Particles encapsulating fusion proteins containing linked epitopes

Abstract

To provide compositions and methods that allow incorporation of multiple disease epitopes into a single therapeutic composition for use in treatment of inflammatory diseases, particularly diseases where multiple epitopes or proteins are involved in pathogenesis.SOLUTION: The present invention provides compositions comprising biodegradable particles that encapsulate two or more epitopes linked together by one or more linkers that are susceptible to cleavage by specific proteases. The present invention further provides methods for inducing antigen-specific tolerance and protective immune responses and for treating inflammatory diseases, such as autoimmune diseases, allergies, cancers, or infectious diseases.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 274,711, filed January 4, 2016, the entire contents of which are incorporated herein by reference.

[0002] Description of text files submitted electronically The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: Computer-readable copy of the Sequence Listing (Filename: COUR-013_01WO_Seqlist.txt, Recording Date: January 4, 2016, File Size: 1.17 MB). [Background technology]

[0003] Inflammatory diseases and disorders are conditions in which an aberrant or otherwise unregulated inflammatory response contributes to the pathogenesis or severity of the disease, and encompass a variety of illnesses including autoimmune diseases, allergies, cancer, and infectious diseases.

[0004] 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 blockers (S1B) such as cyclosporine A (CsA), FK506 (tacrolimus), and corticosteroids. However, these drugs often require high doses to achieve the desired efficacy, and long-term use frequently results in toxic side effects. Furthermore, even in patients who tolerate these drugs, the need for lifelong immunosuppressive therapy carries the risk of severe side effects, nephrotoxicity, and metabolic disorders. Furthermore, nonspecific immunosuppression generally inhibits both pathological immune responses (e.g., autoreactivity) and beneficial immune responses, such as those elicited against cancer cells and infectious pathogens. As a result, patients undergoing immunosuppressive therapy are at increased risk for the development of various cancers and severe infectious diseases.

[0005] Similarly, cancer treatments typically result in broad, non-specific immune activation in an attempt to eradicate cancer cells. However, these broad activation strategies result in damage and even death of healthy, non-cancerous or non-malignant tissues. Therefore, there is a need in the art for improved therapeutic agents that can effectively control immune responses in an antigen-specific manner. Such therapeutic agents would enable targeted treatment of inflammatory diseases such as autoimmune diseases and cancer, thereby minimizing negative side effects associated with broad, non-specific activation or inhibition of the immune system.

[0006] 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 involves the collection, separation, and processing of peripheral blood cells with disease-specific autoantigens and ethylene carbodiimide (ECDI)-binding reagents under sterile conditions. These peptide-bound cells are then reinfused into the donor / patient. This process is expensive, must be performed under closely monitored conditions by skilled practitioners, and the number of facilities capable of performing this procedure is limited. The use of red blood cells as a donor cell type expands the potential sources to include allogeneic donors, dramatically increasing the supply of source cells and potentially increasing the number of suitable delivery facilities to include any setting where blood transfusion is permitted, but significant drawbacks remain, including a limited supply of source cells and the need for blood type matching to minimize immune responses to donor cells.

[0007] Recently, peptide-linked particles have been described that eliminate the requirement for a source cell supply and avoid the tissue-type requirements of prior approaches (see U.S. Patent Publication No. 2012-0076831, incorporated herein by reference in its entirety). Nevertheless, the use of antigens bound to the outside of particles has been associated with increased anaphylaxis and poses significant chemical, manufacturing, and administrative challenges. However, encapsulating antigens within particles can avoid these adverse events. Surprisingly, size and charge can be altered to control the phenotype of the elicited immune response, inducing either an enhanced tolerogenic or regulatory response against a specific antigen (e.g., in the context of autoimmunity) or an enhanced protective immune response (e.g., in the context of cancer).

[0008] While particles encapsulating a single epitope or protein have been created, many pathologies are associated with multiple proteins. Furthermore, a single disease may have several immunogenic epitopes within each antigen. For example, multiple sclerosis (MS) is thought to involve an inflammatory response directed against multiple sites on several different self-proteins, including at least proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), and myelin basic protein (MBP). However, the primary target antigen is not known with certainty. Furthermore, target antigens vary between patients, and T cell reactivity to different antigens changes over time in a process known as epitope spreading. Therefore, encapsulation of the entire protein is necessary to ensure coverage of all possible antigenic epitopes to which a particular MS patient may be reactive at any given time. However, encapsulation is extremely challenging given the size and physical properties of these large proteins. Therefore, particles encapsulating multiple epitopes associated with a particular disease are likely to enhance the therapeutic efficacy of such particles. However, different protein properties such as solubility or isoelectric point add complexity to producing particles that encapsulate more than one protein or epitope, often resulting in variable and difficult to control encapsulation efficiencies.

[0009] Additionally, methods for inducing antigen-specific immune activation, for example against tumor antigens, have also been developed, including the use of multiple epitope constructs expressing MAGE3 and HPV in the treatment of head and neck squamous cell carcinoma (see U.S. Pat. Nos. 8,263,560 and 7,842,480). Furthermore, a multi-epitope system using RNA-lipoplexes (RNA-LPX) has also been described, employing individual RNA vectors encoding four tumor antigens (NY-ESO-1, MAGE-A3, tyrosinase, and TPTE) encapsulated in liposomes (Kranz et al. (See, e.g., J. et al., Nature, V. 534, pp. 396-401, 2016). Administration of these RNA-LPXs induced a systemic IFNα response and amplified T cell responses to the encoded antigen. However, this system cannot overcome the difficulties associated with encapsulating multiple independent components into a single particle. Variations in encapsulation efficiency can result in disproportionate incorporation of one component relative to another. Furthermore, the use of nucleic acid-based vaccines requires the use of endogenous transcription and translation pathways. Each of these factors increases the variability of the system, altering the relative expression of the encoded proteins and reducing the therapeutic efficacy of the composition. Therefore, there is a need in the art for compositions and methods that enable the incorporation of multiple disease epitopes into a single therapeutic composition used to treat inflammatory diseases, particularly diseases in which multiple epitopes or proteins are involved in the pathogenesis. Summary of the Invention

[0010] The present invention provides biodegradable particles encapsulating two or more epitopes linked together by one or more linkers. Linkers are amino acid sequences susceptible to cleavage by specific proteases, allowing for control of antigen presentation by major histocompatibility complex (MHC)-I or MHC-II, further enhancing control of the resulting immune response. By combining epitopes in a single protein, it is possible to deliver epitopes in a controlled ratio to one another. This allows for the production of particles capable of inducing tolerance to multiple epitopes. Such particles are useful for ameliorating inflammatory diseases associated with more than one epitope, such as autoimmune diseases or allergies. The incorporation of immunomodulators and agonists, such as TLR agonists, is also useful for the use of such particles in the treatment of cancer.

[0011] Some aspects of the present invention provide biodegradable particles comprising one or more encapsulated fusion proteins, each of which comprises two or more antigenic epitopes, the two or more antigenic epitopes separated by a linker, the linker comprising an amino acid sequence susceptible to specific cleavage, and the biodegradable particles have a negative zeta potential. In some embodiments, the biodegradable particles have a zeta potential of about -100 mV to about 0 mV. In further embodiments, the biodegradable particles have a zeta potential of about -50 mV to about -40 mV. In further embodiments, the biodegradable particles have a zeta potential of about -75 mV to about -50 mV. In still further embodiments, the biodegradable particles have a zeta potential of about -50 mV.

[0012] In some embodiments, the biodegradable particles comprise poly(lactide-co-glycolide) (PLG). In further embodiments, the biodegradable particles comprise PLG in a copolymer ratio of polylactic acid:polyglycolic acid of about 50:50. In some embodiments, the surface of the biodegradable particles is carboxylated. In further embodiments, carboxylation is achieved by using poly(ethylene-maleic anhydride) (PEMA), polyacrylic acid, or sodium cholate.

[0013] In some embodiments, the biodegradable particles have a diameter of about 0.1 μm to about 10 μm. In some embodiments, the biodegradable particles have a diameter of about 0.3 μm to about 5 μm. In some embodiments, the biodegradable particles have a diameter of about 0.5 μm to about 3 μm. In some embodiments, the biodegradable particles have a diameter of about 0.5 μm to about 1 μm. In some embodiments, the biodegradable particles have a diameter of about 0.5 μm. In some embodiments, the biodegradable particles have a diameter of about 0.6 μm.

[0014] Some aspects of the present invention provide biodegradable particles comprising one or more encapsulated fusion proteins, each of which comprises two or more antigenic epitopes, the two or more antigenic epitopes separated by a linker, and the biodegradable particles have a negative zeta potential. In some embodiments, the linker comprises an amino acid sequence susceptible to specific cleavage by a protease located in the phagolysosome of a cell or site susceptible to specific cleavage by a protease located in the cytosol of the cell. In some embodiments, the linker comprises an amino acid sequence susceptible to specific cleavage by a protease located in the phagolysosome of a cell or site susceptible to specific cleavage by a protease located in the cytosol of the cell.

[0015] In some embodiments, the site susceptible to specific cleavage by a protease located in the phagolysosome is susceptible to cleavage by a furin or cathepsin protease. In further embodiments, the site susceptible to specific cleavage by a protease located in the phagolysosome is susceptible to cleavage by a furin protease. In further embodiments, the site susceptible to specific cleavage by a protease located in the phagolysosome is susceptible to cleavage by a cathepsin protease. In still further embodiments, the site susceptible to specific cleavage by a protease located in the phagolysosome is one or more of cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin W, or cathepsin Z ... This is pusin L.

[0016] In a further embodiment, the site susceptible to specific cleavage by a protease located in the cytosol is susceptible to cleavage by a furin or cathepsin protease. In a further embodiment, the site susceptible to specific cleavage by a protease located in the cytosol is susceptible to cleavage by cathepsin S. In a further embodiment, the amino acid sequence of the linker comprises a site susceptible to specific cleavage by cathepsin L and a site susceptible to specific cleavage by cathepsin S. In yet a further embodiment, the amino acid sequence of the linker is Gly-Ala-Val-Val-Arg-Gly-Ala (SEQ ID NO: 5141).

[0017] Some aspects of the present invention provide biodegradable particles containing one or more encapsulated fusion proteins, each of which contains two or more antigenic epitopes, separated by a linker, the linker comprising an amino acid sequence susceptible to specific cleavage, and the biodegradable particles have a negative zeta potential. In some embodiments, the two or more antigenic epitopes include an autoimmune antigen, an antigen expressed on a tissue to be transplanted into a subject, an antigen derived from an enzyme for enzyme replacement therapy, or an antigen derived from an allergen. In further embodiments, the two or more antigenic epitopes each comprise at least a portion of a protein, the portions being derived from the same protein. In further embodiments, the two or more antigenic epitopes each comprise at least a portion of a protein, the portions being derived from different proteins. In some embodiments, the different proteins are associated with the same autoimmune disorder, the same tissue to be transplanted into a subject, or the same allergen.

[0018] Some embodiments of the present invention provide biodegradable particles comprising one or more encapsulated fusion proteins, each of which comprises two or more antigenic epitopes, the two or more antigenic epitopes being separated by a linker, the linker comprising an amino acid sequence susceptible to specific cleavage, and the biodegradable particles having a negative zeta potential. In some embodiments, the two or more antigenic epitopes are each 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, α-gliadin, gliadine, insulin, proinsulin, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), human tropomyosin isoform 5, bahiagrass pollen (BaGP), peach allergen Pru p3, αS-1 caeine milk allergen, Apig1 celeriac allergen, Bere1 Brazil nut allergen, B-lactoglobulin milk allergen, bovine serum albumin, Cor a 1.04 The composition comprises at least a portion of a protein selected from the group consisting of hazelnut allergen, myelin-associated glycoprotein, aquaporin, alpha 3 chain of type IV collagen, ovalbumin egg allergen, Advate, antihemophilic factor, Kogenate, Eloctate, recombinant factor VIII fusion protein, Refacto, Novo VIIa, recombinant factor VII, eptacog alfa, Helixate, Monanine, coagulation factor IX, Wilate, Ceredase, alglucerase, Cerezyme, imiglucerase, Elelso, taliglucerase alfa, Fabrazyme, agalsidase beta, Aldurazyme, -I-iduronidase, Myozyme, acid glucosidase, Elaprace, iduronate-2-sulfatase, Naglazyme arylsulfatase B, or N-acetylgalactosamine-4-sulfatase.

[0019] In some embodiments, the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 2-1294. In further embodiments, the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 1295-1724; SEQ ID NOs: 1726-1766; SEQ ID NOs: 4986-5140; and discontinuous epitopes derived from SEQ ID NO: 1725.

[0020] In some embodiments, the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 1767-1840; SEQ ID NOs: 1842-1962; SEQ ID NOs: 1964-2027; SEQ ID NOs: 2029-2073; SEQ ID NOs: 2075-2113; SEQ ID NOs: 2115-2197; SEQ ID NOs: 2199-2248; SEQ ID NOs: 2250-2259; SEQ ID NOs: 2261-2420; SEQ ID NOs: 2422-2486; SEQ ID NOs: 2489-2505, and discontinuous epitopes derived from SEQ ID NOs: 1841, 1963, 2028, 2074, 2114, 2198, 2260, 2249, 2421, 2487, and 2488.

[0021] In some embodiments, the two or more antigenic epitopes are selected from the group consisting of discontinuous epitopes derived from SEQ ID NOs: 2506-3260; 3262-3693; and 3261. In some embodiments, the two or more antigenic epitopes are selected from the group consisting of discontinuous epitopes derived from SEQ ID NOs: 3694-3857; 3860-4565; and 3857, 3858, and 3859. In some embodiments, the two or more antigenic epitopes are selected from the group consisting of discontinuous epitopes derived from SEQ ID NOs: 4566-4576; 4578-4610; 4612-4613; and SEQ ID NOs: 5018-5039; and 4357, 4577, and 4611.

[0022] In some embodiments, two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4614-4653. In some embodiments, two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4654-4694; SEQ ID NOs: 4696-4894; SEQ ID NOs: 4896-4901; and discontinuous epitopes derived from 4695 and 4895. In some embodiments, two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4902-4906. In some embodiments, two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4907-4914. In some embodiments, two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4915-4917. In some embodiments, two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4918-4941. In some embodiments, two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4942-4952. In some embodiments, the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4953 to 4963. In some embodiments, the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4964 to 4974.

[0023] Some aspects of the present invention provide biodegradable particles containing one or more encapsulated fusion proteins, each of which contains two or more antigenic epitopes, the two or more antigenic epitopes separated by a linker, the linker comprising an amino acid sequence susceptible to specific cleavage, and the biodegradable particles have a negative zeta potential. In some embodiments, the two or more antigenic epitopes are derived from a therapeutic antibody, or an antigen-binding fragment thereof, such as an Fc fragment. In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a humanized monoclonal antibody, a human monoclonal antibody, a chimeric antibody, a single-chain antibody, an antigen-binding fragment region (Fab), a single-chain variable fragment (scFv), a small modular immunopharmaceutical (SMIP), or a single-chain antigen-binding domain.

[0024] In further embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of α4β1 integrin, Bacillus anthracis, BL(γS), C5, CD3, CD11a, CD20, CD25, CD30, In further embodiments, the antibody or antigen-binding fragment thereof binds to CD33, CD52, CD59, CTLA4, EGFR, GD2, GPIIb, IIIa, HER2, IgE, IL-1β, IL-5, IL12 / 23, PCSK9, PD1, RANK, RSV-F protein, TNFα, or VEGF-A. In further embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of abciximab, adalimumab, adotrastuzumab emtansine, alemtuzumab, basiliximab, bevacizumab, belimumab, blinatumomab, brentuximab vedotin, canakinumab, catumaxomab, cetuximab, certolizumab pegol, daclizumab, denosumab, dinutuximab, eculizumab, efalizumab, evolocumab, gemtuzumab ozogamicin, golimumab ib, ibritumomab tiuxetan, ipilimumab, infliximab, motavizumab, muronomab, natalizumab, nivolumab, obinutuzumab, ofatumumab, omalizumab, panitumumab, palivizumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, raxibacumab, rituximab, secukinumab, siltuximab, trastuzumab, tocilizumab, tositumomab-I-131, ustekinumab, or vedolizumab.

[0025] In some embodiments of the invention, the two or more antigenic epitopes are derived from a variant of a therapeutic antibody or antigen-binding fragment thereof lacking functional complementarity-determining regions (CDRs). In further embodiments, the variant of the antibody or antigen-binding fragment thereof lacking functional CDRs is a monoclonal antibody, a humanized monoclonal antibody, a human monoclonal antibody, a chimeric antibody, a single-chain antibody, a fragment antigen-binding region (Fab), a single-chain variable fragment (scFv), a small modular immunopharmaceutical (SMIP), or a single-chain antigen-binding domain.

[0026] Some aspects of the present invention provide biodegradable particles comprising one or more encapsulated fusion proteins, each of which comprises two or more antigenic epitopes, the two or more antigenic epitopes being separated by a linker, the linker comprising an amino acid sequence susceptible to specific cleavage, and the biodegradable particles having a negative zeta potential. In some embodiments, one of the one or more fusion proteins comprises the antigenic epitope MOG. 1-20 , MBP 13-32 , MOG 35-55 , MBP 146-170 , P.L.P. 139-154 , MBP 111-129 , and MBP 83-99 In a further embodiment, one of said one or more fusion proteins comprises One contains the antigenic epitopes SEQ ID NO: 1350, SEQ ID NO: 4986, and SEQ ID NO: 4987.

[0027] Some aspects of the present invention provide pharmaceutical compositions comprising the biodegradable particles described herein. In further aspects, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. In further aspects, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0028] Some aspects of the present invention provide methods for inducing antigen-specific tolerance in a subject, comprising administering an effective amount of a biodegradable particle described herein. In some aspects, the method for inducing antigen-specific tolerance in a subject comprises administering to the subject an effective amount of biodegradable particles comprising one or more encapsulated fusion proteins, each of the one or more fusion proteins comprising two or more antigenic epitopes, the two or more antigenic epitopes being separated by a linker, the linker comprising an amino acid sequence susceptible to specific cleavage, and the biodegradable particles having a negative zeta potential. In some embodiments, an effective amount of the biodegradable particles is administered to the subject orally, intravenously, sublingually, bucally, intestinal, topically, rectally, subcutaneously, nasally, intraosseously (i.e., intraosseous injection), intraperitoneally, intrathecally, transdermally, or transmucosally. In further embodiments, an effective amount of the biodegradable particles is administered to the subject intravenously or subcutaneously. In further embodiments, an effective amount of the biodegradable particles is administered to the subject intravenously. In a further embodiment, an effective amount of the biodegradable particles is administered subcutaneously to the subject.

[0029] In some embodiments, an effective amount of the biodegradable particles described herein is used to treat a disease or The composition is administered to a subject to treat or prevent a condition. In some embodiments, the disease or condition is selected from the group consisting of autoimmune diseases, lysosomal storage diseases, enzyme deficiencies, inflammatory diseases, allergies, transplant rejection, and hyperimmune responses. In further embodiments, the disease or condition is selected from the group consisting of multiple sclerosis, type 1 diabetes, asthma, food allergies, environmental allergies, celiac disease, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), mucopolysaccharidosis, gangliosidosis, low alkaline phosphatase, cholesterol ester storage disease, hyperuricemia, growth hormone deficiency, renal anemia, hemophilia, hemophilia A, hemophilia B, von Willebrand disease, Gaucher disease, Fabry disease, Hurler disease, Pompe disease, Hunter disease, Maroteaux-Lamy disease, and conditions triggered by an antigen in a subject resulting in an exaggerated response to the antigen.

[0030] In some embodiments, the disease or condition is multiple sclerosis, and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 2-1294.

[0031] In some embodiments, the disease or condition is celiac disease, and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 1295-1724; SEQ ID NOs: 1726-1766; SEQ ID NOs: 4986-5140; and discontinuous epitopes derived from SEQ ID NO: 1725.

