Methods and compositions for enhancing CD4+ regulatory T cells

Abstract

Methods and related compositions are provided for administering immunosuppressants and therapeutic macromolecules to enhance CD4+ regulatory T cells. [Solution] Provided is a composition comprising (i) a synthetic nanocarrier attached to an immunosuppressant, and (ii) a synthetic nanocarrier attached to the immunosuppressant for use in a method comprising increasing the number or proportion of CD4+ regulatory T cells by administering the synthetic nanocarrier attached to the immunosuppressant to a subject, wherein the therapeutic polymer is not co-formulated with the synthetic nanocarrier attached to the immunosuppressant prior to administration.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 61 / 819,517, filed May 3, 2013; U.S. Provisional Application No. 61 / 881,851, filed September 24, 2013; U.S. Provisional Application No. 61 / 881,913, filed September 24, 2013; U.S. Provisional Application No. 61 / 881,921, filed September 24, 2013; U.S. Provisional Application No. 61 / 907,177, filed November 21, 2013; U.S. Provisional Application No. 61 / 948,313, filed March 5, 2014; and U.S. Provisional Application No. 61 / 948,384, filed March 5, 2014, under 35 U.S.C. § 119, the entire contents of each of which are incorporated herein by reference.

[0002] Field of the Invention The present invention relates to administering an immunosuppressive agent and a therapeutic polymer for enhancing CD4+ regulatory T cells, such as those specific for a therapeutic polymer. The methods and compositions provided herein enable the development of a tolerogenic immune response, particularly the production or development of CD4+ regulatory T cells. Accordingly, the methods and compositions provided can be used to generate a tolerogenic immune response in a subject in which administration of a therapeutic polymer can result in an unwanted immune response. The methods and compositions are preferably used for a subject who would benefit from enhancement of CD4+ regulatory T cells.

Background Art

[0003] Background of the Invention Therapeutic treatments, such as protein or enzyme replacement therapy, often result in an unwanted immune response to the specific therapeutic agent. Such unwanted immune responses can be reduced through the use of immunosuppressive drugs. However, conventional immunosuppressive drugs are broad acting. In addition, to maintain immunosuppression, immunosuppressive drug therapy is generally a lifelong proposition. Unfortunately, the use of broad acting immunosuppressive agents is associated with the risk of serious side effects such as tumors, infections, nephrotoxicity and metabolic disorders. Accordingly, new tolerogenic therapies would be beneficial.

Summary of the Invention

[0004] Summary of the present invention In one aspect, the present invention provides a method for increasing the number or proportion (or ratio) of CD4+ regulatory T cells (such as those specific to the therapeutic polymer) by administering a synthetic nanocarrier attached to an immunosuppressant and a therapeutic polymer, wherein the therapeutic polymer is not co-formulated with the synthetic nanocarrier attached to the immunosuppressant before administration. In one embodiment of any of the methods provided herein, an immunosuppressant and a synthetic nanocarrier attached to a therapeutic polymer are administered to a subject in combination. In any other embodiment of the methods provided herein, when the therapeutic polymer is not co-formulated with a synthetic nanocarrier prior to administration, the administration follows a protocol previously demonstrated to result in an increased number or proportion (or ratio) of CD4+ regulatory T cells. In any other embodiment of the methods provided herein, the method further includes determining the protocol.

[0005] In any other embodiment of the methods provided herein, the method further comprises assessing the number or proportion (or ratio) of CD4+ regulatory T cells in a subject before and / or after administration. In any other embodiment of the methods provided herein, the increased number or proportion (or ratio) of CD4+ regulatory T cells is at least a 2-fold, 3-fold, 4-fold, 5-fold, or 6-fold increase compared to the number or proportion of CD4+ regulatory T cells before administration. In any other embodiment of the methods provided herein, administration is by intravenous, intraperitoneal, or subcutaneous administration.

[0006] In any other embodiment of the methods provided herein, the method further includes recording the increase in the number or percentage (or ratio) of CD4+ regulatory T cells after administration. In any other aspect of any method provided herein, the immunosuppressant includes statins, mTOR inhibitors, TGF-β signaling agents, corticosteroids, mitochondrial function inhibitors, P38 inhibitors, NF-κβ inhibitors, adenosine receptor agonists, prostaglandin E2 agonists, phosphodiesterase 4 inhibitors, HDAC inhibitors, or proteasome inhibitors. In any other aspect of any method provided herein, the mTOR inhibitor is rapamycin.

[0007] In any other aspect of the methods provided herein, the therapeutic polymer is a therapeutic protein or a therapeutic polynucleotide. In any other aspect of the methods provided herein, the therapeutic protein is for protein supplementation in protein-fortified therapy. In any other aspect of the methods provided herein, the therapeutic protein includes intravenous or injectable therapeutic proteins, enzymes, enzyme cofactors, hormones, blood or blood coagulation factors, cytokines, interferons, growth factors, monoclonal antibodies, polyclonal antibodies, or proteins associated with Pompe disease. In any other aspect of the methods provided herein, the intravenous or injectable therapeutic protein includes tocilizumab, alpha-1 antitrypsin, hematide, albuinterferon alpha-2b, rucin, tesamorelin, ocrelizumab, belimumab, pegroticase, taliglucerase alpha, agalsidase alpha, or veraglucerase alpha. In any other embodiment of the methods provided herein, the enzyme comprises an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. In any other embodiment of the methods provided herein, the enzyme comprises an enzyme for enzyme replacement therapy for lysosomal storage disorders. In any other embodiment of the methods provided herein, the enzyme for replacement therapy for lysosomal storage disorders comprises imiglucerase, α-galactosidase A (α-galA), agalsidase beta, acid α-glucosidase (GAA), alglucosidase alpha, LUMIZYME, MYOZYME, arylsulfatase B, laronidase, ALDURAZYME, idursulfase, ELAPRASE, arylsulfatase B, pegroticase, pegsticase, or NAGLAZYME.

[0008] In another embodiment of any one of the methods provided herein, the cytokine includes a lymphokine, an interleukin, a chemokine, a type 1 cytokine, or a type 2 cytokine. In any other embodiment of the methods provided herein, the blood or blood coagulation factors include factor I, factor II, tissue factor, factor V, factor VII, factor VIII, factor IX, factor X, factor Xa, factor XII, factor XIII, von Willebrand factor, prekallikrein, high molecular weight kininogen, fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z-related protease inhibitor (ZPI), plasminogen, alpha-2-antiplasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI1), plasminogen activator inhibitor-2 (PAI2), cancer procoagulant, or epoetin alpha. In any other embodiment of the methods provided herein, the load of immunosuppressant attached to the synthetic nanocarrier is between 0.1% and 50% on average across the synthetic nanocarrier. In any other embodiment of the methods provided herein, the load is between 0.1% and 20%.

[0009] In any other embodiment of the methods provided herein, the synthetic nanocarrier comprises lipid nanoparticles, polymer nanoparticles, metal nanoparticles, surfactant emulsions, dendrimers, buckyballs, nanowires, virus-like particles, or peptide or protein particles. In any other embodiment of the methods provided herein, the synthetic nanocarrier comprises lipid nanoparticles. In any other embodiment of the methods provided herein, the synthetic nanocarrier comprises liposomes. In any other embodiment of the methods provided herein, the synthetic nanocarrier comprises metal nanoparticles. In any other embodiment of the methods provided herein, the metal nanoparticles comprise gold nanoparticles. In any other embodiment of the methods provided herein, the synthetic nanocarrier comprises polymer nanoparticles. In any other embodiment of the methods provided herein, the polymer nanoparticles comprise polymers that are non-methoxy-terminated pluronic polymers. In any other embodiment of the methods provided herein, the polymer nanoparticles comprise polyesters, polyesters attached to polyethers, polyamino acids, polycarbonates, polyacetals, polyketals, polysaccharides, polyethyl oxazolines, or polyethyleneimines. In any other embodiment of the methods provided herein, the polyester comprises poly(lactic acid), poly(glycolic acid), poly(lactic acid-co-glycolic acid), or polycaprolactone. In any other embodiment of the methods provided herein, the polymer nanoparticles comprises polyester and polyester attached to a polyether. In any other embodiment of the methods provided herein, the polyether comprises polyethylene glycol or polypropylene glycol.

[0010] In any other embodiment of the methods provided herein, the average particle size distribution obtained using dynamic light scattering of synthetic nanocarriers has a diameter greater than 100 nm. In any other embodiment of the methods provided herein, the diameter is greater than 150 nm. In any other embodiment of the methods provided herein, the diameter is greater than 200 nm. In any other embodiment of the methods provided herein, the diameter is greater than 250 nm. In any other embodiment of the methods provided herein, the diameter is greater than 300 nm. In any other embodiment of the methods provided herein, the aspect ratio of the synthetic nanosupport is greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or 1:10.

[0011] In another aspect, a method for manufacturing any one of the compositions or kits provided herein is provided. In one embodiment, the manufacturing method comprises generating a dose or dosage form of a therapeutic polymer and generating a dose or dosage form of an immunosuppressant. In another embodiment of any of the manufacturing methods provided, the step of generating a dose or dosage form of an immunosuppressant comprises attaching the immunosuppressant to a synthetic nanocarrier. In another embodiment of any of the manufacturing methods provided, the method further comprises combining the dose or dosage form of the immunosuppressant and the dose or dosage form of the therapeutic polymer in a kit. In another embodiment of any of the manufacturing methods provided herein, the therapeutic polymer is not formulated concurrently with the immunosuppressant.

[0012] In another aspect, the use of any one of the compositions or kits provided herein is provided for the manufacture of a pharmaceutical for increasing the number or proportion (or ratio) of CD4+ regulatory T cells, such as therapeutic polymer-specific CD4+ regulatory T cells, in a subject. In one embodiment, the composition or kit comprises an immunosuppressant and a therapeutic polymer that are not concurrently formulated. In another embodiment of any one of the uses provided herein, the immunosuppressant is attached to a synthetic nanocarrier. In another aspect, any one of the compositions provided herein is provided for use in any one of the methods provided herein. In one embodiment, the method comprises administering an immunosuppressant and a therapeutic polymer, wherein the therapeutic polymer is not co-formulated with the immunosuppressant prior to administration. In another embodiment, the immunosuppressant is attached to a synthetic nanocarrier. In yet another embodiment, the administration is a co-administration. In another aspect, a method is provided for producing a pharmaceutical product instead of enhancing the number or proportion (or ratio) of CD4+ regulatory T cells, such as therapeutic polymer-specific CD4+ regulatory T cells. In one embodiment, the pharmaceutical product comprises an immunosuppressant and a therapeutic polymer, which are not concurrently prescribed. In another embodiment of any one of the production methods provided herein, the immunosuppressant is attached to a synthetic nanocarrier. [Brief explanation of the drawing]

[0013] Simple explanation of the diagram [Figure 1] Figure 1 shows the percentage of CD4+ and CD25+Fox3p+ (regulatory T cells) assessed by flow cytometry after administration of the indicated treatment. [Figure 2] Figure 2 shows the reduction in antibody formation against polyethylene glycol (PEG) after administration of the indicated treatment. [Modes for carrying out the invention]

[0014] Detailed description of the present invention Before describing the present invention in detail, it should be understood that the invention is not limited to the materials or process parameters specifically exemplified, as these may naturally vary. It should also be understood that the terminology used herein is solely for describing specific aspects of the invention and is not intended to limit the use of alternative terminology for describing the invention. All publications, patents, and patent applications cited herein, either above or below, are incorporated herein by reference in their entirety for all purposes.

[0015] When used herein and in the accompanying claims, unless otherwise expressly specified, the singular forms "a," "an," and "the" encompass multiple references. For example, a reference to "a polymer" encompasses a mixture of two or more such molecules or a mixture of different molecular weights of a single polymer species; a reference to "synthetic nanocarrier" encompasses a mixture of two or more such synthetic nanocarriers or multiple such synthetic nanocarriers; a reference to "RNA molecule" encompasses a mixture of two or more such RNA molecules or multiple such RNA molecules; a reference to "immunosuppressant" encompasses two or more such materials or multiple such immunosuppressant molecules, etc. When used herein, the term “comprise,” or variations such as “comprises” or “comprising,” should be read as indicating the inclusion of any enumerated integer (e.g., feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g., multiple features, elements, characteristics, property, method / process step or limitation), and not as indicating the exclusion of any other integer or group of integers. Therefore, when used herein, the term “comprising” is inclusive and does not exclude additional, unenumerated integers or methods / process steps.

[0016] In one aspect of any one of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of" or "consisting of". The phrase "consisting essentially of" is used herein to require the specified integer(s) or steps, together with those that do not materially affect the nature or function of the claimed invention. As used herein, the term "consisting of" is used to indicate the presence of only the recited integer(s) (e.g., feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g., plurality of features, elements, characteristics, properties, method / process steps or limitations).

[0017] A. Introduction As described above, current conventional immunosuppressive agents are broad acting and generally result in a global systemic downregulation of the immune system. The methods and compositions provided herein enable more targeted immune effects and, in particular, surprisingly enable an increase in the production of CD4+ regulatory T cells such as therapeutic polymer-specific CD4+ regulatory T cells. It has been found that an increased number or proportion (or ratio) of therapeutic polymer-specific CD4+ regulatory T cells can be achieved by performing the described methods or by administering the compositions provided herein. Thus, such methods and compositions can result in a decrease in unwanted immune responses associated with the administration of therapeutic polymers and / or can be beneficial for subjects that require treatment with therapeutic polymers. The inventors have unexpectedly and surprisingly discovered that the above problems and limitations can be overcome by practicing the invention disclosed herein. The invention is illustrated by the following examples. Herein, the invention will be described in more detail below.

[0018] B. Definitions "Administering" or "administration" or "administer" means providing a material to a subject in a pharmacologically useful manner. The term is intended to encompass causing to be administered in some embodiments. "Causing to be administered" means causing, prompting, encouraging, assisting, inducing, or directing another party to administer the material. "Effective amount" means, in the context of a composition or dosage form for administration to a subject, an amount of the composition or dosage form that produces or enhances the development of one or more desired immune responses in the subject, such as a tolerogenic immune response, for example, the production or development of CD4+ regulatory T cells specific for a therapeutic polymer. Thus, in some embodiments, an effective amount is an amount of a composition provided herein that produces one or more desired immune responses, such as an increase in the number or percentage (or ratio) of CD4+ regulatory T cells. An effective amount can be for in vitro or in vivo purposes. For in vivo purposes, the amount can be what a clinician would consider to have clinical utility for a subject in whom an undesired immune response could occur as a result of administration of a therapeutic polymer.

[0019] An effective amount can be involved in reducing the level of an undesired immune response, but in some embodiments, it is involved in completely preventing an undesired immune response. An effective amount can also be involved in delaying the occurrence of an undesired immune response. An effective amount can also be an amount of a composition provided herein that results in an increase in the production or development of CD4+ regulatory T cells, such as CD4+ regulatory T cells specific for a therapeutic polymer. Specifically, the increase in production or development can be an increase in the number or percentage (or ratio) of such cells. An effective amount can also be an amount that results in a desired therapeutic endpoint or desired therapeutic result. An effective amount preferably results in a tolerogenic immune response in the subject to an antigen, such as a therapeutic polymer. Any of the above achievements can be monitored by routine methods.

[0020] In some embodiments of any one of the compositions and methods provided, the effective amount is an amount that causes a desired immune response to persist in a subject for at least one week, at least two weeks, or at least one month. In other embodiments of any one of the compositions and methods provided, the effective amount is an amount that causes a measurable desired immune response, such as a measurable reduction in the immune response (e.g., to a specific antigen), for at least one week, at least two weeks, or at least one month. The effective dose will naturally depend, within the knowledge and experience of the healthcare professional, on the specific subject being treated; the severity of the condition, disease, or disorder; individual patient parameters including age, physical condition, size, and weight; the duration of treatment; the characteristics of any concomitant therapies; and the specific route of administration and related factors. These factors are well known to those skilled in the art and can be addressed with routine experimental methods. Generally, it is preferable to use the maximum dose, i.e., the safest dose in accordance with sound medical judgment. However, it will be understood to those skilled in the art that patients may insist on a lower dose, or a tolerable dose, for medical, psychological, or virtually any other reason.

[0021] Generally, the dose of the immunosuppressant and / or therapeutic polymer in the composition of the present invention refers to the amount of the immunosuppressant and / or therapeutic polymer. Alternatively, the dose may be administered based on the number of synthetic nanocarriers that provide a desired amount of the immunosuppressant and / or antigen. "Antigen-specific" refers to an immune response that is either brought about by the presence of an antigen or a part thereof, or that generates molecules that specifically recognize or bind to an antigen. For example, if the immune response is antigen-specific antibody production, antibodies that specifically bind to the antigen are produced. Another example is the production of CD4+ regulatory T cells, which may be CD4+ regulatory T cells that bind to antigen-presenting cells (APCs) of therapeutic macromolecules capable of presenting an antigen when presented by an APC.

