , CYP2D6 318-332 , CYP2D6 313-332 , CYP2D63 93-412 , CYP2D6 192-206 , CYP2D 65-19 , CYP2D6 293-307 (for DRB1*0301), CYP2D6 219-233 , CYP2D6 237-251 , CYP2D6 15-29 (for DRB3*0202), CYP2D6 235-249 , CYP2D6 317-331 , CYP2D6 293-307 (for DRB4*0101), CYP2D6 428-442 , CYP2D6 237-251 , CYP2D6 14-28 (for DRB5*0101), CYP2D6 199-213 , CYP2D6 450-464 , CYP2D6 301-315 (for DRB1*0401), CYP2D6 452-466 , CYP2D6 59-73 , CYP2D6 130-144 , CYP2D6 193-212 , CYP2D6 305-324 , and CYP2D6 15-29(relative to DRB1*0701). In certain embodiments, the polypeptide derived from CYP2D6 is any one or more of SEQ ID NOs: 13-37.

[0027] In certain embodiments, the ubiquitous autoantigen used in the nanoparticle compositions described herein is a polypeptide derived from soluble liver antigen (SLA). 334-348 , SLA 196-210 , SLA 115-129 , SLA 373-386 , SLA 186-197 (for DRB1*0301), SLA 342-256 , SLA 110-124 , SLA 299-313 (for DRB3*0202), SLA 49-63 , SLA 260-27 4. SLA 119-133 (for DRB4*0101), SLA 86-100 , SLA 26-40 , SLA 331-345 (for DRB5*0101), SLA 317-331 , SLA 171-185 , SLA 417-431 (for DRB1*0401), SLA 359-373 , SLA 215-229 , and SLA 111-125 (relative to DRB1*0701). In certain embodiments, the polypeptide derived from SLA is any one or more of SEQ ID NOs: 53-72.

[0028] In certain embodiments, the ubiquitous autoantigen used in the nanoparticle compositions described herein is a polypeptide derived from actin (ACTB). In certain embodiments, the polypeptide derived from ACTB is ACTB. 202-216 , ACTB 170-184 , ACTB 245-259 (for DRB1*0301), ACTB 187-201 , ACTB 172-186 , ACTB 131-145 (for DRB3*0202), ACTB131-145 , ACTB 171-185 , ACTB 129-143 (for DRB4*0101), ACTB 164-178 , ACTB 25-39 and ACTB 323-337 In certain embodiments, the polypeptide derived from ACTB is any one or more of ACTB*1, ACTB*2, ACTB*3, ACTB*4, ACTB*5, ACTB*6, ACTB*7, ACTB*8, ACTB*9, ACTB*10, ACTB*11, ACTB*12, ACTB*13, ACTB*14, ACTB*15, ACTB*16, ACTB*17, ACTB*18, ACTB*19, ACTB*19, 146-160 , ACTB 18-32 , and ACTB 171-185 In certain embodiments, the polypeptide derived from ACTB is any one or more of SEQ ID NOs: 38-52.

[0029] In certain embodiments, the ubiquitous autoantigen used in the nanoparticle compositions described herein is a polypeptide derived from formimidoyltransferase-cyclodeaminase (FTCD). In certain embodiments, the polypeptide derived from FTCD is FTCD 439-453 , FTCD 381-395 , FTCD 297-311 (for DRB3*0202), FTCD 525-539 , FTCD 218-232 , FTCD 495-509 (for DRB1*0301), FTCD 262-276 , FTCD 300-314 , FTCD 259-273 (for DRB4*0101), FTCD 490-504 , FTCD 389-403 , and FTCD 295-309 In certain embodiments, the polypeptide derived from FTCD is any one or more of: FTCD 271-285 , FTCD 498-512 , and FTCD 301-315 In certain embodiments, the polypeptide derived from FTCD is any one or more of SEQ ID NOs: 73-87.

[0030] In certain embodiments, the ubiquitous autoantigen used in the nanoparticle compositions described herein is a polypeptide derived from myeloperoxidase (MPO). In certain embodiments, the MPO-derived polypeptide is 322-336 , MPO 714-728 , MPO 617-631 (for DRB3*0202), MPO 504-518 , MPO 462-476 , MPO 617-631 (for DRB1*0301), MPO 444-458 , MPO 689-703 , MPO 248-262 (for DRB4*0101), MPO 511-525 , MPO 97-111 , and MPO 616-630 (relative to DRB5*0101). In certain embodiments, the polypeptide derived from MPO is any one or more of SEQ ID NOs: 88-99.

[0031] Additional ubiquitous autoantigens are listed in Table 5 at the end of this disclosure. In certain embodiments, the ubiquitous autoantigen used in the nanoparticle compositions described herein is derived from a protein or polypeptide listed in Table 5. In certain embodiments, the ubiquitous autoantigen is selected from the group consisting of Mdh1, Actg1, Vim, Ldha, Gapdh, Ywhaz, Fabp3, Atox1, Prdx1, Txndc17, Ncl, Hnrnpf, Cops9, Lsm5, Pcna, Hnrnpa2b1, Tkt, Rbbp4, Rbbp7, Nme1, Rack1, Tfrc, Gab1, Lifr, Egfr, Tfrc, S100a6, Fadd, Cnrip1, Eps15l1, Nptp, Hs and polypeptides derived from any one or more of: pel, Bax, Hspa9, Gstp1, Ndufab1, Mdh2, Hspd1, Atp5f1a, Hspd1, Atp5f1e, Arf3, Arf4, Arf5, Dpy30, Pitpnb, Ap1b1, Arl1, Prrc1, Copz1, Sar1b, Pgrmc1, Cyp2f2, Atp2a2, Fkbp2, Cyb5a, Erp44, Canx, Hsp90b1, Vcp, and Lman1. In certain embodiments, the ubiquitous autoantigen used in the nanoparticle compositions described herein is derived from a human homolog of a protein or polypeptide listed in Table 5. In certain embodiments, the homolog or human homolog is a protein or polypeptide exhibiting at least about 75%, 80%, 85%, 90%, 95%, or 98% identity to a protein listed in Table 5.

[0032] tissue specific antigen The nanoparticle compositions and methods described herein utilize ubiquitous autoantigens that are not tissue-specific antigens. Many autoimmune or inflammatory diseases involve immune responses against tissue-specific antigens. This presents a manufacturing challenge for drugs to treat autoimmune or inflammatory diseases, because each disease requires a specific drug that targets that antigen. Alternatively, non-specific immune inhibitors can be used, but these have significant systemic side effects.

[0033] Tissue-specific antigens are often expressed by tissue or cell types affected by autoimmune diseases; for example, a major pathological consequence of multiple sclerosis is demyelination of nervous system tissues; as a result, tissue-specific antigens for multiple sclerosis are primarily restricted to the nervous system (e.g., myelin basic protein). Tissue-specific antigens are antigens associated with specific cells or cell types. Tissue-specific antigens may perform specialized functions or contribute to specialized tissue structures. In certain embodiments, a tissue-specific antigen has expression restricted to any one of the following tissues: nervous tissue, kidney tissue, heart tissue, lung tissue, liver tissue, small intestine tissue, colon tissue, stomach tissue, muscle tissue, connective tissue, and vascular tissue. In certain embodiments, a tissue-specific antigen is restricted to expression in any one of the following cell types: beta cells, alpha cells, B lymphocytes, T lymphocytes, Schwann cells, and adrenal cortical cells.

[0034] Many tissue-specific antigens may be expressed at very low levels in other cell or tissue types, but the primary source of expression is one specific cell or tissue type. For example, a single cell or tissue type exhibiting cell- or tissue-type-specific expression of a particular gene may express that gene at least 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, or more at the mRNA or protein level compared to any other unrelated cell type. In addition, some tissue-specific antigens may acquire ectopic expression of the cell-specific antigen under pathogenic conditions or due to exogenous stimuli. It is intended that the tissue specificity of an antigen is not lost simply because a different cell type may acquire ectopic expression under pathological or exogenous conditions. For example, although insulin is a tissue-specific antigen produced by beta cells, due to genetic instability, some tumors (known as insulinomas) may express insulin; under these types of circumstances, insulin is still considered tissue-specific.

[0035] Tissue-specific antigens that are not ubiquitous autoantigens are primarily antigens associated with particular tissue-specific autoimmune or inflammatory diseases.

[0036] In certain embodiments, the autoimmune or inflammatory disease is multiple sclerosis. In certain embodiments, the tissue-specific antigen that is not a ubiquitous autoantigen is myelin basic protein, myelin-associated glycoprotein, myelin oligodendrocyte protein (MOG), proteolipid protein, oligodendrocyte myelin oligoprotein, myelin-associated oligodendrocyte basic protein, oligodendrocyte-specific protein, heat shock protein, oligodendrocyte-specific protein, NOGO A, glycoprotein Po, peripheral myelin protein 22, and / or a polypeptide derived from 2'3'-cyclic nucleotide 3'-phosphodiesterase.

[0037] In certain embodiments, the autoimmune or inflammatory disease is diabetes mellitus type 1. In certain embodiments, the tissue-specific antigen that is not a ubiquitous autoantigen is a polypeptide derived from preproinsulin, proinsulin, islet-specific glucose-6-phosphatase (IGRP), glutamic acid decarboxylase (GAD), islet cell autoantigen 2 (ICA2), and / or insulin.

[0038] In certain embodiments, the autoimmune or inflammatory disease is pemphigus foliaceus (PF) or pemphigus vulgaris (PV). In certain embodiments, the tissue-specific antigen that is not a ubiquitous autoantigen is a polypeptide derived from desmoglein 3 (DG3) and / or desmoglein 1 (DG1).

[0039] In certain embodiments, the autoimmune or inflammatory disease is neuromyelitis optica spectrum disorder (NMO). In certain embodiments, the tissue-specific antigen that is not a ubiquitous autoantigen is a polypeptide derived from aquaporin 4 (AQP4).

[0040] In certain embodiments, the autoimmune or inflammatory disease is arthritis. In certain embodiments, the tissue-specific antigen that is not a ubiquitous autoantigen is selected from the group consisting of heat shock proteins, immunoglobulin-binding proteins, heterogeneous nuclear RNPs, annexin V, calpastatin, type II collagen, glucose-6-phosphate isomerase, elongation factor human cartilage gp39, mannose-binding lectin, citrullinated vimentin, type II collagen, fibrinogen, alpha-enolase, anti-carbamylated protein (anti-CarP), peptidylarginine deiminase type 4 (PAD4), BRAF, and furan. The polypeptides are derived from fibrinogen gamma chain, inter-alpha trypsin inhibitor heavy chain H1, alpha-1-antitrypsin, plasma protease C1 inhibitor, gelsolin, alpha 1-B glycoprotein, ceruloplasmin, inter-alpha trypsin inhibitor heavy chain H4, complement factor H, alpha 2 macroglobulin, serum amyloid, C-reactive protein, serum albumin, fibrogen beta chain, serotransferrin, alpha 2HS glycoprotein, vimentin, and / or complement C3.

[0041] In certain embodiments, the autoimmune or inflammatory disease is allergic asthma. In certain embodiments, the tissue-specific antigen that is not a ubiquitous autoantigen is a polypeptide derived from DERP1 and / or DERP2.

[0042] In certain embodiments, the autoimmune or inflammatory disease is inflammatory bowel disease. In certain embodiments, the tissue-specific antigen that is not a ubiquitous autoantigen is a polypeptide derived from Bacteroides integrase, flagellin, flagellin 2 (Fla-2 / Fla-X), or an uncharacterized E. coli protein (YIDX).

[0043] In certain embodiments, the autoimmune or inflammatory disease is systemic lupus erythematosus. In certain embodiments, the tissue-specific antigen that is not a ubiquitous autoantigen is polypeptide H4, H2B, H1', ​​dsDNA, RNP, Smith (Sm), Sjogren's syndrome-associated antigen A (SS-A) / Ro, Sjogren's syndrome-associated antigen B (SS-B) / La, and / or a histone. In some embodiments, SS-A includes, but is not limited to, RO60 and RO52. In some embodiments, histone includes, but is not limited to, H4, H2B, and H1.

[0044] In certain embodiments, the autoimmune or inflammatory disease is atherosclerosis. In certain embodiments, the tissue-specific antigen that is not a ubiquitous autoantigen is a polypeptide derived from apolipoprotein B (ApoB) and / or apolipoprotein E (ApoE).

[0045] In certain embodiments, the autoimmune or inflammatory disease is chronic obstructive pulmonary disease (COPD). In certain embodiments, the tissue-specific antigen that is not a ubiquitous autoantigen is a polypeptide derived from elastin.

[0046] In certain embodiments, the autoimmune or inflammatory disease is psoriasis. In certain embodiments, the tissue-specific antigen that is not a ubiquitous autoantigen is a polypeptide derived from human adamis-like protein 5 (ATL5), cathelicidin antimicrobial peptide (CAP18), and / or ADAMTS-like protein 5 (ADMTSL5).

[0047] In certain embodiments, the autoimmune or inflammatory disease is uveitis. In certain embodiments, the tissue-specific antigen that is not a ubiquitous autoantigen is an arrestin, a human retinal S antigen, and / or a polypeptide derived from interphotoreceptor retinoid-binding protein (IRBP).

[0048] In certain embodiments, the autoimmune or inflammatory disease is Sjogren's syndrome. In certain embodiments, the tissue-specific antigen that is not a ubiquitous autoantigen is a polypeptide derived from (SS-A) / Ro, (SS-B) / La, RO60, RO52, and / or muscarinic receptor 3 (MR3).

[0049] In certain embodiments, the autoimmune or inflammatory disease is scleroderma. In certain embodiments, the tissue-specific antigen that is not a ubiquitous autoantigen is a polypeptide derived from the centromeric autoantigen centromere protein C (CENP-C), DNA topoisomerase I (TOP1), and / or RNA polymerase III.

[0050] In certain embodiments, the autoimmune or inflammatory disease is antiphospholipid syndrome. In certain embodiments, the tissue-specific antigen that is not a ubiquitous autoantigen is a polypeptide derived from beta-2-glycoprotein 1 (BG2P1 or APOH).

[0051] In certain embodiments, the autoimmune or inflammatory disease is stiff man syndrome. In certain embodiments, the tissue-specific antigen that is not a ubiquitous autoantigen is a polypeptide derived from GAD65.

[0052] Antigen-major histocompatibility complex (MHC) The nanoparticle complexes of the present disclosure comprise a nanoparticle core coupled to a ubiquitous autoantigen-MHC, with or without layers and / or coatings. The individual MHC polypeptide and antigen (e.g., polypeptide) components form a complex via covalent or non-covalent bonds (e.g., via hydrogen, ionic, or hydrophobic bonds). Preparation of such complexes may require varying degrees of manipulation, and such methods are well known in the literature. In some embodiments, the antigen component can be non-covalently associated with the pocket portion of the MHC component, for example, by mixing the MHC component with the antigen component. This relies on the natural binding affinity between the MHC and the antigen. Alternatively, in some embodiments, the MHC component can be covalently associated with the antigen component using standard procedures, for example, but not limited to, by the introduction of known coupling agents or photoaffinity labels (see, e.g., Hall et al., Biochemistry 24:5702-5711 (1985)). In certain aspects, antigenic components may be operatively coupled to MHC components via peptide linkage or other methods discussed in the literature, for example, by attachment onto glycoproteins via carbohydrate groups, including, but not limited to, carbohydrate moieties on the alpha and / or beta chains. In particular embodiments, antigenic components may be attached to the N- or C-terminus of the appropriate MHC molecule. Alternatively, in certain embodiments, MHC complexes may be formed recombinantly by incorporating the sequence of the antigenic component into the sequence encoding the MHC, such that both retain their functionality.

[0053] Multiple ubiquitous autoantigen-MHC complexes can be coupled to the same nanoparticle core, and these complexes, MHCs, and / or antigens can be identical or different from one another.