[0032] In some embodiments, the disease or condition is type 1 diabetes, and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 1767-1840; SEQ ID NOs: 1842-1962; SEQ ID NOs: 1964-2027; SEQ ID NOs: 2029-2073; SEQ ID NOs: 2075-2113; SEQ ID NOs: 2115-2197; SEQ ID NOs: 2199-2248; SEQ ID NOs: 2250-2259; SEQ ID NOs: 2261-2420; SEQ ID NOs: 2422-2486; SEQ ID NOs: 2489-2505; and discontinuous epitopes derived from SEQ ID NOs: 1841, 1963, 2028, 2074, 2114, 2198, 2260, 2249, 2421, 2487, and 2488.

[0033] In some embodiments, the disease or condition is rheumatoid arthritis, and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of discontinuous epitopes derived from SEQ ID NOs: 2506-3260; SEQ ID NOs: 3262-3693; and 3261.

[0034] In some embodiments, the disease or condition is systemic lupus, and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 3694-3857; SEQ ID NOs: 3860-4565; and discontinuous epitopes from 3857, 3858, and 3859.

[0035] In some embodiments, the disease or condition is Goodpasture's syndrome, and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4566-4576; SEQ ID NOs: 4578-4610; SEQ ID NOs: 4612-4613; and SEQ ID NOs: 5018-5039; and discontinuous epitopes derived from 4357, 4577, and 4611.

[0036] In some embodiments, the disease or condition is uveitis, and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4614-4653.

[0037] In some embodiments, the disease or condition is thyroiditis, and Each of the fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4654-4694; SEQ ID NOs: 4696-4894; SEQ ID NOs: 4896-4901; and discontinuous epitopes derived from 4695 and 4895.

[0038] In some embodiments, the disease or condition is myositis, and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4902-4906.

[0039] In some embodiments, the disease or condition is vasculitis, and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4907-4914.

[0040] In some embodiments, or the condition is pancreatitis, and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4915-4917.

[0041] In some embodiments, the disease or condition is Crohn's disease, and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4918-4941.

[0042] In some embodiments, the disease or condition is ulcerative colitis, and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4942-4952.

[0043] In some embodiments, the disease or condition is psoriasis, and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4953-4963.

[0044] In some embodiments, the disease or condition is reactive arthritis, and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4964-4974.

[0045] Some aspects of the present invention provide methods for reducing suppressive neutrophil accumulation in a subject, comprising administering to the subject an effective amount of a biodegradable particle described herein. In some embodiments, the subject has cancer. In some embodiments, the two or more antigenic epitopes are selected from the group consisting of CD19, CD20, BCMA, CD22, CLL1, CD33, CEA, CD123, CS1, EGFR, PSMA, EphA2, MCSP, ADAM17, PSCA, TPTE, HPU16, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, Ep-CAM, EphA3, Her2 / neu, telomerase, and mesothelial cell proliferation. The protein comprises at least a portion of a protein selected from the group consisting of thelin, SAP-1, survivin, BAGE family proteins, CAGE family proteins, GAGE ​​family proteins, MAGE family (e.g., MAGE-A3), SAGE family proteins, XAGE family proteins, CT9, CT10, NY-ESO1 / LAGE-1, PRAME, SSX-2, MelanA / MART-1, Cp100 / pmel17, tyrosinase, TRP-1 / TRP-2, P. polypeptide, MC1R, prostate-specific antigen, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII, and MUC1.

[0046] Some embodiments of the present invention provide a method for administering to a subject an effective amount of one or more encapsulated fusion proteins.

[0013] The present invention provides a method for increasing tissue regeneration in a subject, comprising administering biodegradable particles comprising one or more fusion proteins each comprising two or more antigenic epitopes, the two or more antigenic epitopes separated by a linker, the linker comprising an amino acid sequence susceptible to specific cleavage, and the biodegradable particles having a negative zeta potential. In some embodiments, the particles increase epithelial cell regeneration in a colitis patient. In further embodiments, each of the one or more fusion proteins encapsulated in the particles comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4918-4941 and SEQ ID NOs: 4942-4952. In some embodiments, the particles increase remyelination in a multiple sclerosis patient. In further embodiments, each of the one or more fusion proteins encapsulated in the particles comprises two or more antigenic epitopes derived from myelin basic protein and / or myelin oligodendrocyte glycoprotein. In a further embodiment, each of the one or more fusion proteins encapsulated in a particle comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 2-1294.

[0047] Some aspects of the present invention provide methods for reducing the incidence and / or severity of an immune response to a therapeutic protein by a subject, comprising administering to the subject an effective amount of biodegradable particles comprising one or more encapsulated fusion proteins, each of the one or more fusion proteins comprising two or more antigenic epitopes, the two or more antigenic epitopes separated by a linker, the linker comprising an amino acid sequence susceptible to specific cleavage, and the biodegradable particles having a negative zeta potential. In some embodiments, the subject is receiving enzyme replacement therapy for the treatment of a disease selected from the group consisting of hemophilia, hemophilia A, hemophilia B, von Willebrand disease, Gaucher disease, Fabry disease, Hurler disease, Pompe disease, Hunter disease, mucopolysaccharidosis, gangliosidosis, low alkaline phosphatase, cholesterol ester storage disease, hyperuricemia, growth hormone deficiency, renal anemia, and Maroteaux-Lamy disease.

[0048] In further embodiments, the antigenic epitope comprises one or more enzymes selected from the group consisting of Advate, antihemophilic factor, Kogenate, Eloctate, recombinant factor VIII fusion protein, Refacto, Novo VIIa, recombinant factor VII, eptacog alfa, Helixate, Monanine, coagulation factor IX, Wilate, Ceredase, alglucerase, Cerezyme, imiglucerase, Elelso, taliglucerase alfa, Fabrazyme, agalsidase beta, Aldurazyme, -I-iduronidase, Myozyme, acid glucosidase, Elaprace, iduronate-2-sulfatase, Naglazyme arylsulfatase B, and N-acetylgalactosamine-4-sulfatase. In further embodiments, the antigenic epitope comprises one or more proteins selected from the group consisting of interferon alpha, interferon alpha-2a, interferon beta Ib, interferon beta Ia, insulin, DNAase, Neupogen, Epogen, Procrit (epotein alpha), Aranesp (second generation Procrit), Intron A (interferon alpha-2b), IL-2 (Proleukin), IL-Ira, BMP-7, TNF-alpha Ia, tPA, PDGF, interferon gamma-Ib, uPA, GMCSF, Factor VII, Factor VIII, Betaferon (interferon beta-Ia), somatotropin, and Rebif (interferon beta-Ia).

[0049] In some embodiments, the therapeutic protein is an antibody, or antigen-binding fragment thereof, Fc fragment. In further embodiments, the antibody, or antigen-binding fragment thereof, Fc fragment is selected from the group consisting of abciximab, adalimumab, adotrastuzumab emtansine, alemtuzumab, basiliximab, bevacizumab, belimumab, blinatumomab, brentuximab vedotin, canakinumab, catumaxomab, cetuximab, and sebetuximab. These include lutolizumab pegol, daclizumab, denosumab, dinutuximab, eculizumab, efalizumab, evolocumab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan, ipilimumab, infliximab, motavizumab, muronomab, natalizumab, nivolumab, obinutuzumab, ofatumumab, omalizumab, panitumumab, palivizumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, raxibacumab, rituximab, secukinumab, siltuximab, trastuzumab, tocilizumab, tositumomab-I-131, ustekinumab, and vedolizumab.

[0050] Some aspects of the present invention provide methods for enhancing or inducing a protective immune response in a subject, comprising administering an effective amount of a biodegradable particle described herein. In some embodiments, the method comprises administering to a subject an effective amount of biodegradable particles comprising one or more encapsulated fusion proteins, each of which comprises two or more antigenic epitopes, the two or more antigenic epitopes being separated by a linker, the linker comprising an amino acid sequence susceptible to specific cleavage, and the biodegradable particles having a negative zeta potential. In some embodiments, the biodegradable particles are administered to a subject orally, intravenously, sublingually, bucally, intestinal, topically, rectally, subcutaneously, nasally, intraosseously (i.e., intraosseous injection), intraperitoneally, intrathecally, transdermally, or transmucosally. In some embodiments, the biodegradable particles are administered to a subject intravenously or subcutaneously. In further embodiments, the biodegradable particles are administered to a subject intravenously. In further embodiments, the biodegradable particles are administered to a subject subcutaneously.

[0051] Some aspects of the present invention provide methods for increasing or inducing a protective immune response in a subject, comprising administering an effective amount of a biodegradable particle described herein to the subject, wherein the biodegradable particle is administered to the subject to treat or prevent a disease or condition. In some embodiments, the disease or condition is cancer or an infectious disease. In some embodiments, the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, such as liposarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, leiomyosarcoma, chordoma, lymphangiosarcoma, lymphangioendothelial sarcoma, rhabdomyosarcoma, fibrosarcoma, myxosarcoma, chondrosarcoma, neuroendocrine tumor, mesothelioma, synovioma, schwannoma, meningioma, adenocarcinoma, melanoma, leukemia, and lymphoid tumors. In some embodiments, the two or more antigenic epitopes are each selected from the group consisting of CD19, CD20, BCMA, CD22, CLL1, CD33, CEA, CD123, CS1, EGFR, PSMA, EphA2, MCSP, ADAM17, PSCA, TPTE, HPU16, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, Ep-CAM, EphA3, and Her2 / ne. The antibody or antibody fragments may comprise at least a portion of a protein selected from the group consisting of u, telomerase, mesothelin, SAP-1, survivin, BAGE family proteins, CAGE family proteins, GAGE ​​family proteins, MAGE family (e.g., MAGE-A3), SAGE family proteins, XAGE family proteins, CT9, CT10, NY-ESO1 / LAGE-1, PRAME, SSX-2, MelanA / MART-1, Cp100 / pmel17, tyrosinase, TRP-1 / TRP-2, P. polypeptide, MC1R, prostate-specific antigen, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII, and MUC1.

[0052] In some embodiments, the infectious disease is a bacterial infection, a fungal infection, a parasitic infection, or a viral infection. In some embodiments, the viral infection is selected from the group consisting of a herpesvirus infection, a hepatitis virus infection, a West Nile virus infection, a flavivirus infection, an influenza virus infection, a rhinovirus infection, a papillomavirus infection, a paramyxovirus infection, a parainfluenza virus infection, and / or a retrovirus infection. In some embodiments, the bacterial The infection is selected from the group consisting of a staphylococcal infection, a streptococcal infection, a mycobacterial infection, a bacillary infection, a salmonella infection, a vibrio infection, a spirochete infection, and a neisseria infection.

[0053] Some embodiments of the present invention provide biodegradable particles comprising encapsulated one or more fusion proteins, each of which is selected from the group consisting of MOG 1-20 , MBP 13-32 , MOG 35-55 , MBP 146-170 , P.L.P. 139-154 , MBP 111-129 , and MBP 83-99 wherein the two or more antigenic epitopes are separated by a linker, the linker comprising an amino acid sequence susceptible to specific cleavage; the biodegradable particles have a diameter of about 200 nm to 1000 nm; and the biodegradable particles have a negative zeta potential of less than -30 mV.

[0054] Some embodiments of the present invention provide methods of treating multiple sclerosis in a subject, comprising administering an effective amount of biodegradable particles comprising one or more encapsulated fusion proteins, each of which is a fusion protein selected from the group consisting of MOG, PG ... 1-20 , MBP 13-32 , MOG 35-55 , MBP 146-170 , P.L.P. 139-154 , MBP 111-129 , and MBP 83-99The biodegradable particles comprise two or more antigenic epitopes selected from the group consisting of: (I) (II) (III) (IV ... [Brief explanation of the drawings]

[0055] [Figure 1] 1 shows an exemplary fusion protein encapsulated in a biodegradable particle. [Figure 2] The concept of multiple peptides is illustrated in a table showing the tolerogenic and control antigens, amino acid sequences, and sample samples (Figure 2A). Also shown is a diagram highlighting the difference between single encapsulation peptides (top) and multiple encapsulation peptides (bottom) (Figure 2B). [Figure 3] Physical analysis including size distribution (FIG. 3A) and zeta potential (FIG. 3B) of particles encapsulating PLP139-151 as determined by light scattering is shown. [Figure 4] Figure 4 shows the results of an in vitro proliferation assay of PLG nanoparticles using DO11.10 transgenic T cells. Figure 4A shows the results for cells treated with nanoparticles alone, and Figure 4B shows the results for cells treated with nanoparticles and 1 μg of Ova323. [Figure 5] Figure 5 shows the results of an in vitro proliferation assay of PLG nanoparticles using transgenic DO11.10 T cells. Results are shown for nanoparticles alone (Figure 5A), cells containing only nanoparticles (Figure 5B), cells containing nanoparticles and 1 μg of Ova323 (Figure 5C), and cells containing nanoparticles and 1 μg / mL of αCD28 (Figure 5D). [Figure 6] A list of particle batches encapsulating tolerogenic antigens individually (PLP139-151, PLP178-191, MBP84-104, and MOG92-106) or together (all tolerogenic) is shown. [Figure 7] A list of particle batches encapsulating control peptides individually (Ova323-339, PLP56-70, VP1233-250, and VP270-86) or together (all controls) is shown. [Figure 8] Figure 8 shows the effect of PLG particles encapsulating control (OVA323-339-PLG) or tolerogenic (PLP139-151-PLG) peptides on the splenic regulatory type 1 T cell (TR1) population. Figure 8A shows the percentage of LAG3+FoxP3- cells and the percentage of antigen-specific TR1 cells (LAG3+FoxP3-) that were also IFNγ+IL-10+ in mice injected with PLG particles encapsulating control or tolerogenic peptides on days 3 (left) and 5 (right). Figure 8B shows the number of LAG3+FoxP3- cells in mice treated with control or peptides encapsulating tolerogenic peptides. Figure 8C shows the number of LAG3+FoxP3-IFNγ+IL-10+ cells in mice treated with control or peptides encapsulating tolerogenic peptides. [Figure 9] Data from a separate but overlapping experiment are shown in Figure 8. These data differ from those shown in Figure 8 in that TR1 is not confirmed as FoxP3- in this graph. [Figure 10] Flow cytometry analysis of splenic regulatory T cell populations after transfer of naive 5B6 (PLP139-151 TCR transgenic mouse) lymphocytes into naive SJL mice and treatment with either PLP139-151-PLG particles or control OVA323-339-SE PLG particles is shown. All results are gated on the CD90.1 / Thy1.1 (PLP139-151 TCR+) population. [Figure 11]CD4+ cells from donor 5B6 were transferred into naive SJL mice and treated with either PLP139-151-SE PLG particles or control OVA323-339-SE PLG particles (d3 and d5). EAE was induced in separate cohorts (day 5 post-EAE and day 17 post-EAE). Flow cytometry analysis of the numbers of antigen-specific T cells (Figure 11A), proliferating antigen-specific T cells (Figure 11B), antigen-specific regulatory T cells (Figure 11C), and non-antigen-specific regulatory T cells (Figure 11D) is shown. Unless otherwise indicated, all results are gated on the CD90.1 / Thy1.1 (PLP139-151 TCR+) population (Figure 11D). [Figure 12] CD4+ cells from DO11 (OVA323-339 TCR transgenic mouse) donors were transferred into naive Balb / c RAG KO mice, and the increase in antigen-specific regulatory T cells after treatment with either OVA323-339-SE PLG particles or control PLP139-151-SE PLG particles is shown. Flow cytometry analysis of the percentage (FIG. 12A) and number (FIG. 12B) of regulatory CD25+FoxP3+ T cell populations is shown. All results are gated on the DO11 TCR+ population. [Figure 13] Flow cytometry analysis of the percentage (FIG. 13A) and number (FIG. 13B) of IFNγ-producing antigen-specific regulatory T cells is shown, demonstrating IFNγ-producing antigen-specific regulatory T cells after transfer of CD4+ cells from DO11 (OVA323-339 TCR transgenic mouse) donors into naive Balb / c RAG KO mice and treatment with either OVA323-339-SE PLG particles or control PLP139-151-SE PLG particles. [Figure 14] Flow cytometry analysis of percentage (FIG. 14A) and number (FIG. 14B) of the TR1 population after CD4+ cells from DO11 (OVA323-339 TCR transgenic mouse) donor were transferred into naive Balb / c RAG KO mice and treated with either OVA323-339-SE PLG particles or control PLP139-151-SE PLG particles is shown. [Figure 15] Figure 15 shows the expansion of antigen-specific regulatory T cell populations after injection of OVA323-339-SE PLG particles or control PLP139-151-SE PLG particles. Flow cytometry results are shown for Ki67+ DO11 TCR+ CD4+ cells (Figure 15A), CD25+ FoxP3+ gated on DO11 TCR+ CD4+ cells (Figure 15B), and Ki67+ CD49b+ LAG3+ cells (Figure 15C, from a separate experiment, not confirmed as FoxP3-). [Figure 16] The amino acid sequence of the PLP139-Ova323 fusion peptide linked by a cathepsin-specific cleavage site is shown. [Figure 17] 17A and 17B show the results of an in vitro proliferation assay using the PLP139-Ova323 fusion peptide and DO11 (FIG. 17A) or 5B6 (FIG. 17B) cells. [Figure 18] Characterization of three attempts at encapsulation of the PLP139-Ova323 fusion peptide is shown. [Figure 19] 19A and 19B show the results of an in vitro proliferation assay of DO11.10 transgenic T cells treated with PLG(PLP139-Ova323) particles, PLG(Ova323) particles, or PLG(PLP139) particles. Results are shown for cells treated with nanoparticles alone (FIG. 19A), nanoparticles and 1 μg of Ova323 (FIG. 19B), and nanoparticles, 1 μg of Ova323, and 1 μg / mL of αCD28 (FIG. 19C). [Figure 20] 20A and 20B show the results of an in vitro proliferation assay using 5B6 transgenic T cells treated with PLG(PLP139-Ova323) particles, PLG(Ova323) particles, or PLG(PLP139) particles. Results are shown for cells treated with nanoparticles alone (FIG. 20A), nanoparticles and 1 μg of PLP139 (FIG. 20B), and nanoparticles, 1 μg of PLP139, and 1 μg / mL of αCD28 (FIG. 20C). [Figure 21] 1 is a table listing particle batches encapsulating the PLP139-Ova323 fusion peptide. [Figure 22]Induction of EAE after administration of PLP139-Ova323 fusion peptide is shown, as indicated by disease score (FIG. 22A) and mean ear swelling (FIG. 22B). [Figure 23] The amino acid sequences of the four-epitope fusion peptides are shown: EAE-1 binding epitope, PLP139:PLP178:MOG92:MBP (Figure 23A), and control binding epitope, OVA323:PLP56:VP1-233:VP2-70 (Figure 23B). [Figure 24] 1 shows the characterization of nanoparticles encapsulating a tolerogenic antigen fusion peptide (EAE-1 binding epitope). [Figure 25] 1 is a table listing particle batches encapsulating a tolerogenic fusion peptide (EAE-1 binding epitope) or a negative control fusion peptide. [Figure 26] The encapsulation efficiency of single and combined EAE-specific epitopes is shown. [Figure 27] The effect of encapsulated EAE-1 binding epitopes (EAE-1 tolerance treatment) compared to encapsulated control binding epitopes (control binding epitope tolerance), OVA, and PLP139 on EAE disease scores is shown. [Figure 28] 28A and 28B show that FALK peptide induces EAE (FIG. 28A) and T cell proliferation (FIG. 28B). [Figure 29] Potential fusion peptides containing neoepitopes, immunomodulators, and TLR agonists are shown. [Figure 30] An overview of the development of conjugated tumor epitope proteins, their subsequent nanoparticle encapsulation, and patient administration protocols is presented. [Figure 31] Figure 31 shows predicted results of functional in vitro assays in which peripheral blood monocytes were treated with encapsulated NyEso1 with or without anti-PD1. Assays for the effect on T cell proliferation (Figure 31A), IFNγ production (Figure 31B), and IFNα production (Figure 31C) are shown. [Figure 32] 1 shows predicted survival curves for mice treated with encapsulated NyEso1 with or without anti-PD1 in a mouse model of melanoma. [Figure 33] Exemplary in vitro functional assays of PBMCs treated with encapsulated conjugated NY-ESO-1:Mage-A3:TPTE:tyrosinase fusion protein are shown, along with predicted results of a T cell proliferation assay (FIG. 33A) and IFNγ production (FIG. 33B). [Figure 34] Potential fusion peptides containing epitopes found in multiple diseases and disorders are shown. DETAILED DESCRIPTION OF THE INVENTION

[0056] The inventors of the present invention have discovered that nanoparticles encapsulating fusion proteins consisting of multiple peptide epitopes connected by cleavable linkers with specific protease sites can induce antigen-specific immune tolerance and thus control immune responses in a number of disease models. In one embodiment, such particles can reduce immune responses to one or more of the peptide epitopes of the fusion protein, and can also inhibit immune responses to more than one These particles are particularly useful in the treatment of diseases or conditions characterized by an excessive inflammatory immune response associated with many antigenic epitopes, e.g., autoimmune diseases or allergies. In another embodiment, such particles are capable of inducing a protective immune response against one or more of the peptide epitopes of the fusion protein, and are particularly useful in the treatment of diseases or conditions characterized by the absence of an immunogenic response, e.g., cancer.

