Highly active myeloid therapy

Abstract

Disclosed herein are tumor-associated macrophage-targeting nanoparticles and wafers that deliver a drug combination payload for treating cancer.

Description

[0001] Highly Active Myeloid Therapy

[0002] TECHNICAL FIELD

[0003] Disclosed herein are tumor-associated macrophage-targeting nanoparticlesand wafers that deliver a drug combination payload for treating cancer.

[0004] BACKGROUND

[0005] The biology of tumor-associated macrophages (TAM) has received increasing attention over the last decade as immunotherapies have taken center stage in cancer treatment. The emerging consensus is that TAM are abundant, are recruited primarily from bone marrow (myeloid origin) and the vast majority of these cells are immunosuppressive and support and enable tumor growth. Conversely, a much smaller subtype of TAM (“Ml -like’’; M9) have antitumor effects. However, consensus on which TAM phenotypes have optimal anti-tumor function remains uncertain. Therefore, manipulation and reprogramming of TAM could enable improved disease control in patients where TAM are abundant and functionally active.

[0006] Various therapeutic options exist to modulate TAM function (myeloid therapeutics), including elimination, containment, or suppression of tumor-promoting macrophages, amplification or activation of anti -tumor macrophages, subtype switching, or any combinations. Most therapeutic approaches today are based on antibodies targeting cell surface proteins (CSF1-R. CD40, TREM2, CD47) or small molecules targeting growth factors (CSFR1).

[0007] A less explored option is to harness the phagocytic function of TAM to efficiently deliver small-molecule immune modulators and thus improve the therapeutic index of those drugs. However, this approach often has limited efficacy and / or exhibits unacceptable systemic toxicity. The diverging efficacy data are further evidence that TAM function and reprogrammability is not fully understood. In addition, there is emerging evidence that TAMs are plastic, have multiple phenotypic states, and have redundant wiring mechanisms to escape therapeutic pressures. SUMMARY

[0008] Provided herein is a nanoparticle comprising a. a cyclodextrin; b. a first payload; c. a second payload; and d. a third payload.

[0009] Provided herein is a wafer comprising a. a cyclodextrin; b. a first payload; c. a second payload; and d. a third payload.

[0010] Also provided herein is a pharmaceutical composition comprising a nanoparticle of the present invention and one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition is a wafer.

[0011] Also provided herein is a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a nanoparticle of the present invention, or a pharmaceutical composition of the present invention.

[0012] Other embodiments include those described in the Detailed Description and / or in the claims.

[0013] Additional Definitions

[0014] To facilitate understanding of the disclosure set forth herein, a number of additional terms are defined below. Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, and phannacology described herein are those well-known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary7skill in the art to which this disclosure belongs. Each of the patents, applications, published applications, and other publications that are mentioned throughout the specification and the attached appendices are incorporated herein by reference in their entireties.

[0015] The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation, for example, within experimental variability and / or statistical experimental error, and thus the number or numerical range may vary up to ±10% of the stated number or numerical range.

[0016] The term “acceptable” with respect to a formulation, composition or ingredient, as used herein, means having no persistent detrimental effect on the general health of the subject being treated.

[0017] The term "inhibit" or "inhibition of means to reduce by a measurable amount, or to prevent entirely (e.g., 100% inhibition). The terms “antagonist” and “antagonism” are used interchangeably with “inhibit”.

[0018] The phrase “therapeutically effective amount” means an amount of compound that, when administered to a subj ect in need of such treatment, is sufficient to (i) treat the indicated disease or disorder, (ii) attenuate, ameliorate, or eliminate one or more symptoms of the particular disease or disorder, or (iii) delay the onset of one or more symptoms of the particular disease or disorder described herein.

[0019] The term “pharmaceutical composition” as used herein is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) that make up the excipient or carrier.

[0020] The term “pharmaceutically acceptable excipient” means a pharmaceutically- acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, carrier, solvent, or encapsulating material. In one embodiment, each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Remington: The Science and Practice of Pharmacy. 21st ed. Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed. ; Rowe et al. , Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed. ; Gibson Ed. ; CRC Press LLC: Boca Raton, FL, 2009.

[0021] The term “payload” as used herein refers to a molecule that that exerts a biological effect on its target. For example, an agonist or inhibitor of the activity of a protein.

[0022] As used herein, the term “subject” refers to any animal, including mammals such as primates (e.g., humans), mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, primates, and humans. In some embodiments, the subject is a human. In some embodiments, the subject has experienced and / or exhibited at least one symptom of the disease or disorder to be treated and / or prevented.

[0023] As used herein, terms "treat" or "treatment" refer to therapeutic or palliative measures. Beneficial or desired clinical results include, but are not limited to, alleviation, in whole or in part, of symptoms associated with a disease or disorder or condition, diminishment of the extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state (e.g.. one or more symptoms of the disease), and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0024] The details of one or more embodiments of this disclosure are set forth in the accompanying drawings and the description below. Other features and advantages of the present disclosure will be apparent from the description and drawings, and from the claims.

[0025] DESCRIPTION OF DRAWINGS

[0026] Fig. 1A. depicts a proof-of-concept scheme consisting of a myeloid cell targeting carbohydrate nanoparticle containing a triple therapeutic payload to affect the wiring of tumor- associated macrophages.

[0027] Fig IB. depicts that targeting three pathways (TLR (red dot), non-canonical NF-KB (dark blue dot), and JAK / STAT (light blue dot) is synergistic and drives macrophages toward a unique phenotype resulting in high IL-12 production and T cell priming. Overall pathways towards an Ml phenotype are activated (green check marks) and pathways towards an immunosuppressive M2 phenotype (red stop sign) are turned off. The increased IL- 12 production, jumpstarts an effective anti-tumor T cell response.

[0028] Fig. 2A. depicts the synthetic strategy to form ~17 nm nanoparticles consisting of succinyl-P-cyclodextrin (s-P-CD) cross-linked by L-lysine linkers via EDC / NHS chemistry. Each nanoparticle contains an average of -900 s-P-CD units and forms spherical nanoparticles capable of carrying small-molecule payloads.

[0029] Fig. 2B. Different views of repeating CD units, including the guest-host interaction of a model therapeutic payload (Ruxolitinib, Compound 3).

[0030] Fig. 2C. Structures of all prototypical small-molecule (SM) modulators potentially affecting macrophage function (see B on how the lipophilic moieties fit into the s-P-CD). Fig. 2D. The images show the different nanoparticles with payload combinations investigated in this research. The numbers in each nanoparticle refer to the compounds from panel C. Yellow particles have only one payload, orange particles dual payload, and pink nanoparticles have triple payloads. The same scheme is used for subsequent figures.

[0031] Fig. 3A. depicts dynamic light scattering (DLS) of empty (“0”) or triple-loaded HAMT (“1+2+3”; see Table SI for detail). Drug loading at 0.3 pmol per mg of particle does not change the nanoparticle size (Z-Av.) or poly dispersity index (PDI).

[0032] Fig. 3B. depicts transmission electron microscopy (TEM) of multiple HAMT particles stained with aqueous uranyl acetate (2%).

[0033] Fig. 3C. depicts turbidity assay to measure drug loading into the CANDI nanoparticle assessed by an increase in absorbance. We determined the loading capacity for HAMT to be -0.19 mg of payloads 1+2+3 (0.5 pmol) per mg of CANDI nanoparticle. For subsequent biological studies, we loaded 0.11 mg (0.3 pmol) of 1+2+3 per mg of CANDI.

[0034] Fig. 3D. depicts the release kinetics of triple small molecules from HAMT using a closed-dialysis system with a porous membrane (3 kDa) in PBS (lx) at 37 °C (shown in picture). The cumulative release (%) of each drug was determined as the integrated area of the peak corresponding to each compound (diode array, UV) at nine time points (t = 0, 1, 1.5, 2, 2.5, 4, 5, 6 and 24 h) compared to the total amount of compound found in CANDI loaded with 1+2+3 (no membrane). Aliquots were analyzed by liquid chromatography coupled to a mass spectrometer (LC-MS) with a UV detector, koff values were determined by fitting each curve to a one-phase decay. All three compounds show similar release kinetics and achieve a plateau at t = 4 h. All experiments were done in triplicates (n = 3).

[0035] Fig. 4A. depicts the isolation and differentiation scheme of bone marrow-derived macrophages (BMDM) from IL-12 eYFP reporter mice with subsequent in vitro stimulation assay.

[0036] Fig. 4B. depicts the Stimulation of BMDM with different nanoparticle formulations (see Fig. 2 sand Table SI; single treatments: 0.5 pM LCL-161 (Compound 1), 0.32 pM R848 (Compound 2), 0.65 pM Ruxolitinib (Compound 3); same concentrations for combination therapies) leads to induction of IL- 12 in vitro. Dual therapy (“Compound 1 + Compound 2”) can be significantly boosted by adding Ruxolitinib (Compound 3) into the same nanoparticle (HAMT, “Compound 1 + Compound 2 + Compound 3”). Each data point represents a separate FOV with the % of eYFP positive cells of an average of -1500 cells; * = p = 0.0131; **** = p < 0.0001; 100 ng / mL LPS. 50 ng / mL IFNy. Fig. 4C. depicts representative microscopy images showing eYFP positive cells (corresponding to IL- 12 induction) after cell stimulation assay in vitro (normalized contrast in 488 nm channel, 100 ms exposure time) as shown in panel B. Scale bar = 200 pm.

[0037] Fig. 5A. depicts the treatment overview of colorectal MC38 murine model. MC38 tumor cells (2 x 106) were injected into the flank of immunocompetent C57BL / 6J mice (day 0). The first HAMT treatment (5 mg triple-loaded nanoparticle, 100 pL PBS (0.5x), red triangle) was injected after tumor growth on day 8, followed by a second treatment on day 12. Complete responders were re-challenged on day 49 and 60.

[0038] Fig. 5B. depicts tumor growth curves of control mice receiving the empty nanoparticle (CANDIE, n = 18).

[0039] Fig. 5C. depicts tumor growth curves of mice treated with HAMT (n = 15). 4 mice with residual tumors due to partial response (those above dashed line) were sacrificed on days 13 and 19 so that tumors could be processed for flow cytometry.

[0040] Fig. 5D. depicts survival curves for the two different cohorts show significantly longer survival for treated animals (p < 0.0001; n = 21 mice total).

[0041] Fig. 5E. depicts a re-challenge experiment in n = 13 mice previously treated with HAMT and complete tumor regression. Note that MC38 tumor-bearing mice previously treated with HAMT (green curves, n = 5) are immune to further tumor growth, suggesting T cell memory response (n = 8 control mice).

[0042] Figs. 5F and 5G. depicts the treatment overview of flank melanoma (F) and metastatic (G) B16-F10 murine tumor model. In F, 0.5 x 106 fluorescent B16-F10 H2B-mApple cells were injected into the flanks of C57BL / 6J mice (n = 41, day 0). After 7 days of tumor growth, mice received a total of four intravenous doses of HAMT (5 mg triple-loaded nanoparticle, 100 pl PBS (0.5x), red triangle) over a period of 2 to 3 weeks. Additional anti-PDl treatment (200 pg, 50 pl PBS, green triangle) was administered intraperitoneally on the same days as the first two HAMT treatments. In G, 0.2 x 106 fluorescent B16-F10 H2B-mApple cells were injected into mice via tail-vein injection (n = 12, day 0). Mice received a total of three intravenous HAMT or combination treatments with anti-PDl antibody (amount same as in F) over a period of 2 weeks before sacrificing animals for lung clearing and imaging.

[0043] Fig. 5H. depicts tumor growth curves of treatment groups of B16-F10 flank melanoma mice. Control mice (n = 12) received PBS injections, other cohorts anti-PDl antibody alone (n = 5), HAMT (n = 12) or HAMT + anti-PDl combination (n = 12) treatment. Fig. 51. depicts a survival curve of B16-F10 flank tumor model (n = 26 mice) shows significantly longer survival for treated animals: Median survival for untreated mice was 19 days, anti-PDl treatment alone 28 days, HAMT alone treatment 31 days, and combination treatment 62 days with 3 mice that showed complete response after therapy.

[0044] Fig. 6. Dorsal window chambers were implanted into IL-12 eYFP mice, followed by tumor cell injection into the window chambers (105 MC38 H2B-Apple cells, red). Imaging was performed serially before drug administration (pre) and after drug administration (t = 10 min to 5 h), and again 24 and 48 hours after systemic HAMT administration. Immediately after systemic administration, HAMT was detected in tumor microvessels, followed by cellular uptake 1 to 5 hours after intravenous administration. Note the marked induction of IL- 12 after HAMT administration. Scale bar = 40 pm.

[0045] Fig. 7A depicts the synthesis of s-0-CD (DS 2.5) from 0-CD.

[0046] Fig. 7B depicts the liquid chromatographs and mass-to-charge ratio (ES-) of different s-P-CD analogs (DS 2, 3, and > 4) obtained from the condensation reaction of P-CD with succinic acid at different time scales (1-24 h).

[0047] Fig. 7C depicts pictures of solutions containing s-P-CD (DS 2.5) and s-P-CD (DS > 4) in water at 4 °C to display enhanced water solubility and resistance to aggregation.

[0048] Fig. 7D depicts 1H-NMR spectrum of soluble s-P-CD (DS 2.5) and equations used to determine the average degree of substitution based on two conventional methods, DSi and DS2.

[0049] Fig. 8A depicts a schematic depiction of the CD units showing top: protons found on the outside (Hi, H2, H4) and inner (H3, H5 and He) cavity, middle: cross-section of the CD units and bottom: expected protons to yield chemical shift upon loading with hydrophobic molecules (H3, H5and H6).

[0050] Fig. 8B. 'H-NMR spectra of free and loaded s-P-CD (26 mM) in D2O (0.7 mL) with LCL-161 (1, 26 mM) with a fixed d6-DMSO concentration (10%). The included table in panel B shows in red the values in ppm for the protons that undergo a chemical shift upon loading.

[0051] Fig. 8C. Picture of translucent solutions of s-P-CD containing different LCL-161 concentrations and a turbid solution of LCL-161 without any s-P-CD (red asterisk).

[0052] Fig. 8D. 1H-NMR titrations of s-P-CD in D2O with LCL-161 up to an equimolar concentration (26 mM) show ing the presence of aromatic protons on dissolution of LCL-161 by inclusion-complex formation with s-P-CD.

[0053] Fig. 9A depicts 2D-ROESY NMR analysis of s-p-CD (DS 2.5, 26 mM) in D2O loaded with LCL-161 (1, 26 mM) to determine which functional groups undergo the strongest interaction with the inner cavity protons of s-0-CD (H3, H5 and He). The strongest binding was correlated with the cyclohexyl group (orange), methyl group H5, and the weakest for the parafluorophenol group (green).

[0054] Fig. 9B depicts 2D-ROESY NMR analysis of s-p-CD (DS 2.5, 26 mM) in D2O loaded with R848 (2, 26 mM) to determine which functional groups undergo the strongest interaction with the inner cavity protons of s-p-CD (H3, H5 and Hg). The strongest binding was correlated from the aliphatic carbon chains H1-H2 and a smaller iteration from the aromatic H9 to the Hr, succinyl of the CD unit. Signal integrals were normalized to the reference proton Hi in s-0- CD.

[0055] Fig. 10A depicts 2D-R0ESY NMR analysis of s-p-CD (DS 2.5, 26 mM) in D2O loaded with A. Ruxolitinib (3, 26 mM) to determine which functional groups undergo the strongest interaction with the inner cavity protons of s-0-CD (H3, H5 and Hg). The strongest binding was correlated with the cyclopentyl group (green) and the weakest for the indole pyrimidine aromatic group (orange).

[0056] Fig. 10B depicts 2D-R0ESY NMR analysis of s-p-CD (DS 2.5, 26 mM) in D2O loaded with Upadacitinib (4, 26 mM) to determine which functional groups undergo the strongest interaction with the inner cavity7protons of s-0-CD (H3, H5 and Hg). The strongest binding was correlated from the aliphatic carbon chains (green) and the indole pyrimidine aromatic group (orange). Signal integrals were normalized to the reference proton Hi in s-0-CD.

[0057] Fig. 11A depicts an example of a stopped-flow plot displaying the decrease in absorbance (A = 532 nm) of a turbid solution containing a fixed concentration of payload (LCL- 161, 5 mM, PBS) after rapid mixing with a solution of s-0-CD (equivalents = 0.75, 1.5, 3 and 4).

[0058] Fig. 1 IB depicts stopped-flow plots depicting the individual association rates for LCL- 161, R848 and Ruxolitinib as depicted in panel A using three or four concentrations of s-0- CD. The individual association rates were calculated by fitting the corresponding absorbance plot to a two phase decay.

[0059] Fig. 11C depicts the dissociation and association constant were determined as follows: Kd = koir / kon and K;I= kon / koff. All experiments were performed in triplicates (n = 3).

[0060] Fig. 12A depicts intracellular staining of BMDMs for IL-12 (p40 / 70) 24 h after stimulation shows significantly higher levels of IL-12 positive cells, reaching up to -30% IL- 12 positive BMDMs for HAMT (p = 0.0003 vs. control) and -15% IL-12 positive cells for double therapy “1+2” (p = 0.0063 vs. HAMT). Fig. 12B depicts a histogram showing a clear shift towards cells with higher fluorescence intensity of IL-12-PE upon stimulation. This increase in efficacy from dual to triple loading represents a major impetus for the HAMT design.

[0061] Fig. 13 CA DI1647uptake into iMACs. Fluorescence imaging of iMACs treated with C ANDIA1647. Hoechst was used as a nuclear stain and imaged in the 405 nm channel (DAPI). CANDI '1 47particles were imaged in the 647 nm channel. Cell morphology was assessed in the brightfield (white light) channel. Merged composites and zoomed-in regions of interest (dashed white squares) were generated to show cellular detail. The composite images clearly show efficient CANDI uptake into nearly all cells. Scale bar = 200 pm.

[0062] Fig. 14A depicts representative fluorescence imaging of BMDCs and merged composites generated with the brightfield (white light) and 488 nm channel of different conditions: PBS, HAMT, or CANDI monotherapies (see Fig. 2 and Table SI; single treatments: 0.5 pM LCL-161, 0.32 pM R848, 0.65 pM Ruxolitinib; same concentrations for HAMT). Note the marked IL-12 induction and cluster formation in BMDCs treated with HAMT. Scale bar = 200 pm. The lower panel shows higher magnification images, as identified by the dashed white squares. Scale bar = 50 pm.

[0063] Fig. 14B depicts flow cytometry data of BMDCs show that HAMT treatment leads to up to 50% IL-12 positive cells. In contrast, single therapy with R848 (2) leads to a maximum of 30% positivity, and LCL-161 (1) and Ruxolitinib (3) alone do not significantly increase IL- 12 levels in vitro in BMDCs. HAMT (“1+2+3”), however, increases the amount of IL-12 positive cells to above 40% in this assay (p = 0.0041 v. PBS).

[0064] Fig. 15 depicts cell viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H- tetrazolium bromide, MTT assay) data that was obtained by incubating the immortalized macrophage cell line (iMAC) with HAMT at different concentrations. The estimated in vivo concentration is indicated by the vertical dashed line (0. 1 mg / mL, > 80% viability).

[0065] Fig. 16 depicts selected enzyme levels indicative of toxicity of the free drug combo (0.25 mg LCL-161, 0.1 mg R848, 0.2 mg Ruxolitinib in 20 pL DMSO; equivalent dose to HAMT group) given systemically but not when given as nanoparticle formulation HAMT (n = 12 mice; note that only 1 of 3 mice of the “free drug” group survived after injection). Dashed lines: reference range of normal enzyme levels as provided by our pathology laboratory.

[0066] Fig. 17A depicts liver histology assessed by hematoxylin-eosin staining following administration of empty nanoparticle CANDIE(5 mg nanoparticle; 100 pL PBS (0.5x)). Fig. 17B depicts liver histology assessed by hematoxylin-eosin staining following administration of HAMT (5 mg triple-loaded nanoparticle with 0.25 mg LCL-161 (Compound 1), 0. 1 mg R848 (Compound 2), 0.2 mg Ruxolitinib (Compound 3); 100 pL PBS (0.5x)).

[0067] Fig. 17C depicts liver histology assessed by hematoxylin-eosin staining following administration of PBS (100 pL).

[0068] Fig. 17D depicts liver histology assessed by hematoxylin-eosin staining following administration of free drug (0.25 mg LCL-161 (Compound 1). 0.1 mg R848 (Compound 2), 0.2 mg Ruxolitinib ((Compound 3); 20 pL DMSO). Note the extensive hepatocyte damage and tissue disintegration with free drug ( ellow arrows).

[0069] Fig 18A. depicts that whole body excretion was primarily via urine and feces. Taking into account radioactive decay, the remaining whole body radioactivity was 25% injected dose (ID) at 21 hours, similar to other carbohydrate nanopreparations of similar size. Major nanoparticle uptake w as observed in the macrophage-rich organs of the reticuloendothelial system such as the liver. This is characteristic of most materials with high molecular w eights, such as antibodies, proteins, and nanoparticles. Tumoral uptake in whole tumors varied across animals and ranged from 6.54 to 1.32 %IDGT. Uptake in remaining organs (lung, heart, brain, digestive tract, muscle, fat and reproductive organs) was low.

[0070] Fig. 18B depicts that autoradiography of removed MC38 tumors shows64Cu-CANDI accumulation in peripheral zones, which contain the highest amounts of tumor-associated macrophages (TAMs). In these focal areas, uptake was similar to values observed in the liver.

[0071] Fig. 19A depicts lungs that were harvested on day 14 and cleared using cardiac perfusion to show intrapulmonary melanoma deposits as dark melanotic deposits. Note that HAMT treatment alone and HAMT + anti-PDl resulted in significantly fewer pulmonary lesions in mice compared to control mice. A total of n = 12 mice were used (n = 4 mice per group).

[0072] Fig. 19B depicts that quantification of tumor burden in confocal microscopy images of cleared lungs shows significantly reduced tumors in HAMT-treated mice (p = 0.0121) compared to control mice without treatment. Combination treatment of HAMT + anti-PDl led to the lowest tumor burden (p < 0.0001). Each data point represents the number of tumors identified per mouse (n = 12 mice total).

[0073] Fig. 19C depicts a graph showing mouse body weight variations throughout the entire therapy. No significant decrease in body weight was observed due to treatment toxicity. Fig. 20A depicts the intracellular localization of the nanoparticle in the tumor microenvironment. MC38 bearing mice were injected with HAMT-AF647 and Dextran-Pacific blue. Note that HAMT is almost exclusively associated with cells that also internalize dextran. There was no uptake into primary tumor cells. Scale bar = 20 pm.

[0074] Fig. 20B depicts that HAMT-containing cells also contain 2M MW Dextran-PB, a known macrophage marker. Nearly all HAMT-containing cells show IL-12 induction, as shown by the green IL- 12 eYFP signal.

[0075] Fig. 21 depicts flow cytometry analysis of immune cell subsets in MC38 tumors 24 h after intravenous injection of CANDI. Analysis shows unique uptake of CANDI into myeloid cell populations but not into lymphoid cell populations.

[0076] Fig. 22A depicts flow cytometry of functional T cell status in HAMT treated mice to determine the effects of treatment on T cells. HAMT treatment results in fewer exhausted T cells, which are still capable of mounting an effective anti-tumor response.

[0077] Fig. 22B depicts flow cytometry experiment of in vitro stimulated IFNy-GFP reporter CD8+T cells. Upon stimulation (aCD3 / CD28) of T cells with IL-12 but not IL-10 (both 20 ng / mL), T cells start producing the pro-inflammatory cytokine IFNy. This mechanism highlights the importance of high IL-12 levels within the TME as activated T cells further lead to anti-tumor immunity' and activation of DCs.

[0078] Fig. 23A depicts western blot analysis of mechanism of action for TLR 7 / 8 agonist R848. Stimulation of iMACs with R848 (0.96 pM; LPS + IFNy (100 ng / mL and 50 ng / mL respectively) as control) for 30 min leads to phosphorylation of p38 MAPK (Thrl80 / Tyrl82) indicating an activation of NF-KB signaling within the cells. Similarly, stimulation leads to (p- )ERKl / 2 and p-IKKa (data not shown).

[0079] Fig. 23B depicts western blot analysis of NIK levels in iMACs stimulated with cIAP inhibitor LCL-161. Stimulation of iMACs with LCL-161 (1.5 pM; TNFa (10 ng / mL) as control) for 15 min show s increase in NIK levels indicating an activation of non-canonical NF- KB signaling as constitutive proteosomal degradation of NIK is inhibited and signaling therefore activated.

[0080] Fig. 23C depicts how Ruxolitinib (Compound 3) could act synergistic to NF-kB modulation, we hypothesized that it could reverse IL-10 mediated effects of NF-KB induced IL-12 production. Therefore iMACs w'as treated with IL-10 (10 ng / ml) for 30 min showing marked phosphorylation of STAT3 (Tyr 705) as expected. Ruxolitinib (Compound 3, 2 pM) was able to inhibit this phosphorylation completely. Fig. 24A depicts that bulk RNA sequencing results in BMDMs treated with HAMT. These results show highly up-regulated IL- 12 levels after HAMT treatment, whereas IFN- related markers do not increase significantly when compared to LPS + IFNy as control. The Z- score (-1 to +1) was assessed as the relative expression of genes compared to other treatment groups of the same row.

[0081] Fig. 24B depicts temporal analysis of selected cytokines following HAMT stimulation as determined by cytokine array panel.