[0054] major histocompatibility molecules The ubiquitous autoantigens described herein are associated with MHC molecules (to form ubiquitous autoantigen-MHC) and coupled to nanoparticles. The antigen is bound to the binding groove of the MHC molecule. MHC molecules primarily bind antigens that are polypeptides, but the polypeptides may contain modifications such as lipidation, glycosylation, and phosphorylation. The MHC molecule can be an MHC class I molecule (MHC1) or an MHC class II molecule (MHCII). MHC class I molecules have closed binding grooves on both sides, allowing them to bind polypeptides of 8 to 10 amino acid residues. MHC class II molecules, including those described herein, bind polypeptides of at least 8 amino acid residues in length, but have open binding grooves on both sides, allowing them to bind longer peptides of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 amino acid residues or longer.

[0055] For use in human individuals, the MHC molecules utilized herein are human (also known as human leukocyte antigens, abbreviated as "HLA"). In certain embodiments, the MHC class I molecule is a classical or non-classical MHC class I molecule, HLA-A, HLA-B, HLA-C, HLA-E, CD1d, or a fragment or biological equivalent thereof. In certain embodiments, the MHC class II molecule is HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQB1, or HLA-DPB1, or a fragment or biological equivalent thereof. In some embodiments, the antigen-MHC (pMHC) can be a single-chain construct. In some embodiments, the pMHC can be a two-chain construct. In the case of MHC class II, the beta chain of HLA will generally be non-covalently linked to an appropriate alpha chain, with the alpha chain pairing with the beta chain to form a two-chain heterodimer. In general, the alpha chains of MHC class II, such as the DR alpha chain, exhibit a fairly low degree of polymorphism.

[0056] Because the MHC class II complex is a heterodimer comprising an alpha chain and a beta chain, the heterodimer can be problematic under some conditions or is inherently unstable in some situations. When utilizing MHC class II molecules in the methods or compositions herein, the MHC molecule can further comprise a knob-in-hole architecture. Typically, the alpha or beta chain is an antibody C modified to include a bulge. H 2 and C H The antibody C3 domain is fused to the corresponding alpha or beta chain of the heterodimer, but the corresponding other alpha or beta chain is modified to include a depression. H 2 and C H It is fused into three domains.

[0057] As used herein, "knob-in-hole" or "knob-into-hole" refers to a polypeptide architecture requiring a protuberance (or "knob") at the interface of a first polypeptide and a corresponding recess (or "hole") at the interface of a second polypeptide, such that the protuberance can be positioned within the recess to promote heterodimer formation. A protuberance can be constructed by replacing a small amino acid side chain at the interface of a first polypeptide with a larger one (e.g., phenylalanine or tyrosine). A recess of identical or similar size to the protuberance can be created at the interface of a second polypeptide by replacing the side chain of a large amino acid with a smaller one (e.g., alanine or threonine). Protuberances and recesses can be produced by synthetic means, e.g., by modifying a nucleic acid encoding the polypeptide or by peptide synthesis, using routine methods known to those skilled in the art. In some embodiments, the interface of the first polypeptide is located on an Fc domain of the first polypeptide, and the interface of the second polypeptide is located on an Fc domain of the second polypeptide. Knob-in-hole heterodimers and methods for their preparation and use are disclosed in U.S. Patent Nos. 5,731,168, 5,807,706, 5,821,333, 7,642,228, 7,695,936, 8,216,805, and 8,679,785, and Merchant et al., Nature Biotechnology, 1998, 16:677-681, all of which are incorporated by reference in their entireties.

[0058] Alternatively or additionally, any of the antigens described herein may contain a cysteine ​​residue (engineered or natural) that interacts with a cysteine ​​residue in the MHC class II alpha or beta chain, commonly known as a cysteine ​​trap.

[0059] Cysteine ​​trapping can be used to stabilize the heterodimers described herein. Cysteine ​​trapping involves forming a covalently linked polypeptide complex from unbound polypeptide partners. In some embodiments, cysteine ​​trapping involves introducing cysteines at strategically selected positions within the interaction interface of the polypeptide partners to form a stabilized polypeptide complex. In some embodiments, cysteine ​​trapping can stabilize the polypeptide complex to favor a specific conformation and prevent dissociation. Cysteine ​​trapping is also referred to as disulfide trapping or disulfide bridging. Examples of cysteine ​​trapping methods and applications are reviewed in Kufareva, et al., Methods Enzymol. 570:389-420 (2016). In the context of MHC, cysteines are engineered into polypeptides known or suspected to associate with the binding groove of MHC class II dimers. The cysteine ​​is then engineered to be in or near the binding groove so that when the polypeptide associates with the binding groove, the binding groove cysteine ​​is in close proximity to the polypeptide cysteine ​​and can form a disulfide linkage therewith.

[0060] In one aspect, provided herein is an isolated heterodimer comprising at least one first polypeptide and at least one second polypeptide, wherein the first and second polypeptides meet at an interface, the interface of the first polypeptide comprises an engineered protuberance positionable in an engineered recess of the interface of the second polypeptide, and (i) the first polypeptide comprises an MHC class II α1 domain, an MHC class II α2 domain, or a combination thereof, and the second polypeptide comprises an MHC class II β1 domain, an MHC class II β2 domain, or a combination thereof, or (ii) the first polypeptide comprises an MHC class II β1 domain, an MHC class II β2 domain, or a combination thereof, and the second polypeptide comprises an MHC class II α1 domain, an MHC class II α2 domain, or a combination thereof. The first polypeptide, the second polypeptide, or both, can comprise an antibody CH3 domain fused thereto. Optionally, the first polypeptide, the second polypeptide, or both, comprise an MHC (α chain or β chain) and a C H Antibody C located between domain 3 H In certain embodiments, the first polypeptide comprises two domains. H 3 domains and antibody C H In certain embodiments, the second polypeptide comprises at least one mutation selected from the list consisting of S354C, T366W, and both S354C and T366W (EU numbering). H 3 domains and antibody C H The three domains comprise at least one mutation selected from the list consisting of Y349C, T366S, L368A, Y407V (EU numbering), and any combination thereof. In a further embodiment, the isolated heterodimer comprises a ubiquitous autoantigen optionally covalently linked to either the first or second polypeptide. Optionally, the ubiquitous autoantigen comprises a cysteine ​​residue that interacts with a cysteine ​​residue on either the first or second polypeptide to form a cysteine ​​trap.

[0061] In one aspect, one polypeptide of the heterodimer comprises an MHC class II α1 domain, an MHC class II α2 domain, or a combination thereof, and at least one engineered bulge. In some embodiments, the at least one engineered bulge is not located in either the MHC class II α1 domain or the MHC class II α2 domain. In some embodiments, the engineered bulge is located in an antibody C fused to the polypeptide. H In some embodiments, the polypeptide is located in the MHC class II α2 domain and a C α3 domain with an engineered bulge. H Antibody C located between domain 3 H In certain embodiments, the polypeptide optionally comprises an antibody C H 3 domains, and the antibody CH3 domain comprises at least one mutation selected from the list consisting of S354C, T366W, and both S354C and T366W (EU numbering). In a further embodiment, the polypeptide comprises a ubiquitous autoantigen. Optionally, the ubiquitous autoantigen comprises a cysteine ​​residue that interacts with a cysteine ​​residue in either the MHC α1 or β1 domain to form a cysteine ​​trap.

[0062] In one aspect, one polypeptide of the heterodimer comprises an MHC class II β1 domain, an MHC class II β2 domain, or a combination thereof, and at least one engineered bulge. In some embodiments, the at least one engineered bulge is not located in either the MHC class II β1 domain or the MHC class II β2 domain. In some embodiments, the engineered bulge is located in an antibody C fused to the polypeptide. H In some embodiments, the polypeptide is located in the MHC class II β2 domain and a C β3 domain with an engineered bulge. H Antibody C located between domain 3 H In certain embodiments, the polypeptide optionally comprises an antibody C H 3 domains and antibody C HThe MHC α1 domain comprises at least one mutation selected from the list consisting of S354C, T366W, and both S354C and T366W (EU numbering). In a further embodiment, the polypeptide comprises a ubiquitous autoantigen. Optionally, the ubiquitous autoantigen comprises a cysteine ​​residue that interacts with a cysteine ​​residue in either the MHC α1 or β1 domain to form a cysteine ​​trap.

[0063] In one aspect, one polypeptide of the heterodimer comprises an MHC class II α1 domain, an MHC class II α2 domain, or a combination thereof, and at least one engineered cavity. In some embodiments, the at least one engineered cavity is located in neither the MHC class II α1 domain nor the MHC class II α2 domain. In some embodiments, the engineered cavity is located in an antibody C fused to the polypeptide. H In some embodiments, the polypeptide is located in the MHC class II α2 domain and a C α3 domain with an engineered cleft. H Antibody C located between domain 3 H In certain embodiments, the polypeptide optionally comprises an antibody C H 3 domains and antibody C H The three domains comprise at least one mutation selected from the list consisting of Y349C, T366S, L368A, Y407V (EU numbering), and any combination thereof. In a further embodiment, the polypeptide comprises a ubiquitous autoantigen. Optionally, the ubiquitous autoantigen comprises a cysteine ​​residue that interacts with a cysteine ​​residue in either the MHC α1 or β1 domain to form a cysteine ​​trap.

[0064] In one aspect, one polypeptide of the heterodimer comprises an MHC class II β1 domain, an MHC class II β2 domain, or a combination thereof, and at least one engineered cavity. In some embodiments, the at least one engineered cavity is located in neither the MHC class II β1 domain nor the MHC class II β2 domain. In some embodiments, the engineered cavity is located in an antibody C fused to the polypeptide. H In some embodiments, the polypeptide is located in the MHC class II β2 domain and a C β3 domain with an engineered cleft. H Antibody C located between domain 3 H In certain embodiments, the polypeptide optionally comprises an antibody C H 3 domains and antibody C H The three domains comprise at least one mutation selected from the list consisting of Y349C, T366S, L368A, Y407V (EU numbering), and any combination thereof. In a further embodiment, the polypeptide comprises a ubiquitous autoantigen. Optionally, the ubiquitous autoantigen comprises a cysteine ​​residue that interacts with a cysteine ​​residue in either the MHC α1 or β1 domain to form a cysteine ​​trap.

[0065] Figures 14A and 14B show non-limiting embodiments of engineered uaMHCs containing an engineered depression and an engineered protuberance. Figure 14A shows a human MHC class II alpha chain fused to immunoglobulin CH2 and CH3 domains (SEQ ID NO: 100). The CH3 domain contains an engineered knob formed by two amino acid substitutions, S354C and T366W (SEQ ID NO: 104). The alpha chain contains an optional c-terminal cysteine ​​to allow conjugation to a functionalized linker, although this c-terminal cysteine ​​may alternatively be included on the beta chain. Figure 14B shows a human MHC class II beta chain fused to immunoglobulin CH2 and CH3 domains (SEQ ID NO: 101). The CH3 domain contains an engineered hole formed by four amino acid substitutions, Y349C, T366S, L368A, and Y407V (SEQ ID NO: 105). The beta chain also contains a ubiquitous autoantigen (PDC-E2) covalently coupled to the beta chain by a peptide linker. 122-135, SEQ ID NO: 8). The ridge-pit interactions favor assembly, allowing for purification of the intact uaMHC heterodimer using standard immunoglobulin purification techniques. Once purified, the uaMHC can be coupled to a suitable nanoparticle via a functionalized linker molecule (e.g., a functionalized PEG molecule). In certain embodiments, the MHC heterodimer comprises the amino acid sequence set forth in SEQ ID NOs: 100 and 101. In certain embodiments, the MHC heterodimer comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 100 and 101. In certain embodiments, the alpha chain of the MHC heterodimer comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 102. In certain embodiments, the beta chain of the MHC heterodimer comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 103. In certain embodiments, the MHC heterodimer comprises an alpha chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 102 and a beta chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 103. In certain embodiments, the MHC heterodimer comprises an alpha chain and a beta chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 102 and 103, respectively, and any one or more of the CH2 and CH3 domains of SEQ ID NO: 104, the CH2 and CH3 domains of SEQ ID NO: 105, and / or the ubiquitous autoantigen are identical to those disclosed in SEQ ID NO: 101 (individually disclosed as SEQ ID NO: 8). In certain embodiments, the alpha chain and beta chain are at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 102 and 103, respectively, and the ubiquitous autoantigen is identical to that disclosed in SEQ ID NO: 101. In certain embodiments, the alpha chain of the MHC heterodimer comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 104.In certain embodiments, the beta chain of the MHC heterodimer comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 105. In certain embodiments, the alpha chain of the MHC heterodimer comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 104, while preserving the specific knob or hole mutations. In certain embodiments, the beta chain of the MHC heterodimer comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 105, while preserving the specific knob or hole mutations. Those skilled in the art will appreciate that the alpha and beta chains of human MHC are highly polymorphic and can tolerate a relatively high degree of variability. Additionally, one skilled in the art would be able to substitute the alpha and beta chains of the MHC shown in SEQ ID NOs: 102 and 103, respectively, with suitable ubiquitous autoantigens capable of binding to different HLA alleles and specific replacement alleles. Such pairings of HLA alleles and ubiquitous autoantigens are disclosed elsewhere in this application, for example, in the sequence listing at the end of this application.

[0066] nanoparticles The ubiquitous autoantigen-MHC is coupled to a nanoparticle core (uaMHC-NP). The nanoparticles can be made from a variety of materials. In certain embodiments, the nanoparticles are non-liposomal and / or have a solid core. In certain embodiments, the solid core can be a metal or metal oxide. In certain embodiments, the solid core can be iron, iron oxide, or gold. The solid core has a density of about 2.0 g / cm. 3 , about 3.0g / cm 3 , about 4.0g / cm 3 , about 5.0g / cm 3 , about 6.0g / cm 3 , or about 7.0 g / cm 3 In one particular embodiment, the density of the solid core is about 4.0 g / cm 3 ~about 8.0g / cm 3In certain embodiments, the density of the solid core is about 5.0 g / cm 3 ~about 8.0g / cm 3 In certain embodiments, the density of the solid core is about 5.0 g / cm 3 ~Approx. 7.0g / cm 3 In certain embodiments, the density of the solid core is about 5.0 g / cm 3 ~About 6.0g / cm 3 is.

[0067] The nanoparticle core of the uaMHC-NP can comprise, consist essentially of, or even consist of a solid core, metal core, dendrimer core, polymeric micelle nanoparticle core, nanorod, fullerene, nanoshell, core-shell, protein-based nanostructure, lipid-based nanostructure, etc. In some embodiments, the nanoparticle core is bioabsorbable and / or biodegradable. In some embodiments, the nanoparticle core is a dendrimer nanoparticle core comprising, alternatively consisting essentially of, or even consisting of highly branched macromolecules having tree-like structures growing from the core. In further embodiments, the dendrimer nanoparticle core can comprise, alternatively consisting essentially of, or even consisting of poly(amidoamine)-based dendrimers or poly-L-lysine-based dendrimers. In certain embodiments, the nanoparticle core is a polymeric micelle core comprising, alternatively consisting essentially of, or even consisting of amphiphilic block copolymers assembled into nanoscale core-shell structures. In a further aspect, the polymeric micelle core comprises, alternatively consists essentially of, or even consists of, polymeric micelles formed using polyethylene glycol-diastearoylphosphatidylethanolamine block copolymers. In a further aspect, the nanoparticle core comprises, alternatively consists essentially of, or even consists of metals. In another aspect, the nanoparticle core is not a liposome. Additional examples of core materials include, but are not limited to, standard and specialty glass, silica, polystyrene, polyester, polycarbonate, acrylic polymers, polyacrylamide, polyacrylonitrile, polyamide, fluoropolymers, silicone, cellulose, silicon, metals (e.g., iron, gold, silver), minerals (e.g., ruby), nanoparticles (e.g., gold nanoparticles, colloidal particles, metal oxides, metal sulfides, metal selenides, and magnetic materials such as iron oxide), and composites thereof. In some embodiments, the iron oxide nanoparticle core comprises iron(II,III) oxide. The core can be of uniform composition or can be a composite of two or more classes of materials depending on the desired properties.In certain aspects, metal nanoparticles will be used. Such 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, binary alloys thereof, ternary alloys thereof, and intermetallic compounds thereof. See U.S. Pat. No. 6,712,997, which is incorporated herein by reference for its disclosure. In certain embodiments, the composition of the core and layers (described below) can vary so long as the nanoparticles are biocompatible and bioabsorbable. The core can be of uniform composition or can be a composite of two or more classes of materials depending on the desired properties. In certain aspects, metal nanospheres will be used. Such metal nanoparticles can be formed from Fe, Ca, Ga, etc. In certain embodiments, the nanoparticles comprise, alternatively consist essentially of, or even consist of a core comprising a metal or metal oxide, such as gold or iron oxide.