[0057] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0058] As used herein, the term "and / or" is used in this disclosure to mean either "and" or "or," unless the context dictates otherwise.

[0059] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations thereof, such as "comprising" or "comprises," will be understood to refer to the inclusion of a stated element or integer or group of elements or integers, but not to the exclusion of any other element or integer or group of elements or integers.

[0060] All publications and patents mentioned in and / or listed below in this application are hereby incorporated by reference in their entirety.

[0061] fusion proteins Certain embodiments of the present invention are based, at least in part, on the novel discovery that particles encapsulating multiple antigens or epitopes can induce tolerance to each of these antigens when the antigens are linked together in a fusion protein by a cleavable linker. In some embodiments, the linker is an amino acid sequence containing a specific protease site and can be designed to enable processing by either the class I pathway or the class II pathway. In such embodiments, epitopes encapsulated in particles bound on the same fusion protein can be processed by both the class I pathway and the class II pathway. Thus, an epitope processed by the class I pathway can be combined with an epitope processed by the class II pathway in the encapsulated fusion protein.

[0062] In some embodiments described herein, the fusion protein is encapsulated by a biodegradable particle. The terms "fusion protein," "fusion peptide," "fusion polypeptide," and "chimeric peptide" are used interchangeably herein to refer to a polypeptide chain created by the joining of two or more nucleotide sequences that originally encode different proteins or different portions of the same protein. Antigen fragments suitable for incorporation into the fusion proteins described herein include any fragment of the full-length peptide that retains the function of producing the desired antigen-specific tolerance function of the present invention. A "fragment" refers to a portion of a protein, preferably containing at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference sequence of the protein.

[0063] Fusion proteins can be produced by various means understood in the art (e.g., gene fusion, chemical conjugation, etc.). The polypeptides forming the fusion protein are typically joined C-terminally to N-terminally, but they may also be joined C-terminally to C-terminally, N-terminally to N-terminally, or N-terminally to C-terminally. The polypeptides of the fusion protein can be in any order. The two proteins can be fused either directly or via an amino acid linker. The peptide linker sequence allows each polypeptide to have its own secondary and tertiary structure. A linker sequence may be used to separate the first and second polypeptide components by a distance sufficient to ensure proper folding. Amino acid sequences that may be useful as linkers include those described in Maratea et al., Gene 40:39-46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262 (1986); U.S. Patent No. 4,935,233, and U.S. Patent No. 4,751,180, each of which is incorporated herein by reference in its entirety. Linker sequences generally may be from 1 to about 50 amino acids in length. In some embodiments, a linker sequence is not necessary and / or used, 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.

[0064] In preferred embodiments, individual antigens or epitopes are linked via amino acid linkers containing protease cleavage sites specific for intracellular proteases (e.g., proteases present in the phagolysosome or cytosol of a cell). In some embodiments, individual antigens or epitopes are linked by linkers containing the same protease cleavage site. In some embodiments, individual antigens or epitopes are linked by linkers containing different protease cleavage sites. In further embodiments, one or more of the linker sequences in the fusion protein may contain one or more protease cleavage sites.

[0065] Cleavage of the fusion protein by proteases located in the phagolysosome or cytosol induces cleavage products (e.g., individual peptide epitopes) for presentation as class I or class II antigens. Class I antigen presentation is mediated by cytosolic proteases and major histocompatibility complex (MHC)-I, which promotes intracellular protein presentation. Thus, MHC1 molecules typically present as autoantigens or foreign proteins as a result of intracellular infection. Antigens presented in the context of MHC1 are expressed by CD8 + T cells Class II antigen presentation is mediated by phagocytosis of extracellular antigens, which are degraded by proteases present in the phagolysosome. Extracellular antigens are presented in the context of MHC II and are typically recognized by CD4 + Recognized by T cells This recognition can trigger multiple downstream immune responses, e.g., Th1, Th2, Th17, Th22, or regulatory T cell responses, depending on the nature of the antigen, the activation state of the antigen-presenting cell, and the local cytokine microenvironment.

[0066] Therefore, the introduction of a specific cleavage site allows for the control of downstream immune response phenotypes. For example, in the context of autoimmunity, it may be desirable to introduce a cleavage site for a protease present in the phagolysosome to increase the likelihood that the epitope present in the fusion protein will be present on MHCII, thereby triggering a regulatory or tolerogenic response. Alternatively, in the context of cancer therapeutics, it may be desirable to introduce a cleavage site for a protease present in the cytosol to increase the likelihood that the epitope present in the fusion protein will be present on MHCII, thereby triggering a cytotoxic response that kills cancer cells.

[0067] The cleavage site can be specific for any type of protease, such as serine proteases, cysteine ​​proteases (e.g., cathepsins), metalloproteases, aspartic acid proteases, and others. In some embodiments, the cleavage site is specific for cathepsin and / or furin proteases located in the phagolysosome. In some embodiments, the cleavage site is specific for one or more of the cathepsin proteases located in the phagolysosome, such as cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin W, or cathepsin Z. In certain embodiments, the cleavage site is specific for cathepsin L. In some embodiments, the cleavage site is specific for cathepsin L. is specific for cathepsin and / or furin proteases located in the cytosol. In certain embodiments, the cleavage site is specific for cathepsin S. In further embodiments, the fusion protein comprises cleavage sites specific for cathepsin S and cathepsin L. In certain embodiments, the linker sequence is Gly-Ala-Val-Val-Arg-Gly-Ala (SEQ ID NO: 5141).

[0068] As used herein, "antigen" or "antigenic portion" refers to any portion, e.g., a peptide, that is recognized by a host's immune system. Examples of antigenic portions include, but are not limited to, autoantigens, enzymes, and / or bacterial or viral proteins, peptides, drugs, or components. An antigen may contain one or more epitopes. As used herein, "epitope" refers to the portion of an antigen that is recognized by an antibody or T-cell receptor. Not all epitopes are linear epitopes; epitopes may be discontinuous, conformational epitopes. The number of discontinuous epitopes associated with autoimmune or inflammatory diseases and / or disorders is unknown. In some embodiments, the fusion proteins of the invention comprise epitopes or antigens previously described by PCT Application Publication No. WO2015 / 023796, U.S. Patent Publication No. US2015-0283218, and U.S. Patent Publication No. US2015-0190485, each of which is incorporated by reference in its entirety. Sequence identifiers used herein correspond in number to the sequence identifiers in U.S. Patent Publication No. US2015-0190485.

[0069] In certain embodiments of the present invention, the antigen or epitope is not expressed in the same form as it is expressed in the subject being treated, but is a fragment or derivative thereof. Inducible 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 can 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. The terms insulin peptide, collagen peptide, and myelin basic protein peptide, etc., as used herein, refer not only to intact subunits but also to allotypic 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 to the respective molecules that are analogs, and the homologous regions of the derivatives share with the respective parent molecules the ability to induce tolerance to the target antigen.

[0070] It should be recognized that the tolerogenic region of an inducing antigen is often distinct from the immunodominant epitope, e.g., for stimulating antibody and / or T cell responses. A tolerogenic region is generally a region that can be presented in specific cellular interactions involving T cells. A tolerogenic region may be present and can induce tolerance when the intact antigen is presented. Some antigens contain cryptic tolerogenic regions, in that processing and presentation of native antigens does not usually result in tolerance. Details of cryptic antigens and their identification can be found in International Patent Publication No. WO 94 / 27634.

[0071] In certain embodiments of the invention, the fusion protein is comprised of two, three, or more antigens or epitopes. It may be desirable to implement these embodiments when multiple target antigens are present.

[0072] Antigens can be prepared by a number of techniques known in the art, depending on the nature of the molecule. Polynucleotide, polypeptide, and carbohydrate antigens can be isolated from cells of the species being treated in which they are abundant. Short peptides can be prepared by amino acid synthesis. Longer proteins of known sequence can be prepared by synthesizing the coding sequence or by PCR amplifying the coding sequence from a natural source or vector, and then expressing the coding sequence in a suitable bacterial or eukaryotic host cell.

[0073] In some embodiments, the antigen or epitope is derived from a therapeutic antibody, or antigen-binding fragment thereof, Fc fragment. In some embodiments, the antigen is derived from a mutated therapeutic antibody, or antigen-binding fragment thereof, lacking functional complementarity-determining regions (CDRs). In such embodiments, the antibody or antigen-binding fragment thereof may comprise a monoclonal antibody, a humanized monoclonal antibody, a human monoclonal antibody, a chimeric antibody, a single-chain antibody, a fragment antigen-binding region (Fab), a single-chain variable fragment (scFv), a small modular immunopharmaceutical (SMIP), or a single-chain antigen-binding domain. In some embodiments, the therapeutic antibody or antigen-binding fragment thereof binds to α4β1 integrin, Bacillus anthracis, BL(γS), C5, CD3, CD11a, CD20, CD25, CD30, CD33, CD52, CD59, CTLA4, EGFR, GD2, GPIIb, IIIa, HER2, IgE, IL-1β, IL-5, IL12 / 23, PCSK9, PD1, RANK, RSV-F protein, TNFα, or VEGF-A.

[0074] In some embodiments, the antigen is selected from the group consisting of abciximab, adalimumab, adotrastuzumab emtansine, alemtuzumab, basiliximab, bevacizumab, belimumab, blinatumomab, brentuximab vedotin, canakinumab, catumaxomab, cetuximab, certolizumab pegol, daclizumab, denosumab, dinutuximab, eculizumab, efalizumab, evolocumab, gemtuzumab ozogamicin, golimumab, ibritumomab Derived from therapeutic antibodies such as tiuxetan, ipilimumab, infliximab, motavizumab, muronomab, natalizumab, nivolumab, obinutuzumab, ofatumumab, omalizumab, panitumumab, palivizumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, raxibacumab, rituximab, secukinumab, siltuximab, trastuzumab, tocilizumab, tositumomab-I-131, ustekinumab, or vedolizumab.

[0075] 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, produced by detergent solubilization or mechanical disruption of cells or tissues followed by clarification. Antigens may also be obtained by subcellular fractionation, particularly enrichment of plasma membranes by techniques such as differential centrifugation, optionally followed by detergent solubilization and dialysis. Other separation techniques, such as affinity or ion-exchange chromatography of solubilized membrane proteins, are also suitable.

[0076] In one embodiment, the antigenic peptide or protein is an autoantigen, alloantigen, neoantigen, cancer antigen, or transplantation antigen. In yet another specific embodiment, the autoantigen is selected from the group consisting of myelin basic protein, collagen or fragments thereof, DNA, nuclei and nuclear proteins, mitochondrial proteins, and pancreatic beta cell proteins. In some embodiments, the one or more fusion proteins comprise the antigenic epitope MOG. 1-20 , MBP 13-32 , MOG 35-55 , MBP 146-170 , P.L.P. 139-154 , MBP 111-129 and / or MBP 83-99 In some embodiments, the antigenic peptide or protein comprises: In some embodiments, the antigen is one or more antigens selected from the group consisting of SEQ ID NOs: 1295-1724, 1726-1766, and 4986-5140.

[0077] The present invention provides for the induction of tolerance to autoantigens for the treatment of autoimmune diseases by administering antigens to which tolerance is desired. For example, autoantibodies against myelin basic protein (MBP) have been 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.

[0078] As another non-limiting example, a subject 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 subject to the intended transplant suppresses or reduces subsequent rejection. Reduction or elimination of long-term anti-rejection therapy can be achieved by implementing the present invention. 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.

[0079] In a further example, sensitization of a subject to industrial pollutants or chemicals, such as those that may be encountered at work, presents a risk of an immune response. Pre-tolerizing the subject's immune system to chemicals / pollutants, particularly in the form of chemicals / pollutants that react with the subject's endogenous proteins, may be desirable to prevent the development of a subsequent occupational immune response.

[0080] Allergens are other antigens for which tolerance of the immune response is also desired. In one embodiment, the antigen is gliaden or a gliaden epitope. In a further embodiment, the antigen is A-gliadene or an A-gliadene epitope. In some embodiments, the antigen is a mixture of gliaden or gliaden epitopes. In a further embodiment, the gliaden or gliaden epitope comprises one or more of SEQ ID NOs: 4983-4985.

[0081] 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, the gene encoding collagen can be used as a gene module that expresses antigens to treat rheumatoid arthritis (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).

[0082] As another example, autoantibodies against myelin oligodendrocyte glycoprotein (MOG) have been observed in autoimmune encephalomyelitis, as well as in many other CNS diseases and 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 enables the treatment of multiple sclerosis and related autoimmune disorders of the central nervous system.

[0083] Further examples of candidate autoantigens for use in the treatment of autoimmune diseases 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, histones, glycoprotein gp70, pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PCD-E2), hair follicle antigen, α-gliadin, gliadin, insulin, proinsulin, and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). , human tropomyosin isoform 5, bahiagrass pollen (BaGP), peach allergen Pru p3, αS-1 caeine milk allergen, Apig1 celeriac allergen, Bere1 Brazil nut allergen, B-lactoglobulin milk allergen, bovine serum albumin, Cor a 1.04 hazelnut allergen, myelin-associated glycoprotein, aquaporin, α3 chain of type IV collagen, ovalbumin egg allergen, Advate, antihemophilic factor, Kogenate, Eloctate, recombinant factor VIII fusion protein, Refacto, Novo VIIa, recombinant factor VII, eptacog alfa, Helixate, Monanine, coagulation factor IX, Wilate, Ceredase, alglucerase, Cerezyme, imiglucerase, Elelso, taliglucerase alfa, Fabrazyme, agalsidase beta, Aldurazyme, -I-iduronidase, Myozyme, acid glucosidase, Elaprace, iduronate-2-sulfatase, Naglazyme arylsulfatase B, or N-acetylgalactosamine-4-sulfatase pancreatic beta cell antigen, insulin, and GAD for treating insulin-dependent diabetes mellitus; type 11 collagen, human cartilage 39 (HCgp39), and gpl30-RAPS used to treat rheumatoid arthritis; Examples of antigens include 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) for treating Graves' disease; nuclear antigens, histones, glycoprotein gp70, and ribosomal proteins for treating systemic lupus erythematosus; pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PCD-E2) for treating primary biliary cirrhosis; hair follicle antigens for treating alopecia areata; and human tropomyosin isoform 5 (hTM5) for treating ulcerative colitis. In some embodiments, the antigen is selected from SEQ ID NOs: 2-1294.

[0084] Combinations can be humanized for their ability to promote tolerance by performing experiments using isolated cells or in animal models.

[0085] In some embodiments, the tolerogenic composition of the present invention contains an apoptosis signaling molecule (e.g., in addition to the fusion protein). In some embodiments, the apoptosis signaling molecule binds and / or associates with the surface of the carrier. In some embodiments, the apoptosis signaling molecule enables the carrier to be recognized as an apoptotic body by host antigen-presenting cells, e.g., host reticuloendothelial cells, thereby allowing relevant peptide epitopes to be presented in a tolerance-inducing manner. Without being bound by theory, this is presumed to prevent upregulation of molecules involved in immune cell stimulation, e.g., MHC class I / II and costimulatory molecules. These apoptosis signaling molecules may also serve as phagocytosis markers. For example, apoptosis signaling molecules suitable for the present invention are described in U.S. Pat. No. 8,198,020, 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.

[0086] In some embodiments, molecules suitable as apoptosis signaling molecules act to enhance the tolerance of related peptides. Furthermore, carriers linked to apoptosis signaling molecules can be bound by Clq in apoptotic cell recognition (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 rapamycin, phosphatidylserine, annexin-1, annexin-5, milk fat globule-EGF-factor 8 (MFG-E8), or thrombopoietin. and the family of thrombospondins (e.g., thrombospondin-(TSP-1)). Various molecules suitable for use as apoptosis signaling molecules with the present invention are discussed, for example, in U.S. Patent Publication No. 2012 / 0076831, which is incorporated herein by reference in its entirety.

[0087] In some embodiments, the fusion protein comprises one or more immune agonists. Immune agonist, as used herein, refers to a molecule that activates a specific immune signaling pathway, particularly an immunogenic signaling pathway. In some embodiments, the immune agonist activates a pattern recognition receptor, such as a Toll-like receptor (TLR), a C-type lectin receptor (CLR), a NOD-like receptor, a RIG-like receptor, or others. In certain embodiments, the agonist is a TLR agonist, such as a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR9, or TLR10 agonist. In such embodiments, the immune agonist promotes the generation of an immunogenic response against one or more epitopes contained within the fusion protein. Such embodiments are particularly useful in the context of vaccines and cancer immunotherapy.

[0088] By way of example, and not intended to be limiting, a hypothetical exemplary fusion protein is shown in FIG. 1 and contains the multiple sclerosis (MS)-associated epitope MOG. 1-20 , MOG 35-55 , MBP 13-32 , MBP 83-99 , MBP 111-129 , MBP 146-170 , and PLP 139-154 The fusion protein is constructed by linking these seven polypeptide epitopes together using specific linkers. These linkers are repetitive amino acid sequences that are susceptible to cleavage by specific proteases. The protein has a common isoelectric point (PI) and solubility. When encapsulated into particles, the particles encapsulate the polypeptide epitopes in equal proportions to each other.

[0089] biodegradable particles Certain embodiments are directed to biodegradable particles encapsulating fusion proteins comprising two or more peptides, antigens, or epitopes connected by an amino acid linker sequence containing a specific protease site. Certain embodiments contemplate that these particles are surprisingly effective at inducing tolerance to some or all of the bound peptides, antigens, or epitopes of the fusion protein. Certain embodiments contemplate that the manufacture of these biodegradable particles is improved compared to biodegradable particles encapsulating more than one unbound peptide, antigen, and / or epitope in a fusion protein.

[0090] As used herein, "particle" refers to any non-tissue-derived composition, which may be a sphere or spherical entity, a bead, or a liposome. The terms "particle," "immunomodulating 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. 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 within these embodiments are those having a spherical shape.

[0091] As used herein, "negatively charged particles" refers to particles that have been modified to have a net surface charge of less than zero.

[0092] "Carboxylated particles" or "carboxylated beads" or "carboxylated spheres" include any particles that have been modified to contain carboxyl groups on their surface. In some embodiments, the addition of carboxyl groups increases phagocyte / monocyte uptake of particles from the circulation, for example, through interaction with scavenger receptors such as MARCO. Carboxylation of the particles can be achieved using any compound that adds carboxyl groups, including, but not limited to, poly(acrylic acid), poly(ethylene-maleic anhydride) (PEMA), poly(vinyl alcohol), and sodium cholate.

[0093] In some embodiments, the antigenic peptide molecule is bound to the carrier particle (e.g., immunomodified particle) by a conjugate molecule and / or linker group. In some embodiments, the binding of the antigenic peptide and / or apoptosis signal molecule to the carrier particle (e.g., PLG particle) comprises 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. In some embodiments, the antigenic peptide is conjugated or bound to the carrier particle to produce an antigen-conjugated particle (see PCT Application No. PCT / US2016 / 068423, the contents of which are incorporated herein by reference in their entirety).

[0094] In one embodiment, the buffer solution contacting the immunomodified particles can have a basic pH. Suitable basic pHs 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.

[0095] In one embodiment, the buffer in contact with the immunomodified particles may have an acidic pH. Suitable acidic pHs for the acidic solution include 4, 4.1, 4.2, 4.5, 5, 5.5, 6, and 6.5.

