Delivery of immunosuppressants having specified pharmacodynamic effective-life and antigen for induction of immune tolerance

Abstract

To provide methods that offer immunosuppressants and therapeutic macromolecules that are administered within a pharmacodynamically effective window of the immunosuppressants to induce immune tolerance to the therapeutic macromolecules.SOLUTION: A method comprises: administering an immunosuppressant to a subject at an administration dose that provides an administration pharmacodynamic effective-life, with respect to a therapeutic macromolecule, the duration of which pharmacodynamic effective-life ranges from a minimum of 20 hours to a maximum of 1 month; and administering the therapeutic macromolecule to the subject within the duration of the administration pharmacodynamic effective-life of the immunosuppressant; wherein the therapeutic macromolecule and immunosuppressant are not attached to one another, and the therapeutic macromolecule is not attached to a synthetic nanocarrier. The method allows shifting the immune response in favor of tolerogenic immune response development specific to the therapeutic macromolecule.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. 35 Section 119 of U.S. Provisional Application Nos. 61 / 819,517, filed May 3, 2013, 61 / 881,851, filed September 24, 2013, 61 / 881,913, filed September 24, 2013, 61 / 881,921, filed September 24, 2013, 61 / 907,177, filed November 21, 2013, 61 / 948,313, filed March 5, 2014, and 61 / 948,384, filed March 5, 2014, the contents of each of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE PRESENT APPLICATION The present invention relates to a method of providing an immunosuppressant and a therapeutic macromolecule, administered within a pharmacodynamically effective window of the immunosuppressant to induce immune tolerance to the therapeutic macromolecule, which allows shifting the immune response for the development of a tolerogenic immune response specific to the therapeutic macromolecule. The method provided herein can be used to generate a tolerogenic immune response in a subject in which administration of the therapeutic macromolecule results, or is expected to result, in an undesirable immune response. [Background technology]

[0003] 2. Background of the Invention Therapeutic treatments such as protein or enzyme replacement therapy often result in undesired immune responses to the particular therapy. Such undesired immune responses can be reduced through the use of immunosuppressants. However, conventional immunosuppressants are broad-spectrum acting. Furthermore, to maintain immunosuppression, immunosuppressant therapy is generally a lifelong proposition. Unfortunately, the use of broad-spectrum acting immunosuppressants is associated with the risk of serious side effects such as tumors, infections, nephrotoxicity and metabolic disorders. Therefore, new tolerogenic therapies would be beneficial. Summary of the Invention

[0004] SUMMARY OF THE PRESENT APPLICATION In one aspect, a method is provided that includes administering an immunosuppressant to a first class of subjects at a dosage that provides a pharmacodynamic effective period for the therapeutic macromolecule, and administering the therapeutic macromolecule to the subject within the pharmacodynamic effective period of the immunosuppressant. In one embodiment, the pharmacodynamic effective period has a duration ranging from a minimum of 20 hours to a maximum of one month. In one embodiment, the therapeutic macromolecule and the immunosuppressant are not attached to each other. In one embodiment, the therapeutic macromolecule is not attached to a synthetic nanocarrier. In one embodiment of any one of the methods provided herein, the method further comprises determining an administration dose of the immunosuppressant based on a test dose of the immunosuppressant. In one embodiment of any one of the methods provided herein, in the second class of subjects, the test dose has a therapeutic macromolecule having a test pharmacodynamic effective period ranging from a minimum of 20 hours to a maximum of 1 month.

[0005] In another aspect, any one of the immunosuppressant compositions provided herein is provided for use in a method of inducing tolerance to a therapeutic macromolecule. In one embodiment of any one of the methods provided herein, the method comprises (a) administering an immunosuppressant to a subject at a dose sufficient to elicit a pharmacodynamic effective period ranging from 20 hours to 1 month, and (b) administering a therapeutic macromolecule to the subject within said pharmacodynamic effective period. In one embodiment of any one of the methods provided herein, the therapeutic macromolecule and the immunosuppressant are not attached to each other. In one embodiment of any one of the methods provided herein, the therapeutic macromolecule is not attached to a synthetic nanocarrier.

[0006] In another aspect, a use of any one of the immunosuppressant compositions provided herein is provided for the manufacture of a medicament for use in any one of the methods provided herein, such as a method of inducing tolerance to a therapeutic macromolecule in a subject. In one embodiment, the method comprises (a) administering an immunosuppressant to a subject at a dose sufficient to elicit a pharmacodynamic effective period ranging from 20 hours to 1 month; and (b) administering a therapeutic macromolecule to said subject within said pharmacodynamic effective period. In one embodiment of any one of the methods provided herein, the therapeutic macromolecule and the immunosuppressant are not attached to each other. In another embodiment of any one of the methods provided herein, the therapeutic macromolecule is not attached to a synthetic nanocarrier.

[0007] In one embodiment of any one of the methods, immunosuppressants or uses provided herein, the immunosuppressant comprises an immunosuppressant attached to a synthetic nanocarrier, an implantable osmotic pump, a synthetic nanocarrier comprising a bispecific antibody or an implantable polymeric depot material. In one embodiment of any one of the methods, immunosuppressants or uses provided herein, the immunosuppressant comprises an immunosuppressant attached to a synthetic nanocarrier. In one embodiment of any one of the methods, immunosuppressants or uses provided herein, the synthetic nanocarrier comprises a lipid nanoparticle, a polymeric nanoparticle, a metal nanoparticle, a surfactant-based emulsion, a dendrimer, a buckyball, a nanowire, a virus-like particle, a protein particle, or a nanoparticle comprising a combination of nanomaterials, optionally such nanoparticles are lipid-polymer nanoparticles.

[0008] In one embodiment of any one of the methods, immunosuppressants or uses provided herein, the immunosuppressant is a statin; an mTOR inhibitor; a TGF-βc agent; a TGF-β receptor agonist; a histone deacetylase inhibitor; a corticosteroid; an inhibitor of mitochondrial function; a P38 inhibitor; an NF-κβ inhibitor, dexamethasone; TCPA-1; IKK VII; an adenosine receptor agonist; a prostaglandin E2 agonist; a phosphodiesterase inhibitor; a proteasome inhibitor; a kinase inhibitor; a G-protein coupled receptor agonist; a G-protein coupled receptor antagonist; a glucocorticoid; a retinoid; a cytokine inhibitor; a cytokine receptor inhibitor; a cytokine receptor activator; a peroxisome proliferator-activated receptor antagonist; a peroxisome proliferator-activated receptor agonist; a histone deacetylase inhibitor; a calcineurin inhibitor. In one embodiment of any one of the methods, immunosuppressants or uses provided herein, the immunosuppressant comprises an mTOR inhibitor. In one embodiment of any one of the methods, immunosuppressants or uses provided herein, the mTOR inhibitor comprises rapamycin.

[0009] In one embodiment of any one of the methods, immunosuppressants or uses provided herein, the loading of the synthetic nanocarrier in the synthetic nanocarrier ranges between 0.0001 wt% and 50 wt%, based on the total dry recipe weight of the material in the synthetic nanocarrier (wt / wt). In one embodiment of any one of the methods, immunosuppressants or uses provided herein, the loading of the immunosuppressant in the synthetic nanocarrier ranges between 0.1 wt% and 10 wt%, based on the total dry recipe weight of the material in the synthetic nanocarrier (wt / wt). In one embodiment of any one of the methods, immunosuppressants or uses provided herein, the therapeutic macromolecule comprises a therapeutic protein or a therapeutic polynucleotide. In one embodiment of any one of the methods, immunosuppressants or uses provided herein, the therapeutic protein comprises an enzyme, an enzyme cofactor, a hormone, a blood clotting factor, a cytokine, a growth factor, a monoclonal antibody or a polyclonal antibody.

[0010] In one embodiment of any one of the methods, immunosuppressants or uses provided herein, the administration pharmacodynamic effective period has a duration ranging from a minimum of 20 hours to a maximum of 2 weeks. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the administration pharmacodynamic effective period has a duration ranging from a minimum of 20 hours to a maximum of 1 week. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the administration pharmacodynamic effective period has a duration ranging from a minimum of 24 hours to a maximum of 2 days. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the test pharmacodynamic effective period has a duration ranging from a minimum of 20 hours to a maximum of 2 weeks. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the test pharmacodynamic effective period has a duration ranging from a minimum of 20 hours to a maximum of 1 week. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the test pharmacodynamic effective period has a duration ranging from a minimum of 24 hours to a maximum of 2 days.

[0011] In one embodiment of any one of the methods, immunosuppressants or uses provided herein, the administered dose of the immunosuppressant is determined based on a test dose of the immunosuppressant along with the use of allometric or isometric scaling techniques. In one embodiment of any one of the methods, immunosuppressants or uses provided herein, the first class of subject and the second class of subject are the same class of subject.In one embodiment of any one of the methods, immunosuppressants or uses provided herein, the first class of subject and the second class of subject are different classes of subject.

[0012] In one embodiment of any one of the methods, immunosuppressants or uses provided herein, the synthetic nanocarriers comprise lipid nanoparticles. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the synthetic nanocarriers comprise liposomes. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the synthetic nanocarriers comprise metal nanoparticles. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the metal nanoparticles comprise gold nanoparticles. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the synthetic nanocarriers comprise polymeric nanoparticles. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the polymeric nanoparticles comprise a polymer that is a non-methoxy terminated pluronic polymer. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the polymeric nanoparticles comprise a polyester, a polyester attached to a polyether, a polyamino acid, a polycarbonate, a polyacetal, a polyketal, a polysaccharide, a polyethyloxazoline, or a polyethyleneimine. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the polyester comprises poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid) or polycaprolactone. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the polymeric nanoparticles comprise a polyester and a polyester attached to a polyether. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the polyether comprises polyethylene glycol or polypropylene glycol.

[0013] In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the mean of the particle size distribution obtained using dynamic light scattering of the synthetic nanocarriers is greater than 100 nm in diameter. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the diameter is greater than 150 nm. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the diameter is greater than 200 nm. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the diameter is greater than 250 nm. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the diameter is greater than 300 nm. In another embodiment of any one of the methods, immunosuppressants or uses provided herein, the aspect ratio of the synthetic nanocarriers is greater than 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7 or 1:10.

[0014] In another aspect, a method of manufacturing any one of the immunosuppressant and / or therapeutic polymer compositions provided herein is provided. In one embodiment, the method of manufacturing includes producing a dose or dosage form of the therapeutic polymer and producing a dose or dosage form of the immunosuppressant. In another embodiment of any one of the methods of manufacturing provided, the step of producing a dose or dosage form of the immunosuppressant includes attaching the immunosuppressant to a synthetic nanocarrier. In another embodiment of any one of the methods of manufacturing provided, the method further includes combining the dose or dosage form of the immunosuppressant and the dose or dosage form of the therapeutic polymer in a kit.

[0015] In another aspect, any one of the manufacturing methods can be for manufacturing a medicament for performing any one of the methods provided herein, and such manufacturing methods are also provided. In one embodiment, the manufacturing method is for manufacturing a medicament for inducing tolerance to a therapeutic macromolecule. In another embodiment, the manufacturing method is for manufacturing a therapeutic macromolecule medicament for administering an immunosuppressant and the therapeutic macromolecule for the pharmacodynamic effective period of the immunosuppressant. In another embodiment of any one of the manufacturing methods provided herein, the immunosuppressant is attached to a synthetic nanocarrier. [Brief description of the drawings]

[0016] Brief explanation of the figure [Figure 1] FIG. 1 shows IgG responses to encapsulated rapamycin administered with OVA and KLH. [Diagram 2] FIG. 2 shows the protocol and results from various rapamycin doses. [Diagram 3] FIG. 3 demonstrates the effect of administration of synthetic nanocarriers attached to rapamycin within the pharmacodynamic window. [Figure 4] FIG. 4 demonstrates the effect of synthetic nanocarriers attached to rapamycin or nanocarriers attached to GSK1059615 administered simultaneously with encapsulated protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description of the Invention Before describing the invention in detail, it is to be understood that the invention is not limited to particularly exemplified materials or process parameters, since such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to limit the use of alternative terminology to describe the invention. All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety for all purposes.

[0018] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. For example, a reference to "a polymer" includes a mixture of two or more such molecules or a mixture of different molecular weights of a single polymer type, a reference to "synthetic nanocarrier" encompasses a mixture of two or more such synthetic nanocarriers or a plurality of such synthetic nanocarriers, a reference to "RNA molecule" includes a mixture of two or more such RNA molecules or a plurality of such RNA molecules, a reference to "immunosuppressant" includes two or more such materials or a plurality of such immunosuppressant molecules, etc.

[0019] As used herein, the term "comprise" or variations such as "comprises" or "comprising" should be read as indicating the inclusion of any recited integer (e.g., a feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g., a plurality of features, elements, features, properties, method / process steps or limitations) and should not be read as indicating the exclusion of any other integer or group of integers. Thus, as used herein, the term "comprising" is inclusive and does not exclude additional, unrecited integers or method / process steps.

[0020] In certain embodiments of any one of the compositions and methods provided herein, "comprising" may be substituted with "consisting essentially of" or "consisting of." The phrase "consisting essentially of" is used herein to require the specified entity(ies) 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 denote the presence of only the recited entity (e.g., a feature, element, characteristic, property, method / process step or limitation) or group of entities (e.g., a plurality of features, elements, characteristics, properties, method / process steps or limitations).

[0021] A. Introduction The methods provided herein allow for improved therapeutic efficacy as a result of administration of a therapeutic macromolecule during the pharmacodynamically effective window (or pharmacodynamically effective period) of the immunosuppressant. The immunosuppressant, in some embodiments, is administered by attachment to a synthetic nanocarrier. The methods and compositions provided herein help maximize the benefit of immunosuppressant therapy when administering a therapeutic macromolecule to a subject.

[0022] The inventors have unexpectedly and surprisingly discovered that the above problems and limitations can be overcome by implementing the invention disclosed herein. In particular, the inventors have unexpectedly discovered that it is possible to provide a method comprising administering an immunosuppressant to a subject at an administration dose that provides a pharmacodynamic effective period for the therapeutic macromolecule, the administration dose having a period ranging from a minimum of 20 hours to a maximum of one month, and administering the therapeutic macromolecule to a subject within the pharmacodynamic effective period of the immunosuppressant, wherein the immunosuppressant and the therapeutic macromolecule are not linked to each other, and the therapeutic macromolecule is not linked to a synthetic nanocarrier. Related compositions are also provided herein. The invention is illustrated in the following examples, which illustrate various compositions useful in the practice of the invention and also provide data illustrating the concepts underlying the pharmacodynamic effective periods in certain embodiments of the invention. The present invention will now be described in more detail below.

[0023] B. Definition "Administered dose" or "administered dose of immunosuppressant" refers to a dose of the immunosuppressant suitable for administration. In some embodiments, the administered dose is based on a test dose of the immunosuppressant. In some embodiments, the administered dose may be determined based on information from the test dose of the immunosuppressant along with application of allometric scaling techniques. See, for example, I. Mahmood, "Interspecies Pharmacokinetic Scaling: Principles and Application of Allometric Scaling", Pine House Publishers 2005. In some embodiments, particularly when the first class of subjects and the second class of subjects are from the same species, the administered dose may be determined based on information from the test dose of the immunosuppressant along with application of isometric scaling techniques. In some embodiments, the administered dose may be determined by direct experimentation in the second class of subjects, rather than simply predicted using allometric or isometric scaling as described above, based on the test dose.

[0024] "Administering" or "administration" or "administering" means providing a material to a subject in a pharmacologically useful manner. In embodiments, "administering" or "administration" or "administering" includes "causing to administer." "Causing to administer" means directly or indirectly causing, encouraging, encouraging, assisting, inducing, or directing another party to administer the material.

[0025] "Administration pharmacodynamic effective period" refers to the pharmacodynamic effective period of the immunosuppressant determined for the therapeutic macromolecule at the administered dose of the immunosuppressant and in the first class of subjects. In some embodiments, the recited immunosuppressant may have an administration pharmacodynamic effective period for the therapeutic macromolecule having a period ranging from a minimum of 20 hours to a maximum of 1 month, preferably from a minimum of 20 hours to a maximum of 2 weeks, preferably from a minimum of 20 hours to a maximum of 1 week, preferably from a minimum of 20 hours to a maximum of 5 days, preferably from a minimum of 20 hours to a maximum of 3 days, preferably from a minimum of 24 hours to a maximum of 2 days. Preferably, the therapeutic macromolecule is administered for the pharmacodynamic effective period, and thus in some embodiments, it can be administered simultaneously or immediately after the administration of the immunosuppressant, provided that the administration of the therapeutic macromolecule is performed before the end of the pharmacodynamic effective period.

[0026] "Effective amount", in the context of a composition or dose for administration to a subject, refers to an amount or dose of a composition that produces one or more desired responses in the subject, such as the development of a tolerogenic immune response (e.g., reduced proliferation, activation, induction, survival, recruitment of B cells specific for a therapeutic macromolecule, or reduced production of antibodies specific for a therapeutic macromolecule). In some embodiments, an effective amount is a therapeutically effective amount. Thus, in some embodiments, an effective amount is an amount or dose of any of the compositions provided herein that produces one or more of the desired immune and / or therapeutic effects provided herein. This amount can be for in vitro or in vivo purposes. For in vitro purposes, the amount can be an amount that a clinician would consider to have a clinical effect for a patient in need of administration of a therapeutic macromolecule and / or antigen-specific immune tolerogenicity thereto.

