Nanoparticle Formulations of STING Agonists RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Applications serial number 62/775,596, filed December 5, 2018; 62/678,542, filed May 31, 2018; and 62/710,423, filed February 16, 2018. FIELD OF DISCLOSURE

Disclosed are nanoparticles comprising STING agonists, and compositions thereof, which activate in a host the innate immune defense system and induce expression of pattern recognition receptors. Also disclosed are methods of using the nanoparticles and compositions thereof in the treatment of a proliferative disease (e.g., cancer) or a microbial infection. BACKGROUND

Nanoparticle formulations improve the pharmaceutical properties of a drug by, for example, enhancing its circulation half-life and facilitating the drugs accumulation in a target tissue.

Nanoparticle-based formulations of immunomodulatory drugs can be used to elicit antitumor immunity; efficient and targeted delivery of immunomodulatory and immunostimulatory molecules to appropriate cells is important for the successful development of immune therapeutic drugs and vaccines. Compared to conventional approaches, nanoparticle formulations can protect the antigen/adjuvant payloads from the surrounding biological environment, thus increasing their half-life and minimizing their systemic toxicity.

Cyclic dinucleotides (CDNs) have been demonstrated to be highly effective immunomodulatory molecules capable of robust activation of interferon regulatory factor 3 (IRF3) and NF-KB. CDNs are known ligands for STING localized in the endoplasmic reticulum. Ligand binding and activation of STING result in the activation of IFN signaling cascade. Several chemically distinct classes of CDNs including 3’5-linked cyclic di-GMP and cyclic di-AMP, as well as, 2,5 linked cGAMP have been demonstrated to directly bind STING and subsequently initiate TBK1-IRF3 and NFkB signaling axis of the IFN signaling cascade. Importantly, the STING pathway of cytosolic DNA sensing has been shown to play an important mechanistic role in innate immune sensing, driving type I IFN production in cancer and in the context of immune- oncology applications, including therapeutics and diagnostics.

To date there are no FDA-approved nanoparticle formulations of STING molecules, presenting an unmet need. SUMMARY OF THE INVENTION

Disclosed are nanoparticles and compositions thereof that activate in a host the innate immune defense system and induce expression of pattern recognition receptors, as well as methods of using them for the treatment of a microbial infection or a proliferative disease (e.g., cancer).

In certain aspects, the present disclosure provides a nanoparticle, comprising:

i) a polymer shell comprising a plurality of monomers and an active agent; or ii) a core conjugated to an active agent, wherein the core is a metal; or

iii) a biomolecule conjugated to an active agent; or

iv) an active agent and a liposome, wherein the liposome further comprises a membrane; or

v) an active agent and a lipid bilayer, wherein the lipid bilayer is comprised of a plurality of bilayer units; or

vi) an active agent and a surfactant; or

vii) a nanocrystal comprising an active agent;

wherein the active agent is compound of formula (I)

Formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

Z ¹ is either S or O; each of B ¹ and B ² is independently a purinyl nucleobase or pyrimidinyl nucleobase; each of X ¹ and X ² is independently O or S;

each of Y ¹ and Y ² is independently O, S, or NR ⁵ ;

each of L ¹ and L ² is independently absent, C1-C6 alkyl or C1-C6 heteroalkyl, wherein each C1-C6 alkyl and C1-C6 heteroalkyl is optionally substituted with R ⁶ ;

each of R ¹ and R ² is independently hydrogen, halo, -CN, C1-C20 alkyl (e.g., C1-C6 alkyl), or OR ⁷ ; each of R ³ and R ⁴ is independently hydrogen, C1-C20 alkyl (e.g., C1-C6 alkyl), C1-C20 heteroalkyl (e.g., C ₁ -C ₆ heteroalkyl), cycloalkyl, heterocyclyl, OC(O)OC ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), aryl, or heteroaryl, wherein each C ₁ -C ₂₀ alkyl, C ₁ -C ₂₀ heteroalkyl, cycloalkyl, heterocyclyl, aryl, OC(O)O C ₁ -C ₂₀ alkyl (e.g., C _1-6 alkyl), and heteroaryl is optionally substituted with 1-5 R ⁸ ;

each R ⁵ is independently hydrogen or C1-C20 alkyl (e.g., C1-C6 alkyl);

R ⁶ is halo, -CN, C1-C20 alkyl (e.g., C1-C6 alkyl), OR ⁷ , oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C1-C20 alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-5 R ⁹ ;

R ⁷ is hydrogen, C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C ₁ -C ₂₀ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-5 R ⁹ ;

each R ⁸ is independently C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), O-aryl, OC(O)NR ₅ -C ₁ -C ₂₀ alkyl (e.g., C1-C6 alkyl), S(O)2NR5-aryl, NR5C(O)-aryl, N(R5)2C(O)-aryl, C(O)-aryl, C(O)- heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, OC(O)-C1-C20 alkyl (e.g., C1-C6),

OC(O)O-C1-C20 alkyl (e.g., C1-C6), wherein each C1-C20 alkyl, O-aryl, OC(O)NR5-C1- C20 alkyl, S(O)2NR5-aryl, NR5C(O)-aryl, CH2NR5C(O)-aryl, C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, OC(O)-C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ ), OC(O)O-C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ ), is optionally substituted by 1-5 R ⁹ ; and

each R ⁹ is independently C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), halo, -CN, OH, O-C ₁ -C ₂₀ alkyl, O-C ₁ - C20 heteroalkyl, O-aryl, O-heteroaryl.

In certain aspects, the present disclosure provides a nanoparticle comprising:

i) a polymer shell comprising a plurality of monomers and an active agent; or ii) a core conjugated to an active agent, wherein the core is a metal; or

iii) a biomolecule conjugated to an active agent; or iv) an active agent and a liposome, wherein the liposome further comprises a membrane; or

v) an active agent and a lipid bilayer, wherein the lipid bilayer is comprised of a plurality of bilayer units; or

vi) an active agent and a surfactant; or

vii) a nanocrystal comprising an active agent;

wherein the active agent is compound of Formula (II):

Formula (II)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

Z ² is either S or O;

each of B ³ and B ⁴ is independently a purinyl nucleobase or pyrimidinyl nucleobase;

each of X ³ and X ⁴ is independently O or S;

each of Y ³ and Y ⁴ is independently O, S, or NR ¹⁴ ;

each of L ³ and L ⁴ is independently absent, C ₁ -C ₆ alkyl or C ₁ -C ₆ heteroalkyl, wherein each alkyl and heteroalkyl is optionally substituted with R ¹⁵ ;

each of R ¹⁰ and R ¹¹ is independently hydrogen, halo, -CN, C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), or OR ¹⁶ ;

each of R ¹² and R ¹³ is independently hydrogen, C1-C20 alkyl (e.g., C1-C6 alkyl), C1-C20

heteroalkyl (e.g., C1-C6 heteroalkyl), OC(O)OC1-C20 alkyl (e.g., C1-C6 alkyl), cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each alkyl, heteroalkyl, cycloalkyl,

heterocyclyl, aryl, and heteroaryl is optionally substituted with one or more R ¹⁷ ;

R ¹⁴ is hydrogen or C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl); R ¹⁵ is halo, -CN, C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), OR ¹⁶ , oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted with one or more R ¹⁸ ;

R ¹⁶ is hydrogen, C1-C20 alkyl (e.g., C1-C6 alkyl), cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted with one or more R ¹⁸ ;

each R ¹⁷ is independently C1-C20 alkyl (e.g., C1-C6 alkyl), C1-C20 heteroalkyl, C(O)-C1-C20 alkyl, OC(O)-C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), C(O)O-C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), OC(O)O- C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), C(O)N(R ¹⁴ )-C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), N(R ¹⁴ )C(O)- C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), OC(O)N(R ¹⁴ )-C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), O-aryl, O- heteroaryl, C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, C(O)O-aryl, OC(O)-heteroaryl, C(O)O-heteroaryl, C(O)O-aryl, C(O)O-heteroaryl, C(O)N(R ¹⁴ )-aryl, C(O)N(R ¹⁴ )- heteroaryl, N(R ¹⁴ )C(O)-aryl, N(R ¹⁴ )2C(O)-aryl, or N(R ¹⁴ )C(O)-heteroaryl, S(O)2N(R ¹⁴ )- aryl, wherein each alkyl, heteroalkyl, aryl, and heteroaryl is optionally substituted by one or more R ¹⁸ ; and

each R ¹⁸ is independently C ₁ -C ₂₀ alkyl, O-C ₁ -C ₂₀ alkyl, C ₁ -C ₂₀ heteroalkyl, halo, -CN, OH, oxo, aryl, heteroaryl, O-aryl, or O-heteroaryl. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-1B are graphs showing the evaluation of percent (%) IRF induction by compound 1 and compound 11 administration compared with 2’,3’-cGAMP in wild type THP1 cells (FIG.1A) and THP1 cells in which STING has been knocked out (FIG.1B).

FIGS. 2A-2B are graphs showing the evaluation of percent (%) NF-κβ induction by compound 1 and compound 11 compared with 2’,3’-cGAMP in wild type THP1 cells (FIG. 2A) and THP1 cells in which STING has been knocked out (FIG.2B).

FIGS. 3A-3B are graphs showing the percent (%) cell death caused by compound 1 and compound 11 compared with 2’,3’-cGAMP in wild type THP1 cells (FIG.3A) and THP1 cells in which STING has been knocked out (FIG.3B).

FIG.4 is a graph depicting the percent (%) cell death caused by compound 4 in wild type THP1 cells. FIG. 5 is a graph depicting the percent (%) IRF induction by compound 4 in wild type THP1 cells.

FIGS. 6A-6B are graphs depicting the percent (%) IRF induction (FIG. 6A) and percent (%) NF-κβ induction in THP1 cells caused by compound 1 administration.

FIGS. 7A-7B are graphs indicating the level of ISG54 ISRE-luc activity (FIG. 7A) and NF-κβ-luc activity (FIG. 7B) of compound 1, compound 2, compound 3, and 2’,3’-cGAMP at varying concentrations as fold increase over DMSO in HEK293 cells.

FIG. 8 is a graph indicating the level of ISG54 ISRE-luc activity of compound 1, compound 2, compound 3, and 2’,3’-cGAMP at varying concentrations as fold increase over DMSO in HEK293 cells.

FIG. 9 is a chart showing the IRF-type I interferon activity in THP1 cells upon administration of exemplary compounds of the present disclosure.

FIG.10A, FIG.10B and FIG.10C show IRF induction by exemplary compounds.

FIG.11A, FIG.11B and FIG.11C show IRF induction by exemplary compounds.

FIGS. 12A-12B are bar graphs showing RSV infection and RSV percentage infection when RSVA2 infected A459 cells were treated with vehicle (DMSO) or Compound 1. FIG.12A shows RSV infection (RSV titer) calculated by performing a viral plaque assay when RSV infected cells were treated with vehicle (DMSO), 50 ^M, 100 ^M or 200 ^M of Compound 1. FIG. 12B shows RSV percentage infection calculated based on the viral titer shown in FIG.12A when RSV infected cells were treated with vehicle (DMSO), 50 ^M, 100 ^M or 200 ^M of Compound 1 (FIG.12B).100% infection represents RSV infection in vehicle treated cells.

FIGS. 13A-13G are graphs showing the effect on HCV RNA replication in THP-1 calls when treated with increasing concentrations of Compound 1.

FIGS.14A-14B are graphs showing the cell viability of Vero and A549 cells measured by MTT method when treated with increasing concentrations of DMSO (FIG. 14A) or Compound 1 (FIG.14B).

FIG. 15 is a bar graph showing the virus yield of Junin virus at 24 and 48 hours post infection in A549 cells when treated with Compound 1. Virus yield diminished 1 log in A549 infected cells treated with Compound 1 compared to untreated A549 infected cells, both at 24 and 48 hours post infection (h p.i.). FIG. 16 is a bar graph showing the virus yield of Dengue virus serotype 2 (DSV2) at 24 and 48 hours post infection in A549 cells when treated with Compound 1. Virus yield diminished 1 log in A549 cells infected with DSV2 compared to untreated A549 infected cells at 24 hours post infection (h p.i.). At 48 hours post infection, no significant difference was found.

FIGS. 17A-17B are bar graphs showing the percent (%) IRF induction (FIG. 17A) and percent (%) NF-κB (FIG. 17B) when THP1 dual cell are treated with different concentrations of Compound (Cmd) 1, Cmd 1A, and Cmd 1B. The THP1 dual cells carry both the secreted embryonic alkaline phosphatase (SEAP) reporter gene which is under the control of an IFN-β minimal promoter fused to five copies of the NF-κB consensus transcription response element and the Lucia reporter gene which is under the control of an ISG54 minimal promoter.