[0096] In some embodiments of the present invention, the immunomodulatory particles contain copolymers. These copolymers can have various molar ratios. In some embodiments, the copolymer ratio suitable for the carrier particles described herein is 50:50. In further embodiments, the copolymer ratio suitable for the carrier particles described herein 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 other embodiments, the copolymer can be periodic, statistical, linear, or branched (including star, brush, or comb copolymers). In some embodiments, the copolymer ratio can 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.

[0097] 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 yet a further embodiment, the particle is encapsulated within a liposome.

[0098] Although it is not necessary for each particle to be of uniform size, particles should generally be of a size sufficient to trigger phagocytosis in antigen-presenting cells or other MPS cells. Preferably, particles are of microscopic or nanoscale size to enhance solubility, avoid complications that may be caused by aggregation in vivo, and promote pinocytosis. Particle size can be a factor in uptake from the interstitial space into areas of lymphocyte maturation. Particles having a diameter of about 0.1 μm to about 10 μm can induce phagocytosis. Thus, in one embodiment, the particles have a diameter within these limits. In another embodiment, the particles have a diameter of about 0.3 μm to about 5 μm. In yet another embodiment, the particles have a diameter of about 0.5 μm to about 3 μm. In a further embodiment, the particles have a diameter of about 0.2 μm to about 1 μm. In further embodiments, the particles have a diameter of about 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 1.0 μm, 1.5 μm, 2.0 μm, 2.5 μm, 3.0 μm, 3.5 μm, 4.0 μm, 4.5 μm, or about 5.0 μm. In a specific embodiment, 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. In some embodiments, the total weight of the particles is less than about 5,000 kDa, 1,000 kDa, 500 kDa, 400 kDa, 300 kDa, 200 kDa, 100 kDa, 50 kDa, 20 kDa, or less than about 10 kDa. The particles in the composition do not need to be of uniform diameter. For 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 is also useful.

[0099] 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 still 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 some embodiments, the particles have a zeta potential of -80 mV to -30 mV. In further embodiments, the particles have a zeta potential of -80 mV to -20 mV. In further embodiments, the particles have a zeta potential of -80 mV to -10 mV. In further embodiments, the particles have a zeta potential of -70 mV to -30 mV. In further embodiments, the particles have a zeta potential of -70 mV to -20 mV. In further embodiments, the particles have a zeta potential of -70 mV to -10 mV. In further embodiments, the particles have a zeta potential of -60 mV to -30 mV. In further embodiments, the particles have a zeta potential of -60 mV to -20 mV. In further embodiments, the particles have a zeta potential of -60 mV to -10 mV. In further embodiments, the particles have a zeta potential of -50 mV to -30 mV. In further embodiments, the particles have a zeta potential of between -50 mV and -20 mV. In further embodiments, the particles have a zeta potential of between -50 mV and -10 mV. In further embodiments, the particles have a zeta potential of between -50 mV and -40 mV. In further embodiments, the zeta potential is less than about -30 mV.

[0100] In some embodiments, the charge (e.g., positive, negative, neutral) of the carrier particles is selected to confer specific benefits to the application (e.g., physiological compatibility, beneficial surface-peptide interactions, etc.). In some embodiments, the carrier particles have a net neutral or negative charge (e.g., to reduce non-specific binding to cell surfaces, which generally bear a net negative charge). In certain embodiments, the carrier particles may 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 particles may be conjugated to an antigen-specific peptide (e.g., to increase the likelihood of a tolerance response). In some embodiments, the carrier particles have multiple binding sites (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or more) to expose multiple copies of a peptide or multiple different peptides on the surface. In some embodiments, the carrier particles display a single type of antigenic peptide. In some embodiments, the carrier particles display multiple different antigenic peptides on the surface. In some embodiments, the carrier particle surface displays functional groups for covalent attachment of a selected moiety (e.g., an antigenic peptide). In some embodiments, the functional groups on the carrier particle surface provide sites for non-covalent interaction with a selected moiety (e.g., an antigenic peptide). In some embodiments, the carrier particles have a surface to which a conjugate moiety can be adsorbed without forming a chemical bond.

[0101] In some embodiments, the particles are non-metallic. In these embodiments, the particles may be formed from a polymer. In a preferred embodiment, the particles are biodegradable in the subject. In this embodiment, the particles can be provided to the subject over multiple doses without accumulation of the particles in the subject. Examples of suitable particles include polystyrene particles, PLGA particles, citrate particles, and diamond particles.

[0102] 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 play a role in lymphatic uptake. The particle surface may be coated with a material to prevent or reduce nonspecific interactions. As demonstrated 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 (containing 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 in relation to lymphatic uptake. Biodegradable polymers may be used to constitute 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 aqueous solution. As used herein, the term "degradation" refers to becoming soluble through a decrease in molecular weight or through the conversion of hydrophobic groups to hydrophilic groups. Polymers with ester groups, such as polylactic acid and polyglycolide, are generally subject to spontaneous hydrolysis.

[0103] The particles of the present invention may contain additional components.For example, the carrier may have a contrast agent incorporated or conjugated thereto.An example of a carrier nanosphere with a contrast agent that is currently commercially available 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 different sizes to emit from blue to infrared when excited by a single ultraviolet wavelength. (Bruchez, et al., Science, 1998, 281, 2013; Niemeyer, C.M. Angew. Chem. Int. Ed. 2003, 42, 5796; Waggoner, A. Methods Enzymol. 1995, 246, 362; Brus, L.E.J. 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 toxic, unstable reagents. (Etienne, et al., Appl. Phys. Lett. 87, 181913, 2005)

[0104] The particles can be formed from a wide variety 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 straight-chain or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked polyesters of alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxyhydroxy acids, or straight-chain or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked polyanhydrides of alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxydicarboxylic acids. Furthermore, the carrier particles may be quantum dots or may consist 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) may also be used. For example, the carrier particles may comprise materials including polyglycolic acid polymer (PGA), polylactic acid polymer (PLA), polysebacic acid polymer (PSA), poly(lactic-co-glycolic) acid copolymer (PLGA or PLG, these terms are interchangeable), [rho]oly(lactic-co-sebacic) acid copolymer (PLSA), poly(glycolic-co-sebacic) acid copolymer (PGSA), and the like.

[0105] 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 hydroxycarboxylic 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 aforementioned materials to provide reactive groups for conjugation to antigenic 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 may also be used in the carrier particles of the present invention. For example, non-biodegradable polymers of acrylates, ethylene-vinyl acetate, acyl-substituted cellulose acetate, non-degradable urethanes, styrene, vinyl chloride, vinyl fluoride, vinyl imidazole, chlorosulfonated olefins, ethylene oxide, vinyl alcohol, TEFLON® (DuPont, Wilmington, Del.), and nylon may be used.

[0106] Suitable beads currently available commercially include polystyrene beads such as FluoSpheres (Molecular Probes, Eugene, Oreg.).

[0107] 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 bound to an antigen for induction of antigen-specific tolerance. In some embodiments, at least a portion of the delivery scaffold is microporous. In some embodiments, the antigen-bound 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.

[0108] 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., a PEG overlayer, as in the PPS-PEG system recently developed and characterized for systemic (rather than 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 certain elastic materials 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; the term sulfide polymer refers to polymers with sulfur in the polymer backbone. Other rubbery polymers that can be used are polyesters with a glass transition temperature of less than about 37°C under hydrated conditions. Hydrophobic cores can be advantageously used with hydrophilic overlayers because the core and overlayer tend not to mix, which can lead to the overlayer expanding 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 can 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 hydrophobic drug loading.

[0109] 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, the high stealth properties of the particles may also reduce internalization by phagocytes in areas with immature lymphocytes. The challenge of balancing these competing characteristics has been met, and 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 ordinary 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.

[0110] The nanoparticles may incorporate functional groups for further reaction. Functional groups for further reaction include electrophiles or nucleophiles, which are suitable for reacting with other molecules. Examples of nucleophiles are primary amines, thiols, and hydroxyls. Examples of electrophiles are succinimidyl esters, aldehydes, isocyanates, and maleimides.

[0111] A variety of means well known in the art can be used to conjugate antigenic peptides and proteins to carriers. These methods include any standard chemistry that does not destroy or significantly limit the biological activity of the antigenic peptides and proteins and allows a sufficient number of antigenic peptides and proteins to be conjugated to the carrier in an orientation that allows the antigenic peptide or protein to interact with its cognate T cell receptor. Generally, methods that conjugate the C-terminal region of the antigenic peptide or protein, or the C-terminal region of an antigenic peptide or protein fusion protein, to the carrier are preferred. The exact chemistry will, of course, depend on the nature of the carrier material, the presence or absence of a C-terminal fusion to the antigenic peptide or protein, and / or the presence or absence of a conjugated moiety.

[0112] Functional groups may be located on the particle as needed due to availability. One location may be a side group or terminus on the core polymer, or on a polymer that is a layer on the core, or on a polymer that is otherwise tethered to the particle. For example, examples are included herein describing PEG stabilizing nanoparticles that can be easily functionalized for specific cell targeting or protein and peptide drug delivery.

[0113] 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, ECDI is thought to perform two major functions to induce tolerance: (a) chemically attaching proteins / peptides to cell surfaces via catalysis of peptide bond formation between free amino and free carboxyl groups, and (b) inducing carriers to mimic apoptotic cell death, allowing them to be selected by host antigen-presenting cells in the spleen and induce tolerance. It is this non-immunogenic presentation to host T cells that directly induces anergy in autoreactive cells. Furthermore, ECDI serves as a potent stimulus for inducing specific regulatory T cells.

[0114] In one series of embodiments, the antigenic peptide and protein are attached to the carrier via a covalent chemical bond. For example, a reactive group or moiety near the C-terminus of the antigen (e.g., a C-terminal carboxyl group, or a hydroxyl, thiol, or amine group on an amino acid side chain) may be directly conjugated to a reactive group or moiety on the surface of the carrier (e.g., a hydroxyl or carboxyl group on PLA or PGA, a terminal amine or carboxyl group on a dendrimer, or a hydroxyl, carboxyl, or phosphate group on a phospholipid) by direct chemical reaction. Alternatively, a conjugation moiety may be present that covalently conjugates both the antigenic peptide and the protein to the carrier, thereby binding them together.

[0115] Reactive carboxyl groups on the surface of the carrier may be coupled to free amines (e.g., from Lys residues) on antigenic peptides or proteins by reacting them with, for example, 1-ethyl-3-[3,9-dimethylaminopropyl]carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide ester (NHS). Similarly, the same chemistry may 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 may be covalently coupled 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.

[0116] In another embodiment, the antigen may be conjugated to the carrier by non-covalent binding between a ligand bound to the antigenic peptide or protein and an anti-ligand attached to the carrier. For example, a biotin ligase recognition sequence tag may be attached to the C-terminus of the antigenic peptide or protein, and this tag may be biotinylated by biotin ligase. The biotin may then serve as a ligand to non-covalently conjugate the antigenic peptide or protein to avidin or streptavidin adsorbed or otherwise bound to the surface of the carrier as an anti-ligand. Alternatively, if the antigenic peptide or protein is fused to an immunoglobulin domain bearing 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 bind the antigenic peptide or protein. It can serve as an anti-ligand for non-covalently conjugating proteins to carriers. Metal ion chelation techniques (e.g., poly-His tags at the C-terminus of antigenic peptides or proteins or antigenic peptide or protein fusion proteins, and Ni +Antigen peptides and proteins are non-covalently attached to the carrier, including carriers coated with PEG. Other means that can be used to effect covalent conjugation are well known in the art, and these methods may be substituted for the methods described herein.

[0117] Conjugation of the nucleic acid moiety to the platform molecule can be accomplished 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 attached to the platform using standard synthetic chemistry techniques. The linking group can be attached to the nucleic acid moiety using standard synthetic chemistry techniques. The practitioner has numerous options for the antigen to be 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, and is the target of the unwanted immunological response, an antigen present or given to the subject receiving the treatment to which tolerance is desired.

[0118] 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 materials, provided that when combined with a mucosally binding component, they have the ability to induce tolerance according to the present invention.

[0119] In some embodiments, the present invention provides carriers (e.g., immunomodified 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 experimental autoimmune encephalomyelitis (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 induce T cells to initiate early events associated with T cell activation, but cannot induce T cells to acquire effector function. For example, administration of a composition of the present invention can give rise to T cells with a quasi-activated phenotype, such as upregulation of CD69 and / or CD44, but which do not exhibit effector function, as suggested by a lack of IFN-γ or IL-17 synthesis. In some embodiments, administration of a composition of the present invention can induce naive antigen-specific T cells, e.g., upregulation of CD25 + Foxp 3 + have a subactivation phenotype without converting to a regulatory phenotype, such as one having a Give rise to T cells.

[0120] 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 the chemical moieties and / or functional groups on a carrier (e.g., a particle) will vary depending on the chemistry of the carrier, the desired use, etc.

[0121] In some embodiments, the carrier contains one or more biological or chemical agents attached to, adsorbed to, encapsulated in, and / or contained throughout the carrier. In some embodiments, the chemical or biological agents are encapsulated in the particle and / or contained throughout the particle. 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, the different agents are configured for different release rates. For example, a first agent may be released over a period of several hours, and a second agent may be released 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 a biological or chemical agent. In some embodiments, the sustained release provides for release of a biologically active amount of the agent over a period of at least 30 days (e.g., 40, 50, 60, 70, 80, 90, 100, 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 can be selected for the particular cell type of interest and / or the amount of ingrowth desired.

[0122] Surprisingly, it has been found that encapsulating antigens, biological agents, and / or chemical agents into the particles of the present invention induces immune tolerance and has several advantages. First, encapsulated particles have a slower cytokine response. Second, when multiple antigens, biological agents, and / or chemical agents are used, encapsulation eliminates the competition between these various molecules that may occur when 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 facilitates the use of complex protein antigens or organ homogenates (e.g., pancreatic homogenate for type 1 diabetes or peanut extract for peanut allergy). Finally, encapsulating antigens, biological agents, and / or chemical agents within the particles, instead of conjugating them to the particle surface, maintains a net negative charge on the particle surface.

[0123] 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 have demonstrated the ability to incorporate peptides (PLPs) into these particles. 139-151 Conjugation of peptides has been demonstrated. showed that they are effective in preventing disease onset and in inducing immunological tolerance (e.g., SJL / J PLP in multiple sclerosis). 139-151 / CFA-induced R-EAE in mice The 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, the particles are functionalized to promote internalization without activating the cells (e.g., phosphatidylserine loaded onto PLG microparticles). In some embodiments, the 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 promote tolerance induction via energy and / or deletion and activation of regulatory T cells.

[0124] composition In some embodiments, biodegradable particles encapsulating the fusion proteins described herein can be formulated into a composition. As used herein, the term "composition" refers to a formulation of one or more particles encapsulating one or more fusion proteins that can be administered to a subject and / or cells. In some embodiments, a composition can consist of multiple particles, each of which encapsulates the same fusion protein. In some embodiments, a composition can consist of multiple particles, each of which encapsulates two or more different fusion proteins. For example, a composition may consist of a plurality of particles, each encapsulating one of two, three, four, five, six, seven, eight, nine, ten, or more different fusion proteins. In some embodiments, the composition optionally further comprises one or more additional therapeutic agents. Alternatively, the particles of the present invention may be administered to a patient in need thereof in combination with the administration of 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 Food and Drug Administration approval that may eventually be approved for the treatment of any disorder characterized by an uncontrolled inflammatory immune response or bacterial or viral infection. It should also be understood that the particles of the present invention may be present in free form for treatment, or, if desired, as a pharmaceutically acceptable derivative thereof.

[0125] Composition formulations may include derivatives, prodrugs, solvates, stereoisomers, racemates, and / or tautomers of the particles described herein, along with any acceptable carriers, diluents, and / or excipients. A "therapeutic composition" or "pharmaceutical composition" (used interchangeably herein) is a composition of particles encapsulating one or more fusion proteins described herein that can be administered to a patient and / or cells and can produce a particular physiological result (e.g., antigen-specific tolerance).

[0126] As used herein, a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent, or emulsifier that is suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergenicity, or other problem or complication. Examples of 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; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and any other compatible substances used in pharmaceutical formulations. Except insofar as any conventional media and / or agent is incompatible with the particles and / or fusion proteins of the present disclosure, its use in the therapeutic compositions is contemplated.

[0127] "Pharmaceutically acceptable" includes both acid addition salts and base addition salts. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of a protein), including those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, No. 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, Salts formed with lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0128] Wetting agents, emulsifying agents, and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0129] Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; fat-soluble antioxidants such as ascorbic acid palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl bile acid, α-tocopherol, etc.; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0130] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as 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, flavoring agents, and aromatic agents. In some embodiments, the present invention provides methods for inducing a particular physiological effect (e.g., modulation of immune responses / induction of antigen-specific tolerance) in a subject, comprising administering an effective amount of a biodegradable particle or composition described herein. Accordingly, in one aspect, tolerogenic immunomodulating particles are provided. Such tolerogenic particles comprise at least two or more antigenic epitopes to which tolerance induction is desired (e.g., autoantigens, allergens, and / or transplantation antigens), separated by a linker (e.g., a protease-specific linker). In another aspect, activating immunomodulating particles are provided. In one embodiment, such immunostimulatory particles comprise two or more antigenic epitopes to which a protective immune response is desired, separated by a linker (e.g., a protease-specific linker). (e.g., a tumor antigen and / or an infectious agent). In certain embodiments, the activated immunomodulated particle further comprises an immunoactivating agent. In one embodiment, the immunoactivating agent is a TLR agonist. In certain embodiments, the immunoactivating agent is a TLR7, TLR3, or TLR9 agonist. In a further embodiment, the immunoactivating agent is a TLR7 agonist.

[0131] The composition may be formulated in a specific manner suitable for the desired route of administration and / or to achieve the desired result. "Administration" refers to introducing or delivering the biodegradable particles or a composition thereof to a subject, or contacting the biodegradable particles or a composition thereof with a cell or a sample. The term "sample" refers to a volume and / or mass obtained, provided, and / or subjected to analysis. In some embodiments, a sample includes a tissue sample, a cell sample, a bodily fluid sample, etc. In some embodiments, a sample is collected from a subject (e.g., a human or animal subject). In some embodiments, a tissue sample includes a portion of tissue collected from any internal organ, cancerous, precancerous, or noncancerous tumor, skin, hair (including hair roots), eye, muscle, bone marrow, cartilage, white adipose tissue, or brown adipose tissue. In some embodiments, a bodily fluid sample includes a buccal swab, blood, umbilical cord blood, saliva, semen, urine, peritoneal fluid, pleural effusion, cerebrospinal fluid, lung lavage fluid, tears, sweat, etc. Those skilled in the art will understand that in some embodiments, a "sample" is a "primary sample" in that it is obtained directly from a subject. In some embodiments, a "sample" is the result of processing of the primary sample, e.g., to remove certain potentially contaminating components and / or to isolate and / or purify certain components of interest.

[0132] Administration can be by injection, irrigation, inhalation, ingestion, electroosmosis, hemodialysis, iontophoresis, and other methods known in the art. The particles and compositions of the present invention can be administered via any acceptable route, including, but not limited to, oral, intravenous, sublingual, buccal, enteral, topical, rectal, subcutaneous, nasal, intraosseous (e.g., intraosseous injection), intraperitoneal, intrathecal, transdermal, or transmucosal. In certain embodiments, the particles of the present invention are administered intravenously or subcutaneously.

[0133] The frequency of administration can be determined based on the desired physiological results, the nature of the disorder to be treated and / or prevented, the severity of the disorder, and the subject's response to the formulation. In some embodiments, the composition is administered at least once. In further embodiments, the administration is administered more than once, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times within a given period. The dosage and / or frequency of each administration can be adjusted as needed based on the patient's condition and physiological response. When the composition is administered more than once, each administration can be administered by the same actor and / or in the same geographic location. Alternatively, each administration can be administered by a different actor and / or in a different geographic location.