[0027] An effective amount may involve reducing the level of an undesired immune response, but in some embodiments, it involves preventing an undesired immune response altogether. An effective amount may also involve delaying an undesired immune response from occurring. An effective amount may also be an amount that produces a desired therapeutic endpoint or a desired therapeutic outcome. In other embodiments, an effective amount may involve improving the level of a desired response, such as a therapeutic endpoint or outcome. An effective amount preferably results in a tolerogenic immune response in a subject against an antigen, such as a therapeutic macromolecule. Achievement of any of the above may be monitored by routine methods.

[0028] In some embodiments of any one of the methods provided, the effective amount is an amount in which the desired response persists in the subject. In other embodiments of any of the compositions and methods provided, the effective amount is an amount that produces a measurable desired response over a period of time. The effective amount will of course depend on the specific subject being treated; the severity of the condition, disease or disorder; individual patient parameters including age, physical condition, size and weight; duration of treatment; the nature of concurrent treatment (if any); the specific route of administration and the like factors within the knowledge and experience of the medical practitioner. These factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. In general, it is preferable to use the maximum dose, i.e., the highest safe dose according to sound medical judgment. However, those skilled in the art will understand that a patient may insist on a lower dose, or a tolerated dose, for medical reasons, psychological reasons, or virtually any other reason.

[0029] Generally, the dose of the immunosuppressant and / or therapeutic macromolecule in the composition of the present invention refers to the amount of the immunosuppressant and / or therapeutic macromolecule. Alternatively, the dose can be administered based on the number of synthetic nanocarriers that provide the desired amount of the immunosuppressant. "Antigen" refers to a B cell antigen or a T cell antigen. "Type(s) of antigen" refers to molecules that share the same or substantially the same antigenic characteristics. In some embodiments, the antigen may be a protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, polysaccharide, or is contained or expressed in a cell. In some embodiments, such as when the antigen is not well defined or characterized, the antigen may be contained in a cell or tissue preparation, cell debris, cell exosomes, conditioned medium, etc.

[0030] "Antigen-specific" refers to any immune response that is brought about by the presence of an antigen or a portion thereof, or that generates a molecule that specifically recognizes or binds to the antigen. In some embodiments, when the antigen includes a therapeutic macromolecule, antigen-specific can mean therapeutic macromolecule-specific. For example, when the immune response is antigen-specific antibody production, an antibody that specifically binds to the antigen (e.g., therapeutic macromolecule) is produced. As another example, when the immune response is the proliferation and / or activity of antigen-specific B cells or CD4+ T cells, the proliferation and / or activity is brought about by the recognition of the antigen or a portion thereof, alone or in complex with MHC molecules, B cells, etc. "Assessing an immune response" refers to any measurement or determination of the level, presence or absence, reduction, increase, etc., of an 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 of the methods provided herein or otherwise known in the art. Any one of the methods provided herein may include a step of assessing the immune response.

[0031] "Attach" or "attached" or "link" or "linked" (etc.) means to chemically associate one entity (e.g., moiety) with another. In some embodiments, the attachment is covalent, meaning that the attachment occurs in the context of the presence of a covalent bond between the two entities. In non-covalent embodiments, the non-covalent attachment is mediated by non-covalent interactions, including, but not limited to, charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bond interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and / or combinations thereof. In some embodiments, encapsulation is a form of attachment. In some embodiments, the therapeutic macromolecule and the immunosuppressant are not attached to each other, meaning that the therapeutic macromolecule and the immunosuppressant are not subjected to a process specifically intended to chemically associate one with the other. In some embodiments, the therapeutic polymer and / or immunosuppressant are 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.

[0032] "Average," as used herein, refers to the arithmetic mean value unless otherwise noted. "Class of subject" refers to a grouping of subjects that share one or more common characteristics (biological classification, eating habits, sleep habits, physical presence in immune system biology, etc.). Classes do not have to follow only standard biological classifications. Determining outcomes in a class of subjects can be useful for predicting achievable outcomes in another class of subjects (e.g., using animal models in predicting or exploring human disease).

[0033] "Determining" or "determining" means ascertaining a factual situation. Determining can be accomplished in a number of ways, including but not limited to conducting an experiment or making a prediction. By way of example, the dose of an immunosuppressant or therapeutic macromolecule can 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 can 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 embodiments, "determining" or "determining" includes "having determined". "Having determined" means having, encouraging, encouraging, assisting, inducing or directing an entity to ascertain a factual situation, or acting in cooperation with that entity, including directly or indirectly, or explicitly or implicitly.

[0034] "Dosage form" means 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. "Dose" refers to a specific quantity of a pharmacologically and / or immunologically active material to be administered to a subject over a given period of time. "Encapsulate" means to enclose 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 forms, no more than 50%, 40%, 30%, 20%, 10% or 5% (weight / weight) is exposed to the local environment. Encapsulation is distinct from absorption, which places most or all of a substance on the surface of the synthetic nanocarrier, leaving the substance exposed to the local environment outside the synthetic nanocarrier.

[0035] "Generating" means causing, directly or indirectly, an effect such as a physiological or immunological response (eg, a tolerogenic immune response) by itself. "Identifying a subject" is any action or set of actions that allows a clinician to recognize a subject as a subject who may benefit from a method, composition, or kit provided herein. Preferably, the identified subject is in need of a therapeutic benefit from a therapeutic macromolecule as provided herein, and / or is experiencing or expected to experience (or is at risk of experiencing) an anti-therapeutic macromolecule specific antibody response. The action or set of actions may be direct in itself or indirect. In one embodiment of any one of the methods provided herein, the method further comprises identifying a subject in need of a method, composition, or kit provided herein.

[0036] "Immunosuppressant" refers to a compound that causes APC to have an immunosuppressive effect (e.g., a tolerogenic effect) or to suppress T cells or B cells. Immunosuppressant effect generally refers to the production or expression by APC of cytokines or other factors that reduce, inhibit or prevent undesired immune responses or promote desirable immune responses, such as regulatory immune responses. When APC acquires (under immunosuppressant effect) immunosuppressive function on immune cells that recognize the antigen presented by this APC, the immunosuppressant effect is said to be specific to the antigen presented. Without being bound to any particular theory, it is believed that the immunosuppressant effect is the result of the immunosuppressant being delivered to the APC, preferably in the presence of the antigen. In one embodiment, the immunosuppressant causes the APC to promote a regulatory phenotype in one or more immune effector cells. For example, the regulatory phenotype may be characterized by the inhibition of the production, induction, stimulation or mobilization of antigen-specific CD4+ T cells or B cells, the inhibition of the production of antigen-specific antibodies, the production, induction, stimulation or mobilization of Treg cells (e.g., CD4+CD25highFoxP3+ Treg cells), etc. This may be the result of the conversion of CD4+ T cells or B cells to a regulatory phenotype. This may also be the result of the induction of FoxP3 in other immune cells, such as CD8+ T cells, macrophages and iNKT cells. In one embodiment, the immunosuppressant is one that affects the response of APCs after processing antigen. In another embodiment, the immunosuppressant is not one that interferes with the processing of antigen. In a further embodiment, the immunosuppressant is not an apoptotic signaling molecule. In another embodiment, the immunosuppressant is not a phospholipid.

[0037] 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; inhibitors of mitochondrial function, such as rotenone; P38 inhibitors; NF-κβ inhibitors, such as 6Bio, dexamethasone, TCPA-1, and 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 agonists. These include, but are not limited to, oxidized ATP 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 P2X receptor blockers. Immunosuppressants also include IDO, vitamin D3, cyclosporines such as cyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol, azathioprine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sanglifehrin A, salmeterol, mycophenolate mofetil (MMF), aspirin and other COX inhibitors, niflumic acid, estriol, methotrexate, and triptolide. In some embodiments, the immunosuppressant may include any of the agents provided herein.

[0038] An immunosuppressant may be a compound that directly provides an immunosuppressant effect on APCs, or a compound that provides an immunosuppressant effect indirectly (i.e., after being processed in some manner after administration). Thus, immunosuppressants include 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 the synthetic nanocarrier. In a preferred embodiment, the immunosuppressant is an additional element to the material that constitutes 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 an additional compound attached to the one or more polymers. As another example, in one embodiment where the synthetic nanocarrier is composed of one or more lipids, the immunosuppressant is also an additional compound attached to the one or more lipids. In some embodiments, such as when the material of the synthetic nanocarrier also provides an immunosuppressant effect, the immunosuppressant is an additional element present in the material of the synthetic nanocarrier that provides the immunosuppressant effect.

[0039] In some embodiments of any one of the methods, compositions, or kits provided herein, the immunosuppressant is in a form such as a nanocrystalline form, whereby the form of the immunosuppressant itself is particulate or particle-like. In some embodiments, such forms mimic viruses or other foreign pathogens. Many drugs are nanosized, 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 Resesarch; Vol. 1, No. 3; pp682-694). As used herein, "drug nanocrystal" refers to a form of a drug (e.g., an immunosuppressant) that is not encapsulated in a carrier or matrix material. In some embodiments, the drug drug nanocrystal comprises 90%, 95%, 98%, or 99% or more of the drug. Methods for producing drug nanocrystals include, but are not limited to, milling, high pressure homogenization, precipitation, spray drying, rapid expansion of supercritical solutions (RESS), Nanoedge® technology (Baxter Healthcare) and Nanocrystal Technology™ (Elan Corporation). In some embodiments, surfactants or stabilizers may be used for steric or electrostatic stability of drug nanocrystals. In some embodiments, nanocrystals or nanocrystalline forms of immunosuppressants can be used to increase the solubility, stability and / or bioavailability of immunosuppressants, especially insoluble or labile immunosuppressants. In some embodiments, administration of nanocrystalline forms of immunosuppressants induces tolerance to therapeutic macromolecules.

[0040] Other exemplary immunosuppressants include, but are not limited to, small molecule drugs, natural products, antibodies (e.g., antibodies against CD20, CD3, CD4), biologic drugs, carbohydrate drugs, nanoparticles, liposomes, RNAi, antisense nucleic acids, aptamers, methotrexate, NSAIDs; fingolimod; natalizumab; alemtuzumab; anti-CD3; tacrolimus (FK506); cytokines and growth factors such as TGF-β and IL-10; and the like. In any one of the aspects or embodiments provided herein, the immunosuppressant can be attached to a synthetic nanocarrier or delivered in the form of an implantable osmotic pump, ALZET® implantable osmotic pump, implantable depot material, or bispecific antibodies (anti-CD22+Ag, anti-GITR+Ag, or anti-LAG3+Ag), and the like. Further immunosuppressants are known to those skilled in the art, and the invention is not limited in this respect.

[0041] The "load" of an immunosuppressant when attached to a synthetic nanocarrier is the amount of immunosuppressant attached to the synthetic nanocarrier based on the total dry recipe weight of material in the synthetic nanocarrier (wt / wt). Generally, the loading is calculated as an average across a population of synthetic nanocarriers. In one embodiment, the loading of the immunosuppressant, averaged across the synthetic nanocarriers, ranges from 0.0001% to 99% by weight. In another embodiment, the loading of the immunosuppressant ranges from 0.01% to 50% by weight. In another embodiment, the loading is from 0.1% to 20% by weight. In a further embodiment, the loading of the immunosuppressant ranges from 0.1% to 10% by weight. In yet a further embodiment, the loading of the immunosuppressant ranges from 1% to 10% by weight. In yet a further embodiment, the loading is from 7% to 20% by weight. In yet another embodiment, the immunosuppressant loading is, on average across a population of synthetic nanocarriers, at least 0.1 wt%, at least 0.2 wt%, at least 0.3 wt%, at least 0.4 wt%, at least 0.5 wt%, at least 0.6 wt%, at least 0.7 wt%, at least 0.8 wt%, at least 0.9 wt%, at least 1 wt%, at least 2 wt%, at least 3 wt%, at least 4 wt%, at least 5 wt%, at least 6 wt%, at least 7 wt%, at least 8 wt%, at least 9 wt%, at least 10 wt%, at least 12 wt%, at least 14 wt%, at least 16 wt%, at least 18 wt%, at least 19 wt%, at least 20 wt%, at least 22 wt%, at least 24 wt%, at least 26 wt%, at least 28 wt%, at least 29 wt%, at least 30 wt%, at least 31 wt%, at least 32 wt%, at least 33 wt%, at least 34 wt%, at least 35 wt%, at least 36 wt%, at least 37 wt%, at least 38 wt%, at least 39 wt%, at least 40 ... In the range of 11% by weight, at least 12% by weight, at least 13% by weight, at least 14% by weight, at least 15% by weight, at least 16% by weight, at least 17% by weight, at least 18% by weight, at least 19% by weight, at least 20% by weight, at least 25% by weight, at least 30% by weight, at least 40% by weight, at least 50% by weight, at least 60% by weight, at least 70% by weight, at least 80% by weight, at least 90% by weight, at least 95% by weight, at least 96% by weight, at least 97% by weight, at least 98% by weight or at least 99% by weight.In still further embodiments, the immunosuppressant 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% by weight, on average across the population of synthetic nanocarriers. In some of the above embodiments, the immunosuppressant loading is not more than 25% by weight, on average across the population of synthetic nanocarriers. In some embodiments, the loading is calculated as may be described in the examples or as otherwise known in the art.

[0042] In some embodiments, when the form of the immunosuppressant is a particle or particle-like per se, such as a nanocrystalline immunosuppressant, the loading of the immunosuppressant is the amount (weight / weight) of immunosuppressant in the particle, etc. In such embodiments, the loading can reach 97%, 98%, 99% or more.

[0043] "Maximum dimension of a synthetic nanocarrier" means the longest dimension of the nanocarrier measured along any axis of the synthetic nanocarrier. "Minimum dimension of a synthetic nanocarrier" means 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 cube-like 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. In one embodiment, the minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is 100 nm or greater.

[0044] In one embodiment, the maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample based on the total number of synthetic nanocarriers in the sample is 5 μm or less. Preferably, the minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample based on the total number of synthetic nanocarriers in the sample is greater than 110 nm, more preferably greater than 120 nm, more preferably greater than 130 nm, and more preferably still greater than 150 nm. The aspect ratio of the maximum dimension to the minimum dimension of the synthetic nanocarriers can vary depending on the embodiment. For example, the aspect ratio of the maximum dimension to the minimum dimension of the synthetic nanocarriers can vary from 1:1 to 1,000,000:1, preferably from 1:1 to 100,000:1, more preferably from 1:1 to 10,000:1, more preferably from 1:1 to 1000:1, even more preferably from 1:1 to 100:1, and even more preferably from 1:1 to 10:1. Preferably, at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, have a maximum dimension that 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.

[0045] In preferred embodiments, at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample based on the total number of synthetic nanocarriers in the sample have a minimum dimension of 100 nm or more, more preferably 120 nm or more, more preferably 130 nm or more, more preferably 140 nm or more, and even more preferably 150 nm or more. Measurements of synthetic nanocarrier dimensions (e.g., effective dimensions) may be obtained in some embodiments 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 may be diluted from an aqueous buffer into 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 inside or transferred to a cuvette suitable for DLS analysis. The cuvette may then be placed in the DLS, equilibrated to a controlled temperature, and then scanned for a sufficient 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. The effective diameter or mean value of the distribution is then reported. Determination of the effective size of high aspect ratio or non-spheroidal synthetic nanocarriers may require augmentative techniques, such as electron microscopy, to obtain a more accurate measurement. "Dimensions" or "size" or "diameter" of synthetic nanocarriers refers to the mean value of the particle size distribution, as obtained, for example, using dynamic light scattering.

[0046] "Non-methoxy terminated polymer" refers to a polymer having at least one end terminating at a site other than methoxy. In some embodiments, the polymer has at least two ends terminating at a site other than methoxy. In other embodiments, the polymer has no ends terminating at methoxy. "Non-methoxy terminated Pluronic polymer" refers to a polymer other than a linear Pluronic polymer having methoxy at both ends. The polymeric nanoparticles provided herein can include non-methoxy terminated polymers or non-methoxy terminated Pluronic polymers. "Pharmaceutically acceptable excipient" or "Pharmaceutically acceptable carrier" refers to a pharmacologically inactive material used together with a pharmacologically active material to formulate a composition. Pharmaceutically acceptable excipients include various materials known in the art, including, but not limited to, sugars (e.g., glucose, lactose, etc.), preservatives such as antibacterial agents, reconstitution aids, coloring agents, saline (e.g., phosphate buffered saline), and buffers.

[0047] "Pharmacodynamic effectiveness period" refers to a first dose-dependent period following administration of an immunosuppressant to a subject, during which administration of the antigen results in antigen-specific tolerance in a measurable subject during a second period beginning 5 days to 3 months after administration of the antigen. In embodiments, the pharmacodynamic effectiveness period can be measured as the average of individual pharmacodynamic effectiveness periods measured in one or more subjects, a first class of subjects, etc., and can be measured a single time or multiple times in each measured subject. "Providing" refers to an action or set of actions taken by an individual that supplies an item or set of items or a method required to carry out the invention. The action or set of actions may be taken directly or indirectly by oneself.

[0048] "Providing a subject" refers to any action or set of actions that allows a clinician to contact a subject and administer the composition provided herein to the subject or perform the method provided herein on the subject.Preferably, the subject is one that requires administration of a therapeutic macromolecule and antigen-specific tolerance thereto.The action or set of actions can be taken directly or indirectly by itself.In one embodiment of any one of the methods provided herein, the method further comprises providing a subject. "Subject" means animals, including warm-blooded animals such as humans and primates; birds; pets or farm animals such as cats, dogs, sheep, goats, cows, horses, and pigs; laboratory animals such as mice, rats, and guinea pigs; fish; reptiles; zoo and wild animals; and the like. "Synthetic nanocarrier(s)" refers to a discrete object not found in nature that possesses at least one dimension less than or equal to 5 microns 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.