FIGS. 18A-18D are graphs showing the induction of IRF (FIGS. 18A-18B) and NF-κB (FIGS. 18C-18D) by Cmd 1. The results in FIGS. 18A-18D indicate that Cmd 1 is taken up by cells without the use of transfection agents.

FIGS.19A-19B are graphs showing the induction of IRF by Cmd 3, and indicate that Cmd 3 is taken up by cells without the use of transfection agents.

FIGS. 20A-20D are graphs showing the induction of IRF (FIGS. 20A-20B) and NF-κB (FIGS. 20C-20D) by Cmd 12, and indicate that Cmd 12 is taken up by cells without the use of transfection agents.

FIGS. 21A-21D are graphs showing the induction of IRF (FIGS. 21A-21B) and NF-κB (FIGS. 21C-21D) by Cmd 13, and indicate that Cmd 13 is taken up by cells without the use of transfection agents.

FIGS. 22A-22D are graphs showing the induction of IRF (FIGS. 22A-22B) and NF-κB (FIGS. 22C-22D) by Cmd 14, and indicate that Cmd 14 is taken up by cells without the use of transfection agents.

FIGS. 23A-23D are graphs showing the induction of IRF (FIGS. 23A-23B) and NF-κB (FIGS. 23C-23D) by Cmd 15, and indicate that Cmd 15 is taken up by cells without the use of transfection agents.

FIGS.24A-24B are bar graphs comparing the relative induction of IRF (FIG. 24A) and NF-κB (FIG. 24B) by Cmd 1, Cmd 3, Cmd 12, Cmd 13, Cmd 14, and Cmd 15 at various concentrations. FIGS. 25A-25B are graphs showing the stability of Cmd 1 in serum (FIG. 25A) and in microsomes (FIG. 25B). In FIGS. 25A-25B, Peak 1 and Peak 2 represent Cmds 1-A and 1-B, respectively.

FIGS. 26A-26B are graphs showing the stability of Cmd 15 in serum (FIG. 26A) and in microsomes (FIG. 26B). In FIGS. 26A-26B, Peak 1 and Peak 2 represent Cmds 15-A and 15-B, respectively.

FIGS. 27A-27B are bar graphs comparing the induction of IRF (FIG. 27A) and NF-κB (FIG.27B) by Cmd 15 and its isomers, Cmd 15-A and Cmd 15-B.

FIG. 28 is a bar graph showing the induction of apoptosis through % cytoxicity of THP1 cells when treated with various concentrations of Cmd 15 and its isomers, Cmd 15-A and Cmd 15- B.

FIGS.29A-29B are graphs showing the effect on type 1 IFN signaling in cells when treated with various concentrations of Cmd 1 and 2’,3’-cGAMP. As shown in FIGs.29A-29B, the binding of Cmd 1 to STING activates type 1 IFN signaling, similar to the activation of type 1 IFN signaling observed with 2’,3’-cGAMP.

FIG.30 is a bar graph showing the effect on type 1 IFN signaling in mouse macrophages when treated with various concentrations of Cmd 1 and 2’,3’-cGAMP. As shown in FIG 30, Cmd 1 is highly active in mouse macrophages in activating type 1 IFN signaling, similar to the activation of type 1 IFN signaling observed with 2’,3’-cGAMP.

FIGS. 31A-31B are graphs showing the effect on type 1 IFN signaling when Human monocytes (THP1 cells) and Mouse macrophages (RAW) are treated with various concentrations. As shown in FIGs. 31A-31B, Cmd 1, Cmd 5, Cmd 12, Cmd 13, Cmd 14, and Cmd 15 are more active in human monocytes (FIG. 31A) and mouse macrophages (FIG. 31B) than the natural STING ligand 3’,3’-cGAMP.

FIGS.32A-32B are graphs showing the induction of type I IFN signaling in HEK293 (FIG. 32A) and THP1 (FIG. 32B) cells treated with Cmd 1 and its isomers Cmd 1A (Cmd 1-PK1) and Cmd 1B (Cmd 1-PK2).

FIGS.33A-33B are bar graph showing the effects on type III interferon (IL-29) production in THP1 cells treated with various concentrations of Cmd 1 and Cmd 15. As shown in FIGS.33A- 33B, Cmd 1 and Cmd 15 induce type III interferon (IL-29) production in THP1 cells (FIG.33A), and indicating that both Cmd 1 and Cmd 15 are taken up by cells without use of a transfection reagent (FIG.33B).

FIGS.34A-34B are graphs comparing the induction of type I IFN signaling in THP1 cells treated with Cmd 1, Cmd 13, Cmd 15.

FIGS. 35A-35B are bar graphs comparing the induction of IRF (FIG. 35A) and NF-κB (FIG.35B) when THP dual cells were treated with various concentrations of DMSO, Cmd 15, or Cmd 16.

FIGS. 36A-36F show the potency comparison of an exemplary compound (Cmd 1) vs. a natural STING ligand, 2’-3’ cGAMP.

FIGS.37A-37B show that an exemplary compound has STING-dependent activity.

FIG.38 shows IRF induction by exemplary compounds.

FIGS. 39A-39B show that exemplary compounds engage with STING and activate STING-dependent type I IFN and NF-κB signaling in HEK293 cells.

FIG.40 shows NF-K ^ induction by exemplary compounds.

FIGS.41A-41E show that an exemplary compound causes cell death by apoptosis through the modulation of BAX and BCL-2 levels.

FIGS. 42A-42B show the selective induction of apoptosis by Cmd 1 in acute monocytic leukemia cell line (THP1) vs. PBMCs.

FIGS.43A-43B show that an exemplary compound (Cmd 1) causes selective and enhanced induction of ISG and PRR-associated genes in acute monocytic leukemia cell line (THP1) compared to primary cells PBMCs. Gene expression analysis was conducted in THP1 and PBMCs.

FIGS.44A-44B show that an exemplary compound inhibits tumor cell growth.

FIGS. 45A-45B show that an exemplary compound has STING-dependent IRF activity but does not cause NF-kB induction.

FIG.46 shows that an exemplary compound activates IRF signaling in THP1 cells.

FIGS.47A-47D show that an exemplary compound has similar activity as natural STING ligand 2’-3’ cGAMP.

FIG.48 shows that an exemplary compound directly binds to STING.

FIGS.49A-49B show that an exemplary compound has STING dependent IRF activity but does not cause NF-kB induction.

FIG.50 shows that an exemplary compound directly binds to STING. FIGS.51A-51B show that an exemplary compound has STING dependent activity.

FIGS.52A-52D show that an exemplary compound has similar potency as natural STING ligand 2’-3’ cGAMP.

FIGS. 53A-53B shows that an exemplary compound has enhanced activity in acute monocytic leukemia cell line (THP1) compared to primary cells PBMCs.

FIGS.54A-54C show IRF induction by an exemplary compound.

FIGS.55A-55C show IRF induction by an exemplary compound.

FIGS. 56A-56B are graphs showing the evaluation of percent (%) IRF induction (FIG. 56A) and percent (%) NF-κB (FIG.56B) by Cmd 1, Cmd 1A, and Cmd 1B in THP1 dual cells that carry both the secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of an IFN-β minimal promoter fused to five copies of the NF-κB consensus transcription response element and Lucia reporter gene under the control of an ISG54 minimal promoter.

FIGS. 57A-57D are graphs showing the induction of IRF (FIGS. 57A-57B) and NF-κB (FIGS. 57C-57D) by Cmd 1, and indicate that Cmd 1 is taken up by cells without the use of transfection agents.

FIGS.58A-58B are graphs showing the induction of IRF by Cmd 8, and indicate that Cmd 8 is taken up by cells without the use of transfection agents.

FIGS. 59A-59D are graphs showing the induction of IRF (FIGS. 59A-59B) and NF-κB (FIGS. 59C-59D) by Cmd 12, and indicate that Cmd 12 is taken up by cells without the use of transfection agents.

FIGS. 60A-60D are graphs showing the induction of IRF (FIGS. 60A-60B) and NF-κB (FIGS. 60C-60D) by Cmd 13, and indicate that Cmd13 is taken up by cells without the use of transfection agents.

FIGS. 61A-61D are graphs showing the induction of IRF (FIGS. 61A-61B) and NF-κB (FIGS. 61C-61D) by Cmd 14, and indicate that Cmd14 is taken up by cells without the use of transfection agents.

FIGS. 62A-62D are graphs showing the induction of IRF (FIGS. 62A-62B) and NF-κB (FIGS. 62A-62D) by Cmd 15, and indicate that Cmd 15 is taken up by cells without the use of transfection agents.

FIGS.63A-63B are charts comparing the relative induction of IRF (FIG.63A) and NF-κB (FIG.63B) by Cmd 1, Cmd 8, Cmd 12, Cmd 13, Cmd 14, and Cmd 15. FIGS. 64A-64B are graphs showing the stability of Cmd 1 in serum (FIG. 64A) and in microsomes (FIG. 64B). In FIG. 75B, Peak 1 and Peak 2 represent Cmds 1-A and 1-B, respectively.

FIGS. 65A-65B are graphs showing the stability of Cmd 15 in serum (FIG. 65A) and in microsomes (FIG. 65B). In FIG. 65B, Peak 1 and Peak 2 represent Cmds 1-A and 1-B, respectively.

FIGS. 66A-66B are charts comparing the induction of IRF (FIG. 66A) and NF-κB (FIG. 66B) by Cmd 1 and its isomers, Cmd 1-A and Cmd 1-B.

FIG.67 is a chart showing the induction of apoptosis through % cytoxicity of THP1 cells by Cmd 15 and its isomers, Cmd 1-A and Cmd 1-B.

FIGS.68A-68B show that Cmd 1 binds to STING to activate type 1 IFN signaling, similar to 2’,3’-cGAMP.

FIG.69 is a chart showing that Cmd 1 is highly active in mouse macrophages in activating type 1 IFN signaling, similar to 2’,3’-cGAMP.

FIGS.70A-70B are graphs that show that Cmd 1, Cmd 5, Cmd 12, Cmd 13, Cmd 14, and Cmd 15 are more active against the natural STING ligand 3’,3’-cGAMP in human monocytes (FIG.70A) and mouse macrophages (FIG.70B).

FIGS. 71A-71B are graphs that show the induction of type I IFN signaling in HEK293 (FIG.71A) and THP1 (FIG.71B) cells by Cmd 1 and its isomers Cmd 1A and Cmd 1B.

FIGS.72A-72B are charts showing that Cmd 1 and Cmd 15 induce type III interferon (IL- 29) production in THP1 cells (FIG.72A), and that both Cmd 1 and Cmd 15 are taken up by cells without use of a transfection reagent (FIG.72B).

FIGS.73A-73B are graphs comparing the induction of type I IFN signaling in THP1 cells by Cmd 1, Cmd 13, Cmd 15 as STING agonists.

FIGS. 74A-74B are charts comparing the induction of IRF (FIG. 74A) and NF-κB (FIG. 74B) by Cmd 15 and Cmd 16.

FIGS. 75A-75B show that Cmd1 is capable of activating the major STING-HAQ polymorphic variant in humans.

FIG. 76 shows that residues R238 and Y167 in STING Laboratory-generated loss-of- function STING mutants (STING-R238A and STING-Y167A) are critical for Cmd 1 as well as cGAMP activation of STING-dependent IFN response. FIG.77 shows IRF-type I IFN activity by Cmd1 in co-cultured tumor/THP1 cell system. FIGS.78A-78B show that Cmd1 inhibits tumor cell growth in tumor cells and THP1 cells using high-content image-based approach and is STING dependent.

FIGS.79A-79B show that Cmd 1 causes apoptosis acute monocytic leukemia cells. FIGS.80A-80E show that Cmd 1 induces apoptosis in the mouse lymphoma cell line A20. FIGS.81A-81B show that Cmd1 causes apoptosis of mouse melanoma cells.

FIGS.82A-82D show that Cmd1 inhibits mouse A20 B cell lymphoma tumor cells. FIG.83 shows anti-tumor activity of Cmd 1 using high-content image-based approach. FIGS.84A-84F show that the induction of cell death by Cmd 1 is STING-mediated. FIGS.85A-85C show the results of a gel shift assay indicating that Cmd 1 binds to STING. A close structural analog of Cmd 1 carrying a fluorescent substituent was synthesized for Gel Shift Assay. FIG.85A shows 250 µM of Cmd 1 analog with 20 µM to 0 µM of STING. FIG.85B shows 10 µM of STING with 1 mM to 0 mM of Cmd 1 analog. FIG.85C shows an immunoblot to detect STING.