[0082] Fig. 24C depicts that comprehensive cytokine array panel showing expression of selected cytokines by BMDMs at different stimulation timepoints (8 h, 24 h, 36 h). Acute inflammatory' cytokines and myeloid activation markers are highly expressed.

[0083] Fig. 25A depicts that iMAC cells were incubated with different uptake inhibitors, and cellular uptake of C ANDIAl fi47was assessed using live-cell microscopy.

[0084] Fig. 25B depicts similar experiments as in Fig. 25A but with flow cytometry read-out. Chlorpromazine (10 pg / mL) inhibits Clathrin-mediated endocytosis (Rho-GTPase); Wortmannin (0.1 pg / mL) inhibits micropinocytosis / phagocytosis (PI3K); Imipramine (1.5 pg / mL) inhibits macropinocytosis; E1PA (Ethylisopropylamiloride; 7.5 pg / mL) inhibits macropinocytosis via Na / H exchange; Fucoidan (0.5 mg / mL) inhibits the scavenger receptor. The biggest effects were observed with scavenger receptor inhibition through Fucoidan.

[0085] Fig. 26A depicts overview of the triple strategy in inhibiting (JAK, cIAP) and agonizing (TLR) pathways in TAM. The final effect is a sustained and high local IL- 12 elevation, much higher than is possible by endogenous stimuli.

[0086] Fig. 26B depicts that sustained levels of IL-12 are necessary to maximize the effector T cell response against cancer. Endogenously, this is achieved by IFNy signaling. Here, we show that agonizing the TAM / DC pathways in Fig. 26A pharmacologically enhances IL-12 production and improves the efficacy of PD-1 checkpoint blockade.

[0087] Fig. 27 depicts images from a thirty minute time-lapse movie of MC38 H2B-apple tumor-bearing mice on the IL-12 eYFP background. Images were acquired 24 hours after systemic administration of HAMT-AF647. Note the drug distribution in both sessile (macrophages) and mobile (DC) cells in the tumor microenvironment. HAMT-AF647 colocalizes with 2M dextran-PB. All movies have the same scale bar = 40 pm.

[0088] Fig. 28 depicts an exemplary' synthesis of the nanoparticles of the present invention.

[0089] Fig. 29 depicts dynamic light scattering data for the nanoparticles of the present invention. Fig. 30 Cellular effects of the CANDI wafer. The CANDI wafer material exerts important effects on myeloid-derived cells both in vitro and in vivo. Fig. 30A. To determine cellular uptake of the wafer material (CANDI450), bone marrow-derived myeloid (BMDM) cells were differentiated into macrophages using M-CSF. After 7 days, macrophages were incubated with 0.01 mg CANDI450-AF647 / ml for 42 hrs and cells were imaged by confocal microscopy. Note the uptake of the CANDI450 material into vesicular structures, presumably perinuclear lysosomes. DAPI = blue, CANDI450 = red. CellBrite membrane stain = green; scale bar: 10 pm. Fig. 30B. Mechanism of cellular CANDI uptake. iMAC cells were incubated with different uptake inhibitors, and cellular uptake of CANDI-AF647 was assessed using flow cytometry. Chlorpromazine inhibits clathrin-mediated endocytosis (Rho-GTPase). Wortmannin inhibits micropinocytosis / phagocytosis (PI3K). Imipramine inhibits macropinocytosis. EIPA inhibits macropinocytosis via Na / H exchange. The biggest effects were observed with chlorpromazine. Fig. 30C. Summary' of TME cells staining positive for wafer material. Each column represents a mouse. Note that the material is mostly present in CD1 lb positive TAM as opposed to other cells. Fig. 30D. Schematic overview of the effects of the triple drug on myeloid function. The combined effect is an immunostimulatory one. Fig. 30E. Cytokine induction in BMDM exposed to drug-loaded CANDI wafer as determined by ELISA. Note the high expression of IL12, CCL5, and the down regulation of SPP135. F. Prominent in vitro induction of IL12 in BMDM derived macrophages. Scale bar: 50 pm. G. Bulk RNAseq up-regulation of IL12, CCL5, MARCO, and other immunostimulatory genes. Note the down-regulation of TREM2, MRC 1, and CLEC7a.

[0090] Fig. 31A and Fig. 31B. Synthetic spatial maps of myeloid cell distribution in CT-2A. The white grids represent 63 separate fields of view imaged to acquire the entire tissue specimen. Note that -25% of all cells in CT-2A GBM represent macrophages. Fig. 3 IB. High- resolution maps show the distribution of different immune cells in GBM specimen (each FOV is 512 pm x 512 pm; scale bar: 100 pm). Fig. 31C. scRNAseq data from CT-2A dataset30 shows the prominence of the macrophage / monocyte pool. Fig. 3 ID. Gene set enrichment analysis: the CT-2A TAM pool is characterized by high NFkB signaling and hypoxia, contributing to the highly immunosuppressive environment. Fig. 3 IE. Schematic overview of the study. Intracranially implanted murine GBM were surgically resected, and the resection cavity was filled with a triple drug-loaded CANDI w afer. The release of the wafer payload converts the immunosuppressive pro-tumorigenic myeloid compartment into an anti-tumor one (“myeloid immunoconversion”). The converted myeloid cells produce cytokines that result in T-cell recruitment (via IL12. CCL5, and others) and further anti-tumor effects.

[0091] Fig. 32 CANDI wafer material. Fig. 32A. Bissucinyl P-cyclodextrin (bsCD) was crosslinked with lysine to yield a gel-like material (E) that was subsequently lyophilized into implantable solid but porous wafers. Fig. 32B. The cyclodextrin units in the wafer act as hostguest units (see Fig. 32C) and are loaded with immune stimulatory small molecules (JAKi, cIAPi, TLR7 / 8 agonist) such as those shown in Fig. 32D and which stimulate IL 12 production in TAM27. Fig. 32E. Gel-like core material following cross-linking. Fig. 32F. Lyophilized gel material ready for implantation into the GBM resection cavity. Fig. 32G. Scanning electron microscopy of the wafer surface showing a porous solid material. Fig. 32H. MRI was used to monitor the in vivo degradation of covalently Gd-labeled wafer material (n=3). Fig. 321. Release rates of the small molecule compounds from the wafer material (n=3). The release reaches a maximum within ~7 days. Additional release in vivo depends on the degradation of the wafer material, which occurs over several days.

[0092] Fig. 33 Phannacodynamics revealed by serial intravital imaging. Fig. 33A. Brain windows were implanted into mice to allow for senal imaging of tumor growth, resection, and monitoring of drug effects of implanted CANDI wafers. Experiments were performed in Mer- TK-GFP mice (Fig. 33B) and IL12-eYFP mice (Fig. 33C). B. Three days after wafer-AF647 implantation, wafer material could be detected in Mer-TK-GFP positive macrophages which had accumulated at the periphery of the wafers. Fig. 33C. Using IL12-eYFP reporter mice, we measured temporal IL12-eYFP induction in the tissue surrounding the wafer. The highest IL12 concentrations occur between 1 and 5 days after implantation. Of note is the fact that only microwafers and not full-sized ones could be implanted under the windows, leading to a faster degradation time. Scale bar: 100 pm. Fig. 33D. Note that the locally high IL12 levels do not cause an increase in systemic IL12 levels. The gray shaded area represents the normal level. Fig. 33E. Quantitation of IL12-eYFP levels in the window model. Note that the wafer in the window model was much smaller, and hence, the IL12 induction effects are shorter than those of the GBM resection model.

[0093] Fig. 34 Therapeutic efficacy of surgery with myeloid immunoconversion. Fig. 34A. Study overview. Following implantation, GBMs were resected, and wafers were implanted on day 12. Tumors were monitored by serial bioluminescence and or MR imaging up to 96 days after tumor implantation. Tumors were processed for histology, flow, cytometry, and other studies at various times after resection. The dark blue line in the surgical curve reflects animals (n=18) whose GBM were surgically resected without wafer implantation. The light blue curve shows resected GBM with wafer implantation (n=21). Eighty days after tumor inoculation, approximately half of the animals were alive and appeared functionally intact. Fig. 34B. Similar experiment as in Fig. 34A but with additional combination therapies, including radiation and temozolomide (standard-of-care). Note the higher survival. MRI imaging (Fig. 34C) typically showed a residual surgical cavity filled with fluid but completely absent of tumor as determined by histology; scale bar: 2 mm.( Fig. 34D).

[0094] Fig. 35 Immune cell analysis in TME. Fig. 35A. Immunohistochemistry on native CT- 2A tumors (top panel; n=3), 6 days after subtotal resection and wafer implantation (middle panel; n=6) and 90 days after surgery (bottom panel; n=4). Analysis shows abundant macrophages in the TME while other immune cells are scant, consistent with the known immunosuppressive environment. Following resection and wafer implantation, the myeloid cell compartment increases, and so does the CD8 and CD4 recruitment. Ninety days after surgery, there are hardly any immune cells remaining at the prior GBM site. Fig. 35B. Representative synthetic images corresponding to the three different conditions and two different time points (FOV : 512 pmx 512 pm; scale bar: 100 pm). Fig. 35C. Temporal analysis of CD8, CD4, and total immune cells over time.

[0095] Fig. 36 Overview of myeloid cell screening approach. Fig. 36A) TAM are bone marrow -derived, abundant in many cancers, and mostly immunosuppressive and thus pro- tumorigenic. Fig. 36B and Fig. 36C This phenotype is driven primarily by IL10 and hypoxia (SPP1) signaling. We hypothesized that it should be possible to polarize macrophages to an antitumorigenic phenotype by increasing CXCL9 signaling. Yet, no effective pharmaceutical strategies have emerged to do this effectively while retaining TAM specificity. Fig. 36D In this research, we used BMDM from Rex3 reporter mice (expressing CXCL9-RFP) to screen for small-molecules and combinations that could induce CXCL9 in myeloid cells. Top hits from the screen were then encapsulated into TAM-avid nanoparticles to elicit a CXCL9 phenotype in vivo.

[0096] Fig. 37 Small-molecule compounds tested. Fig. 37A Summary of the chemical structure of the 21 small-molecule compounds tested. The colored dots rep-resent individual compounds for identification across figures. Fig. 37B Different classes of compounds are considered according to the current understanding of CXCL9 regulation in macrophages. Fig. 37C Summary of single compound screens, dual compound screens, and triple compound screens. The colors of the dots represent the molecular structures shown in panel A. Fig. 38 Screening results. Three separate screens were performed, each one informing the design of the next screen. Fig. 38A Screen 1 was performed without IFNg stimulation of BMDM and explored CXCL9 TAM expression after single or dual agent exposure, as shown. Note the lack of efficacy of any of the compounds, indicating that baseline levels of IFNg are likely required for pharmacological CXCL9 induction. Screen 2 repeated the same screen but with baseline stimulation of IFNg. Note that some of the dual combinations yielded elevated levels of CXCL9. Screen 3 largely explored triple drug combinations with IFNg stimulation. Note the highest CXCL9 expression of combinations involving RBN2397, CRX527, R848, and / or MSA-2. In parallel experiments, drug toxicity was determined. Drugs or combinations with a limited therapeutic window are shown in light brown. Given these results, the top hit emerging from the screen was the triple combination of RBN2397+MSA-2+R848 which was then formulated into the CANDI400 formulation shown in subsequent figures. Fig. 38B Representative images (550 nm channel, RFP) obtained from screens 2 (dual compound R848+RBN2397) and screen 3 (Bottom triple compound RBN2397+MSA-2+R848). The black inserts represent the negative controls without drugs and IFNg stimulation alone. Scale bar 200 pm.

[0097] Fig. 39 Formulation of small-molecule hits into TAM-avid nanoparticle (CANDI400). Fig. 39A Synthetic strategy to form ^17 nm nanoparticles consisting of sbCD cross-linked by L-lysine linkers via EDC / NHS chemistry. Fig. 39B. Transmission electron microscopy images revealed that the nanoparticles retained a spherical form and were able to carry the proposed three small-molecule payloads. Fig. 39C. Synthesis of MSA-2p from MSA-2. The left bottom graph indicates the percent of free / bound drug to sbCD, determined as turbidity value (measured at 550 nm, average of 500 s time-lapse, N = 3). Bulk studies using MSA-2p in buffer (pH 7.4) showed that complexation to sbCD leads to a significant reduction in the hydrolysis rates of MSA-2p (right). Fig. 39D. Turbidity’ assay to measure loading and stability' of the CANDI400 formulation assessed by an increase in absorbance at 550 nm. We detemrined the loading capacity for CANDI400 to be ^0. 15 mg of payloads per mg of CANDI nanoparticle. E. Release kinetics of the triple small-molecule combo from CANDI400 using a porous membrane (3 kDa) in PBS (1 *)at 37°C. The release rates for R848, MSA-2, and RBN2397 are depicted as the dissociation rates (kofl)and complex half-life (t 1 / 2), respectively determined in a 7 h time lapse (N = 3).

[0098] Fig. 40 Cellular properties of CANDI400. Fig. 40A BMDM obtained from Rex3 reporter mice were incubated with CANDI400 for 24 h and then observed by microscopy. Cellular nuclei were stained with SYTO™ 11 Green (green, 473 nm channel), CANDI400 was revealed by labeling the NP with AF647 (white. 633 nm channel). CXCL9 (red) and CXCL10 (cyan) were imaged by endogenous fluorescent protein expression (RFP, 559 nm channel and BFP, 405 nm channel respectively). Note the high CXCL9 levels in all cells containing CANDI400. Scale bar: 20 pm. Fig. 40B Comparative measurements of key cytokines and TAM activation makers using flow cytometry. Note the high CXCL9 expression with CANDI400, even higher than with IFNg and LPS stimulation of cells. Fig. 40C Dose-response curve with increasing concentration of CANDI400, revealing EC50 values of 3.4 ng mL-1 for CXCL9 and 3.2 ng ml1for CXCL10, respectively. Fig. 40D Cellular toxicity using the MTT assay. Note the lack of toxicity at drug concentrations <0.1 mg mL-1. C and D were used to estimate drug concentrations for in vivo experiments (green-shaded area).

[0099] Fig. 41 Intravital microscopy of CXCL9 induction in a tumor mouse model. A Serial imaging of the TME in the colorectal MC38-H2B-GFP tumor model. Eight days after tumor implantation, CANDI400 was administered by tail vein, and serial repeat imaging was performed. C AND 1400 was labeled with AF647 and contained the triple small-molecule cocktail (RBN2397, MSA-2p, and R848) to induce CXCL9 in myeloid cells in the TME. The top row (scale bars 50 pm) shows MC38-H2B-GFP tumor cells (green, 488 nm channel), the middle row CANDI400AF647 (white, 647 nm channel), and the lower row CXCL9-RFP (red, 550 nm channel). Immediately after intravenous injection of CANDI400, the nanotherapeutic drug is largely confined to vessels. Within several hours, the C ANDI is later taken up by TAM. Note the high CXCL9 induction within 24 h after systemic CANDI400 administration.

[0100] Fig. 42 SPP1 expression in tumor-associated macrophages (TAM). Fig. 42A TAM are often abundant and are recruited from the bone marrow via monocytes. SPP1 levels increase as monocytes differentiate into TAM. TAM can be targeted with systemically administered nanopreparations, a strategy that allows high local drug concentrations and multi- pharmacological pathway modulation. Fig. 42B Note the negative correlation of TAM SPP1 with clinical outcomes in patients with HNSCC1. Fig. 42C SPPl / Sppl is induced by many different factors and conditions, usually pro-inflammatory conditions. The effects of Sppl expression (osteopontin) and secretion are generally pro-tumorigenic and immunosuppressive. Fig. 42D In vivo imaging of Sppl in an MC38 mouse model. Note the abundance of Sppl- positive cells in the tumor microenvironment. E. Temporal Sppl induction. Bone marrow cells were obtained from Sppl-TdTomato reporter mice and differentiated into macrophages using MCSF. The signal plateaus around 6-8 days. Representative Sppl-tdTomato images were obtained on day 1 (left), day 3 (middle), and day 5 (right). Note the increase in the Sppl signal.

[0101] Fig. 43 Small molecule screen to identify compounds capable of inhibiting Sppl production. Fig. 43 A Experimental outline. Bone marrow was harvested from Sppl-tdTomato reporter mice, and cells were differentiated into macrophages for 5 days. Cells were incubated with different concentrations (L: low, 0.1 pM; M: medium, 0.5 pM; H: high, 1 pM) of inhibitors on day 1, and Sppl signal was determined on day 5. Fig. 43B List of small molecule compounds tested. The list was chosen based on prior reports on pathway interactions of Sppl (hypoxia26, MIF21, PPARG27 and putative direct inhibition. The color coding reflects normalized MFI as shown in Fig. 43 A. Most putative Sppl modulators did not affect the high Sppl expression levels (red). Compounds that showed a dose-dependent effect (blue) included TLR agonists (R848), CSFIRi (PLX3397 and PLX5622), and TNFa inhibitor (Shikonin). Fig. 43C Dose-response of different small molecule modulators in decreasing SPPL Bone marrow was harvested from Sppl-tdTomato reporter mice, and cells were differentiated into macrophages for 5 days. Cells were incubated with different concentrations of inhibitors on day 1, and SPP1 signal was determined on day 5. Note the Sppl decrease as a function of drug dose. The green bar represents the approximate in vivo dose. Images on the right are representative examples of Sppl-tdTomato at baseline and at EC so. Scale bar: 100 pm. Fig. 43D Drug effects (decrease in Sppl) for representative single, dually combined, or triple lead compounds. High Sppl inhibition was observed with dual and triple combinations. The graph on the right shows the cellular toxicity of the same small molecule combinations. Based on these data, we incorporated the top hits into CANDI460 for TAM delivery (Fig. 45)

[0102] Fig. 44 Characterization of triple-drug loaded CANDI460 nanoformulation. Fig. 44A CANDI was synthesized by cross-linking bis-succinyl cyclodextrin with lysine into ~16 nm nanoparticles. Fig. 44B TEM of CANDi shows small uniform nanoparticles measuring ~18 nm in diameter. Fig. 44C Payload of CANDI460. Fig. 44D Biological characterization of CANDI460 in IMAC. Shown are dose-dependent cellular uptake and decrease of Sppl as a function of dose (0.08 mg drug / mg CANDI). E. Cellular uptake of CANDI-460 (white) in Sppl-tdTomato expressing BMDM. Scale bar: 100pm. F. Cellular toxicity of CANDI460 in iMAC. The green shaded area show's the expected in vivo concentrations. Note the favorable therapeutic window'. For high-resolution cellular uptake. Note the down-regulation of Sppl and up-regulation of CXCL9, and CCL5. Fig. 45 Efficacy in MC38 model. Fig. 45A MC38 tumors were implanted into the flank of mice on day 0. CANDI460 (5 mg / mouse) or control (CANDI empty) was administered IV on days 8,10, 12 after tumor implantation. Tumor sizes were monitored over time by caliper measurements. Fig. 45B Tumor volume changes plotted as a function of time with either three IV doses of CANDI460 or control. Note that of the 12 animals treated with CANDI460, 9 were tumor-free at 30 days. These tumor-free mice were resistant to MC38 re-challenge. Fig. 45C Survival curves. Fig. 45D MC38 re-challenge experiment. Survivors (n=6) were reimplanted with MC38 after one month. Fig. 45E Representative photographs of the tumor implantation site. H&E staining of MC38 tumors treated with CANDI460 or CANDI empty control. Note that no residual tumor exists in the representative example of this CANDI460-treated mouse. Scale bar: 100 pm.

[0103] Fig. 46 Serial In vivo microscopic imaging. Fig. 46A Dorsal window chamber model of MC38-H2BGFP tumors implanted in Sppl-tdTomato mice. Tumors were imaged 8 days after implantation when the MC38 tumors had grown into solid masses. At this time point (Day 0 pre), there was diffuse infiltration of the TME with highly Sppl -positive cells. B-C. Both the number (Fig. 46B) and the Sppl mean fluorescence intensity (MFI, Fig. 46C) per cell was higher than on subsequent days after treatment with an IV dose of CANDI460. Furthermore, tumors became smaller within 2 days of treatment. Scale bar: 100 pm.

[0104] DETAILED DESCRIPTION

[0105] Tumor-associated macrophages (TAM) interact with cancer and stromal cells and are integral in sustaining many cancer-promoting features. Therapeutic manipulation of TAM could therefore improve clinical outcomes and synergize with immuno- and other cancer therapies. While different nano-carriers have been used to target TAM, a knowledge gap exists on which TAM pathways to target and what payloads to deliver for optimal anti-tumor effects. We hypothesized that a multi-part combination involving the Janus Tyrosine Kinase (JAK), non-canonical nuclear factor kappa light chain enhancer of activated B cells (NF-KB). and tolllike receptor (TLR) pathways could lead to a highly active myeloid therapy (HAMT). Thus, we devised a screen to determine drug combinations that yield maximum IL- 12 production from myeloid cells to treat the otherwise highly immunosuppressive myeloid environments in tumors. Here we show the extraordinary' efficacy of a triple small-molecule combination in a T AM-targeted nanoparticle for eradicating murine tumors, jumpstarting a highly efficient anti- tumor response by adopting a unique anti-tumor TAM phenotype and synergizing with other immunotherapies. The HAMT therapy represents anew approach in immunotherapy and leads to durable responses in murine cancer models.

[0106] Also described herein is the design and synthesis of a carbohydrate nano-carrier able to encapsulate multiple different small-molecule drugs into a single formulation to i) concentrate drug combinations in tumor-associated macrophages and ii) to affect multiple immune modulatory pathways in targeted cells. We found that this multi-target engagement is a highly effective method to modulate TAM function and is much more effective and better tolerated than single-agent therapeutics since the latter would require much higher doses. The reasoning behind developing HAMT is similar to the success stories of highly effective retroviral combination therapies. The combination of three drugs allows stimulation or antagonism of multiple pathways, likely eliminating resistance mechanisms (e.g. counteractive IL-10 effects during IL-12 production), and the use of much lower drug doses of each component. This method of simultaneous targeting of linked immune stimulatory and counter-regulatory pathways in one therapeutic particle can potently reprogram TAM towards anti-tumor phenotypes.

[0107] A variety of different approaches have previously been used to deliver IL-12 to tumors using protein, viral vectors, and mRNA. Many of these prior approaches have failed clinically for efficacy or toxicity reasons while murine work supports the efficacy of IL-12-based therapies. However, many of these prior approaches rely upon the direct delivery or production of IL-12 through therapeutic vectors. The challenge then is to develop more efficient and locally active IL-12 therapeutics. Immune signaling is complex but involves multiple regulatory' pathways acting in concert to provide strong immune stimulation. For example, T cell stimulation requires at least three discrete signals such as T cell receptor (signal 1), co- stimulation (signal 2), and cytokines (signal 3). These signals are provided in context by activated antigen-presenting cells such as dendritic cells or macrophages, and perhaps the failure of earlier IL- 12 modulating therapeutics w as due to the IL- 12 signal acting in isolation. The work described here is fundamentally different from the above approaches in that we stimulate local myeloid cells rather than deliver exogenous proteins, fusion proteins, proteinencoding sequences, or engineered cells. This approach has multiple advantages, including simplicity, much higher efficacy, and importantly, multi-pathway modulation to more effectively stimulate myeloid cells, all while lacking measurable toxicity'. Oddly, the HAMT approach using a specific triple-drug combination did not enhance other co-stimulation (CD80 / 86). cytokines (IL-15, IL-18, IL-27), and chemokines (Cxcl9 / 10) and was largely devoid of ISGs (Fig. 24); nonetheless conferred potent anti-tumor phenotypes in vivo. A possible explanation for this observation could be that normally TAM antigen presentation to cytotoxic T cells drives T cell exhaustion, activation of IL- 12 in TAM without enhanced antigen presentation could therefore be a feature of HAMT therapy. HAMT therapy could also have an effect on naive T cell priming for immune cold tumors whereby, defective antigen presentation early on results in diminished T cell responses. Our data with CD8+T cells shows that HAMT treatment reduces terminally exhausted T cell numbers in tumors, so in fact, it does not trigger exhaustion. T cell-driven effects seen in our models likely rely upon efficient antigen-presenting cell (macrophage and dendritic cell) activation within the tumor microenvironment as HAMT therapy does not accumulate within T cells nor tumor cells in vivo (Fig. 21). In summary, these multiple mechanisms are likely the reason why the featured approach is superior to simple IL-12 delivery, which is often modest and by itself may lead to T cell exhaustion since T cell stimulatory signals in isolation do not produce robust T cell responses.

[0108] While we tested multiple different triple combinations, we found that loading CAND1 particles with LCL-161 (Compound 1), R848 (Compound 2), and ruxolitinib (Compound 3) (HAMT) lead to the most stable nanoparticle formulation and strongest IL-12 induction. The cIAP inhibitor LCL-161 essentially works through the non-canonical NF-KB pathway, while the R848 agonist works through the canonical NF-KB pathway by stimulating TLR7 / 8. From a host-guest chemistry point of view, we observed a two-phase drug release behavior which was expected for a drug delivery systems that utilize macromolecular host-guest interactions such as small-molecule payload (guest) complexing to cyclodextrins (host). The two-phase release can be often desirable to provide an initial rapid therapeutic effect followed by a slower sustained and prolonged release. The initial rapid-release phase could be mainly attributed to weaker host-guest interactions occurring on the particles surface. There, the molecules can undergo fast adsorption-desorption cycles and are prone to competitive displacement from other targets present in solution. From our 2D-R0ESY NMR experiments, we confirmed that the small-molecule payloads in HAMT undergo multiple orientations during complexation with the cavity of s- -CDs (Fig. 9 and Fig. 10). It was observed that cyclohexyl group in LCL- 161 and cyclopentyl in ruxolitinib, as well as ethylene glycol groups found in R848, displayed the strongest spatial interactions with the protons found in the hydrophobic cavity of the s- - CDs and induced a chemical shift in Haand Hb (Fig. 9 and Fig. 10). The slower, prolonged release of the payloads can be attributed to molecules found closer to the particles core governed by strong inclusion complexes or those that are surrounded by higher number of free s-P-CDs.

