Peptide conjugated particles

Abstract

To provide therapeutic agents for eliminating the need to synthesize and couple large panels of peptides.SOLUTION: The present invention provides compositions comprising peptide-coupled biodegradable poly(lactide-co-glycolide) (PLG) particles. In particular, PLG particles are surface-functionalized to allow coupling of peptide molecules to the surface of the particles (e.g., for use in eliciting immunological tolerance).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Application Description This application claims the benefit of U.S. Provisional Patent Application No. 61 / 662,687, filed June 21, 2012, which is incorporated herein by reference in its entirety.

[0002] Government support This invention was made with government support under R01 EB013198 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. [Background technology]

[0003] Inflammatory diseases and disorders are conditions in which an abnormal or otherwise unregulated inflammatory response contributes to the pathogenesis or severity of the disease. Examples include autoimmune diseases such as type 1 diabetes and celiac disease.

[0004] Many of these diseases are characterized by the infiltration of mononuclear cells at sites of tissue damage or other insults. Examples of mononuclear cells that have been observed in these infiltrates include lymphocytes, particularly T lymphocytes, as well as cells of the mononuclear phagocyte system (MPS cells), such as monocytes, macrophages, dendritic cells, microglial cells, and other cells.

[0005] Many of the cells observed in mononuclear cell infiltrates are suspected to play a role in these aberrant inflammatory responses. For example, in diseases such as multiple sclerosis, CD4+ T cells have been shown to play a central role in pathological autoimmune responses. At earlier points in T cell activation, dendritic cells and other MPS cells may contribute to CD4+ T cell activation. MPS cells may also contribute to inflammation through phagocytosis, although it is unclear whether such cells are able to do so in the absence of CD4+ T cells, at least in some inflammatory diseases.

[0006] Peripheral blood monocytes can be classified into one of two groups based on the expression or absence of specific cell surface molecules. Specifically, human "resident" or "mature" monocytes are considered to have a CD14loCD16+ phenotype (their mouse counterparts are CX3CR1hiCCR2-Gr1-). Another group of cells, "inflammatory" or "immature" monocytes, are considered to have a CD14+CD16- phenotype (their mouse counterparts are CX3CR1loCCR2+Gr1+). (Geissmann F. et al. 2003 Immunity 19:71-82)

[0007] Importantly, although the latter are understood to be "inflammatory" in the sense that they are observed migrating from bone marrow-derived peripheral blood cells into inflamed tissues, these cells have not been shown to cause inflammation either directly or through the action of other cells. Furthermore, the various MPS cells that can form when these cells differentiate have also not been shown to cause inflammation.

[0008] Conventional clinical strategies for general long-term immunosuppression in disorders associated with undesired immune responses are based on the long-term administration of broad-acting immunosuppressants, such as signal transduction inhibitors (S1) such as cyclosporine A (CsA), FK506 (tacrolimus), and corticosteroids. These drugs, when used at high doses for extended periods, can have toxic side effects. Furthermore, even in patients who can tolerate these drugs, the need for lifelong immunosuppressant therapy carries a significant risk of serious side effects, including tumors, severe infections, nephrotoxicity, and metabolic disorders.

[0009] Methods for inducing antigen-specific tolerance, including cell-binding of antigens or peptides, have been developed. For example, in one method, peptide-induced cell-binding tolerance involved the collection, isolation, and treatment of peripheral blood cells with disease-specific autoantigens and ethylene carbodiimide (ECDI)-binding reagents under sterile conditions, followed by reinfusion into the donor / patient. This process is expensive, must be performed under closely monitored conditions by skilled artisans, and the number of facilities capable of performing this procedure is limited. Using red blood cells as the donor cell type expands the potential sources to include allogeneic donors, dramatically increasing the supply of source cells and potentially expanding the delivery of this therapeutic agent to any setting where transfusion is permitted. These approaches have significant limitations, such as the supply of source cells and the need for tissue type matching to minimize immune responses against donor cells. Furthermore, local processing of cells that bind autoantigens via EDCI presents significant quality control challenges. Furthermore, these approaches require at least some knowledge of the pathological antigen to which immune tolerance is sought.

[0010] Recently, peptide-conjugated particles have been described that eliminate the requirement for source cell supply and circumvent the tissue-type requirements of previous approaches. See International Publication No. WO 2010 / 085509, which is incorporated herein by reference in its entirety. However, these approaches still rely on antigen-specific immune tolerance.

[0011] Antigen-specific tolerance is generally not ideal because specific antigens / epitopes are generally unknown in human diseases. Furthermore, to enable antigen-specific approaches, it is necessary to determine which antigens each individual patient recognizes, since antigens may vary from subject to subject, or to conjugate potential peptide libraries to particles prior to administration. Both the synthesis and individual conjugation of these peptides are time-consuming and expensive. Therefore, there is a need for therapeutic agents that solve both of these problems, thereby eliminating the need for a tissue-matched cell source and, at the same time, the need to synthesize and conjugate large peptide panels. Summary of the Invention

[0012] In some embodiments, the present invention provides compositions (e.g., for inducing antigen-specific tolerance) comprising carrier particles (e.g., PLG particles) attached to antigenic peptides. In certain embodiments, the carrier particles are poly(lactide-co-glycolide) (PLG) particles.

[0013] In some embodiments, the present invention provides compositions comprising an antigen bound to carrier particles having a negative zeta potential. In some embodiments, the zeta potential of the particles is about -100 mV to about 0 mV. In some embodiments, the zeta potential of the particles is about -50 mV to about -40 mV. In some embodiments, the particles are copolymers having a molar ratio of about 80:20 to about 100:0. In some embodiments, the copolymer ratio may be, but is not limited to, polystyrene:poly(vinyl carboxylate) / 80:20, polystyrene:poly(vinyl carboxylate) / 90:10, poly(vinyl carboxylate):polystyrene / 80:20, poly(vinyl carboxylate):polystyrene / 90:10, polylactic acid:polyglycolic acid / 80:20, or polylactic acid:polyglycolic acid / 90:10. In still other embodiments, the particles are polystyrene particles, carboxylated polystyrene particles, or poly(lactic acid-co-glycolic acid) particles. In some embodiments, the particles are poly(lactic-co-glycolic acid) particles.

[0014] In some embodiments, the particles have a diameter of about 0.1 μm to about 10 μm. In some embodiments, the particles have a diameter of about 0.3 μm to about 5 μm. In some embodiments, the particles have a diameter of about 0.5 μm to about 3 μm. In some embodiments, the particles have a diameter of about 0.5 μm to about 1 μm. In some embodiments, the particles have a diameter of about 0.5 μm.

[0015] In further embodiments, the antigen comprises at least a portion of an autoimmune antigen, an antigen expressed on tissue to be transplanted into the subject, or an allergen. In some embodiments, the antigen is selected from the group consisting of myelin basic protein, acetylcholine receptor, endogenous antigen, myelin oligodendrocyte glycoprotein, pancreatic beta cell antigen, insulin, glutamic acid decarboxylase (GAD), type 11 collagen, human cartilage gp39, fp130-RAPS, proteolipid protein, fibrillarin, small nucleolar protein, thyroid stimulating factor receptor, histone, glycoprotein gp70, pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PCD-E2), hair follicle antigen, A-gliadin, and human tropomyosin isoform 5, bahiagrass pollen (BaGP), peach allergen Pru p 3, αs-1 casein milk allergen, Apig1 celeriac allergen, Bere1 Brazil nut allergen, B-lactoglobulin milk allergen, bovine serum albumin, Cor a 1.04 Contains at least a portion of the hazelnut allergen, or the ovalbumin egg allergen.

[0016] In further embodiments, the antigen comprises an autoimmune antigen, an antigen expressed on tissue to be transplanted into the subject, or an allergen. In non-limiting embodiments, the antigen is selected from the group consisting of, for example, myelin basic protein, acetylcholine receptor, endogenous antigen, myelin oligodendrocyte glycoprotein, pancreatic beta cell antigen, insulin, glutamic acid decarboxylase (GAD), type 11 collagen, human cartilage gp39, fp130-RAPS, proteolipid protein, fibrillarin, small nucleolar protein, thyroid stimulating factor receptor, histone, glycoprotein gp70, pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PCD-E2), hair follicle antigen, A-gliadin, or human tropomyosin isoform 5, bahiagrass pollen (BaGP), peach allergen Pru p 3, αs-1 casein milk allergen, Apig1 celeriac allergen, Bere1 Brazil nut allergen, B-lactoglobulin milk allergen, bovine serum albumin, Cor a 1.04 Contains hazelnut allergen, or ovalbumin egg allergen.

[0017] In some embodiments, the antigen is bound to the particle by a conjugate molecule. In some embodiments, the antigen is bound to the particle by a linker. In some embodiments, the conjugate molecule is ethylene carbodiimide (ECDI). In some embodiments, the antigen is bound to the exterior of the particle, which has a negative zeta potential. In some embodiments, the antigen is encapsulated within the particle, which has a negative surface zeta potential.

[0018] In some embodiments, the particles are biodegradable. In some embodiments, the particles are surface functionalized. In some embodiments, the particles are surface functionalized with a carboxylic acid.

[0019] In some embodiments, the present invention provides a method for inducing antigen-specific tolerance in a subject, the method comprising administering to the subject an effective amount of a composition comprising antigen-bound particles, the particles having a negative zeta potential, and the particles and the antigen induce tolerance to the antigen in the subject. In some embodiments, the administration is performed to treat or prevent a disease or condition. In some embodiments, the administration is performed before or after the onset of a disease or condition caused by the antigen. In some embodiments, the disease or condition is selected from the group consisting of autoimmune diseases, inflammatory diseases, allergies, transplant rejection, and hyperimmune responses. In some embodiments, the disease or condition is selected from the group consisting of multiple sclerosis, type 1 diabetes, asthma, food allergies, environmental allergies, celiac disease, and conditions caused by the antigen in the subject to reduce an overreaction to the antigen. In some embodiments, the method further comprises repeating the administration of the composition to the subject.

[0020] In some embodiments, the composition is administered intravenously.

[0021] In some embodiments, the present invention further provides a process for preparing immune-modified particles having a negative zeta potential, the process comprising contacting immune-modified particle precursors with a buffer under conditions effective to form immune-modified particles having a negative zeta potential. In some embodiments, the immune-modified particle precursors are formed by copolymerization. In some embodiments, the buffer has a basic pH. In some embodiments, the buffer is sodium bicarbonate, potassium bicarbonate, lithium bicarbonate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, or lithium dihydrogen phosphate.

[0022] In some embodiments, the present invention provides a composition comprising an antigen encapsulated within the core of a surface-functionalized liposome. In a further embodiment, the liposome is composed of a 30:30:40 ratio of phosphatidylcholine:phosphatidylglycerol:cholesterol. In a further embodiment, the antigen comprises an autoimmune antigen, an antigen expressed on a tissue to be transplanted into a subject, or an allergen. [Brief explanation of the drawings]

[0023] [Figure 1] (A) Micrograph of poly(lactide-co-glycolide) (PLG) particles. (B) and (C) show dynamic light scattering analysis of surface-functionalized poly(lactide-co-glycolide) particles. Surface-functionalized poly(lactide-co-glycolide) particles were analyzed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water at a count rate of 2.5 × 10 counts per second. The population of surface-functionalized poly(lactide-co-glycolide) particles generally had a Z-average particle size of 567 nm, a peak diameter of 670 nm, and a polydispersity index of 0.209, although variations of 5–15% were observed between batches. [Figure 2] We demonstrate that PLG nanoparticles induce antigen-specific tolerance. The immunodominant proteolipid protein PLP139-151 epitope (PLG-PLP139-151) was used to induce tolerance for the prevention of recurrent experimental autoimmune encephalitis (R-EAE). Mice were treated with either PLP139-151-PLGA (N = 5), OVA323-339-PLGA (N = 5), or unconjugated PLGA (N = 5) on day -7 relative to the time of immunization (day 0). Peak disease was typically observed around days 12-14, and mice were scored for clinical disease. Particles containing no peptide or particles modified with the control peptide OVA323-339 did not prevent disease induction. However, PLGA particles modified with PLP139-151 all showed a clinical score of 0 (no disease observed), except for a low clinical score of <1 on days 20-30. [Figure 3A] This figure shows that the type of administered particle affects the development of EAE in a mouse model. A) The mean clinical score of EAE animals is shown, and B) the mean cumulative score is shown. Mice were treated with either OVA323-339-PLS (N=5), OVA323-339-PLGAPHOSPOREX (N=5), OVA323-339-PLGAPEMA (N=5), PLP139-151-PLA (N=5), PLP139-151-PLGAPHOSPOREX (N=5), or PLP139-151-PLGPEMA (N=5) on day -7 relative to the time of immunization (day 0). Peak disease was typically observed around days 12-14, and mice were scored for clinical disease. Particles of any of the compositions modified with the control peptide OVA323-339 did not prevent disease induction. However, PLP139-151-conjugated PLG beads were more effective in downregulating the induction of R-EAE than PLP139-151-conjugated commercial (Phosphorex) PLG or polystyrene. [Figure 3B] Same as above. [Figure 4] Figure 2 shows that mice treated with soluble OVA on day 28 exhibited a decrease in body temperature compared to animals treated with OVA-PLG particles, with no decrease in body temperature observed within 1 hour of particle delivery. [Figure 5] This shows that administration of PLP-PLG during remission does not cause any anaphylaxis-related deaths. EAE was induced in 6-8 week-old female SJL / J mice by subcutaneous injection of PLP139-151 in CFA, and the onset of clinical disease was monitored and recorded (B). On day 21 after disease induction, mice were intravenously injected with soluble PLP139-151 (open squares), soluble OVA323-339 (open circles), or the same peptides conjugated to PLG nanoparticles (filled circles). Animal body temperature was monitored and recorded every 10 minutes for 1 hour after injection (A). [Figure 6]The optimal dose of PLP139-151-PLG administered intravenously 7 days before disease induction is shown. Clinical disease onset was measured in SJL / J mice treated with OVA323-339-PLG (A). Six- to eight-week-old female SJL / J mice were intravenously injected with PLG nanoparticles conjugated with either PLP139-151 (squares) or OVA323-339 (circles). Seven days later (B), 25 days later (C), or 50 days later (D), EAE was induced by subcutaneous injection of PLP139-151 in CFA. Animals in panel B were followed for 100 days for clinical disease. Eight days after disease induction, delayed-type hypersensitivity (DTH) was performed in a subset of mice shown in panel B (E). The ears of representative animals (OVA323-339-PLG and PLP139-151-PLG) selected from the PLP139-151 / CFA-primed group in panel B were challenged with the PLP139-151 epitope and the OVA323-339 control peptide. After 24 hours, ear swelling was assessed as a measure of DTH, and the pre-challenge response was subtracted. Six- to eight-week-old female SJL / J mice were intravenously injected with PLG nanoparticles conjugated with PLP178-191 (triangles), OVA323-339 (circles), or PLP139-151 (squares), or with unconjugated particles alone (circles with outlines) (F). Seven days later, EAE was induced by subcutaneous injection of PLP178-191 in CFA, and disease was monitored at the indicated time points. [Figure 7] Figures A-D show that preventive tolerance was most efficient when PLG-PLP139-151 particles were administered either intravenously or intraperitoneally. Animals treated with intravenously administered PLP139-151-PLG did not develop disease and had an average clinical score of 0 at most time points. [Figure 8] AF show that administration of OVA323-339-PLG particles inhibited Th1 and Th17 responses in treated animals. [Figure 9]A–C show reduced immune cell infiltration within the spinal cord of animals treated with PLP139–151-PLG, which resembles native tissue more closely than tissue from animals treated with OVA323–339-PLG. Animals treated with OVA323–339-PLG showed positive staining for CD45, CD4, and CD11b, whereas animals treated with PLP139–151-PLG showed minimal staining for these factors. [Figure 10] Figures A–C show that administration of PLP139–151-PLG particles inhibits blood-brain barrier (BBB) ​​disruption and macrophage activation in the spinal cord of treated mice. Animals were treated with either complete Freund's adjuvant (CFA), OVA323–339 PLG particles, or PLP139–151-PLG particles. Clinical scores and the incidence of EAE were determined (B), and spinal cords were observed by in vivo imaging (A and C). [Figure 11A] 1 shows spinal cords of treated mice with in vivo imaging. [Figure 11B] 1 shows spinal cords of treated mice with in vivo imaging. [Figure 11C] 1 is a graph showing quantification of image data. [Figure 11D] 1 is a graph showing quantification of image data. [Figure 11E] 1 is a graph showing quantification of image data. [Figure 11F] 1 is a graph showing quantification of image data. [Figure 12] We demonstrate that administration of PLG particles encapsulating PLP139-151 inhibits the induction of R-EAE in mice. The ability to encapsulate autoantigens allows for the use of complex mixtures of proteins or even organ homogenates, which is not possible with surface binding, thus enabling greater antigen coverage and therefore more effective epitope spreading. [Figure 13] 1 shows that animals treated with PLP139-151-PLG particles and anti-CD25 antibody occasionally exhibited higher mean clinical scores than animals treated with PLP139-151-PLG particles and control IgG antibody. [Figure 14]Therapeutic tolerance induced by PLP139-151-PLG particles in active and adoptive EAE was shown. Adoptive EAE was induced in 6-8 week-old female SJL / J mice by adoptive transfer of 2.5 x 106 PLP139-151-activated blasts. Two days (A) and 14 days (C) after disease induction, mice were intraperitoneally injected with PLP139-151 (squares) or OVA323-339 (circles) peptides conjugated to 500 nm PLG nanoparticles. Clinical disease scores were compared with those after treatment with antigen-bound splenocytes (A). On day 42, brain tissue and spinal cord tissue were harvested from PLP139-151- or OVA323-339-tolerized mice for histological analysis. Sections from mice in panel A were stained for PLP protein and CD45 (B). A spinal cord section from the mouse in panel (C) was stained with Luxol Fast Blue (D). Areas of demyelination and cellular infiltration are indicated by arrows. [Figure 15] Graphs depicting the mean clinical scores of mice with active and adoptive EAE after treatment with either SP or PLG particles conjugated to OVA323-339 or PLP139-151. Ten days (A) and two days (B) after disease induction, mice were intraperitoneally injected with PLP139-151-SP, PLP139-151-PLG, or OVA323-339-SP, or OVA323-339-PLG peptides conjugated to 500 nm nanoparticles, and the mean clinical scores were determined. In both cases, administration of PLP139-151-PLG particles induces tolerance in mice. [Figure 16]We show that CNS immune cell infiltration was also dramatically reduced in PLP-PLG-tolerant mice. Two days after EAE induction by adoptive transfer, SJL / J mice were intraperitoneally injected with 500 nm PLG nanoparticles conjugated with PLP139-151 (squares) or OVA323-339 (circles). At the peak of disease (day 14), brains and spinal cords were removed, and the numbers of lymphocytes (B), APCs (C), microglia (D), peripheral dendritic cells (E), myeloid dendritic cells (F), and macrophages (G) were enumerated by flow cytometry. The gating strategy for these populations is shown in (A). CNS cell preparations were stimulated with PMA and ionomycin for 5 h before intracellular staining for IL-17A and IFN-γ (H). [Figure 17] Administration of the PLP139-151 peptide encapsulated in PLG particles induces tolerance when the particles are administered with PBS, but administration of an anti-PD-1 antibody reduces this tolerance. [Figure 18] Administration of the PLP139-151 peptide encapsulated in PLG particles induces tolerance when the particles are administered with PBS. Administration of anti-CD40 antibodies reduces this tolerance, but this reduction in tolerance is reversed by the addition of anti-IL-12 antibodies. [Figure 19] A to G show that prophylactic administration of OVA-PLG reduced the secretion of IL-4, IL-5, IL-13, and IL-10, and reduced serum OVA IgE and pulmonary eosinophil levels. [Figure 20] We demonstrate that OVA encapsulated within PLG particles prophylactically inhibits OVA-specific ex vivo recall responses from mediastinal lymph nodes. In animals treated with OVA-PLG, lymph node proliferation observed after restimulation with 25 μg of OVA was reduced (A). Furthermore, treatment with OVA-PLG reduced cytokine release after restimulation with OVA. In mice treated with OVA-PLG, levels of IL-4, IL-5, IL-13, and IL-10 were reduced (B). [Figure 21]We show that therapeutic administration of OVA-PLG reduced the secretion of IL-4, IL-5, IL-13, and IL-10, and reduced serum OVA IgE and pulmonary eosinophil levels. [Figure 22] These results demonstrate that OVA encapsulated in PLG particles therapeutically down-regulates OVA-specific Th2 cytokines in bronchoalveolar lavage fluid better than OVA-conjugated PLG particles. Mice were treated intraperitoneally with OVA / alum at a dose of 10 μg / mouse on days 0 and 14. On days 28 and 42, mice were intravenously administered either OVA bound to PLG particles or OVA encapsulated within PLG particles. On days 56-58, mice were treated three times with aerosolized OVA. The graphs show cytokine secretion when animals were treated with either OVA bound to PLG particles (A) or OVA encapsulated within PLG particles (B). [Figure 23] Blood glucose levels in type 1 diabetic animals after treatment with p31-PLG particles. Administration of p31 peptide-conjugated PLG resulted in lower blood glucose levels compared to those observed after administration of MOG35-55 peptide-conjugated particles (A and B). The percentage of IFNγ-secreting cells observed in animals was also reduced in mice treated with p31-PLG compared to mice treated with MOG35-55 peptide-PLG (C). [Figure 24] (A-B) Tolerance induced by p31-PLG requires Tregs. Type 1 diabetes was induced in mice by adoptive transfer. Two hours after the transfer of activated cells to NOD SCID mice, the mice were tolerized with either p31-PLG or MOG35-55PLG particles. Depletion of Tregs suppresses tolerance induced by administration of p31-PLG particles. [Figure 25]These results show that administration of insulin-conjugated PLG particles significantly increased the proportion of mice that remained diabetes-free over 300 days (69.6% compared to 22.7%; p=0.0027). NOD mice were treated intravenously at 6, 8, and 10 weeks of age with either BSA (N=22) or insulin (N=23)-conjugated PLG particles. Mice were then assayed for the development of diabetes. [Figure 26] The percentage of CD45.1 donor cells observed in recipient mice is shown. Female CD45.2 mice were tolerized with either OVA-PLG or Dby-PLG on day -7. Mice were irradiated with 200 rads on day -1 and then transplanted with 1 x 10, 5 x 10, or 1 x 10 bone marrow cells from male CD45.1 mice on day 0. Recipient mice were then tolerized with either OVA-PLG, Dby-SP, or Dby-PLG on day 1, and blood was collected for FACS analysis of chimerism. [Figure 27] The percentage of donor CD45.1 cells in recipient mice after tolerization with either OVA-PLG, Dby-SP, or Dby-PLG on day 1 is shown. One positive control mouse showed no significant engraftment (approximately 10%). All negative control mice did not engraft donor cells. One Dby-SP mouse showed no significant engraftment (approximately 10%). Two OVA-PLG mice engrafted donor cells (approximately 10%), and one showed complete rejection by week 16. One Dby-PLG mouse began to show rejection at week 12, reaching 10% by week 16. The Dby-PLG groups ranged from 10% to 56% engraftment by week 16. OVA-PLG mice demonstrated 1) natural engraftment, 2) sequence homology between OVA323 and Dby, or 3) immunogenic properties of the particles. Dby-PLG allows for greater engraftment than Dby-SP and OVA-PLG. [Figure 28]The timing of tolerance influences the percentage of CD45.1 cells in recipient mice. The positive control shows less engraftment (approximately 4%) than expected (approximately 10%). One negative control mouse had 5% engraftment in all three OVA-PLG groups, and one mouse in the day -7 and +1 groups showed engraftment (12%). Day 1 tolerance is more clinically relevant than day -7 tolerance. [Figure 29] Coumarin-6 PLGA particles, either conjugated to antigen or free of antigen, were detectable at 3 hours post-administration but undetectable at 24 hours post-administration. Particles were detectable at 3 hours post-administration but undetectable at 24 hours post-administration. Spleen (left column), liver (middle column), and lung (left column) sections of mice injected with intraperitoneal fluorescent PLGA / PEMA microparticles compared with uninjected naive mice (top row) at 3 hours post-injection (middle row) and 24 hours post-injection (bottom row), counterstained with DAPI. [Figure 30] Figure 1 shows that PLGA particles were co-localized with F4 / 80+ cells in the liver 6 and 15 hours after administration. [Figure 31] Twenty-four hours after intravenous injection, marginal zone macrophages preferentially take up particles conjugated with TAMRA-labeled PLP139-151. The highest proportion of PLP139-151+ cells is found in marginal zone macrophages. [Figure 32] The mean daily clinical scores are shown against the number of days of priming with PLP139-151 / CFA. Induction of immunological tolerance using surface-functionalized poly(lactide-co-glycolide) particles containing soluble PLP139-151 in the core inhibited PLP139-151 / CFA-induced R-EAE in SJL / J mice. [Figure 33] 1 shows that mice treated with encapsulated OVA-PLG showed a significant reduction in eosinophil accumulation. [Figure 34] Figure 1 shows that mice treated with encapsulated OVA-PLG showed a significant decrease in serum IgE levels compared to untreated or control-treated animals. [Figure 35] Dynamic light scattering analysis of surface-functionalized poly(lactide-co-glycolide) particles containing soluble PLP139-151 within their cores is shown. The surface-functionalized poly(lactide-co-glycolide) particles were analyzed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water at a count rate of 1.792 × 10 counts per second. The population of surface-functionalized poly(lactide-co-glycolide) particles had a Z-average particle size of 584 nm, a peak diameter of 679 nm, and a polydispersity index of 0.162. These results are representative of six batches synthesized according to the protocol described above. [Figure 36] Surface-functionalized poly(lactide-co-glycolide) particles containing soluble PLP139-151 within their cores were characterized by zeta-potential measurements. Surface-functionalized poly(lactide-co-glycolide) particles were analyzed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water at a count rate of 6.67 × 10 counts per second. The population of surface-functionalized poly(lactide-co-glycolide) particles had a peak zeta potential of -48.9 mV and a zeta deviation of 5.14 mV. These results are representative of six batches synthesized according to the protocol described above. [Figure 37] Dynamic light scattering analysis of surface-functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin within their cores is shown. Surface-functionalized poly(lactide-co-glycolide) particles were analyzed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water at a count rate of 1.822 × 10 counts per second. The population of surface-functionalized poly(lactide-co-glycolide) particles had a Z-average particle size of 569.7 nm, a peak diameter of 700.3 nm, and a polydispersity index of 0.230. These results are representative of three batches synthesized according to the protocol described above. [Figure 38]Surface-functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin within their cores were characterized by zeta-potential measurements. Surface-functionalized poly(lactide-co-glycolide) particles were analyzed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water at a count rate of 2.67 × 10 counts per second. The population of surface-functionalized poly(lactide-co-glycolide) particles had a peak zeta potential of -52.2 mV and a zeta deviation of 5.38 mV. These results are representative of three batches synthesized according to the protocol described above. [Figure 39] This graph demonstrates that surface-functionalized liposomes containing soluble PLP139-151 peptide within the core induce immunological tolerance in a mouse model of multiple sclerosis. Animals were treated with either surface-functionalized liposomes containing soluble PLP139-151 peptide within the core (circles) or surface-functionalized liposomes containing soluble OVA323-339 peptide (squares). The mean clinical scores of those animals receiving PLP139-151 peptide liposomes were lower than those of animals receiving OVA323-339 peptide liposomes. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present inventors have discovered that antigen-bound nanoparticles can induce tolerance and reduce immune responses to autoimmune diseases. These particles can induce tolerance regardless of whether they are bound to the surface of the particle or encapsulated within it. Therefore, these particles may be useful in treating any disease or condition characterized by an excessive inflammatory immune response, such as autoimmune diseases.