[0068] In another aspect, provided herein is a uaMHC-NP comprising at least one ubiquitous autoantigen-MHC as described herein and a nanoparticle, wherein the nanoparticle is non-liposomal and has an iron oxide core.

[0069] In another aspect, provided herein is a uaMHC-NP comprising at least one ubiquitous autoantigen-MHC as described herein and a nanoparticle, wherein the nanoparticle is non-liposomal and has a gold core.

[0070] In another aspect, provided herein is a uaMHC-NP comprising at least one ubiquitous autoantigen-MHC herein and a nanoparticle, wherein the nanoparticle is non-liposomal and has an iron oxide core, and the at least one ubiquitous autoantigen-MHC is covalently linked to the nanoparticle via a linker.

[0071] In some aspects, the nanoparticle core has a diameter selected from the group consisting of about 1 nm to about 100 nm, about 1 nm to about 75 nm, about 1 nm to about 50 nm, about 1 nm to about 25 nm, about 5 nm to about 100 nm, about 5 nm to about 50 nm, about 5 nm to about 40 nm, about 5 nm to about 30 nm, about 5 nm to about 25 nm, or about 5 nm to about 20 nm. In some embodiments, the nanoparticle core has a diameter of about 10 nm to about 100 nm, about 10 nm to about 50 nm, about 10 nm to about 40 nm, about 10 nm to about 30 nm, about 10 nm to about 25 nm, or about 10 nm to about 20 nm. In certain embodiments, the nanoparticle core has a diameter of about 1 nm, 2 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, or greater than 50 nm. In certain embodiments, the nanoparticle core has a diameter of less than about 100 nm, 75 nm, 50 nm, 40 nm, 30 nm, 20 nm, or 15 nm.

[0072] In some embodiments, the nanoparticle core is a dendrimeric nanoparticle core comprising, alternatively consisting essentially of, or even consisting of highly branched macromolecules with tree-like structures growing from the core. In further embodiments, the dendrimeric nanoparticle can comprise, alternatively consisting essentially of, or even consisting of poly(amidoamine)-based dendrimers or poly-L-lysine-based dendrimers. In certain embodiments, the nanoparticle core is a polymeric micelle core comprising, alternatively consisting essentially of, or even consisting of amphiphilic block copolymers assembled into nanoscale core-shell structures. In further embodiments, the polymeric micelle core can comprise, alternatively consisting essentially of, or even consisting of polymeric micelles formed using polyethylene glycol-diastearoylphosphatidylethanolamine block copolymers. The dendrimeric or polymeric micelle core can further comprise an outer coating or outer layer as described herein.

[0073] In certain embodiments, specific synthetic routes to dendrimer nanoparticles or nanoparticles with dendrimer nanoparticle cores may involve the extraction of metal ions into the interior of the dendrimer followed by chemical reduction to yield nearly monodisperse particles with dimensions less than 3 nm. For example, in the method disclosed in Crooks et al., "Synthesis, Characterization, and Applications of Dendrimer-Encapsulated Nanoparticles," The Journal of Physical Chemistry B(109):692-704 (2005), the resulting dendrimer core component not only serves as a template for preparing nanoparticles but also stabilizes them, allowing for tunable solubility and providing a means for immobilizing nanoparticles on solid supports.

[0074] The nanoparticle core typically consists of a substantially spherical core and, optionally, one or more layers or coatings. The core can vary in size and composition as described herein. In addition to the core, the particle can have one or more layers to provide functionality suitable for the intended application. The thickness of the layers, if any, can vary as needed for a specific application. For example, the layers can impart useful optical properties.

[0075] Layers can also impart chemical or biological functionality (referred to herein as chemically or biologically active layers). These layers are typically applied onto the outer surface of the particle to impart functionality to the pMHC-NP. The layer or layers can typically be about 0.001 micrometer (1 nanometer) to about 10 micrometers or more (depending on the desired particle diameter), or within the ranges of about 1 nm to 5 nm, about 1 nm to about 10 nm, about 1 nm to about 40 nm, about 15 nm to about 25 nm, or about 20 nm, and ranges therebetween.

[0076] The layer or coating can comprise, alternatively consist essentially of, or even consist of biodegradable sugar polymers or other polymers. Examples of biodegradable layers include, but are not limited to, dextran, poly(ethylene glycol), poly(ethylene oxide), mannitol, polylactide (PLA)-based, polyglycolide (PGA)-based, polycaprolactone (PCL)-based poly(esters), PHB-PHV class poly(hydroxyalkanoates), and other modified polysaccharides, such as starch, cellulose, and chitosan. Additionally, the nanoparticles can include a layer with a surface suitable for providing chemical functionality for chemical binding or coupling sites.

[0077] Antigen-MHC surface valency and density The ubiquitous autoantigen-MHC described herein is coupled to nanoparticles at a particular valency. Valency refers to the number of pMHC per nanoparticle core. In certain embodiments, the valency of the nanoparticles can be in the range of about 1 pMHC / nanoparticle core to about 6,000 pMHC / nanoparticle core. In certain embodiments, the valency of the nanoparticles can be in the range of about 10 pMHC / nanoparticle core to about 6,000 pMHC / nanoparticle core. In certain embodiments, the valency of the nanoparticles can be in the range of about 50 pMHC / nanoparticle core to about 6,000 pMHC / nanoparticle core. In certain embodiments, the valency of the nanoparticles can be in the range of about 1 pMHC / nanoparticle core to about 5,000, about 4,000, about 3,000, about 2,000, or about 1,000 pMHC / nanoparticle core. In certain embodiments, the valency of the nanoparticles can be in the range of about 10 pMHC / nanoparticle core to about 5000, about 4000, about 3000, about 2000, or about 1000 pMHC / nanoparticle core. In certain embodiments, the valency of the nanoparticles can be in the range of about 50 pMHC / nanoparticle core to about 5000, about 4000, about 3000, about 2000, or about 1000 pMHC / nanoparticle core. In certain embodiments, the valency of the nanoparticles can be in the range of about 1 pMHC / nanoparticle core to about 1000 pMHC / nanoparticle core, or about 10:1 to about 1000:1, or about 11:1 to about 1000:1, or about 12:1 to about 1000:1. In certain embodiments, the valency (antigen-MHC to nanoparticle core) can be in the range of about 10:1 to about 500:1, or about 11:1 to about 500:1, or about 12:1 to about 500:1. In certain embodiments, the valency (antigen-MHC to nanoparticle core) can be in the range of about 10:1 to about 200:1, or about 11:1 to about 200:1, or about 12:1 to about 200:1. In certain embodiments, the valency (antigen-MHC to nanoparticle core) can be in the range of about 10:1 to about 150:1, or about 11:1 to about 150:1, or about 12:1 to about 150:1. In certain embodiments, the valency (antigen-MHC to nanoparticle core) can be in the range of about 10:1 to about 100:1, or about 11:1 to about 100:1, or about 12:1 to about 100:1.In certain embodiments, the valency (antigen-MHC to nanoparticle core) can be within the range of about 10:1 to about 200:1, about 20:1 to about 200:1, about 30:1 to about 200:1, about 40:1 to about 200:1, or about 50:1 to about 200:1. In certain embodiments, the valency (antigen-MHC to nanoparticle core) can be within the range of about 10:1 to about 150:1, about 20:1 to about 150:1, about 30:1 to about 200:1, about 40:1 to about 150:1, or about 50:1 to about 150:1. In certain embodiments, the valency (antigen-MHC to nanoparticle core) can be in the range of about 10:1 to about 100:1, about 20:1 to about 100:1, about 30:1 to about 100:1, about 40:1 to about 100:1, or about 50:1 to about 100:1.

[0078] In some embodiments, the nanoparticle core has a defined valency per unit surface area of ​​the core (also referred to herein as "density"). In these embodiments, the pMHC density / nanoparticle is about 0.025 pMHC / 100 nm 2 ~about 100pMHC / 100nm 2 Nanoparticle core surface area, or alternatively, approximately 0.406 pMHC / 100 nm 2 ~about 50pMHC / 100nm 2 , or alternatively about 0.05 pMHC / 100 nm 2 ~about 25pMHC / 100nm 2 In certain embodiments, the pMHC density per nanoparticle is about 0.4 pMHC / 100 nm 2 ~about 25pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~about 20pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~about 15pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~about 14pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~approximately 13pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~about 12pMHC / 100nm 2, or about 0.4 pMHC / 100 nm 2 ~approximately 11.6pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~about 11.5pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~about 11pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~approximately 10pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~about 9pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~approximately 8pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~about 7pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~approximately 6pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~about 5pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~about 4pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~about 3pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~about 2.5pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~about 2pMHC / 100nm 2 , or about 0.4 pMHC / 100 nm 2 ~about 1.5pMHC / 100nm 2 is.

[0079] In another embodiment, the nanoparticles have a molecular weight of about 0.22 pMHC / 100 nm 2 ~approximately 10pMHC / 100nm 2 , or about 0.22 pMHC / 100 nm 2 ~about 9pMHC / 100nm 2 , or about 0.22 pMHC / 100 nm 2~approximately 8pMHC / 100nm 2 , or about 0.22 pMHC / 100 nm 2 ~about 7pMHC / 100nm 2 , or about 0.22 pMHC / 100 nm 2 ~approximately 6pMHC / 100nm 2 , or about 0.22 pMHC / 100 nm 2 ~about 5pMHC / 100nm 2 , or about 0.22 pMHC / 100 nm 2 ~about 4pMHC / 100nm 2 , or about 0.22 pMHC / 100 nm 2 ~about 3pMHC / 100nm 2 , or about 0.22 pMHC / 100 nm 2 ~about 2pMHC / 100nm 2 , or about 0.22 pMHC / 100 nm 2 ~about 1.5pMHC / 100nm 2 In some embodiments, the nanoparticles may have a pMHC density of about 0.22 pMHC / 100 nm 2 ~approximately 10pMHC / 100nm 2 , or 0.24pMHC / 100nm 2 ~about 9pMHC / 100nm 2 , or about 0.26 pMHC / 100 nm 2 ~approximately 8pMHC / 100nm 2 , or about 0.28 pMHC / 100 nm 2 ~about 7pMHC / 100nm 2 , or about 0.24 pMHC / 100 nm 2 ~about 4pMHC / 100nm 2 , or about 0.5 pMHC / 100 nm 2 ~about 3pMHC / 100nm 2 , or about 0.6 pMHC / 100 nm 2 ~about 1.5pMHC / 100nm 2 In a further embodiment, the nanoparticles have a pMHC density of about 0.4 pMHC / 100 nm 2 ~about 1.3pMHC / 100nm 2 , or alternatively about 0.5 pMHC / 100 nm 2 ~about 0.9pMHC / 100nm 2, or alternatively about 0.6 pMHC / 100 nm 2 ~approx. 0.8pMHC / 100nm 2 has a pMHC density of

[0080] Linker In certain aspects, the ubiquitous autoantigen-MHC can be coupled to the nanoparticle core by one or more of a covalent bond, a non-covalent bond, or cross-linking, and optionally via a linker. In aspects involving one or more linkers, the linkers on a single nanoparticle core can be the same or different from one another. In some embodiments, the ubiquitous autoantigen-MHC comprises at least one ubiquitous autoantigen-MHC described herein and a nanoparticle, provided that the nanoparticle is non-liposomal and has a metal or metal oxide core, and the at least one ubiquitous autoantigen-MHC is covalently linked to the nanoparticle via a linker comprising polyethylene glycol having a molecular weight of less than 5 kilodaltons (kD). In some embodiments, the polyethylene glycol has a molecular weight of less than 1 kD, 2 kD, 3 kD, 4 kD, 5 kD, 6 kD, 7 kD, 8 kD, 9 kD, or 10 kD. In some embodiments, the polyethylene glycol is functionalized with maleimide. In some embodiments, the polyethylene glycol has a molecular weight of about 1 kD to about 5 kD, about 2 kD to about 5 kD, or about 3 kD to about 5 kD. In some embodiments, the polyethylene glycol is functionalized with maleimide. In certain embodiments, the end of the linker that contacts the solid core is embedded in the solid core. In further aspects, the linker can be less than 5 kD in size and is optionally polyethylene glycol. The linker can be any of the linkers listed in Table 1.

[0081] [Table 1]

[0082] To couple substrates or particles of ubiquitous autoantigen-MHC to nanoparticles, the following techniques can be applied.

[0083] Binding can be achieved by chemical modification of the substrate or particle, which typically involves the creation of "functional groups" on the surface, said functional groups being capable of binding to MHC complexes, and / or by linking so-called "linking molecules", either covalently or non-covalently, to the optionally chemically modified surface of the surface or particle, followed by reaction of the MHC or MHC complex with the resulting particle.

[0084] The functional group or the linking molecule carrying it may be selected from an amino group, a carbonate group, a thiol, a thioether, a disulfide, a guanidino, a hydroxyl group, an amine group, a vicinal diol, an aldehyde, an alpha haloacetyl group, an organyl mercury, an ester group, an acid halide, an acid thioester, an acid anhydride, an isocyanate, an isothiocyanate, a sulfonic acid halide, an imidoester, a diazoacetate, a diazonium salt, a 1,2-diketone, a phosphoric acid, a phosphate ester, a sulfonic acid, an azolide, an imidazole, an indole, an N-maleimide, an alpha beta unsaturated carbonyl compound, an aryl halide, or a derivative thereof.

[0085] Non-limiting examples of other higher molecular weight linking molecules are nucleic acid molecules, polymers, copolymers, polymeric coupling agents, silica, proteins, and chain molecules with surfaces of opposite polarity to the substrate or particle. Nucleic acids can provide linkage to affinity molecules that themselves contain nucleic acid molecules but have a complementary sequence to the linking molecule.

[0086] In some embodiments, the linking molecule comprises polyethylene glycol. In some embodiments, the linking molecule comprises polyethylene glycol and maleimide. In some embodiments, the polyethylene glycol is a C1-C3 alkoxy group, -R 10 NHC(O)R-, -R 10 C(O)NHR-, -R 10 OC(O)R-, -R 10 C(O)OR-, where each R is independently H or C1-C6 alkyl, and each R 10is independently a bond or C1-C6 alkyl.

[0087] pMHC can be coupled to nanoparticles by various methods, including, but not limited to, conjugation to NPs generated using PEG linkers bearing a terminal -NH or -COOH group, which can be achieved by amide bond formation in the presence of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC). First, NPs bearing a -COOH group are dissolved in 20 mM MES buffer, pH 5.5. N-hydroxysulfosuccinimide sodium salt (Sulfa-NHS, Thermo Scientific, Waltham, MA, final concentration 10 mM) and EDC (Thermo Scientific, Waltham, MA, final concentration 1 mM) are added to the NP solution. After stirring at room temperature for 20 minutes, the NP solution is added dropwise to a solution containing pMHC monomers dissolved in 20 mM borate buffer (pH 8.2). The mixture is stirred for an additional 4 hours. To conjugate MHC to NH2-functionalized NPs, pMHC was first dissolved in 20 mM MES buffer (pH 5.5) containing 100 mM NaCl. Sulfur-NHS (10 mM) and EDC (5 mM) were then added to the MHC solution. The activated MHC molecules were then added to the NP solution in 20 mM borate buffer (pH 8.2) and stirred at room temperature for 4 hours.

[0088] To conjugate MHC to maleimide-functionalized NPs, pMHC was first incubated with tributylphosphine (TBP, 1 mM) for 4 hours at room temperature. Then, pMHC engineered to encode a free carboxy-terminal Cys residue was mixed with NPs in 40 mM phosphate buffer, pH 6.0, containing 2 mM EDTA and 150 mM NaCl, and incubated overnight at room temperature. The MHC of pMHC was covalently attached to the NPs via carbon-sulfur bond formation between the maleimide group and the Cys residue.