[0022] "Assessing the immune response" means measuring or determining the level, presence, absence, reduction, or increase of the immune response in vitro or in vivo. Such measurement or determination may be performed on one or more samples obtained from a subject. Such assessment may be performed by any method provided herein or otherwise known in the art. The assessment may assess the number or proportion of CD4+ regulatory T cells, such as those specific to therapeutic polymers, in a sample from a subject. "To adhere," "to be attached," "to link," or "to be linked" (etc.) means to chemically associate one entity (e.g., site) with another. In some embodiments, adhesion is covalent, meaning that adhesion occurs in the context of the presence of a covalent bond between the two entities. In non-covalent embodiments, non-covalent adhesion is mediated by non-covalent interactions, including but not limited to charge interactions, affinity interactions, metal coordination, physicoadsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions and / or combinations thereof. In some embodiments, encapsulation is a form of adhesion. In some embodiments, therapeutic polymers and immunosuppressants are not adhered to each other, meaning that the therapeutic polymer and immunosuppressant are not subjected to a process specifically intended to chemically associate one with the other. In one embodiment, the therapeutic polymer and / or immunosuppressant is not attached to the synthetic nanocarrier, meaning that the therapeutic polymer (and / or immunosuppressant) and the synthetic nanocarrier are not subjected to a process specifically intended to chemically associate one with the other.

[0023] When used herein, "mean" refers to the arithmetic mean unless otherwise noted. "Concurrent formulation" means that the materials shown are processed to produce a filled pharmaceutical dosage form in which the materials are in close physical contact or chemically attached by covalent or non-covalent bonds. In this specification, "not concurrently formulated" means that the materials shown (e.g., therapeutic polymers and immunosuppressants (or synthetic nanocarriers attached to immunosuppressants)) are not in close physical contact or chemically attached. In some embodiments, the therapeutic polymers and immunosuppressants (or synthetic nanocarriers attached to immunosuppressants) are not concurrently formulated prior to administration to the subject, as described herein. When "combination" is applied to two or more materials and / or agents (also referred to herein as components), it is intended to define the materials to which those two or more materials / agents are related. Components may be individually identified, for example, as the first component, the second component, the third component, etc. The terms "combined" and "combining" in this context shall be interpreted accordingly.

[0024] The relationship between two or more materials / agents in a combination may be physical or non-physical. Examples of physically related materials / agents include: • Compositions (e.g., unit formulations) containing two or more materials / agents in a mixture (e.g., within the same unit dose); • Compositions comprising two or more materials / agents that are chemically / physicochemically linked (e.g., crosslinking, molecular aggregation, or bonding to a common vehicle site); • Compositions comprising two or more materials / agents that are chemically / physicochemically co-packaged (e.g., arranged on or within lipid vesicles, particles (e.g., micro or nanoparticles), or emulsion droplets); • A pharmaceutical kit, pharmaceutical pack, or patient pack in which two or more materials / agents are co-packaged or co-presented (for example, as part of a series of unit doses).

[0025] Examples of materials / agents that are non-physically related and combined include: A material (e.g., a unit formulation) comprising at least one of two or more materials / agents, along with instructions for use for an improvised association of at least one compound / agent to form a physical association of the two or more materials / agents; • Materials (e.g., unit formulations) that include at least one of two or more materials / agents, along with instructions for use for combination therapy with those two or more materials / agents; • Materials comprising at least one of two or more materials / agents, along with instructions for use to administer to a patient population in which the other (one or more) of those two or more materials / agents have been administered (or are being administered); A material comprising at least one of two or more materials / agents in an amount or form specifically adapted for use in combination with the other (one or more) of those two or more materials / agents.

[0026] As used herein, the term “combination therapy” is intended to define a treatment involving the use of a combination of two or more materials / agents (as defined above). Thus, references in this application to “combination therapy,” “combination,” and the “combined” use of materials / agents may refer to materials / agents administered as part of the same overall treatment regimen. Consequently, the pharmacologics of each of the two or more materials / agents may differ, and each may be administered simultaneously or at different times. Thus, it will be seen that the materials / agents of a combination may be administered sequentially (e.g., before or after) or simultaneously, in the same pharmaceutical formulation (i.e., together) or in different pharmaceutical formulations (i.e., separately). Simultaneously in the same formulation means as a unified formulation, while simultaneously in different pharmaceutical formulations means non-unified. The pharmacologics of each of the two or more materials / agents in combination therapy may also differ with respect to the route of administration.

[0027] "Combined administration" means administering two or more materials / agents to a subject in a manner that is time-correlated, preferably time-sufficiently correlated to provide modulation in the immune response, and more preferably administered in combination with the other two or more materials / agents. In some embodiments, combined administration may encompass the administration of two or more materials / agents within a specified period, preferably within one month, more preferably within one week, even more preferably within one day, and even more preferably within one hour. In some embodiments, the materials / agents may be administered in combination repeatedly, i.e., in combination on more than one occasion, such as the combined administration provided in the examples.

[0028] "To determine" or "to decide" means to confirm the facts. Determining can be carried out in a number of ways, including but not limited to conducting experiments or making predictions. For example, the dose of an immunosuppressant or therapeutic polymer may be determined by starting with a test dose and using known scaling techniques (such as allometric or isometric scaling) to determine the dose for administration. Such methods may also be used to determine the protocols provided herein. In another embodiment, the dose may be determined by testing various doses in a subject, i.e., through direct experimentation based on experience and guide data. In some embodiment, "to determine" or "to decide" includes "to cause to determine." "To cause to determine" means to cause, urge, encourage, assist, guide or direct an entity to confirm the facts, or to act in cooperation with that entity, directly or indirectly, explicitly or implicitly.

[0029] “Dosage form” means a pharmacologically and / or immunologically active material in a medium, carrier, vehicle, or device suitable for administration to a subject. Any one of the compositions or doses provided herein may be in a dosage form. "Dosage" refers to the specific quantity of pharmacologically and / or immunologically active material to be administered to a subject over a given period of time. "Encapsulation" means sealing off at least a portion of a substance within a synthetic nanocarrier. In some embodiments, the substance is completely encapsulated within the synthetic nanocarrier. In other embodiments, most or all of the encapsulated substance is not exposed to the local environment outside the synthetic nanocarrier. In other embodiments, exposure to the local environment does not exceed 50%, 40%, 30%, 20%, 10%, or 5% (by weight / weight). Encapsulation is distinct from absorption (absorption places most or all of a substance on the surface of the synthetic nanocarrier, exposing the substance to the local environment outside the synthetic nanocarrier).

[0030] "Enhancing the number or proportion of CD4+ regulatory T cells" means increasing the number or proportion (or ratio) of such cells in a single or multiple subject (such as the total number of cell types, like the total number of T cells or CD4+ T cells), as determined by taking a sample from the subject and then analyzing the sample using an appropriate test method. In some embodiments, by carrying out the methods provided herein or by following the administration of the compositions described herein, the proportion of CD4+ regulatory T cells, such as those specific to the therapeutic polymer, increases by at least two, three, four, five, or six times.

[0031] CD4+ regulatory T cells can be characterized as CD4+CD25+Fox3p+ cells. The number or proportion of CD4+ regulatory T cells can be assessed by any method described herein or known in the art. For example, CD4+ regulatory T cells in the peripheral blood of a subject can be quantified by obtaining a peripheral blood sample from the subject and assessing gene expression, the presence of proteins, and / or the localization of one or more molecules associated with CD4+ regulatory T cells, including, without limitation, CD25, Foxp3, CCR4, CCR8, CCR5, CTLA4, CD134, CD39, and / or GITR. Any of the aforementioned molecules can be assessed by transcriptional analysis such as quantitative RT-PCR, Northern blotting, microarrays, fluorescence in situ hybridization, or RNA-seq. Proteins can be detected by Western blotting, immunofluorescence microscopy, flow cytometry, or ELISA. Cell surface molecules such as CD25, CCR4, CCR8, CCR5, CTLA4, CD134, CD39, and / or GITR can be evaluated by flow cytometry, cell surface staining, immunofluorescence microscopy, ELISA, etc.

[0032] In some embodiments, CD4+ regulatory T cells are detected based on an anergistic phenotype (e.g., lack of proliferation after TCR stimulation). In some embodiments, CD4+ regulatory T cells are identified based on resistance to activation-induced cell death or sensitivity to cytokine-deficiency-induced death. In some embodiments, CD4+ regulatory T cells can be identified based on the methylation status of the gene encoding Foxp3. For example, it has been found that a portion of the Foxp3 gene is demethylated in CD4+ regulatory T cells, which can be detected by DNA methylation analysis by PCR or other DNA-based methods. CD4+ regulatory T cells can be further identified or quantified based on the production of immunosuppressive cytokines, including IL-9, IL-10, or TGF-β. Therapeutic polymer-specific CD4+ regulatory T cells can be identified and quantified by any method known in the art, e.g., by stimulating cells ex vivo with antigen-presenting cells loaded with antigens derived from therapeutic polymers and assessing the activation of CD4+ regulatory T cells, or by evaluating the T cell receptors of CD4+ regulatory T cells. The number or proportion (or ratio) of therapeutic polymer-specific CD4+ regulatory T cells can be indirectly quantified by evaluating the function or activity of one or more activated CD4+ regulatory T cells after administration of the therapeutic polymer or its antigenic moiety.

[0033] "To cause" means to directly or indirectly trigger an action such as an immune response (e.g., a tolerance-induced immune response). "Identifying a target" is any action or set of actions that causes a clinician to recognize a target as one for which an effect can be obtained from the methods, compositions, or kits provided herein. Preferably, the identified target is one that requires a tolerogenic immune response provided herein, such as a target requiring increased production or development of CD4+ regulatory T cells, such as the production or development of therapeutic polymer-specific CD4+ regulatory T cells. The action or set of actions may be direct or indirect. In one embodiment of any of the methods provided herein, the method further includes identifying a target that requires the methods, compositions, or kits provided herein.

[0034] "Immunosuppressant" refers to a compound that imparts an immunosuppressive effect (e.g., a tolerance-induced effect) to an APC, or causes T cells or B cells to be suppressed. Immunosuppressive effect generally refers to the production or expression by an APC of cytokines or other factors that reduce, inhibit, or prevent an undesirable immune response, or promote a desirable immune response, such as a regulatory immune response (e.g., the production or development of CD4+ regulatory T cells). When an APC acquires immunosuppressive function (under the immunosuppressive effect) against immune cells that recognize the antigen presented by that APC, the immunosuppressive effect is said to be specific to the presented antigen. Without adhering to any specific theory, the immunosuppressive effect is thought to be a result of the delivery of the immunosuppressant to the APC, preferably in the presence of the antigen. In one embodiment, the immunosuppressant promotes a regulatory phenotype in one or more immune effector cells of an APC. For example, a regulatory phenotype may be characterized by inhibition of the production, induction, stimulation, or recruitment of antigen-specific CD4+ T cells or B cells, inhibition of the production of antigen-specific antibodies, or the production, induction, stimulation, or recruitment of Treg cells (e.g., CD4+CD25highFoxP3+Treg cells). This may result from the conversion of CD4+ T cells or B cells to a regulatory phenotype. This may also result from the induction of FoxP3 in other immune cells such as CD8+ T cells, macrophages, and iNKT cells. In one embodiment, the immunosuppressant affects the APC response after antigen treatment. In another embodiment, the immunosuppressant does not interfere with antigen treatment. In a further embodiment, the immunosuppressant is not an apoptosis signaling molecule. In yet another embodiment, the immunosuppressant is not a phospholipid.

[0035] Immunosuppressants include: statins; mTOR inhibitors such as rapamycin or rapamycin analogs; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase inhibitors such as trichostatin A; corticosteroids; mitochondrial function inhibitors such as rotenone; P38 inhibitors; NF-κβ inhibitors such as 6Bio, dexamethasone, TCPA-1, IKK VII; adenosine receptor agonists; prostaglandin E2 agonists (PGE2) such as misoprostol; phosphodiesterase inhibitors such as phosphodiesterase 4 inhibitors (PDE4) such as rolipram; proteasome inhibitors; kinase inhibitors; G protein-coupled receptor agonists; G protein-coupled receptor antagonists; glucocorticoids; retinoids; cytokine inhibitors; cytokine receptor inhibitors; cytokine receptor inhibitors. Examples include, but are not limited to, activators; peroxisome proliferator-activated receptor antagonists; peroxisome proliferator-activated receptor agonists; histone deacetylase inhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3KB inhibitors such as TGX-221; autophagy inhibitors such as 3-methyladenine; aryl hydrocarbon receptor inhibitors; proteasome inhibitor I (PSI); and oxidized ATP such as P2X receptor blockers. Other immunosuppressants include IDO, vitamin D3, cyclosporine such as cyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol, azathioprine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sangliferin A, salmeterol, mycophenolate mofetil (MMF), aspirin and other COX inhibitors, niflumic acid, estriol, and triptolide. In some embodiments, the immunosuppressant may comprise any of the agents provided herein.

[0036] The immunosuppressant may be a compound that directly provides an immunosuppressive effect to APC, or a compound that provides an immunosuppressive effect indirectly (i.e., after being treated in some way after administration). Therefore, the immunosuppressant encompasses any prodrug form of the compounds provided herein. In one embodiment of any one of the methods, compositions, or kits provided herein, the immunosuppressant provided herein is attached to a synthetic nanocarrier. In a preferred embodiment, the immunosuppressant is an element added to the material constituting the structure of the synthetic nanocarrier. For example, in one embodiment where the synthetic nanocarrier is composed of one or more polymers, the immunosuppressant is a compound added to and attached to one or more polymers. As another example, in one embodiment where the synthetic nanocarrier is composed of one or more lipids, the immunosuppressant is again a compound added to and attached to one or more lipids. In some embodiments, such as when the material of the synthetic nanocarrier also provides an immunosuppressive effect, the immunosuppressant is an element present in addition to the material of the synthetic nanocarrier that provides the immunosuppressive effect.

[0037] Other exemplary immunosuppressants include, but are not limited to, small molecule drugs, natural products, antibodies (e.g., antibodies against CD20, CD3, and CD4), biologic drugs, carbohydrate drugs, nanoparticles, liposomes, RNAi, antisense nucleic acids, aptamers, methotrexate, NSAIDs; fingolimod; natalizumab; alemtuzumab; anti-CD3; tacrolimus (FK506); and others. Further immunosuppressants are known to those skilled in the art, and the present invention is not limited in this respect. In one embodiment of any method, composition, or kit provided herein, the immunosuppressant is in a form such as a nanocrystalline form, thereby the form of the immunosuppressant itself being particles or particle-like. In some embodiments, such forms mimic viruses or other foreign pathogens. Many drugs have been nano-sized, and suitable methods for producing such drug forms will be known to those skilled in the art. Drug nanocrystals, such as nanocrystalline rapamycin, are known to those skilled in the art (Katteboinaa, et al. 2009, International Journal of PharmTech Research; Vol. 1, No. 3; pp682-694). As used herein, “drug nanocrystal” means a form of a drug (e.g., an immunosuppressant) that does not include a carrier or matrix material. In some embodiments, drug nanocrystals contain 90%, 95%, 98%, or 99%, or more, of the drug. Methods for generating drug nanocrystals include, but are not limited to, milling, high-pressure homogenization, precipitation, spray drying, supercritical solution rapid expansion (RESS), Nanoedge® technology (Baxter Healthcare), and Nanocrystal Technology® (Elan Corporation). In some embodiments, surfactants or stabilizers may be used for the steric or electrostatic stability of the drug nanocrystals. In some embodiments, nanocrystals or nanocrystalline forms of immunosuppressants may be used to increase the solubility, stability, and / or bioavailability of immunosuppressants, particularly insoluble or labile immunosuppressants. In some embodiments, co-administration of a nanocrystalline form of a therapeutic polymer with an immunosuppressant results in an increase in the number or proportion (or ratio) of CD4+ regulatory T cells, such as those specific to the therapeutic polymer.

[0038] The "loading amount" when attached to a synthetic nanocarrier is the amount (weight / weight) of immunosuppressants and / or therapeutic polymers attached to the synthetic nanocarrier, based on the total dry formulation weight of the material in the entire synthetic nanocarrier. Generally, such loading amount is calculated as an average across a population of synthetic nanocarriers. In one embodiment, the loading amount is between 0.1% and 99% on average across the synthetic nanocarriers. In another embodiment, the loading amount is between 0.1% and 50%. In yet another embodiment, the loading amount is between 0.1% and 20%. In yet another embodiment, the loading amount is between 0.1% and 10%. In yet another embodiment, the loading amount is between 1% and 10%. In yet another embodiment, the loading amount is between 7% and 20%. In yet another embodiment, the load is, on average across the population of synthetic nanocarriers, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In a further embodiment, the loading is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% on average across the population of synthetic nanocarriers. In some embodiments of the above, the loading is not greater than 25% on average across the population of synthetic nanocarriers.In one embodiment, the load capacity is calculated as may be described in the examples, or otherwise as is known in the art.