FIGS.86A-86P show analysis of IRF3 & NF-kB pathways after Cmd 1 treatment: FIGS. 86A-86P show immunoblots in which THP-1 cells were treated with 5 µM Cmd 1 or 2’-3’ cGAMP.

FIGS. 87A-87C show images in which THP-1 derived macrophages were treated with Cmd 1 or DMSO control for 2 hrs (FIG.87A), 4 hrs (FIG.87B), or 6 hrs (FIG.87C) and analyzed for nuclear translocation. Cells were imaged on IXM (Molecular Devices) (40x) and were analyzed using ImageJ.

FIGS. 88A-88B show the evaluation of IFN secretion and gene expression after Cmd 1 treatment. FIG. 88A is a graph showing the fold induction of gene expression in THP-1 cells treated with 5 uM of either Cmd 1 or 2’-3’-cGAMP. Gene expression was evaluated by Taqman Assays. Fold Induction was calculated by ΔΔct method. In FIG. 88B, THP-1 cells were treated with 1 uM of Cmd 1 and secretion of certain cytokines was evaluated by on Quansys Biosciences’ (Logan, UT) Q-Plex ^TM Human Custom, IFN, and IL-1 Family multiplexed ELISA arrays.

FIG. 88C-88D show the induction of apoptosis-related genes and ISGs by an exemplary compound (Cmd 1) compared with 2’3’-cGAMP in A20 mouse B cell lymphoma tumor cells. In FIG. 88D, a higher BAX/BCL2 ratio in cells administered Cmd 1 promotes apoptosis via upregulation of caspase 3. FIGS.89A-89G are graphs showing the induction of various cytokines by Cmd 1 in wild type THP1 cells as determined by multiplex ELISA.

FIGS. 90A-90D are graphs showing that an exemplary compound (Cmd 1) strongly activates the IRF-type I and type III IFN response.

FIG.91 is a chart showing that an exemplary compound (Cmd 1) activates human natural killer (NK) cells and induces IFN-γ production.

FIGS. 92A-92B show that an exemplary compound (Cmd 1) potently inhibits lymphoma tumor growth in the syngeneic A20 lymphoma model.

FIGS. 93A-93D are graphs showing that an exemplary compound (Cmd 1) administered in combination with cyclophosphamide results in tumor-free survival in a syngeneic A20 lymphoma mouse model.

FIGS.94A-94B show that monotherapy of Cmd 1 and combination therapy of Cmd 1 plus cyclophosphamide significantly improve the survival rate of mice in the syngeneic A20 lymphoma mouse model. Note that in FIG.94B, VS1 refers to Cmd 1.

FIGS. 95A-95D are images showing immunohistochemistry data on tissues taken from mice treated with Cmd 1. The images show that the anti-tumor activity of Cmd 1 correlates with the induction of the innate and adaptive immune response.

FIGS. 96A-96B show that an exemplary compound (Cmd 1) is highly effective in inhibiting tumor growth in the syngeneic CT26 colon cancer model.

FIGS.97A-97B show that monotherapy of Cmd 1 and combination therapy of Cmd 1 plus an anti-CTLA4 antibody significantly improve the survival rate of mice in the syngeneic CT26 colon cancer mouse model. Note that in FIG.97B, VS1 refers to Cmd 1.

FIG. 98 shows that mice that are found to be tumor-free following treatment with either Cmd 1 or Cmd1+cyclocphosphamide experience no tumor growth compared with control upon re- challenging the mice with tumor cells (A20 lymphoma tumor challenge study).

FIGS. 99A-99H are images showing immunohistochemistry data using an anti-CD38 antibody on tumor tissue collected from mice treated with vehicle (FIGS. 99A-99D) or Cmd 1 (FIGS. 99E-99H) in the syngeneic A20 lymphoma model. The images show that Cmd 1 induces migration of CD8 T into the tumor site.

FIGS. 100A-100H are images showing immunohistochemistry data using an anti- granzyme B antibody on tumor tissue collected from mice treated with vehicle (FIGS.100A-100D) or Cmd 1 (FIGS.100E-100H) in the syngeneic A20 lymphoma model. The images show that Cmd 1 induces migration of NK cells into the tumor site.

FIGS. 101A-101H are images showing immunohistochemistry data using an anti-F4/80 antibody on tumor tissue collected from mice treated with vehicle (FIGS.101A-101D) or Cmd 1 (FIGS.101E-101H) in the syngeneic A20 lymphoma model. The images show that Cmd 1 induces migration of macrophages into the tumor site.

FIGS.102A-102G show administration of Cmd 1 to a panel of normal cell lines, indicating that Cmd 1 is non-cytotoxic.

FIGS. 103A-103D show that palmitoylation of STING is involved in Cmd 1-induced activation of NF-κB (FIGS. 103A-103B) and the IRF-type I interferon response in THP1 cells (FIGS.103C-103D).

FIG.104 is a graph showing that intraperitoneal administration of Cmd 1 causes significant decline in tumor volume in the syngeneic mouse metastatic breast cancer model as described in Example 17.

FIG. 105 is a graph showing the results of the oral dosage study, indicating that all participating subjects are within acceptable body weight ranges.

FIG. 106 is a graph showing the anti-tumor activity of Cmd 1, Cmd 1A, and Cmd 21 in the syngeneic mouse A20 lymphoma model. All compounds showed considerable tumor growth inhibition compared with the vehicle.

FIGS. 107A-107B are graphs showing the abscopal antitumoral activity of Cmd 1 when administered intratumorally in CT26 colon cancer model. FIG. 107A shows tumor volume of tumor in left flank and FIG.107B shows tumor volume of tumor on right flank over 13 days post initiation treatment. Cmd 1 showed considerable tumor growth inhibition compared with the vehicle.

FIG. 108 is a graph showing effects on tumor growth at a dose of vehicle and 10 ^g, 30 ^g, and 100 ^g of Cmd 1 in a CT26 colon cancer model. Cmd 1 showed considerable tumor growth inhibition at all three doses compared with the vehicle.

FIG.109 is a graph showing effects on tumor growth of Cmd 1 and vehicle in a 4T1 breast cancer model. Cmd 1 showed considerable tumor growth inhibition compared with the vehicle. FIGS. 110A-110D are bar graphs percent induction of CD8+ T cells, CD4+ T cells, and MDSCs by Cmd 1 in spleen, lymph nodes and blood on day 19 measured by flow cytometry. Cmd 1 showed increase in CD8+ T cells, CD4+ T cells, and MDSCs when compared with the vehicle.

FIG. 111 is a graph showing anti-tumor activity of vehicle Cmd 1, Cmd 1A (isomer of Cmd 1), and Cmd 21. The graph shows that Cmd 1, Cmd 1A and Cmd 21 inhibit mouse A20 B cell lymphoma tumor cells.

FIGS.112 is a Kaplan-Meier plot showing that Cmd 1, Cmd 1A, and Cmd 21 significantly improve the survival rate of mice in the A26 lymphoma model.

FIG. 113 is a graph showing effects on tumor growth of intratumoral administration of Cmd 1, Cmd 21, and Cmd 25 in a CT26 colorectal carcinoma model. Cmd 1, Cmd 21, and Cmd 25 showed considerable tumor growth inhibition compared with the vehicle.

FIG. 114 is a graph showing effects on tumor growth of intratumoral administration of vehicle, vehicle and Ethanol. Cmd 1, Cmd 1A, and Cmd 1B in a CT26 colorectal carcinoma model. Cmd 1, Cmd 1A, and Cmd 1B showed considerable tumor growth inhibition compared with the vehicle.

FIG.115A-115B is a graph showing the stability of Cmd 1 in Rabbit serum (FIG. 116A) and in Human microsomes (FIG.115B).

FIG. 116 are luminescence images showing effects on tumor growth of intraperitoneal administration of Cmd 1 in a 4T1 breast cancer syngeneic mouse model. Cmd 1 showed considerable tumor growth inhibition at all three doses compared with the vehicle.

FIG. 117 depicts exemplary design principles for nanoparticle-based, tumor-cytotoxic vehicles. Different types of nanoparticles can serve as vehicles for targeted delivery of tumor- cytotoxic drugs. Some of the key parameters and distinguishing features are size, surface chemistry, structural stability, circulating half-life, vascular permeability, extravasation, and retention into the tumor due to impaired lymphatic drainage, tumor cell binding, and internalization via receptor- or transporter-mediated interactions, and intracellular delivery of payload in response to intracellular stimuli.

Figure 118 depicts SZ14 cells treated with either compound 2* or compound 4* and digitonin for 5.5 hours. ISG54 ISRE-luciferase activity was determined and normalized to DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant). Figure 119 depicts SZ14 cells treated with compound 3* and digitonin for 6 hours. ISG54 ISRE-luciferase activity was determined and normalized to DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant).

Figure 120A depicts NK-92 cells were stimulated with compound 4* alone in the absence of IL-2 for 23 hours. Levels of IFN-γ in culture supernatants were quantified using ELISA, and results were shown as pg/mL. Cells were also treated with control DMSO as well as cultured with medium in presence/absence of IL-2

Figure 120B depicts that the growth of NK-92 cells is IL-2 dependent. The presence of IL-2 is able to induce the production of IFN-γ.

Figure 121 depicts RAW-Lucia-ISG-WT and RAW-Lucia-ISG-STING KO cells were stimulated with compound 2* or compound 4*, alone for 19 hours. Activity of secreted luciferase (IRF-type I IFN activity) in cell culture supernatant was measured using Invivogen’s Quanti-luc. Data is shown as fold induction over DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant). EC50 values were calculated using XLfit. Neither compound induced IRF activity in STING KO cells.

Figure 122 depicts human endothelial cells isolated from lung microvascular, liver sinusoidal microvascular, and large intestine microvascular were plated into 96-well plate and stimulated with compound 4* alone. Cell culture supernatants were collected at 6 and 23 hours post-treatment. Levels of RANTES in culture supernatants were quantified using ELISA and results were shown as pg/mL. Concentrations (1.25, 2.5, 5, and 10 micromolar) increase from left to right within each of the six bar graphs.

Figure 123 depicts THP1-Dual-KI-STING-R232 cells stimulated with compound 4* alone for 20 hours. Activities of secreted luciferase (IRF-type I IFN activity) (top panels) and NF-ĸB (bottom panels) in cell culture supernatant were measured using Invivogen’s Quanti-luc and Quanti-blue, respectively. Data are shown as fold induction over DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant). EC ₅₀ values were calculated using XLfit. THP1-Dual-KI-STING-R232 reporter cell line was generated from THP-Dual-STING KO cells by inserting STING-R232 (STING-WT) to rescue the STING signaling pathway. Compound did not induce IRF activity in STING KO cells.

Figure 124 depicts THP1-Dual-WT cells stimulated with compound 4* alone for 20 hrs. Activities of secreted luciferase (IRF-type I IFN activity) (top panels) and NF-ĸB (bottom panels) in cell culture supernatant were measured using Invivogen’s Quanti-luc and Quanti-blue, respectively. Data are shown as fold induction over DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant). EC ₅₀ values were calculated using XLfit. THP1-Dual- WT cells express STING-HAQ variant. Compound did not induce IRF activity in STING KO cells.

Figure 125A depicts THP1-Dual-WT cell stimulated in triplicate with compound 4* alone for 20 hours. Activities of IRF-driven secreted luciferase in cell culture supernatant were measured using Invivogen’s Quanti-luc and Quanti-blue, respectively. Data are shown as fold increase over DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant).

Figure 125B depicts THP1-Dual-WT cell stimulated in triplicate with compound 4* alone for 20 hours. Activities of NF-κB in cell culture supernatant were measured using Invivogen’s Quanti-luc and Quanti-blue, respectively. Data are shown as fold increase over DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant).

Figure 125C depicts THP1-Dual-WT cell stimulated in triplicate with compound 4* alone for 20 hours. Levels of RANTES in culture supernatants were quantified using ELISA and results were shown as pg/mL.

Figure 125D depicts THP1-Dual-WT cell stimulated in triplicate with compound 4* alone for 20 hours. Levels of IL-29 in culture supernatants were quantified using ELISA and results were shown as pg/mL.

Figure 126A THP1-Dual (WT) cells were stimulated with compound 3* alone (top panel) or compound/lipo mixture (bottom panel) for 20 hours. Activities of secreted luciferase (IRF-type I IFN activity) in cell culture supernatant were measured using Invivogen’s Quanti-luc and Quanti- blue, respectively. Data are shown as fold induction over DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant). EC ₅₀ values were calculated using XLfit.

Figure 126B depicts THP1-Dual (WT) cells were stimulated with compound 3* alone (top panel) or compound/lipo mixture (bottom panel) for 20 hours. Activities of sNF-ĸB in cell culture supernatant were measured using Invivogen’s Quanti-luc and Quanti-blue, respectively. Data are shown as fold induction over DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant). EC50 values were calculated using XLfit.