[0025] As used herein, "particle" refers to any composition derived from a non-tissue source, which may be a sphere or spherical entity, a bead, or a liposome. The terms "particle," "immunomodulatory particle," "carrier particle," and "bead" may be used interchangeably depending on the context. Furthermore, the term "particle" may be used to encompass beads and spheres.

[0026] As used herein, "negatively charged particles" refers to particles that have been modified to have a net surface charge of less than zero.

[0027] "Carboxylated particles" or "carboxylated beads" or "carboxylated spheres" include any particles modified to contain carboxyl groups on their surface. In some embodiments, the addition of carboxyl groups enhances phagocyte / monocyte uptake of the particles from the circulation, for example, through interaction with scavenger receptors such as MARCO. Carboxylation of particles can be achieved using any compound that adds carboxyl groups, including, but not limited to, poly(ethylene-maleic anhydride) (PEMA).

[0028] As used herein, "antigenic moiety" refers to any moiety, for example, a peptide, that is recognized by the host's immune system. Examples of antigenic moieties include, but are not limited to, autoantigens and / or bacterial or viral proteins, peptides, or components. Without being bound by theory, carboxylated beads themselves may be recognized by the immune system, but carboxylated beads without anything attached thereto are not considered "antigenic moieties" for the purposes of the present invention.

[0029] As used herein, "bare beads" or "bare particles" or "bare spheres" refer to beads, particles, or spheres that are not carboxylated.

[0030] As used herein, "pro-inflammatory mediator" or "pro-inflammatory polypeptide" refers to a polypeptide or fragment thereof that induces, maintains, or prolongs inflammation in a subject. Examples of pro-inflammatory mediators include, but are not limited to, cytokines and chemokines.

[0031] The particles may have any particle shape or configuration. However, in some embodiments, it is preferable to use particles that are less likely to aggregate in vivo. Exemplary particles in these embodiments are those having a spherical shape.

[0032] Another aspect of the present invention relates to a composition comprising an immunomodified particle having a negative zeta potential and not containing an antigen moiety. In a further embodiment, the present invention provides a composition comprising an immunomodified particle having a negative zeta potential bound to an antigen. In a further embodiment, the antigen is bound to the outside of the particle. In a further embodiment, the antigen is encapsulated within the particle.

[0033] Another aspect of the present invention relates to a process for preparing immunomodified particles having a negative zeta potential and not containing antigen moieties. The process comprises contacting an immunomodified particle precursor with a buffer under conditions effective to form an immunomodified particle having a negative zeta potential. In some embodiments of the present invention, the immunomodified particle precursor is formed via copolymerization. The particle microstructure can depend on the method of copolymerization.

[0034] In some embodiments, the antigenic peptide molecule is bound to the carrier particle (e.g., immunomodified particle) by a conjugate molecule and / or a linker group. In some embodiments, binding the antigenic peptide and / or apoptosis signaling molecule to the carrier (e.g., PLG particle) involves one or more covalent and / or non-covalent interactions. In some embodiments, the antigenic peptide is attached to the surface of the carrier particle having a negative zeta potential. In some embodiments, the antigenic peptide is encapsulated within the carrier particle having a negative zeta potential.

[0035] In one embodiment, the buffer solution contacting the immunomodified particles can have a basic pH. Suitable basic pH values ​​for the basic solution include 7.1, 7.5, 8.0, 8.5, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, and 13.5. The buffer solution can also be made with any suitable base and its conjugates. In some embodiments of the present invention, the buffer solution can include, but is not limited to, sodium bicarbonate, potassium bicarbonate, lithium bicarbonate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, or lithium dihydrogen phosphate, and conjugates thereof.

[0036] In one embodiment of the present invention, the immunomodified particles contain copolymers. These copolymers can have various molar ratios. Suitable copolymer ratios for the immunomodified particles of the present invention can be 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0. In another embodiment, the copolymer can be a periodic, statistical, linear, or branched (including star, brush, or comb) copolymer. In some embodiments, the ratio of the copolymers may be, but is not limited to, polystyrene:poly(vinyl carboxylate) / 80:20, polystyrene:poly(vinyl carboxylate) / 90:10, poly(vinyl carboxylate):polystyrene / 80:20, poly(vinyl carboxylate):polystyrene / 90:10, polylactic acid:polyglycolic acid / 80:20, or polylactic acid:polyglycolic acid / 90:10.

[0037] In one embodiment, the particle is a liposome. In a further embodiment, the particle is a liposome composed of the following lipids in the following molar ratio - 30:30:40 phosphatidylcholine:phosphatidylglycerol:cholesterol. In a further embodiment, the particle is encapsulated within a liposome.

[0038] While each particle need not be uniform in size, particles generally must be large enough to induce phagocytosis in antigen-presenting cells or other MPS cells. Preferably, particles are microscopic or nanoscale in size to enhance solubility, avoid potential complications caused by in vivo aggregation, and promote pinocytosis. Particle size can be a factor in uptake from the interstitial space into areas of lymphocyte maturation. Particles with diameters of about 0.1 μm to about 10 μm can induce phagocytosis. Thus, in one embodiment, particles have diameters within these limits. In another embodiment, particles have diameters of about 0.3 μm to about 5 μm. In yet another embodiment, particles have diameters of about 0.5 μm to about 3 μm. In further embodiments, the particles have a size of about 0.1 μm, or about 0.2 μm, or about 0.3 μm, or about 0.4 μm, or about 0.5 μm, or about 1.0 μm, or about 1.5 μm, or about 2.0 μm, or about 2.5 μm, or about 3.0 μm, or about 3.5 μm, or about 4.0 μm, or about 4.5 μm, or about 5.0 μm. In certain embodiments, the particles have a size of about 0.5 μm. In some embodiments, the total weight of the particles is less than about 10,000 kDa, less than about 5,000 kDa, or less than about 1,000 kDa, 500 kDa, 400 kDa, 300 kDa, 200 kDa, 100 kDa, 50 kDa, 20 kDa, or 10 kDa. The particles in the composition do not need to be of uniform diameter. As an example, a pharmaceutical formulation may contain multiple particles, some of which are about 0.5 μm and some of which are about 1.0 μm. Any mixture of particle sizes within these given ranges would also be useful.

[0039] The particles of the present invention can have a specific zeta potential. In certain embodiments, the zeta potential is negative. In one embodiment, the zeta potential is less than about -100 mV. In one embodiment, the zeta potential is less than about -50 mV. In certain embodiments, the particles have a zeta potential of -100 mV to 0 mV. In further embodiments, the particles have a zeta potential of -75 mV to 0 mV. In further embodiments, the particles have a zeta potential of -60 mV to 0 mV. In further embodiments, the particles have a zeta potential of -50 mV to 0 mV. In further embodiments, the particles have a zeta potential of -40 mV to 0 mV. In further embodiments, the particles have a zeta potential of -30 mV to 0 mV. In further embodiments, the particles have a zeta potential of -20 mV to +0 mV. In further embodiments, the particles have a zeta potential of -10 mV to -0 mV. In certain embodiments, the particles have a zeta potential of between −50 mV and −40 mV.

[0040] In some embodiments, the charge (e.g., positive, negative, neutral) of the carrier is selected to confer specific benefits to the application (e.g., physiological compatibility, beneficial surface-peptide interactions, etc.). In some embodiments, the carrier has a net neutral or negative charge (e.g., to reduce nonspecific binding to cell surfaces, which generally bear a net negative charge). In certain embodiments, the carrier can be conjugated, either directly or indirectly, to an antigen to which tolerance is desired (also referred to herein as an antigen-specific peptide, antigenic peptide, autoantigen, inducible antigen, or tolerizing antigen). In some cases, the carrier has multiple binding sites (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10...20...50...100, or more) to expose multiple copies of an antigen-specific peptide or multiple different peptides on the surface (e.g., to increase the likelihood of a tolerance response). In some embodiments, the carrier displays a single type of antigenic peptide. In some embodiments, the carrier displays multiple different antigenic peptides on its surface. In some embodiments, the carrier surface presents functional groups for covalent attachment of a selected moiety (e.g., an antigenic peptide). In some embodiments, the functional groups on the carrier surface provide sites for non-covalent interaction with a selected moiety (e.g., an antigenic peptide). In some embodiments, the carrier has a surface to which a conjugated moiety can be adsorbed without forming a chemical bond.

[0041] In some embodiments, the particles are non-metallic. In these embodiments, the particles may be formed from polymers. In a preferred embodiment, the particles are biodegradable within an individual. In this embodiment, the particles can be provided to an individual over multiple doses without accumulation of particles within the individual. Examples of suitable particles include polystyrene particles, PLGA particles, and diamond particles.

[0042] Preferably, the particle surface is made of a material that minimizes nonspecific or undesirable biological interactions. Interactions between the particle surface and the interstitium may be a factor that plays a role in lymphatic uptake. The particle surface may be coated with a material to prevent or reduce nonspecific interactions. As evidenced by improved lymphatic uptake after subcutaneous injection, steric stabilization by coating particles with a hydrophilic layer, such as poly(ethylene glycol) (PEG) and its copolymers, e.g., PLURONICS (comprising copolymers of poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol)), can reduce nonspecific interactions with interstitial proteins. All of these facts demonstrate the significance of the particle's physical properties with respect to lymphatic uptake. Biodegradable polymers may be used to fabricate all or part of the polymer and / or particle and / or layer. Biodegradable polymers may undergo degradation, for example, as a result of functional groups reacting with water in solution. As used herein, the term "degradation" refers to becoming soluble either by a decrease in molecular weight or by converting hydrophobic groups to hydrophilic groups. Polymers containing ester groups, such as polylactide and polyglycolide, are generally subject to spontaneous hydrolysis.

[0043] The particles of the present invention may contain additional components. For example, the carrier may have a contrast agent incorporated or conjugated to the carrier. An example of a carrier nanosphere with a contrast agent currently on the market is Kodak X-sight nanosphere. Inorganic quantum-confined luminescent nanocrystals known as quantum dots (QDs) have emerged as an ideal donor for FRET applications: their high quantum yield and adjustable size-dependent Stokes shift allow them to emit light of different sizes from blue to infrared when excited by a single ultraviolet wavelength. (Bruchez, et al., Science, 1998, 281, 2013; Niemeyer, CM Angew. Chem. Int. Ed. 2003, 42, 5796; Waggoner, A. Methods Enzymol. 1995, 246, 362; Brus, LEJ Chem. Phys. 1993, 79, 5566). Quantum dots, such as hybrid organic / inorganic quantum dots based on a class of polymers known as dendrimers, can be used in biological labeling, imaging, and optical biosensing systems (Lemon, et al., J. Am. Chem. Soc. 2000, 122, 12886). Unlike the synthesis of traditional inorganic quantum dots, the synthesis of these hybrid quantum dot nanoparticles does not require high temperatures or highly toxic, unstable reagents (Etienne, et al., Appl. Phys. Lett. 87, 181913, 2005).