[0039] In some embodiments, when the form of the immunosuppressant is itself a particle or particle-like, such as a nanocrystalline immunosuppressant, the loading of the immunosuppressant is the amount of immunosuppressant in the particles (weight / weight). In such embodiments, the loading can reach 97%, 98%, 99%, or more. The "maximum dimension of the synthetic nanocarrier" refers to the longest dimension of the nanocarrier measured along any axis of the synthetic nanocarrier. The "minimum dimension of the synthetic nanocarrier" refers to the smallest dimension of the synthetic nanocarrier measured along any axis of the synthetic nanocarrier. For example, in a spheroidal synthetic nanocarrier, the maximum and minimum dimensions of the synthetic nanocarrier will be substantially the same and will be the size of its diameter. Similarly, in a cuboidal synthetic nanocarrier, the minimum dimension of the synthetic nanocarrier will be the smallest of its height, width, or length, while the maximum dimension of the synthetic nanocarrier will be the largest of its height, width, or length.

[0040] In one embodiment, based on the total number of synthetic nanocarriers in the sample, the minimum dimensions of at least 75%, preferably at least 80%, and more preferably at least 90% of the synthetic nanocarriers in the sample are 100 nm or larger. In one embodiment, based on the total number of synthetic nanocarriers in the sample, the maximum dimensions of at least 75%, preferably at least 80%, and more preferably at least 90% of the synthetic nanocarriers in the sample are 5 μm or smaller. Preferably, based on the total number of synthetic nanocarriers in the sample, the minimum dimensions of at least 75%, preferably at least 80%, and more preferably at least 90% of the synthetic nanocarriers in the sample are greater than 110 nm, more preferably greater than 120 nm, more preferably greater than 130 nm, and more preferably even greater than 150 nm. The aspect ratio between the maximum and minimum dimensions of the synthetic nanocarriers may vary depending on the embodiment. For example, the aspect ratio of the maximum dimension of the synthetic nanosupport to the minimum dimension can vary from 1:1 to 1,000,000:1, preferably 1:1 to 100,000:1, more preferably 1:1 to 10,000:1, more preferably 1:1 to 1000:1, even more preferably 1:1 to 100:1, and still more preferably 1:1 to 10:1. Preferably, based on the total number of synthetic nanosupports in the sample, the maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanosupports in the sample is 3 μm or less, more preferably 2 μm or less, more preferably 1 μm or less, more preferably 800 nm or less, more preferably 600 nm or less, and even more preferably 500 nm or less.

[0041] In a preferred embodiment, based on the total number of synthetic nanocarriers in the sample, the minimum dimensions of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample are 100 nm or larger, more preferably 120 nm or larger, more preferably 130 nm or larger, more preferably 140 nm or larger, and even more preferably 150 nm or larger. Measurement of the synthetic nanocarrier dimensions (e.g., effective dimensions) may, in some embodiments, be obtained by suspending the synthetic nanocarriers in a liquid (usually aqueous) medium and using dynamic light scattering (DLS) (e.g., using a Brookhaven ZetaPALS instrument). For example, a suspension of synthetic nanocarriers can be diluted from an aqueous buffer to pure water to achieve a final synthetic nanocarrier suspension concentration of approximately 0.01–0.1 mg / mL. The diluted suspension may be prepared directly in a cuvette suitable for DLS analysis, or transferred thereto. The cuvette is then placed in DLS, equilibrated to a controlled temperature, and then scanned for a sufficient amount of time to obtain a stable and reproducible distribution based on appropriate inputs regarding the viscosity of the medium and the refractive index of the sample. Next, the effective diameter or the average value of the distribution is reported. Determining the effective size of high-aspect-ratio or non-rotating elliptical synthetic nanosupports may require augmentative techniques such as electron microscopy to obtain more accurate measurements. The "dimensions," "size," or "diameter" of the synthetic nanosupports refer to the average value of the particle size distribution, obtained, for example, using dynamic light scattering.

[0042] "Non-methoxy-terminated polymer" means a polymer having at least one end portion terminated at a site other than methoxy. In some embodiments, the polymer has at least two end portions terminated at sites other than methoxy. In other embodiments, the polymer does not have end portions terminated at methoxy. "Non-methoxy-terminated pluronic polymer" means a polymer other than a linear pluronic polymer having methoxy at both ends. Polymer nanoparticles provided herein may include non-methoxy-terminated polymers or non-methoxy-terminated pluronic polymers. "Pharmacologically acceptable excipients" or "pharmaceutically acceptable carriers" means pharmacologically inert materials used together with pharmacologically active materials to formulate a composition. Pharmaceutically acceptable excipients include, but are not limited to, various materials known in the art, such as sugars (e.g., glucose, lactose, etc.), preservatives such as antimicrobial agents, reconstitution aids, colorants, salines (e.g., phosphate-buffered salines), and buffers.

[0043] A “protocol” refers to a pattern of administration to a subject and encompasses any of the administration regimens of one or more substances to a subject. A protocol consists of elements (or variables); therefore, a protocol contains one or more elements. Such elements of a protocol may include dosage, frequency of administration, route of administration, duration of administration, rate of administration, interval between administrations, any combination of the above, etc. In some embodiments, such a protocol may be used to administer one or more compositions of the present invention to one or more test subjects. The immune response in these test subjects may then be assessed to determine whether the protocol is effective in producing a desired or desired level of immune response or therapeutic effect. Any therapeutic and / or immune effect may also be assessed. One or more elements of a protocol may be transferred to a human protocol after being demonstrated in a test subject, such as a non-human subject. For example, dosages demonstrated in a non-human subject may be scaled as elements of a human protocol using established techniques such as allometric scaling or other scaling methods. Whether the protocol had the desired effect can be determined using either the methods provided herein or other methods known in the art. For example, a sample may be obtained from a subject to which the composition provided herein has been administered according to a specific protocol in order to determine whether specific immune cells, cytokines, antibodies, etc., were reduced, generated, activated, etc.

[0044] In a preferred embodiment, the number or proportion (or ratio) of CD4+ regulatory T cells, such as those specific to the therapeutic polymer, is determined. A typical protocol has been previously demonstrated to result in an increased number or proportion (or ratio) of CD4+ regulatory T cells (by comparing the number or proportion of CD4+ regulatory T cells before administration according to the protocol in one or more test subjects) when the therapeutic polymer is not co-prescribed with an immunosuppressant (an immunosuppressant not attached to a synthetic nanocarrier) prior to administration. Useful methods for detecting the presence and / or number of immune cells include, but are not limited to, flow cytometry (e.g., FACS), ELISpot, proliferation response, cytokine production, and immunohistochemistry. Antibodies and other conjugates for specific staining of immune cell markers are commercially available. Such kits typically include staining reagents for antigens that enable FACS-based detection, separation, and / or quantification of a desired cell population from a heterogeneous population of cells. In a certain embodiment, some of the compositions provided herein are administered to another subject using one or more, or all or substantially all, of the elements comprising the protocol. In some embodiments, protocols have been demonstrated to be specific to therapeutic polymers and to induce the development or production of CD4+ regulatory T cells with immunosuppressants such as the therapeutic polymers provided herein.

[0045] "Providing" means an action or set of actions taken by an individual to supply an article or set of articles or a method required to carry out the present invention. The action or set of actions may be taken directly or indirectly by the individual. "Providing a subject" means either an action or set of actions in which a clinician brings a subject into contact with the subject and administers the composition provided herein to the subject, or performs the method provided herein on the subject. Preferably, the subject is one that requires increased production or development of CD4+ regulatory T cells, such as antigen-specific immune tolerance or therapeutic polymer-specificity. The action or set of actions may be taken directly or indirectly by the subject itself. In one embodiment of any of the methods provided herein, the method further includes providing a subject.

[0046] "Recording" means that the methods or compositions provided herein directly or indirectly cause activity, either in written or electronic form, to be noted or expected to be noted, resulting in increased production or development of CD4+ regulatory T cells, such as those specific to the therapeutic polymer. In some embodiments, recording occurs when an immunosuppressant combined with the therapeutic polymer is administered to a subject in accordance with the methods provided herein or at some point thereafter. "Written form" as used herein means any record on a medium such as paper. "Electronic form" as used herein means any record on an electronic medium. "Subjects" refers to animals including warm-blooded animals such as humans and primates; birds; pets or livestock such as cats, dogs, sheep, goats, cows, horses, and pigs; laboratory animals such as mice, rats, and guinea pigs; fish; reptiles; zoo animals and wild animals; etc. "Synthetic nanocarrier(single or multiple)" means a discrete object not found in nature that has at least one dimension of 5 microns or less in size. Albumin nanoparticles are generally included as synthetic nanocarriers, but in certain embodiments, synthetic nanocarriers do not include albumin nanoparticles. In some embodiments, synthetic nanocarriers do not include chitosan. In other embodiments, synthetic nanocarriers are not lipid-based nanoparticles. In further embodiments, synthetic nanocarriers do not include phospholipids.

[0047] The synthetic nanocarrier may be, but is not limited to, a nanoparticle developed using a combination of nanomaterials such as one or more lipid nanoparticles (also referred herein as lipid nanoparticles, i.e., nanoparticles whose structural components are predominantly lipids), polymer nanoparticles, metal nanoparticles, surfactant emulsions, dendrimers, buckyballs, nanowires, virus-like particles (i.e., particles composed mainly of viral structural proteins but not infectious or having low infectiousness), peptide or protein-based particles (also referred herein as protein particles, i.e., particles whose structural components are predominantly peptides or proteins) (such as albumin nanoparticles) and / or lipid polymer nanoparticles. The synthetic nanocarrier may have a variety of different shapes, including but not limited to spheroidal, cubic, pyramidal, elliptical, cylindrical, donut-shaped, etc. The synthetic nanocarrier according to the present invention includes one or more surfaces.

[0048] Exemplary synthetic nanocarriers that can be adapted for use in the practice of the present invention include: (1) biodegradable nanoparticles disclosed in U.S. Patent No. 5,543,158 (Gref et al.), (2) polymer nanoparticles of published U.S. Patent Application No. 20060002852 (Saltzman et al.), (3) lithographically constructed nanoparticles of published U.S. Patent Application No. 20090028910 (DeSimone et al.), (4) disclosures of International Patent Publication No. 2009 / 051837 (von Andrian et al.), (5) nanoparticles disclosed in published U.S. Patent Application No. 2008 / 0145441 (Penades et al.), (6) protein nanoparticles disclosed in published U.S. Patent Application No. 20090226525 (de los Rios et al.), and (7) published U.S. Patent Application No. 20060222652 (Sebbel et al. (8) Virus-like particles disclosed in (1) al., (9) Virus-like particles with nucleic acid attachment disclosed in published U.S. Patent Application No. 20060251677 (Bachmann et al.), (10) Virus-like particles disclosed in International Publication No. 2010047839A1 or 2009106999A2, (11) Nanoprecipitated nanoparticles disclosed in P. Paolicelli et al., "Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles", Nanomedicine. 5(6): 843-853 (2010), (12) Apoptotic cells, apoptotic bodies, or synthetic or semi-synthetic mimics disclosed in U.S. Patent Publication No. 2002 / 0086049, or (13) Look et al., "Nanogel-based delivery of mycophenolic acid ameliorates systemic lupus This includes the findings disclosed in "erythematosus in mice," J. Clinical Investigation 123(4): 1741-1749 (2013).In one embodiment, synthetic nanosupports may have aspect ratios greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.

[0049] A synthetic nanocarrier according to the present invention having a minimum dimension of about 100 nm or less, preferably 100 nm or less, does not contain a surface having a complement-activating hydroxyl group, or alternatively contains a surface essentially composed of sites that are not complement-activating hydroxyl groups. In a preferred embodiment, a synthetic nanocarrier according to the present invention having a minimum dimension of about 100 nm or less, preferably 100 nm or less, does not contain a surface substantially activating complement, or alternatively contains a surface essentially composed of sites that do not substantially activate complement. In a more preferred embodiment, a synthetic nanocarrier according to the present invention having a minimum dimension of about 100 nm or less, preferably 100 nm or less, does not contain a surface activating complement, or alternatively contains a surface essentially composed of sites that do not activate complement. In some embodiments, the synthetic nanocarrier excludes virus-like particles. In some embodiments, the synthetic nanocarrier may have an aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.

[0050] A "therapeutic polymer" refers to any protein, carbohydrate, lipid, or nucleic acid that, when administered to a subject, may have a therapeutic effect. In some embodiments, administration of a therapeutic polymer to a subject may result in an undesirable immune response. As described herein, administration of a therapeutic polymer in combination with an immunosuppressant may improve the production or development of therapeutic polymer-specific CD4+ regulatory T cells, etc., and / or the therapeutic efficacy of the therapeutic polymer, by reducing the undesirable immune response to the immunosuppressant. In some embodiments, the therapeutic polymer may be a therapeutic polynucleotide or a therapeutic protein.

[0051] "Therapeutic polynucleotide" means any polynucleotide or polynucleotide-based treatment that, when administered to a subject, may have a therapeutic effect. Such treatments include gene silencing. Examples of such constructs for such treatments are known in the art and include, but are not limited to, naked RNA (including messenger RNA, modified messenger RNA, and RNAi forms). Other examples of therapeutic polynucleotides are listed separately herein. Therapeutic polynucleotides may be produced in, on, or by cells, or may be obtained using cell-free methods or entirely synthetic in vitro methods. Subjects therefore include any subject that requires treatment by any of the foregoing. Such subjects include subjects that would undergo any of the foregoing.

[0052] "Therapeutic protein" means any protein or protein-based treatment that, when administered to a subject, may have a therapeutic effect. Such treatments include protein replacement therapy and protein supplementation therapy. Such therapies also include the administration of exogenous or foreign proteins, antibody therapy, and cell or cell-based therapy. Examples of therapeutic proteins include, but are not limited to, enzymes, enzyme cofactors, hormones, blood coagulation factors, cytokines, growth factors, monoclonal antibodies, antibody-drug conjugates, and polyclonal antibodies. Other examples of therapeutic proteins are listed separately herein. Therapeutic proteins may be produced in, on, or by cells, obtained from, or administered in the form of such cells. In some embodiments, therapeutic proteins are produced in, on, or by mammalian cells, insect cells, yeast cells, bacterial cells, plant cells, transgenic animal cells, transgenic plant cells, etc. Therapeutic proteins may be produced by genetic recombination in such cells. Therapeutic proteins may be produced in, on, or by virally transformed cells. The subject therefore includes any subject that requires treatment by any of the aforementioned methods. Such subject includes any subject that would be subject to any of the aforementioned methods.

[0053] "APC-presentable antigens of therapeutic polymers" refers to antigens associated with therapeutic polymers (i.e., therapeutic polymers, or fragments thereof that can generate an immune response to therapeutic polymers (e.g., production of antibodies specific to the therapeutic polymer)). Generally, antigen-presenting cell (APC)-presentable antigens of therapeutic polymers can be presented for recognition by immune system cells (e.g., cells of the immune system such as antigen-presenting cells including, but not limited to, dendritic cells, B cells, or macrophages). APC-presentable antigens of therapeutic polymers can also be presented for recognition by T cells, for example. Such antigens can be recognized by T cells and trigger an immune response in T cells through the presentation of an antigen epitope bound to a class I or class II major histocompatibility complex (MHC) molecule. APC-presentable antigens of therapeutic polymers generally include proteins, polypeptides, peptides, polynucleotides, lipoproteins, or are contained in, on, or expressed in cells. The therapeutic polymer antigens, in some embodiments, comprise MHC class I-restricted epitopes and / or MHC class II-restricted epitopes and / or B-cell epitopes. Preferably, a tolerogenic immune response specific to the therapeutic polymer is brought about by the methods, compositions, or kits provided herein. In some embodiments, a population of synthetic nanocarriers does not contain APC-presentable antigens of the added therapeutic polymer, meaning that substantial amounts of APC-presentable antigens of the therapeutic polymer are not intentionally added to the synthetic nanocarriers during the manufacturing of the synthetic nanocarriers.

[0054] "Undesirable immune response" means any undesirable immune response that results from exposure to an antigen and promotes or exacerbates a disease, disorder or condition (or its signs) provided herein, or is a sign of such disease, disorder or condition provided herein. Such immune responses generally have an adverse effect on the health of the subject or are a sign of an adverse effect on the health of the subject. C. Composition Compositions for use in the administration of therapeutic polymers to enhance the production or development of CD4+ regulatory T cells, such as immunosuppressants and those specific to therapeutic polymers, as well as related methods and kits, are provided herein. Such compositions, kits, and methods are useful for subjects requiring therapeutic polymer therapy, such as those who would receive therapeutic polymer therapy. A wide variety of synthetic nanocarriers can be used in accordance with the present invention. In some embodiments, the synthetic nanocarriers are spherical or spheroidal. In some embodiments, the synthetic nanocarriers are flat or plate-shaped. In some embodiments, the synthetic nanocarriers are cubic or cubic in shape. In some embodiments, the synthetic nanocarriers are oval or oval. In some embodiments, the synthetic nanocarriers are cylindrical, conical, or pyramidal.

