阅读说明：本技术 用于降低的或增强的药效学作用的致耐受性合成纳米载体和治疗性大分子 (Tolerogenic synthetic nanocarriers and therapeutic macromolecules for reduced or enhanced pharmacodynamic effects ) 是由 罗伯托·A·马尔多纳多 岸本·隆·慧 于 2014-05-02 设计创作，主要内容包括：本发明涉及用于降低的或增强的药效学作用的致耐受性合成纳米载体和治疗性大分子。公开了提供治疗性大分子特异性的药效学作用的组合物和方法。所述作用可由与免疫抑制剂剂量相组合的治疗性大分子的降低的剂量产生。所述作用还可用这样的组合物来增强。(The present invention relates to tolerogenic synthetic nanocarriers and therapeutic macromolecules for reduced or enhanced pharmacodynamic effects. Compositions and methods are disclosed that provide pharmacodynamic effects specific to therapeutic macromolecules. The effect may result from a reduced dose of the therapeutic macromolecule in combination with an immunosuppressant dose. The effect may also be enhanced with such compositions.)

1. A method, comprising:

providing an immunosuppressant dose, wherein the immunosuppressant dose is linked to a synthetic nanocarrier; and

concomitantly administering a reduced pharmacodynamically effective dose of the therapeutic macromolecule and the immunosuppressant dose to a subject in which an anti-therapeutic macromolecule antibody response is expected to occur;

wherein the concomitant administration is according to a protocol that has been demonstrated to produce a pharmacodynamic effect at the reduced pharmacodynamically effective dose of the therapeutic macromolecule following concomitant administration with the immunosuppressant dose as compared to administration of the therapeutic macromolecule when not concomitant administration with the immunosuppressant dose and an anti-therapeutic macromolecule antibody response is present.

2. The method of claim 1, wherein the method further comprises determining the protocol.

3. The method of claim 1 or 2, wherein the method further comprises determining the reduced pharmacodynamically effective dose.

4. The method of any one of claims 1 to 3, wherein the method further comprises assessing the pharmacodynamic effect in the subject prior to and/or after the administering.

5. The method of any one of claims 1 to 4, wherein the administering is performed by intravenous, intraperitoneal, or subcutaneous administration.

6. A method, comprising:

providing an immunosuppressant dose, wherein the immunosuppressant dose is linked to a synthetic nanocarrier; and

administering a reduced pharmacodynamically effective dose of a therapeutic macromolecule concomitantly with the immunosuppressant dose;

wherein the reduced pharmacodynamically effective dose of the therapeutic macromolecule is less than the following pharmacodynamically effective dose of the therapeutic macromolecule: (A) administering the therapeutic macromolecule in the presence of an anti-therapeutic macromolecule antibody response, and (B) not administering the therapeutic macromolecule concomitantly with the immunosuppressant dose.

7. The method of claim 6, wherein the method further comprises determining the reduced pharmacodynamically effective dose.

8. The method of claim 6 or 7, wherein the method further comprises assessing pharmacodynamic effects in the subject prior to and/or after the administering.

9. The method of any one of claims 6 to 8, wherein the administering is performed by intravenous, intraperitoneal, or subcutaneous administration.

10. A method, comprising:

providing an immunosuppressant dose, wherein the immunosuppressant dose is linked to a synthetic nanocarrier; and

concomitantly administering a pharmacodynamically effective dose of the therapeutic macromolecule and an immunosuppressant dose to a subject in which an anti-therapeutic macromolecule antibody response is expected to occur;

wherein the concomitant administration is according to a protocol that has been demonstrated to enhance the pharmacodynamic effect of the therapeutic macromolecule following concomitant administration with the immunosuppressant dose as compared to administration of the therapeutic macromolecule when not concomitant administration with the immunosuppressant dose and each has an anti-therapeutic macromolecule antibody response.

Technical Field

The present invention relates to immunosuppressant doses (in some embodiments, linked to synthetic nanocarriers) for concomitant administration with therapeutic macromolecules and related methods. The compositions and methods allow for highly effective pharmacodynamic action specific to the therapeutic macromolecule. Thus, the provided compositions and methods are useful for generating a pharmacodynamic response in a subject even at reduced doses of a therapeutic macromolecule. The compositions and methods provided herein can also be concomitantly repeated to produce the desired pharmacodynamic and immunological effects.

Background

Therapeutic treatments (e.g., protein or enzyme replacement therapy) often result in an undesirable immune response to a particular therapeutic agent. In these cases, cells in the immune system recognize the therapeutic as foreign and attempt to neutralize or destroy it just as they attempt to destroy infectious organisms, such as bacteria and viruses. Such an undesirable immune response may neutralize the efficacy of the therapeutic treatment or cause hypersensitivity to the therapeutic agent. These undesirable responses can be reduced by the use of immunosuppressive drugs. However, conventional immunosuppressive drugs are widely used, and the use of widely used immunosuppressive agents is associated with the risk of serious side effects such as tumors, infections, nephrotoxicity, and metabolic disorders. Therefore, new treatments would be beneficial.

Disclosure of Invention

In one aspect, a method is provided that includes providing an immunosuppressant dose, wherein in some embodiments, the immunosuppressant dose is linked to a synthetic nanocarrier; and administering the reduced pharmacodynamically effective dose of the therapeutic macromolecule to the subject concomitantly with the immunosuppressant dose. In one embodiment, the concomitant administration is according to a protocol that has been demonstrated to produce a pharmacodynamic effect at a reduced pharmacodynamically effective dose of the therapeutic macromolecule following concomitant administration with the immunosuppressive dose, as compared to administration of the therapeutic macromolecule when not concomitant administration with the immunosuppressive dose and each present an anti-therapeutic macromolecule antibody response. In another embodiment of any one of the methods provided, the reduced pharmacodynamically effective dose of the therapeutic macromolecule is less than (a) a pharmacodynamically effective dose of the therapeutic macromolecule administered in the presence of an anti-therapeutic macromolecule antibody response, and (B) a pharmacodynamically effective dose of the therapeutic macromolecule administered without concomitant administration of an immunosuppressant dose.

In another aspect, a method is provided that includes providing an immunosuppressant dose, wherein in some embodiments, the immunosuppressant dose is linked to a synthetic nanocarrier; and concomitantly administering a pharmacodynamically effective dose of the therapeutic macromolecule with a dose of the immunosuppressant. In one embodiment, the concomitant administration is according to a regimen that has been demonstrated to enhance the pharmacodynamic effect of the therapeutic macromolecule following concomitant administration with the immunosuppressive agent dose as compared to administration of the therapeutic macromolecule when not concomitant administration with the immunosuppressive agent dose and each present an anti-therapeutic macromolecule antibody response.

In another aspect, a method is provided that includes providing an immunosuppressant dose, wherein in some embodiments, the immunosuppressant dose is linked to a synthetic nanocarrier; concomitantly administering a pharmacodynamically effective dose of the therapeutic macromolecule with a dose of the immunosuppressive agent; and recording the enhanced pharmacodynamic effect following concomitant administration.

In another aspect, a method is provided that includes providing a therapeutic macromolecule that produces or is expected to produce an anti-therapeutic macromolecule antibody upon repeated dosing in one or more subjects; and providing an immunosuppressant dose, wherein the immunosuppressant dose is linked to a synthetic nanocarrier. In some embodiments, the method comprises concomitantly repeating administration of the therapeutic macromolecule to the subject with the immunosuppressant dose at the same or a lower dose. In some embodiments, the concomitant administration is according to a regimen that has been demonstrated to maintain the pharmacodynamic effect of the therapeutic macromolecule during two or more doses of the therapeutic macromolecule to the subject.

In one embodiment of any one of the methods provided, the method further comprises determining the protocol. In another embodiment of any one of the methods provided, the method further comprises determining a pharmacodynamically effective dose, e.g., a reduced or increased pharmacodynamically effective dose. In another embodiment of any one of the methods provided, the method further comprises assessing pharmacodynamic effects in the subject prior to and/or after the administering. In another embodiment of any one of the methods provided, the concomitant administration is repeated one or more times. In another embodiment of any one of the methods provided, the administering is performed by intravenous, intraperitoneal, or subcutaneous administration. In another embodiment of any one of the methods provided, the subject is at risk for an anti-therapeutic macromolecular antibody response. In another embodiment of any one of the methods provided, the subject is a subject in whom an anti-therapeutic macromolecular response is expected to occur.

In another aspect, a composition or kit (kit) is provided comprising an immunosuppressant dose, wherein in some embodiments the immunosuppressant is linked to a synthetic nanocarrier; and a reduced pharmacodynamically effective dose of the therapeutic macromolecule.

In another aspect, a composition or kit is provided comprising a reduced pharmacodynamically effective dose of a therapeutic macromolecule for use in any of the methods provided herein in combination with an immunosuppressant dose, wherein in some embodiments, the immunosuppressant is linked to a synthetic nanocarrier.

In one embodiment of any one of the compositions or kits provided, the composition or kit is for use in any one of the methods provided herein. In one embodiment of any one of the compositions or kits provided, the composition or kit further comprises a pharmaceutically acceptable carrier.

In one embodiment of any one of the methods or compositions or kits provided, the therapeutic macromolecule is not linked to a synthetic nanocarrier. In another embodiment of any one of the methods or compositions or kits provided, the therapeutic macromolecule is linked to a synthetic nanocarrier. In another embodiment of any one of the methods or compositions or kits provided, the synthetic nanocarriers do not comprise a therapeutic macromolecular APC presentable antigen.

In one embodiment of any one of the compositions or kits provided, the immunosuppressant dose and therapeutic macromolecule are separately contained in a container. In another embodiment of any one of the compositions or kits provided, the immunosuppressant dose and therapeutic macromolecule are contained in separate containers. In another embodiment of any one of the compositions or kits provided, the immunosuppressant dose and therapeutic macromolecule are contained in the same container.

In one embodiment of any one of the methods or compositions or kits provided, the reduced pharmacodynamically effective dose of the therapeutic macromolecule is at least 30% lower than a pharmacodynamically effective dose of the therapeutic macromolecule that is (a) administered in the presence of an anti-therapeutic macromolecule antibody response, and (B) administered without concomitant administration of an immunosuppressant dose. In another embodiment of any one of the methods or compositions or kits provided, the reduced pharmacodynamically effective dose is reduced by at least 40%. In another embodiment of any one of the methods or compositions or kits provided, the reduced pharmacodynamically effective dose is reduced by at least 50%.

In one embodiment of any one of the methods or compositions or kits provided, the immunosuppressant dose comprises a statin, an mTOR inhibitor, a TGF- β signaling agent, a corticosteroid, an inhibitor of mitochondrial function, a P38 inhibitor, an NF- κ β inhibitor, an adenosine receptor agonist, a prostaglandin E2 agonist, a phosphodiesterase4inhibitor, an HDAC inhibitor or a proteasome inhibitor.

In one embodiment of any one of the methods or compositions or kits provided herein, the therapeutic macromolecule comprises a therapeutic protein in any one of the methods or compositions or kits provided herein, or in another embodiment of any one of the methods or compositions or kits provided herein, the therapeutic protein is for use in protein replacement or protein supplementation therapy, in another embodiment of any one of the methods or compositions or kits provided herein, the therapeutic protein comprises an infusible or injectable therapeutic protein, enzyme, cofactor, hormone, blood factor or clotting factor, cytokine, interferon, growth factor, monoclonal antibody, polyclonal antibody, or protein associated with dermatosis (Pomace' S disease), in another embodiment of any one of the methods or compositions or kits provided herein, the infusible or injectable therapeutic protein comprises Tortula globalpina (Tocilimab), α -1 antitrypsin, Hedgelide, interferon, or peroxidase, or thrombin, or peroxidase, or a.

In one embodiment of any one of the methods or compositions or kits provided, the loading of immunosuppressive agent linked to the synthetic nanocarriers is 0.1% to 50% based on the average of all synthetic nanocarriers. In another embodiment of any one of the methods or compositions or kits provided, the loading is 0.1% to 20%.

In one embodiment of any one of the methods or compositions or kits provided, the synthetic nanocarriers comprise lipid nanoparticles, polymer nanoparticles, metal nanoparticles, surfactant-based emulsions (surfactant-based emulsions), dendrimers (dendrimers), buckyballs (buckyballs), nanowires (nanowires), virus-like particles, or peptide or protein particles. In another embodiment of any one of the methods or compositions or kits provided, the synthetic nanocarriers comprise lipid nanoparticles. In another embodiment of any one of the methods or compositions or kits provided, the synthesis isThe nanocarrier comprises a liposome. In another embodiment of any one of the methods or compositions or kits provided, the synthetic nanocarriers comprise metal nanoparticles. In another embodiment of any one of the methods or compositions or kits provided, the metal nanoparticle comprises a gold nanoparticle. In another embodiment of any one of the methods or compositions or kits provided, the synthetic nanocarriers comprise polymeric nanoparticles. In another embodiment of any one of the methods or compositions or kits provided, the polymeric nanoparticle comprises a polymer that is a non-methoxy-terminated pluronic (pluronic) polymer. In another embodiment of any one of the methods or compositions or kits provided, the polymeric nanoparticle comprises a polyester, a polyether-linked polyester, a polyamino acid, a polycarbonate, a polyacetal, a polyketal, a polysaccharide, a polyethyl

Oxazoline or polyethyleneimine. In another embodiment of any one of the methods or compositions or kits provided, 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 or compositions or kits provided, the polymeric nanoparticle comprises a polyester and a polyester linked to a polyether. In another embodiment of any one of the methods or compositions or kits provided, the polyether comprises polyethylene glycol or polypropylene glycol.

In one embodiment of any one of the methods or compositions or kits provided, the average of the particle size distribution obtained using dynamic light scattering of synthetic nanocarriers is a diameter greater than 100 nm. In another embodiment of any one of the methods or compositions or kits provided, the diameter is greater than 150 nm. In another embodiment of any one of the methods or compositions or kits provided, the diameter is greater than 200 nm. In another embodiment of any one of the methods or compositions or kits provided, the diameter is greater than 250 nm. In another embodiment of any one of the methods or compositions or kits provided, the diameter is greater than 300 nm.