[0134] In some embodiments, an effective amount of the particles and / or compositions described herein is administered to a subject. The terms "subject" and "patient" are used interchangeably herein and refer to animals (e.g., mammals, pigs, fish, birds, insects, etc.) suitable for treatment with an effective amount of the particles and / or compositions described herein. In some embodiments, the subject is a mammal, such as a primate, human, or rabbit; livestock, such as cattle, sheep, goats, cows, pigs, etc.; poultry, such as chickens, ducks, geese, turkeys, etc.; domestic animals, such as dogs and cats; rodents, such as mice, rats, or hamsters. In some embodiments, particularly in a research environment, the subject is a mouse. In some embodiments, the subject is a human.

[0135] The term "effective amount" refers to the amount of biodegradable particles or An effective amount refers to the minimum amount of a particle or composition. For example, an effective amount may be the minimum amount necessary to induce antigen-specific tolerance or otherwise regulate an immune response. As described herein, regulation of an immune response may be humoral and / or cellular and is measured as described herein using standard techniques in the art. The effective amount of a given particle or composition will depend on a variety of factors, including the nature of the disorder being treated and the severity of the disorder; the activity of the particular particle(s) of the composition(s) used; the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and excretion rate of the particle(s) or composition(s) used; the duration of treatment; drugs used in combination with or concurrently with the particle(s) or composition(s) used; the judgment of the prescribing physician or veterinarian; the size and physical characteristics of the particle or composition; and similar factors known in the art. Useful dosage ranges for the particles or compositions described herein can be, for example, about any of the following: 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 the number of particles. For example, a useful dosage of carrier, expressed as the amount of carrier delivered, can be, for example, about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 The number of particles per dose may be 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 89 th Edition, 1990, Mack Publishing Co., Easton, Pa., USA., which is incorporated herein by reference in its entirety.

[0136] In some embodiments, compositions of the invention find use 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, U.S. Patent No. 6,281,256, which are incorporated by reference in their entireties). In some embodiments, particles are associated with, adsorbed to, embedded in, or conjugated to 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, a 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 is made of a material described herein.

[0137] In some embodiments, microporous scaffolds are provided (e.g., for implanting biological materials (e.g., cells, tissues, etc.) into a subject). In some embodiments, microporous scaffolds having drugs (e.g., extracellular matrix proteins, exendin-4) and biological materials (e.g., pancreatic islet cells) thereon are provided. In some embodiments, scaffolds are used in the treatment of diseases (e.g., type 1 diabetes) and related methods (e.g., diagnostic methods, research methods, drug screening). In some embodiments, scaffolds are provided having carrier particles described herein on and / or within the scaffold. In some embodiments, scaffolds are made from materials conjugated to antigens (e.g., PLG conjugated to antigens).

[0138] In some embodiments, the scaffold and / or delivery system comprises one or more layers and / or has one or more chemical and / or biological entities / agents (e.g., particles conjugated to proteins, peptides, small molecules, cells, tissues, etc.); see, e.g., U.S. Patent Publication No. 2009 / 0238879, incorporated herein by reference in its entirety. In some embodiments, the particles described herein comprise a scaffold and a delivery system. In some embodiments, the microporous scaffold is co-administered with a scaffold delivery system to induce the induction of immunological tolerance to the combined material. In some embodiments, the microporous scaffold is administered to a subject with particles described herein on or in the scaffold. In some embodiments, the particles described herein are bound to a scaffold delivery system. In some embodiments, the scaffold delivery system comprises any of the carrier particles described herein.

[0139] It should also be understood that the particles and compositions of the present invention can be used in combination therapy, i.e., the particles and compositions can be combined with, or administered simultaneously with, before, or after, one or more other desired therapeutic agents or medical procedures. The particular therapeutic combination (e.g., combination of therapeutic compounds and / or procedures) to be used in a combined administration regimen takes 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 treatments used can achieve the desired effect for the same disorder (e.g., a compound of the present invention can be administered simultaneously with another anti-inflammatory agent), or they can achieve different effects (e.g., control of any adverse effects).

[0140] 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 agents). 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 therapy includes analgesics, antiemetics, and antiemetics.

[0141] In some embodiments, the compositions described herein are administered in conjunction with (e.g., simultaneously with, before, or after) an implant (e.g., a device) and / or graft (e.g., tissue, cell, organ) to mediate, neutralize, control, and / or mitigate an immune response associated with the implant and / or graft.

[0142] How to use In some embodiments, the present invention provides a method for inducing or otherwise regulating an existing immune response in a subject, preferably a mammal, more preferably a human, comprising administering to the subject a particle or composition described herein. As used herein, the term "immune response" includes both innate and adaptive immune responses (e.g., T cell-mediated and / or B cell-mediated immune responses). Generally, innate and adaptive immune responses are distinguished by the level of antigen specificity. For example, cells directly involved in adaptive immune responses (e.g., T cells and B cells) express T cell receptors (TCRs) and B cell receptors specific for particular antigens. Adaptive immune receptors are thus activated and respond to specific antigens (e.g., specific epitopes or components of larger antigens). In contrast, cells of the innate immune system express TLRs, CLRs, NLRs, RLRs, and other innate immune receptors (e.g., pattern recognition receptors (PRRs)). PRRs are germline-encoded, unrearranged receptors that recognize a wide variety of antigens (e.g., CLRs generally recognize carbohydrate moieties, while RLRs recognize viral nucleic acids). Thus, receptors of the innate immune system are activated and respond to a wide range of antigens and are not considered to be antigen-specific.

[0143] Cells involved in the immune response include lymphocytes, e.g., B cells and T cells (CD4 + , CD8 +, Th1, Th2, Th17, T regulatory cells; antigen-presenting cells (APCs) (including professional APCs, e.g., dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional APCs, e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells; and myeloid cells, e.g., macrophages, eosinophils, mast cells, basophils, and other granulocytes. Exemplary immune cells include: The immune response may include T cell responses, such as T cell proliferation, T cell expansion, cytokine production, chemokine production, and T cell-mediated cytotoxicity (e.g., CD8 + to cytotoxic T cells (CTL) The term immune response includes immune responses that indirectly or directly mediate T cell activation or T cell suppression, such as APC migration, proliferation, and activation, and mechanisms of antigen presentation. The term immune response also includes immune responses that are indirectly influenced by T cell activation, e.g., antibody production (humoral response), and activation of cytokine-responsive cells, e.g., macrophages, dendritic cells, neutrophils, mast cells, basophils, B cells, T cells themselves, and structural cells, e.g., epithelial cells, endothelial cells, and / or other stromal cells. In some embodiments, the particles of the present invention are effective in reducing trafficking of inflammatory cells to sites of inflammation.

[0144] "Regulating an immune response" may refer to regulating any aspect of an immune response, or to regulating multiple aspects. In some embodiments, methods for regulating an immune response as provided herein include modulating an immunogenic, pro-inflammatory, or otherwise activating immune response (e.g., by the use of activated immunomodulated particles). In such embodiments, the methods provided herein encompass specifically inducing a TH1, TH2, or TH17 response, reducing or suppressing a regulatory T cell response, or a combination of those responses. Induction of a TH1 response can involve, for example, increasing the expression of IFNγ and / or IL-12, and / or increasing the population of TH1 cells (e.g., IFNγ, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-49, IL-49, IL-49, IL-49, IL-49, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-49, IL-4 + , IL-12+ , and / or T-bet + Increase the number or proportion of cells Induction of a TH2 response includes, for example, increasing the expression of IL-4, IL-5, IL-10, IL-13, or any combination thereof. Typically, an increased TH2 response includes increased expression of at least one of IL-4, IL-5, IL-10, or IL-13; more typically, an increased TH2 response includes increased expression of at least two of IL-4, IL-5, IL-10, or IL-13, and most typically, an increased TH2 response includes increased expression of at least three of IL-4, IL-5, IL-10, or IL-13, but ideally, an increased TH2 response includes increased expression of all of IL-4, IL-5, IL-10, and IL-13. Induction of a TH2 response also includes increasing the population of TH2 cells (e.g., increasing the expression of IL-4, IL-5, IL-10, and IL-13). + , IL-5 + , IL-10 + , IL-13 + , and / or GATA3 + Increase the number or percentage of cells Induction of a TH17 response can include, for example, increasing the expression of TGF-β, IL-6, IL-21, IL-23, or any combination thereof, as well as the effective levels of IL-17, IL-21, and IL-22. Induction of a TH17 response can also include increasing the population of TH17 cells (e.g., increasing the expression of IL-17, IL-21, and IL-22). + , IL-21 + , IL-22 + , and / or RORγt + Reducing a regulatory T cell response can include reducing the expression of TGFβ, IL-10, or any combination thereof. Reducing a regulatory T cell response can also include reducing the population of T regulatory cells (e.g., increasing the number or proportion of T cells). + , IL-10 + , and / or FoxP3 + Number or percentage of cells (reducing

[0145] In some embodiments, methods for controlling an immune response as provided herein include modulating a regulatory, tolerogenic, or otherwise suppressive immune response (e.g., by the use of tolerogenic immunomodulating particles). In such embodiments, the methods provided herein encompass specifically reducing a TH1, TH2, or TH17 response, increasing a regulatory T cell response, or a combination of those responses. Reducing a TH1 response can involve, for example, reducing the expression of IFNγ and / or IL-12, and / or reducing the population of TH1 cells (e.g., IFNγ + , IL-12 + , and / or T-bet + TH2 response (reducing the number or proportion of TH2 cells) Reducing a TH2 response includes, for example, reducing the expression of IL-4, IL-5, IL-10, IL-13, or any combination thereof. Typically, reducing a TH2 response involves reducing the expression of IL-1, IL-2, IL-3, IL-4, IL-5, IL-10, IL-13, or any combination thereof. more typically, a reduction in a TH2 response involves a reduction in expression of at least two of IL-4, IL-5, IL-10, or IL-13, and most typically, a reduction in a TH2 response involves a reduction in expression of at least three of IL-4, IL-5, IL-10, or IL-13, but ideally, a reduction in a TH2 response involves a reduction in expression of all of IL-4, IL-5, IL-10, and IL-13. A reduction in a TH2 response also involves a reduction in the population of TH2 cells (e.g., IL-4, IL-5, IL-10, or IL-13). + , IL-5 + , IL-10 + , IL-13 + , and / or GATA3 + The present invention may include reducing the number or proportion of TH17 cells. Reducing includes, for example, reducing the expression of TGF-β, IL-6, IL-21, IL-23, or any combination thereof, as well as the effective levels of IL-17, IL-21, and IL-22. Reducing a TH17 response also includes reducing the population of TH17 cells (e.g., IL-17).+ , IL-21 + , IL-22 + , and / or RORγt + Induction of a regulatory T cell response can include increasing the expression of TGFβ and / or IL-10. Induction of a regulatory T cell response can also include increasing the population of T regulatory cells (e.g., increasing the expression of TGFβ and / or IL-10). + , IL-10 + , and / or FoxP3 + Increasing the number or proportion of cells.

[0146] As used herein, the term "tolerance" or "immunological tolerance" refers to a state of unresponsiveness of the immune system. Immunological tolerance is essential for preventing aberrant (e.g., reactivity to self-antigens in the context of autoimmunity) and / or excessive immune responses. "Specific" immunological tolerance occurs when immunological tolerance is preferentially elicited to a specific antigen over others. "Nonspecific" immunological tolerance occurs when immunological tolerance is elicited indiscriminately to antigens that elicit inflammatory immune responses. "Semispecific" immunological tolerance occurs when immunological tolerance is elicited semi-discriminately to antigens that elicit pathogenic immune responses, but not to other antigens that elicit protective immune responses. In certain embodiments, the present invention provides a method for inducing antigen-specific tolerance in a subject, comprising administering an effective amount of a biodegradable particle or composition described herein. As used herein, "antigen-specific tolerance" refers to the insensitivity and / or unresponsiveness of T cells to TCR-mediated stimulation by a specific antigen.

[0147] Immunological tolerance is the result of both central and peripheral tolerance. Central tolerance refers to the positive and negative selection of T cells in the thymus, which results in the selection of functional antigen-specific T cells (positive selection) and the elimination of autoreactive T cells (negative selection). Peripheral tolerance refers to tolerance mechanisms present in the periphery (e.g., bone marrow, lymph nodes, spleen, and / or mucosal surfaces). Peripheral tolerance mechanisms prevent aberrant responses by autoreactive T cells that escape thymic deficiency and prevent excessive activation of the immune response to foreign antigens. Peripheral tolerance encompasses various mechanisms, including T cell anergy, activation-induced T cell death, and immunosuppression mediated by regulatory T cells.

[0148] As used herein, the term "anergy" refers to the insensitivity of T cells to T cell receptor (TCR)-mediated stimulation. Such insensitivity is antigen-specific and generally persists after exposure to the antigenic peptide has ceased. 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, re-exposure of the cells to the same antigen, even if the re-exposure occurs in the presence of costimulatory molecules, renders the T cells unable to produce cytokines (e.g., IL-2) and subsequently unable to proliferate. Thus, the inability to produce cytokines prevents proliferation. However, anergic T cells can proliferate when cultured with cytokines (e.g., IL-2). T cell anergy is a condition characterized by the inability of T cells to produce cytokines (e.g., IL-2) as measured by ELISA or by proliferation assays using indicator cell lines. This can be observed by the lack of IL-2 production by cells. Alternatively, a reporter gene construct can be used. For example, anergic T cells cannot initiate the transcription of the DL-2 gene, which is induced by a heterologous promoter under the control of the 5'IL-2 gene enhancer or by a multimer of the API sequence found within the enhancer (Kang et al. 1992 Science. 257:1134).

[0149] The terms "regulatory T cells," "T regulatory cells," and "Tregs" are used interchangeably herein and refer to T cells that suppress or prevent the induction of an immune response. The term Tregs can refer to both endogenous Tregs (e.g., Tregs that arise from the thymus as suppressor cells) and inducible Tregs (e.g., Tregs that differentiate into suppressor cells in response to peripheral stimuli). Inducible Tregs can be divided into multiple subpopulations based on their expression of transcription factors, FoxP3, cell surface markers, and cytokine production. In some embodiments, Tregs are classified into Tr1, Th3, CD8, and CD8+ subpopulations. + Suppressor cells and others In certain embodiments, Tregs may refer to inducible Treg populations such as CD4 + FoxP3 - LAG3 + IFNγ + IL-10 + This may refer to Tr1 cells, which are defined as In some embodiments, Treg-mediated suppression of an immune response can be antigen-specific or non-antigen-specific. In some embodiments, Treg-mediated suppression of an immune response can be the result of cytokine production by Tregs (e.g., production of IL-10 and / or TGFβ) or can be due to the production of another immunosuppressive mediator.

[0150] Tregs play an important role in mediating and maintaining 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 an animal model of EAE, activation of APCs by innate immune receptors such as Toll-like receptors (TLRs) has been shown to destroy self-tolerance, resulting in the induction of EAE (Waldner et al. (2004) J. Clin. Invest. 113:990-997). Furthermore, Tregs can prevent excessive immune activation associated with beneficial immune responses, such as those generated in response to viral or bacterial infections. Therefore, controlling these immune responses can prevent excessive damage to healthy cells or tissue debris.

[0151] In some embodiments, immunological tolerance can be measured by, for example, a reduction in the level of a specific immune response, such as one mediated at least in part by antigen-specific effector T lymphocytes, B lymphocytes, antibodies, or equivalents thereof; a delay in the initiation or progression of a specific immune response; or a reduced risk of initiation or progression of a specific immune response. Immunological tolerance can be determined by methods based on the proportion of treated subjects compared to untreated subjects, and T cell and / or B cell proliferation and / or activation, cytokine production, antibody production can be determined by methods known in the art (e.g., in vitro proliferation assays, flow cytometry, ELISA, Western blot, etc.).

[0152] In some embodiments, inducing an antigen-specific immune response comprises inducing an increase in tolerogenic activity. In some embodiments, the increase in tolerogenic activity comprises an increase and / or proliferation of Tregs. In some embodiments, the increase in tolerogenic activity comprises an increase in the production of regulatory cytokines, such as IL-10 and / or TGFβ. 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 regulatory cells at the target site is thought to be TGF-β (Miller et al., Proc. Natl. Immunol. 2012; 10:111-113). 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 an activated immune response secrete cytokines such as IL-1, IL-2, IL-6, and IFNγ. Thus, the ability of an antigen to induce a tolerogenic or immunogenic response can be assessed by measuring its ability to stimulate the production of immunoregulatory cytokines (e.g., TGFβ and / or IL-10) relative to immune stimulatory cytokines (e.g., IFNγ, IL-2, IL-6, IL-17, etc.).

[0153] In certain embodiments, the present invention relates to the priming of immune tolerance in a subject not previously tolerized by therapeutic intervention. In some embodiments, the present invention relates to methods for reducing the incidence and / or severity of an aberrant immune response to a therapeutic protein in a subject. These embodiments generally involve multiple administrations of a combination of an antigen and a mucosal-binding component. Typically, at least three administrations, frequently at least four administrations, and sometimes at least six administrations are administered during priming to achieve long-lasting results, although the subject 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 time between administrations is generally between one day and three weeks, and typically between about three days and two weeks. Generally, the same antigen and mucosal-binding component are present at the same concentrations, and administrations are to the same mucosal surface, although variations in any of these variables may be accommodated during the treatment course.

[0154] In some embodiments, the methods of the present invention involve inducing a protective immune response against a specific antigen, such as a target antigen. Such methods are particularly useful in the context of cancer therapeutics and infectious diseases. In such embodiments, the methods involve administering particles encapsulating a fusion protein containing two or more target antigens (e.g., tumor antigens) separated by a protease-specific linker. In certain embodiments, the particles encapsulating the binding epitopes further comprise an immune agonist. The immune agonist may comprise any of proteins, haptens, toxins, lipids, and / or nucleic acids and can act as an adjuvant to generate an antigen-specific immune response against the target antigen. The immune agonist may include haptens such as biotin, dinitrophenol, urushiol, fluorescein, and others. In some embodiments, the immune agonist may include nucleic acids, including single-stranded (ss) and double-stranded RNA and DNA, as well as modified forms thereof. In some embodiments, the immune agonist is a toxin. In some embodiments, the immune agonist is a protein such as an immune-activating cytokine (e.g., IL-2, IL-12, IFNγ, IFNα, IFNβ, TNFα, etc.); a chemokine that can recruit T cells, antigen-presenting cells, and / or granulocytes; or an antibody or fragment thereof that binds to and inhibits an immune checkpoint receptor (e.g., PD1, PDL1, CTLA4, LAG3, TIM3, or A2aR).

[0155] In some embodiments, the immune agonist is an agonist of a CLR (e.g., DEC-205, DC-SIGN, DCIR, CLEC-1, Dectin-1, Dectin-2, or DLEC), a TLR (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, or TLR13), an NLR (e.g., NOD1, NOD2, NAIP, NLRC4, NLRC3, NLPR1, NLPR3, NLRP10), an RLR (e.g., MDA or RIG1), STING, or an inflammasome (e.g., NLPR3 or AIM2). In further embodiments, the immune agonist is a TLR7 or TLR9 agonist. In further specific embodiments, the immune agonist is a CD8 + Activation of CTL In further embodiments, the immune agonist causes cytolysis of cells expressing a target antigen. In some embodiments, the target antigen is CD19, CD20, BCM A, CD22, CLL1, CD33, CEA, CD123, CS1, EGFR, PSMA, EphA2, MCSP, ADAM17, PSCA, TPTE, HPU16, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, Ep-CAM, EphA3, Her 2 / neu, telomerase, mesothelin, SAP-1, survivin, BAGE family proteins, CAGE family proteins, GAGE ​​family proteins, MAGE family (e.g., MAGE-A3), SAGE family proteins, XAGE family proteins, CT9, CT10, NY-ESO1 / LAGE-1, PRAME, SSX-2, MelanA / MART-1, Cp100 / pmel17, tyrosinase, TRP-1 / TRP-2, P. polypeptide, MC1R, prostate-specific antigen, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII, and tumor antigens such as MUC1.

[0156] An increase in antigen-specific immune response can be measured by an increase in antigen-specific effector T cell proliferation, an increase in the production of pro-inflammatory and / or immune stimulatory cytokines (e.g., IFNγ or IFNα), or an increase in cytolysis of cells expressing the target antigen.