[0055] In some embodiments, it is desirable to use a population of synthetic nanocarriers that are relatively uniform in terms of size or shape so that each synthetic nanocarrier has similar properties. For example, based on the total number of synthetic nanocarriers, at least 80%, at least 90%, or at least 95% of the synthetic nanocarriers may have a minimum or maximum dimension that falls within 5%, 10%, or 20% of the average diameter or average dimensions of the synthetic nanocarriers. Synthetic nanocarriers may be solid or hollow and may comprise one or more layers. In some embodiments, each layer may have its own composition and properties relative to the other layers(s). For example, a synthetic nanocarrier may have a core / shell structure in which the core is one layer (e.g., a polymer core) and the shell is a second layer (e.g., a lipid bilayer or lipid monolayer). A synthetic nanocarrier may comprise multiple different layers.

[0056] In some embodiments, the synthetic nanocarrier may optionally contain one or more lipids. In some embodiments, the synthetic nanocarrier may contain liposomes. In some embodiments, the synthetic nanocarrier may contain a lipid bilayer. In some embodiments, the synthetic nanocarrier may contain a lipid monolayer. In some embodiments, the synthetic nanocarrier may contain micelles. In some embodiments, the synthetic nanocarrier may contain a core comprising a polymer matrix surrounded by lipid layers (e.g., lipid bilayer, lipid monolayer, etc.). In some embodiments, the synthetic nanocarrier may contain a nonpolymer core (e.g., metal particles, quantum dots, ceramic particles, bone particles, virus particles, proteins, nucleic acids, carbohydrates, etc.) surrounded by lipid layers (e.g., lipid bilayer, lipid monolayer, etc.). In other embodiments, the synthetic nanosupport may include metal particles, quantum dots, ceramic particles, etc. In some embodiments, the nonpolymer synthetic nanosupport is an aggregate of nonpolymer components, such as an aggregate of metal atoms (e.g., gold atoms).

[0057] In some embodiments, the synthetic nanosupport may optionally contain one or more amphiphilic entities. In some embodiments, the amphiphilic entities may facilitate the creation of synthetic nanosupports with increased stability, improved uniformity, or increased viscosity. In some embodiments, the amphiphilic entities may be associated with the inner surface of lipid membranes (e.g., lipid bilayers, lipid monolayers, etc.). Many amphiphilic entities known in the art are suitable for use in creating synthetic nanosupports according to the present invention. Such amphiphilic entities include phosphoglycerides; phosphatidylcholine; dipalmitoylphosphatidylcholine (DPPC); dioleylphosphatidylethanolamine (DOPE); dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine; cholesterol; cholesterol esters; diacylglycerol; diacylglycerol succinate; diphosphatidylglycerol (DPPG); hexanedecanol; aliphatic alcohols such as polyethylene glycol (PEG); and polyoxyethylene-9 - Lauryl ether; surface-active fatty acids such as palmitic acid or oleic acid; fatty acids; fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides; sorbitan trioleate (Span(registered trademark) 85) glycocollate; sorbitan monolaurate (Span(registered trademark) 20); polysorbate 20 (Tween(registered trademark) 20); polysorbate 60 (Tween(registered trademark) 60); polysorbate 65 (Tween(registered trademark) 65); polysorbate 80 (Tween(registered trademark) 80); polysorbate 85 (Tween(registered trademark) 85);

[0058] Polyoxyethylene monostearate; surfactin; poloxomer; sorbitan fatty acid esters such as sorbitan trioleate; lecithin; lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin; phosphatidylethanolamine (kephalin); cardiolipin; phosphatidic acid; cerebroside; dicetyl phosphate; dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine; hexadecylamine; acetyl palmitate; glycerol ricinoleate; hexadecyl stearate; isopropyl myristate; tyroxapole; poly(ethylene glycol) 5000-phosphatidylethanolamine; poly(ethylene glycol) 400-monostearate; phospholipids; synthetic and / or natural detergents with high surfactant activity; deoxycholates; cyclodextrins; chaotropic salts; ion pairing agents; and combinations thereof. The amphiphilic entity components may be a mixture of different amphiphilic entities. Those skilled in the art will recognize that this is a non-exclusive, exemplary list of substances having surfactant activity. Any of the amphiphilic entities may be used to produce synthetic nanocarriers used in accordance with the present invention.

[0059] In some embodiments, the synthetic nanocarrier may optionally contain one or more carbohydrates. The carbohydrates may be natural or synthetic. The carbohydrates may be derived natural carbohydrates. In certain embodiments, the carbohydrates include, but are not limited to, monosaccharides or disaccharides such as glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cerbiose, mannose, xylose, arabinose, gluconic acid, galactonic acid, mannuronic acid, glucosamine, galatosamine, and neuraminic acid. In certain embodiments, carbohydrates are polysaccharides, including but not limited to pullulan, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen, hydroxyethyl starch, carrageenan, glycone, amylose, chitosan, N,O-carboxymethyl chitosan, algin and alginic acid, starch, chitin, inulin, konjac, glucomannan, pustulan, heparin, hyaluronic acid, curdlan and xanthan gum. In certain embodiments, the synthetic nanocarrier does not contain (or is specifically excluded from) carbohydrates such as polysaccharides. In certain embodiments, carbohydrates may include carbohydrate derivatives such as sugar alcohols, including but not limited to mannitol, sorbitol, xylitol, erythritol, maltitol and lactitol.

[0060] In some embodiments, the synthetic nanocarrier may comprise one or more polymers. In some embodiments, the synthetic nanocarrier comprises one or more polymers that are non-methoxy-terminated pluronic polymers. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (by weight / weight) of the polymers constituting the synthetic nanocarrier are non-methoxy-terminated pluronic polymers. In some embodiments, all of the polymers constituting the synthetic nanocarrier are non-methoxy-terminated pluronic polymers. In some embodiments, the synthetic nanocarrier comprises one or more polymers that are non-methoxy-terminated polymers. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (by weight / weight) of the polymer constituting the synthetic nanocarrier is a non-methoxy-terminated polymer. In some embodiments, all of the polymers constituting the synthetic nanocarrier are non-methoxy-terminated polymers. In some embodiments, the synthetic nanocarrier comprises one or more polymers that do not contain a pluronic polymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (by weight / weight) of the polymer constituting the synthetic nanocarrier is free of pluronic polymers. In some embodiments, all of the polymer constituting the synthetic nanocarrier is free of pluronic polymers. In some embodiments, such polymers may be surrounded by a coating layer (e.g., liposomes, lipid monolayers, micelles, etc.). In some embodiments, various elements of the synthetic nanocarrier may be attached to the polymer.

[0061] Immunosuppressants can be attached to synthetic nanocarriers by one of many methods. Generally, attachment may result from binding between the immunosuppressant and the synthetic nanocarrier. This binding may result in the immunosuppressant being attached to the surface of the synthetic nanocarrier and / or contained within (encapsulated) the synthetic nanocarrier. However, in some embodiments, the immunosuppressant is encapsulated by the synthetic nanocarrier not as a result of binding to the synthetic nanocarrier, but rather as a result of the structure of the synthetic nanocarrier. In a preferred embodiment, the synthetic nanocarrier comprises a polymer provided herein, and the immunosuppressant is attached to the polymer.

[0062] When adhesion occurs as a result of binding between an immunosuppressant and a synthetic nanocarrier, the adhesion may occur via linking sites. These linking sites may be any site through which the immunosuppressant and / or therapeutic polymer are bound to the synthetic nanocarrier. Such sites may include covalent bonds, such as amide or ester bonds, and separate molecules that bind the immunosuppressant to the synthetic nanocarrier (covalently or noncovalently). Such molecules may include linkers, polymers, or units thereof. For example, a linking site may include a charged polymer to which the immunosuppressant is electrostatically bound. Alternatively, a linking site may include a polymer or units thereof to which it is covalently bound. In a preferred embodiment, the synthetic nanosupport comprises a polymer provided herein. These synthetic nanosupports may be entirely polymeric or a mixture of the polymer and other materials.

[0063] In some embodiments, polymers of synthetic nanocarriers are related to each other to form a polymer matrix. In some of these embodiments, components such as immunosuppressants may be covalently related to one or more polymers of the polymer matrix. In some embodiments, the covalent relationship is mediated by a linker. In some embodiments, components may be noncovalently related to one or more polymers of the polymer matrix. For example, in some embodiments, components may be encapsulated within the polymer matrix, thereby surrounded, and / or dispersed throughout it. Alternatively or additionally, components may be related to one or more polymers of the polymer matrix by hydrophobic interactions, charge interactions, van der Waals forces, etc. A wide variety of polymers and methods for forming polymer matrices are conventionally known.

[0064] The polymer may be a natural or non-natural (synthetic) polymer. The polymer may be a homopolymer or a copolymer containing two or more monomers. In terms of arrangement, the copolymer may be random, blocky, or a combination of random and blocky arrangements. Typically, the polymer according to the present invention is an organic polymer. In some embodiments, the polymer comprises polyester, polycarbonate, polyamide, or polyether, or units thereof. In other embodiments, the polymer comprises poly(ethylene glycol) (PEG), polypropylene glycol, poly(lactic acid), poly(glycolic acid), poly(lactic acid-co-glycolic acid), or polycaprolactone, or units thereof. In some embodiments, the polymer is preferably biodegradable. Therefore, in these embodiments, if the polymer comprises polyether such as poly(ethylene glycol) or polypropylene glycol or units thereof, the polymer is preferably biodegradable, comprising a block copolymer of polyether and a biodegradable polymer. In other embodiments, the polymer does not comprise polyether or units thereof, such as poly(ethylene glycol) or polypropylene glycol or units thereof, by itself.

[0065] Other examples of polymers suitable for use in the present invention include, but are not limited to, polyethylene, polycarbonate (e.g., poly(1,3-dioxan-2one)), polyacid anhydride (e.g., poly(sebacic anhydride)), polypropyl fumarate, polyamide (e.g., polycaprolactam), polyacetal, polyether, polyester (e.g., polylactide, polyglycolide, polylactide-co-glycolide, polycaprolactone, polyhydroxy acid (e.g., poly(β-hydroxyalkanoate))), poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polyurea, polystyrene and polyamine, polylysine, polylysine-PEG copolymer and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymer. In some embodiments, polymers according to the present invention include, but are not limited to, polyesters (e.g., polylactic acid, poly(lactic acid-co-glycolic acid), polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2one)); polyacid anhydrides (e.g., poly(sebacic acid anhydride)); polyethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; and polycyanoacrylates, and polymers approved for human use by the U.S. Food and Drug Administration (FDA) under 21 C. FR §177.2600.

[0066] In some embodiments, the polymer may be hydrophilic. For example, the polymer may contain anionic groups (e.g., phosphate groups, sulfate groups, carboxylate groups); cationic groups (e.g., quaternary amine groups); or polar groups (e.g., hydroxyl groups, thiol groups, amine groups). In some embodiments, a synthetic nanosupport containing a hydrophilic polymer matrix generates a hydrophilic environment within the synthetic nanosupport. In some embodiments, the polymer may be hydrophobic. In some embodiments, a synthetic nanosupport containing a hydrophobic polymer matrix generates a hydrophobic environment within the synthetic nanosupport. The choice of hydrophilic or hydrophobic polymer may affect the properties of the material incorporated (e.g., attached) within the synthetic nanosupport.

[0067] In some embodiments, the polymer may be modified with one or more sites and / or functional groups. Various sites or functional groups may be used in accordance with the present invention. In some embodiments, the polymer may be modified with polyethylene glycol (PEG), carbohydrates, and / or acyclic polyacetals derived from polysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Specific embodiments may be made using the general teachings of U.S. Patent No. 5,543,158 (Gref et al.) or International Publication No. 2009 / 051837 (Von Andrian et al.). In some embodiments, the polymer may be modified with lipid or fatty acid groups. In some embodiments, the fatty acid groups may be one or more of butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidonic acid, behenic acid, or lignoceric acid. In some embodiments, the fatty acid groups may be one or more of palmitoleic acid, oleic acid, vaccenic acid, linoleic acid, alpha-linoleic acid, gamma-linoleic acid, arachidonic acid, gadoleic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, or erucic acid.

[0068] In some embodiments, the polymer may be a polyester comprising copolymers containing lactic acid and glycolic acid units, such as poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide), collectively referred herein as "PLGA"; homopolymers containing glycolic acid units, collectively referred herein as "PGA"; and homopolymers containing lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred herein as "PLA". In some embodiments, exemplary polyesters include, for example, polyhydroxy acids; PEG copolymers and copolymers of lactide and glycolide (e.g., PLA-PEG copolymer, PGA-PEG copolymer, PLGA-PEG copolymer, and derivatives thereof). In some embodiments, polyesters include, for example, poly(caprolactone), poly(caprolactone)-PEG copolymer, poly(L-lactide-co-L-lysine), poly(serine ester), poly(4-hydroxy-L-proline ester), poly[α-(4-aminobutyl)-L-glycolic acid] and derivatives thereof.

[0069] In some embodiments, the polymer is PLGA. PLGA is a biocompatible and biodegradable copolymer of lactic acid and glycolic acid, and various forms of PLGA are characterized by the lactic acid:glycolic acid ratio. The lactic acid may be L-lactic acid, D-lactic acid, or D,L-lactic acid. The degradation rate of PLGA can be adjusted by changing the lactic acid:glycolic acid ratio. In some embodiments, the PLGA used according to the present invention is characterized by lactic acid:glycolic acid ratios of approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, or approximately 15:85.

[0070] In some embodiments, the polymer may be one or more acrylic polymers. In certain embodiments, the acrylic polymer includes, for example, copolymers of acrylic acid and methacrylic acid, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), alkyl methacrylate amide copolymers, poly(methyl methacrylate), poly(methacrylic anhydride), methyl methacrylate, polymethacrylate, poly(methyl methacrylate) copolymers, polyacrylamide, aminoalkyl methacrylate copolymers, glycidyl methacrylate copolymers, polycyanoacrylate, and combinations of one or more of the polymers described above. The acrylic polymer may include well-polymerized copolymers of acrylic and methacrylate esters with a low quaternary ammonium group content.

[0071] In some embodiments, the polymer may be a cationic polymer. Generally, cationic polymers have the ability to enrich and / or protect negatively charged strands of nucleic acids. Poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; Kabanov et al., 1995, Bioconjugate Chem., 6:7), poly(ethyleneimine) (PEI; Boussif et al., 1995, Proc. Natl. Acad. Sci., USA, 1995, 92:7297) and poly(amidoamine) dendrimers (Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al., 1996, Bioconjugate Chem., 7:703; Haensler et al., 1993, Bioconjugate Chem., Amine-containing polymers, such as those described in 4:372, are positively charged at physiological pH and form ion pairs with nucleic acids. In some embodiments, the synthetic nanosupport may not contain (or may exclude) cationic polymers.

[0072] In some embodiments, the polymer may be a biodegradable polyester having cationic side chains (Putnam et al., 1999, Macromolecules, 32:3658; Barrera et al., 1993, J, Am. Chem. Soc., 115:11010; Kwon et al., 1989, Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633; Zhou et al., 1990, Macromolecules, 23:3399). Examples of these polyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J. Am. Chem. Soc., 115: 11010), poly(serine ester) (Zhou et al., 1990, Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam et al., 1999, Macromolecules, 32:3658; Lim et al., 1999, J. Am. Chem. Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam et al., 1999, Macromolecules, 32:3658; Lim et al., 1999, J. Am. Chem. Soc., 121:5633).

[0073] The properties of these and other polymers and their preparation methods are well known in the art (e.g., U.S. Patent Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al., 2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc., 123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J. Control. Release, 62:7; Uhrich et al., 1999, Chem. Rev., 99:3181). More generally, various methods for synthesizing specific preferred polymers are described in Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles of Polymerization by Odian, John Wiley & Sons, Fourth Edition, 2004; Contemporary Polymer Chemistry by Allcock et al., Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and U.S. Patents No. 6,506,577, No. 6,632,922, No. 6,686,446, and No. 6,818,732.

[0074] In some embodiments, the polymer may be linear or branched polymer. In some embodiments, the polymer may be a dendrimer. In some embodiments, the polymer may be substantially crosslinked with each other. In some embodiments, the polymer may not be substantially crosslinked. In some embodiments, the polymer may be used in accordance with the invention without undergoing a crosslinking step. It should be further understood that the synthetic nanocarrier may include block copolymers, graft copolymers, or blends, mixtures, and / or adducts of any of the aforementioned and other polymers. Those skilled in the art will recognize that the polymers listed herein represent a non-exclusive, exemplary list of polymers that may be used in accordance with the invention.