In one embodiment of any one of the methods or compositions or kits provided, the synthetic nanocarriers have an aspect ratio (aspect ratio) of greater than 1: 1, 1: 1.2, 1: 1.5, 1: 2, 1: 3, 1: 5, 1: 7, or 1: 10.

In another aspect, a method of making any one of the compositions or kits provided herein is provided. In one embodiment, the method of making comprises generating a dose or dosage form of a therapeutic macromolecule and generating a dose or dosage form of an immunosuppressive agent. In some embodiments, the dose or dosage form of the therapeutic macromolecule is a reduced, pharmacodynamically effective dose of the therapeutic macromolecule. In another embodiment of any one of the methods of making provided, the step of generating a dose or dosage form of the immunosuppressant comprises attaching the immunosuppressant to a synthetic nanocarrier. In another embodiment of any one of the methods of making provided, the method further comprises combining a dose or dosage form of an immunosuppressive agent with a dose or dosage form of a therapeutic macromolecule in a kit.

In another aspect, there is provided a use of any one of the compositions or kits provided herein for the manufacture of a medicament for reducing an anti-therapeutic macromolecular antibody response in a subject. In one embodiment, the composition or kit comprises an immunosuppressant and a therapeutic macromolecule, wherein the therapeutic macromolecule may be provided at a reduced pharmacodynamically effective dose of the therapeutic macromolecule. In another embodiment of any one of the uses provided herein, the immunosuppressant is attached to a synthetic nanocarrier. In another embodiment of any one of the uses provided herein, the use is for carrying out any one of the methods provided herein.

In another aspect, any of the compositions or kits provided herein can be used in any of the methods provided herein. In one embodiment, the composition or kit comprises one or more doses or dosage forms of a therapeutic macromolecule and/or one or more doses or dosage forms of an immunosuppressive agent. In one embodiment, the dose of the therapeutic macromolecule is a reduced pharmacodynamically effective dose. In another embodiment, the immunosuppressant is attached to a synthetic nanocarrier.

In another aspect, a method of preparing a medicament intended for reducing an anti-therapeutic macromolecular antibody response is provided. In one embodiment, the medicament comprises an immunosuppressive agent and/or a therapeutic macromolecule, wherein the therapeutic macromolecule may be at a reduced pharmacodynamically effective dose. In another embodiment of any one of the methods of making provided herein, the immunosuppressant is attached to a synthetic nanocarrier.

Drawings

Figure 1 shows the levels of circulating antigen-specific antibody production under concomitant administration provided herein.

Figure 2 shows the levels of circulating antigen-specific antibody production under concomitant administration provided herein.

Figure 3 provides anti-OVA antibody titers under concomitant administration provided herein.

Figure 4 provides anti-KLH antibody titers under concomitant administration provided herein.

Figure 5 shows the antibody recall response against FVIII 1 month after the last nanocarrier and FVIII administration.

Figures 6A and 6B show the efficacy of nanocarrier and FVIII administration in hemophilia a mice.

Fig. 7A and 7B show the immune response against HUMIRA in mice treated with HUMIRA/adalimumab with or without nanocarriers linked to rapamycin.

Fig. 8 shows anti-KLH antibody titers in mice treated with Keyhole Limpet Hemocyanin (KLH) with or without nanocarriers linked to rapamycin.

Figure 9 shows anti-OVA antibody titers in mice treated with Ovalbumin (OVA) with or without nanocarriers linked to rapamycin.

Figure 10 shows anti-krysteke xxa antibody titers in mice treated with krysteke xxa with or without nanocarriers attached to rapamycin.

Figure 11 shows antibody titers in mice treated with OVA and KLH in the presence or absence of nanocarriers linked to rapamycin.

Fig. 12A and 12B show immune responses against KLH in mice treated with KLH with or without nanocarriers linked to rapamycin.

Fig. 13A and 13B show the immune response against HUMIRA/adalimumab in mice treated with HUMIRA/adalimumab with or without nanocarriers linked to rapamycin.

Fig. 14 provides one exemplary scheme for implementing the methods provided herein.

Figure 15 illustrates the beneficial effects of the methods provided herein with respect to the administration of treatment with HUMIRA.

Fig. 16 provides one exemplary scheme for practicing the methods provided herein.

Figure 17 illustrates the beneficial effects of the methods provided herein with respect to the administration of therapy with HUMIRA.

Figure 18 demonstrates that anti-protein antibody responses were reduced as a result of two different immunosuppressive agents attached to synthetic nanocarriers.

Figure 19 illustrates another exemplary protocol and benefits for practicing the methods provided herein with respect to treatment with HUMIRA.

Detailed Description

Before the present invention is described in detail, it is to be understood that this invention is not limited to the particular illustrative materials or process parameters, as 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 be limiting.

All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety for all purposes.

As used in this specification and the appended claims, the term "a" or "an" unless the content clearly dictates otherwise, means one or more. For example, reference to "a polymer" includes a mixture of two or more such molecules or a mixture of single polymer species of different molecular weights, reference to "a synthetic nanocarrier" includes a mixture of two or more such synthetic nanocarriers or a plurality of such synthetic nanocarriers, reference to "an RNA molecule" includes a mixture of two or more such RNA molecules or a plurality of such RNA molecules, reference to "an immunosuppressant" includes a mixture of two or more such materials or a plurality of such immunosuppressant molecules, and the like.

As used herein, the term "comprises/comprising" or variations thereof, is understood to imply the inclusion of a stated integer or group of integers (e.g. features, elements, features, characteristics, method/process steps or limitations), but not the exclusion of any other integer or group of integers. Thus, the term "comprising" as used herein is inclusive and does not exclude additional unrecited integers or method/process steps.

In some embodiments of any of the compositions and methods provided herein, the term "comprising" may be substituted for "consisting essentially of or" consisting of. The phrase "consisting essentially of" as used herein requires the specified integers or steps as well as those that do not significantly affect the characteristics or function of the claimed invention. The term "consisting of" as used herein is intended to refer only to the presence of the referenced integer (e.g., feature, element, feature, characteristic, method/process step or limitation) or group of integers (e.g., features, elements, features, characteristics, method/process steps or limitations).

A. Introduction to the design reside in

The methods, compositions, or kits provided herein can be used to enhance the pharmacodynamic effect of a therapeutic macromolecule in a subject who has developed an antibody response to the therapeutic macromolecule. Thus, the methods or compositions or kits provided herein can be used to improve the pharmacodynamic effects of a therapeutic macromolecule (which would otherwise be attenuated by an anti-therapeutic macromolecule antibody response). Without being limited to a particular theory, it is believed that the provided methods, compositions, or kits can be used to reduce an undesirable humoral immune response against a therapeutic macromolecule. In some embodiments, the methods, compositions, or kits can be used to tolerize a subject to a therapeutic macromolecule, reducing an undesirable immune response that would otherwise result when the therapeutic macromolecule is administered without concomitant administration of the immunosuppressant dose provided, which may be repeated with concomitant administration. Such an undesirable immune response may result in enhanced clearance of the therapeutic macromolecule or other interference with the therapeutic activity of the therapeutic macromolecule. Thus, as a result of the reduction in the undesired immune response, the pharmacodynamic effects of the therapeutic macromolecule may be enhanced with the methods, compositions, or kits provided herein and/or the same level of effect may be achieved using a reduced dose of the therapeutic macromolecule. Thus, as another result of the reduction in the undesired immune response, repeated doses of the therapeutic macromolecule may be administered to the subject.

It has been unexpectedly and surprisingly found that concomitant administration of an immunosuppressant (preferably, in some embodiments, when linked to a synthetic nanocarrier) to a therapeutic macromolecule in the presence of an anti-therapeutic macromolecule antibody response can produce an enhanced pharmacodynamic effect. For example, the above combinations may help neutralize anti-therapeutic macromolecule-specific antibodies that interfere with the desired therapeutic effect of the therapeutic macromolecule. In some embodiments, the methods, compositions, or kits provided herein not only reduce an undesired immune response to a therapeutic macromolecule, but also allow for enhancement of a desired therapeutic effect of the therapeutic macromolecule (as a result of the undesired immune response to the therapeutic macromolecule) that is otherwise attenuated when the therapeutic macromolecule is administered alone. Thus, the methods, compositions, or kits provided herein can provide a subject with the therapeutic benefit of a therapeutic macromolecule without increasing the dose of the therapeutic macromolecule, which would typically be increased in order to compensate for an undesired immune response against the therapeutic macromolecule when administered without the benefits of the invention provided herein. Surprisingly, the methods, compositions, or kits provided herein even allow for administration of reduced therapeutic macromolecular doses to a subject to achieve the same therapeutic benefit.

Since the provided methods, compositions, or kits can be used to counteract an undesired immune response generated during therapeutic treatment with a therapeutic macromolecule, the present invention can be used to achieve an enhanced pharmacodynamic effect or to use a reduced pharmacodynamically effective dose in a subject in which an undesired immune response to a therapeutic macromolecule is generated or expected to be generated. In one embodiment of any one of the methods provided herein, the subject may be a subject at risk of such an undesired immune response.

Now, the present invention will be described in more detail hereinafter.

B. Definition of

By "administering" is meant providing a substance to a subject in a pharmacologically useful manner. In some embodiments, the term is intended to include "causing administration". By "causing administration" is meant directly or indirectly causing, promoting, encouraging, assisting, inducing or directing the administration of a substance by another party.

In the context of a composition or dose for administration to a subject, "effective amount" refers to the amount of the composition or dose that produces one or more desired responses in the subject, such as a tolerogenic immune response (e.g., a reduction in proliferation, activation, induction, survival, recruitment of therapeutic macromolecule-specific B cells, or a reduction in production of therapeutic macromolecule-specific antibodies). In some embodiments, the effective amount is a pharmacodynamically effective amount. Thus, in some embodiments, an effective amount is any amount of a composition or dose provided herein (or compositions or doses provided herein) that produces one or more of the desired pharmacodynamic effects, therapeutic effects, and/or immune responses provided herein. This amount can be used for in vitro or in vivo purposes. For in vivo purposes, the amount may be that amount deemed clinically beneficial by a clinician to a subject in need of administration of the therapeutic macromolecule and/or antigen-specific immune tolerance thereto.

An effective amount may relate to reducing the level of an undesired immune response, but in some embodiments it relates to completely preventing an undesired immune response. An effective amount may also involve delaying the onset of an undesired immune response. An effective amount can also be an amount that produces a desired therapeutic endpoint or desired therapeutic result. In other embodiments, an effective amount may relate to enhancing the level of a desired response (e.g., therapeutic endpoint or outcome). Preferably, the effective amount elicits a tolerogenic immune response in the subject against the antigen (e.g., a therapeutic macromolecule). The implementation of any of the above can be monitored by conventional methods.

In some embodiments of any one of the methods provided, the effective amount is an amount wherein a response is desired in the subject for at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or longer. In still other embodiments of any one of the compositions or methods provided, an effective amount is an amount that produces a measurable desired response over at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or more.

Of course, the effective amount will depend on the particular subject being treated; the severity of the condition, disease or disorder; individual patient parameters including age, physical condition, size and weight; the duration of the treatment; the nature of concurrent therapy (if any); specific route of administration and similar factors within the knowledge and experience of a health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with only routine experimentation. It is generally preferred to use the maximum dose, i.e., the highest safe dose according to sound medical judgment. However, one of ordinary skill in the art will appreciate that patients may insist on using lower or tolerable doses for medical reasons, psychological reasons, or virtually any other reason.

In general, the dosage of the immunosuppressant and/or therapeutic macromolecule in the compositions of the invention refers to the amount of immunosuppressant and/or therapeutic macromolecule. Alternatively, the dose may be administered based on the amount of synthetic nanocarriers that provide the desired amount of immunosuppressant and/or therapeutic macromolecule.

An "anti-therapeutic macromolecule antibody response" or "anti-therapeutic macromolecule-specific antibody response" is the production of anti-therapeutic macromolecule-specific antibodies or the induction of a process for producing such antibodies as a result of administration of a therapeutic macromolecule. In some embodiments, such a response counteracts the therapeutic effect of the therapeutic macromolecule.

"antigen" means a B cell antigen or a T cell antigen. By "type of antigen" is meant 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 contained in or expressed within a cell. In some embodiments, for example 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, or the like.

"antigen-specific" refers to any immune response generated by the presence of the antigen or a portion thereof or any immune response that generates a molecule that specifically recognizes or binds the antigen. In some embodiments, when the antigen comprises a therapeutic macromolecule, antigen-specificity may mean that the therapeutic macromolecule is specific. For example, when the immune response is antigen-specific antibody production (e.g., therapeutic macromolecule-specific antibody production), an antibody is produced that specifically binds the antigen (e.g., therapeutic macromolecule). As another example, when the immune response is antigen-specific B cell or CD4+ T cell proliferation and/or activity, the proliferation and/or activity is caused by recognition of the antigen or a portion thereof, alone or in complex with MHC molecules, B cells, and the like.

By "assessing a pharmacodynamic effect" is meant any measurement or determination of the level, presence or absence, reduction, increase, etc., of a pharmacodynamic effect in vitro or in vivo. Such measurements or determinations may be made on one or more samples obtained from a subject. Such assessment may be performed in any of the methods provided herein or otherwise known in the art.

A subject "at risk" is a subject that a health practitioner believes has a likelihood of having a disease, disorder or condition, or that a health practitioner believes has a likelihood of experiencing an undesired anti-therapeutic macromolecular antibody response as provided herein and would benefit from the compositions and methods provided herein. In one embodiment of any one of the methods, compositions, or kits provided herein, the subject is a subject at risk of having an anti-therapeutic macromolecule antibody response against a therapeutic macromolecule. In another embodiment of any one of the methods, compositions, or kits provided herein, the subject is a subject expected to have an anti-therapeutic macromolecular antibody response to a therapeutic macromolecule.

"attached" or "linked" or "coupled" (etc.) means that one entity (e.g., a moiety) is chemically bound to another entity. In some embodiments, the linkage is covalent, meaning that the linkage occurs in the presence of a covalent bond between the two entities. In some non-covalent embodiments, the non-covalent attachment is mediated by non-covalent interactions including, but not limited to: charge interactions, affinity interactions, metal coordination, physisorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and/or combinations thereof. In some embodiments, encapsulation is a form of ligation.