[0109] It was surprising that JAK inhibition through ruxolitinib significantly improved the efficacy of IL-12 induction at much lower doses. Stimulation of macrophages with TLR agonists results in the secretion of TNFa, IL-6, and IL-12, which is also controlled by multiple feedback pathways. Importantly, macrophages also produce IL- 10, inhibiting pro- inflammatory cytokine production via the JAK / STAT3-dependent pathway. Prior work has shown that ruxolitinib can block the IL-10-mediated feedback inhibition on cytokine transcription in macrophages. Overall, these results suggest that inhibition of JAKs may increase the inflammatory potential of macrophages stimulated with TLR agonists.

[0110] The current research shows that combinatorial loading of myeloid-avid nano-carriers offers an attractive venue for more efficient and potentially complementary cancer therapy. A particularly interesting observation was that the pharmacologic manipulation resulted in TAM phenotypes orthogonal to biological systems. HAMT-induced macrophage re-programming can be neither defined as theilMl” or ”M2” axis. Instead, we show that this approach leads to a unique activated TAM phenotype that confers anti-tumor functions. This TAM phenotype is characterized by high IL- 12 production, surface expression of macrophage receptor with collagenous structure (MARCO), DC-SIGN, and SIGNR7; however, interferon-stimulated genes (ISG) were largely absent in this TAM phenotype, suggesting that IL- 12 expression can occur independently of ISG responses. Since the Ml and M2 phenotype classification does not capture the optimal anti-tumor TAM phenotype, we argue that IL-12 induction by myeloid cells should be a guiding principle of anti-tumor TAM re-programming. Using high throughput, image-based screening we identified HAMT as a therapeutic combination that most strongly induces IL- 12 and exhibits potent efficacy in multiple pre-clinical cancer models. This platform for TAM re-programming agent selection has the potential to fine-tune desirable effector functions while avoiding undesirable features such as compensatory7immune suppression (e.g. PD-L1). While we show remarkable efficacy in multiple murine models, we anticipate future research and optimization to result in further improvements. For example, broader screens might identify further synergistic combinations, while radiometric loading could be optimized to titrate macrophage activation. Finally, it is possible to target CANDI formulations to more specific myeloid cell subtypes for further enhanced efficacy. Nanoparticles

[0111] Provided herein is a nanoparticle comprising a cyclodextrin; and a. a first payload; b. optionally a second payload; and c. a third pay load.

[0112] Also provided herein is a nanoparticle comprising a cyclodextrin; and a. a first payload; b. a second payload; and c. a third payload.

[0113] In some embodiments, the nanoparticle has a diameter of about 10 to about 20 nm. In some embodiments, the nanoparticle has a diameter of about 15 nm to about 20 nm. In some embodiments, the nanoparticle has a diameter of about 17 nm. In some embodiments, the nanoparticle has a diameter of 16 nm to 18 nm. In some embodiments, the nanoparticle has a diameter of about 16 nm.

[0114] In some embodiments, the nanoparticle has a surface to volume ratio of about 0.3 to about 0.4. In some embodiments, the nanoparticle has a surface to volume ratio of about 0.35. In some embodiments, the nanoparticle has a surface to volume ratio of 0.34 to 0.36.

[0115] In some embodiments, the cyclodextrin is crosslinked. In some embodiments, the cyclodextrin is crosslinked with a crosslinker. In some embodiments, the crosslinker comprises at least two nucleophilic groups (e.g., -OH or -NH2) or at least two -C(=O)OH groups. In some embodiments, the cyclodextrin is crosslinked with a crosslinker comprising aspartic acid, alanine, cysteine, arginine, / .-serine, diamine, ethylene glycol, polyethylene glycol, triethanolamine, diamine, Nl-(2-(4-(2-Aminoethyl)piperazin-l-yl)ethyl)ethane-l,2-diamine, ethanolamine, diethanolamine, / / -lysine, or / .-lysine. In some embodiments, the cyclodextrin is crosslinked with a crosslinker comprising -lysine. In some embodiments, the ratio of the cyclodextrin to crosslinker is about 1 to about 4.

[0116] In some embodiments, the cyclodextrin is carboxymethyl cyclodextrin. In some embodiments, the cyclodextrin is P-cyclodextnn. In some embodiments, the cyclodextrin is mono substituted P-cyclodextrin. In some embodiments, the cyclodextrin is bi substituted P- cyclodextrin. In some embodiments, the cyclodextrin is tri substituted P-cyclodextrin. In some embodiments, the cyclodextrin is succinyl-P-cyclodextrin. In some embodiments, the succinyl- P-cyclodextrin has a degree of substitution of about 2 to about 3. In some embodiments, the succinyl-p-cyclodextrin has a degree of substitution of about 2.5.

[0117] In some embodiments, the cyclodextrin is bis-succinyl-P-cyclodextrin.

[0118] In some embodiments, the cyclodextrin is alpha cyclodextrin. In some embodiments, the cyclodextrin is gamma cyclodextrin.

[0119] In some embodiments, the amount of pay load per mg of empty nanoparticles is about 0. 10 to about 0.20. In some embodiments, the amount of payload per mg of empty nanoparticles is about 0.1 1 , about 0. 12, or about 0.15.

[0120] In some embodiments, the first, second, and third payloads are different. In some embodiments, the first, second, and third payloads are directed to different biological targets. In some embodiments, the first, second, and third payloads comprise at least one agonist and at least one inhibitor. In some embodiments, a. the first payload is an inhibitor; b. the second payload is an inhibitor; and c. the third payload is an agonist. In some embodiments, a. the first payload is an inhibitor; b. the second payload is an agonist; and c. the third payload is an agonist.

[0121] In some embodiments, the nanoparticle does not comprise a second payload.

[0122] In some embodiments, the first and third payloads are different. In some embodiments, the first and third payloads are directed to different biological targets. In some embodiments, a. the first payload is an inhibitor; and c. the third payload is an agonist.

[0123] In some embodiments, the first payload is a cIAP inhibitor. In some embodiments, the cIAP inhibitor is birinapant, GDC-0152, BV-6, or LCL-161. In some embodiments, the cIAP inhibitor is LCL-161.

[0124] In some embodiments, the first payload is a PARP7 inhibitor. In some embodiments, the PARP7 inhibitor is RBN2397.

[0125] In some embodiments, the first payload is a CSF1R inhibitor. In some embodiments, the CSF1R inhibitor is PLX3397.

[0126] In some embodiments, the second payload is a JAK / STAT inhibitor. In some embodiments, the JAK / STAT inhibitor is ruxolitinib, upadacitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, ritlecitinib, pacricitinib, fedratinib. filgotinib, abrocitinib, momelotinib, cerdulatinib, lestaurtinib, gandotinib, BP-1-102, or STX-0119. In some embodiments, the JAK / STAT inhibitor is ruxolitinib, upadacitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, ritlecitinib, pacricitinib, fedratinib, filgotinib, abrocitinib, momelotinib, cerdulatinib, lestaurtinib, or gandotinib. In some embodiments, the JAK / STAT inhibitor is ruxolitinib or upadacitinib. In some embodiments, the JAK / STAT inhibitor is ruxolitinib. In some embodiments, the JAK / STAT inhibitor is upadacitinib. In some embodiments, the JAK / STAT inhibitor is BP- 1-102.

[0127] In some embodiments, the second payload is a STING agonist. In some embodiments, the STING agonist is MSA2.

[0128] In some embodiments, the STING agonist is a prodrug of MSA2. In some embodiments, the STING agonist is 4a,7,8-trimethoxy-2,3,4a.9b- tetrahydrodibenzo{b,d}thiophene-l ,4-dione (MSA-2p).

[0129] In some embodiments, the second payload is a HIF-1 inhibitor. In some embodiments, the HIF-1 inhibitor is BAY 87-2243.

[0130] In some embodiments, the third payload is a TLR agonist. In some embodiments, the third payload is a TLR7 / 8 agonist. In some embodiments, the TLR7 / 8 agonist is imiquimod, resiquimod (R848), PF-4878691, vesatolimod, AZD8848, motolimod, selgantolimod, NKTR- 262, RG-7854, DSP-0509, BDB-001, BDC-1001, LHC-165, SHR-165, JNJ-2150, JNJ-4964, RO-7119929, VX-1463, BNT-411, or APR-0003. In some embodiments, the TLR7 / 8 agonist is imiquimod, resiquimod (R848), vesatolimod, motolimod, or selgantolimod. In some embodiments, the TLR7 / 8 agonist is resiquimod (R848). In some embodiments, the TLR7 / 8 agonist is CRX527.

[0131] In some embodiments, a. the first payload is a cIAP inhibitor; b. the second payload is a JAK / STAT inhibitor; and c. the third payload is a TLR7 / 8 agonist.

[0132] In some embodiments, a. the first payload is LCL-161; b. the second payload is ruxolitinib; and c. the third payload is resiquimod (R848).

[0133] In some embodiments, a. the first payload is LCL-162; b. the second payload is Upalitinib; and c. the third payload is resiquimod (R848).

[0134] In some embodiments, a. the first payload is LCL-162; b. the second payload is BP-1-102; and c. the third payload is resiquimod (R848).

[0135] In some embodiments, a. the first payload is a PARP7 inhibitor; b. the second payload is a STING agonist; and c. the third payload is a TLR7 / 8 agonist.

[0136] In some embodiments, a. the first payload is RBN2397; b. the second payload is MSA2; and c. the third payload is resiquimod (R848).

[0137] In some embodiments, a. the first payload is RBN2397; b. the second payload is MSA2; and c. the third payload is CRX527.

[0138] In some embodiments, a. the first payload is a PARP7 inhibitor; b. the second payload is a HlF-1 inhibitor; and c. the third payload is a TLR7 / 8 agonist.

[0139] In some embodiments, a. the first payload is RBN2397; b. the second payload is BAY 87-2243; and c. the third payload is resiquimod (R848).

[0140] In some embodiments, a. the first payload is a RBN2397; b. the second payload is MSA2; and c. the third payload is resiquimod (R848).

[0141] In some embodiments, a. the first payload is a RBN2397; b. the second payload is 4a,7,8-trimethoxy-2,3,4a,9b- tetrahydrodibenzo{b,d}thiophene-1.4-dione (MSA-2p); and c. the third payload is resiquimod (R848).

[0142] In some embodiments, a. the first payload is PLX3397; c. the third payload is resiquimod (R848). In some embodiments, the first, second, and third payloads are selected from In some embodiments, the first and third payloads and the optional second payload are selected from

[0143]

[0144] In some embodiments, the nanoparticle comprises a surface and core. In some embodiments, the surface of the nanoparticle is chemically modified. In some embodiments, the surface of the nanoparticle is modified to improve pharmacokinetics. In some embodiments, the surface of the nanoparticle is modified to improve targeting. In some embodiments, the surface of the nanoparticle is modified with antibodies. In some embodiments, the surface of the nanoparticle is modified to target antibodies.

[0145] Also provided herein is a wafer comprising a nanoparticle of the present invention.

[0146] In some embodiments, the nanoparticles are aggregated. In some embodiments the nanoparticles are crosslinked. In some embodiments, the nanoparticles are aggregated and crosslinked to form a polymer-like wafer material.

[0147] In some embodiments, the wafer has a mass of about 5-15 mg in its dry fonn. In some embodiments, the wafer has a mass of about 10 mg in its dry form. In some embodiments, the wafer has a volume of about 4-6 mm3in its dry form. In some embodiments, the wafer comprises about 0.22 mg of the first payload, the second payload, and the third payload. In some embodiments, the wafer comprises about 0.1 mg of the first payload, about 0.08 mg of the second payload, and about 0.04 mg of the third payload.

[0148] Also provided herein is a wafer prepared according to a process, wherein the process comprises:

[0149] A) mixing cyclodextrin with lysine to form unloaded nanoparticles; B mixing the unloaded nanoparticles with a first payload, optionally a second payload, and a third payload to form loaded nanoparticles;

[0150] C) lyophilizing the loaded nanoparticles to form a powder; and

[0151] D) pressing the white powder to form the wafer.

[0152] Also provided herein is a pharmaceutical composition comprising a nanoparticle of the present invention and one or more pharmaceutically acceptable excipients.

[0153] Methods of T reatment

[0154] Some embodiments provide a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a nanoparticle as described herein.

[0155] In some embodiments, the nanoparticles as described herein (e.g., with a first, second, and third payload) provide greater efficacy than administration of the first, second, and third payloads as a non-fixed combination.

[0156] In some embodiments, the subject has been identified or diagnosed as having a cancer (e.g., as determined using a regulatory agency-approved, e.g., FDA-approved, assay or kit). In some embodiments, the subject has a clinical record indicating that the subject has a cancer (and optionally the clinical record indicates that the subject should be treated with a nanoparticle as described herein).

[0157] Provided herein is a method of treating cancer in a subject in need of such treatment, the method comprising administering to the subject a therapeutically effective amount of a nanoparticle as described herein. For example, provided herein are methods for treating cancer in a subject in need of such treatment, the method comprising a) detecting a cancer in a sample from the subject; and b) administering a therapeutically effective amount of a nanoparticle as described herein.

[0158] Also provided herein are methods for treating a subject diagnosed with (or identified as having) a cancer that include administering to the subject a therapeutically effective amount of a nanoparticle as described herein.

[0159] Also provided herein are methods for treating a subject identified or diagnosed as having a cancer that include administering to the subject a therapeutically effective amount of a nanoparticle as described herein. In some embodiments, the subject that has been identified or diagnosed as having a cancer through the use of a regulatory agency-approved, e.g., FDA- approved test or assay for identifying such a cancer, in a subject or a biopsy sample from the subject. In some embodiments, the test or assay is provided as a kit.

[0160] The term "regulatory agency" refers to a country's agency for the approval of the medical use of pharmaceutical agents with the country. For example, a non-limiting example of a regulatory7agency is the U.S. Food and Drug Administration (FDA).

[0161] In some embodiments, the cancer is adrenal cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, glioblastoma, kidney cancer, penile cancer, oral cancer, liver cancer, leukemia, melanoma, mesothelioma, lung cancer, skin cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, testicular cancer, or thyroid cancer. In some embodiments, the cancer is colorectal carcinoma, murine melanoma, adult acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, or hairy cell leukemia.

[0162] In some embodiments, the cancer is a glioblastoma multiforme.

[0163] Also provided herein is a method of inhibiting cell proliferation, in vitro or in vivo, the method comprising contacting a cell with an effective amount of a nanoparticle as described herein.

[0164] Further provided herein is a method of increase cell death, in vitro or in vivo, the method comprising contacting a cell with an effective amount of a nanoparticle as described herein.

[0165] Also provided herein is a method of increasing tumor cell death in a subj ect, the method comprising administering to the subject an effective amount of a nanoparticle as described herein, in an amount effective to increase tumor cell death.

[0166] In some embodiments, the nanoparticles used in the methods of treatment described herein are in the form of a wafer, as described herein.

[0167] EXAMPLES

[0168] Example 1. Synthesis of CANDI Nanoparticles

[0169] Materials

[0170] All reagents and solvents were purchased from Thermo Fischer or Sigma- Aldrich and used as received. Small molecules (LCL-161 (Compound 1), R848 (Compound 2), ruxolitinib (Compound 3), upadacitinib (Compound 4), BP-1-102 (Compound 5), STX-0119 (Compound 6)) were purchased from MedChem Express, dissolved in DMSO accordingly and used without any further purification. MilliQ water was obtained from the Waters filtration system. Synthesis of succinyl- -cyclodextrin (s-f-CD)

[0171] P-cyclodextrin (Sigma, 1.3 g, 1.2 mmol) was dried at 60 °C for 72 h. In a pressure vessel (50 mL) charged with a magnetic stirrer, succinic acid (Sigma, 1 g, 8.2 mmol) was dissolved in water (1 mL) followed by sodium hypophosphite monohydrate (Sigma, 65 mg, 0.6 mmol), and the previously dried P-cyclodextrin (1.3 g). The reaction was heated to 120 °C under constant stirring and monitored by LC-MS (ELSD signal) until reaching the desired degree of substitution (t = 24 h). The clear solution was cooled to room temperature and triturated with ethanol (50 mL, 200-proof). The solids were sonicated and washed with abundant ethanol (-150 mL), filtered, and dried at 60 °C for at least 24 h. The desired succinyl-P-cyclodextrin (DS -2.5, 1.3 g) was obtained as white crystals (75% yield).XH NMR (400 MHz, D2O) 5 = 5.05 (s, 7H, Hl) reference proton, 4.56-5.53 (m, 2H, Ha), 4.26-4.21 (m, 2H, Hb), 4.04-3.98 (m, 2H, H5’), 3.94-3.90 (m, 12H, H3 & H5), 3.85-3.75 (m, 10H, H6), 3.65-3.56 (m, 21H, H2 & H4), 2.68-2.63 (m, 10H, Hsuc) ppm.

[0172] Synthesis of empty CANDI nanoparticle (CANDIE)

[0173] Succinyl-P-cyclodextrin (DS -2.5, 250 mg, 1.0 eq to carboxylate) was dissolved in MES buffer (6 mL, 50 mM, pH = 6.5) and activated with N-(3-(dimethylamino)propyl)-N'- ethyl carbodiimide hydrochloride (EDC) (Fisher; 1.5 g, 10.0 eq to carboxylate) and N- hydroxysuccinimide (NHS) (Sigma; 550 mg, 5.0 eq to carboxylate) for 30 min at 25 °C under constant stirring in a closed 20 mL scintillation vial charged with a magnetic stirrer. A solution of L-lysine (Sigma; 35 mg, 0.25 eq to carboxylate) in MES buffer (1.5 mL) was added in a drop-wise manner, and the reaction was allowed to stir for 18 h at 25 °C. The particles were precipitated with absolute ice-cold ethanol (70 mL, 99.9%), yielding a white precipitate that was decanted and dissolved in water (14 mL). The particles were punfied with 10 kDa MWCO centrifugal filters (Amicon; 10,000 g for 8 min), and lyophilized for 48 h. The dry particles (-320 mg) were stored as solids at -20 °C until further use.

[0174] Synthesis of Filled CANDI nanoparticle

[0175] A solution of CANDI nanoparticles (CANDIE; 5 mg) in PBS (0. lx, 90 pL) was used for payload loading to a final DMSO concentration of 10%. The solutions were vortexed rapidly until the complete dissolution of the drugs. All solutions were filtered through a 0.22 pm sterile filter (VWR) and used immediately for characterization, in vitro assays, or stored at -20 °C until further use. See Table 1A for a list of synthesized nanoparticles.

[0176] Table 1A

[0177] Synthesis of64Cu-CANDI

[0178] CANDIEparticles (60 mg) were dissolved in carbonate buffer (0.1 M, pH = 8.5, 500 pL 0.5x) and DOTA-NHS ester (30 mg, Macrocyclics) was added and stirred at 37 °C for 45 min in a thermocycler (600 rpm). The DOTA-functionalized particles were purified by buffer exchange into water against 10 kDa MWCO centrifugal filters (Ami con; 10,000 ref for 5 min; 300 pL water per wash, 3-4x) and lyophilized for 24 h to obtain a white powder (50 mg). The DOTA-CANDI (50 mg) were dissolved in citrate buffer (1 mL, pH = 4.5) and ~6 mCi of radioactive64CuCb in 0.1 NHC1 was added. The CANDI particles were labelled at 70 °C for 45 min in a thermomixer (700 rpm). After labelling, the particles were purified by buffer exchange into water against 10 kDa MWCO centrifugal filters (Ami con; 10,000 ref for 5 min; 300 pL water per wash, 4x). The total radioactivity for the washed64Cu-CANDI was measured (2.9 mCi in 1 mL, 50% loading efficiency). The total dose was divided into 9 injectable doses (each 100 pL of 300 pCi).

[0179] Synthesis ofCANDIAF647

[0180] Lyophilized CANDIEwas dissolved in carbonate buffer (0. 1 M, pH = 8.5) and AF647 succinimidyl ester (ThermoFisher, 2 mg / mL in DMSO) was added to achieve a final concentration of 50 pM. The reaction was stirred for 45 min at 37 °C in a thermocycler (600 rpm). The labeled nanoparticles were purified by buffer exchange into water against 10 kDa MWCO centrifugal filters (Amicon; 10.000 ref for 5 min; 300 pL water per wash, 3-4x) and the final products were diluted with water or PBS to a final concentration of 50 mg / mL and filtered through a 0.22 pm sterile filter (VWR) prior to use.

[0181] Example 2. Characterization of CANDI Nanoparticles

[0182] Turbidity assay

[0183] A turbidity assay to measure the loading efficiency of individual or combination of drugs (Fig. 3C) was developed. In essence, nanoparticle loading via guest-host interactions resulted in the immediate dissolution assessed by the loss of turbidity. On average, loading of Compound 1, Compound 2, Compound 3, and Compound 4 alone or combined up to 0.19 mg (0.5 pmol) per mg of CANDI particle yielded stable formulations (71.4% of the particles theoretical loading capacity).

[0184] CANDIEstock solutions (0-100 mg / mL, 0.5x PBS) were prepared at pH = 7.4 and small molecules were dissolved in DMSO (200 mM LCL-161, R848, ruxolitinib, upadacitinib; 100 mM BP-1-102) to prepare payload stocks. Loading of CANDI particles with payloads (8 mM and 4 mM, respectively, 10% DMSO) was quantified by absorbance scan measurement (Labs = 350-700 nm) after thoroughly mixing the solutions. Total loading of the payload was determined by the complete loss of absorbance. Data were normalized to payload-free control and experiments were performed in triplicates (n = 3). Turbidity Score was determined as the difference in absorbance (550 run) values between the samples and the control (CANDI-100). Values were obtained in the range of < 1 (no turbidity’)- > 1 (moderate aggregation), and > 10 (very high turbidity')- Turbidity' results are shown in Table 2A.

[0185] Table 2A

[0186] Loading assessment by nuclear magnetic resonance (NMR)

[0187] ID- and 2D-1H-NMR were performed spectroscopy to monitor the changes in chemical shifts of both the cyclodextrin and the payloads when forming inclusion complexes. Most payloads, excluding R848 (Compound 2), underwent significant precipitation when dissolved with D2O (3-26 mM, 10% 6-DMSO) in the absence of s- -CD. Upon inclusion-complexation of the payloads, we were able to identify and correlate the relative binding strength of the different functional groups contained in each molecule to determine the payloads possible binding orientations (Fig. 9 and Fig. 10).

[0188] NMR spectra were recorded on a Bruker Avance UltraShield 400 MHz spectrometer. 'H NMR chemical shifts are reported in ppm relative to SiMe4 (4 = 0) and were referenced internally concerning residual protons (<5 = 4.79 for D2O). Peak assignments, calculated chemical shifts and peak integrals are based on reference solvent peaks. Two-dimensional Rotating Frame Overhauser Enhancement Spectroscopy (2D-ROESY) experiments were performed to assess the interactions between dipolarly coupled hydrogens, and integrals were normalized to the reference hydrogen (H1) of the s-P-CD. All experiments were performed in D2O (0.7 mL) at a fixed s- -CD concentration (26 mM) with 10% (CDs SO.

[0189] Liquid chromatography-mass spectrometry (LCMS)

[0190] High-performance liquid chromatography-mass spectrometry7analysis (HPLC-MS, LCMS) was performed on a Waters instrument equipped with a Waters 2424 ELS Detector, Waters 2998 UV-Vis Diode array Detector, and a Waters 3100 Mass Detector. Separations employed an HPLC-grade water / acetonitrile (0.1 % formic acid) solvent gradient with XTerra MS C18 Column, 125 A, 5 pm, 4.6 mm X 50 mm column; Waters XBridge BEH C18 Column, 130A, 3.5 pm, 4.6 mm X 50 mm.

[0191] Loading Capacity

[0192] The total amount of payload (mg or pmols) loadable in one mg of particle (CANDI). Assuming complete cross-linking and semi-quantitative yield of CANDI synthesis (> 95% weight recovery), we estimated the following values to determine the loading capacity7: s-P-CD per particle = -900 s-P-CD content per mg CANDI = 87%, maximal theoretical loading capacity per mg of CANDI (0.7 pmols). For HAMT = LCL-161, R848, ruxolitinib 1+2+3 molar ratios (1.6: 1:2); -0.19 mg of payloads (0.5 pmol) per mg of CANDI nanoparticle by turbidity assessment (Fig. 3).

[0193] Absorance Spectroscopy

[0194] We performed UV-Vis spectroscopy and assessed changes in turbidity7of loaded CANDI solutions to confirm the degree of payload solubility7and to determine their loading capacity as a measurement of amount of payload per particle (Fig. 3 and Table 2B). All absorbance spectra were performed with a multimode microplate reader (Tecan, Spark 500) using 96-w ell transparent bottom black polystyrene microplates (Coming).

[0195] Table 2B

[0196] Fluorescent Spectroscopy

[0197] All fluorescent spectra were performed with a multimode microplate reader (Tecan, Spark 500) using 96-well transparent bottom black polystyrene microplates (Coming).