[0075] In some embodiments, the synthetic nanosupports do not contain polymer components. In some embodiments, the synthetic nanosupports may include metal particles, quantum dots, ceramic particles, etc. In some embodiments, the non-polymer synthetic nanosupports are aggregates of non-polymer components, such as aggregates of metal atoms (e.g., gold atoms). Compositions according to the present invention may contain elements such as immunosuppressants in combination with pharmaceutically acceptable excipients such as preservatives, buffers, and saline or phosphate-buffered saline. Compositions may be prepared using conventional pharmaceutical manufacturing and compounding techniques to arrive at useful dosage forms. In one embodiment, a composition such as one containing an immunosuppressant is suspended in a sterile saline solution for injection together with a preservative. In one embodiment, when preparing synthetic nanosupports as carriers, a method of attaching components to the synthetic nanosupports may be useful. If the components are small molecules, it may be advantageous to attach them to a polymer before assembling the synthetic nanosupports. In one embodiment, it may also be advantageous to prepare synthetic nanosupports having surface groups that are used to attach the components to the synthetic nanosupports through the use of their surface groups, rather than attaching the components to a polymer and then using this polymer composite to construct the synthetic nanosupports.

[0076] In certain embodiments, the attachment may be a covalent linker. In one embodiment, an immunosuppressant according to the present invention may be covalently attached to an external surface via a 1,2,3-triazole linker formed by a 1,3-dipole cyclic addition reaction between an immunosuppressant containing an azide group on the surface of a nanocarrier and an alkyne group, or by a 1,3-dipole cyclic addition reaction between an immunosuppressant containing an alkyne on the surface of a nanocarrier and an azide group. Such a cyclic addition reaction is preferably carried out in the presence of a Cu(I) catalyst, together with a suitable Cu(I) ligand and a reducing agent for reducing a Cu(II) compound to a catalytically active Cu(I) compound. This Cu(I)-catalyzed azide-alkyne cyclic addition (CuAAC) is also sometimes referred to as a click reaction.

[0077] In addition, covalent linkages may include covalent linkers such as amide linkers, disulfide linkers, thioether linkers, hydrazone linkers, hydrazide linkers, imines or oxime linkers, urea or thiourea linkers, amidine linkers, amine linkers, and sulfonamide linkers. Amidolinkers are formed via amide bonds between an amine on one component, such as an immunosuppressant, and a carboxylic acid group on a second component, such as a nanosupport. The amide bonds in the linker can be created using either conventional amide bond formation reactions with suitably protected amino acids and activated carboxylic acids (such as N-hydroxysuccinimide activated esters).

[0078] Disulfide linkers can be created, for example, through the formation of disulfide (SS) bonds between two sulfur atoms in the form of R1-SS-R2. Disulfide bonds can be formed by thiol exchange between a component containing a thiol / mercaptan group (-SH) and another activated thiol group on a polymer or nanosupport, or between a nanosupport containing a thiol / mercaptan group and a component containing an activated thiol group.

[0079] Triazole linkers, specifically in the following forms: [ka] 1,2,3-triazoles (wherein R1 and R2 may be either chemical entity) are produced by a 1,3-dipole cyclic addition reaction of an azide attached to a first component, such as a nanocarrier, with a terminal alkyne attached to a second component, such as an immunosuppressant. The 1,3-dipole cyclic addition reaction is carried out with or without a catalyst, preferably using a Cu(I) catalyst, thereby linking the two components through 1,2,3-triazole functionality. This chemistry is described in detail by Sharpless et al., Angew. Chem. Int. Ed. 41(14), 2596, (2002) and Meldal, et al, Chem. Rev., 2008, 108(8), 2952-3015, and is often referred to as the "click" reaction or CuAAC.

[0080] In one embodiment, a polymer containing azide or alkyne groups at the ends of polymer chains is prepared. This polymer is then used to prepare a synthetic nanosupport such that multiple alkyne or azide groups are located on the surface of the nanosupport. Alternatively, the synthetic nanosupport may be prepared by another route and subsequently functionalized with alkyne or azide groups. The components are prepared in the presence of either alkyne (if the polymer contains azide) or azide (if the polymer contains alkyne) groups. The components are then reacted with the nanosupport via a 1,3-dipole cyclic addition reaction with or without a catalyst, thereby covalently attaching the components to the particles through a 1,4-disubstituted 1,2,3-triazole linker.

[0081] Thioether linkers are made, for example, by the formation of sulfur-carbon (thioether) bonds in the form of R1-S-R2. Thioethers are made by alkylation of a thiol / mercaptan (-SH) group on one component with an alkylating group such as a halide or epoxide on a second component. Thioether linkers can also be formed by Michael addition of a thiol / mercaptan group on one component to an electron-deficient alkene group on a second component containing a maleimide group or a vinyl sulfone group as a Michael acceptor. In another method, thioether linkers can be prepared by a radical thiol-ene reaction between a thiol / mercaptan group on one component and an alkene group on a second component.

[0082] Hydrazone linkers are produced by the reaction of a hydrazide group on one component with an aldehyde / ketone group on a second component. Hydrazide linkers are formed by the reaction of a hydrazine group on one component with a carboxylic acid group on a second component. Such reactions generally involve chemistry similar to that used for amide bond formation when a carboxylic acid is activated by an activating reagent. Imines or oxime linkers are formed by the reaction of an amine or N-alkoxyamine (or aminooxy) group on one component with an aldehyde or ketone group on a second component.

[0083] Urea or thiourea linker is prepared by the reaction of an amine group on one component with an isocyanate or thioisocyanate group on a second component. Amidine linkers are prepared by the reaction of an amine group on one component with an imide ester group on a second component. Amine linkers are produced by an alkylation reaction between an amine group on one component and an alkylating group, such as a halide, epoxide, or sulfonate ester group, on a second component. Alternatively, amine linkers may also be produced by reductive amination of an amine group on one component and an aldehyde or ketone group on a second component using a suitable reducing reagent, such as sodium cyanoborohydride or sodium triacetoxyborohydride.

[0084] A sulfamide linker may be produced by the reaction of an amine group on one component with a sulfonyl halide (sulfonyl chloride, etc.) group on a second component. Sulfone linkers are created by Michael addition of a nucleophile to a vinyl sulfone. Either the vinyl sulfone or the nucleophile may be on the surface of the nanosupport or attached to a component. The components may also be compounded onto the nanosupport via non-covalent compounding methods. For example, a negatively charged immunosuppressant may be compounded onto a positively charged nanosupport via electrostatic adsorption. Components containing metal ligands may also be compounded onto a metal complex-containing nanosupport via metal-ligand complexes.

[0085] In one embodiment, the components may be attached to a polymer, such as polylactic acid-block polyethylene glycol, before the assembly of the synthetic nanocarrier, or the synthetic nanocarrier may be formed so that it has reactive or activatable groups on its surface. In the latter case, the components may be prepared to have groups compatible with the adhesion chemistry presented by the surface of the synthetic nanocarrier. In another embodiment, the peptide components may be attached to the VLP or liposome using a suitable linker. The linker is a compound or reagent capable of linking two molecules together. In one embodiment, the linker may be a homobifunctional or heterobifunctional reagent as described in Hermanson 2008. For example, a VLP or liposome synthetic nanocarrier containing carboxyl groups on its surface may be treated with a homobifunctional linker, dihydrazide adipic acid (ADH), in the presence of EDC to form a corresponding synthetic nanocarrier having an ADH linker. The resulting ADH-linked synthetic nanocarrier is then complexed with an acid-containing peptide component via the other end of the ADH linker on the nanocarrier to generate the corresponding VLP or liposomal peptide complex.

[0086] For a detailed description of available compounding methods, see Hermanson GT, "Bioconjugate Techniques," 2nd Edition, Published by Academic Press, Inc., 2008. In addition to covalent adhesion, the components may be attached by adsorption to a pre-formed synthetic nanosupport or by encapsulation during synthetic nanosupport formation.

[0087] Any immunosuppressant provided herein may be used in the provided method or composition and, in some embodiments, may be attached to a synthetic nanocarrier. Examples of immunosuppressants include: statins; mTOR inhibitors such as rapamycin or rapamycin analogs; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase (HDAC) inhibitors; corticosteroids; mitochondrial function inhibitors such as rotenone; P38 inhibitors; NF-κβ inhibitors; adenosine receptor agonists; prostaglandin E2 agonists; phosphodiesterase inhibitors such as phosphodiesterase 4 inhibitor; proteasome inhibitors. Immunosuppressants include, but are not limited to, kinase inhibitors, G protein-coupled receptor agonists, G protein-coupled receptor antagonists, glucocorticoids, retinoids, cytokine inhibitors, cytokine receptor inhibitors, cytokine receptor activators, peroxisome proliferator-activated receptor antagonists, peroxisome proliferator-activated receptor agonists, histone deacetylase inhibitors, calcineurin inhibitors, phosphatase inhibitors, and oxidized ATP. Other immunosuppressants include, but are not limited to, IDO, vitamin D3, cyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol, azathioprine, 6-mercaptopurine, aspirin, niflumic acid, estriol, tripolide, interleukins (e.g., IL-1, IL-10), cyclosporine A, cytokines, or cytokine receptor-targeting siRNAs.

[0088] Examples of statins include atorvastatin (LIPITOR®, TORVAST®), cerivastatin, fluvastatin (LESCOL®, LESCOL®XL), lovastatin (MEVACOR®, ALTOCOR®, ALTOPREV®), mevastatin (COMPACTIN®), pitavastatin (LIVALO®, PIAVA®), rosuvastatin (PRAVACHOL®, SELEKTINE®, LIPOSTAT®), rosuvastatin (CRESTOR®), and simvastatin (ZOCOR®, LIPEX®). Examples of mTOR inhibitors include rapamycin and its analogues (e.g., CCL-779, RAD001, AP23573, C20-methallylrapamycin (C20-Marap), C16-(S)-butylsulfonamiderapamycin (C16-BSrap), C16-(S)-3-methylindolerapamycin (C16-iRap)) (Bayle et al., Chemistry & Biology 2006). Examples include 13:99-107), AZD8055, BEZ235 (NVP-BEZ235), chrysophanic acid (chrysophanol), defololimus (MK-8669), everolimus (RAD0001), KU-0063794, PI-103, PP242, temsirolimus, and WYE-354 (available from Selleck, Houston, TX, USA).

[0089] Examples of TGF-β signaling agents include TGF-β ligands (e.g., activin A, GDF1, GDF11, bone morphogenetic protein, nodal, TGF-β) and their receptors (e.g., ACVR1B, ACVR1C, ACVR2A, ACVR2B, BMPR2, BMPR1A, BMPR1B, TGFβRI, TGFβRII), R-SMADS / co-SMADS (e.g., SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD8) and ligand inhibitors (e.g., follistatin, noggin, colzine, DAN, lefty, LTBP1, THBS1, decorin). Examples of mitochondrial function inhibitors include atractyroside (dipotassium salt), bongukrecic acid (triammonium salt), carbonyl cyanide m-chlorophenylhydrazone, caloboxy atractyroside (e.g., from Atractylis), CGP-37157, (-)-deguerin (e.g., from Mundulea sericea), F16, hexokinase II VDAC-binding domain peptide, oligomycin, rotenone, Ru360, SFK1, and valinomycin (e.g., from Streptomuces fulvissimus) (EMD4 Biosciences, USA).

[0090] Examples of P38 inhibitors include SB-203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole), SB-239063 (trans-1-(4-hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxypyrimidine-4-yl)imidazole), SB-220025 (5-(2-amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole), and ARRY-797. Examples of NF (e.g., NK-κβ) inhibitors include IFRD1, 2-(1,8-naphthyridine-2-yl)-phenol, 5-aminosalicylic acid, BAY11-7082, BAY11-7085, CAPE (phenethyl caffeate ester), diethyl maleate, IKK-2 inhibitor IV, IMD0354, lactacystin, MG-132[Z-Leu-Leu-Leu-CHO], and NFκB activating inhibitor III. Examples include NF-κB activating inhibitor II, JSH-23, parthenolide, phenylarsine oxide (PAO), PPM-18, pyrrolidinedithiocarbamate ammonium salt, QNZ, RO106-9920, locaglamide, locaglamide AL, locaglamide C, locaglamide I, locaglamide J, locaglamol, (R)-MG-132, sodium salicylate, triptolide (PG490), and wederolactone.

[0091] Examples of adenosine receptor agonists include CGS-21680 and ATL-146e. Examples of prostaglandin E2 agonists include E-prostanoid 2 and E-prostanoid 4. Examples of phosphodiesterase inhibitors (non-selective and selective inhibitors) include caffeine, aminophylline, IBMX (3-isobutyl-1-methylxanthine), paraxanthine, pentoxifylline, theobromine, theophylline, methylated xanthine, vinpocetine, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), anagrelide, enoximon (PERFAN®), milrinone, levosimendon, and mesembrine. Examples include ibudilast, picramiralast, luteolin, dorotaberine, roflumilast (DAXAS®, DALIRESP®), sildenafil (REVATION®, VIAGRA®), tadalafil (ADCIRCA®, CIALIS®), vardenafil (LEVITRA®, STAXYN®), udenafil, avanafil, icariin, 4-methylpiperazine, and pyrazolopyrimidine-7-1.

[0092] Examples of proteasome inhibitors include bortezomib, disulfiram, epigallocatechin-3-gallate, and salinosporamide A. Examples of kinase inhibitors include bevacizumab, BIBW2992, cetuximab (ERBITUX®), imatinib (GLEEVEC®), trastuzumab (HERCEPTIN®), gefitinib (IRESSA®), ranibizumab (LUCENTIS®), pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, panitumumab, vandetanib, E7080, pazopanib, and mblitinib.

[0093] Examples of glucocorticoids include hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate (DOCA), and aldosterone. Examples of retinoids include retinol, retinal, tretinoin (retinoic acid, RETIN-A®), isotretinoin (ACCUTANE®, AMNESTEEM®, CLARAVIS®, SOTRET®), alitretinoin (PANRETIN®), etretinate (TEGISON®) and its metabolite acitretin (SORIATANE®), tazarotene (TAZORAC®, AVAGE®, ZORAC®), bexarotene (TARGRETIN®), and adapalene (DIFFERIN®).

[0094] Examples of cytokine inhibitors include IL1ra, IL1 receptor antagonists, IGFBP, TNF-BF, uromodulin, alpha-2-macroglobulin, cyclosporine A, pentamidine, and pentoxifylline (PENTOPAK®, PENTOXIL®, TRENTAL®). Examples of peroxisome proliferator-activated receptor antagonists include GW9662, PPARγ antagonist III, G335, and T0070907 (EMD4 Biosciences, USA). Examples of peroxisome proliferator-activated receptor agonists include pioglitazone, ciglitazone, clofibrate, GW1929, GW7647, L-165,041, LY171883, PPARγ activator, Fmoc-Leu, troglitazone, and WY-14643 (EMD4 Biosciences, USA).

[0095] Examples of histone deacetylase inhibitors include hydroxamic acids (or hydroxamates) such as trichostatin A, cyclic tetrapeptides (e.g., trapoxin B), and depsipeptides; benzamides; electrophilic ketones; aliphatic acid compounds such as phenyl butyrate and valproic acid; hydroxamic acids such as vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat (LBH589); benzamides such as entinostat (MS-275), CI994, and mosetinostat (MGCD0103); nicotinamides; NAD derivatives; dihydrocoumarins; naphthopyranes, and 2-hydroxynaphaldehyde.

[0096] Examples of calcineurin inhibitors include cyclosporine, pimecrolimus, voclosporine, and tacrolimus. Examples of phosphatase inhibitors include BN82002 hydrochloride, CP-91149, kallikrin A, cantharidinic acid, cantharidin, cypermethrin, ethyl-3,4-dehostatin, fostriesin sodium salt, MAZ51, methyl-3,4-dehostatin, NSC95397, norcantharidin, ammonium okadaate salt from prorocentrum concavum, okadaic acid, potassium okadaate salt, sodium okadaate salt, phenylarsine oxide, various phosphatase inhibitor mixtures, protein phosphatase 1C, protein phosphatase 2A inhibitor protein, protein phosphatase 2A1, protein phosphatase 2A2, and sodium orthovanadate.

[0097] In some embodiments of any one of the methods, compositions, or kits provided herein, the therapeutic polymers described herein are also attached to synthetic nanocarriers. In other embodiments, the therapeutic polymers are not attached to any synthetic nanocarriers. In some embodiments of any of these embodiments, the therapeutic polymers may be delivered in the form of the therapeutic polymers themselves or in the form of fragments or derivatives thereof. Therapeutic polymers may include therapeutic proteins or therapeutic polynucleotides. Therapeutic proteins include, but are not limited to, intravenously administered therapeutic proteins, enzymes, enzyme cofactors, hormones, blood coagulation factors, cytokines and interferons, growth factors, monoclonal and polyclonal antibodies (e.g., those administered to the subject as replacement therapy), and proteins associated with Pompe disease (e.g., acid glucosidase alfa, rhGAA (e.g., myozyme and lumizyme (Genzyme))). Therapeutic proteins also include proteins involved in the blood coagulation cascade. Examples of therapeutic proteins include, but are not limited to, factor VIII, factor VII, factor IX, factor V, von Willebrand factor, von Herdebrand factor, tissue plasminogen activator, insulin, growth hormone, erythropoietin alfa, VEGF, thrombopoietin, lysozyme, and antithrombin. Therapeutic proteins also include adipokines such as leptin and adiponectin. Other examples of therapeutic proteins are listed below and separately herein.