In some embodiments, the therapeutic macromolecule and the immunosuppressant are not linked to each other, meaning that the therapeutic macromolecule and immunosuppressant have not undergone a process specifically intended to chemically associate with each other. In some embodiments, the therapeutic macromolecule and/or immunosuppressant is not attached to the synthetic nanocarrier, meaning that the therapeutic macromolecule (and/or immunosuppressant) and synthetic nanocarrier have not undergone a process to specifically chemically associate with each other.

The term "average" as used herein refers to an arithmetic average unless otherwise indicated.

When applied to two or more materials and/or agents (also referred to herein as components), the "combination" is intended to define a material in which the two or more materials/agents are associated. The components may be individually identified, such as a first component, a second component, a third component, and so forth. The terms "combined" and "combination" in this case are to be interpreted accordingly.

The association of two or more materials/agents in a combination may be physical or non-physical. Examples of physically associated combination materials/agents include:

a composition (e.g. a single formulation) comprising two or more materials/agents in admixture (e.g. within the same unit dose);

compositions comprising materials in which two or more materials/agents are chemically/physicochemically linked (e.g. by cross-linking, molecular aggregation or association with common carrier moieties);

a composition comprising a material in which two or more materials/agents are chemically/physicochemically co-packaged (e.g. arranged on or in lipid vesicles, particles (e.g. microparticles or nanoparticles) or emulsion droplets);

a kit, pharmaceutical pack or patient pack in which two or more materials/agents are co-packaged or co-present (e.g. as part of a set of unit doses);

examples of non-physically associated combination materials/agents include:

a material (e.g. a non-unitary formulation) comprising at least one of the two or more materials/agents, accompanied by instructions for temporarily associating the at least one compound/agent to form a physical association of the two or more materials/agents;

a material (e.g. a non-single agent) comprising at least one of the two or more materials/agents, with instructions for use of the two or more materials/agents for combination therapy;

a material comprising at least one of the two or more materials/agents, accompanied by instructions for administration to a patient population to which the other of the two or more materials/agents has been administered (or is being administered);

a material comprising at least one of the two or more materials/agents in an amount or form specifically suitable for use in combination with the other of the two or more materials/agents.

The term "combination therapy" as used herein is intended to define a treatment which includes the use of a combination of two or more materials/agents (as defined below). Thus, references to "combination therapy", "combination" and "combined" use of materials/agents in this application may refer to materials/agents that are administered as part of the same overall treatment regimen. Thus, the respective dosimetry of two or more materials/agents may differ: each may be administered at the same time or at different times. Thus, it is to be understood that the materials/agents of the combination may be administered sequentially (e.g., before or after) or simultaneously (either in the same pharmaceutical formulation (i.e., together) or in different pharmaceutical formulations (i.e., independently)). When in the same formulation, simultaneously as a single formulation; while in different pharmaceutical formulations, are not unitary at the same time. The dosimetry of each of the two or more materials/agents in the combination therapy may also vary with respect to the route of administration.

By "concomitantly" is meant that two or more materials/agents are administered to a subject in a manner that is correlated in time, preferably sufficiently correlated in time to provide modulation of a physiological or immunological response, and even more preferably two or more materials/agents are administered in combination. In some embodiments, concomitant administration may include administration of two or more materials/agents within a specified time, preferably within 1 month, more preferably within 1 week, still more preferably within 1 day, and even more preferably within 1 hour. In some embodiments, the materials/agents may be administered concomitantly, reproducibly; i.e. more than one concomitant application, as may be provided in the examples.

"determining" means ascertaining the actual relationship. The determination may be accomplished in a variety of ways, including but not limited to performing experiments or making predictions. For example, the dose of the immunosuppressant or therapeutic macromolecule can be determined as follows: the administered dose is determined starting with the test dose and using known scaling techniques (e.g., isovelocity scaling or isovelocity scaling). This can also be used to determine the protocol as provided herein. In another embodiment, the dosage may be determined by testing multiple dosages in a subject, i.e., by direct experimentation based on empirical and instructional data. In some embodiments, "determining" comprises "causing a determination. "cause determination" means to cause, promote, encourage, assist, induce or direct an entity to ascertain an actual relationship or to act synergistically with an entity to make it ascertain an actual relationship; including directly or indirectly, or explicitly or implicitly.

By "dosage form" is meant a pharmacologically and/or immunologically active material in a medium, carrier, vehicle, or device suitable for administration to a subject. Any of the compositions or dosages provided herein can be in dosage form.

"dosage" refers to the specific amount of pharmacologically and/or immunologically active material administered to a subject over a given period of time.

By "encapsulating" is meant encapsulating at least a portion of the substance within a synthetic nanocarrier. In some embodiments, the substance is fully encapsulated within the synthetic nanocarrier. In other embodiments, most or all of the encapsulated substance is not exposed to the local environment external to the synthetic carrier. In other embodiments, no more than 50%, 40%, 30%, 20%, 10%, or 5% (weight/weight) is exposed to the local environment. Encapsulation is distinct from adsorption, which places most or all of a substance on the surface of a synthetic carrier and exposes the substance to a local environment outside the synthetic nanocarrier.

By "producing" is meant self-directly or indirectly eliciting an effect such as a physiological or immunological response (e.g., a tolerogenic immune response).

An "identified subject" is any activity or set of activities that allows a clinician to identify the subject as one that may benefit from the methods, compositions, or kits provided herein. Preferably, the identified subject is a subject in need of therapeutic benefit from a therapeutic macromolecule as provided herein and in which an anti-therapeutic macromolecule-specific antibody response has occurred or is expected to occur. The activity or set of activities may itself be taken directly or indirectly. In one embodiment of any one of the methods provided herein, the method further comprises identifying a subject in need of the methods, compositions, or kits provided herein.

By "immunosuppressive agent" is meant a compound that causes an APC to have an immunosuppressive effect or a T cell or B cell to be inhibited (e.g., tolerogenic effect). Immunosuppression generally refers to the production or expression by an APC of cytokines or other factors that reduce, inhibit or prevent an undesired immune response or promote a desired immune response (e.g., a regulatory immune response). When an APC acquires immunosuppressive function (belonging to an immunosuppressive action) on an immune cell that recognizes an antigen presented by the APC, the immunosuppressive action is considered to be specific to the presented antigen. Without being bound by any particular theory, it is believed that: immunosuppression is the result of delivery of an immunosuppressive agent to the APC, preferably in the presence of an antigen. In one embodiment, the immunosuppressive agent causes the APC to promote a regulatory phenotype in one or more immune effector cells. For example, the regulatory phenotype may be characterized by: inhibition of antigen-specific CD4+ T or B cell production, induction, stimulation or recruitment, inhibition of antigen-specific antibody production, Treg cell production, induction, stimulation or recruitment (e.g., CD4+ CD25 high FoxP3+ Treg cells), and the like. This may be the result of the conversion of CD4+ T cells or B cells into a regulatory phenotype. This may also be the result of induction of FoxP3 in other immune cells (CD8+ T cells, macrophages, and iNKT cells). In one embodiment, the immunosuppressive agent affects the response of the APC after it processes the antigen. In another embodiment, the immunosuppressive agent does not interfere with processing of the antigen. In another embodiment, the immunosuppressive agent is not an apoptosis-signaling molecule. In another embodiment, the immunosuppressive agent is not a phospholipid.

Immunosuppressants include, but are not limited to, 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-. kappa. β inhibitors such as 6Bio, dexamethasone, TCPA-1, IKKVII, adenosine receptor agonists, prostaglandin E2 agonists (prostaglandin E2 agonst, PGE2) such as misoprostol, phosphodiesterase inhibitors such as phosphodiesterase4 inhibitors (phosphodiesterase4 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 agonists, peroxisome of the peroxisome, peroxisome of which include adenosine receptor kinase inhibitors, phosphoenolpyruvate inhibitors such as adenosine monophosphate, phosphokinase inhibitors, inhibitors such as adenosine receptor inhibitors, phosphokinase inhibitors, inhibitors of the enzyme receptor agonists, PGE 3 inhibitors, adenosine receptor inhibitors.

The immunosuppressant may be a compound that directly provides an immunosuppressive effect on an APC or it may be a compound that indirectly provides an immunosuppressive effect (i.e., is processed in some manner after administration). Thus, immunosuppressive agents include prodrug forms of any of the compounds provided herein.

In some embodiments of any one of the methods, compositions, or kits provided herein, the immunosuppressant provided herein is linked to a synthetic nanocarrier. In some preferred embodiments, the immunosuppressive agent is a component other than the material that makes up the structure of the synthetic nanocarrier. For example, in one embodiment, when the synthetic nanocarriers are comprised of one or more polymers, the immunosuppressant is a compound other than and attached to the one or more polymers. As another example, in one embodiment, when the synthetic nanocarriers are comprised of one or more lipids, the immunosuppressant is still in addition to and attached to the one or more lipids. In some embodiments, e.g., when the material from which the nanocarriers are synthesized also elicits immunosuppressive effects, the immunosuppressive agent is a component that elicits immunosuppressive effects that is present in addition to the material from which the nanocarriers are synthesized.

Other exemplary immunosuppressive agents include, but are not limited to, small molecule drugs, natural products, antibodies (e.g., antibodies to CD20, CD3, CD 4), biologic-based drugs, carbohydrate-based drugs, nanoparticles, liposomes, RNAi, antisense nucleic acids, aptamers, methotrexate, NSAIDs, fingolimod, natalizumab, alemtuzumab, anti-CD 3, tacrolimus (FK506), cytokines and growth factors, such as TGF- β and IL-10, and the like.

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 is itself particulate or particle-like. In some embodiments, such forms mimic viruses or other foreign pathogens. Many drugs have been nanocrystallized and suitable methods for producing such drug forms are known to those of ordinary skill in the art. Drug nanocrystals (e.g., nanocrystalline rapamycin) are known to those of ordinary skill in the art (Katteboinaa, et al, 2009, International Journal of PharmTechResearch; Vol.1, No. 3; pp.682-694). As used herein, "drug nanocrystal" refers to a form of a drug (e.g., an immunosuppressant) that does not comprise a carrier or matrix material. In some embodiments, the drug nanocrystals comprise 90%, 95%, 98%, or 99% or more drug. Methods for producing drug nanocrystals include, but are not limited to: grinding, high pressure homogenization, precipitation, spray drying, Rapid Expansion of Supercritical Solutions (RESS),Technology (Baxter Healthcare) and nanocrystalline Technology^TM(Elan Corporation). In some embodiments, surfactants or stabilizers may be used for steric or electrostatic stabilization of the drug nanocrystals. In some embodiments, nanocrystalline or nanocrystalline forms of an immunosuppressant may be used to improve the solubility, stability and/or bioavailability of an immunosuppressant, particularly an insoluble or unstable immunosuppressant. In some embodiments, concomitant administration of a reduced pharmacodynamically effective dose of a therapeutic macromolecule with a nanocrystalline form of an immunosuppressive agent results in a reduced anti-therapeutic macromolecule antibody response.

When attached to a synthetic nanocarrier, the "loading" is the amount (weight/weight) of immunosuppressant and/or therapeutic macromolecule attached to the synthetic nanocarrier based on the total dry formulation weight of material in the entire synthetic nanocarrier. Generally, such loadings are calculated as the average of the entire population of synthetic nanocarriers. In one embodiment, the loading is from 0.1% to 99% based on the average of all synthetic nanocarriers. In another embodiment, the loading is from 0.1% to 50%. In another embodiment, the loading of immunosuppressant and/or therapeutic macromolecule is from 0.1% to 20%. In another embodiment, the loading of immunosuppressant and/or therapeutic macromolecule is from 0.1% to 10%. In yet another embodiment, the loading of immunosuppressant and/or therapeutic macromolecule is 1% to 10%. In yet another embodiment, the loading of immunosuppressive agent is 7% to 20%. In yet another embodiment, the population of synthetic nanocarriers, based on an average across the population of synthetic nanocarriers, the loading of the immunosuppressant/therapeutic macromolecule is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19% at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In yet another embodiment, the loading of the immunosuppressant/therapeutic macromolecule 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% based on the average across the population of synthetic nanocarriers. In some of the above embodiments, the loading of immunosuppressive and/or therapeutic macromolecules is no more than 25% based on the average of the entire population of synthetic nanocarriers. In some embodiments, the loading may be calculated as described in the examples or as otherwise known in the art.

In some embodiments, when the form of the immunosuppressant is itself particulate or particulate-like (e.g., nanocrystalline immunosuppressant), the loading of immunosuppressant is the amount (weight/weight) of immunosuppressant in the particulate or the like. In such some embodiments, the loading may approach 97%, 98%, 99% or more.