[0049] Synthetic nanocarriers may be, but are not limited to, nanoparticles developed using one or more lipid-based nanoparticles (also referred to herein as lipid nanoparticles, i.e., nanoparticles whose structural material is predominantly lipid), polymeric nanoparticles, metal nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles (i.e., particles that are primarily composed of viral structural proteins but are not or less infectious), peptide- or protein-based particles (also referred to herein as protein particles, i.e., particles whose structural material is predominantly peptides or proteins) (such as albumin nanoparticles) and / or combinations of nanomaterials such as lipid-polymer nanoparticles. Synthetic nanocarriers may be of a variety of different shapes, including, but not limited to, spheroidal, cubic, pyramidal, elliptical, cylindrical, donut-shaped, etc.

[0050] Synthetic nanocarriers according to the present invention comprise one or more surfaces. Exemplary synthetic nanocarriers that may be adapted for use in the practice of the present invention include (1) biodegradable nanoparticles as disclosed in U.S. Pat. No. 5,543,158 (Gref et al.), (2) polymeric nanoparticles as disclosed in published U.S. Pat. App. No. 20060002852 (Saltzman et al.), (3) lithographically constructed nanoparticles as disclosed in published U.S. Pat. App. No. 20090028910 (DeSimone et al.), (4) nanoparticles as disclosed in International Patent Publication No. 2009 / 051837 (von Andrian et al.), (5) nanoparticles as disclosed in published U.S. Pat. App. No. 2008 / 0145441 (Penades et al.), (6) protein nanoparticles as disclosed in published U.S. Pat. App. No. 20090226525 (de los Rios et al.), (7) protein nanoparticles as disclosed in published U.S. Pat. App. No. 20060222652 (Sebbel et al.), and (8) nanoparticles as disclosed in published U.S. Pat. App. No. 20060222652 (Sebbel et al.). al.), (8) nucleic acid-attached virus-like particles disclosed in published U.S. Patent Application No. 20060251677 (Bachmann et al.), (9) virus-like particles disclosed in WO 2010047839A1 or WO 2009106999A2, (10) 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), (11) apoptotic cells, apoptotic bodies, or synthetic or semi-synthetic mimetics disclosed in U.S. Patent Publication No. 2002 / 0086049, or (12) Look et al., "Nanogel-based delivery of mycophenolic acid ameliorates systemic lupus erythematosus in mice", J. Clinical Investigation 123(4): 1741-1749 (2013).In certain embodiments, synthetic nanocarriers may possess an aspect ratio of greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.

[0051] Synthetic nanocarriers according to the invention having a minimum dimension of about 100 nm or less, preferably 100 nm or less, do not include a surface with hydroxyl groups that activate complement, or alternatively include a surface that consists essentially of sites that are not hydroxyl groups that activate complement. In a preferred embodiment, synthetic nanocarriers according to the invention having a minimum dimension of about 100 nm or less, preferably 100 nm or less, do not include a surface that substantially activates complement, or alternatively include a surface that consists essentially of sites that do not substantially activate complement. In a more preferred embodiment, synthetic nanocarriers according to the invention having a minimum dimension of about 100 nm or less, preferably 100 nm or less, do not include a surface that activates complement, or alternatively include a surface that consists essentially of sites that do not activate complement. In some embodiments, the synthetic nanocarriers exclude virus-like particles. In some embodiments, the synthetic nanocarriers 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.

[0052] "Test dose" or "test dose of immunosuppressant" means the dose of an immunosuppressant for testing. "Test pharmacodynamic effective period" refers to the pharmacodynamic effective period of the immunosuppressant determined for a test dose of the immunosuppressant therapeutic macromolecule and a second class of subjects. In some embodiments, the recited immunosuppressant may have a test pharmacodynamic effective period with respect to the therapeutic macromolecule having a period ranging from a minimum of 20 hours to a maximum of 1 month, preferably from a minimum of 20 hours to a maximum of 2 weeks, preferably from a minimum of 20 hours to a maximum of 1 week, preferably from a minimum of 20 hours to a maximum of 5 days, preferably from a minimum of 20 hours to a maximum of 3 days, preferably from a minimum of 24 hours to a maximum of 2 days.

[0053] A "therapeutic macromolecule" refers to any protein, carbohydrate, lipid, or nucleic acid that can be administered to a subject and have a therapeutic effect. In some embodiments, administration of a therapeutic macromolecule to a subject can result in an unwanted immune response, including the production of anti-therapeutic macromolecule-specific antibodies. As described herein, in some embodiments, administration of a therapeutic macromolecule can improve the therapeutic efficacy of the therapeutic macromolecule, such as by reducing an unwanted immune response thereto. In some embodiments, the therapeutic macromolecule can be a therapeutic polynucleotide or a therapeutic protein.

[0054] "Therapeutic polynucleotide" refers to any polynucleotide or polynucleotide-based therapy that can be administered to a subject and have a therapeutic effect. Such therapy includes gene silencing. Examples of such therapy 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 described elsewhere herein. Therapeutic polynucleotides may be produced in, on, or by cells, and may be obtained using cell-free methods or fully synthetic in vitro methods. Subjects therefore include any subject in need of treatment by any of the above. Such subjects include subjects who will receive any of the above.

[0055] "Therapeutic protein" refers to any protein or protein-based therapy that can be administered to a subject to have a therapeutic effect. Such therapies include protein replacement therapy and protein supplementation therapy. Such therapies also include administration of exogenous or foreign proteins, antibody therapy, and cell or cell-based therapy. Therapeutic proteins include, but are not limited to, enzymes, enzyme cofactors, hormones, blood clotting factors, cytokines, growth factors, monoclonal antibodies, antibody-drug conjugates, and polyclonal antibodies. Examples of other therapeutic proteins are described elsewhere 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, and the like. Therapeutic proteins may be recombinantly produced in such cells. Therapeutic proteins may be produced in, on, or by virally transformed cells. A subject therefore includes any subject in need of treatment with any of the foregoing. Such subjects include subjects who will be undergoing any of the foregoing.

[0056] C. Compositions Useful in Carrying Out the Method Provided herein are methods and related compositions useful for reducing the occurrence of undesired immune responses and promoting the occurrence of tolerogenic immune responses specific to therapeutic macromolecules. The methods and compositions help maximize the benefits of administering immunosuppressants to therapeutic macromolecules of immunosuppressants by administering the therapeutic macromolecules for a pharmacodynamic effective period. The methods of the present invention can be carried out on subjects in which a tolerogenic immune response to the therapeutic macromolecules is desired. Such subjects include those who will be administered the therapeutic macromolecules. Of particular utility in carrying out the present invention are immunosuppressants that have a pharmacodynamic effective period that ranges from a minimum of 20 hours to a maximum of one month in relation to the therapeutic macromolecules. A variety of immunosuppressants may be used in carrying out the present invention.

[0057] In certain embodiments, the recited immunosuppressants may be in the form of a device such as an implantable osmotic pump. One such implantable osmotic pump that may be used in the practice of the present invention is the ALZET® brand implantable osmotic pump (available from Durect Corporation, Cupertino CA). The ALZET® brand osmotic pump is a small implantable osmotic pump that can continuously deliver drugs, hormones and other test agents at a controlled rate for 1 day to 4 weeks without the need for external connections, frequent handling or repeated dosing. These infusion pumps can be used for systemic administration when implanted under the skin or in the body. They can be attached to catheters for intravenous, intracerebral or intraarterial infusion or targeted delivery, where the effect of the drug or test agent is localized to a specific tissue or organ. The pumps are powered by the osmotic pressure difference between the pump and the animal's body fluids, and therefore do not require an external power source. The ALZET pumps are used to target delivery to a wide variety of sites, including the spinal cord, spleen, liver, organ or tissue transplants and wound healing sites. See, e.g., SM Stepkowski et al., "Inhibition of host-versus-graft and graft-versus-host responses after small bowel transplantation in rats by rapamycin." Transplantation (1992) 53(-2-):258-264. Additional information regarding ALZET® brand osmotic pumps is available at alzet.com.

[0058] In some embodiments, the recited immunosuppressants may be in the form of bispecific antibodies (BsAbs) with one variable region designed to target a receptor for an antigen (such as a BCR) that selectively binds to a macromolecule of interest on an immune cell and the other variable region designed to bind to a target on an immune cell involved in stimulating tolerance (such as a cell surface receptor). In another embodiment, the BsAb consists of one variable region (anti-idiotypic region or antibody) designed to target the variable region of another antibody that binds to the macromolecule of interest and the other variable region designed to bind to a target on an immune cell involved in stimulating tolerance (such as a cell surface receptor). The combination of two binding specificities can improve the selectivity and efficacy of the BsAb immunosuppressant and can induce immune tolerance to the macromolecule of interest. In some embodiments, immunosuppressant BsAb targets include, but are not limited to, CD19, CD20, CD21, CD22 (Ref: MR Clatworthy, American Journal of Transplantation 2011; 11: 1359-1367) on B cells, and GITR (glucocorticoid-induced tumor necrosis factor (TNF) receptor family related gene). GITR is a type I transmembrane protein with homology to TNF receptor family members. GITR is expressed at low levels on resting CD4+ and CD8+ T cells and upregulated following T cell activation. Ligation of GITR provides a costimulatory signal that enhances the proliferation and efficacy or function of both CD4+ and CD8+ T cells, especially in the setting of suboptimal T cell receptor (TCR) stimulation. Additionally, GITR is continuously expressed at high levels on regulatory T cells (Tregs) and has been explored as a potential target to overcome Treg suppression. Signaling through GITR using GITR ligands neutralizes the suppressive effects of Tregs and enhances autoreactive and alloreactive T cell responses, exacerbating autoimmunity. Another BsAb target may include LAG3 (also known as CD223).CD223 binds to MHC class II with higher affinity than CD4, and this interaction is believed to be involved in the negative control of T cell activation and homeostatic proliferation. Furthermore, CD223 is expressed by CD4+CD25+ regulatory T cells, suggesting that CD223 may be involved in their regulatory function in addition to other such targets known in the art.

[0059] In certain embodiments, the recited immunosuppressant may be in the form of an implantable polymeric depot material. In some embodiments, the implantable polymeric depot material comprises a solid matrix of a microporous, biocompatible, biodegradable, thermoplastic polymer, a rate modifying agent, and a bioactive material. The matrix is ​​formed in solution or suspension to form an injectable liquid. The matrix controls the rate and extent of release of the bioactive agent from the matrix. The process by which the depot is partially formed is responsible for the development of the rate and release control. The interaction of the liquid composition with an aqueous medium, either in situ in the body or outside the body, to solidify the polymer system results in a desired controlled release profile, at least in part, as a function of the diversity of components and the concentration of the various components. Exemplary implantable polymeric depot materials can be found in U.S. Pat. No. 5,702,716 (Dunn et al.), entitled "Polymeric compositions useful as controlled release implants," and U.S. Pat. No. 6,130,200 (Brodbeck et al.), entitled "Gel compositions and methods."

[0060] The listed immunosuppressants can also be administered in the form of synthetic nanocarriers that contain the immunosuppressant. 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 planar. In some embodiments, the synthetic nanocarriers are cubic or cuboidal. In some embodiments, the synthetic nanocarriers are ovoid or elliptical. In some embodiments, the synthetic nanocarriers are cylindrical, conical, or pyramidal. In some embodiments, it is desirable to use a population of synthetic nanocarriers that are relatively uniform in size or shape, such that each synthetic nanocarrier has similar properties. For example, at least 80%, at least 90%, or at least 95% of the synthetic nanocarriers, based on the total number of synthetic nanocarriers, may have a minimum or maximum dimension that falls within 5%, 10%, or 20% of the average diameter or average dimension of the synthetic nanocarriers.

[0061] Synthetic nanocarriers may be solid or hollow and may include one or more layers. In some embodiments, each layer has a unique composition and unique properties relative to the other layer(s). For example, synthetic nanocarriers may have a core / shell structure, where 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). Synthetic nanocarriers may include multiple different layers.

[0062] In some embodiments, synthetic nanocarriers optionally include one or more lipids. In some embodiments, synthetic nanocarriers may include liposomes. In some embodiments, synthetic nanocarriers may include lipid bilayers. In some embodiments, synthetic nanocarriers may include lipid monolayers. In some embodiments, synthetic nanocarriers may include micelles. In some embodiments, synthetic nanocarriers may include a core comprising a polymer matrix surrounded by a lipid layer (e.g., a lipid bilayer, a lipid monolayer, etc.). In some embodiments, synthetic nanocarriers may include a non-polymer core (e.g., a metal particle, a quantum dot, a ceramic particle, a bone particle, a virus particle, a protein, a nucleic acid, a carbohydrate, etc.) surrounded by a lipid layer (e.g., a lipid bilayer, a lipid monolayer, etc.). In other embodiments, synthetic nanocarriers may include metal particles, quantum dots, ceramic particles, etc. In some embodiments, non-polymeric synthetic nanocarriers are aggregates of non-polymeric components, such as aggregates of metal atoms (e.g., gold atoms).

[0063] In some embodiments, synthetic nanocarriers may optionally include one or more amphiphilic entities. In some embodiments, amphiphilic entities may facilitate the production of synthetic nanocarriers with increased stability, improved homogeneity, or increased viscosity. In some embodiments, amphiphilic entities may be associated with the inner surface of a lipid membrane (e.g., lipid bilayer, lipid monolayer, etc.). Many amphiphilic entities known in the art are suitable for use in making synthetic nanocarriers according to the present invention. Such amphiphilic entities include, but are not limited to, phosphoglycerides; phosphatidylcholine; dipalmitoylphosphatidylcholine (DPPC); dioleylphosphatidylethanolamine (DOPE); dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine; cholesterol; cholesterol esters; diacylglycerol; diacylglycerol succinate; diphosphatidylglycerol (DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol (PEG); 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® 85) glycocholate; sorbitan monolaurate (Span® 20); polysorbate 20 (Tween® 20);

[0064] Polysorbate 60 (Tween® 60); Polysorbate 65 (Tween® 65); Polysorbate 80 (Tween® 80); Polysorbate 85 (Tween® 85); Polyoxyethylene monostearate; Surfactin; Poloxomer; Sorbitan fatty acid esters such as sorbitan trioleate; Lecithin; Lysolecithin; Phosphatidylserine; Phosphatidylinositol; Sphingomyelin; Phosphatidylethanolamine (cephalin); Cardiolipin; Phosphatidic acid; Cerebrosides; Dicetylphos Examples of amphiphilic entities include, but are not limited to, phosphate; dipalmitoyl phosphatidylglycerol; stearylamine; dodecylamine; hexadecylamine; acetyl palmitate; glycerol ricinoleate; hexadecyl stearate; isopropyl myristate; tyloxapol; poly(ethylene glycol) 5000-phosphatidylethanolamine; poly(ethylene glycol) 400-monostearate; phospholipids; synthetic and / or natural detergents with high surfactant activity; deoxycholate; cyclodextrin; chaotropic salts; ion pairing agents; and combinations thereof. The amphiphilic entity component may be a mixture of different amphiphilic entities. Those skilled in the art will recognize that this is a non-exhaustive, exemplary list of substances with surfactant activity. Any amphiphilic entity may be used to generate synthetic nanocarriers for use in accordance with the present invention.

[0065] In some embodiments, the synthetic nanocarrier may optionally include one or more carbohydrates. The carbohydrate may be natural or synthetic. The carbohydrate may be a derived natural carbohydrate. In certain embodiments, the carbohydrate comprises a monosaccharide or disaccharide, including, but not limited to, glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellobiose, mannose, xylose, arabinose, gluconic acid, galactonic acid, mannuronic acid, glucosamine, galatosamine, and neuraminic acid. In certain embodiments, the carbohydrate is a polysaccharide, including, but not limited to, pullulan, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen, hydroxyethyl starch, carrageenan, glycon, amylose, chitosan, N,O-carboxymethyl chitosan, algin and alginic acid, starch, chitin, inulin, konjac, glucomannan, pustulan, heparin, hyaluronic acid, curdlan and xanthan. In some embodiments, the synthetic nanocarrier does not include (or specifically excludes) carbohydrates such as polysaccharides. In certain embodiments, the carbohydrate may include carbohydrate derivatives, such as sugar alcohols, including, but not limited to, mannitol, sorbitol, xylitol, erythritol, maltitol and lactitol.

[0066] In some embodiments, the synthetic nanocarriers may include one or more polymers. In some embodiments, the synthetic nanocarriers include 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% (w / w) of the polymers that make up the synthetic nanocarriers are non-methoxy-terminated Pluronic polymers. In some embodiments, all of the polymers that make up the synthetic nanocarriers are non-methoxy-terminated Pluronic polymers. In some embodiments, the synthetic nanocarriers include 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% (weight / weight) of the polymers that make up the synthetic nanocarrier are non-methoxy terminated polymers. In some embodiments, all of the polymers that make up the synthetic nanocarrier are non-methoxy terminated polymers. In some embodiments, the synthetic nanocarrier comprises one or more polymers that do not include 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% (weight / weight) of the polymers that make up the synthetic nanocarrier do not contain Pluronic polymers. In some embodiments, all of the polymers that make up the synthetic nanocarrier do not contain 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 a polymer.

