Compositions and methods for low dose systemic drug delivery

Abstract

Provided herein are compositions comprising protein encapsulated nanoparticles, and methods of making said compositions. In an aspect, provided herein are compositions comprising cytokine encapsulated nanoparticles, and methods of making said compositions. Further provided herein are methods of monitoring an immunophenotype of a subject and associated immunodiagnostic assays and immunodiagnostic compositions.

Description

WSGR Ref. 59910-701.601COMPOSITIONS AND METHODS FOR LOW DOSE SYSTEMIC DRUG DELIVERYBACKGROUND

[0001] Immunostimulation is an important mechanism that 1) can prevent malignant cells from proliferating and / or forming metastases and 2) can clear viral and bacterial infections. Immunotherapy cancer treatment can involve monoclonal antibody blockade of specific immune regulatory checkpoints, including the Programmed Death-Ligand 1 (PD-L1) and Cytotoxic T-Lymphocyte- Associated Protein 4 (CTLA-4) axes.

[0002] Historically, the focus has been on the tumor microenvironment and the immune response. However, a systemic response can provide a lasting immunological response and cure disease.

[0003] Preclinical studies have shown that interleukins can induce antitumor responses against many malignancies. Interleukin- 12 (IL-12), an immunostimulatory cytokine with antitumor activity that is maximized when given systemically, can induce such antitumor responses. While high-dose IL-12 administration can have toxic side effects, low doses of IL-12 can be considered safe.

[0004] Poly(D, L-lactic acid-co-glycolic acid) (PLGA) drug delivery vectors are FDA approved and can elute a wide variety of substances as the polymer breaks down. For therapeutic purposes, encapsulating peptides, potentially including immunostimulatory interleukin proteins, within PLGA nanospheres can allow for systemic delivery and tissue deposition without the need for toxic loading doses. In order to be used in systemic settings, nanospheres can achieve safe and effective blood-borne travel through the macro- and micro-vasculature of an organism.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This application was made with government support under grant numbers P20GM121322 and P20GM109098 awarded by the National Institute of Health. The government has certain rights in this invention.SUMMARY

[0006] In one aspect, disclosed herein is a method of treating a disease in a subject in need thereof, the method comprising: administering a first dose of poly(D, L-lactic acid-co-glycolic acid)(PLGA) nanospheres encapsulating a first dose of IL-12 to a subject; performing an immunodiagnostic assay on the subject, wherein the immunodiagnostic assay comprises measuring an expression level of IFN-y, IL-10, CCL4 (MIP-ip), IFN-a, CXCL9 (MIG),WSGR Ref. 59910-701.601CXCL10 (IP- 10), TNF-a, IL-6, VEGF, IL-4, CCL3 (MIP-la), CCL2 (MCP-1), GM-CSF, or a combination thereof and comparing to the result of the immunodiagnostic assay to a first immunodiagnostic assay performed before the administering of a; and administering a second dose of poly(D, L-lactic acid-co-glycolic acid)(PLGA) nanospheres encapsulating a second dose IL- 12 to a subject, wherein the second dose of the nanospheres or the second dose of the IL- 12 is based on the results of b, and wherein the second dose of the nanospheres or the second dose of the IL- 12 is the same as the first dose of the nanospheres or IL- 12 or different than the first dose of the nanospheres or IL-12. In some embodiments, the first dose of the nanospheres is from about O. OOlmg to about 0.099mg. In some embodiments, the first dose of the nanospheres is O. OOlmg. In some embodiments, the first dose of the IL-12 is from about 0.16ng / kg / day to about 16 ng / kg / day. In some embodiments, the first dose of the IL-12 is 0.16ng / kg / day. In some embodiments, the second dose of the nanospheres or the second dose of the IL-12 is increased or decreased relative to the first dose of the nanospheres or the first dose of the IL- 12 if the expression level of one or more of fFN-y, IL-10, CCL4 (MfP-ip), IFN-a, CXCL9 (MIG), CXCL10 (IP- 10), TNF-a, IL-6, VEGF, IL-4, CCL3 (MIP-la), CCL2 (MCP-1), or GM-CSF as measured by the immunodiagnostic assay increases or decreases relative to the first immunodiagnostic assay. In some embodiments, the second dose of the nanospheres or the IL-12 is maintained relative to the first dose of the nanospheres or the first dose of the IL- 12 if the expression level of one or more of fFN-y, IL-10, CCL4 (MfP-ip), IFN-a, CXCL9 (MIG), CXCL10 (IP- 10), TNF-a, IL-6, VEGF, IL-4, CCL3 (MIP-la), CCL2 (MCP-1), or GM-CSF as measured by the immunodiagnostic assay increases decreases, or remains the same relative to the first immunodiagnostic assay. In some embodiments, the second dose of the nanospheres or the IL- 12 is increased relative to the first dose of the nanospheres or the first dose of the IL- 12 if the expression level of one or more of fFN-y, IL- 10, CCL4 (MfP-ip), IFN-a, CXCL9 (MIG), CXCL10 (IP- 10), TNF-a, IL-6, VEGF, IL-4, CCL3 (MIP-la), CCL2 (MCP-1), or GM-CSF as measured by the immunodiagnostic assay decreases relative to the first immunodiagnostic assay. In some embodiments, the immunodiagnostic assay or the first immunodiagnostic assay comprises measuring an expression level of IFN-y, IL-10, IL-6, TNF-a, or a combination thereof. In some embodiments, the immunodiagnostic assay or the first immunodiagnostic assay comprises measuring an expression level of CCL3 (MIP-la), CCL2 (MCP-1), CCL4 (MfP-ip), CXCL9 (MIG), CXCL10 (IP- 10), or a combination thereof. In some embodiments, the method of administering the nanospheres or the IL-12 comprises intravenous administration. In some embodiments, performing the immunodiagnostic assay or the first immunodiagnostic assay on the subject comprises collecting a sample from the subject. In some embodiments, the sample comprises blood. In some embodiments, the blood comprises peripheral blood. In someWSGR Ref. 59910-701.601embodiments, the sample comprises peripheral blood mononuclear cells. In some embodiments, the immunodiagnostic assay comprises spectral flow cytometry, cytokine analysis, chemokine analysis, RNA sequencing, gene set enrichment analysis (GSEA), histology, or a combination thereof. In some embodiments, he spectral flow cytometry comprises a panel of antibodies. In some embodiments, the antibodies comprise a fluorophore. In some embodiments, the antibody panel comprises a panel as disclosed in Table 9, or any combination thereof. In some embodiments, performing the immunodiagnostic assay on the sample comprises measuring one or more markers disclosed in Table 10, or any combination thereof. In some embodiments, the immunodiagnostic assay comprises measuring an expression level of Fcgrl, Gbp2, Gbp3, Gbp5, Gbp7, Irfl, Irf7, Irf9, Oasl2, Statl, Socsl, or a combination thereof. In some embodiments, the immunodiagnostic assay comprises measuring an expression level of Stat3, Ccl2, Cxcl9, CxcllO, Statl, Gbp2, Gbp3, Irfl, PD-L1, Lag3, Socs3, Itgam, or a combination thereof. In some embodiments, the immunodiagnostic assay comprises measuring an expression level of Itgam, Ly6cl, Ly6g, Nos2, Argl, and PD-L1 (CD274), CD4, CD8, Foxp3, CD 19, Ncrl, CTLA-4, Lag3, Havcr2, or a combination thereof. In some embodiments, the immunodiagnostic assay comprises measuring an expression level of II 12rb 1, Il 12rb2, Psme2, Anxa2, Stat4, or a combination thereof. In some embodiments, the immunodiagnostic assay comprises measuring an expression level of 1118, Icaml, Ccr5, Stat3, Cxcl2, Ptgs2, or a combination thereof. In some embodiments, the immunodiagnostic assay comprises measuring an expression level of Statl, Gbp2, Gpb3, Ifng, Cxcl9, CxcllO, Lag3, Socsl, Argl, Ly6g, Itgam, CD 19, or a combination thereof. In some embodiments, the immunodiagnostic assay comprises measuring an expression level of Statl, Gbp2, Gpb3, Irfl, Cxcl9, CxcllO, Ccl2, PD-L1, Argl, Itgam, CD4, Ncrl, or a combination thereof. In some embodiments, the immunodiagnostic assay comprises measuring an expression level of Alasl, Argl, Ccl2, Ccl3, Ccl4, CD19, PD-L1, CD4, CD8a, CTLA-4, CxcllO, Cxcl9, Eeflg, G6pd, Gapdh, Gbp2, Gbp3, Havcr2, Hprt, Ifng, 1110, Il 12rb 1, Il lb, I12rg, 116, Irfl, Itgam, Itgax, Lag3, Ly6g, Ncrl, Nos2, Polrlb, Polr2a, Rpll9, Sdha, Socsl 1, Socs3, Statl, Stat3, Stat4, or a combination thereof. In some embodiments, the immunodiagnostic assay comprises measuring an expression level of Abcbll, Abcb4, Abcc2, Abcc3, Apexl, Btg2, Casp3, Ccngl, Cd36, Cdknla, Cypla2, Cyplbl, Fasn, Fmol, Gadd45a, Gclc, Gpxl, Gsr, Hmoxl, Icaml, Lpl, Mt2, Nqol, Ppara, Rbl, Rbpl, Serpinel, Srebfl, Thrsp, Txnrdl, or a combination thereof. In some embodiments, the subject comprises a mammal.

[0007] In another aspect, disclosed herein is a method of administering a dose of poly(D, L-lactic acid-co-gly colic acid)(PLGA) nanospheres encapsulating IL- 12 to a subject wherein the dose of the nanospheres is from about O. OOlmg to about 0.099mg. In some embodiments, the dose of the nanospheres comprises O. OOlmg. In some embodiments, the subject has a disease. InWSGR Ref. 59910-701.601some embodiments, the disease comprises cancer. In some embodiments, the cancer comprises a tumor. In some embodiments, the tumor comprises a solid tumor. In some embodiments, the subject comprises a mammal.

[0008] In another aspect, disclosed herein is a method of administering a dose of IL-12 to a subject wherein the 11-12 is encapsulated in poly(D, L-lactic acid-co-glycolic acid)(PLGA) nanospheres and the dose of the IL-12 is from about 0.16ng / kg / day to about 16 ng / kg / day. In some embodiments, the dose of the IL-12 comprises 0.16ng / kg / day. In some embodiments, the subject has a disease. In some embodiments, the disease comprises cancer. In some embodiments, the cancer comprises a tumor. In some embodiments, the tumor comprises a solid tumor.

[0009] In another aspect, disclosed herein is a method treating a cancer in a subject in need thereof, the method comprising encapsulating IL12 in poly(D, L-lactic acid-co-glycolic acid)(PLGA) nanospheres and administering a dose of the nanospheres to the subject, wherein the dose of the nanospheres is from about 0.001 mg to about 0.099 mg. In some embodiments, the dose comprises about 0.001 mg of the nanosphere. In some embodiments, the administering comprises intravenous administration. In some embodiments, the cancer comprises a tumor. In some embodiments, the tumor comprises a solid tumor. In some embodiments, the subject comprises a mammal.

[0010] In another aspect, disclosed herein is a method of treating a cancer in a subject in need thereof, the method comprising encapsulating IL12 in poly(D, L-lactic acid-co-glycolic acid)(PLGA)nanospheres and administering a dose of the IL-12 to the subject, wherein the dose of the IL-12 is from about 0.16ng / kg / day to about 16 ng / kg / day. In some embodiments, the dose of the IL-12 comprises 0.16ng / kg / day. In some embodiments, the administering comprises intravenous administration. In some embodiments, the cancer comprises a tumor. In some embodiments, the tumor comprises a solid tumor. In some embodiments, the subject comprises a mammal.INCORPORATION BY REFERENCE

[0011] All publications, patents, and patent applications mentioned in this specification 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 publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.WSGR Ref. 59910-701.601BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The novel features of the 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 of which:

[0013] FIG. 1. FIGS. 1A and IB show distribution of fluorescein isothiocyanate-labeled bovine serum albumin protein in a PLGA nanosphere. FIG. 1C shows a biphasic protein elution curve from PLGA nanospheres, due to initial release of adsorbed protein on the surface of the nanospheres, followed by controlled release of protein entrapped with the PLGA nanospheres. FIG. ID shows the mechanism of biphasic protein release from PLGA nanospheres.

[0014] FIG. 2 shows fluorophore distribution over a 67-minute period in BALB / c mice inoculated with 1 mg / kg of Alexa Fluor® 647-loaded nanospheres dissolved in sterile saline by either intraperitoneal (mouse on left) or intravenous (mouse on right) injection.

[0015] FIG. 3 shows the effect of sonication on IL- 12, where IL- 12 was ultrasonicated at three different wattages for 10, 20, 30, 40, or 60 seconds.

[0016] FIG. 4. FIGS. 4 A and 4B show scanning electron microscopy (SEM) images of unloaded (blank) poly(lactide-co-glycolide) acid (PLGA) nanospheres lyophilized without 25 mM trehalose at 1 l,000X (FIG. 3a) and 13,000X (FIG. 3b), respectively. FIG. 4C shows scanning electron microscopy (SEM) images of unloaded (blank) PLGA nanospheres lyophilized with 25 mM trehalose at 5,000X magnification.

[0017] FIG. 5. FIG. 5 A shows PLGA nanospheres loaded with FITC-conjugated bovine serum albumin visualized for protein incorporation via confocal microscopy. FIGS. 5B and 5C show scanning electron microscopy (SEM) images of lyophilized recombinant mouse IL-12-loaded PLGA nanospheres at 8,000X and 25,000X magnification, respectively.

[0018] FIG. 6. FIGS. 6A and 6B show size distribution analyses of unloaded (blank) PLGA acid nanospheres and recombinant mouse IL-12 (IL-12)-loaded PLGA nanospheres run at 1:50 and 1:14 dilution factors in water at 25 degrees Celsius, respectively.

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

[0019] FIG. 7. FIGS. 7 A and 7B shows the estimated total amount of protein eluted over time from recombinant mouse IL-12-loaded PLGA nanospheres in terms of total protein (FIG. 7 A) and protein per 100,000 particles (FIG. 7B). FIG. 7C shows the encapsulation efficiency (EE) of recombinant mouse IL-12-loaded nanospheres calculated using the area under the curveWSGR Ref. 59910-701.601(AUCs) of each elution profile for three different particle concentrations (500 million particles / mL, 750 million particles / mL, and 1 billion particles / mL).

[0020] FIG. 8. FIGS. 8 A to 8C show IL-12-loaded nanospheres prepared using sonication under varying conditions of sonication power and sonication time.

[0021] FIG. 9 shows the elution profiles of IL-12 from nanospheres prepared using sonication under varying conditions of sonication power and sonication time.

[0022] FIG. 10 shows the elution profile of IL-12 from PLGA nanospheres prepared using high speed stirring, using a nanosphere concentration of 750 million particles / mL, as a function of stirring speed.

[0023] FIG. 11 shows the effect of trehalose and a magnesium compound on IL- 12 elution from PLGA nanospheres.

[0024] FIG. 12 shows the effect of fetal bovine serum (FBS) on IL- 12 elution from PLGA nanospheres.

[0025] FIG. 13 shows the effect of surfactants, alone or in combination with FBS, on IL- 12 elution from PLGA nanospheres.

[0026] FIG. 14. FIGS. 14A and 14B show the percent elution of the protein over time where the nanospheres were prepared under varying conditions and the elution as a percent of total elution, respectively.

[0027] FIG. 15. FIGS. 15A-B show an overview of the experimental design used in an IL- 12 loaded nanosphere toxicity study and FIGS. 15C-P show the results of spectral flow cytometry experiments measuring various markers following the experiments outlined in FIGS 15A-B. FIG. 15A shows an overview of the experimental design used in an IL-12 loaded nanosphere toxicity study. The resulting systemic immune response from various IL-12 treatment strategies in healthy 7-8-week male / female BALB / c mice was analyzed via seven serial blood sampling timepoints. Timepoints included baseline (Tl), 12-h (T2), day 4 (T3), day 8 (T4), day 11 (T5), day 15 (T6), and day 18 (T7), at which time mice were humanely euthanized for full necropsy following cardiac puncture. Experimental mice received either weekly (IL 12ns) or daily (MTD) injections at indicated timepoints. At day 18 (T7, euthanasia), peripheral blood was collected via cardiac puncture. This was followed by necropsy to harvest heart, liver, spleen, lungs, and kidneys for histopathological analysis. NanoString nCounter analysis with a custom pro-inflammatory panel was also performed on RNA isolated from the formalin-fixed, paraffin embedded liver, spleen, and lung specimens. FIG. 15B shows an overview of the experimental procedures used following the blood sampling outlined in FIG. 15 A. At each sampling timepoint, peripheral blood was collected via cheek bleed (~80 mL) for analysis via an immune diagnostic platform (IDP) consisting of PBMC spectral flow cytometric analysis, plasmaWSGR Ref. 59910-701.601cytokine / chemokine analysis, and bulk PBMC RNA-sequencing. FIGS. 15C-P show the results of spectral flow cytometry experiments measuring various markers following the experiments outlined in FIGS 15A-B. PBMCs isolated into single cell suspension at serial blood sampling timepoints [baseline (Tl), 12-h (T2), day 4 (T3, n = 7 for saline male), day 8 (T4, n = 7 for saline male), day 11 (T5, n = 7 for saline male and 10 mg female), day 15 (T6, n = 7 for saline male and 10 mg female), and day 18 (T7, n = 7 for saline male and 10 mg female)] were analyzed by spectral flow cytometry using the appended gating strategy (FIG. 28). Flow cytometric analysis at each timepoint (T), expressed as a percent (%) of all live cells, revealed immunological differences between experimental groups for both the neutrophil (FIGS. 15C-15I) and polymorphonuclear (PMN)-myeloid derived suppressor cell (MDSC) populations (FIGS. 15 J- 15P). Neutrophils (FIG. 15C) were analyzed for expression of iNOS (FIG. 15D), Arg-1 (FIG. 15E), PD-L1 (FIG. 15F), CD66b (FIG. 15G), CD80 (FIG. 15H), and MHC-II (FIG. 151). Polymorphonuclear myeloid derived suppressor cells (PMN-MDSC) (FIG. 15 J) were also analyzed for expression of iNOS (FIG. 15K), Arg-1 (FIG. 15L), PD-L1 (FIG. 15M), CD66b (FIG. 15N), CD80 (FIG. 150), and MHC-II (FIG. 15P) (male mice - left, female mice - right). Each data point represents the average percent (%) of all (live) cells for each cellular population. Unless indicated above, an n = 8 mice per group per sex were included in the analysis. Where visible, error bars represent standard error of the mean (SE). The x axis represents the timepoint of blood collection while the y axis indicates the % of all (live) cells.

[0028] FIG. 16. FIGS. 16A-R show the results of peripheral blood plasma and bulk peripheral blood mononuclear cell (PBMC) RNA-seq analyses at each timepoint of the IL- 12 loaded nanosphere toxicity study. Plasma was isolated at each serial blood sampling timepoint [baseline (Tl, n = 7 for MTD male), 12-h (T2), day 4 (T3, n = 7 for 10 mg IL12ns male, MTD male, saline male, 0.1 mg IL12ns female), day 8 (T4, n = 7 for 0.001 mg IL12ns male, saline male, 0.1 mg IL12ns female), day 11 (T5, n = 7 for saline male and 10 mg female), day 15 (T6, n = 7 for saline male and 10 mg female), and day 18 (T7, n = 6 for saline male, n = 7 for 10 mg female)]. Plasma cytokines including IFN- y (FIG. 16A), IL-10 (FIG. 16B), IL-6 (FIG. 16C), and tnf-a (FIG. 16D), as well as chemokines CCL2 (MCP-1) (FIG. 16E), CCL3 (MIP-la) (FIG. 16F), CCL4 (MIP-ip) (FIG. 16G), CXCL9 (MIG) (FIG. 16H), and CXCL10 (IP-10) (FIG. 161), were quantified (pg / mL) using the BioLegend LEGENDplex Cytokine Release Syndrome Panel (male mice - left, female mice - right). Bulk PBMC RNA-seq was also utilized to evaluate gene expression of Ifng (FIG. 16 J), 1110 (FIG. 16K), 116 (FIG. 16L), and Tnf (FIG.16M), Ccl2 (FIG. 16N), Ccl3 (FIG. 160), Ccl4 (FIG. 16P), Cxcl9 (FIG. 16Q), and CxcllO (FIG. 16R) (male mice - left, female mice - right). An n = 3 mice per group per sex were included in the bulk PBMC RNA-seq analysis at each timepoint. Where visible, error barsWSGR Ref. 59910-701.601represent standard error of the mean (SE). The x axis represents the timepoint of blood collection while the y axis indicates either concentration (pg / mL) or gene expression (reads per kilobase of exon model per million mapped - RPKM).

[0029] FIG. 17. FIGS. 17A-B show the results of Gene set enrichment analysis (GSEA) of peripheral blood plasma and bulk peripheral blood mononuclear cells for four Reactome gene sets including Interleukin- 12 signaling, Interferon gamma signaling, Interleukin- 10 signaling, and TNF signaling of mice included in the IL-12 loaded nanosphere toxicity study. FIG. 17A shows GSEA for male mice and FIG. 17B shows GSEA results for female mice. The normalized enrichment score (NES) and false discovery rate (FDR) are presented on each enrichment score (ES) graph.

[0030] FIG. 18. FIGS. 18A-K show the results of Core enrichment gene analysis of peripheral blood plasma and bulk peripheral blood mononuclear cells at each timepoint of the IL- 12 loaded nanosphere toxicity study for genes within the interferon gamma (IFNG) signaling Reactome. RPKMs for individual core enrichment genes within the interferon gamma (IFNG) signaling Reactome including Fcgrl (FIG. 18A), Gbp2 (FIG. 18B), Gbp3 (FIG. 18C), Gbp5 (FIG. 18D), Gbp7 (FIG. 18E), Irfl (FIG. 18F), Irf7 (FIG. 18G), Irf9 (FIG. 18H), Oasl2 (FIG. 181), Statl (FIG. 18J), and Socsl (FIG. 18K) were analyzed (male mice - left, female mice -right). An n = 3 mice per group per sex were included in the bulk PBMC RNA-seq analysis at each timepoint. Where visible, error bars represent standard error of the mean (SE). The x axis represents the timepoint of blood collection while the y axis indicates gene expression (reads per kilobase of exon model per million mapped - RPKM).

[0031] FIG. 19. FIGS 19A-H show histopathology results of mice following the IL-12 loaded nanosphere toxicity study, including histology of the liver, kidney, lung, heart, and spleen. FIGS. 19A-D show the results of male mice, and FIGS. 19E-H show the results of female mice. Representative H& E images (FIG. 19 A, FIG. 19E) highlight differences in portal (liver), cortical (kidney), interstitial (lung), endomyocardial (heart), and stromal (spleen) inflammation (left). Violin plots display the differences in total pathologic (TOTAL PATH, FIG.19B, FIG. 19F), total necrosis (FIG. 19C, FIG. 19G), and total inflammation (FIG. 19D, FIG. 19H) scores amongst experimental groups. Results from statistical analysis via a one-way ANOVA are presented (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).

[0032] FIG. 20. FIGS. 20A-L show the results of NanoString nCounter analysis for differential gene expression of murine liver specimens using a 42 gene panel on the NanoString nCounter following the IL-12 loaded nanosphere toxicity study. NanoString nCounter differential gene expression analysis of murine liver specimens are shown for Stat3 (FIG. 20 A), Ccl2 (FIG. 20B), Cxcl9 (FIG. 20C), CxcllO (FIG. 20D), Statl (FIG. 20E), Gbp2 (FIG. 20F),WSGR Ref. 59910-701.601Gbp3 (FIG. 20G), Irfl (FIG. 20H), PD-L1 (FIG. 201), Lag3 (FIG. 20 J), Socs3 (FIG. 20K), and Itgam (FIG. 20L) expression amongst experimental groups in comparison to saline control. Log2 normalized expressions for each experimental group (male mice - left, female mice -right), with an n = 3 mice per group per sex, were analyzed by NanoString nSolver Analysis Software 4.0 and graphed using GraphPad Prism9 (version 9.4.1). Benjamini-Yekutieli adjusted p values were then calculated for each experimental group in comparison to saline controls (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001) as determined by NanoString Advanced Analysis Software.

[0033] FIG. 21. FIGS. 21A-D show characterization of IL- 12 PLGA nanospheres (IL 12ns) used in the IL-12 loaded nanosphere toxicity study. FIG. 21 A depicts a representative scanning electron microscopy image of IL12ns. The Scale bar is 10 pm in length, with each bar representing 1 pm. FIG. 21B shows the results of an IL12ns elution profile study as determined by a 14-day gel elution study and analysis of eluted recombinant murine (rm)IL-12 by ELISA. The x-axis indicates the timepoint (days) of collection whereas the y-axis indicates recombinant murine IL-12 eluted (pg). The table represents the average (AVG - pg), standard error of the mean (SE - pg), and standard error of the mean expressed as a percent (SE - %) for each elution day and total 14-day elution. FIG 21C shows the Concentration (mg / mL), yield, and encapsulation efficiency (EE) of each IL12ns batch (n = 6) generated for the IL-12 toxicity study, presented as average (AVG) and standard error of the mean (SE). FIG. 2 ID shows results of Dynamic light scattering (DLS) analysis, including the Zeta average (Z-Ave - nm), poly dispersity index (PDI), Zeta potential (Zeta - mV), and pH of IL 12ns fabrications (n = 4).

[0034] FIG. 22. FIGS 22A-B show the appended gating strategy used for spectral flow cytometry analysis of Peripheral blood mononuclear cells (PBMCs) collected from subjects of the IL-12 loaded nanosphere toxicity study.

[0035] FIG. 23. FIGS. 23A-N show the results of bulk PBMC RNA-seq gene expression of both myeloid-associated Itgam (FIG. 23 A), Ly6cl (FIG. 23B), Ly6g (FIG. 23C), Nos2 (FIG. 23D), Argl (FIG. 23E), and PD-L1 (CD274) (FIG. 23F) and lymphoid-associated CD4 (FIG.23G), CD8 (FIG. 23H), Foxp3 (FIG. 231), CD19 (FIG. 23J), Ncrl (FIG. 23K), CTLA-4 (FIG.23L), Lag3 (FIG. 23M), and Havcr2 (FIG. 23N) (male mice - left, female mice - right). An n = 3 mice per group per sex were included in the bulk PBMC RNA-seq analysis at each timepoint. Where visible, error bars represent standard error of the mean (SE). The x-axis represents the timepoint of blood collection while the y-axis indicates gene expression (reads per kilobase of exon model per million mapped - RPKM).

[0036] FIG. 24. FIGS. 24A-K show the RPKMs for individual core enrichment genes within the Interleukin- 12 signaling Reactome including II 12rb 1 (FIG. 24 A), Il 12rb2 (FIG. 24B), Psme2WSGR Ref. 59910-701.601(FIG. 24C), Anxa2 (FIG. 24D), and Stat4 (FIG. 24E) were analyzed. RPKMs for individual core enrichment genes within the Interleukin- 10 signaling Reactome including 1118 (FIG. 24F), Icaml (FIG. 24G), Ccr5 (FIG. 24H), Stat3 (FIG. 241), Cxcl2 (FIG. 24 J), and Ptgs2 (FIG. 24K) were also analyzed (male mice - left, female mice - right). An n = 3 mice per group per sex were included in the bulk PBMC RNA-seq analysis at each timepoint. Where visible, error bars represent standard error of the mean (SE). The x-axis represents the timepoint of blood collection while the y-axis indicates gene expression (reads per kilobase of exon model per million mapped - RPKM).

[0037] FIG. 25. FIGS. 25A-L show the results of NanoString nCounter analysis for differential gene expression of murine spleens using a 42 gene panel on the NanoString nCounter following the IL-12 loaded nanosphere toxicity study. NanoString nCounter differential gene expression analysis of murine spleen specimens are shown for Statl (FIG. 25 A), Gbp2 (FIG. 25B), Gpb3 (FIG. 25C), Ifng FIG. 25 (D), Cxcl9 (FIG. 25E), CxcllO (FIG.25F), Lag3 (FIG. 25G), Socsl (FIG. 25H), Argl (FIG. 251), Ly6g (FIG. 25J), Itgam (FIG. 25K), and CD 19 (FIG. 25L) of euthanasia-harvested spleens. Log2 normalized expressions for each experimental group (male mice - left, female mice - right), with an n = 3 mice per group per sex, were analyzed by NanoString nSolver Analysis Software 4.0 and graphed using GraphPad Prism9 (version 9.4.1). Benjamini-Yekutieli adjusted p values were then calculated for each experimental group in comparison to saline controls (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001) as determined by NanoString Advanced Analysis Software.

[0038] FIG. 26. FIGS. 26A-L show the results of NanoString nCounter analysis for differential gene expression of murine Lungs using a 42 gene panel on the NanoString nCounter following the IL-12 loaded nanosphere toxicity study. NanoString nCounter differential gene expression analysis of murine lung specimens are shown for Statl (FIG. 26A), Gbp2 (FIG. 26B), Gpb3 (FIG. 26C), Irfl (FIG. 26D), Cxcl9 (FIG. 26E), CxcllO (FIG. 26F), Ccl2 (FIG. 26G), PD-L1 (FIG. 26H), Argl (FIG. 261), Itgam (FIG. 26J), CD4 (FIG. 26K), and Ncrl (FIG.26L) for euthanasia-harvested lungs. Log2 normalized expressions for each experimental group (male mice - left, female mice - right), with an n = 3 mice per group per sex, were analyzed by NanoString nSolver Analysis Software 4.0 and graphed using GraphPad Prism9 (version 9.4.1). Benjamini-Yekutieli adjusted p values were then calculated for each experimental group in comparison to saline controls (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001) as determined by NanoString Advanced Analysis Software.

[0039] FIG. 27. FIGS. 27A-E show additional histopathology results of mice following the IL-12 loaded nanosphere toxicity study, including histology of the liver at T7 following 0.001 mg IL12ns administration (FIG. 27 A), histology of the liver and heart at T7 following MTDWSGR Ref. 59910-701.601administration of bolus IL12 (FIG. 27B), histology of the liver and kidney at T7 following 0.1 mg IL12ns administration (FIG. 27C), histology of the liver and spleen at T7 following 10 mg IL12ns administration (FIG. 27D), and histology of the liver at T7 following MTD administration of bolus IL12 (FIG. 27E).

[0040] FIG. 28. FIGS. 28A-B show results depicting core enrichment genes identified from IFNG, IL12, and IL10 GSEA Reactome analyses. Bar graphs displaying the rank metric for all core enrichment genes as determined by GSEA at timepoint 3 (T3) are shown for males (FIG.28A) and females (FIG. 28B).DETAILED DESCRIPTION

[0041] As used in the specification and claims, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.

[0042] A “nanosphere,” or “nanoparticle” can be an ultrafine particle. Such a particle can be made from a variety of materials, including but not limited to Poly(D, L-lactic acid-co-glycolic acid) (PLGA).

[0043] The publication titled “Nanosphere pharmacodynamics improves safety of immunostimulatory cytokine therapy,” iScience. 2024 Jan 9;27(2):108836.doi: 10.1016 / j.isci.2024.108836, as well as all accompanying supplemental information is incorporated by reference herein. US patent publication number 20230070180 is incorporated by reference herein.