"maximum dimension of a synthetic nanocarrier" means the maximum dimension of the nanocarrier as measured along any axis of the synthetic nanocarrier. "minimum dimension of a synthetic nanocarrier" means the minimum dimension of the synthetic nanocarrier as measured along any axis of the synthetic nanocarrier. For example, for a spherical synthetic nanocarrier, the largest and smallest dimensions of the synthetic nanocarrier are substantially the same and are the dimensions of their diameters. Similarly, for a cuboidal synthetic nanocarrier, the smallest dimension of the synthetic nanocarrier is the smallest of its height, width, or length, while the largest dimension of the synthetic nanocarrier is the largest of its height, width, or length. In one embodiment, at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample have a smallest dimension equal to or greater than 100nm, based on the total number of synthetic nanocarriers in the sample. In one embodiment, at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample have a largest dimension that is equal to or less than 5 μm, based on the total number of synthetic nanocarriers in the sample. Preferably, at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample have a smallest dimension greater than 110nm, more preferably greater than 120nm, more preferably greater than 130nm, and more preferably still greater than 150nm, based on the total number of synthetic nanocarriers in the sample. The aspect ratio of the largest dimension and the smallest dimension of the synthetic nanocarriers may vary depending on the embodiment. For example, the aspect ratio of the largest dimension to the smallest dimension of the synthetic nanocarriers may be 1: 1 to 1,000,000: 1, preferably 1: 1 to 100,000: 1, more preferably 1: 1 to 10,000: 1, more preferably 1: 1 to 1000: 1, still more preferably 1: 1 to 100: 1 and yet more preferably 1: 1 to 10: 1. Preferably, at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample have a maximum dimension equal to or less than 3 μm, more preferably equal to or less than 2 μm, more preferably equal to or less than 1 μm, more preferably equal to or less than 800nm, more preferably equal to or less than 600nm, and more preferably also equal to or less than 500nm, based on the total number of synthetic nanocarriers in the sample. In some preferred embodiments, at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample have a minimum dimension equal to or greater than 100nm, more preferably equal to or greater than 120nm, more preferably equal to or greater than 130nm, more preferably equal to or greater than 140nm, and more preferably also equal to or greater than 150nm, based on the total number of synthetic nanocarriers in the sample. In some embodiments, a measurement of the size (e.g., effective diameter) of the synthetic nanocarriers can be obtained as follows: the synthetic nanocarriers are suspended in a liquid medium (typically an aqueous medium) and Dynamic Light Scattering (DLS) is used (e.g., using a brookhaven zetapals instrument). For example, a suspension of synthetic nanocarriers can be diluted from an aqueous buffer into pure water to obtain a final synthetic nanocarrier suspension concentration of about 0.01mg/mL to 0.1 mg/mL. The diluted suspension can be prepared directly in a suitable cuvette for DLS analysis or the diluted suspension can be transferred to a suitable cuvette for DLS analysis. The cuvette can then be placed in a DLS, allowed to equilibrate to a controlled temperature, and then scanned for sufficient time to obtain a stable and reproducible profile based on appropriate inputs for the viscosity of the medium and the refractive index of the sample. Then, the average of the effective diameter or distribution is reported. Determining the effective size of high aspect ratio or non-spherical synthetic nanocarriers may require magnification techniques (e.g., electron microscopy) to obtain more accurate measurements. The "size" or "diameter" of the synthetic nanocarriers means the average value of the particle size distribution obtained, for example, using dynamic light scattering.

By "non-methoxy-terminated polymer" is meant a polymer at least one end of which terminates in a moiety other than a methoxy group. In some embodiments, the polymer has at least two ends that terminate in a moiety other than a methoxy group. In other embodiments, the polymer does not have methoxy-terminated ends. By "non-methoxy-terminated pluronic polymer" is meant a polymer other than a linear pluronic polymer having methoxy groups at both ends. The polymeric nanoparticles provided herein can comprise a non-methoxy-terminated polymer or a non-methoxy-terminated pluronic polymer.

By "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is meant a pharmacologically inert material used in conjunction with a pharmacologically active material to formulate a composition. Pharmaceutically acceptable excipients include a variety of materials known in the art, including but not limited to: sugars (e.g., glucose, lactose, etc.), preservatives (e.g., antimicrobials), reconstitution aids, colorants, saline (e.g., phosphate buffered saline), and buffers.

By "pharmacodynamic effect" of a "pharmacodynamic response" is meant any physiological or immunological response resulting from administration of a therapeutic macromolecule. Such a response may be a desired response, such as a response related to the therapeutic effect. In some embodiments, it has been found that the methods, compositions, or kits provided herein produce an enhanced pharmacodynamic effect, e.g., an enhanced therapeutic effect, when administered in the presence of an anti-therapeutic macromolecular antibody response. In some cases, enhanced pharmacodynamic effects may be obtained using a therapeutic macromolecular dose that is the same or lower than the therapeutic macromolecular dose administered when an immunosuppressant dose provided herein is not concomitantly administered in the presence of an anti-therapeutic macromolecular antibody response. Whether a material or/and agent is pharmacodynamically effective can be assessed by standard methods. In some embodiments, the pharmacodynamic effect of using the provided methods, compositions, or kits in the presence of an anti-therapeutic macromolecular antibody response is compared to the pharmacodynamic effect of administering a therapeutic macromolecule otherwise but still in the presence of an anti-therapeutic macromolecular antibody response. In some embodiments, the therapeutic macromolecule is compared against pharmacodynamic effects when administered alone in the presence of an anti-therapeutic macromolecule antibody response. In general, pharmacodynamic effects are assessed when administered in the presence of an anti-therapeutic macromolecular antibody response, as it is desirable that the methods, compositions or kits effectively overcome such a response. Thus, the pharmacodynamic effect is determined as such a response is occurring.

"regimen" means the mode of administration to a subject and includes any dosing regimen for one or more substances in a subject. A solution consists of elements (or variables), and thus a solution contains one or more elements. Such elements of a regimen may include the amount administered, frequency of administration, route of administration, duration of administration, rate of administration, interval of administration, combinations of any of the foregoing, and the like. In some embodiments, such regimens may be used to administer one or more compositions of the invention to one or more test subjects. The immune response in these test subjects can then be evaluated to determine whether the regimen is effective to produce the desired pharmacodynamic effect or the desired level of pharmacodynamic effect. Any other therapeutic and/or immunological effect may be assessed in addition to or instead of the aforementioned immune response. One or more elements of a regimen may have been previously proven in a test subject (e.g., a non-human subject) and subsequently converted to a human regimen. For example, the dose administered as demonstrated in a non-human subject may be scaled to be an element of a human regimen using known techniques such as isovelocity scaling or other scaling methods. Regardless of whether the protocol has the desired effect, it can be determined using any method provided in the art or otherwise known in the art. For example, a sample can be obtained from a subject to whom a composition provided herein has been administered according to a particular protocol to determine whether a particular immune cell, cytokine, antibody, etc., is reduced, produced, activated, etc. Methods that can be used to detect the presence and/or quantity of immune cells include, but are not limited to: flow cytometry (e.g., FACS), ELISpot, proliferative responses, cytokine production, and immunohistochemistry. Antibodies and other binding agents for specific staining of immune cell markers are commercially available. Such kits typically comprise staining reagents for the antigen that allow FACS-based detection, isolation and/or quantification of the desired cell population from a heterogeneous cell population. In some embodiments, the compositions provided herein are administered to another subject using one or more elements, or all or substantially all elements, comprised by the regimen. In some embodiments, the regimen has been demonstrated to result in a reduction in antibody response to the therapeutic macromolecule and/or to produce an improved pharmacodynamic effect.

"providing" means an activity or set of activities performed by an individual to supply a desired item or group of items or methods for practicing the present invention. The activity or set of activities may itself be taken directly or indirectly.

A "providing a subject" is any activity or set of activities that causes a clinician to contact the subject and administer a composition provided herein to the subject or subject it to a method provided herein. Preferably, the subject is a subject in need of therapeutic macromolecular administration and antigen-specific immune tolerance thereto. The activity or set of activities may itself be taken directly or indirectly. In one embodiment of any one of the methods provided herein, the method further comprises providing the subject.

By "recording an enhanced pharmacodynamic effect" is meant recording in any written or electronic form a therapeutic macromolecular dose that achieves an enhanced pharmacodynamic effect in the presence of an actual, expected or suspected anti-therapeutic macromolecular antibody response, or that directly or indirectly causes such activity in anticipation of such recording occurring. In general, in these cases, based on the information obtained with the administration provided herein, it has been expected that if not administered with (e.g., administered alone with) the immunosuppressant dose provided herein, the therapeutic macromolecule dose would not achieve an enhanced pharmacodynamic effect in the presence of an anti-therapeutic macromolecule antibody response. For example, in these cases, it is expected that the effectiveness of the therapeutic macromolecule would be diminished if it were not administered with an immunosuppressant dose, but that an enhanced pharmacodynamic effect is observed with the concomitant administration provided herein. In some cases, the recording occurs at the time of administration of the immunosuppressant dose in combination with the therapeutic macromolecule dose to the subject or at some point thereafter. In some of these embodiments, the therapeutic macromolecule dose is reduced (or does not exceed) compared to the therapeutic macromolecule dose when administered without an immunosuppressant dose in the presence of an anti-therapeutic macromolecule antibody response. As used herein, "handwritten form" refers to any recording on a medium, such as paper. As used herein, "electronic form" refers to any recording on an electronic medium. Any of the methods provided herein can further comprise the step of recording a treatment and/or immune response in a subject receiving treatment according to the methods provided herein.

By "reduced pharmacodynamically effective dose" is meant a reduced therapeutic macromolecular weight that achieves a similar pharmacodynamic effect when administered concomitantly with an immunosuppressant dose as provided herein, as compared to the therapeutic macromolecular weight when administered without an immunosuppressant dose (e.g., when the therapeutic macromolecule is administered alone). As used herein, a similar pharmacodynamic effect is a level of effect within one log of another level measured in the same manner. Preferably, the difference in similar pharmacodynamic effects is no more than 5-fold. Still more preferably, the difference in similar pharmacodynamic effects is no more than 2-fold.

By "subject" is meant an animal, including warm-blooded mammals, such as humans and primates; (ii) poultry; domestic or farm animals, such as cats, dogs, sheep, goats, cattle, horses and pigs; laboratory animals such as mice, rats and guinea pigs; fish; a reptile; zoo and wild animals; and the like.

By "synthetic nanocarriers" is meant discrete objects that do not occur in nature and have at least one dimension that is less than or equal to 5 microns in size. Albumin nanoparticles are typically included as synthetic nanocarriers, however in certain embodiments, synthetic nanocarriers do not include albumin nanoparticles. In some embodiments, the synthetic nanocarriers do not comprise chitosan. In other embodiments, the synthetic nanocarriers are not lipid-based nanoparticles. In other embodiments, the synthetic nanocarriers do not comprise phospholipids.

The synthetic nanocarriers can be, but are not limited to, one or more of the following: lipid-based nanoparticles (also referred to herein as lipid nanoparticles, i.e., nanoparticles whose majority of the material making up their structure is lipid), polymeric nanoparticles, metallic nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles (i.e., particles composed primarily of viral structural proteins that are non-infectious or have low infectivity), peptide-or protein-based particles (also referred to herein as protein particles, i.e., particles whose majority of the material making up their structure is a peptide or protein) (e.g., albumin nanoparticles), and/or nanoparticles developed using combinations of nanomaterials (e.g., lipid-polymer nanoparticles). Synthetic nanocarriers can be in a variety of different shapes, including but not limited to: spherical, cubic, pyramidal, rectangular, cylindrical, toroidal, and the like. The synthetic nanocarriers according to the invention comprise one or more surfaces. Exemplary synthetic nanocarriers that can be suitable for use in the practice of the invention include: (1) biodegradable nanoparticles disclosed in U.S. Pat. No.5,543,158 to Gref et al, (2) Polymer nanoparticles in Saltzman et al, published U.S. patent application 20060002852, (3) lithographically-constructed nanoparticles in published U.S. patent application 20090028910 to Desimone et al, (4) the disclosure of WO2009/051837 to von Andrian et al, (5) nanoparticles disclosed in U.S. patent application 2008/0145441 to Penades et al, (6) protein nanoparticles disclosed in U.S. patent application 20090226525 to de los Rios et al, (7) virus-like particles disclosed in U.S. patent application 20060222652 to Sebbel et al, (8) nucleic acid-linked virus-like particles disclosed in U.S. patent application 20060251677 to Bachmann et al, (9) virus-like particles disclosed in WO2010047839A1 or WO2009106999A2, (10) P.Paolicel et al, "Surface-modified PLGA-based Nanoparticles which can be used as efficient Association and Deliver Virus-like particles" Nanomedicine.5 (6): 843-853(2010), (11) apoptotic cells, apoptotic bodies, or synthetic or semisynthetic mimetics as disclosed in U.S. publication 2002/0086049, or (12) Look et al, Nanogel-based delivery of mycophenolic acid amides systems systemic lupus erythematosus in mice "J.clinical Investigation 123 (4): 1741-1749 (2013). In some embodiments, the aspect ratio of the synthetic nanocarriers may be greater than 1: 1, 1: 1.2, 1: 1.5, 1: 2, 1: 3, 1: 5, 1: 7, or greater than 1: 10.

Synthetic nanocarriers according to the invention that have a smallest dimension equal to or less than about 100nm, preferably equal to or less than 100nm, do not comprise a surface with complement-activating hydroxyl groups, or alternatively comprise a surface that consists essentially of moieties that are not complement-activating hydroxyl groups. In a preferred embodiment, synthetic nanocarriers according to the invention that have a smallest dimension equal to or less than about 100nm, preferably equal to or less than 100nm, do not comprise a surface that significantly activates complement, or alternatively comprise a surface that consists essentially of portions that do not significantly activate complement. In a more preferred embodiment, synthetic nanocarriers according to the invention that have a smallest dimension equal to or less than about 100nm, preferably equal to or less than 100nm, do not comprise a complement-activating surface, or alternatively comprise a surface that consists essentially of a fraction that does not activate complement. In some embodiments, the synthetic nanocarriers do not comprise virus-like particles. In some embodiments, the aspect ratio of the synthetic nanocarriers may be greater than 1: 1, 1: 1.2, 1: 1.5, 1: 2, 1: 3, 1: 5, 1: 7, or greater than 1: 10.

"therapeutic macromolecule" refers to any protein, carbohydrate, lipid, or nucleic acid that can be administered to a subject and has a therapeutic effect. In some embodiments, administration of the therapeutic macromolecule to the subject may elicit an undesirable immune response, including the production of antibodies specific for the therapeutic macromolecule. As described herein, concomitant administration of a therapeutic macromolecule with an immunosuppressant dose can enhance the therapeutic effectiveness of the therapeutic macromolecule, e.g., by reducing an undesired immune response thereto. In some embodiments, the therapeutic macromolecule may be a therapeutic polynucleotide or a therapeutic protein.

By "therapeutic polynucleotide" is meant any polynucleotide or polynucleotide-based therapy that can be administered to a subject and has a therapeutic effect. Such treatments include gene silencing. Examples of such treatments are known in the art and include, but are not limited to: naked RNA (including messenger RNA, modified messenger RNA, and forms of RNAi). Examples of other therapeutic polynucleotides are also provided elsewhere herein. The therapeutic polynucleotide may be produced in, on or by a cell, and may also be obtained using cell-free in vitro methods or obtained by entirely in vitro synthetic methods. Thus, a subject includes any subject in need of treatment with any of the aforementioned therapeutic polynucleotides. Such subjects include those that will receive any of the aforementioned therapeutic polynucleotides.