[0067] The immunosuppressant may be attached to the synthetic nanocarrier by any of a number of methods. In general, the attachment may be the result of 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 being contained (encapsulated) within the synthetic nanocarrier. However, in some embodiments, the immunosuppressant is encapsulated by the synthetic nanocarrier as a result of the structure of the synthetic nanocarrier rather than binding to the synthetic nanocarrier. In a preferred embodiment, the synthetic nanocarrier comprises a polymer provided herein and the immunosuppressant is attached to the polymer. When the attachment occurs as a result of binding between the immunosuppressant and the synthetic nanocarrier, the attachment can occur via a linkage site. The linkage site can be any site through which the immunosuppressant is bound to the synthetic nanocarrier. Such sites include covalent bonds, such as amide or ester bonds, as well as separate molecules that bind (covalently or non-covalently) the immunosuppressant to the synthetic nanocarrier. Such molecules include linkers, or polymers or units thereof. For example, the linkage site can include a charged polymer to which the immunosuppressant is electrostatically bound. As another example, the linkage site can include a polymer or units thereof to which it is covalently bound.

[0068] In a preferred embodiment, the synthetic nanocarriers comprise the polymers provided herein. These synthetic nanocarriers may be entirely polymeric or may be a mixture of polymers and other materials. In some embodiments, the polymers of the synthetic nanocarriers are associated with each other to form a polymer matrix. In some of these embodiments, a component, such as an immunosuppressant, can be covalently associated with one or more polymers of the polymer matrix. In some embodiments, the covalent association is mediated by a linker. In some embodiments, a component can be non-covalently associated with one or more polymers of the polymer matrix. For example, in some embodiments, a component can be encapsulated within, surrounded by, and / or dispersed throughout the polymer matrix. Alternatively or additionally, a component can be associated with 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 known in the art.

[0069] The polymer may be a natural or non-natural (synthetic) polymer. The polymer may be a homopolymer or a copolymer comprising two or more monomers. In terms of sequence, the copolymer may be random, block or may contain a combination of random and block sequences. 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-co-glycolic acid) or polycaprolactone, or units thereof. In some embodiments, the polymer is preferably biodegradable. Thus, in these embodiments, when the polymer comprises a polyether, such as poly(ethylene glycol) or polypropylene glycol or units thereof, it is preferred that the polymer comprises a block copolymer of polyether and a biodegradable polymer, so that it is biodegradable. In other embodiments, the polymer does not comprise a polyether, such as poly(ethylene glycol) or polypropylene glycol or units thereof, by itself, or units thereof.

[0070] Other examples of polymers suitable for use in the present invention include, but are not limited to, polyethylene, polycarbonates (e.g., poly(1,3-dioxan-2-one)), polyanhydrides (e.g., poly(sebacic anhydride)), polypropyl fumarate, polyamides (e.g., polycaprolactam), polyacetals, polyethers, polyesters (e.g., polylactides, polyglycolides, polylactide-co-glycolides, polycaprolactones, polyhydroxy acids (e.g., poly(β-hydroxyalkanoates)), poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polyureas, polystyrenes, and polyamines, polylysine, polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymers.

[0071] In some embodiments, polymers in accordance with the present invention include polymers approved for human use by the United States Food and Drug Administration (FDA) under 21 C.FR § 177.2600, including, but not limited to, polyesters (e.g., polylactic acid, poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2-one)); polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; and polycyanoacrylates.

[0072] In some embodiments, the polymer can be hydrophilic. For example, the polymer can include 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, synthetic nanocarriers that include a hydrophilic polymer matrix generate a hydrophilic environment within the synthetic nanocarrier. In some embodiments, the polymer can be hydrophobic. In some embodiments, synthetic nanocarriers that include a hydrophobic polymer matrix generate a hydrophobic environment within the synthetic nanocarrier. The selection of the hydrophilic or hydrophobic nature of the polymer can have an impact on the properties of the material incorporated (e.g., attached) within the synthetic nanocarrier. In some embodiments, the polymer can be modified with one or more sites and / or functional groups. A variety of sites or functional groups can be used according to the present invention. In some embodiments, the polymer can be modified with polyethylene glycol (PEG), carbohydrates, and / or acyclic polyacetals derived from polysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Particular embodiments can be made using the general teachings of U.S. Pat. No. 5,543,158 (Gref et al.) or WO 2009 / 051837 (Von Andrian et al.).

[0073] In some embodiments, the polymer can be modified with lipid or fatty acid group.In some embodiments, the fatty acid group can 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 group can 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.

[0074] In some embodiments, the polymer may be a polyester, including copolymers containing lactic acid and glycolic acid units, such as poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide), collectively referred to herein as "PLGA"; and homopolymers containing glycolic acid units, collectively referred to 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 to 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 copolymers, PGA-PEG copolymers, PLGA-PEG copolymers, and derivatives thereof). In some embodiments, polyesters include, for example, poly(caprolactone), poly(caprolactone)-PEG copolymers, 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.

[0075] In some embodiments, the polymer is PLGA. PLGA is a biocompatible and biodegradable copolymer of lactic acid and glycolic acid, and the various forms of PLGA are characterized by the ratio of lactic acid:glycolic acid. Lactic acid can be L-lactic acid, D-lactic acid or D,L-lactic acid. The degradation rate of PLGA can be adjusted by changing the ratio of lactic acid:glycolic acid. In some embodiments, the PLGA used according to the present invention is characterized by a lactic acid:glycolic acid ratio of approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, or approximately 15:85.

[0076] 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), methacrylic acid alkylamide copolymers, poly(methyl methacrylate), poly(methacrylic anhydride), methyl methacrylate, polymethacrylate, poly(methyl methacrylate) copolymers, polyacrylamide, aminoalkyl methacrylate copolymers, glycidyl methacrylate copolymers, polycyanoacrylate, and combinations comprising one or more of the above polymers.The acrylic polymer may include fully polymerized copolymers of acrylic and methacrylic acid esters with low content of quaternary ammonium groups.

[0077] In some embodiments, the polymer can be a cationic polymer. Generally, cationic polymers are capable of condensing and / or protecting negatively charged strands of nucleic acids. Amine-containing polymers such as 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., 4:372) are positively charged at physiological pH and form ion pairs with nucleic acids. In some embodiments, synthetic nanocarriers may not include (or may exclude) cationic polymers.

[0078] In some embodiments, the polymer can be a biodegradable polyester with 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).

[0079] The properties of these and other polymers and methods for their preparation are well known in the art (e.g., U.S. Pat. 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 certain suitable 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. Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

[0080] In some embodiments, the polymers may be linear or branched polymers. In some embodiments, the polymers may be dendrimers. In some embodiments, the polymers may be substantially crosslinked to one another. In some embodiments, the polymers may be substantially free of crosslinks. In some embodiments, the polymers may be used in accordance with the present invention without undergoing a crosslinking step. It should further be understood that the synthetic nanocarriers may include block copolymers, graft copolymers, 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-exhaustive list of exemplary polymers that may be used in accordance with the present invention. In some embodiments, synthetic nanocarriers do not include polymeric components. In some embodiments, synthetic nanocarriers may include metal particles, quantum dots, ceramic particles, etc. In some embodiments, non-polymeric synthetic nanocarriers are aggregates of non-polymeric components, such as aggregates of metal atoms (e.g., gold atoms).

[0081] The compositions according to the present invention may contain elements in combination with pharma- ceutically acceptable excipients, such as preservatives, buffers, saline or phosphate-buffered saline. The compositions may be made using conventional pharmaceutical manufacturing and compounding techniques to arrive at a useful dosage form. In some embodiments, compositions such as those containing synthetic nanocarriers are suspended in a sterile saline solution for injection together with a preservative. In some embodiments, when preparing synthetic nanocarriers for use as carriers, methods of attaching components to synthetic nanocarriers can be useful. When the components are small molecules, it can be advantageous to attach the components to a polymer prior to assembly of the synthetic nanocarrier. In some embodiments, rather than attaching the components to a polymer and then using this polymer complex to construct the synthetic nanocarrier, it can also be advantageous to prepare synthetic nanocarriers with surface groups that are used to attach the components to the synthetic nanocarrier through the use of those surface groups.

[0082] In certain embodiments, attachment can be achieved by a covalent linker. In some embodiments, components according to the present invention can be covalently attached to the external surface via a 1,2,3-triazole linker formed by 1,3-dipolar cycloaddition reaction between an azide group on the surface of the nanocarrier and a component containing an alkyne group, or by 1,3-dipolar cycloaddition reaction between an alkyne on the surface of the nanocarrier and a component containing an azide group. Such cycloaddition 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 the Cu(II) compound to a catalytically active Cu(I) compound. This Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is also sometimes referred to as a click reaction. In addition, the covalent attachment may involve covalent linkers including amide linkers, disulfide linkers, thioether linkers, hydrazone linkers, hydrazide linkers, imine or oxime linkers, urea or thiourea linkers, amidine linkers, amine linkers, and sulfonamide linkers.

[0083] Amide linkers are formed via an amide bond between an amine on one component, such as an immunosuppressant, and a carboxylic acid group on a second component, such as a nanocarrier. The amide bond in the linker can be made using any of the conventional amide bond forming reactions with a suitably protected amino acid and an activated carboxylic acid (such as an N-hydroxysuccinimide activated ester). Disulfide linkers can be created, for example, through the formation of a disulfide (SS) bond between two sulfur atoms in the form 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 the polymer or nanocarrier, or between a nanocarrier containing a thiol / mercaptan group and a component containing an activated thiol group. Triazole linkers, specifically those of the following form: [ka] 1,2,3-triazoles of the formula (wherein R1 and R2 can be any chemical entity) are made by 1,3-dipolar cycloaddition 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-dipolar cycloaddition reaction is carried out with or without a catalyst, preferably using a Cu(I) catalyst, thereby linking the two components through the 1,2,3-triazole functionality. This chemistry is described in more detail in 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.

[0084] In some embodiments, a polymer containing azide or alkyne groups at the end of the polymer chain is prepared. This polymer is then used to prepare a synthetic nanocarrier in such a manner that multiple alkyne or azide groups are located on the surface of the nanocarrier. Alternatively, a synthetic nanocarrier may be prepared by another route and then functionalized with an alkyne or azide group. A component is prepared with either an alkyne (if the polymer contains azide) or azide (if the polymer contains alkyne) group present. The component is then reacted with the nanocarrier through a 1,3-dipolar cycloaddition reaction with or without a catalyst, thereby covalently attaching the component to the particle through a 1,4-disubstituted 1,2,3-triazole linker.

[0085] Thioether linkers are made by the formation of a sulfur-carbon (thioether) bond, for example of the form 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 that contains a maleimide or vinyl sulfone group as a Michael acceptor. In another manner, thioether linkers can be prepared by radical thiol-ene reaction of a thiol / mercaptan group on one component with an alkene group on a second component.

[0086] Hydrazone linkers are made by 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, generally using chemistry similar to the formation of an amide bond when the carboxylic acid is activated with an activating reagent. Imine or oxime linkers are formed by reaction of an amine or N-alkoxyamine (or aminooxy) group on one component with an aldehyde or ketone group on a second component. Urea or thiourea linkers are prepared by reaction of an amine group on one component with an isocyanate or thioisocyanate group on a second component. Amidine linkers are prepared by reaction of an amine group on one component with an imidoester group on a second component.

[0087] Amine linkers are made by the alkylation reaction of an amine group on one component with an alkylating group such as a halide, epoxide or sulfonate ester group on a second component. Alternatively, amine linkers may also be made by reductive amination of an amine group on one component with an aldehyde or ketone group on a second component using a suitable reducing reagent such as sodium cyanoborohydride or sodium triacetoxyborohydride. Sulfonamide linkers may be made by reaction of an amine group on one component with a sulfonyl halide (such as a sulfonyl chloride) group on a second component. The sulfone linker is made by Michael addition of a nucleophilic group to a vinyl sulfone. Either the vinyl sulfone or the nucleophilic group can be on the surface of the nanocarrier or attached to a moiety.

[0088] Components may also be conjugated to nanocarriers via non-covalent conjugation methods. For example, negatively charged immunosuppressants may be conjugated to positively charged nanocarriers through electrostatic adsorption. Components containing metal ligands may also be conjugated to nanocarriers containing metal complexes via metal-ligand complexes. In some embodiments, the components may be attached to a polymer, such as polylactide-block-polyethylene glycol, prior to assembly of the synthetic nanocarrier, or the synthetic nanocarrier may be formed to have reactive or activatable groups on its surface. In the latter case, the components may be prepared to have groups compatible with the attachment chemistry presented by the surface of the synthetic nanocarrier. In other embodiments, the peptide components may be attached to the VLP or liposome using a suitable linker. A linker is a compound or reagent that can attach 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 liposomal synthetic nanocarrier containing carboxyl groups on its surface may be treated with the homobifunctional linker, adipic acid dihydrazide (ADH), in the presence of EDC to form the corresponding synthetic nanocarrier with an ADH linker. The resulting ADH-tethered synthetic nanocarriers are then conjugated to a peptide component containing an acid group via the other end of the ADH linker on the nanocarrier to generate the corresponding VLP or liposome-peptide complex.

[0089] For a detailed description of available conjugation methods, see Hermanson GT "Bioconjugate Techniques", 2nd Edition Published by Academic Press, Inc., 2008. In addition to covalent attachment, components may be attached by adsorption to preformed synthetic nanocarriers or by encapsulation during the formation of the synthetic nanocarriers. Any of the immunosuppressants provided herein can be used in accordance with the present invention, for example, by use in nanocrystalline form, by loading into an ALZET® osmotic pump, or by attachment to synthetic nanocarriers (i.e., immunosuppressants attached to synthetic nanocarriers).

[0090] Any immunosuppressant provided herein may be used in the provided methods or compositions, and in some embodiments may be attached to synthetic nanocarriers. Immunosuppressants include statins; mTOR inhibitors, such as rapamycin or rapamycin analogs; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase (HDAC) inhibitors; corticosteroids; inhibitors of mitochondrial function, such as rotenone; P38 inhibitors; NF-κβ inhibitors; adenosine receptor agonists; prostaglandin E2 agonists; phosphodiesterase inhibitors, such as phosphodiesterase 4 inhibitors; proteasome inhibitors. Immunosuppressants include, but are not limited to, inhibitors, 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. Immunosuppressants also include IDO, vitamin D3, cyclosporin A, aryl hydrocarbon receptor inhibitors, resveratrol, azathioprine, 6-mercaptopurine, aspirin, niflumic acid, estriol, triporide, interleukins (e.g. IL-1, IL-10), cyclosporin A, siRNAs targeting cytokines or cytokine receptors, and the like.

[0091] 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®).

[0092] 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, 13:99-107), AZD8055, BEZ235 (NVP-BEZ235), chrysophanic acid (chrysophanol), deforolimus (MK-8669), everolimus (RAD0001), KU-0063794, PI-103, PP242, temsirolimus and WYE-354 (available from Selleck, Houston, TX, USA).

[0093] 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, chordin, DAN, lefty, LTBP1, THBS1, decorin).

[0094] Examples of inhibitors of mitochondrial function include atractyloside (dipotassium salt), bongkrekic acid (triammonium salt), carbonyl cyanide m-chlorophenylhydrazone, carboxyatractyloside (e.g. from Atractylis sp.), CGP-37157, (-)-deguelin (e.g. from Mundulea sericea), F16, hexokinase II VDAC binding domain peptide, oligomycin, rotenone, Ru360, SFK1 and valinomycin (e.g. from Streptomuces fulvissimus) (EMD4Biosciences, USA). 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-methoxy-pyrimidin-4-yl)imidazole), SB-220025 (5-(2 amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole)) and ARRY-797.

[0095] Examples of NF (e.g., ΝΚ-κβ) inhibitors include IFRD1, 2-(1,8-naphthyridin-2-yl)-phenol, 5-aminosalicylic acid, BAY11-7082, BAY11-7085, CAPE (caffeic acid phenethyl ester), diethyl maleate, IKK-2 inhibitor IV, IMD0354, lactacystin, MG-132 [Z-Leu-Leu-Leu-CHO], and NFκB activation inhibitor III. , NF-κB activation inhibitor II, JSH-23, parthenolide, phenylarsine oxide (PAO), PPM-18, pyrrolidine dithiocarbamic acid ammonium salt, QNZ, RO106-9920, rocaglamide, rocaglamide AL, rocaglamide C, rocaglamide I, rocaglamide J, rocaglaol, (R)-MG-132, sodium salicylate, triptolide (PG490), and wedelolactone.

[0096] 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 xanthines, vinpocetine, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), anagrelide, enoximone (PERFAN®), milrinone, levosimendon, mesembrine. , ibudilast, piclamilast, luteolin, drotaverine, roflumilast (DAXAS™, DALIRESP™), sildenafil (REVATION®, VIAGRA®), tadalafil (ADCIRCA®, CIALIS®), vardenafil (LEVITRA®, STAXYN®), udenafil, avanafil, icariin, 4-methylpiperazine, and pyrazolopyrimidine-7-one.

[0097] 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 mubritinib.

[0098] 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®).

[0099] Examples of cytokine inhibitors include IL1ra, IL1 receptor antagonist, 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 (EMD4Biosciences, USA). Examples of peroxisome proliferator-activated receptor agonists include pioglitazone, ciglitazone, clofibrate, GW1929, GW7647, L-165,041, LY171883, PPARγ activators, Fmoc-Leu, troglitazone and WY-14643 (EMD4Biosciences, USA).

[0100] Examples of histone deacetylase inhibitors include hydroxamic acids (or hydroxamates) such as trichostatin A, cyclic tetrapeptides (such as 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 mocetinostat (MGCD0103), nicotinamide, derivatives of NAD, dihydrocoumarins, naphthopyranones and 2-hydroxynaphaldehydes.