[0089] Click chemistry can be used to conjugate ncMHC or avidin to NPs functionalized with azide groups. In this reaction, the MHC or avidin molecule is first incubated with a suitable reagent containing a dibenzocyclooctyl (DBCO) functional group, such as DBCO-NHS (Click Chemistry Tools, Scottdale, AZ) reagent, at room temperature for 2 hours. Molecules containing free DBCO can be removed by overnight dialysis. The MHC-DBCO or avidin-DBCO conjugate is then incubated with SFP-Z for 2 hours, resulting in the formation of a triazole bond between the ncMHC or avidin molecule and the NP.

[0090] Unconjugated pMHC in various MHC-NP conjugation reactions can be removed by large-scale dialysis using methods known in the art. A non-limiting example is dialysis against PBS, pH 7.4, at 4°C through a 300 kD molecular weight cutoff membrane (Spectrum Labs). Alternatively, pMHC-conjugated IONPs can be purified by magnetic separation. Conjugated NPs can be concentrated by ultrafiltration through an Amicon Ultra-15 unit (100 kD MWCO) and stored in PBS.

[0091] The particle surface can be chemically modified, for example, by the attachment of phosphonic acid derivatives bearing functional reactive groups. One example of such a phosphonic acid or phosphonic acid ester derivative is imino-bis(methylenephosphono)carbonic acid, which can be synthesized according to the Mannich-Medlitzer reaction. This attachment reaction can be carried out on substrates or particles directly obtained from the preparation process or after pretreatment (e.g., with trimethylsilyl bromide). In the first case, the phosphoric acid (ester) derivative may, for example, displace components of the reaction medium that remain attached to the surface. This displacement can be enhanced at higher temperatures. On the other hand, trimethylsilyl bromide is thought to dealkylate alkyl-containing phosphorus-based complexing agents, thereby creating new attachment sites for the phosphonic acid (ester) derivative. The phosphonic acid (ester) derivative or the linking molecule attached to it can exhibit the same functional groups as those described above. A further example of surface treatment of substrates or particles involves heating in a diol, such as ethylene glycol. It should be noted that this treatment may be redundant if the synthesis has already been carried out in a diol. Under these circumstances, the directly obtained synthesis is likely to exhibit the required functional groups. However, this treatment is applicable to substrates or particles formed in N- or P-containing complexing agents. When such substrates or particles are subjected to post-treatment with ethylene glycol, components of the reaction medium (e.g., complexing agents) that remain surface-bound can be displaced and / or dealkylated by diols.

[0092] The N-containing complexing agent that remains attached to the particle surface can also be replaced by a primary amine derivative having a second functional group. The surface of the substrate or particle can also be coated with silica. Because silica reacts easily with organic linkers such as triethoxysilane and chlorosilane, silica allows for relatively simple chemical conjugation of organic molecules. The particle surface can also be coated with a homopolymer or copolymer. Examples of polymerizable coupling agents are N-(3-aminopropyl)-3-mercaptobenzamidine, 3-(trimethoxysilyl)propylhydrazide, and 3-trimethoxysilylpropylmaleimide. Other non-limiting examples of polymerizable coupling agents are listed herein. These coupling agents can be used alone or in combination, depending on the type of copolymer to be produced as the coating.

[0093] Another surface modification technique that can be used with substrates or particles containing oxide-based transition metal compounds is the conversion of the oxide-based transition metal compounds to the corresponding oxychlorides using chlorine gas or organic chlorinating agents. These oxychlorides are capable of reacting with nucleophiles, such as hydroxyl and amino groups, often found on biomolecules. This technique allows for direct conjugation with proteins, for example, via the amino groups of lysine side chains. Conjugation with proteins after surface modification with oxychlorides can also be achieved using bifunctional linkers such as maleimidopropionic acid hydrazide.

[0094] In non-covalent linking techniques, chain-type molecules with polarity opposite to that of the substrate or particle surface are particularly suitable. Examples of linking molecules that can be non-covalently linked to core / shell nanoparticles involve anionic, cationic, or zwitterionic surfactants, acidic or basic proteins, polyamines, polyamides, polysulfones, or polycarboxylic acids. The required linkage can be generated by hydrophobic interactions between the substrate or particle and an amphiphilic reagent with functional reactive groups. Particularly useful are chain-type molecules with amphiphilic properties that can be crosslinked with each other, such as phospholipids or derivatized polysaccharides. Absorption of these molecules onto the surface can be achieved by co-incubation. Binding of affinity molecules to substrates or particles can also be based on non-covalent self-assembly. One example involves a simple detection probe with biotin as the linking molecule and an avidin- or streptavidin-coupled molecule.

[0095] Protocols for coupling reactions of functional groups to biological molecules can be found, for example, in the book "Bioconjugate Techniques" (Greg T. Hermanson, Academic Press 1996). Biological molecules (e.g., MHC molecules or derivatives thereof) can be covalently or non-covalently coupled to linking molecules according to standard organic chemistry procedures such as oxidation, halogenation, alkylation, acylation, addition, substitution, amidation, etc. These methods for coupling covalent or non-covalent linking molecules can be applied before or after coupling of the linking molecules to substrates or particles. Furthermore, direct binding of molecules to correspondingly pretreated substrates or particles (e.g., with trimethylsilyl bromide), which, based on this pretreatment, exhibit a modified surface (e.g., a more charged or polar surface), can be carried out by incubation.

[0096] Nanoparticle synthesis Nanoparticles may be formed by contacting an aqueous phase containing pMHC complexes and polymers with a non-aqueous phase, followed by evaporation of the non-aqueous phase to cause coalescence of particles from the aqueous phase, as taught in U.S. Pat. Nos. 4,589,330 or 4,818,542. Certain polymers for such preparations are natural or synthetic copolymers or polymers including gelatin agar, starch, arabinogalactan, albumin, collagen, polyglycolic acid, polylactic acid, glycolide-L(-)lactide, poly(epsilon caprolactone), poly(epsilon caprolactone-co-lactic acid), poly(epsilon caprolactone-co-glycolic acid), poly(β-hydroxybutyric acid), poly(ethylene oxide), polyethylene, poly(alkyl-2-cyanoacrylate), poly(hydroxyethyl methacrylate), polyamide, poly(amino acids), poly(2-hydroxyethyl DL-aspartamide), poly(ester urea), poly(L-phenylalanine / ethylene glycol / 1,6-diisocyanatohexane), and poly(methyl methacrylate). Specifically, certain polymers are polyesters such as polyglycolic acid, polylactic acid, glycolide-L(-)lactide poly(epsilon caprolactone), poly(epsilon caprolactone-co-lactic acid), poly(epsilon caprolactone-co-glycolic acid), etc. Solvents useful for dissolving the polymer include water, hexafluoroisopropanol, methylene chloride, tetrahydrofuran, hexane, benzene, or hexafluoroacetone sesquihydrate.

[0097] The uaMHC described herein can be coupled to the nanoparticles via the aforementioned layer, or, if the layer is not present, a linker molecule. In certain embodiments, such a linker molecule comprises, consists essentially of, or consists of polyethylene glycol (PEG), dextran, or mannitol. In certain embodiments, such a linker molecule comprises, consists essentially of, or consists of polyethylene glycol (PEG). In certain embodiments, such a linker molecule comprises, consists essentially of, or consists of dextran. Such layers and linkers can be functionalized or derivatized with groups capable of forming covalent bonds with the uaMHC. The reaction can be any suitable reaction, including, but not limited to, amine-to-amine, sulfhydryl-to-sulfhydryl, amine-to-sulfhydryl, carboxyl-to-amine, or sulfhydryl-to-carboxyl. In certain embodiments, the uaMHC is coupled to the linker or layer by reaction of an NHS ester with a primary amine (e.g., a lysine residue) on the uaMHC. In certain embodiments, the uaMHC is coupled to a linker or layer by reaction of an imidoester with a primary amine (e.g., a lysine residue) on the uaMHC. In certain embodiments, the uaMHC is coupled to a linker or layer by reaction of an amide group with a sulfhydryl group (e.g., a cysteine ​​residue) on the uaMHC. In certain embodiments, the uaMHC is coupled to a linker or layer by reaction of a maleimide group with a sulfhydryl group (e.g., a cysteine ​​residue) on the uaMHC. In certain embodiments, the uaMHC is coupled to a linker or layer by reaction of a maleimide group with a primary amine group on the uaMHC. In certain embodiments, the uaMHC is coupled to a linker or layer by reaction of a haloacetyl group with a sulfhydryl group on the uaMHC. In certain embodiments, the uaMHC is coupled to a linker or layer by reaction of a haloacetyl group with a primary amine group on the uaMHC.In certain embodiments, the uaMHC is coupled to a linker or layer by reaction of a pyridyldithiol with a sulfhydryl group on the uaMHC. In certain embodiments, the uaMHC is coupled to a linker or layer by reaction of a pyridyldithiol with a primary amine group on the uaMHC. In certain embodiments, the uaMHC is coupled to a linker or layer by reaction of a carbodiimide with a primary amine group on the uaMHC. In certain embodiments, the uaMHC is coupled to a linker or layer by a heterobifunctional linker. In certain embodiments, the uaMHC is coupled to a linker or layer by reaction of a maleimide / hydrazide with a sulfhydryl group on the uaMHC. In certain embodiments, the uaMHC is coupled to a linker or layer by reaction of a pyridyldithiol / hydrazide with a sulfhydryl group on the uaMHC. In certain embodiments, the crosslinker is a photoreactive crosslinker.

[0098] Gold nanoparticles (GNPs) were synthesized using the chemical reduction of gold chloride with sodium citrate, as described in Perrault, SD et al. (2009) Nano Lett 9:1909-1915. Briefly, 2 mL of 1% HAuCl4 (Sigma-Aldrich) was added to 100 mL HO under vigorous stirring, and the solution was heated in an oil bath. 6 mL (for 14 nm GNPs) or 2 mL (for 40 nm GNPs) of 1% Na citrate was added to the boiling HAuCl4 solution, which was stirred for an additional 10 min and then cooled to room temperature. GNPs were stabilized by the addition of 1 μM of a thiol-PEG linker (Nanocs, MA) functionalized with -COOH or -NH2 groups as MHC acceptors. The PEGylated GNPs were washed with water to remove free thiol-PEG, concentrated, and stored in water for further analysis. NP density is determined spectrophotometrically and calculated according to Beer's law.

[0099] SFP series iron oxide NPs (SFP IONPs) can be produced by thermal decomposition of iron acetate in an organic solvent in the presence of a surfactant, followed by PEGylation to render them soluble in aqueous buffers (Xie, J. et al. (2007) Adv Mater 19:3163; Xie, J. et al. (2006) Pure Appl. Chem. 78:1003-1014; Xu, C. et al. (2007) Polymer International 56:821-826). Briefly, 2 mMol Fe(acac)3 (Sigma Aldrich, Oakville, ON) is dissolved in 10 mL of a mixture of benzyl ether and oleylamine and heated to 100 °C for 1 hour under reflux under the protection of a nitrogen blanket, followed by 300 °C for 2 hours. The synthesized NPs are precipitated by the addition of ethanol and resuspended in hexane. For IONP PEGylation, 100 mg of various 3.5 kD DPA-PEG linkers (Jenkem Tech USA) were dissolved in a mixture of CHCl3 and HCON(CH3)2 (dimethylformamide (DMF)). The NP solution (20 mg Fe) was then added to the DPA-PEG solution and stirred at room temperature for 4 hours. The PEGylated SFP NPs were precipitated by the addition of hexane and then resuspended in water overnight. Traces of aggregates were removed by high-speed centrifugation (20,000 × g, 30 min), and the monodisperse SFP NPs were stored in water for further characterization and pMHC conjugation. The iron concentration in the IONP product was determined spectrophotometrically in 2 N HCl at A410. Based on the molecular structure and diameter of SFP NPs (Fe3O4, 8 + 1 nm diameter) (Xie, J. et al. (2007) Adv Mater 19:3163, Xie, J. et al. (2006) Pure Appl. Chem. 78:1003-1014), an SFP solution containing 1 mg of iron can produce 5 × 10 14 It is estimated to contain NPs.

[0100] Nanoparticles can also be made by thermal decomposition or heating of nanoparticle precursors. In one embodiment, the nanoparticles are metal nanoparticles or metal oxide nanoparticles. In one embodiment, the nanoparticles are iron oxide nanoparticles. In one embodiment, the nanoparticles are gold nanoparticles. In one embodiment, provided herein are nanoparticles prepared in accordance with the present technology. In one embodiment, provided herein are methods for making iron oxide nanoparticles comprising the thermal decomposition reaction of iron acetylacetonate. In one embodiment, the resulting iron oxide nanoparticles are water-soluble. In one aspect, the iron oxide nanoparticles are suitable for protein conjugation. In one embodiment, the method comprises a single-step thermal decomposition reaction.

[0101] In one embodiment, the pyrolysis is carried out in the presence of functionalized PEG molecules. Certain non-limiting examples of functionalized PEG linkers are shown in Table 1.

[0102] In one aspect, the pyrolysis comprises heating iron acetylacetonate. In one embodiment, the pyrolysis comprises heating iron acetylacetonate in the presence of functionalized PEG molecules. In one embodiment, the pyrolysis comprises heating iron acetylacetonate in the presence of benzyl ether and functionalized PEG molecules. Without being bound by theory, in one embodiment, functionalized PEG molecules are used as the reducing reagent and surfactant. The methods of making nanoparticles provided herein simplify and improve upon conventional methods that use surfactants that are difficult or cannot be completely substituted for PEG molecules to impart water solubility to the particles. Traditionally, surfactants can be expensive (e.g., phospholipids) or toxic (e.g., oleic acid or oleylamine). In another aspect, without being bound by theory, the methods of making nanoparticles achieve high molecular purity and water solubility by avoiding the need for traditional surfactants.

[0103] In one embodiment, the thermal decomposition involves iron acetylacetonate and benzyl ether and is in the absence of conventional surfactants other than those utilized herein.

[0104] In one embodiment, the pyrolysis temperature is from about 80° C. to about 300° C., or from 80° C. to about 200° C., or from about 80° C. to about 150° C., or from about 100° C. to about 250° C., or from about 100° C. to about 200° C., or from about 150° C. to about 250° C., or from about 150° C. to about 250° C. In one embodiment, the pyrolysis is carried out for about 1 to about 2 hours.

[0105] In one embodiment, the method for producing iron oxide nanoparticles includes a purification step using, for example, a Miltenyi Biotec LS magnetic column.

[0106] In one embodiment, the nanoparticles are stable in phosphate buffered saline (PBS) at about 4° C. without any detectable degradation or aggregation. In one embodiment, the nanoparticles are stable for at least six months.

[0107] In one aspect, provided herein is a method for preparing a nanoparticle complex, comprising contacting pMHC with the iron oxide nanoparticles provided herein. Without being bound by theory, the pMHC encodes a cysteine ​​at its carboxy terminus, which can react with a maleimide group in the functionalized PEG at about pH 6.2 to about pH 6.5 for about 12 to about 14 hours.

[0108] In one embodiment, the method for producing the nanoparticle conjugates includes a purification step using, for example, a Miltenyi Biotec LS magnetic column.

[0109] Regulatory immune cell types The uaMHC-NP complexes of the present disclosure reprogram or differentiate autoreactive T cells into T regulatory cells or TR1 cells. In certain embodiments, TR1 cells express IL-10. In certain embodiments, TR1 cells secrete IL-10. In certain embodiments, TR1 cells express CD49b. In certain embodiments, TR1 cells express LAG-3. T cells with these phenotypic characteristics are useful for treating inflammatory or autoimmune conditions in individuals. In certain embodiments, the uaMHC-NP complexes are useful in methods of reprogramming or differentiating autoreactive T cells into TR1 cells in individuals after administration. The methods generate antigen-specific TR1 cells.

[0110] The ubiquitous autoantigen-MHC of the present disclosure is useful for generating B regulatory cells. In certain embodiments, the ubiquitous autoantigen-MHC of the present disclosure is utilized in a method for generating B cells that express high levels of CD1d, CD5, and / or secrete IL-10. B-regs are also identified by expression of Tim-1. In certain embodiments, the uaMHC-NP complex is useful in a method for inducing B regulatory cells in an individual after administration. This method generates antigen-specific B regulatory cells.