By "therapeutic protein" is meant any protein or protein-based therapy that can be administered to a subject and has a therapeutic effect. Such therapies include protein replacement or protein supplementation therapies. Such treatments also include the administration of foreign or foreign proteins, antibody therapies and cell or cell-based therapies. Therapeutic proteins include, but are not limited to: enzymes, enzyme cofactors, hormones, clotting factors, cytokines, growth factors, monoclonal antibodies, antibody-drug conjugates, and polyclonal antibodies. Examples of other therapeutic proteins are also provided elsewhere herein. The therapeutic protein may be produced in, on or by a cell, and may be obtained from such a cell or administered in the form of such a cell. In some embodiments, the therapeutic protein is produced in, on, or by a mammalian cell, insect cell, yeast cell, bacterial cell, plant cell, transgenic animal cell, transgenic plant cell, or the like. The therapeutic protein may be recombinantly produced in such cells. The therapeutic protein may be produced in, on, or from a virally transformed cell. Thus, a subject may include any subject in need of treatment with any of the aforementioned therapeutic proteins. Such subjects include those that will receive any of the aforementioned therapeutic proteins.

By "therapeutic macromolecular APC presentable antigen" is meant an antigen associated with a therapeutic macromolecule (i.e., a therapeutic macromolecule or a fragment thereof that can generate an immune response against the therapeutic macromolecule (e.g., generation of antibodies specific for the therapeutic macromolecule)). In general, a therapeutic macromolecular Antigen Presenting Cell (APC) may present an antigen that is recognized by the immune system (e.g., a cell in the immune system, such as a cell presented by an antigen presenting cell, including but not limited to a dendritic cell, B cell, or macrophage). Therapeutic macromolecular APC presentable antigens can be presented for recognition by, for example, T cells. Such antigens can be recognized by T cells and trigger an immune response in T cells by presenting epitopes of the antigen bound to a class I or class II Major Histocompatibility Complex (MHC). Therapeutic macromolecular APC presentable antigens generally include proteins, polypeptides, peptides, polynucleotides, lipoproteins, or are contained within or expressed within, expressed on, or expressed by a cell. In some embodiments, the therapeutic macromolecular antigen is linked to a synthetic nanocarrier and comprises MHC class I and/or MHC class II and/or B cell epitopes. Preferably, one or more tolerogenic immune responses specific for the therapeutic macromolecule are elicited using the methods, compositions, or kits provided herein. In some embodiments, the population of synthetic nanocarriers does not comprise added therapeutic macromolecular APC presentable antigen, meaning that no significant amount of therapeutic macromolecular APC presentable antigen is intentionally added to the population during synthetic nanocarrier preparation.

An "undesired immune response" refers to any undesired immune response that is caused by exposure to an antigen, promotes or exacerbates a disease, disorder, or condition (or symptoms thereof) provided herein, or that is a symptom of a disease, disorder, or condition provided herein. Such an immune response is generally an adverse effect on or a symptom of an adverse effect on the health of the subject. Undesirable immune responses include antigen-specific antibody production, antigen-specific B cell proliferation and/or activity, or antigen-specific CD4+ T cell proliferation and/or activity. In general, these undesirable immune responses may be specific to the therapeutic macromolecule and counteract the beneficial effects expected from administration of the therapeutic macromolecule. Thus, in some embodiments, the undesired immune response is an anti-therapeutic macromolecular antibody response.

C. Compositions and related methods

Provided herein are compositions comprising an immunosuppressive agent and a therapeutic macromolecule, as well as related methods or kits. Such methods, compositions, or kits are useful for enhancing the pharmacodynamic effects of a therapeutic macromolecule in the presence of an anti-therapeutic macromolecule antibody response, for example, by reducing or inhibiting a therapeutic macromolecule-specific undesired immune response that diminishes the therapeutic benefit of the therapeutic macromolecule. Such methods, compositions, or kits may also be useful for allowing repeat dosing of therapeutic macromolecules. Thus, the methods, compositions, or kits provided herein can be used to achieve or enhance a desired therapeutic effect of a therapeutic macromolecule. In some embodiments, such therapeutic effects may be achieved or enhanced at reduced pharmacodynamically effective doses. The provided methods, compositions, or kits can be used in any subject in need of therapeutic benefit of a therapeutic macromolecule.

As described above, it was found that concomitant delivery of an immunosuppressant (preferably, in some embodiments, when linked to a synthetic nanocarrier) to a therapeutic macromolecule in the presence of an anti-therapeutic macromolecule antibody response can result in enhanced pharmacodynamic effects, including enhancement of such effects even at reduced therapeutic macromolecule doses. For example, the methods, compositions, or kits can aid in neutralizing anti-therapeutic macromolecule-specific antibodies that interfere with the desired therapeutic effect of the therapeutic macromolecule. Thus, the methods, compositions, or kits provided herein can result in the enhancement of the desired therapeutic effect of a therapeutic macromolecule that would otherwise be diminished when the therapeutic macromolecule is not administered with an immunosuppressant dose (e.g., when the therapeutic macromolecule is administered alone). Thus, in some embodiments, the methods, compositions, or kits provided herein allow a subject to obtain therapeutic benefits of a therapeutic macromolecule without increasing the dose of the therapeutic macromolecule, which would typically be increased in order to compensate for an undesired immune response against the therapeutic macromolecule when administered without the benefits of the invention provided herein. Surprisingly, the methods, compositions, or kits provided herein allow for administration of even a reduced therapeutic macromolecule dose to a subject to achieve the same or better therapeutic benefit in the presence of an anti-therapeutic macromolecule antibody response.

A variety of immunosuppressive agents may be used in the practice of the invention, preferably, the immunosuppressive agent is linked to a synthetic nanocarrier. A variety of synthetic nanocarriers can be used according to the invention. In some embodiments, the synthetic nanocarriers are spheres or spheroids. In some embodiments, the synthetic nanocarriers are flat or platy. In some embodiments, the synthetic nanocarriers are cubic or cubic. In some embodiments, the synthetic nanocarriers are oval or elliptical. In some embodiments, the synthetic nanocarriers are cylinders, cones, or pyramids.

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 may have a maximum dimension or a minimum dimension that falls within 5%, 10%, or 20% of the average diameter or average dimension of the synthetic nanocarriers, based on the total number of synthetic nanocarriers.

The synthetic nanocarriers may be solid or hollow and may comprise one or more layers. In some embodiments, each layer has a unique composition and unique characteristics relative to the other layers. To give but one example, a synthetic nanocarrier may have a core/shell structure, wherein the core is one layer (e.g., a polymer core) and the shell is a second layer (e.g., a lipid bilayer or monolayer). The synthetic nanocarriers may comprise a plurality of different layers.

In some embodiments, the synthetic nanocarriers may optionally comprise one or more lipids. In some embodiments, the synthetic nanocarriers can comprise liposomes. In some embodiments, the synthetic nanocarriers may comprise a lipid bilayer. In some embodiments, the synthetic nanocarriers may comprise a lipid monolayer. In some embodiments, the synthetic nanocarriers may comprise micelles. In some embodiments, the synthetic nanocarriers may comprise a core comprising a polymer matrix surrounded by a lipid layer (e.g., a lipid bilayer, a lipid monolayer, etc.). In some embodiments, the synthetic nanocarriers can comprise a non-polymeric core (e.g., metal particles, quantum dots, ceramic particles, bone particles, viral particles, proteins, nucleic acids, carbohydrates, etc.) surrounded by a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.).

In other embodiments, the synthetic nanocarriers may comprise metal particles, quantum dots, ceramic particles, and the like. In some embodiments, the non-polymeric synthetic nanocarriers are aggregates of non-polymeric components, such as aggregates of metal atoms (e.g., gold atoms).

In some embodiments, the synthetic nanocarriers may optionally comprise one or more amphiphilic entities. In some embodiments, amphiphilic entities can facilitate the production of synthetic nanocarriers with increased stability, increased uniformity, or increased viscosity. In some embodiments, the amphiphilic entity may be associated with the inner surface of a lipid membrane (e.g., a lipid bilayer, a lipid monolayer, etc.). Many amphiphilic entities known in the art are suitable for use in the preparation of synthetic nanocarriers according to the invention. Such amphiphilic entities include, but are not limited to: glycerol phosphate; phosphatidylcholine; dipalmitoyl phosphatidylcholine (DPPC); dioleylphosphatidylethanolamine (DOPE); dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine; cholesterol; a cholesterol ester; a diacylglycerol; diacylglycerol succinate; diphosphatidyl glycerol (DPP)G) (ii) a Cetyl alcohol (hexadecanol); fatty alcohols, such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; surface-active fatty acids, such as palmitic acid or oleic acid; a fatty acid; a fatty acid monoglyceride; a fatty acid diglyceride; a fatty acid amide; sorbitan trioleate (

85) Glycocholate; sorbitan monolaurate (C)

20) (ii) a Polysorbate 20(

20) (ii) a Polysorbate 60 (C)

60) (ii) a Polysorbate 65 (C)

65) (ii) a Polysorbate 80 (C)

80) (ii) a Polysorbate 85(85) (ii) a Polyoxyethylene monostearate; a surfactant; a poloxamer; sorbitan fatty acid esters, such as sorbitan trioleate; lecithin; lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin; phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid; cerebroside; dicetyl phosphate; dipalmitoyl phosphatidylglycerol; stearamide; a dodecylamine; hexadecylamine; acetyl palmitate; glyceryl ricinoleate; cetyl stearate; isopropyl myristate; tyloxapol; poly (ethylene glycol) 5000-phosphatidylethanolamine; poly (ethylene glycol) 400-monostearate; a phospholipid; synthetic and/or natural detergents with high surfactant properties; deoxycholate; a cyclodextrin; chaotropic salts; ion pairAligning agent; 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 an exemplary, non-comprehensive list of surfactant-active substances. Any amphiphilic entity can be used to produce the synthetic nanocarriers used according to the invention.

In some embodiments, the synthetic nanocarriers may optionally comprise one or more carbohydrates. Carbohydrates may be natural or synthetic. The carbohydrate may be a derivatized 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, glucuronic acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine 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, glycoglycoconjugate (glycon), amylose, chitosan, N, O-carboxymethyl chitosan, algin and alginic acid, starch, chitin, inulin, konjac, glucomannan, conchiolin (pulsulan), heparin, hyaluronic acid, curdlan (curdlan) and xanthan gum. In some embodiments, the synthetic nanocarriers do not comprise (or specifically exclude) carbohydrates, such as polysaccharides. In certain embodiments, the carbohydrate may comprise a carbohydrate derivative, such as a sugar alcohol, including but not limited to: mannitol, sorbitol, xylitol, erythritol, maltitol, and lactitol.

In some embodiments, the synthetic nanocarriers may comprise one or more polymers. In some embodiments, the synthetic nanocarriers comprise 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% (weight/weight) of the polymers that make up the synthetic nanocarriers are non-methoxy-terminated pluronic polymers. In some embodiments, all of the polymers comprising the synthetic nanocarriers are non-methoxy-terminated pluronic polymers. In some embodiments, the synthetic nanocarriers comprise 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 nanocarriers are non-methoxy-terminated polymers. In some embodiments, all of the polymers comprising the synthetic nanocarriers are non-methoxy-terminated polymers. In some embodiments, the synthetic nanocarriers comprise one or more polymers that do not comprise 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% (weight/weight) of the polymers that make up the synthetic nanocarriers do not comprise pluronic polymers. In some embodiments, none of the polymers comprising the synthetic nanocarriers comprise pluronic polymers. In some embodiments, such polymers may be surrounded by a coating (e.g., liposomes, lipid monolayers, micelles, etc.). In some embodiments, multiple components in a synthetic nanocarrier can be attached to a polymer.

The immunosuppressants and/or therapeutic macromolecules may be attached to the synthetic nanocarriers by any of a variety of methods. In general, the linkage may be the result of a bond between the immunosuppressant and/or therapeutic macromolecule and the synthetic nanocarrier. Such bonding may result in the attachment of the immunosuppressant and/or therapeutic macromolecule to the surface of the synthetic nanocarrier and/or inclusion (encapsulation) in the synthetic nanocarrier. However, in some embodiments, due to the structure of the synthetic nanocarriers, the immunosuppressants and/or therapeutic macromolecules are encapsulated by the synthetic nanocarriers rather than bonded to the synthetic nanocarriers. In some preferred embodiments, the synthetic nanocarriers comprise a polymer provided herein, and the immunosuppressant and/or therapeutic macromolecule is attached to the polymer.

When the linkage occurs due to bonding between the immunosuppressant and/or therapeutic macromolecule and the synthetic nanocarrier, the linkage can occur via the coupling moiety. The coupling moiety may be any moiety through which the immunosuppressant and/or therapeutic macromolecule is bonded to the synthetic nanocarrier. Such moieties include covalent bonds (e.g., amide or ester bonds) as well as separate molecules that bond (covalently or non-covalently) the immunosuppressant and/or therapeutic macromolecule to the synthetic nanocarrier. Such molecules comprise a linker or a polymer or a unit thereof. For example, the coupling moiety may comprise a charged polymer that is electrostatically bound to the immunosuppressant and/or therapeutic macromolecule. As another example, the coupling moiety may comprise a polymer or unit thereof covalently bonded to the immunosuppressant and/or therapeutic macromolecule.

In some preferred embodiments, the synthetic nanocarriers comprise a polymer provided herein. These synthetic nanocarriers may be entirely polymeric or they may be a mixture of polymers with other materials.

In some embodiments, the polymers in the synthetic nanocarriers associate to form a polymer matrix. In some of these embodiments, a component (e.g., an immunosuppressant or therapeutic macromolecule) can be covalently associated with one or more polymers in the polymer matrix. In some embodiments, the covalent association is mediated by a linker. In some embodiments, the component may be non-covalently associated with one or more polymers in the polymer matrix. For example, in some embodiments, the components may be encapsulated within, surrounded by, and/or dispersed within the polymer matrix. Alternatively or additionally, the component may associate with one or more polymers in the polymer matrix through hydrophobic interactions, charge interactions, van der waals forces, and the like. A variety of polymers and methods for forming polymer matrices therefrom are conventionally known.