[0044] The particles can be formed from a wide range of materials. Preferably, the particles are made of a material suitable for biological use. For example, the particles may be made of glass, silica, polyesters of hydroxycarboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxycarboxylic acids and dicarboxylic acids. More generally, the carrier particles may be made of polyesters of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxyhydroxy acids, or linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxydicarboxylic acid polyanhydrides. Furthermore, the carrier particles may be quantum dots or may be made of quantum dots, such as quantum dot polystyrene particles (Joumaa et al. (2006) Langmuir 22:1810-6). Carrier particles containing a mixture of ester and anhydride linkages (e.g., copolymers of glycolic acid and sebacic acid) can also be used. For example, the carrier particles may comprise materials including polyglycolic acid polymers (PGA), polylactic acid polymers (PLA), polysebacic acid polymers (PSA), poly(lactic-co-glycolic) acid copolymers (PLGA or PLG; these terms are synonymous), [rho]oly(lactic-co-sebacic) acid copolymers (PLSA), poly(glycolic-co-sebacic) acid copolymers (PGSA), etc. Other biocompatible, biodegradable polymers useful in the present invention include polymers or copolymers of caprolactone, carbonate, amide, amino acid, orthoester, acetal, cyanoacrylate, and degradable urethane, as well as copolymers thereof with linear or branched, substituted or unsubstituted alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy- or dicarboxylic acids.Furthermore, biologically important amino acids with reactive side groups, such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or their enantiomers, may be included in copolymers with any of the above materials to provide reactive groups for conjugation to antigen peptides and proteins or conjugate moieties. Biodegradable materials suitable for the present invention include diamond, PLA, PGA, and PLGA polymers. Biocompatible but non-biodegradable materials can also be used in the carrier particles of the present invention. For example, non-biodegradable polymers such as acrylate, ethylene-vinyl acetate, acyl-substituted cellulose acetate, non-degradable urethane, styrene, vinyl chloride, vinyl fluoride, vinyl imidazole, chlorosulfonated olefins, ethylene oxide, vinyl alcohol, TEFLON® (DuPont, Wilmington, Del.), and nylon may be used.

[0045] Suitable beads currently available commercially include polystyrene beads such as FluoSpheres (Molecular Probes, Eugene, Oreg.).

[0046] In some embodiments, the present invention provides a system comprising: (a) a delivery scaffold configured for delivery of a chemical and / or biological agent to a subject; and (b) poly(lactide-co-glycolide) particles conjugated with an antigen for inducing antigen-specific tolerance. In some embodiments, at least a portion of the delivery scaffold is microporous. In some embodiments, the antigen-conjugated poly(lactide-co-glycolide) particles are encapsulated within the scaffold. In some embodiments, the chemical and / or biological agent is selected from the group consisting of proteins, peptides, small molecules, nucleic acids, cells, and particles. In some embodiments, the chemical and / or biological agent comprises cells, and the cells comprise pancreatic islet cells.

[0047] Physical properties also relate to the usefulness of nanoparticles after uptake and retention in areas with immature lymphocytes. These include mechanical properties such as rigidity or rubberiness. Some embodiments are based on a rubbery core, e.g., a poly(propylene sulfide) (PPS) core, with a hydrophilic overlayer, e.g., as in PEG, as in the PPS-PEG system recently developed and characterized for systemic (but not targeted or immune) delivery. The rubbery core contrasts with a substantially rigid core, as in polystyrene or metal nanoparticle systems. The term rubbery refers to a specific elastic material other than natural or synthetic rubber, a term familiar to those skilled in the polymer art. For example, crosslinked PPS can be used to form a hydrophobic rubbery core. PPS is a polymer that degrades under oxidative conditions to polysulfoxide and ultimately polysulfone, transitioning from a hydrophobic rubber to a hydrophilic, water-soluble polymer. Other sulfide polymers may also be adapted for use, and the term sulfide polymer refers to polymers containing sulfur in the polymer backbone. Other rubbery polymers that can be used are polyesters with a glass transition temperature under hydration conditions of less than about 37°C. Hydrophobic cores can be advantageously used with hydrophilic overlayers because the core and overlayer tend not to mix and the overlayer tends to expand sterically away from the core. Core refers to a particle having a layer thereon. Layer refers to a material that covers at least a portion of the core. The layer may be adsorbed or covalently bonded. The particle or core may be solid or hollow. Rubbery hydrophobic cores have an advantage over rigid hydrophobic cores, such as crystalline or glassy (as in polystyrene) cores, in that particles with rubbery hydrophobic cores can achieve higher loadings of hydrophobic drugs.

[0048] Another physical property is surface hydrophilicity. Hydrophilic materials can have a water solubility of at least 1 gram per liter when not crosslinked. Steric stabilization of particles with hydrophilic polymers can improve interstitial uptake by reducing nonspecific interactions; however, increased particle stealth may also reduce internalization by phagocytes in areas with immature lymphocytes. While the challenge of balancing these competing properties has been addressed, this application demonstrates the creation of nanoparticles for effective lymphatic delivery to DCs and other APCs in lymph nodes. Some embodiments include a hydrophilic component, e.g., a layer of hydrophilic material. Examples of suitable hydrophilic materials are one or more of polyalkylene oxides, polyethylene oxides, polysaccharides, polyacrylic acids, and polyethers. The molecular weight of the polymer in the layer can be adjusted to provide a degree of steric hindrance that is useful in vivo, for example, from about 1,000 to about 100,000 or even higher; one of skill in the art will readily appreciate that all ranges and values ​​within the explicitly stated ranges are contemplated, e.g., 10,000-50,000.

[0049] The nanoparticles may incorporate functional groups for further reaction. Functional groups for further reaction include electrophiles or nucleophiles: these favor reaction with other molecules. Examples of nucleophiles are primary amines, thiols, and hydroxyls. Examples of electrophiles are succinimidyl esters, aldehydes, isocyanates, and maleimides.

[0050] A variety of means well known in the art can be used to conjugate antigen peptides and proteins to carriers.These methods include any standard chemistry that does not destroy or significantly limit the biological activity of antigen peptides and proteins, and can conjugate a sufficient number of antigen peptides and proteins to carriers in an orientation that allows the antigen peptides or proteins to interact with their cognate T cell receptors.Generally, preferred methods are those that conjugate the C-terminal region of antigen peptides or proteins, or the C-terminal region of antigen peptide or protein fusion proteins, to carriers.The exact chemistry naturally depends on the nature of the carrier material, the presence or absence of C-terminal fusion to antigen peptides or proteins, and / or the presence or absence of conjugated moieties.

[0051] Functional groups can be located on particles as needed due to availability.One location can be a side group or terminal on core polymer, or on the polymer that is a layer on core, or on the polymer that is otherwise anchored to particle.For example, the example described herein describes the PEG stabilization of nanoparticles, which can be easily functionalized for specific cell targeting or protein and peptide drug delivery.

[0052] Conjugates such as ethylene carbodiimide (ECDI), hexamethylene diisocyanate, propylene glycol diglycidyl ether containing two epoxy residues, and epichlorohydrin can be used to immobilize peptides or proteins on carrier surfaces. Without being bound by theory, ECDIs are thought to perform two major functions for inducing tolerance: (a) they chemically attach proteins / peptides to cell surfaces through catalysis of peptide bond formation between free amino and free carboxyl groups, and (b) they induce carriers to mimic apoptotic cell death, thereby being selected by host antigen-presenting cells in the spleen and inducing tolerance. It is this non-immunogenic presentation to host T cells that directly induces anergy in autoreactive cells. ECDIs also serve as a potent stimulus for inducing specific regulatory T cells.

[0053] In a series of embodiments, antigen peptides and proteins are bound to carriers via covalent chemical bonds.For example, the reactive group or moiety near the C-terminus of antigen (for example, the C-terminal carboxyl group, or the hydroxyl group, thiol group, or amine group of amino acid side chain) can be directly conjugated to the reactive group or moiety on the surface of carriers (for example, the hydroxyl or carboxyl group of PLA or PGA, the terminal amine or carboxyl group of dendrimer, or the hydroxyl group, carboxyl group, or phosphate group of phospholipid) by direct chemical reaction.Alternatively, there can be a conjugation moiety that covalently conjugates both antigen peptides and proteins with carriers, thereby binding them together.

[0054] Reactive carboxyl groups on the surface of the carrier can be bound to free amines (e.g., from Lys residues) on antigenic peptides or proteins by, for example, reacting with 1-ethyl-3-[3,9-dimethylaminopropyl]carbodiimide hydrochloride (EDC) or hydroxysuccinimide ester (NHS). Similarly, the same chemistry can be used to conjugate free amines on the surface of the carrier with free carboxyls (e.g., from the C-terminus, or Asp or GIu residues) on antigenic peptides or proteins. Alternatively, free amines on the surface of the carrier can be covalently bound to antigenic peptides and proteins, or antigenic peptide or protein fusion proteins, using sulfo-SIAB chemistry essentially as described in Arano et al. (1991) Chem. 2:71-6.

[0055] In another embodiment, the antigen can be conjugated to the carrier by non-covalent binding between the ligand bound to the antigen peptide or protein and the anti-ligand attached to the carrier. For example, a biotin ligase recognition sequence tag can be attached to the C-terminus of the antigen peptide or protein, and this tag can be biotinylated by biotin ligase. Biotin can then function as a ligand to non-covalently conjugate the antigen peptide or protein with avidin or streptavidin adsorbed or otherwise bound to the surface of the carrier as an anti-ligand. Alternatively, if the antigen peptide and protein are fused with an immunoglobulin domain having an Fc region, as described above, the Fc domain can act as a ligand, and protein A covalently or non-covalently bound to the surface of the carrier can function as an anti-ligand to non-covalently conjugate the antigen peptide or protein to the carrier. Other means that can be used to non-covalently conjugate antigenic peptides and proteins to carriers are well known in the art, including metal ion chelation techniques (e.g., using a poly-His tag at the C-terminus of an antigenic peptide or protein or antigenic peptide or protein fusion protein, and a Ni+-coated carrier), and these methods may be substituted for the methods described herein.

[0056] Conjugation of the nucleic acid moiety to the platform molecule can be achieved in any number of ways, but typically requires one or more crosslinkers and functional groups on the nucleic acid moiety and the platform molecule. The linking group is added to the platform using standard synthetic chemistry techniques. The linking group can be added to the nucleic acid moiety using standard synthetic techniques. The practitioner has many options for the antigen used in the combination of the present invention. The inducing antigen present in the combination contributes to the specificity of the induced tolerogenic response. It may or may not be the same as the target antigen, which is the antigen present or given to the subject to be treated, that is the target of the undesired immunological response and to which tolerance is desired.

[0057] The inducing antigens of the present invention may be polypeptides, polynucleotides, carbohydrates, glycolipids, or other molecules isolated from biological sources, or may be chemically synthesized small molecules, polymers, or derivatives of biological substances, provided that when combined with a mucosally binding component, they have the ability to induce tolerance in accordance with the present invention.

[0058] In some embodiments, the present invention provides carriers (e.g., immunomodulatory particles) coupled to one or more peptides, polypeptides, and / or proteins. In some embodiments, carriers such as those described herein (e.g., PLG carriers) are effective in inducing antigen-specific tolerance and / or preventing the onset of immune-related diseases (such as EAE in a mouse model) and / or reducing the severity of existing immune-related diseases. In some embodiments, the compositions and methods of the present invention can cause T cells to initiate early events associated with T cell activation but cannot cause T cells to acquire effector function. For example, administration of a composition of the present invention can result in T cells with a quasi-activated phenotype, such as upregulation of CD69 and / or CD44, but which do not exhibit effector function, as indicated by a lack of synthesis of IFN-γ or IL-17. In some embodiments, administration of a composition of the present invention results in T cells with a quasi-activated phenotype that do not undergo a transition from naive antigen-specific T cells to a regulatory phenotype, such as one with a CD25+ / Foxp3+ phenotype.

[0059] In some embodiments, the surface of a carrier (e.g., a particle) comprises chemical moieties and / or functional groups that allow for attachment (e.g., covalently, non-covalently) of antigenic peptides and / or other functional elements to the carrier. In some embodiments, the number, orientation, spacing, etc. of chemical moieties and / or functional groups on a carrier (e.g., a particle) will vary depending on the chemistry of the carrier, the desired application, etc.

[0060] In some embodiments, the carrier contains one or more biological or chemical agents attached to, adsorbed on, encapsulated in, and / or contained throughout the carrier. In some embodiments, the chemical or biological agents are encapsulated within particles and / or contained throughout the carrier. The present invention is not limited by the nature of the chemical or biological agents. Such agents include, but are not limited to, proteins, nucleic acid molecules, small molecule drugs, lipids, carbohydrates, cells, cellular components, etc. In some embodiments, two or more (e.g., three, four, five, etc.) different chemical or biological agents are contained on or within the carrier. In some embodiments, the agents are configured for a specific release rate. In some embodiments, multiple different agents are configured for different release rates. For example, a first agent may release over a period of hours and a second agent may release over a longer period (e.g., days, weeks, months, etc.). In some embodiments, the carrier, or a portion thereof, is configured for sustained release of the biological or chemical agent. In some embodiments, the sustained release provides for release of a biologically active amount of agent over a period of at least 30 days (e.g., 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 180 days, etc.). In some embodiments, the carrier, or a portion thereof, is configured to be sufficiently porous to allow cellular ingrowth into the pores. The size of the pores may be selected for the particular cell type of interest and / or the amount of ingrowth desired.

[0061] The encapsulation of antigens, biological agents, and / or chemical agents into the particles of the present invention has surprisingly been found to induce immune tolerance and has several advantages. First, encapsulated particles have a slower cytokine response. Second, when using multiple antigens, biological agents, and / or chemical agents, encapsulation eliminates the competition between these various molecules that may occur when the agents are attached to the particle surface. Third, encapsulation allows for the incorporation of more antigens, biological agents, and / or chemical agents into the particles. Fourth, encapsulation allows for the easier use of complex protein antigens or organ homogenates (e.g., pancreatic homogenate in type 1 diabetes or peanut extract in peanut allergy). Finally, encapsulation of antigens, biological agents, and / or chemical agents within particles, instead of conjugation to the particle surface, maintains a net negative charge on the particle surface.

[0062] In certain embodiments, the present invention provides a carrier having cells or other biological or chemical agents thereon (or therein). When cells are used, the carrier is not limited to a specific type of cell. In some embodiments, the carrier has pancreatic islet cells thereon. In some embodiments, a microporous carrier further has ECM proteins and / or exendin-4 thereon. The carrier is not limited to a specific type. In some embodiments, the carrier has regions of different porosity (e.g., pore size, pore depth, and / or pore density). In some embodiments, the carrier has pharmaceutical agents, DNA, RNA, extracellular matrix proteins, exendin-4, etc. thereon (or therein). In certain embodiments, the present invention provides a method for transplanting pancreatic islet cells using such a carrier. In certain embodiments of the present invention, the inducing antigen is a single isolated molecule or a recombinantly produced molecule. To treat conditions in which the target antigen is dispersed in various locations within the host, it is generally necessary for the inducing antigen to be identical to or immunologically related to the target antigen. Examples of such antigens include most polynucleotide antigens and certain carbohydrate antigens (such as blood group antigens).

[0063] Any suitable antigen may find use within the scope of the present invention. In some embodiments, the inducing antigen contributes to the specificity of the induced tolerogenic response. The inducing antigen may or may not be the same as the target antigen, which is the antigen present or given to the subject being treated that is the target of the unwanted immunological response and to which tolerance is desired.

[0064] If the target antigen is selectively expressed in a particular organ, cell, or tissue type, the practitioner again has the option of using an inducing antigen that is identical to or immunologically related to the target antigen. However, there is also the additional option of using an antigen that is a bystander to the target. This is an antigen that may not be immunologically related to the target antigen but is preferentially expressed in the tissue where the target antigen is expressed. The working theory behind the effectiveness of bystander suppression is that suppression is an active cell-mediated process that downregulates the effector arm of the immune response in target cells. Suppressor cells are specifically stimulated by the inducer antigen at mucosal surfaces and home to tissue sites where the bystander antigen is selectively expressed. Then, through interaction or cytokine-mediated mechanisms, the localized suppressor cells downregulate nearby effector cells (or their inducers), regardless of what they are responding to. When effector cells are specific for a target different from the inducing antigen, the result is a bystander effect. For further details on bystander responses and a list of tolerogenic peptides that have this effect, the reader is referred to International Patent Publication WO 93 / 16724. The bystander theory means that one of skill in the art need not identify or isolate the particular target antigen to which tolerance is desired in order to practice the present invention. The practitioner need only be able to obtain at least one molecule selectively expressed at the target site to be used as the inducing antigen.

[0065] In certain embodiments of the present invention, the inducing antigen is not in the same form as that expressed in the individual receiving treatment, but is a fragment or derivative thereof. Inducing antigens of the present invention include peptides based on molecules of appropriate specificity but adapted by fragmentation, residue substitution, labeling, conjugation, and / or fusion with peptides having other functional properties. Adaptation may be performed for any desired purpose, including, but not limited to, eliminating any undesirable properties, such as toxicity or immunogenicity, or enhancing any desirable properties, such as mucosal binding, mucosal penetration, or stimulation of the tolerogenic arm of the immune response. Terms such as insulin peptide, collagen peptide, and myelin basic protein peptide, as used herein, refer not only to the complete subunits, but also to allotypes and synthetic variants, fragments, fusion peptides, conjugates, and other derivatives that contain regions of homology (preferably 70% identical, more preferably 80% identical, and even more preferably 90% identical at the amino acid level) of at least 10, and preferably 20, consecutive amino acids of the respective molecules that are analogs, and which regions of homology of the derivatives share the ability to induce tolerance to the target antigen with their respective parent molecules.

[0066] It should be recognized that the tolerogenic region of an inducing antigen is often different from the immunodominant epitope that stimulates antibody response. A tolerogenic region is generally a region that can be presented in specific cell interactions involving T cells. A tolerogenic region may exist, and when the complete antigen is presented, tolerance can be induced. Certain antigens contain latent tolerogenic regions, in that the processing and presentation of natural antigens do not usually induce tolerance. Details of latent antigens and their identification can be found in International Patent Publication WO 94 / 27634.

[0067] In certain embodiments of the invention, two, three, or more inducing antigens are used. It may be desirable to implement these embodiments when multiple target antigens are present, or to provide multiple bystanders of the target. For example, both insulin and glucagon can be mixed with a mucosally binding component in the treatment of diabetes. It may also be desirable to provide a cocktail of antigens that covers several possible alternative targets. For example, a cocktail of histocompatibility antigen fragments can be used to tolerize a subject facing a future transplant with an allograft of unknown phenotype. Allovariant regions of human leukocyte antigens are well known in the art (e.g., Immunogenetics 29:231, 1989). In another example, a mixture of allergens can serve as an inducing antigen for the treatment of atopy.

[0068] Depending on the nature of the molecule, inducing antigens can be prepared by many techniques known in the art.Enriched polynucleotides, polypeptides, and carbohydrate antigens can be isolated from the cells of the species to be treated.Short peptides are conveniently prepared by amino acid synthesis.Longer proteins of known sequence can be prepared by synthesizing coding sequences or PCR amplifying coding sequences from natural sources or vectors, and then expressing the coding sequences in suitable bacterial or eukaryotic host cells.