[0198] Dynamic Light Scattering

[0199] Particle size for all nanoparticle formulations was determined using dynamic light scattering (DLS) at 5 mg / mL in PBS (lx) measured in DTS1170 cuvettes (Malvern) at 25 °C. Fig. 3 and Table 2C summarize the size distribution for the particle formulation characterized by dynamic light scattering (DLS) for empty, mono- and combinational therapies. The empty nanoparticle (CANDIE) had an average size of 16.7±2.73 nm. Loading of single payloads to saturation (0.5 pmols per mg of particle) led to small fluctuations in particle diameter: 18.8±0.65 nm (Compound 1), 18.3±1.63 nm (Compound 2), 16.9±3.1 nm (Compound 3), and 21.1±4.5 nm (Compound 4) with respect to the empty nanoparticle (Table 2C). Conversely, loading experiments at isotonic conditions and at a physiological pH of 7.4 with payloads 5 and 6 resulted in unsuccessful dissolution and sedimentation, indicating sub-optimal loading and inefficient inclusion-complex formation with s-[3-CD.

[0200] Particles loaded with two and three components showed similar results in size (1+2 d = 17.3+2.3 nm, 1+2+3 d = 17.3+1.8 nm, and 1+2+4 d = 18.5+11 nm) and poly dispersity index

[0201] (PDI, 1+2 = 0.258, 1+2+3 = 0.338 and 1+2+4 = 0.390, Table 2C).

[0202] Table 2C

[0203] Zeta Potential

[0204] Particle size and surface charge for all nanoparticle formulations were determined by dynamic light scattering (DLS) and zeta potential measured on a Malvern Zetasizer APS at 5 mg / mL in PBS (lx) and 2 mg / mL in PBS (O.lx), respectively measured in DTS1170 cuvettes (Malvern) at 25 °C.

[0205] Drug release kinetics

[0206] Kinetics of drug release was performed in a closed dialysis set-up employing a 3 kDa molecular weight cut-off membrane (Pur-A-Lyzer™ Midi Dialysis Kit). Solutions of CANDIE(50 mg) were loaded with LCL-161 (2.5 mg), R848 (1 mg), and ruxolitinib (2 mg) in PBS (lx, 1 mL) containing 10% DMSO and were dialyzed against PBS (lx, 6 mL) at 37 °C under constant stirring (600 rpm). The percentage of eluted molecules was quantified by analysis of the liquid chromatographs at different time points (t = 0, 1, 1.5, 2, 2.5, 4, 5, 6, and 24 h), injecting a total of 60 pL aliquots into an LC-MS and subsequently replacing the system with additional 60 pL of PBS. Each payload was identified by its unique retention time (R848 = 0.78 min, Ruxolitinib = 0.95 min, and LCL-161 = 1.01 min) and mass-to-charge ratio (ES‘: R848 = 313. Ruxolitinib = 305 M- and LCL-161 = 499). The cumulative drug release was determined as the ratio of the integrated area under the curve for each eluted peak to the total area under the curve of chromatographs obtained from the non-dialyzed solutions. All experiments were performed in triplicates (n = 3).

[0207] Due to the excellent loading ability and particle size of the Compound 1, Compound 2, Compound 3 combination, we determined the cumulative drug release for each pharmaceutical component by LC-MS using a closed-dialysis set-up. Analysis of all time points resulted in a similar average half-life (ti / 2 = 1-1.5 h) for all three payloads reaching a release plateau after 4 h (Fig. 3).

[0208] Transmission Electron Microscopy

[0209] HAMT particles were freshly prepared (50 mg / mL, PBS lx) and diluted with water to a final concentration of 0. 1 mg / mL. The particle solution was charged on a TEM grid for 1 min and treated with a 2% aqueous uranyl acetate solution for 15 min, followed by three washing steps with ultra-pure water (x3). Imaging was performed in a transmission electron microscope (JEOL 2100). Transmission electron microscopy (TEM) showed nanoparticles as single structures without any apparent sign of aggregation, especially when kept at physiological conditions (lx PBS, Fig. 3).

[0210] Binding Affinity

[0211] We quantified the binding affinity7(Ka) of each drug towards the s-P-CD host in stopped-flow experiments (Fig. 11 and Fig. 3). The binding affinity of each pay load in HAMT was determined from the association (kon) and dissociation (koff) rates at physiological pH (PBS). Using a fixed concentration of each payload, we measured the change in absorbance (X = 532 nm) as a function of time in the absence or presence of solutions containing s-P-CD (kon, Fig. 11). The fastest binding rate w as determined for LCL-161 (kon= 902 M^s’1), followed by R848 (kon = 18.8 M 's1) and ruxolitinib (kon= 8.8 M-1s_1, Fig. 11). The dissociation rate (koff) was estimated from the cumulative release of each component in a closed-dialysis setup (kOff = 0.81, 0.74 and 0.48 s1for LCL-161, ruxolitinib and R848 respectively, Fig. 11). We determined that LCL-161 exhibited remarkably strong binding affinity (K., = 3.81xl06M'1) to s-P-CD, comparable to known adamantyl-containing payloads. R848 and ruxolitinib also exhibited moderately strong binding affinities (Ka= 4.75xl04M'1and 3.28x 104M'1) within the expected range of previously studied -CD-complexed systems (Fig. 11).

[0212] Example 3. Nanoparticle Optimization

[0213] The nanoparticles of the present invention were synthesized using succinyl- -CD with a DS of 2.5 (a mixture of di-succinyl-P-CD and tri-succinyl-P-CD) (Fig. 7A). Using the succinyl-P-CD with a DS of 2.5. resulted in optimal w ater solubility and stability (Fig. 7B-D).

[0214] Multiple cross-linking reaction conditions were screened by varying the amount of L- lysine linker (0.5-0.25 eq to carboxylate). The 1 :4 succinyl-P-CD to L -lysine ratio yielded stable particles with a ~17 nm hydrodynamic diameter (Fig. 2A).

[0215] The 17 nm s-P-CD nanoparticle of the present invention had a ~2-fold higher surface area to volume ratio compared to a 37 nm analog synthesized with penta-succinyl-P-CD (0.35 for 17 nm nanoparticle vs. 0.16 for 37 nm nanoparticle). The higher surface area to volume ratio facilitates drug loading.

[0216] Example 4. Cell experiments

[0217] Immortalized cell lines

[0218] The immortalized murine bone marrow-derived macrophages (iMACs) were acquired from Charles L. Ev avoid (Ragon Institute, Harvard University ). B16-F10 cells were obtained from ATCC. MC38 cells were obtained from Kerafast (ENH204-FP). Fluorescent versions of MC38 and B16F10 for intravital microscopy were made using an H2B-mApple vector. Specifically, cells were transfected with pLVX-H2B-mApple lentiviral vector (Clonetech) in the presence of 10 pg mU1polybrene (Santa Cruz Biotech). They were then selected using 3 pg mL’1puromycin.

[0219] All cell lines were cultured following standard cell culture protocols (Table 4A). Briefly, iMACs and B16-F10 cells were plated and grown in Dulbecco’s Modified Eagle Medium (DMEM, Coming) supplemented with 10% Fetal Bovine Serum (Coming) and 1% Penicillin Streptomycin (Coming) at 37 °C and 5% CO2 and MC38 cells were cultured in [sco e's Modification of DMEM (Coming). Upon reaching confluency, cells were split using 0.05% Trypsin / 0.53 mM EDTA (Coming), and all in vitro assays were performed after the cells reached 90% confluency. Prior to cell culture application, all CANDI preparations were filtered through a 0.22 pm sterile filter (VWR).

[0220] Table 4A

[0221] Bone marrow -derived cells

[0222] Murine bone marrow-derived cells were isolated from IL-12 eYFP reporter or wild type C57BL / 6J mice. To obtain the whole bone marrow, femurs were prepared and flushed with sterile PBS using syringes and a 28-gauge needle. RBC Lysis Buffer (BioLegend) was then used according to manufacturer’s instructions to lyse red blood cells. The remaining cells were counted using a Neubauer chamber and seeded into either transparent (NEST, flow cytometry analysis) or black (Ibidi, glass bottom for imaging) 96 well plates at a density of 1 x 105cells per well. Bone marrow-derived macrophages (BMDMs) were differentiated by adding 50 ng / mL recombinant murine M-CSF (BioLegend) to cell culture media for 7 days. To obtain bone-marrow derived dendritic cells (BMDCs), 300 ng / mL recombinant mouse Flt3L (BioLegend) and 50 ng / mL GM-CSF were added into RPMI 1640 with / .-glutamine cell culture media (optimized with 25 mM HEPES, 10% FCS, nonessential amino acids, sodium pyruvate, B-ME and penicillin / streptomycin) for 9 days. New media was added every' 3-4 days.

[0223] Cell viability assay

[0224] For testing immortalized cell lines, iMACs were seeded in 96 well plates at a density of 8000 cells per well and incubated for 24 h at 37 °C and 5% CO2 before use. Stock solutions of compounds in DMSO (200 mM) and in different CANDI nanoparticles were prepared and then diluted in cell culture medium to desired concentrations (0.6 pg / mL to 12.5 mg / rnL. DMSO 0.5%). Cells were incubated for 2.5 h with nanoparticles before the medium was exchanged. Cells were further incubated for 48 h at 37 °C and 5% CO2 before adding MTT solution (5 g / L in FluoroBrite DMEM, 10% final) to each well. After 3 h, the supernatant was carefully removed, and metabolized formazan was dissolved with isopropyl alcohol. Plates were shaken at 500 rpm on a microplate shaker (VWR) for 30 min, and the absorbance of each well was measured ( / .abs = 550 nm). Duplicates of triplicates were sampled for each concentration, and IC50 values were calculated.

[0225] Live-cell microscopy

[0226] Cells were treated with various combinations of nanoparticles loaded with small molecules (0-0.5 pM. DMSO < 0.5%) for 24 h by adding prepared stock solutions to cell culture media. Before imaging, cells were stained with Hoechst 33342 (15 pg / mL, Thermo Fisher) according to the manufacturer’s protocol. Cells were imaged in a 96-well plate. Fluorescence microscopy was performed using an 1X81 inverted fluorescence microscope (Olympus, Tokyo, Japan) equipped with a motorized stage (Renishaw, Wotton-under-Edge, England, UK) and fitted with an ORCA-Fusion Digital CMOS camera (Hamamatsu Photonics, Hamamatsu, Japan). Using CellSens Dimension 3.1.1 software (Olympus), multiple fields of view were acquired for each sample with a UPlanSApo xlO (numerical aperture (NA) 0.75, Olympus) or a UPlanSApo x40 air objective (NA 0.95, Olympus). In addition to brightfield, five fluorescent channels were acquired: DAPI (345 / 455), GFP (489 / 508), YFP (550 / 565), CY3 (550 / 565), and CY5 (625 / 670) were excited with the appropriate optical filters.

[0227] Flow cytometry

[0228] Bone marrow-derived cells of IL-12 eYFP reporter mice were stimulated o / n with the respective drug combinations, then trypsinized and washed with PBS. Next, the cells were stained using AquaAmine LiveDead Fixable viability stain (Thermo Fisher) diluted in PBS, followed by treatment with Fc block (BioLegend) and fluorochrome-conjugated antibodies (Table 4B and 4C) diluted in FACS buffer (lx PBS, 2 mM EDTA, 2% FBS). For intracellular cytokine staining of primary bone marrow-derived cells, samples were incubated for 6 hours with GolgiPlug (BD Biosciences, 1 pL / rnL of culture media) after stimulations. Cells were then surface stained and, if necessary, fixed and permeabilized using the BD Cytofix / Cy toperm kit (BD) according to the manufacturer’s protocol and stained for intracellular cytokines. For flow cytometry measurements, cells were resuspended in a FACS buffer. All conditions were measured in triplicates in Attune NxT flow cytometer (Thermo Fisher), and the data was analyzed using FlowJo 10 software (TreeStar).

[0229] Table 4B

[0230] Table 4C RNA sequencing

[0231] Bone marrow-derived macrophages were isolated and differentiated as previously described. Cells were then stimulated for 24 h with desired treatments (PBS; HAMT; LPS / IFNy) to induce activation and RNA was isolated using the RNeasy Plus Micro Kit (Qiagen). Final RNA concentration was determined by absorbance (Nanodrop), and samples were stored at -80 °C until shipment for sequencing (NovoGene).

[0232] Cell-based screens identify hits

[0233] Since the ~17 nm CANDI nanoparticle had not been tested biologically, we first determined whether it had macrophage affinity. We utilized an immortalized mouse macrophage cell line (iMAC) for these experiments and incubated these macrophages with different amounts of a fluorescently labeled CAND1A1'647analog. Flow cytometry (data not shown) and fluorescence microscopy (Fig. 13) showed high uptake and internalized punctate structures in -99% of all iMACs.

[0234] Having prepared different nanoparticle combinations, we next wanted to determine their effects on macrophage phenoty pes (Fig. 4). Since IL-12 is a well-known myeloid cell- produced factor with potent anti-tumor activity, we tested the ability of drug-loaded CANDI to induce IL- 12 in macrophages derived from IL- 12 eYFP reporter mice by culturing whole bone marrow with recombinant macrophage colony-stimulating factor (M-CSF). The different amounts of drugs w ere chosen based on therapeutic efficacy, synergy with each other, CANDI payload capacity and results from in vitro IL-12 induction screens. Four different JAK / STAT inhibitors (ruxolitinib (Compound 3), upadacitinib (Compound 4). BP-1-102 (Compound 5), STX-0119 (Compound 6)) were tested, differing in their JAK1 / JAK2 / STAT3 selectivity, pharmacological profile and clinical translation. Different cIAP inhibitors are also known and had previously been screened by us, yielding LCL-161 as a top candidate. Finally, R848 is a prototype TLR7 / 8 agonist but has not progressed beyond early-stage clinical data in 2013. Fig. 4 summarizes the results from these screens.

[0235] Empty nanoparticles showed no IL-12 positive cells, demonstrating the low background of the IL- 12 assay, while the positive control using LPS / IFNy resulted in an average of 5% IL-12 positive cells per well (p = 0.0078). Similar results were observed with R848 (2) loaded monotherapy particles. Dual loading of nanoparticles induced approximately 10% of cells to produce IL-12 (p < 0.0001). This number increased to -30% for triple-loaded therapy (Fig. 4). The highest effect was identified for HAMT, which was significantly higher than for any other combination or monotherapy (p < 0.0001). These results w ere also confirmed by flow cytometry analysis of bone marrow-derived macrophages (BMDMs) differentiated from C57BL / 6J wild type mice (Fig. 14). Given these results, we performed subsequent in vivo experiments with this particular HAMT preparation. We also confirmed that HAMT up- regulates IL-12 in bone marrow-derived dendritic cells (BMDCs, Fig. 14) using the in vitro IL-12 induction assay by fluorescence microscopy. The strong induction of IL-12 in HAMT- treated cells was quantified by flow cytometry resulting in -50% total IL- 12 positive BMDCs. In contrast, cells treated with single-loaded CANDI particles were significantly lower, resulting in 30% for R848 (2) and almost no IL- 12 induction for LCL-161 (1) and ruxolitinib (3) relative to the control (PBS). We also performed dose-dependent toxicity experiments identifying 0.1 mg / mL as a highly efficient and non-toxic dose (Fig. 15). Example 5. In vivo experiments

[0236] Mouse models

[0237] All mice (n = 123) were bred and housed under specific pathogen-free conditions at the Massachusetts General Hospital (MGH). Experiments were approved by the MGH Institutional Animal Care and Use Committee (IACUC) and were performed in accordance with MGH IACUC regulations. IL-12p40-eYFP mice (n = 17) were used for IL-12 induction experiments. C57BL / 6J mice (n = 106) were utilized for MC38 and B1 -F10 tumor implantations (Table 4A).

[0238] Table 4A

[0239] Biodistribution experiments

[0240] Mice (C57BL / 6J; n = 6) received tail-vein injections of ~100 pCi64Cu-CANDI with HAMT under anesthesia (2% isoflurane with 2 L / min O2). Whole-body biodistribution studies were performed 24 h after administration. Mice were euthanized and perfused with PBS through a left ventricle prior to organ harvesting. Excised organs were weighed and subjected to radioactivity measurement using a y counter (1480 Wizard 3-in., PerkinElmer, Waltham, MA). Biodistribution data were obtained after corrections of radioisotope decay and residual activity at the injection site and expressed as percent injected dose per gram tissue (%IDGT)). Autoradiography of tissues was performed using a storage phosphor screen in a cassette (GE Healthcare) for ~ 60 h and read with a Sapphire Biomolecular Imager (Azure Biosystems).

[0241] Toxicity experiments

[0242] Wild type C57BL / 6J mice (n = 12) were injected with different drugs via the tail vein. Twenty-four hours later mice were sacrificed. Blood was drawn for comprehensive testing (serum analytes, blood counts, metabolic parameters). Mice were then perfused with PBS though a left ventricle before collection of the liver for histology. Livers were fixed in formalin solution (10%) overnight before washing in ethanol (70%), embedding in paraffin, and processing for hematoxylin-eosin staining.

[0243] Tumor cell implantation

[0244] MC38 and B16-F10 cells were implanted at 2 * 106cells and 0.5 x io6cells, respectively, in the flank of C57BL / 6J mice, and tumors were allowed to grow for at least 1 week before treatment. Tumor size was at least 50 mm3before the initiation of therapy for the MC38 model. For the metastatic model, Bl 6-F10 cells were injected intravenously at 0.2 x 106cells in sterile PBS via tail vein injection.

[0245] Drug treatment

[0246] All CANDI preparations were administered by tail-vein injection (100 pL PBS 0.5x, pH = 7.4) containing 5 mg nanoparticle. Before injection, all solutions were sterilized by filtration through a 0.22 pm sterile centrifugal filter (VWR), vortexed, and used promptly or frozen at -20 °C.

[0247] Intravital microscopy

[0248] Dorsal windows were implanted into IL-12 eYFP reporter mice. All confocal images were collected using a customized Olympus FV1000 confocal microscope (Olympus America). A 2x (XLFluor, NA 0.14), a 4x (UPlanSApo, NA 0.16), and an XLUMPlanFL N 20x (NA 1.0) water immersion objective were used for imaging (Olympus America). MC38 H2B-apple tumor cells, H AMTAI 647. and vascular probes were excited sequentially using a 405 nm, a 473 nm, a 559 nm, and a 633 nm diode laser, respectively, in combination with a DM- 405 / 488 / 559 / 635 nm dichroic beam splitter. Emitted light was further separated by beam splitters (SDM-473, SDM-560, and SDM-640) and emission filters BA430-455, BA490-540, BA575-620, and BA655-755 (Olympus America). Confocal laser power settings were carefully optimized to avoid photobleaching, phototoxicity, or damage to the tissues. FIJI (ImageJ, 2.9.0 / 1.53t) was used for image analysis. HAMTAI M7was administered as a single injection containing a mixture of HAMT 1+2+3 (5 mg per injection, 100 pL) and CANDIA1 647(5 mg, 100 pL).

[0249] Statistical analysis All statistical data analyses were performed using GraphPad Prism 9 software, and results are expressed as mean ±standard deviation. We used a 2-tailed Student's t-test and oneway ANOVA followed by Bonferroni’s multiple comparison tests for normally-distributed datasets. We performed non-parametric Mann-Whitney or Kuskal-Wallis tests when variables were not normally distributed. For survival analysis, p values were computed using the Log Rank test, p values > 0.05 were considered insignificant (n.s.), and p values < 0.05 were considered significant. * p value < 0.05, ** p value < 0.01. *** p value < 0.001. **** p value < 0.0001.

[0250] Pharmacological behavior

[0251] To better understand the in vivo behavior of the CANDI-300 series, we performed biodistribution experiments with a64Cu-labeled version (Fig. 18). Whole body excretion was primarily via urine and feces. Taking into account the very small size of the nanoparticle (17 nm), the remaining whole body radioactivity was -25% injected dose within 1 day after administration and similar to other carbohydrate nanopreparations of similar size. Major CANDI uptake was observed in the macrophage-rich organs of the reticuloendothelial system, such as the liver. This is characteristic of most materials with high molecular weights, such as antibodies, proteins, and nanoparticles. Tumoral uptake in whole tumors varied across animals and ranged from 6.54 to 1.32 %IDGT. Uptake in remaining organs (lung, heart, brain, digestive tract, muscle, fat and reproductive organs) was low. Autoradiography of removed MC38 tumors shows CANDI accumulation in peripheral zones, which contained the highest amounts of TAM. In these focal areas, uptake was similar to values observed in the liver.

[0252] Within tumor tissues, CANDI was almost exclusively associated with macrophages that also internalize dextran, a known macrophage marker (Fig. 20), and other myeloid cells like dendritic cells (Fig. 21). Nearly all HAMT-containing cells showed IL- 12 induction (Fig. 20). We further investigated the mechanism of cellular CANDI uptake by performing typical uptake experiments in the presence of specific inhibitors (Fig. 25). The biggest effects were observed with scavenger receptor inhibition (Fucoidan).

[0253] Finally, we determined the toxicity of HAMT at the cellular (Fig. 15) and whole-body levels (Fig. 16 and Fig. 17). At the in vivo dose chosen (0.25 mg LCL-1 1 , 0.1 mg R848, 0.2 mg ruxolitinib), we did not observe any significant enzyme or electrolyte abnormalities. However, when the drug combo was given as free drugs (0.25 mg LCL-1 1, 0. 1 mg R848, 0.2 mg ruxolitinib in DMSO; equivalent dose to HAMT group), only 1 of 3 mice survived and showed extensive liver function (Fig. 16) and morphology (Fig. 17) abnormalities. These findings were not observed with the HAMT preparation.

[0254] Anticancer effects

[0255] We next determined the in vivo efficacy of HAMT in different mouse tumor models. We first performed in vivo efficacy in the subcutaneous implanted MC38 colorectal mouse model. For these experiments. 2x l06cells were implanted into the flank of recipient mice. At days 8 and 12, with established tumors, HAMT was given systemically through intravenous injection (1 = 0.05 mg, 2 = 0.02 mg, and 3 = 0.04 mg, a total of 0.3 pmol payloads per mg of CANDI). Tumor sizes were measured three times a week by calipers. Empty CANDI nanoparticles were used as a control and compared directly to HAMT therapy. The data show no therapeutic effect on tumor growth for the empty nanoparticle control (Fig. 5). In contradistinction, systemic HAMT eradicated tumors in two-thirds of the mice and lead to a long-term durable response. The remainder -30% of mice showed a partial response, with significant tumor growth delay compared to control-treated mice (Fig. 5). Interestingly, when complete responders were re-challenged on contralateral flanks with tumor cells at ~2 months following the initial tumor rejection, they were protected from future tumor growth, demonstrating that a long-term memory response to tumor cells persists and is driven by HAMT immunotherapy (Fig. 5). These results indicate that memory T cell effect can be induced by HAMT therapy resulting in long-lasting durable immunity. Immune stimulatory therapies such as HAMT potently activate professional antigen presenting cells (APCs) such as macrophages and dendritic cells. These cells are known to be required for generating robust T cell responses. Likewise, optimally stimulated APCs are known to stimulate effective T cell responses. From these experiments, we observed that the degree of T cell exhaustion in the TME of HAMT treated mice is far lower than the control. Finally, PD1 expressing CD8+T cells that are not terminally exhausted remain sensitive to anti-PDl therapy (Fig. 22).

[0256] In the metastatic model (Fig. 5), we injected Bl 6-F 10 melanoma cells intravenously on day zero and gave three courses of HAMT intravenously over the next week, with or without anti-PDl therapy. On day 14, animals were sacrificed, and lungs were removed after cardiac perfusion to determine metastatic tumor burden (Fig. 19). The data show greatly diminished tumor grow th in animals that had received HAMT (p = 0.0121) or a combination of HAMT with anti-PDl (p < 0.0001) therapy without signs of toxicity. Mechanism of action

[0257] In the next set of experiments, we explored how the HAMT therapies were so effective in vivo. We first turned to intravital microscopy (IVM) to identify which cells take up the nanomaterial. IVM data showed that 24 hours after administration, the CANDI particles mostly localized in TAM. Fig. 20 shows that the nanoparticles co-localize with systemically injected 2M MW dextran-pacific blue (PB), a standard marker for macrophages. These results were confirmed by flow cytometry analysis of immune cells in MC38 tumors 24 hours after injection of fluorescent C ANDIAI 647showing nanoparticle uptake into myeloid cells but not tumor cells or T and B cells (Fig. 21).

[0258] We next determined in vivo whether there was IL-12 induction within the tumor microenvironment as suggested by in vitro experiments. Intravital imaging data in IL- 12 eYFP reporter mice shows that this was indeed the case (Fig. 6). The baseline tumor microenvironment is mainly devoid of IL-12 signals. However, within 48 hours of HAMT treatment, there was a marked up-regulation of IL- 12 in the entire tumor microenvironment observed in all animals tested (Movie 1). We also confirmed that the HAMT treatment cohort had fewer terminally exhausted T cells (PD-1+TlM-3+) in the tumor microenvironment, suggesting a more effective T cell anti-tumor response (Fig. 22).