Figure 127 depicts THP1 dual & STING KO THP1 dual cells grown in complete media were treated with various concentrations of compound 2* or DMSO control with Lipofectamine LTX. Dual cells carry both secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of an IFN-b minimal promoter fused to five copies of the NF-kB consensus transcriptional response element to measure NF-kB activity and Lucia reporter gene under the control of an ISG54 minimal promoter to measure IRF activity. After 20 hours incubation, IRF activity was assessed using QUANTI-luc to measure levels of Lucia and NF-kB activity was determined by measure SEAP levels at 620-655 nm. % induction was calculated from fold change in luminescence/absorbance compared to DMSO treated sample.

Figure 128 depicts SZ14 reporter cells (a HEK293-derived type I IFN inducible reporter cell line) that were treated with compound and digitonin. ISG54 ISRE-luciferase activity was determined and normalized to DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant).

Figure 129 depicts THP1-Dual-WT cells in a 96-well plate that were stimulated in triplicate with 8 concentrations of compound 4* for 20 hours. Activities of IRF-driven secreted luciferase and NF-κB-driven SEAP in cell culture supernatant were measured using Invivogen’s Quanti-luc and Quanti-blue, respectively. Data is shown as fold increase over DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant). Compound 4* did not induce IRF activity in THP1-STING KO cells.

Figure 130 depicts the treatment of groups of 5 Balb/C mice (female, 8 weeks of age) which were intravenously injected via tail vein with saline control or compound 4* at 9 mg/kg. Serum, spleen, and liver samples were collected at 2, 4, and 24 hours post-treatment. Levels of RANTES (A, C, E) and TNF-α (B, D, F) were measured using ELISA. Results are displayed as pg/mL for serum samples and pg/g of tissue for spleen and liver samples.

Figure 131 depicts the treatment groups of 5 Balb/C mice (female, 10 weeks of age) which were intravenously injected via tail vein with saline control or compound 3 at 9 mg/kg. Serum, spleen, and liver samples were collected at 2 and 24 hrs post-treatment. Levels of RANTES and TNF-α were measured using ELISA and results are shown as pg/mL for serum samples and pg/g of tissue for spleen and liver samples.

Figure 132 depicts the response summary in the study to determine the efficacy of compound 4* in a CT26 murine colon carcinoma model using female balb/c mice. Mice in the treatment groups took at least 10 days longer to reach the endpoint when compared to the vehicle group. Figure 133 depicts tumor growth inhibition in the study to determine the efficacy of compound 4* in a CT26 murine colon carcinoma model using female balb/c mice. Mice in the treatment groups displayed at least 89% increase tumor growth inhibition when compared to the vehicle group.

Figure 134 depicts the individual times to endpoint for mice in the study to determine the efficacy of compound 4* in a CT26 murine colon carcinoma model using female balb/c mice. Mice in the treatment groups took longer to reach the endpoint when compared to the vehicle group.

Figure 135 depicts the tumor volume distribution on day 18 in the study to determine the efficacy of compound 4* in a CT26 murine colon carcinoma model using female balb/c mice. Mice in the treatment groups possessed significantly smaller tumor volumes when compared to the vehicle group.

Figure 136 depicts the Kaplan-Meier plot for the study to determine of the study to determine the efficacy of compound 4* in a CT26 murine colon carcinoma model using female balb/c mice. The treatment groups had a greater percent of mice remaining at day 21 than the vehicle group.

Figure 137 depicts compound 4* administered intravenously at 1 mg/kg and 3 mg/kg to mice in a CT26 colon cancer model. Tumor growth was slowed in the compound 4 group(s) compared to the vehicle.

Figure 138 depicts cells stably expressing both or either one of the reporters to measure IRF and NF-kB activity were treated with a range of concentrations of compound 1* or DMSO control for 20 hours. IRF activity was assessed using QUANTI-luc to measure levels of Lucia and NF-kB activity was determined by measure SEAP levels at 620-655 nm. % induction was calculated from fold change in luminescence/absorbance compared to DMSO treated sample. EC ₅₀ values are generated by curve fit in Xlfit. Compound 1 did not induce IRF activity or NF-κβ in STING KO cells. Compound 1* possess STING dependent activity.

Figure 139 depicts cells stably expressing both or either one of the reporters to measure IRF and NF-kB activity were treated with a range of concentrations of compound 2* or DMSO control for 20 hours. IRF activity was assessed using QUANTI-luc to measure levels of Lucia and NF-kB activity was determined by measure SEAP levels at 620-655 nm. % induction was calculated from fold change in luminescence/absorbance compared to DMSO treated sample. EC ₅₀ values are generated by curve fit in Xlfit. Compound 2* did not induce IRF activity or NF-κβ in STING KO cells. Compound 2* possess activity against wild type STING, R71H-G230A-R293Q (HAQ) variants of hSTING and wt-mSTING.

Figure 140 depicts cryopreserved mouse bone marrow derived dendritic cells (DCs) and macrophages treated with a range of concentrations of compound 2* or DMSO control for 20 hours. Cell pellets were harvested to collect total RNA. The gene expression levels of ISGs were measured by Taqman Assays. Compound 2* induces ISG expression in mouse bone marrow derived DCs and macrophages.

Figure 141 depicts cells stably expressing both or either one of the reporters to measure IRF and NF-kB activity were treated with a range of concentrations of compound 3* or DMSO control for 20 hours. IRF activity was assessed using QUANTI-luc to measure levels of Lucia and NF-kB activity was determined by measure SEAP levels at 620-655 nm. % induction was calculated from fold change in luminescence/absorbance compared to DMSO treated sample. EC50 values are generated by curve fit in Xlfit. Compound 3* did not induce IRF activity or NF-κβ in STING KO cells. Compound 3* possess activity against wild type STING, STING-HAQ and wt- mSTING.

Figure 142 depicts freshly isolated peripheral blood mononuclear cells (PBMCs) treated with a range of concentrations of compound 3* or DMSO control for 20 hours. Supernatants were collected to measure IFNb and TNFa secretion by Verikine-Human IFN beta Serum ELISA kit and Human TNF Alpha ELISA kit respectively. The amount of cytokine releases into supernatant was calculated by a standard curve. Compound 3* induces IFNβ and TNFα secretion in PBMCs after treatment.

Figure 143 depicts compound 3* administered intravenously at 3 mg/kg to mice in a CT26 colon cancer model. Tumor growth was slowed in the compound 3* group when compared to the vehicle.

Figure 144 depicts cells stably expressing both or either one of the reporters to measure IRF and NF-kB activity were treated with a range of concentrations of compound 4* or DMSO control for 20 hours. IRF activity was assessed using QUANTI-luc to measure levels of Lucia and NF-kB activity was determined by measure SEAP levels at 620-655 nm. % induction was calculated from fold change in luminescence/absorbance compared to DMSO treated sample. EC50 values are generated by curve fit in Xlfit. Compound 4* did not induce IRF activity or NF-κβ in STING KO cells. Compound 4* possess activity against wild type STING, R71H-G230A-R293Q (HAQ), R232H variants of hSTING, MYD88 knock out cells and wt-mSTING.

Figure 145 depicts cells stably expressing both or either one of the reporters to measure IRF and NF-kB activity were treated with a range of concentrations of compound 4* or DMSO control for 20 hours. IRF activity was assessed using QUANTI-luc to measure levels of Lucia and NF-kB activity was determined by measure SEAP levels at 620-655 nm. % induction was calculated from fold change in luminescence/absorbance compared to DMSO treated sample. EC50 values are generated by curve fit in Xlfit. RIG-I is not implicated in the mechanism of action of compound 4*.

Figure 146 depicts freshly isolated PBMCs that were treated with a range of concentrations of compound 4* or DMSO control for 20 hours. Supernatants were collected to measure IFNb and TNFa secretion by Verikine-Human IFN beta Serum ELISA kit and Human TNF Alpha ELISA kit respectively. The amount of cytokine releases into supernatant was calculated by a standard curve. Compound 4* induces IFNβ and TNFα secretion in PBMCs after treatment.

Figure 147 depicts SEM images of nanoparticles containing STING agonists (Batch - SF18000484) at varying magnification levels. Image properties: magnification: 500x, HV: 2.00k V, WD: 9.6mm, spot: 2.5, mode: SE, HFW: 298 µm; magnification: 5,000x, HV: 2.00 kV, WD: 10.0 mm, spot: 2.5, mode: SE, HFW: 29.8 µm; magnification: 20,000x, HV: 2.00 kV, WD: 9.7 mm, spot: 2.5, mode: SE, HFW: 7.46 µm; and magnification: 60,000x, HV: 2.00 kV, WD: 9.7 mm, spot: 2.5, mode: SE, HFW: 2.49 µm.

Figure 148 depicts SEM images of nanoparticles containing STING agonists (Batch - SF18000578) at varying magnification levels. Image properties: magnification: 500x, HV: 10.00 kV, WD: 10.0 mm, spot: 2.5, mode: SE, HFW: 298 µm; magnification: 20,000x, HV: 200kV, WD: 9.9 mm, spot: 2.5, mode: SE, HFW: 7.46 µm; magnification: 60,000x, HV: 200kV, WD: 9.9 mm, spot: 2.5, mode: SE, HFW: 2.49 µm; and magnification: 100,000x, HV: 10.00 kV, WD: 9.9 mm, spot: 2.5, mode: SE, HFW: 1.49 µm.

Figure 149 depicts SEM images of nanoparticles containing STING agonists (Batch - SF18000578) at varying magnification levels. Image properties: magnification: 20,000x, HV: 200kV, WD: 10.0 mm, spot: 2.5, mode: SE, HFW: 7.46 µm; and magnification: 60,000x, HV: 15.00 kV, WD: 10.1 mm, spot: 2.5, mode: SE, HFW: 2.49 µm. Figure 150 depicts SEM images of nanoparticles containing STING agonists (Batch - SF18000759) at varying magnification levels. Image properties: magnification: 60,000x, HV: 10.00 kV, WD: 10.0 mm, spot: 2.5, mode: SE, HFW: 2.49 µm; and magnification: 100,000x, HV: 10.00 kV, WD: 9.9 mm, spot: 2.5, mode: SE, HFW: 1.49 µm.

Figure 151 depicts SEM images of nanoparticles containing STING agonists (Batch - SF18000759) at varying magnification levels. Image properties: magnification: 60,000x, HV: 10.00 kV, WD: 10.0 mm, spot: 2.5, mode: SE, HFW: 2.49 µm; and magnification: 100,000x, HV: 10.00 kV, WD: 9.9 mm, spot: 2.5, mode: SE, HFW: 1.49 µm.

Figure 151 depicts SEM images of nanoparticles containing STING agonists. Image properties: magnification: 2000x, HV: 10.00 kV, WD: 10.0 mm, spot: 2.5, mode: SE, HFW: 74.6 µm; and magnification: 2000x, HV: 10.00 kV, WD: 9.9 mm, spot: 2.5, mode: SE, HFW: 74.6 µm.

Figure 152A depicts the % IRF induction in THF-1 cells treated with certain nanoparticle formulations (e.g., NP1, NP2, and NP3).

Figure 152B depicts the % IRF induction in THF-1 cells treated with certain nanoparticle formulations (e.g., NP1, NP2, and NP3).

Figure 152C depicts the % IRF induction in RAW cells treated with certain nanoparticle formulations (NP1, NP2, and NP3).

Figure 152D depicts the % IRF induction in RAW cells treated with certain nanoparticle formulations (NP1, NP2, and NP3).

Figure 153A depicts the measurement of maturation of dendritic cells (mDCs) after treating immature dendritic cells (iDCs) with nanoparticles and Cmd 1 alone. CD14+ monocytes were isolated from PBMCs by negative selection by EasySep™ Human Monocyte Isolation Kit (Stemcell Technologies). The isolated CD14+ monocytes were differentiated into immature dendritic cells by adding 50 ng/mL human GM-CSF (Biolegend) and 100 ng/mL human IL-4 (Biolegend) for 6 days followed by treatment with different concentration of nanoparticles (NP1) or compound alone or LPS and 2’-3’ cGAMP as positive controls. 20 hrs after treatment, cells were harvested and stained with surface antibodies CD209-APC and CD83-FITC (Biolegend) followed by flow cytometric analysis. % cells were shown above from each treatment.