[0111] Pharmaceutical Compositions and Administration Provided herein are pharmaceutical compositions of ubiquitous autoantigen-MHC-NPs useful for the treatment and prevention of disease, which compositions comprise, alternatively consist essentially of, or even consist of the nanoparticle conjugates described herein and a carrier.

[0112] Compositions of the present disclosure can be conventionally administered parenterally, such as by intravenous, subcutaneous, or intramuscular injection. Additional formulations suitable for other modes of administration include oral formulations. Oral formulations contain commonly employed excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders and contain about 10% to about 95%, preferably about 25% to about 70%, of the active ingredient. The preparation of aqueous compositions containing antigen-MHC-nanoparticle complexes that modify the immune status of a subject will be known to those of skill in the art in light of the present disclosure. In one embodiment, the ubiquitous autoantigen-MHC-nanoparticle complexes are administered systemically. In a specific embodiment, a composition comprising a ubiquitous autoantigen-MHC-NP complex or multiple ubiquitous autoantigen-MHC-N complexes can be administered intravenously.

[0113] Typically, the ubiquitous autoantigen-MHC-NPs described herein are administered in a therapeutically effective and immunomodulating amount compatible with the dosage formulation. The dosage will depend on the subject being treated. The precise amount of active ingredient required for administration will depend on the judgment of the practitioner. However, preferred dosage ranges are on the order of tens to hundreds of nanograms or micrograms of antigen / MHC / nanoparticle complex per administration. Suitable regimes for initial administration and booster administration vary, but typically involve an initial administration followed by subsequent administrations.

[0114] The mode of application can be varied. Any conventional method of administering a vaccine is applicable. These may include oral administration in a solid physiologically acceptable carrier or a physiologically acceptable dispersion, parenteral administration by injection, etc. The dosage of the antigen / MHC / nanoparticle complex will depend on the route of administration and will vary depending on the size and health of the subject. The ubiquitous autoantigen-MHC-NPs can be administered by any suitable route, including intravenously, subcutaneously, intradermally, intramuscularly, rectally, or intraperitoneally. In certain embodiments, the autoantigen-MHC-NPs are administered parenterally. In certain embodiments, the autoantigen-MHC-NPs are administered intravenously. In certain embodiments, the autoantigen-MHC-NPs are administered subcutaneously.

[0115] In many cases, it will be desirable to administer multiple doses of ubiquitous autoantigen-MHC-NP, at about, at least about, or at most about 3, 4, 5, 6, 7, 8, 9, 10, or more doses. Administration will typically range from 1, 2, 3, 4, 5, 6, or 7 days to 12 weeks apart, more commonly at 1-2 week intervals. To maintain immune system status, periodic boosters may be desirable, such as every other day, twice a week, weekly, once a week, once every two weeks, or monthly, or for 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, or 5 years, typically 2 years. Following this course of administration, the autoreactive immune response, cognate T R Assays for T cell activity can be performed.

[0116] In certain embodiments, a single dose of ubiquitous autoantigen-MHC-NP, not including a nanoparticle core or any bioabsorbable / biocompatible outer layer, comprises from about 0.001 mg / kg to about 2.0 mg / kg, or from about 0.001 mg / kg to about 1.5 mg / kg, or from about 0.001 mg / kg to about 1.4 mg / kg, or from about 0.001 mg / kg to about 1.3 mg / kg, or from about 0.001 mg / kg to about 1.2 mg / kg, or from about 0.001 mg / kg to about 1.1 mg / kg, or from about 0.001 mg / kg to about 1.0 mg / kg. In some embodiments, a single dose comprises from about 0.004 mg / kg to about 1.014 mg / kg, or from about 0.02 mg / kg to about 0.811 mg / kg, or from about 0.041 mg / kg to about 0.608 mg / kg, or from about 0.061 mg / kg to about 0.507 mg / kg, or from about 0.081 mg / kg to about 0.405 mg / kg, or from about 0.121 mg / kg to about 0.324 mg / kg, or from about 0.162 mg / kg to about 0.243 mg / kg. In some embodiments, a single dose comprises from about 0.004 mg / kg to about 1.015 mg / kg, or from about 0.004 mg / kg to about 1.0 mg / kg, or from about 0.004 mg / kg to about 0.9 mg / kg, or from about 0.004 mg / kg to about 0.8 mg / kg, or from about 0.004 mg / kg to about 0.7 mg / kg, or from about 0.004 mg / kg to about 0.6 mg / kg, or from about 0.004 mg / kg to about 0.5 mg / kg, or from about 0.004 mg / kg to about 0.4 mg / kg, or from about 0.004 mg / kg to about 0.3 mg / kg, or from about 0.004 mg / kg to about 0.2 mg / kg, or from about 0.004 mg / kg to about 0.1 mg / kg. As used herein, mg / kg refers to milligrams of ubiquitous autoantigen-MHC or ubiquitous autoantigen without regard to MHC components administered per kg of subject body weight.

[0117] hepatitis disease The ubiquitous autoantigen-MHC of the present disclosure is useful for treating hepatic inflammatory disorders. Hepatic inflammatory disorders include diseases or disorders resulting from liver inflammation and may involve autoantibodies, inflammatory cell infiltrates (including T cells, natural killer T cells, macrophages, and / or monocytes). Hepatic inflammatory disorders may also involve activation of liver-resident macrophages (Kupffer cells). In certain embodiments, the ubiquitous autoantigen-MHC of the present disclosure is useful in methods for treating or ameliorating hepatic inflammatory disorders selected from the group consisting of hepatitis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), cirrhosis, and pyogenic liver abscess. In certain embodiments, the ubiquitous autoantigen-MHC of the present disclosure is useful in methods for treating pyogenic liver abscess. In certain embodiments, the ubiquitous autoantigen-MHC of the present disclosure is useful in methods for treating or ameliorating nonalcoholic steatohepatitis (NASH). In certain embodiments, the ubiquitous autoantigen-MHC of the present disclosure is useful in methods of treating or ameliorating non-alcoholic fatty liver disease (NAFLD). In certain embodiments, the ubiquitous autoantigen-MHC of the present disclosure is useful in methods of treating or ameliorating cirrhosis.

[0118] Pharmaceutically acceptable stabilizers, excipients, and diluents In some embodiments, the uaMHC-NPs are formulated as pharmaceutical compositions. Pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active agent into a preparation for pharmaceutically use. The appropriate formulation depends on the chosen route of administration. Compendia for pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed. (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), which are incorporated herein by reference for such disclosures.

[0119] The pharmaceutical composition may also include a surfactant, dispersant, and / or viscosity adjusting agent. These agents include materials that can control the diffusion and uniformity of a drug through a liquid medium or a granulation or blending method. In some embodiments, these agents also enhance the effectiveness of a coating or erosion matrix. Exemplary diffusion enhancers / dispersing agents include, for example, hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG, tyloxapol, polyvinylpyrrolidone (PVP, commercially known as Plasdone®), and carbohydrate-based dispersing agents, such as hydroxypropylcellulose (e.g., HPC, HPC-SL, and HPC-L), hydroxypropylmethylcellulose (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M), sodium carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate stearate (HPMCAS), amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), vinylpyrrolidone / vinyl acetate copolymer (S630), 4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamer (e.g., Pluronic F68®, F88®, and F108® (which are block copolymers of ethylene oxide and propylene oxide, and Poloxamer 188), and poloxamines (e.g., Tetronic 908® (also known as Poloxamine 908®, which is a tetrafunctional block copolymer derived from the sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Corporation, Parsippany, NJ)))), polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone / vinyl acetate copolymer (S-630), polyethylene glycol (e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 4000 to about 5400), sodium carboxymethylcellulose, methylcellulose, polysorbate-80, sodium alginate, gums, such as Examples of suitable surfactants include ragweed gum and acacia gum, guar gum, xanthan (including xanthan gum), sugars, cellulosic materials such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, polysorbate 8O, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, carbomer, polyvinyl alcohol (PVA), alginate, chitosan, and any combination thereof. Plasticizers such as cellulose or triethylcellulose can also be used as dispersing agents. In some cases, the pharmaceutical composition contains 0.01% to 0.5% (w / v) of a surfactant. In some cases, the pharmaceutical composition contains 0.01%, 0.05%, 0.1%, 0.2%, 0.4%, or 0.5% (w / v) of a surfactant.

[0120] In certain embodiments, the uaMHC-NPs described herein are included in a pharmaceutical composition with a solubilizer, emulsifier, or dispersant. In certain embodiments, the solubilizer may enable highly concentrated solutions of uaMHC-NPs, at least about 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, or greater than 20 mg / mL. The carbomer in the aqueous pharmaceutical composition acts as an emulsifier and viscosity modifier. In certain embodiments, the pharmaceutically acceptable excipient comprises or consists of a carbomer. In certain embodiments, the carbomer comprises or consists of carbomer 910, carbomer 934, carbomer 934P, carbomer 940, carbomer 941, carbomer 1342, or a combination thereof. The cyclodextrin in the aqueous pharmaceutical composition acts as a solubilizer and stabilizer. In certain embodiments, the pharmaceutically acceptable excipient comprises or consists of a cyclodextrin. In certain embodiments, the cyclodextrin comprises or consists of alpha cyclodextrin, beta cyclodextrin, gamma cyclodextrin, or a combination thereof. The lecithin in the pharmaceutical composition may function as a solubilizer. In certain embodiments, the solubilizer comprises or consists of lecithin. The poloxamer in the pharmaceutical composition acts as an emulsifier, solubilizer, and dispersant. In certain embodiments, the pharmaceutically acceptable excipient comprises or consists of a poloxamer. In certain embodiments, the poloxamer comprises or consists of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, or a combination thereof. The polyoxyethylene sorbitan fatty acid ester in the pharmaceutical composition acts as an emulsifier, solubilizer, surfactant, and dispersant. In certain embodiments, the pharmaceutically acceptable excipient comprises or consists of a polyoxyethylene sorbitan fatty acid ester.In certain embodiments, the polyoxyethylene sorbitan fatty acid ester comprises or consists of polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, polysorbate 120, or a combination thereof. Polyoxyethylene stearate in the pharmaceutical composition acts as an emulsifier, solubilizer, surfactant, and dispersant. In certain embodiments, the pharmaceutically acceptable excipient comprises or consists of polyoxyethylene stearate. In certain embodiments, the polyoxyethylene stearate comprises or consists of polyoxyl 2 stearate, polyoxyl 4 stearate, polyoxyl 6 stearate, polyoxyl 8 stearate, polyoxyl 12 stearate, polyoxyl 20 stearate, polyoxyl 30 stearate, polyoxyl 40 stearate, polyoxyl 50 stearate, polyoxyl 100 stearate, polyoxyl 150 stearate, polyoxyl 4 distearate, polyoxyl 8 distearate, polyoxyl 12 distearate, polyoxyl 32 distearate, polyoxyl 150 distearate, or a combination thereof. The sorbitan ester in the pharmaceutical composition functions as an emulsifier, solubilizer, nonionic surfactant, and dispersant. In certain embodiments, the pharmaceutically acceptable excipient comprises or consists of a sorbitan ester. In certain embodiments, the sorbitan ester comprises or consists of sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan stearate, sorbitan trioleate, sorbitan sesquioleate, or a combination thereof. In certain embodiments, solubility can be achieved with a protein carrier. In certain embodiments, the protein carrier comprises recombinant human albumin.

[0121] In certain embodiments, the uaMHC-NP complexes of the present disclosure are included in a pharmaceutical composition comprising one or more pharmaceutically acceptable stabilizers, excipients, carriers, and diluents. In certain embodiments, the uaMHC-NP complexes of the present disclosure are administered suspended in a sterile solution. In certain embodiments, the solution comprises 0.9% NaCl. In certain embodiments, the solution further comprises one or more of a buffering agent such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris), a surfactant such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188, a polyol / disaccharide / polysaccharide such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40, an amino acid such as glycine or arginine, an antioxidant such as ascorbic acid, methionine, or a chelating agent such as EDTA or EGTA. In certain embodiments, the uaMHC-NP complexes of the present disclosure are lyophilized for transport / storage and reconstituted prior to administration. In certain embodiments, lyophilized uaMHC-NP complex formulations include bulking agents such as mannitol, sorbitol, sucrose, trehalose, and dextran 40. Lyophilized formulations may be contained in glass vials. When formulated, uaMHC-NP complexes, whether reconstituted or not, may be buffered at a specific pH, generally below 7.0. In certain embodiments, the pH may be 4.5-6.5, 4.5-6.0, 4.5-5.5, 4.5-5.0, or 5.0-6.0. In certain embodiments, uaMHC-NP complexes may be formulated for intravenous injection. In certain embodiments, uaMHC-NP complexes may be formulated for oral ingestion. In certain embodiments, uaMHC-NP complexes may be formulated for parenteral administration, intramuscular injection, subcutaneous injection, or other tissue injection. In certain embodiments, the uaMHC-NP complexes can be formulated and / or administered without any immunological adjuvants or other compounds or polypeptides intended to increase or decrease the immune response. [Example]

[0122] The following illustrative examples represent embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0123] Example 1 - Generation and expansion of TR1-like CD4+ T cells by PBC-associated pMHC class II-NP NOD.c3c4 mice, carrying the antidiabetogenic B6-derived chromosome 3 and 4 regions, spontaneously develop a form of autoimmune cholangiopathy similar to human PBC. See Irie, J., et al. J. Exp. Med. 203, 1209-1219. Like >90% of patients, these mice develop pathogenic T and B cell responses against the E2 and E3BP components of the pyruvate dehydrogenase (PDC) complex. See Kita, H. et al. J. Clin. Invest. 109, 1231-1240. In NOD.c3c4 mice and, in humans, this autoimmune response promotes destruction of biliary epithelial cells, leading to cholestasis, small bile duct proliferation, and ultimately liver failure.

[0124] To design PBC-associated pMHC class II-based nanomedicines, we investigated the effects of NOD / NOD.c3c4 mouse MHC class II molecules (IA) in silico. g7 We searched for 15-mer peptides of murine PDC-E2 that could bind to two such epitopes (PDC-E2 166-181 and PDC-E2 82-96 ) encoding IA g7 The base pMHC was chosen for the experiment. T1D-associated IA was used as a negative control. g7The conjugated BDC2.5 mimotope was used. These conjugates were produced in lentivirally transduced Chinese hamster ovary (CHO) cells, purified by sequential nickel and strep-tag affinity chromatography, and covalently coated via the free carboxy-terminal cysteine ​​onto iron oxide nanoparticles generated by thermal decomposition of iron(III) acetylacetonate (Fe(acac)3) in the presence of maleimide-functionalized polyethylene glycol, as described by Singha, S. et al., Nature Nanotechnology 12, 701-710.

[0125] In pMHC tetramer staining studies, as shown in Figure 1A (inverted triangles, middle and right panels), NOD.c3c4 (but not NOD) mice expressed PDC-E2 at gradually increasing levels with age. 166-181 / IA g7 and PDC-E2 82-96 / IA g7 In contrast, as shown in Figure 1A (triangles, middle and right panels), unlike NOD mice, NOD.c3c4 mice express negligible levels of the T1D-associated BDC2.5mi / IA. g7 These results suggest that the progression of hepatic autoimmunity in NOD.c3c4 mice is accompanied by an increase in the size and / or circulating activity of PDC-E2-specific CD4+ T cells, an outcome consistent with the propensity of these two strains for PBC versus T1D.

[0126] To confirm whether PBC-associated pMHC class II-NPs can trigger the formation and expansion of PDC-E2-specific TR1-like CD4+ T cells in NOD.c3c4 mice, we first investigated the effect of PDC-E2 166-181 / IA g7 We treated 15-week-old NOD.c3c4 mice (when disease was well established) with pMHC-expressing NPs or control NPs (Cys-NPs) intravenously twice weekly for up to 13.5 weeks. As shown in Figure 1B (squares, left and middle panels), treatment significantly reduced PDC-E2 expression compared with mice treated with bare NPs or untreated NOD mice.166-181 / IA g7 This triggered a rapid increase in the frequency of circulating tetramer+ CD4+ T cells (within 2.5 weeks). As shown in Figure 1C, in mice tested at the end of follow-up, the PDC-E2 expression of cognate CD4+ T cells was significantly increased. 166-181 / IA g7 The NP-induced spread was systemic, with increased frequency observed in the spleen, bone marrow, liver, and liver-regional lymph nodes (portal lymph nodes and celiac lymph nodes), but not in non-regional lymph nodes (as opposed to mesenteric lymph nodes (MLN)).