[0069] In certain embodiments of the invention, the combination comprises a complex mixture of antigens obtained from cells or tissues, one or more of which serve as inducing antigens. The antigens may be in the form of whole cells, either intact or treated with fixatives such as formaldehyde, glutaraldehyde, or alcohol. The antigens may also be in the form of cell lysates, prepared by detergent solubilization or mechanical disruption of cells or tissues followed by clarification. Antigens may also be obtained by enrichment of plasma membranes by techniques such as subcellular fractionation, particularly differential centrifugation, followed by optional detergent solubilization and dialysis. Other separation techniques, such as affinity or ion-exchange chromatography of solubilized membrane proteins, are also suitable.

[0070] In one embodiment, the antigenic peptide or protein is an autoantigen, an alloantigen, or a transplantation antigen. In yet another specific embodiment, the autoantigen is selected from the group consisting of myelin basic protein, collagen or fragments thereof, DNA, nuclear and nucleolar proteins, mitochondrial proteins, and pancreatic beta cell proteins.

[0071] The present invention provides for the induction of tolerance to autoantigens for the treatment of autoimmune diseases by administering the antigen to which tolerance is desired. For example, autoantibodies against myelin basic protein (MBP) are observed in patients with multiple sclerosis; therefore, MBP antigen peptides or proteins delivered using the compositions of the present invention can be used in the present invention to treat and prevent multiple sclerosis.

[0072] As another non-limiting example, an individual who is a candidate for a transplant from a fraternal twin may suffer from rejection of the transplanted cells, tissue, or organ because the transplanted antigen is foreign to the recipient. Prior tolerance of the recipient individual to the intended transplant inhibits or reduces subsequent rejection. By practicing the present invention, reduction or elimination of long-term anti-rejection therapy may be achieved. In another example, many autoimmune diseases are characterized by cellular immune responses to endogenous or self-antigens. Tolerance of the immune system to endogenous antigens is desirable for disease control.

[0073] In a further example, sensitization of an individual to industrial pollutants or chemicals, such as may be encountered in the workplace, presents a risk for an immune response. Prior tolerance of an individual's immune system to chemicals, particularly in the form of chemicals that react with the individual's endogenous proteins, may be desirable to prevent the subsequent occupational development of an immune response.

[0074] Allergens are other antigens to which tolerance of the immune response is also desired. In one embodiment, the antigen is gliadin. In a further embodiment, the antigen is A-gliadin.

[0075] In particular, even in diseases where pathogenic autoantigens are unknown, bystander suppression can be induced using antigens that exist in close anatomical proximity.For example, autoantibodies against collagen have been observed in rheumatoid arthritis, and therefore, to treat rheumatoid arthritis, collagen-encoding genes can be used as gene modules that express antigens (see, for example, Choy (2000) Curr Opin Investig Drugs 1:58-62).In addition, tolerance to β cell autoantigens can be used to prevent the onset of type 1 diabetes (see, for example, Bach and Chatenoud (2001) Ann Rev Immunol 19:131-161).

[0076] As another example, autoantibodies against myelin oligodendrocyte glycoprotein (MOG) have been observed in autoimmune encephalomyelitis, as well as in many other CNS diseases, and even multiple sclerosis (see, e.g., Iglesias et al. (2001) Glia 36:22-34). Thus, the use of constructs expressing the MOG antigen in the present invention allows for the treatment of multiple sclerosis, as well as related autoimmune disorders of the central nervous system.

[0077] Further examples of candidate autoantigens for use in treating autoimmune diseases include pancreatic beta cell antigens, insulin, and GAD for treating insulin-dependent diabetes; type 11 collagen, human cartilage protein, and phospholipase A (GPA) for treating rheumatoid arthritis; 39 (HCgp39) and gpl30-RAPS; myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG, see above) for treating multiple sclerosis; fibrillarin and small nucleolar proteins (snoRNPs) for treating scleroderma; thyroid-stimulating factor receptor (TSH-R) used in treating Graves' disease; nuclear antigens, histones, glycoprotein gp70, and ribosomal proteins used in treating systemic lupus erythematosus; pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PCD-E2) used in treating primary biliary cirrhosis; hair follicle antigens used to treat alopecia areata; and human tropomyosin isoform 5 (hTM5) used in treating ulcerative colitis.

[0078] Combinations can be tested for their ability to promote tolerance by performing experiments with isolated cells or in animal models.

[0079] In some embodiments, the tolerogenic composition of the present invention contains an apoptosis signaling molecule (e.g., in addition to the antigenic peptide or other antigenic molecule). In some embodiments, the apoptosis signaling molecule binds to and / or associates with the surface of the carrier. In some embodiments, the apoptosis signaling molecule causes the carrier to be recognized as an apoptotic body by the host's antigen-presenting cells (such as cells of the host's reticuloendothelial system), allowing the associated peptide epitope to be presented in a tolerance-inducing manner. Without being bound by theory, this is presumed to prevent the upregulation of molecules involved in immune cell stimulation, such as MHC class I / II and costimulatory molecules. These apoptosis signaling molecules may also function as phagocytosis markers. For example, apoptosis signaling molecules suitable for the present invention are described in U.S. Patent Application Publication No. 20050113297, which is incorporated herein by reference in its entirety. Molecules suitable for the present invention include molecules that target phagocytes, including macrophages, dendritic cells, monocytes, and neutrophils.

[0080] In some embodiments, molecules suitable as apoptosis signaling molecules act to enhance the tolerance of associated peptides. Furthermore, carriers bound to apoptosis signaling molecules may be bound by Clq in the recognition of apoptotic cells (Paidassi et al., (2008) J. Immunol. 180:2329-2338; incorporated herein by reference in its entirety). For example, molecules that may be useful as apoptosis signaling molecules include phosphatidylserine, annexin-1, annexin-5, milk fat globule-EGF-factor 8 (MFG-E8), or the thrombospondin family (e.g., thrombospondin-1 (TSP-1)). Various molecules suitable for use as apoptosis signaling molecules in conjunction with the present invention are discussed, for example, in U.S. Patent Application No. 2012 / 0076831, incorporated herein by reference in its entirety).

[0081] In some embodiments, the apoptosis signaling molecule may be conjugated to an antigen-specific peptide. In some cases, the apoptosis signaling molecule and the antigen-specific peptide are conjugated by creating a fusion protein. For example, the fusion protein may comprise at least one antigen-specific peptide (or a fragment or variant thereof) linked to at least one apoptosis signaling molecule (or a fragment or variant thereof). With respect to creating a fusion protein, the terms "fusion protein," "fusion peptide," "fusion polypeptide," and "chimeric peptide" are used interchangeably. Suitable fragments of antigen-specific peptides include any fragment of the full-length peptide that retains the function of generating the desired antigen-specific tolerance function of the present invention. Fusion proteins may be created by various means understood in the art (e.g., gene fusion, chemical conjugation, etc.). The two proteins may be fused either directly or via an amino acid linker. While the polypeptides forming the fusion protein are typically linked C-terminally to N-terminally, they can also be linked C-terminally to C-terminally, N-terminally to N-terminally, or N-terminally to C-terminally. The polypeptides of the fusion protein may be in any order. A peptide linker sequence can be used to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structure. Amino acid sequences that can be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262 (1986); U.S. Patent Nos. 4,935,233 and 4,751,180, each of which is incorporated herein by reference in its entirety. Linker sequences can generally be from 1 to about 50 amino acids in length. In some embodiments, for example, when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate functional domains and prevent steric hindrance, a linker sequence is not necessary and / or is not used.

[0082] A surrogate for tolerogenic activity is the ability of an intact antigen or fragment to stimulate the production of appropriate cytokines at the target site. The immunoregulatory cytokine released by T suppressor cells at the target site is thought to be TGF-β (Miller et al., Proc. Natl. Acad. Sci. USA 89:421, 1992). Other factors that may be produced during tolerance are the cytokines IL-4 and IL-10, and the mediator PGE. In contrast, lymphocytes in tissues undergoing active immune destruction secrete cytokines such as IL-1, IL-2, IL-6, and γ-IFN. Therefore, the effectiveness of a candidate inducing antigen can be assessed by measuring its ability to stimulate the appropriate types of cytokines.

[0083] Taking this into consideration, syngeneic animals can be used as donors for in vitro cell assays to rapidly screen for tolerogenic epitopes of inducing antigens, effective mucosal-binding components, effective combinations, or effective modes and schedules of mucosal administration. Animals are treated at the mucosal surface with the test composition and, at one point, challenged with the target antigen parenterally in Freund's complete adjuvant. Splenocytes are isolated and cultured ex vivo in the presence of the target antigen at a concentration of approximately 50 μg / mL. To map the location of tolerogenic epitopes, the target antigen may be replaced by a candidate protein or subfragment. Cytokine secretion into the culture medium can be quantified by standard immunoassays.

[0084] The ability of cells to suppress the activity of other cells can be determined using cells isolated from animals immunized with the target antigen or by generating cell lines reactive to the target antigen (Ben-Nun et al., Eur. J. Immunol. 11:195, 1981; incorporated herein by reference in its entirety). In one variation of this experiment, suppressor cell populations are mildly irradiated (approximately 1000-1250 rads) to prevent proliferation, and the suppressors are co-cultured with responder cells, followed by quantification of the responder's proliferative activity using tritiated thymidine incorporation (or MTT). In another variation, suppressor and responder cell populations are cultured in the upper and lower chambers of a dual-chamber transwell culture system (Costar, Cambridge Mass.), allowing the populations to be co-incubated within 1 mm of each other and separated by a polycarbonate membrane (WO 93 / 16724). This approach does not require irradiation of the suppressor cell population, as the proliferative activity of the responders can be measured separately.

[0085] In an embodiment of the present invention, if the target antigen is already present in an individual, there is no need to isolate the antigen or pre-combine it with the mucosal-binding component.For example, the antigen may be expressed in a specific manner in an individual as a result of a pathological condition (such as inflammatory bowel disease or celiac disease) or by the digestion of food allergens.Testing is carried out by administering the mucosal-binding component in one or more doses or formulations and determining its ability to promote tolerization to the antigen in situ.

[0086] The effectiveness of compositions and administration modes for treating specific diseases can also be demonstrated in corresponding animal disease models.Depending on the model used, the therapeutic ability to alleviate or delay the symptoms of disease is monitored at the level of the circulating biochemical and prominent immunological characteristics of disease, the immunohistology of affected tissue, and gross clinical characteristics.Non-limiting examples of animal models that can be used for testing are included in the following section.

[0087] The present invention contemplates modulating tolerance by modulating TH1, TH2, or TH17 responses, or a combination of these responses. Modulating a TH1 response includes, for example, altering the expression of interferon-gamma. Modulating a TH2 response includes, for example, altering the expression of any combination of IL-4, IL-5, IL-10, and IL-13. Typically, an increase (decrease) in a TH2 response involves an increase (decrease) in the expression of at least one of IL-4, IL-5, IL-10, or IL-13; more typically, an increase (decrease) in a TH2 response involves an increase (decrease) in the expression of at least two of IL-4, IL-5, IL-10, or IL-13; most typically, an increase (decrease) in a TH2 response involves an increase (decrease) in the expression of at least three of IL-4, IL-5, IL-10, or IL-13, but ideally, an increase (decrease) in a TH2 response involves an increase (decrease) in the expression of all of IL-4, IL-5, IL-10, and IL-13. Modulating TH17 includes, for example, altering the expression of TGF-β, IL-6, IL-21, and IL23, and influencing the levels of IL-17, IL-21, and IL-22.

[0088] Other suitable methods for assessing the effectiveness of the compositions and methods of the present invention are understood in the art, for example, as described in U.S. Patent Application No. 2012 / 0076831, which is incorporated herein by reference in its entirety.

[0089] Certain embodiments of the present invention relate to priming immune tolerance in individuals not previously tolerized by therapeutic intervention. These embodiments generally involve multiple administrations of a combination of an antigen and a mucosal-binding component. To achieve long-lasting results, typically at least three administrations, frequently at least four administrations, and occasionally at least six administrations are administered during priming, although subjects may show signs of tolerance early in the treatment course. In most cases, each dose is administered as a bolus, although sustained-release formulations capable of mucosal release are also suitable. When multiple administrations are administered, the interval between administrations is generally between one day and three weeks, typically about three days to two weeks. Generally, the same antigen and mucosal-binding component are present at the same concentration, and administrations are administered to the same mucosal surface, although variations in any of these variables can be accommodated during the treatment course.

[0090] Other embodiments of the invention relate to boosting or extending the durability of previously established immune tolerance. These embodiments generally require a single administration or short-term treatment at a time when established tolerance has waned or is at risk of waning. Boosting generally occurs one month to one year, typically two to six months, after the priming or previous boost. The invention also includes embodiments involving regular maintenance of tolerance, with administration schedules administered twice weekly, weekly, biweekly, or any other regular schedule.

[0091] The particles of the present invention may be administered at any dose effective to reduce an inflammatory immune response in a subject in need thereof or to treat a bacterial or viral infection in a subject in need thereof. In certain embodiments, about 10 to about 10 particles are provided to an individual. In further embodiments, about 10 to about 10 particles are provided. In further embodiments, about 10 to about 10 particles are provided. In further embodiments, about 10 to about 10 particles are provided. In a preferred embodiment, the preferred dose is 0.1% solids / ml. Thus, for 0.5 μm beads, the preferred dose is about 4×10 beads, for 0.05 μm beads, the preferred dose is about 4×10 beads, and for 3 μm beads, the preferred dose is about 2×10 beads. However, any dose effective to treat the particular condition being treated is encompassed by the present invention.

[0092] The present invention is useful for the treatment of immune-related disorders such as autoimmune diseases, transplant rejection, and allergic reactions. The substitution of synthetic biocompatible particle systems for inducing immune tolerance can provide ease of manufacturing, wide availability of therapeutic agents, increased sample-to-sample uniformity, an increased number of potential treatment sites, and dramatically reduced potential for allergic responses to carrier cells.

[0093] As used herein, the term "immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxicity. Furthermore, the term immune response includes immune responses indirectly influenced by T cell activation, e.g., antibody production (humoral response), and activation of cytokine-responsive cells, e.g., macrophages. Immune cells involved in immune responses include lymphocytes, such as B cells and T cells (CD4+, CD8+, Th1 and Th2 cells); antigen-presenting cells (e.g., professional antigen-presenting cells, such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen-presenting cells, such as keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes); natural killer cells; and myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the modified particles of the present invention are effective in reducing the trafficking of inflammatory cells to the site of inflammation.

[0094] As used herein, the terms "anergy," "tolerance," or "antigen-specific tolerance" refer to the insensitivity of T cells to T cell receptor-mediated stimulation. Such insensitivity is generally antigen-specific and persists even after exposure to antigenic peptides has ceased. For example, anergy in T cells is characterized by the lack of cytokine production, such as IL-2. T cell anergy occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD3-mediated signal) in the absence of a second signal (costimulatory signal). Under these conditions, if the cells are re-exposed to the same antigen (even if the re-exposure occurs in the presence of costimulatory molecules), they are unable to produce cytokines and subsequently unable to proliferate. Therefore, the inability to produce cytokines prevents proliferation. However, anergic T cells can proliferate when cultured with cytokines (e.g., IL-2). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes, as measured by ELISA or a proliferation assay using an indicator cell line. Alternatively, reporter gene constructs may be used. For example, anergic T cells are unable to initiate transcription of the DL-2 gene, driven by a heterologous promoter under the control of the 5'IL-2 gene enhancer or by multimers of API sequences that can be found within the enhancer (Kang et al. 1992 Science. 257:1134).

[0095] As used herein, the term "immunological tolerance" refers to a method performed on a proportion of treated subjects compared to untreated subjects: a) a reduction in the level of a specific immunological response (believed to be mediated, at least in part, by antigen-specific effector T lymphocytes, B lymphocytes, antibodies, or equivalents thereof); b) a delay in the onset or progression of a specific immunological response; or c) a reduction in the risk of the onset or progression of a specific immunological response. "Specific" immunological tolerance occurs when immunological tolerance is selectively induced to a particular antigen compared to others. "Nonspecific" immunological tolerance occurs when immunological tolerance is indiscriminately induced to antigens that evoke inflammatory immune responses. "Semi-specific" immunological tolerance occurs when immunological tolerance is semi-discriminately induced to antigens that evoke pathogenic immune responses, but not to other antigens that evoke protective immune responses.

[0096] Tolerance to self-antigens and autoimmune diseases is achieved through various mechanisms, including negative selection of autoreactive T cells in the thymus and peripheral tolerance of autoreactive T cells that escape thymic deletion and are found in the periphery. Examples of mechanisms that provide peripheral T cell tolerance include "ignorance" of self-antigens, anergy or unresponsiveness to self-antigens, cytokine immune deviation, and activation-induced cell death of autoreactive T cells. Furthermore, regulatory T cells have been shown to mediate peripheral tolerance. See, for example, Walker et al. (2002) Nat. Rev. Immunol. 2:11-19; Shevach et al. (2001) Immunol. Rev. 182:58-67. In some situations, peripheral tolerance to self-antigens is lost (or destroyed), resulting in an autoimmune response. For example, in animal models of EAE, activation of antigen-presenting cells (APCs) by TLR innate immune receptors has been shown to break self-tolerance and ultimately induce EAE (Waldner et al. (2004) J. Clin. Invest. 113:990-997).

[0097] Thus, in some embodiments, the present invention provides methods for increasing antigen presentation while suppressing or reducing TLR7 / 8, TLR9, and / or TLR7 / 8 / 9-dependent cellular stimulation. As described herein, administration of certain modified particles results in antigen presentation by DCs or APCs while suppressing TLR7 / 8, TLR9, and / or TLR7 / 8 / 9-dependent cellular responses associated with immunostimulatory polynucleotides. Such suppression can include a reduction in the levels of one or more TLR-associated cytokines.

[0098] As discussed above, the present invention provides novel compounds that have biological properties useful in the treatment of disorders mediated by Mac-1 and LFA-1.

[0099] Thus, in another aspect of the present invention, pharmaceutical compositions are provided that include immunomodulatory particles and, optionally, a pharmaceutically acceptable carrier. In certain embodiments, these compositions optionally further include one or more additional therapeutic agents. Alternatively, the modified particles of the present invention may be administered to a patient in need thereof in combination with one or more other therapeutic agents. For example, the additional therapeutic agent for co-administration with the compounds of the present invention or for inclusion in a pharmaceutical composition comprising the compounds of the present invention may be an approved anti-inflammatory agent or any one of a number of drugs currently undergoing approval by the U.S. Food and Drug Administration for eventual approval for the treatment of any disorder characterized by an uncontrolled inflammatory immune response or bacterial or viral infection. It should also be understood that some of the modified particles of the present invention may be present for treatment in free form or, if desired, as a pharmaceutically acceptable derivative thereof.

[0100] As used herein, the pharmaceutical compositions of the present invention further comprise pharmaceutically acceptable carriers, including any solvents, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, suitable for the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for their preparation. Except insofar as any conventional carrier medium is incompatible with the compounds of the present invention, for example, by producing any undesirable biological effects or otherwise interacting adversely with any other component(s) of the pharmaceutical composition, its use is contemplated within the scope of the present invention. Some examples of materials that can function as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil and soybean oil; propylene glycol; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic, compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0101] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.In addition to active compounds, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.In addition to inert diluents, oral compositions may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings, and fragrances.

[0102] The particles of the invention may be administered orally, intranasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously, hi one embodiment, the particles of the invention are administered intravenously.