[0044] PLGA drug delivery vectors, e.g., PLGA nanospheres, are FDA approved and can elute a wide variety of substances as the polymer coating breaks down into Krebs cycle intermediates. Drug solubility, bioavailability, and stability can all be altered by the organic coating allowing for large shifts in the pharmacokinetic and pharmacodynamics properties of the encapsulated substrate. For tumor and infection therapeutic purposes, encapsulating IL-12 within PLGA nanospheres can allow for systemic delivery and tissue deposition without the need for toxic loading doses. The negatively charged surface can be repelled by the glycocalyx, and along with their smaller size allows for increased deposition in the interstitial space where they can elute their contents undisturbed. To date, however, the successful encapsulation of IL-12 within submicron scale PLGA particles has not been achieved. Nanospheres can provide safe and effective blood-borne travel through the microvasculature of an organism (capillaries can be approximately 4-9 microns in diameter) with minimal risk of forming emboli.

[0045] A PLGA nanosphere with an encapsulated protein can be created by: preparing an oil phase by dissolving from 2.5% w / v to 17% w / v of PLGA in an organic solvent; preparing anWSGR Ref. 59910-701.601aqueous phase by dissolving from 1% w / v to 3% w / v of polyvinyl alcohol in an aqueous solvent; suspending a protein in an aqueous medium; adding the aqueous medium to the oil phase to form a first emulsion, and homogenizing the first emulsion; adding the first emulsion to the 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 PLGA nanospheres containing the protein from the aqueous solution.I. PROTEINS

[0046] 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.

[0047] 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 following the D helix. The cytokine can lack a substantial C-terminal extension, and may be a granulocytemacrophage colony-stimulating factor, a granulocyte colony-stimulating factor, interferon alpha-1, interferon beta, interferon gamma interferon kappa, interferon tau-1, interferon omega- 1, or an interleukin (IL) selected from the group consisting of IL-2, IL-3, IL-4, IL-5, IL-5, IL-6, IL-7, IL-9, IL-10, IL- 11, the alpha chain of 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.

[0048] In various embodiments, the cytokine can be an immunological cytokine that either enhances cellular immune responses; or enhances antibody responses. If the cytokine is an immunological cytokine that enhances cellular immune responses, the cytokine can be selected from the group consisting of tumor necrosis factor-alpha (TNF-a), interferon-gamma (TFN-y), and interleukin- 12 (which can stimulate the production of IFN-y and TNF-a). If the cytokine is an immunological cytokine that enhances antibody responses, the cytokine can be selected from the group consisting of transforming growth factor beta (TGF-f3), IL-4, IL-10, and IL-13.

[0049] In various embodiments, the protein to be encapsulated in a nanosphere can be a globular protein. Suitable globular proteins can include serum albumin proteins; enzymes, e.g., esterases; hormones, e.g., insulin; and transporter proteins.

[0050] In various embodiments, PLGA nanospheres can be used to encapsulate pharmaceutical active ingredients; vitamins; nutraceutical active ingredients, e.g., phytochemicals; and organic dyes or contrast agents. PLGA nanospheres can be used for controlled release of a variety of drugs, including poorly soluble Class III and Class IV drugs.WSGR Ref. 59910-701.601A. Administration of IL12ns

[0051] In some embodiments, a nanosphere comprises a protein as disclosed herein. In some embodiments, a protein loaded nanosphere is administered to a subject. In some embodiments, an IL-12 loaded nanosphere is administered to a subject. In some embodiments, 0.001 mgs to 13 mgs of IL-12 loaded nanospheres are administered to a subject. In some embodiments, 0.001 mgs to 0.005 mgs, 0.001 mgs to 0.01 mgs, 0.001 mgs to 0.05 mgs, 0.001 mgs to 0.1 mgs, 0.001 mgs to 1 mg, 0.001 mgs to 2 mgs, 0.001 mgs to 5 mgs, 0.001 mgs to 10 mgs, 0.001 mgs to 11 mgs, 0.001 mgs to 12 mgs, 0.001 mgs to 13 mgs, 0.005 mgs to 0.01 mgs, 0.005 mgs to 0.05 mgs, 0.005 mgs to 0.1 mgs, 0.005 mgs to 1 mg, 0.005 mgs to 2 mgs, 0.005 mgs to 5 mgs, 0.005 mgs to 10 mgs, 0.005 mgs to 11 mgs, 0.005 mgs to 12 mgs, 0.005 mgs to 13 mgs, 0.01 mgs to 0.05 mgs, 0.01 mgs to 0.1 mgs, 0.01 mgs to 1 mg, 0.01 mgs to 2 mgs, 0.01 mgs to 5 mgs, 0.01 mgs to 10 mgs, 0.01 mgs to 11 mgs, 0.01 mgs to 12 mgs, 0.01 mgs to 13 mgs, 0.05 mgs to 0.1 mgs, 0.05 mgs to 1 mg, 0.05 mgs to 2 mgs, 0.05 mgs to 5 mgs, 0.05 mgs to 10 mgs, 0.05 mgs to 11 mgs, 0.05 mgs to 12 mgs, 0.05 mgs to 13 mgs, 0.1 mgs to 1 mg, 0.1 mgs to 2 mgs, 0.1 mgs to 5 mgs, 0.1 mgs to 10 mgs, 0.1 mgs to 11 mgs, 0.1 mgs to 12 mgs, 0.1 mgs to 13 mgs, 1 mg to 2 mgs, 1 mg to 5 mgs, 1 mg to 10 mgs, 1 mg to 11 mgs, 1 mg to 12 mgs, 1 mg to 13 mgs, 2 mgs to 5 mgs, 2 mgs to 10 mgs, 2 mgs to 11 mgs, 2 mgs to 12 mgs, 2 mgs to 13 mgs, 5 mgs to 10 mgs, 5 mgs to 11 mgs, 5 mgs to 12 mgs, 5 mgs to 13 mgs, 10 mgs to 11 mgs, 10 mgs to 12 mgs, 10 mgs to 13 mgs, 11 mgs to 12 mgs, 11 mgs to 13 mgs, or 12 mgs to 13 mgs of IL- 12 loaded nanospheres are administered to a subject. In some embodiments, 0.001 mgs, 0.005 mgs, 0.01 mgs, 0.05 mgs, 0.1 mgs, 1 mg, 2 mgs, 5 mgs, 10 mgs, 11 mgs, 12 mgs, or 13 mgs of IL-12 loaded nanospheres are administered to a subject. In some embodiments, at least 0.001 mgs, 0.005 mgs, 0.01 mgs, 0.05 mgs, 0.1 mgs, 1 mg, 2 mgs, 5 mgs, 10 mgs, 11 mgs, or 12 mgs of IL- 12 loaded nanospheres are administered to a subject.II. METHODS OF MAKING NANOPARTICLES

[0052] To create the oil phase, from 2.5% w / v to 17% w / v of PLGA can be dissolved in an organic solvent. The PLGA may contain from 50% to 90% lactide, from 65% to 90% lactide, or from 75% to 90% lactide. The organic solvent can be a halogenated C1-C3 organic solvent, e.g., di chloromethane, chloroform, or 1,1,1 -tri chloroethane; a C2-C3 nitrile solvent, e.g., acetonitrile or propionitrile; or a C2-C5 alkyl ester solvent, e.g. ethyl acetate or butyl acetate; or a C3 to C5 ketone solvent, e.g., 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 the organic solvent at room temperature (RT) with stirring, where the stirring can be at 300 to 600 RPM, 350 to 550 RPM, or 425 to 500 RPM.WSGR Ref. 59910-701.601

[0053] In various embodiments, the solvent selection can be made based on a lactide content in the PLGA. If the PLGA contains 75% to 90% lactide, the organic solvent can be a halogenated C1-C3 organic solvent, C2-C3 nitrile solvent, or a C2-C5 alkyl ester solvent, or a C3 to C5 ketone solvent. If the PLGA contains less than 75% lactide, the organic solvent can be a halogenated Cl- C3 organic solvent, acetonitrile, or a C3 to C4 ketone solvent.

[0054] To create the aqueous phase of the emulsion, from 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.

[0055] Next, a protein, e.g., IL-12 or bovine serum albumin, can 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 to 20,000 RPM; 12,000 to 19,500 RPM; 13,000 to 19,000 RPM; 15,000 to 18,000 RPM, or 16,000 to 17,500 RPM, to produce a first emulsion.Alternatively, the protein suspension can be added to the PLGA-containing oil phase with sonication to produce the first emulsion.

[0056] The first emulsion can be then added to the PVA aqueous phase with rapid stirring, e.g., 10,000 to 20,000 RPM; 12,000 to 19,500 RPM; 13,000 to 19,000 RPM; 15,000 to 18,000 RPM, or 16,000 to 17,500 RPM; with homogenization; or with sonication to produce a second emulsion. The organic solvent can be then evaporated from the second emulsion. PLGA nanoparticles containing the protein from the aqueous medium can be recovered by centrifugation, and flash- frozen in liquid nitrogen and / or lyophilized.

[0057] Without being bound by any theory, the use of sonication over time can increase formation of nanoparticles by increasing the number of nanoparticles, decreasing the size of said nanoparticles, and increasing the uniformity of the nanoparticles. Further, higher sonication wattage can also increase the formation of nanoparticles. Proteins, however, can dissociate when exposed to sonication over time, particularly at higher sonication wattage over longer periods of time. Thus, as disclosed here, the parameters of sonication wattage and time in the creation of protein loaded nanoparticles can be optimized for individual protein variations.

[0058] When the second emulsion can be formed, protein can become wound up in strands of the PLGA matrix polymer, which can coalesce and precipitate into a sphere as the organic solvent can be removed. During this process, protein can become both entrapped within the polymer matrix (Figure 1 A, where the protein can be fluorescein isothiocyanate-labeled bovine serum albumin [BSA-FITC]) and adsorbed to the outer surface (Figure IB), producing a characteristic biphasic elution curve shown in FIG. 1C. The burst phase, which can occur between baseline and two days, can be due to the adsorbed protein on the surface of theWSGR Ref. 59910-701.601nanospheres being released upon resuspension in aqueous medium (FIG. ID). The controlled release phase can be due to entrapped protein (FIG. ID), and the protein can be released slowly over time as the PLGA hydrolyzes.

[0091] For purposes of comparison, the above process can be carried out using a buffered saline solution instead of a protein suspension, and adding this saline solution to the PLGA-containing oil phase to produce the first emulsion. Upon adding this first emulsion to a PVA aqueous phase, protein-free blank nanoparticles can be produced.

[0059] Blank nanospheres and IL-12-loaded PLGA nanospheres can be synthesized using the above techniques. The morphology of blank and protein-loaded PLGA nanospheres can be determined via scanning electron microscopy to be spherical in shape with a mean particle diameter of 50 nm to 500 nm, 100 to 250 nm, 100 to 150 nm, or 175 to 225 nm. In various embodiments, IL-12-loaded PLGA nanospheres can have a diameter of 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 nm. Blank, i.e., protein-free nanospheres can have a diameter of 175 to 225 nm. Zeta potential.

[0060] The zeta potentials of both blank and IL-12 loaded PLGA nanospheres were also determined in a deionized water medium. The zeta potential can be the potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particle, and can range from -15 to -25 mV, respectively, following the introduction of 12.5 - 25 ug recombinant mouse IL-12 (rmIL-12) for protein loading during synthesis. As the magnitude of the zeta potential increases, the stability of the nanosphere dispersion can increase.B. Encapsulation efficiency

[0061] Protein can be eluted from the nanospheres in a nanosphere release buffer. The amount of IL- 12 (percent) encapsulated and released (encapsulation efficiency, EE) by the nanospheres can be estimated using the area under the curve (AUC) of each elution profile, the particle concentration (PC; particles / mL), the total volume of synthesized particles (V, mL), and the total mass of IL-12 (mg) added during synthesis via the following equation:

[0062]

[0063] Elution can be measured by dispersing from 200 million particles / mL to 100 billion parti cles / mL, from 300 million parti cles / mL to 50 billion parti cles / mL, from 400 million particles / mL to 20 billion particles / mL, or from 500 million particles / mL to 1 billion particles / mL of protein-loaded PLGA nanospheres in a nanosphere release buffer, and analyzing the resulting dispersion for protein concentrations released over time. The protein concentrationsWSGR Ref. 59910-701.601can be bioactive. In the case of IL-12, the total amount of rmIL-12 eluted from the aforementioned particle concentrations can be determined by area under the curve (AUC) analysis to be from 1500 to 4,000 pg. The amount of IL-12 eluted per 100,000 nanospheres can be determined to be 0.3 to 0.45 pg / 100,000 nanospheres, respectively, with the most efficient elution kinetics being obtained at a concentration of 750 million particles / mL and the least efficient elution kinetics being obtained at a concentration of 1 billion particles / mL for the particles made via the homogenization method. Based on equation (1), the average encapsulation efficiency (EE) was determined to range from 0.4% to 0.5%. The highest EE was obtained at a concentration of 750 million particles / mL.

[0064] To determine whether the synthesized nanospheres could indeed encapsulate protein and not merely adsorb it to the outer wall, PLGA nanospheres containing fluorescein isothiocyanate- labeled bovine serum albumin were synthesized. The resulted nanospheres were imaged via confocal microscopy to visualize the internal structure. Analysis confirmed that labeled BSA was successfully incorporated within the nanospheres.III. SYSTEMIC DISTRIBUTION

[0065] To determine whether the contents of PLGA nanospheres distribute systemically and without causing injury following various administration routes, PLGA nanospheres loaded with the fluorescent dye Alexa Fluor® 647 can be injected into female mice, either intravenously through the tail vein or intraperitoneally, and monitored via in vivo imaging systems. Both routes of administration can result in systemic distribution of nanosphere contents, as depicted in FIG. 2, without any signs of morbidity or mortality.IV. WATER-INSOLUBLE PAYLOADS

[0066] Similar techniques can be used to encapsulate a water-insoluble free base or salt of a drug, or a water-insoluble dye or contrast agent, in a PLGA nanosphere, where the term “waterinsoluble” means that the drug or salt is less soluble in water than in the organic solvent dissolving the PLGA polymer. Similar techniques can be used to encapsulate a soluble drug, dye, or contrast agent, where controlled release by a PLGA polymer is desired to provide a therapeutically safe and effective dose while avoiding toxic side effects from rapid initial release.

[0067] To create the oil phase, from 2.5% w / v to 17% w / v of PLGA can be dissolved in an organic solvent, e.g., a halogenated C1-C3 organic solvent; a C2-C3 nitrile solvent; a C2-C5 alkyl ester solvent; or a C3 to C5 ketone solvent. To create the aqueous phase of the emulsion, from 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.WSGR Ref. 59910-701.601

[0068] Next, a water-insoluble free base or salt of a drug, a water-insoluble dye, or a contrast agent, can be suspended in an aqueous medium, and the resulting protein suspension can be added to the PLGA-containing oil phase, which can be subjected to rapid stirring or sonication to produce a first emulsion.

[0069] The first emulsion can then be added to the PVA aqueous phase with rapid stirring or sonication to produce a second emulsion. The organic solvent can then be evaporated from the second emulsion. PLGA nanoparticles containing the drug, dye, or contrast agent from the aqueous medium can be recovered by centrifugation, and can be lyophilized.V. ADDITIVES

[0070] Various further modifications to the process for nanosphere preparation can increase encapsulation efficiency, and change the elution profile of the nanospheres. Preparation of nanospheres using a protein solution made by suspending 12.5 micrograms of IL-12 in 1.2 mL DPBS containing 1.5% w / v trehalose can produce nanospheres with delayed-release elution profile. The initial burst phase can be delayed to second day after the start of the elution study, but encapsulation efficiency can be decreased.

[0071] Preparation of nanospheres using a protein solution made by suspending 12.5 micrograms of IL-12 in 1.2 mL DPBS containing 2% w / v Mg(OH)2can produce nanospheres with decreased encapsulation efficiency.

[0072] Preparation of nanospheres using a protein solution made by suspending 12.5 micrograms of IL-12 in 1.2 mL DPBS containing 3% to 15%, 5% to 12%, 8% to 12%, or about 10% whole serum, serum albumin, fetal serum, or fetal serum albumin that is species specific to the patient can produce nanospheres with delayed release. The initial burst phase can be delayed to the second day after the start of the elution study, and encapsulation efficiency can be increased. If the IL-12 suspension is incubated with fetal serum for 24 hours prior to nanosphere preparation, the burst phase can be delayed to the third day after the start of the elution study. Further incubation with whole serum, serum albumin or fetal serum for 48 hours also prolongs the elution of the burst phase and increases encapsulation efficiency. For in vitro studies, any type of serum, serum albumin (including synthetically manufactured serum albumin), fetal serum, or fetal serum albumin can be used, e.g. fetal bovine serum or fetal murine serum.Alternatively, human serum can be used. For in vivo studies, where nanospheres can be administered to a human or non-human patient, selection of serum or serum albumin can be specific to the species to be treated. For treatment of cattle, nanospheres made by suspending IL-12 in DPBS containing fetal bovine serum can be used. For treatment of mice, nanospheres should be made with DPBS containing fetal murine serum. For treatment of humans,WSGR Ref. 59910-701.601nanospheres can be made with DPBS containing human serum. Administering nanospheres treated with serum or fetal serum from one species to a different species can cause graft vs. host disease. Also, if using whole serum or collected native human albumin, a cross match process can be performed for each patient receiving these products, as can be the case for any blood product. In some cases, at least a portion of the therapeutic substance (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 placement of the composition in a solution. In some cases, least a portion of the therapeutic substance (e.g., IL-12) can elute from the composition (e.g., nanospheres) about 24 hr to about 48 hr, about 48 hr to about 72 hr, about 72 hr to about 192 hr, about 72 hr to about 168 hr, about 96 hr to about 168 hr, about 120 hr to about 168 hr, about 144 hr to about 192 hr after placement of the composition in a solution.

[0073] The presence of surfactants during nanosphere preparation can increase encapsulation efficiency. The surfactants can be incorporated into the PLGA-containing oil phase, or into the PVA / water phase. Suitable surfactants include oil-soluble sorbitan fatty acid esters (Span 20, Span 40, Span 60, and Span 80, for example), and / or water-soluble polyoxyethylene sorbitan fatty acid esters (Tween 20, Tween 40, Tween 60, and Tween 80, for example). In various embodiments, nanospheres can be prepared with a PLGA-containing oil phase containing 4% to 20% w / w of a Span surfactant, a PVA / water phase containing 2% to 10% w / v of a Tween surfactant, or both. For example, the oil phase can contain 4% to 20% w / w Span 60, 10% to 16% w / w Span 60, or about 14% w / w Span 60; the PVA / water phase can 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 can contain 4% to 20% w / w Span 60 and the PVA / water phase can contain 2% to 10% w / v Tween 80.

[0074] The presence of both a 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 DPBS containing 10% fetal serum can produce nanospheres with an IL- 12 encapsulation efficiency of between 2% and 10%, between 4% and 8%, or between 5% and 7%. Preparation of nanospheres using a protein solution made by suspending 12.5 micrograms of IL- 12 in 1.2 mL DPBS containing 10% fetal serum which has been incubated in the DPBS for 24 hours can produce nanospheres with an IL- 12 encapsulation efficiency of between 10% and 50%, between 20% and 45%, or between 30% and 40%.Preparation of nanospheres using a protein solution made by suspending 12.5 micrograms of IL-12 in 1.2 mL DPBS containing both 10% fetal serum and a surfactant can produce nanospheres with an IL-12 encapsulation efficiency of between 50% and 95%, between 60% and 85%, or between 70% and 80%. In various embodiments, use of a protein solution containing a cytokine,WSGR Ref. 59910-701.601fetal serum, and a surfactant synergistically increases encapsulation efficiency of the cytokine in PLGA nanospheres.VI. IMMUNOPHENOTYPING

[0075] Various embodiments disclosed herein relate to a technique allowing a medical professional to systemically analyze the immune system of a patient from a blood draw or from two to three drops of blood obtained using a finger stick blood draw obtained using a lancet and Microtainer®. The blood draw can be done at home, in the office of a medical professional; in a clinic; or in a hospital.

[0076] In various embodiments, the blood draw can be done at home, in the office of a medical professional; in a clinic; or in a hospital. The blood draw can be analyzed at a testing site or a medical facility.

[0077] The blood sample can be analyzed for the level of an immunochemical naturally present in the body of the patient, and / or for the level of an immunomodulatory drug administered to the patient. The blood sample can be analyzed for the level of an immunochemical naturally present in the body of the patient, as a function of time, allowing a medical professional to observe the effect of a treatment regimen on the immunochemical levels. Blood draws can be taken and analyzed at regular intervals, allowing a medical professional to assess changes in the immune status, or immunophenotype, over the course of treatment.

[0078] If a patient undergoing treatment for a first disease which affects the immune system is, after the initiation of treatment, diagnosed with a second disease, i.e., a medical comorbidity, either a causative agent of the second disease or a symptom of the second disease can affect: the clinical outcome of treatment of the first disease, the change in the patient’s immunophenotype over time, the stage or level of the first disease, and / or the efficacy of treatment administered for the first disease.

[0079] Similarly, a change in disease stage or disease level of the first disease, e.g., progression of a cancer from stage 2 to stage 3, can affect the patient’s immunophenotype as a function of time, protocols for treatment of the first disease, and the clinical outcome of treatment of the first disease.

[0080] The database would thus include information on treatment of a variety of diseases with immunomodulating drugs, and allow predictions on how a particular patient presenting with that disease will respond to a given immunomodulating therapy.

[0081] Use of such a database allows a medical professional to predict the response of a patient’s immune system to a given disease state, and to predict changes in the patient’s immunophenotype over the course of a disease or treatment. The database will be useful in theWSGR Ref. 59910-701.601treatment, surveillance, and diagnosis of many diseases including cancer, autoimmune diseases, and infections. As immunomodulating agents become more common, such a database provides the ability to assess where the status of the immune system at a specific time in the pathogenesis of a disease, and to predict which immunomodulating treatments would be most effective in treating that disease.

[0082] Each blood sample obtained from a patient’s blood draw or finger stick blood draw can be analyzed for the patient’s immunophenotype at the time of the blood draw. The results of the analysis, together with information as to the patient’s disease state and current treatment, if any, can be compared to data present in the database to develop a treatment plan which is most likely to effectively modulate the patient’s immunophenotype. Blood draws can be taken and analyzed at regular intervals, allowing a medical professional to assess changes in the immune status, or immunophenotype, over the course of treatment. Data from the current patient can be added to the database, potentially improving assessments of other patients. This has massive commercial potential for analysis of a patient’s blood with complex immunophenotyping, whether the analysis is for cancer, infection, or autoimmune diseases. The database that can be created with cross referencing of diseases with immunophenotyping will be a powerful tool to treat patients in the future. The database will be a living database that generates constantly updating information on immunophenotyping and status of treatment. Immunophenotyping of a patient’s blood sample involves the ability to analyze and assess the data appropriately. This system will allow the appropriate dosing, treatment, and corrections to be made across several disease states. As the collection of data increases the growing database will also be able help direct medical professionals to diagnosis, treat, and dose immunotherapeutics in the broadest sense.

[0083] Although the various exemplary embodiments have been described in detail with particular reference to certain exemplary aspects thereof, it should be understood that the disclosure can be capable of other embodiments and its details can be capable of modifications in various obvious respects. As is readily apparent to those skilled in the art, variations and modifications can be affected while remaining within the spirit and scope of the 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 only by the claims.

[0084] In some embodiments, an immunophenotype is determined by measuring and / or analyzing one or more markers as disclosed in Table 10 or any combination thereof.WSGR Ref. 59910-701.601

[0085] Table 10. List of immunophenotypic markers or gene IDs, alternative names, and their descriptionsMarker / Gene ID DescriptionAbcbll ATP-binding cassette, sub-family B (MDR / TAP), member 11 Abcb4 ATP-binding cassette, sub-family B (MDR / TAP), member 4Abcc2 ATP-binding cassette, sub-family C (CFTR / MRP), member 2 Abcc3 ATP-binding cassette, sub-family C (CFTR / MRP), member 3Alasl 5°-aminolevulinate synthase 1Anxa2 Annexin A2Apexl APEX nuclease (multifunctional DNA repair enzyme) 1Arg-1 (Argl) Arginase 1B2M Beta-2-microglobulinBtg2 BTG family, member 2Casp3 Caspase 3, apoptosis-related cysteine peptidaseCCL2 (MCP-1) C-C motif chemokine ligand 2 (Monocyte Chemoattractant Protein- 1) CCL3 (MIP-la) C-C motif chemokine ligand 3 (Macrophage Inflammatory Protein- 1 Alpha) CCL4 (MIP-lb) C-C motif chemokine ligand 4 (Macrophage Inflammatory Protein- 1 Beta) CCL5 (RANTES) C-C Motif Chemokine Ligand 5Ccngl Cyclin G1CCR2 (CD 192) C-C Motif Chemokine Receptor 2CCR5 (CD195) Chemokine (C-C Motif) Receptor 5CD119 (IFNGR1) Interferon gamma receptor 1CDllb (Itgam) Integrin subunit alpha MCDllc (Itgax) Integrin subunit alpha XCD127 (IL-7Ra) Interleukin 7 receptor alphaCD132 (IL2RG) Interleukin-2 receptor subunit gammaCD152 (CTLA-4) Cytotoxic T-lymphocyte-associated protein 4CD159a (NKG2A) NK group 2, member ACD183 (CXCR3) C-X-C motif chemokine receptor 3CD19 B-Lymphocyte Surface Antigen B4CD223 (LAG3) Lymphocyte-activation gene 3CD226 (DNAM-1) DNAX Accessory Molecule- 1CD25 (IL2Ra) Interleukin-2 receptor alpha chainCD274 (PD-L1) Programmed death-ligand 1CD279 (PD-1) Programmed cell death protein 1CD335 (NKp46 orNcrl) Natural cytotoxicity triggering receptor 1Cd36 CD36 molecule (thrombospondin receptor)CD4 Cluster of differentiation 4CD66b (CEACAM8) Carcinoembryonic antigen-related cell adhesion molecule 8CD69 Cluster of Differentiation 69CD80 (B7-1) Cluster of Differentiation 80 (B7, type I)CD86 (B7-2) Cluster of Differentiation 86 (B7, type 2)CD8a Cluster of Differentiation 8aCdknla Cyclin-dependent kinase inhibitor 1A (p21, Cipl)WSGR Ref. 59910-701.601Csfl (M-CSF) Colony Stimulating Factor 1 (Macrophage Colony Stimulating Factor) CXCL10 (IP 10) C-X-C motif chemokine ligand 10 (Interferon gamma-induced protein 10) CXCL2 (MIP-2a) C-X-C motif chemokine ligand 2 (Macrophage Inflammatory Protein 2-Alpha) CXCL9 (MIG) C-X-C motif chemokine ligand 9 (Monokine induced by gamma interferon) Cypla2 Cytochrome P450, family 1, subfamily A, polypeptide 2Cyplbl Cytochrome P450, family 1, subfamily B, polypeptide 1Eeflg Eukaryotic translation elongation factor 1 gammaFasn Fatty acid synthaseFcer2a (CD23) Fc Epsilon Receptor IIFcgrl High affinity immunoglobulin gamma Fc receptorFmol Flavin containing monooxygenase 1FoxP3 Forkhead box P3Fprl Formyl peptide receptor 1G6pd Glucose-6-phosphate dehydrogenaseGadd45a Growth arrest and DNA-damage-inducible, alphaGapdh Glyceraldehyde-3-phosphate dehydrogenaseGBP2 Interferon-induced guanylate-binding protein 2GBP3 Interferon-induced guanylate-binding protein 3GBP4 Interferon-induced guanylate-binding protein 4GBP5 Interferon-induced guanylate-binding protein 5GBP6 Interferon-induced guanylate-binding protein 6GBP7 Interferon-induced guanylate-binding protein 7Gclc Glutamate-cysteine ligase, catalytic subunitGM-CSF Granulocyte-macrophage colony-stimulating factorGpxl Glutathione peroxidase 1Gsr Glutathione reductaseHavcr2 (Tim-3) Hepatitis A Virus Cellular Receptor 2 (T cell Immunoglobulin Mucin Domain 3) Hmoxl Heme oxygenase 1Hprt Hypoxanthine phosphoribosyltransferase 1Icaml Intercellular adhesion molecule 1Icaml (CD54) Intercellular Adhesion Molecule 1IFN-a Interferon alphaIFN-g (Ifng) Interferon gammaIL-10 (1110) Interleukin 10IL-4 Interleukin 4IL-6 (116) Interleukin 6I112rbl Interleukin 12 receptor subunit beta 1I112rb2 Interleukin 12 receptor subunit beta 2IL18 Interleukin 18Il lb Interleukin 1 BetaIllr2 (CD121b) Interleukin 1 Receptor Type 2Illrn Interleukin 1 Receptor AntagonistiNOS (Nos2) Inducible nitric oxide synthase (Nitric oxide synthase 2)IRF1 Interferon regulatory factor 1IRF4 Interferon regulatory factor 4WSGR Ref. 59910-701.601IRF7 Interferon regulatory factor 7Jakl Janus Kinase 1Ki67 (Mki67) Antigen KI-67 (Marker of Proliferation Ki -67)Lmnbl Lamin BlLox-1 Lectin-Like Oxidized Low-Density Lipoprotein Receptor 1Lpl Lipoprotein lipaseLy6C (Ly6cl) Lymphocyte antigen 6 complex (Lymphocyte antigen 6 complex, locus Cl Ly6G (Ly6g) Lymphocyte antigen 6 complex, locus GMHC-II Major histocompatibility complex class IIMt2 Metallothionein 2ANqol NAD(P)H dehydrogenase, quinone 1Oasl 2’-5’-Oligoadenylate Synthetase 1AOaslg 2’-5’-Oligoadenylate Synthetase 1GOasl2 2 -5 -Oligoadenylate synthetase-like 2Polrlb RNA polymerase I subunit BPolr2a RNA polymerase II subunit APpara Peroxisome proliferator-activated receptor alphaPsme2 Proteasome activator subunit 2Ptafr Platelet Activating Factor ReceptorPtgs2 (COX-2) Prostaglandin-Endoperoxide Synthase 2 (Cyclooxygenase 2)Rbl Retinoblastoma 1Rbpl Retinol binding protein 1, cellularRpll9 Ribosomal protein L19Sdha Succinate dehydrogenase complex flavoprotein subunit ASerpinel Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1Socsl Suppressor of cytokine signaling 1Socs3 Suppressor of cytokine signaling 3Srebfl Sterol regulatory element binding transcription factor 1Statl Signal transducer and activator of transcription 1Stat2 Signal transducer and activator of transcription 2Stat3 Signal transducer and activator of transcription 3Stat4 Signal transducer and activator of transcription 4Stat5a Signal transducer and activator of transcription 5AThrsp Thyroid Hormone ResponsiveTnf(TNF-a) Tumor necrosis factor (alpha)Tnfrsfla TNF Receptor Superfamily Member 1ATnfrsflb TNF Receptor Superfamily Member IBTxnrdl Thioredoxin reductase 1Tyk2 Tyrosine Kinase 2VEGF Vascular endothelial growth factorWSGR Ref. 59910-701.601VII. ADDITIONAL METHODSA. Immunodiagnostic Methods

[0086] In some aspects, disclosed herein are immunodiagnostic methods. In some embodiments, an immunodiagnostic method as disclosed herein is used in combination with a method of treatment or administration of a composition as disclosed herein. In some embodiments, an immunodiagnostic method comprises administering a nanosphere as disclosed herein to a subject, collecting a sample from the subject, and performing an immunodiagnostic assay on the sample. In some embodiments, an immunodiagnostic assay comprises spectral flow cytometry, cytokine analysis, chemokine analysis, RNA sequencing, gene set enrichment analysis (GSEA), histology, or a combination thereof.1) Sample

[0087] In some embodiments, a sample comprises blood. In some embodiments, a sample comprise PBMCs.