[0157] In further embodiments, methods for treating specific diseases or disorders are provided. As used herein, "treating" and "treatment" refer to an improvement in a disease or the symptoms of a disease, and may be a measurable or observable improvement, or an improvement in the overall health of a subject. In certain embodiments, treating a specific disease or disorder refers to inducing antigen-specific tolerance or otherwise increasing a regulatory immune response to reduce or ameliorate pathological inflammation (e.g., associated with autoimmune diseases).

[0158] In some embodiments, the invention relates to the use of the particles and compositions described herein before the onset of disease. In other embodiments, the invention relates to the use of the particles and compositions described herein to inhibit disease once it has progressed. In some embodiments, the invention relates to ameliorating disease in a subject. Ameliorating disease in a subject is meant to include treating, preventing, or suppressing disease in a subject.

[0159] In some embodiments, the present invention relates to preventing disease relapse. For example, unwanted immune responses can be generated against certain regions of a peptide (such as antigenic determinants). Disease relapses associated with unwanted immune responses can occur due to immune attack against different regions of the peptide. T cell responses in some immune response disorders, including MS and other Th1 / 17-mediated autoimmune diseases, can be dynamic and evolve over the course of relapsing-remitting and / or chronic progressive disease. The dynamic nature of the T cell repertoire impacts treatment of specific diseases, as targets can change as the disease progresses. Previously, prior knowledge of response patterns was required to predict disease progression. The present invention provides compositions that can counteract the function of "epitope spreading," a dynamically changing disease effect. A known model of relapse is the immune response against proteolipid protein (PLP), a model of multiple sclerosis (MS). The initial immune response can be generated by a response to PLP139-15. Subsequent disease development can result from a recurrent immune response to PLP[pi]s-iβi. The compositions of the invention are particularly useful for treating MS and other autoimmune diseases in which disease-causing epitopes are present in multiple proteins (e.g., PLP, MBP, and MOG) or multiple disease-causing epitopes are present on a single protein, making encapsulation of the entire protein otherwise impossible.

[0160] In certain embodiments, the subject suffers from a disease associated with unwanted immune activation, such as an allergic disease or condition, allergy, and asthma. A subject with an allergic disease or asthma is one with recognizable symptoms of an existing allergic disease or asthma. For example, tolerance can be induced in such subjects by particles complexed with specific foods (e.g., peanut proteins, etc.), injected substances (e.g., bee venom proteins, etc.), or inhaled substances (e.g., ragweed pollen proteins, pet dander proteins, etc.) that induce an allergic response.

[0161] In certain embodiments, the subject suffers from a disease associated with unwanted immune activation, such as an autoimmune disease or an inflammatory disease. A subject with an autoimmune or inflammatory disease is one with recognizable symptoms of an existing autoimmune or inflammatory disease. For example, tolerance can be induced in such a subject by particles complexed with the relevant autoantigen that drives the particular autoimmune disease.

[0162] In certain embodiments, the subject suffers from diseases related to enzyme replacement therapy.For example, to prevent the patient from developing a neutralizing antibody response to the recombinantly produced enzyme administered to treat the specific defect, the particles can be used to induce tolerance in such subjects with the enzyme that the patient with genetic defect cannot produce (for example, tolerance to human factor VIII in patients with hemophilia due to genetic defect in the ability to produce factor VIII).

[0163] In certain embodiments, the subject 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 therapeutics for autoimmune diseases).

[0164] 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 1 diabetes, type 2 diabetes, multiple sclerosis (MS), immune-mediated infertility such as premature ovarian failure, 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.

[0165] Autoimmune diseases also include, but are not limited to, Hashimoto's thyroiditis, polyglandular autoimmune syndrome types 1 and 2, 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, monoclonal antibody-linked immunosorbent assay (MACE)-associated encephalopathy (MACE-associated encephalopathy), and unilateral hemoglobinuria. These conditions also include chronic neuropathy with rheumatoid gammopathies, 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 vasculitis of the central nervous system, thromboangiitis obliterans, sarcoidosis, glomerulonephritis, and cold syndrome. These conditions are well known in the medical field and are described, for example, in Harrison's Principles of Internal Medicine, 14th ed., Fauci AS et al., eds., New York rk: McGraw-Hill, 1998.

[0166] Animal models for the study of autoimmune disease are known in the art.Animal models for the study of autoimmune disease are known in the art.For example, the animal model that is considered to be most similar to human autoimmune disease comprises animal strains that naturally develop certain diseases at a high incidence.Examples of such models include, but are not limited to, non-obese diabetic (NOD) mice that develop diseases similar to type 1 diabetes, as well as animals that are susceptible to lupus-like diseases, such as New Zealand hybrids, MRL-Fas lpr and BXSB mice Animal models in which autoimmune diseases are induced include, but are not limited to, EAE (a mouse model of multiple sclerosis), collagen-induced arthritis (CIA, a mouse model of rheumatoid arthritis), and experimental autoimmune uveitis (EAU, a mouse 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-1 receptor antagonist knockout mice for rheumatoid arthritis.

[0167] In certain embodiments, the subject is suffering from an infectious disease. A subject with a bacterial, fungal, parasitic, or viral infection is a subject with recognizable symptoms of an existing bacterial, fungal, parasitic, or viral infection. Infectious pathogens include, but are not limited to, bacterial, fungal, parasitic, and viral pathogens. Examples of such infectious agents include: Staphylococcus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Streptococcus, Neisseriaceae, Cocci, Enterobacteriaceae, Enterococcus, Vancomycin-resistant Enterococcus, Cryptococcus, Histoplasmosis, Aspergillus, Pseudomonadaceae, Vibriales, Campylobacter, Pasteurellaceae, Bordetella, Francisella, Brucella, Legionellaceae, Bacteroidales, Gram-negative bacilli, Clostridium, Corynebacterium, Propionibacterium, Gram-positive bacilli, Bacillus anthracis, Actinomyces, Nocardia, Mycobacterium, Treponema, Borrelia, Leptospira, Mycoplasma, Ureaplasma, Rickettsia, Chlamydia, Candida, and systemic purpura. Mycoses, opportunistic fungal infections, protozoa, nematodes, trematodes, cestodes, adenoviruses, herpesviruses (including, for example, herpes simplex virus and Epstein-Barr virus, and varicella-zoster virus), poxviruses, papovaviruses, hepatitis viruses (including, for example, hepatitis B virus and hepatitis C virus), papillomaviruses, orthomyxoviruses (including, for example, influenza A, influenza B, and influenza C), paramyxoviruses, coronaviruses, picornaviruses, reoviruses, togaviruses, flaviviruses, bunyaviridae, rhabdoviruses, rotaviruses, respiratory syncytial virus, human immunodeficiency virus, and retroviruses. Exemplary infectious diseases include, but are not limited to, candidiasis, candidemia, aspergillosis, streptococcal pneumonia, streptococcal skin and oropharyngeal disease, gram-positive sepsis, tuberculosis, mononucleosis, influenza, respiratory disease caused by respiratory syncytial virus, malaria, schistosomiasis, and trypanosomiasis.

[0168] In some embodiments, the viral infection is a herpesvirus infection, a hepatitis virus infection, a West Nile virus infection, a flavivirus, an influenza virus infection, a rhinovirus infection, a papillomavirus infection, a paramyxovirus infection, a parainfluenza virus infection, and / or a retrovirus infection. Preferred viruses are those that infect the central nervous system of a subject. Most preferred viruses are those that cause encephalitis or meningitis.

[0169] In some embodiments, the bacterial infection is a staphylococcal infection, a streptococcal infection, a mycobacterial infection, a bacillary infection, a salmonella infection, a vibrio infection, a spirochete infection, or a neisseria infection. Preferred are bacteria that infect the central nervous system of a subject. Most preferred are those that cause encephalitis or meningitis.

[0170] Another embodiment of the present invention relates to transplantation. Transplantation refers to the transfer of a sample or graft from a donor subject to a recipient subject, and is frequently performed on human recipients who require 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.

[0171] A serious potential complication of any transplant arises 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 factors according to accepted clinical procedures. The immunological attack may be the result of a pre-existing immunological response (such as pre-formed antibodies) or a response initiated around the time of transplantation (such as the production of TH cells). Antibodies, TH cells, or TC cells may engage with each other and with various effector molecules and cells in any combination. However, the antigens involved in the immune response are generally unknown, making it difficult to design antigen-specific therapies or induce antigen-specific tolerance.

[0172] 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 established when the transplant is placed; however, if this is not possible, treatment may be initiated simultaneously with or after transplantation. Regardless of when it is initiated, transplantation is generally continued at regular intervals for at least the first month after transplantation. Booster administration may not be necessary if sufficient graft adaptation occurs, but may be resumed if any evidence of graft rejection or inflammation is observed. Of course, the tolerization procedure of the present invention may be combined with other forms of immunosuppression to achieve even lower levels of risk.

[0173] In some embodiments, the present invention provides a method for treating cancer in a subject. As used herein, "cancer" refers to or describes a physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, leiomyosarcoma, chordoma, lymphangiosarcoma, lymphangioendothelial sarcoma, rhabdomyosarcoma, fibrosarcoma, myxosarcoma, and chondrosarcoma), neuroendocrine tumors, mesothelioma, synovioma, schwannoma, meningioma, adenocarcinoma, melanoma, and leukemia, or lymphatic tumors. More specific examples of such cancers include squamous cell carcinoma (e.g., squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung, lung cancer including small cell lung cancer, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, anal cancer, penile cancer, testicular cancer, esophageal cancer, biliary tract tumor, Ewing's tumor, basal cell carcinoma, adenocarcinoma, and sweat gland cancer. , sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom's hypergammaglobulinemia, myelodysplastic disorders, heavy chain disease, neuroendocrine tumors, schwannoma, and other cell tumors, and head and neck cancer.

[0174] In some embodiments, the present invention provides a method for treating allergies in a subject. "Allergy," as used herein, includes all immune responses mediated by IgE and responses that mimic IgE-mediated responses. Allergies are induced by allergens, including proteins, peptides, carbohydrates, and combinations thereof, that provoke IgE or IgE-like immune responses. Allergies include food allergies (e.g., nut, milk, egg, fish, shellfish, wheat, or soy allergies). Exemplary food allergens include the Ara h1, Ara h2, and Ara h3 epitopes of peanut; the 15 kd antigen of celery; the apple antigen Mal d1; the Pru p3 of peach; and the α-gliadin and γ-gliadin epitopes of gluten. Allergies also include other environmental allergies (e.g., pollen, insect stings, dust, mold, fungal allergies, etc.). Exemplary environmental allergens include urushiol from poison ivy and oak; house dust antigens; birch pollen components Bet v1 and Bet v2; timothy grass pollen allergen Phl p1; rye grass Lol p3, Lol pI, or Lol pV; bermudagrass Cyn d1; house dust mite allergens Der p1, Der p2, or Derf1; bee venom phospholipase A2; and cedar pollen.

[0175] Various modifications, rearrangements, 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 should not 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. [Example]

[0176] The following examples are provided to further illustrate the advantages and features of the present invention and are not intended to limit the scope of the disclosure.

[0177] Example 1. Characterization of PLG particles encapsulating peptide epitopes Experiments were conducted to determine the physical and functional properties of PLG particles encapsulating different peptide epitopes. A list of tolerogenic and control epitopes is shown in Figure 2. As an example, PLP 139-151 PLG particles encapsulating the α-glucan were generated and their size distribution and zeta potential were analyzed by light scattering. The PLP was ranked 1st. 139-151 The peak of the size distribution of PLG particles encapsulating The Z-average size was 882.6 nm, suggesting the presence of larger species (Figure 3A). Furthermore, PLP was significantly larger than PLP, possibly due to the use of poly(ethylene-co-maleic acid) (PEMA) as an emulsifier. 139-151 The PLG particles encapsulating β-glucan had a very high negative charge (Z potential = −97.5 mV, Figure 3B).

[0178] Next, we determined the effect of PLG nanoparticles encapsulating different peptide epitopes on cell proliferation. 2 x 10 cells from DO11.10 transgenic mice (TCR transgenic mice specific for Ova323) were cultured. 5 Spleen cells, PLP 139-151 (0.737ng / μg) or Ova 323-339 PLG nanoparticles individually encapsulating (1.729ng / μg) Child, or PLP 139-151 and Ova 323-339 The cells were incubated with PLG nanoparticles encapsulating Ova323 (0.615 ng / μg). Nanoparticles and splenocytes were seeded with or without Ova323 (1 μg), grown for 2 days, and then pulsed with thymidine and cultured for an additional 3 days. Exposure of cells to Ova323 in the absence of nanoparticles resulted in increased cell proliferation (Figure 4B). However, Ova 323-339 Individually enclosed nanoparticles, or PLP 139-151 and Ova 323-339Treatment of cells exposed to Ova323 together with nanoparticles encapsulating Ova323 significantly reduced cell proliferation (Figure 4B). Cells not exposed to Ova323 did not proliferate (Figure 4A, negative control).

[0179] Different amounts of Ova 323-339 (1.1ng / μg, 23.1ng / μg, 0.2ng / μg Similar experiments were performed using nanoparticles encapsulating Ova323 (g) or full-length Ova (Figure 5). When the Ova323 peptide was not added to the culture, the highest amount of Ova 323-339 along with nanoparticles encapsulating peptide (23.1 ng / μg) or full-length Ova Only the cells incubated with the highest amount of Ova proliferated (Fig. 5B). 323-339 The peptide-encapsulating nanoparticles were activated by adding Ova323 to the cultures on day 2. However, lower doses of Ova did not inhibit the proliferation induced by Ova (Fig. 5C). 323-339 Higher doses of nanoparticles encapsulating (1.1 ng / μg and 0.2 ng / μg) , reduced the level of cell proliferation induced by Ova323 (Figure 5C). Addition of αCD28 to the cultures did not significantly affect cell proliferation, suggesting that proliferation in response to Ova323 was antigen-specific. These results suggest that nanoparticles encapsulating peptide epitopes can modulate antigen-induced cell proliferation.

[0180] Furthermore, various peptide epitopes can be encapsulated into nanoparticles. For example, Figures 6 and 7 show the encapsulation efficiency of tolerogenic and control peptide epitopes, respectively. Peptide epitopes can be encapsulated individually or together. The batch was aliquoted into pre-weighed test tubes, dried, and weighed to determine the mass of particles (mg / tube) in the pre-weighed tubes. μg peptide / mg particles was determined using a 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) protein quantification assay.

[0181] Example 2. PLP 139-151 Nanoparticles encapsulating peptide epitopes stimulate type 1 regulatory T cell recruitment Adjust the group To assess the effects of nanoparticles encapsulating peptide epitopes on different cell populations, we performed experiments using ex vivo assays. Briefly, on day -2, PLP 139-151 TCR transgenic mice (5B6 mice, donor, Thy1.1 + ) from 3.5x10 6 CD4 + The cells were transferred intravenously into naive 6- to 8-week-old female SJL recipient mice. On day 0, SJL recipient mice were transfected with PLP. 139-151 -S PLG nanoparticles encapsulating E or OVA 323-339 -PLG encapsulating SE (control particles) The recipient mice were injected with either one of the nanoparticles. On days 3 and 5 after injection, spleens were harvested from the recipient mice and the regulatory T cell population was analyzed by flow cytometry (Figure 8). All populations were CD90.1 / Thy1.1 + Gating on cells and PLP 139-151 -TCR + group was selected.

[0182] OVA on days 3 (Fig. 8A, left panel) and 5 (Fig. 8A, right panel). 323-339 -SE PLG or PLP 139-151 Lag3 in mice injected with -SE PLG + FoxP3 - Cells were first gated to determine the proportion of type 1 regulatory T cells (TR1). On the third day, Lag3 + FoxP3 - No differences were observed in the populations. However, by day 5, PLP 139-151 In animals injected with -PLG, OVA 323-339 -SE PLG control compared to Lag3 + FoxP3 -These cells also expressed a large proportion of IFNγ. + IL-10 + Lag3 indicates that they have the TR1 phenotype (Fig. 8A, lower panel). + FoxP3 - Quantification of the number of cells PLP 139-151 -PLG-treated mice and OVA 323-339 However, there was no difference between the PLP- and SE-treated mice. 139-151 -PLG-treated mice The OVA 323-339 -SE PLG-treated controls compared to antigen-specific TR1 cells (LA G3 + FoxP3 - IFNγ + IL-10 + ) showed a significant increase in the number of PLP 139-151 - SE PLG or OVA 323-339 -SE PLG injection on days 3, 5, and 7 Similar experiments were performed using the collected spleens (Fig. 9). 139-151 The number of TR1 cells was significantly increased in mice treated with -PLG. The data demonstrate that PLG nanoparticles encapsulating peptide epitopes increase the number of regulatory T cells, suggesting a potential therapeutic role in tolerance induction.

[0183] Another experiment was performed following the same protocol to analyze splenic regulatory T cells by flow cytometry. CD90.1 / Thy1.1 (PLP) was analyzed as described above. 139-151 TCR + Cells in this population were gated and analyzed for expression of regulatory T cell markers CD25, FoxP3, Helios, NP1, and IFNγ. 139-151 - Injection of PLG inhibits CD25 + FoxP3+, Helios + NP1 + , and IFNγ+ These results further support the conclusion that PLG nanoparticles encapsulating peptide epitopes have a potential therapeutic role in tolerance induction.

[0184] The ability of PLG nanoparticles encapsulating peptide epitopes to modulate disease activity was tested in EAE, a mouse model of multiple sclerosis. Briefly, on day -2, 3.5x106 cells from the 5B6 donor were injected. 6 CD4 + The cells were transferred intravenously into naive 6- to 8-week-old female SJL mice. On day 0, SJL recipient mice were transfected with PLP. 139-151 -SE PLG or control OVA 323-339 -SE PLG injection. +3 days after injection Spleens were harvested from mice on days +1 and +5. EAE was induced in another cohort on day 7, and spleens were harvested from these mice on days 5 and 17 post-induction for analysis. Splenic T cell populations were then analyzed by flow cytometry. Cell populations were CD90.1 / Thy1.1 (PLP 139-151 TCR + ) population was gated (Figure 11A). As shown, at later time points in both EAE and non-EAE mice, PLP 139-151 -SE PLG injection, but OVA 323-339 -SE PLG-injected controls compared with antigen The number of specific T cells was significantly increased (Fig. 11A). 139-151 -SE P Injection of LG with OVA 323-339 -SE PLG-injected controls compared to the antigen-increasing The number of antigen-specific T cells (Fig. 11B) and the number of antigen-specific regulatory T cells (Fig. 11C) were significantly increased. No significant difference was observed in the number of non-antigen-specific regulatory T cells (Fig. 11D). These results suggest that PLP 139-151 -SE PLG increases the proliferation of antigen-specific T regulatory cells These results suggest that nanoparticles encapsulating peptide epitopes mediate tolerogenic responses in models of autoimmunity.

[0185] Example 3. OVA 323-339 Nanoparticles encapsulating peptide epitopes stimulate type 1 regulatory T cell recruitment Adjust the group OVA 323-339 2.5x10 from TCR donor (DO11 mouse) 6 CD4 + Cells Complementary experiments were performed by intravenously transferring the OVA-1 ... 323-339 -SE PLG or PLP 139-151 After injection, mice were injected with either DO11-TCR or DO11-SE PLG (control). Spleens were harvested from the mice and the regulatory T cell population was analyzed by flow cytometry (FIG. 12). The cell population was DO11-TCR. + The group was gated. As shown , P.L.P. 139-151 -OVA compared with SE PLG-injected controls 323-339 -CD25 from PLG-injected animals + FoxP3 + CD4 + Percentage (Fig. 12A) and total number of T cells In a similar experiment, the percentage (Fig. 13A) and number (Fig. 13B) of antigen-specific regulatory T cells producing IFNγ were also significantly increased by PLP. 139-151 -S E OVA compared with PLG-injected controls 323-339 - in PLG-injected animals IFNγ increased significantly. + There was a significant difference in the number of non-regulatory T cells between the experimental and control groups. Furthermore, the increase in the proportion (FIG. 14A) and number (FIG. 14B) of the TR1 population from the DO11 donor was not observed with 5B6 CD90.1 isolated from SJL mice. + A similar trend to that observed in TR1 cells was observed.