[0103] The effective amount and administration method of the present invention for modulating an immune response may vary based on the individual, the condition being treated, and other factors apparent to one of skill in the art. Factors to be considered include the route of administration and the number of doses administered. Such factors are known in the art, and making such determinations without undue experimentation is well within the skill of one of ordinary skill in the art. A suitable dosage range is one that provides the desired modulation of immunity. Useful dosage ranges of the carrier, expressed as the amount of carrier delivered, may be, for example, approximately 0.5-10 mg / kg, 1-9 mg / kg, 2-8 mg / kg, 3-7 mg / kg, 4-6 mg / kg, 5 mg / kg, 1-10 mg / kg, or 5-10 mg / kg. Alternatively, dosages may be administered based on particle number. For example, useful dosages of the carrier, expressed as the amount of carrier delivered, may be, for example, approximately 10, 10, 10, 10, 10, or more particles per dose. The absolute amount administered to each patient depends on pharmacological properties such as bioavailability, clearance rate, and route of administration. Details of pharmaceutically acceptable carriers, diluents, and excipients, as well as methods for preparing pharmaceutical compositions and formulations, are provided in Remmington's Pharmaceutical Sciences 18th Edition, 1990, Mack Publishing Co., Easton, Pa., USA, which is incorporated herein by reference in its entirety.

[0104] The effective amount and method of administration of a particular carrier formulation may vary based on the individual patient, the desired outcome and / or type of disorder, the stage of the disease, and other factors apparent to one of skill in the art. The route(s) of administration useful in a particular application will be apparent to one of skill in the art. Routes of administration include, but are not limited to, topical, dermal, transdermal, transmucosal, epidermal, parenteral, gastrointestinal, and nasopharyngeal and pulmonary (including transbronchial and transalveolar). A suitable dosage range is one that provides sufficient IRP-containing composition to achieve a tissue concentration of approximately 1-50 μM as measured by blood levels. The absolute amount administered to each patient will depend on pharmacological properties such as bioavailability, clearance rate, and route of administration.

[0105] The present invention provides suitable carrier formulations for topical use, including, but not limited to, physiologically acceptable implants, ointments, creams, rinses, and gels. Exemplary routes of dermal administration are the least invasive routes, such as transdermal penetration, epidermal administration, and subcutaneous injection.

[0106] Transdermal administration is achieved by applying a cream, rinse, gel, etc., which allows the carrier to penetrate the skin and enter the bloodstream. Compositions suitable for transdermal administration include, but are not limited to, pharmaceutically acceptable suspensions, oils, creams, and ointments, which are applied directly to the skin or incorporated into a protective carrier, such as a transdermal device (so-called "patch"). Examples of suitable creams, ointments, etc. can be found, for example, in the Physician's Desk Reference. Transdermal penetration can also be achieved by iontophoresis, for example, using commercially available patches that continuously deliver products through intact skin for periods of several days or more. The use of this method allows for controlled penetration of pharmaceutical compositions at relatively high concentrations, allows for the infusion of concomitant drugs, and allows for the simultaneous use of absorption enhancers.

[0107] Parenteral administration routes include, but are not limited to, electrophoresis (iontophoresis) or direct injection, such as direct injection into a central venous line, intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. Carrier formulations suitable for parenteral administration are generally formulated in USP water or water for injection and may further include pH buffers, salt bulking agents, preservatives, and other pharmaceutically acceptable excipients. Immunoregulatory polynucleotides for parenteral injection may be formulated in pharmaceutically acceptable sterile isotonic solutions such as saline and phosphate-buffered saline for injection.

[0108] Alimentary routes of administration include, but are not limited to, ingestion and rectal routes, and can include, for example, the use of a pharmaceutically acceptable powder, pill, or liquid for ingestion, and a suppository for rectal administration.

[0109] Nasopharyngeal and pulmonary administration is achieved by inhalation and includes delivery routes such as intranasal, transbronchial, and transalveolar routes. The present invention includes formulations of carriers suitable for administration by inhalation, including, but not limited to, liquid suspensions for forming aerosols, and powder forms for dry powder inhalation delivery systems. Devices suitable for administration by inhalation of carrier formulations include, but are not limited to, atomizers, vaporizers, nebulizers, and dry powder inhalation delivery devices.

[0110] Injectable preparations, for example, sterile injectable aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersants or wetting agents and suspending agents.Sterile injectable preparations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, for example, as solutions in 1,3-butanediol.Among acceptable vehicles and solvents that can be used are water, Ringer's solution, USP, and isotonic sodium chloride solution.In addition, sterile, fixed oils are commonly used as solvents or suspending media.For this purpose, any non-irritating, fixed oil can be used, including synthetic mono- or diglycerides.Fatty acids, such as oleic acid, can also be used to prepare injectable solutions.

[0111] Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0112] To prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by the use of liquid suspensions or crystalline or amorphous materials with poor water solubility. Consequently, the rate of absorption of the drug depends on its dissolution rate, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0113] In some embodiments, the synthetic biodegradable particles of the present invention offer ease of manufacture, broad availability of therapeutic agents, and increased treatment sites. In certain embodiments, surface-functionalized biodegradable poly(lactide-co-glycolide) particles with a high density of surface carboxylate groups, synthesized using the surfactant poly(ethylene-alt-maleic anhydride), provide a carrier that offers numerous advantages over other carrier particles and / or surfaces. Experiments conducted during the development of embodiments of the present invention demonstrated the conjugation of peptides (e.g., PLP139-151 peptide) to these particles. Particles bearing such peptides have been shown to be effective in preventing disease development and in inducing immunological tolerance (e.g., in the SJL / J PLP139-151 / CFA-induced R-EAE mouse model of multiple sclerosis). Peptide-conjugated carriers of the present invention offer numerous advantages over other tolerance-inducing structures. In some embodiments, the particles are biodegradable and therefore do not persist in the body for extended periods. The time for complete degradation can be controlled. In some embodiments, particles are functionalized to promote internalization without activating cells (e.g., phosphatidylserine loaded onto PLG microspheres). In some embodiments, particles incorporate targeting ligands for specific cell populations. In some embodiments, anti-inflammatory cytokines such as IL-10 and TGF-β are included on or within the particles to limit activation of cell types that internalize the particles and to promote tolerance induction via energy and / or deletion and activation of regulatory T cells.

[0114] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the modified particles are mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants, such as glycerol; d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicate silicas, and sodium carbonate; e) solution retardants, such as paraffin; f) absorption accelerators, such as quaternary ammonium compounds; g) humectants, such as, for example, cetyl alcohol and glycerol monostearate; h) absorbents, such as kaolin and bentonite clay; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0115] Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar and high molecular weight polyethylene glycols. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may optionally contain opacifying agents and may be of a composition that releases the active ingredient(s) only, or preferentially, in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar and high molecular weight polyethylene glycols.

[0116] The modified particles may also be in microencapsulated form with one or more excipients as described above.Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings, release-controlling coatings, and other coatings well known in the pharmaceutical formulation art.In such solid dosage forms, the active compound may be mixed with at least one inert diluent, such as sucrose, lactose, and starch.In addition, such dosage forms can contain additional substances other than inert diluents, such as tableting lubricants and other tableting aids, as is common practice.In the case of capsules, tablets, and pills, the dosage forms can also contain buffering agents.They may optionally contain opacifying agents, and they may be of a composition that releases only the modified particles, or selectively in a specific part of the digestive tract, optionally in a delayed manner.Examples of embedding compositions that can be used include polymeric substances and waxes.

[0117] The present invention encompasses pharmaceutically acceptable topical formulations of the modified particles of the present invention. As used herein, the term "pharmaceutically acceptable topical formulation" refers to any formulation pharmaceutically acceptable for intradermal administration of the modified microparticles of the present invention by application of the formulation to the epidermis. In certain embodiments of the present invention, the topical formulation comprises a carrier system. Pharmaceutically effective carriers include, but are not limited to, solvents (e.g., alcohols, polyalcohols, water), creams, lotions, ointments, oils, plasters, liposomes, powders, emulsions, microemulsions, and buffer solutions (e.g., hypotonic or buffered saline), or any other carrier known in the art for topical administration of pharmaceuticals. A more complete list of carriers known in the art is provided by standard reference texts in the art, such as Remington's Pharmaceutical Sciences, 16th Edition, 1980 and 17th Edition, 1985, both published by Mack Publishing Company, Easton, Pa., the disclosures of which are incorporated herein by reference in their entireties. In certain other embodiments, the topical formulations of the present invention may comprise an excipient. Any pharmaceutically acceptable excipient known in the art can be used to prepare the pharmaceutically acceptable topical formulation of the present invention.Examples of excipients that can be included in the topical formulation of the present invention include, but are not limited to, preservatives, antioxidants, moisturizers, emollients, buffers, solubilizers, other penetrants, skin protectants, surfactants, and propellants, and / or additional therapeutic agents used in combination with modified particles.Suitable preservatives include, but are not limited to, alcohols, quaternary amines, organic acids, parabens, and phenols.Suitable antioxidants include, but are not limited to, ascorbic acid and its esters, sodium bisulfite, butylated hydroxytoluene, butylated hydroxyanisole, tocopherol, and chelating agents such as EDTA and citric acid.Suitable moisturizers include, but are not limited to, glycerin, sorbitol, polyethylene glycol, urea, and propylene glycol.Suitable buffers for use with the present invention include, but are not limited to, citric acid, hydrochloric acid, and lactic acid buffers. Suitable solubilizers include, but are not limited to, quaternary ammonium chloride, cyclodextrin, benzyl benzoate, lecithin, and polysorbate. Suitable skin protectants that can be used in the topical formulations of the present invention include, but are not limited to, vitamin E oil, allantoin, dimethicone, glycerin, petrolatum, and zinc oxide.

[0118] In certain embodiments, the pharmaceutically acceptable topical formulation of the present invention comprises at least the modified particles of the present invention and a penetration enhancer.The selection of topical formulation depends on several factors, including the condition to be treated, the physicochemical characteristics of the compound of the present invention and other excipients present, their stability in the formulation, available manufacturing equipment, and cost constraints.As used herein, the term " penetration enhancer " preferably refers to an agent that can transport pharmacologically active compounds through the stratum corneum into the epidermis or dermis with little or no systemic absorption.A variety of compounds have been evaluated for their effectiveness in promoting the penetration rate of drugs through the skin. See, for example, Percutaneous Penetration Enhancers, Maibach HI and Smith HE (eds.), CRC Press, Inc., Boca Raton, Fla. (1995), which reviews the use and testing of various skin penetration enhancers, and Buyuktimkin et al., Chemical Means of Transdermal Drug Permeation Enhancement in Transdermal and Topical Drug Delivery Systems, Gosh TK, Pfister WR, Yum SI (Eds.), Interpharm Press Inc., Buffalo Grove, Ill. (1997). In certain exemplary embodiments, penetrating agents for use with the present invention include, but are not limited to, triglycerides (e.g., soybean oil), aloe compositions (e.g., aloe vera gel), ethyl alcohol, isopropyl alcohol, octylphenyl polyethylene glycol, oleic acid, polyethylene glycol 400, propylene glycol, N-decylmethyl sulfoxide, fatty acid esters (e.g., isopropyl myristate, methyl laurate, glycerol monooleate, and propylene glycol monooleate), and N-methylpyrrolidone.

[0119] In certain embodiments, the composition may be in the form of an ointment, paste, cream, lotion, gel, powder, solution, spray, inhalant, or patch. In certain exemplary embodiments, the formulation of the composition according to the present invention is a cream, which may further contain saturated or unsaturated fatty acids such as stearic acid, palmitic acid, oleic acid, palmito-oleic acid, cetyl alcohol, or oleyl alcohol, with stearic acid being particularly preferred. Creams of the present invention may also contain a nonionic surfactant, such as polyoxy-40 stearate. In certain embodiments, the active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers, if necessary. Ophthalmic formulations, ear drops, and eye drops are also contemplated within the scope of the present invention. Furthermore, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms are made by dissolving or dispersing the compound in a suitable medium. As mentioned above, penetration enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0120] The modified particles can be administered by aerosol. This can be achieved by preparing an aqueous aerosol, liposomal preparation, or solid particles containing the modified particles. Non-aqueous (e.g., fluorocarbon propellant) suspensions can also be used.

[0121] Usually, aqueous aerosol is prepared by formulating the aqueous solution or suspension of drug with conventional pharmaceutically acceptable carrier and stabilizer.Carrier and stabilizer vary according to the requirements of specific compound, but typically comprise nonionic surfactant (Tween, Pluronic or polyethylene glycol), harmless protein such as serum albumin, sorbitan ester, oleic acid, lecithin, amino acid such as glycine, buffer, salt, sugar or sugar alcohol.Aerosol is generally prepared from isotonic solution.

[0122] It should also be understood that the modified particles and pharmaceutical compositions of the present invention can be formulated and used in combination therapy, i.e., the compounds and pharmaceutical compositions can be formulated with one or more other desired therapeutic agents or medical procedures, or can be administered simultaneously, before, or after. The specific combination of therapies (therapeutics or procedures) to be used in a combined administration regimen will take into account the compatibility of the desired therapeutic agents and / or procedures and the desired therapeutic effect to be achieved. It should also be understood that the therapies used may achieve the desired effect for the same disorder (e.g., the compounds of the present invention may be administered simultaneously with another anti-inflammatory agent), or may achieve different effects (e.g., control of any adverse effects).

[0123] In certain embodiments, pharmaceutical compositions containing the modified particles of the present invention further comprise one or more additional therapeutically active ingredients (e.g., anti-inflammatory and / or palliative). For purposes of the present invention, the term "palliative" refers to treatment that focuses on reducing the symptoms of a disease and / or the side effects of a treatment regimen, but is not curative. For example, palliative treatment includes analgesics, antiemetics, and antiemetics.

[0124] The present invention provides methods of modulating an immune response in an individual, preferably a mammal, more preferably a human, comprising administering to the individual a modified particle as described herein. Methods of immunomodulation provided by the present invention include, but are not limited to, suppressing and / or inhibiting innate or adaptive immune responses, including immune responses stimulated by immunostimulatory polypeptides or viral or bacterial components.

[0125] The modified particles are administered in an amount sufficient to modulate the immune response. As described herein, modulation of the immune response can be humoral and / or cellular, and is measured using standard techniques in the art and as described herein.

[0126] In some embodiments, the compositions described herein are administered together with (e.g., simultaneously with, before, or after) implants (e.g., devices) and / or grafts (e.g., tissues, cells, organs) to mediate, neutralize, modulate, and / or attenuate the immune response associated therewith.

[0127] In certain embodiments, the individual suffers from a disorder associated with undesired immune activation, such as an allergic disease or condition, allergy, and asthma. An individual with an allergic disease or asthma is an individual with a recognizable symptom of an existing allergic disease or asthma. For example, tolerance can be induced in such individuals by particles complexed with specific foods (e.g., peanut protein, etc.), injected substances (e.g., bee venom protein, etc.), or inhaled substances (e.g., ragweed pollen protein, pet dander protein, etc.) that induce allergic reactions.

[0128] In certain embodiments, the individual suffers from a disorder associated with unwanted immune activation, such as an autoimmune disease or an inflammatory disease. An individual with an autoimmune or inflammatory disease is an individual with a pre-existing, recognizable symptom of an autoimmune or inflammatory disease. For example, tolerance can be induced in such individuals by particles complexed with the relevant autoantigen that drives the specific autoimmune disease.

[0129] In certain embodiments, the individual suffers from a disease associated with enzyme replacement therapy. For example, tolerance can be induced in such an individual by particles complexed with an enzyme that the patient with the genetic deficiency cannot produce, to prevent the patient from forming a neutralizing antibody response to the recombinantly produced enzyme administered to treat the particular deficiency (e.g., tolerance to human factor VIII in patients with hemophilia due to a genetic defect in the ability to make factor VIII).

[0130] In certain embodiments, the individual suffers from a disorder related to the treatment of the disease. In the case of recombinant antibodies, for example, tolerance is induced to humanized antibodies used in therapeutic settings to prevent patients from forming neutralizing antibodies to the antibody therapeutic (tolerance to humanized immune subset-depleting antibodies or anti-cytokine antibodies used as treatments for autoimmune diseases).

[0131] Autoimmune diseases can be classified into two broad categories: organ-specific and systemic. Autoimmune diseases include, but are not limited to, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), type I diabetes, type II diabetes, multiple sclerosis (MS), immune-mediated infertility such as premature menopause, scleroderma, Sjogren's disease, vitiligo, alopecia (baldness), polyglandular deficiency, Graves' disease, hypothyroidism, polymyositis, pemphigus vulgaris, pemphigus foliaceus, inflammatory bowel diseases including Crohn's disease and ulcerative colitis, autoimmune hepatitis including those associated with hepatitis B virus (HBV) and hepatitis C virus (HCV), hypopituitarism, graft-versus-host disease (GvHD), myocarditis, Addison's disease, autoimmune skin diseases, uveitis, pernicious anemia, celiac disease, and hypoparathyroidism.

[0132] In addition, autoimmune diseases include, but are not limited to, Hashimoto's thyroiditis, type I and type II autoimmune polyglandular syndrome, paraneoplastic pemphigus, bullous pemphigoid, dermatitis herpetiformis, linear IgA disease, epidermolysis bullosa acquisita, erythema nodosum, pemphigoid of pregnancy, cicatricial pemphigoid, essential mixed cryoglobulinemia, chronic bullous disease of childhood, hemolytic anemia, thrombocytopenic purpura, Goodpasture's syndrome, autoimmune neutropenia, myasthenia gravis, Eaton-Lambert myasthenic syndrome, stiff-person syndrome, acute disseminated encephalomyelitis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy with conduction block, Also includes chronic neuropathy with monoclonal globulinemia, opsoclonus-myoclonus syndrome, cerebellar degeneration, encephalomyelitis, retinopathy, primary biliary sclerosis, sclerosing cholangitis, gluten-sensitive enteropathy, ankylosing spondylitis, reactive arthritis, polymyositis / dermatomyositis, mixed connective tissue disease, Behçet's syndrome, psoriasis, polyarteritis nodosa, allergic vasculitis and granulomatosis (Churg-Strauss disease), polyangiitis overlap syndrome, hypersensitivity vasculitis, Wegener's granulomatosis, temporal arteritis, Takayasu's arteritis, Kawasaki disease, isolated central nervous system vasculitis, thromboangiitis obliterans, sarcoidosis, glomerulonephritis, and cold illness. These conditions are well known in the medical arts and are described, for example, in Harrison's Principles of Internal Medicine, 14th ed., Fauci AS et al., eds., New York: McGraw-Hill, 1998.

[0133] Animal models for studying autoimmune diseases are known in the art. For example, animal models that are considered most similar to human autoimmune diseases include animal strains that naturally develop a particular disease at a high incidence. Examples of such models include, but are not limited to, non-obese diabetic (NOD) mice, which develop a disease similar to type 1 diabetes, and animals prone to lupus-like diseases, such as New Zealand hybrid, MRL-Faslpr, and BXSB mice. Animal models in which autoimmune diseases are induced include, but are not limited to, experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, collagen-induced arthritis (CIA), a model of rheumatoid arthritis, and experimental autoimmune uveitis (EAU), a model of uveitis. Animal models of autoimmune diseases have also been created by genetic engineering, including, for example, IL-2 / IL-10 knockout mice for inflammatory bowel disease, Fas or Fas ligand knockout mice for SLE, and IL-I receptor antagonist knockout mice for rheumatoid arthritis.

[0134] In certain embodiments, the individual is suffering from a bacterial or viral infection. An individual with a bacterial or viral infection is one who has recognizable symptoms of an existing bacterial or viral infection.