[0088] In some embodiments, a sample is collected from a subject. In some embodiments, a subject can be a patient, for example, a cancer patient or a patient suspected of having cancer or other diseases requiring immunosurveillance. The subject can be a mammal, e.g., a human, and can be male or female. The subject can be an animal. The subject can be a mouse.

[0089] The sample can be obtained from a subject by, for example, a health care provider, including a physician, physician assistant, nurse, veterinarian, dentist, chiropractor, paramedic, dermatologist, oncologist, gastroenterologist, orthopedist, or surgeon.

[0090] The sample can be a biologic sample. The sample can be a whole blood sample. The sample can be a whole peripheral blood sample. The sample can be bone marrow, solid tumor sample, cerebrospinal fluid, plasma, serum, or lymph.

[0091] The sample can be associated with information regarding the sample or subject. The information can include a description of the sample, e.g., the time the sample was taken, the date the sample was taken, a subject (e.g., patient) identification number, type of sample, or other properties of the sample. The sample can include information regarding the subject from whom the sample is taken. For example, this information can include height, weight, eye color, hair color, age, ethnicity, gender; clinical information, e.g., blood pressure, LDL cholesterol levels, HDL cholesterol levels, and triglyceride levels, heart rate; personal medical history, including cancer treatments already received; family medical history of the subject; and information on molecular markers.

[0092] The sample can be obtained by any technique used by those skilled in the art to obtain a blood sample. The sample can be obtained by removing a fluid through a needle, suchWSGR Ref. 59910-701.601as in venipuncture. The sample can be obtained by phlebotomy. The sample can be obtained by using a lancet to obtain a drop of blood from a finger of a subject. The sample can be collected in a container. For example, the container can be a K2E vacutainer. The container can be labelled with the information described provided herein. The container can comprise an additive. The additive can be, e.g., sodium citrate, a clot activator, a gel, sodium heparin, lithium heparin, EDTA (e.g., potassium EDTA), sodium fluoride, sodium oxalate, or potassium oxalate. In some cases, the container does not comprise an additive. The container can comprise an anticoagulant.

[0093] The volume of the sample can be less than, or about 20 mL, 15 mL, 10 mL, 9 mL, 8 mL, 7 mL, 6 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, 900 pL, 800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 150 pL, 100 pL, 75 pL, 50 pL, or 25 pL. The volume of the sample can be more than 20 mL, 15 mL, 10 mL, 9 mL, 8 mL, 7 mL, 6 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, 900 pL, 800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 150 pL, 100 pL, 75 pL, 50 pL, or 25 pL. The volume of the sample can be from about 50 pL to about 1.5 mL, from about 100 pL to about 5 mL, from about 100 pL to about 500 pL, from about 75 pL to about 200 pL, from about 1 mL to about 10 mL, or from about 5 mL to about 20 mL. In some cases, the sample can be a volume of less than 20mL. The volume of the sample can be the volume of the sample obtained from a subject or the volume of the sample used in an assay provided herein.

[0094] In some cases, the sample, e.g., a whole blood or peripheral blood sample, can be subjected to a processing step that removes or eliminates red blood cells in a way that generates a sample that contains a full population of immune cells with minimal cell damage. In some cases, the red blood cells are removed by lysis, e.g., by treating with red blood cell lysis buffer following the obtaining of the sample. The red blood cell removal or elimination can occur in a hospital, research laboratory, laboratory, and other locations similarly equipped to handle the processing of biologic samples. The red blood cell lysis buffer can be from Miltenyi. The lysis buffer can be an ammonium chloride (NH4C1) based solution (pH 7.5).

[0095] In some cases, the red blood cells are removed using a Ficoll gradient using, e.g., Sepmate, CPT, or Ficoll Paque reagent. The red blood cells can be removed, e.g., based on size or a cell surface marker. The red blood cells can be removed, e.g., by immunological based separation in which a red blood cell is contacted with an affinity ligand. The red blood cells can be removed, e.g., by density-based centrifugation, with or without hydrophilic polysaccharide addition. The red blood cells can be removed, e.g., by cell buoyancy based on contacting the red blood cells with buoyancy activated microbubbles. The red blood cells can be removed, e.g., byWSGR Ref. 59910-701.601methods including hydrodynamic filtration, dielectrophoresis, acoustic transduction, hydrogenperoxide-powered pumping, simple sedimentation, agglutination, or passive filtration.

[0096] In some cases, the health care provider can use a sample collection procedure to obtain the immune cells without subjecting the sample to separative polysaccharide gradients that remove polymorphonuclear cells and can induce damage of specific cellular epitopes.

[0097] A cell pellet can be obtained following removal or elimination of red blood cells. White blood cells can remain in the pellet.

[0098] The cell pellet can be washed one or more times to yield washed cells. The cell pellet can be washed, e.g., with a cell culture medium, e.g., by resuspension. The cell culture medium can be a natural medium or an artificial medium. The cell culture medium can be a liquid or a gel. The cell culture medium can comprise amino acids, vitamins, inorganic salts, glucose, hormones, or attachment factors. Natural cell culture media can include coagulant or clot-based media, e.g., plasma separated from heparinized blood, serum, or fibrinogen, tissue extracts, e.g., extracts of chicken embryos, liver, spleen, or bone marrow extracts, or biological fluids, e.g., plasma, serum, lymph, amniotic fluid, or pleural fluid. Artificial media can include serumcontaining media, serum-free media, xeno-free media, protein-free media, or chemically defined media. Artificial media can include, e.g., medium 199, CMRL1066, Basal medium Eagle (BME), Minimum essential medium, Eagle’s minimum essential medium (EMEM), Dulbecco’s modified minimum essential medium (DMEM), a-MEM, Iscove’s modified DMEM, NCTC109, Ham’s F-10, Ham’s F-12, Kaighn’s modified Ham’s F-12, RPMI 1640, MCDB202, MCDB301, MCDB153, MCDB110, MCDB402, MCDB170, MCDB131, DMEM / F-12, RPMI 1640 / DMEM / F-12, Waymouth’s MB752 / 1, Trowell's T-8, Leibovitz’s L-15, Fischer's Medium, Neurobasal medium, McCoy's 5A Medium, or any combination of these artificial media. The cell culture medium can comprise a serum. The serum can be bovine serum, fetal bovine serum, chicken serum, goat serum, horse serum, sheep serum, newborn calf serum, porcine serum, or rabbit serum. The percentage of the serum in the medium can be about 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, or 25%. The percentage of serum in the medium can be about 0.5% to about 5%, or about 1% to about 15%. The percentage of serum in the medium can be less than 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, or 25%. The percentage of serum in the medium can be more than 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, or 25%. The percentage of the serum in the medium in different washes can be different, e.g., if can be about 5% to about 15% (e.g., about 8%, 9%, 10%, 11%, 12%) in the first wash) and about 0.1% to about 5% in the subsequent washes (e.g., about 1%, about 1.5%, about 2%, about 2.5%).WSGR Ref. 59910-701.601

[0099] The one or more washes can occur at temperatures of about 4°C or about 4°C to about 10°C or at about 25°C. The cell pellet can be washed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The cell culture medium can be the same for each of the one or more washes. The cell culture medium can be different for each of the one or more washes. The washed cell pellet can be centrifuged following each wash.2) Spectral Flow Cytometry

[0100] In some embodiments, an immunodiagnostic assay comprises spectral flow cytometry, e.g., as outlined in Examples 18-19. Performing spectral flow cytometry can include placing fixed and stained cells into spectral flow cytometry compatible containers. The spectral flow cytometry compatible container can include a 96-well plate. Spectral flow cytometry can differ from conventional flow cytometry as it can use prisms to capture all the emitted light from laser excited fluorophores across a set of detectors, or an array of channels, rather than detecting emitted photons that are collected into individual detectors. This means that while conventional flow cytometry can effectively detect signals from specific fluorophores over defined wavelengths, spectral flow cytometry can instead collect the entire spectral profile of fluorophores from multiple lasers.

[0101] Spectral flow cytometry can enable the individual resolution of fluorophores with similar emission spectra, therefore, the number of markers possible in a multicolor panel can be greatly expanded to over 40+.

[0102] Spectral flow cytometry can include a panel of cell binding and cell labeling agents that comprises the panel of cell binding and cell labeling agents disclosed above. The panel of cell labeling agents can include a panel with emission spectra that do not overlap. The high-resolution of spectral flow cytometry combined with the emission spectra of the cell labeling agent panel can enable the detection of isolated immune cells. The detection of isolated immune cells can include detection of leukocytes, lymphocytes, monocytes, neutrophils, eosinophils, basophils, and dendritic cells.

[0103] In some embodiments, spectral flow cytometry comprises an antib ody / fluorophore panel. In some embodiments, an antib ody / fluorophore panel comprises a panel as shown in Table 9, or any combination thereof.WSGR Ref. 59910-701.601

[0104] TABLE 9: Spectral Flow Cytometry Antibody / Fluorophore Panel Antibodies Source IdentifierBUV395 Mouse Anti-Ki-67, Clone B56 BD Biosciences 564071; RRID: AB 2738577 BUV563 Hamster Anti-Mouse CD69, BD Biosciences 741234; RRID: AB 2870786 Clone Hl.2F3BUV737 Rat Anti-Mouse I-A / I-E, BD Biosciences 748845; RRID: AB 2873248 CloneM5 / 114.15.2 (also known asM5 / 114)BV480 Rat Anti-Mouse CD25, Clone BD Biosciences 566120; RRID: AB 2739522 PC61BB515 Rat Anti-Mouse CD223, Clone BD Biosciences 564672; RRID: AB 2738884 C9B7WBB700 Hamster Anti-Mouse CD279 BD Biosciences 566514; RRID: AB 2869777 (PD-1), Clone J43APC-R700 Hamster Anti-Mouse BD Biosciences 565778; RRID: AB 2739350 CD152, Clone UC 10-4F 10-11BB700 Rat Anti-Mouse CD86, Clone BD Biosciences 742120; RRID: AB 2871388 GL1BUV737 Rat Anti -Mouse CD19, Clone BD Biosciences 612781; RRID: AB 2870110 1D3BV605 Rat Anti-Mouse CD119, Clone BD Biosciences 745111; RRID: AB 2742716 GR20Brilliant Violet 421 anti-mouse CD366 BioLegend 119723; RRID: AB 2616908 (Tim-3) Antibody, Clone RMT3-23Brilliant Violet 750 anti-mouse CD4 BioLegend 100467; RRID: AB 2734150 Antibody, Clone GK1.5Spark Blue 550 anti-mouse CD8a BioLegend 100780; RRID: AB 2819773 Antibody, Clone 53-6.7PerCP anti-mouse CD19 Antibody, BioLegend 115532; RRID: AB 2072926 Clone 6D5PE / Dazzle 594 anti-mouse CD183 BioLegend 155914 (CXCR3)Antibody, Clone S18001APE / Cyanine7 anti-mouse CD226 BioLegend 128812; RRID: AB 2566629 (DNAM-l)Antibody, Clone 10E5Zombie NIR Fixable Viability Kit BioLegend 423105Brilliant Violet 785 anti-mouse BioLegend 124331; RRID: AB 2629659 CD274(B7-H1, PD-L1) Antibody,Clone 10F.9G2Brilliant Violet 711 anti-mouse Ly-6G BioLegend 127643; RRID: AB 2565971 Antibody, Clone 1A8Alexa Fluor 700 anti-mouse I-A / I-E BioLegend 107622; RRID: AB 493727 Antibody, Clone Clone M5 / 114.15.2Brilliant Violet 570anti-mouse Ly- BioLegend 128029; RRID: AB 10896061 6CAntibody, Clone HK1.4WSGR Ref. 59910-701.601PerCP anti-mouse CD11c Antibody, BioLegend 117326; RRID: AB 2129643 Clone N418CF594 CEACAM8 antibody, Biorbyt orb213728-CF594polyclonalCD80 Antibody, anti-mouse, PE-Vio Miltenyi Biotech 130-116-398; RRID: AB 2727516 770, REAfmty, Clone REA983Arginase 1 / ARGl / liver Arginase Novus NBP1-32731AF405Antibody [Alexa Fluor 405], polyclonalMouse NKG2A / CD159a APC- R& D Systems FAB6867A; RRID: AB 10972604 conjugatedAntibody, Clone 705829Invitrogen CD1 lb Monoclonal ThermoFisher Scientific 69-0112-82; RRID: AB 2637406 Antibody(Ml / 70), eFluor 506,eBioscienceInvitrogen CD127 Monoclonal ThermoFisher Scientific 64-1271-82; RRID: AB 2744868 Antibody(A7R34), Super Bright 645,eBioscienceInvitrogen CD335 (NKp46) ThermoFisher Scientific 46-3351-82; RRID: AB 1834441 Monoclonal Antibody (29A1.4),PerCP-eFluor 710, eBioscienceInvitrogen FOXP3 Monoclonal ThermoFisher Scientific 12-4771-82; RRID: AB 529580 Antibody(NRRF-30), PE, eBioscienceInvitrogen CD1 lb Monoclonal ThermoFisher Scientific 69-0112-82; RRID: AB 2637406 Antibody(Ml / 70), eFluor 506,eBioscienceInvitrogen iNOS Monoclonal ThermoFisher Scientific 53-5920-82; RRID: AB 2574423 Antibody (CXNFT), Alexa Fluor 488,eBioscience3) Additional Immunodiagnostic Assays

[0105] In some cases, an immunodiagnostic assay comprises one or more additional analyses. In some embodiments, the one or more additional analyses can be one or more of RNA sequencing, gene-set enrichment analysis, proteomics, or cytokine analysis, chemokine analysis, or RNA, DNA, or protein analysis, histology, or a combination thereof, e.g., as outlined in Examples 18-19.

[0106] In some embodiments, an immunodiagnostic assay comprises measuring an expression level of one or more cytokine, chemokines, or a combination thereof.

[0107] In some embodiments, an immunodiagnostic assay comprises measuring an expression level of any marker disclosed in Table 10, or any combination thereof.

[0108] In some embodiments, an immunodiagnostic assay comprises measuring an expression level of about 2 to about 24 of the markers disclosed in Table 10. In some embodiments, an immunodiagnostic assay comprises measuring an expression level of about 2WSGR Ref. 59910-701.601to about 4, about 2 to about 6, about 2 to about 8, about 2 to about 10, about 2 to about 12, about 2 to about 14, about 2 to about 16, about 2 to about 18, about 2 to about 20, about 2 to about 22, about 2 to about 24, about 4 to about 6, about 4 to about 8, about 4 to about 10, about 4 to about 12, about 4 to about 14, about 4 to about 16, about 4 to about 18, about 4 to about 20, about 4 to about 22, about 4 to about 24, about 6 to about 8, about 6 to about 10, about 6 to about 12, about 6 to about 14, about 6 to about 16, about 6 to about 18, about 6 to about 20, about 6 to about 22, about 6 to about 24, about 8 to about 10, about 8 to about 12, about 8 to about 14, about 8 to about 16, about 8 to about 18, about 8 to about 20, about 8 to about 22, about 8 to about 24, about 10 to about 12, about 10 to about 14, about 10 to about 16, about 10 to about 18, about 10 to about 20, about 10 to about 22, about 10 to about 24, about 12 to about 14, about 12 to about 16, about 12 to about 18, about 12 to about 20, about 12 to about 22, about 12 to about 24, about 14 to about 16, about 14 to about 18, about 14 to about 20, about 14 to about 22, about 14 to about 24, about 16 to about 18, about 16 to about 20, about 16 to about 22, about 16 to about 24, about 18 to about 20, about 18 to about 22, about 18 to about 24, about 20 to about 22, about 20 to about 24, or about 22 to about 24 of the markers disclosed in Table 10. In some embodiments, an immunodiagnostic assay comprises measuring an expression level of about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, about 20, about 22, or about 24 of the markers disclosed in Table 10. In some embodiments, an immunodiagnostic assay comprises measuring an expression level of at least about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, about 20, or about 22 of the markers disclosed in Table 10. In some embodiments, an immunodiagnostic assay comprises measuring an expression level of at most about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, about 20, about 22, or about 24 of the markers disclosed in Table 10.

[0109] In some embodiments, an immunodiagnostic assay comprises measuring an expression level of about 2 to about 116 of the markers disclosed in Table 10. In some embodiments, an immunodiagnostic assay comprises measuring an expression level of about 2 to about 116 of the markers disclosed in Table 10. In some embodiments, an immunodiagnostic assay comprises measuring an expression level of about 2, or about 116 of the markers disclosed in Table 10. In some embodiments, an immunodiagnostic assay comprises measuring an expression level of at least about 2 of the markers disclosed in Table 10.

[0110] In some embodiments, an immunodiagnostic assay comprises measuring an expression level of IFN-y, IL- 10, CCL4 (MIP-ip), IFN-a, CXCL9 (MIG), CXCL10 (IP- 10), TNF-a, IL-6, VEGF, IL-4, CCL3 (MIP-la), CCL2 (MCP-1), GM-CSF, or any combination thereof.WSGR Ref. 59910-701.601

[0111] In some embodiments, an immunodiagnostic assay comprises measuring an expression level of IFN-y, IL-10, IL-6, TNF-a or any combination thereof. In some embodiments, an immunodiagnostic assay comprises measuring an expression level of any 1, 2, 3, or 4 of IFN-y, IL- 10, IL-6, or TNF-a.

[0112] In some embodiments, an immunodiagnostic assay comprises measuring an expression level of any 1, 2, 3, 4, or 5 of CCL3 (MIP-la), CCL2 (MCP-1), CCL4 (MIP-ip), or CXCL9 (MIG), CXCL10 (IP- 10).

[0113] In some embodiments, an immunodiagnostic assay comprises measuring an expression level of any 1-11 of Fcgrl, Gbp2, Gbp3, Gbp5, Gbp7, Irfl, Irf7, Irf9, Oasl2, Statl, Socsl, or a combination thereof.

[0114] In some embodiments, an immunodiagnostic assay comprises measuring an expression level of Stat3, Ccl2, Cxcl9, CxcllO, Statl, Gbp2, Gbp3, Irfl, PD-L1, Lag3, Socs3, Itgam, or a combination thereof.

[0115] In some embodiments, an immunodiagnostic assay comprises measuring an expression level of Itgam, Ly6cl, Ly6g, Nos2, Argl, and PD-L1 (CD274), CD4, CD8, Foxp3, CD19, Ncrl, CTLA-4, Lag3, Havcr2, or a combination thereof.

[0116] In some embodiments, an immunodiagnostic assay comprises measuring an expression level of II 12rb 1, Il 12rb2, Psme2, Anxa2, Stat4, or a combination thereof.

[0117] In some embodiments, an immunodiagnostic assay comprises measuring an expression level of 1118, Icaml, Ccr5, Stat3, Cxcl2, Ptgs2, or a combination thereof.

[0118] In some embodiments, an immunodiagnostic assay comprises measuring an expression level of Statl, Gbp2, Gpb3, Ifing, Cxcl9, CxcllO, Lag3, Socsl, Argl, Ly6g, Itgam, CD 19, or a combination thereof.

[0119] In some embodiments, an immunodiagnostic assay comprises measuring an expression level of Statl, Gbp2, Gpb3, Irfl, Cxcl9, CxcllO, Ccl2, PD-L1, Argl, Itgam, CD4, Ncrl, or a combination thereof.

[0120] In some embodiments, an immunodiagnostic assay comprises measuring an expression level of Alas 1, Argl, Ccl2, Ccl3, Ccl4, CD 19, PD-L1, CD4, CD8a, CTLA-4, CxcllO, Cxcl9, Eeflg, G6pd, Gapdh, Gbp2, Gbp3, Havcr2, Hprt, Ifng, 1110, Il 12rb 1, Il lb, I12rg, 116, Irfl, Itgam, Itgax, Lag3, Ly6g, Ncrl, Nos2, Polrlb, Polr2a, Rpll9, Sdha, Socsl 1, Socs3, Statl, Stat3, Stat4, or a combination thereof.

[0121] In some embodiments, an immunodiagnostic assay comprises measuring an expression level of Abcbll, Abcb4, Abcc2, Abcc3, Apexl, Btg2, Casp3, Ccngl, Cd36, Cdknla, Cypla2, Cyplbl, Fasn, Fmol, Gadd45a, Gclc, Gpxl, Gsr, Hmoxl, Icaml, Lpl, Mt2, Nqol, Ppara, Rbl, Rbpl, Serpinel, Srebfl, Thrsp, Txnrdl, or a combination thereof.WSGR Ref. 59910-701.601B. Methods of Treatment

[0122] In some embodiments, a composition or pharmaceutical composition as disclosed herein can be administered to a subject in need thereof. In some embodiments, a composition or pharmaceutical composition as disclosed herein can be administered to a subject as a method of treating a disease and / or or condition in a subject in need thereof.1) AdministrationA) Route of Administration

[0123] In some embodiments, a composition or pharmaceutical composition as disclosed herein is administered to a subject. In some embodiments, the composition is formulated for systemic or local administration. In some embodiments, the composition is formulated for intravenous administration.B) Dose of Administration

[0124] In some embodiments, a composition comprising a nanosphere comprises IL-12 as disclosed herein. In some embodiments, IL-12 is administered to a subject at a dosage. In some embodiments, the dosage comprises about 0.16 ngs / kg / day to about 13 ngs / kg / day of IL-12. In some embodiments, the dosage comprises about 0.16 ngs / kg / day to about 0.2 ngs / kg / day, about 0.16 ngs / kg / day to about 0.5 ngs / kg / day, about 0.16 ngs / kg / day to about 1 ng / kg / day, about 0.16 ngs / kg / day to about 2 ngs / kg / day, about 0.16 ngs / kg / day to about 5 ngs / kg / day, about 0.16 ngs / kg / day to about 10 ngs / kg / day, about 0.16 ngs / kg / day to about 11 ngs / kg / day, about 0.16 ngs / kg / day to about 12 ngs / kg / day, about 0.16 ngs / kg / day to about 13 ngs / kg / day, about 0.2 ngs / kg / day to about 0.5 ngs / kg / day, about 0.2 ngs / kg / day to about 1 ng / kg / day, about 0.2 ngs / kg / day to about 2 ngs / kg / day, about 0.2 ngs / kg / day to about 5 ngs / kg / day, about 0.2 ngs / kg / day to about 10 ngs / kg / day, about 0.2 ngs / kg / day to about 11 ngs / kg / day, about 0.2 ngs / kg / day to about 12 ngs / kg / day, about 0.2 ngs / kg / day to about 13 ngs / kg / day, about 0.5 ngs / kg / day to about 1 ng / kg / day, about 0.5 ngs / kg / day to about 2 ngs / kg / day, about 0.5 ngs / kg / day to about 5 ngs / kg / day, about 0.5 ngs / kg / day to about 10 ngs / kg / day, about 0.5 ngs / kg / day to about 11 ngs / kg / day, about 0.5 ngs / kg / day to about 12 ngs / kg / day, about 0.5 ngs / kg / day to about 13 ngs / kg / day, about 1 ng / kg / day to about 2 ngs / kg / day, about 1 ng / kg / day to about 5 ngs / kg / day, about 1 ng / kg / day to about 10 ngs / kg / day, about 1 ng / kg / day to about 11 ngs / kg / day, about 1 ng / kg / day to about 12 ngs / kg / day, about 1 ng / kg / day to about 13 ngs / kg / day, about 2 ngs / kg / day to about 5 ngs / kg / day, about 2 ngs / kg / day to about 10 ngs / kg / day, about 2 ngs / kg / day to about 11 ngs / kg / day, about 2 ngs / kg / day to about 12 ngs / kg / day, about 2 ngs / kg / day to about 13 ngs / kg / day, about 5 ngs / kg / day to about 10WSGR Ref. 59910-701.601ngs / kg / day, about 5 ngs / kg / day to about 11 ngs / kg / day, about 5 ngs / kg / day to about 12 ngs / kg / day, about 5 ngs / kg / day to about 13 ngs / kg / day, about 10 ngs / kg / day to about 11 ngs / kg / day, about 10 ngs / kg / day to about 12 ngs / kg / day, about 10 ngs / kg / day to about 13 ngs / kg / day, about 11 ngs / kg / day to about 12 ngs / kg / day, about 11 ngs / kg / day to about 13 ngs / kg / day, or about 12 ngs / kg / day to about 13 ngs / kg / day of IL-12. In some embodiments, the dosage comprises about 0.16 ngs / kg / day, about 0.2 ngs / kg / day, about 0.5 ngs / kg / day, about 1 ng / kg / day, about 2 ngs / kg / day, about 5 ngs / kg / day, about 10 ngs / kg / day, about 11 ngs / kg / day, about 12 ngs / kg / day, or about 13 ngs / kg / day of IL-12. In some embodiments, the dosage comprises at least about 0.16 ngs / kg / day, about 0.2 ngs / kg / day, about 0.5 ngs / kg / day, about 1 ng / kg / day, about 2 ngs / kg / day, about 5 ngs / kg / day, about 10 ngs / kg / day, about 11 ngs / kg / day, or about 12 ngs / kg / day of IL-12.

[0125] In some embodiments, the dosage comprises about 14 ngs / kg / day to about 50 ngs / kg / day of IL-12. In some embodiments, the dosage comprises about 14 ngs / kg / day to about 15 ngs / kg / day, about 14 ngs / kg / day to about 16 ngs / kg / day, about 14 ngs / kg / day to about 17 ngs / kg / day, about 14 ngs / kg / day to about 18 ngs / kg / day, about 14 ngs / kg / day to about 20 ngs / kg / day, about 14 ngs / kg / day to about 25 ngs / kg / day, about 14 ngs / kg / day to about 30 ngs / kg / day, about 14 ngs / kg / day to about 35 ngs / kg / day, about 14 ngs / kg / day to about 40 ngs / kg / day, about 14 ngs / kg / day to about 45 ngs / kg / day, about 14 ngs / kg / day to about 50 ngs / kg / day, about 15 ngs / kg / day to about 16 ngs / kg / day, about 15 ngs / kg / day to about 17 ngs / kg / day, about 15 ngs / kg / day to about 18 ngs / kg / day, about 15 ngs / kg / day to about 20 ngs / kg / day, about 15 ngs / kg / day to about 25 ngs / kg / day, about 15 ngs / kg / day to about 30 ngs / kg / day, about 15 ngs / kg / day to about 35 ngs / kg / day, about 15 ngs / kg / day to about 40 ngs / kg / day, about 15 ngs / kg / day to about 45 ngs / kg / day, about 15 ngs / kg / day to about 50 ngs / kg / day, about 16 ngs / kg / day to about 17 ngs / kg / day, about 16 ngs / kg / day to about 18 ngs / kg / day, about 16 ngs / kg / day to about 20 ngs / kg / day, about 16 ngs / kg / day to about 25 ngs / kg / day, about 16 ngs / kg / day to about 30 ngs / kg / day, about 16 ngs / kg / day to about 35 ngs / kg / day, about 16 ngs / kg / day to about 40 ngs / kg / day, about 16 ngs / kg / day to about 45 ngs / kg / day, about 16 ngs / kg / day to about 50 ngs / kg / day, about 17 ngs / kg / day to about 18 ngs / kg / day, about 17 ngs / kg / day to about 20 ngs / kg / day, about 17 ngs / kg / day to about 25 ngs / kg / day, about 17 ngs / kg / day to about 30 ngs / kg / day, about 17 ngs / kg / day to about 35 ngs / kg / day, about 17 ngs / kg / day to about 40 ngs / kg / day, about 17 ngs / kg / day to about 45 ngs / kg / day, about 17 ngs / kg / day to about 50 ngs / kg / day, about 18 ngs / kg / day to about 20 ngs / kg / day, about 18 ngs / kg / day to about 25 ngs / kg / day, about 18 ngs / kg / day to about 30 ngs / kg / day, about 18 ngs / kg / day to about 35 ngs / kg / day, about 18 ngs / kg / day to about 40 ngs / kg / day, about 18 ngs / kg / day to about 45 ngs / kg / day, about 18 ngs / kg / day to about 50WSGR Ref. 59910-701.601ngs / kg / day, about 20 ngs / kg / day to about 25 ngs / kg / day, about 20 ngs / kg / day to about 30 ngs / kg / day, about 20 ngs / kg / day to about 35 ngs / kg / day, about 20 ngs / kg / day to about 40 ngs / kg / day, about 20 ngs / kg / day to about 45 ngs / kg / day, about 20 ngs / kg / day to about 50 ngs / kg / day, about 25 ngs / kg / day to about 30 ngs / kg / day, about 25 ngs / kg / day to about 35 ngs / kg / day, about 25 ngs / kg / day to about 40 ngs / kg / day, about 25 ngs / kg / day to about 45 ngs / kg / day, about 25 ngs / kg / day to about 50 ngs / kg / day, about 30 ngs / kg / day to about 35 ngs / kg / day, about 30 ngs / kg / day to about 40 ngs / kg / day, about 30 ngs / kg / day to about 45 ngs / kg / day, about 30 ngs / kg / day to about 50 ngs / kg / day, about 35 ngs / kg / day to about 40 ngs / kg / day, about 35 ngs / kg / day to about 45 ngs / kg / day, about 35 ngs / kg / day to about 50 ngs / kg / day, about 40 ngs / kg / day to about 45 ngs / kg / day, about 40 ngs / kg / day to about 50 ngs / kg / day, or about 45 ngs / kg / day to about 50 ngs / kg / day of IL-12. In some embodiments, the dosage comprises about 14 ngs / kg / day, about 15 ngs / kg / day, about 16 ngs / kg / day, about 17 ngs / kg / day, about 18 ngs / kg / day, about 20 ngs / kg / day, about 25 ngs / kg / day, about 30 ngs / kg / day, about 35 ngs / kg / day, about 40 ngs / kg / day, about 45 ngs / kg / day, or about 50 ngs / kg / day of IL-12. In some embodiments, the dosage comprises at least about 14 ngs / kg / day, about 15 ngs / kg / day, about 16 ngs / kg / day, about 17 ngs / kg / day, about 18 ngs / kg / day, about 20 ngs / kg / day, about 25 ngs / kg / day, about 30 ngs / kg / day, about 35 ngs / kg / day, about 40 ngs / kg / day, or about 45 ngs / kg / day of IL-12.

