Protein-loaded PLGA nanospheres

Abstract

The present disclosure provides compositions comprising nanoparticles encapsulating proteins and methods for making the compositions. In one embodiment, the compositions can include a drug delivery vector and a therapeutic agent, wherein the composition elutes at least 1.0 pg of therapeutic agent per 100,000 particles of drug delivery vector over a period of time under conditions of a drug delivery vector release buffer, the therapeutic agent, drug delivery vector, and drug delivery vector release buffer forming a solution, the solution being centrifuged, and an aliquot stored at about 1-10°C, and the amount of eluted therapeutic agent determined by ELISA assay. The present disclosure further describes methods for controlling the immune phenotype in patients suffering from a disease affecting the immune system.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 944,191, filed December 5, 2019, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING GOVERNMENT-SPONSORED RESEARCH This application was made with government support under Grant Nos. 2U54GM104942-02, S10OD016165, P30GM103488 and P20GM103434 awarded by the National Institutes of Health / National Institute of General Medical Sciences. The federal government has certain rights in this invention.

[0003] Field The various embodiments disclosed herein relate generally to the preparation of protein-containing nanospheres.

[0004] Various embodiments disclosed herein relate to immunophenotyping for immunoassays. [Background technology]

[0005] background Immune stimulation is an important mechanism that can 1) inhibit the proliferation of malignant cells and / or the formation of tumor metastases, and 2) eliminate viral and bacterial infections. Immunotherapy cancer treatments can include monoclonal antibody blockade of specific immunoregulatory checkpoints, including the programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) axis.

[0006] Historically, the focus has been on the tumor microenvironment and immune response. However, a systemic response can result in a sustained immunological response and cure the disease. Many groups have focused on immunophenotyping treatments to help describe what is immunologically occurring in patients in real time. This information will play a critical role as immunotherapeutic agents for many diseases become more mainstream, and developing a living database of these responses will significantly advance this process.

[0007] Preclinical studies have shown that interleukins can induce antitumor responses against a number of malignancies. Interleukin-12 (IL-12), an immunostimulatory cytokine with antitumor activity that is maximized when administered systemically, can induce such antitumor responses. Although high doses of IL-12 can have toxic side effects, low doses of IL-12 are considered safe.

[0008] Poly(D,L-lactic-co-glycolic acid) (PLGA) drug delivery vectors have been approved by the FDA and are capable of eluting a wide range of substances as the polymer degrades. For therapeutic purposes, encapsulating peptides, potentially including immunostimulatory interleukin proteins, within PLGA nanospheres may enable systemic delivery and tissue deposition without the need for toxic payloads. Used in a systemic context, nanospheres can achieve safe and effective blood-mediated transport through macrovascular and microvascular systems. Summary of the Invention [Means for solving the problem]

[0009] Abstract In light of the current need for improved cytokine administration methods, a brief summary of various exemplary embodiments is presented. Some simplifications and omissions may be made in the following summary, which is intended to highlight and introduce aspects of certain embodiments disclosed herein, but is not intended to limit the scope of the disclosure. Detailed descriptions of various embodiments adequate to enable those skilled in the art to make and use the concepts disclosed herein follow in subsequent sections.

[0010] According to various embodiments disclosed herein, proteins can be encapsulated in nanospheres. This encapsulation method can be performed by creating a double emulsion, where a first aqueous phase can be emulsified in an oil phase, which is emulsified in a second aqueous phase. The protein can be a cytokine, which can be a small protein (approximately 5-20 kDa) that plays a role in cell signaling. Suitable cytokines include the following: Interleukins produced by T helper cells Lymphokines produced by lymphocytes Monokine produced exclusively by monocytes Interferon in the antiviral response colony-stimulating factors to support cell growth in semi-solid medium, and Chemokines mediate chemoattraction between cells Includes:

[0011] In various embodiments, the protein encapsulated in the nanospheres can be a cytokine having a three-dimensional structure with a bundle of 4ac helices, and the cytokine can be an interferon, an interleukin, such as interleukin-2 or interleukin-12, or a non-immunological cytokine, including erythropoietin and thrombopoietin. The protein encapsulated in the nanospheres can be interleukin-12 (IL-12).

[0012] The oil phase can be prepared by dissolving 2.5% w / v to 17% w / v of poly(lactic-co-glycolic acid) (PLGA) in an organic solvent. The first aqueous phase can be made by suspending a protein in an aqueous medium. Finally, the second aqueous phase can be made by dissolving polyvinyl alcohol (PVA) in water.

[0013] The PLGA can contain 50% to 90% lactide, and the organic solvent can be a halogenated C1-C3 organic solvent, a C2-C3 nitrile solvent, a C2-C5 alkyl ester solvent, a C3-C5 ketone solvent, or a mixture thereof. The PLGA can contain 75% to 90% lactide. The PLGA can contain 50% to 75% lactide, and the organic solvent can be a halogenated C1-C3 organic solvent, acetonitrile, a C3-C4 ketone solvent, or a mixture thereof. In various embodiments, the PLGA can contain 50% to 90% lactide, and the organic solvent can be acetonitrile, acetone, ethyl acetate, or dichloromethane.

[0014] The protein-containing aqueous medium can be added to the oil phase with stirring to form a first emulsion. The first emulsion can be added to the PVA-containing aqueous phase with stirring to form a second emulsion. The organic solvent can then be evaporated from the second emulsion to form an aqueous solution. The protein-containing PLGA nanospheres from the second aqueous phase can be recovered from the aqueous solution.

[0015] In one aspect, the present disclosure describes a composition comprising poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres and a therapeutic agent, wherein at least a portion of the therapeutic agent can be eluted from the composition more than 72 hours after placing the composition in solution.

[0016] In various embodiments, at least a portion of the therapeutic substance can be eluted from the composition after 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, or 336 hours after placing the composition in solution. At least a portion of the therapeutic substance can be eluted from the composition between 72 and 288 hours after placing the composition in solution. The therapeutic substance can be IL-12. The solution can be mammalian serum and about 100 units / mL penicillin-streptomycin (Pen-Strep) in phosphate buffered saline (DPBS).

[0017] In one aspect, the present disclosure describes a composition comprising poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres and IL-12, wherein the IL-12 can be incorporated into the PLGA nanospheres with an encapsulation efficiency of at least 2%.

[0018] In various embodiments, IL-12 can be incorporated into PLGA nanospheres with an encapsulation efficiency of at least 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. IL-12 can be incorporated into PLGA nanospheres with an encapsulation efficiency of about 2% to about 40%. The IL-12 can be bioactive IL-12.

[0019] In one aspect, the present disclosure describes a composition comprising a drug delivery vector and a therapeutic substance, wherein the composition can elute at least 1.0 pg of therapeutic substance per 100,000 particles of drug delivery vector under conditions of a drug delivery vector release buffer, and the composition continues to elute the therapeutic substance for more than 3 days, wherein the therapeutic substance, drug delivery vector, and drug delivery vector release buffer constitute a solution, the solution is centrifuged, and an aliquot is stored at about 1-10°C, and the amount of eluted therapeutic substance is determined by ELISA assay. The composition can include a surfactant. The surfactant can be Tween® 80 and Span® 60. The composition can include

[0020] In various embodiments, the drug delivery vector can comprise poly(D,L-lactic-co-glycolic acid) (PLGA). The therapeutic agent can be a protein. The protein can be a cytokine. The cytokine can be IL-12. The drug delivery vector release buffer can comprise about 10% Fetal Bovine Serum Qualified Heat Inactivated (HI-FBS) and about 100 units / mL penicillin-streptomycin (Pen-Strep) in phosphate buffered saline (DPBS). The composition can further comprise species-specific whole serum, species-specific engineered serum albumin, or species-specific whole fetal serum.

[0021] In one aspect, the present disclosure describes a composition comprising protein-loaded poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres, wherein the nanospheres can comprise a diameter of about 100-1000 nm, a surfactant, and species-specific, whole serum, engineered, or native serum albumin.

[0022] In various embodiments, the protein can include IL-12. The surfactant can include Tween® 80 and Span® 60.

[0023] In one aspect, the present disclosure provides a method of making protein-encapsulated nanospheres, comprising the steps of: determining a protein dissociation rate with increasing time and / or sonication wattage; comparing the protein dissociation rate with a rate of nanosphere formation with increasing time and / or sonication wattage; determining the time and sonication wattage at which the protein dissociation rate intersects with the rate of nanosphere formation; preparing a first phase by dissolving PLGA in a solvent containing a first surfactant; and preparing a second surfactant by dissolving PLGA in a solvent containing a second surfactant. The method includes the steps of preparing a second phase by dissolving alcohol in water containing the agent and mammalian serum, suspending the components in the aqueous medium, forming a first emulsion containing the aqueous medium and the first phase, forming a second emulsion containing the first emulsion and the second phase, and sonicating the second emulsion for a time and sonication power determined at the intersection, evaporating the solvent from the second emulsion to form an aqueous solution, and recovering PLGA nanospheres containing the components from the aqueous solution.

[0024] In one aspect, the present disclosure describes a method for encapsulating a component in nanospheres, the method comprising the steps of preparing a first phase by dissolving poly(lactic-co-glycolic acid) (PLGA) in a solvent, preparing a second phase by dissolving an alcohol in water, suspending the component in an aqueous medium, forming a first emulsion comprising the aqueous medium and the first phase, forming a second emulsion comprising the first emulsion and the second phase, evaporating the solvent from the second emulsion to form an aqueous solution, and recovering PLGA nanospheres containing the component from the aqueous solution.

[0025] In various embodiments, the components can include a protein. The protein can include IL-12. The components can include a surfactant and mammalian serum. The PLGA can include 50% to 90% lactide, and the solvent can be selected from the group consisting of a halogenated C1-C3 organic solvent, a C2-C3 nitrile solvent, a C2-C5 alkyl ester solvent, a C3-C5 ketone solvent, and a mixture thereof. The solvent can be acetonitrile, acetone, ethyl acetate, or dichloromethane. The PLGA can include 75% to 90% lactide. The PLGA can include 50% to 75% lactide, and the solvent can be selected from the group consisting of a halogenated C1-C3 organic solvent, acetonitrile, a C3-C4 ketone solvent, and a mixture thereof. The method can include adding an aqueous medium to a first phase to form a first emulsion and stirring the first emulsion with a tissue homogenizer at a speed of 13,000 RPM to 20,000 RPM, and adding the first emulsion to a second phase to form a second emulsion and stirring the second emulsion with a tissue homogenizer at a speed of 13,000 RPM to 20,000 RPM.The method can include adding an aqueous medium to the first phase to form the first emulsion and stirring the first emulsion by sonication, and adding the first emulsion to the second phase to form a second emulsion and stirring the second emulsion by sonication. The method may include agitating the first emulsion for a period of 5 to 30 seconds at a power level of 30 to 50 W and agitating the second emulsion for a period of 5 to 30 seconds at a power level of 30 to 50 W. The protein may be a cytokine or a globular protein. The protein may be a cytokine selected from the group consisting of interleukins, lymphokines, monokines, interferons, colony-stimulating factors, and chemokines.The cytokine can be selected from the group consisting of interleukins and non-immunological cytokines. The protein can be a cytokine having an N-terminal signal sequence, a four-helix bundle including four helices labeled A-D, and no C-terminal extension after the D helix. The cytokine can be granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interferon alpha-1, interferon beta, interferon gamma, interferon kappa, interferon tau-1, interferon omega-1, or the alpha chain of an interleukin selected from the group consisting of IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-12, IL-13, IL-15, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-26, and IL-27. The protein can be an immunological cytokine that either a) enhances a cellular immune response or b) enhances an antibody response. The cytokine can be an immunological cytokine that enhances a cellular immune response selected from the group consisting of TNFα, IFN-γ, and interleukin-12. The cytokine can be IL-12. The cytokine can be an immunological cytokine that enhances an antibody response selected from the group consisting of TGF-β, IL-4, IL-10, and IL-13. The first and second emulsions can each be stirred using a tissue homogenizer at a speed of 13,000 RPM to 20,000 RPM, and IL-12 is incorporated into the PLGA nanospheres at an encapsulation efficiency of about 0.5% to about 2.1%. The first and second emulsions can each be agitated by sonication at a power level of 30W to 50W for a period of 5 to 30 seconds, and IL-12 is incorporated into the PLGA nanospheres with an encapsulation efficiency of about 4.5% to about 10%. The second phase can contain polyvinyl alcohol and mammalian serum. The first phase can contain a first surfactant, and / or the second phase can contain a second surfactant.The first surfactant can be a sorbitan fatty acid ester, and / or the second surfactant can be a polyoxyethylene sorbitan fatty acid ester. The second phase can contain polyvinyl alcohol and fetal serum.

[0026] In various embodiments, a first portion of the protein can be adsorbed to the surface of the nanosphere, and a second portion of the protein can be incorporated into a PLGA matrix in the core of the nanosphere, and the nanosphere can include at least one additive selected from the group consisting of mammalian serum albumin, trehalose, and first and second surfactants. The nanosphere can include mammalian serum albumin and a surfactant.

[0027] In various embodiments, the present disclosure describes a dosage form comprising a plurality of nanospheres produced by the described methods, each nanosphere comprising a PLGA matrix and a protein, wherein a first portion of the protein can be adsorbed to the surface of the nanosphere and a second portion of the protein is incorporated into the PLGA matrix in the core of the nanosphere. The protein can be IL-12, and the IL-12 can be incorporated into the nanosphere with an encapsulation efficiency of at least 2%.

[0028] In one aspect, the present disclosure describes a method for encapsulating a protein in nanospheres, the method comprising: preparing an oil phase by dissolving 2.5% w / v to 17% w / v of poly(lactic-co-glycolic acid) (PLGA) in an organic solvent, optionally containing a first surfactant; preparing an aqueous phase containing polyvinyl alcohol and at least one additive selected from the group consisting of mammalian serum, trehalose, and a second surfactant, and suspending the protein in the aqueous medium; adding the aqueous medium to the oil phase to form a first emulsion and stirring the first emulsion; adding the first emulsion to the aqueous phase to form a second emulsion and stirring the second emulsion; evaporating the organic solvent from the second emulsion to form an aqueous solution; and recovering the protein-containing poly(lactic-co-glycolic acid) nanospheres from the aqueous solution.