[0098] Examples of therapeutic proteins used in enzyme replacement therapy for patients with lysosomal storage disorders include, but are not limited to, imiglucerase (e.g., CEREZYME®) for the treatment of Gaucher disease, α-galactosidase A (α-galA) (e.g., agalsidase beta, FABRYZYME®) for the treatment of Fabry disease, acid α-glucosidase (GAA) (e.g., acid glucosidase alpha, LUMIZYME®, MYOZYME®) for the treatment of Pompe disease, arylsulfatase B (e.g., laronidase, ALDURAZYME®, idursulfase, ELAPRASE®, arylsulfatase B, NAGLAZYME®), pegroticase (KRYSTEXXA), and pegsticase for the treatment of mucopolysaccharidosis.

[0099] Examples of enzymes include oxidoreductase, transferase, hydrolase, lyase, isomerase, asparaginase, uricase, glycosidase, asparaginase, uricase, protease, nuclease, collagenase, hyaluronidase, heparinase, heparanase, lysine, and ligase. The therapeutic protein may also contain enzymes, toxins, or other proteins or peptides isolated or derived from any bacterial, fungal, or viral source.

[0100] Examples of hormones include melatonin (N-acetyl-5-methoxytryptoamine), serotonin, thyroxine (or tetraiodothyronine) (thyroid hormone), triiodothyronine (thyroid hormone), epinephrine (or adrenaline), norepinephrine (or noradrenaline), dopamine (or prolactin inhibitor hormone), anti-Müllerian hormone (or Müllerian inhibitor or hormone), adiponectin, and adrenocorticotropic hormone (or corticotropic hormone). Tropins), angiotensinogen and angiotensin, antidiuretic hormone (or vasopressin, arginine vasopressin), atrial natriuretic peptide (or atriopeptin), calcitonin, cholecystokinin, corticotropin-releasing hormone, erythropoietin, follicle-stimulating hormone, gastrin, ghrelin, glucagon, glucagon-like peptide (GLP-1), GIP, gonadotropin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, Examples include human placental lactogens, growth hormone, inhibin, insulin, insulin-like growth factor (or somatomedin), leptin, luteinizing hormone, melanocyte-stimulating hormone, orexin, oxytocin, parathyroid hormone, prolactin, relaxin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone (or thyrotropin), thyrotropin-releasing hormone, cortisol, aldosterone, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, estradiol, estrone, estriol, progesterone, calcitriol (1,25-dihydroxyvitamin D3), calcidiol (25-hydroxyvitamin D3), prostaglandins, leukotrienes, prostacyclins, thromboxanes, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptides, renin, and enkephalins.

[0101] Examples of blood or blood coagulation factors include factor I (fibrinogen), factor II (prothrombin), tissue factor, factor V (proaccelerin, instability factor), factor VII (stabilization factor or proconvertin), factor VIII (antihemophilic globulin), factor IX (Christmas factor or plasma thromboplastin component), factor X (Stuart-Prower factor), factor Xa, factor XI, factor XII (Hagemann factor), factor XIII (fibrin stabilizing factor), von Willebrand factor, prekallikrein (Fletcher factor), and high molecular weight kininogen (HMW). Examples include Fitzgerald factor (K), fibronectin, fibrin, thrombin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z-related protease inhibitors (ZPIs), plasminogen, alpha-2-antiplasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI1), plasminogen activator inhibitor-2 (PAI2), cancer procoagulant or epoetin alpha (Epogen, Procrit). Examples of cytokines include type 1 cytokines such as lymphokines, interleukins and chemokines, and IFN-γ, as well as type 2 cytokines such as TGF-β and IL-4, and IL-10 and IL-13.

[0102] Examples of growth factors include adremedullin (AM), angiopoietin (Ang), autologous motility stimulant, bone morphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth and differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), liver cancer-derived growth factor (HDGF), insulin-like growth factor (IGF), These include migration-stimulating factors, myostatin (GDF-8), nerve growth factor (NGF) and other neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), Wnt signaling pathway, placental growth factor (P1GF), fetal bovine somatotropin (FBS), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-7.

[0103] Examples of monoclonal antibodies include avagovomab, absiximab, adalimumab, adecatumumab, aferimomab, aftuzumab, alacizumab pegol, ALD, alemtuzumab, altumomab pentetate, and anatumomab mafenatox. mafenatox), anlukinzumab, antithymocyte globulin, apolizumab, alsitumomab, aselizumab, atorizumab (tocilizumab), atorimumab, bapineozumab, basiliximab, bavituximab, bectumomab, belimumab, benralizumab, vertilimumab, becylesomab, bevacizumab, bisilomab, vibatuzumab meltansine Mertansine), blinatumomab, brentuximab vedotin, briakinumab, canakinumab, cantuzumab meltansine, capromab pendetide, catumakisomab, sedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox, sixtumumab, clenoliximab, cribatuzumab tetraxetan, conatumumab, dasetuzumab, dacrizumab, daratumumab, denosumab, detumomab, dorlimomab aritox aritox), dorlixizumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab, enrimomab pegol, epitumomab cituxetan, epratuzumab, erulizumab, ertumaxomab, etalacizumab, exbivirumab,

[0104] Fanolesomab, Faralimomab, Farletuzumab, Felvizumab, Fezakinumab, Figitumumab, Fontrizumab, Foravirumab, Fresolimumab, Galiximab, Gantenerumab, Gambirimomab, Gemtumab Ozogamicin, GC1008, Gilentuximab, Glembatumumab Vedotin Vedotin, golimumab, gomiliximab, ibalizumab, ibritumomab, tiuxetan, igobomab, imusilomab, infliximab, intetumumab, inorimomab, inotuzumab, ozogamicin, ipilimumab, iratumumab, keriximab, rabetsumab, lebrikizumab, remalesomab, reldelimumab, lexatumumab, ribivirumab, lintuzumab, lobotuzumab, meltansine (Lorvotuzumab) Mertansine), Lucatumumab, Lumiliximab, Mapatumumab, Maslimomab, Matuzumab, Mepolizumab, Meterimumab, Miratuzumab, Minretumomab, Mitsumomab, Morolimmab, Motabizumab, Muromonab-CD3, Nacolomab tafenatox, Naptumomab estafenatox, Natalizumab, Nevacumab, Necitumumab, Nerelimomab, Nimotuzumab, Nofetumomab merpentan Merpentan), ocrelizumab, odulimomab, ofatumumab, olalatumab, omalizumab, opportuzumab monatox, olegobomab, otelixizumab, pagibaximab, palivizumab, panitumumab, panobacumab, pascolizumab, pemtumomab, pertuzumab, pexerizumab,

[0105] Pentumomab, Priliximab, Pritumumab, Rafivirumab, Ramucirumab, Ranibizumab, Laxibakumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Robatumumab, Lontalizumab, Roberizumab, Luprizumab, Satumomab pendetide pendetide), sevilumab, sibrotuzumab, cifarimumab, siltuximab, ciprizumab, solanezumab, sonepcizumab, sontuzumab, stamrumab, thresomab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefivazumab, telimomab aritox Examples include aritox, tenatumomab, teneriximab, teprizumab, tisilimumbab (tremelimumab), tigatuzumab, tocilizumab (atlizumab), tralizumab, tocitumomab, trastuzumab, tremelimumab, tucotzumab cermoloukin, tuvirumab, urtoxazumab, ustekinumab, bapaliximab, vedolizumab, bertuzumab, bepalimomab, bicilizumab, borosiximab, botumumab, zaltumumab, zanolimumab, ziralimumab, and zolimomab aritox. Monoclonal antibodies further include anti-TNF-α antibodies.

[0106] Examples of intravenous or injectable therapeutic proteins include, for example, tocilizumab (Roche / Actemra®), alpha-1 antitrypsin (Kamada / AAT), hematide® (Affymax and Takeda, synthetic peptide), albumin interferon alpha-2b (Novartis / Zalbin®), lucin® (Pharming Group, C1 inhibitor replacement therapy), tesamorelin (Theratechnologies / Egrifta, synthetic growth hormone-releasing factor), ocrelizumab (Genentech, Roche and Biogen), belimbab (GlaxoSmithKline / Benlysta®), and pegroticase (Savient). Examples include Pharmaceuticals / Krystexxa(trademark), pegcicase, taliglucerase alpha (Protalix / Uplyso), agalsidase alpha (Shire / Replagal(registered trademark)), bellaglucerase alpha (Shire), and keyhole limpet hemocyanin (KLH). Additional therapeutic proteins include, for example, genetically modified proteins such as Fc fusion proteins, bispecific antibodies, multispecific antibodies, nanobodies, antigen-binding proteins, antibody fragments, and protein complexes such as antibody-drug conjugates.

[0107] Examples of therapeutic polynucleotides include, but are not limited to, nucleic acid aptamers such as pegaptanib (Makgen, a pegylated anti-VEGF aptamer), antisense therapeutics such as antisense polynucleotides or oligonucleotides (e.g., the antiviral drugs fomivirsen or mipomersen, antisense therapeutics that target messenger RNA for apolipoprotein B to reduce cholesterol levels); small interfering RNAs (siRNAs) (e.g., Dicer substrate siRNA molecules (DsiRNAs), which are 25-30 base pair asymmetric double-stranded RNAs that mediate RNAi with extremely high potency); or modified messenger RNAs (mmRNAs) such as those disclosed in U.S. Patent Application No. 2013 / 0115272 (Fougerolles et al.) and the published U.S. Patent Application No. 2012 / 0251618 (Schrum et al.). Additional therapeutic polymers useful in accordance with aspects of the present invention will be obvious to those skilled in the art, and the present invention is not limited in this respect.

[0108] In some embodiments, components such as therapeutic polymers or immunosuppressants may be isolated. “Isolated” means an element that has been separated from its original environment and is present in a quantity sufficient to allow its identification or use. This means, for example, that the element can be (i) selectively produced by expression cloning or (ii) purified by chromatography or electrophoresis. The isolated element may be substantially pure, but does not need to be. Since the isolated element may be miscible with pharmaceutically acceptable excipients in the pharmaceutical preparation, the element may be included in the preparation in a very small weight percentage. The element is still isolated in the sense that it has been separated from substances that may be related in a biological system, i.e., isolated from other lipids or proteins. Any of the elements provided herein may be isolated and incorporated into a composition, or used in a method in an isolated form.

[0109] D. Methods for preparing and using compositions, and related methods. One aspect of the present invention relates to determining a protocol for the administration method provided herein. The protocol may be determined by changing the frequency, dose, and other aspects of the administration of the therapeutic polymer and immunosuppressant, and subsequently assessing, based on such changes, the number or proportion (or ratio) of CD4+ regulatory T cells, such as therapeutic polymer-specific ones, and / or any desirable or undesirable immune response. A preferred protocol for carrying out the present invention is to increase the number or proportion (or ratio) of CD4+ regulatory T cells, such as therapeutic polymer-specific CD4+ regulatory T cells.

[0110] Synthetic nanosupports can be prepared using a variety of methods known in the art. For example, synthetic nanosupports can be formed by methods such as nanoprecipitation, flow focusing using fluid channels, spray drying, single and double emulsion solvent distillation, solvent extraction, phase separation, milling, microemulsion procedures, microfabrication, nanofabrication, sacrificial layers, simple and composite coacervation, and other methods well known to those skilled in the art. Alternatively or additionally, aqueous and organic solvent synthesis for monodisperse semiconductors or conductive, magnetic, organic and other nanomaterials has been described (Pellegrino et al., 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; Trindade et al., 2001, Chem. Mat., 13:3843). Additional methods are described in the literature (see, for example, Doubrow, Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy," CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5: 13; Mathiowitz et al., 1987, Reactive Polymers, 6:275; Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755; U.S. Patents 5578325 and 6007845; P. Paolicelli et al., "Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles" Nanomedicine. 5(6): 843-853 (2010)).

[0111] As appropriate, see C. Astete et al., "Synthesis and characterization of PLGA nanoparticles" J. Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K. Avgoustakis "Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide) Nanoparticles: Preparation, Properties and Possible Applications in Drug Delivery" Current Drug Delivery 1: 321-333 (2004); C. Reis et al., "Nanoencapsulation I. Methods for preparation of drug-loaded polymeric nanoparticles" Nanomedicine 2:8-21 (2006); P. Paolicelli et al., "Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles" Nanomedicine. Various methods may be used to encapsulate various materials in synthetic nanosupports, including, but not limited to, 5(6):843-853 (2010). Other methods may be used, including, but not limited to, the method disclosed in U.S. Patent No. 6,632,671 (Unger, issued October 14, 2003), which is suitable for encapsulating materials in synthetic nanosupports.

[0112] In certain embodiments, synthetic nanosupports are prepared by a nanoprecipitation process or spray drying. The conditions used for preparing the synthetic nanosupports may be varied to produce particles of a desired size or properties (e.g., hydrophobicity, hydrophilicity, external morphology, "adhesion", shape, etc.). The method of preparing the synthetic nanosupports and the conditions used (e.g., solvent, temperature, concentration, airflow rate, etc.) may depend on the composition of the material and / or polymer matrix to which the synthetic nanosupports are to be attached. If the synthetic nanosupports prepared by any of the above methods have a size range that falls outside the desired range, the synthetic nanosupports may be separated into particles, for example, using a sieve.

[0113] The elements (i.e., components) of the synthetic nanosupport may be attached to the entire surface of the synthetic nanosupport by, for example, one or more covalent bonds, or by using one or more linkers. Additional methods for functionalizing the synthetic nanosupport may be adapted from published U.S. Patent Application No. 2006 / 0002852 (Saltzman et al.), published U.S. Patent Application No. 2009 / 0028910 (DeSimone et al.), or published International Patent Application No. WO / 2008 / 127532Al (Murthy et al.). Alternatively or additionally, synthetic nanocarriers may be attached to components directly or indirectly via non-covalent interactions. In the non-covalent embodiment, non-covalent attachment is mediated by non-covalent interactions including, but not limited to, charge interactions, affinity interactions, metal coordination, physicoadsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions and / or combinations thereof. Such attachment may be arranged on the external or internal surface of the synthetic nanocarrier. In some embodiments, encapsulation and / or absorption are forms of attachment. In some embodiments, the synthetic nanocarrier may be combined with the antigen by mixing in the same vehicle or delivery system.

[0114] The compositions provided herein include inorganic or organic buffers (e.g., phosphoric acid, sodium or potassium salts of carboxylic acids, acetic acid or citrate) and pH adjusters (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citric acid or acetic acid, amino acids and their salts), antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10 nonylphenol, sodium deoxycholate), and solubilating and / or cryopreserving / freezing / lyophilization stabilizers (cryo / lyo). It may also contain stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic pressure regulators (e.g., salts or sugars), antimicrobial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity modifiers (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose), and cosolvents (e.g., glycerol, polyethylene glycol, ethanol).

[0115] Compositions according to the present invention may contain pharmaceutically acceptable excipients. Compositions can be prepared using conventional pharmaceutical manufacturing and compounding techniques to obtain useful dosage forms. Suitable techniques for use in the practice of the present invention can be found in Handbook of Industrial Mixing: Science and Practice, Edited by Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004 John Wiley & Sons, Inc.; and Pharmaceutics: The Science of Dosage Form Design, 2nd Ed. Edited by ME Auten, 2001 Churchill Livingstone. In one embodiment, the composition is suspended in sterile saline solution for injection together with a preservative. It should be understood that the compositions of the present invention may be prepared in any preferred manner, and the present invention is not limited in any way to compositions that can be produced using the methods described herein. The selection of an appropriate manufacturing method may require attention to the properties of the specific parts involved.

[0116] In some embodiments, the composition is manufactured under sterile conditions or ultimately sterilized. This can ensure that the resulting composition is sterile and non-infectious, thus improving safety compared to non-sterile compositions. This provides a valuable safety measure, in particular, when the recipient of the composition is immunodeficient, suffers from an infection, and / or is susceptible to infection. In some embodiments, the composition may be lyophilized and stored for extended periods without loss of activity, depending on the formulation strategy, in a suspension or as a lyophilized powder.