[0126] In some embodiments, the dosage comprises about 1,570 ngs / kg / day to about 1,630 ngs / kg / day of IL-12. In some embodiments, the dosage comprises about 1,570 ngs / kg / day to about 1,580 ngs / kg / day, about 1,570 ngs / kg / day to about 1,590 ngs / kg / day, about 1,570 ngs / kg / day to about 1,600 ngs / kg / day, about 1,570 ngs / kg / day to about 1,610 ngs / kg / day, about 1,570 ngs / kg / day to about 1,620 ngs / kg / day, about 1,570 ngs / kg / day to about 1,630 ngs / kg / day, about 1,580 ngs / kg / day to about 1,590 ngs / kg / day, about 1,580 ngs / kg / day to about 1,600 ngs / kg / day, about 1,580 ngs / kg / day to about 1,610 ngs / kg / day, about 1,580 ngs / kg / day to about 1,620 ngs / kg / day, about 1,580 ngs / kg / day to about 1,630 ngs / kg / day, about 1,590 ngs / kg / day to about 1,600 ngs / kg / day, about 1,590 ngs / kg / day to about 1,610 ngs / kg / day, about 1,590 ngs / kg / day to about 1,620 ngs / kg / day, about 1,590 ngs / kg / day to about 1,630 ngs / kg / day, about 1,600 ngs / kg / day to about 1,610 ngs / kg / day, about 1,600 ngs / kg / day to about 1,620 ngs / kg / day, about 1,600 ngs / kg / day to about 1,630 ngs / kg / day, about 1,610 ngs / kg / day to about 1,620 ngs / kg / day, about 1,610 ngs / kg / day to about 1,630 ngs / kg / day, or about 1,620 ngs / kg / day to about 1,630 ngs / kg / day of IL-12. In some embodiments, the dosage comprises about 1,570 ngs / kg / day, about 1,580 ngs / kg / day, about 1,590 ngs / kg / day, about 1,600 ngs / kg / day, about 1,610 ngs / kg / day, about 1,620 ngs / kg / day, or about 1,630 ngs / kg / day of IL-12. In some embodiments, the dosage comprises at least about 1,570 ngs / kg / day, about 1,580 ngs / kg / day,WSGR Ref. 59910-701.601about 1,590 ngs / kg / day, about 1,600 ngs / kg / day, about 1,610 ngs / kg / day, or about 1,620 ngs / kg / day of IL-12. In some embodiments, the dosage comprises at most about 1,580 ngs / kg / day, about 1,590 ngs / kg / day, about 1,600 ngs / kg / day, about 1,610 ngs / kg / day, about 1,620 ngs / kg / day, or about 1,630 ngs / kg / day of IL-12.

[0127] In some embodiments, the dosage comprises about 0.16 ngs / kg / day to about 1,600 ngs / kg / day of IL-12. In some embodiments, the dosage comprises about 0.16 ngs / kg / day to about 1,600 ngs / kg / day of IL-12. In some embodiments, the dosage comprises about 0.16 ngs / kg / day, or about 1,600 ngs / kg / day of IL-12. In some embodiments, the dosage comprises at least about 0.16 ngs / kg / day of IL-12.2) Subjects

[0128] The compositions disclosed herein can be used in a method of treating a subject with a condition or a disease. The compositions disclosed herein can be used in a method of treating a subject with cancer.

[0129] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a variety of mammalian species utilized in research for various purposes, including medical, biological, and veterinary investigations. In some embodiments, the mammal is a mouse, a rat, a guinea pig, a rabbit, a hamster, a ferret, a cat, a pig, a dog, or a non-human primate. In some embodiments, the non-human primate can be rhesus macaques, marmosets, and / or chimpanzees. In some embodiments, the subject is a human.3) Diseases or Conditions

[0130] The present disclosure discloses a method of treating a subject with a disease or a condition. In some embodiments, a disease or condition comprises cancer, an autoimmune disease, and infections. In some embodiments, a cancer comprises a tumor. In some embodiments, a tumor comprises a solid tumor.VIII. CERTAIN EMBODIMENTS AND ENUMERATED EMBODIMENTS

[0131] According to various embodiments disclosed herein, a protein can be encapsulated in a nanosphere. This encapsulation process can be performed by making a double emulsion, where a first water phase can be emulsified in an oil phase, and the oil phase is emulsified in a second water phase. The protein can be a cytokine, which can be a small protein (-5-20 kDa) that plays a role in cell signaling. Suitable cytokines include: interleukins, produced by T-helper cells; lymphokines, produced by lymphocytes; monokines, produced exclusively by monocytes; interferons, involved in antiviral responses; colony stimulating factors, which support the growth of cells in semisolid media; and chemokines, which mediate chemoattraction between cells. InWSGR Ref. 59910-701.601various embodiments, the protein encapsulated in the nanosphere can be cytokines with three-dimensional structures having a bundle of four α-helices, which can be interferons, interleukins, e.g., interleukin-2 or interleukin- 12, or non-immunological cytokines, including erythropoietin and thrombopoietin. The protein encapsulated in the nanosphere can be interleukin- 12 (IL-12). The oil phase can be prepared by dissolving from 2.5% w / v to 17% w / v of poly(lactic acid- co-glycolic acid) (PLGA) in an organic solvent. A first aqueous phase can be made by suspending a protein in an aqueous medium. Finally, a second aqueous phase can be made by dissolving polyvinyl alcohol (PVA) in water.

[0013] The PLGA can comprise from 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 to C5 ketone solvent, or a mixture thereof. The PLGA can comprise from 75% to 90% lactide. The PLGA can comprise from 50% to 75% lactide, and the organic solvent can be a halogenated Cl- C3 organic solvent, acetonitrile, a C3 to C4 ketone solvent, or a mixture thereof. In various embodiments, the PLGA comprises from 50% to 90% lactide, and the organic solvent can be acetonitrile, acetone, ethyl acetate, or dichloromethane. The protein-containing aqueous medium can be added to the oil phase to form a first emulsion, with agitation. The first emulsion can be added to the PVA-containing aqueous phase to form a second emulsion, with agitation. The organic solvent can then be evaporated from the second emulsion to form an aqueous solution; and PLGA nanospheres containing the protein from the second aqueous phase can be recovered from the aqueous solution. In an aspect, this disclosure describes a composition comprising poly(D, L-lactic acid-co- glycolic acid)(PLGA) nanospheres and a therapeutic substance, wherein at least a portion of the therapeutic substance can elute from the composition more than 72 hours after placement of the composition in a solution. In various embodiments, at least a portion of the therapeutic substance can elute from the composition more than 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336 hours after placement of the composition in a solution. At least a portion of the therapeutic substance can elute from the composition between 72 and 288 hours after placement of the composition in a solution. The therapeutic substance can be IL-12. The solution can be a mammalian serum and about 100 units / mL Penicillin-Streptomycin (Pen-Strep) in Phosphate-Buffered Salt Solution (DPBS). In an aspect, this disclosure describes a composition comprising poly(D, L-lactic acid-co- glycolic acid)(PLGA) nanospheres and IL- 12, wherein IL- 12 can be incorporated into the PLGA nanospheres with an encapsulation efficiency of at least 2%. In various embodiments, IL-12 can be incorporated into the PLGA nanospheres with an encapsulation efficiency of at least 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, 100%. IL-12 can be incorporated into the PLGA nanospheres with an encapsulation efficiency of from about 2% to about 40%. The IL-12 can be bioactive IL-12. In an aspect, this disclosure describes a composition comprising a drug deliveryWSGR Ref. 59910-701.601vector and a therapeutic substance, wherein the composition can elute at least 1.0 pg of the therapeutic substance per 100,000 particles of the drug delivery vector under conditions of a drug delivery vector release buffer, wherein the composition continues to elute therapeutic substance over more than 3 days, wherein the therapeutic substance, drug delivery vector and drug delivery vector release buffer comprise a solution, wherein the solution is centrifuged and a portion stored at about 1 to 10° C, and wherein the elution of the therapeutic substance is determined by ELISA assay. The composition can comprise a surfactant. The surfactant can be Tween 80 and Span 60. In various embodiments, the drug delivery vector can comprise poly(D, L-lactic acid-co- glycolic acid)(PLGA). The therapeutic substance 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 Salt Solution (DPBS). species specific whole serum, species specific engineered serum albumin or species specific whole fetal serum. In an aspect, this disclosure describes a composition comprising protein loaded poly(D, L- lactic acid-co-glycolic acid)(PLGA) nanospheres, wherein the nanospheres can comprise a diameter of about 100 to 1000 nm, a surfactant, and a species specific whole serum, engineered or native serum albumin. In various embodiments, the protein can comprise IL-12. The surfactant can comprise Tween 80 and Span 60. In an aspect, this disclosure describes a method of making a protein encapsulated nanosphere, comprising determining the rate of dissociation of the protein with increasing time and / or increasing sonication wattage, comparing the rate of dissociation of the protein with a rate of formation of the nanosphere with increasing time and / or sonication wattage, determining a time and sonication wattage at a point of intersection between the rate of dissociation of the protein and the rate of formation of the nanosphere, preparing a first phase by dissolving of PLGA in a solvent with a first surfactant, preparing a second phase by dissolving an alcohol in water with a second surfactant and a mammalian serum, 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 and sonicating the second emulsion for the time and sonication power determined at the point of intersection, evaporating the solvent from the second emulsion to form an aqueous solution, and recovering PLGA nanospheres containing the component from the aqueous solution. In an aspect, this disclosure describes a method of encapsulating a component in a nanosphere, comprising preparing a first phase by dissolving of poly(lactic acid- 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 theWSGR Ref. 59910-701.601first 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. In various embodiments, the component can comprise a protein. The protein can comprise IL-12. The component can comprise a surfactant and a mammalian serum. The PLGA can comprise from 50% to 90% lactide, and the solvent can be selected from the group consisting of halogenated C1-C3 organic solvents, C2-C3 nitrile solvents, C2-C5 alkyl ester solvents, C3 to C5 ketone solvents, and mixtures thereof. The solvent can be acetonitrile, acetone, ethyl acetate, or di chloromethane. The PLGA can comprise from 75% to 90% lactide. The PLGA can comprise from 50% to 75% lactide, and the solvent can be selected from the group consisting of halogenated C1-C3 organic solvents, acetonitrile, C3 to C4 ketone solvents, and mixtures thereof. The method can comprise adding the aqueous medium to the first phase to form the first emulsion, and agitating the first emulsion with a tissue homogenizer at a rate 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 rate of 13,000 RPM to 20,000 RPM. The method can comprise adding the aqueous medium to the first phase to form the first emulsion, and agitating the first emulsion by sonication and adding the first emulsion to the second phase to form the second emulsion, and agitating the second emulsion by sonication. The method can comprise agitating the first emulsion comprises sonication at a power level of 30 W to 50 W, for a period of time of 5 sec to 30 sec, and agitating the second emulsion comprises sonication at a power level of 30 W to 50 W, for a period of time of 5 sec to 30 sec. The protein can be a cytokine or a globular protein. 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 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 comprising four helices labeled A through D, and no C-terminal extension following the D helix. The cytokine can be a granulocyte-macrophage colony-stimulating factor, a granulocyte colony-stimulating factor, interferon alpha- 1, interferon beta, interferon gamma interferon kappa, interferon tau-1, interferon omega-1, or an interleukin selected from the group consisting of IL-2, IL-3, IL-4, IL-5, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, the 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. The protein can be an immunological cytokine that either: a) enhances cellular immune responses, or b) enhances antibody responses. The cytokine can be an immunological cytokine that enhances cellular immune responses, selected from the group consisting of TNFa, IFN-y, and interleukin- 12. The cytokine can be IL-12. The cytokine can be an immunological cytokine that enhances antibody responses, selected from theWSGR Ref. 59910-701.601group consisting of TGF-b, IL-4, IL-10, and IL-13. The first and second emulsions can each be agitated with a tissue homogenizer at a rate of 13,000 RPM to 20,000 RPM, and IL-12 is incorporated into the PLGA nanospheres with an encapsulation efficiency of about 0.5% to about 2.1%. The first and second emulsions can each be agitated with sonication at a power level of 30 W to 50 W, for a period of time of 5 sec to 30 sec, 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 the first surfactant, and / or the second phase can contain the 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. In various embodiments, a first portion of the protein can be adsorbed onto a surface of the nanosphere, a second portion of the protein can be incorporated into the PLGA matrix at a core of the nanosphere, and the nanosphere can comprise at least one additive selected from the group consisting of mammalian serum albumin, trehalose, the first surfactant, and the second. The nanosphere can comprise mammalian serum albumin and a surfactant. In various embodiments, this disclosure describes a dosage form comprising a plurality of nanospheres produced by the methods described, each nanosphere comprising a PLGA matrix and a protein, wherein a first portion of the protein can be adsorbed onto a surface of the nanosphere, and a second portion of the protein is incorporated into the PLGA matrix at a 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%. In an aspect, this disclosure describes method of encapsulating a protein in a nanosphere, comprising preparing an oil phase by dissolving from 2.5% w / v to 17% w / v of poly(lactic acid- 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; suspending the protein in an aqueous medium, adding the aqueous medium to the oil phase to form a first emulsion, and agitating 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, and recovering poly(lactic acid-co-glycolic acid) nanospheres containing the protein from the aqueous solution. In an aspect, this disclosure describes method of controlling an immunophenotype in a patient suffering from a disease which impacts the immune system, comprising (a) determining a disease state of a patient, where the disease state includes a diagnosis and an initial immunophenotype, (b) comparing the disease state of the patient to a plurality of disease states within a database, where each disease state in the database includes a diagnosis, an initial immunophenotype, and aWSGR Ref. 59910-701.601treatment protocol, and (c) based on the comparing step (b), selecting a treatment protocol from the database, where the treatment protocol involves administering an immunomodulating drug. In various embodiments, the method can comprise (d) administering the immunomodulating drug to the patient, (e) after step (d), monitoring the patient’s immunophenotype as a function of time, and adjusting administration of the immunomodulating drug if the patient’s immunophenotype falls outside a desired range. In various embodiments, the protein-containing aqueous medium can be added to the oil phase to form a first emulsion, with agitation by a tissue homogenizer at a rate of 13,000 RPM to 20,000 RPM. The first emulsion can be added to the PVA-containing aqueous phase to form a second emulsion, with agitation by a tissue homogenizer at a rate of 13,000 RPM to 20,000 RPM. The organic solvent can then be evaporated from the second emulsion to form an aqueous solution; and PLGA nanospheres containing the protein from the second aqueous phase can be recovered from the aqueous solution. 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 cytokines with a three-dimensional structures having a bundle of four α-helices, e.g., interleukins, e.g., interleukin-2 or interleukin-12, or non-immunological cytokines, including erythropoietin and thrombopoietin. In various embodiments, the protein in the protein-containing aqueous medium can be IL- 12. IL-12 can be incorporated into PLGA nanospheres by agitating the first and second emulsions with a tissue homogenizer at a rate of 13,000 RPM to 20,000 RPM. IL-12 can be incorporated into the resulting PLGA nanospheres with an encapsulation efficiency of about 0.5% to about 2.1%. In various embodiments, the protein-containing aqueous medium can be added to the oil phase to form a first emulsion, with agitation by ultrasonication. The first emulsion can be added to the PVA-containing aqueous phase to form a second emulsion, with agitation by sonication.Agitation during formation of either or both of the first and second emulsions can comprise sonication at a power level of 30 W to 50 W, 30 W to 40 W, or 40 W to 50 W, for a period of time of 5 sec to 30 sec, 10 sec to 30 sec, 10 sec to 20 sec, or 10 sec to 15 sec. The organic solvent can then be evaporated from the second emulsion to form an aqueous solution; and PLGA nanospheres containing the protein from the second aqueous phase can then be recovered from the aqueous solution. In various embodiments, the protein-containing aqueous medium can be added to the oil phase to form a first emulsion, with agitation by a tissue homogenizer at a rate of 13,000 RPM to 20,000 RPM. The first emulsion can be added to the PVA-containing aqueous phase to form a second emulsion, with agitation by sonication at a power level of 30 W to 50 W, 30 W to 40 W, or 40 W to 50 W, for a period of time of 5 sec to 30 sec. The organic solvent can then be evaporated from the second emulsion to form an aqueous solution; andWSGR Ref. 59910-701.601PLGA nanospheres containing the protein from the second aqueous phase can be recovered from the aqueous solution. 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 at a power level of 30 W to 50 W, for a period of time of 10 sec to 20 sec. 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%. Various embodiments disclosed herein are directed to a nanosphere comprising a poly(lactic acid-co-glycolic acid) matrix and a protein, where a first portion of the protein can be adsorbed onto a surface of the nanosphere and a second portion of the protein can be incorporated into the poly(lactic acid-co-glycolic acid) matrix at a core of the nanosphere. The nanosphere can be produced by making a double emulsion, where a first, protein-containing, water phase can be emulsified in an oil phase, and the oil phase can be then emulsified in a second water phase. The protein can be a cytokine, such as IL-12. The IL-12 can be incorporated into the nanosphere with an encapsulation efficiency of about 0.5% to about 85%, about 1% to about 70%, about 2% to 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 the 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%. Various embodiments disclosed herein relate to a method of encapsulating a protein in a nanosphere, by preparing an oil phase by dissolving from 2.5% w / v to 17% w / v of poly(lactic acid- co-gly colic acid) (PLGA) in an organic solvent containing an optional first surfactant; 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 an 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 agitated, and the first emulsion can be added to the aqueous phase to form a second emulsion, which can then be agitated. The organic solvent can be evaporated from the second emulsion to form an aqueous solution; and poly(lactic acid-co-glycolic acid) nanospheres containing the protein can be recovered from the aqueous solution. The aqueous phase can contain polyvinyl alcohol and mammalian serum, e.g., fetal serum. The aqueous phase can contain polyvinyl alcohol and the 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. Various embodiments disclosed herein relate to a nanosphere comprising a poly(lactic acid- co-glycolic acid) matrix and a protein, where a first portion of the protein can be adsorbed onto a surface of the nanosphere;WSGR Ref. 59910-701.601and a second portion of the protein can be incorporated into the poly(lactic acid-co-glycolic acid) matrix at a core of the nanosphere. The nanosphere can further comprise at least one additive selected from the group consisting of mammalian serum albumin, trehalose, and a surfactant. The nanosphere can comprise mammalian serum albumin, mammalian recombinant / native albumin, and a surfactant. The nanosphere can comprise mammalian whole serum, mammalian recombinant / native albumin, a first surfactant, and a second surfactant. In many disease states, e.g., cancer and autoimmune disorders, the human immune system can be in constant flux. In order to treat such diseases, it can be beneficial for a medical practitioner to assess the immune system of a patient in real time, and to follow the immune system status over time. When cancer, infections, and / or autoimmune disorders are being treated with immunomodulating agents, regardless of whether they are immunosuppressive or immunostimulating, it can be beneficial for the medical practitioner to be able to follow the impact of such agents on the immune system. Various embodiments disclosed herein relate to a method allowing systemic analysis of the immune system from a blood draw or a finger stick blood draw that can be analyzed at a testing site. The response of the immune system to a disease state, e.g., cancer or an autoimmune disease, can be analyzed at a selected time, and the immune response to a treatment protocol can be followed over the course of a disease or treatment. This diagnostic method can be useful in the treatment, surveillance, and diagnosis of many diseases, including cancer, autoimmune diseases, and infections. As immunomodulating agents become more common, the method can provide the medical practitioner with the ability to assess the status of the immune system at a specific time in the pathogenesis of a disease, and can allow prediction as to which immomodulating treatment can be most effective in combating the disease. This can increase the overall effectiveness of treatment, and can allow improved assessment of the immune status of the patient in the disease process. This information can also be organized in a living database of immune profiles across disease specific categories that can allow practitioners to be more informed on the treatments they will be giving based on previous experiences. Various embodiments disclosed herein relate to a method of controlling an immunophenotype in a patient suffering from a disease which impacts the immune system, including steps of determining an initial immunophenotype, or immune status, of a patient; and either: administering a first drug which stimulates the immune system if the initial immunophenotype shows immunosuppression; or administering a second drug which suppresses the immune system if the initial immunophenotype shows overstimulation of the immune system. After administering the selected drug, the patient’s immunophenotype can be monitored as a function of time; and administration of the first and / or second drug can be adjusted if the patient’s immunophenotype falls outside a desired range. Various embodiments disclosed hereinWSGR Ref. 59910-701.601relate to a method of controlling an immunophenotype in a patient suffering from a disease which impacts the immune system, by determining a disease state of a patient, where the disease state includes a diagnosis and an initial immunophenotype; and comparing the disease state of the patient to a plurality of disease states within a database, where each disease state in the database includes a diagnosis, an initial immunophenotype, and a treatment protocol. Based on the comparison between the patient’s disease state and disease states and treatment protocols from the database, a treatment protocol can be selected from the database, where the treatment protocol involves administering an immunomodulating drug. The method can include administering the immunomodulating drug to the patient; monitoring the patient’s immunophenotype as a function of time after administering the drug; and adjusting administration of the immunomodulating drug if the patient’s immunophenotype falls outside a desired range. Immunophenotyping of a patient’s blood sample can involve the ability to analyze and assess the data appropriately. This system can allow the appropriate dosing, treatment, and corrections to be made across several disease states. As the collection of data increases the growing database can also be able help direct medical professionals to diagnosis, treat, and dose immunotherapeutics in the broadest sense.

[0132] Certain enumerated embodiments

[0133] 1. A composition comprising poly(D, L-lactic acid-co-glycolic acid)(PLGA) nanospheres and a therapeutic substance, wherein at least a portion of the therapeutic substance elutes from the composition more than 72 hours after placement of the composition in a solution.

[0134] 2 The composition of embodiment 1, wherein at least a portion of the therapeutic substance elutes from the composition more than 96 hours after placement of the composition in a solution.

[0135] 3. The composition of embodiment 1, wherein at least a portion of the therapeutic substance elutes from the composition more than 120 hours after placement of the composition in a solution.

[0136] 4. The composition of embodiment 1, wherein at least a portion of the therapeutic substance elutes from the composition between 72 and 288 hours after placement of the composition in a solution.

[0137] 5. The composition of embodiment 1, wherein the therapeutic substance is IL-12.

[0138] 6. The composition of embodiment 1, wherein the solution is a mammalian serum and about 100 units / mL Penicillin-Streptomycin (Pen-Strep) in Phosphate-Buffered Salt Solution (DPBS).WSGR Ref. 59910-701.601

[0139] 7 A composition comprising poly(D, L-lactic acid-co-glycolic acid)(PLGA) nanospheres and IL- 12, wherein IL- 12 is incorporated into the PLGA nanospheres with an encapsulation efficiency of at least 2%.

[0140] 8. The composition of embodiment 7, wherein IL-12 is incorporated into the PLGA nanospheres with an encapsulation efficiency of at least 10%.

[0141] 9. The composition of embodiment 7, wherein IL-12 is incorporated into the PLGA nanospheres with an encapsulation efficiency of at least 20%.

[0142] 10. The composition of embodiment 7, wherein IL-12 is incorporated into the PLGA nanospheres with an encapsulation efficiency of at least 40%.

[0143] 11. The composition of embodiment 7, wherein IL-12 is incorporated into the PLGA nanospheres with an encapsulation efficiency of from about 2% to about 40%.

[0144] 12. The composition of embodiment 7, wherein the IL-12 is bioactive IL-12.

[0145] 13. A composition comprising a drug delivery vector and a therapeutic substance, wherein the composition elutes at least 1.0 pg of the therapeutic substance per 100,000 particles of the drug delivery vector under conditions of a drug delivery vector release buffer, wherein the composition continues to elute therapeutic substance over more than 3 days, wherein the therapeutic substance, drug delivery vector and drug delivery vector release buffer comprise a solution, wherein the solution is centrifuged and a portion stored at about 1 to 10° C, and wherein the elution of the therapeutic substance is determined by ELISA assay.

[0146] 14. The composition of embodiment 13, wherein the drug delivery vector comprises poly(D, L- lactic acid-co-glycolic acid)(PLGA).

[0147] 15. The composition of embodiment 13, wherein the therapeutic substance is a protein.

[0148] 16. The composition of embodiment 14, wherein the protein is a cytokine.

[0149] 17. The composition of embodiment 16, wherein the cytokine is IL-12.

[0150] 18. The composition of embodiment 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 Salt Solution (DPBS).

[0151] 19. The composition of embodiment 13, further comprising a surfactant.

[0152] 20. The composition of embodiment 19, wherein the surfactant comprises Tween 80 and Span 60.

[0153] 21. The composition of embodiment 13, further comprising species specific whole serum, species specific engineered serum albumin or species specific whole fetal serum.WSGR Ref. 59910-701.601

[0154] 22. A composition comprising: protein loaded poly(D, L-lactic acid-co-glycolic acid)(PLGA) nanospheres, wherein the nanospheres comprise a diameter of about 100 to 1000 nm; a surfactant; and a species specific whole serum, engineered or native serum albumin.

[0155] 23. The composition of embodiment 22, wherein the protein comprises IL-12.

[0156] 24. The composition of embodiment 22, wherein the surfactant comprises Tween 80 and Span 60.

[0157] 25. A method of making a protein encapsulated nanosphere, comprising: determining the rate of dissociation of the protein with increasing time and / or increasing sonication wattage; comparing the rate of dissociation of the protein with a rate of formation of the nanosphere with increasing time and / or sonication wattage; determining a time and sonication wattage at a point of intersection between the rate of dissociation of the protein and the rate of formation of the nanosphere; preparing a first phase by dissolving of PLGA in a solvent with a first surfactant; preparing a second phase by dissolving an alcohol in water with a second surfactant and a mammalian serum; 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 and sonicating the second emulsion for the time and sonication power determined at the point of intersection; evaporating the solvent from the second emulsion to form an aqueous solution; and recovering PLGA nanospheres containing the component from the aqueous solution

[0158] 26. A method of encapsulating a component in a nanosphere, comprising, preparing a first phase by dissolving of poly(lactic acid- 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.

[0159] 27. The method of embodiment 26, wherein the component comprises a protein.

[0160] 28. The method of embodiment 27, wherein the protein comprises IL-12.

[0161] 29. The method of embodiment 27, wherein the component further comprises a surfactant and a mammalian serum.

[0162] 30. The method of embodiment 26, wherein the PLGA comprises from 50% to 90% lactide; and the solvent is selected from the group consisting of halogenated C1-C3 organic solvents, C2-C3 nitrile solvents, C2-C5 alkyl ester solvents, C3 to C5 ketone solvents, and mixtures thereof.WSGR Ref. 59910-701.601

[0163] 31. The method of embodiment 30, wherein the solvent is acetonitrile, acetone, ethyl acetate, or dichloromethane.

[0164] 32. The method of embodiment 26, wherein the PLGA comprises from 75% to 90% lactide.

[0165] 33. The method of embodiment 26, wherein the PLGA comprises from 50% to 75% lactide; and the solvent is selected from the group consisting of halogenated C1-C3 organic solvents, acetonitrile, C3 to C4 ketone solvents, and mixtures thereof.

[0166] 34. The method of embodiment 26, wherein the method comprises adding the aqueous medium to the first phase to form the first emulsion, and agitating the first emulsion with a tissue homogenizer at a rate 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 rate of 13,000 RPM to 20,000 RPM.

[0167] 35. The method of embodiment 26, wherein the method comprises adding the aqueous medium to the first phase to form the first emulsion, and agitating the first emulsion by sonication; and adding the first emulsion to the second phase to form the second emulsion, and agitating the second emulsion by sonication.

[0168] 36. The method of embodiment 35, wherein: agitating the first emulsion comprises sonication at a power level of 30 W to 50 W, for a period of time of 5 sec to 30 sec; and agitating the second emulsion comprises sonication at a power level of 30 W to 50 W, for a period of time of 5 sec to 30 sec.

[0169] 37. The method of embodiment 36, wherein: agitating the first emulsion comprises sonication for a period of time of 10 sec to 20 sec; and agitating the second emulsion comprises sonication for a period of time of 10 sec to 20 sec.

[0170] 38. The method of embodiment 27, wherein the protein is a cytokine or a globular protein.

[0171] 39. The method of embodiment 38, wherein the protein is a cytokine selected from the group consisting of interleukins, lymphokines, monokines, interferons, colony stimulating factors, and chemokines.

[0172] 40. The method of embodiment 39, wherein the cytokine is selected from the group consisting of interleukins and non-immunological cytokines.

[0173] 41. The method of embodiment 38, wherein the protein is a cytokine having an N-terminal signal sequence, a four-helix bundle comprising four helices labeled A through D, and no C-terminal extension following the D helix.

[0174] 42. The method of embodiment 41, wherein the cytokine is a granulocytemacrophage colony- stimulating factor, a granulocyte colony-stimulating factor, interferonWSGR Ref. 59910-701.601alpha- 1, interferon beta, interferon gamma interferon kappa, interferon tau-1, interferon omega-1, or an interleukin selected from the group consisting of IL-2, IL-3, IL-4, IL-5, IL-5, IL-6, IL-7, IL-9, IL- 10, IL-11, IL- 12, the 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.

[0175] 43. The method of embodiment 38, wherein the protein is an immunological cytokine that either: a) enhances cellular immune responses; or b) enhances antibody responses.

[0176] 44. The method of embodiment 43, wherein the cytokine is an immunological cytokine that enhances cellular immune responses, selected from the group consisting of TNFa, IFN-y, and interleukin-12.

[0177] 45. The method of embodiment 44, wherein the cytokine is IL-12.

[0178] 46. The method of embodiment 43, wherein the cytokine is an immunological cytokine that enhances antibody responses, selected from the group consisting of TGF-b, IL-4, IL-10, and IL-13.

[0179] 47. The method of embodiment 45, wherein: the first and second emulsions are each agitated with a tissue homogenizer at a rate of 13,000 RPM to 20,000 RPM; and IL-12 is incorporated into the PLGA nanospheres with an encapsulation efficiency of about 0.5% to about 2.1%.

[0180] 48. The method of embodiment 45, wherein: the first and second emulsions are each agitated with sonication at a power level of 30 W to 50 W, for a period of time of 5 sec to 30 sec; and IL-12 is incorporated into the PLGA nanospheres with an encapsulation efficiency of about 4.5% to about 10%.

[0181] 49. A nanosphere produced by the method of embodiment 26, the nanosphere comprising a PLGA matrix and a protein, wherein: a first portion of the protein is adsorbed onto a surface of the nanosphere; a second portion of the protein is incorporated into the PLGA matrix at a core of the nanosphere.

[0182] 50. The nanosphere of embodiment 49, wherein: the protein is IL-12; and the IL-12 is incorporated into the nanosphere with an encapsulation efficiency of about 0.5% to about 10%.

[0183] 51. The method of embodiment 26, wherein the second phase contains polyvinyl alcohol and mammalian serum.

[0184] 52. The method of embodiment 51, wherein: the first phase contains the first surfactant; and / or the second phase contains the second surfactant.

[0185] 53. The method of embodiment 52, wherein: the first surfactant is a sorbitan fatty acid ester; and / or the second surfactant is a polyoxyethylene sorbitan fatty acid ester.WSGR Ref. 59910-701.601

[0186] 54. The method of embodiment 26, wherein the second phase contains polyvinyl alcohol and fetal serum.

[0187] 55. A nanosphere produced by the method of embodiment 53, the nanosphere comprising a PLGA matrix and a protein, wherein: a first portion of the protein is adsorbed onto a surface of the nanosphere; a second portion of the protein is incorporated into the PLGA matrix at a core of the nanosphere; and the nanosphere comprises at least one additive selected from the group consisting of mammalian serum albumin, trehalose, the first surfactant, and the second.

[0188] 56. The nanosphere of embodiment 55, wherein the nanosphere comprises mammalian serum albumin and a surfactant.