Figure 153B depicts the measurement of maturation of dendritic cells (mDCs) after treating immature dendritic cells (iDCs) with nanoparticles and Cmd 1 alone. CD14+ monocytes were isolated from PBMCs by negative selection by EasySep™ Human Monocyte Isolation Kit (Stemcell Technologies). The isolated CD14+ monocytes were differentiated into immature dendritic cells by adding 50ng/mL human GM-CSF ( Biolegend) and 100ng/mL human IL-4 (Biolegend) for 6 days followed by treatment with different concentration of nanoparticles (NP1) or compound alone or LPS and 2’-3’ cGAMP as positive controls.20hrs after treatment, cells were harvested and stained with surface antibodies CD209-APC and CD86-PE (Biolegend) followed by flow cytometric analysis. % cells were shown above from each treatment.

Figure 154A depicts the measurement of cytokine release by Peripheral Blood Mononuclear cells (PBMCs) after treatment with nanoparticles and Cmd 1 alone: 200,000 PBMCs seeded per well in 96 well plate were induced with different concentrations of either nanoparticles (NP3) or Cmd 1 alone for 20 hrs. The secretion of RANTES into cell culture media was measured by ELISA. A RANTES human cytokine ELISA Kit from R & D Systems were used in these expriments. Representative data from one PBMCs donor is shown above.

Figure 154B depicts the measurement of cytokine release by Peripheral Blood Mononuclear cells (PBMCs) after treatment with nanoparticles and Cmd 1 alone: 200,000 PBMCs seeded per well in 96 well plate were induced with different concentrations of either nanoparticles (NP3) or Cmd 1 alone for 20hrs. The secretion of TNF-α into cell culture media was measured by ELISA. A TNF-α human cytokine ELISA Kit from R & D Systems was used in these expriments. Representative data from one PBMCs donor is shown above.

Figure 154C depicts the measurement of cytokine release by Peripheral Blood Mononuclear cells (PBMCs) after treatment with nanoparticles and Cmd 1 alone: 200,000 PBMCs seeded per well in 96 well plate were induced with different concentrations of either nanoparticles (NP3) or Cmd 1 alone for 20hrs. The secretion of IFN-β into cell culture media was measured by ELISA. A IFN-β VeriKine-HS Human Interferon Beta Serum ELISA Kit (PBL Assay Science) was used in these expriments. Representative data from one PBMCs donor is shown above. DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

"About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.

As used herein, the term“acquire” or“acquiring” as the terms are used herein, refer to obtaining possession of a physical entity (e.g., a sample, e.g., blood sample or liver biopsy specimen), or a value, e.g., a numerical value, by“directly acquiring” or“indirectly acquiring” the physical entity or value.“Directly acquiring” means performing a process (e.g., an analytical method) to obtain the physical entity or value. “Indirectly acquiring” refers to receiving the physical entity or value from another party or source (e.g., a third party laboratory that directly acquired the physical entity or value). Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process which includes a physical change in a substance, e.g., a sample, performing an analytical method, e.g., a method as described herein, e.g., by sample analysis of bodily fluid, such as blood by, e.g., mass spectroscopy, e.g., LC-MS.

As used herein, the terms“induce” or“induction of” refer to the increase or enhancement of a function, e.g., the increase or enhancement of the expression of a pattern recognition receptor (e.g, STING). In some embodiments,“induction of PRR expression” refers to induction of transcription of PRR RNA, e.g., STING RNA (e.g., mRNA, e.g., an increase or enhancement of), or the translation of a PRR protein, e.g., the STING protein (e.g., an increase or enhancement of). In some embodiments, induction of PRR expression (e.g., STING expression) refers to the increase or enhancement of the concentration of a PRR RNA, e.g., or STING RNA (e.g., mRNA) or the STING protein, e.g., in a cell. In some embodiments, induction of PRR expression (e.g., STING expression) refers to the increase of the number of copies of PRR RNA, e.g., STING RNA (e.g., mRNA) or PRR protein, e.g., the STING protein, e.g., in a cell. In some embodiments, to induce expression of a PRR (e.g., STING) may refer to the initiation of PRR RNA (e.g., STING RNA (e.g., mRNA)) or transcription or PRR protein (e.g., STING protein) translation. In some embodiments, to induce expression of a PRR (e.g., STING) may refer to an increase in the rate of PRR RNA (e.g., STING RNA (e.g., mRNA)) transcription or an increase in the rate of PRR protein (e.g., STING protein) expression.

As used herein, the terms“activate” or“activation” refer to the stimulation or triggering of a function, e.g., of a downstream pathway, e.g., a downstream signaling pathway. In some embodiments, activation of a pattern recognition receptor (PRR) (e.g., STING) refers to the stimulation of a specific protein or pathway, e.g., through interaction with a downstream signaling partner (e.g., IFN-β promoter stimulator 1 (IPS-1), IRF3, IRF7, NF-κB, interferons (e.g., IFN-α or IFN-β) and/or cytokines). In some embodiments, activation is distinct from the induction of expression of a PRR. In some embodiments, a PRR may be activated without resulting in an induction of PRR expression (e.g., expression of STING). In some embodiments, activation may include induction of expression of a PRR (e.g., STING). In some embodiments, activation of a PRR may trigger the induction of expression of a PRR (e.g., STING) by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more compared to a reference standard (e.g., basal expression levels of a PRR (e.g., STING)).

As used herein, an amount of a compound, conjugate, nanoparticle, or substance effective to treat a disorder (e.g., a disorder described herein),“therapeutically effective amount,”“effective amount” or“effective course” refers to an amount of the compound, substance, nanoparticle, or composition which is effective, upon single or multiple dose administration(s) to a subject, in treating a subject, or in curing, alleviating, relieving or improving a subject with a disorder (e.g., a microbial infection) beyond that expected in the absence of such treatment.

As used herein, the terms“prevent” or“preventing” as used in the context of a disorder or disease, refer to administration of an agent to a subject, e.g., the administration of a nanoparticle of the present disclosure to a subject, such that the onset of at least one symptom of the disorder or disease is delayed as compared to what would be seen in the absence of administration of said agent.

As used herein, the terms“reference treatment” or“reference standard” refer to a standardized level or standardized treatment that is used as basis for comparison. In some embodiments, the reference standard or reference treatment is an accepted, well known, or well characterized standard or treatment in the art. In some embodiments, the reference standard describes an outcome of a method described herein. In some embodiments, the reference standard describes a level of a marker (e.g., a level of induction of a PRR, e.g., STING) in a subject or a sample, e.g., prior to initiation of treatment, e.g., with a nanoparticle or composition described herein. In some embodiments, the reference standard describes a measure of the presence of, progression of, or severity of a disease or the symptoms thereof, e.g., prior to initiation of treatment, e.g., with a nanoparticle or composition described herein. As used herein, the term“subject” is intended to include human and non-human animals. Exemplary human subjects include a human patient having a disorder, e.g., a disorder described herein, or a normal subject. The term“non-human animals” includes all vertebrates, e.g., non- mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, domesticated and/or agriculturally useful animals, e.g., sheep, dogs, cats, cows, pigs. In exemplary embodiments of the disclosure, the subject is a woodchuck (e.g., an Eastern woodchuck (Marmota monax)).

As used herein, the terms“treat” or“treating” a subject having a disorder or disease refer to subjecting the subject to a regimen, e.g., the administration of a nanoparticle of the disclosure, or a composition thereof, such that at least one symptom of the disorder or disease is cured, healed, alleviated, relieved, altered, remedied, ameliorated, or improved. Treating includes administering an amount effective to alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder or disease, or the symptoms of the disorder or disease. The treatment may inhibit deterioration or worsening of a symptom of a disorder or disease.

As used herein, the term“Cmd” refers to the word“compound” or“Compound”, and all of the terms are used interchangeably.

Numerous ranges, e.g., ranges for the amount of a drug administered per day, are provided herein. In some embodiments, the range includes both endpoints. In other embodiments, the range excludes one or both endpoints. By way of example, the range can exclude the lower endpoint. Thus, in such an embodiment, a range of 250 to 400 mg/day, excluding the lower endpoint, would cover an amount greater than 250 that is less than or equal to 400 mg/day.

The term“alkyl,” as used herein, refers to a monovalent saturated, straight- or branched-chain hydrocarbon such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C ₁ -C ₁₂ alkyl, C ₁ -C ₁₀ alkyl, and C ₁ -C ₆ alkyl, respectively. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, and the like.

The terms“alkenyl” and“alkynyl” are art-recognized and refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond, respectively. Exemplary alkenyl groups include, but are not limited to, -CH=CH2 and -CH2CH=CH2.

The term“alkylene” refers to the diradical of an alkyl group. The terms“alkenylene” and“alkynylene” refer to the diradicals of an alkenyl and an alkynyl group, respectively.

The term“methylene unit” refers to a divalent -CH ₂ - group present in an alkyl, alkenyl, alkynyl, alkylene, alkenylene, or alkynylene moiety.

The term“carbocyclic ring system”, as used herein, means a monocyclic, or fused, spiro- fused, and/or bridged bicyclic or polycyclic hydrocarbon ring system, wherein each ring is either completely saturated or contains one or more units of unsaturation, but where no ring is aromatic.

The term“carbocyclyl” refers to a radical of a carbocyclic ring system. Representative carbocyclyl groups include cycloalkyl groups (e.g., cyclopentyl, cyclobutyl, cyclopentyl, cyclohexyl and the like), and cycloalkenyl groups (e.g., cyclopentenyl, cyclohexenyl, cyclopentadienyl, and the like).

The term“aromatic ring system” is art-recognized and refers to a monocyclic, bicyclic or polycyclic hydrocarbon ring system, wherein at least one ring is aromatic.

The term“aryl” refers to a radical of an aromatic ring system. Representative aryl groups include fully aromatic ring systems, such as phenyl, naphthyl, and anthracenyl, and ring systems where an aromatic carbon ring is fused to one or more non-aromatic carbon rings, such as indanyl, phthalimidyl, naphthimidyl, or tetrahydronaphthyl, and the like.

The term“heteroalkyl” refers to an“alkyl” moiety wherein at least one of the carbone molecules has been replaced with a heteroatom such as O, S, or N.

The term“heteroaromatic ring system” is art-recognized and refers to monocyclic, bicyclic or polycyclic ring system wherein at least one ring is both aromatic and comprises a heteroatom; and wherein no other rings are heterocyclyl (as defined below). In certain instances, a ring which is aromatic and comprises a heteroatom contains 1, 2, 3, or 4 independently selected ring heteroatoms in such ring.

The term“heteroaryl” refers to a radical of a heteroaromatic ring system. Representative heteroaryl groups include ring systems where (i) each ring comprises a heteroatom and is aromatic, e.g., imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, thiophenyl pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl; (ii) each ring is aromatic or carbocyclyl, at least one aromatic ring comprises a heteroatom and at least one other ring is a hydrocarbon ring or e.g., indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one, 5,6,7,8-tetrahydroquinolinyl and 5,6,7,8-tetrahydroisoquinolinyl; and (iii) each ring is aromatic or carbocyclyl, and at least one aromatic ring shares a bridgehead heteroatom with another aromatic ring, e.g., 4H-quinolizinyl. In certain embodiments, the heteroaryl is a monocyclic or bicyclic ring, wherein each of said rings contains 5 or 6 ring atoms where 1, 2, 3, or 4 of said ring atoms are a heteroatom independently selected from N, O, and S.

The term“heterocyclic ring system” refers to monocyclic, or fused, spiro-fused, and/or bridged bicyclic and polycyclic ring systems where at least one ring is saturated or partially unsaturated (but not aromatic) and comprises a heteroatom. A heterocyclic ring system can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted.

The term“heterocyclyl” refers to a radical of a heterocyclic ring system. Representative heterocyclyls include ring systems in which (i) every ring is non-aromatic and at least one ring comprises a heteroatom, e.g., tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl; (ii) at least one ring is non-aromatic and comprises a heteroatom and at least one other ring is an aromatic carbon ring, e.g., 1,2,3,4-tetrahydroquinolinyl, 1,2,3,4-tetrahydroisoquinolinyl; and (iii) at least one ring is non-aromatic and comprises a heteroatom and at least one other ring is aromatic and comprises a heteroatom, e.g., 3,4-dihydro-1H-pyrano[4,3-c]pyridine, and 1,2,3,4-tetrahydro-2,6-naphthyridine. In certain embodiments, the heterocyclyl is a monocyclic or bicyclic ring, wherein each of said rings contains 3-7 ring atoms where 1, 2, 3, or 4 of said ring atoms are a heteroatom independently selected from N, O, and S.

The term“saturated heterocyclyl” refers to a radical of heterocyclic ring system wherein every ring is saturated, e.g., tetrahydrofuran, tetrahydro-2H-pyran, pyrrolidine, piperidine and piperazine.

“Partially unsaturated” refers to a group that includes at least one double or triple bond. A “partially unsaturated” ring system is further intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic groups (e.g., aryl or heteroaryl groups) as herein defined. Likewise,“saturated” refers to a group that does not contain a double or triple bond, i.e., contains all single bonds.