[0259] Fig. 23 shows pathway analysis of single compounds in HAMT therapy by western blotting. As expected, TLR agonism through R848 led to p38 MAPK (Thrl80 / Tyrl82) activation. LCL-161 showed an increase in NIK levels, indicating activation of non-canonical NF-KB signaling as constitutive proteosomal degradation of NIK is inhibited and signaling therefore activated. Finally, IL-10 is a know n negative regulator of IL-12 signaling. Therefore, we hypothesized that IL- 10 w ould diminish activating signals to macrophages. This was indeed shown by blocking the phosphorylation of STAT3 (Tyr 705) with ruxolitinib in the presence of IL- 10 (Fig. 23).

[0260] In order to determine the effects of pathway modulation on TAM, we next performed cytokine analysis (Fig. 24). These data showed that acute inflammatory7cytokines (e.g. IL-12 and TNFa) and myeloid activation markers (MCP-1, MIP-la, and MIP-ip) were highly up- regulated. Finally, we performed RNA sequencing on HAMT -stimulated bone marrow-derived macrophages (Fig. 24), which showed a TAM phenotype characterized by an over-expression of IL- 12, surface expression of macrophage receptor with collagenous structure (MARCO), DC-SIGN, and SIGNR7. Interestingly, interferon-stimulated genes (ISG) were largely absent in this TAM phenotype, suggesting that IL- 12 expression can occur independently of ISG responses. Furthermore, interferon-inducible inhibitor}' mechanisms such as Pdll (Cd274), Idol, and Ido2 were absent in HAMT-treated macrophages. The mechanism of action is summarized in Fig. 26.

[0261] Example 6. Particle Synthesis

[0262] All reagents and solvents were procured from Thermo Fisher or Sigma-Aldrich and employed without further purification. Small-molecules, namely 2-Fucosyllactose. 2-NP, Baicalein, Eganelisib, Entinostat, KIN-1408, LCL161, MSA-2, Picro-side, Pidotimod, R848, Ruxolitinib, SR-717, and Tilorone, were purchased from MedChemExpress; CRX527, MP LA, M-TriDAP, and Murabutide were obtained from InvivoGen, while RBN2397 was acquired from AmBeed. The compounds were dissolved in dimethyl sulfoxide (DMSO) as appropriate and were utilized without further processing. MilliQ water was sourced from the Waters filtration system.

[0263] Particle Synthesis

[0264] Bis-succinyl cyclodextrm (sbCD) to produce smaller nanoparticles (17 nm vs 37 nm) was employed. sbCD de novo with a well-defined degree of substitution (DS) of 2.5 addressing variability and high cost observed in commercially available products is synthesized. The resulting compound was then used to prepare CANDI nanoparticles, activated with EDC and NHS in MES buffer. Briefly, L-lysine was added drop-wise, and the reaction was allowed to stir for 18 h. The particles were precipitated with ice-cold ethanol, purified, and characterized by DLS and Zeta potential before storage at -20 °C.

[0265] Synthesis of MSA-2 (4-(5, 6-Dimethoxybenzo{b}Thiophen-2-yl)~ 4-Oxobutanoic Acid)

[0266] Succinic anhydride (77 mg, 0.77 mmol, 3.0 equiv.) and AICE (69 mg, 52 mmol, 2.0 equiv.) were dissolved in anhydrous DCM (3 mL) under argon and stirred at 0 °C for 30 min. Separately, 5,6-dimethoxy benzothiophene (50 mg, 26 mmol, 1.0 equiv.) was dissolved in anhydrous DCM and added dropwise to the first mixture over 30 min. The reaction was stirred at 43 °C overnight. The dark green reaction was poured into ice water, and the pH was adjusted to ten using 1 M NaOH. The filtrate was collected and acidified to pH 2 using 1 pm HCL. The resulting brown precipitate was collected by gravity filtration, rinsed with water and DCM, dried, and collected to afford the desired product as a brown solid, 42 mg (55%). 1HNMR (400 MHz, DMSO-D6) 3 12.20 (s, 1H), 8.21 (s, 1H), 7.60 (s, 1H), 7.48 (s, 1H), 3.86 (s, 3H). 3.84 (s, 3H). 3.26 (t, J = 6.4 Hz. 2H), 2.60 (t, J = 6.4 Hz, 2H). 13C NMR (101 MHz, DMSO-D6) 8 192.5, 173.7. 150.8, 148.5, 140.6, 135.6, 132.5, 130.4, 106.6, 104.3, 55.9, 55.6, 33.1, 27.9. ESIMS for C14H14O5S {M-H}-: Calc’d: 293.05, Found: 293.27. {2M-H}-: Calc’d: 587.11, Found: 587.45.

[0267] Synthesis of MSA-2p (4a, 7,8-Trimethoxy-2,3,4a,9b-Tetrahydrodibenzo{b,d}Thiophene-l,4- Dione)

[0268] In an oven-dried scintillation vial (20 mL) equipped with a magnetic stirrer have suspended a mixture of MSA-2 (150 mg, mmol, equiv), and EDC (150 mg) in anhydrous acetonitrile: methanol mixture (3: 1, 15 mL). The reaction was allowed to stir for 1 h at 25 °C and was monitored by LCMS. Upon full conversion to MSA-2p, the solvents were evaporated and the crude was subjected to normal-phase column chromatography (Hex: EtOAc 5%^40%). The pure fractions were collected and evaporated to yield MS A-2p as a transparent oil that crystallized overnight under vacuum (67 mg, mmol, 43% yield). 1H NMR (400 MHz, DMSO-D6) 8 7.55 (s, 1H), 7.40 (s, 1H. Hl), 7.34 (s, 1H, H2), 3.82 (s, 3H, H3), 3.80 (s, 3H, H4), 3.23 (s, 3H, H5), 2.93-2.76 (m, 1H, H7&H8), 2.74-2.56 (m, 2H, H7, H8), 2.50 (m, 1H, H7, H8). 13CNMR (101 MHz, DMSO-D6) 8 175.29, 148.52, 148.15, 139.33, 132.25, 131.84, 122.36, 107.92, 105.77, 104.51, 55.80, 55.61, 51.40, 35.84, 28.22. ESI-MS or CI5HI6O5S {M- H}+: Calc’d: 309.0718, found: {M-H}+: 309.2200.

[0269] Synthesis of 2D216

[0270] Compound 2D216 from activation of 4-(Piperidine-l-sulfonyl)-benzoic acid (50 mg, 0.19 mmol) using HATU (78 mg, 0.21 mmol) followed by amide formation with 4-(2,5- Dimethylphenyl)thiazol-2-ylamine (42 mg, 0.21 mmol) in the presence of triethylamine (28 mg, 0.28 mmol) is synthesized. Normal-phase silica gel column chromatography yielded compound 2D216 (65 mg, yield = 77%) as an off-white, pink solid. 1H NMR (400 MHz, CHLOROFORM-d) 8 12.14 (br. s„ 1H), 7.80 (d, J = 8.31 Hz, 2H), 7.63 (d, J = 8.56 Hz, 2H), 7.14 (s. 1H), 7.00 (s, 1H), 6.98 (d, J = 7.58 Hz, 1H), 6.87-6.93 (m, 1H), 2.93-3.01 (m. 3H), 2.29 (s, 3H), 2.24 (s, 2H), 1.61-1.68 (m, 4H), 1.39-1.46 (m, 2H). LCMS for C23H26N3O3S2 {M+H}+ calculated 456.13, found 456.71.

[0271] Example 7. Particle Characterization

[0272] Particle Size Particle size and surface charge for all nanoparticle formulations were determined by DLS and zeta poten-tial measured on a Malvern Zetasizer APS at 5 mg mL1in PBS (0.5x) and 2 mg mL1in PBS (O. l x), respectively measured in DTS1 170 cu-vettes (Malvern) at 25 °C. All absorbance and fluorescent spectra (e.g, CANDIAF647 analogs) were performed with a multimode microplate reader (Tecan, Spark 500) using 96-well transparent bottom black polystyrene mi-croplates (Coming).

[0273] Small-Molecule Loading of Nanoparticles

[0274] A solution of empty CANDI (CANDIE; 5 mg) in PBS (0.5*,90pL) was used for payload loading to a final DMSO concentration of 10%. The following nanoparticle compounds were prepared: CANDI400 containing MSA-2p (0.26 mg), RBN2397 (0.1 mg), and R848 (0.22 mg). The solutions were vortexed rapidly until the complete dissolution of the drugs. All solutions were filtered through a 0.22 pm sterile filter (VWR) and used immediately for characterization, in vitro assays, or stored at -20 °C until further use.

[0275] Turbidity Assay

[0276] CANDIE stock solutions (2.5 mg ml.1.0.5 / PBS) were prepared at pH 7.4 and the small-molecules were dissolved in DMSO (140 mM R848, 170 mM MSA-2 Prodrug and 40 mM RBN2397) to prepare payload stocks. Loading of CANDI particles with payloads (0.1-52 mM, respectively, 10% DMSO) was quantified by absorbance scan measurement ( / Lbs = 400- 700 nm) after thoroughly mixing the solutions. Total loading of the payload was determined by the complete loss of absorbance. Data were normalized to payload-free control and experiments were performed in triplicates (N = 3).

[0277] Loading Assessment by Nuclear Magnetic Resonance (NMR) and Liquid Chromatography- Mass Spectrometry (LCMS)

[0278] NMR spectra were recorded on a Bruker Avance UltraShield 400 MHz spectrometer. 1H NMR chemical shifts were reported in ppm relative to SiMe4 (8 = 0) and were referenced internally concerning residual pro-tons (8 = 4.79 for D2O). Peak assignments, calculated chemical shifts, and peak integrals were based on reference solvent peaks. 2D Rotating Frame Overhauser Enhancement Spectroscopy (2D-ROESY) experiments were performed to assess the interactions between dipolarly coupled hydrogens, and integrals were normalized to the reference hydrogen (Hl) of the sbCD. All experiments were performed in D2O(0.5mL) at a fixed sCD concentration (26 mM) with 10% (CDs SO. High-performance liquid chromatography-mass spectrometry analysis (HPLCMS) was per-formed on a Waters instrument equipped with a Waters 2424 ELS Detec-tor, Waters 2998 UV-Vis Diode array Detector, and a Waters 3100 Mass Detector. Separations employed an HPLC-grade water / acetonitrile (0.1%formic acid) solvent gradient with XTerra MS Cl 8 Column, 125 A, 5 pm, 4.6 x 50 mm column; Waters XBridge BEH C18 Column. 130 A, 3.5 pm. 4.6 x 50 mm.

[0279] MSA-2p and MSA-2 Loading Experiments

[0280] A stock of MSA-2p (3. 14 mg) was prepared in DMSO (70 pL). A solution of sbCD (64 mg in 500 pL water) was prepared. Turbidity experiments were per-formed in a 394-well plate using 50 pL volume measuring absorbance point at 550 nm even' »100 s for 5 cycles with 60 s shaking between cycles. The average values from the individual cycles gave the best approximation of the state of the turbid suspensions. Using a fixed concentration of 0.13 mg per well, ranges of sbCD concentration equaling 0. 0.224, 0.448, 0.896, 1.79, 3.58, and 7.16 equivalents of MSA-2p are titrated. To obtain the curve for MSA-2 an initial concentration stock of 3.0 mg in 70 pL DMSO was used. All points were averages of three independent replicates (n = 3).

[0281] MSA-2p Hydrolysis Experiments

[0282] A stock of MSA-2p (3 mg) in DMSO (750 pL) was prepared for hydrolysis experiments in phosphate aqueous buffers (pH 4-8) at a 10% concentration. A solution of sbCD (6-36 mg in 800 pL buffer) was prepared and mixed with the MSA-2p stock (90%). All experiments were performed by sequential reading of an absorbance scan between 300-500 nm, a single point at 325 nm followed by 5 s quick mixing to ensure homogeneous mixing. Each experiment was performed in triplicates in time lapses of 85 cycles (2000 s). The percent of MSA-2p converting to MSA-2 (%) was calculated by correlating the total absorbance value at 325 nm to the relative MSA-2 to MSA-2p concentration quantified as the area under each peak by LCMS.

[0283] Drug Release Kinetics

[0284] Kinetics of drug release were performed in a closed dialysis set-up employing a 3 kDa molecular weight cut-off membrane (Pur-A-Lyzer Midi Dialysis Kit). Solutions of the empty nanoparticle CANDIE (50 mg) were loaded with R848 (2.2 mg), MSA-2p (2.6 mg), and RBN2397 (1.0 mg) in PBS (l x, 1 mL) containing 10% DMSO and were dialyzed against PBS (l x,5mL) at 37°C under constant stirring (600 rpm). The percentage of eluted molecules was quantified by analysis of the liquid chromatographs at different time points (t = 0, 1, 5, 10, 25, 45, 60, 90, 120, 180, 210, 240, 270, 300, 330, 360, 390 min), injecting atotal of 90 pL aliquots into an LC-MS and subsequently replacing the system with additional 100 pL of PBS. Each payload was identified by its unique retention time (R848 = 0.90 min, MSA-2 = 1.57 min, RBN2397 = 1.87 min) and mass-to-charge ratio (R848 = 314 {M}+ ES+. MSA-2 = 293 {M}- ES-, RBN2397 = 522 {M}- ES- MSA-2 Prodrug = 309 {M}+ ES+). The cumulative drug release was determined as the ratio of the integrated area under the curve for each eluted peak to the total area under the curve of chromatographs obtained from the non-membrane controls. All experiments were performed in triplicates (N = 3).

[0285] Transmission Electron Microscopy

[0286] CANDI400 particles were freshly prepared (50 mg mL-1, PBS l x) and diluted with water to a final concentration of 0.1 mg mL1. The particle solution was charged on a TEM grid for 1 min and treated with a 2% aqueous uranyl acetate solution for 15 min. followed by three washing steps with ultra-pure water (x3). Imaging was performed in a transmission electron microscope (JEOL 2100).

[0287] Nanoparticle Tracking Analysis (NTA)

[0288] A stock of pharmaceutically loaded CANDI400 was prepared in freshly filtered PBS (50 mg rnL-1). Next, the total particle count, size distribution, and homogeneity of four dilutions (x62.5, xl25, x250, x500, N = 3) are estimated. The particle counts obtained from each concentration were multiplied by the dilution factor and averaged to obtain the total particle count depicted in Figure S6 (Supporting Information). All experiments and analyses were performed using a Panalytical NanoSight LN10 (Malvern) nanoparticle characterization system. All nanoparticle tracking analyses (NTA) were done with identical experiment settings.

[0289] Example 8. In Vitro and In Vivo Experiments

[0290] Optical Screens in Freshly Isolated Target Cells

[0291] Pharmacological modulation of TAM in vivo has been challenging due to several reasons: first, the lack of cost-effective methods to combinatorically screen drugs that can polarize TAM; second, the still limited knowledge of key regulators of TAM programming; and third, the availability of efficient and selective delivery vehicles with high drug payloads. To address these challenges, we have developed optically resolved screening approaches that more efficiently and rapidly identity’ therapeutic combinations that induce CXCL9 and other interferon-stimulated genes (ISG) (Fig. 36). In this study, we performed the screening in primary isolated bone marrow-derived cells (BMDC) from CXCL9 red fluorescent protein (RFP) and CXCL10 blue fluorescent protein (BFP) reporter mice to identify potential smallmolecule com-pound hits capable of inducing RFP expression. Freshly obtained BMDC from these mice were cultured with macrophage colony-stimulating factor (M-CSF) for 7 days, at which time they ty pically have low levels of baseline CXCL9 expression. Upon the addition of potential modulators fluorescence microscopy and flow cytometry can be used to measure increases in RFP (Fig. 36).

[0292] Creation of a Mini-Library for Screening

[0293] There is an emerging realization that combination therapies are needed to: i) improve treatment efficacy, ii) lower the dose of single immune-stimulatory agonists, and iii) circumvent immune cell resistance mechanisms. Based on the hypothesis that certain smallmolecule combinations may indeed affect distinct cellular programs when delivered specifically to TAM, we curated a small collection of drugs (Fig. 37). We focused primarily on known modulators of several major pathways (IFNg, NFkB, TLR, STAT1, and interleukin 10 (IL10). As no direct single agonist of IFNg signaling nor CXCL9 has been reported, we adapted our imaging readout approach to measure dozens of therapeutic combinations at varying dose levels for their ability' to boost CXCL9 in macrophages. The selection of these compounds was largely driven by the current understanding of TAM signaling (Fig. 37) and the potential of a given compound to be useable clinically. Our collections contained 21 individual smallmolecules and another ~20 combinations, resulting in screens with ~40 different drug combinations.

[0294] Screening Identifies Drug Combinations that Induce CXCL9

[0295] Fig. 38 summarizes the primary screening results. All screenings were carried out in triplicates, yielding consistent and re-producible results with minimal variation. The first screening, performed as a control, examined whether any single compound or dual combination thereof could induce CXCL9 production in non-stimulated baseline macrophages. As expected, the screening did not identify any significant hits capable of elevating CXCL9 expression in vitro.

[0296] The second screening paralleled the first, yet it incorporated baseline IFNg stimulation to mimic native cytokine exposure within the tumor environment, if any. IFNg is primarily secreted by T-cells and NK cells, instigating macrophage and dendritic cell responses via the interferon-gamma receptor (IFNGR) and JAK / STAT pathways. This time, most single agents induced a mild increase in CXCL9 response, and certain dual combinations induced a moderate increase. However, specific pairings, particularly those including the RIG-1 -like receptor agonist KIN-1408, resulted in cellular toxicity and did not induce CXCL9.

[0297] Based on these results, we performed a third screening using triple drug combinations to determine whether CXCL9 expression could be further enhanced. Certain combinations with KIN- 1408 and CRX527 (an LPS mimetic) were associated with toxicity at the doses applied, leading to their exclusion from sub-sequent testing. The screening identified several triple combinations that achieved exceptionally high CXCL9 expression. The key finding resulting from the third screen was the combination consisting of RBN2397 (a PARP7 inhibitor), MSA- 2 (a STING agonist), and R848 (a TLR7 / 8 agonist) capable of increasing CXCL9 expression ~8 fold. Interestingly, the expression of IL12 was also enhanced by this triple combination (Fig. 38). Subsequent experiments were then performed to incorporate this po-tent combination into a TAM-avid nanotherapeutic formulation (CANDI400).

[0298] CANDI400 Formulation and Efficacy

[0299] We first validated the propensity of our hit drug combination to form strong complexes with the bisuccinyl cyclodextrin (sbCD) monomers of CANDI nanoparticle. Initially, we looked for changes in chemical shifts and equimolar complexation in nuclear magnetic resonance (NMR) titration experiments with R848, RBN2397, and MSA-2 individually. These payloads were mixed in a D2O solution containing a fixed concentration of the host, sbCD. From these experiments, we concluded that R848 had a very high binding affinity toward sbCD and potentially multiple binding orientations which favored its high solubility. RBN2397 had optimal complexation at a three-host per guest complexation ratio. Potentially accounting for two aromatic and one aliphatic binding moieties. Throughout all measured conditions, however, MSA-2 showed very poor affinity to sbCD and remained a stable turbid suspension regardless of the presence or concentration of sbCD, hindering our NMR comparative studies in D2O.

[0300] For this reason, we opted to design a more lipophilic pro-drug of MSA-2 with enhanced inclusion complexation ability with sbCD, termed MSA-2p. The synthesis of MSA-2p was achieved in moderate yields activating MSA-2 with l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC). Using an anhydrous mixture of acetonitrile and methanol resulted in the highest yields (56%). MSA-2p was the main product from this reaction (>85% conversion) and interestingly, the structure differed significantly from the other MSA- 2 prodrug analogs. MSA-2p is a peculiar cyclic lactone locked in a chiral conformation as shown on the aliphatic region of the 1H-NMR spectra.

[0301] Solution experiments with MSA-2p showed a fast decrease in turbidity when quasi- equimolar concentrations of sbCD were present, indicating a strong affinity between MSA-2p and sbCD. In the absence of the host, MSA-2p had low water solubility (<3.6 mg mL-1, Fig. 39). We then examined the hydrolysis rate of MSA-2p to MSA-2. We used the spectrally unique 325 nm absorbance peak to quantify the formation of MSA-2 in buffered solutions. We studied the hydrolysis rates of MSA-2p from absorbance scans acquired at equilibrium and assessed that the complexation of MSA-2p to sbCD had a significant effect on the hydrolysis rates. When dissolved in phosphate-buff ered saline (PBS), regardless of pH, MSA-2p exhibited very fast hydrolysis (0.22% s-1). Adding an increasing amount of sbCD to six equivalents dropped the hydrolysis rate to (0.018% s-1 ~11.8-fold). These results indicate that the strong binding with sbCD hinders the nucleophilic attack of the water molecule, suggesting that the inclusion complexation inserts around the cyclic chiral lactone.

[0302] In the next set of experiments, we performed nanoparticle characterization experiments of the triple-loaded CANDI400 formulation. Upon loading, the nanoparticle size was 19.8 ± 1.1 nm, and the zeta potential was -6.15 mV. Transmission electron microscopy revealed spherical polymeric structures with matching size distribution to our dynamic light scattering (DLS) measurements. We determined the drug loading capacity to be around 0.15 mg of drug combo (R848, RBN2397, and MSA-2p) per mg of particle before it reaches saturation. In a closed-dialysis set-up mimicking physiological conditions, we determined the release rates for each drug in the CANDI400 formulation. The release of each released drug was quantified simultaneously using an optimized liquid chromatography method coupled to mass spectrometry (LCMS), which resulted in similar dissociation rates koff (0.46, 0.39, and 0. 19 h1 for R848, MSA-2, and RBN2397) and a complexation half-life of ti / 2 = 1.5, 1.8 and 3.6 h respectively (Fig. 39).

[0303] A dose-response curve was obtained with BMDM from CXCL9-RFP mice, yielding an effective ECso of 3.3 ng mL '.As shown in Fig. 40, there was uniform CANDI400 uptake and concomitant CXCL9 production in BMDM. Additional cytokine screening experiments were conducted to ascertain that cellular cytokine production was due to the CANDI payload and not the nanoparticle drug carrier itself. At this dose, there was no discernible cellular toxicity with CANDI400. These results confirmed the efficacy of the triple combination nanoparticle to increase CXCL9 production. Subsequent experiments were therefore conducted to show in vivo efficacy.

[0304] Intravital Imaging Reveals Drug Action in the Tumor Microenvironment and Antitumor Efficacy

[0305] We first determined the vascular half-life of CANDI400 by serial imaging of the microvasculature in the mouse ear. This showed a vascular half-life of approximately 2.3 h. Cellular uptake could be identified as early as Ih but was more pronounced by 4 h after IV administration. To determine whether the CANDI400 formulation indeed accumulated within TAM in vivo following systemic administration and elicited CXCL9 expression, we performed intravital microscopic imaging in live mice. CXCL9-RFP transgenic mice were implanted with dorsal window chambers into which MC38-GFP tumors were grown. After 8 to 10 days, we performed se-rial microscopic examinations of the TME both before and after intravenous systemic administration of CANDI400 labeled with AF647 (Fig. 41). Baseline expression of CXCL9 pre-treatment was very low, with sparse cellular expression within the TME. Remarkably, within 24 to 48 h postinfusion of CANDI400. we observed a considerable induction of CXCL9 throughout the tumor. Using a fluorescent analog of our particles labeled with AF647, we confirmed that these CXCL9-expressing cells had incorporated the nano formulation (Fig. 41).

[0306] To determine whether these changes translate to antitumor efficacy, we performed tumor growth experiments in mouse models. We observed remark-able efficacy in the MC38 tumor model, with all tumors disappearing after two systemic administrations of CANDI400. 60 days after tumor inoculation, all mice that received CANDI400 were still alive, in contrast to a median survival time of 28 days in the control group. Discussion

[0307] The expression of CXCL9. related chemokines (CXCL10), and other ISG is primarily driven by IFNGR signaling through IFNg produced by lymphocytes (e.g., through IL12, IL18, or antigen stimulation). Within macrophages, IFNg response is mediated by JAK1 / 2, STAT1 signaling, which leads to transcription factor binding to gamma interferon activation site (GAS) elements and activation of IFNg programs in macrophages. Additional pathway s are being revealed by ongoing research. For example, in a recent study. CRISPR-Cas9 screening identified numerous positive regulators of CXCL9, including Dnttipl , Prdml4, Zfp431, Klf6, Aridla, Socsl and Smarcdl. Interestingly, epigenetic regulation through the SWI / SNF-PRC2 axis (e.g., by inhibiting EED or Irfl) led to up-regulation of CXCL9. Thus, additional CXCL9 drug targets may become available in the future.