[0127] In contrast, T1D-associated BDC2.5 / IA g7 Treatment of NOD.c3c4 mice with pMHC-coated NPs did not trigger the formation or expansion of TR1 cells (Figure 1B, right panel and Figure 1D). This result is consistent with the nanomedicine acting exclusively on autoantigen-experienced T cells, and since NOD.c3c4 mice do not develop islet inflammation, antigen-activated BDC2.5mi / IA is not a predictor of T cell proliferation. g7 They are not expected to harbor autoreactive CD4+ T cells.

[0128] In experiments with additional cohorts of mice, PDC-E2 expression of cognate CD4+ T cells was observed. 166-181 / IA g7 -NP-induced expansion is PDC-E2 82-96 / IA g7 The peptide-specific expression of PDC-E2 in these mice was demonstrated without any detectable expansion of reactive CD4+ T cells (Fig. 1E, top row). 166-181 / IA g7 The expansion of specific CD4+ T cells was significantly enhanced by PDC-E2 in all organs examined compared to levels detected in age-matched untreated mice. 82-96 / IA g7 This was accompanied by a significant reduction in the frequency of reactive CD4+ T cells (Fig. 1E, top row). 166-181 / IA g7 Specific CD4+ T cell subsets somehow express their PDC-E2 in response to endogenous autoantigen exposure. 82-96 / IAg7 This suggests that the proliferation of the reactive counterpart was inhibited.

[0129] As expected, PDC-E2 expanded in these mice, as shown in Figure 1F and Figures 2A and 2B. 166-181 / IA g7 Tetramer+ CD4+ T cells expressed the TR1 cell markers LAG-3, CD49b, and LAP. Furthermore, unlike their tetramer-negative counterparts, splenic tetramer+ CD4+ T cells from these mice expressed PDC-E2 166-181 In response to peptide-pulsed bone marrow-derived DCs (but not BDC2.5), the TR1 cytokine IL-10 was produced, but not IFNγ, IL-2, IL-4, IL-9, or IL-17 (Figure 1G). 82-96 / IA g7 Similar results were obtained in mice treated with NPs exhibiting the cognate PDC-E2 gene, as shown in Figures 1B (center) and 1E (bottom) and Figures 2A and 2B. 82-96 / IA g7 Significant expansion of reactive TR1-like CD4+ T cells and PDC-E2 166-181 / IA g7 A significant reduction in the frequency of reactive CD4+ T cells was observed, suggesting that this outcome is not unique to any particular epitope on PDC-E2.

[0130] In summary, as described above for T1D-associated, EAE-associated, and CIA-associated pMHC class II-NP in the corresponding disease models, the PDC-E2 peptide / IA was administered in NOD.c3c4 mice. g7 These data demonstrate that -NP efficiently triggers the formation and expansion of cognate TR1-like CD4+ T cells.

[0131] Example 2 - Reversal of established PBC by disease-associated pMHC class II-NP Compared to age-matched NOD mice, 6- to 8-week-old NOD.c3c4 mice begin to exhibit elevated serum alanine aminotransferase (ALT) levels, microscopic biliary epithelial proliferation, biliary leukocyte infiltration, extensive biliary involvement (near-maximal portal triad involvement), and macroscopic enlargement of the common bile duct (CBD) (Figures 3A and 3C). Figure 3B shows an exemplary scoring matrix for quantifying microscopic analysis. By approximately 15-16 weeks of age, these signs worsen, and mice begin to exhibit increased total serum bilirubin (TB) levels (Figure 3A), high titers of anti-mitochondrial / PDC-E2-specific autoantibodies (absent in NOD mice, Figure 3E), and macroscopic signs of liver disease (gallbladder) (Figure 3D). All of these disease signs peaked in severity at approximately 24 weeks of age (Figures 3A-3D), coinciding with extensive infiltration of the biliary epithelium by CD4+ and CD8+ T cells (Figure 3F), high titers of antinuclear autoantibodies (ANA) (Figure 3E), and an almost three-fold increase in liver weight (Figure 3D).

[0132] We investigated the role of PDC-E2 in 15-week-old NOD.c3c4 mice (the age at which liver autoimmunity is well established in these mice). 166-181 / IA g7 -NP, PDC-E2 82-96 / IA g7 -NP and BDC2.5 / IA g7 The therapeutic potential of pMHC-NP was tested. Mice received a 20 μg dose of pMHC-NP or an equivalent dose of control (Cys-conjugated NP, Cys-NP) once every two weeks for 9 to 13.5 weeks. 166-181 / IA g7 Treatment with PDC-E2 NP resulted in significant reductions in serum ALT and TB levels (Figure 4A), biliary involvement, biliary epithelial proliferation, and leukocyte infiltration (Figure 4B), common bile duct diameter and macroscopic score (Figure 4C), liver weight and macroscopic liver score (Figure 4D), and abdominal circumference (Figure 4E). The autoantibody titers found at the start of therapy were not reduced by treatment, but were significantly higher in PDC-E2 NP than in control NP. 166-181 / IA g7 -NP, PDC-E2 82-96 / IA g7In mice treated with pMHC-NP, the progression of autoantibody formation was clearly attenuated, as evidenced by the significant reduction in titers of anti-PDC-E2 and antinuclear autoantibodies (Figure 4F). 82-96 / IA g7 -NP) and T1D-related (but PBC-unrelated) counterparts (BDC2.5 / IA g7 Additional testing with ribosomal inhibitors (RIs) confirmed the disease specificity of these compounds (Figures 4C and 4D).

[0133] Similar results were obtained when treatment was initiated at the peak of disease severity (24 weeks of age). 166-181 / IA g7 Analysis of mice (38-44 weeks old) 14-20 weeks after therapy with -NPs revealed a systemic expansion of TR1-like CD4+ T cells (Figure 4G and Figure 2C), and the magnitude of disease signs (Figures 4H and 4I) was significantly less than that seen at the age when therapy was initiated, suggesting that resolution of liver inflammation by PBC-specific nanomedicines promotes repair of existing liver damage.

[0134] Example 3 – Continuous vs. Intermittent Treatment pMHC-NP therapy triggers the formation and expansion of cognate TR1 cells systemically, leading to their accumulation in most lymphoid organs and even at sites of inflammation (see Figures 1-3). Furthermore, because these TR1 cells circulate in the blood, their presence in the bloodstream can be used as a biomarker to determine the need for retreatment. To investigate this and to determine whether the level of circulating cognate TR1-like CD4+ T cells can actually be used to guide treatment decisions, we used PDC-E2 166-181 / IA g7 In NOD.c3c4 mice treated with α-NP, we withdrew from treatment from 15 to 24 weeks of age. We then measured the PDC-E2 levels in peripheral blood. 166-181 / IA g7The percentage of tetramer+ CD4+ T cells was measured every two weeks. Animals whose percentage of tetramer+ cells fell to approximately 50% of their baseline values ​​were re-treated. When the tetramer+ values ​​recovered at the next scheduled measurement, the animals were again withdrawn from treatment. This cycle was repeated until the mice reached 50 weeks of age. Although considerable variability was observed among mice (Figure 5A), in most animals, the percentage of tetramer+ TR1 cells in peripheral blood gradually declined to approximately 50% of their baseline values ​​within 4–6 weeks of treatment withdrawal. Retreatment of these animals rapidly restored these values ​​(Figure 5B). Intermittent treatment did not impair the pharmacodynamic effects of treatment (systemic expansion of TR1-like CD4+ T cells) (Figures 5C and 5D), nor the therapeutic efficacy, including reductions in common bile duct diameter / macroscopic score and liver weight / macroscopic score (Figure 5E), compared with untreated mice or mice continuously treated from 24 weeks of age to 38–44 weeks of age.

[0135] Example 4 – pMHC-NP vs. standard of care Ursodeoxycholic acid (UDCA, a hydrophilic bile acid) is the standard of care for PBC. See Charatcharoenwitthaya, P. et al. Long-term survival and impact of ursodeoxycholic acid treatment for recurrent primary biliary cirrhosis after liver transplantation. Liver Transpl. 13, 1236–1245. UDCA has anticholestatic properties and stimulates hepatic bile secretion, thus protecting cholangiocytes from the toxic effects of hydrophobic bile acids. When given early in the disease process, it is effective in approximately 50% of patients but is ineffective in advanced stages of PBC.

[0136] Oral administration of UDCA to 6-week-old NOD.c3c4 mice via a UDCA-supplemented diet for 9 consecutive weeks had a therapeutic effect on PBC progression, as evidenced by reduced liver scores and liver weights, but not ALT, CBD scores, or CBD diameter, compared with untreated mice (Figure 6A), and reduced biliary involvement and bile duct proliferation, but not leukocyte infiltration, compared with untreated mice (Figure 6B). However, when UDCA was given at an advanced stage of disease (24 weeks of age), no such therapeutic effect was observed except for a highly significant reduction in CBD diameter compared with untreated animals, likely due to its anticholestastic effect (Figures 6D and 6E).

[0137] In contrast, the PDC-E2 166-181 / IA g7 -NP treatment had a highly significant therapeutic effect in both 6- and 24-week-old animals, as evidenced by a significant reduction in the severity of all measured measures (Figures 6A-6E). As expected, this was associated with a systemic expansion of cognate TR1-like CD4+ T cells (Figure 6F).

[0138] Example 5 - IL-10, TGFb, and CD4+ T cells are required for disease suppression TR1-like pMHC-NP-expanded pDC-E2 166-181 / IA g7 To confirm whether CD4+ T cell-mediated disease reversal is mediated by the TR1 cytokines IL-10 and / or TGFb, we investigated the effects of PDC-E2 on 15-week-old NOD.c3c4 mice treated for 5 weeks with blocking anti-IL10 mAb, anti-TGFb mAb, or rat IgG. 166-181 / IA g7 The immunological and therapeutic effects of PDC-E2 were compared. 166-181 / IA g7 PDC-E2 did not significantly inhibit the expansion of specific TR1-like CD4+ T cells (Figure 7A), but suppressed their therapeutic effect compared to age-matched NOD.c3c4 mice treated with rat IgG (Figures 7B and 7C). 166-181 / IA g7Purified splenic CD4+ T cells from -NP-treated NOD.c3c4 mice were able to transfer disease suppression to NOD.scid.c3c4 hosts reconstituted with splenocytes from diseased NOD.c3c4 mice, as shown in Figures 7D-7F. 166-181 / IA g7 Treatment of the host with -NP enhanced this effect.

[0139] Example 6 - Therapy-induced suppression of pro-inflammatory properties of local and nearby APCs PDC-E2 166-181 / IA g7 To confirm whether the reversal of PBC by -NP is associated with specific suppression of disease-amplifying APCs, we investigated PDC-E2 166-181 / IA g7 We compared the cytokine and chemokine profiles of portal (draining) versus mesenteric (non-draining) lymph node CD11b cells and hepatic Kupffer cells isolated from PDC-E2- and control NP-treated animals. LPS-challenged CD11b cells from portal lymph nodes of control NP-treated animals secreted a wide range of pro-inflammatory cytokines and chemokines at significantly higher levels than their mesenteric lymph node counterparts (Figure 7G). Conversely, PDC-E2 166-181 / IA g7 Portal lymph node CD11b cells from PDC-E2-NP-treated mice secreted these pro-inflammatory mediators at significantly lower levels than both their mesenteric lymph node counterparts and CD11b cells isolated from control NP-treated animals (Figure 7G). 166-181 / IA g7 Kupffer cells isolated from the livers of -NP-treated mice secreted significantly lower levels of some of these mediators (Figure 7H). Thus, the systemic expansion of PDC-E2-specific TR1 CD4+ cells in NOD.c3c4 mice by treatment with PBC-associated pMHC class II-NPs is associated with a dramatic inhibition of the pro-inflammatory properties of local and nearby APC types, likely due to increased uptake of liver-derived PDC-E2 autoantigenic material.

[0140] Example 7 – PBC-related nanomedicines promote local formation of regulatory B cells Pancreatic beta cell-specific TR1 CD4 T cells promote B cell recruitment to the pancreas and its draining lymph nodes, as well as the local formation of antidiabetogenic IL-10-producing Breg cells. To confirm whether this is also the case for PDC-E2-specific TR1 CD4 T cells in the context of PBC, we investigated the effects of PDC-E2 on the expression of B cells in the pancreas and its draining lymph nodes. 166-181 / IA g7 B cells and pDC-E2 in the liver, portal vein, and mesenteric lymph nodes of -NP-treated mice 166-181 / IA g7 We investigated whether there was a statistically significant correlation between the absolute numbers of specific TR1 cells. 166-181 / IA g7 The liver and portal vein, but not the mesenteric lymph nodes, of -NP-treated mice contained significantly more PDC-E2 than those from control NP-treated animals. 166-181 / IA g7 Furthermore, the numbers of tetramer+ cells and B cells in both the liver and portal lymph nodes were statistically correlated, and such correlation was consistent with PDC-E2. 166-181 / IA g7 However, it was not observed in the mesenteric lymph nodes of PDC-E2-NP-treated mice (Figure 7J). 166-181 / IA g7 Enhanced recruitment of cognate TR1 cells to the liver and liver-draining lymph nodes of -NP-treated mice promotes local accumulation of B cells.

[0141] PDC-E2 166-181 / IA g7 To confirm whether B cells in the liver and portal lymph nodes of PDC-E2-NP-treated mice could be enriched for Breg cells, we compared the ability of corresponding B cells to produce IL-10 in response to LPS stimulation. 166-181 / IA g7 B cells in the liver and portal vein, but not in the mesenteric lymph nodes, of -NP-treated mice produced significant levels of IL-10, whereas neither B cells in the liver nor B cells in the portal lymph nodes of control NP-treated animals produced IL-10 (Figure 7K). Thus, TR1 CD4+ T cell-enhanced recruitment of B cells to the liver and draining lymph nodes is associated with the local formation of IL-10-producing B cells.

[0142] To further demonstrate the direct relationship between TR1 cell recruitment and Breg cell formation, we investigated the effect of PDC-E2 166-181 / IA g7 PDC-E2 accumulates in the spleen, liver, and portal (but not mesenteric) lymph nodes of treated mice 166-181 / IA g7 Specific TR1 cells were cloned into PDC-E2 cells derived from NOD.Il10-eGFP reporter mice. 166-181 We confirmed the ability of PDC-E2-specific TR1 cells to promote the differentiation of peptide-pulsed conventional (IL-10 / eGFP-) B cells into CD1dhigh / CD5+ / eGFP+ progeny. As shown in Figures 7K-7M, we observed clear Breg cell formation in the spleen, liver, and portal lymph nodes (containing cognate TR1 cells), but not in the mesenteric lymph nodes (lacking cognate TR1 cells). Collectively, these results suggest that PDC-E2-specific TR1 cells promote the recruitment and differentiation of conventional B cells into Breg-like cells.

[0143] Example 8 – Humanized mice with PBC DRB4*0101 and DRB1*0801 have been associated with PBC in several studies. To confirm the HLA haplotype diversity in PBC, we performed high-resolution HLA-DRB1 typing of 154 Spanish PBC patients. 40.3% of patients expressed DRB1*0701, 25% expressed DRB1*0301+, and 14% expressed DRB1*0801+. Because haplotypes carrying DRB1*0701+ and other DRB1 alleles carry the oligomeric HLA-DRB4 locus, we also typed these patients for DRB4*0101. 61.7% of all PBC patients carried the DRB4*0101 allele.

[0144] Several T cell epitopes derived from PDC-E2 that bind to two of these HLA-DRB types (DRB4*0101 and DRB1*0801) are 249-262 (GDLLAEIETDKATI, DRB4*0101 binder), PDC-E2 122-135(GDLIAEVETDKATV, also DRB4*0101 binder), PDC-E2 249-263 (GDLLAEIETDKATIG, DRB1*0801), and PDC-E2 629-643 (AQWLAEFRKYLEKPI, DRB1*0801). Therefore, we 122-135 / DRB4*0101 complex, PDC-E2 249-262 / DRB4*0101 complex, and PDC-E2 629-643 We expressed and purified the / DRB1*0801 complexes and generated iron oxide nanoparticles representing each of these complexes described.