[0189] 57. A dosage form, comprising a plurality of nanospheres produced by the method of embodiment 26, each nanosphere comprising a PLGA matrix and a protein, wherein: a first portion of the protein is adsorbed onto a surface of the nanosphere; a second portion of the protein is incorporated into the PLGA matrix at a core of the nanosphere.

[0190] 58. The dosage form of embodiment 57, wherein: the protein is IL-12; and the IL-12 is incorporated into the nanosphere with an encapsulation efficiency of at least 2%

[0191] 59. A method of encapsulating a protein in a nanosphere, comprising: preparing an oil phase by dissolving from 2.5% w / v to 17% w / v of poly(lactic acid- co-gly colic 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; suspending the protein in an aqueous medium; adding the aqueous medium to the oil phase to form a first emulsion, and agitating 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; and recovering poly(lactic acid-co-glycolic acid) nanospheres containing the protein from the aqueous solution.

[0192] 60. A method of controlling an immunophenotype in a patient suffering from a disease which impacts the immune system, comprising: a) determining an initial immunophenotype of a patient; b) if the initial immunophenotype shows immunosuppression, administering a first drug which stimulates the immune system; or if the initial immunophenotype shows overstimulation of the immune system, administering a second drug which suppresses the immune system; c) after step (b), monitoring the patient’s immunophenotype as a function of time; and d) adjusting administration of the first and / or second drug if the patient’s immunophenotype falls outside a desired range.WSGR Ref. 59910-701.601

[0193] 61. A method of controlling an immunophenotype in a patient suffering from a disease which impacts the immune system, comprising: a) determining a disease state of a patient, where the disease state includes a diagnosis and an initial immunophenotype; b) comparing the disease state of the patient to a plurality of disease states within a database, where each disease state in the database includes a diagnosis, an initial immunophenotype, and a treatment protocol; c) based on the comparing step (b), selecting a treatment protocol from the database, where the treatment protocol involves administering an immunomodulating drug.

[0194] 62. The method of embodiment 61, further comprising: d) administering the immunomodulating drug to the patient; e) after step (d), monitoring the patient’s immunophenotype as a function of time; and f) adjusting administration of the immunomodulating drug if the patient’s immunophenotype falls outside a desired range.IX. DEFINITIONS

[0195] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0196] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0197] As used in the specification and claims, the singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.

[0198] The terms “subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtainedWSGR Ref. 59910-701.601in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0199] The term “zzz vivo" is used to describe an event that takes place in a subject’s body.

[0200] The term “zzz vitro" is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed.

[0201] As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.

[0202] As used herein, the terms “treatment” or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0203] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.X. EXAMPLES

[0204] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0205] In the following examples, dichloromethane (DCM, #320269), PLGA (Resomer RG 756 S, 75% lactide, #719927), NaCl (#7647-14-5), and poly(vinyl alcohol) (#341584) were purchased from Sigma Aldrich (St. Louis, Mo.). Fluorescein isothiocyanate-labeled bovineWSGR Ref. 59910-701.601serum 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, Mass.). Recombinant mouse IL-12 (p70, rmIL-12, #577008) and Mouse IL-12 ELISA MAX deluxe ELISA kits (#433606) were purchased from Biolegend (San Diego, Calif.). Gibco Fetal Bovine Serum Qualified Heat Inactivated US Origin (HI-FBS, #MT35011CV) and Dulbecco's Phosphate-Buffered Salt Solution 1* (DPBS, #21031CV) were purchased from Fisher Scientific (Pittsburgh, Pa.). Female BALB / c mice (6-8 weeks of age) (#000651) were purchased from the Jackson Laboratory (Bar Harbor, Me.).

[0206] Part A. IL-12 Stability Studies.

[0207] Example 1. Stability of IL-12 in Acidic and Basic Solutions

[0208] To determine if IL- 12 could handle the acidic nanoenvironment of a PLGA nanosphere, the recovery of bioactive recombinant mouse IL- 12 following a three hour exposure to solutions of varying pH was tested. Specifically, IL- 12 was incubated for 3 hours in a solution of pH 1, a solution of pH 3, a solution of pH 7.4, a solution of pH 11, and a solution of pH 13. The results are shown in Table 1. The percentage of active IL-12 recovered, relative to an initial concentration, was determined using enzyme-linked immunosorbent assay (ELISA) to determine the amount of protein in its native, and hence biologically-active, conformation.

[0209] TABLE 1. IL-12 stability as a function of pH.PH IL- 12 Recovery (%)1 0% ± 0%3 39.7% ± 8.44%7.4 100% ± 0%11 96.6% ± 6.08%13 0% ± 0%

[0210] As seen in Table 1, IL-12 has good stability at a pH value of 7.4 to 11, and can be subject to denaturation outside this range.

[0211] Example 2. Stability of IL-12 in Organic Solvents

[0212] The double emulsion method of synthesizing PLGA nanospheres can involve dissolving the PLGA in an organic solvent. Therefore, the stability of IL-12 was tested in several aprotic solvents of varying polarity. The organic solvents tested, in order of increasing hydrophilicity, were dichloromethane (DCM), ethyl acetate (EA), and acetone (AC). While DCM dissolves PLGA well, it is also the most hydrophobic and hence can have the greatest effect on the biological activity of IL-12. PLGA can be poorly soluble in acetone, but this solvent is the most polar and therefore can affect the bioactivity of IL-12 the least.WSGR Ref. 59910-701.601

[0213] IL-12 was added to a 1:1 solution of solvent and phosphate-buffered salt solution, and stirred to completely remove the organic solvent; the remaining bioactive IL- 12 concentrations were then determined using ELISA and expressed as a percentage, relative to an initial concentration. The results are shown in Table 2. Interestingly, AC had the most detrimental effect on the protein with only 43% (SE=1.06%) recovered, while EA showed the highest recovery at 63% (SE=0.77%); DCM left approximately half of the protein in its native form (49%, SE=0.55).

[0214] TABLE 2. IL-12 stability as a function of organic solvent.Solvent IL- 12 Recovery (%)DCM 49% ± 0.55%EA 63% ± 0.77%AC 43% ± 1.06%

[0215] Unless otherwise indicated, DCM was used to create nanosphere batches in further examples. Although use of DCM as a solvent led to reduced protein recovery, when compared to ethyl acetate, DCM dissolves PLGA better and results in better nanosphere morphology.

[0216] Example 3. Stability of IL-12 Upon Sonication

[0217] To form emulsions when synthesizing PLGA nanospheres, IL-12 can be subjected to sonication twice. Ultrasonication during nanosphere preparation can result in more uniform and smaller nanospheres. However, sensitive proteins like IL-12 become denatured when exposed to intense agitation.

[0218] Therefore, various sonication wattages and times were tested to determine suitable conditions for IL-12-loaded PLGA nanosphere synthesis. IL-12 was suspended in a phosphate-buffered salt solution, and sonicated at 30 Watts, 40 Watts, or 50 Watts. The duration of sonication was 10, 20, 30, 40, or 60 seconds and compared to baseline. The bioactive IL- 12 concentrations remaining after sonication were then determined using ELISA and expressed as a percentage, relative to an initial concentration. IL- 12 was found to be more sensitive to time than wattage, as protein recovery past 30 seconds was less than 10% for all wattages, as seen in FIG.3. The scanning electron microscopy (SEM) images of various IL-12-loaded batches are shown in FIGS. 4 A to 4C. The box in FIG. 3 indicates the combination of times / wattages that recover the most protein, i.e., sonication at 30 Watts to 50 Watts for 5 to 30 sec.

[0219] Unless otherwise indicated, nanospheres made by sonication in subsequent examples were prepared using: Sonication at 30 Watts for 10 to 20 sec; Sonication at 40 Watts for 10 to 15 sec; or Sonication at 50 Watts for 10 to 15 sec.WSGR Ref. 59910-701.601

[0220] Part B. Studies on PLGA Nanospheres.

[0221] Example 4. Synthesis of PLGA Nanospheres

[0222] To create an oil phase, 800 mg of PLGA was dissolved in 32 ml DCM at room temperature for two hours using a magnetic stir bar at 500 RPM.

[0223] To create an aqueous phase of the emulsion, 2400 mg PVA and 96 mg NaCl were dissolved in 120 ml deionized water and microwaved for 10 second bursts in a standard kitchen microwave on setting HIGH until clear. The aqueous phase was then cooled on ice.

[0224] The first emulsion (wl) was made by suspending a material to be encapsulated in 1.2 mL DPBS and added the resulting suspension to the oil phase, which was stirred at 17,500 RPM using a tissue homogenizer for 6 minutes. The stirring was performed on ice. As a control, blank particles were made with no encapsulation material suspended in the DPBS.

[0225] The second emulsion (w2) was formed by slowly pouring the first emulsion into 120 ml of the aqueous phase. During addition of the first emulsion, the aqueous phase was stirred with the tissue homogenizer at 17,500 RPM. Following addition of the first emulsion, stirring was continued for a total of 8 minutes. The resulting suspension was then stirred for 16 hours with a magnetic stir bar at 750 RPM to evaporate the organic solvent.

[0226] Once the solvent was evaporated, the resulting solution was centrifuged at 3500 RPM three times; following each centrifugation step, a pellet of nanospheres can be recovered and resuspended, while the supernatant produced during centrifugation can be collected and stored on ice. The nanospheres were then washed twice via ultracentrifugation at 20,000 RPM for 40 minutes at 4 degrees Celsius, flash-frozen in liquid nitrogen, and stored at -20 degrees Celsius.

[0227] The nanospheres can then be lyophilized under vacuum to remove water from the nanospheres.

[0228] The properties of nanospheres obtained are summarized in Table 3. Nanospheres containing FITC-labeled BSA, described in Table 3, were imaged via confocal microscopy to visualize the internal structure. As shown in FIG. 5A, Z-stacking analysis confirmed that FITC-labeled BSA was successfully incorporated within the nanospheres.

[0229] The morphology of both blank (FIGS. 4 A to 4C) and IL- 12 loaded (FIGS. 5B and 5C) PLGA nanospheres was determined via scanning electron microscopy (SEM) to be spherical in shape, with a mean particle diameter of 201.7±6.7 nm (FIG. 6A) and 138.1±10.8 nm (FIG. 6B), respectively. Zeta potentials of both blank and loaded PLGA nanospheres were also determined, with a decrease in the magnitude of the voltage from -21.3±0.808 mV (FIG. 6C) to -15.1±1.249 mV (FIG. 6D), respectively.

[0230] Nanospheres containing the Alexa 647 dye and fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC) were also successfully prepared using the above procedure.WSGR Ref. 59910-701.601

[0231] TABLE 3. Nanosphere Properties.Encapsulation Material Amount / 1.2 mL DPBS Nanosphere Diameter Zeta Potential (mV) Nanosphere Concentration (Particles / mL) Nothing — 201.7 ± 6.7 -21.3 ± 0.81 6.49 x 109± 6.19 x 108Alexa 647 5 mg — — —BSA-FITC 5 mg — — —IL- 12 0.025 mg 138.1 ± 10.8 -15.1 ± 1.25 1.66 x 109± 4.45 x108

[0232] Example 5. Nanosphere IL-12 Elution Profile

[0233] PLGA nanospheres encapsulating IL-12 were obtained by the process of Example 4. Three different concentrations (500 million particles / mL, 750 million particles / mL, and 1 billion partici es / mL) of IL-12-loaded PLGA nanospheres were prepared in 500 pl of nanosphere release buffer (NRB, 10% HI-FBS and 100 units / ml Pen-Strep in DPBS). The suspensions were then centrifuged for 15 minutes at 4° C. to pellet the nanospheres, after which a 250 ul aliquot of the supernatant was removed and stored at 4° C. While on ice, 250 pl NRB was then added to the pellet to bring the total volume back up to 500 pl, which was resuspended and incubated at 37° C. with constant agitation (750 RPM) for 24 hours. This process was repeated for a total of 12 samples over 12 days (1 time point sample / 24 hours). Each aliquot was stored at 4 degrees Celsius for at least 24 hours to equilibrate with the release buffer before determining IL- 12 concentration via ELISA. The aliquots were analyzed via ELISA for bioactive IL- 12 concentrations released over time.

[0234] The total amount of IL- 12 eluted was determined by ELISA and subsequent area under the curve (AUC) analysis to be: 1907.66 ± 162.00 pg at a nanosphere concentration of 500 million particles / mL; 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.

[0235] The amount of IL-12 eluted per 100,000 nanospheres was determined to be: 0.3815 ± 0.03240 pg / 100,000 particles at a nanosphere concentration of 500 million particles / mL; 0.4440 ± 0.02169 pg / 100,000 particles at a nanosphere concentration of 750 million particles / mL; and 0.3416 ± 0.08489 pg / 100,000 particles at a nanosphere concentration of 1 billion particles / mL.

[0236] The 750 million particles / mL sample reported the most efficient elution kinetics of the three concentrations tested.

[0237] As seen in FIG. 7 A, IL- 12 concentration eluted as a function of time at a nanosphere concentration of 750 million particles / mL is significantly greater than IL-12 concentration eluted as a function of time at a nanosphere concentration of 500 million particles / mL. However, thereWSGR Ref. 59910-701.601can be little difference between IL- 12 elution as a function of time at a nanosphere concentration of 750 million particles / mL and IL-12 elution as a function of time at a nanosphere concentration of 1 billion particles / mL. Also as seen in FIG. 7A, IL-12 nanoparticles show a biphasic elution profile, with a burst phase lasting about 2 days and a sustained release phase lasting about from about day 3 to day 11. Moreover, the total amount of IL- 12 eluted over time at a nanosphere concentration of 750 million particles / mL can be about 75% greater than the total amount of IL-12 eluted 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% greater than the total amount of IL-12 eluted at a concentration of 750 million particles / mL. As seen in FIG. 7B, IL-12 concentration eluted per 100,000 particles, as a function of time, can be greater at a nanosphere concentration of 750 million particles / mL than at a concentration of either 500,000 particles / mL or 1 billion particles / mL.

[0238] As shown in FIG. 7C, the encapsulation efficiency (EE) of recombinant mouse IL-12 (rmIL-12)-loaded nanospheres was calculated using the area under the curve (AUCs) of each elution profile for the three different particle concentrations investigated, using Equation 1. The amount of IL- 12 eluted per 100,000 nanospheres was determined to be 0.44% at a nanosphere concentration of 500 million particles / mL, 0.50% at a nanosphere concentration of 750 million particles / mL, and 0.39% at a nanosphere concentration of 1 billion particles / mL. The average encapsulation efficiency (EE) can be 0.443%±0.0551%. Due to differing elution kinetics from the tested concentrations, EE was reported for all three concentrations and subsequently averaged to reflect overall EE.

[0239] Based on the above data, increasing the nanosphere concentration above 750 million particles / mL cannot offer a significant increase in the elution profile of the drug, possibly due to reduced encapsulation efficiency at high particle concentrations. However, this can be due to the constraints of eluting particles in vitro in a small volume (500 pL) of elution solvent. Elution in a larger volume, e.g., a larger volume of an in vitro elution solvent or an in vivo blood supply, can provide an increased elution profile as the nanosphere concentration increases above 750 million particles / mL.

[0240] Example 6. In Vivo Alexa 647-Loaded PLGA Nanosphere Substrate Biodistribution

[0241] PLGA nanospheres loaded with the fluorescent dye Alexa 647, described in Table 1, were injected into female BALB / c mice. In a first group of mice, the dye-loaded PLGA nanospheres were injected intravenously through the tail vein. In a second group of mice, the dye-loaded PLGA nanospheres were injected intraperitoneally. Dye distribution was monitoredWSGR Ref. 59910-701.601via IVIS imaging over a 76-minute period. At 35 minutes post-injection, both routes of administration resulted in systemic distribution of nanosphere contents, as depicted in FIG. 2. Intravenous injection resulted in generalized distribution of labeled dye through the body of the subject mouse. Intraperitoneal injection resulted in localized distribution of labeled dye within the abdominal cavity of the subject mouse, with the most intense distribution of the dye within the peritoneal cavity. Distribution occurred without any signs of morbidity or mortality.

[0242] Part C. Studies on PLGA Nanospheres with Sonication.

[0243] Example 7. Synthesis of PLGA Nanospheres with Sonication

[0244] To create an oil phase, 250 mg of PLGA was dissolved in 1.51 ml DCM at room temperature. 1 g PVA was dissolved in 100 ml deionized water to create an aqueous phase. The aqueous phase was then cooled on ice. The first emulsion (wl) was made by suspending 12.5 micrograms of IL-12 in about 20±6 microliters DPBS and adding the resulting suspension to the oil phase with sonication at 30 to 50 Watts for 10 to 20 sec.

[0245] The second emulsion (w2) was formed by slowly adding the first emulsion into 5 ml of the aqueous phase. During addition of the first emulsion, the aqueous phase was subjected to sonication at 30 to 50 Watts for 10 to 30 sec. The resulting suspension was then stirred for 3 hours with a magnetic stir bar at 1,000 RPM to evaporate the organic solvent.

[0246] Once the solvent was evaporated, nanospheres were recovered from the resulting solution via three ultracentrifugation treatments at 10,000 g for 15 minutes at 4 degrees Celsius, flash-frozen in liquid nitrogen, and stored at -20 degrees Celsius.

[0247] The nanospheres can then be lyophilized under vacuum to remove water from the nanospheres.

[0248] FIG. 8A shows IL-12-loaded nanospheres prepared using sonication at a sonication power of 30 Watts and a sonication time ranging from 10 sec to 20 sec (Batches 30W10S, 30W15S, and 30W20S of Table 2).

[0249] FIG. 8B shows IL-12-loaded nanospheres prepared using sonication at a sonication power of 40 Watts and a sonication time ranging from 10 sec to 15 sec (Batches 40W10S and 40W15S of Table 2).

[0250] FIG. 8C shows IL-12-loaded nanospheres prepared using sonication at a sonication power of 50 Watts and a sonication time ranging from 10 sec to 15 sec (Batches 50W10S and 50W15S of Table 2).

[0251] As seen in Table 4, sonication with 30 Watts to 50 Watts power for 10 to 20 sec generally produces IL- 12 loaded PLGA nanospheres with an encapsulation efficiency of about 5% to about 10%, and a zeta potential of between -30 and -40 mV.WSGR Ref. 59910-701.601

[0252] TABLE 4. Impact of Sonication Conditions on Nanosphere Properties Sonication Power Particle Batch Sonication Time Encapsulation Zeta Potential (mV) Efficiency %30 W 30W10S 10 sec 9.67 ± 4.25 -36.15 ± 2.2530W15S 15 sec 7.60 ± 3.45 -38.15 ± 4.75 30W20S 20 sec 4.84 ± 0.80 -31.75 ± 6.35 40 W 40W10S 10 sec 6.23 ± 1.29 -31.55 ± 8.3540W15S 15 sec 6.92 ± 0.44 -33.55 ± 7.35 50 W 50W10S 10 sec 8.80 ± 1.88 -36.35 ± 2.8550W15S 15 sec 5.20 ± 3.10 -35.50 ± 4.90

[0253] Example 8. Elution of PLGA Nanospheres Prepared with Sonication

[0254] The IL-12-loaded nanospheres shown in Table 2 were tested for elution characteristics by the procedure of Example 5. Protein concentration per day was determined by ELISA, and the total protein released was determined using the area under the raw elution curve.

[0255] Elution curves obtained by the above procedure are shown in FIG. 9. The elution curve for each batch of Table 2 shows a biphasic curve with an initial burst phase and a sustained release phase. The burst phase can be due to the adsorbed protein on the surface of the nanospheres being released upon resuspension in aqueous medium, while the controlled release phase can be due to protein entrapped within the PLGA matrix. As seen in FIG. 8, for a constant sonication period, e.g., 10 sec., the initial rate of drug release during the burst phase decreases as the sonication power increases. In a batch sonicated for 10 sec at 30 W power (30W10S), between 60% and 65% of the IL-12 can be eluted within one day. In a batch sonicated for 10 sec at 50 W power (50W10S), less than 50% of the IL-12 can be eluted within one day. This suggests that the percentage of protein within the PLGA matrix in the nanosphere core increases as the sonication power increases.

[0256] Part D. Impact of Stirring Speed on PLGA Nanospheres.

[0257] Example 9. Stirring Speed

[0258] To create an oil phase, 800 mg of PLGA was dissolved in 32 ml DCM at room temperature for two hours using a magnetic stir bar at 500 RPM.

[0259] To create an aqueous phase of the emulsion, 2400 mg PVA and 96 mg NaCl were dissolved in 120 ml deionized water and microwaved for 10 second bursts in a standard kitchen microwave on setting HIGH until clear. The aqueous phase was then cooled on ice.

[0260] An IL- 12 suspension was made by suspending 25 micrograms of IL- 12 in 1.2 mL DPBS. Nanospheres were prepared as described in Example 4.

[0261] To observe the impact of stirring speed on encapsulation efficiency of IL-12 in a PLGA nanosphere, and elution profile of IL-12 from PLGA nanospheres, stirring duringWSGR Ref. 59910-701.601preparation of the first and second emulsions was done 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. Encapsulation efficiency as a function of agitation speed is shown in Table 5.

[0262] TABLE 5. Encapsulation Efficiency as a function of stirring speed.Setting on Tissue Homogenizer Agitation Speed (RPM) Encapsulation Efficiency %3 13,125 0.683.5 15,312 0.504 17,500 2.064.5 19,688 0.945 21,875 0.705.5 24,063 0.506 26,250 0.15

[0263] As seen in FIG. 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 gives a high encapsulation efficiency, and a total drug release over a 12-day period of about 4500 pg IL-12. Stirring at about 13,000 to about 20,000 RPM gives an acceptable drug release over a 12-day period of about 3500 pg IL-12 or greater. Stirring at greater than about 22,000 RPM gives a low encapsulation efficiency, and a total drug release over a 12-day period of less than 3,000 pg IL-12. The optimum stirring speed for producing IL-12 loaded PLGA nanospheres can be about 17,500 RPM.

[0264] Part F. Effect of Nanosphere Additives on Release of a Soluble Cytokine.

[0265] Example 11. Release Profiles of Nanospheres Prepared with Nanosphere Additives

[0266] Seven batches of nanospheres containing the drug IL- 12 were prepared. The elution profile of these batches was obtained by following the procedure of Example 5, with sonication at 50 Watts power for 10 sec.

[0267] 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 about 20±6 microliters DPBS and a PVA / water phase. No additives not listed in Example 7 were included. Upon elution of the first batch by the method of Example 5, the drug was released with an initial burst phase lasting 1 to 2 days, and a peak IL-12 concentration of-P65,000 pg / mL, reached on the second day, as shown in FIG. 11.

[0268] The 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 about 20±6 microliters DPBS containing 1.5% w / v trehalose was used. For the second batch, the drug was released with anWSGR Ref. 59910-701.601initial burst phase lasting 3 days, and a peak IL-12 concentration of-P75,000 pg / mL, reached on the third day. As seen in FIG. 11, the presence of trehalose in the protein solution used to make nanospheres significantly increased the overall drug release (measured as area under the curve), compared to drug release from nanospheres made without additional additives.

[0269] The 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 about 20±6 microliters DPBS containing 2% w / v Mg(OH)2 was used. Upon elution of the third batch by the method of Example 5, the drug was released with an initial burst phase lasting 1 day, and a peak IL-12 concentration of-P10,000 pg / mL. As seen in FIG. 11, the presence of Mg(OH)2 in the protein solution used to make nanospheres decreased the overall drug release, compared to drug release from nanospheres made without additional additives.

[0270] For the fourth batch, nanospheres were made by following the procedure of Example 7, using a protein solution made by suspending 12.5 micrograms of IL- 12 in about 20±6 microliters DPBS containing 10% fetal bovine serum (FBS). Upon elution by the method of Example 5, the drug was released with an initial burst phase lasting 3 days, and a peak IL-12 concentration of -Pl 15,000 pg / mL, reached on the second day. The presence of 10% FBS in the protein solution significantly increased the overall drug release (measured as area under the curve), compared to drug release from nanospheres made without additional additives, as seen in FIG. 12.

[0271] For the fifth batch, nanospheres were made by following the procedure of Example 7, using a protein solution made by suspending 12.5 micrograms of IL-12 in about 20±6 microliters DPBS containing 10% fetal bovine serum (FBS), where the protein solution was incubated for 24 hours prior to production of nanospheres. Upon elution by the method of Example 5, the drug was released with an initial burst phase lasting 3 days, and a peak IL-12 concentration of from -Pl 05,000 pg / mL to -Pl 10,000 pg / mL, reached on the third day. As seen in FIG. 14A, the presence of 10% incubated FBS in the protein solution significantly increased the overall drug release from the nanospheres (measured as area under the curve), compared to 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 of FIG. 14B, the incubation of the protein solution for 48 hours prior to the production of nanospheres led to a decrease in total release of IL-12, and lowered the encapsulation efficiency.

[0272] For the sixth batch, nanospheres were made by following the procedure of Example 7, using: a PVA / water phase containing 4% w / v Tween 80; and an oil phase containing 14% w / v Span 60. Upon elution by the method of Example 5, the drug was released with an initial burst phase lasting 1 day, and a peak IL-12 concentration of from -P120,000 pg / mL to -P125,000WSGR Ref. 59910-701.601pg / mL. As seen in FIG. 13, the presence of Tween 80 and Span 60 surfactants in the protein solution increased the peak drug release from the nanospheres by about a factor of 2, compared to peak drug release from nanospheres made without additional additives. The presence of Tween 80 also substantially increased overall drug release.

[0273] For the seventh batch, nanospheres were made by following the procedure of Example 7, using: 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. Upon elution by the method of Example 5, the drug was released with an initial burst phase lasting 1 day, and a peak IL- 12 concentration of about -580,000 pg / mL. As seen in FIG. 13, the presence of both FBS and surfactants in the protein solution increased the peak drug release from the nanospheres by about a factor of about 9, compared to a 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 about a factor of about 4.5, compared to a peak drug release of -115,000 pg / mL from nanospheres made with FBS alone; and increased the peak drug release from the nanospheres by about a factor of about 4.5, compared to a peak drug release of -120,000 pg / mL from nanospheres made with Tween 80 alone.

[0274] TABLE 6 Effect of Nanosphere Additives on Drug Release and Encapsulation Efficiency.Drug ReleaseAdditives Total (pg) Peak (pg / mL) Per 100,000 EE (%) Zeta Potential (mV)Particles (Pg)None 289135.30 65.000.5782 8.8 -36.35 Trehalose 207386.3905 75.000 0.414 5.79 -38.95 Mg(OH)2 26065.70647 10.000 0.0521 2.19 -22.4FBS 285394.6775 -115,000 0.571 6.67 -36.6FBS (incubated 654806.6577 -110,000 1.31 35.71 -39.7for 24 h)Surfactant* 419307.8952 -120.000 0.839 17.39 -32.5 Surfactant* + FBS 1341061.532 -580,000 2.68 87.54 -35.6*Tween 80 + Span 60

[0275] 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 the overall drug release upon elution of the drug IL- 12 from nanospheres can be increased by making the nanospheres in the presence of an additive selected from the group consisting of trehalose, FBS, FBS with 24 hours incubation, a surfactant, or a mixture thereof. Also, FBS with 24 hours incubation, a surfactant, or a mixture of FBS and a surfactant lead to dramatic increases in encapsulation efficiency. The peak drugWSGR Ref. 59910-701.601release and the overall drug release upon elution can be synergistically increased by making the nanospheres in the presence of both FBS and a surfactant.

[0276] As seen in FIGS. 14A and 14B, batches of nanospheres wherein the solutions were sonicated at 50W for 10 seconds (50W10S), FBS with no incubation (“baseline”) (B), FBS with 24 hour incubation (24H), and FBS with 48 incubation (48H) were compared as percent elution (FIG. 14A) and percent of total elution (FIG. 14B). Percent of total allows for accurate comparison of the elution profile versus modified batches. The values for these figures are seen in Table 7. A comparison of the 50W10S elution to the baseline and incubated batches is shown in Table 8.

[0277] TABLE 7 Gel elution totals from 10 mg over 14 days and Encapsulation Efficiencies.TOTAL (pg / mL) TOTAL (ng / mL) (ng / mL) / batch EE %Baseline FBS.l 302893.6686 302.8936686 2398.917855 9.59567142 Baseline FBS.2 430181.3177 430.1813177 2529.466148 10.11786459 Baseline FBS AVG 366537.4931 366.5374931 2464.192001 9.856768006 Incubation: 24.1 h 717214.2512 717.2142512 1738.527345 6.954109379 Incubation: 24.2 h 964011.074 964.011074 2336.762843 9.347051374 Incubation: 24 h AVG 840612.6626 840.6126626 2037.645094 8.150580376 Incubation: 48.1 h 475557.718 475.557718 1339.170534 5.356682136 Incubation: 48.2 h 660407.1071 660.4071071 1859.706414 7.438825655 Incubation: 48 h AVG 567982.4126 567.9824126 1599.438474 6.397753896 MSA / SURF.l 1630803.835 1630.803835 7032.026137 28.12810455MSA / SURF.2 1217626.815 1217.626815 4928.953348 19.71581339 MSA / SURF AVG 1424215.325 1424.215325 5980.489742 23.92195897 FBS / SURF.l 1149762.568 1149.762568 10393.85362 41.57541446 FBS / SURF.2 1370583.004 1370.583004 10175.20823 40.7008329 FBS / SURF AVG 1260172.786 1260.172786 10284.53092 41.13812368WSGR Ref. 59910-701.601

[0278] TABLE 8. Comparison of elution values.T TESTS v 50W10S DAY 1-5Baseline 0.37672959Inc 24 H 0.06955482Inc 48 H 0.03852793MSA / SURF 0.03007545FBS / SURF 0.02631816T TESTS B v 24 / 48 DAY 1-5Inc 24 H 0.03282131Inc 48 H 0.00907923T TESTS 24 v 48 DAY 1-5Inc 24 H vine 48 H 0.02050595

[0279] Part G. Assessing Immunotoxicity and Pharmacodynamics of IL-12 Loaded PLGA Nanosphere Delivery in Healthy Male and Female BALB / c mice

[0280] Example 12. Overview and Design of IL-12 Loaded PLGA Nanosphere Immunotoxicity and Pharmacodynamic Study

[0281] Systemic administration of interleukin (IL)-12 induces potent anti-tumor immune responses in preclinical cancer models through the systemic activation of effector immune cells and release of proinflammatory cytokines. IL-12-loaded PLGA nanospheres (IL12ns) are hypothesized to improve therapeutic efficacy and thwart unwanted side effects observed in previous human clinical trials. Through the investigation of peripheral blood and local tissue immune responses in healthy BALB / c mice, the immune-protective pharmacodynamics of IL12ns were suggested. Nanospheres increased pro-inflammatory plasma cytokines / chemokines (IFN-y, IL-6, TNF-a, and CXCL10) without inducing maladaptive transcriptomic signatures in circulating peripheral immune cells. Gene expression profiling revealed activation of pro-inflammatory signaling pathways in systemic tissues, the likely source of these effector cytokines. These data support that nanosphere pharmacodynamics, including shielding IL- 12 from circulating immune cells, depositing peripherally in systemic immune tissues, and then slowly eluting bioactive cytokine, thereafter, are essential to safe immunostimulatory therapy.