[0308] We identified a triple-drug combination that works synergistically in upregulating CXCL9. The combination in-volves RBN2397, a PARP7 inhibitor in clinical trials, a STING prodrug (MSA-2), and R848, a TLR 7 / 8 agonist. The ADP -ribosyltransferase PARP7 modulates protein function by conjugating ADP-ribose to the side chains of acceptor amino acids. PARP7 is expressed in various cells and can affect tumor growth through multiple mechanisms. For example, RBN2397-mediated inhibition has been show n to induce both cancer cell-autonomous effects and antitumor immunity via enhanced type I IFN signaling. It negatively regulates tank binding kinase 1 activity, which restrains phosphory lation and activation of the transcription factor IRF3. inhibiting androgen-induced ADP-ribosylation of the androgen receptor in prostate cancer and trapping PARP7 yvithin the nucleus. MSA-2 is an orally available non-nucleotide STING agonist. Activation of STING by cyclic dinucleotide (CDN) ligands in human monocy tes induces a type I IFN response and production of pro-inflammatory cytokines as-sociated with the induction of massive cell death. We show that a CANDI encapsulated prodrug formulation using prodrug MSA-2p had similar effects in BMDM. Finally, R848 is a TLR7 / 8 agonist that activates the canonical NFkB pathway leading to IL12 secretion, which then stimulates T-cells to produce IFNg. As we based our screening on the compounds’ ability to increase CXCL9 in IFNg-exposed macrophages, this latter mechanism is important for triggering IFNg in vivo and maximizing the full efficacy of the compounds identified. Despite the different mechanisms of action of the small-molecule modulators, the unifying theme was that they acted synergistically, presumably in part because they were delivered to TAM in an efficient manner. Immortalized Cell Lines

[0309] The immortalized murine bone marrow-derived macrophages (iMACs) were acquired from Charles L. Evavold (Ragon Institute, Harvard University) and used to assess toxicity. Briefly, iMAC cells were plated and grown in Dulbecco’s Modified Eagle Medium (DMEM, Coming) supplemented with 10% Fetal Bovine Serum (FBS, Coming) and 1% Penicillin Streptomycin (Coming) at 37 °C and 5% CO2 and MC38 cells were cultured in Iscove’s Modification of DMEM (Coming). Upon reaching confluency, cells were split using 0.05% Trypsin / 0.53 mM EDTA (Coming), and all in vitro assays were performed after the cells reached 90% confluency. Prior to cell culture application, all CANDI preparations were filtered through a 0.22 pm sterile filter (VWR).

[0310] Bone Marrow -Derived Cells

[0311] Murine BMDC were isolated from CXCL9-RFP / CXCL10-BFP reporter mice, IL12- eYFP reporter mice, or wild-type C57BL / 6J mice. BMDC of reporter mice were employed for flow cytometry and live-cell microscopy analyses, while BMDC of wild-type cells were utilized to evaluate cytokine induction. To obtain the whole bone marrow, femurs were prepared and flushed with sterile PBS using sy-ringes and a 28-gauge needle. RBC Eysis Buffer (BioLegend) was then used according to the manufacturer’s instmctions to lyse red blood cells. The remaining cells were counted using a Neubauer chamber and seeded into either transparent (NEST, flow cytometry analysis) or black (ibidi, glass bottom for imaging) 96 well plates at a density of 1.25 * 105 cells per well. For cytokine assays, cells were seeded into transparent 6- well plates (Coming) at a density of 1 x 106 cells per well. BMDM were differenti-ated by adding 50 ng mL-1 recombinant murine M-CSF (BioLegend) to cell culture media for 7 days. New media was added every 3-4 days.

[0312] Cytokine Screen

[0313] To determine the effect of nanoparticle drug loading on broader cytokine induction, wild-type C57BL / 6J BMDM were seeded and stimulated with 50 ng mL-1 IFNg. Subsequently, CANDI400 formulation (5 pgmL-1) or empty control nanoparticles were added for 24 h. The conditioned media were then collected for cytokine array analysis. Cytokine array analysis was performed using the Proteome Profiler Mouse Cytokine Array Kit, Panel A (R&D, ARY006) according to the manufacturer’s instmctions. Images of the membranes were obtained (Azure Sapphire Biomolecular Imager) and quantified using ImageJ. Toxicity) iMACs were seeded in 96 well plates at a density of 15 x 103 cells per well and incubated for 24 h at 37 °C and 5% CO2 before use. Stock solutions of different CANDI nanoparticles were prepared and then diluted in cell culture medium to desired concentrations (0.09 pgmL1to 10 mg mL1. DMSO 0.5%). Cells were incubated for 2.5 h with nanoparticles before the medium was exchanged. Cells were further incubated for 48 h at 37 °C and 5% CO2 before adding MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) solution (5 g L"1in FluoroBrite DMEM, 10%final) to each well. After 3 h, the supernatant was carefully removed, and metabolized formazan was dissolved with isopropyl alcohol. Plates were shaken at 500 rpm on a microplate shaker (VWR) for 30 min, and the absorbance of each well was measured ( / labs = 550 nm). Triplicates were obtained for each concentration tested, and IC50 values were calculated from means.

[0314] Flow Cytometry.

[0315] Flow cytometry was used to assess cytokine in-duction and characterize the TME. BMDM of CXCL9-RFP / CXCL10-BFP reporter mice or IL12-eYFP reporter mice were stimulated o / n with the respective drug combinations, then trypsinized and washed with PBS. Next, the cells were stained using AquaAmine LiveDead Fixable viability stain (Thermo Fisher) diluted in PBS, followed by treatment with Fc block (BioLegend) and fluorochrome- conjugated antibodies diluted in FACS buffer (lx PBS, 2 mM EDTA, 2%FBS). For flow cytometry measurements, cells were resuspended in a FACS buffer. All conditions were measured in triplicates in Attune NxT flow cytometer (Thermo Fisher), and the data was analyzed using FlowJo 10 software (TreeStar).

[0316] Dose-Response

[0317] To determine the dose-response of the triple labeled nanoparticle, a stock solution of CANDI400 nanoparticles was prepared and subsequently diluted in cell culture medium to achieve the desired concentrations (0.08 pg mL-1 to 1 mg mL-f , DMSO 0.5%). CXCL9- RFP / CXCL10-BFP BMDM from reporter cells were incubated overnight in the nanoparticle- spiked media before removing the media. Cells were then prepared for microscopy and flow cytometry analysis. Live-cell microscopy.

[0318] Live-cell microscopy was performed to deter-mine the cytokine production in BMDM of reporter mice with or without IFNg stimulation. Harvested cells were treated with various combinations of small-molecules (0-10 pm. DMSO <0.5%) for 24 h by adding prepared stock solutions to cell culture media. Cells were imaged in a 96- ell plate. Before imaging, cells were stained with Hoechst 33 342 (15 pgmL1. Thermo Fisher) or SYTO 11 Green Fluorescent Nucleic Acid Stain (2.5 pp. Thermo Fisher) according to the manufacturer’s protocol. Fluorescence microscopy was performed using an 1X81 inverted fluorescence microscope (Olympus, Tokyo, Japan) equipped with a motorized stage (Renishaw. Wotton-under-Edge, England, UK) and fitted with an ORCA-Fusion Digital CMOS camera (Hamamatsu Photonics, Hamamatsu, Japan). Using CellSens Dimension 3.1.1 software (Olympus), multiple fields of view were acquired for each sample with a UPlanSApo *10 (numerical aperture (NA) 0.75, Olympus) or a UPlanSApo x40 air objective (NA 0.95, Olympus). In addition to brightfield, four fluo-rescent channels were acquired DAPI (345 / 455 nm), GFP (489 / 508 nm), YFP (550 / 565 nm), CY3 (550 / 565 nm), and CY5 (625 / 670 nm) were ex-cited with the appropriate optical filters.

[0319] Mouse Models

[0320] All animals were bred and housed under specific pathogen-free conditions at the Massachusetts General Hospital. Experiments were approved by the MGH Institutional Animal Care and Use Committee (IACUC) and were performed in accordance with MGH IACUC regulations. CXCL9-RFP / CXCL10-BFP mice (N = 9) were employed for the assessment of CXCL9 induction. IL12p40-eYFP mice (N = 2) were used for IL12 induction experiments. Female C57BL / 6J mice (N = 18) were utilized for MC38 tumor growth experiments.

[0321] Intravital Microscopy

[0322] Mice-bearing dorsal window' chambers with MC38-mTAG-GFP were performed to determine the kinetics of CXCL9 induction in the TME (Fig. 40). Dorsal window' chambers were implanted into Rex3 mice using well-established techniques. Fluorescent tumor cells (MC38-H2B-GFP) were implanted in the window chambers as previously described and allowed to grow' for 7-21 days before imaging experiments, with tumor growth monitored regularly. In additional experiments, the vascular half-life of CANDI400 by serial imaging of the microvascular in the mouse ear were determined. All confocal images were collected using a customized Olympus FV1000 confocal microscope (Olympus America). A 2x (XLFluor, NA 0.14), a 4x (UPlanSApo. NA 0.16), and an XLUMPlanFL N 20x (NA 1.0) water immersion objective were used for imaging (Olympus America). Fusion-protein CXCL10-BFP, tumor cells (MC38-H2B-GFP) and SYTO 11 Green, fusion-protein CXCL9-RFP, and CANDIAF647 were excited sequentially using a 405, a 473, a 559, and a 633 nm diode laser in combination with a DM- 405 / 488 / 559 / 635 nm dichroic beam splitter. Emitted light was further separated by beam splitters (SDM-473, SDM-560, and SDM-640) and emission filters BA430-455, BA490-540, BA575-620, and BA655-755 (Olympus America). Confocal laser power settings were carefully optimized to avoid photobleaching, phototoxicity, or tissue damage. Fiji (ImageJ, 2.9.0 / 1.53t) was used for image analysis.

[0323] Drug Treatment

[0324] CANDI400 was administered by tail-vein injection (100 pL PBS 0.5x, pH 7.4) containing 5 mg nanoparticle (0.22 mg of R848, 0.26 mg of MSA-2 Prodrug, 0.10 mg of RBN2397). Before injection, the solution was sterilized by filtration through a 0.22 pm sterile centrifugal filter (VWR), vortexed, and used promptly or frozen at -20 °C.

[0325] Example 9. Materials and Methods

[0326] Materials

[0327] All reagents and solvents were obtained from Thermo Fisher or Sigma-Aldrich and used without additional purification. The small molecules, including Lificiguat, IDF 1174, CAY10585, Chiysin, OPN expression inhibitor 1, Andrographolide, Mesalazine, Entrectinib, CPSI-1306, Iguratimod, Losartan, Parecoxib, Troglitazone. Indomethacin, Resiquimod, PLX5622, PLX3397, Shikonm, RN-1734, Tiypthantnn, GSK-3484862, Procainamide, Tofacitinib, Baricitinib, and Curcumin, were purchased from MedChemExpress. The compounds were dissolved in dimethyl sulfoxide (DMSO) as needed and were used without further treatment. MilliQ water was sourced from the Waters filtration system.

[0328] Particle synthesis

[0329] We synthesized sbCD from scratch with a defined degree of substitution of 2.5, to address the variability and high cost associated with commercially available reagents. This sbCD was used to create small nanoparticles with an average diameter of 16 nm (poly dispersity index, PDI=0.198). To obtain crosslinked bsCD nanoparticles (CANDI), L-lysine was added dropwise to sbCD. and the reaction mixture was stirred for 18 hours. The resulting particles were precipitated using ice-cold ethanol, then purified and characterized using DLS and Zeta potential analysis before being stored at -20 °C.

[0330] Fluorescent CANDI Analogs

[0331] Lyophilized CANDIE was dissolved in a 0. 1 M carbonate buffer at pH 8.5, and AF647 succinimidyl ester (ThermoFisher, 2 mg / mL in DMSO) was added to reach a concentration of 50 pM. The mixture was stirred at 37 °C for 45 minutes in a therrnocycler at 600 rpm. The labeled nanoparticles were then purified using buffer exchange with water, passing through 10 kDa MWCO centrifugal filters (Ami con; 10,000 rpm for 5 minutes; 300 pL water per wash, repeated 4-5 times). The final products were diluted with water or PBS to a concentration of 50 mg / mL and filtered through a 0.22 pm sterile filter (VWR) before use.

[0332] Small-molecule loading of nanoparticles

[0333] For payload loading, a solution of empty CANDI (5 mg) in 90 pL of PBS (lx) was prepared in 10% DMSO. The following nanoparticle formulation was developed: CANDI460 containing R848 (0.25 mg) and PLX3397 (0.142 mg). 10 pL of 2M NaOH and 20 pL of IM HC1 were added, and the mixtures were rapidly vortexed until the drugs were fully dissolved. The solution was then filtered through a 0.22 pm sterile filter (VWR) and either used immediately for characterization and in vitro assays or stored at -20 °C for later use.

[0334] Example 10. Characterization

[0335] The particle size and surface charge of all nanoparticle formulations was measured using dynamic light scattering (DLS) and zeta potential analysis on a Malvern Zetasizer APS. Measurements were conducted at a concentration of 2 mg / mL in PBS using DTS1170 cuvettes (Malvern) at 25 °C.

[0336] Turbidity assay

[0337] This assay was used to determine drug loading. CANDIE solutions (0.39-100 mg / mL in lx PBS) were prepared at pH 7.4, and small molecules were dissolved in DMSO (0.0638 mg R848 and 0.0362 mg PLX3397 per well) to create payload stocks. The payloads were then added to buffered CANDI. Loading efficiency was assessed by measuring absorbance at 550 nm ( abs) after thorough mixing. Complete absorbance loss indicated full payload loading. Data were normalized against a payload-free control, with all experiments conducted in triplicate (N = 3).

[0338] Drug Release Kinetics

[0339] The kinetics of drug release were evaluated using a closed dialysis setup with a 3 kDa molecular weight cutoff membrane (Pur-A-Lyzer Midi Dialysis Kit). Solutions of CANDI400 (50 mg) were prepared with PLX3397 (0.56 mM) and R848 (1 .3 mM) in 1 mL of phosphate- buffered saline (PBS, l x) containing 10% dimethyl sulfoxide (DMSO). These solutions were dialyzed against 5 mL of DMSO containing 20% PBS (1 x) at 37 °C, under constant stirring at 600 rpm. At predetermined time points, 100 pL aliquots were withdrawn from the dialysis setup and analyzed using liquid chromatography -mass spectrometry (LC-MS). Each aliquot was replaced with an equal volume of fresh DMSO (20% PBS) to maintain constant volume. The identification of PLX3397 and R848 was based on their characteristic retention times (0.92 min and 0.78 min, respectively) and their mass-to-charge ratios (PLX3397: 421, R848: 315) in positive electrospray ionization mode (ES+). The percentage of eluted molecules was quantified by integrating the area under the curve (AUC) for each chromatographic peak, and the cumulative drug release was calculated as the ratio of the AUC of each eluted peak to the total AUC of chromatographs obtained from non-dialyzed solutions. All experiments were conducted in triplicates (N = 3).

[0340] Transmission Electron Microscopy

[0341] CANDI460 particles were prepared (50 mg mL’1, PBS lx) and diluted with water to a final concentration of 0. 1 mg mL'1. The particle solution was charged on a TEM grid for 1 min and treated with a 2% aqueous uranyl acetate solution for 15 min, followed by three washing steps with ultra-pure water (x3). Imaging was performed in a transmission electron microscope (JEOL 2100).

[0342] Example 11. In Vitro and In Vivo Experiments

[0343] Induction of Sppl expression in TAM

[0344] TAM are abundant in many solid tumors and have generally been associated with negative outcomes, therapeutic resistance and tumor invasion. Yet, macrophages are plastic, and different phenotypes have been identified by single cell RNAseq. Notably, a recent study identified TAM-associated SPP1 expression as a key negative predictor of patient clinical outcome in various cancer types (Fig. 42A and Fig. 42B). Analysis of published single cell RNAseq datasets confirms this finding in several large human cancer cohorts.

[0345] SPPl / Sppl production in TAM can be regulated by several factors (Fig. 42C), including tumor necrosis factor (TNF), interleukin 6 (IL-6), interleukin lb (IL-lb), lipopolysaccharide (LPS), colony-stimulating factor 2 (CSF2, GM-CSF), macrophage migration inhibitory factor (MIF) and hypoxia or inversely by interferon regulatory factor 8 (IRF8). In turn, SPP1 expression leads to the production of osteopontin, a molecule with pleiotropic effects, and globally promoting different hallmarks of cancer (Fig. 42C).

[0346] To investigate Sppl expression induced in TAM, initially we used SppltdTomatoreporter mice in which MC38-H2BGFPtumor cells were implanted in dorsal skinfold window chambers. We found large numbers of SppltdTomato-positive macrophages (positive for F4 / 80 and CD1 lb) in these tumors, as illustrated on day 7 after tumor implantation (Fig. 42D). Next we studied the temporal kinetics of Sppl expression in macrophages in vitro by using bone marrow cells from SppltdTomat° reporter mice, and which were differentiated into macrophages over 8 days using well-established protocols. We found that Sppl expression levels increased with cell differentiation, reaching a plateau at around day 6. At this point, all bone marrow-derived macrophages showed extensive Sppl expression (Fig. 42E). Taken together, these data indicate the usefulness of the Sppl,dTomatoreporter system to study the emergence of SppltdTomato- positive macrophages both in vivo and in vitro, and thus potentially how the induction of Sppl expression could be therapeutically inhibited, as explored below.

[0347] Cell-based screen identifies TAM Sppl inhibitors

[0348] Having shown the temporal induction of Sppl in myeloid cells, we next set up a phenotypic screen to test the effects of putative small molecule modulators of SPPl. For this screen, we prioritized the use of primary cells isolated from donor mice over immortalized cell lines to more accurately represent TAM as seen in vivo. Due to this constraint, we opted for a focused approach, testing a curated set of 26 compounds identified from the literature and known to be associated with Sppl modulation. The selection was based on established pathway interactions with Sppl, such as hypoxia, MIF, and peroxisome proliferator-activated receptor gamma (PPARG), as well as potential direct inhibitors.

[0349] Specifically, bone marrow cells from SppltdTomatoreporter mice were harvested, differentiated into macrophages for 5 days, incubated with different concentrations (L: low, 0. 1 pM; M: medium, 0.5 pM; H: high, 1 pM) of inhibitors on day 1 for 24 hours, and analyzed for SppltdTomatosignal on day 5 (Fig 43A). Despite the limited availability of primary cells, we successfully screened at least 104 cells per condition in duplicate at three different doses for each compound. This targeted strategy allowed us to effectively identify promising Sppl inhibitors with relevance to TAM biology7. Of interest, most putative Sppl modulators actually did not decrease or prevent Sppl expression levels in this screen, whereas compounds that showed a dose-dependent Sppl inhibition included TLR agonists (R848, p=0.0196), CSFIRi (PLX3397, p=0.0117 and PLX5622, p=0.01 4), and an indirect TNFa inhibitor (shikonin, p=0.0398) (Fig 43B). With all three compounds there was complete Sppl inhibition as a function of dose (Fig. 43C).

[0350] Next, we tested whether the compounds could have additive or synergistic effects. We performed similar screens but with single, dual, and triple combinations of small molecule drugs (Fig. 43D). Our data show that dual (R848 and PLX3397) and triple (R848 and PLX3397 and shikonin) combinations were more potent in Sppl inhibition over single drugs. To determine the therapeutic windows (ED50 over LC50) of these three compound classes, we performed toxicity experiments. We show broad therapeutic windows in the low pM range for R848 and PLX3397, but not for shikonin. Based on this information, we next designed a TAM delivery7system to bring the top two hits (R848 and PLX3397) to TAM in vivo, which we named CANDI460 as described below. We performed molecular modeling to determine whether R848 and PLX3397 would fit into the CANDI cavity, which they do. Using Autodock Vina, we docked R848 and PLX3397 into the central cavity of cyclodextrin. We found that in the highest-scoring docking poses, R848 maintains four hydrogen bonding interactions with the alcohols in the cavity, and PLX3397 maintains two hydrogen bonding interactions.

[0351] CANDI460 nanoformulation: characterization and impact on Sppl High macrophages

[0352] The dually loaded, TAM-avid CANDI nanoformulation was synthesized by7crosslinking bis-succinyl cyclodextrin using EDC (l-ethyl-3-(3-dimethy7laminopropyl) carbodiimide hydrochloride) chemistry. The resulting nanoparticles had an average particle size of ~16 nm (Fig. 44A) and were monodispersed (Fig. 44B). The CANDI was well internalized into SPP1+ bone marrow-derived macrophages and appeared in lysosomal cellular compartments. CANDI could be loaded with R848 and PLX3397 at ~ 0.08mg small molecule per mg CANDI. The resulting CANDI460 (R848 0.25mg, PLX 0. 142mg, size) was then used for additional efficacy and cellular toxicity studies using bone marrow-derived macrophages. The results show efficient (Fig. 44D and Fig. 44E) and safe (Fig. 44F) drug effects up to 0.2 mg / mL, well below the in vivo dose.

[0353] To evaluate the effects of CANDI460 on macrophage phenotypes, we conducted bulk RNA sequencing on bone marrow-derived macrophages treated or not with the nanoformulation. As anticipated, CANDI460 treatment significantly down-regulated the expression of Sppl (Fig. 44G). Notably, the treatment also resulted in decreased expression of the mannose receptor Mrcl (also known as CD206), which is linked to alternative activation of macrophages, and Trem2, a membrane protein associated with tumor promotion and considered a candidate therapeutic target2. Furthermore, CANDI460 treatment triggered the upregulation of several inflammatory' genes, including Cxcl9 and interleukin 1112a, which are key players in promoting adaptive antitumor immunity’ (Fig. 44G). These findings indicate that CANDI460 can induce multiple phenotypic changes in TAM, which may contribute to enhanced tumor control.

[0354] Therapeutic antitumor efficacy

[0355] To determine whether these changes translate into anti-tumor efficacy, we conducted tumor growth experiments in the MC38 mouse model (Fig. 45 A). The results showed significant antitumor effects following systemic administration of CANDI460. On day 15 after treatment, tumor volumes in the CANDI460 group were significantly reduced (82± 155mm3) compared to the control group (740±344mm3) (p<0.0001) (Fig. 45B). Flow cytometry showed increased CD8 T-cell infiltration and decreased pro-tumorigenic TAM.

[0356] Furthermore, 60 days after tumor inoculation, 9 of 12 mice (75%) in the CANDI460 group were still alive, compared to 0 of 12 mice (0%) in the control group. The median survival time was only 30 days in the control group and was not reached after 60 days in the CANDI460 group (Fig. 45C). Furthermore, the mice still alive showed a lack of tumor burden, both macroscopically (Fig. 45E) and microscopically (Fig. 45E). These results indicate that CANDI460 not only targets SPP1 expression in TAM but can also potently control tumor progression in mice and promote anti-tumor immune response. Furthermore, cured mice were immune to tumor re-challenge (Fig. 45D).

[0357] To corroborate these results, we performed antitumor efficacy studies in a second model. Using the B16F10 melanoma model, we again observed remarkable findings. In the CANDI460 group, 2 of 5 mice survived, whereas in the group treated with CANDI460+ anti- PD1, 4 of 7 mice were cured. Intravital imaging reveals drug action in the tumor microenvironment

[0358] We initially used intravital imaging to determine the pharmacokinetics and dynamics of CANDI460 using serial imaging in the MC38-TagBFP2 window chamber model. We observed a vascular half-life of approximately 2 hours, similar to other CANDI formulations. Cellular accumulation in the TME was most pronounced at 24 hours after intravenous administration. At this time, the material primarily localized to tumor macrophages as determined by MerTK-GFP imaging and corroborative flow cytometry.

[0359] To assess the pharmacodynamic effects of CANDI460, we performed additional longitudinal tumor imaging in SppltdTomatoreporter mice. Specifically, we performed serial microscopic examinations of the tumor microenvironment 8 days after tumor implantation, both before and after intravenous systemic administration of CANDI460 (Fig. 46A). As expected, baseline expression of Sppl was high pre-treatment. However, within 48 hours postinfusion of CANDI460, we observed a significant reduction of Sppl expression in TAM as evidenced by the number of Sppl -positive cells (Fig. 46B, p<0.0001) and the Sppl signal intensity per cell (Fig. 46C, p=0.01). Importantly, these changes were accompanied by tumor shrinkage, which was quite pronounced.

[0360] Discussion

[0361] TAM are often the most abundant cell ty pes in tumors, with many of them being pro- tumorigenic. Recent scRNA sequencing studies have shed light on the composition of human myeloid subsets in cancers and other inflammatory' diseases. One of the overarching discoveries of these studies was i) the high SPP1 expression in undesirable macrophages and ii) the virtual exclusiveness of SPP1 with other biomarkers of favorable macrophage subtypes (e.g., CXCL9). SPPl / Sppl is a secreted phosphorylated protein produced by different cell types. It is expressed at low levels in circulating monocytes and can be induced during their differentiation into macrophages, particularly' in some of those found in tumors and sites of inflammation. Our knowledge of SPP1 function and induction originates mostly from work in cell lines including THP-1, RAW and HL-60 cells but has recently been substantiated by large scale scRNA sequencing studies in human tissues. One of these studies found stereotyped gene expression programs revealing seven conserved TAM states in which only two preferentially expressed SPP1 (with or without MTH1) and almost mutually exclusive with antitumori genic programs (e.g., CXCL9). These unique and opposed expression profiles, as well as the emerging knowledge of SPP1 downstream effects, form the basis of the current therapeutic strategy design.

[0362] Sppl has a diverse range of effector functions, mediated in part through integrin binding. This enables Sppl to affect multiple cell types and their functions. Such broad activity suggests potential therapeutic modulation. Notably, mouse studies have provided revealing data, demonstrating that Sppl knock-out mice have lower TAM infdtration and tumor growth compared to wild-type counterparts. Various therapeutic strategies for Sppl inhibition have been explored, including the use of siRNA, shRNA, aptamers, antibodies, and small -molecule inhibitors but efficacy has generally been modest. Interestingly, in our screening efforts, we found that many of the previously identified small molecules (e.g., andrographolide) were inefficient, prompting a broader comparative screen.

[0363] Unexpectedly, we identified that the TLR7 / 8 agnostic R848 (also named resiquimod) and the CSFR1 inhibitors PLX3397 (also named pexidartinib) and PLX5622 had the highest efficacy in silencing Sppl expression. R848, an imidazoquinoline, stimulates the NFkB pathway through TLR7 / 8 MyD88-dependent signaling, though its link to Sppl downregulation has not been reported. PLX3397, a CSFR1 inhibitor, has been extensively studied for its role in reducing TAM recruitment and reprogramming of these cells, particularly when combined with other immunotherapies. In thyroid cancer, the effects of CSF1R inhibition by PLX3397 and inhibition of Sppl have been noted; however, the specific signaling pathways remain to be elucidated. In general, mechanistic studies are warranted to further elucidate how R848 and PLX3397 contribute to Sppl down-regulation in TAM.