The polymer may be a natural polymer or a non-natural (synthetic) polymer. The polymer may be a homopolymer or a copolymer containing two or more monomers. With respect to sequence, the copolymer can be random, block, or comprise a combination of random and block sequences. Generally, the polymers according to the invention are organic polymers.

In some embodiments, the polymer comprises a 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, preferably, the polymer is biodegradable. Thus, in these embodiments, preferably, if the polymer comprises a polyether (e.g., poly (ethylene glycol), polypropylene glycol, or units thereof), the polymer comprises a block copolymer of the polyether and the biodegradable polymer such that the polymer is biodegradable. In other embodiments, the polymer comprises not only a polyether or units thereof, for example poly (ethylene glycol) or polypropylene glycol or units thereof.

Other examples of polymers suitable for use in the present invention include, but are not limited to: polyethylene, polycarbonates (e.g. poly (1, 3-di)

Alk-2-ones)), polyanhydrides (e.g., poly (sebacic dianhydride)), polypropylmaleites, polyamides (e.g., polycaprolactam), polyacetals, polyethers, polyesters (e.g., polylactide, polyglycolide, polylactide-glycolide copolymers, polycaprolactone, polyhydroxy acids (e.g., poly (β -hydroxyalkanoate))), poly (orthoesters), polycyanoacrylates, polyvinyl alcohol, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polyureas, polystyrene, and polyamines, polylysines, polylysine-PEG copolymers and poly (ethyleneimine), poly (ethyleneimine) -PEG copolymers.

In some embodiments, a polymer package according to the present inventionIncluding polymers that have been approved by the U.S. food and Drug Administration, FDA for use in humans according to 21c.f.r. § 177.2600, including but not limited to: polyesters (e.g., polylactic acid, polylactic-co-glycolic acid, polycaprolactone, polypentanolide, poly (1, 3-di-poly (lactic acid-co-glycolic acid)) (poly (caprolactone)) (poly (valerolactone)), and poly (1, 3-di (lactic acid-co-Alk-2-one)); polyanhydrides (e.g., poly (sebacic dianhydride)); polyethers (e.g., polyethylene glycol); a polyurethane; polymethacrylates; a polyacrylate; and polycyanoacrylates.

In some embodiments, the polymer may be hydrophilic. For example, the polymer can comprise anionic groups (e.g., phosphate groups, sulfate groups, carboxylate groups); cationic groups (e.g., quaternary amine groups); or polar groups (e.g., hydroxyl, thiol, amine). In some embodiments, synthetic nanocarriers comprising a hydrophilic polymer matrix create a hydrophilic environment within the synthetic nanocarriers. In some embodiments, the polymer may be hydrophobic. In some embodiments, synthetic nanocarriers comprising a hydrophobic polymer matrix create a hydrophobic environment within the synthetic nanocarriers. The choice of hydrophilicity or hydrophobicity of the polymer can affect the nature of the material incorporated (e.g., attached) in the synthetic nanocarrier.

In some embodiments, the polymer may be modified with one or more moieties and/or functional groups. A variety of moieties or functional groups can be used in accordance with the present invention. In some embodiments, the polymer may be modified with polyethylene glycol (PEG), carbohydrates, and/or polysaccharide-derived acyclic polyacetals (Papisov, 2001, ACS Symposium Series, 786: 301). Certain embodiments may be performed using the general teachings of Gref et al, U.S. patent No.5543158 or Von Andrian et al, WO 2009/051837.

In some embodiments, the fatty acid groups may be one or more of butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, or lignoceric acid.

In some embodiments, the polymer may be a polyester, including copolymers containing lactic acid and glycolic acid units, such as polylactic-co-glycolic acid and polylactide-co-glycolide, collectively referred to herein as "PLGA", and homopolymers containing glycolic acid units (referred to herein as "PGA") and homopolymers containing lactic acid units, such as poly-L-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, the polymer may be PLGA. PLGA is a biocompatible, biodegradable copolymer of lactic acid and glycolic acid, and various PLGA forms are characterized by the ratio of lactic acid to glycolic acid. The lactic acid may be L-lactic acid, D-lactic acid or D, L-lactic acid. The degradation rate of PLGA can be adjusted by varying the ratio of lactic acid to glycolic acid. In some embodiments, the PLGA used according to the present invention is characterized in that: the ratio of lactic acid to glycolic acid is about 85: 15, about 75: 25, about 60: 40, about 50: 50, about 40: 60, about 25: 75, or about 15: 85.

In some embodiments, the polymer may be one or more acrylic polymers. In certain embodiments, acrylic polymers include, for example: acrylic and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly (acrylic acid), poly (methacrylic acid), alkylamide methacrylate copolymers, poly (methyl methacrylate), poly (methacrylic anhydride), methyl methacrylate, polymethacrylates, poly (methyl methacrylate) copolymers, polyacrylamides, aminoalkyl methacrylate copolymers, glycidyl methacrylate copolymers, polycyanoacrylates, and combinations comprising one or more of the foregoing polymers. The acrylic polymer may comprise a fully polymerized copolymer of an acrylate and a methacrylate with a low content of quaternary ammonium groups.

In some embodiments, the polymer may be a cationic polymer. Generally, the cationic polymer is capable of condensing and/or protecting the negatively charged chains of the nucleic acid. Amine-containing polymers such as poly (lysine) (Zanner et al, 1998, adv. drug Del. Rev., 30: 97; and 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; and Haensler et al, 1993, Bioconjugate chem., 4: 372) are positively charged at physiological pH and form ion pairs with nucleic acids. In some embodiments, the synthetic nanocarriers may not comprise (or may exclude) cationic polymers.

In some embodiments, the polymer may be a degradable polyester bearing 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; and Zhou et al, 1990, Macromolecules, 23: 3399). Examples of such polyesters include poly-L-lactide-L-lysine copolymer (Barrera et al, 1993, J.Am. chem. Soc., 115: 11010), poly (serine esters) (Zhou et al, 1990, Macromolecules, 23: 3399), poly (4-hydroxy-L-proline ester) (Putnam et al, 1999, Macromolecules, 32: 3658; and Lim et al, 1999, J.Am. chem. Soc., 121: 5633) and poly (4-hydroxy-L-proline ester) (Putnam et al, 1999, Macromolecules, 32: 3658; and Lim et al, 1999, J.Am. chem. Soc., 121: 5633).

The characteristics of these and other polymers and methods for their preparation are well known in the art (see, 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; and Uhrich et al, 1999, m.Rev., 99: 3181). More generally, various methods for synthesizing certain suitable polymers are described in the following: circumcise Encyclopedia of Polymer science and Polymeric Amines and Ammonium Salts, edited by Goethals, Pergamon Press, 1980; principles of Polymerization, Odian, John Wiley & Sons, fourth edition, 2004; contextual Polymer Chemistry, Allcock et al, Prentice-Hall, 1981; deming et al, 1997, Nature, 390: 386; and U.S. patents 6,506,577, 6,632,922, 6,686,446 and 6,818,732.

In some embodiments, the polymer may be a linear polymer or a branched polymer. In some embodiments, the polymer may be a dendrimer. In some embodiments, the polymers may be substantially crosslinked to each other. In some embodiments, the polymer may be substantially free of cross-linking. In some embodiments, polymers may be used according to the present invention without undergoing a crosslinking step. It is also understood that the synthetic nanocarriers may comprise block copolymers, graft copolymers, blends, mixtures, and/or adducts of the foregoing and other polymers. Those skilled in the art will recognize that the polymers listed herein represent an exemplary, non-comprehensive list of polymers that may be used in accordance with the present invention.

In some embodiments, the synthetic nanocarriers do not comprise a polymeric component. In some embodiments, the synthetic nanocarriers can comprise metal particles, quantum dots, ceramic particles, and the like. In some embodiments, the non-polymeric synthetic nanocarriers are aggregates of non-polymeric components, such as aggregates of metal atoms (e.g., gold atoms).

The compositions according to the invention may comprise the components in combination with pharmaceutically acceptable excipients such as preservatives, buffers, saline and phosphate buffered saline. The compositions can be prepared using conventional pharmaceutical preparation and compounding techniques to achieve a useful dosage form. In one embodiment, compositions (e.g., those comprising synthetic nanocarriers) are suspended in sterile saline solution with a preservative for injection.

In some embodiments, methods for attaching components to synthetic nanocarriers are useful when preparing synthetic nanocarriers as supports. If the component is a small molecule, it may be advantageous to attach the component to a polymer prior to assembly into a synthetic nanocarrier. In some embodiments, it is also advantageous that synthetic nanocarriers having surface groups can be prepared that can be used to attach components to synthetic nanocarriers by using these surface groups, rather than attaching components to polymers and then using the polymer conjugates in constructing synthetic nanocarriers.

In certain embodiments, the linkage may be a covalent linker. In some embodiments, the component according to the present invention may be covalently linked to the outer surface via a1, 2, 3-triazole linker formed by a1, 3-dipolar cycloaddition reaction of an azide group on the surface of the nanocarrier with an alkyne-containing component or by a1, 3-dipolar cycloaddition reaction of an alkyne and an azide-containing component on the surface of the nanocarrier. Such cycloaddition reaction is preferably carried out in the presence of a cu (i) catalyst and suitable cu (i) ligands and reducing agents to reduce the cu (ii) compounds to catalytically active cu (i) compounds. This cu (i) catalyzed azide-alkyne cycloaddition (CuAAC) may also be referred to as a click reaction.

Additionally, the covalent linkage can comprise a covalent linker including an amide linker, a disulfide linker, a thioether linker, a hydrazone linker, a hydrazide linker, an imine or oxime linker, a urea or thiourea linker, an amidine linker, an amine linker, and a sulfonamide linker.

The amide linker is formed by an amide bond between an amine on one component and a carboxylic acid group of a second component (e.g., nanocarrier). The amide bond in the linker can be formed using any conventional amide bond formation reaction using an appropriately protected amino acid and an activated carboxylic acid (e.g., an N-hydroxysuccinimide activated ester).

Disulfide linkers are made by forming a disulfide (S-S) bond between two sulfur atoms of the form, for example, R1-S-S-R2. The disulfide bond may be formed by subjecting a component containing a thiol/thiol group (-SH) to thiol exchange with another activated thiol group on the polymer or the nanocarrier, or by subjecting a nanocarrier containing a thiol/thiol group to thiol exchange with a component containing an activated thiol group.

Triazole linkers, in particular wherein R1 and R2 may be any chemical entity

The form of 1, 2, 3-triazole is formed by a1, 3-dipolar cycloaddition reaction of an azide attached to a first component (e.g., a nanocarrier) and a terminal alkyne attached to a second component (e.g., an immunosuppressant or therapeutic macromolecule). The 1, 3-dipolar cycloaddition reaction is carried out in the presence or absence of a catalyst, preferably a cu (i) catalyst, which links the two components by means of a1, 2, 3-triazole function. This chemistry is described in detail by Sharpless et al, angelw.chem.int. 41(14) edition, 2596 (2002) and Meldal et al, chem.rev., 2008, 108(8), 2952-.

In some embodiments, a polymer is prepared that includes an azide or alkyne group at the end of the polymer chain. Such polymers are then used to prepare synthetic nanocarriers in such a way that multiple alkyne or azide groups are located at the surface of the synthetic nanocarriers. Alternatively, synthetic nanocarriers can be prepared by another route and subsequently functionalized with alkynes or azides. The component is prepared in the presence of an alkyne (if the polymer comprises an azide) group or an azide (if the polymer comprises an alkyne) group. This component is then reacted with the nanocarrier via a1, 3-dipolar cycloaddition reaction in the presence or absence of a catalyst that links the component to the particle through a1, 4-disubstituted 1, 2, 3-triazole linker.

The thioether linker is constituted by a sulfur-carbon (thioether) bond formed in the form of, for example, R1-S-R2. Thioethers may be formed by alkylation of a mercapto/thiol group (-SH) on one component with an alkylating group (e.g., halide or epoxide) on a second component. Thioether linkers can also be formed by Michael addition of a thiol/thiol group on one component to an electron deficient alkenyl group on a second component containing a maleimide group or a vinylsulfone group as a Michael acceptor. In another approach, thioether linkers can be prepared by a free radical mercapto-ene reaction of a mercapto/thiol group on one component with an ene group on a second component.

The hydrazone linker is formed by reacting a hydrazide group on one component with an aldehyde/ketone group on a second component.

The hydrazide linker is formed by reacting a hydrazine group on one component with a carboxylic acid group on a second component. Such reactions are generally carried out using chemistry similar to amide bond formation in which a carboxylic acid is activated by an activating reagent.

Imine or oxime linkers are formed by reacting 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 reacting amine groups on one component with isocyanate or thioisocyanate groups on a second component.

The amidine linker is prepared by reacting an amine group on one component with an imidate group on a second component.

The amine linker is formed by the alkylation reaction of an amine group on one component with an alkylating group (e.g., halide, epoxide, or sulfonate group) on a second component. Alternatively, the amine linker may also be formed by reductive amination of an amine group on one component with an aldehyde or ketone group on a second component in the presence of a suitable reducing agent (e.g., sodium cyanoborohydride or sodium triacetoxyborohydride).

The sulfonamide linker is formed by reacting an amine group on one component with a sulfonyl halide (e.g., sulfonyl chloride) group on a second component.

The sulfone linker is formed by the Michael addition of a nucleophile to a vinyl sulfone. The vinyl sulfone or nucleophile may be on the surface of the nanocarrier or attached to the component.

Conjugation of the component to the nanocarrier can also be by non-covalent conjugation methods. For example, a negatively charged therapeutic macromolecule or immunosuppressant can be conjugated to a positively charged nanocarrier by electrostatic adsorption. The metal ligand-containing component can be conjugated to a nanocarrier containing a metal complex via a metal-ligand complex.