[0282] The impact of immune surveillance and cancer immunoediting theories on the development of immunotherapies has been limited. The cornerstone of these theories is that an immunosurveillance-like state must be maximized to overcome the constitutive immunosuppression seen in cancer patients. In essence, immunotherapies would then promote the elimination of tumor cells through enhanced effector immune responses driven by overcoming this unbalanced cancer-immune cell equilibrium. Targeted immunotherapeutic treatments have been proposed to avoid immunologic toxicity (immunotoxicity) whileWSGR Ref. 59910-701.601maintaining therapeutic efficacy. Unfortunately, pleiomorphic tumors are often capable of downregulating the presentation of the associated target antigens. These tumor-immune escape mechanisms thereby limit effective immune responses and overall efficacy. Even if these immunosuppressive interactions are blocked, the immune system often requires an additional stimulus to create the pro-inflammatory and supra-normal immune response required for tumor cell cytotoxicity. Systemic administration of interleukin (IL)-12 has been proposed to induce these desired anti-tumor immune responses.

[0283] IL-12 is an immunostimulatory cytokine. This pro-inflammatory cytokine is a key regulator of cell-mediated immune responses and is produced endogenously by dendritic cells, monocytes, and Ml-like macrophages. Importantly, preclinical studies have consistently suggested that IL-12’ s antitumor effects are most evident when given systemically as opposed to locally via intratumoral delivery. This finding supported by observed increases in systemic immune cell activation, production of proinflammatory effector cytokines, and indirect inhibition of pro-tumoral angiogenesis with systemic vs non-systemic administration.

[0284] While these initial preclinical data were promising, clinical trials using intravenous (i.v.) recombinant human IL-12 (rhIL-12) concluded that bolus IL-12 injection exhibited limited therapeutic efficacy. This limited efficacy was associated with signs of clinical toxicity including lymphopenia and elevated liver enzymes. Signs of immunotoxicity, evidenced by the reduction in serum IFN-g levels with continued treatment, were also evident. Importantly, dosing strategies for cytokine-based immunotherapies developed in the late 1990s were influenced by approaches deployed for cytotoxic chemotherapy at this time, namely, maximal tolerable dosing strategies. While still important to monitor for signs of clinical toxicity during treatment, a maximal tolerable dosing (MTD) strategy likely does not translate to treatment with immunotherapies.

[0285] Strategies to both reduce the required loading dose and monitor the immune-related changes associated with immunostimulatory therapies are critical for their clinical efficacy. To address these barriers, IL-12-loaded PLGA nanospheres (IL12ns) were developed (using methods described in above examples 1-16) that release their contents systemically in a slow and controlled manner and administered to healthy BALB / c mice.

[0286] The purpose of this study was to assess the toxicity of the IL12ns vector system in healthy BALB / c mice through repeated blood sampling and organ histological analysis. Both systemic and tissue-resident immune responses to IL12ns therapy, delivered at three doses (10, 0.1, and 0.001 mg of IL-12ns; corresponding to approximately 1600, 16, and 0.16 ng / kg / of IL-12 per day), were compared to a daily IL-12 bolus MTD strategy (10,000 ng / kg / of IL-12 per day - positive control) and saline (negative) control. Systemic immunophenotyping with anWSGR Ref. 59910-701.601immune diagnostic platform (IDP) occurred at designated serial blood sampling timepoints (T) including baseline (Tl), 12-h (T2), day 4 (T3), day 8 (T4), day 11 (T5), day 15 (T6), and day 18 (T7). This analysis was followed by both traditional histopathological analysis and targeted gene-expression profiling of euthanasia-harvested tissues (FIGS. 15A-P).

[0287] Example 13: IL-12 Loaded PLGA Nanosphere Immunotoxicity and Pharmacodynamic Study: Methods

[0288] Synthesis and Characterization of PLGA IL12ns

[0289] IL12ns were prepared via the double-emulsion solvent evaporation (DESE) method with ultrasonication as previously described in Examples 1-16. Briefly, under sterile conditions, the primary emulsion was formed using an Omni International (Kennesaw, GA) Sonic Ruptor 250 microprobe sonicator set at 50% power to agitate 150 pL of aqueous (aq) solution (including 12.5 pg rmIL-12), 250 mg Resomer RG 503 H, Poly(D, L-lac-tide-co-glycolide), and 14% w / w Sorbitan Monostearate dissolved in 1.5 mL dichloromethane (DCM) for 10 s in a Pyrex Vista test tube held on ice bath. The 150 pL aq solution contained 136 pL of 10% w / v mouse serum albumin (MSA) and 14 pL of rmIL-12 (BioLegend). The primary emulsion was transferred to a second Pyrex Vista test tube containing 1% w / v polyvinyl alcohol (PVA) and 4% w / v Polysorbate 80 in deionized water, and the solution was sonicated on ice at 50% for an additional 10 s to create the double emulsion. The double emulsion was subsequently stirred for a minimum of 3 h at 1000 rpm to allow organic solvent evaporation, washed via resuspension and ultracentrifugation at 47,807 g three times for 40 min, flash-frozen in liquid nitrogen, lyophilized using the Labconco (Fort Scott, KS) 700802000 FreeZone 8L -50°C Benchtop Freeze Dryer, and stored in a vacuum desiccator. IL 12ns batches were prepared weekly to ensure quality controlled batches were injected at dosing timepoints.

[0290] The morphology of the IL 12ns was determined by scanning electron microscopy (SEM) (Nanocomposix, San Diego, CA) Hitachi (Tokyo, Japan) S-4700 FE-SEM. To determine IL12ns elution profile, a 0.6% Lonza (Allendale, NJ) SeaPlaque Agarose gel was prepared. Briefly, IL12ns were prepared by resuspending 1 mL of lyophilized particle in D-PBS before aliquoting 10 mg of IL12ns into a 1.5 mL tube. IL12ns were spun for 15 min at 10,000 x g at 4°C before pouring off supernatant, leaving pelleted particles. Upon cooling the gel to 37°C, 10 mg of IL12ns were resuspended in 1 mL of SeaPlaque agarose gel and plated into a 24-well standard cell culture plate. Agarose / particle was then gelled at 4°C for minimum of 15 min before incubating in standard cell culture conditions (37°C, 5% CO2) for an additional 15 min. One mL of release buffer [10% fetal bovine serum (FBS) in D-PBS with 1% penicillin / streptomycin] was added on top of the gel to begin the elution study. Samples were collected every 24 h by gently removing the supernatant above the gel (now containing elutedWSGR Ref. 59910-701.601rmIL-12) and replacing 1 mL of release buffer. This collected supernatant was then analyzed by enzyme-linked immunosorbent assay (ELISA) for rmIL-12 concentration using murine IL- 12 ELISA Max Deluxe kit (BioLegend). Concentration (mg / mL) from each IL 12ns batch was calculated by weighing dried IL 12ns product after lyophilizing 1 mL of suspended IL 12ns in a pre-weighed 1.5 mL tube for 12 h. IL12ns yield (mg / mL) was calculated by multiplying this concentration (mg / mL) by the total volume of which the IL 12ns batch was resuspended (4 mL). Encapsulation efficiency (EE) of IL12ns was determined as disclosed herein. Dynamic light scattering (DLS) analysis was performed on a Malvern Panalytical (Worcestershire, UK) Zetasizer Nano ZS instrument. All values are presented as an average ± standard error of the mean (SE) (n = 4 or 6).

[0291] Spectral Flow Cytometric Analysis

[0292] Peripheral blood mononuclear cells (PBMCs) were analyzed via a 16-marker myeloid and 23-marker lymphoid panel on the Cytek Biosciences (Fremont, CA) Aurora at each peripheral blood sampling timepoint using the antibodies outlined in the Table 9 and the appended gating strategy FIGS. 22A-B. The lymphoid and myeloid panels were designed using a Cytek Full Spectrum Viewer to achieve a complexity score of 5.63 and 4.55, respectively, before full panel titration using calculated staining indexes. Briefly, blood was prepared into a PBMC single cell suspension using Miltenyi Biotec (Gaithersburg, MD) Red Blood Cell Lysis lOx solution with a modified laboratory protocol. For each staining day, a staining plate contained individual PBMCs for full staining with all dyes and antibodies, as well as PBMCs from euthanized instrument control animals (n = 2) to generate no stain controls, single-stain samples for each fluorophore in the staining panel (sometimes performed on compensation beads), and fluorescence minus one (FMO) controls for specific fluorophores. A live / dead master mix sufficient for the number of samples plus one was prepared and added to each sample, live / dead single stain control, and all necessary FMO controls before incubation in the dark for 30 min at 4°C. Following a wash by adding 100 mL of PBS to each well before centrifugation at 1200 rpm at 4°C for 5 min, an Fc blocking master mix with volume sufficient for the number of samples plus one using 0.4 mL of Jackson Immunoresearch (West Grove, PA) ChromPure Rat IgG, 0.4 mL of ChromPure Mouse IgG, and 19.2 mL D-PBS was added to each sample well before incubation in the dark for 30 min at 4°C. Samples were again washed before cell surface staining with a freshly prepared surface stain master mix sufficient for the number of full-stain samples plus one containing 50 mL Brilliant Staining Buffer and each surface-staining antibody. To FMO control wells, each antibody-fluorophore conjugate in the staining panel minus one was added to instrument control PBMCs. Samples were then incubated in the dark for 30 min at 4°C before two washing steps as previously described. Then, 200 mL of preparedWSGR Ref. 59910-701.601fixation reagent was added to all sample wells and incubated in the dark for 30 min at 25°C. Samples were then washed by adding 200 mL of lx permeabilization buffer to each well and centrifuging at 1200 rpm, 25°C, for 5 min. Following decantation, an intracellular stain master mix sufficient for the number of samples plus one containing 30 mL permeabilization buffer and each intracellular-staining antibody was added to each full-stain sample well, appropriate singlestain, and FMO control wells. Samples were again incubated in the dark for 30 min at 4°C before washing two times by adding 200 mL of lx permeabilization buffer to each well and centrifuging at 1200 rpm, 25°C, for exactly 5 min. Finally, cells were resuspended in 200 mL D-PBS and transferred to a clean 96-well plate that was then sealed and packed for overnight shipping to UVA Flow Cytometry Core RRID: SCR 017829, Charlottesville, VA) for spectral flow cytometric analysis on the Cytek Aurora (Cytek Biosciences) equipped with 355nm, 405nm, 488nm, 561nm, and 633nm lasers. Acquisition was performed using the automated sample loader set to acquire 200 mL of each sample stained and prepared in 96-well plates. For analysis, data were unmixed using SpectroFlo 3.0 software (Cytek Biosciences) (FIG. 15B). Unmixed flow cytometry standard (FCS) files were analyzed using FCS Express [version 7, De Novo Software (Pasadena, CA)] and pre-determined gating strategies. Plots and statistics (% of all live cells) were exported using the Batch Export function from FCS Express to Microsoft Excel (Redmond, VA) before further statistical analysis via JMP (JMP Statistical Discovery, Cary, NC) [version Prol6] and graphical representation using GraphPad (San Diego, CA) Prism [version 9.4.1],

[0293] Plasma Analysis

[0294] Peripheral plasma cytokine and chemokine analyses at all blood collection timepoints were conducted using the BioLegend LEGENDplex Cytokine Release Syndrome Panel for quantification of IFN-g, IL-10, CCL4 (MIP-ip), IFN-a, CXCL9 (MIG), CXCL10 (IP-10), tnf-a, IL-6, VEGF, IL-4, CCL3 (MIP-la), CCL2 (MCP-1), and GM-CSF concentration according to manufacturer specifications for a v-bottom 96-well plate. Briefly, following collection of peripheral blood in 100 mL Microvette EDTA tubes, plasma was isolated from 50 mL of peripheral blood by centrifuging for 20 min at 1,000 x g within 30 min of blood collection. Plasma was then stored at -80°C for later analysis. Each plasma sample underwent one freeze / thaw cycle and was processed at a 1:2 dilution according to manufacturer specifications. Multiplexed-plasma samples were then read on the Life Technologies Attune Nxt (ThermoFisher Scientific) at the UVA Flow Cytometry Core Facility (RRID: SCR 017829). Samples were gated, processed, and analyzed using the online BioLegend LEGENDplex Data Analysis Software [version 2022-07-15] before statistical analysis via JMP [version Pro 16] and graphical representation using GraphPad Prism9 [version 9.4.1] (FIG. 15B).WSGR Ref. 59910-701.601

[0295] RNA extraction, library preparation, and next-generation sequencing of PBMCs

[0296] Total RNA was isolated from PBMCs collected at all sampling timepoints following preparation into single cell suspension using the Qiagen (Germantown, MD) RNeasy Plus Mini Kit. Total RNA sample quality was assessed for purity by the Thermo / Spectronic BioMate3 (Spectropho-tomer, ThermoScientific) using standard OD 260 / 280 range before being stored in 30 mL RNase free water at -80°C. Samples were then processed by the UVA Genome Analysis and Technology Core, RRID: SCR_018883 using Standard Operating Procedures for an n = 3 for each group (10 mg IL12ns, 0.1 mg IL12ns, MTD, and saline control) for both males and females at all sampling timepoints for a total of 168 total RNA samples. Total RNA quality was checked using the Agilent (Santa Clara, CA) Tape Station 4200 along with either the RNA (5067-5576, 5067-5577, 5067-5578) or RNA HS (5067-5579, 5067-5580) kit as appropriate to the RNA concentration provided and as described by the manufacturer’s protocols. RNA-seq libraries were prepared using New England Biolab’s (Ipswich, MA) NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490L), Globin & rRNA Depletion Kit (E7750X), Ultra II Directional RNA Library Prep Kit for Illumina (E7760L), and Multiplex Oligos for Illumina (E6440L) according to the manufacturer’s instructions. Libraries were checked for quality, size, and concentration using the Agilent Tape Station 4200 High Sensitivity D5000 kit (5067-5592, 5067-5593) and Qubit 3.0 ThermoFisher dsDNA HS Assay Kit (Q32854). Libraries were pooled at equimolar concentrations and sequenced using Illumina’s NextSeq 2000 on one P2-100 (20046811) kit and four P3-100 (20040559) kits according to the manufacturer’s instructions (FIG. 15B).

[0297] Bulk PBMC RNA-seq analysis

[0298] Following concatenation of FASTQ files each sequencing run (n = 2), alignment of paired-end short reads to the reference mouse genome (mm 10) was performed with subread v2.0.1. Reads aligned to RefSeq transcripts were summarized at the gene level by feature counts. The gene expression level was measured by reads per kilobase of exon model per million mapped (RPKM). Differentially expressed (DE) genes were predicted by EdgeR3 with FDR <0.05, fold change >1.5, and CPM (count per million; log2) > 0. The resulting logFc’s were then used for targeted Gene Set Enrichment Analysis (GSEA) [version GSEA 4.3.2] for analysis of four Reactome gene sets, including Interleukin- 12 signaling, Interferon gamma signaling, Interleukin- 10 signaling, and TNF signaling. Unbiased GSEA of both the Reactome and Hallmark gene sets were also performed. RPKMs for those related / matching genes from flow cytometric and cytokine / chemokine panels, as well as the above Reactome pathways, were then analyzed using JMP Pro 16, with statistical significance set at p < 0.01.WSGR Ref. 59910-701.601

[0299] Necropsy and histopathology assessment

[0300] At day 18 euthanasia (T7) (FIG. 15 A), following cardiac puncture exsanguination, mice were humanely euthanized via cervical dislocation under anesthesia. Necropsy for collection of heart, liver, spleen, lungs, and kidneys was then performed. Each tissue was immediately placed in 5 mL of FormaFix (prepared per manufacturer protocol) before serially transecting, paraffin embedding, slicing with a microtome at 10 mm, and placement on glass slides. Tissues were analyzed by two independent board-certified pathologists at both the WVU School of Medicine (WVUSOM) and University of Pittsburgh Medical Center (UPMC), Departments of Pathology, as well as a Veterinarian Medical Doctor at the University of Pittsburgh School of Medicine, blinded to both group and outcome.

[0301] For evaluation of the liver, specimens were examined for the following: portal inflammation (predominant cell type, granulomas present), lobular inflammation (predominant location, predominant cell type, granulomas present), bile duct inflammation (cholangitis), steatosis (predominantly microvesicular steatosis), cholestasis, hepatocyte single cell necrosis, hepatocyte grouped (confluent) necrosis, hepatocyte ballooning (hydropic change), hepatocyte mitotic activity, and other hepatocyte changes (e.g., dysplasia, pigment deposition, nuclear or cytoplasmic inclusions). If present, bile duct injury (nuclear displacement, eosinophilia, vacuolization), ductopenia, bile ductular reaction / proliferation, sinusoidal congestion, sinusoidal dilatation, vascular thrombosis, vascular inflammation, fibrosis, or tumor was specified.

[0302] For the spleen, specimens were examined for hypoplasia, increased apoptosis, white pulp atrophy, red pulp telangiectasis, red pulp atrophy, red pulp congestion, increased erythrophagocytosis, fibrosis, increased pigment, increased macrophage aggregates, increased plasma cells, white pulp hyperplasia, red pulp adipocyte hypercellularity, increased mast cells, peri-splenic inflammation, stromal cell hyperplasia, and increased extramedullary hematopoiesis (EMH).

[0303] For the kidneys, specimens were examined for the following: cortical interstitial inflammation (predominant cell type, granulomas present), medullary interstitial inflammation (predominant cell type, granulomas present), tubular inflammation (tubulitis) (predominant cell type, involved tubule location), glomerulitis (predominant cell type), arterial inflammation (arteritis), tubular degen eration / necrosis (indicating the areas involved with tubular injury), tubular vacuolization, vacuolization pattern, tubular casts, cast type, tubular dilatation, interstitial renal hemorrhage, renal infarct, vascular thrombosis, vascular necrosis, vascular smooth muscle vacuolization, glomerulopathy, or glomerulonephritis.

[0304] For the lung, specimens were examined for interstitial inflammation (predominant inflammatory cell type, granulomas present), airway (bronchial / bronchiolar) inflammation,WSGR Ref. 59910-701.601alveolar injury (if inflammation is present, predominant cell type, indicate granuloma presence), vascular thrombosis, vascular inflammation (vasculitis), vascular necrosis, pulmonary fibrosis, and presence of tumor.

[0305] For evaluation of the heart, specimens were analyzed for endomyocardial inflammation (predominant cell type, granuloma present), myocardial necrosis, myocardial fibrosis, and coronary artery changes (including thrombosis, arteritis, atherosclerosis).

[0306] For quantification of histopathological findings, total pathology (total path), total necrosis, and total inflammation scores were calculated. Presence of the histopathological finding was indicated as a “1”, with absence of the finding indicated as a “0”. The total necrosis score was calculated by summing both necrotic and fibrotic histopathological findings for each specimen. The total inflammation score was calculated by summing those non-necrotic and non-fibrotic histopathological findings. The total pathology score equals the summation of both total necrosis and inflammation scores. Data were then transformed to generate non-zero findings in Microsoft Excel using the below formula where N represents number of animals within each group observed:r,,,....,, of stooathcfoqical findings) 4-1 Transformed Hfstopatholoqfcat rinding —! - —: - - — - N+2

[0307] Following this data transformation, histopathological findings were then normalized to background by dividing the total pathology (total path), total necrosis, and total inflammation scores for each experimental animal by the average total pathology (total path), total necrosis, or total inflammation score for the respective matched-sex saline control. This normalization was completed for each of the three independent pathological assessments and accounts for any subjectivity in analysis by three independent reviewers. Normalized scores for male and female experimental groups were then plotted within violin plots using GraphPad Prism9 [version 9.4.1],

[0308] RNA extraction from formalin-fixed, paraffin embedded liver, spleen, and lung specimens

[0309] A total of 72 blocks of FFPE mouse tissue (24 liver, lung, and spleen specimens) representing an n = 3 for each group (10 mg IL12ns, 0.1 mg IL12ns, MTD, and saline control), both male and female, were used for RNA extraction. Four slices of 10 mm thickness from each selected block were deparaffinized using DL-Limonene (MilliporeSigma, Burlington, MA) and digested with proteinase K (Qiagen). RNA was then isolated using Qiagen RNeasy Plus Mini Kit according to manufacturer protocol. The final volume of extracted RNA was 14 pL RNA concentration and purity were assessed using a NanoDrop instrument. Sample concentration wasWSGR Ref. 59910-701.601measured at 260 nm and 280 nm. The ratio of optical density 260 / 280 and 260 / 230 were used to test for protein and phenol contamination, respectively.

[0310] NanoString nCounter analysis of formalin-fixed, paraffin-embedded liver, spleen, and lung specimens

[0311] A custom 42-marker nCounter CodeSet panel including NanoString (Seattle, WA) probes for Alasl, Argl, Ccl2, Ccl3, Ccl4, CD19, PD-L1, CD4, CD8a, CTLA-4, CxcllO, Cxcl9, Eeflg, G6pd, Gapdh, Gbp2, Gbp3, Havcr2, Hprt, Ifng, 1110, Il 12rb 1, Il lb, I12rg, 116, Irfl, Itgam, Itgax, Lag3, Ly6g, Ncrl, Nos2, Polrlb, Polr2a, Rpll9, Sdha, Socsll, Socs3, Statl, Stat3, Stat4, and Tnf was designed for targeted gene expression analysis of FFPE-isolated RNA from liver, spleen, and lung specimens collected at euthanasia. An additional custom 30-marker nCounter CodeSet Plus panel including probes for Abcbl 1, Abcb4, Abcc2, Abcc3, Apexl, Btg2, Casp3, Ccngl, Cd36, Cdknla, Cypla2, Cyplbl, Fasn, Fmol, Gadd45a, Gclc, Gpxl, Gsr, Hmoxl, Icaml, Lpl, Mt2, Nqol, Ppara, Rbl, Rbpl, Serpinel, Srebfl, Thrsp, Txnrdl was also designed for targeted gene expression analysis of the same FFPE-isolated RNA from liver specimens for analysis of drug-induced liver toxicity. Alasl, Eeflg, G6pd, Gapdh, Hprt, I12rg, Polrlb, Polr2a, Rip 19, and Sdha were included as reference or housekeeping genes for each tissue analyzed. NanoString probes are made with target-specific sequences and tag-specific sequences at 5' and 3' tailing ends. The RNA samples (150 ng) were incubated for 24 h at 65°C in a hybridization buffer containing the CodeSet (reporter and capture probes). Hybridized samples were processed using the Prep Station high sensitivity protocol, 3 h per 12-sample cartridge. The Prep Station purifies the RNA / probe complexes and places them in a cartridge where they are immobilized and aligned for data collection. Data acquisition was carried out using the NanoString nCounter Digital Analyzer with the ‘Max’ Field of View (FOV) setting to acquire 555 images per sample in a 5-h scan per cartridge as previously described.

[0312] The NanoString probes used for these analyses are listed in Table 14.Table 14. NanoString probesNanoString nCounter custom CodeSet NanoString NM 021022.2: 1660Probes, AbcbllNanoString nCounter custom CodeSet NanoString NM 008830.1:1945Probes, Abcb4NanoString nCounter custom CodeSet NanoString NM 013806.2:4600Probes, Abcc2NanoString nCounter custom CodeSet NanoString NM 029600.3:2730Probes, Abcc3NanoString nCounter custom CodeSet NanoString NM 020559.2: 1034Probes, AlaslWSGR Ref. 59910-701.601NanoString nCounter custom CodeSet NanoString NM 009687.2:289 Probes, ApexlNanoString nCounter custom CodeSet NanoString NM 007482.3:626 Probes, ARG1NanoString nCounter custom CodeSet NanoString NM_007570.2:50 Probes, Btg2NanoString nCounter custom CodeSet NanoString NM 009810.2: 630 Probes, Casp3NanoString nCounter custom CodeSet NanoString NM 011333.3:415 Probes, CCL2NanoString nCounter custom CodeSet NanoString NM 011337.2:219 Probes, CCL3NanoString nCounter custom CodeSet NanoString NM 013652.2:200 Probes, CCL4NanoString nCounter custom CodeSet NanoString NM_009831.2:545 Probes, CcnglNanoString nCounter custom CodeSet NanoString NM 009844.2: 1697 Probes, CD19NanoString nCounter custom CodeSet NanoString NM 007643.3: 1520 Probes, Cd36NanoString nCounter custom CodeSet NanoString NM 013488.2:422 Probes, CD4NanoString nCounter custom CodeSet NanoString NM 001081110.2:355 Probes, CD8aNanoString nCounter custom CodeSet NanoString NM 007669.4: 1670 Probes, CdknlaNanoString nCounter custom CodeSet NanoString NM 009843.3: 1475 Probes, CLTA-4NanoString nCounter custom CodeSet NanoString NM 021274.2: 194 Probes, CXCL10NanoString nCounter custom CodeSet NanoString NM_008599.4:112 Probes, CXCL9NanoString nCounter custom CodeSet NanoString NM 009993.3:975 Probes, Cypla2NanoString nCounter custom CodeSet NanoString NM_009994.1:114 Probes, CyplblNanoString nCounter custom CodeSet NanoString NM 026007.4:338 Probes, EeflgNanoString nCounter custom CodeSet NanoString NM 007988.3:6560 Probes, FasnNanoString nCounter custom CodeSet NanoString NM 010231.3:514 Probes, FmolNanoString nCounter custom CodeSet NanoString NM 008062.2:2030 Probes, G6pdNanoString nCounter custom CodeSet NanoString NM 007836.1:654 Probes, Gadd45aWSGR Ref. 59910-701.601NanoString nCounter custom CodeSet NanoString NM_008084.1:755 Probes, GapdhNanoString nCounter custom CodeSet NanoString NM_010260.1:1996 Probes, GBP2NanoString nCounter custom CodeSet NanoString NM 018734.3: 1894 Probes, GBP3NanoString nCounter custom CodeSet NanoString NM_010295.2:1102 Probes, GclcNanoString nCounter custom CodeSet NanoString NM 008160.5:315 Probes, GpxlNanoString nCounter custom CodeSet NanoString NM 010344.4: 1507 Probes, GsrNanoString nCounter custom CodeSet NanoString NM 134250.2: 134 Probes, HAVCR2NanoString nCounter custom CodeSet NanoString NM 010442.2:610 Probes, HmoxlNanoString nCounter custom CodeSet NanoString NM_013556.2:30 Probes, HprtNanoString nCounter custom CodeSet NanoString NM 010493.2:2195 Probes, IcamlNanoString nCounter custom CodeSet NanoString NM 008337.3:402 Probes, IFNGNanoString nCounter custom CodeSet NanoString NM 010548.2:250 Probes, IL 10NanoString nCounter custom CodeSet NanoString NM 008353.2:2765 Probes, IL12RB1NanoString nCounter custom CodeSet NanoString NM_008361.3:108 Probes, IL1BNanoString nCounter custom CodeSet NanoString NM 013563.3:566 Probes, I12rgNanoString nCounter custom CodeSet NanoString NM_031168.1:200 Probes, IL6NanoString nCounter custom CodeSet NanoString NM_008390.2:845 Probes, IRF1NanoString nCounter custom CodeSet NanoString NM_008401.2:155 Probes, ILGAMNanoString nCounter custom CodeSet NanoString NM 021334.2:327 Probes, ITGAXNanoString nCounter custom CodeSet NanoString NM 008479.1:1700 Probes, LAG3NanoString nCounter custom CodeSet NanoString NM_008509.2:802 Probes, LplNanoString nCounter custom CodeSet NanoString XM_909927.2:91 Probes, Ly6gNanoString nCounter custom CodeSet NanoString NM 008630.2: 105 Probes, Mt2WSGR Ref. 59910-701.601NanoString nCounter custom CodeSet NanoString NM 010746.3:995 Probes, NCR1NanoString nCounter custom CodeSet NanoString NM 010927.3: 1541 Probes, NOS2NanoString nCounter custom CodeSet NanoString NM 008706.5:430 Probes, NqolNanoString nCounter custom CodeSet NanoString NM 021893.2:515 Probes, PD-L1NanoString nCounter custom CodeSet NanoString NM 009086.2:2795 Probes, PolrlbNanoString nCounter custom CodeSet NanoString NM 001291068.1:2768 Probes, Polr2aNanoString nCounter custom CodeSet NanoString NM 011144.2: 1345 Probes, PparaNanoString nCounter custom CodeSet NanoString NM 009029.2: 1590 Probes, RblNanoString nCounter custom CodeSet NanoString NM 011254.5:709 Probes, RbplNanoString nCounter custom CodeSet NanoString NM_009078.1:20 Probes, Rpll9NanoString nCounter custom CodeSet NanoString NM 023281.1:250 Probes, SdhaNanoString nCounter custom CodeSet NanoString NM 008871.2: 1822 Probes, SerpinelNanoString nCounter custom CodeSet NanoString NM 009896.2:440 Probes, SOCS1NanoString nCounter custom CodeSet NanoString NM 007707.3: 1952 Probes, SOCS3NanoString nCounter custom CodeSet NanoString NM_011480.1:1145 Probes, SrebflNanoString nCounter custom CodeSet NanoString NM 001205313.1:430 Probes, STAT1NanoString nCounter custom CodeSet NanoString NM 213659.2: 1360 Probes, STAT3NanoString nCounter custom CodeSet NanoString NM 011487.4: 1816 Probes, STAT4NanoString nCounter custom CodeSet NanoString NM_009381.2:760 Probes, ThrspNanoString nCounter custom CodeSet NanoString NM 013693.2:514 Probes, TNFNanoString nCounter custom CodeSet NanoString NM 015762.2:2245 Probes, TxnrdlWSGR Ref. 59910-701.601

[0313] Quantification and Statistical Analysis

[0314] Statistical power analysis determined that a minimum of eight mice (per sex) was sufficient to detect immunophenotypic differences in NK cells via spectral flow cytometry between treated and untreated mice, to obtain 80% power, a desired p value of 0.05, and an anticipated standard deviation (STDEV) of 33%. First, for spectral flow cytometric, cytokine, and chemokine analyses, data were analyzed using a two-factor analysis of variance (ANOVA) with interaction stratified by each sex and marker. A Bonferroni correction was applied separately within each flow cytometric, cytokine, or chemokine analysis to account for the large number of significance tests completed. Bonferroni correction factor was calculated using a multiple of the number of cellular population / markers (myeloid - 112, lymphoid - 134, NK cells - 159 markers, cytokine / chemokine - 13), timepoints (7), and sex (2) for each analysis. Only those cellular populations / markers deemed statistically significant in the above analysis were subsequently analyzed using a one-factor ANOVA with group as the factor of interest. Those analyses were performed separately for each timepoint, sex, and cellular population / marker. Post hoc pairwise means comparisons were done using Tukey’s HSD procedure. A further Bonferroni correction was used to adjust for the large number of inferences tested, with statistical significance for all remaining cellular populations / markers set at p < 0.0001. Error bars represent standard error of the mean (SE). RNA-Seq data generated from the study were deposited to GEO with an accession # of GSE241939. For bulk PBMC RNA-seq analysis, those GSEAs with an NES >1.6 and FDR <0.05 were considered statistically significant (version GSEA _4.3.2). Additionally, a one-factor ANOVA with post hoc pairwise means comparison by Tukey’s HSD procedure was used to compare gene expression (RPKM) between experimental groups, with significance set at p < 0.01. For histopathological analysis, the total pathology (total path), total necrosis, and total inflammation scores were compared between experimental groups using a one-way ANOVA with statistical significance set at p < 0.05 [GraphPad Prism9 (version 9.4.1)]. NanoString nCounter analysis was performed using NanoString nSolver Analysis Software 4.0. Following selection and annotation, samples were processed using background thresholding, with a threshold count value of 20, positive control normalization, and CodeSet content normalization using housekeeping genes to compute a normalization factor. Low count data were omitted at this threshold count value of 20 with an observation frequency of 0.99. Housekeeping selection for each specimen was made using Geonorm standards within the nCounter Advanced Analysis software (version 2.0.134). Differentially expressed genes between treatment groups and saline controls were calculated using the nCounter Advanced Analysis software. To estimate differential expression, either the mixed negative binomial model using the mle function to run the Wald test, the simplified negative binomial model using theWSGR Ref. 59910-701.601glm.nb function, or the log-linear model using the Im function were deployed to determine estimated log fold-change. This estimated log fold-change and Benjamini-Yekutieli adjusted p value (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001) were then calculated for each experimental group in comparison to saline controls. Log2 normalized expressions for each experimental group are graphed using GraphPad Prism9 (version 9.4.1). Each analysis was completed on both sexes independently. Statistical parameters are reported in the accompanying figure legend.