[0364] To enhance the targeted delivery of small molecules to TAM after systemic injection, we used a cyclodextrin-adjuvant nanoparticle-drug deliver}' system. Earlier iterations of this platform were engineered to induce IL-12 or CXCL9 in TAM. Since prior scRNAseq data had shown mutually exclusive CXCL9 and SPP1 expression in TAM, we initially tested whether the CXCL9-modulating CANDI preparation (CANDI400) could affect Sppl levels. However, we observed only minor effects, prompting us to design the screening strategy featured here.

[0365] Immortalized cell lines

[0366] The immortalized murine bone marrow-derived macrophages (iMACs) used to evaluate toxicity were obtained from Charles L. Evavold at the Ragon Institute, Harvard University, as described by Evavold et al. (2021, Cell, 184, 4495). The iMAC and MC38 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Coming) with 10% Fetal Bovine Serum (Coming) and 1% Penicillin Streptomycin (Coming) at 37°C with 5% CO2. When the cells reached confluency. they were split using 0.05% Trypsin / 0.53 mM EDTA (Coming), and all in vitro assays were conducted when cells were 90% confluent. Before being used in cell culture, all CANDI preparations were filtered through a 0.22 pm sterile filter (VWR).

[0367] Bone marrow-derived cells

[0368] Murine bone marrow-derived cells (BMDCs) were isolated from Sppl-tdTomato reporter mice and wild-type C57BL / 6J mice (see below). BMDCs from the reporter mice were used for flow cytometry and live-cell microscopy, while those from wild-type mice were used for cytokine induction evaluation. To collect bone marrow, femurs were flushed with sterile PBS using syringes and a 27-gauge needle. Red blood cells were lysed using RBC Lysis Buffer (BioLegend) as per the manufacturer’s guidelines. The remaining cells were counted with a Neubauer chamber and plated into 96-well plates — transparent plates (NEST) for flow cytometry or black plates (ibidi, glass bottom) for imaging — at a density of 1. 1 x 10A5 cells per well. Bone marrow-derived macrophages (BMDMs) were differentiated by adding 50 ng / mL recombinant murine M-CSF (BioLegend) to the culture media over 5 days. The treatment was applied on day 1 for 24 hours, with fresh media added on day 4. For RNA sequencing, cells were plated into transparent 6-well plates (Coming) at a density of 1 x 10A6 cells per well. Cells were stimulated with 50 ng / mL recombinant murine M-CSF (BioLegend) for 7 days before treatment. Fresh media was added on day 4. RNA was purified with the RNeasy Mini Kit (Qiagen) according to the manual.

[0369] Live-Cell Microscopy

[0370] Cells were exposed to a range of small molecules (0-1 pM, DMSO < 0.5%) for 24 hours by adding pre-prepared stock solutions to the culture media. Prior to imaging, cells were stained with Hoechst 33342 (15 pg / mL, Thermo Fisher) following the manufacturer’s instructions. Imaging was conducted in a 96-well plate using an 1X81 inverted fluorescence microscope (Olympus. Toky o, Japan) equipped with a motorized stage (Renishaw. Wotton- under-Edge, England, UK) and an ORCA-Fusion Digital CMOS camera (Hamamatsu Photonics, Hamamatsu, Japan). Multiple fields of view w ere captured for each sample with either a UPlanS Apo xlO objective (NA 0.75, Olympus) or aUPlanSApo 40x air objective (NA 0.95, Olympus), using CellSens Dimension 3.1.1 software (Olympus). Along with bright-field images, two fluorescent channels were recorded: DAPI (345 / 455) and TdTomato (550 / 585), using the corresponding optical filters for excitation.

[0371] Flow Cytometry

[0372] Bone-marrow-derived cells from Sppl -TdTomato reporter mice were stimulated with specific drug combinations, then trypsinized and washed with PBS. The cells were first stained with AquaAmine LiveDead Fixable viability stain (Thermo Fisher) diluted in PBS, followed by treatment with Fc block (BioLegend) and fluorochrome-conjugated antibodies prepared in FACS buffer (l x PBS, 2 mM EDTA, 2% FBS). For How cytometry analysis, the cells were resuspended in FACS buffer. All samples were measured using an Attune NxT flow- cytometer (Thermo Fisher), and the data was analyzed with FlowJo 10 software (TreeStar).

[0373] Toxicity iMACs were seeded in 96-well plates at a density of 15 x 103cells per well and incubated for 24 hours at 37 °C with 5% CO2 before use. A stock solution of CANDI460 was prepared and diluted in cell culture medium to the desired concentrations (0.153 pg / mL to 10 mg / mL, DMSO 0.5%). Cells were incubated with nanoparticles for 24 hours, after which the medium was replaced with FluoroBrite DMEM containing AlamarBlue (Invitrogen; 10% final). Cells were then incubated at 37 °C and 5% CO2 for an additional hour. The fluorescence of each well was measured (Lex = 550 nm, Lem = 590 nm). Triplicates were performed for each concentration tested, and IC50 values were calculated from the means.

[0374] Dose response

[0375] To assess the dose-response of the dual-labeled nanoparticle, a stock solution of CANDI460 nanoparticles was prepared and then diluted in cell culture medium to achieve the desired concentrations (6.1 ng / mL to 1.6 mg / mL, DMSO 0.5%). Sppl-tdTomato BMDM reporter cells w ere incubated with the nanoparticle-spiked media on day 1 for 24 hours, with fresh media added on day 4. The cells were then prepared for fluorescence microscopy to determine Sppl-dtTomato expression.

[0376] Mouse models

[0377] All animals were bred and housed under specific pathogen-free conditions at the Massachusetts General Hospital. Experiments were approved by the MGH Institutional Animal Care and Use Commitee (IACUC) and were performed in accordance with MGH IACUC regulations. The main model was the S ppi -IRES -tdTomato mouse, a CRISPR / Cas9 generated mutant carrying a tdTomato reporter inserted after the stop codon in exon 7 of the Sppl gene. The Sppl-IRES-TdTomato model is available as B6J.Sppltml(tdTomato)Msasn / J (stock 33731) from the Jackson Laboratory'. We also crossed Sppl-IRES-TdTomato mice with Mert- TK-GFP mice to define the intratumoral SPP 1 cell population.

[0378] Tumor Cell Implantation

[0379] MC38 and B16-F10 cells were injected into the flanks of C57BL / 6J mice at concentrations of 2.5 x 106 cells and 0.5 x 106 cells, respectively. The tumors were given a minimum of one week to develop before treatment began and reached a size of at least 50 mm3before therapy was initiated.

[0380] Intravital microscopy

[0381] Imaging of mice bearing dorsal window chambers with MC38-mTAG-BFP or MC38- H2B-GFP was performed to determine the kinetics of Sppl modulation in the tumor microenvironment (Fig 46). Dorsal window chambers were implanted into reporter mice using well-established techniques. Fluorescent tumor cells (MC38-H2B-GFP or MC38-mTAG-BFP) were implanted in the window chambers as previously described and allowed to grow- for 7-10 days before imaging experiments, with tumor growth monitored regularly. In additional experiments, we determined the vascular half-live of CANDI460 by serial imaging of the microvascular in the mouse ear.

[0382] All confocal images were collected using a customized Olympus FV1000 confocal microscope (Olympus America). A 2x (XLFluor, NA 0.14), a 4x (UPlanSApo, NA 0.16), and an XLUMPlanFL N 20x (NA 1.0) water immersion objective were used for imaging (Olympus America). Fusion-protein MC38-Tag2-BFP, macrophage host cells (MerTK-GFP), fusionprotein Sppl-tomato and CANDIAF647 were excited sequentially using a 405 nm, a 473 nm, a 559 nm, and a 633 nm diode laser in combination with a DM-405 / 488 / 559 / 635 nm dichroic beam spliter. Emited light was further separated by beam spliters (SDM-473, SDM-560, and SDM-640) and emission filters BA430-455, BA490-540, BA575-620, and BA655-755 (Olympus America). Confocal laser power setings were carefully optimized to avoid photobleaching, phototoxicity, or tissue damage. Fiji (ImageJ, 2.9.0 / 1.53t) was used for image analysis. Flow cytometry

[0383] Mice with MC38 tumors were treated with one dose of CANDI460, and tumors w ere harvested after 4 days. Subsequently, the tumors w'ere processed in RPMI medium with 0.2 mg / ml and then passed through 40pm filters. Next, the cells were stained with AquaAmine LiveDead Fixable viability stain (Thermo Fisher), which was diluted in PBS. Following this, they were treated with an Fc block (BioLegend) and then stained with fluorochrome- conjugated antibodies, which were diluted in FACS buffer (lx PBS, 2 mM EDTA, 2% FBS). For How cytometry' analysis, the cells were resuspended in FACS buffer. Each condition was assessed in triplicate or quadruplicate using an Attune NxT flow cytometer (Thermo Fisher), and the resulting data were analyzed with FlowJo 10 software (TreeStar).

[0384] Drug treatment

[0385] CANDI460 was administered via tail-vein injection using 100 pL of normal saline containing 5 mg of nanoparticles per mouse, loaded with 0.25 mg of R848 and 0.142 mg of PLX3397. Before injection, the solution was sterilized using a 0.22 pm sterile centrifugal filter (VWR), vortexed, and used immediately.

[0386] Histology

[0387] MC38-WT tumors were harvested and fixed in 10% formalin solution. The tumors were paraffin-embedded and sectioned at 5 pm. The sections were then deparaffinized and rehydrated before immunofluorescence staining. Heat-induced antigen retrieval was performed using Retrievagen A pH6.0 (550524, BD Bioscience), and the sections were permeabilized with 0.3% Triton X-100 in PBS for 10 minutes at room temperature. After the sections were blocked with 4% normal goat serum in PBS, Sppl antibody (EPR21138; ab218237, Abeam 1 : 100) w'as incubated at 4 °C overnight. The sections were incubated with a biotinylated goat anti-rabbit IgG antibody followed by streptavidin Dy Light 594 (BA-1000 and SA-5594, Vector Laboratories, 1: 100 and 1 :600 respectively), and the nuclei were stained with DAPI (D21490, Thermo Fisher Scientific 1 :3000). After Sppl staining, the coverslips were removed and H&E staining was performed according to manufacturer’s protocol (ab245880. Abeam). All the slides w'ere scanned using the NanoZoomer 2.0RS scanner (Hamamatsu) for analysis. Example 12. Synthesis of CANDI Wafers

[0388] Materials

[0389] All reagents and solvents were purchased from Thermo Fischer or Sigma- Aldrich and used as received. Small molecules (R848, LCL-161, and ruxolitinib) were purchased from MedChem Express and used as is. MilliQ water obtained from Waters filtration system.

[0390] Synthesis of CANDI wafer material

[0391] Bis succinyl-P-cyclodextrin (275 mg, 1.0 eq. to carboxylate) was dissolved in MES buffer (3 ml, 50 mM, pH = 5) and activated with N-(3-(dimethylamino)propyl)-N'-ethyl carbodiimide hydrochloride (EDC) (Fisher; E2 g, 10.0 eq. to carboxylate) and N- hydroxysuccinimide (NHS) (Sigma; 228.5 mg, 5.0 eq. to carboxylate) for 10 min at 25 °C. A solution containing L-lysine (Sigma; 200 mg, 0.5 eq. to carboxylate) in MES buffer (0.35 ml) was added rapidly under vigorous stirring and the reaction was allowed to stir for 24 h at 25 °C. The resulting viscous gel was added drop-wise to an ice-cold absolute ethanol solution (50 ml) and subsequently centrifuged for 5 min at 800 ref yielding a white precipitate decanted and dissolved in water (6 ml). The opaque and viscous gel was then dialyzed for 48 h in water and consecutively lyophilized for 48 h to yield the unloaded wafer material in powder form, which was characterized by DLS (2 mg / mL, IX PBS) and zeta potential (2 mg / mL, 0.1X PBS) and stored at -20 °C.

[0392] Small-molecule loading and wafer preparation

[0393] R848A (0.04 mg), LCL-161 (0.1 mg), and ruxolitinib (0.08 mg) were dissolved in DMSO (5 pl) and a solution of the unloaded wafer material (10 mg) in water (95 pl) was used for payload loading to a final DMSO concentration of 5%. After vigorous vortexing, the resulting small-molecule loaded CANDI wafer material was lyophilized for 48 h, yielding a white powder material, which was subsequently pressed into the wafer shape.

[0394] Fluorescent labeling of the CANDI wafer

[0395] Lyophilized CANDI wafer material (20 mg) was dissolved in water (1 mL) to which AF647 succinimidyl ester (Fisher; 2 mg rnL-1 in dimethylsulfoxide) was added. The reaction was incubated for 2 h at 37 °C in a thermocycler (Eppendorf, 550 rpm). The resulting fluorescent wafer material particles were purified by dialyzing for 48 h in water and subsequently lyophilized for 48 h before being pressed into the wafer shape. CANDI-wafer-Gd Conjugate

[0396] CANDI wafer (100 mg) was dissolved in dichloromethane (DCM, 5 mL) and activated using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 60 mg, 386.5 pmol) andN-hydroxysuccinimide (NHS, 79 mg, 686.3 pmol) overnight. The resulting solution was precipitated using diethyl ether. The activated CANDI was then dissolved in a mixture of DCM (5 mL) and dimethyl sulfoxide (DMSO, 3 mL). followed by the addition of Gd-DO3A- butylamine (24 mg, 36 pmol) for 24 hours. Subsequently, the conjugated CANDI-Gd was reprecipitated using diethyl ether and subjected to lyophilization. The resulting product was dissolved in acetonitrile (CAN), and unreacted Gd-DO3A-butylamine was removed by filtration using a 3.5 k Ami con filter, followed by lyophilization. The MR imaging with implanted Gd-wafers was conducted in triplicates (N=3 mice).

[0397] Example 13. Characterization of CANDI Wafers

[0398] Drug release kinetics

[0399] The kinetics of drug release were investigated using a closed dialysis system with a 3 kDa molecular weight cut-off membrane (Pur-A-Lyzer Midi Dialysis Kit). CANDI wafers (60 mg) loaded with R848 (0.24 mg), LCL-161 (0.6 mg), and ruxolitinib (0.48 mg) were placed in phosphate-buffered saline (PBS, IX, 1 mL) and dialyzed against PBS (IX, 5 mL) at 23 °C under continuous shaking. The percentage of eluted molecules was quantified by analyzing liquid chromatographs at specified time points (t = 0, 0.33, 0.5. 1, 2, 5. 8, 24, 55, 76, 98. 122, 144 h). For analysis, 90 pL aliquots were injected into an LC-MS, and the system was replenished with an additional 90 pL of PBS after each injection. Each payload (R848, ruxolitinib, and LCL-161) was identified by its distinctive retention time (R848 = 0.78 min, ruxolitinib = 0.95 min, and LCL-161 = 1.01 min) and mass-to-charge ratio (ES-: R848 = 313, ruxolitinib = 305, and LCL-161 = 499). The cumulative drug release was determined by calculating the ratio of the integrated area under the curve for each eluted peak to the total area under the curve of chromatographs obtained from non-membrane controls. All experiments were conducted in distinct triplicates (N=3) to ensure reproducibility and reliability of the results.

[0400] Scanning electron microscopy

[0401] The surface morphology of the CANDI wafer was examined by field emission scanning electron microscopy (FE-SEM) (Zeiss Gemini 360. USA). The secondary electron (SE2) detector obtained images with 10 keV electron beam. Elemental analysis was performed using energy-dispersive X-ray spectroscopy (EDX).

[0402] Example 14. In vivo and In vitro Studies

[0403] Cell models

[0404] The primary glioma cell line model for this study was CT-2A, a syngeneic GBM model histologically similar to human GBM46. Cells were obtained from Dr. Samuel Rabkin (CT- 2A), Dr. Xandra Breakefield (CT-2A-mCherry-luc) and Dr. Katy Yang (CT-2A-H2B- mApple). We used CT-2A mCherry-luc cells for pharmacokinetics experiments, MRI experiments, bioluminescence imaging, and survival studies. For IVM and histology experiments, we used CT-2A H2B-mApple. In separate control experiments, we determined that all CT-2A subclones had similar growth rates in vitro and in vivo. A second GBM cell line was SB-28 obtained from Dr Hideho Okada61. In this model, there is invasive GBM into normal brain parenchyma while immunofluorescence shows a heterogeneous but never abundant T-cell infiltration61. Murine CT-2A and SB-28 were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Coming) containing 4.5 g / 1 glucose and 10% FCS. Cells were detached from plastic with accutase (Sigma- Aldrich).

[0405] The immortalized murine bone marrow-derived macrophages (iMACs) were acquired from Charles L. Evavold (Ragon Institute, Harvard University) and used to assess toxicity (Fig. 30). iMAC cells were plated and grown in DMEM. supplemented with 10% Fetal Bovine Serum (Coming) and 1% Penicillin Streptomycin (Coming) at 37 °C. Upon reaching confluency, cells were split using 0.05% Trypsin / 0.53 mM EDTA (Coming), and all in vitro assays were performed after the cells reached 90% confluency. The cell lines were tested mycoplasma-negative.

[0406] Mouse models

[0407] We used various mouse models to study different aspects of drug delivery7, IL-12 induction, and therapeutic efficacy. All experiments were approved by the MGH Institutional Animal Care and Use Committee (IACUC) and performed according to MGH IACUC regulations. A total of N = 142 mice were used. This includes immunocompetent C57BL / 6J wild-ty pe mice for GBM implantations (N = 138, C57BL / 6J, Strain Number: 000664, JAX, Bar Harbor, ME), MerTK-GFP63 mice for co-localization IVM studies (N = 1; Strain Number: 036071, JAX, Bar Harbor, ME), and IL-12-eYFP mice for GBM implantations, IVM mechanistic studies and bone marrow harvesting (N = 3; B6.129-IL-12btml.lLky / J, Strain Number: 006412, JAX, Bar Harbor, ME).

[0408] Tumor implantation

[0409] We used 10-12 week old C57BL / 6J mice, anesthetized them with isoflurane, shaved their heads and immobilized the cranium in a stereotactic frame (Kopf, Tujunga, CA). The surgical site was sterilized with two cycles of betadine-isopropanol. Using a Dremel with a burr (Fine Science Tools, 19007-07) a trepanation was performed and 5 x 104 CT-2A (2.5 x 104 SB-28) cells diluted in 2 pl sterile PBS (Sigma-Aldrich) were stereotactically implanted into the right cerebral cortex (coordinates: 2 mm right lateral of the bregma and 2 mm posterior to the coronal suture with an injection depth of 0.7 mm below the dural surface) using a 10 pl Hamilton micro-syringe driven by a fine step stereotactic device (Kopf)- CT-2A mCherry-luc cells were used for therapeutic efficacy studies, whereas CT-2A H2B-mApple cells were utilized for IVM studies and multiplexed FAST-profiling of GBM.

[0410] Tumor resection and wafer implantation

[0411] On day 12 after tumor implantations, animals were immobilized in a stereotactic frame (Kopf, Tujunga, CA), and the surgical site was sterilized with two cycles of betadine- isopropanol. A right paramedian rostral-caudal curvilinear incision was made, and subcutaneous layers of the scalp, including the periosteum, were removed. Under 10-20X magnification, with a radius of 2 mm around the initial tumor injection site burr holes were made using a Dremel with a burr (Fine Science Tools, 19007-07). The burr holes were connected using fine micro scissors, and the bone flap was consecutively lifted off with fine forceps resulting in a craniectomy. After peeling away the dura mater, tumors were resected, leaving behind a residual tumor volume and creating a cavity for consecutive wafer implantation. The resection cavity was then either left empty or drug-loaded wafers were placed using fine forceps. The skin was closed with interrupted sutures.

[0412] Histology /

[0413] Frozen tissue sections were cut to 5 pm thickness and then processed for immunofluorescence. Frozen tissue sections were thawed, rehydrated, and blocked with Intercept Blocking buffer (EI-COR) for 30 min before antibody staining. Tissue sections were incubated with antibodies for 1-2 h and washed with PBS for 10 min three times. An Olympus BX-63 microscope was used for image acquisition (Metamorph software version 7.10.4). CellProfiler version 4.1.8 was used for the analysis of tissue section images. A custom Python script was used to generate synthetic image maps.

[0414] Intravital microscopy

[0415] All experiments were approved by the MGH Institutional Animal Care and Use Committee (IACUC) and performed according to MGH IACUC regulations. Cranial window s were implanted with modifications according to established methods. The head of 10-14 week old mice was shaved, animals were immobilized in a stereotactic frame (Kopf, Tujunga, CA), and the skull was sterilized with two cycles of betadine-isopropanol. A large oval skin area was removed, from behind the ears to between the eyes, surrounding the lambda and bregma sutures. The periosteum was pushed to the side, and all tissue on top of the skull was scraped off.

[0416] The rim of the 5 mm circular section of the head, excluding the lambda and bregma, was sanded down using a Dremel with a burr (Fine Science Tools) and removed to provide an opening to the brain. Using stereotaxic positioning. 2 pl of Optimum (Thermo Fisher Scientific) with 105 CT-2A-H2B-mApple cells were injected at about 1 mm depth near the middle of the opening, avoiding vasculature. Gelfoam and saline w ere used to remove blood during surgery and after injection. A drop of saline and an 8 mm round cover glass were placed onto the opening. Super glue was used to attach only the rim of the cover glass to the skull, avoiding any contact between the adhesive and the brain. Then, dental cement was used to cement the cover glass onto the skull, cover skull areas without skin, and form an elevated rim for water immersion imaging.

[0417] All confocal images were collected using a customized Olympus FV1000 confocal microscope (Olympus America). A 2x (XLFluor, NA 0.14), a 4x (UPlanSApo, NA 0.16), and an XLUMPlanFL N 20x (NA 1.0) water immersion objective were used for imaging (Olympus America). CT-2A H2B-apple tumor cells, CANDIAF647, and vascular probes were excited sequentially using a 405 nm, a 473 nm, a 559 nm, and a 633 nm diode laser, respectively, in combination with a DM-405 / 488 / 559 / 635 nm dichroic beam splitter. Emitted light was further separated by beam splitters (SDM-473, SDM-560, and SDM-640) and emission filters BA430- 455, BA490-540, BA575-620, and BA655-755 (Olympus America). Confocal laser powder settings were carefully optimized to avoid photobleaching, phototoxicity, or damage to the brain. All images were processed using Fiji (ImageJ2. Vers.2.3 / 1.53f). Flow cytometry

[0418] Tissues were isolated, mechanically dissociated using surgical scissors, and digested using Collagenase IV at 0.2 mg / ml in RPMI 1640 at 37 °C for 45 minutes with vigorous shaking. After digestion, tissues were filtered through a 40 pM cell strainer and resuspended in protein-free PBS. Cells were stained using AquaAmine Live Dead Fixable viability7stain (Thermo Fisher) and then washed with PBS. Cells were then resuspended in FACS buffer (PBS with 2 mM EDTA and 2% Fetal Calf Serum) and stained with Fc block (Biolegend) and fluorochrome-conjugated antibodies. Sample data were acquired using an Attune NxT flow cytometer (Thermo Fisher), and data were analyzed using FlowJo 10 software (TreeStar).

[0419] Bioinformatic analysis of published scRNAseq data sets

[0420] Mouse data was downloaded viaZenodo (zenodo.org / records / 6654420). Human GBM Full aggregate data from newly diagnosed patients was downloaded via Brain Immune Atlas (www.brainimmuneatlas.org / download.php). Seurat v4 (Butler et al., 2018; Hafemeister and Satija, 2019) was used for single cell RNA-seq data analysis. We constructed a Seurat object using the feature-barcode matrix for each sample. A series of quality filters were applied to the data to remove low-quality cell barcodes; possible debris with too few genes expressed (< 300); possible more than one cell with too many genes expressed (> 6,000-10,000 according to the sample); possible dead cell or a sign of cellular stress and apoptosis with too high proportion of mitochondrial gene expression over the total transcript counts (> 20%). Each sample was scaled and normalized using Seurat’s ‘NormalizeData’ and 'Seal eData’ functions. We then merged all samples and repeated the same scaling and normalization method. For human data, all cells in the merged Seurat object were integrated using ‘IntegrateData’ and the top 30 PCA dimensions. Data was clustered via Seurat's TindNeighbors’ and TindClusters’ (with parameters: resolution = 0.5) functions. The resulting merged and normalized matrix was used for the subsequent analysis. Differentially expressed genes were identified by FindMarkers function comparing cells belonging to one subty pe to the rest. Wilcoxon statistical test was used. log2FC > 0.25 and FDR < 0.05 was used to filter DEGs. Clusters were annotated as described in the original papers. Macrophage and microglia-specific markers were used for further characterization. Gene set enrichment analysis was performed with ‘enricher’ function. The hallmark collection of MSigDB repository was tested for this analysis with the following parameters: pAdjustMethod = "BH",pvalueCutoff = 0.05, qvalueCutoff = 0.05. Bulk RNA seq

[0421] To determine the effects of the wafer on phagocytic cells, bulk RNAseq was performed. Bone marrow-derived macrophages were isolated and differentiated as previously described. Cells were then stimulated for 24 h with drug loaded wafer material to induce activation. RNA was isolated using the RNeasy Plus Mini Kit (Qiagen). Final RNA concentration was determined by absorbance (Nanodrop), and samples were stored at -80 °C until shipment for sequencing (NovoGene).