The term“nucleobase” as used herein, is a nitrogen-containing biological compound found linked to a sugar within a nucleoside—the basic building blocks of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The primary, or naturally occurring, nucleobases are cytosine (DNA and RNA), guanine (DNA and RNA), adenine (DNA and RNA), thymine (DNA) and uracil (RNA), abbreviated as C, G, A, T, and U, respectively. Because A, G, C, and T appear in the DNA, these molecules are called DNA-bases; A, G, C, and U are called RNA-bases. Adenine and guanine belong to the double-ringed class of molecules called purines (abbreviated as R). Cytosine, thymine, and uracil are all pyrimidines. Other nucleobases that do not function as normal parts of the genetic code are termed non-naturally occurring.

As described herein, compounds of the disclosure may contain“optionally substituted” moieties. In general, the term“substituted”, whether preceded by the term“optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an“optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at each position. Combinations of substituents envisioned under this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term“stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

As used herein, the definition of each expression, e.g., alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

As described herein, compounds of the disclosure may contain“optionally substituted” moieties. In general, the term“substituted”, whether preceded by the term“optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an“optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at each position. Combinations of substituents envisioned under this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term“stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

The term“monoclonal antibody” refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against a variety of different antigenic determinants. The term“monoclonal antibody” includes 33 antibody fragments (such as Fab, Fab′, F(ab′)2, Fd, Fv), single chain (scFv) mutants, fusion proteins including an antibody portion, and any other modified immunoglobulin molecule including an antigen recognition site as well as both intact and full-length monoclonal antibodies, but are not limited thereto. Additionally,“monoclonal antibody” refers to such antibodies made in any number of methods including but not limited to hybridoma, phage selection, recombinant expression, and transgenic animals.

The term“humanized antibody” refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. In general, humanized antibodies are human immunoglobulins in which residues from complementary determining region (CDR) are replaced by residues from CDR of a non-human species (e.g., mouse, rat, rabbit, and hamster) having the desired specificity, affinity, and capability (ref: Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some instances, Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species having the desired specificity, affinity, and/or binding capability. The humanized 34 antibody may be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or binding capability. In general, the humanized antibody includes substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDRs that correspond to the non-human immunoglobulin whereas all or substantially all of the framework regions (FRs) have those of a human immunoglobulin consensus sequence. The humanized antibody may also include at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent No.5,225,539.

The term“human antibody” as used herein refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human using any technique known in the art. This definition of the human antibody includes intact or fulllength antibodies, fragments thereof, and/or antibodies including at least one human heavy and/or light chain polypeptide such as, for example, an antibody including murine light chain and human heavy chain polypeptides.

The term“chimeric antibody” refers to an antibody wherein an amino acid sequence of an immunoglobulin molecule is derived from two or more species. In general, variable regions of both light and heavy chains correspond to variable regions of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc) with the desired specificity, affinity, and capability, while constant regions are homologous to the sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in that species.

The term“targeting moiety” as used herein refers to a moiety (e.g., an antibody, a protein a hormone, a hormone derivative, a folic acid, a folic acid derivative, a biotin, a small molecule, an oligopeptide, a sigma-2-ligand, or a sugar) that serves to target or direct the conjugate to a particular location (e.g., cell type, or diseased tissue) or interaction (e.g., a specific binding event). Pattern Recognition Receptors

The disclosure presented herein features methods for the activation and induction of PRR expression (e.g., STING expression) in a subject, e.g., a subject with a microbial infection (e.g., a viral infection, bacterial infection, fungal infection, or parasitic infection) or a proliferative disease (e.g., cancer).

Pattern recognition receptors (PRRs) are a broad class of proteins which recognize pathogen-associated molecular patterns (PAMPs) conserved within pathogenic invaders. PAMPs are typically products of biosynthetic pathways that are essential to the survival and/or infectivity of the pathogen, e.g., lipopolysaccharides, glycoproteins, and nucleic acids. Recognition of PAMPs by their cognate PRRs activates signaling pathways that result in the production of immune defense factors such as pro-inflammatory and anti-inflammatory cytokines, type I interferons (IFN-α, IFN-β), and/or interferon stimulated genes (ISGs). It is well known that induction of innate immune signaling also results in the activation of T cell responses as well as the induction of adaptive immunity. These downstream immune effects are essential for clearance of the virus through apoptosis and killing of infected cells through cytotoxic T lymphocytes and other defense mechanisms. It is also well known that interferons act on ISRE (interferon response elements) that can trigger the production of ISGs, which play an important role in antiviral cellular defense.

The stimulator of interferon genes (STING) is a cytosolic microbial-derived DNA sensor that has been shown to be particularly sensitive to double-stranded DNA and cyclic dinucleotides (e.g., cyclic di-GMP) (Burdette, D. L. and Vance, R. E. (2013) Nat Immunol 14:19-26). Two molecules of STING form a homodimer mediated by an α-helix present in the C-terminal dimerization domain, and molecular binding studies have revealed that each STING dimer binds one molecule of microbial nucleic acids, e.g., DNA or a cyclic dinucleotide. Upon ligand binding, STING activates the innate immune response through interaction with RIG-I and IPS-1, resulting in interferon production (e.g., IFN-α and IFN-β) and other downstream signaling events. Since its discovery, STING has been shown to function as a critical sensor of viruses (e.g., adenovirus, herpes simplex virus, hepatitis B virus, vesicular stomatitis virus, hepatitis C virus), bacteria (e.g., Listeria monocytogenes, Legionella pneumopholia, Mycobacterium tuberculosis) and protozoa (Plasmodium falciparum, Plasmodium berghei). In addition, STING has been shown to play a major role in the innate immune response against tumor antigens, driving dendritic cell activation and subsequent T cell priming in several cancers (Woo, S.R. et al. Trends in Immunol (2015) 36:250-256).

Another class of PRRs includes RIG-I, which is the founding member of a family of PRRs termed RIG-I-like receptors (RLRs) that primarily detect RNA derived from foreign sources. It is a critical sensor of microbial infection (e.g., viral infection) in most cells and is constitutively expressed at low levels in the cytosol. After ligand binding, the expression of RIG-I is rapidly enhanced, leading to increased RIG-I concentrations in the cell (Jensen, S. and Thomsen, A.R. J Virol (2012) 86:2900-2910; Yoneyama M. et al. Nat Immunol (2004) 5:730-737). RIG-I is an ATP- dependent helicase containing a central DExD/H box ATPase domain and tandem N-terminal caspase-recruiting domains (CARDs) that mediate downstream signaling. The C-terminus of RIG- I comprises an ssRNA/dsRNA-binding domain that when unbound acts to silence CARD function at the N-terminus. Without wishing to be bound by theory, it is believed that upon recognition of target RNA structures, two N-terminal CARDs are exposed, allowing for interaction with the CARD of a downstream binding partner, IFN-β promoter stimulator 1 (IPS-1), also known as mitochondrial antiviral signaling molecule (MAVS) and CARDIF. This interaction in turn triggers further downstream signaling, such as induction of IRF3, IRF7, NF-κB, IFNs, and cytokine production that results in the initiation of the host immune response.

Other RLRs are homologous to RIG-I and function in a similar manner, including MDA5, LGP2, and RNase L. MDA5 is highly homologous to RIG-I, and has been shown to be crucial for triggering a cytokine response upon infection with picornaviruses (e.g., encephalomyocarditis virus (EMCV), Theiler’s virus, and Mengo virus), Sendai virus, rabies virus, West Nile virus, rabies virus, rotavirus, murine hepatitis virus, and murine norovirus. LPG2 lacks a CARD domain found in RIG-I and MDA5, which is responsible for direct interaction with IPS-1 to initiate downstream signaling. As such, LPG2 is believed to behave as a modulator of the innate immune response in conjunction with other CARD-bearing RLRs such as RIG-I and MDA5.

Another class of PRRs encompasses the nucleotide-binding and oligomerization domain (NOD)-like receptors, or NLR family (Caruso, R. et al, Immunity (2014) 41:898-908), which includes the microbial sensor NOD2. NOD2 is composed of an N-terminal CARD, a centrally- located nucleotide-binding oligomerization domain, and a C-terminal leucine rich repeat domain that is responsible for binding microbial PAMPs, such as bacterial peptidoglycan fragments and microbial nucleic acids. Ligand binding activates NOD2 and is believed to drive interaction with the CARD-containing kinase RIPK2, which in turn activates a number of downstream proteins including NF-κB, MAPK, IRF7, and IRF3, the latter of which results in the induction of type 1 interferons. NOD2 is expressed in a diverse set of cell types, including macrophages, dendritic cells, paneth cells, epithelial cells (e.g., lung epithelial cells, intestinal epithelia), and osteoblasts. NOD2 has been established as a sensor of infection by variety of pathogenic invaders, such as protozoa (e.g., Toxoplasma gondii and Plasmodium berghei), bacteria (e.g., Bacillus anthracis, Borrelia burgdorferi, Burkholderia pseudomallei, Helicobacter hepaticus, Legionella pneumophilia, Mycobacterium tuberculosis, Propionibacterium acne, Porphyromonas gingivalis, Salmonella enterica, and Streptococcus pneumonia), and viruses (e.g., respiratory syncytial virus and murine norovirus-1) (Moreira, L. O. and Zamboni, D. S. Front Immunol (2012) 3:1-12). Recent work has shown that mutation of NOD2 may contribute to inflammatory diseases such as Crohn’s disease, resulting in an aberrant inflammatory response upon stimulation.

Nanoparticle formulations

Nanoparticle formulations improve the pharmaceutical properties of drugs, for example they enhance the circulation half-life of the drugs by protecting them from degradation, and facilitating their accumulation in tumors. The accumulation at the tumor sites can occur through enhanced permeability and retention (EPR) effects, these effects result from the abnormal vascular architecture of the tumor site and impaired lymphatic drainage.

A variety of nanoparticle formulations have been used to deliver cytostatic and/or cytotoxic drugs to sites of tumor growth. Efficient and targeted delivery of Nanoparticles depends on the molecular size, particles larger than 7 nm in hydrodynamic diameter evade renal filtration and urinary excretion, whilst certain particles that are larger than 100 nm are rapidly eliminated from circulation by phagocytosis. In some cases, grafting polyethylene glycol or other polymers to surface of the nanoparticle can lengthen the circulation time by inhibiting phagocyte uptake. It is also possible to incorporate a tumor targeting moiety into the nanoparticle, thus facilitating delivery to the target site. For example, incorporation of ligands such as Herceptin, folate, or transferrin facilitate the delivery and uptake of nanoparticles by tumor cells.

One important consideration is the structural stability of nanoparticles in serum; some organic nanoparticles can be eliminated by the circulation shortly after administration. For example, some polymeric micelles may not be able to maintain their structural integrity due to rapid dilution and structural dissociation post i.v. injection. To overcome this problem, strategies employing nanoparticles sensitive to certain stimuli (e.g., pH, redox, temperature, and UV light) have been employed. These strategies can improve the physiochemical properties, thus impacting bio-distribution, pharmacokinetics, and toxicology of tumor-targeting nanomedicines.

The development of nanoparticle based formulations of immunomodulatory drugs can also be used to elicit antitumor immunity. Efficient and targeted delivery of immunomodulatory and immunostimulatory molecules to appropriate cells and tissues is key to successful development of immune therapeutic drugs including therapeutic and prophylactic vaccines. Such formulations can promote delivery to antigen presenting cells (APCs), or even directly trigger the activation of TAA-specific T-cells. Nanoparticles can also be employed to deliver antigens to APCs in vivo, thus eliciting an adaptive immune responses. Consequently, antigens and adjuvants bound to and/or encapsulated within such nanoparticles have been shown to trigger T- and B-cell responses of increased magnitude when compared to soluble antigens.

Cyclic dinucleotides (CDNs) have been demonstrated to be highly effective immunomodulatory molecules capable of robust activation of interferon regulatory factor 3 (IRF3) and NF-KB. CDNs are known ligands for STING localized in the endoplasmic reticulum. Ligand binding and activation of STING result in the activation of IFN signaling cascade. Several chemically distinct classes of CDNs including 3’5-linked cyclic di-GMP and cyclic di-AMP, as well as, 2,5 linked cGAMP have been shown to directly bind STING and subsequently initiate TBK1-IRF3 and NFkB signaling axis of the IFN signaling cascade.

The present disclosure relates to methods of activating and/or inducing the expression of PRRs (e.g., STING) in a subject, in particular for the treatment of a microbial infection or a proliferative disease (e.g., cancer). In some aspects, the method comprises administration of a nanoparticle described herein or composition thereof. It is to be noted that induction of any PRR with these compounds can stimulate interferon and/or NF-KB production which can induce the expression of a variety of PRRs which are inducible genes by feedback mechanism.