[0315] Additional reagents, resources, assays, and sources of deposited data used for the IL-12 loaded PLGA nanosphere immunotoxicity study are disclosed in Tables 11-13.

[0316] Table 11. Chemicals, Peptides, Recombinant proteins, Experimental Animals, Oligonucleotides, Software and Algorithms, and Additional ResourcesREAGENT or RESOURCE SOURCE IDENTIFIERBD Horizon Brilliant Stain BD Biosciences 566349BufferRecombinant murine IL-12 (p70) BioLegend 577008(carrier free)Corning Regular Fetal Bovine Fisher Scientific MT35011CVSerumCorning Cell Culture Buffers: Fisher Scientific 21031CVDulbecco’s Phosphate-BufferedSalt Solution IXGibco Fetal Bovine Serum, Fisher Scientific 10-082-147certified heat inactivated, UnitedStatesChromPure Rat IgG, Whole Jackson ImmunoResearch 012-000-003MoleculeChromPure Mouse IgG, Whole Jackson ImmunoResearch 015-000-003MoleculeSeaPlaque Agarose Lonza 50100Dichloromethane Millipore Sigma 320269Resomer RG 503 H, Poly(D, L- Millipore Sigma 719870lactide-co-glycolide)Albumin from mouse serum Millipore Sigma A3559Red Blood Cell Lysis Solution Miltenyi Biotech 130-094-183(10x)Sorbitan Monosterate, NF Spectrum Chemical SPA63Polyvinyl Alcohol, USP Spectrum Chemical Pl 180Polysorbate 80, NF Spectrum Chemical PO138Gibco RPMI 1640 Medium ThermoFisher Scientific 21875034Invitrogen eBioscienceTM ThermoFisher Scientific 00-5523-00F oxP3 / T ranscriptionF actorStaining Buffer SetWSGR Ref. 59910-701.601DL-Limonene Sigma Aldrich 8.14546Experimental models: The Jackson Laboratory 000651Organisms / strains: BALB / cmice: 7-8-week-old male andfemaleOligonucleotides: NEBNext New England Biolabs Inc. E6440LMultiplex Oligos for Illumina(96Unique Dual Index Primer Pairs)Subread v2.0.1 Liao et al. 2013 subread, sourceforge.netRefS eq Liao et al. 2014 ncbi.nlm.nih.gov / refseq / EdgeR3 Mortazavi et al. 2008130 bioconductor.org / packages / release / bioc / html / edgeR.html GSEA 4.3.2 Robinson et al. 2010 gsea-msigdb.org / gsea / index.jspFCS Express [version 7] De Novo Software deno vo software.comBioLegend LEGENDplex Data BioLegend legendplex.qognit.com / user / login?next = home AnalysisSoftware version 2022- 07-15Spectrofl 3.0 software Cytek Biosciences cytekbio.com / pages / spectro-flCytek Full Spectrum Viewer Cytek Biosciences spectrum.cytekbio.comNanoString nCounter Advanced NanoString NanoString.com / products / analysis-solutions / nsolver- AnalysisSoftware (version advanced-analy sis-software / 2.0.134)NanoString nSolver Analysis NanoString nanostring.com / products / analysis-solutions / ncounter- Software 4.0 analy sis-solutionsPrism9 (version 9.4.1) GraphPad graphpad.comJMP Pro 16 JMP Statistical Discovery jmp.com / en_us / software / predictive-analytics- software.htmlGoldenrod Animal Lancet, 4mm Braintree Scientific GR4MMSterile, 5 trays of 200Micro vette 100100 UL, round Braintree Scientific MV- 100bottom, inner tube PotassiumEDTAPyrex Vista Test Tubes, 153 125 Carolina Biological Supply 721172mm CompanyInvitrogen OneComp eBeads ThermoFisher Scientific 01-1111-42Compensation BeadsInvitrogen UltraComp eBeads ThermoFisher Scientific 01-2222-42Compensation BeadsWSGR Ref. 59910-701.601

[0317] Table 12. Commercial AssaysASSAY SOURCE IDENTIFIER RNA Agilent Tape Station 4200 High Agilent 5067-5576, 5067-5577, 5067- Sensitivity D5000 kit 5578,5067-5592, 5067-5593 RNA HS Agilent Tape Station4200 Agilent 5067-5579, 5067-5580,5067-5592, High Sensitivity D5000 kit 5067-5593Mouse IL- 12 (p70) ELISA MAX BioLegend 433606deluxe ELISA kitsLEGENDplex MU Cytokine BioLegend 741024ReleaseSyndrome Panel (13-plex)w / VbPNextSeq 1000 / 2000 P2 Reagents (100 Illumina 20046811Cycles) v3NextSeq 2000 P3 Reagents (100 Illumina 20040559Cycles)XT GX CodeSet FRSMK DR NanoString 116003421 (lot -C 10552X1) XT GXA P1CS-096 NanoString 116003421 (lot -C 10552X1) nCounter Master Kit NanoString 100054NEBNext Ultra II Directional New England Biolabs Inc. E7760LRNALibrary Prep Kit for IlluminaNEBNext Poly(A) mRNA Magnetic New England Biolabs Inc. E7490LIsolation ModuleNEBNext Globin & rRNA Depletion New England Biolabs Inc. E7750XKit (Human / Mouse / Rat)RNeasy Plus Mini Kit Qiagen Sciences Inc. 74136Invitrogen Qubit dsDNA HS and BR ThermoFisher Scientific Q32854Assay Kits

[0318] Table 13. Deposited DataInterferon gamma signaling Reactome (Garapati40) reactome. org / content / detai 1 / R-HSA- 877300Interleukin- 12 signaling Reactome (Jupe and Duenas) reactome. org / content / detai 1 / R-HSA- 9020591Interleukin- 10 signaling Reactome (Jupe) reactome. org / content / detai 1 / R-HSA- 6783783TNF signaling Reactome (Shamovsky) reactome. org / content / detai 1 / R-HSA- 75893.7Chemical and Drug Induced Liver Comparative Toxicogenomic Database ctdbase.org / detail. go?type = Toxicity (Davis et al.) disease&acc = MESH: D056486 Raw and Processed RNA-seq data This paper GSE241939WSGR Ref. 59910-701.601

[0319] Example 14: IL-12 Loaded PLGA Nanosphere Immunotoxicity and Pharmacodynamic Study: Results

[0320] Characterization of IL12ns

[0321] Scanning electron microscopy (SEM) confirmed successful synthesis of IL12ns by the double-emulsion solvent evaporation (DESE) method with modification (FIG. 21A). IL12ns elution studies confirmed the release of recombinant murine IL- 12 over a 14-day period with minimal elution profile variability, as evidenced by an average standard error of the mean per day (expressed as %) of 8.10% (FIG. 21B). IL12ns batches also exhibited consistent and reproducible concentrations (38.08 ± 2.97 mg / mL), yields (152.30 ± 11.86 mg / mL), and encapsulation efficiencies (EE, 35.47 ± 4.79%) (n = 6) (FIG. 21C). Dynamic light scattering (DLS) analysis reported a mean diameter of 632.53 ± 65.16 nm and poly dispersity index (PDI) of 0.641 ± 0.038 (n = 4) (FIG. 21D). Based upon in vitro elution studies, the 10, 0.1, and 0.001 mg IL12ns doses delivered approximately 1600, 16, and 0.16 ng / kg of recombinant murine IL-12 per day, respectively.

[0322] Spectral Flow Cytometric Analysis of Peripheral Blood Mononuclear Cells (PBMCs)

[0323] Peripheral blood sampling occurred at designated timepoints (T) including baseline (Tl), 12-h (T2), day 4 (T3), day 8 (T4), day 11 (T5), day 15 (T6), and day 18 (T7). Flow cytometric analysis at each timepoint (T), expressed as a percent (%) of all live cells, revealed immunological differences between experimental groups for both the neutrophil (FIGS. 15C-15I) and polymorphonuclear (PMN)-myeloid derived suppressor cell (MDSC) populations (FIGS. 15J-15P). The immunophenotypic markers or genes of interest, and their associated descriptors, identified in this study can be found in Table 10. In males, there were significant increases (p < 0.0001) in the % neutrophils for the 10 mg IL 12ns group versus all other experimental groups at T4 and T6, with similar trends also persisting at T2. This elevation in neutrophils for the 10 mg IL 12ns group increased overtime, with the largest % neutrophils evident at T6 (57.7% higher than saline control, 40.5% higher than MTD). This finding was also evident within female mice, particularly at IL12ns-dosing timepoints T4 and T6. Trending increases in % neutrophils for the MTD group compared to saline control were also evident at timepoints T2 and T3 (FIG 15C).

[0324] Neutrophils were then analyzed for expression of iNOS, Arg-1, PD-L1, CD66b, CD80, and MHC-II (FIGS, 15D-15I). Within males, the % iNOS+ neutrophils were significantly increased (p < 0.0001) for the 10 mg IL12ns group over all other experimental groups at T2, T4, and T6 (exception - T2 and T6, versus 0.1 mg IL12ns group). A significant (p < 0.0001) or trending increase in this cellular population continued for 10 mg IL12ns over allWSGR Ref. 59910-701.601other experimental groups at T7. This finding was also consistent within female mice (FIG. 15D). Analysis revealed a significant increase (p < 0.0001) in % Arg-1+ neutrophils for the 10 mg IL12ns group in comparison to all other experimental groups at T2 and T4 within male mice. A similar significant increase (p < 0.001) was present in female mice (FIG 15E). At T3, T4, T5, and T7, there were significant increases (p < 0.0001) within the % PD-L1+ neutrophil population for the MTD group versus all other experimental groups in males, with a significant increase also evident at T2 within females. While still significantly elevated, levels of this cellular population drop from a peak (approximately 20% of all cells) at T2 to less than 5% of all cells at T7 (FIG 15F). Analysis of % CD66b+ neutrophils showed no differences across experimental groups at all timepoints examined (FIG. 15G). However, the % CD80+neutrophils displayed a significant increase (p < 0.0001) in the 10 mg IL12ns group at T2 and T4, with similar findings persisting within female mice (FIG. 15H). Finally, a significant (female, p < 0.0001) or trending (male) increase in % MHC-II+ neutrophils was evident for the 10 mg IL12ns group over all other experimental groups at T5, with additional trending increases also evident at T7 for female mice (FIG. 151). Overall, it appears that while the MTD strategy initiates the release of dysfunctional and immunosuppressive neutrophils positive for PD-L1, IL12ns drives transient increases in phagocytic, inflammatory neutrophils capable of initiating pro-inflammatory immune responses.

[0325] Analysis of PMN-MDSCs revealed potential hyper-sensitivity to the bolus MTD strategy. First, both male and female mice displayed transient changes in the overall PMN-MDSC population across the timepoints examined (FIG. 15 J). However, when analyzing the PMN-MDSC population for iNOS, Arg-1, PD-L1, CD66b, CD80, and MHC-II expression (FIGS. 15K-15P), differences were observed. First, a significant increase (p < 0.0001) in % iNOS+ PMN-MDSCs was evident for the MTD group over 10 mg IL12ns and 0.1 mg IL12 ns at T2 and T5, respectively. However, at T7, there were significant increases (p < 0.0001) within the 10 mg IL12ns over 0.001 mg IL12ns, MTD, and saline control groups. A similar trend was also evident within females, albeit with additional significant increases (p < 0.0001) in MTD versus saline control at T3 (FIG. 15K). Further, while there was a significant increase of % Arg-1+ PMN-MDSCs at T2 for the MTD group over 10 mg IL12ns, there was again an eventual shift by T7 resulting in a significant increase (p < 0.0001) of this population within the 10 mg IL12ns group over 0.001 mg IL12ns, MTD, and saline control groups for both males and females (FIG.15L). There were significant increases (p < 0.0001) in % PD-L1+ PMN-MDSCs for the MTD group versus all experimental groups from T2 through T7. Similar findings were apparent in female mice, with significant increases (p < 0.0001) in this population evident at T3, T4, T5, and T7 for the MTD group (FIG. 15M). Further, the % CD66b+ PMN-MDSCs displayed aWSGR Ref. 59910-701.601significant increase (p < 0.0001) within the male MTD group over 10 mg, 0.1 mg IL12ns, and saline control groups at T2, T4, and T5, with a trending increase over 0.001 mg IL12ns evident at the same timepoints. This finding was also apparent for female mice at T2 through T5, revealing the generation of an activated PMN-MDSC population with the MTD (FIG. 15N). Similarly, within female mice, there was a significant increase (p < 0.0001) in % CD80+PMN-MDSCs for the MTD group when compared to 10, 0.1 mg IL12ns as well as saline control groups at T3 and T5. While an increase was also evident in male MTD mice, the % CD80+PMN-MDSCs was only significantly increased for the MTD group in comparison to 10 mg IL12 ns at T4 (FIG. 150). Finally, there was a significant increase (p < 0.0001) in the % MHC-II+ PMN-MDSCs for the MTD group at T7 in both male and female mice (FIG. 15P). Ultimately, the MTD strategy drives an increase in % PD-L1+, CD66b+, and MHC-II+ PMN-MDSCs. Increased antigen presentation alongside immune checkpoint inhibitor expression on PMN-MDSCs was previously associated with T cell suppression through heightened T-cell-PMN-MDSC interactions, a likely maladaptive effect of IL-12 overstimulation with the MTD strategy.

[0326] Bulk PBMC-RNA-seq Analysis - Myeloid and Lymphoid Gene Expression

[0327] Total RNA isolated from PBMCs was evaluated using next-generation bulk RNA-sequencing (RNA-seq). There were, on average, 24.6 ± 14.5 million reads per sample with a 91.9 ± 0.02% mapping rate to the murine mmlO genome. Following the generation of reads per kilobase of transcript per million reads mapped (RPKM) matrices, gene expression differences between experimental groups were analyzed. First, correlating genes of the myeloid flow cytometric panel were evaluated. There was a significant increase (p < 0.01) in PD-L1 expression for the male MTD group versus all other experimental groups at T2 (exception - 10 and 0.1 mg IL12ns), T3, T4 (exception - 10 mg IL12ns), T5, T6, and T7. A similar trend was also apparent in female mice, with a significant increase evident for the MTD group versus all experimental groups at T2 through T6 (FIG 23F). Evidence of IFN-g-driven peripheral immune activation with MTD (PD-L1) was also evident (FIGS. 23A-23F).145

[0328] Differences within lymphoid-related gene expression, including but not limited to CD4, CD8a, Foxp3, CD19, and Ncrl (NKp46), were also examined. These data also provided further support to the previous flow cytometric analysis which suggested that elevated IL-12 dosing (10 mg IL 12ns and MTD) had limited effect on peripheral T-lymphocyte cell populations. These dosing strategies were further associated with decreases in CD19+B-lymphocytes and initial yet unstainable increases in NK cells (both 10 mg IL12ns and MTD) (FIGS. 23G-23K). Further analysis of lymphoid immune exhaustion genes including CTLA-4, Lag3, and hepatitis A virus cellular receptor 2 (Havcr2, Tim-3) revealed increased gene expression with MTD (FIGS 23L-23N). There were significant increases (p < 0.01) in Lag3WSGR Ref. 59910-701.601gene expression for the MTD group versus both 0.1 mg IL12ns and saline control at T3. This finding was apparent, however, only trending in the female MTD group at both T2 and T3 (FIG 23M). There were also significant (p < 0.01) or trending increases in Havcr2 expression within the MTD group at T2 (versus 10 mg IL12ns and saline), T3 (versus 0.1 mg IL12ns and saline), and T6 (versus all other groups). This result was also observed in female MTD mice, (p = 0.025) at T4 versus the 10 mg IL12ns group (FIG. 23N). Overall, bulk PBMC RNA-seq analysis conveys prominent immunological changes for the lymphoid functional markers examined and suggests the generation of a hyporesponsive immune signature in peripheral lymphoid cells with the MTD strategy.

[0329] Plasma Proteomic Analysis - Cytokines

[0330] Differences in the plasma cytokines IFN-y, IL- 10, IL-6, and tnf-a were also measured following IL-12 dosing (FIGS. 16A-16D). There were significant increases (p < 0.0001) in IFN-y concentration (pg / mL) for the male MTD group at T3 versus all other groups, with trending increases versus 0.1 mg, 0.001 mg IL12ns, and saline at T4. There was also a significant increase (p < 0.0001) over 0.1 and 0.001 mg IL12ns as well as saline control groups at T6. The increase at T2 was not sustained, as evidenced by an over 4-fold decrease in IFN- y levels by T7. In contrast, the 10 mg IL12ns group maintained its peak IFN- y levels with repeated dosing at IL12ns dosing timepoints T2, T4, and T6. Interestingly, the female 10 mg IL12ns group also experienced significant (versus 0.1 mg IL12ns, p < 0.0001) or trending (versus 0.001 mg IL12ns, p = 0.0001) increases in IFN- y levels at T2 (FIG. 16A). There were no significant differences in IL- 10 concentration (pg / mL) measured amongst groups at all sampling timepoints (FIG. 16B). This finding is likely related to the highly transient nature of IL- 10 and difficulty quantifying this cytokine within plasma / serum samples.

[0331] Plasma proteomic analysis also revealed a significant increase (p < 0.0001) of IL-6 within male 10 mg IL12ns versus all other experimental groups at T2, T4, T6, and T7 (exception - only trending increase versus 0.1 mg IL12 ns at T2 and versus saline control at T7). A similar finding was also evident in female mice, however, the increase for the 10 mg IL12ns group at T7 was merely trending versus all other groups. Additionally, there was a significant increase (p < 0.0001) in IL-6 concentration in the female 10 mg IL12ns group when compared to all other experimental groups at T5 (FIG. 16C). Similarly, a significant increase (p < 0.0001) in tnf-a concentration was evident in the 10 mg IL 12ns group versus all other experimental groups at T2, T4, and T6, further indicating the activation of acute inflammatory responses with 10 mg IL12ns dosing. Additional trending and significant increases for the 10 mg IL 12ns and MTD groups were also measured versus all other experimental groups at T5 (FIG. 16D). This observation suggests that increased tnf-a could be, in part, associated with large-dose IL-12 therapy.WSGR Ref. 59910-701.601

[0332] Plasma Proteomic Analysis Chemokines

[0333] Plasma samples were also analyzed for concentration of circulating pro-inflammatory chemokines CCL2 (MCP-1), CCL3 (MIP-la), CCL4 (MIP-ip), CXCL9 (MIG), and CXCL10 (IP-10) (FIGS. 16E-16I). A significant increase (p < 0.0001) in CCL2 concentration was measured within the female 10 mg IL12ns group versus all other experimental groups at T2, T4, and T6. Comparable increases were also evident in males (FIG. 16E). Similarly, there were significant increases (p < 0.0001) in CCL3 concentration for the male 10 mg IL12ns group when compared to all other experimental groups at T2, T4, and T6 (again, timepoints associated with IL12ns dosing). In females, a similar significant increase (p < 0.0001) was evident at T4 and T6 (FIG. 16F). Significant increases (p < 0.0001) in CCL4 concentration were also measured within the male 10 mg IL12ns group versus all other experimental groups at T2 and T6. Similar findings were again evident in female mice (FIG. 16G).

[0334] Additionally, CXCL9 concentration (pg / mL) was significantly increased (p < 0.0001) for male MTD group versus all other groups from T3 through T7, with additional significant (versus 0.1 and 0.001 mg IL12ns, p < 0.0001) and trending (versus saline control, p = 0.0003) increases evident at T2. The peak in CXCL9 at T3 was not sustained with MTD, with a near 3-fold decrease toward baseline levels by T7. These findings were consistent in female mice and may indicate exhaustive IL-12 stimulation within peripheral blood (FIG. 16H). Furthermore, significant increases (p < 0.0001) in CXCL10 were evident in both the male MTD and 10 mg IL12ns groups versus both 0.1, 0.001 mg IL12ns and saline control groups at T2. At T3 and T5, there was a significant increase (p < 0.0001) in CXCL10 concentration for the MTD versus all other experimental groups. However, at T4 and T6, significant (p < 0.0001) or trending increases in the 10 mg IL 12ns group emerged. While these findings were also demonstrated in the female groups, there remained a significant increase (p < 0.0001) in CXCL10 for the MTD group compared to all other experimental groups at T7 (FIG. 161).

[0335] Bulk PBMC RNA-seq analysis - Cytokine and chemokine gene expression

[0336] Circulating PBMCs were also analyzed for expression of the cytokine-related genes Ifing, 1110, 116, and Tnf (FIGS. 16J-16M). Within males, there was a significant (versus 10 mg IL 12ns) or trending (versus saline control) increase in Ifng gene expression in the MTD group at T3. Similarly, the female MTD group displayed a significant increase (p < 0.01) in Ifng gene expression over all other groups at this same timepoint (FIG. 16J). This result correlated with a significant increase (p < 0.01) in 1110 gene expression for both the male and female MTD groups versus all other groups at T3. This significant increase in 1110 expression was then maintained at T4 (trending versus 0.1 mg IL12ns and saline), ), T5, T6, and T7 for the male MTD group in comparison to all other groups. A similar finding was evident in females, with trending increasesWSGR Ref. 59910-701.601in 1110 expression evident at T4 and T6 for the MTD group in comparison to both 0.1 mg IL12ns and saline control groups (FIG. 16K). Furthermore, analysis revealed a trending increase in 116 gene expression for the MTD group versus both the 0.1 mg IL12ns and saline control groups at T5 in males (FIG. 16L). Additionally, Tnf analysis in male mice revealed trending increases at T2 (MTD versus saline control) and T4 (10 mg versus 0.1 mg IL12ns) for both MTD and 10 mg IL12ns groups, respectively. Similar findings were evident in females, where a trending increase in 116 expression was evident for the MTD group in comparison to the 0.1 mg IL12ns group at T2 (FIG. 16M). These gene expression results for both 116 and Tnf contrast with the proteomic cytokine analysis previously highlighted. While circulating levels of both IL-6 and TNF-a are increased in the 10 mg IL12ns group, RNA isolated from PBMCs suggests minimal gene expression changes in circulating immune cells.

[0337] Analysis of chemokine-related genes Ccl2, Ccl3, Ccl4, Cxcl9, and CxcllO also revealed important trends (FIGS 16N-16R). Within males, a significant increase (p < 0.01) in Ccl2 gene expression was evident for the 10 mg IL12ns group versus all other groups at T3. Analysis in females revealed significant (p < 0.01, versus 10 and 0.1 mg IL12ns) or trending (versus saline control) increases in Ccl2 expression within the MTD group at T5 (FIG. 16N). Additionally, a trending decrease in Ccl3 expression was measured within the male 10 mg IL12ns group at T4 versus saline control. Similarly, in females, there were significant (T2) or trending (T4) decreases in Ccl3 gene expression for the 10 mg IL12ns group in comparison to all other groups (exception - no decrease versus 0.1 mg IL12 ns at T2) (FIG. 160). While there were no differences in Ccl4 gene expression amongst all experimental groups in males, there were trending or significant (p < 0.01) increases in Ccl4 expression for the female MTD group versus 10 mg IL12ns (T4, T6) and saline control (T3) groups (FIG. 16P). In both males and females, there was a significant increase (p < 0.01) in Cxcl9 gene expression within the MTD group versus all other groups at T2 through T6, with a trending increase also evident for the male MTD group at T7 (FIG. 16Q). Similarly, CxcllO gene expression in males revealed a significant increase (p < 0.01) in gene expression for the MTD group versus all other groups at T2 through T7. Additional significant (versus 0.1 mg IL12ns) or trending (versus saline) increases for the 10 mg IL 12ns group were evident at T3. For females, a significant increase (p < 0.01) in CxcllO gene expression was also evident for the MTD group versus all other experimental groups at T2 through T6 (FIG. 16R). While CXCL10 plasma levels were elevated with IL12ns treatment, those circulating immune cells ultimately lack CxcllO gene expression. This finding may suggest the initiation of tissue-resident pro-inflammatory signal transduction as opposed to stimulation within the peripheral blood (as seen with the MTD strategy).WSGR Ref. 59910-701.601

[0338] Bulk PBMC RNA-seq analysis - GSEA

[0339] A targeted gene set enrichment analysis (GSEA) was performed on the Reactome gene sets Interferon gamma signaling (IFNG), Interleukin- 12 signaling (IL12), Interleukin- 10 signaling (IL10), and Tumor necrosis factor (TNF) signaling. This assessment was conducted at the timepoint of maximal gene expression differences between treatment groups and saline control (T3). For both male and female MTD groups, GSEA Reactome analysis revealed significant upregulation of the IFNG, IL12, and IL10 signaling pathways in comparison to matched-sex saline controls (FIGS. 17A-B). 10 mg or 0.1 mg IL12ns groups both showed significant upregulation of the IL 10 signaling Reactome at T3 versus the saline control (FIG 17A). A similar relationship was also apparent in female mice. (FIG 17B). An unbiased GSEA using both Hallmark (Table SI) and Reactome gene sets (Table S2) further supported the modulation of these isolated pathways. Both male and female MTD groups displayed statistically significant upregulation of the Reactome signature pyroptosis, or caspase-1 mediated inflammatory cell death (q = 0.0498 and 0.0225, respectively, Table S2). These data further suggest overt immunological toxicity with the bolus MTD strategy even at early dosing timepoints (T3). The core enrichment genes identified from these GSEA Reactome analyses (FIGS. 28A-B) were then assessed for expression changes at all peripheral blood sampling timepoints T1-T7.

[0340] Bulk PBMC RNA-seq analysis - IFNG signaling reactome

[0341] RPKMs for individual core enrichment genes within the IFNG signaling reactome were also analyzed (FIGS. 18A-18K). Significant increases in Fcgrl expression were evident in the male MTD group versus all other groups at T3 through T5, T6, and T7. Similar increases were evident in the female MTD group (FIG. 18 A). Furthermore, analysis of the interferon-induced Gbp2, Gbp3, Gbp5, and Gbp7 revealed similar expression changes for males and females (FIGS. 18B-18E). Overall, the MTD group exhibited significant (p < 0.01) or trending increases in Gbp expression versus all other experimental groups from T3 through T7, with additional significant increases evident at T2 (Gbp5, Gbp7). The 10 mg IL12ns group displayed a significant increase in Gbp2 expression at T2 (versus saline), T3, T4, T6, and T7 (versus 0.1 mg IL12ns and saline) (FIG. 18B). For Gbp7, the 10 mg IL12ns group also exhibited significant or trending increases at T2 (versus saline), T4, T5 (versus 0.1 mg IL12ns and saline), T6 (versus saline), and T7 (versus 0.1 mg IL12ns and saline) (FIG. 18E).

[0342] Additionally, analysis revealed significant increases (p < 0.01) in Irfl gene expression for the male MTD group at T2, T3, T4, T5, and T6 versus all groups, as well as at T7 versus both the 0.1 mg IL12ns and saline control groups. Significant or trending increases were also evident for the 10 mg IL12ns group at T2, T3, and T4 (versus 0.1 mg IL12ns and salineWSGR Ref. 59910-701.601control), as well as T6 and T7 (versus saline control) (FIG. 18F). Further, Irf7 expression displayed significant (p < 0.01) or trending increases within both male and female MTD groups in comparison to all other experimental groups from T2 through T7 (FIG. 18G). Similarly, Irf9 expression was significantly increased within the male MTD group in comparison to the saline control group at T2, T3, T6, and T7. Additional significant increases (p < 0.01) were also evident within the male 10 mg IL12ns group versus saline at T2, T3, T4, with trending increases evident at T3 (versus 0.1 mg IL12ns), T6, and T7 (versus saline). Overall, similar findings were also apparent for female experimental groups (FIG. 18H).

[0343] Both male and female MTD groups exhibited significant increases in Oasl2 gene expression at T2 through T7 versus all other experimental groups. Additional trending or significant increases were apparent in the male 10 mg IL 12ns group at T2 (versus saline), T3, T4, T5, and T7 (versus saline and 0.1 mg IL12ns) (FIG. 181). Expression of the signal transducer of the IFNG pathway, Statl, was significantly upregulated at T2 through T6 for the male MTD group versus all other groups. A significant or trending increase in Statl gene expression was also apparent within the 10 mg IL12ns group at T2, T3, T4, T6, and T7. Similar trends were evident within the female MTD and 10 mg IL 12ns groups, further suggesting IFNG signaling activation in these groups (FIG. 18 J). Socsl was significantly upregulated in the male MTD group at T2 (versus 0.1 mg IL12ns and saline), T3 (versus all other groups), T4 (versus 0.1 mg IL12ns and saline), as well as T5 and T6 (versus all other groups). Similar trends persist within female MTD mice, with a significant increase (p < 0.01) in Socsl gene expression evident at T2, T3, T5, and T6 (exception 10 mg IL12ns) versus all other groups. Additional significant or trending increases within the 10 mg or 0.1 mg IL12ns group versus saline controls were apparent at early dosing timepoints T3 (both groups) and T4 (10 mg IL12ns) within males, as well as T2 (10 mg 1112ns) within females (FIG. 18K). The lack of markedly increased Socsl expression at later timepoints with IL 12ns dosing likely suggests a more controlled pro-inflammatory signature within this group.

[0344] Bulk PBMC RNA-seq analysis - IL12 signaling Reactome

[0345] RPKMs for individual genes of the Interleukin- 12 signaling Reactome were also analyzed (FIGS. 24A-24E). Importantly, Il 12rb 1 gene expression appeared sensitive to peripheral immune stimulation with bolus IL- 12 delivery (MTD). Here, there was a significant increase (p < 0.01) in II 12rb 1 gene expression within the male MTD group at T2 (versus saline), T3 (versus 0.1 mg IL12ns, saline), and T5 (versus all other groups), with trending increases evident at T2 versus both 10 and 0.1 mg IL12ns groups, T3 versus 10 mg IL12ns, and T7 versus all other groups. Similar trends were also evident for female MTD mice (FIG. 24A). Analysis of Stat4 expression indicated possible overstimulation and suppression of IL- 12 signaling withWSGR Ref. 59910-701.601elevated IL-12 dosing strategies (10 mg IL12ns, MTD). In females, there were significant decreases (p < 0.01) in Stat4 gene expression within the 10 mg IL12ns group at T4 (versus 0.1 mg IL12ns) and T6 (versus 0.1 mg IL12ns and saline control). This result was accompanied by significant decreases in Stat4 expression for the female MTD group at T4 and T6 (versus 0.1 mg IL12ns), as well as T7 (versus both 0.1 mg IL12ns and saline control) (FIG. 24E). Overall, these data suggest that increased II 12rb 1 expression alongside a compensatory downregulation of Stat4 expression may indicate peripheral overstimulation with IL-12 delivery.