[0145] To examine the translational significance of these observations, we analyzed NOD.scid / Il2rg IgG reconstituted with PBMCs from 11 DRB4*0101+ and 5 DRB1*0801+ PBC patients. - / - We tested the ability of these three human PBMC-associated pMHC class II-NPs to expand cognate TR1-like CD4+ T cells in NSG (NSG) hosts (PBL-NSG mice, Tables 2, 3, and 4). PBMC-infused NSG hosts were then treated 8–10 times with 20 μg intravenous pMHC-NPs (twice weekly for 5 weeks). One mouse did not engraft, and the other three died of GvHD before the end of treatment. As a control, we infused a second mouse per donor and treated it with control (non-pMHC-coated NPs). Cognate CD4+ T cell expansion was analyzed in the spleen, liver, portal vein / peritoneal cavity, and axillary lymph nodes. We found that PDC-E2 mice significantly increased cognate CD4+ T cells compared with untreated controls. 122-135 All 6 of 6 PBL-NSG mice treated with / DRB4*0101-NP, PDC-E2 249-262 Five out of six PBL-NSG mice treated with / DRB4*0101-NP, and PDC-E2 629-643We observed an expansion of tetramer+CD49b+LAG-3+CD4+ T cells in the spleen and / or liver and LNs in 4 of 5 PBL-NSG mice treated with / DRB1*0801-NP (Tables 2, 3, and 4). Treatment-responsive mice had significantly higher percentages and absolute numbers of tetramer+ cells in the spleen, liver, and lymph nodes than control NP-treated or non-responsive mice (Figures 9A and 9B), and these cells expressed the TR1 markers CD49b and LAG-3 (Figure 9C).

[0146] [Table 2]

[0147] [Table 3]

[0148] [Table 4]

[0149] Example 9 – Disease specificity versus organ specificity Given the large autoantigenic burden in organs such as the liver compared to smaller organs such as the endocrine pancreas, and the fact that PDC-E2 is an autoantigen expressed in virtually all cell types, PBC-related nanomedicines (i.e., PDC-E2 166-181 / IA g7 Our results raise the question of whether IFN-NPs are disease (PBC) specific, or organ (liver) specific, or can reduce distal liver inflammation.

[0150] Primary sclerosing cholangitis (PSC) is a chronic cholestatic disease characterized by intrahepatic and extrahepatic bile duct inflammation, resulting in fibrous obstructive cholangitis with periductal fibrosis around medium and large bile ducts and degenerative changes in the bile duct epithelium, progressing to portal cirrhosis, biliary cirrhosis, and ultimately liver cirrhosis. Human PSC is often associated with inflammatory bowel disease and is frequently associated with atypical perinuclear antineutrophil cytoplasmic autoantibodies (pANCA), but not antimitochondrial autoantibodies. See Fickert, P. et al., Characterization of animal models for primary sclerosing cholangitis (PSC). J. Hepatol. 60, 1290-1303. Abcb4 gene knockout mice spontaneously develop sclerosing cholangitis, which is caused by cholangiocyte damage due to impaired biliary phospholipid secretion, in a morphology strikingly similar to human PSC. See Pollheimer, MJ & Fickert, P. Animal models in primary biliary cirrhosis and primary sclerosing cholangitis. Clin. Rev. Allergy Immunol. 48, 207-217, doi:10.1007 / s12016-014-8442-y (2015).

[0151] Autoimmune hepatitis (AIH) is characterized by portal vein mononuclear cell infiltration of the liver parenchyma, associated with the presence of antinuclear and / or antismooth muscle autoantibodies (type 1 AIH) or anti-liver-renal microsomal type 1 or anti-liver cytosolic type 1 autoantibodies (type 2 AIH), which specifically target microsomal cytochrome P450IID6 (CYP2D6) or formiminotransferase cyclodeaminase (FTCD), respectively. See Longhi, MS et al., Aetiopathogenesis of autoimmune hepatitis. J. Autoimmun. 34, 7-14. Recently, infection of NOD mice with a replication-deficient adenovirus encoding the human liver autoantigen formiminotransferase cyclodeaminase (Ad-FTCD) has been shown to trigger a form of chronic autoimmune hepatitis similar to type 2 AIH. See Hardtke-Wolenski, M. et al. Genetic predisposition and environmental danger signals initiate chronic autoimmune hepatitis driven by CD4+ T cells. Hepatology 58, 718-728.

[0152] Damage to large bile ducts and liver parenchyma underlying PSC and AIH is due to the release of PDC-E2 and PDC-E2 166-181 / IA g7 -NP therapy may trigger the priming of cognate autoreactive CD4+ T cells capable of responding to therapy, in which case the therapy may involve the TR1-like PDC-E2 166-181 / IA g7 The expansion of specific CD4+ T cells and the recognition of local and proximal PDC-E2-loaded APCs should trigger the suppression of local inflammation. Alternatively, the amount of PDC-E2 shed into the inflammatory environment of PSC and / or AIH may be related to the PDC-E2 166-181 / IA g7 Empirical CD4+ T cell development and therefore PDC-E2 166-181 / IA g7 -The immunological and therapeutic response to NP may be inadequate.

[0153] To test this possibility, we used NOD.Abcb4 - / - PDC-E2 triggers expansion of cognate TR1-like CD4+ T cells and reverses PSCs in mice 166-181 / IA g7 The ability of PDC-E2-NP to inhibit the growth of erythrocytes was first investigated. Notably, as shown in Figures 10A-10B and 11A and 11B, 166-181 / IA g7 -NP triggered a systemic expansion of cognate TR1-like CD4+ T cells and reversed established disease in these animals compared with control NP-treated controls.

[0154] Next, we investigated whether this also applies to Ad-FTCD-induced AIH. We found that PDC-E2 expression was significantly elevated in Ad-FTCD-infected NOD mice. 166-181 / IA g7 -mFTCD along with NP (PBC related) 58-72 / IA g7 -NP and CYPD 398-412 / IA g7 We compared the pharmacodynamic and therapeutic activities of the three compounds, Ad-FTCD-NP (AIH-associated). All three compounds triggered the formation and expansion of cognate TR1-like CD4+ T cells to a similar extent in these animals compared with untreated Ad-FTCD-infected animals (Figure 10C and Figures 12A and 12B), which was accompanied by a significant reduction in liver inflammation, as shown in Figures 10C and 10D, and serum ALT levels, as shown in Figure 10E.

[0155] This ability of ubiquitous autoantigen-based pMHC nanomedicines to alleviate liver autoimmunity in an organ-specific, rather than disease-specific, manner is also relevant for CYPD 398-412 / IA g7 This was also present in NOD.c3c4 mice treated with NP (Figures 12A-12C). In fact, the latter was associated with a significant increase in the expansion of cognate TR1 cells compared with PDC-E2. 166-181 / IA g7The beta cell-specific BDC2.5 / IA-NP was as efficient as β-cell-specific BDC2.5 / IA-NP (Figure 12A) and attenuated PBC in 15-week-old mice (Figure 12B). In contrast, in 10-week-old prediabetic NOD mice, the beta cell-specific BDC2.5 / IA-NP g7 -NP, IGRP 4-22 / IA g7 -NP or IGRP 128-145 / IA g7 Unlike the -NP, the PDC-E2 166-181 / IA g7 -NP and CYPD 398-412 / IA g7 -NP also failed to trigger the expansion of cognate TR1 CD4+ T cells (Figure 12C), likely because the content of PDC-E2 and CYPD in beta cells is insufficient to prime the activation of cognate CD4+ T cells.

[0156] Collectively, these observations suggest that upon hepatocellular injury (AIH) or biliary epithelial cell injury (PBC and PSC), abundant levels of PDC-E2 (mitochondrial), CYPD2D6, and FTCD antigens (Golgi-resident or cytoplasmic, respectively) are delivered to local and nearby APCs, enabling priming of autoreactive CD4+ T cells, generation of cognate TR1 cells by pMHC-NPs, and suppression of local and nearby autoantigen-loaded APCs.

[0157] Example 10 – Therapeutic efficacy in other PBC models The NOD.c3c4 model does not adequately recapitulate the immunopathology of human PBC, which is characterized by a female prevalence, progression to liver fibrosis, and the absence of liver cyst formation. B6 mice carrying a deletion of the IFNγ 3' untranslated region adenylate uridylate-rich element (ARE) (ARE-Del+ / -) have a disregulated Ifng locus and develop a form of PBC resembling the human disease, primarily affecting women and accompanied by upregulation of TBA, production of anti-PDC-E2 autoantibodies, portal vascular and hepatic lobule inflammation, bile duct injury, granuloma formation, and fibrosis. PDC-E2 166-181 / IA g7Figures 13A-C show that treatment of (NODxB6.ARE-Del- / -)F1 mice with -NPs suppressed the upregulation of TBA and ALT levels, hepatic inflammation, and fibrosis compared with mice treated with control NPs.

[0158] Example 11 - Selection methods utilized herein mouse NOD / LtJ, BALB / c, C57BL / 6, NOD.scid.Il2rg (NSG), NOD.c3c4, and FVB / N.Abcb4 (Abcb4 or ATP-binding cassette transporter, subfamily B, member 4) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). IFNγARE-Del B6 mice were obtained from H. Young (NIH, Bethesda, MD). NOD.c3c4.scid mice were generated by backcrossing F1 (NOD.c3c4 × NOD.scid) mice with NOD.c3c4 mice for five generations, followed by interbreeding mice heterozygous for the scid mutation and homozygous for the B6 chromosome 3 and 4 interval derived from NOD.c3c4 mice. NOD.Abcb4 mice were derived by backcrossing the mutant Abcb4 allele from FVB / N-Abcb4 mice onto a NOD / Ltj background for six generations, followed by interbreeding. (NODxB6.IFNγARE-Del)F1 mice were generated by intercrossing IFNγARE-Del mice with NOD / LtJ mice. NOD.Il10tm1Flv (Tiger) mice were derived by backcrossing the Il10tm1Flv allele from C57BL / 6.Il10tm1Flv mice (Jackson Lab) onto a NOD / Ltj background for 10 generations. RIP-DTR.NOD transgenic mice were generated by backcrossing an X-linked rat insulin promoter-driven human diphtheria toxin receptor (RIP-DTR) transgene from transgenic B6 mice into the NOD background for over 10 generations.

[0159] Cell lines, pathogens, and tumors CHO-S, BSC-1, MDCK, 293T, B16 / F10, and CT26 cell lines were purchased from ATCC (Manassas, VA). Listeria monocytogenes was obtained from DMX Corporation (Philadelphia, PA).

[0160] Antibodies and flow cytometry FITC-, PE-, APC-, PerCP-, or biotin-conjugated mAbs against mouse CD4 (RM4-5), CD5 (53-7.3), CD19 (1D3), B220 (RA36B2), and CD49b (HMa2) and streptavidin-PerCP were purchased from BD Biosciences (San Diego, CA). Anti-murine LAG-3 mAb (C9B7W) was purchased from eBioscience (San Diego, CA). Anti-latency-associated TGF-β (LAP) antibody (TW7-16B4) was from BioLegend (San Diego, CA). PE-conjugated pMHC class II tetramers were generated using biotinylated pMHC monomers. pMHC class II tetramer staining and phenotypic marker analysis were performed essentially as described with minor modifications. Briefly, after avidin incubation (15 min at room temperature), blood leukocytes and single cell suspensions derived from spleen, lymph nodes, liver mononuclear cells, and bone marrow cells were first incubated with pMHC tetramers (5 μg ml ) in FACS buffer (0.05% sodium azide and 1% FBS in PBS) at 37°C. -1 ) for 60 min, followed by FITC-conjugated anti-mouse CD4 (5 μg ml) at 4°C. -1 ) and PerCP-conjugated anti-mouse B220 (2 μg ml -1The cells were stained for 30 min in a 100% PBS-based FACS channel (as a "dump" channel). After washing, the cells were fixed (1% paraformaldehyde in PBS) and analyzed on a FACScan, FACSaria, or BD LSRII flow cytometer. For phenotypic analysis, cells were incubated with anti-FcR Abs and then stained with cell surface marker antibodies diluted 1:100 in FACS buffer (4°C for anti-CD49b and anti-LAP Abs, and 37°C for anti-LAG-3 Abs), followed by pMHC tetramers, FITC-conjugated anti-mouse CD4 (5 μg ml -1 ), and PerCP-conjugated anti-mouse B220. Once stained, cells were washed, fixed, and analyzed by flow cytometry. All analyses were performed using FlowJo software.

[0161] The following mAbs were used to analyze NSG-transferred human T cells: FITC-conjugated anti-CD4 (OKT4, BioLegend), APC-conjugated anti-CD19 (HIB19, BD Biosciences, San Jose, CA), PerCP-conjugated polyclonal goat anti-LAG-3 IgG (R&D Systems, Minneapolis, MN), biotin-conjugated anti-CD49b (AK7, Pierce Antibodies, Thermo Fisher Scientific, Waltham, MA), and eFluor 450-conjugated streptavidin (eBioscience). Briefly, avidin (0.25 mg ml in FACS buffer) was used. -1 Splenocytes and pancreatic lymph node cells were incubated with tetramer (5 μg ml ) at room temperature for 30 min, washed, and incubated at 37°C with tetramer (5 μg ml -1) for 1 h, washed, and incubated with FITC-conjugated anti-CD4 (2 / 100), APC-conjugated anti-CD19 (5 / 100, used as a "dump" channel), PerCP-conjugated anti-LAG-3 (8 / 100), and biotin-conjugated anti-CD49b (4 / 100) for 45 min at 4 °C. After washing, cells were incubated with eFluor 450-conjugated streptavidin for 30 min at 4 °C, washed, fixed with 1% PFA in PBS, and analyzed by hCD4 + / hCD19 - Cells within the gate were analyzed using a FACSCanto II (BD Bioscience).

[0162] pMHC monomers and peptides Recombinant pMHC class II monomers were purified from the supernatant of CHO-S cells transduced with lentiviruses encoding monocistronic messages in which the peptide-MHCb and MHCc chains of the complex were separated by a ribosomal skipping P2A sequence. The peptide was tethered to the amino terminus of the MHCb chain via a flexible GS linker, and the MHCc chain was engineered to encode a BirA site, a 6xHis tag, a twin Strep tag, and a free Cys at its carboxy terminus. Secreted self-assembled pMHC class II complexes were purified by sequential nickel and Strep-Tactin® chromatography and used for coating onto NPs or processed for biotinylation and tetramer formation as described above. Epitopes encoded by murine monomer constructs were selected based on predicted MHCII binding capacity by RANKPEP (http: / / imed.med.ucm.es / cgi-bin / rankpep_mif.cgi) using a threshold score of 7.54. PDC-E2 166-181Although hPDC-E2 had a score below the threshold, it was selected for the experiment because it is contained within one of the lipoyl-binding domains of the antigenic target of AMA, PDC-E2. For CYPD and FTCD epitope prediction, we used a second online algorithm (GPS-MBA) (http: / / mba.biocuckoo.org / ), and selected peptides predicted by both RANKPEP and GPS-MBA for the experiment. hPDC-E2 122-135 , hPDC-E2 249-262 (both contained within the lipoyl-binding domain of PDC-E2), and hPDC-E2 629-643 The sequences of various epitopes were identified in PDC-E2. 166-181 / IA g7 (LAEIETDKATIGFEVQ), PDC-E2 82-96 / IA g7 (EKPQDIEAFKNYTLD), FTCD 58-72 / IA g7 (VVEGALHAARTASQL), CYPD 398-412 / IA g7 (LITNLSSALKDETVW), 2.5mi / IA g7 (AHHPIWARMDA), hPDC-E2 122-135 / DRB4*0101(GDLIAEVETDKATV), hPDC-E2 249-262 / DRB4*0101(GDLLAEIETDKATI), and hPDC-E2 629-643 / DRB1*0801(AQWLAEFRKYLEKPI). Synthetic PDC-E2 166-181 , 2.5mi, and mMOG 36-55 The (EVGWYRSPFSRVVHLYRNGK) peptide was purchased from Genscript (Piscataway, NJ). The amino acid residue numbers of each peptide correspond to those found in the mature form of the corresponding antigen.