In certain aspects, the present disclosure provides a nanoparticle comprising:

i) a polymer shell comprising a plurality of monomers and an active agent; or ii) a core conjugated to an active agent, wherein the core is a metal; or

iii) a biomolecule conjugated to an active agent; or

iv) an active agent and a liposome, wherein the liposome further comprises a membrane; or

v) an active agent and a lipid bilayer, wherein the lipid bilayer is comprised of a plurality of bilayer units; or

vi) an active agent and a surfactant; or

vii) a nanocrystal comprising an active agent;

wherein the active agent is a compound of formula (I)

Formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

Z ¹ is either S or O;

each of B ¹ and B ² is independently a purinyl nucleobase or pyrimidinyl nucleobase;

each of X ¹ and X ² is independently O or S;

each of Y ¹ and Y ² is independently O, S, or NR ⁵ ;

each of L ¹ and L ² is independently absent, C ₁ -C ₆ alkyl or C ₁ -C ₆ heteroalkyl, wherein each C ₁ -C ₆ alkyl and C ₁ -C ₆ heteroalkyl is optionally substituted with R ⁶ ;

each of R ¹ and R ² is independently hydrogen, halo, -CN, C1-C20 alkyl (e.g., C1-C6 alkyl), or OR ⁷ ; each of R ³ and R ⁴ is independently hydrogen, C1-C20 alkyl (e.g., C1-C6 alkyl), C1-C20 heteroalkyl (e.g., C1-C6 heteroalkyl), cycloalkyl, heterocyclyl, OC(O)OC1-C20 alkyl (e.g., C1-C6 alkyl), aryl, or heteroaryl, wherein each C1-C20 alkyl, C1-C20 heteroalkyl, cycloalkyl, heterocyclyl, aryl, OC(O)O C ₁ -C ₂₀ alkyl (e.g., C _1-6 alkyl), and heteroaryl is optionally substituted with 1-5 R ⁸ ;

each R ⁵ is independently hydrogen or C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl);

R ⁶ is halo, -CN, C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), OR ⁷ , oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C1-C20 alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-5 R ⁹ ;

R ⁷ is hydrogen, C1-C20 alkyl (e.g., C1-C6 alkyl), cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C1-C20 alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-5 R ⁹ ; each R ⁸ is independently C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), O-aryl, OC(O)NR ₅ -C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), S(O) ₂ NR ₅ -aryl, NR ₅ C(O)-aryl, N(R ₅ ) ₂ C(O)-aryl, C(O)-aryl, C(O)- heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, OC(O)-C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ ),

OC(O)O-C1-C20 alkyl (e.g., C1-C6), wherein each C1-C20 alkyl, O-aryl, OC(O)NR5-C1- C20 alkyl, S(O)2NR5-aryl, NR5C(O)-aryl, CH2NR5C(O)-aryl, C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, OC(O)-C1-C20 alkyl (e.g., C1-C6), OC(O)O-C1-C20 alkyl (e.g., C1-C6), is optionally substituted by 1-5 R ⁹ ; and

each R ⁹ is independently C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), halo, -CN, OH, O-C ₁ -C ₂₀ alkyl, O-C ₁ - C ₂₀ heteroalkyl, O-aryl, O-heteroaryl.

In certain embodiments, the compound is a compound of Formula (I-a):

Formula (I-a) or a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

In certain embodiments the compound of Formula I is represented by Formulas (I-b), (I- c), (I-d), or (I-e):

Formula (I-b) Formula (I-c)

Formula (I-d) Formula (I-e) or a pharmaceutically acceptable salt thereof.

In some embodiments, B ¹ is a purinyl nucleobase. In some embodiments, B ² is a pyrimidinyl nucleobase. In some embodiments, B ¹ is a purinyl nucleobase and B ² is a pyrimidinyl nucleobase.

In some embodiments, each of B ¹ or B ² is selected from:

In some embodiments, one of B ¹ or B ² is selected from a naturally occurring nucleobase and the other of B ¹ or B ² is a modified nucleobase. In some embodiments, B1 is adenosinyl or guanosinyl. In some embodiments, B2 is cytosinyl, thyminyl, or uracilyl. In some embodiments, B ¹ is adenosinyl or guanosinyl and B ² is cytosinyl, thyminyl, or uracilyl. In some embodiments, each of B ¹ and B ² is independently uracilyl.

In some embodiments, each of R ¹ and R ² is independently hydrogen, halo, or OR ⁶ . In some embodiments, each of R ¹ and R ² is independently halo (e.g., fluoro). In some embodiments, each of R ¹ and R ² is not hydrogen or OR ⁷ .

In some embodiments, X ¹ is O. In some embodiments, X ² is O. In some embodiments, each of X ¹ and X ² is O.

In some embodiments, Y ¹ is O or S. In some embodiments, Y ² is O or S. In some embodiments, each of Y ¹ and Y ² is independently O or S. In some embodiments, one of Y ¹ or Y ² is O and the other of Y ¹ or Y ² is S. In some embodiments, each of Y ¹ or Y ² is independently S. In some embodiments, each of Y ¹ or Y ² is independently O.

In some embodiments, L ¹ is C ₁ -C ₆ alkyl (e.g., CH ₂ ). In some embodiments, L ² is C ₁ -C ₆ alkyl (e.g., CH ₂ ). In some embodiments, each of L ¹ and L ² is independently C ₁ -C ₆ alkyl (e.g., CH2).

In some embodiments, R ³ is hydrogen, aryl, or heteroaryl, wherein aryl and heteroaryl is optionally substituted with 1-5 R ⁸ . In some embodiments, R ³ is aryl or heteroaryl, each of which is optionally substituted with 1-5 R ⁸ . In some embodiments, R ³ is phenyl substituted with 1 R ⁸ .

In some embodiments, R ⁴ is independently hydrogen, aryl, or heteroaryl, wherein aryl and heteroaryl is optionally substituted with 1-5 R ⁸ . In some embodiments, R ⁴ is aryl or heteroaryl, each of which is optionally substituted with 1-5 R ⁸ . In some embodiments, R ⁴ is phenyl substituted with 1 R ⁸ . In some embodiments, each of R ³ and R ⁴ is independently hydrogen, aryl, or heteroaryl, wherein aryl and heteroaryl is optionally substituted with 1-5 R ⁸ .

In some embodiments, R ³ is aryl or heteroaryl, each of which is optionally substituted with 1-5 R ⁸ , and R ⁴ is hydrogen.

In some embodiments, R ³ is phenyl substituted with 1 R ⁸ and R ⁴ is hydrogen. In some embodiments, each of R ³ and R ⁴ is independently phenyl substituted with 1 R ⁸ .

In some embodiments, each of Y ¹ and Y ² is O; and each of R ³ and R ⁴ is independently hydrogen. In some embodiments, Y ² is O; and R ⁴ is hydrogen. In some embodiments, each of Y ¹ and Y ² is independently S and each of R ³ and R ⁴ is independently substituted with 1 R ⁸ . In some embodiments, Y ¹ is S and R ³ is substituted with 1 R ⁸ .

In some embodiments, R ⁸ is OC(O)-aryl optionally substituted by 1-5 R ⁹ (e.g., 1 R ⁹ ). In some embodiments, R ⁸ is -C(O)-aryl optionally substituted by 1-5 R ⁹ (e.g., 1 R ⁹ ). In some embodiments, R ⁹ is O-C1-C12 alkyl (e.g., O-CH2(CH2)8CH3). In some embodiments, R ⁹ is O-C1- C10 alkyl (e.g., O-CH2(CH2)8CH3). In some embodiments, R ⁹ is O-C1-C8 alkyl (e.g., O- CH ₂ (CH ₂ ) ₆ CH ₃ ). In some embodiments, R ⁹ is O-C ₁ -C ₆ alkyl (e.g., O-CH ₂ (CH ₂ ) ₄ CH ₃ ).

In some embodiments, the compound is represented by Formula (I-f):

Formula (I-f) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: each of B ¹ and B ² is independently a purinyl nucleobase or pyrimidinyl nucleobase;

each of X ¹ and X ² is independently O or S;

each of Y ¹ and Y ² is independently O, S, or NR ⁵ ; each of L ¹ and L ² is independently absent, C ₁ -C ₆ alkyl or C ₁ -C ₆ heteroalkyl, wherein each C ₁ -C ₆ alkyl and C ₁ -C ₆ heteroalkyl is optionally substituted with R ⁶ ;

each of R ¹ and R ² is independently halo;

each of R ³ and R ⁴ is independently hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, cycloalkyl,

heterocyclyl, aryl, or heteroaryl, wherein each C1-C6 alkyl, C1-C6 heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted with 1-5 R ⁸ ;

R ⁵ is hydrogen or C1-C6 alkyl;

R ⁶ is halo, -CN, C ₁ -C ₆ alkyl, OR ⁷ , oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C ₁ -C ₆ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-5 R ⁹ ;

R ⁷ is hydrogen, C1-C6 alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C1-C6 alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-5 R ⁹ ; each R ⁸ is independently C1-C6 alkyl, C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, or OC(O)- heteroaryl, wherein each C1-C6 alkyl, C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, or OC(O)- heteroaryl is optionally substituted by 1-5 R ⁹ ; and

each R ⁹ is independently C ₁ -C ₂₀ alkyl, halo, -CN, OH, O-C ₁ -C ₂₀ alkyl, O-C ₁ -C ₂₀ heteroalkyl, O- aryl, or O-heteroaryl.

In some embodiments, the compound is represented by Formula (I-g):

Formula (I-g) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

each of B ¹ and B ² is independently a purinyl nucleobase or pyrimidinyl nucleobase;

each of X ¹ and X ² is independently O; each of Y ¹ and Y ² is independently O or S;

each of L ¹ and L ² is independently absent or C ₁ -C ₆ alkyl;

each of R ¹ and R ² is independently halo or OH;

each of R ³ and R ⁴ is independently hydrogen or aryl optionally substituted with 1-5 R ⁸ ;

each R ⁸ is independently OC(O)-aryl optionally substituted by 1-5 R ⁹ ; and

each R ⁹ is independently O-C1-C20 alkyl. In some embodiments, the compound of Formula (I) is selected from those depicted in Table 1:

In other aspects, the present disclosure provides a nanoparticle, comprising:

i) a polymer shell comprising a plurality of monomers and an active agent; or ii) a core conjugated to an active agent, wherein the core is a metal; or

iii) a biomolecule conjugated to an active agent; or

iv) an active agent and a liposome, wherein the liposome further comprises a membrane; or

v) an active agent and a lipid bilayer, wherein the lipid bilayer is comprised of a plurality of bilayer units; or

vi) an active agent and a surfactant; or

vii) a nanocrystal comprising an active agent;

wherein the active agent is compound of Formula (II):

Formula (II)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

Z ² is either S or O;

each of B ³ and B ⁴ is independently a purinyl nucleobase or pyrimidinyl nucleobase;

each of X ³ and X ⁴ is independently O or S;

each of Y ³ and Y ⁴ is independently O, S, or NR ¹⁴ ;

each of L ³ and L ⁴ is independently absent, C1-C6 alkyl or C1-C6 heteroalkyl, wherein each alkyl and heteroalkyl is optionally substituted with R ¹⁵ ;

each of R ¹⁰ and R ¹¹ is independently hydrogen, halo, -CN, C1-C20 alkyl (e.g., C1-C6 alkyl), or OR ¹⁶ ; each of R ¹² and R ¹³ is independently hydrogen, C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), C ₁ -C ₂₀ heteroalkyl (e.g., C ₁ -C ₆ heteroalkyl), OC(O)OC ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each alkyl, heteroalkyl, cycloalkyl,

heterocyclyl, aryl, and heteroaryl is optionally substituted with one or more R ¹⁷ ;

R ¹⁴ is hydrogen or C1-C20 alkyl (e.g., C1-C6 alkyl);

R ¹⁵ is halo, -CN, C1-C20 alkyl (e.g., C1-C6 alkyl), OR ¹⁶ , oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted with one or more R ¹⁸ ;

R ¹⁶ is hydrogen, C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted with one or more R ¹⁸ ;

each R ¹⁷ is independently C1-C20 alkyl (e.g., C1-C6 alkyl), C1-C20 heteroalkyl, C(O)-C1-C20 alkyl, OC(O)-C1-C20 alkyl (e.g., C1-C6 alkyl), C(O)O-C1-C20 alkyl (e.g., C1-C6 alkyl), OC(O)O- C1-C20 alkyl (e.g., C1-C6 alkyl), C(O)N(R ¹⁴ )-C1-C20 alkyl (e.g., C1-C6 alkyl), N(R ¹⁴ )C(O)- C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), OC(O)N(R ¹⁴ )-C ₁ -C ₂₀ alkyl (e.g., C ₁ -C ₆ alkyl), O-aryl, O- heteroaryl, C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, C(O)O-aryl, OC(O)-heteroaryl, C(O)O-heteroaryl, C(O)O-aryl, C(O)O-heteroaryl, C(O)N(R ¹⁴ )-aryl, C(O)N(R ¹⁴ )- heteroaryl, N(R ¹⁴ )C(O)-aryl, N(R ¹⁴ ) ₂ C(O)-aryl, or N(R ¹⁴ )C(O)-heteroaryl, S(O) ₂ N(R ¹⁴ )- aryl, wherein each alkyl, heteroalkyl, aryl, and heteroaryl is optionally substituted by one or more R ¹⁸ ; and

each R ¹⁸ is independently C1-C20 alkyl, O-C1-C20 alkyl, C1-C20 heteroalkyl, halo, -CN, OH, oxo, aryl, heteroaryl, O-aryl, or O-heteroaryl.