[0101] Examples of calcineurin inhibitors include cyclosporine, pimecrolimus, voclosporin and tacrolimus. Examples of phosphatase inhibitors include BN82002 hydrochloride, CP-91149, calyculin A, cantharidic acid, cantharidin, cypermethrin, ethyl-3,4-dephostatin, fostriecin sodium salt, MAZ51, methyl-3,4-dephostatin, NSC95397, norcantharidin, okadaic acid ammonium salt from prorocentrum concavum, okadaic acid, okadaic acid potassium salt, okadaic acid sodium salt, phenylarsine oxide, various phosphate inhibitor cocktails, protein phosphatase 1C, protein phosphatase 2A inhibitor protein, protein phosphatase 2A1, protein phosphatase 2A2, and sodium orthovanadate.

[0102] In some embodiments, the therapeutic macromolecule may be delivered in the form of the therapeutic macromolecule itself or a fragment or derivative thereof. Therapeutic macromolecules may include therapeutic proteins and therapeutic polynucleotides. Therapeutic proteins include, but are not limited to, infusible therapeutic proteins, enzymes, enzyme cofactors, hormones, blood clotting factors, cytokines and interferons, growth factors, monoclonal and polyclonal antibodies (e.g., administered to a subject as replacement therapy), and proteins associated with Pompe disease (e.g., acid glucosidase alpha, rhGAA (e.g., Myozyme and Lumizyme (Genzyme)). Therapeutic proteins also include proteins involved in the blood clotting cascade. Therapeutic proteins include, but are not limited to, Factor VIII, Factor VII, Factor IX, Factor V, von Willebrand factor, von Heldebrand factor, tissue plasminogen activator, insulin, growth hormone, erythropoietin alpha, VEGF, thrombopoietin, lysozyme, antithrombin, and the like. Therapeutic proteins also include adipokines, such as leptin and adiponectin. Other examples of therapeutic proteins are described below and elsewhere herein.

[0103] Examples of therapeutic proteins used in enzyme replacement therapy of subjects with lysosomal storage disorders include, but are not limited to, imiglucerase (e.g., CEREZYME™) for the treatment of Gaucher disease, a-galactosidase A (a-galA) (e.g., agalsidase beta, FABRYZYME™) for the treatment of Fabry disease, acid alpha glucosidase (GAA) (e.g., acid glucosidase alpha, LUMIZYME™, MYOZYME™) for the treatment of Pompe disease, and arylsulfatase B (e.g., laronidase, ALDURAZYME™, idursulfase, ELAPRASE™, arylsulfatase B, NAGLAZYME™), pegloticase (KRYSTEXXA), and pegsiticase for the treatment of mucopolysaccharidoses.

[0104] Examples of enzymes include oxidoreductases, transferases, hydrolases, lyases, isomerases, asparaginases, uricase, glycosidases, asparaginases, uricases, proteases, nucleases, collagenases, hyaluronidases, heparinases, heparanases, lysines, and ligases. Therapeutic proteins may also include enzymes, toxins, or other proteins or peptides isolated or derived from any bacterial, fungal, or viral source.

[0105] Examples of hormones include melatonin (N-acetyl-5-methoxytryptamine), serotonin, thyroxine (or tetraiodothyronine) (thyroid hormone), triiodothyronine (thyroid hormone), epinephrine (or adrenaline), norepinephrine (or noradrenaline), dopamine (or prolactin-inhibiting hormone), anti-Müllerian hormone (or Müllerian inhibitory factor or hormone), adiponectin, adrenocorticotropic hormone (or corticotropic hormone), and the like. tropin), angiotensinogen and angiotensin, antidiuretic hormone (or vasopressin, arginine vasopressin), atrial natriuretic peptide (or atriopeptide), 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, These include human placental lactogen, 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, prostacyclin, thromboxane, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, renin, and enkephalins.

[0106] Examples of blood or blood clotting factors include factor I (fibrinogen), factor II (prothrombin), tissue factor, factor V (proaccelerin, labile factor), factor VII (stable 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 (Hageman factor), factor XIII (fibrin-stabilizing factor), von Willebrand factor, prekallikrein (Fletcher factor), high molecular weight kininogen (HMW kininogen), and factor VIII (antihemophilic globulin). K) (Fitzgerald factor), fibronectin, fibrin, thrombin, 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 alfa (Epogen, Procrit).

[0107] Examples of cytokines include lymphokines, interleukins and chemokines, type 1 cytokines such as IFN-γ, TGF-β and type 2 cytokines such as IL-4, IL-10 and IL-13. Examples of growth factors include adrenomedullin (AM), angiopoietin (Ang), autocrine motility stimulating factor, 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 differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin-like growth factor (IGF), These include migration stimulating factor, 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.

[0108] Examples of monoclonal antibodies include abagovomab, abciximab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab pegol, ALD, alemtuzumab, altumomab pentetate, anatumomab mafenatoxin, and anatumomab mafenatox, anrukinzumab, antithymocyte globulin, apolizumab, arcitumomab, aselizumab, tocilizumab, atorolimumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, biciromab, bivatuzumab mertansine mertansine, Blinatumomab, Brentuximab vedotin, Briakinumab, Canakinumab, Cantuzumab mertansine, Capromab pendetide, Catumaxomab, Cedelizumab, Certolizumab pegol, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clenoliximab, Crivatuzumab tetraxetan, Conatumumab, Dacetuzumab, Daclizumab, Daratumumab, Denosumab, Detumomab, Dorlimomab aritoxin aritox, dorlixizumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab, enlimomab pegol, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, exbivirumab,

[0109] Fanolesomab, Faralimomab, Farletuzumab, Felvizumab, Fezakinumab, Figitumumab, Fontolizumab, Foravirumab, Fresolimumab, Galiximab, Gantenerumab, Gambilimomab, Gemtumab ozogamicin, GC1008, Girentuximab, Glembatumumab vedotin vedotin, Golimumab, Gomiliximab, Ibalizumab, Ibritumomab Tiuxetan, Igovomab, Imusiromab, Infliximab, Intetumumab, Inolimomab, Inotuzumab Ozogamicin, Ipilimumab, Iratumumab, Keliximab, Labetuzumab, Lebrikizumab, Remaresomab, Lerdelimumab, Lexatumumab, Ribivirumab, Lintuzumab, Lovotuzumab Mertansine mertansine, lucatumumab, rumiliximab, mapatumumab, maslimomab, matuzumab, mepolizumab, methelimumab, milatuzumab, minretumomab, mitumomab, morolimumab, motavizumab, muromonab-CD3, nacolomab tafenatox, naptumomab estafenatox, natalizumab, nebacumab, necitumumab, nerelimomab, nimotuzumab, nofetumomab merpentane merpentan), Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Omalizumab, Oportuzumab monatox, Olegovomab, Otelixizumab, Pagibaximab, Palivizumab, Panitumumab, Panobacumab, Pascolizumab, Pemtumomab, Pertuzumab, Pexelizumab,

[0110] Pintumomab, Priliximab, Pritumumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Robatumumab, Rontalizumab, Rovelizumab, Ruplizumab, Satumomab pendetide pendetide, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Siplizumab, Solanezumab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox, Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab, Teplizumab, Ticilimumab (Tremelimumab), Tigatuzumab, Tocilizumab (Atrizumab), Toralizumab, Tositumomab, Trastuzumab, Tremelimumab, Tucotuzumab Celmoleukin, Tuvirumab, Urtoxazumab, Ustekinumab, Vapaliximab, Vedolizumab, Veltuzumab, Bepalimomab, Vicilizumab, Volociximab, Votumumab, Zalutumumab, Zanolimumab, Ziralimumab, and Zolimomab aritox. Monoclonal antibodies further include anti-TNF-α antibodies.

[0111] Examples of infusion therapies or injectable therapeutic proteins include, for example, tocilizumab (Roche / Actemra®), alpha-1 antitrypsin (Kamada / AAT), Hematide® (Affymax and Takeda, synthetic peptide), albinterferon alpha-2b (Novartis / Zalbin™), lucin® (Pharming Group, C1 inhibitor replacement therapy), tesamorelin (Theratechnologies / Egrifta, synthetic growth hormone releasing factor), ocrelizumab (Genentech, Roche and Biogen), belimumab (GlaxoSmithKline / Benlysta®), pegloticase (Savient), and others. Pharmaceuticals / Krystexxa™), pegsiticase, taliglucerase alfa (Protalix / Uplyso), agalsidase alfa (Shire / Replagal®), velaglucerase alfa (Shire) and keyhole limpet hemocyanin (KLH).

[0112] Additional therapeutic proteins include, for example, engineered proteins such as Fc fusion proteins, bispecific antibodies, multispecific antibodies, nanobodies, antigen binding proteins, antibody fragments and protein conjugates such as antibody drug conjugates. Therapeutic polynucleotides include, but are not limited to, nucleic acid aptamers such as pegaptanib (Macugen, 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 published U.S. Patent Application No. 2012 / 0251618 (Schrum et al.).

[0113] Additional therapeutic macromolecules useful in accordance with aspects of the present invention will be apparent to those of skill in the art, and the present invention is not limited in this respect. In some embodiments, a component, such as a therapeutic macromolecule or an immunosuppressant, may be isolated. "Isolated" refers to an element that is separated from its native environment and is present in sufficient quantity 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. An isolated element may be substantially pure, but need not be. An isolated element may be mixed with a pharma- ceutically acceptable excipient in a pharmaceutical preparation, so that the element may be included in a very small weight percentage in the preparation. The element is still isolated in that it has been separated from substances with which it may be associated in a biological system, i.e., from other lipids or proteins. Any of the elements provided herein may be isolated and included in a composition or used in a method in an isolated form.

[0114] D. Methods of Making and Using the Compositions and Related Methods In some embodiments where the immunosuppressant listed comprises an implantable osmotic pump, the immunosuppressant can be prepared using an ALZET® implantable osmotic pump containing a solution of the immunosuppressant. Such implantable pumps can be implanted subcutaneously for controlled release of the immunosuppressant into the local subcutaneous space resulting in systemic administration due to absorption of the compound by local capillaries, or intraperitoneally for controlled release of the immunosuppressant into the portal circulation of the liver. Implantable pumps can also be used via catheters for controlled intravenous delivery of the immunosuppressant into the venous or arterial circulation. They can be attached to catheters for intravenous, intracerebral or intraarterial infusion, or for targeted delivery, where the effect of the drug or test agent is localized to a specific tissue or organ. See, for example, SM Stepkowski et al., "Inhibition of host-versus-graft and graft-versus-host responses after small bowel transplantation in rats by rapamycin." Transplantation (1992) 53(-2-):258-264. Additional information regarding the set-up and use of ALZET® brand osmotic pumps, including an extensive literature database, is available at alzet.com.

[0115] In some embodiments where the immunosuppressant listed comprises a bispecific antibody (BsAb), the bispecific antibody can be prepared by genetic or biological methods such as fusing two different hybridoma cell lines or chemical methods such as crosslinking two antibody molecules via a suitable linker by methods known in the art. Several methods have been developed to generate BsAb. BsAb can be generated biologically by fusing two hybridoma cell lines to produce a quadroma capable of secreting BsAb. BsAb can also be generated genetically, and various genetic techniques have been used to generate bispecific molecules. The third method to generate BsAb is by chemical means, using various homobifunctional and heterobifunctional chemical linkers (see Bispecific Antibodies, Springer, 2011, edited by Roland E. Kontermann). Further information regarding the forms of BsAbs can be found in the literature, including, but not limited to, M. Peipp et al., "Bispecific antibodies targeting cancer cells" Biochemical Society Transaction pp. 507-511 vol. 30 part 4 (2002). BsAbs can be administered to patients by subcutaneous or intravenous injection, similar to the methods used for monoclonal antibodies.

[0116] In certain embodiments, the recited immunosuppressants may be in the form of an implantable polymeric depot material, which may be formulated and administered via conventional particles such as those found in U.S. Patent No. 5,702,716 (Dunn et al.), entitled "Polymeric compositions useful as controlled release implants," and U.S. Patent No. 6,130,200 (Brodbeck et al.), entitled "Gel compositions and methods." In some embodiments, the immunosuppressant is attached to a synthetic nanocarrier. Synthetic nanocarriers can be prepared using a variety of methods known in the art. For example, synthetic nanocarriers can be formed by methods such as nanoprecipitation, flow focusing using fluidic channels, spray drying, single and double emulsion solvent distillation, solvent extraction, phase separation, milling, microemulsion procedures, microfabrication, nanofabrication, sacrificial layers, simple and complex coacervation, and other methods known to those skilled in the art. Alternatively or additionally, aqueous and organic solvent syntheses for monodisperse semiconducting or conductive, magnetic, organic and other nanomaterials have 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, e.g., 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. Patent Nos. 5,578,325 and 6,007,845; P. Paolicelli et al., "Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles" Nanomedicine. 5(6): 843-853 (2010)).

[0117] As appropriate, please refer to 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 materials may be encapsulated in synthetic nanocarriers using a variety of methods, including, but not limited to, those disclosed in U.S. Patent No. 6,632,671 (Unger, issued October 14, 2003), suitable for encapsulating materials in synthetic nanocarriers.

[0118] In certain embodiments, synthetic nanocarriers are prepared by a nanoprecipitation process or spray drying. The conditions used to prepare synthetic nanocarriers can be varied to yield particles of desired size or characteristics (e.g., hydrophobicity, hydrophilicity, external morphology, "stickiness", shape, etc.). The method of preparation of synthetic nanocarriers and the conditions used (e.g., solvent, temperature, concentration, air flow rate, etc.) can depend on the composition of the material and / or polymer matrix to be attached to the synthetic nanocarriers. If synthetic nanocarriers prepared by any of the above methods have a size range outside the desired range, such synthetic nanocarriers may be sized, for example, using sieves. The elements (i.e., components) of the synthetic nanocarrier may be attached, for example, via one or more covalent bonds all over the synthetic nanocarrier or may be attached using one or more linkers. Additional methods for functionalizing synthetic nanocarriers 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 WO / 2008 / 127532A1 (Murthy et al.).

[0119] Alternatively or additionally, the synthetic nanocarriers may be attached to the components directly or indirectly through non-covalent interactions. In non-covalent embodiments, the non-covalent linkage is mediated by non-covalent interactions, including but not limited to charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bond interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions and / or combinations thereof. Such linkages may be arranged on the exterior or interior surfaces of the synthetic nanocarriers. In some embodiments, encapsulation and / or absorption are forms of linkage. In some embodiments, the synthetic nanocarriers may be combined with immunosuppressants or therapeutic macromolecules by blending in the same vehicle or delivery system.

[0120] The compositions provided may contain inorganic or organic buffers (e.g., sodium or potassium salts of phosphates, carboxylates, acetates or citric acids) and pH adjusting agents (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citric acid or acetate, 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), dissolution and / or freeze / lyophilization stabilizers (cryo / lyo stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmolality adjusting agents (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity adjusting agents (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and cosolvents (e.g., glycerol, polyethylene glycol, ethanol).

[0121] The composition according to the present invention may include pharma- ceutically acceptable excipients. The composition may be made using conventional pharmaceutical manufacturing and compounding techniques to arrive at a useful dosage form, or may be made using special techniques (such as in the case of ALZET® pumps or bispecific antibodies). Techniques suitable for use in the practice of the present invention may 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 some embodiments, the composition is in a sterile saline solution for injection together with a preservative. It should be understood that the compositions of the present invention may be made in any suitable manner, and the invention is in no way limited to compositions that may be produced using the methods described herein. Selection of an appropriate manufacturing method may require attention to the nature of the particular moiety involved.

[0122] In some embodiments, the immunosuppressant, therapeutic macromolecule or composition comprising such material is manufactured under sterile conditions or terminally sterilized.This can ensure that the resulting material or composition is sterile and non-infectious, thus improving safety when compared with non-sterile compositions.This provides a valuable safety measure, especially when the subject receiving the listed material or composition has immune deficiency, suffers from infectious disease, and / or is susceptible to infection.In some embodiments, the material or composition can be lyophilized and stored for long periods without losing activity, in suspension or as lyophilized powder, depending on the formulation strategy. Administration according to the present invention may be by a variety of routes, including, but not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, transmucosal, transdermal, transcutaneous or intradermal routes. In a preferred embodiment, administration is via the subcutaneous route of administration. The compositions referred to herein may be manufactured and prepared for administration, and in some embodiments, co-administration, using conventional methods.

[0123] The compositions of the invention can be administered in an effective amount, such as an effective amount as described elsewhere herein. The dosage form dose may contain varying amounts of the immunosuppressant or therapeutic macromolecule according to the invention. The amount of immunosuppressant or therapeutic macromolecule present in the dosage form may vary according to the characteristics of the therapeutic macromolecule, the immunosuppressant, the therapeutic effect to be achieved, and other such parameters. In some embodiments, dose ranging studies may be performed to establish the optimal therapeutic amount of the immunosuppressant or therapeutic macromolecule to be present in any dosage form. In some embodiments, the immunosuppressant or therapeutic macromolecule is present in the dosage form in an amount effective to generate a tolerogenic immune response to the therapeutic macromolecule, which is then administered to the subject. It may be possible to determine the amount of the immunosuppressant or therapeutic macromolecule effective to generate a tolerogenic immune response in the subject using conventional dose ranging studies and techniques. The administration of the immunosuppressant or therapeutic macromolecule may occur at various frequencies.