[0029] In one aspect, the present disclosure describes a method of controlling immune phenotype in a patient suffering from a disease affecting the immune system, the method comprising: (a) determining the patient's disease state, wherein the disease state comprises a diagnosis and an initial immune phenotype; (b) comparing the patient's disease state to a plurality of disease states in a database, wherein each disease state in the database comprises a diagnosis, an initial immune phenotype, and a treatment protocol; and (c) selecting a treatment protocol from the database based on the comparison of step (b), wherein the treatment protocol administers an immune modulating drug.

[0030] In various embodiments, the method can include (d) administering an immunomodulatory drug to the patient; (e) after step (d), monitoring the patient's immunophenotype as a function of time, and adjusting the administration of the immunomodulatory drug if the patient's immunophenotype falls outside a desired range.

[0031] In various embodiments, a protein-containing aqueous medium can be added to an oil phase while stirring at a speed of 13,000 RPM to 20,000 RPM with a tissue homogenizer to form a first emulsion. The first emulsion can be added to a PVA-containing aqueous phase while stirring at a speed of 13,000 RPM to 20,000 RPM with a tissue homogenizer to form a second emulsion. The organic solvent can then be evaporated from the second emulsion to form an aqueous solution. Protein-containing PLGA nanospheres from the second aqueous phase can be recovered from the aqueous solution.

[0032] In various embodiments, the protein in the protein-containing aqueous medium can be a cytokine. Suitable cytokines include interleukins, lymphokines, monokines, interferons, colony-stimulating factors, and chemokines. The protein can be a cytokine having a three-dimensional structure with a bundle of 4ac helices, such as an interleukin, for example, interleukin-2 or interleukin-12, or a non-immunological cytokine, including erythropoietin and thrombopoietin.

[0033] In various embodiments, the protein in the protein-containing aqueous medium can be IL-12. IL-12 can be incorporated into PLGA nanospheres by stirring the first and second emulsions using a tissue homogenizer at a speed of 13,000 RPM to 20,000 RPM. IL-12 can be incorporated into the resulting PLGA nanospheres at an encapsulation efficiency of about 0.5% to about 2.1%.

[0034] In various embodiments, a protein-containing aqueous medium can be added to an oil phase while stirring with sonication to form a first emulsion. The first emulsion can be added to a PVA-containing aqueous phase while stirring with sonication to form a second emulsion. Stirring during the formation of one or both of the first and second emulsions can include sonication at a power level of 30W-50W, 30W-40W, or 40W-50W for a period of 5-30 seconds, 10-30 seconds, 10-20 seconds, or 10-15 seconds. The organic solvent can then be evaporated from the second emulsion to form an aqueous solution. Protein-containing PLGA nanospheres from the second aqueous phase can then be recovered from the aqueous solution.

[0035] In various embodiments, a protein-containing aqueous medium can be added to an oil phase while stirring at a speed of 13,000 RPM to 20,000 RPM using a tissue homogenizer to form a first emulsion. A second emulsion can be formed by adding the first emulsion to a PVA-containing aqueous phase while stirring by sonication at a power level of 30W to 50W, 30W to 40W, or 40W to 50W for a period of 5 seconds to 30 seconds. The organic solvent can then be evaporated from the second emulsion to form an aqueous solution. Protein-containing PLGA nanospheres from the second aqueous phase can be recovered from the aqueous solution.

[0036] In various embodiments, the protein in the protein-containing aqueous medium can be IL-12. IL-12 can be incorporated into PLGA nanospheres by sonication for a period of 10 to 20 seconds at a power level of 30 to 50 W. IL-12 can be incorporated into the resulting PLGA nanospheres with an encapsulation efficiency of about 2% to about 85%, about 4.5% to about 70%, about 5% to 60%, about 7% to about 50%, about 8% to about 40%, about 10% to 30%, or about 5% to 10%.

[0037] Various embodiments disclosed herein are directed to nanospheres comprising a poly(lactic-co-glycolic acid) matrix and a protein, wherein a first portion of the protein can be adsorbed to the surface of the nanosphere and a second portion of the protein can be incorporated into the poly(lactic-co-glycolic acid) matrix in the core of the nanosphere. The nanospheres can be produced by creating a double emulsion, in which a first protein-containing aqueous phase can be emulsified in an oil phase, which can then be emulsified in a second aqueous phase. The protein can be a cytokine, such as IL-12. IL-12 can be incorporated into the nanospheres with an encapsulation efficiency of about 0.5% to about 85%, about 1% to about 70%, about 2% to about 60%, about 3% to about 50%, about 4% to about 40%, about 5% to 30%, about 0.5% to 10%, about 1% to 8%, or about 2% to 5%. IL-12 can be incorporated into PLGA nanospheres with an encapsulation efficiency of at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100%.

[0038] Various embodiments disclosed herein relate to a method for encapsulating proteins in nanospheres by preparing an oil phase by dissolving 2.5% w / v to 17% w / v of poly(lactic-co-glycolic acid) (PLGA) in an organic solvent, optionally containing a first surfactant. The method includes preparing an aqueous phase containing polyvinyl alcohol and at least one additive selected from the group consisting of mammalian whole serum, recombinant / native mammalian albumin, trehalose, and a second surfactant, and suspending the protein in the aqueous medium. The first surfactant can be a sorbitan fatty acid ester. The aqueous medium can be added to the oil phase to form a first emulsion. The first emulsion can be stirred, and the first emulsion can be added to the aqueous phase to form a second emulsion, which can then be stirred. The organic solvent can be evaporated from the second emulsion to form an aqueous solution. The protein-containing poly(lactic-co-glycolic acid) nanospheres can be recovered from the aqueous solution. The aqueous phase can contain polyvinyl alcohol and mammalian serum, such as fetal serum. The aqueous phase can contain polyvinyl alcohol and a second surfactant, where the first surfactant can be a sorbitan fatty acid ester and the second surfactant can be a polyoxyethylene sorbitan fatty acid ester.

[0039] Various embodiments disclosed herein relate to nanospheres comprising a poly(lactic-co-glycolic acid) matrix and a protein, wherein a first portion of the protein can be adsorbed to the surface of the nanosphere and a second portion of the protein can be incorporated into the poly(lactic-co-glycolic acid) matrix in the core of the nanosphere. The nanospheres can further comprise at least one additive selected from the group consisting of mammalian serum albumin, trehalose, and a surfactant. The nanospheres can comprise mammalian serum albumin, mammalian recombinant / native albumin, and a surfactant. The nanospheres can comprise mammalian whole serum, mammalian recombinant / native albumin, a first surfactant, and a second surfactant.

[0040] In many disease states, for example, cancer and autoimmune disorders, human immune system can be in a state of constant change.To treat such diseases, it can be beneficial for doctors to evaluate patient's immune system in real time, and track the immune system status over time.When cancer, infection and / or autoimmune disorders are treated with immunomodulatory agents, it can be beneficial for doctors to be able to track the effect of such agents on immune system, regardless of whether immunomodulatory agents are immunosuppressive or immunostimulatory.

[0041] Various embodiments disclosed herein relate to methods that allow for systemic analysis of the immune system from a blood or fingerstick draw that can be analyzed in a laboratory. The immune system's response to a disease state, e.g., cancer or autoimmune disease, can be analyzed at selected times, and the immune response to a treatment protocol can be tracked over the course of the disease or treatment.

[0042] This diagnostic method can be useful in the treatment, monitoring, and diagnosis of numerous diseases, including cancer, autoimmune diseases, and infections. As immune-modulating agents become more common, this method can provide physicians with the ability to assess the state of the immune system at specific times in the pathogenesis of a disease, allowing for predictions as to which immune-modulating treatments will be most effective in addressing the disease. This can increase the overall effectiveness of treatments and allow for improved assessment of a patient's immune status during the course of a disease. This information can also be organized into a living database of immune profiles across disease-specific categories, which can provide physicians with more information about the treatments they administer based on previous experience.

[0043] Various embodiments disclosed herein include a method of regulating the immune phenotype in a patient suffering from a disease affecting the immune system, comprising: determining the patient's initial immune phenotype or immune status; and if the initial immune phenotype indicates immunosuppression, administering a first agent that stimulates the immune system; or If the initial immune phenotype indicates overstimulation of the immune system, administering a second drug that suppresses the immune system. Either step The present invention relates to a method, including:

[0044] After administration of the selected drug, the patient's immunophenotype can be monitored as a function of time, and if the patient's immunophenotype falls outside the desired range, administration of the first and / or second drug can be adjusted.

[0045]

[0003] Various embodiments disclosed herein relate to a method of controlling the immune phenotype in a patient suffering from a disease affecting the immune system by determining the patient's disease state, the disease state comprising a diagnosis and an initial immune phenotype, and comparing the patient's disease state to a plurality of disease states in a database, each disease state in the database comprising a diagnosis, an initial immune phenotype, and a treatment protocol. Based on the comparison between the patient's disease state(s) and a treatment protocol from the database, a treatment protocol can be selected from the database, where the treatment protocol comprises administering an immune modulating drug. The method can include administering the immune modulating drug to the patient, monitoring the patient's immune phenotype as a function of time after administration of the drug, and adjusting the administration of the immune modulating drug if the patient's immune phenotype falls outside a desired range.

[0046] Immunophenotyping of patient blood samples can include the ability to appropriately analyze and evaluate the data. This system can allow for appropriate administration, treatment, and correction across several disease states. As data collection increases, the expanded database can also, in the broadest sense, allow direct medical professionals to aid in diagnosis, treatment, and administration of immunotherapeutic agents.

[0047] Incorporation by Reference All publications, patents, and patent applications identified herein are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or supersede any such conflicting material.

[0048] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also referred to herein as "Figure" and "FIG."). [Brief explanation of the drawings]

[0049] [Figure 1A-1B] 1A and 1B show the distribution of fluorescein isothiocyanate-labeled bovine serum albumin protein in PLGA nanospheres.

[0050] [Figure 1C] Figure 1C shows a biphasic protein elution curve from PLGA nanospheres, with an initial release of protein adsorbed on the surface of the nanospheres followed by a controlled release of protein entrapped by the PLGA nanospheres.

[0051] [Figure 1D] FIG. 1D shows the mechanism of biphasic protein release from PLGA nanospheres.

[0052] [Figure 2] Figure 2 shows fluorophore distribution over a 67-minute period in BALB / c mice inoculated with 1 mg / kg of Alexa Flour® 647-loaded nanospheres dissolved in sterile saline by injection either intraperitoneally (mouse on the left) or intravenously (mouse on the right).

[0053] [Figure 3] FIG. 3 shows the effect of sonication on IL-12 sonicated for 10, 20, 30, 40 or 60 seconds at three different wattages.

[0054] [Figure 4A-4B]Figures 4A and 4B show scanning electron microscope (SEM) images of unloaded (blank) poly(lactide-co-glycolide) acid (PLGA) nanospheres lyophilized without 25 mM trehalose at 11,000X (Figure 3a) and 13,000X (Figure 3b), respectively.

[0055] [Figure 4C] Figure 4C shows a scanning electron microscope (SEM) image at 5,000X magnification of unloaded (blank) PLGA nanospheres lyophilized with 25 mM trehalose.

[0056] [Figure 5A] Figure 5A shows PLGA nanospheres loaded with FITC-conjugated bovine serum albumin, with protein uptake visualized by confocal microscopy.

[0057] [Figure 5B-5C] 5B and 5C show scanning electron microscope (SEM) images of lyophilized recombinant murine IL-12-loaded PLGA nanospheres at 8,000X and 25,000X magnification, respectively.

[0058] [Figures 6A-6B] Figures 6A and 6B show the size distribution analysis of unloaded (blank) PLGA acid nanospheres and recombinant murine IL-12 (IL-12)-loaded PLGA nanospheres at dilution factors of 1:50 and 1:14 in water, respectively, at 25 degrees Celsius.

[0059] [Figure 6C-6D] 6C and 6D show the zeta potential distribution of unloaded blank and IL-12 loaded PLGA nanospheres, respectively, at a dilution factor of 1:50 in water at 25 degrees Celsius.

[0060] [Figures 7A-7B]Figures 7A and 7B show the estimated total amount of protein eluted over time from PLGA nanospheres loaded with recombinant murine IL-12 in terms of total protein (Figure 7A) and protein per 100,000 particles (Figure 7B).

[0061] [Figure 7C] Figure 7C shows the encapsulation efficiency (EE) of recombinant murine IL-12-loaded nanospheres calculated using the area under the curve (AUC) of each elution profile for three different particle concentrations (0.5 billion particles / mL, 0.75 billion particles / mL, and 1 billion particles / mL).

[0062] [Figures 8A-8C] 8A-8C show IL-12-loaded nanospheres prepared using sonication under various conditions of sonication power and sonication time.

[0063] [Figure 9] FIG. 9 shows the elution profile of IL-12 from nanospheres prepared using sonication under various conditions of sonication power and sonication time.

[0064] [Figure 10] FIG. 10 shows the elution profile of IL-12 from PLGA nanospheres prepared using high-speed stirring as a function of stirring rate using a nanosphere concentration of 750 million particles / mL.

[0065] [Figure 11] FIG. 11 shows the application of IL-12 derived from PLGA nanospheres to treat metastatic osteosarcoma.

[0066] [Figure 12] FIG. 12 shows the effect of trehalose and magnesium compounds on IL-12 elution from PLGA nanospheres.

[0067] [Figure 13] FIG. 13 shows the effect of fetal bovine serum (FBS) on IL-12 elution from PLGA nanospheres.