In some embodiments, the components may be attached to a polymer (e.g., a polylactic acid-ethylene glycol block copolymer) prior to assembly of the synthetic nanocarriers, or the synthetic nanocarriers may be formed to have reactive or activatable groups on their surfaces. In the latter case, the components may be prepared using groups that are chemically compatible with the attachment presented by the surface of the synthetic nanocarriers. In other embodiments, the peptide component may be linked to the VLP or liposome using a suitable linker. A linker is a compound or agent capable of linking two molecules together. In one embodiment, the linker may be a homobifunctional or heterobifunctional agent as described in Hermanson 2008. For example, a VLP or liposome comprising carboxyl groups on the surface can be treated with a homobifunctional linker Adipic Dihydrazide (ADH) in the presence of EDC to form a corresponding synthetic nanocarrier with an ADH linker. The resulting ADH-linked 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 produce corresponding VLP or liposomal peptide conjugates.

For a detailed description of the conjugation methods that can be used, see Hermanson G T "bioconjugate technology", 2 nd edition, published by Academic Press, Inc.2008. In addition to covalent attachment, the components may be attached to the preformed synthetic nanocarriers by adsorption or they may be attached by encapsulation during formation of the synthetic nanocarriers.

Any of the immunosuppressive agents provided herein can be used in the methods or compositions provided, and in some embodiments, can be linked to a synthetic nanocarrier, immunosuppressive agents include, but are not limited to, 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-kappa β inhibitors, adenosine receptor agonists, prostaglandin E2 agonists, phosphodiesterase inhibitors such as phosphodiesterase4 inhibitors, proteasome inhibitors, kinase inhibitors, G protein-coupled receptor agonists, G protein-coupled receptor antagonists, glucocorticoids, retinoids, cytokine siRNAs, 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, immunosuppressive agents also include IDO, vitamin D3, cyclosporin aryl receptor inhibitors, resveratrol inhibitors, interleukin A receptor inhibitors, interleukin A receptor agonists, interleukin A6, interleukin A targeting factor, interleukin A-10, or the like.

Examples of statins include: atorvastatinCerivastatin, fluvastatin

XL), lovastatin

Mevastatin

Pitavastatin

Rosuvastatin

RosuvastatinAnd simvastatin

Examples of mTOR inhibitors include: rapamycin and its analogs (e.g., CCL-779, RAD001, AP23573, C20-methallyl rapamycin (C20-Marap), C16- (S) -butylsulfonylamino rapamycin (C16-Bsrap), C16- (S) -3-methylindole rapamycin (C16-iRap) (Bayle et al Chemistry & Biology 2006, 13: 99-107)), AZD8055, BEZ235(NVP-BEZ235), rhein (chrysophanol), desfomolimus (MK-8669), everolimus (RAD0001), KU-0063794, PI-103, PP242, temsirolimus, and WYE-354 (available from Selleck, Houston, TX, USA).

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, TGF β RI, TGF β RII), R-SMAD/co-SMADS (e.g., SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD8) and ligand inhibitors (e.g., follistatin, noggin, chordin), DAN, lefty, LTBP1, THBS1, Decorin).

Examples of inhibitors of mitochondrial function include atractyloside (dipotassium salt), mellitic acid (triammonium salt), carbonyl cyanide metachlorophenylhydrazone, carboxyatractyloside (e.g., from Atractylis lancea (Atractylis gummifera)), CGP-37157, (-) -deguelin (e.g., from mullein (mullea sericea)), F16, hexokinase II VDAC binding domain peptides, oligomycin, rotenone, Ru360, SFK1, and valinomycin (e.g., from streptomyces fulvisimus (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.

Examples of NF (e.g., NK- κ β) inhibitors include IFRD1, 2- (1, 8-naphthyridin-2-yl) phenol, 5-aminosalicylic acid, BAY 11-7082, BAY 11-7085, CAPE (caffeic acid phenethyl ester), diethyl maleate, IKK-2 inhibitor IV, IMD0354, lactacystin, MG-132[ Z-Leu-Leu-Leu-CHO ], NF κ B activation inhibitor III, NF κ B activation inhibitor II, JSH-23, parthenolide, phenylarsonium oxide (PAO), PPM-18, pyrrolidinedithiocarbamate ammonium salt, QNZ, RO 106-9920, melinamide AL, melinamide C, melinamide I, melinamide J, clomipraminol (rocaglaol), (R) -MG-132, sodium salicylate, lactone (PG490), and triptolide (wectone).

Examples of adenosine receptor agonists include CGS-21680 and ATL-146 e.

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), accessory xanthine, pentoxifylline, theobromine, theophylline, methylated xanthine, vinpocetine, EHNA (erythro-9- (2-hydroxy-3-nonyl) adenine), anagrelide, enoximone (PERFAN)^TM) Miinolone, levosimendan, heliotropine (mesembrine), ibudilast, piracetam, luteolin, drotaverine, roflumilast (DAXAS)^TM、DALIRESP^TM) Sildenafil, sildenafil

Tadalafil

Vardenafil

Udenafil, avanafil, icariin (icarin), 4-methylpiperazine and pyrazolopyrimidine-7-1.

Examples of proteasome inhibitors include: bortezomib, disulfiram, epigallocatechin-3-gallate and salinosporamide A.

Examples of kinase inhibitors include bevacizumab, BIBW 2992, cetuximabImatinibTrastuzumab

Gefitinib

RalizumabPegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, panitumumab, vandetanib, E7080, pazopanib (pazopanib), and xylitinib (mubritinib).

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-

) IsotretinoinAliretin A acid

Etriatexate (tegispon)^TM) And its metabolite Avermectin ATazaroteneBexarotene

And adapalene

Examples of cytokine inhibitors include IL1ra, IL1 receptor antagonists, IGFBP, TNF-BF, uromodulin, α -2-macroglobulin, cyclosporin A, pentamidine and pentoxifylline

Examples of peroxisome proliferator activated receptor antagonists include GW9662, PPAR γ antagonist III, G335, and T0070907(EMD4Biosciences, USA).

Examples of peroxisome proliferator activated receptor agonists include pioglitazone, ciglitazone, clofibrate, GW1929, GW7647, L-165, 041, LY 171883, PPAR γ activators, Fmoc-Leu, troglitazone and WY-14643(EMD4Biosciences, USA).

Examples of histone deacetylase inhibitors include hydroxamic acids (or hydroxamates) such as trichostatin a, cyclotetrapeptides (e.g., trapoxin B) and depsipeptides, benzamides, electrophilic ketones, aliphatic acid compounds (e.g., phenylbutyric acid and valproic acid), hydroxamic acids (e.g., vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat (LBH589)), benzamides such as entinostat (MS-275), CI994, and mocetinostat (MGCD0103), nicotinamide, derivatives of NAD, dihydrocoumarins, naphthopyrones, and 2-hydroxynaphthaldehyde.

Examples of calcineurin inhibitors include cyclosporine, pimecrolimus, cyclosporine (voclosporine), and tacrolimus.

Examples of phosphatase inhibitors include BN82002 hydrochloride, CP-91149, calyxin spongio-carcinogenin a (calyculin a), cantharidinic acid, cantharidin, cypermethrin, ethyl-3, 4-difosstatin, forskocin sodium salt, MAZ51, methyl-3, 4-difosstatin, NSC 95397, norcantharidin, ammonium okadaic acid salt from protozobium vulgare (prorocentrum concavum), okadaic acid, potassium okadaic acid salt, sodium okadaic acid salt, phenylarsonic oxide, a mixture of various phosphatase inhibitors, protein phosphatase 1C, protein phosphatase 2A inhibitor protein, protein phosphatase 2A1, protein phosphatase 2A2, and sodium orthovanadate.

In some embodiments of any one of the methods, compositions, or kits provided, a therapeutic macromolecule described herein is also linked to a synthetic nanocarrier. In other embodiments, the therapeutic macromolecule is not attached to any synthetic nanocarriers. In some embodiments of any of these cases, the therapeutic macromolecule may be delivered as the therapeutic macromolecule itself or as a fragment or derivative thereof.

Therapeutic proteins include, but are not limited to, infusible therapeutic proteins, enzymes, enzyme cofactors, hormones, clotting factors, cytokines and interferons, growth factors, monoclonal and polyclonal antibodies (e.g., administered to a subject as a replacement therapy), and proteins associated with pompe disease (e.g., acid glucosidase α) (e.g., Myozyme and lumizyme (genzyme)).

Examples of therapeutic proteins for use in enzyme replacement therapy in a subject having a lysosomal storage disease include, but are not limited to: imidamidase (e.g., CEREZYME) for the treatment of Gaucher's disease^TM) Alpha-galactosidase A (a-gal A) for the treatment of Fabry disease (Fabry disease) (e.g., acaccharidase β, Fabryzyme)^TM) Acid α -glucosidase GAA (e.g., acid glucosidase α, LUMIZYME) for the treatment of Pompe disease^TM、MYOZYME^TM) Arylsulfatase B (e.g., Raronidase, ALDURAZYME) for the treatment of mucopolysaccharidosis (Mucopysaccharidose)^TMIduronidase, ELAPRASE^TMArylsulfatase B, NAGLAZYME^TM) Pegylated recombinant uricase (KRYSTEXXA) and pegylated recombinant candida uricase.

Examples of enzymes include: oxidoreductases, transferases, hydrolases, lyases, isomerases, asparaginases, uricases, glycosidases, asparaginases, uricases, proteases, nucleases, collagenases, hyaluronidase, heparanase, lysin (lysin) and ligases.

Therapeutic proteins may also include any enzyme, toxin, or other protein or peptide isolated or derived from a bacterial, fungal, or viral source.

Examples of hormones include: melatonin (N-acetyl-5-methoxytryptamine), serotonin, thyroxine (or tetraiodothyronine) (thyroid hormone), triiodothyronine (thyroid hormone), epinephrine (or adrenal hormone), norepinephrine (or norepinephrine), dopamine (or prolactin-inhibiting hormone), anti-mullerian hormone (or mullerian tube-inhibiting factor or hormone), adiponectin, corticotropin (or corticotropin), angiotensinogen and angiotensin, anti-diuretic hormone (or vasopressin, arginine vasopressin), atrial natriuretic peptide (or atrial natriuretic peptide), calcitonin, cholecystokinin, corticotropin-releasing hormone, erythropoietin, follicle stimulating hormone, gastrin, ghrelin, glucagon-like peptide (GLP-1), GIP, gonadotropin releasing hormone, growth hormone releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin-like growth factor (or growth regulator), 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), calcifediol (25-hydroxyvitamin D3), prostaglandins, leukotrienes, prostacyclins, thromboxanes, prolactin releasing hormone, Lipotropin, natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, renin, and enkephalin.

Examples of blood factors or coagulation factors include factor I (fibrinogen), factor II (prothrombin), tissue factor, factor V (pro-accelerin, labile factor), factor VII (stabilizing factor, pre-convertin), factor VIII (anti-hemophilia globulin), factor IX (Krestamas factor (Christmas factor) or the plasmaphromokinase component), factor X (Stuart-Prower factor), factor Xa, factor XI, factor XII (Hageman factor), factor XIII (fibrin stabilizing factor), von Willebrand factor, prekallikrein (Flett factor), High Molecular Weight Kininogen (HMWK) (Figertald factor)), fibronectin, fibrin, thrombin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z-related inhibitors (3556), plasminogen activator (Prothrombin-2), plasminogen activator (Prothrombin-kinase inhibitor), plasminogen activator (plasminogen activator), plasminogen activator (plasminogen-kinase inhibitor), plasminogen activator (plasminogen-3683), plasminogen activator (plasminogen-plasminogen activator), plasminogen (plasminogen-kinase inhibitor), plasminogen activator (plasminogen-36632), plasminogen activator (plasminogen, thromboplastin inhibitor), thromboplastin inhibitor, thromboplastin-2, plasminogen, thromboplastin inhibitor, thromboplastin-2, and plasminogen activator (thromboplastin inhibitor).

Examples of cytokines include lymphokines, interleukins, and chemokines, type 1 cytokines (e.g., IFN-. gamma., TGF- β), and type 2 cytokines (e.g., IL-4, IL-10, and IL-13).

Examples of Growth factors include Adrenomedullin (AM), angiogenin (Ang), autotaxin, Bone Morphogenetic Protein (BMP), Brain-derived neurotrophic factor (BDNF), Epidermal Growth Factor (EGF), Erythropoietin (EPO), Fibroblast Growth factor (Fibroblast Growth factor, FGF), Glial cell line-derived neurotrophic factor (glian cell-derived neurotrophic factor, GDNF), Granulocyte colony stimulating factor (Granulocyte colony stimulating factor, G-CSF), Granulocyte macrophage colony stimulating factor (GF-CSF, GM-CSF), GM-CSF, Growth factor-9 (VEGF-9), Growth factor (VEGF-3, Fibroblast Growth factor, VEGF-derived Growth factor, Growth factor-Growth factor, VEGF-derived Growth factor, Growth factor-Growth factor, Growth factor-Growth factor, Growth factor-Growth factor, Growth factor-Growth factor, Fibroblast Growth factor.

Examples of the monoclonal antibody include apyrazumab, abciximab, adalimumab, adolimumab, aphidimomab, afzezumab, afzeolizumab (afuttuzumab), perizezumab, ALD, alemtuzumab, adozezumab, rituximab, etc.

Examples of therapeutic proteins for infusion therapy or injectable include, for example: tubizumab (Roche @)

α -1 antitrypsin (Kamada/AAT),

(Affymax and Takeda, synthetic peptides), Albumin α -2b (Albiteferon alfa-2b) (Novartis/Zalbin^TM)、(Pharming Group, C1 inhibitor replacement therapy), temerelin (theratetechnologies/Egrifta, synthetic growth hormone releasing factor), ocrelizumab (Genentech, Roche and Biogen), belimumab (GlaxoSmithKline @

) PEGylated recombinant uricase (Savient Pharmaceuticals/Krystex xxa)^TM) Pegylated recombinant Candida uricase, Talinulase α (Protalix/Uplyso), and arabinosidase α (Shire @)

) Glucocerebrosidase α (shine) and keyhole limpet Lindbie hemocyanin (KLH).