[0346] Bulk PBMC RNA-seq analysis - IL10 signaling reactome

[0347] RPKMs for core enrichment genes of the Interleukin- 10 signaling Reactome were also examined (FIGS. 24F-24K). The IFN-g associated genes of this pathway, including 1118 (IFN-g-inducing factor) (FIG. 24F), Icam (FIG 24G), and Ccr5 (FIG. 24H), were more significantly upregulated within the MTD group in comparison to IL12ns, further affirming peripheral immune activation of the IFNG pathway with unencapsulated IL-12 delivery. STAT3 is a regulator at the crossroads of both inflammatory and anti-inflammatory responses. The IL-10 / STAT3-mediated anti-inflammatory immune signaling works in opposition of the pro-inflammatory IL-6 / STAT3 pathway. Here, in males, Stat3 gene expression was significantly upregulated within the 10 mg IL12ns group at T2 (versus saline), T3 (versus saline), T4 (versus all other groups), and T7 (versus saline). In females, Stat3 was significantly upregulated in both the 10 mg IL12ns and MTD groups in comparison to the saline control at T6 (FIG. 241). Similar gene expression increases were also apparent for Cxcl2, an acute inflammatory chemokine (FIG.24J). Significant decreases in Ptgs2 expression were evident with 10 mg IL12ns dosing (FIG. 24K), suggesting a lack of suppressive PGE2production and sustained inflammatory capacity with IL12ns therapy. Overall, while the IL10 signaling Reactome was significantly upregulated in both IL 12ns and MTD strategies at T3 (FIG. 17A-B), the individual core enrichment genes driving this significance are distinctly different. Significance within the MTD group is driven by expression of compensatory 1110 and Ifng-associated genes. In contrast, genes driving significance within the IL12ns dosing groups are associated with pro-inflammatory signal transduction, however, without compensatory 1110 gene expression (FIG. 28A-B).

[0348] Histopathologic analysis

[0349] Full necropsy for histopathological analysis by two independent board-certified pathologists and a veterinarian medical doctor was performed at T7 (euthanasia). Representative Hematoxylin and Eosin (H& E) stained sections highlighting differences in portal (liver), cortical (kidney), interstitial (lung), endomyocardial (heart), and stromal (spleen) inflammation amongst both male (FIG 19 A) and female (FIG 19E) experimental groups are presented. Additional pathological findings from these analyses can be found in FIGS. 27A-E. Quantification ofWSGR Ref. 59910-701.601pathological findings into a total pathology, total inflammation, and total necrosis scores revealed striking differences between experimental groups (FIGS. 19B-19D, and 19F-19H). In males, statistical analysis revealed significant increases in total pathology score for both 10 mg IL12ns and MTD group in comparison to 0.001 mg IL12ns and matched-sex saline control groups (FIG. 19B). Additionally, there was a significant increase in total pathology score for the MTD group versus 0.1 mg IL12ns (FIG. 19B). In females, both the 10 mg IL12ns and MTD groups exhibited significant increases in total pathology score in comparison to all other experimental groups (FIG. 19F). In both males and females, there were no differences in total pathologic necrosis measured between experimental groups (FIGS. 19C and 19G), suggesting these differences were due to histologically determined inflammation. Accordingly, in both males and females, analysis revealed significant increases in total inflammation for the 10 mg IL12ns and MTD groups in comparison to all other experimental groups (FIGS. 19D and 19H).

[0350] These results indicate IL12ns can be effectively delivered to healthy BALB / c mice and increase tissue-level inflammatory responses.

[0351] NanoString nCounter gene expression analysis - Liver

[0352] Gene expression profiles of formalin-fixed, paraffin embedded (FFPE) liver specimens harvested at euthanasia (T7) were examined using a 42 gene panel on the NanoString nCounter. Following normalization by housekeeping genes Eeflg, Gapdh, Hprt, Polr2a, Rpll9, and Sdha (both male and female), differentially expressed genes, in comparison to the saline control, were analyzed (FIGS. 20A-L). Stat3 was significantly increased in both male (p < 0.01) and female (p < 0.05) 10 mg IL12ns groups, with a trending increase also evident from the female MTD group (p = 0.0651) (FIG 20A). Further, both the 10 mg IL12ns (male - p < 0.01, female - p < 0.05) and MTD (male and female - p < 0.01) groups displayed significant increases in Ccl2 expression, indicating pro-inflammatory immune cell recruitment (FIG. 20B).

[0353] Both Cxcl9 and CxcllO displayed increased gene expression in both male and female 10 mg IL 12ns and MTD groups, however, their expression was more significantly upregulated in the MTD group (FIGS. 20C and 20D). Statl gene expression was significantly upregulated in both the 10 mg IL12ns (male - p < 0.01) and MTD (male - p < 0.0001, female - p < 0.001) groups (FIG. 20E). Significant or trending increases in Gbp2 and Gbp3 expression were also observed for both groups (FIGS. 20F and 20G). While Irfl expression was increased with IL12ns dosing, only the MTD group displayed a significant increase in comparison to the saline control (male - p < 0.01, female - p < 0.001) (FIG. 20H). Furthermore, significant (male) or trending (female) increases in PD-L1 gene expression was evident for the 10 mg IL12ns group. Analysis of the MTD group, however, revealed significant increases in both PD-L1 (male and female, p < 0.01) and Lag3 (female, p < 0.05) expression (FIGS. 201 and 20J). SustainedWSGR Ref. 59910-701.601expression of LAG-3 is associated with reduced cytolytic and cytokine-producing functions of infiltrating T cells, while co-expression of LAG-3 and PD-1 / PD-L1 exemplifies T cell dysfunction in tumor patients. Significant elevation of both checkpoint molecules with the MTD strategy suggests immunotoxic overstimulation. Finally, the 10 mg IL12ns group displayed significant increases (p < 0.05) in both Socs3 (males) and Itgam (males and females) expression (FIGS. 20K- 20L). Upregulation of SOCS3, an integral regulator responsible for maintaining controlled inflammation downstream of both pro-inflammatory STAT1 / STAT3 (IL-6) and STAT4 (IL-12) signal transduction, suggests healthy inflammatory signaling with IL12ns dosing. Overall, the MTD strategy appears to promote immune exhaustion, as evidenced by increased co-expression of immune checkpoint genes PD-L1 and Lag3.

[0354] NanoString nCounter gene expression analysis - Spleen

[0355] Gene expression profiles of FFPE splenic specimens harvested at euthanasia (T7) were also examined using the same 42 gene panel on the NanoString nCounter. Following normalization using housekeeping genes Alasl, Eeflg, G6pd, Gapdh, Hprt, Polr2a, Rpll9, and Sdha (male spleens) or Eeflg, G6pd, I12rg, Polr2a, Rpll9, and Sdha (female spleens), differentially expressed genes, in comparison to the saline control, were analyzed (FIG. 25A-L). There were minimal differentially expressed genes present within harvested spleens. Of note, the MTD group displayed significant (male - p < 0.01) or trending (female - p = 0.0676) increases in Statl expression (FIG. 25A). This Statl expression was associated with significant increases (p < 0.01) in both Gbp2 and Cxcl9 in this same group (FIGS. 25B and 25E). While IFNG-related genes were increased with IL12ns dosing (in comparison to the saline control), these levels were not statistically significant. In males, the 10 mg IL12ns dosing strategy was associated with trending increases in both Ly6g and Itgam expression, with a trending decrease in CD 19 expression also apparent (FIGS. 25J-25L). Increases in Ifng-associated genes were apparent, with the greatest increases evident with the MTD strategy.

[0356] NanoString nCounter gene expression analysis - Lung

[0357] Gene expression profiles of FFPE lung specimens collected at euthanasia (T7) were also analyzed. Lungs were selected due to the previously observed cytokine-induced acute lung injury in the setting of systemic inflammatory response syndrome (SIRS) or cytokine storm. Following normalization using housekeeping genes Eeflg, G6pd, Hprt, I12rg, Polr2a, Rpll9, and Sdha (male) or Eeflg, G6pd, Hprt, Polr2a, Rpll9, and Sdha (female), differentially expressed genes, in comparison to the saline control, were further analyzed (FIGS 26A-L). Again, there were a minimal number of differentially expressed genes present amongst groups. Importantly, the female 10 mg IL12ns (p = 0.0527) and MTD (p < 0.001) groups displayed trending or significant increases in Statl gene expression, respectively (FIG. 26A). This increase wasWSGR Ref. 59910-701.601associated with significant (p < 0.01) or trending (p = 0.0512) increases in Gbp2 and Gbp3 expression within the female MTD group, respectively (FIGS 26B and 26C). Furthermore, the female MTD group also displayed trending increases in both Cxcl9 (FIG 26E) and PD-L1 (FIG 26H), indicating signs of exhaustive pro-inflammatory signaling within lung tissues. Lastly, the 10 mg IL 12ns group displayed a significant increase (p < 0.05) in Itgam expression, however, only within female mice. Overall, these data suggest that the IL 12ns dosing strategy may not cause SIRS-associated acute lung injury in a short-duration treatment setting, as measured by the lack of pro-inflammatory signaling within this tissue. MTD, in contrast, increased both pro-inflammatory Ifng signaling alongside the immune checkpoint molecule PD-L1 within harvested lungs. These changes ultimately correlate with the previously described systemic immune findings with MTD strategy and indicate development of SIRS-like inflammatory insults as seen in the setting of acute lung injury.

[0358] Example 15: IL-12 Loaded PLGA Nanosphere Immunotoxicity and Pharmacodynamic Study: Discussion and Conclusions

[0359] The purpose of this study was to assess the immunotoxicity of IL12ns in healthy male and female BALB / c mice while examining the pharmacodynamics of nanosphere cytokine delivery. While histopathological and NanoString nCounter analysis supports that neither MTD nor IL12ns therapy drove pathological, drug-induced necrosis within examined tissues, the deployment of a rigorous immunodiagnostic platform (IDP) detailed numerous immunological differences between these dosing strategies. At the forefront of these differences was pathologic peripheral overstimulation with the MTD strategy, as evidenced by signs of immune exhaustion and inflammatory cell death. Accordingly, findings from the IDP can be grouped into three, often overlapping, categories: (1) immunologic markers associated with undesirable peripheral immune activation, (2) immunologic markers that may allow for IL 12ns dose titration in a clinical setting, and (3) immunologic markers which support the necessity of the IL12ns vector delivery system and the proposed pharmacodynamic mechanism of action.

[0360] First, IDP analysis revealed numerous markers associated with peripheral immune overstimulation, as measured within the bolus MTD group. It is well understood that upon exposure to IFN-y expression of numerous immune inhibitory molecules, such as PD-L1, are upregulated. Expression of CD86, CEACAM-1, and class II MHC molecules, the ligands for immunosuppressive checkpoint inhibitors CTLA-4, TIM-3, and LAG3, respectively, are also increased in response to IFN-y. Accordingly, our study reports an increase in neutrophil PD-L1 expression alongside peripheral IFN-y and associated chemokines with the bolus MTD strategy. PD-L1+ neutrophils have been associated with decreased cytotoxicity in the setting of cancer and worsening severity of autoimmune disorders. Further, the MTD strategy was also associatedWSGR Ref. 59910-701.601with increases in % PD-L1+, % CD66b+, % CD80+, and % MHC-II+ PMN-MDSCs. This observation likely indicates bone marrow release of an immature, granulocytic myeloid population with heightened immunosuppressive capacity in the setting of IL-12 overstimulation. Additionally, while IL-12 stimulus initially drives NK cell activation, high-dose administration of IL-12 was associated with the induction of NK cell apoptosis and decreasing levels of STAT4 mRNA. Increases in Ki67+ NK cells with high-dose IL-12 administration, previously associated with autoimmunity and its severity, may further indicate the initiation of pathological overstimulation with unencapsulated IL-12 therapy.

[0361] Moreover, plasma cytokine and chemokine changes, as depicted in the MTD group, recapitulate the previous immunotoxicity evident in human clinical trials. Decreases in circulating IFN-y, and subsequent declines in both CXCL9 and CXCL10, were observed with repeated unencapsulated IL-12 stimulation in the MTD group. Maintaining elevated IFN-y levels throughout IL- 12 treatment was rare, however, associated with anti -turn or responses in previous human clinical trials. In support of this assertion, the activation of compensatory mechanisms and generation of an exhaustive, hyporesponsive phenotype were also observed with MTD delivery. The early increase in plasma IFN-y was associated with increases in PBMC 1110 gene expression. MTD also increased expression of lymphoid immune exhaustion genes (CTLA-4, Lag3, and Havcr2 or Tim-3), suggesting the development of a hyporesponsive gene signature within peripheral immune cells. The sustained expression of Socsl, a negative regulator of IFN-y signaling responsible for preventing damaging pro-inflammatory immune responses, with the MTD strategy further suggests compensatory downregulation of IFN-y signaling in peripheral immune cells.

[0362] Secondly, immunologic markers for IL 12ns dose titration were also identified.Peripheral blood assessments made 12 h post-IL12ns injection (T2, T4, and T6) were associated with two distinct cellular changes. Specifically, the 10 mg IL12ns group exhibited increases in myeloid lineage cells (particularly neutrophils) positive for iNOS (NOS2) and, at early timepoints, Arg-1 (ARG). Of note, elevated levels of polymorphonuclear (PMN) cells was observed to be associated with disruption of tumor vessels in the setting of systemic IL-12 therapy. Activated neutrophils, however, can inhibit T-lymphocyte functions through release of Arg-1 and subsequent depletion of the integral amino acid L-arginine. These cells can also directly inhibit T cell activation by cell-cell interactions through the PD-L1 / PD-1 axis, which is upregulated in response to IFN-y stimulation. Therefore, observing PMN-related increases in peripheral blood should still be considered a positive immune response to systemic IL- 12 therapy. Coincidentally, B-lymphocytes experienced an opposite trend. The associated declines in % CD 19+ B-lymphocytes are likely the result of B cell recruitment to peripheral sites ofWSGR Ref. 59910-701.601inflammation, acquisition of a more mature phenotype, alongside a bone marrow shift favoring myelopoiesis in the setting of an acute inflammatory response as seen in infection.

[0363] As previously indicated, IL12ns dosing was also associated with elevated plasma levels of the inflammatory cytokines IL-6 and TNF-a. IL-6 stimulates the production of most acute phase proteins in the acute-phase reaction. Importantly, the detrimental effects of IL-6 in the setting of chronic inflammation are associated with persistent, not transient, elevation of this cytokine. In this study, IL-6 plasma cytokine levels were increased 12 h post-10 mg IL12ns dosing timepoints (T2, T4, T6), however, returned to baseline thereafter (T3, T5, T7). While an elevation in IL-6 confirms successful initiation of a desired tissue-resident inflammatory response with IL12ns dosing, monitoring its levels over the course of therapy will likely be useful for the prevention of chronic inflammation and development of SIRS-like immunophenotype. Similar consideration must also be made for the complementary pleiotropic inflammatory cytokine, TNF-a.

[0364] Lastly, the combination of plasma protein and transcriptomic analysis further indicates support for the proposed pharmacodynamic mechanism of IL12ns. While plasma proteomic measurements encompass those cytokines and chemokines released from numerous cellular sources, bulk RNA-seq analysis was performed on total RNA isolated from a PBMC single cell suspension. This distinction ultimately allows discernment of a plasma protein’s source (cells of tissue or peripheral blood) and the likely location of signaling pathway activation. Intriguingly, while plasma protein analysis indicated drastic increases in circulating IL-6 levels with 10 mg IL12ns dosing (not evident within the MTD group), bulk PBMC RNA-seq analysis revealed limited to no differences in 116 expression amongst experimental groups. These findings suggest that the source of this acute phase protein is likely tissue-resident cells (hepatocytes of the liver, fibroblasts at sites of inflammation), responding to IL12ns tissue deposition and activation, however not by activation of cells within the periphery (blood immune cells / endothelial cells). A similar relationship of increased plasma protein with minimal differences in PBMC gene expression was apparent for TNF-a and inflammatory chemokines CCL2, CCL3, and CCL4.

[0365] Additionally, previous literature suggests that CXCL10, in direct response to IFN-y, is secreted predominantly by immune cells (leukocytes, activated neutrophils, monocytes), followed by fibroblasts, keratinocytes, epithelial, and endothelial cells. Peripheral blood plasma analysis revealed that while plasma levels of this chemokine are elevated within the MTD group, these levels were eventually exceeded by the 10 mg IL12ns group at later dosing timepoints. Bulk PBMC RNA-seq analysis demonstrated that while these elevated CXCL10 chemokine levels (from plasma) are associated with increased CxcllO gene expression (from circulatingWSGR Ref. 59910-701.601PBMCs) in the MTD group, only plasma CXCL10 levels are increased with 10 mg IL12ns dosing. The associated CxcllO gene expression increase in PBMCs was not measured with IL12ns dosing. This finding ultimately suggests that IL12ns can effectively deliver IL-12 to tissue-resident immune cells, stimulate pro-inflammatory responses as measured by circulating cytokine and chemokine levels, and limit peripheral activation of circulating immune cells and the potential development of SIRS or cytokine storm.

[0366] Due to the relatively short half-life of IL- 12 and ubiquitous expression of its receptor, large loading doses of the cytokine are likely required for delivery to secondary / tertiary lymphoid organs and tumor microenvironments. Here, the bolus (unencapsulated) MTD delivery strategy recapitulated the immune findings from previous human clinical trials: peripheral immune stimulation with repeated bolus IL- 12 dosing induces initial pro-inflammatory responses that eventually result in the development of an exhausted, immunotoxic immune profile. Conversely, our data suggests that IL 12ns prevent aberrant peripheral blood activation by shielding IL-12 from peripheral immune cells. This protective effect is likely due to the near immediate tissue deposition of the PLGA vector system and slow, controlled cytokine elution thereafter. This efficient vector delivery system presumably reduces the overall dose required for desired tissue delivery and anti-tumor efficacy.

[0367] This phenomenon was ultimately supported by our NanoString nCounter gene expression profiling of harvested liver, spleen, and lung specimens. Here, IL12ns drove tissueresident increases in pro-inflammatory IFNG and IL-6 gene expression alongside the chemokines associated with immune cell recruitment. Importantly, this tissue-resident pro-inflammatory signaling from IL 12ns was accomplished without inducing overt immune exhaustion locally nor within peripheral blood. While the MTD strategy vastly increased expression of many pro-inflammatory genes within analyzed tissues, these increases (particularly in liver and lung) were accompanied by compensatory checkpoint inhibitor expression indicative of an exhaustive immunophenotype. The increases in pro-inflammatory cytokine / chemokine signatures without co-expression of these inhibitory molecules, even days (T7) after the last IL 12ns dose (T6), emphasizes the importance of slow and controlled IL- 12 release after IL12ns deposition as a pharmacodynamic feature of this vector system. While there were limited differentially expressed genes within lung specimens, the lack of both pro-inflammatory and immune checkpoint inhibitor expression (as seen with MTD) further supports that IL12ns can prevent SIRS or cytokine storm-associated pathologies such as cytokine-induced acute lung injury. Ultimately, this multi-organ gene expression analysis further supports that IL12ns can drive healthy and sustained pro-inflammatory signaling systemically within tissues while preventing overt immunologic toxicity in peripheral blood.WSGR Ref. 59910-701.601

[0368] In conclusion, the combined proteomic, transcriptomic, and histopathological analyses ultimately support the proposed mechanistic efficacy of the IL 12ns vector delivery system. These immunological responses, seen in the IL12ns cohorts, correlate with the efficacious, anti -turn or immune adaptations known to be important in systemic IL- 12 therapy including (1) the ability to drive effector signaling within systemic immune organs, as measured by increases in proinfl ammatory cytokines (IFN-y, IL-6), (2) production of anti-angiogenic chemokines (CXCL9, CXCL10), and (3) activation of polymorphonuclear cells (neutrophils) for destruction of tumor blood vessels.20 These responses occur all while avoiding the clinical toxicity seen with the MTD strategy.

[0369] While certain preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. WSGR Ref. 59910-701.601CLAIMSWhat is claimed is:

1. A method of treating a disease in a subject in need thereof, the method comprising: a. administering a first dose of poly(D, L-lactic acid-co-glycolic acid)(PLGA) nanospheres encapsulating a first dose of IL- 12 to a subject;b. performing an immunodiagnostic assay on the subject, wherein the immunodiagnostic assay comprises measuring an expression level of IFN-y, IL- 10, CCL4 (MIP-ip), IFN-a, CXCL9 (MIG), CXCL10 (IP- 10), TNF-a, IL-6, VEGF, IL-4, CCL3 (MIP-la), CCL2 (MCP-1), GM-CSF, or a combination thereof and comparing to the result of the immunodiagnostic assay to a first immunodiagnostic assay performed before the administering of a; andc. administering a second dose of poly(D, L-lactic acid-co-glycolic acid)(PLGA) nanospheres encapsulating a second dose IL- 12 to a subject, wherein the second dose of the nanospheres or the second dose of the IL-12 is based on the results of b, and wherein the second dose of the nanospheres or the second dose of the IL- 12 is the same as the first dose of the nanospheres or IL- 12 or different than the first dose of the nanospheres or IL- 12.

2. The method of claim 1, wherein the first dose of the nanospheres is from about O. OOlmg to about 0.099mg.

3. The method of claim 2, wherein the first dose of the nanospheres is O. OOlmg.

4. The method of claim 1, wherein the first dose of the IL-12 is from about 0.16ng / kg / day to about 16 ng / kg / day.

5. The method of claim 4, wherein the first dose of the IL-12 is 0.16ng / kg / day.

6. The method of any one of claims 1-5, wherein the second dose of the nanospheres or the second dose of the IL-12 is increased or decreased relative to the first dose of the nanospheres or the first dose of the IL-12 if the expression level of one or more of IFN-y, IL- 10, CCL4 (MIP-ip), IFN-a, CXCL9 (MIG), CXCL10 (IP- 10), TNF-a, IL-6, VEGF, IL-4, CCL3 (MIP-la), CCL2 (MCP-1), or GM-CSF as measured by the immunodiagnostic assay increases or decreases relative to the first immunodiagnostic assay.

7. The method of any one of claims 1-5, wherein the second dose of the nanospheres or the IL- 12 is maintained relative to the first dose of the nanospheres or the first dose of the IL-12 if the expression level of one or more of IFN-y, IL-10, CCL4 (MIP-ip), IFN-a, CXCL9 (MIG), CXCL10 (IP- 10), TNF-a, IL-6, VEGF, IL-4, CCL3 (MIP-la), CCL2WSGR Ref. 59910-701.601(MCP-1), or GM-CSF as measured by the immunodiagnostic assay increases decreases, or remains the same relative to the first immunodiagnostic assay.

8. The method of any one of claims 1-5, wherein the second dose of the nanospheres or the IL-12 is decreased relative to the first dose of the nanospheres or the first dose of the IL- 12 if the expression level of one or more of IFN-y, IL-10, CCL4 (MIP-ip), IFN-a, CXCL9 (MIG), CXCL10 (IP- 10), TNF-a, IL-6, VEGF, IL-4, CCL3 (MIP-la), CCL2 (MCP-1), or GM-CSF as measured by the immunodiagnostic assay increases relative to the first immunodiagnostic assay.

9. The method of any one of claims 1-5, wherein the second dose of the nanospheres or the IL-12 is increased relative to the first dose of the nanospheres or the first dose of the IL- 12 if the expression level of one or more of IFN-y, IL-10, CCL4 (MIP-ip), IFN-a, CXCL9 (MIG), CXCL10 (IP- 10), TNF-a, IL-6, VEGF, IL-4, CCL3 (MIP-la), CCL2 (MCP-1), or GM-CSF as measured by the immunodiagnostic assay decreases relative to the first immunodiagnostic assay.

10. The method of any one of claims 1-9 wherein the immunodiagnostic assay or the first immunodiagnostic assay comprises measuring an expression level of IFN-y, IL-10, IL-6, TNF-a, or a combination thereof.

11. The method of any one of claims 1-9 wherein the immunodiagnostic assay or the first immunodiagnostic assay comprises measuring an expression level of CCL3 (MIP-la), CCL2 (MCP-1), CCL4 (MIP-ip), CXCL9 (MIG), CXCL10 (IP- 10), or a combination thereof.

12. The method of any one of claims 1-11, wherein the method of administering the nanospheres or the IL-12 comprises intravenous administration.

13. The method of any one of claims 1-11, wherein performing the immunodiagnostic assay or the first immunodiagnostic assay on the subject comprises collecting a sample from the subject.

14. The method of claim 13, wherein the sample comprises blood.

15. The method of claim 14, wherein the blood comprises peripheral blood.

16. The method of any one of claims 13-15, wherein the sample comprises peripheral blood mononuclear cells.

17. The method of any one of claims 1-16, wherein the immunodiagnostic assay comprises spectral flow cytometry, cytokine analysis, chemokine analysis, RNA sequencing, gene set enrichment analysis (GSEA), histology, or a combination thereof.

18. The method of claim 17, wherein the spectral flow cytometry comprises a panel ofantibodies.WSGR Ref. 59910-701.60119. The method of claim 18, wherein the antibodies comprise a fluorophore.

20. The method of claim 19, wherein the antibody panel comprises a panel as disclosed in Table 9, or any combination thereof.

21. The method of claim any one of claims 1-20, wherein performing the immunodiagnostic assay on the sample comprises measuring one or more markers disclosed in Table 10, or any combination thereof.

22. The method of any one of claims 1-21, wherein the immunodiagnostic assay comprises measuring an expression level of Fcgrl, Gbp2, Gbp3, Gbp5, Gbp7, Irfl, Irf7, Irf9, Oasl2, Statl, Socsl, or a combination thereof.

23. The method of any one of claims 1-21, wherein the immunodiagnostic assay comprises measuring an expression level of Stat3, Ccl2, Cxcl9, CxcllO, Statl, Gbp2, Gbp3, Irfl, PD-L1, Lag3, Socs3, Itgam, or a combination thereof.

24. The method of any one of claims 1-21, wherein the immunodiagnostic assay comprises measuring an expression level of Itgam, Ly6cl, Ly6g, Nos2, Argl, and PD-L1 (CD274), CD4, CD8, Foxp3, CD19, Ncrl, CTLA-4, Lag3, Havcr2, or a combination thereof.

25. The method of any one of claims 1-21, wherein the immunodiagnostic assay comprises measuring an expression level of II 12rb 1, Il 12rb2, Psme2, Anxa2, Stat4, or a combination thereof.

26. The method of any one of claims 1-21, wherein the immunodiagnostic assay comprises measuring an expression level of 1118, Icaml, Ccr5, Stat3, Cxcl2, Ptgs2, or a combination thereof.

27. The method of any one of claims 1-21, wherein the immunodiagnostic assay comprises measuring an expression level of Statl, Gbp2, Gpb3, Ifing, Cxcl9, CxcllO, Lag3, Socsl, Argl, Ly6g, Itgam, CD 19, or a combination thereof.

28. The method of any one of claims 1-21, wherein the immunodiagnostic assay comprises measuring an expression level of Statl, Gbp2, Gpb3, Irfl, Cxcl9, CxcllO, Ccl2, PD-L1, Argl, Itgam, CD4, Ncrl, or a combination thereof.

29. The method of any one of claims 1-21, wherein the immunodiagnostic assay comprises measuring an expression level of Alasl, Argl, Ccl2, Ccl3, Ccl4, CD 19, PD-L1, CD4, CD8a, CTLA-4, CxcllO, Cxcl9, Eeflg, G6pd, Gapdh, Gbp2, Gbp3, Havcr2, Hprt, Ifng, 1110, 1112rb 1, Il lb, I12rg, 116, Irfl, Itgam, Itgax, Lag3, Ly6g, Ncrl, Nos2, Polrlb, Polr2a, Rpll9, Sdha, Socsl 1, Socs3, Statl, Stat3, Stat4, or a combination thereof.

30. The method of any one of claims 1-21, wherein the immunodiagnostic assay comprises measuring an expression level of Abcbll, Abcb4, Abcc2, Abcc3, Apexl, Btg2, Casp3, Ccngl, Cd36, Cdknla, Cypla2, Cyplbl, Fasn, Fmol, Gadd45a, Gclc, Gpxl, Gsr,WSGR Ref. 59910-701.601Hmoxl, Icaml, Lpl, Mt2, Nqol, Ppara, Rbl, Rbpl, Serpinel, Srebfl, Thrsp, Txnrdl, or a combination thereof.

31. The method of claims any one of claims 1-30, wherein the subject comprises a mammal.

32. A method of administering a dose of poly(D, L-lactic acid-co-glycolic acid)(PLGA) nanospheres encapsulating IL-12 to a subject wherein the dose of the nanospheres is from about O. OOlmg to about 0.099mg.

33. The method of claim 32, wherein the dose of the nanospheres comprises O. OOlmg.

34. The method of claims 32 or 33, wherein the subject has a disease.

35. The method of claim 34, wherein the disease comprises cancer.

36. The method of claim 35, wherein the cancer comprises a tumor.

37. The method of claim 36, wherein the tumor comprises a solid tumor.

38. The method of any one of claims 32-37, wherein the subject comprises a mammal.

39. A method of administering a dose of IL-12 to a subject wherein the 11-12 is encapsulated in poly(D, L-lactic acid-co-glycolic acid)(PLGA) nanospheres and the dose of the IL- 12 is from about 0.16ng / kg / day to about 16 ng / kg / day.

40. The method of claim 39, wherein the dose of the IL-12 comprises 0.16ng / kg / day.

41. The method of claims 39 or 40, wherein the subject has a disease.

42. The method of claim 41, wherein the disease comprises cancer.

43. The method of claim 42, wherein the cancer comprises a tumor.

44. The method of claim 43, wherein the tumor comprises a solid tumor.

45. The method of any one of claims 39-44, wherein the subject comprises a mammal.

46. A method of treating a cancer in a subject in need thereof, the method comprising encapsulating IL12 in poly(D, L-lactic acid-co-glycolic acidj(PLGA) nanospheres and administering a dose of the nanospheres to the subject, wherein the dose of the nanospheres is from about 0.001 mg to about 0.099 mg.

47. The method of claim 46, wherein the dose comprises about 0.001 mg of the nanosphere.

48. A method of treating a cancer in a subject in need thereof, the method comprising encapsulating IL12 in poly(D, L-lactic acid-co-glycolic acid)(PLGA)nanospheres and administering a dose of the IL- 12 to the subject, wherein the dose of the IL- 12 is from about 0.16ng / kg / day to about 16 ng / kg / day.

49. The method of claim 48, wherein the dose of the IL-12 comprises 0.16ng / kg / day.

50. The method of any one of claims 32-49, wherein the administering comprises intravenous administration.

51. The method of any one of claims 46-49, wherein the cancer comprises a tumor.

52. The method of claim 51, wherein the tumor comprises a solid tumor.WSGR Ref. 59910-701.60153. The method of any one of claims 46-52, wherein the subject comprises a mammal.

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