[0068] [Figure 14] FIG. 14 shows the effect of surfactants alone and in combination with FBS on the amount of IL-12 eluted from PLGA nanospheres.

[0069] [Figure 15A] 15A and 15B show the protein elution rate over time and the amount eluted as a percentage of the total elution amount, respectively, when nanospheres were prepared under various conditions. [Figure 15B] 15A and 15B show the protein elution rate over time and the amount eluted as a percentage of the total elution amount, respectively, when nanospheres were prepared under various conditions.

[0070] [Figure 16] FIG. 16 shows a schematic illustrating the experimental design.

[0071] [Figure 17] FIG. 17 shows recombinant murine IL-12 (rmIL-12)-induced T cell exhaustion of helper, cytotoxic, and regulatory T lymphocyte subsets in peripheral blood.

[0072] [Figure 18] FIG. 18 shows rmIL-12-induced polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) myeloid proliferation in peripheral blood.

[0073] [Figure 19] FIG. 19 shows IL-12-induced reduction in circulating natural killer (NK) cells.

[0074] [Figure 20A]Figures 20A and 20B show the mean percentage of NKp46+ natural killer (NK) cells in the peripheral blood of healthy versus diseased mice compared across seven time points over 12 weeks at the time of amputation, and mice stratified by the percentage of NK cells in the peripheral blood, respectively. [Figure 20B] Figures 20A and 20B show the mean percentage of NKp46+ natural killer (NK) cells in the peripheral blood of healthy versus diseased mice compared across seven time points over 12 weeks at the time of amputation, and mice stratified by the percentage of NK cells in the peripheral blood, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0075] Detailed Description As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "sample" includes multiple samples, including mixtures thereof.

[0076] "Nanospheres" or "nanoparticles" can be ultrafine particles. Such particles can be made from a variety of materials, including, but not limited to, poly(D,L-lactic-co-glycolic acid) (PLGA).

[0077] PLGA drug delivery vectors are FDA-approved and can release a wide range of substances once the polymer coating degrades into Krebs cycle intermediates. Drug solubility, bioavailability, and stability can all be modified by organic coatings, allowing for significant shifts in the pharmacokinetic and pharmacodynamic properties of encapsulated substances. For tumor and infection treatments, encapsulating IL-12 within PLGA nanospheres could enable systemic delivery and tissue deposition without the need for toxic loading doses. Their negatively charged surface is repelled by the glycocalyx, and their smaller size, coupled with their smaller size, allows for increased deposition in the interstitial space, where the glycocalyx can leach without disrupting its contents. However, to date, IL-12 has not been successfully encapsulated within submicron-scale PLGA particles. Nanospheres can achieve safe and effective blood transport through the organism's microvasculature (capillaries can be approximately 4-9 microns in diameter) while minimizing the risk of embolization.

[0078] The PLGA nanospheres with encapsulated proteins were: Prepare the oil phase by dissolving 2.5% w / v to 17% w / v PLGA in an organic solvent. Prepare an aqueous phase by dissolving 1% w / v to 3% w / v polyvinyl alcohol in an aqueous solvent. Suspending the protein in an aqueous medium Adding an aqueous medium to the oil phase to form a first emulsion and homogenizing the first emulsion. Adding the first emulsion to an aqueous phase to form a second emulsion and homogenizing the second emulsion. evaporating the organic solvent from the second emulsion to form an aqueous solution; and Recovering protein-containing PLGA nanospheres from aqueous solutions It can be generated by

[0079] protein In various embodiments, the protein can be a cytokine selected from the group consisting of interleukins, lymphokines, monokines, interferons, colony-stimulating factors, and chemokines. The cytokine can be an interleukin or a non-immunological cytokine.

[0080] The cytokine can have an N-terminal signal sequence, a four-helix bundle comprising four helices labeled A through D, and an optional C-terminal extension after the D helix. The cytokine can lack a substantial C-terminal extension and can be granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interferon alpha-1, interferon beta, interferon gamma, interferon kappa, interferon tau-1, interferon omega-1, or the alpha chain of an interleukin (IL) selected from the group consisting of IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-26, and IL-27.

[0081] In various embodiments, the cytokine can be an immunological cytokine that either enhances a cellular immune response or an antibody response. If the cytokine is an immunological cytokine that enhances a cellular immune response, the cytokine can be selected from the group consisting of tumor necrosis factor-alpha (TNFoc), interferon-gamma (IFN-'), and interleukin-12 (which can stimulate the production of IFN-' and TNFoc). If the cytokine is an immunological cytokine that enhances an antibody response, the cytokine can be selected from the group consisting of transforming growth factor beta (TGF-f3), IL-4, IL-10, and IL-13.

[0082] In various embodiments, the protein encapsulated in the nanospheres can be a globular protein. Suitable globular proteins can include serum albumin proteins; enzymes, such as esterases; hormones, such as insulin; and transporter proteins.

[0083] In various embodiments, PLGA nanospheres can be used to encapsulate active pharmaceutical ingredients, vitamins, nutraceutical active ingredients such as phytochemicals, and organic dyes or imaging agents. PLGA nanospheres can be used for the controlled release of a variety of drugs, including poorly soluble Class III and Class IV drugs.

[0084] How to make nanoparticles To produce the oil phase, 2.5% w / v to 17% w / v of PLGA can be dissolved in an organic solvent. The PLGA can contain 50% to 90% lactide, 65% to 90% lactide, or 75% to 90% lactide. The organic solvent can be a halogenated C1-C3 organic solvent, such as dichloromethane, chloroform, or 1,1,1-trichloroethane; a C2-C3 nitrile solvent, such as acetonitrile or propionitrile; a C2-C5 alkyl ester solvent, such as ethyl acetate or butyl acetate; or a C3-C5 ketone solvent, such as acetone or pentanone. The organic solvent can be a semipolar solvent with a dipole moment between 1.1 and 3.5. The oil phase can be made by dissolving PLGA in an organic solvent at room temperature (RT) with stirring, where the stirring can be 300-600 RPM, 350-550 RPM, or 425-500 RPM.

[0085] In various embodiments, solvent selection can be based on the lactide content in PLGA. When PLGA contains 75% to 90% lactide, the organic solvent can be a halogenated C1-C3 organic solvent, a C2-C3 nitrile solvent, a C2-C5 alkyl ester solvent, or a C3-C5 ketone solvent. When PLGA contains less than 75% lactide, the organic solvent can be a halogenated C1-C3 organic solvent, acetonitrile, or a C3-C4 ketone solvent.

[0086] To form the aqueous phase of the emulsion, 1% w / v to 3% w / v of polyvinyl alcohol (PVA) can be dissolved in an aqueous solvent, which can be water or a buffered saline solution, e.g., phosphate buffered saline.

[0087] A protein, such as IL-12 or bovine serum albumin, can then be suspended in an aqueous medium, which can be a buffered saline solution, e.g., phosphate-buffered saline, and the resulting protein suspension can be added to the PLGA-containing oil phase, which can be subjected to rapid stirring, e.g., 10,000-20,000 RPM; 12,000-19,500 RPM; 13,000-19,000 RPM; 15,000-18,000 RPM, or 16,000-17,500 RPM, to produce a first emulsion. Alternatively, the protein suspension can be added to the PLGA-containing oil phase while being sonicated to produce a first emulsion.

[0088] The first emulsion can then be added to the PVA aqueous phase with rapid stirring (e.g., 10,000-20,000 RPM; 12,000-19,500 RPM; 13,000-19,000 RPM; 15,000-18,000 RPM; or 16,000-17,500 RPM), homogenization, or sonication to produce a second emulsion. The organic solvent can then be evaporated from the second emulsion. The protein-containing PLGA nanoparticles from the aqueous medium can be recovered by centrifugation, flash-frozen in liquid nitrogen, and / or lyophilized.

[0089] Without being bound by any theory, the use of ultrasonic treatment over time can increase nanoparticle formation by increasing the number of nanoparticles, decreasing the size of the nanoparticles, and improving nanoparticle uniformity. Higher ultrasonic treatment wattages can also increase nanoparticle formation. However, proteins can dissociate when exposed to ultrasonic treatment over time, especially at higher ultrasonic treatment wattages for longer periods of time. Therefore, as disclosed herein, the ultrasonic treatment wattage and time parameters can be optimized for individual protein types in the production of protein-loaded nanoparticles.

[0090] Once the second emulsion is formed, the protein can become wrapped around the PLGA matrix polymer chains, which can coalesce and precipitate into spheres when the organic solvent is removed. During this process, the protein can become both entrapped within the polymer matrix (Figure 1A; in this case, the protein can be fluorescein isothiocyanate-labeled bovine serum albumin [BSA-FITC]) and adsorbed to the outer surface (Figure 1B), generating the characteristic biphasic elution curve shown in Figure 1C. The burst phase, which can occur between baseline and 2 days, can be attributed to the release of protein adsorbed to the nanosphere surface upon resuspension in aqueous media (Figure 1D). The controlled release phase can be attributed to the entrapped protein (Figure 1D), which can be slowly released over time as the PLGA hydrolyzes.

[0091] For comparison purposes, the above process can be performed using a buffered saline solution instead of a protein suspension, which is added to the PLGA-containing oil phase to form a first emulsion, which can be added to the PVA aqueous phase to form blank, protein-free nanoparticles.

[0092] Blank nanospheres and IL-12-loaded PLGA nanospheres can be synthesized using the techniques described above. The geometry of the blank and protein-loaded PLGA nanospheres can be determined by scanning electron microscopy to be spherical with average particle diameters of 50-500 nm, 100-250 nm, 100-150 nm, or 175-225 nm. In various embodiments, the IL-12-loaded PLGA nanospheres can have diameters of 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nm. Blank, i.e., protein-free, nanospheres can have diameters of 175-225 nm.

[0093] Zeta potential The zeta potential of both blank and IL-12-loaded PLGA nanospheres was also determined in deionized water. Zeta potential, which is the potential difference between the dispersion medium and the immobilized layer of fluid bound to the dispersed particles, can range from -15 to -25 mV after the introduction of 12.5 to 25 μg of recombinant murine IL-12 (rmIL-12) when protein was loaded during synthesis. As the magnitude of the zeta potential increases, the stability of the nanosphere dispersion may increase.

[0094] Encapsulation Efficiency The protein can be eluted from the nanospheres in nanosphere release buffer. The amount (percent) of IL-12 encapsulated and released by the nanospheres (encapsulation efficiency, EE) was calculated using the area under the curve (AUC) of each elution profile, the particle concentration (PC; particles / mL), the total amount of particles synthesized (V, mL), and the total weight (mg) of IL-12 added during synthesis, using the following formula:

number

[0095] Elution can be measured by dispersing protein-loaded PLGA nanospheres in nanosphere release buffer at concentrations of 200 million particles / mL to 100 billion particles / mL, 300 million particles / mL to 50 billion particles / mL, 400 million particles / mL to 20 billion particles / mL, or 500 million particles / mL to 1 billion particles / mL and analyzing the released protein concentration over time. The protein concentration may be bioactive. In the case of IL-12, the total amount of rmIL-12 eluted from the above particle concentrations can be determined by area under the curve (AUC) analysis to be 1500 to 4,000 pg. The amount of IL-12 released per 100,000 nanospheres was determined to be 0.3–0.45 pg / 100,000 nanospheres, respectively. For particles prepared by the homogenization method, the most efficient release rate was obtained at a concentration of 750 million particles / mL, and the least efficient release rate was obtained at a concentration of 1 billion particles / mL. Based on Equation (1), the average encapsulation efficiency (EE) was determined to be in the range of 0.4%–0.5%. The highest EE was obtained at a concentration of 750 million particles / mL.

[0096] To determine whether the synthesized nanospheres could actually encapsulate proteins and not simply adsorb proteins to the outer wall, PLGA nanospheres containing fluorescein isothiocyanate-labeled bovine serum albumin were synthesized. The resulting nanospheres were imaged by confocal microscopy to visualize the internal structure. Analysis confirmed that the labeled BSA was successfully incorporated into the nanospheres.

[0097] Distribution throughout the body To determine whether the contents of PLGA nanospheres are distributed throughout the body without causing injury following various routes of administration, PLGA nanospheres loaded with the fluorescent dye Alexa Fluor® 647 can be injected into female mice either intravenously via the tail vein or intraperitoneally and monitored with an in vivo imaging system. Both routes of administration can result in the distribution of nanosphere contents throughout the body without any signs of morbidity or mortality, as illustrated in Figure 2.

[0098] Water-insoluble payload Similar techniques can be used to encapsulate water-insoluble free bases or salts of drugs, or water-insoluble dyes, or imaging agents in PLGA nanospheres, where the term "water-insoluble" means that the drug or salt is less soluble in water than in the organic solvent that dissolves the PLGA polymer. Similar techniques can be used to encapsulate soluble drugs, dyes, or imaging agents, where controlled release by the PLGA polymer is desirable to provide a therapeutically safe and effective dose while avoiding toxic side effects due to rapid initial release.

[0099] To produce the oil phase, 2.5% w / v to 17% w / v of PLGA can be dissolved in an organic solvent, such as a halogenated C1-C3 organic solvent; a C2-C3 nitrile solvent; a C2-C5 alkyl ester solvent; or a C3-C5 ketone solvent. To produce the aqueous phase of the emulsion, 1% w / v to 3% w / v of polyvinyl alcohol (PVA) can be dissolved in an aqueous solvent, which can be water or a buffered saline solution, such as phosphate buffered saline.

[0100] A water-insoluble free base or salt of a drug, a water-insoluble dye, or an imaging agent can then be suspended in an aqueous medium, and the resulting protein suspension can be added to the oil phase containing the PLGA, which can be subjected to rapid stirring or sonication to produce a first emulsion.

[0101] The first emulsion can then be added to a PVA aqueous phase with rapid stirring or sonication to produce a second emulsion. The organic solvent can then be evaporated from the second emulsion. The drug, dye, or contrast agent-containing PLGA nanoparticles from the aqueous medium can be recovered by centrifugation and lyophilized.