[0124] Another aspect of the disclosure relates to kits. In some embodiments, the kit comprises an immunosuppressant dose that provides a pharmacodynamic effective period. In some embodiments, the kit further comprises a dose of a therapeutic macromolecule. The immunosuppressant dose and the therapeutic macromolecule dose may be contained in separate containers or in the same container in the kit. In some embodiments, the container is a vial or an ampoule. The immunosuppressant dose and the therapeutic macromolecule dose may be contained in separate containers or in the same container in the kit. In some embodiments, the container is a vial or an ampoule. In some embodiments, the therapeutic macromolecule dose and / or the immunosuppressant dose may be contained in a solution separate from the container(s) such that the therapeutic macromolecule dose and / or the immunosuppressant dose are added to the container at a later time. In some embodiments, the therapeutic macromolecule dose and / or the immunosuppressant dose may be in lyophilized form, each in a separate container or in the same container, such that they are reconstituted at a later time. In some embodiments, the kit further comprises instructions for reconstitution, mixing, administration, etc. In some embodiments, the instructions include a description of the methods described herein. The instructions may be in any suitable form, such as, for example, a printed insert or label. In some embodiments, the kit further comprises one or more syringes.

[0125] example Example 1: Rapamycin containing nanocarriers material Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA 01702; product catalog # R1017). PLGA of approximately 25,000 Da was purchased from Lakeshore Biochemicals (756 Tom Martin Drive Birmingham, AL 35211). Product code 5050 DLG 2.5A. PLA-PEG-OMe block copolymer with approximately 5,000 Da methyl ether terminated PEG block and 48,000 Da PLA block 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% hydrolyzed, viscosity 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027. Part number 1.41354).

[0126] method The solutions were prepared as follows: Solution 1: 75 mg / mL PLGA, 25 mg / mL PLA-PEG-OMe and 12.5 mg / mL 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 polyvinyl alcohol in 100 mM pH 8 phosphate buffer. Solution 3: 70 mM phosphate buffer, pH 8.

[0127] An oil-in-water emulsion was made by mixing solution 1 (1 mL) and solution 2 (3 mL) in a small glass pressure tube and sonicating for 60 seconds at 30% amplitude using a Branson Digital Sonifier 250. The emulsion was added to an open 50 mL beaker containing solution 3 (30 mL) and stirred at room temperature for 2 hours to allow the dichloromethane to evaporate and form the nanocarriers in suspension. The nanocarrier suspension was then transferred to a centrifuge tube, centrifuged at 75,600 rcf for 40 minutes, the supernatant was removed, and a portion of the suspended nanocarriers was washed by resuspending the pellet in phosphate buffered saline. This washing procedure was repeated, and then the pellet was resuspended in PBS 1× to achieve a nanocarrier suspension with a nominal concentration of 10 mg / mL based on polymer. The suspension was stored frozen at −20° C. until use. Nanocarrier size was determined by dynamic light scattering. The amount of rapamycin in the nanocarriers was determined by HPLC analysis. The total dry nanocarrier mass per mL of suspension was determined by pycnometry. [Table 1]

[0128] Example 2: Polymeric nanocarriers containing polymer-rapamycin conjugates (prophetic) Step 1: Preparation of PLGA-rapamycin conjugate: PLGA polymer with acid end groups (7525 DLG1A, acid value 0.46 mmol / g, Lakeshore Biomaterials; 5 g, 2.3 mmol, 1.0 eq) is dissolved in 30 mL of dichloromethane (DCM). N,N-dicyclohexylcarbodiimide (1.2 eq, 2.8 mmol, 0.57 g) is added, followed by rapamycin (1.0 eq, 2.3 mmol, 2.1 g) and 4-dimethylaminopyridine (DMAP) (2.0 eq, 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 give PLGA-rapamycin as a white solid (approximately 6.5 g).

[0129] Step 2: PLGA-rapamycin-containing nanocarriers are prepared according to the procedure described in Example 1 below: Solutions for nanocarrier formation are prepared as follows: Solution 1: 100 mg / mL PLGA-rapamycin in methylene chloride. The solution is prepared by dissolving PLGA-rapamycin in pure methylene chloride. Solution 2: 100 mg / mL PLA-PEG in methylene chloride. The solution is prepared by dissolving PLA-PEG in pure methylene chloride. Solution 3: 50 mg / mL polyvinyl alcohol in 100 mM pH 8 phosphate buffer.

[0130] The primary water-in-oil emulsion is first prepared. W1 / O1 is prepared by combining solution 1 (0.75 mL) and solution 2 (0.25 mL) in a small pressure tube and sonicating for 40 seconds at 50% amplitude using a Branson Digital Sonifier 250. The secondary emulsion (W1 / O1 / W2) is then prepared by combining solution 3 (3.0 mL) with the primary W1 / O1 emulsion, vortexing for 10 s, and sonicating for 60 seconds at 30% amplitude using a Branson Digital Sonifier 250. The W1 / O1 / W2 emulsion is added to a beaker containing 70 mM pH 8 phosphate buffer solution (30 mL) and stirred at room temperature for 2 hours to allow the methylene chloride to evaporate and form nanocarriers. A portion of the nanocarriers is washed. The nanocarrier suspension is transferred to a centrifuge tube and centrifuged at 75,600×g and 4° C. for 35 minutes, the supernatant is removed and the pellet is resuspended in phosphate-buffered saline. The washing procedure is repeated and the pellet is resuspended in phosphate-buffered saline for a final nanocarrier dispersion of approximately 10 mg / mL.

[0131] Example 3: Preparation of gold nanocarriers (AuNCs) containing rapamycin (prophetic) Step 1. Preparation of HS-PEG-rapamycin: A solution of PEG acid disulfide (1.0 eq), rapamycin (2.0-2.5 eq), DCC (2.5 eq) and DMAP (3.0 eq) in dry DMF is stirred overnight at rt. Insoluble dicyclohexylurea is removed by filtration and the filtrate is added to isopropyl alcohol (IPA) to precipitate the PEG-disulfide-di-rapamycin ester, washed with IPA and dried. The polymer is then treated with tris(2-carboxyethyl)phosphine hydrochloride in DMF to reduce the PEG disulfide to the thiol PEG rapamycin ester (HS-PEG-rapamycin). The resulting polymer is recovered by precipitation from IPA as previously described, dried and analyzed by H NMR and GPC.

[0132] Step 2. Formation of gold NCs (AuNCs): A 500 mL solution of 1 mM HAuCl4 in water is heated to reflux for 10 min with vigorous stirring in a 1 L round bottom flask with condenser. A 50 mL solution of 40 mM trisodium citrate is then rapidly added to the stirred solution. The resulting deep wine red solution is kept at reflux for 25-30 min, the heat is removed and the solution is allowed to cool to room temperature. The solution is then filtered through a 0.8 μm membrane filter to obtain the AuNC solution. The AuNCs are characterized using visible spectroscopy and transmission electron microscopy. The AuNCs are approximately 20 nm in diameter, capped with citrate and have a peak absorption at 520 nm.

[0133] Step 3. AuNC complex with HS-PEG-rapamycin: 150 μl of a solution of HS-PEG-rapamycin (10 μM in 10 mM pH 9.0 carbonate buffer) is added to 1 mL of 20 nm diameter citrate-capped gold nanocarriers (1.16 nM) to generate a thiol:gold molar ratio of 2500:1. The mixture is stirred under argon at room temperature for 1 h to allow complete exchange of thiols with citrates on the gold nanocarriers. The AuNCs with PEG-rapamycin on their surface are then purified by centrifugation at 12,000 g for 30 min. The supernatant is decanted and the pellet containing AuNC-S-PEG-rapamycin is then pellet washed with 1x PBS buffer. The purified gold-PEG-rapamycin nanocarriers are then resuspended in a suitable buffer for further analysis and bioassays.

[0134] Example 4: Effect of encapsulated rapamycin in initiating an antibody response I. IgG measurement IgG antibody levels were generally measured as follows: Blocker Casein in PBS (Thermo Fisher, Cat #37528) was used as the diluent. 0.05% Tween-20 in PBS was used as the wash buffer, prepared by adding 10 mL of Tween-20 (Sigma, Cat #P9416-100 mL) to 2 liters of 10x PBS stock (PBS: OmniPur® 10X PBS Liquid Concentrate, 4 L, EMD Chemicals, Cat #6505) and 18 liters of deionized water. Keyhole limpet hemocyanin (KLH) at a stock concentration of 10 mg / mL or ovalbumin (OVA) protein at a stock concentration of 5 mg / mL were used as coating materials. Both materials were diluted to 5 μg / ml and used as working concentrations. Each well of the assay plate was coated with 100 μl of diluted KLH or OVA per well, and the plate was sealed with sealing membrane (VWR, catalog #60941-120) and incubated overnight at 4°C. Costar 9017 96-well flat bottom plates were used as assay plates (Costar 9017). Low-binding polypropylene 96-well plates or tubes were used as set-up plates in which samples were prepared before being transferred to the assay plates. The set-up plates did not contain any antigens, and therefore serum antibodies did not bind to the plates during sample set-up. The set-up plates were used for sample preparation to minimize binding that would have occurred during sample preparation or pipetting if antigen-coated plates were used to prepare samples. Prior to preparing samples in the set-up plates, the wells were covered with diluent to block any non-specific binding, and the plates were sealed and incubated overnight at 4°C.

[0135] The assay plate was washed three times with wash buffer, and the wash buffer was aspirated completely from the wells after the final wash. After washing, 300 μl of diluent was added to each well of the assay plate(s) to block non-specific binding, and the plate was incubated at room temperature for at least 2 hours. Serum samples were diluted 1:40 in the appropriate wells of the set-up plate. A standard was used as a positive control. For KLH, a mouse anti-KLH IgG antibody was used at a starting dilution of 1 μg / mL, then diluted 3-fold across the plate. For OVA, a mouse anti-OVA IgG antibody was used at a starting dilution of 0.5 μg / mL, then diluted 3-fold across the plate.

[0136] Once all samples were prepared in the setup plate, the plate was sealed and stored at 4°C until blocking of the assay plate was complete. The assay plate was washed three times with wash buffer, and the wash buffer was aspirated completely after the final wash. After washing, 100 μl of diluent was added to columns 2-12 of the assay plate. A pipette was used to transfer the samples from the setup plate to the assay plate. Prior to transfer, the samples were mixed by pipetting 150 μl of diluted serum up and down three times. After mixing, 150 μl of each sample was transferred from the setup plate and added to each assay plate.

[0137] Once the starting dilution of each sample was transferred from the setup plate to the assay plate, the serial dilutions were pipetted onto the assay plate as follows: 50 μl of each serum sample was removed using a pipette and mixed with 100 μl of diluent that had been added previously. This step was repeated across the plate. After the final row of dilutions were pipetted, 50 μl of fluid was removed from the last row of wells and discarded, resulting in a final volume of 100 μl per well of the assay plate. Once the sample dilutions were prepared in the assay plate, the plate was incubated at room temperature for at least 2 hours.

[0138] After incubation, the plates were washed three times with wash buffer. The detection antibody (goat anti-mouse anti-IgG, HRP conjugated) was diluted 1:1500 in diluent (0.33 μg / ml) and 100 μl of diluted antibody was added to each well. The plates were incubated for 1 hour at room temperature and then washed five times with wash buffer, and the wash buffer was completely aspirated from the wells after the last wash. After washing, the detection substrate was added to the wells. Equal parts of Substrate A and Substrate B (BD Biosciences TMB Substrate Reagent Set, Cat#555214) were combined just before adding to the assay plate, and 100 μl of the mixed substrate solution was added to each well and incubated in the dark for 10 minutes. After 10 minutes, the reaction was stopped by adding 50 μl stop solution (2N H2SO4) to each well. The optical density (OD) of the wells immediately after adding the stop solution was assessed at 450 nm in a plate reader and subtracted at 570 nm. Data analysis was performed using Molecular Device's software SoftMax Pro v 6.2.2. A graph was prepared by fitting a four-parameter logistic curve with dilution on the x-axis (log scale) and OD values ​​on the y-axis (linear scale) to obtain half-maximal values ​​(EC 50 ) was determined. The plate template at the top of the layout was adjusted to reflect the dilutions of each sample (one per row).

[0139] II. Preparation of particulate ovalbumin protein (pOVA) material Chicken egg ovalbumin (OVA) was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701; product code LS003054). Phosphate buffered saline (PBS) was purchased from Mediatech (9345 Discovery Boulevard, Manassas, VA 20109; product code 21-040-CV). Sodium hydroxide (NaOH, product code 367176) and trifluoroacetic acid (TFA, product code T62200) were purchased from Sigma-Aldrich Corp. (3050 Spruce Street, St. Louis, MO 63103). method The solutions were prepared as follows: Solution 1: 15 mg / mL OVA in PBS. The solution was prepared by dissolving ovalbumin directly in PBS. Solution 2: 1 M NaOH in water. The solution was prepared by dissolving NaOH directly in endotoxin-free water.

[0140] OVA was coacervated by repeatedly raising and lowering the pH of an ovalbumin solution. Solution 1 (10 mL) was added to a 20 mL glass vial containing a magnetic stir bar. While stirring, solution 2 was added dropwise to the vial until the pH of the solution reached 12. TFA was then added dropwise to the vial until the pH of the solution reached 2. This pH raising and lowering was repeated three more times. Solution 2 was then added dropwise to the vial until the pH of the solution reached 7. The size of the coacervated OVA particles was then reduced by high-pressure homogenization. The coacervated OVA suspension was loaded into a Microfluidics LV1 with a G10Z interaction chamber and then homogenized using three passes at 20,000 psi. The resulting particulate OVA (pOVA) with sizes ranging from 190 to 240 nm was stored at -20 °C.

[0141] III. In vivo testing of encapsulated rapamycin in initiating antibody responses Groups of C57BL / 6 animals (n=5) were left untreated and unimmunized (no immunization), untreated and immunized (no treatment), or treated and immunized with rapamycin-containing nanocarriers from Example 1 (rapa-NC) on days 0, 3, and 7. Immunization consisted of injection of a particulate form of ovalbumin (pOVA), injected subcutaneously into the hind paw. The dose of particles for each injection was the equivalent of 100 μg of rapamycin, for a total of 300 μg after three injections. Starting on day 14, untreated and treated animals (no treatment and rapa-NC) were immunized every other week with 10 μg of pOVA prepared as above in the forelimbs (30 μl each) and 50 μg of keyhole limpet hemocyanin (KLH, Sigma Aldrich) injected into the base of the tail (50 μl each side) (days 14, 28, and 42). All animals were bled on the days indicated in FIG. 1 and anti-OVA responses were determined by ELISA. The data demonstrate that encapsulated rapamycin effectively inhibited anti-OVA responses within the pharmacodynamic window provided herein when injected 7 days prior to antigen challenge.

[0142] Example 5: Dose-dependent inhibition of T cell activation by encapsulated rapamycin To test the dose-dependence of rapamycin-mediated inhibition of T cell activation, CD45.1+ animals (B6.SJL) were injected intravenously with different doses (10-50 μg rapamycin and one PBS control) of encapsulated rapamycin (NPs [Rapa] as disclosed in Example 1). These animals share the same genetic background as C57BL / 6 mice, but express a different isoform of the CD45 molecule (CD45.1 instead of CD45.2). After 6 days, cells were isolated by magnetic cell sorting (MACS, Miltenyi) from spleens and lymph nodes of the OTII mouse strain and expressed CD45.2 and MHC CLII (OVA). 323~339T cells expressing a transgenic TCR that recognizes peptides from chicken ovalbumin presented in the context of TLR7 / 8 (or OPII.323) were matched transferred into CD45.1+ treated animals. These cells were also labeled with CFSE (Invitrogen) to follow their proliferation status. The next day (day 7) all animals were injected subcutaneously in the hind leg with OPII.323 mixed with an immunogenic dose of the TLR7 / 8 agonist R4848. On day 11, animals were sacrificed, lymph nodes draining the injection site (popliteal fossa) were excised, and cells were analyzed by flow cytometry to identify transplanted CD45.2+TCRb+CD4+7AAD-T cells (antibody from Biolegend). As shown in Figure 2, injection of as little as 20 μg of rapamycin has an inhibitory effect on the activation and survival of these T cells, even 6 days after injection of the nanocarriers, still within the pharmacodynamic window provided herein. Mounting rapamycin dosing resulted in decreased cell number and proliferation.

[0143] Example 6: Evaluation of immunosuppressants and their tolerogenic immune responses in vivo using implantable osmotic pumps (prophetic) Step 1: Rapamycin is dissolved in a vehicle consisting of dimethylsulfoxide (DMSO) / polyethylene glycol 400 and loaded into an ALZET® implantable osmotic pump (volume 0.2 mL, model 2001, Durect Corp., Cupertino, Calif.). The pump is then implanted subcutaneously (sc) into the right lateral lower abdomen of the mouse according to the ALZET® pump operating procedure. The pump is used to administer rapamycin sc at a dose of 2.5 mg / kg / day for two consecutive days.

[0144] Step 2: Groups of C57BL / 6 animals (n=5) were left untreated and unimmunized (no immunization), untreated and immunized (no treatment), or treated with rapamycin-loaded osmotic pumps as described above implanted subcutaneously in the lower flank of the mice and immunized on days 0, 3, and 7. Immunization consisted of injections of a particulate form of ovalbumin (pOVA). Rapamycin was administered sc for two consecutive days at a dose of 2.5 mg / kg / day using the pump. The dose of rapamycin for each injection was equivalent to 100 μg of rapamycin, totaling 300 μg after three injections. Beginning on day 14, untreated and treated animals (no treatment and rapamycin-loaded pump) are immunized biweekly (days 14, 28 and 42) with 100 μg of therapeutic protein, such as acid glucosidase alpha or tissue plasminogen activator or erythropoietin alpha or dorlixizumab or rituximab, injected into the forelimb. All animals are bled on days 40 and 54 and anti-protein responses are determined by ELISA. Rapamycin administered via an implanted osmotic pump is expected to effectively inhibit anti-protein responses when injected within the pharmacodynamic window provided herein, and these effects are expected to be evident even after priming and two boosts with the protein.