[0117] Administration according to the present invention may be by various routes, including but not limited to subcutaneous, intravenous, intraperitoneal, intramuscular, transmucosal, transdermal, transcutaneous, or intradermal routes. In a preferred embodiment, administration is via a subcutaneous route. The compositions referenced herein can be manufactured and prepared using conventional methods for administration, and in some embodiments for concomitant administration. The compositions of the present invention may be administered in effective amounts, such as the effective amounts specified herein. The dosage of a dosage form may contain varying amounts of immunosuppressants and / or therapeutic polymers according to the present invention. The amount of immunosuppressants and / or therapeutic polymers present in a dosage form may be varied according to the properties of the therapeutic polymers and / or immunosuppressants, the therapeutic effect to be performed, and other such parameters. In some embodiments, dose range studies may be conducted to establish the optimal therapeutic amount of immunosuppressants and / or therapeutic polymers to be present in a dosage form. In some embodiments, the immunosuppressants and / or therapeutic polymers are present in the dosage form in an amount effective to induce a tolerogenic immune response to the therapeutic polymers when administered to a subject. In preferred embodiments, the immunosuppressants and / or therapeutic polymers are present in the dosage form in an amount effective to enhance the production or development of CD4+ regulatory T cells, such as when co-administered to a subject. It may be possible to determine the amount of immunosuppressants and / or therapeutic polymers effective to induce a desired immune response using conventional dose range studies and techniques in subjects. Dosage forms may be administered at various frequencies.

[0118] In some embodiments, the administration of an immunosuppressant, such as one attached to a synthetic nanocarrier, with a therapeutic polymer is performed, for example, before any further administration of the therapeutic polymer. Another aspect of this disclosure relates to kits. In some embodiments, a kit comprises an immunosuppressant and a therapeutic polymer, which in some embodiments are attached to a synthetic nanocarrier. The immunosuppressant and the therapeutic polymer may be contained in separate containers in the kit. In some embodiments, the container is a vial or ampoule. In some embodiments, the therapeutic polymer or immunosuppressant may be contained in a solution separate from the container so that the therapeutic polymer or immunosuppressant is added to the container at a later time. In a preferred embodiment, the therapeutic polymer is not co-formulated with the immunosuppressant before administration. In some embodiments, the therapeutic polymer or immunosuppressant may be in a lyophilized form in separate containers so that they are reconstituted at a later time. In some embodiments, the kit further includes instructions for use for reconstitution, mixing, administration, etc. In some embodiments, the instructions for use encompass a description of the methods described herein. The instructions for use may be in any preferred form, such as a printed insert or label. In some embodiments, the kit further includes one or more syringes or other means for administering the immunosuppressant and the therapeutic polymer.

[0119] example Example 1: Polymer nanocarrier containing polymer-rapamycin composite (predictive) Preparation of PLGA-rapamycin complex: PLGA polymer with acid-terminated groups (7525 DLG1A, acid value 0.46 mmol / g, Lakeshore Biomaterials; 5 g, 2.3 mmol, 1.0 equivalent) is dissolved in 30 mL of dichloromethane (DCM). N,N-dicyclohexylcarbodiimide (1.2 equivalents, 2.8 mmol, 0.57 g) is added, followed by rapamycin (1.0 equivalent, 2.3 mmol, 2.1 g) and 4-dimethylaminopyridine (DMAP) (2.0 equivalents, 4.6 mmol, 0.56 g). The mixture is stirred at rt for 2 days. The mixture is then filtered to remove insoluble dicyclohexylurea. The filtrate is concentrated to a volume of approximately 10 mL and added to 100 mL of isopropyl alcohol (IPA) to precipitate the PLGA-rapamycin complex. The IPA layer is removed, and the polymer is then washed with 50 mL of IPA and 50 mL of methyl t-butyl ether (MTBE). The polymer is then dried under vacuum at 35°C for 2 days to obtain PLGA-rapamycin as a white solid (approximately 6.5 g).

[0120] Prepare a nanocarrier containing PLGA-rapamycin as follows: Prepare the solution for nanosupport formation as follows: Solution 1: 100 mg / ml of PLGA-rapamycin in methylene chloride. Prepare the solution by dissolving PLGA-rapamycin in pure methylene chloride. Solution 2: 100 mg / ml of PLA-PEG in methylene chloride. Prepare the solution by dissolving PLA-PEG in pure methylene chloride. Solution 3: 50 mg / mL of polyvinyl alcohol in 100 mM pH 8 phosphate buffer. First, prepare the primary water-in-oil emulsion. W1 / O1 is prepared by combining solution 1 (0.75 mL) and solution 2 (0.25 mL) in a small pressure tube and sonicating with a Branson Digital Sonifier 250 at 50% amplitude for 40 seconds. Add the W1 / O1 / W2 emulsion to a beaker containing 30 mL of 70 mM pH 8 phosphate buffer solution and stir at room temperature for 2 hours to evaporate the methylene chloride and form the nanocarrier. Wash a portion of the nanocarrier by transferring the suspension to a centrifuge tube, centrifugating at 75600 × g for 35 minutes at 4°C, removing the supernatant, and resuspending the pellet in phosphate buffered saline. Repeat the washing procedure and resuspend the pellet in phosphate buffered saline for a final dispersion of approximately 10 mg / mL of nanocarrier.

[0121] Example 2: Preparation of gold nanocarriers containing rapamycin (AuNC) (predictive) Preparation of HS-PEG-rapamycin: A solution of PEG acid disulfide (1.0 equivalent), rapamycin (2.0-2.5 equivalents), DCC (2.5 equivalents), and DMAP (3.0 equivalents) in dry DMF is stirred overnight at room temperature. Insoluble dicyclohexylurea is removed by filtration, and the filtrate is added to isopropyl alcohol (IPA) to precipitate PEG-disulfide-di-rapamycin ester. The mixture is washed with IPA and dried. The polymer is then treated with tris(2-carboxyethyl)phosphine hydrochloride in DMF to reduce PEG disulfide to thiol PEG-rapamycin ester (HS-PEG-rapamycin). The resulting polymer is recovered from IPA by precipitation, dried as described above, and analyzed by 1H NMR and GPC.

[0122] Formation of gold NC (AuNC): A 500 mL aqueous solution of 1 mM HAuCl4 is heated under reflux for 10 minutes with vigorous stirring in a 1 L round-bottom flask equipped with a condenser. Then, 50 mL of a 40 mM trisodium citrate solution is rapidly added to the stirred solution. The resulting deep wine-red solution is held under reflux for 25-30 minutes to recover the heat, and the solution is cooled to room temperature. The solution is then filtered through a 0.8 μm membrane filter to obtain the AuNC solution. AuNC is characterized by the use of visible spectroscopy and transmission electron microscopy. AuNC has a diameter of approximately 20 nm and is capped with citrate, which has an absorption peak at 520 nm.

[0123] AuNC and HS-PEG-rapamycin complex: Add 1 mL of a 150 μl solution of HS-PEG-rapamycin (10 μM in 10 mM pH 9.0 carbonate buffer) to a gold nanocarrier (1.16 nM) capped with 20 nm diameter citrate, resulting in a molar ratio of thiol to gold of 2500:1. Stir the mixture under argon at room temperature for 1 hour to completely replace the thiol with citrate on the gold nanocarrier. Then, purify the AuNC with PEG-rapamycin on its surface by centrifugation at 12000 g for 30 minutes. Drain the supernatant, and the pellet containing AuNC-S-PEG-rapamycin is washed with 1 × PBS buffer. The purified gold-PEG-rapamycin nanocarrier is then resuspended in a buffer suitable for further analysis or bioassay.

[0124] Example 3: Mesoporous silica nanoparticles containing attached ibuprofen (predictive) Mesoporous SiO2 nanoparticle cores are prepared via a sol-gel process. Hexadecyltrimethylammonium bromide (CTAB) (0.5 g) is dissolved in deionized water (500 mL), and then 2 M NaOH aqueous solution (3.5 mL) is added to the CTAB solution. The solution is stirred for 30 minutes, and then tetraethoxysilane (TEOS) (2.5 mL) is added to the solution. The resulting gel is stirred at 80°C for 3 hours. The formed white precipitate is captured by filtration, then washed with deionized water and dried at room temperature. The remaining surfactant is then extracted from the particles by suspending them overnight in an HCl-ethanol solution. The particles are washed with ethanol, centrifuged, and redispersed under sonication. This washing procedure is repeated two more times. Next, SiO2 nanoparticles are functionalized with amino groups using (3-aminopropyl)triethoxysilane (APTMS). To do this, the particles are suspended in ethanol (30 mL) and APTMS (50 μL) is added to the suspension. The suspension is left to stand at room temperature for 2 hours, then boiled for 4 hours, maintaining a constant volume by periodically adding ethanol. The remaining reactants are removed by 5 cycles of washing with centrifugation and redispersion in pure ethanol.

[0125] In another reaction, metal species with a diameter of 1-4 nm are produced. All the water used in this reaction is first deionized and then distilled from the glass. Add water (45.5 mL) to a 100 mL round-bottom flask. While stirring, add 0.2 M aqueous NaOH solution (1.5 mL), then add a 1% aqueous solution of tetrakis(hydroxymethyl)phosphonium chloride (THPC) (1.0 mL). Two minutes after the addition of the THPC solution, add a 10 mg / mL aqueous solution of chloroauric acid (2 mL) that has been aged for at least 15 minutes. Purify the metal species by dialysis against water. To form a core-shell nanocarrier, the amino-functionalized SiO2 nanoparticles formed above are first mixed with a gold species at room temperature for 2 hours. The gold-decorated SiO2 particles are collected by centrifugation and mixed with aqueous solutions of chloroauric acid and potassium bicarbonate to form a gold shell. The particles are then washed by centrifugation and redispersion in water. Ibuprofen is loaded by suspending the particles in a solution of ibuprofen sodium (1 mg / L) for 72 hours. The free ibuprofen is washed from the particles by centrifugation and redispersion in water.

[0126] Example 4: Liposomes containing cyclosporine A (predictive) Liposomes are formed using thin-film hydration. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (32 μmol), cholesterol (32 μmol), and cyclosporine A (6.4 μmol) are dissolved in pure chloroform (3 mL). This lipid solution is added to a 50 mL round-bottom flask, and the solvent is evaporated at 60°C using a rotary evaporator. The flask is then flushed with nitrogen gas to remove the remaining solvent. Phosphate-buffered saline (2 mL) and five glass beads are added to the flask, and the lipid membrane is hydrated by shaking at 60°C for 1 hour to form a suspension. The suspension is transferred to a small pressure tube and sonicated at 60°C for 4 cycles of 30-segment pulses with a 30-segment delay between each pulse. The suspension is then allowed to stand at room temperature for 2 hours to fully hydrate. The liposomes are centrifuged and then washed by resuspending them in fresh phosphate-buffered saline.

[0127] Example 5: Synthetic nanocarrier containing rapamycin material Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA 01702) (product catalog # R1017). PLGA with 76% lactide and 24% glycolide content and an intrinsic viscosity of 0.69 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211) (product code 7525 DLG 7A). PLA-PEG block copolymer with approximately 5,000 Da PEG blocks and approximately 40,000 Da PLA blocks was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211) (product code 100 DL mPEG 5000 5CE). Polyvinyl alcohol (85-89% hydrolyzed) was purchased from EMD Chemicals (product number 1.41350.1001). method The solution was prepared as follows: Solution 1: 75 mg / mL of PLGA and 25 mg / mL of PLA-PEG in methylene chloride. This solution was prepared by dissolving PLGA and PLA-PEG in pure methylene chloride. Solution 2: Rapamycin, 100 mg / mL in methylene chloride. This solution was prepared by dissolving rapamycin in pure methylene chloride. Solution 3: Polyvinyl alcohol, 50 mg / mL in 100 mM pH 8 phosphate buffer.

[0128] Nanosupports were prepared using an oil-in-water emulsion. Solution 1 (1 mL), Solution 2 (0.1 mL), and Solution 3 (3 mL) were combined in a small pressure tube and sonicated for 60 seconds at 30% amplitude using a Branson Digital Sonifier 250 to prepare an O / W emulsion. This O / W emulsion was added to a beaker containing 30 mL of 70 mM pH 8 phosphate buffer solution and stirred at room temperature for 2 hours to evaporate the methylene chloride and form the nanosupports. The nanosupport suspension was transferred to a centrifuge tube and centrifuged at 75,600 × g and 4°C for 35 min. The supernatant was removed, and a portion of the nanosupports was washed by resuspending the pellet in phosphate buffered saline. The washing procedure was repeated until a final nanosupport dispersion of approximately 10 mg / mL was obtained by resuspending the pellet in phosphate buffered saline.

[0129] The size of the nanocarriers was determined by dynamic light scattering. The amount of rapamycin in the nanocarriers was determined by HPLC analysis. The total dry nanocarrier mass per 1 mL of suspension was determined by specific gravity measurement. [Table 1]

[0130] Example 6: Synthetic nanocarrier containing GSK1059615 material GSK1059615 was purchased from MedChem Express (11 Deer Park Drive, Suite 102D Monmouth Junction, NJ 08852) (product code HY-12036). PLGA with a lactide:glycolide ratio of 1:1 and an intrinsic viscosity of 0.24 dL / g was purchased from Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, AL 35211) (product code 5050 DLG 2.5A). PLA-PEG-OMe block copolymer with approximately 5,000 Da of methyl ether-terminated PEG blocks and an overall intrinsic viscosity of 0.26 DL / g was purchased from Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, AL 35211) (product code 100 DL mPEG 5000 5K-E). Cellgro Phosphate-buffered saline 1X pH 7.4 (PBS 1X) was purchased from Corning (9345 Discovery Blvd. Manassas, VA 20109) (product code 21-040-CV).

[0131] method The solution was prepared as follows: Solution 1: PLGA (125 mg) and PLA-PEG-OMe (125 mg) were dissolved in 10 mL of acetone. Solution 2: GSK1059615 was prepared by dissolving 10 mg in 1 mL of N-methyl-2-pyrrolizinone (NMP). Nanocarriers were prepared by combining Solution 1 (4 mL) and Solution 2 (0.25 mL) in a small glass pressure tube and adding this mixture dropwise with stirring to a 250 mL round-bottom flask containing 20 mL of ultrapure water. This flask was mounted in a rotary evaporation apparatus and acetone was removed under reduced pressure. The nanocarrier suspension was transferred to a centrifuge tube and centrifuged at 75,600 rcf and 4°C for 50 minutes. The supernatant was removed, and a portion of the nanocarriers was washed by resuspending the pellet in PBS 1×. The washing procedure was repeated, and the pellet was resuspended in PBS 1× to obtain a nanocarrier suspension with a nominal concentration of 10 mg / mL based on polymer. The washed nanocarrier solution was then filtered using a Pall 1.2 μm PES membrane syringe filter (part number 4656). The same nanocarrier solution was prepared as described above and stored together with the first one after the filtration step. This homogeneous suspension was stored frozen at -20°C.

[0132] The size of the nanocarriers was determined by dynamic light scattering. The amount of GSK1059615 in the nanocarriers was determined by UV absorption at 351 nm. The total dry mass of nanocarriers per 1 mL of suspension was determined by specific gravity measurement. [Table 2]

[0133] Example 7: Induction of CD4+ regulatory T cells using synthetic nanocarriers material Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA 01702; product code R1017). PLGA with a lactide:glycolide ratio of 3:1 and an intrinsic viscosity of 0.75 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211; product code 7525 DLG 7A). A block copolymer of PLA-PEG-OMe with a PEG block of approximately 5,000 Da terminated with methyl ether and an overall intrinsic viscosity of 0.5 DL / g was purchased from Lakeshore Biochemicals (756 Tom Martin Drive, Birmingham, AL 35211; product code 100 DL mPEG 5000 5CE). EMPROVE® polyvinyl alcohol 4-88, USP (85-89% hydrolysis, viscosity 3.4-4.6 mPa·s) was purchased from EMD Chemicals Inc. (480 South Democrat Road, Gibbstown, NJ 08027; Product Code 1.41350).

[0134] method The solution was prepared as follows: Solution 1: 75 mg / mL of PLGA, 25 mg / mL of PLA-PEG-OMe, and 12.5 mg / mL of rapamycin in methylene chloride. The solution was prepared by dissolving PLGA, PLA-PEG-OMe, and rapamycin in pure methylene chloride. Solution 2: 50 mg / mL of polyvinyl alcohol in 100 mM pH 8 phosphate buffer. Nanosupports were prepared using an oil-in-water emulsion. The O / W emulsion was prepared by combining solution 1 (1.0 mL) and solution 2 (3.0 mL) in a small pressure tube and sonicating them at 30% amplitude for 60 seconds using a Branson Digital Sonifier 250. The O / W emulsion was added to a beaker containing 70 mM pH 8 phosphate buffer and stirred at room temperature for 2 hours to evaporate the methylene chloride and form nanosupports. A portion of the nanosupports was washed by transferring the nanosupport suspension to a centrifuge tube, centrifuging at 75,600 × g for 50 minutes at 4°C, removing the supernatant, and resuspending the pellet in phosphate-buffered saline. After repeating the washing procedure, the pellet was resuspended in phosphate-buffered saline for a final nanosupport dispersion of approximately 10 mg / mL.