[0102] additives Various further modifications to the process for nanosphere preparation can increase encapsulation efficiency and alter the elution profile of the nanospheres. Nanosphere preparation using a protein solution made by suspending 12.5 micrograms of IL-12 in 1.2 mL of DPBS containing 1.5% w / v trehalose can produce nanospheres with a delayed-release elution profile. The initial burst phase can be delayed up to two days after the start of the elution study, but this may result in reduced encapsulation efficiency.

[0103] Preparation of nanospheres using a protein solution made by suspending 12.5 micrograms of IL-12 in 1.2 mL of DPBS containing 2% w / v Mg(OH)2 can produce nanospheres with reduced encapsulation efficiency.

[0104] Nanospheres with delayed release can be produced by preparing nanospheres using a protein solution made by suspending 12.5 micrograms of IL-12 in 1.2 mL of DPBS containing 3%-15%, 5%-12%, 8%-12%, or approximately 10% total serum, serum albumin, fetal serum, or fetal serum albumin, depending on the patient's species. The initial burst phase can be delayed up to two days after the start of the elution study, increasing encapsulation efficiency. If the IL-12 suspension is incubated with fetal serum for 24 hours before nanosphere preparation, the burst phase can be delayed up to three days after the start of the elution study. Further incubation with whole serum, serum albumin, or fetal serum for 48 hours also extends the burst phase elution and increases encapsulation efficiency. For in vitro studies, any type of serum, serum albumin (including synthetically produced serum albumin), fetal serum, or fetal serum albumin, such as fetal bovine serum or fetal mouse serum, can be used. Alternatively, human serum can be used. For in vivo studies, in which nanospheres can be administered to human or non-human patients, the choice of serum or serum albumin can be specific to the species being treated. For bovine treatment, nanospheres made by suspending IL-12 in DPBS containing fetal bovine serum can be used. For mouse treatment, nanospheres should be made using DPBS containing fetal mouse serum. For human treatment, nanospheres can be made using DPBS containing human serum. Administration of nanospheres treated with serum from one species to another or with fetal serum can cause graft-versus-host disease. Similarly, when using whole serum or harvested native human albumin, cross-matching can be performed for each patient receiving these products, as can any blood product. In some cases, at least a portion of the therapeutic agent (e.g., IL-12) can elute from the composition (e.g., nanospheres) more than 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336 hours after placing the composition in solution.In some cases, at least a portion of the therapeutic substance (e.g., IL-12) can be eluted from the composition (e.g., nanospheres) in about 24 hours to about 48 hours, about 48 hours to about 72 hours, about 72 hours to about 192 hours, about 72 hours to about 168 hours, about 96 hours to about 168 hours, about 120 hours to about 168 hours, or about 144 hours to about 192 hours after placing the composition in solution.

[0105] The presence of a surfactant during nanosphere preparation can increase encapsulation efficiency. The surfactant can be incorporated into the PLGA-containing oil phase or the PVA / water phase. Suitable surfactants include oil-soluble sorbitan fatty acid esters (e.g., Span® 20, Span® 40, Span® 60, and Span® 80) and / or water-soluble polyoxyethylene sorbitan fatty acid esters (e.g., Tween® 20, Tween® 40, Tween® 60, and Tween® 80). In various embodiments, nanospheres can be prepared using a PLGA-containing oil phase containing 4%-20% w / w Span® surfactant, a PVA / water phase containing 2%-10% w / v Tween® surfactant, or both. For example, The oil phase may contain 4% to 20% w / w Span® 60, 10% to 16% w / w Span® 60 or about 14% w / w Span® 60. The PVA / aqueous phase may contain 2% to 10% w / v Tween® 80, 3% to 8% w / v Tween® 80, or 4% to 6% w / v Tween® 80, or The oil phase may contain 4% to 20% w / w Span® 60 and the PVA / water phase may contain 2% to 10% w / v Tween® 80.

[0106] The presence of both surfactant and fetal serum can increase encapsulation efficiency. Preparation of nanospheres using a protein solution made by suspending 12.5 micrograms of IL-12 in 1.2 mL of DPBS containing 10% fetal serum can produce nanospheres containing IL-12 with encapsulation efficiencies between 2% and 10%, 4% and 8%, or 5% and 7%. Preparation of nanospheres using a protein solution made by suspending 12.5 micrograms of IL-12 in 1.2 mL of DPBS containing 10% fetal serum, incubated in DPBS for 24 hours, can produce nanospheres containing IL-12 with encapsulation efficiencies between 10% and 50%, 20% and 45%, or 30% and 40%. Preparing nanospheres using a protein solution made by suspending 12.5 micrograms of IL-12 in 1.2 mL of DPBS containing both 10% fetal serum and a surfactant can produce nanospheres containing IL-12 with encapsulation efficiencies of between 50% and 95%, between 60% and 85%, or between 70% and 80%. In various embodiments, using a protein solution containing cytokine, fetal serum, and a surfactant synergistically increases the encapsulation efficiency of cytokines in PLGA nanospheres.

[0107] Immunophenotyping Various embodiments disclosed herein relate to techniques that allow medical professionals to systemically analyze a patient's immune system from a blood draw or from 2-3 drops of blood obtained using a fingerstick blood draw using a lancet and Microtainer®. The blood draw can be performed at home, in a medical professional's office, in a clinic, or in a hospital.

[0108] In various embodiments, the blood draw can occur at home, at a medical professional's office, at a clinic, or at a hospital. The blood draw can be analyzed at a laboratory or medical facility.

[0109] The blood samples can be analyzed for the levels of immunochemicals originally present in the patient's body and / or the levels of immunomodulatory drugs administered to the patient. The blood samples can be analyzed for the levels of immunochemicals originally present in the patient's body as a function of time, allowing a medical professional to observe the effect of a treatment regimen on immunochemical levels. Blood samples can be taken and analyzed at regular intervals, allowing a medical professional to evaluate changes in immune status or immune phenotype over the course of treatment.

[0110] Referring to Figure 11, four major immunophenotypes are considered: Suppression, e.g., immunosuppression induced by cancer, autoimmune disease, or infection Baseline, e.g., immune status of disease-free subjects Stimulation of the immune system above baseline levels, and A life-threatening condition of immune system overactivation known as immune cell exhaustion (TCE), characterized by exhaustion, i.e., lymphocyte anergy, systemic inflammatory response syndrome, and / or organ failure.

[0111] Figure 11 shows the changes in the immune phenotype of osteosarcoma cancer patients during treatment with various immune modulating therapies. The patient's initial immune phenotype is suppressed due to cancer-induced immune suppression. Programmed death-ligand 1 (PD-L1) is often expressed during osteosarcoma, and therefore, antibodies against PD-L1 (anti-PD-L1) can be administered. See line A in Figure 11. Interleukin-12 (IL-12) has been shown to inhibit osteosarcoma tumor growth, and therefore, free IL-12 can be administered as an immune modulator. See line C in Figure 11.

[0112] At time T1, anti-PD-L1 appears to be ineffective in changing the patient's immune phenotype, while IL-12 appears to shift the immune phenotype to stimulation. However, at time T2, as treatment progresses, IL-12 causes toxic side effects and shifts the patient's immune phenotype to exhaustion. At time T2, anti-PD-L1 shifts the patient's immune phenotype back to baseline, combating cancer-induced immunosuppression without stimulating the immune system to fight cancer.

[0113] As can be seen in Figure 11, line B, the combination of anti-PD-L1 and low dose IL-12 administered in PGLA nanospheres can stimulate the immune system to fight cancer without causing immune system overactivation. Thus, by monitoring the immune phenotype over time, physicians can adjust immune-modulating treatment to provide immune stimulation without T-cell exhaustion.

[0114] Data regarding immune response to cancer therapy can be entered into a database. For example, with reference to Figure 11, the following information can be entered into the database for any given patient with a first disease that suppresses or overstimulates the immune system: a) Type of disease b) Pre-treatment immunophenotype c) Treatment for the primary condition d) Changes in immunophenotype as a function of time e) Clinical outcomes f) Medications taken by the patient for a second illness g) Disease stage or level h) medical comorbidities, and i) Age of the patient.

[0115] A change in any one of these parameters can affect the other parameters. For example, if a patient undergoing treatment for a first disease affecting the immune system is diagnosed with a second disease, i.e., a medical comorbidity, after the start of treatment, either the causative agent of the second disease or the symptom of the second disease can be: Clinical outcome of treatment of primary disease Changes in patient immunophenotype over time the stage or level of the first disease, and / or The effectiveness of treatment given for the primary disease may have an impact on

[0116] Similarly, a change in disease stage or disease level of a first disease, for example, progression of cancer from stage 2 to stage 3, can affect the patient's immunophenotype as a function of time, the protocol for treatment of the first disease, and the clinical outcome of treatment of the first disease.

[0117] The database therefore contains information on the treatment of various diseases with immunomodulatory drugs, allowing predictions to be made as to how a particular patient presenting with that disease will respond to a given immunomodulatory therapy.

[0118] The use of such a database would enable medical professionals to predict a patient's immune system's response to a given disease state and to predict changes in a patient's immune phenotype over the course of a disease or treatment. The database would be useful in the treatment, monitoring, and diagnosis of numerous diseases, including cancer, autoimmune diseases, and infections. As immune-modulating agents become more common, such a database would provide the ability to assess the state of the immune system at a particular time in the pathogenesis of a disease and to predict which immune-modulating treatments are likely to be most effective in treating the disease.

[0119] Each blood sample obtained from a patient's blood draw or fingerstick can be analyzed for the patient's immune phenotype at the time of collection. The results of this analysis, along with information about the patient's disease status and current treatment, if any, can be compared to data present in a database to develop a treatment plan most likely to effectively modulate the patient's immune phenotype. Blood samples can be collected and analyzed at regular intervals, allowing medical professionals to assess changes in immune status or immune phenotype over the course of treatment. Data from current patients can potentially be added to the database to improve the assessment of other patients. This has enormous commercial potential for the analysis of patient blood, including complex immune phenotyping, whether the analysis is for cancer, infection, or autoimmune disease. A database that can be generated by cross-referencing diseases with immune phenotyping would be a powerful tool for treating patients in the future. The database would be a living database, generating constantly updated information regarding immune phenotyping and treatment status. Immunophenotyping of patient blood samples includes the ability to appropriately analyze and evaluate data. This system will allow for appropriate dosing, treatment, and correction across several disease states. As more data is collected, the growing database will also be able to aid direct medical professionals in the diagnosis, treatment, and administration of immunotherapeutic agents in the broadest sense.

[0120] While various exemplary embodiments have been described in detail with specific reference to certain illustrative aspects thereof, it should be understood that the present disclosure may enable other embodiments and that its details may enable modifications in various obvious respects. As will be readily apparent to those skilled in the art, variations and modifications can be made while still remaining within the spirit and scope of the present disclosure. Accordingly, the foregoing disclosure, description, and figures are for illustrative purposes only and do not in any way limit the disclosure, which is defined solely by the claims.

[0121] As discussed in Section F, we explored the effect of IL-12-loaded PLGA nanospheres on metastasis in an immunocompetent K7M2 orthotopic mouse model of osteosarcoma. We observed that nIL-12-treated mice exhibited significantly better outcomes than untreated mice, regardless of dose. Furthermore, a reduced rate of metastasis was linked to an increased percentage of NK cells in the peripheral blood at the time of amputation. The observation that specific components of the blood immune phenotype (NK cell percentage >8.21%) can potentially distinguish complete responders from non-responders at such an early clinical time point (amputation) has clear clinical relevance. This provides a solid foundation for the generation of well-informed hypotheses. Indeed, toxicity studies of free rmIL-12 focused on IL-12-induced toxicity specific to BALB / c mice, which could be investigated in conjunction with nIL-12 treatment, i.e., TCE status, PMN-MDSC myelocytosis, and NK cell depletion. This established that free rmIL-12 induces discernible changes in the systemic immune phenotype of BALB / c mice. Furthermore, nIL-12 treatment may be effective in metastatic OS tumors and may reduce the chance of disease recurrence. Furthermore, monitoring the NK cell percentage in peripheral blood can provide information on response to nIL-12 and outcome. This data supports the use of systemic immune phenotyping and experimental immunotherapy in tandem to generate effective immunotherapeutic regimens for metastatic OS.

[0122] While various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be used. [Example]

[0123] In the following examples, dichloromethane (DCM, #320269), PLGA (Resomer RG756S, 75% lactide, #719927), NaCl (#7647-14-5), and poly(vinyl alcohol) (#341584) were purchased from Sigma-Aldrich (St. Louis, MO). Fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC, #A23015), penicillin-streptomycin (Pen-Strep, 10,000 U / ml, #15140122), and Alexa Fluor® 647 carboxylic acid, tris(triethylammonium) salt were purchased from Thermofisher Scientific (Waltham, MA). Recombinant mouse IL-12 (p70, rmIL-12, #577008) and mouse IL-12 ELISA MAX deluxe ELISA kit (#433606) were purchased from Biolegend (San Diego, CA).

[0124] Gibco Fetal Bovine Serum Qualified Heat Inactivated US Origin (HI-FBS, #MT35011CV) and Dulbecco's Phosphate-Buffered Salt Solution 1X (DPBS, #21031CV) were purchased from Fisher Scientific (Pittsburgh, PA).

[0125] Female BALB / c mice (6–8 weeks old) (#000651) were purchased from Jackson Laboratory (Bar Harbor, ME).

[0126] Item A. IL-12 stability study. Example 1 Stability of IL-12 in acidic and basic solutions. To determine whether IL-12 can be handled in the acidic nanoenvironment of PLGA nanospheres, the recovery of bioactive recombinant murine IL-12 after exposure to solutions of various pH values ​​for 3 hours was tested. Specifically, IL-12 was incubated for 3 hours in solutions of pH 1, 3, 7.4, 11, and 13. The results are shown in Table 1. An enzyme-linked immunosorbent assay (ELISA) was used to determine the percentage of recovered active IL-12 relative to the initial concentration, thereby determining the amount of the native protein and, therefore, its bioactive conformation. [Table 1]

[0127] As can be seen in Table 1, IL-12 has good stability at pH values ​​between 7.4 and 11 and can undergo denaturation outside this range.