[0145] Example 7: Evaluation of immunosuppressants and their tolerogenic immune responses in vivo using implantable osmotic pumps (prophetic) Step 1: Store a stock solution of rapamycin (Rapa) in N,N-dimethylacetamide (DMAC) (19 mg / ml) at 4 °C and protect from exposure to light. Dilute the stock solution in a mixture of 10% Tween-80, 20% DMAC and 70% polyethylene glycol 400 (PEG400) to obtain the appropriate Rapa concentration based on the rat's weight. Load the appropriately diluted Rapa into an ALZET® brand implantable osmotic pump (model 2002; Durect Corp., Cupertino, CA), which has been pre-primed for 4-6 h by incubating at 37 °C in sterile saline. Then, insert a delivery cannula into the lumbar vein of the mouse for intravenous delivery of rapamycin.

[0146] Step 2: Groups of C57BL / 6 animals (n=5) were left untreated and unimmunized (no immunization), untreated and immunized (no treatment), or treated with rapamycin-loaded osmotic pumps as described above and immunized on days 0, 3, and 7. Immunization consisted of injection of a particulate form of ovalbumin (pOVA). The pump was used to administer rapamycin via intravenous infusion at a dose of 2.5 mg / kg / day for 2 days. The dose of rapamycin for each infusion was equivalent to 100 μg of rapamycin, totaling 300 μg after three injections. Beginning on day 14, untreated and treated animals (no treatment and rapamycin-loaded pumps) are immunized every other week (days 14, 28, and 42) with 50-100 μg of a therapeutic polynucleotide, such as pegaptanib, mipomersen, or modified messenger RNA (mmRNA), such as those disclosed in US Patent Application No. 2013 / 0115272 (Fougerolles et al.). All animals are bled on days 40 and 54 and anti-protein responses are determined by ELISA. Rapamycin administered via an implanted osmotic pump is expected to effectively inhibit anti-polynucleotide responses when injected within the pharmacodynamic window provided herein, and these effects are expected to be evident even after priming and two boosts with polynucleotide.

[0147] Example 8: Bispecific antibody immunosuppressant using chemically cross-linked anti-GITR antibody and anti-idiotypic antibody against inhibitory factor VIII antibody to induce tolerance to factor VIII protein (Prophetic) Step 1: Preparation of bispecific antibodies F(ab')2 fragments of anti-GITR antibodies, such as epratuzumab and anti-idiotypic antibodies to inhibitory factor VIII antibodies (prepared according to US Patent No. 8,071,094), are prepared using the ImmunoPure F(ab')2 Preparation Kit (Pierce) according to the manufacturer's instructions. The F(ab')2 containing fractions are pooled and concentrated using a 10,000 MWCO centrifugal filter (Millipore). Each F(ab')2 is reduced to a F(ab)-thiol by adding 2-mercaptoethanol to a final concentration of 20 mM for 20-40 min at 30°C. The sample is cooled on ice and then passed over a Sephadex G25 column equilibrated and cooled in 50 mM sodium acetate / 0.5 mM EDTA pH 5.3. The protein containing fractions are pooled. The anti-GITR Fab-thiol is then maleimidized for 30 min in an ice bath by adding 1 / 2 volume of pre-chilled 12 mM cross-linked, o-phenylenedimaleimide (o-PDM) (dissolved in DMF). The anti-GITR-maleimidized-Fab is then passed over a chilled Sephadex G25 column equilibrated in 50 mM sodium acetate / 0.5 mM EDTA pH 5.3. Protein-containing fractions are pooled and immediately added to the anti-idiotypic antibody to the inhibitory factor VIII antibody-thiol-Fab in a 1:1 molar ratio. The reaction is placed under nitrogen and gently stirred at 4 °C for 15-20 h. The pH is adjusted to pH 8.0 using 1 M Tris-HCl pH 8.0, after which 2-mercaptoethanol is added to a final concentration of 20 mM. The reaction is incubated at 30 °C for 20-40 min and then alkylated by adding iodoacetamide to a final concentration of 25 mM. The mixture is then passed over a Superdex 200 column equilibrated in PBS pH 7.4 and fractions are collected. Individual fractions are run on a 10% SDS-PAGE gel under non-reducing conditions and stained with Coomassie Brilliant Blue (Sigma) or Silver Snap II Kit (Pierce).Bispecific antibodies (anti-GITR / anti-idiotypic antibodies to inhibitory factor VIII antibodies) are identified, pooled, and stored for use as immunosuppressants (dose 10 μg / day).

[0148] Step 2. Tolerance induction for factor VIII Groups of C57BL / 6 animals (n=5) were left naive and unimmunized (no immunization), naive and immunized (no treatment) or treated with 10 μg / day of the bispecific antibody described above and immunized on days 0, 3 and 7. Immunization consisted of injections of particulate ovalbumin (pOVA). Starting on day 14, naive and treated animals (naive and bispecific antibody treated) received biweekly immunizations (days 14, 28 and 42) with 150 IU / kg recombinant human anti-hemophilia factor VIII (Factor VIII). All animals are bled on days 40 and 54 and anti-protein responses are determined by ELISA. Bispecific antibodies (anti-GITR / anti-idiotypic antibodies to inhibitory Factor VIII antibodies) are expected to effectively inhibit anti-Factor VIII responses when injected within the pharmacodynamic window provided herein, and these effects are expected to be evident even after priming and two boosts with Factor VIII protein.

[0149] Example 9: Evaluation of immunosuppressants and their tolerogenic effects in vivo using implantable polymeric depot materials (prophetic) Step 1: Gel vehicle preparation The glass vessel is tared on a Mettler PJ3000 top loader balance. Poly(D,L-lactide-co-glycolide) 50:50 RESOMER.RTM. RG502 (PLGA-502) is weighed into the glass vessel. The glass vessel containing PLGA-502 is tared and the corresponding solvent (as listed in Table 2) is added. The amounts expressed as percentages for the various polymer / solvent combinations are listed in Table 1 below. The polymer / solvent mixture is manually stirred with a stainless steel square end spatula resulting in a sticky amber paste-like material containing white polymer particles. The vessel containing the polymer / solvent mixture is sealed and placed in a temperature controlled incubator equilibrated at 39°C. The polymer / solvent mixture is removed from the incubator when it appears as a clear amber homogenous gel. The incubation time interval may range from 1 to 4 days depending on the type of solvent and polymer as well as the solvent and polymer ratio.

[0150] Additional depot gel vehicles are prepared with the following polymers: Poly(D,L-lactide-co-glycolide) 50:50 RESOMER.RTM. L104, PLGA-L104, code no. 33007, Poly(D,L-lactide-co-glycolide) 50:50 RESOMER.RTM. RG206, PLGA-206, code no. 8815, Poly(D,L-lactide-co-glycolide) 50:50 RESOMER.RTM. RG502, PLGA-502, code no. 0000366, Poly(D,L-lactide-co-glycolide) 50:50 RESOMER.RTM. RG502H, PLGA-502H, code no. 260187, Poly(D,L-lactide-co-glycolide) 50:50 RESOMER.RTM. RG503, PLGA-503, code no. 0080765, poly(D,L-lactide-co-glycolide) 50:50 RESOMER.RTM. RG506, PLGA-506, code no. 95051, poly(D,L-lactide-co-glycolide) 50:50 RESOMER.RTM. RG755, PLGA-755, code no. 95037 (Boehringer Ingelheim Chemicals, Inc., Petersburg, Va.) and the following solvents or mixtures: glyceryl triacetate (Eastman Chemical Co., Kingsport, Tenn.), benzyl benzoate ("BB"), ethyl benzoate ("EB"), methyl benzoate ("MB"), triacetin ("TA") and triethyl citrate ("TC") (Aldrich Chemical Co., St Louis, Mo.). When a combination of solvents is used, such as 20% triacetin and 80% benzyl benzoate, the solvent mixture is added directly to a pre-weighed amount of dry polymer. Typical polymer molecular weights range from 14,400 to 39,700 (M.sub.w) [6,400 to 12,200 (M.sub.n)]. Representative gel vehicles are set forth in Table 1 below.

[0151] [Table 2]

[0152] [Table 3]

[0153] The various immunosuppressants (10-20% w / w) listed in Table 2 above are added to the specific clear amber depot gel vehicle and mixed manually until the dry powder is completely wetted. The mixture is then thoroughly mixed by conventional mixing using a Caframo mechanical stirrer equipped with a square-ended metal spatula. The final homogenous gel immunosuppressant implantable polymeric depot material is transferred to a 3, 10 or 30 cc disposable syringe for storage or dispensing.

[0154] Step 2: Evaluation of tolerogenic immune responses in vivo Groups of C57BL / 6 animals (n=5) are left untreated and unimmunized (no immunization), untreated and immunized (no treatment) or treated with the above implantable polymer depot material containing 100 μg of rapamycin and immunized on days 0, 3 and 7. Immunization consists of injection of particulate ovalbumin (pOVA). Polymer depo-rapamycin is implanted into the hind limb. The dose of polymer depo-rapamycin is equivalent to 100 μg of rapamycin, totaling 300 μg after three injections. Starting on day 14, untreated and treated animals (no treatment and rapa-depot) receive biweekly immunizations (days 14, 28 and 42) with 2 mg / kg of a therapeutic protein, such as recombinant tissue plasminogen activator (rtPA), injected into the forelimb. All animals are bled on the indicated days and anti-rtPA responses are determined by ELISA. Gel implants containing rapamycin are expected to effectively inhibit anti-rtPA responses when injected within the pharmacodynamic window provided herein, and these effects are expected to be evident even after priming and two boosts with rtPA.

[0155] Example 10: Pharmacodynamic administration of rapamycin attached to 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 0.69 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211) (product code 7525 DLG 7A). PLA-PEG-OMe block copolymer with methyl ether terminated PEG blocks of approximately 5,000 Da 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% hydrolyzed, viscosity 3.4-4.6 mPa·s) was purchased from EMD Chemicals Inc., 480 South Democrat Road Gibbstown, NJ 08027 (product code 1.41350).

[0156] method Solutions were prepared as follows: Solution 1: 75 mg / mL PLGA, 25 mg / mL PLA-PEG-OMe and 12.5 mg / mL rapamycin in methylene chloride. This solution was prepared by dissolving PLGA, PLA-PEG-OMe and rapamycin in pure methylene chloride. Solution 2: Polyvinyl alcohol, 50 mg / mL in 100 mM pH 8 phosphate buffer. Nanocarriers were prepared using oil-in-water emulsions. O / W emulsions were prepared by combining solution 1 (1.0 mL) and solution 2 (3.0 mL) in a small pressure tube and sonicating for 60 seconds at 30% amplitude using a Branson Digital Sonifier 250. The O / W emulsion was added to a beaker containing 70 mM pH 8 phosphate buffer solution. Three identical emulsions were formed and added to the same beaker along with the first emulsion. These were then stirred at room temperature for 2 hours to allow the methylene chloride to evaporate and form the nanocarriers. A portion of the nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging at 75,600×g and 4° C. for 35 minutes, removing the supernatant, and resuspending the pellet in phosphate buffered saline. The washing procedure was repeated and the pellet was resuspended in phosphate buffered saline to give a final nanocarrier dispersion of approximately 10 mg / mL. The washed nanocarrier solution was then filtered using a 1.2 μm PES membrane syringe filter from Pall, part number 4656. Nanocarrier size was determined by dynamic light scattering. The amount of rapamycin in the nanocarriers was determined by HPLC analysis. The total dry nanocarrier mass per mL of suspension was determined by pycnometry. [Table 4]

[0157] Determining the immunosuppressive window (pharmacodynamic effective period) of rapamycin-loaded nanocarriers C57BL / 6 age-matched (5-6 weeks) females were injected iv into the tail vein with 0.9 mg of nanocarriers at various time points (shown in Figure 3) from 14, 10, 7, 4, 2 and 1 day(s) prior (d-14, -10, -7, -4, -2 and -1, respectively) until the same day of injection of keyhole limpet hemocyanin (d0, 200 μg, KLH). All animals received weekly injections of KLH on days 0, 7 and 14 (200 μg, KLH). Antibody titers in the blood of these animals are determined on day 19. The results in Figure 3 show that injection of rapamycin-containing nanocarriers even 14 days prior to immunization modifies the antibody response to KLH, and that the tolerogenic nanocarriers provide a significant immunosuppressive window during which the onset of the immune response is attenuated. The results demonstrate the ability to reduce antigen-specific immune responses by administering an immunosuppressant and antigen within the pharmacodynamic effective period of the immunosuppressant.

[0158] Example 11: Antigen-specific tolerogenic responses to chicken ovalbumin with encapsulated rapamycin NP[Rapa] Materials and Methods material Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA 01702) (product code# R1017). 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 methyl ether terminated PEG blocks of approximately 5,000 Da and an overall intrinsic viscosity of 0.50 DL / g was purchased from Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, AL 35211) (product code 100DL mPEG 5000 5CE). EMPROVE® polyvinyl alcohol 4-88, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa·s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027) (product code 1.41350). Cellgro Phosphate-buffered saline 1X (PBS1X) was purchased from Corning (9345 Discovery Blvd. Manassas, VA 20109) (product code 21-040-CV).

[0159] method Solutions were prepared as follows: Solution 1: A polymer and rapamycin mixture was prepared by dissolving 75 mg PLGA per mL, 25 mg PLA-PEG-Ome per mL, and 12.5 mg rapamycin per mL in dichloromethane. Solution 2: Polyvinyl alcohol was prepared at 50 mg / mL in 100 mM pH 8 phosphate buffer.

[0160] An O / W emulsion was prepared by combining solution 1 (1.0 mL) and solution 2 (3.0 mL) in a small glass pressure tube and sonicating for 60 seconds at 30% amplitude using a Branson Digital Sonifier 250. This O / W emulsion was added to an open beaker containing 70 mM pH 8 phosphate buffer solution (60 mL). Three additional identical O / W emulsions were prepared and added to the same beaker as the first one. These were then stirred at room temperature for 2 hours to allow the dichloromethane to evaporate and form the nanocarriers. A portion of the nanocarriers was then washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging at 75,600×g and 4° C. for 35 minutes, removing the supernatant, and resuspending the pellet in PBS1×. The washing procedure was repeated, and then the pellet was resuspended in PBS1× to achieve a nanocarrier suspension with a nominal concentration of 10 mg / mL on the polymer basis. An identical formulation was prepared as above in a separate beaker and combined with the first one after the washing step. The mixed nanocarrier solution was then filtered using a Pall 1.2 μm PES membrane syringe filter (part number 4656) and stored at -20°C.

[0161] 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 mL of suspension was determined by pycnometry. [Table 5]

[0162] NP[OVA] Materials and Methods material Ovalbumin protein was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701) (product code LS003054). PLGA with 54% lactide and 46% glycolide content and inherent 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 block copolymer with approximately 5,000 Da methyl ether terminated PEG block and 28,000 Da Mw, inherent viscosity of 0.38 dL / g was purchased from Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, AL 35211) (product code 100 DL mPEG 5000 4CE). EMPROVE® polyvinyl alcohol 4-88, USP, 85-89% hydrolyzed, viscosity 3.4-4.6 mPa.s, was purchased from EMD Chemicals Inc., 480 South Democrat Road Gibbstown, NJ 08027 (product code 1.41350.1001). Cellgro Phosphate-buffered saline 1X (PBS1X) was purchased from Corning, 9345 Discovery Blvd. Manassas, VA 20109 (product code 21-040-CV).

[0163] method Solutions were prepared as follows: Solution 1: 50 mg / mL ovalbumin protein was prepared in 10 mM phosphate buffer pH 8 with 10 wt% sucrose. Solution 2: PLGA was prepared by dissolving 100 mg PLGA per mL dichloromethane in a fume hood. Solution 3: PLA-PEG-OMe was prepared by dissolving 100 mg PLA-PEG-OMe per mL dichloromethane in a fume hood. Solution 4: Polyvinyl alcohol, 65 mg / mL in 100 mM phosphate buffer pH 8.

[0164] A primary (W1 / O) emulsion was first made by mixing solutions 1-3. Solution 1 (0.2 mL), solution 2 (0.75 mL), and solution 3 (0.25 mL) were combined in a small glass pressure tube that had been pre-cooled in an ice-water bath for >4 min and sonicated for 40 s at 50% amplitude using a Branson Digital Sonifier 250 on an ice bath. A secondary (W1 / O / W2) emulsion was then formed by adding solution 4 (3 mL) to the primary emulsion, vortex mixing to create a milky dispersion, and then sonicating for 60 s at 30% amplitude using a Branson Digital Sonifier 250 on an ice bath. This secondary emulsion was added to an open 50 mL beaker containing PBS1× (30 mL). A second identical double emulsion formulation was prepared as above and added to the same 50 mL beaker as the first. The two formulations were stirred at room temperature for 2 h to allow the dichloromethane to evaporate and form the nanocarriers in suspension. A portion of the suspended nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube, spinning at 75,600 rcf for 50 minutes, removing the supernatant, and resuspending the pellet in PBS 1×. This washing procedure was repeated, and then the pellet was resuspended in PBS 1× to achieve a nanocarrier suspension with a nominal concentration of 10 mg / mL on a polymer basis. The suspension was stored frozen at −20° C. until use. [Table 6]

[0165] NP[GSK1059615] Materials and Methods 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 methyl ether terminated PEG blocks of approximately 5,000 Da 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 (PBS1X) was purchased from Corning (9345 Discovery Blvd. Manassas, VA 20109) (product code 21-040-CV).