[0135] A non-limiting list of viral infections that can be treated using the modified particles of the present invention includes herpesvirus infection, hepatitis virus infection, West Nile virus infection, flavivirus infection, influenza virus infection, rhinovirus infection, papillomavirus infection, paramyxovirus infection, parainfluenza virus infection, and retrovirus infection.Preferred viruses are viruses that infect the central nervous system of a subject.Most preferred viruses are viruses that cause encephalitis or meningitis.

[0136] A non-limiting list of bacterial infections treatable using the modified particles of the present invention includes staphylococcal infections, streptococcal infections, mycobacterial infections, bacillus infections, salmonella infections, vibrio infections, spirochete infections, and neisseria infections. Preferred are bacteria that infect the central nervous system of a subject. Most preferred are bacteria that cause encephalitis or meningitis.

[0137] In some embodiments, the present invention relates to the use of the compositions of the present invention before the onset of disease. In other embodiments, the present invention relates to the use of the compositions of the present invention to inhibit progressing disease. In some embodiments, the present invention relates to ameliorating disease in a subject. Ameliorating disease in a subject refers to treating, preventing, or suppressing disease in a subject.

[0138] In some embodiments, the present invention relates to preventing disease relapse. For example, an unwanted immune response may be generated at one region of a peptide (such as an antigenic determinant). Disease relapse associated with an unwanted immune response may result from immune response attack at a different region of the peptide. Because the immunomodulatory particles of the present invention do not contain attached peptide or antigenic moieties, the particles are effective against multiple epitopes. T cell responses in some immune response disorders, including MS and other ThI / 17-mediated autoimmune diseases, can be dynamic and evolve during the course of relapsing-remitting disease and / or chronic progressive disease. The dynamic nature of the T cell repertoire impacts the treatment of specific diseases, as targets may change as the disease progresses. Previously, predicting disease progression required existing knowledge of response patterns. The present invention provides compositions that can counteract "epitope spreading," a dynamically changing disease effect. A known model of relapse is the immune response to proteolipid protein (PLP), a model of multiple sclerosis (MS). The initial immune response may be caused by a response to PLP139-15, and subsequent disease development may be caused by a recurrent immune response to PLP[pi]s-iβi.

[0139] Other embodiments of the present invention relate to transplantation, which refers to the transfer of a tissue sample or graft from a donor individual to a recipient individual and is frequently performed on human recipients in need of the tissue to restore the physiological function provided by the tissue. Transplanted tissues include (but are not limited to) whole organs such as kidneys, livers, hearts, and lungs; organ components such as skin grafts and corneas of the eye; and cell suspensions such as bone marrow cells and cultures of cells selected and expanded from bone marrow or circulating blood, and whole blood transfusions.

[0140] Serious potential complications of any transplant arise from antigenic differences between the host-recipient and the transplanted tissue. Depending on the nature and extent of the differences, there may be a risk of immunological attack of the graft by the host, of the host by the graft, or both. The degree of risk is determined by tracking response patterns in a population of similarly treated subjects with similar phenotypes and correlating various possible contributing factors according to well-accepted clinical procedures. The immunological attack may be the result of a pre-existing immunological response (such as pre-formed antibodies) or one that is initiated around the time of transplantation (such as the production of TH cells). Antibodies, TH cells, or Tc cells may interact with each other and with various effector molecules and effector cells in any combination. However, the antigens involved in the immune response are generally unknown, thus presenting challenges in designing antigen-specific therapies or inducing antigen-specific tolerance.

[0141] Certain embodiments of the present invention relate to reducing the risk of host-versus-graft disease, which causes the recipient to reject a tissue transplant. Treatment can be administered to prevent or reduce the effects of hyperacute, acute, or chronic rejection. Treatment is preferentially initiated well before transplantation so that tolerance is induced when the transplant is placed; however, if this is not possible, treatment may be initiated simultaneously with or after transplantation. Regardless of the time of initiation, treatment generally continues at regular intervals for at least the first month after transplantation. If sufficient graft adaptation occurs, follow-up administration may not be necessary, but can be resumed if there is evidence of either graft rejection or inflammation. Of course, to achieve even lower levels of risk, the tolerization procedures of the present invention may be combined with other forms of immunosuppression.

[0142] In some embodiments, compositions of the invention (e.g., PLG carriers bound to antigen molecules) are used in conjunction with one or more scaffolds, matrices, and / or delivery systems (see, e.g., U.S. Patent Application Publication No. 2009 / 0238879; U.S. Patent No. 7,846,466; U.S. Patent No. 7,427,602; U.S. Patent No. 7,029,697; U.S. Patent No. 6,890,556; U.S. Patent No. 6,797,738; and U.S. Patent No. 6,281,256, which are incorporated by reference in their entireties). In some embodiments, particles (e.g., PLG particles bound to antigens) are associated with, adsorbed to, embedded in, conjugated to, etc. a scaffold, matrix, and / or delivery system (e.g., for delivery of chemical / biological materials, cells, tissues, and / or organs to a subject). In some embodiments, the scaffold, matrix, and / or delivery system (e.g., for delivery of chemical / biological materials, cells, tissues, and / or organs to a subject) comprises and / or consists of a material described herein (e.g., PLG conjugated to one or more antigenic peptides).

[0143] In some embodiments, microporous scaffolds are provided (e.g., for implanting biological material (e.g., cells, tissues, etc.) into a subject). In some embodiments, microporous scaffolds having an agent thereon (e.g., extracellular matrix protein, exendin-4) and microporous scaffolds having biological material (e.g., pancreatic islet cells) are provided. In some embodiments, the scaffolds are used in the treatment of disease (e.g., type 1 diabetes) or related methods (e.g., diagnostic methods, research methods, drug screening). In some embodiments, the scaffolds are provided with a carrier conjugated to an antigen described herein on and / or within the scaffold. In some embodiments, the scaffolds are made from a material conjugated to an antigen (e.g., PLG conjugated to an antigen).

[0144] In some embodiments, the scaffold and / or delivery system comprises one or more layers and / or bears one or more chemical and / or biological entities / agents (e.g., proteins, peptide-conjugated particles, small molecules, cells, tissues, etc.) (see, e.g., U.S. Patent Application Publication No. 2009 / 0238879, incorporated herein by reference in its entirety). In some embodiments, antigen-binding particles are co-administered with the scaffold delivery system to induce induction of immunological tolerance to the scaffold and associated materials. In some embodiments, a microporous scaffold is administered to a subject with particles described herein on or within the scaffold. In some embodiments, the antigen-binding particles are associated with the scaffold delivery system. In some embodiments, the scaffold delivery system comprises antigen-binding particles.

[0145] Various modifications, variations, and alterations of the described features and embodiments will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although specific embodiments have been described, it should be understood that the invention as claimed is not to be unduly limited to such specific embodiments. Indeed, various modifications of the described modes and embodiments that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims. For example, U.S. Patent Application Nos. 2012 / 0076831, 2002 / 0045672, 2005 / 0090008, 2006 / 0002978, and 2009 / 0238879 (each of which is incorporated by reference herein in its entirety), as well as U.S. Patent Nos. 7,846,466; 7,427,602; 7,029,697; 6,890,556; 6,797,738; and 6,281,256 (each of which is incorporated by reference herein in its entirety) provide details, modifications, and variations for use in the various embodiments described herein.

[0146] All publications and patents mentioned in this application and / or listed below are hereby incorporated by reference in their entirety. [Example]

[0147] The following examples are provided to further illustrate the advantages and features of the present invention, but are not intended to limit the scope of the disclosure.

[0148] material and method Generation of chimeric mice Six- to eight-week-old B6.SJL-PtprcaPep3b / BoyJ (CD45.1) mice were irradiated with a single dose of 950 rads. 12 hours later, mice were reconstituted with 10 bone marrow cells from a C57BL / 6-7.2 fms-EGFP donor. For 10 days after irradiation, mice received sulfamethoxazole (Sigma-Aldrich) and trimethoprim (Sigma-Aldrich) in their drinking water. Six weeks after irradiation, mice were infected with WNV as described above. Chimerism was confirmed using flow cytometry and was consistently found to be 96–99% of donor origin, as previously demonstrated (Getts et al., J Neurochem. 103:1019, 2007).

[0149] immunohistology Mice were anesthetized and perfused with 50 mL of sterile PBS. All organs were isolated and snap-frozen in Optimum Cutting Temperature compound (OCT; Tissue-Tek, Tokyo, Japan) except for the heart, which was processed into paraffin blocks (Getts et al., J. Neurochem 103:10919-1030, 2007). Eight-micron tissue sections were cut on a cryostat microtome, air-dried overnight, and then stored at -80°C until needed. Frozen sections were thawed and subjected to histological examination (standard hematoxylin and eosin staining) or immunohistochemistry (Getts et al., J. Exp Med 205:2319-2337, 2008). Antibodies against MARCO, SIGN-R1, and SIGLEC-1 (R&D Systems, MN, USA), CD68 (Abcam, MA, USA), and Ki67 (Abcam) were used as indicated. Images were acquired on an Olympus BX-51 microscope using a DP-70 camera and DP Manager 2.2.1 software (Olympus, Tokyo, Japan).

[0150] Microscopy and image acquisition Images were acquired on an Olympus BX-51 microscope (Olympus, Japan) using a DP-70 camera and DP Manager 2.2.1 software (Olympus).

[0151] Isolation of leukocytes from brain and liver Leukocytes were obtained from mouse brains perfused with PBS by digesting the brains in PBS containing deoxyribonuclease (0.005 g / ml; Sigma-Aldrich) and collagenase IV (0.05 g / ml; Sigma-Aldrich) for 60 min at 37°C, as previously described (Getts et al., J Exp Med. 29:2319, 2007). Digestion was stopped with 10% FCS, and the homogenate was passed through a 70 μm nylon cell strainer (Becton Dickinson, NJ, USA). The pellet obtained after centrifugation at 340 x g for 10 min was resuspended in 30% Percoll (Amersham, Norway) and layered onto 80% Percoll. After centrifugation at 1140 x g for 25 min at room temperature, leukocytes were collected from the 30% / 80% interface. The same protocol is used to obtain white blood cells from the liver, and the tissue is weighed before processing.

[0152] Isolation of leukocytes from spleen, blood, and bone marrow For flow cytometry analysis, the right femur was excised and bone marrow cells were flushed out using a syringe filled with PBS. For the isolation of bone marrow progenitors, femurs and tibiae from at least four mice were used. The cell suspension obtained after flushing was filtered through a 70 μm cell strainer and centrifuged at 340 x g for 5 min. Red blood cells in the resulting pellet were lysed in an NH₄Cl-based red blood cell lysis buffer (BD Pharm Lyse™; BD Pharmingen) and then centrifuged at 340 x g for 5 min. For peripheral blood, blood was collected by cardiac puncture and immediately transferred into citrate buffer (mMol, Sigma Alrich). The resulting suspension was layered onto 70% Percoll and centrifuged at 1140 x g for 20 min at room temperature with the brake off. The interface was collected, and the cells were washed once in PBS and centrifuged at 340 x g. To isolate splenic leukocytes, the spleen was passed through a 70-μm cell strainer and centrifuged at 340 g for 5 min. The red blood cells in the resulting pellet were lysed in an NH4Cl-based red blood cell lysis buffer (BD Pharm Lyse™; BD Pharmingen) and then centrifuged at 340 x g for 5 min.

[0153] Flow cytometry Cells harvested from brain, liver, blood, and bone marrow (as described above) were washed with PBS and blocked with anti-CD16 / CD32 antibody (Biolegend). Viable cells were counted using trypan blue exclusion, and consistently showed >95% cell viability.

[0154] Expression of cell surface molecules was measured, and cell sorting was performed on a FACS ARIA (Becton Dickinson) equipped with argon ion and HeNe lasers. Viable populations were gated by forward and side scatter, followed by identification of the fluorescent population determined by the forward gate. Sorting was performed using specific fluorescence and scatter parameters to identify the population of interest. Sorting stringency was set to achieve >98% purity for the myeloid population.

[0155] The acquired FACS data files were analyzed using the flow cytometry program FlowJo (FlowJo, Ashland, OR, USA). Quantification of cell populations of interest was calculated based on flow cytometry percentages and absolute cell numbers from each organ at the time of analysis.

[0156] Adoption Experiments were conducted during the development of embodiments of the present invention to investigate a second model of active disease, termed adoptive transfer. Instead of immunizing animals with peptide, lymphocytes from the spleens of mice with active disease were transferred to recipients, who then developed disease. Experiments were conducted during the development of embodiments of the present invention to characterize the ability of PLG nanoparticles to inactivate adoptively transferred activated effector cells. Mice treated with particles conjugated with a control peptide or splenocytes showed an increase in clinical score beginning on day 4. Mice treated with PLG-PLP139-151 particles had an average clinical score of 0 on day 2 and at all but two time points through day 40, with an average clinical score of 0.25 at those other time points.

[0157] Multiplex ELISA Multiplex plate ELISA was performed according to the manufacturer's instructions (Quansys Biosciences, Logan, Utah, USA). Briefly, brain, spleen, and liver tissues were homogenized in PBS, spun at 1000 × g for clarification, and stored at -20 °C until assay. Serum samples were also used. Thawed samples and standards were diluted in the provided buffer, and 30 μl of each was seeded into each well containing 16 spots, each containing a capture antibody against a specific soluble protein. The plate was then incubated for 1 hour at 120 rpm on an orbital shaker. The plate was washed three times, and 30 μl of detection antibody was added to each well and incubated for an additional hour. After three washes, strepavidin-HRP was added and incubated for an additional 15 minutes. The plate was then washed six times, and substrate mix was added. The plate was immediately read on a CCD imager (Kodak, Rochester, NY, USA). Plate images were analyzed using Quansys Q-view software (Quansys Biosciences).

[0158] Induction and evaluation of experimental autoimmune encephalitis (EAE) Mice were subcutaneously injected with an emulsion containing 0.1 mg of MOG peptide (MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 1); Auspep, Parkville, Victoria, Australia; >95% HPLC purified) and 2 mg / mL of Mycobacterium tuberculosis (Sigma Aldrich) in Freund's complete adjuvant. Two days later, mice were intraperitoneally administered 500 μl of pertussis toxin (Sigma Aldrich). Mice were monitored for disease progression and graded on the following scale: 1, tail flaccidity and / or weakness of one hind limb; 2, multiple limb weakness, impaired lameness; 3, single limb paralysis; 4, multiple limb paralysis, incontinence; and 5, moribund.

[0159] statistics Graphs were generated and computational statistical analysis was performed in GraphPad Prism and InStat, respectively (both programs from GraphPad software, San Diego, CA, USA). Depending on the data, unpaired two-tailed Student's t-test or one-way ANOVA with Tukey-Kramer post-hoc test was performed, and P < 0.05 was considered significant.

[0160] For correlation analysis between parameters such as weight loss, infiltration, and viral titer, nonlinear regression (curve fitting) using a second-order polynomial equation (Y=A+B*X+C*X^2) was used.

[0161] Example 1 Preparation of negatively charged immunomodulating particles (IMPs) A solution of poly(lactide-co-glycolic acid) (PLG) (2 mL, 20% w / v) in dichloromethane (DCM) was added dropwise to a solution of poly(ethylene-maleic anhydride) (PEMA) (4 mL, 1% w / v) in DO. The mixture was sonicated on ice for 30 seconds at 16 watts using a VC 30 Ultrasonic Processor. The resulting homogenized crude product was then poured into a solution of DO (200 mL containing 0.5% w / v PEMA). The homogenized slurry was stirred overnight using a Bellco Glass, Inc., Bellstir Multi-stir 9 magnetic stirrer at a speed setting of 3.5 (10 W for 10 seconds, 16 W for 10 seconds, and 16 W for 30 seconds).

[0162] result After 3 hours of stirring, particle size analysis was performed using dynamic light scattering in a disposable polystyrene cuvette.

[0163] a. 10 W, 10 sec - Z-average = 499.9 nm - PdI = 0.23, Peak = 634.5 nm b. 16 W, 10 sec - Z-average = 528.9 nm - PdI = 0.227, Peak = 657.5 nm c. 16 W, 30 sec - Z-average = 471.6 nm - PdI = 0.228, peak = 580.5 nm d.16W, 60 seconds - Z-average = 491.1nm - PdI = 0.275, Peak = 600.8nm After the reaction was completed, the resulting crude suspension was purified.

[0164] purification Fresh DO and 10x sodium bicarbonate buffer were chilled overnight at 4°C. Using a 40 μm cell strainer, 36 mL of particle suspension from each batch was filtered into appropriately labeled 50 mL centrifuge tubes containing 4 mL of cold 10x sodium bicarbonate buffer. Approximately six such tubes were generated from each beaker. All tubes were centrifuged at 7000 g for 15 minutes at 4°C, and the supernatant was aspirated. The suspension preparation was repeated using the procedure described above, suspending as much of the particle pellet as possible in 1 mL of cold DO.

[0165] Transfer the resuspended particles to a new tube containing 4 mL of cold 10x sodium bicarbonate buffer (Step 1).

[0166] Particle resuspension was repeated until all particle pellets were successfully resuspended (Step 2).

[0167] The six centrifuge tubes were then combined into one centrifuge tube (50 mL tube), and the tube was filled to 40 mL with the remaining amount of cold D2O (wash 1).

[0168] The tubes were centrifuged at 7000 g for 20 min at 4°C and the supernatant was aspirated.

[0169] Each time, steps 1 and 2 and washing 1 were repeated on the resulting particles at least two more times. Finally, the resulting particle pellet was subjected to flash freezing in liquid nitrogen and freeze-dried in a manifold to obtain negatively charged IMPs.

[0170] Figure 1 shows the dynamic light scattering analysis characterization of surface-functionalized poly(lactide-co-glycolide) particles. Surface-functionalized poly(lactide-co-glycolide) particles were analyzed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water at a count rate of 2.5 × 10 counts per second. The population of surface-functionalized poly(lactide-co-glycolide) particles had a Z-average particle size of 567 nm, a peak diameter of 670 nm, and a polydispersity index of 0.209.

[0171] Table 1 shows measurements for surface-functionalized PLG-PEMA particles. Each batch is slightly different, so the data in the table are representative. However, the values ​​in the table are based on several batches of particles combined. Measurements for double emulsion particles are similar to those in Table 1. [Table 1]

[0172] Example 2 Administration of antigen-conjugated PLGA beads prevents relapsing experimental autoimmune encephalitis To induce tolerance for the prevention of recurrent experimental autoimmune encephalitis (R-EAE), we investigated PLG nanoparticles using the immunodominant proteolipid protein PLP139-151 epitope (PLG-PLP139-151). R-EAE mice were generated as described above.

[0173] The peptides administered to animals were conjugated to particles with an average diameter of 500 nm. On day -7 relative to the time of immunization (day 0), mice were treated with either PLP139-151-PLGA (N = 5), OVA323-339-PLGA (N = 5), or unconjugated PLGA (N = 5). Peak disease was typically observed around days 12-14, and mice were scored for clinical disease. Particles containing no peptide or particles modified with the control peptide, OVA323-339, did not prevent disease induction. However, PLGA particles modified with PLP139-151 all showed a clinical score of 0 (no disease observed), except for a low clinical score of <1 on days 20-30 (Figure 2). Previous studies using unmodified PLG or polystyrene particles did not demonstrate this effective disease reduction, and polystyrene-conjugated particles often caused anaphylaxis.