[0128] Example 2 Stability of IL-12 in organic solvents. The double emulsion method for synthesizing PLGA nanospheres can involve dissolving PLGA in an organic solvent. Therefore, the stability of IL-12 was tested in several aprotic solvents of varying polarity. The organic solvents tested were dichloromethane (DCM), ethyl acetate (EA), and acetone (AC), in order of increasing hydrophilicity. While DCM dissolves PLGA well, it is also the most hydrophobic and therefore may have the greatest effect on IL-12 bioactivity. Although PLGA may be poorly soluble in acetone, this solvent is the most polar and therefore may have the least effect on IL-12 bioactivity.

[0129] IL-12 was added to a 1:1 solution of solvent and phosphate buffered saline and stirred to completely remove the organic solvent. The remaining bioactive IL-12 concentration was then determined using ELISA and expressed as a percentage of the initial concentration. The results are shown in Table 2. Interestingly, AC had the most detrimental effect on the protein, recovering only 43% (SE = 1.06%), while EA showed the highest recovery of 63% (SE = 0.77%). DCM left approximately half of the protein in its original form (49%, SE = 0.55). [Table 2]

[0130] Unless otherwise indicated, DCM was used to generate nanosphere batches in further examples. Although using DCM as a solvent resulted in reduced protein recovery compared to ethyl acetate, DCM dissolves PLGA better and results in good nanosphere geometry.

[0131] Example 3 Stability of IL-12 upon sonication. To form an emulsion, IL-12 can be sonicated twice during the synthesis of PLGA nanospheres. Sonication during nanosphere preparation can result in uniform and smaller nanospheres. However, sensitive proteins such as IL-12 can become denatured when exposed to strong agitation.

[0132] Therefore, various sonication wattages and times were tested to determine the optimal conditions for synthesizing IL-12-loaded PLGA nanospheres. IL-12 was suspended in phosphate-buffered saline and sonicated at 30, 40, or 50 watts. Sonication times were 10, 20, 30, 40, or 60 seconds, and baselines were compared. The remaining bioactive IL-12 concentration after sonication was then determined using ELISA and expressed as a percentage of the initial concentration. As can be seen in Figure 3, IL-12 proved to be more sensitive to time than wattage, as protein recovery after 30 seconds was less than 10% for all wattages. Scanning electron microscope (SEM) images of various IL-12-loaded batches are shown in Figures 4A–4C. The boxes in Figure 3 indicate the time / wattage combinations that resulted in the majority of protein recovery, i.e., sonication combinations from 30 to 50 watts for 5 to 30 seconds.

[0133] Unless otherwise indicated, the nanospheres produced by sonication in the examples that follow were prepared using: Sonicate at 30 watts for 10-20 seconds Sonicate at 40 watts for 10-15 seconds, or Sonicate at 50 watts for 10-15 seconds.

[0134] Item B. PLGA nanospheres review. Example 4 Synthesis of PLGA nanospheres To prepare the oil phase, 800 mg of PLGA was dissolved in 32 ml of DCM at room temperature for 2 hours using a magnetic stir bar at 500 RPM.

[0135] To prepare the aqueous phase of the emulsion, 2400 mg of PVA and 96 mg of NaCl were dissolved in 120 ml of deionized water and microwaved in 10-second bursts on the HIGH setting in a standard kitchen microwave until clear. The aqueous phase was then cooled on ice.

[0136] The first emulsion (w1) was made by suspending the encapsulated material in 1.2 mL of DPBS, adding the resulting suspension to the oil phase, and stirring it for 6 minutes at 17,500 RPM using a tissue homogenizer. Stirring was performed on ice. As a control, blank particles were made using non-encapsulated material suspended in DPBS.

[0137] The second emulsion (w2) was formed by slowly pouring the first emulsion into 120 ml of aqueous phase. During the addition of the first emulsion, the aqueous phase was stirred at 17,500 RPM using a tissue homogenizer. After the addition of the first emulsion, stirring was continued for a total of 8 minutes. The resulting suspension was then stirred using a magnetic stir bar at 750 RPM for 16 hours to allow the organic solvent to evaporate.

[0138] Once the solvent had evaporated, the resulting solution was centrifuged three times at 3500 RPM. After each centrifugation step, the nanosphere pellet was recovered and resuspended, while the supernatant produced during centrifugation was collected and stored on ice. The nanospheres were then washed twice by ultracentrifugation at 20,000 RPM for 40 minutes at 4°C, flash-frozen in liquid nitrogen, and stored at -20°C.

[0139] The nanospheres can then be freeze-dried under vacuum to remove the water from the nanospheres.

[0140] The properties of the resulting nanospheres are summarized in Table 3. The nanospheres containing FITC-labeled BSA listed in Table 3 were imaged by confocal microscopy to visualize their internal structure. As shown in Figure 5A, Z-stack analysis confirmed that FITC-labeled BSA was successfully incorporated inside the nanospheres.

[0141] The geometries of both blank (Figures 4A-4C) and IL-12-loaded (Figures 5B and 5C) PLGA nanospheres were determined by scanning electron microscopy (SEM) to be spherical, with mean particle diameters of 201.7 ± 6.7 nm (Figure 6A) and 138.1 ± 10.8 nm (Figure 6B), respectively. The zeta potentials of both blank and loaded PLGA nanospheres were also determined, decreasing from -21.3 ± 0.808 mV (Figure 6C) to -15.1 ± 1.249 mV (Figure 6D), respectively.

[0142] Nanospheres containing Alexa647 dye and fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC) were also successfully prepared using the above procedure. [Table 3]

[0143] Example 5 Nanosphere IL-12 elution profile. PLGA nanospheres encapsulating IL-12 were obtained by the method described in Example 4. Three different concentrations of IL-12-loaded PLGA nanospheres (0.5 billion particles / mL, 0.75 billion particles / mL, and 1 billion particles / mL) were prepared in 500 μl of nanosphere release buffer (NRB, 10% HI-FBS and 100 units / mL Pen-Strep in DPBS). The suspension was then centrifuged at 4°C for 15 minutes to pellet the nanospheres, after which a 250 μl aliquot of supernatant was removed and stored at 4°C. Next, 250 μl of NRB was added to the pellet on ice to bring the total volume back to 500 μl, which was then resuspended and incubated at 37°C for 24 hours with constant agitation (750 RPM). This process was repeated for a total of 12 samples over 12 days (1 time point sample per 24 hours). Each aliquot was stored at 4°C for at least 24 hours to equilibrate with the release buffer before IL-12 concentration was determined by ELISA. Aliquots were analyzed by ELISA for the concentration of released bioactive IL-12 over time.

[0144] The total amount of eluted IL-12 was determined by ELISA followed by area under the curve (AUC) analysis as follows: At a nanosphere concentration of 500 million particles / mL, 1907.66 ± 162.00 pg 3329.77 ± 162.67 pg at a nanosphere concentration of 750 million particles / mL, and 3415.64 ± 848.94 pg at a nanosphere concentration of 1 billion particles / mL.

[0145] The amount of IL-12 eluted per 100,000 nanospheres was determined to be: At a nanosphere concentration of 500 million particles / mL, 0.3815 ± 0.03240 pg / 100,000 particles At a nanosphere concentration of 750 million particles / mL, the mean values ​​were 0.4440 ± 0.02169 pg / 100,000 particles and At a nanosphere concentration of 1 billion particles / mL, 0.3416 ± 0.08489 pg / 100,000 particles.

[0146] A 750 million particle / mL sample was reported to be the most effective dissolution rate of the three concentrations tested.

[0147] As can be seen in Figure 7A, the IL-12 concentration eluted as a function of time at a nanosphere concentration of 750 million particles / mL was significantly higher than that at a nanosphere concentration of 500 million particles / mL. However, there was little difference between the amount of IL-12 eluted as a function of time at a nanosphere concentration of 750 million particles / mL and that at a nanosphere concentration of 1 billion particles / mL. As can also be seen in Figure 7A, the IL-12 nanoparticles exhibited a biphasic elution profile, with a burst phase lasting approximately 2 days and a sustained-release phase lasting approximately from about days 3 to 11. Furthermore, the total amount of IL-12 eluted over time at a nanosphere concentration of 750 million particles / mL could be approximately 75% higher than that at a nanosphere concentration of 500 million particles / mL. However, the total amount of IL-12 eluted at a nanosphere concentration of 1 billion particles / mL can be only about 2.6% higher than the total amount of IL-12 eluted at a concentration of 750 million particles / mL. As can be seen in Figure 7B, the IL-12 concentration eluted as a function of time per 100,000 particles can be higher at a nanosphere concentration of 750 million particles / mL than at either the 500,000 particles / mL or 1 billion particles / mL concentrations.

[0148] As shown in Figure 7C, the encapsulation efficiency (EE) of nanospheres loaded with recombinant murine IL-12 (rmIL-12) was calculated using the area under the curve (AUC) of each elution profile for the three different particle concentrations tested using Equation 1. The amount of IL-12 eluted per 100,000 nanospheres was determined to be 0.44% at a nanosphere concentration of 0.50 billion particles / mL, 0.50% at a nanosphere concentration of 0.75 billion particles / mL, and 0.39% at a nanosphere concentration of 1 billion particles / mL. The average encapsulation efficiency (EE) was 0.443% ± 0.0551%. Due to the variable elution rates from the tested concentrations, the EE was reported for all three concentrations and then averaged to reflect the total EE.

[0149] Based on the above data, increasing the nanosphere concentration above 750 million particles / mL fails to significantly improve the drug elution profile. This may be due to decreased encapsulation efficiency at higher particle concentrations. However, this may be due to limited in vitro particle elution in small volumes (500 μL) of elution medium. Increasing the nanosphere concentration above 750 million particles / mL may improve the elution profile due to increased elution volumes, e.g., a larger volume of in vitro elution medium or an in vivo blood supply.

[0150] Example 6 Biodistribution of Alexa647-loaded PLGA nanosphere substrates in vivo. Female BALB / c mice were injected with PLGA nanospheres loaded with the fluorescent dye Alexa647, listed in Table 1. In one group of mice, the dye-loaded PLGA nanospheres were injected intravenously via the tail vein. In a second group of mice, the dye-loaded PLGA nanospheres were injected intraperitoneally. Dye distribution was monitored by IVIS imaging over a 76-minute period. Thirty-five minutes after injection, both routes of administration resulted in systemic distribution of the nanosphere contents, as illustrated in Figure 2. Intravenous injection resulted in general distribution of the labeled dye throughout the body of the subject mice. Intraperitoneal injection resulted in localized distribution of the labeled dye within the abdominal cavity of the subject mice, with the dye most concentrated in the peritoneal cavity. Distribution occurred without any signs of morbidity or mortality.

[0151] Item C. Investigation of PLGA nanospheres by ultrasonic treatment. Example 7 Synthesis of PLGA nanospheres by ultrasonication. To produce the oil phase, 250 mg of PLGA was dissolved in 1.51 ml of DCM at room temperature. 1 g of PVA was dissolved in 100 ml of deionized water to produce the aqueous phase. This aqueous phase was then cooled on ice. The first emulsion (w1) was made by suspending 12.5 micrograms of IL-12 in approximately 20 ± 6 microliters of DPBS and adding the resulting suspension to the oil phase while sonicating at 30-50 watts for 10-20 seconds.

[0152] The second emulsion (w2) was formed by slowly adding the first emulsion to 5 ml of aqueous phase. During the addition of the first emulsion, the aqueous phase was sonicated for 10-30 seconds at 30-50 watts. The resulting suspension was then stirred with a magnetic stir bar at 1,000 RPM for 3 hours to evaporate the organic solvent.

[0153] Once the solvent evaporated, the nanospheres were recovered from the resulting solution by three rounds of ultracentrifugation at 10,000 g for 15 min at 4°C, flash-frozen in liquid nitrogen, and stored at −20°C.

[0154] The nanospheres can then be freeze-dried under vacuum to remove the water from the nanospheres.

[0155] Figure 8A shows IL-12-loaded nanospheres prepared using sonication at a sonication power of 30 watts and sonication times ranging from 10 seconds to 20 seconds (batches 30W10S, 30W15S, and 30W20S in Table 2).

[0156] Figure 8B shows IL-12-loaded nanospheres prepared using sonication at a sonication power of 40 watts and sonication times ranging from 10 to 15 seconds (batches 40W10S and 40W15S in Table 2).

[0157] Figure 8C shows IL-12-loaded nanospheres prepared using sonication at a sonication power of 50 watts and sonication times ranging from 10 to 15 seconds (batch 50W10S and 50W15S in Table 2).

[0158] As can be seen in Table 4, sonication for 10-20 seconds at a power of 30-50 watts generally produces IL-12-loaded PLGA nanospheres with encapsulation efficiencies of about 5% to about 10% and zeta potentials between -30 and -40 mV. [Table 4]

[0159] Example 8 Dissolution of PLGA nanospheres prepared by ultrasonic treatment. The elution characteristics of the IL-12-loaded nanospheres shown in Table 2 were tested according to the procedure in Example 5. The daily protein concentration was determined by ELISA, and the total amount of released protein was determined using the raw area under the elution curve.

[0160] The dissolution curves obtained by the above procedure are shown in Figure 9. The dissolution curves for each batch in Table 2 show a biphasic curve with an initial burst phase and a sustained-release phase. The burst phase can be attributed to the release of protein adsorbed on the nanosphere surface upon resuspension in aqueous medium, while the controlled-release phase can be attributed to protein entrapped within the PLGA matrix. As can be seen in Figure 8, for a given sonication period, e.g., 10 seconds, the initial rate of drug release during the burst phase decreased with increasing sonication power. For batches sonicated at 30 W for 10 seconds (30W10S), between 60% and 65% of the IL-12 was dissolved within one day. For batches sonicated at 50 W for 10 seconds (50W10S), less than 50% of the IL-12 was dissolved within one day. This suggests that the percentage of protein within the PLGA matrix in the nanosphere core increases with increasing sonication power.