[0186] We also measured the number of proliferating antigen-specific cells (Fig. 15A), the number of proliferating antigen-specific T regulatory cells (Fig. 15B), and the number of proliferating antigen-specific TR1 cells (Fig. 15C, from a separate experiment, FoxP3 - These results were consistent with those in Example 2. Similar to the 5B6 transfer experiments in mice, these results show that PLG nanoparticles encapsulating peptide epitopes regulate the expansion of regulatory T cell populations, suggesting a role in the induction of tolerance.

[0187] Example 4. Characterization of nanoparticles encapsulating PLP139-Ova323 fusion peptide PLPs linked by a peptide linker containing a cathepsin-specific cleavage site 139-151 and OVA 323-339 A fusion peptide of the epitope was generated (Figure 16, PLP139-Ov a323 fusion peptide). 2 x 10 5 Splenocytes were incubated with various amounts of OVA 323-339 , P.L.P. 139-151 , OVA 323-339 +PLP 139-151 The PLP139-Ova323 fusion peptide inhibited OVA in DO11 cells, and cell proliferation was evaluated. 323-339 and PLP in 5B6 cells 139-151 These results suggest that the fusion protein, like either the epitopes alone or in combination, is capable of modulating cellular responses.

[0188] The physical properties (Z-average diameter, PDI, peak diameter, and zeta potential) of nanoparticles encapsulating the PLP139-Ova323 fusion peptide were determined by dynamic light scattering (DLS) (Figure 18). The Z-average diameter ranged from 1203 to 3316 nm across three runs, and the zeta potential was -95 to -115 mV. The particles were dried on a carbon-coated copper mesh screen, and images of the nanoparticles were also obtained using transmission electron microscopy (TEM) (Figure 18).

[0189] The effect of nanoparticles on cell proliferation is 2x10 5 1-100 μg of DO11 splenocytes Nanoparticles (PLP139-Ova323 fusion, PLP 139-151 , or OVA 323-339 The effect of OVA on the IL-16 expression was assessed by seeding cells with nanoparticles encapsulating PLP139-Ova323 fusion or OVA. Cells were grown for 2 days, then pulsed with thymidine and cultured for an additional 3 days. 323-339 Treatment of cells with either encapsulated nanoparticles was compared with untreated controls. This resulted in increased cell proliferation compared to (Figure 19A). Similar results were observed when 1 μg of Ova323 (Figure 19B) or 1 μg of Ova323 and 1 μg / mL of αCD28 (Figure 19C) were added to the cultures on day 2.

[0190] Complementary experiments were performed using 5B6 splenocytes (Figure 20). 5 Seeding 5B6 splenocytes The cells were seeded and grown for 2 days, then pulsed with thymidine and cultured for an additional 3 days. 139-151 We used nanoparticles that encapsulate either Treatment of infected cells was either untreated control or OVA. 323-339 Treatment using nanoparticles that encapsulate Higher doses resulted in increased cell proliferation compared to cells treated with αCD28 alone (Figure 20A). Similar results were observed when 1 μg of PLP139 (Figure 20B) or 1 μg of PLP139 and 1 μg / mL of αCD28 (Figure 20C) were added to the cultures on day 2. These data demonstrate that the combined epitope-protein fusions are immunogenic and suggest that they have increased immunomodulatory potential compared to single epitopes alone.

[0191] As shown in Figure 21, the fusion protein can be efficiently encapsulated into nanoparticles. The batch was aliquoted into pre-weighed tubes, dried, and weighed to determine the mass of particles (mg / tube) in the pre-weighed tubes. μg peptide / mg particles was determined using the CBQCA protein quantification assay.

[0192] Example 5. Nanoparticles encapsulating PLP139-Ova323 fusion peptide mediate tolerogenic responses in EAE Experiments were conducted to determine the in vivo effects of nanoparticles encapsulating the PLP139-OVA323 fusion protein in a model of EAE. 139-151 Using peptide epitopes or PLP139-OVA323 fusion proteins EAE was induced in SLJ mice by immunization with the PLP139-OVA323 fusion protein (N=5 per group). As shown in Figure 22A, immunization with the PLP139-OVA323 fusion protein was sufficient to induce EAE. The magnitude of the resulting disease score was significantly higher than that of the PLP139-OVA323 fusion protein. 139-151 The EAE scores were comparable to those induced by immunization with the peptide (Figure 22A). Figure 22 → PLP-OVA-bound EAE scores. The PLP-OVA-bound peptide induces disease and, when encapsulated in PLGA nanoparticles, induces tolerance. Importantly, administration of encapsulated PLP-OVA fusion protein (PLP-OVA) resulted in abrogation of EAE disease scores (Figure 22A). Furthermore, this immune modulation was antigen-specific, as the encapsulated PLP-OVA fusion protein only reduced the immune response in SLJ mice immunized with PLP but did not affect the immune response induced by immunization with the MOG peptide (Figure 22B). These results suggest that nanoparticles encapsulating the binding epitope fusion peptide induce tolerogenic immune responses in an antigen-specific manner.

[0193] Example 6. Characterization of nanoparticles encapsulating multi-epitope fusion peptides PLP linked by a peptide linker containing a cathepsin-specific cleavage site 139-151 , P.L.P. 178-191 , MOG 92-106 , and MBP 84-104 A fusion peptide of the epitope was generated (Figure 23A, tolerogenic EAE-1 fusion peptide). In addition, OVA was linked by a peptide linker containing a cathepsin-specific cleavage site. 323-339 , P.L.P. 56 - 70 , V.P. 1 233-250 , and VP2 70-86 A control fusion peptide containing the epitope was also generated (Figure 23B, EAE-1 control fusion peptide). The physical properties (Z-average diameter, PDI, peak diameter, and zeta potential) of nanoparticles encapsulating the tolerogenic fusion peptides were determined by DLS. Images of the particles were obtained using transmission electron microscopy (TEM) after drying the particles on a carbon-coated copper mesh screen. Both DLS and EM data suggest a larger-than-normal size distribution (Figure 24).

[0194] The encapsulation efficiency of the tolerogenic or negative control fusion peptides is shown in Figure 25. The batches were aliquoted into pre-weighed tubes, dried, and weighed to determine the mass of particles (mg / tube) in the pre-weighed tubes. μg peptide / mg particles was determined using CBQCA protein quantification.

[0195] The encapsulation of single epitopes and combined epitopes into PLGA nanoparticles is shown in Figure 26. As determined by one-way ANOVA statistical test (p<0.0001), single myelin-specific T cell epitopes (PLP139, PLP178, MBP84, and MOG92) were encapsulated less efficiently than combined EAE-1 peptides (PLP139:PLP178:MOG92:MBP). Similarly, individual control epitopes (OVA323, PLP56, VP1233, and VP270) were encapsulated less efficiently than combined EAE-control epitopes (OVA323:PLP56:VP1:VP2).

[0196] Example 7. Characterization of nanoparticles encapsulating multi-epitope fusion peptides Experiments were performed to determine the effect of PLGA nanoparticles encapsulating conjugated EAE-1 fusion peptides (PLP139:PLP178:MOG92:MBP) in an EAE model, resulting in a significant reduction in EAE disease compared with an irrelevant control peptide (OVA) or a control binding epitope (OVA323:PLP56:VP1:VP2). Treatment of mice with encapsulated conjugated EAE-1 fusion peptides resulted in a significant reduction in EAE disease compared with the control fusion peptide. Treatment with FALK significantly reduced EAE disease scores compared to treatment with either FALK or an irrelevant control peptide (OVA) (Figure 28). These data demonstrate that fusion peptides containing multiple linked disease epitopes can induce tolerance in an autoimmune model, suggesting their therapeutic potential. Figure 29 shows that the FALK peptide can also induce EAE, suggesting that epitopes found in this protein could also be incorporated into tolerogenic EAE fusion proteins.

[0197] Example 8. Use of encapsulated binding epitope fusion proteins in the treatment of cancer Fusion peptides containing binding epitopes of neoantigens or tumor antigens are also used to treat cancer. Such epitopes are combined with immunomodulators such as PD1 or Toll-like receptor agonists to enhance therapeutic efficacy (see Figure 29). Data show that encapsulated cancer antigens such as Ny-Eso1 delivered with immunomodulators such as anti-PD1 result in T cell proliferation (see exemplary results in Figure 31A), IFNγ production (see exemplary results in Figure 31B), and IFNα production (see exemplary results in Figure 31C). These results indicate that encapsulated cancer antigens mediate immunogenic responses and lead to the production of pro-inflammatory cytokines.

[0198] Furthermore, encapsulated Ny-Eso1 increased the survival rate of mice in a mouse model of melanoma. 0.01 mg / kg infusion of encapsulated NY-ESO-1 (TIMP-NY-ESO-1) was administered twice a month (7 days apart) for 6 months. PD1 / PD-L1 inhibition was achieved with pembrolizumab, nivolumab, or atezolizumab. TIMP-NY-ESO-1 treatment alone was sufficient to extend survival, and its effect was enhanced when combined with anti-PD1 treatment (see exemplary results in Figure 32).

[0199] Fusion peptides containing cancer epitopes (NY-ESO-1, Mage-A3, TPTE, tyrosinase, and HPU16) have been previously described (Kranz et al., Nature, Vol. 534, pp. 396-401, 2016; see US Patent Publication No. 2011-0070252). Fusion proteins containing four cancer epitopes (NY-ESO-1, Mage-A3, TPTE, and tyrosinase) were generated to form NMTT fusion proteins. Various concentrations of encapsulated NMTT (TIMP-NMTT) were incubated with peripheral blood monocytes (PBMCs) from healthy subjects with or without anti-PD1 / PDL1 treatment. Cultures were incubated for 3–5 days, and T cell proliferation was determined using cell-tire glow or tritiated thymidine incorporation. IFNγ concentrations were determined using ELISA. Incubation of PBMCs with TIMP-NMTT in combination with anti-PD1 / PDL1 resulted in increased T cell proliferation compared to controls (exemplary results in Figure 33A). Furthermore, TIMP-NMTT in combination with anti-PD1 / PDL1 resulted in IFNγ production from PBMCs, whereas TIMP-NY-ESO-1 alone had little effect. These results indicate that fusion proteins encapsulating cancer epitopes mediate beneficial immunogenic responses in the context of cancer therapy.

[0200] To determine the clinical efficacy of TIMP-NMTT, tumor antigens encoding TIMP-NMTT are prepared from components manufactured in accordance with GMP in a dedicated pharmacy under GMP. Patients are intravenously injected with weekly escalating doses of TIMP-NMTT encoding the antigens NY-ESO-149, tyrosinase 50, MAGE-A351, and TPTE52 (1.9, 3.6, or 7.2 μg of each antigen). Blood samples for cytokine measurement are collected for ELISPOT and MHC class I dextramer staining analysis before vaccination, 2, 6, and 24 hours after vaccination on day 1, and on days 8 and 15 after vaccination. T cell monitoring is performed before vaccine administration on each vaccination day. Obtain blood samples for screening. Clinically administered TIMP-NMTT vaccine induces systemic INFα and de novo T cell responses in a dose-dependent manner.

[0201] Example 9. Further uses of encapsulated binding epitope fusion proteins Additional fusion proteins containing binding epitopes may be generated. For example, fusion proteins containing epitopes derived from multiple disease types can be generated and used to treat more than one disease. Alternatively, fusion proteins containing disease-related epitopes may be generated that further include a TLR agonist. The inclusion of such an agonist increases the immunogenicity of the protein and enhances therapeutic efficacy. Additional fusion proteins may be generated using epitopes derived from infectious viruses, bacteria, or fungi. Adding a TLR agonist to such constructs can also enhance therapeutic efficacy.

Claims

1. 1. A biodegradable particle comprising one or more encapsulated fusion proteins, each of the one or more fusion proteins comprises two or more antigenic epitopes; the two or more antigenic epitopes are separated by a linker; the linker comprises an amino acid sequence susceptible to specific cleavage; The biodegradable particles have a negative zeta potential.

2. The biodegradable particle of claim 1, wherein the biodegradable particle comprises poly(lactide-co-glycolide) (PLG).

3. 3. The biodegradable particle of claim 1, wherein the biodegradable particle comprises PLG in a copolymer ratio of polylactic acid:polyglycolic acid of about 50:

50.

4. The biodegradable particles according to any one of claims 1 to 3, wherein the surface of the biodegradable particles is carboxylated.

5. The biodegradable particles according to any one of claims 1 to 4, wherein the carboxylation is achieved by using poly(ethylene-maleic anhydride) (PEMA), polyacrylic acid, or sodium cholate.

6. The biodegradable particle according to any one of claims 1 to 5, wherein the biodegradable particle has a zeta potential of about -100 mV to about 0 mV.

7. The biodegradable particle according to any one of claims 1 to 6, wherein the biodegradable particle has a zeta potential of about -50 mV to about -40 mV.

8. The biodegradable particle according to any one of claims 1 to 7, wherein the biodegradable particle has a zeta potential of about -75 mV to about -50 mV.

9. The biodegradable particle according to any one of claims 1 to 8, wherein the biodegradable particle has a zeta potential of about -50 mV.

10. The biodegradable particles according to any one of claims 1 to 9, wherein the biodegradable particles have a diameter of about 0.1 μm to about 10 μm.

11. The biodegradable particles according to any one of claims 1 to 10, wherein the biodegradable particles have a diameter of about 0.3 μm to about 5 μm.

12. The biodegradable particle according to any one of claims 1 to 11, wherein the biodegradable particle has a diameter of about 0.5 μm to about 3 μm.

13. The biodegradable particle according to any one of claims 1 to 12, wherein the biodegradable particle has a diameter of about 0.5 μm to about 1 μm.

14. The biodegradable particle according to any one of claims 1 to 13, wherein the biodegradable particle has a diameter of about 0.5 µm.

15. The biodegradable particle according to any one of claims 1 to 14, wherein the biodegradable particle has a diameter of about 0.6 μm.

16. A biodegradable particle described in any one of claims 1 to 15, wherein the amino acid sequence of the linker includes a site that is susceptible to specific cleavage by a protease located in the phagolysosome of a cell or a site that is susceptible to specific cleavage by a protease located in the cytosol of a cell.

17. The biodegradable particle of claim 16, wherein the amino acid sequence of the linker includes a site susceptible to specific cleavage by a protease located in the phagolysosome of the cell and a site susceptible to specific cleavage by a protease located in the cytosol of the cell.

18. 18. The biodegradable particle of claim 16 or 17, wherein the site susceptible to specific cleavage by a protease located in the phagolysosome is susceptible to cleavage by a furin or cathepsin protease.

19. The biodegradable particle according to any one of claims 16 to 18, wherein the site susceptible to specific cleavage by a protease located in the phagolysosome is susceptible to cleavage by a furin protease.

20. The biodegradable particle according to any one of claims 16 to 18, wherein the site susceptible to specific cleavage by a protease located in the phagolysosome is susceptible to cleavage by a cathepsin protease.

21. The biodegradable particle according to claims 16 to 18 or 20, wherein the site susceptible to specific cleavage by a protease located in the phagolysosome is one or more of cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin W, or cathepsin Z.

22. 22. The biodegradable particle of claim 21, wherein the site susceptible to specific cleavage by a protease located in the phagolysosome is cathepsin L.

23. 18. The biodegradable particle of claim 16 or 17, wherein the site susceptible to specific cleavage by a protease located in the cytosol is susceptible to cleavage by a furin or cathepsin protease.

24. 24. The biodegradable particle of claim 23, wherein the site susceptible to specific cleavage by a protease located in the cytosol is susceptible to cleavage by cathepsin S.

25. A biodegradable particle described in any one of claims 16 to 18 or 20 to 24, wherein the amino acid sequence of the linker includes a site susceptible to specific cleavage by cathepsin L and a site susceptible to specific cleavage by cathepsin S.

26. 26. The biodegradable particle of claim 25, wherein the amino acid sequence of the linker is Gly-Ala-Val-Val-Arg-Gly-Ala (SEQ ID NO: 5141).

27. The biodegradable particle of any one of claims 1 to 26, wherein the two or more antigenic epitopes include an autoimmune antigen, an antigen expressed on a tissue to be transplanted into a subject, an antigen derived from an enzyme for enzyme replacement therapy, or an antigen derived from an allergen.

28. The two or more antigenic epitopes each comprise at least a portion of a protein, 28. The biodegradable particle of claim 27, wherein the moieties are derived from the same protein.

29. 28. The biodegradable particle of claim 27, wherein the two or more antigenic epitopes each comprise at least a portion of a protein, the portions being derived from different proteins.

30. 30. The biodegradable particle of claim 29, wherein the different proteins are associated with the same autoimmune disorder, the same tissue to be transplanted into a subject, or the same allergen.

31. The two or more antigenic epitopes are, respectively, 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, gliadine, insulin, proinsulin, pancreatic islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), human tropomyosin isoform 5, bahiagrass pollen (BaGP), and peach allergen Pru. p3, αS1-caeine milk allergen, Apig1 celeriac allergen, Bere1 Brazil nut allergen, B-lactoglobulin milk allergen, bovine serum albumin, Cor a 1.04 hazelnut allergen, myelin-associated glycoprotein, aquaporin, α3 chain of type IV collagen, ovalbumin egg allergen, Advate, antihemophilic factor, Kogenate, Eloctate, recombinant factor VIII fusion protein, Refacto, Novo 31. The biodegradable particle of any one of claims 27 to 30, comprising at least a portion of a protein selected from the group consisting of Factor VIIa, recombinant Factor VII, eptacog alfa, Helixate, Monanine, coagulation factor IX, Wilate, Ceredase, alglucerase, Cerezyme, imiglucerase, Elelso, taliglucerase alfa, Fabrazyme, agalsidase beta, Aldurazyme, -I-iduronidase, Myozyme, acid glucosidase, Elaprace, iduronate-2-sulfatase, Naglazyme arylsulfatase B, and N-acetylgalactosamine-4-sulfatase.

32. The biodegradable particle according to any one of claims 1 to 27, wherein the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 2 to 1294.

33. The biodegradable particle of any one of claims 1 to 27, wherein the two or more antigenic epitopes are selected from the group consisting of discontinuous epitopes derived from SEQ ID NOs: 1295 to 1724, SEQ ID NOs: 1726 to 1766, SEQ ID NOs: 4986 to 5140, and SEQ ID NO: 1725.

34. The biodegradable particle of any one of claims 1 to 27, wherein the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 1767 to 1840; SEQ ID NOs: 1842 to 1962; SEQ ID NOs: 1964 to 2027; SEQ ID NOs: 2029 to 2073; SEQ ID NOs: 2075 to 2113; SEQ ID NOs: 2115 to 2197; SEQ ID NOs: 2199 to 2248; SEQ ID NOs: 2250 to 2259; SEQ ID NOs: 2261 to 2420; SEQ ID NOs: 2422 to 2486; SEQ ID NOs: 2489 to 2505, and discontinuous epitopes derived from SEQ ID NOs: 1841, 1963, 2028, 2074, 2114, 2198, 2260, 2249, 2421, 2487, and 2488.

35. The biodegradable particle of any one of claims 1 to 27, wherein the two or more antigenic epitopes are selected from the group consisting of discontinuous epitopes derived from SEQ ID NOs: 2506 to 3260; SEQ ID NOs: 3262 to 3693; and 3261.

36. The biodegradable particle of any one of claims 1 to 27, wherein the two or more antigenic epitopes are selected from the group consisting of discontinuous epitopes derived from SEQ ID NOs: 3694 to 3857; SEQ ID NOs: 3860 to 4565; and 3857, 3858, and 3859.

37. The biodegradable particle of any one of claims 1 to 27, wherein the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4566-4576; SEQ ID NOs: 4578-4610; SEQ ID NOs: 4612-4613; and SEQ ID NOs: 5018-5039; and discontinuous epitopes derived from 4357, 4577, and 4611.

38. The biodegradable particle according to any one of claims 1 to 27, wherein the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4614 to 4653.

39. The biodegradable particle of any one of claims 1 to 27, wherein the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4654 to 4694; SEQ ID NOs: 4696 to 4894; SEQ ID NOs: 4896 to 4901; and discontinuous epitopes derived from 4695 and 4895.

40. The biodegradable particle according to any one of claims 1 to 27, wherein the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4902 to 4906.