[0174] Furthermore, the lack of delayed-type hypersensitivity (DTH) responses to both immunized PLP139-151 epitopes demonstrated specific inactivation of myelin-specific CD4+ T cells. Taken together, prophylactic administration with PLG-PLP139-151 on day 7 specifically prevented the onset of EAE, indicating an improved ability of the particles to prevent disease. The scores obtained with the particles were as good as, and possibly better than, those obtained with antigen-loaded splenocytes.

[0175] The type of particle administered also influences the development of EAE in mouse models. Mice were treated with either OVA323-339-PLS (N=5), OVA323-339-PLGAPHOSPOREX (N=5), OVA323-339-PLGAPEMA (N=5), PLP139-151-PLA (N=5), PLP139-151-PLGAPHOSPOREX (N=5), or PLP139-151-PLGPEMA (N=5) on day -7 relative to the time of immunization (day 0). Peak disease was typically observed around days 12-14, and mice were scored for clinical disease. Particles of any of the compositions modified with the control peptide OVA323-339 did not prevent disease induction. However, PLP139-151-conjugated PLG beads were more effective in downregulating the induction of R-EAE than PLP139-151-conjugated commercial (Phosphorex) PLG or polystyrene (Figures 3A and 3B).

[0176] Example 3 Intravenous injection of antigen-conjugated PLG particles does not induce anaphylaxis-induced hypothermia in animals pre-sensitized with OVA / alum Due to the presence of active disease, anaphylaxis to the antigen is a concern; it can cause immediate death and has been described with polystyrene-bound particles. Anaphylaxis is associated with a significant drop in body temperature. To investigate whether intravenous administration of OVA-PLG induces an anaphylaxis-induced drop in body temperature in pre-sensitized animals, mice were immunized with 10 μg of OVA / alum by intraperitoneal injection on day 0. On day 14, mice were again immunized with 10 μg of OVA / alum by intraperitoneal injection and then tolerized with intravenously administered OVA-PLG on day 21. Mice were then tolerized with either OVA-PLG particles or OVA by intravenous administration on day 28.

[0177] As shown in Figure 4, mice treated with soluble OVA on day 28 exhibited a decrease in body temperature compared to animals treated with OVA-PLG particles. No decrease in body temperature was observed within 1 hour of particle delivery.

[0178] Figure 5 shows that administration of PLP-PLG during remission did not cause any anaphylaxis-related deaths. EAE was induced in 6-8 week-old female SJL / J mice by subcutaneous injection of PLP139-151 in CFA, and the onset of clinical disease was monitored and recorded (Figure 5B). On day 21 after disease induction, mice were intravenously injected with soluble PLP139-151 (open squares), soluble OVA323-339 (open circles), or the same peptides conjugated to PLG nanoparticles (filled circles). Animal body temperature was monitored and recorded every 10 minutes for 1 hour after injection (Figure 5A).

[0179] Example 4 Prophylactic administration of PLP-PLG particles induces long-term antigen-specific tolerance Seven days before disease induction, the optimal dose was determined by intravenous administration of increasing concentrations of PLP139-151-PLG, and SJL / J mice were monitored for the development of clinical disease compared with those treated with OVA323-339-PLG (Figure 6A). Six- to eight-week-old female SJL / J mice were intravenously injected with PLG nanoparticles conjugated with either PLP139-151 (squares) or OVA323-339 (circles). Seven days (Figure 6B), 25 days (Figure 6C), or 50 days (Figure 6D), EAE was induced by subcutaneous injection of PLP139-151 in CFA. Animals in panel B were followed for 100 days for clinical disease. Figure 6E shows delayed-type hypersensitivity (DTH) responses in a subset of mice shown in panel B on day 8 after disease induction. The ears of representative animals (OVA323-339-PLG and PLP139-151-PLG) selected from the PLP139-151 / CFA-primed group in panel B were challenged with the PLP139-151 epitope and the OVA323-339 control peptide. After 24 hours, ear swelling was assessed as a measure of DTH, and the pre-exposure response was subtracted. Figure 6F shows that 6- to 8-week-old female SJL / J mice were intravenously injected with PLG nanoparticles conjugated with PLP178-191 (triangles), OVA323-339 (circles), or PLP139-151 (squares), or with unconjugated particles alone (circles with outlines). Seven days later, EAE was induced by subcutaneous injection of PLP178-191 in CFA, and disease was monitored at the indicated time points.

[0180] Example 5 Treatment of relapsing experimental autoimmune encephalitis with antigen-binding particles Experiments were conducted during the development of embodiments of the present invention to investigate the ability of PLG-PLP139-151 particles to treat disease rather than prevent it, and to determine whether the route of administration affected disease development. Mice were immunized with PLP139-151 and adjuvant on day 0. Mice typically achieve a maximum clinical score on days 12-14. In this model, mice were treated on day 10 with either PLG-PLP139-151 particles or control PLG-OVA323-339 particles, either intravenously (iv), intraperitoneally (ip), subcutaneously (sc), or orally. As shown in Figure 7, preventive tolerance was most efficient when PLG-PLP139-151 particles were administered either intravenously or intraperitoneally. Animals treated with intravenously administered PLP139-151-PLG did not develop disease and had an average clinical score of 0 at most time points. This is in contrast to animals treated with PLP139-151 polystyrene particles, where over 70% of the animals were observed to die from anaphylaxis.

[0181] Example 6 Antigen-binding particle tolerance inhibits the induction of antigen-specific Th1 and Th17 responses in active, relapsing experimental autoimmune encephalitis To determine whether administration of antigen-conjugated particles inhibits T helper cell induction, BALB / c mice were intravenously administered either MOG35-55-PLG or OVA323-339-PLG particles on day -7. On day 0, mice were subcutaneously administered OVA323-339-PLG particles and complete Freund's adjuvant (CFA). On day 10, animals were restimulated with either MOG35-55-PLG or OVA323-339-PLG particles, and draining lymph node cells were isolated. On day 10, CPM, as well as levels of IL-17, GM-CSF, IFN-γ, IL-10, and IL-4, were measured. As shown in Figure 8, administration of OVA323-339-PLG particles inhibited Th1 and Th17 responses in treated animals.

[0182] Example 7 PLP-139~151 Tolerance was induced by PLGA particles conjugated with . We conducted a further therapeutic tolerance strategy by delivering PLP139-151-PLG or OVA323-339-PLG to mice. Histological analysis showed that administration of PLP139-151-PLG particles inhibited inflammation and demyelination in the cervical spinal cord. Mice were treated with PLP-PLG or OVA323-339-PLG, and tissues were collected on day 40. Cervical spinal cords were isolated and sectioned to investigate the immune responses in the CNS underlying the pathology of R-EAE and multiple sclerosis. Figure 9 shows reduced immune cell infiltration within the spinal cord of animals treated with PLP139-151-PLG, which resembles native tissue more than tissue from animals treated with OVA323-339-PLG. Animals treated with OVA323~339-PLG showed positive staining for CD45, CD4, and CD11b, whereas animals treated with PLP139~151-PLG showed minimal staining for these factors.

[0183] Administration of PLP139~151-PLG particles also inhibited blood-brain barrier (BBB) ​​disruption and macrophage activation in the spinal cord of treated mice. Animals were treated with either complete Freund's adjuvant (CFA), OVA323~339 PLG particles, or PLP139~151-PLG particles. Clinical scores and the incidence of EAE were determined (Figure 10B), and spinal cord imaging was observed by in vivo imaging (Figures 10A and 11). AngioSense measures blood leakage within the CNS, and ProSense reports activated macrophages (cathepsin activation cleaves the reporter, revealing a fluorescent signal). Bar graphs show the numerical values ​​relative to the signal intensity shown in the brain and SC scans.

[0184] Tolerance can also be induced by antigen-encapsulated particles. Figure 12 shows that administration of PLG particles encapsulating PLP139-151 inhibits the induction of R-EAE in mice. The ability to encapsulate autoantigens allows the use of complex mixtures of proteins or even organ homogenates to achieve greater antigen coverage and thus more effectively accommodate epitope spreading.

[0185] Example 8 Tolerance induced by PLP-139-151-conjugated PLGA particles depends in part on the expansion / activation of regulatory T cells. On day -9, SJL / J mice were treated with anti-CD25 antibody, a common marker of regulatory T cells (Tregs), and then on day -7, they were treated with either OVA323-339PLG particles and anti-CD25 antibody, OVA323-339PLG particles and control IgG antibody, PLP139-151-PLG particles and anti-CD25 antibody, or PLP139-151-PLG particles and control IgG antibody. As shown in Figure 13, animals treated with PLP139-151-PLG particles and anti-CD25 antibody occasionally showed higher mean clinical scores than animals treated with PLP139-151-PLG particles and control IgG antibody. This supports the role of Tregs, or at least CD25-expressing T cells, in inducing tolerance.

[0186] Example 9 In active and adoptive EAE, PLP 139~151 Therapeutic tolerance was induced by -PLG particles. Therapeutic tolerance induced by PLP139-151-PLG particles was compared in active and adoptive EAE. Adoptive EAE was induced in 6-8 week-old female SJL / J mice by adoptive transfer of 2.5 x 106 PLP139-151-activated blasts. Two days (Figure 14A) and 14 days (Figure 14C) after disease induction, mice were intraperitoneally injected with PLP139-151 (squares) or OVA323-339 (circles) peptides conjugated to 500 nm PLG nanoparticles. Clinical disease scores were compared with those after treatment with antigen-bound splenocytes (Figure 14A). On day 42, brains and spinal cords were harvested from PLP139-151- or OVA323-339-tolerized mice for histological analysis. Sections from the mouse in panel A were stained for PLP protein and CD45 (Figure 14B). Spinal cord sections from the mouse in panel C were stained with Luxol Fast Blue (Figure 14D). Areas of demyelination and cellular infiltration are indicated by arrows. The results demonstrate that tolerance is induced by PLP139-151-PLG particles in mice with adoptive EAE.

[0187] Figure 15 shows graphs depicting the mean clinical scores of mice with active and adoptive EAE after treatment with either SP or PLG particles conjugated to OVA323-339 or PLP139-151. Ten days (Figure 15A) and two days (Figure 15B) after disease induction, mice were intraperitoneally injected with PLP139-151-SP, PLP139-151-PLG, or OVA323-339-SP, or OVA323-339-PLG peptides conjugated to 500 nm nanoparticles, and the mean clinical scores were determined. In both cases, administration of PLP139-151-PLG particles ameliorated disease, suggesting the induction of tolerance.

[0188] CNS immune cell infiltration was also dramatically reduced in PLP-PLG-tolerant mice. Two days after EAE induction by adoptive transfer, SJL / J mice were intraperitoneally injected with 500 nm PLG nanoparticles conjugated with PLP139-151 (squares) or OVA323-339 (circles). At the peak of disease (day 14), brains and spinal cords were removed, and the numbers of lymphocytes (Figure 16B), APCs (Figure 16C), microglia (Figure 16D), peripheral dendritic cells (Figure 16E), myeloid dendritic cells (Figure 16F), and macrophages (Figure 16G) were enumerated by flow cytometry. The gating strategy for these populations is shown in (Figure 16A). CNS cell preparations were stimulated with PMA and ionomycin for 5 h before intracellular staining for IL-17A and IFN-γ (Figure 16H).

[0189] Example 10 In adoptively transferred EAE, treatment with anti-PD-1 monoclonal antibody inhibits PLP 139~151 Suppresses tolerance induction by PLG nanoparticles containing PEG To examine the effect of anti-PD-1 antibody treatment on PLP139-151-induced tolerance in mice with adoptive EAE, mice were intravenously administered 3 × 106 PLP139-151-activated T cell blasts on day 0. On day 2, PLP139-151 or OVA323-339 encapsulated in PLG particles was intravenously administered along with either PBS or anti-PD-1 antibody. On days 4, 6, 8, 10, and 12, all animals received 250 μg of anti-PD-1 antibody or PBS.

[0190] As shown in Figure 17, administration of PLP139-151 peptide encapsulated in PLG particles induces tolerance when the particles are administered with PBS, however, administration of anti-PD-1 antibody reduces this tolerance.

[0191] Example 11 Treatment with an agonistic anti-CD40 monoclonal antibody suppresses tolerance induction by PLP139-151-loaded PLG nanoparticles in an IL-12-dependent manner in adoptively transferred EAE mice To examine the effect of agonistic anti-CD40 antibodies on PLP139-151-induced tolerance in mice with adoptive EAE, mice were intravenously administered 3 x 106 PLP139-151-activated T cell blasts on day 0. On day 2, mice were intravenously administered PLP139-151 or OVA323-339 encapsulated in PLG particles. On day 3, animals were administered either a control IgG2a antibody, an anti-CD40 antibody, or an anti-CD40 antibody and an anti-IL-12 antibody.

[0192] As shown in Figure 18, administration of PLP139-151 peptide encapsulated within PLG particles induces tolerance when the particles are administered with PBS. Administration of an agonistic anti-CD40 antibody reduces this tolerance, but this reduction in tolerance is reversed by the addition of an anti-IL-12 antibody.

[0193] Example 12 OVA encapsulated within PLG particles prophylactically inhibits allergic airway inflammation and OVA-specific Th2 responses in vivo. To examine the preventive effect of OVA encapsulated in PLG particles on airway inflammation, mice were treated intravenously with OVA-PLG on day -7. On day 0, mice were injected intraperitoneally with OVA / alum at a dose of 10 μg / mouse. On day 7, mice were again treated intravenously with OVA-PLG, and on day 14, mice were injected intraperitoneally with an additional 10 μg / mouse of OVA / alum. Mice were treated three times with aerosolized OVA from days 28 to 30.

[0194] As shown in Figure 19, prophylactic administration of OVA-PLG reduced the secretion of IL-4, IL-5, IL-13, and IL-10, and reduced serum OVA IgE and pulmonary eosinophil levels.

[0195] OVA encapsulated within PLG particles prophylactically inhibits OVA-specific ex vivo recall responses from mediastinal lymph nodes. As shown in Figure 20A, lymph node proliferation observed after restimulation with 25 μg of OVA was reduced in animals treated with OVA-PLG. Furthermore, treatment with OVA-PLG reduced cytokine release after restimulation with OVA. Figure 20B shows that levels of IL-4, IL-5, IL-13, and IL-10 were reduced in mice treated with OVA-PLG.

[0196] Example 13 OVA encapsulated within PLG particles therapeutically inhibits allergic airway inflammation and OVA-specific Th2 responses in vivo To examine the therapeutic effect of OVA encapsulated in PLG particles on airway inflammation, mice were treated intraperitoneally with OVA / alum at a dose of 10 μg / mouse on days 0 and 14. OVA-PLG was administered intravenously to mice on days 28 and 42. Mice were treated three times with aerosolized OVA on days 56–58.

[0197] As shown in Figure 21, therapeutic administration of OVA-PLG reduced the secretion of IL-4, IL-5, IL-13, and IL-10, and reduced serum OVA IgE and pulmonary eosinophil levels.

[0198] Figure 22 shows that OVA encapsulated in PLG particles therapeutically down-regulates OVA-specific Th2 cytokines in bronchoalveolar lavage fluid better than OVA-conjugated PLG particles. Animals were treated as described above, except that on days 28 and 42, mice were treated with either OVA encapsulated within PLG particles or OVA conjugated to PLG particles. Surprisingly, encapsulated OVA inhibited Th2 cytokine secretion more than OVA peptide conjugated to the surface of PLG particles.

[0199] Example 14 Tolerance induced by chromogranin A p31 peptide-PLG particles inhibits type 1 diabetes Type 1 diabetes was induced in BDC2.5 mice by isolating splenic, axillary, brachial, inguinal, and pancreatic lymph node cells from 3-week-old mice. Isolated cells were cultured and activated in vitro by incubating 2 x 106 cells / mL with 0.5 µM p31 peptide for 96 hours. At time 0, 5 x 106 cells were intravenously transferred into NOD.SCID mice (6-8 weeks old). Three days later, mice were tolerized by intravenous administration of p31 or MOG35-55 peptides conjugated to SP or PLG2.

[0200] Figures 23A and 23B show the blood glucose levels of animals after treatment. Administration of p31 peptide-conjugated PLG resulted in lower blood glucose levels compared to those observed after administration of MOG35-55 peptide-conjugated particles. Figure 23C shows that the percentage of IFNγ-secreting cells observed in animals was also reduced in mice treated with p31-PLG compared to mice treated with MOG35-55 peptide-PLG.

[0201] Tolerance induced by p31-PLG requires Tregs. Type 1 diabetes was induced in mice as described above, and 2 hours after transferring activated cells to NOD SCID mice, the mice were tolerized with either p31-PLG or MOG35-55PLG particles. As shown in Figure 24, depletion of Tregs suppresses tolerance induced by administration of p31-PLG particles.

[0202] Example 15 Tolerance induced by insulin-conjugated PLG particles inhibits the development of spontaneous type 1 diabetes in NOD mice NOD mice were treated intravenously at 6, 8, and 10 weeks of age with either BSA (N=22) or insulin (N=23)-conjugated PLG particles. Mice were then assayed for the development of diabetes, defined as a blood glucose level >250 mg / dL. As shown in Figure 25, administration of insulin-conjugated PLG particles significantly increased the proportion of mice that remained diabetes-free over 300 days (69.6% compared to 22.7%; p=0.0027).

[0203] Example 16 Engraftment dynamics On day -7, female CD45.2 mice were tolerized with either OVA-PLG or the control peptide Dby-PLG (the major HY antigen expressed by male C57BL / 6 mice). On day -1, the mice were irradiated with 200 rads and then transplanted with 1 x 10, 5 x 10, or 1 x 10 bone marrow cells from male CD45.1 mice on day 0. Recipient mice were then tolerized with either OVA-PLG, Dby-SP, or Dby-PLG on day 1, and blood was collected for FACS analysis of chimerism. Figure 26 shows the percentage of CD45.1 donor cells observed in recipient mice.

[0204] Figure 27 shows the percentage of donor CD45.1 cells in recipient mice after tolerization with either OVA-PLG, Dby-SP, or Dby-PLG on day 1. One positive control mouse showed no significant engraftment (approximately 10%). All negative control mice did not engraft donor cells. One Dby-SP mouse showed no significant engraftment (approximately 10%). Two OVA-PLG mice engrafted donor cells (approximately 10%), and one showed complete rejection by week 16. One Dby-PLG mouse began to show rejection at week 12, reaching 10% by week 16. The Dby-PLG groups ranged from 10% to 56% engraftment by week 16. OVA-PLG mice demonstrated 1) natural engraftment, 2) sequence homology between OVA323 and Dby, or 3) immunogenic properties of the particles. Dby-PLG allows for greater engraftment than Dby-SP and OVA-PLG.

[0205] Figure 28 shows the timing at which tolerance affects the percentage of CD45.1 cells in recipient mice. The positive control shows less engraftment (approximately 4%) than expected (approximately 10%). One negative control mouse had 5% engraftment in all three OVA-PLG groups, and one mouse in the day -7 and +1 groups showed engraftment (12%). Day 1 tolerance is more clinically significant than day -7 tolerance.

[0206] Example 17 Coumarin-6 PLGA particles are undetectable 24 hours after administration. Mice were treated with coumarin-6 PLGA particles either conjugated to antigen or free of antigen. As shown in Figure 29, the particles were detectable 3 hours after administration but undetectable 24 hours after administration. Spleen (left column), liver (middle column), and lung (left column) sections of mice injected with intraperitoneal fluorescent PLGA / PEMA microparticles compared to uninjected naive mice (top row) were shown 3 hours after injection (middle row) and 24 hours after injection (bottom row), counterstained with DAPI.