[0161] Item D. Effect of stirring speed on PLGA nanospheres. Example 9 To prepare the oil phase, 800 mg of PLGA was dissolved in 32 ml of DCM using a magnetic stir bar at 500 RPM for 2 hours at room temperature.

[0162] To prepare the aqueous phase of the emulsion, 2400 mg of PVA and 96 mg of NaCl were dissolved in 120 ml of deionized water and microwaved in 10-second bursts on the HIGH setting in a standard kitchen microwave until clear. The aqueous phase was then cooled on ice.

[0163] An IL-12 suspension was prepared by suspending 25 micrograms of IL-12 in 1.2 mL of DPBS.

[0164] Nanospheres were prepared as described in Example 4.

[0165] To observe the effect of stirring speed on the encapsulation efficiency of IL-12 in PLGA nanospheres and the elution profile of IL-12 from PLGA nanospheres, stirring during the preparation of the first and second emulsions was performed at speeds of 13,125 RPM; 15,312 RPM; 17,500 RPM; 19,688 RPM; 21,875 RPM; 24,063 RPM; and 26,250 RPM. The encapsulation efficiency as a function of stirring speed is shown in Table 5. [Table 5]

[0166] As can be seen in Figure 10, the elution profile of IL-12 from PLGA nanospheres prepared using high-speed stirring rather than sonication at a nanosphere concentration of 750 million particles / mL can be highly dependent on stirring speed. Stirring at 17,500 RPM results in high encapsulation efficiency and a total drug release of approximately 4500 pg of IL-12 over a 12-day period. Stirring at approximately 13,000 to approximately 20,000 RPM results in an acceptable drug release of approximately 3500 pg or more of IL-12 over a 12-day period. Stirring greater than approximately 22,000 RPM results in low encapsulation efficiency and a total drug release of less than 3,000 pg of IL-12 over a 12-day period. The optimal stirring speed for producing IL-12-loaded PLGA nanospheres can be approximately 17,500 RPM.

[0167] Item E. Effect of nanospheres on the release of soluble cytokines. Example 10 Release of free IL-12 versus IL-12 encapsulated in nanospheres. Osteosarcoma (OS) induces systemic immunosuppression in mice that can be reversed by the checkpoint blockade anti-PD-L1, as shown in Figure 11. Anti-PD-L1 treatment reverts the systemic immune phenotype to baseline, relieving immunosuppression from OS. However, the baseline state does not provide sufficient immune stimulation to effectively reduce tumor burden in mice with advanced OS (see line A in Figure 11). However, in a phase II study of rituximab / IL-12 combination therapy, in which IL-12 is administered as a free peptide, for relapsed and refractory non-Hodgkin's lymphoma (NHL), patients showed signs of dose-limiting toxicity (DLT). Their immune status or immune phenotype was pushed beyond the level of stimulation into the realm of a life-threatening condition of immune system overactivation known as immune cell exhaustion (see line C in Figure 11). The goal is to stimulate the immune system above baseline levels without overstimulation or immune cell exhaustion.

[0168] However, in the context of anti-PD-L1 checkpoint blockade resulting in disinhibition of activated T cells, slow and sustained delivery of low-dose IL-12 (generally considered safe) from hydrolysis of PLGA nanospheres (IL-12-NS) into the systemic tumor macroenvironment can provide systemic stimulation and effectively reduce tumor burden while still remaining below the exhaustion threshold, as shown in Figure 11, line B. Real-time immunophenotypic monitoring platforms, such as those described herein, are likely to be of great value for this application.

[0169] Thus, the use of biodegradable PLGA nanospheres as a delivery vehicle for soluble drugs can allow for the administration of therapeutically low doses, which can be useful when the difference between the effective and toxic doses of a drug is small.

[0170] Item E. Effect of nanosphere additives on the release of soluble cytokines. Example 11 Seven batches of nanospheres containing the drug IL-12 were prepared, and the dissolution profiles of these batches were obtained by sonication for 10 seconds at 50 watts power, following the procedure in Example 5.

[0171] The first batch was made by following the procedure of Example 7 using a protein solution made by suspending 12.5 micrograms of IL-12 in approximately 20±6 microliters of DPBS and PVA / aqueous phase. No additives not listed in Example 7 were included. As shown in Figure 12, upon dissolution of the first batch by the method of Example 5, there was an initial burst phase of drug release lasting 1-2 days, reaching a peak IL-12 concentration of 65,000 pg / mL on the second day.

[0172] A second batch was made by following the procedure of Example 7, except that a protein solution made by suspending 12.5 micrograms of IL-12 in approximately 20±6 microliters of DPBS containing 1.5% w / v trehalose was used. For the second batch, there was an initial burst phase of drug release that lasted for 3 days, reaching a peak IL-12 concentration of 75,000 pg / mL on day 3. As can be seen in Figure 12, the presence of trehalose in the protein solution used to make the nanospheres significantly increased the total drug release (measured as the area under the curve) compared to the drug release from nanospheres made without any additional additives.

[0173] A third batch was made by following the procedure of Example 7, except that a protein solution made by suspending 12.5 micrograms of IL-12 in approximately 20±6 microliters of DPBS containing 2% w / v Mg(OH) was used. Upon dissolution of the third batch by the method of Example 5, there was an initial burst phase of drug release lasting 1 day, resulting in a peak IL-12 concentration of 10,000 pg / mL. As can be seen in Figure 12, the presence of Mg(OH) in the protein solution used to make the nanospheres reduced the total drug release compared to the drug release from nanospheres made without any additional additives.

[0174] For the fourth batch, nanospheres were prepared by following the procedure of Example 7 using a protein solution made by suspending 12.5 micrograms of IL-12 in approximately 20±6 microliters of DPBS containing 10% fetal bovine serum (FBS). Upon elution by the method of Example 5, there was an initial burst phase of drug release lasting 3 days, reaching a peak IL-12 concentration of 115,000 pg / mL on day 2. As can be seen in Figure 13, the presence of 10% FBS in the protein solution significantly increased the total drug release (measured as the area under the curve) compared to the drug release from nanospheres prepared without any additional additives.

[0175] For the fifth batch, nanospheres were produced by following the procedure of Example 7 using a protein solution made by suspending 12.5 micrograms of IL-12 in approximately 20±6 microliters of DPBS containing 10% fetal bovine serum (FBS), which was incubated for 24 hours before nanosphere generation. Upon elution by the method of Example 5, there was an initial burst phase of drug release lasting 3 days, reaching peak IL-12 concentrations of -P105,000 pg / mL to -P110,000 pg / mL on day 3. As can be seen in Figure 13A, the presence of 10% incubated FBS in the protein solution significantly increased the total drug release (measured as the area under the curve) from the nanospheres compared to the drug release from either nanospheres made without additional additives or nanospheres made using FBS in the absence of an incubation step. As seen in the gel elution in Figure 13B, incubating the protein solution for 48 hours before nanosphere formation resulted in a decrease in the total release of IL-12 and a decrease in encapsulation efficiency.

[0176] For the sixth batch, the nanospheres were: a PVA / aqueous phase containing 4% w / v Tween® 80, and An oil phase containing 14% w / v Span® 60 The product was prepared by following the procedure in Example 7 using Upon elution using the method of Example 5, there was an initial burst phase lasting 1 day, with drug release at peak IL-12 concentrations ranging from -P120,000 pg / mL to -P125,000 pg / mL. As can be seen in Figure 14, the presence of surfactants Tween® 80 and Span® 60 in the protein solution increased the peak drug release from the nanospheres by approximately two-fold compared to the peak drug release from nanospheres made without any additional additives. The presence of Tween® 80 also substantially increased the total drug release.

[0177] For the seventh batch, the nanospheres were: A PVA / water phase containing 10% w / v FBS and 4% w / v Tween® 80; and an oil phase containing 14% w / v Span® 60. The product was prepared by following the procedure in Example 7 using Upon elution by the method of Example 5, there was an initial burst phase lasting 1 day, with drug release at a peak IL-12 concentration of approximately -580,000 pg / mL. As can be seen in Figure 14, the presence of both FBS and surfactant in the protein solution increased the peak drug release from the nanospheres by approximately 9-fold, compared to the peak drug release of -65,000 pg / mL from nanospheres made without additional additives. The presence of both FBS and Tween® 80 also increased the peak drug release from the nanospheres by approximately 9-fold. The peak drug release from the nanospheres was increased by approximately 4.5-fold compared to the peak drug release of -115,000 pg / mL from nanospheres made with FBS alone, and the peak drug release from the nanospheres was increased by approximately 4.5-fold compared to the peak drug release of -120,000 pg / mL from nanospheres made with Tween® 80 alone. [Table 6]

[0178] The peak drug release, total drug release, drug release / 100,000 particles, encapsulation efficiency (EE), and zeta potential for these modified nanosphere batches are recorded in Table 6. As shown in this table, the peak drug release and total drug release upon elution of the drug IL-12 from nanospheres can be increased by fabricating the nanospheres in the presence of an additive selected from the group consisting of trehalose, FBS, FBS with 24-hour incubation, surfactant, or a mixture thereof. Similarly, FBS, surfactant, or a mixture of FBS and surfactant with 24-hour incubation results in a dramatic increase in encapsulation efficiency. The peak drug release and total drug release upon elution can be synergistically increased by fabricating the nanospheres in the presence of both FBS and surfactant.

[0179] As can be seen in Figures 15A and 15B, batches of nanospheres in which the solution was sonicated for 10 seconds at 50 W (50W10S), FBS with no incubation ("baseline") (B), FBS with 24 hour incubation (24H), and FBS with 48 hour incubation (48H) were compared as elution percentages (Figure 15A) and percent of total elution (Figure 15B). The percent of total allows for an accurate comparison of the elution profiles for the modified batches. Values ​​for these figures can be found in Table 7. A comparison of the elution of 50W10S with the baseline and incubated batches is shown in Table 8. [Table 7] [Table 8]

[0180] Section F. Effect of IL-12-loaded poly(lactic-co-glycolic acid) (PLGA) nanospheres on metastasis and healing rates in an immunocompetent K7M2 orthotopic mouse model of osteosarcoma mouse Four- to five-week-old male and female BALB / c mice (stock number: 000651) were obtained from Jackson Laboratory and housed individually in ventilated Allentown cages in a specific pathogen-free facility on corncob bedding with a 12-hour light / dark cycle, automatic lixit water, and ad libitum food. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC).

[0181] Imaging of OS tumor-bearing mice using an in vivo imaging system (IVIS) Animals were imaged with an IVIS Spectrum CT imaging system (PerkinElmer Life Sciences, Waltham, MA) from the first sign of palpable primary tumor and at weekly intervals thereafter using Living Image version 4.5 software to monitor disease burden. In each session, mice received 150 mg / kg i.p. D-luciferin (Caliper Life Sciences, Hopkinton, MA) to visualize metastatic disease in the lungs, and mice received an additional 15 mg / kg intranasal D-luciferin (approximately 30 μL). Images were captured using autoexposure within a predetermined interval of maximum bioluminescence.

[0182] Flow cytometry (FC) Erythrocytes were lysed using Red Blood Cell (RBC) Lysis Solution (Miltenyi Biotec, Auburn, CA). Single cell suspensions were aliquoted and incubated with optimized lymphocyte (L) and myeloid (M) antibody panels according to the manufacturer's instructions. A minimum of 1 x 10 events was analyzed for each sample.

[0183] histology Histology was performed on the lungs of all nIL-12-treated mice that did not have IVIS-positive lung tumor metastases at the time of euthanasia. The metastasis rate in the untreated control group was assessed by a combination of histology and IVIS positivity. After euthanasia, lungs from nIL-12-treated mice were immediately harvested en bloc, placed in neutral buffered formalin, and mounted on a microtome. 50 μm sections were obtained every 250 μm, stained with hematoxylin and eosin, and analyzed by a certified pathologist for the presence or absence of tumor metastases.

[0184] statistical analysis To determine the significance between the categorized clinical data (metastasis rate and disease-free rate), a two-tailed chi-square test was used. To compare the NK population rate in peripheral blood between healthy and diseased nIL-12-treated mice, the mean values ​​of both groups were calculated at each time point, and the standard deviation (SD) was calculated. Statistical significance was determined by an unpaired two-tailed t-test.

[0185] Experimental design After bleeding 100 μL of whole blood from the cheek of 30 male BALB / c mice (red arrow), 1 × 10 6 luc-K7M2 OS tumor cells were inoculated intratibially. The first 12 mice that developed established primary tumors were re-bled in the cheek and subsequently randomized into three nIL-12 treatment groups (low dose: 0.1 mg, medium dose: 1 mg, or high dose: 10 mg) (n=4). After primary tumor confirmation by IVIS, the first dose of nIL-12 (blue arrow) was administered via intraperitoneal (ip) injection. Mice were imaged by IVIS and subsequently administered at weekly intervals, with consecutive cheek bleeds at weeks 5, 6, 8, 10, and 12. Immediately following cheek bleed at week 12, mice were euthanized. At week 5, the tumor-bearing limb was amputated.