[0135] The size of the nanocarriers was determined by dynamic light scattering. The amount of rapamycin in the nanocarriers was determined by HPLC analysis. The total mass of dry nanocarriers per 1 ml of suspension was determined by specific gravity measurement. [Table 3] To monitor and determine the effects of immunosuppressants attached to synthetic nanocarriers on the development of CD4+ regulatory T cells, CD4+ T cells were purified from the transgenic mouse strain OTII using negative selection by magnetically activated cell sorting (MACS). OTII mice are chicken ovalbumin peptide OVA. 323~339 It expresses a T cell receptor specific to [the cell type]. After isolation, 4 × 10⁻⁶ cells were collected. 6 Individual CD4+ OTII cells were identified using the panleukocyte marker CD45.1 (Ptprc a (SJL-Prprc) was transplanted into SJL mice that congenically express ) a (BoyAiTac). Recipient animals were either untreated (PBS injection) or given rapamycin-containing nanocarriers alone (NP[rapa]) or OVA-free. 323~339 Peptide (fOPII.323) or rapamycin-containing nanocarrier and OVA 323~339The treatment on days 1 and 5 involved subcutaneous injection of both the peptide and the combined treatment into the hind limbs.

[0136] On day 10, five days after the second administration of the indicated treatment, the animals were sacrificed, popliteal lymph nodes draining from the injection site were collected, their developmental status was analyzed, and the CD4+ OTII cells transferred to SJL mice were quantified by flow cytometry. As shown in Figure 1, animals that were untreated (PBS) or received only rapamycin-containing nanocarriers (NP[rapa]) had undetectable levels of OTII cells that acquired the regulatory T cell phenotype (CD25+Fox3p+) characterized by staining with anti-CD25 and anti-Foxp3 antibodies. Administration of free OVA peptide (fOPII.323) resulted in a detectable but not statistically significant increase in the proportion of CD25+Fox3p+ cells. In contrast, co-administration of rapamycin-containing nanocarriers with OVA peptide resulted in a robust population of CD25+Fox3p+ cells, demonstrating that this combination treatment induced the majority of transplanted CD4+OTII cells to develop into regulatory T cells (Tregs). These results demonstrate that the immunosuppressants provided herein, when administered in combination with an antigen, can induce the formation of a regulatory immune response, such as an increase in the proportion of antigen-specific CD4+ regulatory T cells.

[0137] Example 8: Evaluation of anti-PEG immune response material Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA 01702; product code R1017). PLGA with a lactide:glycolide ratio of 3:1 and an intrinsic viscosity of 0.75 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211; product code 7525 DLG 7A). A block copolymer of PLA-PEG-OMe with a PEG block of approximately 5,000 Da terminated with methyl ether and an overall intrinsic viscosity of 0.5 DL / g was purchased from Lakeshore Biochemicals (756 Tom Martin Drive, Birmingham, AL 35211; product code 100 DL mPEG 5000 5CE). EMPROVE® polyvinyl alcohol 4-88, USP (85-89% hydrolysis, viscosity 3.4-4.6 mPa·s) was purchased from EMD Chemicals Inc. (480 South Democrat Road, Gibbstown, NJ 08027; Product Code 1.41350).

[0138] method The solution was prepared as follows: Solution 1: 75 mg / mL of PLGA, 25 mg / mL of PLA-PEG-OMe, and 12.5 mg / mL of rapamycin in methylene chloride. The solution was prepared by dissolving PLGA, PLA-PEG-OMe, and rapamycin in pure methylene chloride. Solution 2: 50 mg / mL of polyvinyl alcohol in 100 mM pH 8 phosphate buffer. Nanosupports were prepared using an oil-in-water emulsion. The O / W emulsion was prepared by combining solution 1 (1.0 mL) and solution 2 (3.0 mL) in a small pressure tube and sonicating them at 30% amplitude for 60 seconds using a Branson Digital Sonifier 250. The O / W emulsion was added to a beaker containing 70 mM pH 8 phosphate buffer and stirred at room temperature for 2 hours to evaporate the methylene chloride and form nanosupports. A portion of the nanosupports was washed by transferring the nanosupport suspension to a centrifuge tube, centrifugating at 75,600 × g for 50 minutes at 4°C, removing the supernatant, and resuspending the pellet in phosphate-buffered saline. After repeating the washing procedure, the pellet was resuspended in phosphate-buffered saline for a final nanosupport dispersion of approximately 10 mg / mL.

[0139] The size of the nanocarriers was determined by dynamic light scattering. The amount of rapamycin in the nanocarriers was determined by HPLC analysis. The total mass of dry nanocarriers per 1 ml of suspension was determined by specific gravity measurement. [Table 4] Female C57BL / 6 cattle of the same age (5-6 weeks) were weekly intravenously injected 250 μg of keyhole limpet hemocyanin and polyethylene glycol complex (KLH-PEG) into the tail vein (days 0, 7, 14, 21, 28, 35, 42, and 49). The first five injections (NP [rapamycin], containing 50 μg of rapamycin) were administered with or without 0.47 mg of rapamycin-containing nanocarrier (days 0, 7, 14, 21, and 28). The next three injections consisted of the same amount of KLH-PEG alone. IgM antibody response to PEG was monitored weekly in the blood of these animals (days 12, 20, 34, 40, 47, and 54).

[0140] As shown in Figure 2, five doses of synthetic nanocarriers co-administered intravenously with KLH-PEG in the same solution were effective in preventing antibody formation against PEG. These results indicate that rapamycin-containing nanocarriers co-administered with PEGylated proteins can reduce or prevent antibody formation against PEG. Therefore, the methods of treatment provided herein can be used to reduce undesirable immune responses to therapeutic proteins, such as pegylated therapeutic proteins. The data also demonstrate a dosing schedule for achieving such an effect.

[0141] Example 9: Increase in Factor VIII-specific CD4+ Treg using synthetic nanosupports (predictive). The pilot study will be conducted in non-human primate subjects and in the synthetic nanocarrier of Example 1, using soluble factor VIII along with a synthetic nanocarrier not co-prescribed before administration. Fifty non-human primate subjects will be randomly assigned to five arms: placebo, then four dose levels of the synthetic nanocarrier selected for dose ranges. The dose ranges will be established to select the optimal increase in CD4+ Tregs (CD4+ regulatory T cells) that would be specific to factor VIII. On day 0, all subjects in each active arm will be subcutaneously administered a dose of the synthetic nanocarrier, and within 24 hours of the dose of the synthetic nanocarrier, they will receive an infusion of the standard infusion dose of factor VIII. After two weeks, each animal will be loaded with the standard dose of soluble factor VIII, and the number or percentage of factor VIII-specific CD4+ Tregs will be measured using standard techniques. The lowest dose of the synthetic nanocarrier will be selected from among the four active arms showing a significant increase in factor VIII-specific CD4+ Tregs as the test dose. The test doses of the synthetic nanocarrier are then scaled non-proportionally for administration to human subjects and used in human clinical trials to determine the range of dose levels of the synthetic nanocarrier used with standard doses of soluble factor VIII. Here again, factor VIII and the synthetic nanocarrier are not co-administered before administration. The co-administered doses of the synthetic nanocarrier and factor VIII are then available for normal clinical practice.

[0142] Example 10: Increase in factor VIII-specific CD4+ Treg using a synthetic osmotic pump (predictive). The pilot study was conducted in non-human primate subjects using soluble factor VIII and an osmotic pump (generally prepared according to Example 6, but with rapamycin replacing GSK1059615 in Example 6), with soluble factor VIII and the osmotic pump not pre-administered. Fifty non-human primate subjects were randomly assigned to five arms: placebo, then delivered by an osmotic pump, and four dose levels of GSK1059615 selected for range doses. The dose range was established to select the optimal increase in CD4+ Tregs that would be factor VIII specific. On day 0, all subjects in each active arm were subcutaneously administered a dose of synthetic nanocarrier and received an infusion of the standard infusion dose of factor VIII within 24 hours of the synthetic nanocarrier dose. After two weeks, each animal was loaded with the standard dose of soluble factor VIII, and the number or percentage of factor VIII specific CD4+ Tregs was measured using standard techniques. The lowest dose of GSK1059615 delivered by osmotic pumps from four active arms showing a significant increase in factor VIII-specific CD4+ Tregs will be selected as the test dose.

[0143] The test dose of GSK1059615 delivered by osmotic pump is then scaled non-proportionally for administration to human subjects and used in human clinical trials to determine the range of dose levels of osmotic pump-delivered GSK1059615 used in standard doses of soluble factor VIII. Here again, factor VIII and the osmotic pump are not co-prescribed before administration. The non-co-prescribed doses of osmotic pump-delivered GSK1059615 and factor VIII are then available for normal clinical practice.

[0144] Example 11: Increase in specific CD4+ Tregs using therapeutic polynucleotides (predictive). A pilot study was conducted in non-human primate subjects using asparaginase mMRNA (generally prepared according to U.S. Patent Application No. 2013 / 0115272 (Fougerolles et al.) ("mmRNA")) and the synthetic nanocarrier of Example 1, where the mMRNA and synthetic nanocarrier were not co-prescribed before administration. Fifty non-human primate subjects were randomly assigned to five arms: placebo, then four doses of synthetic nanocarrier selected for their dose range. The dose range was established to select the optimal increase in mmRNA-specific CD4+ Tregs. On day 0, all subjects in each active arm were subcutaneously administered a dose of the synthetic nanocarrier, receiving an infusion of the standard injection dose of factor VIII within 24 hours of the synthetic nanocarrier dose. After two weeks, each animal was loaded with a standard dose of mMRNA, and the number or percentage of mMRNA-specific CD4+ Tregs was measured using standard techniques. The lowest dose of synthetic nanocarrier from between the four active arms that shows a significant increase in mMRNA-specific CD4+ Tregs is selected as the test dose.

[0145] The test doses of the synthetic nanocarrier are then scaled non-proportionally for administration to human subjects to determine the range of doses of the synthetic nanocarrier used in human clinical trials and in conjunction with standard dose levels of mmRNA. Again, the mmRNA and synthetic nanocarrier are not co-prescribed before administration. The co-prescribed doses of the synthetic nanocarrier and mmRNA are then available for normal clinical practice.

[0146] Example 12: Evaluation of anti-PEG immune response (predictive) Female C57BL / 6 animals of the same age (5-6 weeks) were weekly intravenously injected 250 μg of keyhole limpet hemocyanin and polyethylene glycol complex (KLH-PEG) into the tail vein (on days 0, 7, 14, 21, 28, 35, 42, and 49). For the first five injections (on days 0, 7, 14, 21, and 28), 0.47 mg of nanocrystalline rapamycin was administered with or without this compound. The next three injections consisted of the same amount of KLH-PEG alone. IgM antibody responses to PEG were monitored weekly in the blood of these animals (on days 12, 20, 34, 40, 47, and 54). Compared to animals receiving KLH-PEG (without nanocrystalline rapamycin) alone, the reduction in KLH-specific IgM antibody titers in animals receiving a dose of nanocrystalline rapamycin in combination with KLH-PEG indicates that the nanocrystalline form of rapamycin, when administered in combination with PEGylated proteins, can reduce or prevent antibody formation.

[0147] Example 13: Increase in Factor VIII-specific CD4+ Treg using synthetic nanosupports (predictive). The pilot study will be conducted in non-human primate subjects using soluble factor VIII and nanocrystalline rapamycin, with factor VIII and nanocrystalline rapamycin not being co-administered before administration. Fifty non-human primate subjects will be randomly assigned to five arms: placebo, then four dose levels of nanocrystalline rapamycin selected for dose ranges. The dose ranges will be established to select the optimal increase in CD4+ Tregs (CD4+ regulatory T cells) that will be specific to factor VIII. On day 0, all subjects in each active arm will be subcutaneously administered a dose of nanocrystalline rapamycin and will receive an infusion of a standard infusion dose of factor VIII within 24 hours of the nanocrystalline rapamycin dose. After two weeks, each animal will be loaded with a standard dose of soluble factor VIII, and the number or percentage of factor VIII-specific CD4+ Tregs will be measured using standard techniques. The lowest dose of nanocrystalline rapamycin among the four active arms showing a significant increase in factor VIII-specific CD4+ Tregs is selected as the test dose.

[0148] The test dose of nanocrystalline rapamycin is then scaled non-proportionally for administration to human subjects to determine the range of nanocrystalline rapamycin dose levels used in human clinical trials and in standard doses of soluble factor VIII. Again, factor VIII and nanocrystalline rapamycin are not co-prescribed before administration. The non-co-prescribed doses of nanocrystalline rapamycin and factor VIII are then available for normal clinical practice.

Claims

1. (i) A polymer synthetic nanocarrier attached to an mTOR inhibitor, and (ii) Therapeutic polymers A composition comprising a polymer synthetic nanocarrier attached to an mTOR inhibitor for use in a method comprising increasing the number or proportion of CD4+ regulatory T cells by co-administration to the target, The composition wherein the therapeutic polymer is not formulated simultaneously with the polymer synthetic nanocarrier attached to the mTOR inhibitor before the therapeutic polymer is administered in combination with the polymer synthetic nanocarrier attached to the mTOR inhibitor.

2. (a) A polymer synthetic nanocarrier attached to an mTOR inhibitor and a therapeutic polymer are administered to the subject in combination; (b) The composition according to claim 1, further comprising assessing the number or proportion of CD4+ regulatory T cells in a subject before and after co-administration.

3. The composition according to claim 1 or 2, wherein the increased number or proportion of CD4+ regulatory T cells is at least two-fold, three-fold, four-fold, five-fold, or six-fold compared to the number or proportion of CD4+ regulatory T cells before the co-administration.

4. The composition according to any one of claims 1 to 3, wherein the concomitant administration is by intravenous, intraperitoneal, or subcutaneous administration.

5. The composition according to any one of claims 1 to 4, further comprising recording the increase in the number or percentage of CD4+ regulatory T cells after the concomitant administration.

6. The composition according to any one of claims 1 to 5, wherein the mTOR inhibitor is rapamycin or a rapamycin analog.

7. The composition according to any one of claims 1 to 6, wherein the therapeutic polymer is a therapeutic protein or a therapeutic polynucleotide.

8. The composition according to claim 7, wherein the therapeutic protein is (a) for protein supplementation in protein augmentation therapy, and (b) comprises an intravenous or injectable therapeutic protein, enzyme, enzyme cofactor, hormone, blood or blood coagulation factor, cytokine, interferon, growth factor, monoclonal antibody, polyclonal antibody, or protein associated with Pompe disease.

9. (a) Therapeutic proteins that can be administered intravenously or injected include tocilizumab, alpha-1 antitrypsin, hematide, albumin interferon alpha-2b, lucin, tesamorelin, ocrelizumab, belimumab, pegroticase, taliglucerase alpha, agalsidase alpha, or veraglucerase alpha; (b) The enzyme comprises an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase; (c) The enzyme comprises an enzyme for enzyme replacement therapy for lysosomal storage disorders, and optionally the enzyme for enzyme replacement therapy for lysosomal storage disorders comprises imiglucerase, α-galactosidase A (α-galA), agalsidase beta, acid α-glucosidase (GAA), alglucosidase alpha, LUMIZYME, MYOZYME, arylsulfatase B, laronidase, ALDURAZYME, idursulfase, ELAPRASE, arylsulfatase B, pegroticase, pegsticase, or NAGLAZYME; (d) The cytokines include lymphokines, interleukins, chemokines, type 1 cytokines, or type 2 cytokines; or (e) The composition according to claim 8, wherein the blood or blood coagulation factor comprises factor I, factor II, tissue factor, factor V, factor VII, factor VIII, factor IX, factor X, factor Xa, factor XII, factor XIII, von Willebrand factor, prekallikrein, high molecular weight kininogen, fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z-related protease inhibitor (ZPI), plasminogen, alpha-2-antiplasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI1), plasminogen activator inhibitor-2 (PAI2), cancer procoagulant, or epoetin alpha.

10. The composition according to any one of claims 1 to 9, wherein the amount of mTOR inhibitor attached to the polymer synthetic nanocarrier is between 0.1% and 50% on average across the polymer synthetic nanocarrier.

11. The composition according to claim 10, wherein the load capacity is between 0.1% and 20%.

12. The composition according to any one of claims 1 to 11, wherein the polymer synthetic nanocarrier comprises a polymer which is a non-methoxy-terminated Pluronic® polymer.

13. The composition according to claim 12, wherein the polymer synthetic nanocarrier comprises polyester, polyester attached to a polyether, polyamino acids, polycarbonate, polyacetal, polyketal, polysaccharide, polyethyloxazoline, or polyethyleneimine.

14. The composition according to claim 13, wherein the polyester comprises poly(lactic acid), poly(glycolic acid), poly(lactic acid-co-glycolic acid), or polycaprolactone.

15. The composition according to claim 13 or 14, wherein the polymer synthetic nanocarrier comprises polyester and polyester attached to a polyether.

16. The composition according to any one of claims 13 to 15, wherein the polyether comprises polyethylene glycol or polypropylene glycol.

17. (a) The average particle size distribution obtained using dynamic light scattering of polymer synthetic nanosupports has a diameter greater than (i) 100 nm; (ii) 150 nm; (iii) 200 nm; (iv) 250 nm; or (v) 300 nm; and / or (b) The composition according to any one of claims 1 to 16, wherein the aspect ratio of the polymer synthetic nanocarrier is greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or 1:10.

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