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 therapeutic polynucleotides such as antisense polynucleotides or antisense oligonucleotides (e.g., antiviral drugs fomivirsen, or mipramigen, antisense therapeutics that target the messenger RNA of 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 very high potency); or modified messenger RNA (mmRNA) such as those disclosed in U.S. patent application 2013/0115272 to Fougerolles et al and published U.S. patent application 2012/0251618 to Schrum et al.

Other therapeutic macromolecules that may be used according to aspects of the invention will be apparent to those skilled in the art, and the invention is not limited in this respect.

In some embodiments, the components (e.g., therapeutic macromolecules or immunosuppressive agents) can be isolated. "isolated" means that a component is separated from its natural environment and is present in a sufficient amount to allow its identification or use. This means, for example, that the components can be (i) selectively produced by expression cloning or (ii) purified by chromatography or electrophoresis. The separated components may, but need not, be substantially pure. Since the separated components can be mixed in a pharmaceutical formulation together with pharmaceutically acceptable excipients, the components may constitute only a small weight percentage of the formulation. The component is still isolated as long as it has been separated from the substances with which it is associated in a living system, i.e. from other lipids or proteins. Any of the components provided herein can be isolated and included in the composition in isolated form or used in the method.

D. Methods of making and using the compositions and related methods

Some aspects of the invention relate to determining a regimen for the concomitant administration provided herein. The protocol may be determined as follows: the frequency, dosage and other aspects of administering the therapeutic macromolecule and immunosuppressant are varied and the pharmacodynamic effects are then assessed based on these changes. Each administration occurs in the presence of an anti-therapeutic macromolecular antibody response. One preferred protocol for practicing the invention induces the desired pharmacodynamic effect but induces little to no anti-therapeutic macromolecular antibody response.

In some aspects of the invention, the desired pharmacodynamic effect of the therapeutic macromolecule includes stimulating or inhibiting a particular response. In some embodiments, the pharmacodynamic effect involves, but is not limited to: production or degradation of cytokines, chemokines, signaling molecules, or other molecules; inducing proliferation or death of a particular cell type; maturation or localization of a particular cell type; interaction with an enzyme, structural protein, carrier protein, or receptor protein; modulating the activity of an enzyme, structural protein, or receptor protein, and the like. In some embodiments, the pharmacodynamic effect is to reduce the production of cytokines, such as inflammation-associated cytokines (e.g., TNF, IL-1). In some embodiments, the pharmacodynamic effect is a decrease in cytokine activity. In some embodiments, the pharmacodynamic effect is to reduce the production of undesired molecules. In some embodiments, the pharmacodynamic effect is to enhance degradation of undesired molecules (e.g., uric acid crystals). In some embodiments, the pharmacodynamic effect is an activity of an enzyme.

The pharmacodynamic effects of therapeutic macromolecules can be assessed by standard methods. In some aspects of the invention, the pharmacodynamic effect is a reduction in inflammation. The level of inflammation can be assessed by exemplary methods not limited to scoring inflammatory symptoms (e.g., redness or swelling); scoring arthritic symptoms (e.g., motility, pain, or joint damage); scoring for symptoms of anaphylaxis (e.g., swelling, blood pressure, shortness of breath); detecting and/or quantifying cell infiltration by histology, immunohistochemistry, flow cytometry; measuring the concentration of protein or inflammation-associated cytokine (TNF, IL-1) by ELISA, assessing the expression of the gene or inflammation-associated gene by transcriptional analysis; measuring the activity of inflammation-associated cytokines, and the like.

In some aspects of the invention, the pharmacodynamic effect is a reduction or degradation of the undesired molecule. In some embodiments, the pharmacodynamic effect can be assessed by, but is not limited to, quantifying the undesired molecule in a tissue or blood sample by means of, for example, ELISA. In some embodiments, the pharmacodynamic effect can be assessed by quantifying the molecules resulting from the degradation of the undesired molecules by methods such as ELISA. In some aspects of the invention, the pharmacodynamic effect is an activity of an enzyme that was previously absent or insufficiently present. In some such embodiments, the activity of an enzyme can be assessed by detecting the presence or concentration of a product having the enzyme activity.

In some aspects of the invention, a reduced dose of the therapeutic macromolecule is administered to produce a pharmacodynamic effect. A reduced therapeutic macromolecule dose for this purpose includes any therapeutic macromolecule dose that achieves a pharmacodynamic effect in the presence of an anti-therapeutic macromolecule antibody response with concomitant administration of an immunosuppressant dose, which is lower than the dose required to achieve a similar pharmacodynamic effect with a therapeutic macromolecule in the presence of an anti-therapeutic macromolecule antibody response when not concomitantly administered with an immunosuppressant dose. The reduced dose can be determined as follows: the therapeutic macromolecule is administered at a dose in the presence of an anti-therapeutic macromolecule antibody response and concomitantly with the immunosuppressant dose, and the pharmacodynamic effect is assessed. The pharmacodynamic effect can then be compared to that obtained by administering the therapeutic macromolecule in the presence of an anti-therapeutic macromolecule antibody response, without concomitant administration of an immunosuppressive agent dose. Lower doses to achieve similar pharmacodynamic effects are determined by such comparisons as reduced doses.

As previously described, the immunosuppressants can be attached to synthetic nanocarriers. Synthetic nanocarriers can be prepared using a variety of methods known in the art. For example, synthetic nanocarriers can be formed by the following methods and other methods known to those of ordinary skill in the art: for example, nanoprecipitation, flow focusing using fluidic channels, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, milling, microemulsification, microfabrication, nanofabrication, sacrificial layers, simple coacervation, and complex coacervation. Alternatively or additionally, aqueous and organic solvent syntheses for monodisperse semiconductor nanomaterials, electrically conductive nanomaterials, magnetic nanomaterials, organic nanomaterials and other nanomaterials have been described (Pellegrino et al, 2005, Small, 1: 48; Murray et al, 2000, Ann. Rev. Mat. Sci., 30: 545; and Trindade et al, 2001, chem. Mat., 13: 3843). Additional methods have been described in the literature (see, e.g., Doubrow, eds. "Microcapsules and nanoparticies in Medicine and Pharmacy," CRC Press, bocaaton, 1992; Mathiowitz et al, 1987, J.Control. Release, 5: 13; Mathiowitz et al, 1987, Reactive Polymers, 6: 275; and Mathiowitz et al, 1988, J.Appl. Polymer Sci., 35: 755; U.S. Patents 5578325 and 6007845; P.Paoliceli et al, "Surface-modified PLGA-based nanoparticlehaving can Efficiently Association and Deliver Virus-lipolytics" Nanomedicine.5 (6): 843 853 (2010)).

If desired, various materials may be encapsulated in synthetic nanocarriers using a variety of methods including, but not limited to, C.Atte et al, "Synthesis and catalysis of PLGA nanoparticles" J.Biomate.Sci.Polymer Edn, Vol.17, No. 3, pp.247-289 (2006); avgoustakis "granulated Poly (Lactide) and Poly (Lactide-Co-Glycolide) Nanoparticles: preparation, Properties and Possible Applications in Drug Delivery "Current Delivery 1: 321-333 (2004); reis et al, "nanoencapsidation i. methods for the preparation of drug-loaded polymeric nanoparticles" Nanomedicine 2: 8-21 (2006); paolicelli et al, "Surface-modified PLGA-based Nanoparticles which can be used for efficient Association and Deliver Virus-like Nanoparticles" Nanomedicine.5 (6): 843-853(2010). Other methods suitable for encapsulating materials within synthetic nanocarriers may also be used, including but not limited to the method disclosed in U.S. patent 6,632,671 to Unger, issued 10/14/2003.

In certain embodiments, the synthetic nanocarriers are prepared by a nanoprecipitation method or spray drying. The conditions used to prepare the synthetic nanocarriers can be varied to produce particles having a desired size or characteristic (e.g., hydrophobic, hydrophilic, external morphology, "viscous," shape, etc.). The method of preparing the synthetic nanocarriers and the conditions used (e.g., solvent, temperature, concentration, air flow rate, etc.) may depend on the composition of the material and/or polymer matrix to which the synthetic nanocarriers are to be attached.

If the size range of the synthetic nanocarriers prepared by any of the methods described above is outside the desired range, such synthetic nanocarriers can be size selected, for example, using a sieve.

The components (i.e., components) of the synthetic nanocarriers (e.g., antigens, immunosuppressants, etc.) can be attached to the entire synthetic nanocarrier, e.g., by one or more covalent bonds, or can be attached via one or more linkers. Additional methods of functionalizing synthetic nanocarriers can be modified from published U.S. patent application 2006/0002852 to Saltzman et al, published U.S. patent application 2009/0028910 to Desimone et al, or published International patent application WO/2008/127532A1 to Murthy et al.

Alternatively or additionally, the synthetic nanocarriers and the components can be linked directly or indirectly via non-covalent interactions. In some non-covalent embodiments, the non-covalent attachment is mediated by non-covalent interactions including, but not limited to: charge interactions, affinity interactions, metal coordination, physisorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and/or combinations thereof. Such coupling may be arranged on the outer or inner surface of the synthetic nanocarriers. In some embodiments, the encapsulation and/or adsorption is in the form of a linkage. In some embodiments, synthetic nanocarriers and therapeutic macromolecules or other components can be combined by mixing in the same vehicle or delivery system.

The compositions provided herein can include inorganic or organic buffers (e.g., sodium or potassium salts of phosphoric acid, carbonic acid, acetic acid, or citric acid) and pH adjusters (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citric acid or acetic acid, amino acids, and salts thereof), antioxidants (e.g., ascorbic acid, α -tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10 nonylphenol, sodium deoxycholate), solutions and/or freeze/freeze stabilizers (e.g., sucrose, lactose, mannitol, trehalose), permeation modifiers (e.g., salts or sugars), antimicrobials (e.g., benzoic acid, phenol, gentamicin), defoamers (e.g., polydimethylsiloxane), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity modifiers (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose), and cosolvents (e.g., glycerol, polyethylene glycol, ethanol).

The composition according to the invention may comprise pharmaceutically acceptable excipients. The compositions can be prepared using conventional pharmaceutical preparation and compounding techniques to achieve a useful dosage form. Techniques suitable for implementing the present invention can be found in Handbook of industrial Mixing: science and Practice, edited by Edward l.paul, Victor a.atiemo-Obeng and Suzanne m.kresta, 2004 john wiley & Sons, inc; and pharmaceuticals: the science of Dosage Form Design, 2 nd edition, edited by m.e. auten, 2001, churchl Livingstone. In one embodiment, the composition is suspended in sterile saline solution for injection, along with a preservative.

It will be appreciated that the compositions of the invention may be prepared in any suitable manner and the invention is not in any way limited to compositions that may be prepared using the methods described herein. Selection of an appropriate preparation method may require attention to the nature of the particular moiety associated.

In some embodiments, the compositions are prepared under aseptic conditions, or are terminally sterilized. This ensures that the resulting composition is sterile and non-infectious, thereby increasing safety when compared to non-sterile compositions. This provides a valuable safety measure, especially when the subject receiving the composition is immunodeficient, infected and/or susceptible to infection. In some embodiments, the compositions may be freeze-dried and stored in suspension or as lyophilized powders, depending on long-term formulation strategies without loss of activity.

Administration according to the present invention can be by a variety of routes including, but not limited to: subcutaneous, intravenous, intraperitoneal, intramuscular, transmucosal, transdermal, or intradermal routes. In a preferred embodiment, administration is via a subcutaneous route of administration. The compositions mentioned herein may be formulated and prepared for administration, preferably for concomitant administration, using conventional methods.

The compositions of the present invention may be administered in an effective amount (e.g., an effective amount as described elsewhere herein). According to the present invention, the dosage of the dosage form may comprise different amounts of immunosuppressive and/or therapeutic macromolecules. The amount of immunosuppressant and/or therapeutic macromolecule present in the dosage forms of the invention may vary according to: the nature of the therapeutic macromolecule and/or immunosuppressant, the therapeutic benefit to be achieved, and other such parameters. In some embodiments, a dose range study can be conducted to establish an optimal therapeutic amount of immunosuppressant and/or therapeutic macromolecule present in a dosage form. In some embodiments, the immunosuppressant and/or therapeutic macromolecule is present in the dosage form in an amount effective to produce a desired pharmacodynamic effect upon administration to a subject and/or to reduce an immune response against the therapeutic macromolecule. Conventional dose ranging studies and techniques can be used in subjects to determine the amount of immunosuppressant and/or therapeutic macromolecule that is effective to produce the desired result. The dosage forms of the present invention may be administered at a variety of frequencies. In a preferred embodiment, at least one administration of a composition provided herein is sufficient to produce a pharmacologically relevant response. In some more preferred embodiments, at least two administrations or at least three administrations are utilized to ensure a pharmacologically relevant response. In some embodiments, repeated administrations are utilized to ensure a pharmacologically relevant response.

Another aspect of the disclosure relates to a kit. In some embodiments, the kit comprises one or more than one pharmacodynamically effective dose, e.g., a reduced pharmacodynamically effective dose, of the therapeutic macromolecule. In such embodiments, the kit may further comprise one immunosuppressant dose or more than one immunosuppressant dose. The immunosuppressant dose and the pharmacodynamically effective dose can be contained in separate containers in a kit or in the same container in a kit. In some embodiments, the container is a vial or ampoule. In some embodiments, the pharmacodynamically effective dose and/or immunosuppressant dose of the therapeutic macromolecule is contained in a solution separate from the container, such that the pharmacodynamically effective dose and/or immunosuppressant dose of the therapeutic macromolecule can subsequently be added to the container. In some embodiments, the pharmacodynamically effective dose of the therapeutic macromolecule and/or the immunosuppressant dose are each present in a separate container or in the same container in lyophilized form, such that they can be subsequently reconstituted. In some embodiments, the kit further comprises instructions for reconstitution, mixing, administration, and the like. In some embodiments, the instructions comprise instructions for the methods described herein. The instructions may be in any suitable form, for example, as a printed insert or label. In some embodiments, the kit further comprises one or more syringes.