[0186] In vivo induction of free IL-12 toxicity in healthy mice Two groups of four BALB / c mice (two females and two males per group) were cheek-bled by pricking the facial venous plexus with a 4 mm sterile Goldenrod Animal Lancet (Braintree Scientific, Braintree, MA) to collect 100 μL of whole blood for baseline immunophenotyping. Immediately after cheek bleeding, each group received either a low (100 ng) or high (100 μg) dose of free recombinant mouse (rm) IL-12 (eBiolegend, San Diego, CA) in 150 μL of DPBS containing 0.1% mouse serum albumin (MSA, Sigma, St. Louis, MO) via retroorbital (ro) injection. At 24 and 48 hours after ro injection, mice were further cheek-bled for immunophenotyping. nIL-12 synthesis nIL-12 nanospheres were prepared using a double emulsion solvent evaporation technique (33). Briefly, 150 μL of 83.3 mg / mL recombinant mouse IL-12 (rmIL-12, eBiolegend, San Diego, CA) containing 10% mouse serum albumin (MSA, Sigma, St. Louis, MO) in DPBS was added to 250 mg of PLGA resomer RG 503H (Sigma) dissolved in 1.51 mL of dichloromethane (Sigma) containing 14% w / w Span® 60 (Sigma). The resulting emulsion was sonicated for 10 s at 50 W on ice, then added to 5 mL of 1% w / v polyvinyl alcohol (PVA, Sigma) containing 4% w / v Tween® 80 (Sigma) and sonicated again on ice with the same parameters. The resulting mixture was stirred at room temperature for 3 hours and washed four times. The particles were stored at -80°C until use.

[0187] K7M2 syngeneic orthotopic BALB / c mouse model of metastatic OS K7M2 mouse ostomy tumor cells (ATCC CRL-2836, ATCC) were kindly donated by Dr. Kurt Weiss, MD (University of Pittsburgh Medical Center, Pittsburgh, PA) in April 2014 and were nonvirally transfected with the Promega luc2 reporter vector (luc-K7M2) as previously described (34). Luc-K7M2 cells were mycoplasma-free, and their identity was confirmed in 2018 by IDEXX BioResearch Case #6926-2018 (ID3). For orthotopic inoculation, luc-K7M2 cells (1 × 10) suspended in DMEM medium were used. 6 ) was injected intratibially into 30 mice as previously described (34). Four weeks later, 12 mice with the smallest primary tumor and those with primary tumors confirmed by IVIS to be mostly of similar size were randomized into three logarithmic dose treatment groups: 1) low dose (0.1 mg nIL-12), 2) medium dose (1 mg nIL-12), and 3) high dose (10 mg nIL-12). One week after primary tumors were confirmed by IVIS, mice underwent amputation of their tumor-bearing limbs as previously described (34). Twelve weeks after inoculation, mice were euthanized. A schematic of the overall experimental design can be seen in Figure 16.

[0188] Time points for cheek bleeding in OS tumor-bearing mice Prior to tumor cell inoculation (week 0), 30 male BALB / c mice were cheek-bled to collect 100 μL of whole blood for baseline immunophenotyping. Cheek bleeding was repeated at the first sign of palpable primary tumor (week 4), prior to subsequent amputation of the affected limb (week 5), and again at weeks 6 (T1), 8 (T2), 10 (T3), and 12 (euthanasia / EUTH), as shown in Figure 16.

[0189] nIL-12 treatment of OS tumor-bearing mice Twelve tumor-bearing mice were randomized into three n=4 dose groups; 0.1 mg (low), 1 mg (medium), or 10 mg (high) nIL-12 was suspended in 0.1% MSA in sterile DPBS. As shown in Figure 16, mice received the first dose of nIL-12 after palpable primary tumors were confirmed by IVIS, and then at weekly time points thereafter for a total of eight doses. Each nIL-12 dose was administered intraperitoneally (ip) in a total volume of 500 μL.

[0190] Experiment 12. rmIL-12-induced T cell exhaustion of helper T lymphocytes, cytotoxic T lymphocytes, and regulatory T lymphocyte subsets in peripheral blood Twelve BALB / c mice were cheek-bled for 100 μL of whole blood (baseline) and then administered 100 ng (L: low dose) or 100 μg (H: high dose) of recombinant murine (rm) IL-12 via retroorbital (ro) injection. Mice were cheek-bled again at 24 and 48 hours (48 h), and blood samples were immunophenotyped by flow cytometry. CD8 - CD4 + Helper T cells (left, white), CD4 - CD8 + Cytotoxic T cells (middle, gray) and CD8 - CD4 + FOXP3 + CD25 + / - PD1 on regulatory T cells (right, black) + TIM-3 + The degree to which β-actin becomes positive can be assessed and used as an indicator of exhaustion. T cell data from baseline and 48 hour time points are shown in Figure 17. Individual bars (top half) represent groups of means + standard deviations. The table (bottom half) shows groups of means ± standard deviations for each treatment group and T cell subset. Baseline blood samples (n = 4; 2 males and 2 females per group) were compared to the 48 hour samples using an unpaired, two-tailed t-test. * p<0.05, ** p<0.01, *** p<0.001.

[0191] Experiment 13. rmIL-12-induced polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) myeloid proliferation in peripheral blood Twelve BALB / c mice were cheek-bled for 100 μL of whole blood (baseline) followed by retroorbital (ro) injection of 100 ng (L: low dose) or 100 μg (H: high dose) of recombinant murine (rm) IL-12. Mice were cheek-bled again at 24 and 48 hours (48 h), and blood samples were immunophenotyped by flow cytometry. PMN-MDSC data from the baseline and 48 h time points are shown in Figure 18. Individual bars (top half) represent groups of mean values ​​+ standard deviations. Tables (bottom half) show groups of mean values ​​± standard deviations for each treatment group. Baseline blood samples (n = 4; 2 males and 2 females per group) were compared to the 48 h samples using an unpaired, two-tailed t-test. * p<0.05, ** p<0.01, *** p<0.001.

[0192] Experiment 14. IL-12-induced decrease in circulating natural killer (NK) cells Twelve BALB / c mice were cheek-bled for 100 μL of whole blood (baseline) followed by retroorbital (ro) injection of 100 ng (L: low dose) or 100 μg (H: high dose) of recombinant murine (rm) IL-12. Mice were cheek-bled again at 24 and 48 hours (48 h), and blood samples were immunophenotyped by flow cytometry. Figure 19 shows that individual bars (top half) represent groups of means + standard deviations. The table (bottom half) shows groups of means ± standard deviations for each treatment group. Baseline blood samples (n = 4; 2 males and 2 females per group) were compared to the 48 h samples using an unpaired, two-tailed t-test. * p<0.05, ** p<0.01, *** p<0.001.

[0193] Experiment 15. A percentage of blood NK cells greater than 8.21% at amputation separates non-metastatic and metastatic mice at 12 weeks. nIL-12-treated OS tumor-bearing mice were divided into two groups based on disease status at week 12, including n=8 diseased (metastasis and / or local recurrence positive) and n=4 healthy (both metastasis and local recurrence negative) mice. Figure 20 shows: (A) NKp46 in the peripheral blood of healthy (negative tumor burden at week 12, green circles) vs. diseased (positive tumor burden at week 12, red triangles) mice compared over 7 time points for 12 weeks. + Mean (± standard deviation) percentage of natural killer (NK) cells ( * p<0.05; ** , p<0.01; *** , p<0.001, using an unpaired two-tailed t-test), and (B) mice were stratified based on their NK cell percentage in peripheral blood at the time of amputation. Each bar represents the degree to which that individual mouse's NK cell percentage deviated from the median value (8.21%). A one-tailed chi-squared test was used to compare the metastasis rate of mice above the median with that of mice below the median; p met = P value for metastasis rate between groups with higher and lower NK cell percentages than the median at cutoff. In certain embodiments, for example, the following are provided: (Item 1) 1. A composition comprising poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres and a therapeutic agent, at least a portion of the therapeutic substance elutes from the composition more than 72 hours after placing the composition in solution; composition. (Item 2) 10. The composition of claim 1, wherein at least a portion of the therapeutic substance elutes from the composition more than 96 hours after placing the composition in solution. (Item 3) 10. The composition of claim 1, wherein at least a portion of the therapeutic substance elutes from the composition more than 120 hours after placing the composition in solution. (Item 4) 2. The composition of claim 1, wherein at least a portion of the therapeutic substance elutes from the composition between 72 and 288 hours after placing the composition in a solution. (Item 5) 2. The composition of claim 1, wherein the therapeutic agent is IL-12. (Item 6) 2. The composition of claim 1, wherein the solution is mammalian serum and about 100 units / mL of penicillin-streptomycin (Pen-Strep) in phosphate buffered saline (DPBS). (Item 7) 1. A composition comprising poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres and IL-12, IL-12 is incorporated into the PLGA nanospheres with an encapsulation efficiency of at least 2%. composition. (Item 8) 8. The composition of claim 7, wherein IL-12 is incorporated into the PLGA nanospheres with an encapsulation efficiency of at least 10%. (Item 9) 8. The composition of claim 7, wherein IL-12 is incorporated into the PLGA nanospheres with an encapsulation efficiency of at least 20%. (Item 10) 8. The composition of claim 7, wherein IL-12 is incorporated into the PLGA nanospheres with an encapsulation efficiency of at least 40%. (Item 11) 8. The composition of claim 7, wherein IL-12 is incorporated into the PLGA nanospheres at an encapsulation efficiency of about 2% to about 40%. (Item 12) 8. The composition of claim 7, wherein the IL-12 is bioactive IL-12. (Item 13) 1. A composition comprising a drug delivery vector and a therapeutic agent, the composition elutes at least 1.0 pg of the therapeutic substance per 100,000 particles of the drug delivery vector under drug delivery vector release buffer conditions; the composition continues to elute the therapeutic substance for more than 3 days; the therapeutic agent, the drug delivery vector, and the drug delivery vector release buffer constitute a solution; The solution is centrifuged and a portion is stored at about 1 to 10°C; Elution of the therapeutic agent is determined by an ELISA assay. composition. (Item 14) 14. The composition of claim 13, wherein the drug delivery vector comprises poly(D,L-lactic-co-glycolic acid) (PLGA). (Item 15) 14. The composition of claim 13, wherein the therapeutic agent is a protein. (Item 16) 15. The composition of item 14, wherein the protein is a cytokine. (Item 17) 17. The composition of item 16, wherein the cytokine is IL-12. (Item 18) Item 14. The composition of item 13, wherein the drug delivery vector release buffer comprises about 10% Fetal Bovine Serum Qualified Heat Inactivated (HI-FBS) and about 100 units / mL penicillin-streptomycin (Pen-Strep) in phosphate buffered saline (DPBS). (Item 19) 14. The composition of claim 13, further comprising a surfactant. (Item 20) 20. The composition of claim 19, wherein the surfactant comprises Tween® 80 and Span® 60. (Item 21) 14. The composition of claim 13, further comprising species-specific whole serum, species-specific engineered serum albumin, or species-specific whole fetal serum. (Item 22) Protein-loaded poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres, including diameters of approximately 100–1000 nm; surfactants, and Species-specific whole serum, engineered or native serum albumin A composition comprising: (Item 23) 23. The composition of claim 22, wherein the protein comprises IL-12. (Item 24) 23. The composition of claim 22, wherein the surfactant comprises Tween® 80 and Span® 60. (Item 25) 1. A method for making protein-encapsulated nanospheres, comprising: determining the rate of dissociation of said protein with increasing time and / or wattage of sonication; comparing the rate of protein dissociation with the rate of nanosphere formation with increasing time and / or wattage of sonication; determining the time and wattage of sonication at the intersection of the protein dissociation rate and the nanosphere formation rate; preparing a first phase by dissolving PLGA in a solvent containing a first surfactant; preparing a second phase by dissolving an alcohol in water containing a second surfactant and mammalian serum; suspending the components in an aqueous medium; forming a first emulsion comprising the aqueous medium and the first phase; forming a second emulsion comprising the first emulsion and the second phase, and sonicating the second emulsion for the time and sonication power determined at the intersection point; evaporating the solvent from the second emulsion to form an aqueous solution; recovering PLGA nanospheres containing the components from the aqueous solution; A method comprising: (Item 26) 1. A method for encapsulating components in nanospheres, comprising: preparing a first phase by dissolving poly(lactic-co-glycolic acid) (PLGA) in a solvent; preparing a second phase by dissolving an alcohol in water; suspending the components in an aqueous medium; forming a first emulsion comprising the aqueous medium and the first phase; forming a second emulsion comprising the first emulsion and the second phase; evaporating the solvent from the second emulsion to form an aqueous solution; recovering PLGA nanospheres containing the components from the aqueous solution; A method comprising: (Item 27) 27. The method of claim 26, wherein the component comprises a protein. (Item 28) 28. The method of claim 27, wherein the protein comprises IL-12. (Item 29) 28. The method of claim 27, wherein the components further comprise a surfactant and mammalian serum. (Item 30) The PLGA contains 50% to 90% lactide, the solvent is selected from the group consisting of halogenated C1-C3 organic solvents, C2-C3 nitrile solvents, C2-C5 alkyl ester solvents, C3-C5 ketone solvents, and mixtures thereof; Item 27. The method according to item 26. (Item 31) 31. The method of claim 30, wherein the solvent is acetonitrile, acetone, ethyl acetate or dichloromethane. (Item 32) Item 27. The method according to item 26, wherein the PLGA contains 75% to 90% lactide. (Item 33) The PLGA contains 50% to 75% lactide, The solvent is selected from the group consisting of halogenated C1-C3 organic solvents, acetonitrile, C3-C4 ketone solvents, and mixtures thereof. Item 27. The method according to item 26. (Item 34) adding the aqueous medium to the first phase to form the first emulsion, and stirring the first emulsion using a tissue homogenizer at a speed of 13,000 RPM to 20,000 RPM; and adding the first emulsion to the second phase to form the second emulsion; and agitating the second emulsion with a tissue homogenizer at a speed of 13,000 RPM to 20,000 RPM. Item 27. The method according to Item 26, comprising: (Item 35) adding the aqueous medium to the first phase to form the first emulsion, and agitating the first emulsion by ultrasonic treatment; and adding the first emulsion to the second phase to form the second emulsion, and agitating the second emulsion by sonication. Item 27. The method according to Item 26, comprising: (Item 36) agitating the first emulsion comprises sonicating at a power level of 30 W to 50 W for a period of 5 seconds to 30 seconds; agitating the second emulsion comprises sonicating at a power level of 30 W to 50 W for a period of 5 seconds to 30 seconds; Item 36. The method according to item 35. (Item 37) the step of stirring the first emulsion comprises ultrasonic treatment for a period of 10 seconds to 20 seconds; The step of stirring the second emulsion includes ultrasonic treatment for a period of 10 seconds to 20 seconds. Item 37. The method according to item 36. (Item 38) 28. The method of claim 27, wherein the protein is a cytokine or a globular protein. (Item 39) 39. The method of claim 38, wherein the protein is a cytokine selected from the group consisting of interleukins, lymphokines, monokines, interferons, colony-stimulating factors, and chemokines. (Item 40) 40. The method of claim 39, wherein the cytokine is selected from the group consisting of interleukins and non-immunological cytokines. (Item 41) Item 39. The method of item 38, wherein the protein is a cytokine having an N-terminal signal sequence, a four-helix bundle containing four helices labeled A to D, and no C-terminal extension after the D helix. (Item 42) 42. The method of claim 41, wherein the cytokine is granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interferon alpha-1, interferon beta, interferon gamma, interferon kappa, interferon tau-1, interferon omega-1, or the alpha chain of an interleukin selected from the group consisting of IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-12, IL-13, IL-15, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-26, and IL-27. (Item 43) The protein is a) a cell-mediated immune response, or b) antibody response 39. The method according to Item 38, wherein the immunological cytokine enhances either one of the above. (Item 44) 44. The method of claim 43, wherein the cytokine is an immunological cytokine that enhances a cellular immune response selected from the group consisting of TNFα, IFN-γ, and interleukin-12. (Item 45) 45. The method of item 44, wherein the cytokine is IL-12. (Item 46) 44. The method of claim 43, wherein the cytokine is an immunological cytokine that enhances antibody response selected from the group consisting of TGF-β, IL-4, IL-10, and IL-13. (Item 47) The first and second emulsions are each stirred using a tissue homogenizer at a speed of 13,000 RPM to 20,000 RPM; IL-12 is incorporated into the PLGA nanospheres with an encapsulation efficiency of about 0.5% to about 2.1%. Item 46. The method according to item 45. (Item 48) each of the first and second emulsions is stirred by ultrasonic treatment at a power level of 30 W to 50 W for a period of 5 seconds to 30 seconds; IL-12 is incorporated into the PLGA nanospheres at an encapsulation efficiency of about 4.5% to about 10%. Item 46. The method according to item 45. (Item 49) 27. Nanospheres produced by the method of claim 26, comprising a PLGA matrix and a protein, a first portion of the protein is adsorbed to the surface of the nanosphere; a second portion of the protein is incorporated into the PLGA matrix at the core of the nanosphere; Nanosphere. (Item 50) The protein is IL-12 The IL-12 is incorporated into the nanospheres at an encapsulation efficiency of about 0.5% to about 10%. Item 49. The nanosphere according to item 49. (Item 51) 27. The method of claim 26, wherein the second phase comprises polyvinyl alcohol and mammalian serum. (Item 52) the first phase contains a first surfactant, and / or the second phase contains a second surfactant; Item 51. The method according to item 51. (Item 53) the first surfactant is a sorbitan fatty acid ester, and / or The second surfactant is a polyoxyethylene sorbitan fatty acid ester. Item 53. The method according to item 52. (Item 54) 27. The method of claim 26, wherein the second phase contains polyvinyl alcohol and fetal serum. (Item 55) 54. Nanospheres produced by the method of claim 53, comprising a PLGA matrix and a protein, a first portion of the protein is adsorbed to the surface of the nanosphere; a second portion of the protein is incorporated into the PLGA matrix at the core of the nanosphere; the nanospheres comprise at least one additive selected from the group consisting of mammalian serum albumin, trehalose, the first surfactant, and the second surfactant; Nanosphere. (Item 56) 56. The nanosphere of claim 55, wherein the nanosphere comprises mammalian serum albumin and a surfactant. (Item 57) 27. A dosage form comprising a plurality of nanospheres produced by the method of claim 26, wherein each nanosphere comprises a PLGA matrix and a protein; a first portion of the protein is adsorbed to the surface of the nanosphere; a second portion of the protein is incorporated into the PLGA matrix at the core of the nanosphere; Dosage form. (Item 58) The protein is IL-12 the IL-12 is incorporated into the nanospheres with an encapsulation efficiency of at least 2%. The dosage form described in item 57. (Item 59) 1. A method for encapsulating a protein in a nanosphere, comprising: preparing an oil phase by dissolving 2.5% w / v to 17% w / v of poly(lactic-co-glycolic acid) (PLGA) in an organic solvent optionally containing a first surfactant; preparing an aqueous phase containing polyvinyl alcohol and at least one additive selected from the group consisting of mammalian serum, trehalose, and a second surfactant, and suspending the protein in the aqueous medium; adding the aqueous medium to the oil phase to form a first emulsion and stirring the first emulsion; adding the first emulsion to the aqueous phase to form a second emulsion and agitating the second emulsion; evaporating the organic solvent from the second emulsion to form an aqueous solution; recovering the protein-containing poly(lactic-co-glycolic acid) nanospheres from the aqueous solution; A method comprising: (Item 60) 1. A method of regulating the immune phenotype in a patient suffering from a disease affecting the immune system, comprising: a) determining the patient's initial immunophenotype; b) if the initial immune phenotype indicates immunosuppression, administering a first agent that stimulates the immune system; or if the initial immune phenotype indicates overstimulation of the immune system, administering a second drug that suppresses the immune system; c) monitoring the patient's immune phenotype as a function of time after step (b); and d) adjusting the administration of the first and / or second drug if the patient's immunophenotype is outside a desired range. A method comprising: (Item 61) 1. A method of regulating the immune phenotype in a patient suffering from a disease affecting the immune system, comprising: a) determining a disease status of a patient, said disease status including a diagnosis and an initial immunophenotype; b) comparing the disease state of the patient with a plurality of disease states in a database, each disease state in the database including a diagnosis, an initial immunophenotype, and a treatment protocol; c) selecting a treatment protocol from said database based on said comparing step (b), said treatment protocol comprising administering an immunomodulatory drug; and A method comprising: (Item 62) d) administering said immune modulatory agent to said patient; e) after step (d), monitoring the patient's immunophenotype as a function of time; f) adjusting the administration of said immunomodulatory agent if said patient's immunophenotype falls outside a desired range; Item 62. The method of item 61, further comprising:

Claims

1. 1. A method for producing nanospheres encapsulating a therapeutic protein, said method comprising: (i) i) PLGA, ii) an oil-soluble surfactant; iii) a solvent, and iv) A) a therapeutic protein, and B) Species-specific whole serum, species-specific engineered serum albumin, or species-specific native serum albumin a first aqueous phase comprising forming a first emulsion comprising: (ii) i) the first emulsion, and ii) A) an alcohol, and B) Water-soluble surfactant a second aqueous phase comprising forming a second emulsion comprising: (iii) evaporating the solvent from the second emulsion to form an aqueous solution; (iv) recovering the therapeutic protein-encapsulated PLGA nanospheres from the aqueous solution; A method comprising:

2. The method of claim 1, wherein the therapeutic protein comprises IL-12.

3. the PLGA comprises 50% to 90% lactide; The solvent is selected from the group consisting of halogenated C1-C3 organic solvents, C2-C3 nitrile solvents, C2-C5 alkyl ester solvents, C3-C5 ketone solvents, and mixtures thereof. The method of claim 1.

4. 4. The method of claim 3, wherein the solvent is acetonitrile, acetone, ethyl acetate, or dichloromethane.

5. The method of claim 1, wherein the PLGA comprises 75% to 90% lactide.

6. the PLGA comprises 50% to 75% lactide; the solvent is selected from the group consisting of halogenated C1-C3 organic solvents, acetonitrile, C3-C4 ketone solvents, and mixtures thereof; The method of claim 1.

7. agitating the first emulsion with a tissue homogenizer at a speed of 13,000 RPM to 20,000 RPM; and agitating the second emulsion with a tissue homogenizer at a speed of between 13,000 RPM and 20,000 RPM. The method of claim 1 , comprising:

8. agitating the first emulsion by ultrasonic treatment; and agitating the second emulsion by ultrasonic treatment The method of claim 1 , comprising:

9. agitating the first emulsion comprises sonicating at a power level of 30 W to 50 W for a period of 5 seconds to 30 seconds; agitating the second emulsion comprises sonicating at a power level of 30 W to 50 W for a period of 5 seconds to 30 seconds; The method of claim 8.

10. agitating the first emulsion comprises sonicating for a period of 10 seconds to 20 seconds; agitating the second emulsion comprises sonicating for a period of 10 seconds to 20 seconds; 10. The method of claim 9.

11. The method of claim 1 , wherein the therapeutic protein is a cytokine or a globular protein.

12. 12. The method of claim 11, wherein the therapeutic protein is a cytokine selected from the group consisting of interleukins, lymphokines, monokines, interferons, colony-stimulating factors, and chemokines.

13. 13. The method of claim 12, wherein the cytokine is selected from the group consisting of an interleukin and a non-immunological cytokine.

14. 12. The method of claim 11, wherein the therapeutic protein is a cytokine having an N-terminal signal sequence, a four-helix bundle comprising four helices labeled A-D, and no C-terminal extension after the D helix.

15. The cytokine is granulocyte macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interferon alpha-1, interferon beta, interferon gamma, interferon kappa, interferon tau-1, interferon omega-1, or IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, alpha chain of IL-12, IL-13, IL-15, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-26 and IL-27; an interleukin selected from the group consisting of The method of claim 14, wherein

16. the therapeutic protein a) enhance the cell-mediated immune response, or b) Enhance antibody responses The method of claim 11, wherein the immunological cytokine is either one of the following:

17. 17. The method of claim 16, wherein the cytokine is an immunological cytokine that enhances a cellular immune response selected from the group consisting of TNFα, IFN-γ, and interleukin-12.

18. 17. The method of claim 16, wherein the cytokine is an immunological cytokine that enhances an antibody response selected from the group consisting of TGF-β, IL-4, IL-10, and IL-13.

19. each of the first and second emulsions is stirred using a tissue homogenizer at a speed of 13,000 RPM to 20,000 RPM; the therapeutic protein is incorporated into the PLGA nanospheres with an encapsulation efficiency of about 0.5% to about 2.1%; The method of claim 1.

20. each of the first and second emulsions is agitated by ultrasonic treatment at a power level of 30 W to 50 W for a period of 5 seconds to 30 seconds; the therapeutic protein is incorporated into the PLGA nanospheres with an encapsulation efficiency of about 4.5% to about 10%; The method of claim 1.

21. the oil-soluble surfactant is a sorbitan fatty acid ester, and / or The water-soluble surfactant is a polyoxyethylene sorbitan fatty acid ester. The method of claim 1.

22. 22. The method of claim 21, wherein the oil-soluble surfactant comprises sorbitan monostearate and the water-soluble surfactant comprises polyoxyethylene sorbitan monooleate.

23. A composition comprising: poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres loaded with a therapeutic protein, the nanospheres comprising the therapeutic protein; an oil-soluble surfactant; a water-soluble surfactant; and species-specific whole serum, species-specific engineered serum albumin, or species-specific native serum albumin.

24. 24. The composition of claim 23, wherein the therapeutic protein comprises a cytokine or a globular protein.

25. 25. The composition of claim 24, wherein the cytokine is a cytokine selected from the group consisting of interleukins, lymphokines, monokines, interferons, colony-stimulating factors, and chemokines.

26. 26. The composition of claim 25, wherein the cytokine is selected from the group consisting of an interleukin and a non-immunological cytokine.

27. 25. The composition of claim 24, wherein the cytokine has an N-terminal signal sequence, a four-helix bundle comprising four helices labeled A to D, and no C-terminal extension after the D helix.

28. The cytokine is granulocyte macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interferon alpha-1, interferon beta, interferon gamma, interferon kappa, interferon tau-1, interferon omega-1, or IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, alpha chain of IL-12, IL-13, IL-15, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-26 and IL-27; an interleukin selected from the group consisting of 28. The composition of claim 27, wherein:

29. 24. The composition of claim 23, wherein the therapeutic protein comprises IL-12.

30. 24. The composition of claim 23, wherein the oil-soluble surfactant comprises a sorbitan fatty acid ester.

31. 24. The composition of claim 23, wherein the water-soluble surfactant comprises a polyoxyethylene sorbitan fatty acid ester.

32. 31. The composition of claim 30, wherein the sorbitan fatty acid ester comprises sorbitan monostearate.

33. 32. The composition of claim 31, wherein the polyoxyethylene sorbitan fatty acid ester comprises polyoxyethylene sorbitan monooleate.

34. 24. The composition of claim 23, wherein the composition elutes at least 1.0 pg of the therapeutic protein per 100,000 nanospheres.

35. The composition of claim 23, further comprising an alcohol.

36. 1. A method for making nanospheres encapsulating a therapeutic protein, comprising: determining the rate of dissociation of said therapeutic protein with increasing time and / or wattage of sonication; comparing the rate of dissociation of the therapeutic protein with the rate of formation of the nanospheres with increasing time and / or wattage of sonication; determining the time and wattage of sonication at the intersection of the dissociation rate of the therapeutic protein and the formation rate of the nanospheres; Preparing a first phase by dissolving PLGA in a solvent containing an oil-soluble surfactant; preparing a second phase by dissolving an alcohol in water containing a water-soluble surfactant and mammalian serum; suspending the components in an aqueous medium; forming a first emulsion comprising the aqueous medium and the first phase; forming a second emulsion comprising the first emulsion and the second phase, and sonicating the second emulsion for the time and sonication power determined at the intersection point; evaporating the solvent from the second emulsion to form an aqueous solution; recovering PLGA nanospheres containing the components from the aqueous solution; A method comprising:

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