[0422] Cytokine measurements

[0423] To determine the relative expression levels of several cytokines and chemokines in response to the (drug-loaded) wafer, an immunoassay (R&D Proteome Profiler Mouse XL Cytokine Array) was performed with cell culture supemates from bone marrow-derived macrophages that had been stimulated for 24 h with empty or drug-loaded wafer material.

[0424] Drug-loaded wafers were implanted as described under Tumor resection and wafer implantation. Blood-draws were performed on day 1, 2, 4, 8 and 14 after the implantation. 100- 200 pl of whole blood was collected in K2EDTA-coated blood collection tubes (BD Microtainer 365967) and spun down at 2000 ref for 10 min. Serum was stored at -20 °C for downstream analysis. IL-12 p70 levels were measured using a mouse IL-12 p70 quantikine ELISA Kit (R&D Systems M1270). The experiment was conducted with 3 animals per group (N=6; one animal in the CANDI450 treated cohort had to be euthanized on day 10 due to poor body condition from repeated blood draws).

[0425] MR imaging

[0426] MR imaging of GBM-bearing mice was for performed for different reasons: I) to monitor the growth of GBM following implantation; ii) to monitor the evolution following GBM resection and wafer implantation (at baseline (day 13) and after treatment (day 22)) and iii) to image the dissolution of wafer material. All imaging was performed on an animal 4.7-T MR imaging unit (Bruker Pharmascan) under respiration-monitored isoflurane anesthesia. Coronal imaging parameters for pre-and post-contrast enhanced Tl- weighted imaging were as follows: repetition time (TR) = 700 ms, echo time (TE) = 14 ms, matrix size 256 x 256, and slice thickness 0.5 mm. 12 sections were acquired. Imaging parameters for pre-enhanced T2- weighted imaging were as follows: TR = 4,000 ms, TE = 53.3 ms, matrix size 256 x 256, slice thickness 0.5 mm. Twelve sections were acquired. Tumor or wafer volumes were calculated by using the Horos image-processing software for tumor volumetric data via ROI-based 3D analysis of Gd-DTPA enhanced Tl-weighted MR images (Horos, horosproject.org).

[0427] Bioluminescence imaging

[0428] Bioluminescence imaging was performed using an Ami HTX Spectral Instruments Imaging instrument at baseline (day 12) and after treatment (day 19 and 26). Mice received an intraperitoneal injection of luciferin (5 mg / mouse) and were maintained under respiration- monitored isoflurane anesthesia during imaging. Imaging parameters were as follows: 10 seconds and 0.5 seconds exposure, binning levels 1, 2, 4, and 8. Total flux (p / s) was used for signal quantification.

[0429] Statistics

[0430] All statistical data analyses were performed using GraphPad Prism 9 software and results are expressed as mean ± standard deviation or with 90%-CI. Measurements were taken from distinct samples. For normally -distributed datasets, we used 2-tailed Student's t test and one-way ANOVA followed by Bonferroni’s multiple comparison test. When variables were not normally distributed, we performed non-parametric Mann-Whitney or Kuskal-Wallis tests. To analyze the statistical significance of difference for survival we used the log-rank (Mantel- Cox) test, p values > 0.05 were considered not significant (n.s.), p values < 0.05 were considered significant.

[0431] Analysis of macrophages in murine and human GBM

[0432] We initially performed a bioinformatic analysis to determine the proportion and composition of the myeloid compartment in GBM (Fig. 31) based on published datasets. Through this and spatial profiling of murine GBM, we found that macrophages and monocytes make up -25% of all cells in CT-2A. The macrophages are characterized by immunosuppressive markers such as SPP1, and major pathway hallmarks include NFkB signaling. Similar findings were identified in human GBM, albeit with certain differences. While the immunosuppressive macrophage compartment was still large, the microglial compartment was also sizable. Different myeloid cell therapeutics are being evaluated. One newer approach has been the systemic use of macrophage targeting nanomaterials capable of immune modulation and efficient drug delivery to macrophages, which has been tested in various malignancies and showed potent anti-tumor effects. However, since newly diagnosed GBM are routinely surgically resected, we designed a wafer system that could be implanted during surgery and locally release myeloid immunostimulatory compounds over prolonged periods. We created an implantable crosslinked bis-succinyl cyclodextrin material that serves as a “sponge” to hold immunostimulatory small molecules.

[0433] Synthesis and characterization of the immune stimulatory CANDI wafer

[0434] Bissucinyl [3-cyclodextrin (bsCD) was cross-linked with lysine to yield a thick gel-like material (Fig. 32) that was subsequently dialyzed against a 4kDa MW cut-off and then loaded with three immune modulator}' small molecules. The three molecules included a Janus Tyrosine Kinase (JAK) inhibitor (ruxolitinib), a cIAP inhibitor (LCL-161), and a toll- like receptor TLR7 / 8 agonist (R848). This triple combination was identified by a drug screen to maximize IL-12 production in myeloid cells when given systemically. We did not use systemically administered nanomaterials as the blood-brain barrier (BBB) would form a considerable delivery' barrier. Rather, we developed the higher cross-linked material for prolonged degradation / release and pressed the triple drug-loaded material into implantable wafer shapes. Each implantable wafer weighed ~10 mg (volume ~4-6 mm3) in its dried form and contained 0.22 mg of the active drug substance (0.04 mg R848, 0. 1 mg LCL161, and 0.08 mg ruxolitinib).

[0435] Fig. 32E provide an overview of the material surface as determined by scanning electron microscopy (SEM). The pressed wafer material had a porous surface reminiscent of a sponge. Next, we determined the drug release rates of the small molecules from the wafer material. For these studies, we stored drug-loaded wafers in dialysis tubing and determined the release of individual drugs as a function of time. Fig. 321 summarize the results from these studies. In this closed in vitro system, the half-life of release was approximately 45 hours. At 144 hours, only 8% (range 3.7-13.2%) of the drug remained in the wafer material, as determined by tiypsinization experiments. To supplement our understanding of degradation, we also performed in vivo experiments where proteases in the TME may play additional roles in biodegradation. For these studies, we covalently attached Gd-DOTA to cyclodextrin units, implanted these “MRI-wafers” in GBM resection cavities, and performed serial MR imaging of animals (Fig. 32H). These studies show that the wafer material is degraded in vivo with a half-life of 8.9 days. Cellular effects of the CANDI wafer

[0436] We performed a series of in vitro and in vivo studies to determine the pharmacokinetics and pharmacodynamics of the wafer and its payload. First, we determined whether the wafer material was internalized into macrophages or whether the small molecule payloads exerted their effects without bulk internalization. Bone marrow-derived macrophages (BMDM) were incubated with fluorescently labeled CANDI-AF647 wafer and imaged by microscopy (Fig. 30A). These data clearly show the cellular internalization of wafer material, data that was further corroborated by flow cytometry (Fig. 30B and Fig. 30C). Furthermore, similar experiments were conducted in vivo using MerTK-GFP mice (Fig. 33). These cell internalization effects were most pronounced 3-5 days after implantation. We next determined the mechanism of cellular CANDI wafer uptake using typical inhibitors of different pathways (chlorpromazine for clathrin-mediated endocytosis; wortmannin for micropinocytosis / phagocytosis; Imipramine for macropinocytosis; EIPA (5-(N-ethyl-N- isopropyl)amiloride) for macropinocytosis via Na / H exchange; Fig. 30B). The biggest inhibitory effects in uptake were observed with chlorpromazine, arguing for uptake via clathrin-mediated endocytosis.

[0437] To determine the effects of the wafer payload on different TAM pathways (Fig. 30D), we performed cytokine profiling, imaging of IL-12 reporter cells, and RNAseq. Cytokine profiling of w afer-exposed BMDM show ed high expression of both IL- 12a and IL- 12b, CCL5, and other cytokines, as expected. Interestingly, we also saw down-regulation of SPP1, a negatively associated TAM biomarker. In BMDM derived from IL12-eYFP reporter mice, we observed massive IL-12 induction in virtually all cells upon wafer stimulation (Fig. 30F). Finally, bulk RNAseq (Fig. 30G) of wafer-exposed macrophages showed a few interesting observations. First, when an empty wafer was used without a therapeutic payload, no genes were upregulated or downregulated, attesting to the inertness of the host material. When macrophages were exposed to triple drug-loaded wafer material, w e observed many upregulated (n = 177) and downregulated (n = 117) genes. Among the top upregulated genes were: IL-12 (the main mechanism of action). Marco (SCARA2, a scavenger receptor that could be a CANDI entry point), Cd209 (another receptor that could be a CANDI entry point) and H2-M2 (antigen presentation, MHC). Genes such as CD40 and Fscnl, both connected to non- canonical NFkB activation, were also significantly elevated by drug-loaded wafer. Key downregulated genes included: Mrcl (typical M2 marker for mannose receptor), Siglecl (CD169, anti-inflammatory roles in macrophages)36, Trem2 (a M2 marker, also a drug target for TAMs) 37-39, Clec7a (Dectin-1, associations with poor survival and blocking can enhance immunotherapy).

[0438] Antitumor efficacy as mono and combination therapies

[0439] Having elucidated the molecular and cellular effects of the CANDI wafer, in vitro and in vivo, we next set out to determine efficacy in a glioma resection model. As outlined in Fig. 34. GBMs were resected, and the resection cavity was either left alone (control. n=18) or filled with drug-loaded wafers (n=21). Animals were then serially monitored by imaging and overall survival. All control CT-2A-bearing mice whose tumor was resected died within 23 days after surgery (35 days after tumor implantation). This outcome is similar to the clinical scenario where resection alone cannot achieve tumor control. Conversely, when the resection cavity was packed with the drug-loaded CANDI wafer, over half of the animals were alive at day 96, the longest time point investigated. At that time, all surviving animals showed normal grooming behavior and appeared healthy. MR imaging at that time point showed no enhancing tumor but merely a fluid filled resection cavity. Hence, a decision was made to sacrifice the animals at this point and investigate whether there was any residual microscopic tumor deposits left. Autopsies in these animals showed a fluid-filled resection cavity, and HE staining confirmed the complete absence of any residual tumor.

[0440] We also determined whether the CANDI-wafer-induced immunotherapy could be further enhanced by conventional standard-of-care therapy (Fig. 34B). We thus combined the surgical resection with w afer placement, radiation, and temozolomide treatments (n=18), as is done routinely clinically. Our data show- that the treatment effects are indeed synergistic, with nearly 70% of animals surviving 80 days (p = 0.0328). Finally, we tested some of the above in a second GBM model using SB-28 cells, a much more invasive and aggressive model (n=23). Again, we observed a similar therapeutic phenotype with 25% survival at t = 80 days (p=0.0007).

[0441] CANDI wafer induces a local IL12 induction leading to CD8 recruitment and inhibition of brain tumor recurrence

[0442] To interrogate the mechanism by which the CANDI wafer works in vivo, we performed serial intravital microscopy experiments (Fig. 33), spatial biology experiments (Fig. 35), and flow cytometry. Serial intravital microscopy using the brain window' chamber model was performed following the implantation of micro-sized wavers under the window. Given the tight space below the window, these wafers were much smaller than the post surgically implanted ones and were thus resorbed faster. Serial imaging showed a strong local IL 12 response lasting during the duration of the micronized wafers (4-5 days). High local IL 12 concentrations in the brain (Fig. 33E) did not elevate systemic IL12 levels (Fig. 33D), unlike systemic IL12 therapies.

[0443] Immune cell profiling by immunohistochemistry (Fig. 35) of native CT-2A showed a predominately TAM-rich tumor environment essentially largely devoid of CD8 cells. Following subtotal surgical resection and wafer implantation, we noted a significant increase in tumoral CD8 (7.2-fold; p=0.0258) and CD4 (5.6-fold; p=0.0282) levels, which was not observed in surgical resection alone. Temporal analysis of different cohorts also showed a 3- fold increase in TAM (p=0.0017) and a 1.3-fold increase in CD45+ cells in the CANDI wafer cohort, which returned to baseline levels (p<0.0001) after 90 days. Finally, we performed flow cytometry to confirm histology results further. These data showed a 4.7-fold increase in CD8 (p=0.0094) and a 2.3-fold increase in CD4 (p=0.0365) recruitment in wafer-treated animals. We also observed an immune suppressive, wound-healing macrophage phenotype (high CD206 and TREM2 expression), in mice that underwent surgery but no wafer implantation. In mice with wafers, CD206 (p = 0.092) and TREM2 (p = 0.001) were much lower in macrophages. Finally, we show by flow cytometry that wafer material indeed primarily accumulated in TAM. (Fig. 30C).

[0444] Discussion

[0445] Standard-of-care GBM therapies include surgical resection, radiation and chemotherapy. Despite this multipronged approach, recurrences are frequent, and survival remains abysmal. Various immunotherapies have had anecdotal effects but have not changed the overall outcome for patients. There are several reasons for the extraordinary treatment resistance, the highly immunosuppressive microenvironment being an important one. Myeloid cells in GBM rapidly adopt an immunosuppressive phenoty pe. We reasoned that an immunomodulatory wafer material could be constructed to shift immune suppression to stimulation. Here, we show the synthesis, characterization, and therapeutic efficacy of this approach. Using two different mouse models, we show anti-tumor immune responses preventing postoperative glioma recurrence.

[0446] Tumor-associated macrophages (TAM) in GBM are the most common immune cell types contributing to -20-40% of the actual tumor mass. The cells originate from two independent sources: bone marrow-derived monocytes and brain-resident microglia. Most adult microglia are yolk sac-derived, have longevity, limited self-renewal, and are mostly found at the tumor / parenchyma interface. In contrast, inside GBM, the BBB is impaired, and the expression of the monocyte chemoattractant proteins (MCPs) is increased. This leads to a large influx of bone marrow-derived monocytes and their differentiation into immunosuppressive tumor-associated macrophages, often in the perivascular niche, although different subtypes and programs are being recognized. TAMs have been shown to promote GBM growth through positive feedback loops with tumor cells. Conversely, it is also known that certain TAM subtypes are plastic and can acquire antitumor phenotypes. The task then is to provide longterm stimuli so that recruited TAM adopt an antitumor phenoty pe and prime the TME for more effective tumor control. Our wafer approach offers a therapeutically effective strategy to achieve this goal and eradicate GBM.

[0447] We chose a biodegradable material (CANDI) to deliver small molecule immunomodulatory drugs to affect TAM function. We wished to achieve much longer drug release and slower degradation to modulate TAM function during the prolonged post-surgical healing process. As such, we increased the cross-linking of the bis-succinyl P cyclodextrin monomers to yield a gel-like substance that could be drug-loaded and then lyophilized into implantable wafers. The triple-drug payload (R848, LCL-161, ruxolitinib) was chosen on purpose as it had been shown to be much more effective compared to single and dual drug loads. Furthermore, we adjusted the payloads so that cIAP inhibitors would not cause local toxicity’. Collectively, this resulted in an implantable material that degraded within a couple of weeks, releasing the triple small molecule payload from the surface in a time-dependent fashion. We furthermore show' that the prolonged release of immune modulatory drugs can reprogram TAMs to an immune stimulatory phenotype and prevent post-surgical tumor recurrence. IL- 12 produced by TAM and tumor antigen-presenting cells conditions cytolytic immunity' and Interferon gamma locally, which suppresses tumor regrowth. We further show that key T cell co-stimulation pathways are enhanced by drug loaded wafers, and these signals combined with IL-12 bolster antitumor T cell responses. It is possible that post-surgical recurrence observed clinically is in part driven by immune suppressive mechanisms triggered by surgical trauma. We similarly observed immune suppressive macrophage phenotype induction in surgically resected but non drug treated animals. However, in contrast, we find that as opposed to anti-inflammatory treatments, Type 1 polarizing (TH1) cytokine induction inhibits tumor growth recurrence. Nonetheless, we similarly observed that promoting CDS T cell immune surveillance is essential to halt tumor outgrow th. These data argue in favor of a model that post-surgery macrophage phenotypes are important determinants of tumor recurrence.

Claims

WHAT IS CLAIMED IS:

1. A nanoparticle comprising a cyclodextrin; and a. a first pay load; b. optionally a second payload; and c. a third payload,2. A nanoparticle comprising a cyclodextrin; and a. a first pay load; b. a second payload; and c. a third payload.

3. The nanoparticle of Claim 1 or 2, wherein the nanoparticle has a diameter of about 10 to about 20 nm.

4. The nanoparticle of any one of Claims 1-3, wherein the nanoparticle has a diameter of about 15 nm to about 20 nm.

5. The nanoparticle of any one of Claims 1-4, wherein the nanoparticle has a diameter of about 17 nm.

6. The nanoparticle of any one of Claims 1-4, wherein the nanoparticle has a diameter of 16 nm to 18 nm.

7. The nanoparticle of any one of Claims 1-6, wherein the nanoparticle has a surface to volume ratio of about 0.3 to about 0.4.

8. The nanoparticle of any one of Claims 1-6, wherein the nanoparticle has a surface to volume ratio of about 0.35.

9. The nanoparticle of any one of Claims 1-6, wherein the nanoparticle has a surface to volume ratio of 0.34 to 0.36.

10. The nanoparticle of any one of Claims 1-9 wherein the cyclodextrin is crosslinked.

11. The nanoparticle of Claim 10, wherein the cyclodextrin is crosslinked with a crosslinker comprising aspartic acid, alanine, cysteine, arginine, / .-serine, diamine, ethylene glycol, polyethylene glycol, triethanolamine, diamine, Nl-(2-(4-(2-Aminoethyl)piperazin-l- yl)ethyl)ethane-l,2-diamine, ethanolamine, diethanolamine, D-lysine, or£-lysine.

12. The nanoparticle of Claim 10, wherein the cyclodextrin is crosslinked with a crosslinker comprising L-lysine.

13. The nanoparticle of Claim 1 lor 12, wherein the ratio of the cyclodextrin to crosslinker is about 1 to about 4.

14. The nanoparticle of any one of Claims 1-13, wherein the cyclodextrin is succinyl-P-cyclodextrin.

15. The nanoparticle of Claim 14, wherein the succinyl-[3-cyclodextrm has a degree of substitution of about 2 to about 3.

16. The nanoparticle of Claim 14, wherein the succinyl-P-cyclodextrin has a degree of substitution of about 2.5.

17. The nanoparticle of any one of Claims 1-13, wherein the cyclodextrin is bis- succinyl-P-cyclodextrin.

18. The nanoparticle of any one of Claims 2-17, wherein the first, second, and third payloads are different.

19. The nanoparticle of any one of Claims 2-18, wherein the first, second, and third payloads are directed to different biological targets.

20. The nanoparticle of any one of Claims 2-19, wherein the first, second, and third payloads comprise at least one agonist and at least one inhibitor.

21. The nanoparticle of any one of Claims 1-20, wherein the first payload is a cIAP inhibitor.

22. The nanoparticle of Claim 21, wherein the cIAP inhibitor is birinapant, GDC- 0152, BV-6, or LCL-161.

23. The nanoparticle of Claim 21 or 22, wherein the cIAP inhibitor is LCL-161.

24. The nanoparticle of any one of Claims 1-20, wherein the first payload is a CSF1R inhibitor.

25. The nanoparticle of Claim 24, wherein the CSF1R inhibitor is PLX3397.

26. The nanoparticle of any one of Claims 2-24, wherein the second payload is a JAK / STAT inhibitor.

27. The nanoparticle of Claim 26, wherein the JAK / STAT inhibitor is ruxolitinib, upadacitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, ritlecitinib, pacricitinib, fedratinib, filgotinib, abrocitinib, momelotinib, cerdulatinib, lestaurtinib, gandotinib, BP-1- 102, or STX-0119.

28. The nanoparticle of Claim 26 or 27, wherein the JAK / STAT inhibitor is ruxolitinib, upadacitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, ritlecitinib, pacricitinib, fedratinib. filgotinib, abrocitinib, momelotinib, cerdulatinib, lestaurtinib, or gandotinib.

29. The nanoparticle of any one of Claims 26-28, wherein the JAK / STAT inhibitor is ruxolitinib or upadacitinib.

30. The nanoparticle of any one of Claims 26-29, wherein the JAK / STAT inhibitor is ruxolitinib.

31. The nanoparticle of any one of Claims 26-29, wherein the JAK / STAT inhibitor is upadacitinib.

32. The nanoparticle of any one of Claims 2-24, wherein the second payload is a STING agonist.

33. The nanoparticle of Claim 32, wherein the STING agonist is 4a,7,8- trimethoxy-2, 3, 4a,9b-tetrahydrodibenzo{b,d}thiophene-l, 4-dione (MSA-2p).

34. The nanoparticle of any one of Claims 1-33, wherein the third payload is a TLR7 / 8 agonist.

35. The nanoparticle of Claim 34, wherein the TLR7 / 8 agonist is imiquimod, resiquimod (R848), PF-4878691, vesatolimod, AZD8848, motolimod, selgantolimod, NKTR- 262, RG-7854, DSP-0509, BDB-001, BDC-100L LHC-165, SHR-165, JNJ-2150, JNJ-4964, RO-7119929, VX-1463, BNT-411, or APR-0003.

36. The nanoparticle of Claim 34 or 35, wherein the TLR7 / 8 agonist is imiquimod, resiquimod (R848), vesatolimod, motolimod, or selgantolimod.

37. The nanoparticle of any one of Claims 34-36, wherein the TLR7 / 8 agonist is resiquimod (R848).

38. The nanoparticle of any one of Claims 2-20, wherein: a. the first payload is an inhibitor; b. the second payload is an inhibitor; and c. the third payload is an agonist.

39. The nanoparticle of any one of Claims 2-20, wherein: a. the first payload is a cIAP inhibitor; b. the second payload is a JAK / STAT inhibitor; and c. the third payload is a TLR7 / 8 agonist.

40. The nanoparticle of any one of Claims 2-20, wherein: a. the first payload is LCL-161; b. the second payload is ruxolitinib; and c. the third payload is resiquimod (R848).

41. The nanoparticle of any one of Claims 2-20, wherein: a. the first pay load is LCL-162; b. the second payload is Upalitinib; and c. the third payload is resiquimod (R848).

42. The nanoparticle of any one of Claims 2-20, wherein: a. the first payload is LCL-162; b. the second payload is BP-1-102; and c. the third payload is resiquimod (R848).

43. The nanoparticle of any one of Claims 2-20, wherein: a. the first payload is a PARP7 inhibitor; b. the second payload is a STING agonist; and c. the third payload is a TLR7 / 8 agonist.

44. The nanoparticle of any one of Claims 2-20, wherein: a. the first payload is RBN2397; b. the second payload is MSA2; and c. the third payload is resiquimod (R848).

45. The nanoparticle of any one of Claims 2-20, wherein: a. the first payload is a RBN2397; b. the second payload is MSA2; and c. the third payload is CRX527.

46. The nanoparticle of any one of Claims 2-20, wherein: a. the first payload is a PARP7 inhibitor; b. the second payload is a HIF-1 inhibitor; and c. the third payload is a TLR7 / 8 agonist.

47. The nanoparticle of any one of Claims 2-20, wherein: a. the first payload is RBN2397; b. the second payload is BAY 87-2243; and c. the third payload is resiquimod (R848).

48. The nanoparticle of any one of Claims 2-20, wherein: a. the first payload is a RBN2397; b. the second payload is MSA2; and c. the third payload is resiquimod (R848).

49. The nanoparticle of any one of Claims 2-20, wherein: a. the first payload is a RBN2397; b. the second payload is 4a,7,8-trimethoxy-2,3,4a,9b- tetrahydrodibenzo{b,d}thiophene-l, 4-dione (MSA-2p); and c. the third payload is resiquimod (R848).

50. The nanoparticle of any one of claims Claim 1, 3-25, and 34-37, wherein the nanoparticle does not comprise a second payload.

51. The nanoparticle of any one of claims Claim 1, 3-25, 34-37, and 50, wherein: a. the first payload is PLX3397; and c. the third payload is resiquhnod (R848).

52. A wafer comprising the nanoparticle of any one of claims 2-49.

53. The wafer of Claim 52. wherein the wafer has a mass of about 5-15 mg in its dry form.

54. The wafter of Claim 52, wherein the wafer has a mass of about 10 mg in its dry form.

55. The wafer of any one of Claims 52-54, wherein the wafer has a volume of about 4-6 mm3in its dry fonn.

56. The wafer of any one of Claims 52-55, wherein the wafer comprises about 0.22 mg of the first pay load, the second pay load, and the third payload.

57. The wafer of any one of Claims 52-56. wherein the wafer comprises about 0. 1 mg of the first pay load, about 0.08 mg of the second payload, and about 0.04 mg of the third payload.

58. A pharmaceutical composition comprising a nanoparticle of any one of Claims 1-51 and one or more pharmaceutically acceptable excipients.

59. A method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a nanoparticle of any one of Claims 1-51. or the pharmaceutical composition of Claim 58.

60. The method of Claim 59, wherein the cancer is adrenal cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, glioblastoma, kidney cancer, penile cancer, oral cancer, liver cancer, leukemia, melanoma, mesothelioma, lung cancer, skin cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, testicular cancer, or thyroid cancer.

61. The method of Claim 59, wherein the cancer is colorectal carcinoma, murine melanoma, adult acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, or hairy cell leukemia.

62. The method of Claim 59 wherein the cancer is a glioblastoma multiforme.