[0166] method Solutions were 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 at 10 mg in 1 mL of N-methyl-2-pyrrolidinone (NMP).

[0167] Nanocarriers were prepared by combining solution 1 (4 mL) and solution 2 (0.25 mL) in a small glass pressure tube and adding the mixture dropwise, under stirring, to a 250 mL round-bottom flask containing 20 mL of ultrapure water. The flask was attached to a rotary evaporator and the acetone was removed under reduced pressure. A portion of the nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging at 75,600 rcf and 4°C for 50 minutes, removing the supernatant, and resuspending the pellet in PBS1x. The washing procedure was repeated and the pellet resuspended in PBS1x to achieve a nanocarrier suspension with a nominal concentration of 10 mg / mL on a polymer basis. The washed nanocarrier solution was then filtered using a Pall 1.2 μm PES membrane syringe filter (part number 4656). An identical nanocarrier solution was prepared as above and stored with the first one after the filtration step. This homogenous suspension was stored frozen at -20°C.

[0168] 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 nanocarrier mass per mL of suspension was determined by pycnometry. [Table 7] C57BL / 6 age-matched (5–6 weeks) female mice were injected i.v. in the tail vein with 1.1 mg of whole ovalbumin-loaded nanocarriers (NP[OVA]) combined with either saline (non-treated), 1.2 mg of rapamycin-containing nanocarriers (NP[Rapa]), or 8 mg of GSK1059615-containing nanocarriers (NP[GSK1059615]) on days -21 and -14.

[0169] On day 0, all animals were injected sc in the hind leg with 25 μg particulate OVA (pOVA) mixed with 2 μg CpG, followed by 25 μg pOVA alone on days 7 and 14. Antibody titers were measured on day 21. In the absence of any treatment, animals developed a strong immune response to OVA that could be measured by anti-OVA IgG antibody titers. The antibody titers on day 21 shown in FIG. 4 demonstrate that two doses of synthetic tolerogenic nanocarriers (NP[OVA]+NP[Rapa] or NP[GSK1059615]) administered in the same solution with encapsulated OVA were effective in reducing antibody formation to OVA, even after one injection of OVA+CpG and two injections of OVA alone. These results indicate that encapsulated immunosuppressants (such as rapamycin and GSK1059615) can prevent antibody formation to a protein when delivered in combination with that protein.

[0170] Example 12: Pharmacodynamic Effectiveness Period Administration Using Synthetic Nanocarriers (Prophetic) A pilot study is conducted in non-human primate subjects using soluble factor VIII and the synthetic nanocarrier of Example 1. 50 non-human primate subjects are randomly assigned to 5 arms: placebo, then synthetic nanocarrier at 4 dose levels selected for the dose range. The dose range is to establish a pharmacodynamic effective period ranging from 20 hours to 1 month, with a preferred pharmacodynamic effective period target of 1 day. On day 0, subjects in each active arm are all administered a dose of synthetic nanocarrier subcutaneously, and receive a standard infusion dose of factor VIII infusion within 24 hours of the synthetic nanocarrier dose. After 2 weeks, each animal is challenged with a standard dose of soluble factor VIII, and the level of anti-factor VIII IgG antibodies is measured using standard ELISA techniques. The lowest dose of synthetic nanocarrier from among the 4 active arms that shows a significant reduction in anti-factor VIII antibodies is selected as the test dose. The test doses of the synthetic nanocarriers are then allometrically scaled for administration to human subjects and used in human clinical trials to determine a range of administered dose levels of the synthetic nanocarriers using a standard dose of soluble Factor VIII, and the administered doses of the synthetic nanocarriers and Factor VIII are then made available for routine clinical practice.

[0171] Example 13: Pharmacodynamic Effectiveness Period Administration Using Synthetic Osmotic Pumps (Prophetic) A pilot study is conducted in non-human primate subjects using soluble factor VIII and an osmotic pump (generally prepared according to Example 6, but substituting GSK1059615 for rapamycin in Example 6). Fifty non-human primate subjects are randomly assigned to five arms: placebo, then four dose levels of GSK1059615 delivered by the osmotic pump and selected for a dose range. The dose range is to establish a pharmacodynamic effective period ranging from 20 hours to one month with a preferred pharmacodynamic effective period target of one day. On day 0, all subjects in each active arm are administered the dose of synthetic nanocarriers subcutaneously, and receive a standard infusion dose of factor VIII infusion within 24 hours of the synthetic nanocarrier dose. After two weeks, each animal is challenged with a standard dose of soluble factor VIII, and the levels of anti-factor VIII IgG antibodies are measured using standard ELISA techniques. The lowest dose of GSK1059615 delivered by osmotic pump from among the four active arms that shows a significant reduction in anti-Factor VIII antibodies is selected as the test dose. The test dose of GSK1059615 delivered by the osmotic pump is then allometrically scaled for administration to human subjects and used in human clinical trials to determine a range of administered dose levels of GSK1059615 delivered by the osmotic pump using a standard dose of soluble Factor VIII. The administered doses of GSK1059615 delivered by the osmotic pump and Factor VIII are then made available for clinical practice.

[0172] Example 14: Pharmacodynamic Effectiveness Period Administration Using Therapeutic Polynucleotides (Prophetic) A pilot study is conducted in non-human primate subjects using mmRNA encoding asparaginase (prepared generally according to US Patent Application No. 2013 / 0115272 (Fougerolles et al.) ("mmRNA")) and the synthetic nanocarrier of Example 1. Fifty non-human primate subjects are randomly assigned to five arms: placebo, then four doses of synthetic nanocarrier selected for a dose range. The dose range is to establish a pharmacodynamic effective period ranging from 20 hours to one month with a preferred daily pharmacodynamic effective period target. On day 0, all subjects in each active arm are administered the dose of synthetic nanocarrier subcutaneously and receive an infusion of a standard infusion dose of Factor VIII within 24 hours of the synthetic nanocarrier dose. After two weeks, each animal is challenged with a standard dose of mmRNA and the levels of anti-mmRNA antibodies are measured using standard ELISA techniques. The lowest dose of synthetic nanocarrier from among the four active arms that shows a significant reduction in anti-mmRNA antibodies is selected as the test dose. The test doses of the synthetic nanocarriers are then allometrically scaled for administration to human subjects and used in human clinical trials to determine a range of administered doses of the synthetic nanocarriers using standard dose levels of the mmRNA. The administered doses of the synthetic nanocarriers and the mmRNA are then made available for standard clinical practice.

[0173] Example 15: Pharmacodynamic Effectiveness Period Dosing Using Nanocrystalline Immunosuppressants (Prophetic) A pilot study is conducted in non-human primate subjects using the soluble factor VIII and synthetic nanocarriers of Example 1. 50 non-human primate subjects are randomly assigned to five arms: placebo, then four dose levels of nanocrystalline rapamycin selected for dose range. The dose range is to establish a pharmacodynamic effective period ranging from 20 hours to one month with a preferred pharmacodynamic effective period target of one day. On day 0, all subjects in each active arm are administered the dose of nanocrystalline rapamycin subcutaneously and receive a standard infusion dose of factor VIII infusion within 24 hours of the nanocrystalline rapamycin dose. After two weeks, each animal is challenged with a standard dose of soluble factor VIII and the levels of anti-factor VIII IgG antibodies are measured using standard ELISA techniques. The lowest dose of nanocrystalline rapamycin from among the four active arms that shows a significant reduction in anti-factor VIII antibodies is selected as the test dose. The test doses of nanocrystalline rapamycin are then allometrically scaled for administration to human subjects and used in human clinical trials to determine a range of administered dose levels of nanocrystalline rapamycin using standard doses of soluble Factor VIII, and the administered doses of nanocrystalline rapamycin and Factor VIII are then made available for standard clinical practice.

[0174] Example 16: Pharmacodynamic Effectiveness Period Dosing Using Nanocrystalline Immunosuppressants (Prophetic) A pilot study is conducted in non-human primate subjects using mmRNA encoding asparaginase (prepared generally according to US Patent Application 2013 / 0115272 (Fougerolles et al.) ("mmRNA")) and nanocrystalline rapamycin. Fifty non-human primate subjects are randomly assigned to five arms: placebo, then four doses of synthetic nanocarriers selected for a dose range. The dose range is to establish a pharmacodynamic effective period ranging from 20 hours to one month with a preferred daily pharmacodynamic effective period target. On day 0, all subjects in each active arm receive the dose of nanocrystalline rapamycin subcutaneously and receive an infusion of a standard infusion dose of factor VIII within 24 hours of the nanocrystalline rapamycin dose. Two weeks later, each animal is challenged with a standard dose of mmRNA and the levels of anti-mmRNA antibodies are measured using standard ELISA techniques. The lowest dose of nanocrystalline rapamycin from among the four active arms that shows a significant reduction in anti-mmRNA antibodies is selected as the test dose. The test doses of nanocrystalline rapamycin are then allometrically scaled for administration to human subjects and used in human clinical trials to determine a range of administered doses of nanocrystalline rapamycin using standard dose levels of mmRNA. The administered doses of nanocrystalline rapamycin and mmRNA are then made available for standard clinical practice.

Claims

1. administering an immunosuppressant to the subject at a dosage that provides a pharmacodynamic effective period for the therapeutic macromolecule in a first class of subjects, for a pharmacodynamic effective period ranging from a minimum of 20 hours to a maximum of one month; and administering a therapeutic macromolecule to the subject within a pharmacodynamically effective period of administration of the immunosuppressant; A method comprising: The method, wherein the therapeutic polymer and the immunosuppressant are not attached to each other, and the therapeutic polymer is not attached to a synthetic nanocarrier.

2. The method is as follows: Determine the dosage of the immunosuppressant based on the test dose of the immunosuppressant Further comprising: the test dose has a test pharmacodynamic effective period with respect to the therapeutic macromolecule for a pharmacodynamic effective period ranging from a minimum of 20 hours to a maximum of one month in the second class of subjects; The method of claim 1.

3. An immunosuppressant for use in a method of inducing tolerance to a therapeutic macromolecule, the method comprising: (a) administering the immunosuppressant to a subject at a dose sufficient to elicit a pharmacodynamic effective period that is between 20 hours and 1 month; and (b) administering a therapeutic macromolecule to the subject within the pharmacodynamic effective period, wherein the therapeutic macromolecule and the immunosuppressant are not attached to each other, and the therapeutic macromolecule is not attached to a synthetic nanocarrier.

4. Use of an immunosuppressant for the manufacture of a medicament for use in a method comprising: (a) administering to a subject an immunosuppressant at a dose sufficient to elicit a pharmacodynamic effective period ranging from 20 hours to 1 month; and (b) administering to said subject a therapeutic macromolecule within said pharmacodynamic effective period, wherein the therapeutic macromolecule and the immunosuppressant are not attached to each other, and the therapeutic macromolecule is not attached to a synthetic nanocarrier.

5. The method of claim 1 or 2, the immunosuppressant of claim 3 or the use of claim 4, wherein the immunosuppressant comprises a synthetic nanocarrier comprising an immunosuppressant to which the synthetic nanocarrier is attached, an implantable osmotic pump, a bispecific antibody or an implantable polymeric depot material.

6. The method, agent or use according to any one of claims 1 to 5, wherein the immunosuppressant comprises an immunosuppressant having synthetic nanocarriers attached thereto.

7. 7. The method, immunosuppressant or use according to any one of claims 1 to 6, wherein the synthetic nanocarriers comprise lipid nanoparticles, polymeric nanoparticles, metal nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles, protein particles or nanoparticles comprising a combination of nanomaterials, optionally such nanoparticles are lipid-polymer nanoparticles.

8. Immunosuppressants include, but are not limited to, statins; mTOR inhibitors; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase inhibitors; corticosteroids; inhibitors of mitochondrial function; P38 inhibitors; NF-κβ inhibitors, dexamethasone; TCPA-1; IKK VII; adenosine receptor agonists; prostaglandin E2 agonists; phosphodiesterase inhibitors; proteasome inhibitors; 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; phosphatases The method, immunosuppressant or use of any one of claims 1 to 7, comprising: an aryl hydrocarbon receptor inhibitor; a PI3KB inhibitor; an autophagy inhibitor; an aryl hydrocarbon receptor inhibitor; a proteasome inhibitor I (PSI); oxidized ATP; IDO, vitamin D3; cyclosporine; an aryl hydrocarbon receptor inhibitor; resveratrol; azathioprine; 6-mercaptopurine; 6-thioguanine; FK506; sangliferin A; salmeterol; mycophenolate mofetil; aspirin and other COX inhibitors; niflumic acid; estriol; or triptolide.

9. 9. The method, immunosuppressant or use of claim 8, wherein the immunosuppressant comprises an mTOR inhibitor.

10. 10. The method, immunosuppressant or use of claim 9, wherein the mTOR inhibitor comprises rapamycin.

11. 11. The method, immunosuppressant or use according to any one of claims 1 to 10, wherein the loading of the immunosuppressant in the synthetic nanocarriers ranges between 0.0001 wt% and 50 wt%, based on the total dry recipe weight of material in the synthetic nanocarriers (wt / wt).

12. 12. The method, immunosuppressant or use according to claim 11 , wherein the loading of the immunosuppressant in the synthetic nanocarrier ranges between 0.1 wt % and 10 wt %, based on the total dry recipe weight of the material in the synthetic nanocarrier (wt / wt).

13. The method, immunosuppressant or use according to any one of claims 1 to 12, wherein the therapeutic macromolecule comprises a therapeutic protein or a therapeutic polynucleotide.

14. 14. The method, immunosuppressant or use according to claim 13, wherein the therapeutic protein comprises an enzyme, an enzyme cofactor, a hormone, a blood clotting factor, a cytokine, a growth factor, a monoclonal or polyclonal antibody.

15. The method, immunosuppressant or use according to any one of claims 1 to 14, wherein the pharmacodynamic effective period of administration has a duration ranging from a minimum of 20 hours to a maximum of 2 weeks.

16. 16. The method, immunosuppressant or use of claim 15, wherein the pharmacodynamic effective period of administration has a duration ranging from a minimum of 20 hours to a maximum of 1 week.

17. 17. The method, immunosuppressant or use of claim 16, wherein the pharmacodynamic effective period of administration has a duration ranging from a minimum of 24 hours to a maximum of 2 days.

18. 18. The method, immunosuppressant or use according to any one of claims 1 to 17, wherein the test pharmacodynamic effective period has a duration ranging from a minimum of 20 hours to a maximum of 2 weeks.

19. 20. The method, immunosuppressant or use of claim 18, wherein the test pharmacodynamic effective period has a duration ranging from a minimum of 20 hours to a maximum of 1 week.

20. 20. The method, immunosuppressant or use of claim 19, wherein the test pharmacodynamic effective period has a duration ranging from a minimum of 24 hours to a maximum of 2 days.

21. The method, immunosuppressant or use according to any one of claims 1 to 20, wherein the administered dose of the immunosuppressant is determined based on test doses of the immunosuppressant together with the use of allometric or isometric scaling techniques.

22. The method, immunosuppressant or use according to any one of claims 1 to 21, wherein the first class of subjects and the second class of subjects are the same class of subjects.

23. The method, immunosuppressant or use according to any one of claims 1 to 22, wherein the first class of subject and the second class of subject are different classes of subject.

24. 8. The method, immunosuppressant or use of claim 7, wherein the synthetic nanocarrier comprises a lipid nanoparticle.

25. The method, immunosuppressant or use according to claim 7 , wherein the synthetic nanocarrier comprises a liposome.

26. 8. The method, immunosuppressant or use of claim 7, wherein the synthetic nanocarriers comprise metal nanoparticles.

27. 27. The method, immunosuppressant or use of claim 26, wherein the metal nanoparticles comprise gold nanoparticles.

28. 8. The method, immunosuppressant or use of claim 7, wherein the synthetic nanocarriers comprise polymeric nanoparticles.

29. 29. The method, immunosuppressant or use of claim 28, wherein the polymeric nanoparticles comprise a polymer which is a non-methoxy-terminated Pluronic polymer.

30. 30. The method, immunosuppressant or use of claim 28 or 29, wherein the polymeric nanoparticles comprise polyesters, polyesters attached to polyethers, polyamino acids, polycarbonates, polyacetals, polyketals, polysaccharides, polyethyloxazolines or polyethyleneimines.

31. 31. The method, immunosuppressant or use of claim 30, wherein the polyester comprises poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid) or polycaprolactone.

32. 32. The method, immunosuppressant or use of claim 30 or 31, wherein the polymeric nanoparticles comprise polyester and polyester attached to polyether.

33. The method, immunosuppressant or use according to any one of claims 30 to 32, wherein the polyether comprises polyethylene glycol or polypropylene glycol.

34. 34. The method, immunosuppressant or use according to any one of claims 1 to 33, wherein the mean of the particle size distribution obtained using dynamic light scattering of the synthetic nanocarriers is greater than 100 nm in diameter.

35. 35. The method, immunosuppressant or use according to claim 34, wherein the diameter is greater than 150 nm.

36. 36. The method, immunosuppressant or use according to claim 35, wherein the diameter is greater than 200 nm.

37. 37. The method, immunosuppressant or use of claim 36, wherein the diameter is greater than 250 nm.

38. 38. The method, immunosuppressant or use according to claim 37, wherein the diameter is greater than 300 nm.

39. 39. The method, immunosuppressant or use according to any one of claims 1 to 38, wherein the aspect ratio of the synthetic nanocarriers 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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