[0207] Example 18 Nanoparticles associate with macrophages in vivo Analysis of the liver 6 and 15 hours after administration shows that the PLGA particles were co-localized with F4 / 80+ cells in the liver (FIG. 30).

[0208] Twenty-four hours after intravenous injection, marginal zone macrophages preferentially ingest particles conjugated with TAMRA-labeled PLP139-151. As shown in Figure 31, the marginal zone macrophages contain the highest percentage of PLP139-151+ cells.

[0209] Example 19 Inhibition of R-EAE in SJL / J mice using surface-functionalized poly(lactide-co-glycolide) particles containing soluble PLP139-151 within the core Groups of SJL / J mice were intraperitoneally injected with 2.5 mg of 500 nm–700 nm surface-functionalized poly(lactide-co-glycolide) particles containing soluble PLP139–151 peptide within their cores on days −7 and −1 before priming with PLP139–151 / CFA on day 0. Control mice were primed on day 0 but received no particle treatment on days −7 or −1. Mice were observed for clinical signs of R-EAE for an additional 20 days.

[0210] The results, shown in Figure 32, show the mean daily clinical score versus days of priming with PLP139-151 / CFA. Induction of immunological tolerance using surface-functionalized poly(lactide-co-glycolide) particles containing soluble PLP139-151 in the core inhibited PLP139-151 / CFA-induced R-EAE in SJL / J mice.

[0211] Example 20 Inhibition of allergic airway inflammation by surface-functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin Allergic airway inflammation (AIA) was induced in mice. Groups of Balb / c mice were intravenously injected with 2.5 mg of 500 nm to 700 nm surface-functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin or soluble bovine serum albumin (control) within the core on days -7 and +7, prior to priming with ovalbumin / alum on days 0 and +14. On days +28-30, mice were challenged with aerosolized ovalbumin. Mice were then sacrificed, and bronchoalveolar lavage fluid was obtained. Serum levels of ovalbumin-specific IgE were also measured.

[0212] Eosinophil counts in bronchoalveolar lavage fluid indicate the severity of allergic airway inflammation (AAI): higher numbers indicate worsening disease. Serum IgE levels indicate the severity of AAI: higher levels indicate worsening disease.

[0213] Figure 33 shows that mice treated with encapsulated OVA-PLG showed a significant decrease in eosinophil accumulation, and Figure 34 shows that mice treated with encapsulated OVA-PLG showed a significant decrease in serum IgE levels compared to untreated or control-treated animals.

[0214] Induction of immunological tolerance using surface-functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin in the core inhibited ovalbumin / alum-induced allergic airway inflammation in Balb / c mice.

[0215] Example 21 Synthesis of surface-functionalized poly(lactide-co-glycolide) particles encapsulating antigens This example details the formulation and partial characterization of biodegradable poly(lactide-co-glycolide) particles surface-functionalized with a high density of carboxylate groups and containing a soluble antigen within the core, surrounded by a poly(lactide-co-glycolide) shell, for immune induction in autoimmune diseases and treatment of allergies.

[0216] A high density of carboxylate groups was achieved by using poly(ethylene-alt-maleic anhydride (PEMA)), a polymer with carboxylate groups incorporated into its backbone, as a surfactant for the emulsification process.

[0217] As mentioned above, biodegradable poly(lactide-co-glycolide) particles containing soluble PLP139-151 in the core and surface-functionalized with a high density of carboxylate groups are effective in inducing immunological tolerance in the SJL / J PLP139-151 / CFA-induced R-EAE mouse model of multiple sclerosis. Furthermore, biodegradable poly(lactide-co-glycolide) particles containing soluble ovalbumin in the core and surface-functionalized with a high density of carboxylate groups are effective in inducing immunological tolerance in the ovalbumin / alum-induced AAI mouse model of allergic asthma.

[0218] Poly(lactide-co-glycolide) particles containing soluble ovalbumin or bovine serum albumin in the core and surface functionalized with a high density of carboxylate groups were synthesized using a double emulsion-solvent evaporation method as follows: 1. 150 μL of 200 mg / mL ovalbumin or bovine serum albumin in endotoxin-free water was added dropwise to 2 mL of 20% w / v poly(lactide-co-glycolide) in dichloromethane in a 20 mL scintillation vial. 2. The resulting mixture was placed on ice and sonicated using a probe sonicator at 10 watts for 30 seconds. 3.10 mL of 1% w / v poly(ethylene-alt-maleic anhydride) in water was added. 4. The resulting mixture was placed on ice and sonicated for 30 seconds at 16 watts using a probe sonicator. 5. The resulting mixture was poured into 200 mL of 0.5% w / v poly(ethylene-alt-maleic anhydride) in a 600 mL beaker and stirred overnight to harden the particles. 6. The hardened particles were then purified by centrifugation and washed three times with bicarbonate buffer pH 9.6. 7. The purified particles were resuspended in 4% w / v sucrose and 3% w / v D-mannitol in water, flash frozen in liquid nitrogen and lyophilized.

[0219] Figure 35 shows the dynamic light scattering characterization of surface-functionalized poly(lactide-co-glycolide) particles containing soluble PLP139-151 within their cores. The surface-functionalized poly(lactide-co-glycolide) particles were analyzed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water at a count rate of 1.792 × 10 counts per second. The population of surface-functionalized poly(lactide-co-glycolide) particles had a Z-average particle size of 584 nm, a peak diameter of 679 nm, and a polydispersity index of 0.162. These results are representative of six batches synthesized according to the protocol described above.

[0220] Figure 36 shows the characterization of surface-functionalized poly(lactide-co-glycolide) particles containing soluble PLP139-151 within their cores by zeta-potential measurements. Surface-functionalized poly(lactide-co-glycolide) particles were analyzed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water at a count rate of 6.67 × 10 counts per second. The population of surface-functionalized poly(lactide-co-glycolide) particles had a peak zeta potential of -48.9 mV and a zeta deviation of 5.14 mV. These results are representative of six batches synthesized according to the protocol described above.

[0221] Figure 37 shows the dynamic light scattering analysis characterization of surface-functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin within their cores. The surface-functionalized poly(lactide-co-glycolide) particles were analyzed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water at a count rate of 1.822 × 10 counts per second. The population of surface-functionalized poly(lactide-co-glycolide) particles had a Z-average particle size of 569.7 nm, a peak diameter of 700.3 nm, and a polydispersity index of 0.230. These results are representative of three batches synthesized according to the protocol described above.

[0222] Figure 38 shows the characterization of surface-functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin within their cores by zeta-potential measurements. Surface-functionalized poly(lactide-co-glycolide) particles were analyzed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water at a count rate of 2.67 × 10 counts per second. The population of surface-functionalized poly(lactide-co-glycolide) particles had a peak zeta potential of -52.2 mV and a zeta deviation of 5.38 mV. These results are representative of three batches synthesized according to the protocol described above.

[0223] Example 22 Soluble PLP in the core 139~151 Surface-functionalized liposomes containing α-glucan induce immunological tolerance in the murine R-EAE model of multiple sclerosis. The present inventors also discovered that biodegradable liposomal delivery vehicles, surface-functionalized with a high density of negatively charged groups and containing soluble antigens within the core, induce immunological tolerance in the R-EAE mouse model of multiple sclerosis.

[0224] The liposomes used in this study consisted of the following lipids in the following molar ratio: 30:30:40 phosphatidylcholine:phosphatidylglycerol:cholesterol. Groups of SJL / J mice were intraperitoneally injected with 200 nm surface-functionalized liposomes (10 μmol total lipid per mouse) containing soluble PLP139-151 peptide within the core on day -7 following priming with PLP139-151 / CFA on day 0. Control mice were primed on day 0 and then administered 500 nm to 700 nm surface-functionalized liposomes (10 μmol total lipid per mouse) containing soluble OVA323-339 peptide within the core on day -7. Mice were observed for clinical signs of R-EAE for an additional 17 days.

[0225] The results show the mean daily clinical scores versus days of priming with PLP139-151 / CFA. As shown in Figure 39, animals treated with surface-functionalized liposomes containing soluble PLP139-151 peptide in the core had lower clinical scores than animals treated with surface-functionalized liposomes containing soluble OVA323-339 peptide.

[0226] The results of this study demonstrate that biodegradable liposomes containing soluble PLP139-151 in their core and surface-functionalized with a high density of negatively charged groups are effective in inducing immunological tolerance in the SJL / J PLP139-151 / CFA-induced R-EAE mouse model of multiple sclerosis.

[0227] Tolerance induced by antigen-conjugated or antigen-encapsulated particles is antigen-specific, dose-dependent, and long-lasting (>150 days). Tolerance is best induced by intravenous administration of conjugated particles with a diameter of 500 nm to 1 μm and a zeta potential of ≤-5 mV. Tolerance induction relies on uptake of particles with a polyanionic surface by the MARCO scavenger receptor (MARCO) (e.g., carboxylated PS / PLG particles). Tolerance is induced and maintained by a combination of anergy (partially reversed by anti-PD-1 and agonistic anti-CD40 antibodies) and iTregs (partially reversed by anti-CD25 antibodies). The particles of the present invention primarily accumulate in marginal zone macrophages in the liver and spleen (CD11bhi CD11clo MARCO+ Sign-R1+ Siglec-1-).

[0228] Compared with the use of antigen-stimulatory or antigen-directed immature immunogenic dendritic cells or genetically engineered antigen-specific Tregs, there are numerous advantages to using antigen-binding particles for the treatment of autoimmune diseases. The preparation and induction of tolerogens is rapid and simple, using readily available, general-purpose immunogenic carriers that can be manufactured according to GMP standards; there is no need to isolate and expand immature dendritic cells or Tregs ex vivo; there is no need to worry about immature dendritic cells becoming activated during ex vivo manipulation and becoming stimulatory rather than immunogenic, or about Tregs converting to Th1 / 17 after transfer; these immature host marginal zone APCs process and present antigens in an immunogenic manner, allowing host APCs to select relevant immunodominant self-epitopes from PLG particles encapsulating whole autoantigens or tissue extracts (e.g., OVA-encapsulated PLG particles prevent AAD induced by OVA / alum); and the protocol is antigen-specific, safe, and highly efficient, without bystander suppression, and can induce unresponsiveness in both effector T cells (Th1, Th2, Th17, and CD8) involved in epitope spreading as well as naive T cells.

[0229] Synthetic biodegradable particles and liposomes could offer ease of manufacturing, broader availability of therapeutic agents, and increased potential therapeutic sites. To achieve this goal, we specifically designed surface-functionalized biodegradable poly(lactide-co-glycolide) particles with a high density of surface carboxylate groups using the surfactant poly(ethylene-alt-maleic anhydride).

[0230] We also developed surface-functionalized liposomes using a 30:30:40 ratio of phosphatidylcholine:phosphatidylglycerol:cholesterol.

[0231] We further engineered these particles to contain soluble ovalbumin within their cores to avoid chemical contamination and purity challenges surrounding surface conjugation of peptides or proteins. These surface-functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin within their cores are effective in preventing disease development and, therefore, in inducing immunological tolerance in the Balb / c ovalbumin / alum-induced AAI mouse model of allergic asthma. Peptides or proteins conjugated to carboxylate-functionalized poly(lactide-co-glycolide) particles using EDC adhere in a promiscuous manner, resulting in antigen aggregation and particle-antigen-particle aggregation that are difficult to characterize and purify into homogenous populations.

[0232] We purified a homogenous population of surface-functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin within their cores, which does not require surface conjugation of antigen.

[0233] We further demonstrated that biodegradable liposomes containing soluble PLP139–151 in their core and surface-functionalized with a high density of negatively charged groups were effective in inducing immunological tolerance in the SJL / J PLP139–151 / CFA-induced R-EAE mouse model of multiple sclerosis.

[0234] The liposomes and poly(lactide-co-glycolide) particles of the present invention offer numerous advantages, including: 1) Biodegradable particles do not remain in the body for a long time, and the time for complete decomposition can be controlled. 2) Particles and liposomes can be functionalized to promote internalization without activating cells. To this end, we loaded PLG microspheres with phosphatidylserine. 3) Particles and liposomes can also be designed to incorporate targeting ligands for specific cell populations. 4) Anti-inflammatory cytokines such as IL-10 and TGF-β can also be induced to limit activation of cell types that internalize within the particles and promote the induction of tolerance via anergy and / or deletion and activation of regulatory T cells.

[0235] Designer particles are a significant advance over polystyrene particles because the combined functions of this particle or liposome can target tolerance induction from multiple perspectives. Potential clinical applications of this tolerance induction technology include: (1) T cell and antibody-mediated autoimmune diseases (e.g., multiple sclerosis, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus) – Tolerance is induced using particles conjugated to the relevant autoantigens that drive the specific autoimmune disease. (2) Food and lung allergies, skin allergies, and asthma—Tolerance is induced using particles complexed with specific foods (e.g., peanut proteins), injected substances (e.g., bee venom proteins), or inhaled substances (e.g., ragweed pollen proteins, pet dander proteins) that induce an allergic response. (3) Transplant rejection - tolerance is induced to transplant antigens on the donor organ or donor cells before organ transplantation to prevent rejection by the recipient. (4) Enzyme replacement therapy - To prevent patients from forming a neutralizing antibody response to the recombinantly produced enzyme administered to treat the specific defect, tolerance is induced to an enzyme that patients with a genetic defect are unable to produce.

[0236] While particular embodiments of the present invention have been described and illustrated, such embodiments should be considered merely as examples of the invention and not as limitations on the invention as interpreted according to the appended claims.

[0237] All patents, applications, and other references cited herein are incorporated by reference in their entirety.

Claims

1. A composition comprising an antigen bound to carrier particles having a negative zeta potential.

2. 10. The composition of claim 1, wherein the zeta potential of the particles is from about -100 mV to about 0 mV.

3. 3. The composition of claim 2, wherein the zeta potential of the particles is from about −50 mV to about −40 mV.

4. The composition of claim 1, wherein the particles are copolymers having a molar ratio of about 80:20 to about 100:

0.

5. The composition of claim 1, wherein the particles are polystyrene particles, carboxylated polystyrene particles, or poly(lactic-co-glycolic acid) particles.

6. The composition of claim 1 , wherein the particles have a diameter of about 0.1 μm to about 10 μm.

7. The composition of claim 6, wherein the particles have a diameter of about 0.3 μm to about 5 μm.

8. The composition of claim 7, wherein the particles have a diameter of about 0.5 μm to about 3 μm.

9. The composition of claim 8, wherein the particles have a diameter of about 0.5 μm to about 1 μm.

10. 10. The composition of claim 9, wherein the particles have a diameter of about 0.5 μm.

11. The composition of claim 1 , wherein the antigen is bound to the surface of the carrier particle.

12. The composition of claim 1 , wherein the antigen is encapsulated within the carrier particles.

13. The composition of claim 1 , wherein the antigen comprises an autoimmune antigen, an antigen expressed on a tissue to be transplanted into a subject, or an allergen.

14. The antigens include myelin basic protein, acetylcholine receptor, endogenous antigen, myelin oligodendrocyte glycoprotein, pancreatic beta cell antigen, insulin, glutamic acid decarboxylase (GAD), type 11 collagen, human cartilage gp39, fp130-RAPS, proteolipid protein, fibrillarin, small nucleolar proteins, thyroid stimulating factor receptor, histone, glycoprotein gp70, pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PCD-E2), hair follicle antigen, A-gliadin, and human tropomyosin isoform 5, bahiagrass pollen (BaGP), peach allergen Pru p 3, αs-1 casein milk allergen, Apig1 celeriac allergen, Bere1 Brazil nut allergen, B-lactoglobulin milk allergen, bovine serum albumin, Cor a 14. The composition of claim 13, comprising at least a portion of a protein selected from the group consisting of: 1.04 hazelnut allergen, or ovalbumin egg allergen.

15. The antigen may be myelin basic protein, acetylcholine receptor, endogenous antigen, myelin oligodendrocyte glycoprotein, pancreatic β-cell antigen, insulin, glutamic acid decarboxylase (GAD), type 11 collagen, human cartilage gp39, fp130-RAPS, proteolipid protein, fibrillarin, small nucleolar protein, thyroid-stimulating factor receptor, histone, glycoprotein gp70, pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PCD-E2), hair follicle antigen, A-gliadin, or human tropomyosin isoform 5, bahiagrass pollen (BaGP), peach allergen Pru p 3, αs-1 casein milk allergen, Apig1 celeriac allergen, Bere1 Brazil nut allergen, B-lactoglobulin milk allergen, bovine serum albumin, Cor a 14. The composition of claim 13, comprising 1.04 hazelnut allergen, or ovalbumin egg allergen.

16. 10. The method of claim 1, wherein the antigen is attached to the particle by a conjugate molecule. Finished product.

17. 17. The composition of claim 16, wherein the conjugate molecule is ethylene carbodiimide (ECDI).

18. The composition of claim 1 , wherein the particles are biodegradable.

19. The composition of claim 1 , wherein the particles are surface functionalized.

20. 20. The composition of claim 19, wherein the particles are surface functionalized with a carboxylic acid.

21. The composition of any one of claims 1 to 20, further comprising a pharmaceutically acceptable carrier.

22. 1. A method of inducing antigen-specific tolerance in a subject, comprising administering to the subject an effective amount of a composition comprising antigen-bound particles, wherein the particles have a negative zeta potential.

23. 23. The method of claim 22, wherein the administering is for treating or preventing a disease or condition.

24. 24. The method of claim 23, wherein the disease or condition is selected from the group consisting of an autoimmune disease, an inflammatory disease, an allergy, a transplant rejection, and a hyperimmune response.

25. 25. The method of claim 24, wherein the disease or condition is selected from the group consisting of multiple sclerosis, type 1 diabetes, asthma, food allergies, environmental allergies, celiac disease, and conditions caused by an antigen in the subject to reduce an over-reaction to the antigen.

26. 23. The method of claim 22, wherein the particles are polystyrene particles, carboxylated polystyrene particles, or poly(lactic-co-glycolic acid) particles.

27. 27. The method of claim 26, wherein the particles are poly(lactic-co-glycolic acid) particles.

28. 28. The method of claim 27, wherein administration of the poly(lactic-co-glycolic acid) particles results in less anaphylaxis compared to administration of polystyrene particles.

29. 23. The method of claim 22, wherein the composition is administered intravenously.

30. 1. A process for preparing immunomodified particles having a negative zeta potential, comprising: The process comprises contacting an immunomodified particle precursor with a buffer under conditions effective to form the immunomodified particle having a negative zeta potential.

31. The process of claim 30 , wherein the immunomodified particle precursor is formed by copolymerization.

32. 31. The process of claim 30, wherein the buffer has a basic pH.

33. 31. The process of claim 30, wherein the buffer is sodium bicarbonate, potassium bicarbonate, lithium bicarbonate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, or lithium dihydrogen phosphate.

34. A composition comprising an antigen encapsulated within the core of a surface-functionalized liposome.

35. 35. The composition of claim 34, wherein the liposomes are composed of a 30:30:40 ratio of phosphatidylcholine:phosphatidylglycerol:cholesterol.

36. 35. The composition of claim 34, wherein the antigen comprises an autoimmune antigen, an antigen expressed on tissue to be transplanted into the subject, or an allergen.

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