41. The biodegradable particle according to any one of claims 1 to 27, wherein the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4907 to 4914.

42. The biodegradable particle according to any one of claims 1 to 27, wherein the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4915 to 4917.

43. The biodegradable particle according to any one of claims 1 to 27, wherein the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4918 to 4941.

44. The biodegradable particle according to any one of claims 1 to 27, wherein the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4942 to 4952.

45. The biodegradable particle according to any one of claims 1 to 27, wherein the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4953 to 4963.

46. The biodegradable particle according to any one of claims 1 to 27, wherein the two or more antigenic epitopes are selected from the group consisting of SEQ ID NOs: 4964 to 4974.

47. The biodegradable particle of any one of claims 1 to 27, wherein the two or more antigenic epitopes are derived from a therapeutic antibody, an antigen-binding fragment, or an Fc fragment thereof.

48. 48. The biodegradable particle of claim 47, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody, a humanized monoclonal antibody, a human monoclonal antibody, a chimeric antibody, a single-chain antibody, an antigen-binding fragment region (Fab), a single-chain variable fragment (scFv), a small modular immunopharmaceutical (SMIP), or a single-chain antigen-binding domain.

49. The antibody or antigen-binding fragment thereof may bind to any of the following: α4β1 integrin, Bacillus anthracis, B-L(γS), C5, CD3, CD11a, CD20, CD25, CD30, CD33, CD52, CD59, CTLA4, EGFR, GD2, GPIIb, IIIa, HER2, IgE, IgE-1, IgE-2, IgE-3, IgE-4, IgE-5, IgE-6, IgE-7, IgE-8, IgE-9, IgE-11, IgE-12, IgE-13, IgE-14, IgE-15, IgE-16, IgE-17, IgE-18, IgE-19, IgE-20, IgE-21, IgE-22, IgE-23, IgE-2 49. The biodegradable particle of claim 47 or 48, which binds to IL-1β, IL-5, IL12 / 23, PCSK9, PD1, RANK, RSV F protein, TNFα, or VEGF-A.

50. The antibody or antigen-binding fragment thereof may be selected from the group consisting of abciximab, adalimumab, adotrastuzumab emtansine, alemtuzumab, basiliximab, bevacizumab, belimumab, blinatumomab, brentuximab vedotin, canakinumab, catumaxomab, cetuximab, certolizumab pegol, daclizumab, denosumab, dinutuximab, eculizumab, efalizumab, evolocumab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan, ipi 50. The biodegradable particle according to any one of claims 47 to 49, which is limumab, infliximab, motavizumab, muronomab, natalizumab, nivolumab, obinutuzumab, ofatumumab, omalizumab, panitumumab, palivizumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, raxibacumab, rituximab, secukinumab, siltuximab, trastuzumab, tocilizumab, tositumomab-I-131, ustekinumab, or vedolizumab.

51. The biodegradable particle of any one of claims 1 to 27, wherein the two or more antigenic epitopes are derived from a variant of a therapeutic antibody or antigen-binding fragment thereof lacking functional complementarity-determining regions (CDRs).

52. 52. The biodegradable particle of claim 51, wherein the antibody or antigen-binding fragment variant lacking a functional CDR is a monoclonal antibody, a humanized monoclonal antibody, a human monoclonal antibody, a chimeric antibody, a single-chain antibody, an antigen-binding fragment region (Fab), a single-chain variable fragment (scFv), a small modular immunopharmaceutical (SMIP), or a single-chain antigen-binding domain.

53. One of the one or more fusion proteins may comprise an antigenic epitope MOG 1-20 , MBP 13-32 , MOG 35-55 , MBP 146-170 , P.L.P. 139-154 , MBP 111-129 , and MBP 83-99 The biodegradable particle according to any one of claims 1 to 27, comprising:

54. 28. The biodegradable particle of any one of claims 1 to 27, wherein one of the one or more fusion proteins comprises the antigenic epitopes SEQ ID NO: 1350, SEQ ID NO: 4986, and SEQ ID NO: 4987.

55. A pharmaceutical composition comprising the biodegradable particles of any one of claims 1 to 54.

56. 56. The pharmaceutical composition of claim 55, further comprising a pharmaceutically acceptable carrier.

57. 57. The pharmaceutical composition of claim 55 or 56, further comprising a pharmaceutically acceptable excipient.

58. A method for inducing antigen-specific tolerance in a subject, comprising administering an effective amount of a biodegradable particle according to any one of claims 1 to 54.

59. 1. A method of inducing antigen-specific tolerance in a subject, comprising administering to the subject an effective amount of biodegradable particles comprising one or more encapsulated fusion proteins, each of the one or more fusion proteins comprises two or more antigenic epitopes; the two or more antigenic epitopes are separated by a linker; the linker comprises an amino acid sequence susceptible to specific cleavage; The method, wherein the biodegradable particles have a negative zeta potential.

60. 60. The method of claim 59, wherein the effective amount of the biodegradable particles is administered to the subject orally, intravenously, sublingually, bucally, enterally, topically, rectally, subcutaneously, nasally, intraosseously (i.e., intraosseous injection), intraperitoneally, intrathecally, transdermally, or transmucosally.

61. 61. The method of claim 60, wherein the effective amount of the biodegradable particles is administered to the subject intravenously or subcutaneously.

62. 62. The method of claim 61, wherein the effective amount of the biodegradable particles is administered intravenously to the subject.

63. 62. The method of claim 61, wherein the effective amount of the biodegradable particles is administered subcutaneously to the subject.

64. 64. The method of any one of claims 59 to 63, wherein the effective amount of the biodegradable particles is administered to a subject to treat or prevent a disease or condition.

65. 65. The method of claim 64, wherein the disease or condition is selected from the group consisting of an autoimmune disease, a lysosomal storage disease, an enzyme deficiency, an inflammatory disease, an allergy, a transplant rejection, and a hyperimmune response.

66. 65. The method of claim 64, wherein the disease or condition is selected from the group consisting of multiple sclerosis, type 1 diabetes, asthma, food allergies, environmental allergies, celiac disease, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), mucopolysaccharidosis, gangliosidosis, low alkaline phosphatase, cholesterol ester storage disease, hyperuricemia, growth hormone deficiency, renal anemia, hemophilia, hemophilia A, hemophilia B, von Willebrand's disease, Gaucher's disease, Fabry's disease, Hurler's disease, Pompe's disease, Hunter's disease, Maroteaux-Lamy disease, and conditions caused by an antigen of interest resulting in an exaggerated response to the antigen.

67. 65. The method of claim 64, wherein the disease or condition is multiple sclerosis and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 2-1294.

68. 65. The method of claim 64, wherein the disease or condition is celiac disease, and wherein each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 1295-1724; SEQ ID NOs: 1726-1766; SEQ ID NOs: 4986-5140; and discontinuous epitopes derived from SEQ ID NO: 1725.

69. 65. The method of claim 64, wherein the disease or condition is type 1 diabetes, and wherein each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs:1767-1840; SEQ ID NOs:1842-1962; SEQ ID NOs:1964-2027; SEQ ID NOs:2029-2073; SEQ ID NOs:2075-2113; SEQ ID NOs:2115-2197; SEQ ID NOs:2199-2248; SEQ ID NOs:2250-2259; SEQ ID NOs:2261-2420; SEQ ID NOs:2422-2486; SEQ ID NOs:2489-2505; and discontinuous epitopes derived from SEQ ID NOs:1841, 1963, 2028, 2074, 2114, 2198, 2260, 2249, 2421, 2487, and 2488.

70. 65. The method of claim 64, wherein the disease or condition is rheumatoid arthritis and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of discontinuous epitopes derived from SEQ ID NOs: 2506-3260; SEQ ID NOs: 3262-3693; and 3261.

71. the disease or condition is systemic lupus, and each of the one or more fusion proteins 65. The method of claim 64, wherein said sequence comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 3694-3857; SEQ ID NOs: 3860-4565; and discontinuous epitopes derived from 3857, 3858, and 3859.

72. 65. The method of claim 64, wherein the disease or condition is Goodpasture's syndrome, and wherein each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4566-4576; SEQ ID NOs: 4578-4610; SEQ ID NOs: 4612-4613; and SEQ ID NOs: 5018-5039; and discontinuous epitopes derived from 4357, 4577, and 4611.

73. 65. The method of claim 64, wherein the disease or condition is uveitis and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4614-4653.

74. 65. The method of claim 64, wherein the disease or condition is thyroiditis, and wherein each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4654-4694; SEQ ID NOs: 4696-4894; SEQ ID NOs: 4896-4901; and discontinuous epitopes derived from 4695 and 4895.

75. 65. The method of claim 64, wherein the disease or condition is myositis and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4902-4906.

76. 65. The method of claim 64, wherein the disease or condition is vasculitis and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4907-4914.

77. 65. The method of claim 64, wherein the disease or condition is pancreatitis and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4915-4917.

78. 65. The method of claim 64, wherein the disease or condition is Crohn's disease and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4918-4941.

79. 65. The method of claim 64, wherein the disease or condition is ulcerative colitis and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4942-4952.

80. 65. The method of claim 64, wherein the disease or condition is psoriasis and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4953-4963.

81. 65. The method of claim 64, wherein the disease or condition is reactive arthritis and each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4964-4974.

82. 1. A method for reducing suppressive neutrophil accumulation in a subject, comprising administering to the subject an effective amount of biodegradable particles comprising one or more encapsulated fusion proteins, each of the one or more fusion proteins comprises two or more antigenic epitopes; the two or more antigenic epitopes are separated by a linker; the linker comprises an amino acid sequence susceptible to specific cleavage; The method, wherein the biodegradable particles have a negative zeta potential.

83. 83. The method of claim 82, wherein the subject has cancer.

84. The two or more antigen epitopes are CD19, CD20, BCMA, CD22, CLL1, CD33, CEA, CD123, CS1, EGFR, PSMA, EphA2, MCSP, ADAM17, PSCA, TPTE, HPU16, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride channel 2, and cyclin B, respectively. 1 , 9D7, Ep-CAM, EphA3, Her2 / neu, Te 84. The method of claim 83, comprising at least a portion of a protein selected from the group consisting of methicillinase, mesothelin, SAP-1, survivin, a BAGE family protein, a CAGE family protein, a GAGE ​​family protein, a MAGE family (e.g., MAGE-A3) protein, a SAGE family protein, an XAGE family protein, CT9, CT10, NY-ESO1 / LAG-1, PRAME, SSX-2, melan-A / MART-1, Cp100 / pmel17, tyrosinase, TRP-1 / TRP-2, P. polypeptide, MC1R, prostate-specific antigen, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII, and MUC1.

85. 1. A method of increasing tissue regeneration in a subject, comprising administering to the subject an effective amount of biodegradable particles comprising one or more encapsulated fusion proteins, each of the one or more fusion proteins comprises two or more antigenic epitopes; the two or more antigenic epitopes are separated by a linker; the linker comprises an amino acid sequence susceptible to specific cleavage; The method, wherein the biodegradable particles have a negative zeta potential.

86. 86. The method of claim 85, wherein the particles increase epithelial cell regeneration in a colitis patient.

87. 87. The method of claim 86, wherein each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 4918-4941 and SEQ ID NOs: 4942-4952.

88. 85. The method of claim 84, wherein the particles increase remyelination in multiple sclerosis patients.

89. 88. The method of claim 87, wherein each of the one or more fusion proteins comprises two or more antigenic epitopes derived from myelin basic protein and / or myelin oligodendrocyte glycoprotein.

90. 89. The method of claim 88, wherein each of the one or more fusion proteins comprises two or more antigenic epitopes selected from the group consisting of SEQ ID NOs: 2-1294.

91. 1. A method for reducing the incidence and / or severity of an immune response to a therapeutic protein in a subject, comprising administering to the subject an effective amount of biodegradable particles comprising one or more encapsulated fusion proteins, each of the one or more fusion proteins comprises two or more antigenic epitopes; the two or more antigenic epitopes are derived from the Therapeutic protein; the two or more antigenic epitopes are separated by a linker; the linker comprises an amino acid sequence susceptible to specific cleavage; The method, wherein the biodegradable particles have a negative zeta potential.

92. 91. The method of claim 90, wherein the subject is receiving enzyme replacement therapy for the treatment of a disease selected from the group consisting of hemophilia, hemophilia A, hemophilia B, von Willebrand disease, Gaucher disease, Fabry disease, Hurler disease, Pompe disease, Hunter disease, mucopolysaccharidosis, gangliosidosis, low alkaline phosphatase, cholesterol ester storage disease, hyperuricemia, growth hormone deficiency, renal anemia, and Maroteaux-Lamy disease.

93. The antigenic epitopes are those derived from Advate, antihemophilic factor, Kogenate, Eloctate, recombinant factor VIII fusion protein, Refacto, Novo 91. The method of claim 90, comprising one or more enzymes selected from the group consisting of Factor VIIa, recombinant Factor VII, eptacog alfa, Helixate, Monanine, coagulation factor IX, Wilate, Ceredase, alglucerase, Cerezyme, imiglucerase, Elelso, taliglucerase alfa, Fabrazyme, agalsidase beta, Aldurazyme, -I-iduronidase, Myozyme, acid glucosidase, Elaprace, iduronate-2-sulfatase, Naglazyme arylsulfatase B, and N-acetylgalactosamine-4-sulfatase.

94. 91. The method of claim 90, wherein the antigenic epitope comprises one or more proteins selected from the group consisting of interferon alpha, interferon alpha 2a, interferon beta lb, interferon beta la, insulin, DNAase, Neupogen, Epogen, Procrit (epotein alpha), Aranesp (second generation Procrit), Intron A (interferon alpha 2b), IL-2 (Proleukin), IL-Ira, BMP-7, TNF-alpha la, tPA, PDGF, interferon gamma lb, uPA, GMCSF, Factor VII, Factor VIII, Betaferon (interferon beta la), somatotropin, and Rebif (interferon beta la).

95. 91. The method of claim 90, wherein the therapeutic protein is an antibody, an antigen-binding fragment, or an Fc fragment thereof.

96. The antibody, antigen-binding fragment, or Fc fragment thereof may be selected from the group consisting of abciximab, adalimumab, adotrastuzumab emtansine, alemtuzumab, basiliximab, bevacizumab, belimumab, blinatumomab, brentuximab vedotin, canakinumab, catumaxomab, cetuximab, certolizumab pegol, daclizumab, denosumab, dinutuximab, eculizumab, efalizumab, evolocumab, gemtuzumab ozogamicin, golimumab, ibritumomab, 95. The method of claim 94, wherein the agent is butiuxetan, ipilimumab, infliximab, motavizumab, muronomab, natalizumab, nivolumab, obinutuzumab, ofatumumab, omalizumab, panitumumab, palivizumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, raxibacumab, rituximab, secukinumab, siltuximab, trastuzumab, tocilizumab, tositumomab-I-131, ustekinumab, or vedolizumab.

97. 55. A method for increasing or inducing a protective immune response in a subject, comprising administering an effective amount of a biodegradable particle according to any one of claims 1 to 54.

98. 1. A method for increasing or inducing a protective immune response in a subject, comprising administering to the subject an effective amount of biodegradable particles comprising one or more encapsulated fusion proteins, each of the one or more fusion proteins comprises two or more antigenic epitopes; the two or more antigenic epitopes are separated by a linker; the linker comprises an amino acid sequence susceptible to specific cleavage; The method, wherein the biodegradable particles have a negative zeta potential.

99. 99. The method of claim 98, wherein the effective amount of the biodegradable particles is administered to the subject orally, intravenously, sublingually, bucally, enterally, topically, rectally, subcutaneously, nasally, intraosseously (i.e., intraosseous injection), intraperitoneally, intrathecally, transdermally, or transmucosally.

100. 100. The method of claim 99, wherein the effective amount of the biodegradable particles is administered to the subject intravenously or subcutaneously.

101. 101. The method of claim 100, wherein the effective amount of the biodegradable particles is administered intravenously to the subject.

102. 101. The method of claim 100, wherein the effective amount of the biodegradable particles is administered subcutaneously to the subject.

103. 103. The method of any one of claims 97 to 102, wherein the effective amount of the biodegradable particles is administered to a subject to treat or prevent a disease or condition.

104. 104. The method of claim 103, wherein the disease or condition is cancer or an infectious disease.

105. 105. The method of claim 104, wherein the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, e.g., liposarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, leiomyosarcoma, chordoma, lymphangiosarcoma, lymphangioendothelial sarcoma, rhabdomyosarcoma, fibrosarcoma, myxosarcoma, chondrosarcoma, neuroendocrine tumors, mesothelioma, synovioma, schwannoma, meningioma, adenocarcinoma, melanoma, leukemia, lymphoid tumors.

106. The two or more antigen epitopes are CD19, CD20, BCMA, CD22, CLL1, CD33, CEA, CD123, CS1, EGFR, PSMA, EphA2, MCSP, ADAM17, PSCA, TPTE, HPU16, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride channel 2, and cyclin B, respectively. 1 , 9D7, Ep-CAM, EphA3, Her2 / neu, Te 106. The method of claim 105, comprising at least a portion of a protein selected from the group consisting of methicillinase, mesothelin, SAP-1, survivin, a BAGE family protein, a CAGE family protein, a GAGE ​​family protein, a MAGE family (e.g., MAGE-A3) protein, a SAGE family protein, an XAGE family protein, CT9, CT10, NY-ESO1 / LAG-1, PRAME, SSX-2, melan-A / MART-1, Cp100 / pmel17, tyrosinase, TRP-1 / TRP-2, P. polypeptide, MC1R, prostate-specific antigen, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII, and MUC1.

107. 105. The method of claim 104, wherein the infectious disease is a bacterial infection, a fungal infection, a parasitic infection, or a viral infection.

108. The viral infections include herpes virus infections, hepatitis virus infections, West Nile virus infections, flavivirus infections, influenza virus infections, rhinovirus infections, papillomavirus infections, paramyxovirus infections, and parainfluenza infections.

108. The method of claim 107, wherein the infection is selected from the group consisting of a viral infection, and / or a retroviral infection.

109. 108. The method of claim 107, wherein the viral infection is selected from the group consisting of a staphylococcal infection, a streptococcal infection, a mycobacterial infection, a bacillary infection, a salmonella infection, a vibrio infection, a spirochete infection, and a neisseria infection.

110. 1. A biodegradable particle comprising one or more encapsulated fusion proteins, Each of the one or more fusion proteins may comprise a fusion protein selected from the group consisting of MOG 1-20 , MBP 13-32 , MOG 35-55 , MBP 146-170 , P.L.P. 139-154 , MBP 111-129 , and MBP 83-99 and comprising two or more antigenic epitopes selected from the group consisting of: the two or more antigenic epitopes are separated by a linker; the linker comprises an amino acid sequence susceptible to specific cleavage; the biodegradable particles have a diameter of about 200 nm to 1000 nm; The biodegradable particles have a negative zeta potential of less than -30 mV.

111. 1. A method of treating multiple sclerosis in a subject, comprising administering to the subject an effective amount of biodegradable particles comprising one or more encapsulated fusion proteins, Each of the one or more fusion proteins may comprise a fusion protein selected from the group consisting of MOG 1-20 , MBP 13-32 , MOG 35-55 , MBP 146-170 , P.L.P. 139-154 , MBP 111-129 , and MBP 83-99 and comprising two or more antigenic epitopes selected from the group consisting of: the two or more antigenic epitopes are separated by a linker; the linker comprises an amino acid sequence susceptible to specific cleavage; the biodegradable particles have a diameter of about 200 nm to 1000 nm; The method, wherein the biodegradable particles have a negative zeta potential of less than -30 mV.

112. 112. The method of claim 111, wherein the effective amount of the biodegradable particles is administered to a subject orally, intravenously, sublingually, bucally, enterally, topically, rectally, subcutaneously, nasally, intraosseously (i.e., intraosseous injection), intraperitoneally, intrathecally, transdermally, or transmucosally.

113. 113. The method of claim 112, wherein the effective amount of the biodegradable particles is administered to the subject intravenously or subcutaneously.

114. 114. The method of claim 113, wherein the effective amount of the biodegradable particles is administered intravenously to the subject.

115. 114. The method of claim 113, wherein the effective amount of the biodegradable particles is administered subcutaneously to the subject.

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