[0163] Nanoparticles, pMHCII-NP synthesis, and purification pMHC was coated onto PEGylated iron oxide NPs (PFM-NPs) prepared as described in (2). Briefly, PFM-NPs were generated by thermal decomposition of Fe(acac)3 in the presence of 2 kD methoxy-PEG-maleimide. NPs were purified using a magnetic (MACS) column (Miltenyi Biotec, Auburn, CA). pMHC bearing a free cysteine ​​(control) or a free carboxy-terminal Cys was conjugated to maleimide-functionalized PFM overnight at room temperature in 40 mM phosphate buffer, pH 6.0, containing 2 mM EDTA and 150 mM NaCl. pMHC-conjugated NPs were separated from free pMHC using the magnetic column, sterilized by filtration through a 0.2 μm filter, and stored in water or PBS at 4°C. Quality control was performed using transmission electron microscopy, dynamic light scattering, and native and denaturing gel electrophoresis. pMHC content was measured using the Bradford assay (Thermo Fisher Scientific) and SDS-PAGE.

[0164] Ursodeoxycholic acid treatment Cohorts of 5-6- or 24-week-old male and / or female NOD.c3c4 mice were left untreated, fed a diet supplemented with 0.5% UDCA (BOC Sciences, Upton, NY; TestDiet, Richmond, IN), or treated with pMHCII-NP for 14 or 9 weeks, respectively, and then sacrificed for pMHCII tetramer staining, PBC scoring, and biochemical testing.

[0165] pMHCII-NP therapy in NOD.c3c4 mice, (NODxB6.IFNgARE-Del- / -)F1 mice, and NOD / Ltj mice Cohorts of 15-week-old male and / or female NOD.c3c4 mice with established PBC were either left untreated or treated with 20 mg of pMHCII-NP or Cys-NP (iv) twice weekly for 9 weeks unless otherwise indicated. Liver disease scoring involved macroscopic assessment of cyst content (0-5), liver weight, and CBD diameter (0-4), as well as microscopic assessment of biliary involvement (0-4), bile duct proliferation (0-4), and mononuclear cell infiltration (0-4), essentially as described (23). In other experiments, treatment was initiated at the peak of disease (24 weeks of age) and continued twice weekly for 14-20 weeks. Intermittent treatment involved treating mice twice weekly from 15 to 24 weeks of age, then withdrawing them from treatment until the percentage of tetramer+ cells dropped to approximately 50% of the level seen at the time of treatment withdrawal (peripheral blood measurements were taken every two weeks), re-treating the mice twice weekly until the percentage of tetramer+ cells reached the original value, and repeating this cycle until 50 weeks of age.

[0166] For in vivo cytokine-blocking experiments, mAbs (BioXcell, West Lebanon, NH) against HRPN (rIgG1), IL-10 (JES5-2A5), or TGF-β (1D11) were given i.p. twice weekly for 2 weeks at 500 mg, followed by 200 mg for an additional 7 weeks. Mice were randomized to receive cytokine-neutralizing mAb treatment (anti-IL-10 or anti-TGFβ) or HRPN rat IgG1.

[0167] (NODxB6.IFNgARE-Del - / -In experiments involving F1 mice, 10-week-old male and female mice were treated for 5–6 weeks. Liver histopathological scoring was performed as described. Briefly, severity scores were obtained by scoring portal inflammation, lobular inflammation, and granuloma formation on a scale of 0–4, and bile duct injury on a scale of 0–2. The extent of portal inflammation and bile duct injury was scored on a scale of 0–4 based on the ratio of affected to unaffected areas. The extent of lobular inflammation and granuloma formation was scored on a scale of 0–4 based on the number of lesions per specimen. The severity of fibrosis was scored on a scale of 0–6.

[0168] A study in NOD mice involved treating a cohort of 10-week-old prediabetic female NOD / Ltj mice with 20 mg of pMHCII-NP or Cys-NP iv twice weekly for 5 weeks.

[0169] pMHCII-NP therapy for AIH in NOD mice AIH was induced by infecting 5- to 6-week-old female NOD / Ltj mice with an adenovirus encoding human FTCD (Ad-hFTCD, 10 10 Plaque-forming units (PFU) iv). After 4 weeks, cohorts of mice with established AIH were treated with 20 mg of pMHCII-NP or Cys-NP iv twice weekly for 5–6 weeks. Histopathological scoring was performed using the Ishak scale as described above.

[0170] pMHCII-NP therapy in human PBMC-reconstituted NSG hosts PBMCs from HLA-DRB4*0101+ PBC patients (recruited under informed consent approved by the Hospital Clinic's Institutional Review Board) were depleted of CD8+ T cells using anti-CD8 mAb-coated magnetic beads (Miltenyi Biotech, Auburn, CA) and injected i.v. into 8- to 10-week-old NSG hosts (2 × 10 7Mice were either treated with 30–40 mg pMHC-NP twice weekly for 5 consecutive weeks starting on day 5 after PBMC infusion or left untreated. Therapy-induced expansion of cognate CD4+ T cells was measured in the liver, peripheral LN, spleen, and bone marrow (not shown). Mice were considered responders if the percentage of tetramer+ T cells in at least two different organs was higher than the mean ± 10 standard deviations observed in untreated hosts.

[0171] Cytokine secretion assay Spleen and portal / celiac lymph node (PCLN) cell suspensions from pMHCIINP-treated mice were enriched for CD19+ B cells (EasySep™ Mouse CD19 Positive Selection Kit, Stem Cell Technologies, Vancouver, BC) and CD8+ T cells (CD8 Magnetic Particles, BD Biosciences) to deplete CD4+ T cells. Cells were stained with pMHCII tetramer and sorted by flow cytometry. Sorted cells (2–3 × 10 4 ) is 2 μg ml -1 Peptide-pulsed bone marrow-derived DCs (2 × 10 4 ) After 48 hours, supernatants were collected for cytokine content measurement by Luminex®.

[0172] To confirm whether pMHCII-NP therapy promoted the recruitment / generation of IL10-secreting B cells, mesenteric LN, PCLN, and hepatocyte suspensions were enriched for B cells using a CD19 enrichment kit (Stem Cell Technologies). Cells (2–3 × 10 in 200 mL / well) were added. 5 ) were incubated in RPMI-1640 medium containing 10% FCS for 24 h with LPS (1 μg ml -1 The cells were stimulated in duplicate with IL-10 (Sigma). The levels of IL-10 in the supernatants were measured by Luminex®.

[0173] Isolation and in vitro stimulation of lymph node CD11b+ cells and hepatic Kupffer cells LN-derived CD11b cells were incubated with collagenase D (1.25 μg mL -1 ) and DNase I (0.1 μg mL -1 The purified cells (2-3 × 10 cells in 200 mL / well) were obtained by digestion in PBS at 37 °C for 15 min, washing, incubation with anti-FcR Ab, and purification using anti-CD11b mAb-coated magnetic beads (BD Biosciences). 5 ) is LPS (2 μg ml -1 ) for 3 days and supernatants were analyzed for cytokine content using the Luminex® multiplex cytokine assay.

[0174] To isolate Kupffer cells (KCs), livers from treated and untreated mice were minced and digested with 15 ml of 0.05% collagenase solution in HBSS for 20-30 min at 37°C. The resulting cell suspension was filtered through a nylon mesh (0.7 μm) and centrifuged at 50 × g for 3 min at 4°C to remove tissue debris and hepatocytes. Cells in the supernatant were pelleted by centrifugation at 300 × g for 5 min at 4°C. The cell pellet, consisting primarily of non-parenchymal liver immune cells, KCs, sinusoidal endothelial cells, and stellate cells, was resuspended in 33% Percoll® solution and centrifuged at 350 × g for 30 min to isolate mononuclear cells. The pellet was then resuspended in 10% FCS (5 × 10 6 cells ml -1 ) and resuspended in DMEM containing 1-3 x 10 7 Cells / well were seeded into 6-well plates and incubated at 37°C in a 5% CO2 atmosphere for 2-3 hours. Non-adherent cells were removed by gentle washing with PBS. The adherent fraction (enriched in KCs) was harvested by trypsin digestion (5 min, 0.25% trypsin). The resulting cell suspension contained 1-2 x 10 5 96-well plates at 200 mL / well and incubated for 3 days with LPS (2 mg ml -1) and supernatants were analyzed for cytokine content using the Luminex® multiplex cytokine assay.

[0175] Adoptive transfer of suppression Untreated mice or PDC-E2 166-181 / IA g7 Splenic CD4+ T cells (10 7 ) were adoptively transferred into 10-14 week-old sex-matched NOD.c3c4.scid hosts (iv). One day later, recipients were transfected with 4 × 10 blastocysts derived from sex-matched NOD.c3c4 donor mice with established PBCs (>35 weeks old). 7 One cohort of mice infused with CD4+ T cells from pMHCII-NP-treated donors was adoptively transferred with whole splenocytes. 166-181 / IA g7 The recipients were further treated with 12 doses of NP. Recipients were sacrificed 6 weeks later for tetramer staining and PBC scoring.

[0176] In vivo Breg induction assay NOD.Il10 tm1Flv Splenic B cells from (Tiger) mice were enriched using the EasySep Mouse B Cell Isolation Kit (Stem Cell Technologies) and isolated from 2.5 mi of BDC or PDC. 166-181 Peptide (10 μg ml -1 ) for 2 hours at 37°C. The peptide-pulsed B cells were washed twice with PBS, labeled with PKH26 (Sigma), and infused (3 × 10) into pMHC-NP-treated or untreated mice. 6 The host was sacrificed 7 days later, and the spleen, MLN, PCLN, and liver mononuclear cells were labeled with anti-B220-APC mAb and biotinylated anti-CD1d mAb or anti-CD5 mAb, followed by streptavidin-PerCP labeling. PKH26+ B cells were identified as eGFP+ / CD1d by flow cytometry. high and analyzed for the presence of eGFP+ / CD5+ cells.

[0177] Histology and immunohistochemistry Livers were fixed in 10% formalin for 2 days, embedded in paraffin, cut into 5-μm sections, and stained with H&E or picrosirius red. For immunohistochemistry, liver tissues were embedded in Tissue-Tek OCT, sectioned into 30-μm frozen sections, and stored on slides at -80°C. Slides were fixed in chilled acetone, washed in PBS, treated with a 1:10 dilution of 30% H2O2 in PBS, washed in PBS, blocked with 10% normal goat serum in PBS, washed again, and stained with anti-mouse CD4 (GK1.5) or CD8 (Lyt-2) antibodies (1.5 h, 4°C). After washing, slides were stained with biotinylated goat anti-rat secondary antibody (1:200 dilution) and incubated with horseradish peroxidase (HRP)-conjugated streptavidin, followed by 3,3-diaminobenzidine (DAB) substrate. Slides were counterstained with hematoxylin before mounting.

[0178] ALT and TBA assays Serum alanine aminotransferase (ALT) levels were determined using a Thermo Fisher Scientific kit according to the manufacturer's protocol. Briefly, serum samples were mixed with prewarmed (37°C) Infinity™ ALT (GPT) Liquid Stable Reagent at a 1:10 ratio, and OD readings were obtained over 3 min at 37°C, 340 nm wavelength, nanodroplet, and 1 min intervals. The slope was calculated by plotting absorbance versus time using linear regression and multiplying by a factor to obtain serum ALT levels (U / L), as described in the kit. Serum total bile acid (TBA) levels were analyzed using a TBA Enzymatic Cycling Assay Kit (Diazyme, Poway, CA) according to the manufacturer's protocol, with the corrections described.

[0179] Measurement of antinuclear and antimitochondrial autoantibodies The presence of antinuclear autoantibodies (ANA) in serum was confirmed using the NOVA Lite® HEp-2 Slides kit (Inova Diagnostics, San Diego, CA). ANA titers were measured according to a semiquantitative approach. Briefly, serum samples were serially diluted in PBS (at 1:160, 1:320, 1:640, 1:1280, and 1:2560), then applied to pre-fixed Hep-2 substrate slides, washed, stained with FITC-conjugated goat anti-mouse IgG in PBS containing 5% normal donkey serum (1:200 dilution), washed, mounted, and read under a fluorescent microscope.

[0180] Serum levels of anti-mitochondrial PDC-E2 antibodies were determined by ELISA. Briefly, ELISA plates were prepared by ELISA using PDC-E2 protein (5 μg ml -1 The wells were coated overnight at room temperature with 100 mL of 3% fat-free milk (SurModics Inc., Eden Prairie, MN). The plates were washed, blocked with 150 mL of 3% fat-free milk in PBS (pH 7.4), and incubated with serially diluted serum samples (100 mL, 1:250 dilutions prepared with reagent diluent) for 2 h at room temperature. The wells were washed, incubated with 100 mL of HRP-conjugated anti-mouse IgG (1:2000 in reagent diluent) for 2 h at room temperature, and then washed. Finally, the wells were incubated with 100 mL of DAB substrate for 20 min at room temperature in the dark. After stopping the enzyme reaction with 50 mL of 2N H2SO4, absorbance was measured at a wavelength of 450 nm using an ELISA plate reader. Positive antibody activity (PAA) levels were calculated by calculating the mean OD ± 2 SD (positive index) of the control NOD serum samples and dividing the OD value corresponding to the NOD.c3c4 serum sample by the positive index. In this case, a value >1.0 corresponds to PAA.

[0181] statistical analysis Unless otherwise specified, sample size values ​​listed in figure legends correspond to the total number of mice tested pooled from different experiments. Data were compared in GraphPad Prism 6 by Mann-Whitney U test, chi-square, log-rank (Mantel-Cox), Pearson correlation, two-way ANOVA, or multiple t-test analysis with Holm-Sidak correction. A p-value <0.05 was considered statistically significant. Only statistically significant p-values ​​are represented in the figures.

[0182] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be utilized in practicing the invention.

[0183] All publications, patent applications, issued patents, and other documents referenced herein are incorporated by reference herein to the same extent as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated by reference to be incorporated in its entirety. Definitions incorporated by reference and contained herein are excluded to the extent they contradict definitions in the present disclosure.

[0184] [Table 5]

[0185] [Table 6]

[0186] [Table 7]

[0187] [Table 8]

[0188]

Table 9

[0189]

Table 10

[0190]

Table 11

[0191]

Table 12

[0192]

Table 13

Claims

1. A composition for use in the treatment of non-alcoholic steatohepatitis (NASH), comprising a plurality of antigen-major histocompatibility complex (antigen-MHC) molecules coupled to a nanoparticle core having a diameter of 1 nanometer to 100 nanometers, the plurality of antigen-MHC molecules comprising a ubiquitous autoantigen associated with the binding groove of the MHC, the ubiquitous autoantigen being not a liver-specific antigen, and the ubiquitous autoantigen being PDC-E2 122-135.

2. The composition of claim 1 , wherein the MHC molecule is an MHC class II molecule.

3. The composition of claim 1 or 2, wherein the nanoparticle core is a metal or metal oxide.

4. The composition of claim 3 wherein the metal oxide is iron oxide.

5. The composition of any one of claims 1 to 4, wherein the diameter is from 5 nanometers to 50 nanometers.

6. 6. The composition of any one of claims 1 to 5, wherein the plurality of antigen-MHCs are coupled to the nanoparticle core at an antigen-MHC to nanoparticle core ratio of at least 10:

1.

7. The plurality of antigen-MHC nanoparticles have a core surface area of ​​100 nm ２ The composition of any one of claims 1 to 6, wherein the nanoparticles are coupled to the nanoparticle core at a density of 0.4 to 13 antigen-MHC per nanoparticle.

8. 8. The composition of any one of claims 1 to 7, wherein the antigen-MHC is covalently coupled to the nanoparticle core by a dextran linker and / or a polyethylene glycol (PEG) linker having a mass of less than 5 kilodaltons.

9. The composition of any one of claims 1 to 8, further comprising a pharmaceutically acceptable stabilizer, excipient, diluent, or combination thereof.

10. The composition of any one of claims 1 to 9, formulated for intravenous administration.

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