In certain embodiments, the compound is a compound of Formula (II-a), (II-b), (II-c), or (II-d):

Formula (II-a) Formula (II-b)

Formula (II-c) Formula (II-d) or a pharmaceutically acceptable salt thereof.

In some embodiments Z ¹ is S. In some embodiments, Z ¹ is O.

In some embodiments, at least one of B ³ or B ⁴ is a purinyl nucleobase. In some embodiments, each of B ³ or B ⁴ is independently a purinyl nucleobase. In some embodiments, B ³ is a purinyl nucleobase. In some embodiments, B ⁴ is a pyrimidinyl nucleobase. In some embodiments, B ³ is a purinyl nucleobase and B ⁴ is a pyrimidinyl nucleobase.In some embodiments, B ³ is a pyrimidinyl nucleobase. In some embodiments, B ⁴ is a purinyl nucleobase. In some embodiments, B ³ is a pyrimidinyl nucleobase and B ⁴ is a purinyl nucleobase.

In some embodiments, each of B ³ or B ⁴ is selected from a naturally occurring nucleobase or a modified nucleobase. In some embodiments, each of B ³ or B ⁴ is selected from adenosinyl, guanosinyl, cytosinyl, thyminyl, uracilyl, 5’-methylcytosinyl, 5’-fluorouracilyl, 5’- propynyluracilyl and 7-deazaadenosinyl. In me embodiments each of B ¹ or B ² is selected from:

wherein“ ” indicates the linkage of the nucleobase to the ribose ring.

In some embodiments, one of B ³ or B ⁴ is selected from a naturally occurring nucleobase and the other of B ¹ or B ² is a modified nucleobase. In some embodiments, one of B ¹ or B ² is adenosinyl, guanosinyl, thyminyl, cytosinyl, or uracilyl, and the other of B ¹ or B ² is 5’- methylcytosinyl, 5’-fluorouracilyl, 5’-propynyluracilyl, or 7-deazaadenosinyl.

In some embodiments, B ³ is adenosinyl or guanosinyl. In some embodiments, B ⁴ is cytosinyl, thyminyl, or uracilyl. In some embodiments, B ³ is adenosinyl or guanosinyl and B ⁴ is cytosinyl, thyminyl, or uracilyl. In some embodiments, B ⁴ is adenosinyl or guanosinyl. In some embodiments, B ³ is cytosinyl, thyminyl, or uracilyl. In some embodiments, B ⁴ is adenosinyl or guanosinyl and B ³ is cytosinyl, thyminyl, or uracilyl.

In some embodiments, each of B ³ and B ⁴ is independently uracilyl. In some embodiments, each of B ³ and B ⁴ is independently adenosinyl.

In some embodiments, each of R ¹⁰ and R ¹¹ is independently hydrogen, halo, or OR ¹⁶ . In some embodiments, each of R ¹⁰ and R ¹¹ is independently halo (e.g., fluoro). In some embodiments, each of R ¹⁰ and R ¹¹ is not hydrogen or OR ¹⁶ .

In some embodiments, X ³ is O. In some embodiments, X ⁴ is O. In some embodiments, each of X ³ and X ⁴ is independently O.

In some embodiments, Y ³ is O or S. In some embodiments, Y ⁴ is O or S. In some embodiments, each of Y ³ and Y ⁴ is independently O or S. In some embodiments, one of Y ³ or Y ⁴ is O and the other of Y ³ or Y ⁴ is S. In some embodiments, each of Y ³ or Y ⁴ is independently S. In some embodiments, each of Y ³ or Y ⁴ is independently O. In some embodiments, L ³ is C ₁ -C ₆ alkyl (e.g., CH ₂ ). In some embodiments, L ⁴ is C ₁ -C ₆ alkyl (e.g., CH ₂ ). In some embodiments, each of L ³ and L ⁴ is independently C ₁ -C ₆ alkyl (e.g., CH ₂ ).

In some embodiments, R ¹² is hydrogen, aryl, or heteroaryl, wherein aryl and heteroaryl is optionally substituted with 1-5 R ¹⁷ . In some embodiments, R ¹² is aryl or heteroaryl, each of which is optionally substituted with 1-5 R ¹⁷ . In some embodiments, R ¹² is phenyl substituted with 1 R ¹⁷ .

In some embodiments, R ¹³ is independently hydrogen, aryl, or heteroaryl, wherein aryl and heteroaryl is optionally substituted with 1-5 R ¹⁷ . In some embodiments, R ¹³ is aryl or heteroaryl, each of which is optionally substituted with 1-5 R ¹⁷ . In some embodiments, R ¹³ is phenyl substituted with 1 R ¹⁷ .

In some embodiments, each of R ¹² and R ¹³ is independently hydrogen, aryl, or heteroaryl, wherein aryl and heteroaryl is optionally substituted with 1-5 R ¹⁷ . In some embodiments, R ¹² is aryl or heteroaryl, each of which is optionally substituted with 1-5 R ¹⁷ , and R ¹³ is hydrogen. In some embodiments, R ¹² is phenyl substituted with 1 R ¹⁷ and R ¹³ is hydrogen. In some embodiments, each of R ¹² and R ¹³ is independently phenyl substituted with 1 R ¹⁷ .

In some embodiments, each of Y ³ and Y ⁴ is O; and each of R ¹² and R ¹³ is independently hydrogen. In some embodiments, Y ⁴ is O; and R ¹³ is hydrogen. In some embodiments, each of Y ³ and Y ⁴ is independently S and each of R ¹² and R ¹³ is independently substituted with 1 R ¹⁷ . In some embodiments, Y ³ is S and R ¹² is substituted with 1 R ¹⁷ .

In some embodiments, each R ¹⁷ is independently C1-C20 alkyl (e.g., C1-C6 alkyl), C1-C20 heteroalkyl, C(O)-C1-C20 alkyl, OC(O)-C1-C20 alkyl, OC(O)O-C1-C20 alkyl, OC(O)N(R ⁵ )-C1-C20 alkyl, O-aryl, C(O)-aryl, OC(O)-aryl, or C(O)N(R ⁵ )-aryl, wherein each alkyl, heteroalkyl, aryl, and heteroaryl is optionally substituted by one or more R ¹⁸ .

In some embodiments, R ¹⁷ is OC(O)-aryl optionally substituted by 1-5 R ¹⁸ (e.g., 1 R ¹⁸ ). In some embodiments, R ¹⁷ is OC(O)-aryl optionally substituted by 1-5 R ¹⁸ (e.g., 1 R ¹⁸ ).

In some embodiments, R ¹⁸ is O-C ₁ -C ₁₂ alkyl (e.g., O-CH ₂ (CH ₂ ) ₈ CH ₃ ). In some embodiments, R ¹⁸ is O-C1-C10 alkyl (e.g., O-CH2(CH2)8CH3). In some embodiments, R ¹⁸ is O-C1- C8 alkyl (e.g., O-CH2(CH2)6CH3). In some embodiments, R ¹⁸ is O-C1-C6 alkyl (e.g., O- CH2(CH2)4CH3).

In some embodiments, the compound of Formula (II) is selected from those depicted in Table 2:

wherein X is any pharmaceutically acceptable counterion, e.g., lithium, sodium, potassium, calcium, magnesium, aluminum, ammonium, ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et al., supra); and the label“n” indicates that the associated alkyl chain is“normal” (i.e., unbranched). In some embodiments, the compound of Table 2 is not a salt (i.e., the compound is a free acid or free base).

In an embodiment, a compound described herein is in the form of a pharmaceutically acceptable salt. Exemplary salts are described herein, such as ammonium salts. In some embodiments, the compound is a mono-salt. In some embodiments, the compound is a di-salt. In some embodiments, a compound described herein (e.g., a compound in Table 1 or Table 2) is not a salt (e.g., is a free acid or free base).

Without wishing to be bound by theory, a compound of Formula (I) or Formula (II) is a small molecule nucleic acid hybrid (cyclic dinucleotide) compound that combines both antiviral and immune modulating activities. The latter activity mediates, for example, controlled apoptosis of virus-infected hepatocytes via stimulation of the innate immune response, similar to what is also achieved by IFN-α therapy in patients suffering from a viral infection. The mechanism of action of a compound of Formula (I) or Formula (II) entails its host immune stimulating activity, which may induce endogenous IFNs via the activation of a PRR, e.g., RIG-I, NOD2, and STING. Activation may occur by binding of a compound of Formula (I) or Formula (II) to the nucleotide binding domain of a PRR (e.g., STING), as described previously, and may further result in the induction of PRR expression (e.g., STING expression).

The compounds disclosed herein may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included within the scope. Unless otherwise indicated when a compound is named or depicted by a structure without specifying the stereochemistry and has one or more chiral centers, it is understood to represent all possible stereoisomers of the compound. The compounds provided herewith may also contain linkages (e.g., carbon-carbon bonds, phosphorus-oxygen bonds, or phosphorus-sulfur bonds) or substituents that can restrict bond rotation, e.g., restriction resulting from the presence of a ring or double bond. In some embodiments, the compound of Formula (I) or Formula (II) comprises an isomer (e.g., an Rp-isomer or Sp isomer) or a mixture of isomers (e.g., Rp-isomers or Sp isomers) of a compound of Formula (I) or Formula (II).

In some embodiments, the nanoparticles of the disclosure are degradable by an acid, a base, an oxidant, a reductant, ultraviolet light, infrared light, a liposome, or heat, thereby releasing the active agent.

Further exemplary active agents and their methods of use are described in WO/2018/009652 and WO18/009648, the contents of which are hereby incorporated by reference in their entirety.

Polymeric Nanoparticles

In some embodiments, polymeric nanoparticles can be used to deliver the active agents of the disclosure to the site of action. Analogous polymeric nanoparticles can be synthesized by in situ polymerization and other methods known to one of ordinary skill in the art, such as those described in US/2010/0216804, US 8,912,212, and US 8,318,211, the contents of which are hereby incorporated by referenced in their entirety.

In some embodiments, at least one monomer is positively charged. In some embodiments, at least one monomer is independently selected from the group consisting of lactide, glycolide, ethylene glycol, 4-amino-1-butanol, 1 ,2-dioleoyl-3-trimethylammonium-propane chloride salt, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, acrylic acid, methacrylic acid, vinyl alcohol, ethylene, styrene, acrylamide, leuprolide acetate, glutamate, allylamine, 2-(piperidin-1-yl)ethyl methacrylate, 2-(azepan-1-yl)ethyl methacrylate, 2-(azocan-1-yl)ethyl methacrylate, 2-(4- methylpiperidin-1-yl)ethyl methacrylate, 2-(3,5-dimethylpiperidin-1-yl)ethyl methacrylate, 2- (diethylamino)ethyl methacrylate, 2-(ethyl(propyl)amino)ethyl methacrylate, 2- (dipropylamino)ethyl methacrylate, 2-(dibutylamino)ethyl methacrylate, 2-(dipentylamino)ethyl methacrylate, and Poly(ethylene glycol) methacrylate.

In some embodiments, at least one monomer is selected from the group consisting of lactic acid, glycolic acid, and caprolactone. In some embodiments, at least one monomer is lactic acid. In some embodiments, at least one monomer is caprolactone. In some embodiments, at least one monomer is lactic acid and at least one monomer is glycolic acid. In some embodiments, the lactic acid and the glycolic acid are in a ratio of 5:1, 4:1, 3:1, 2:1, or 1:1. In some embodiments, the lactic acid and the glycolic acid are in a ratio of 3:1 or 1:1.

In some embodiments, the polymer shell further comprises at least one cross-linker. In some embodiments, the cross-linker is selected from selected from the group consisting of 1,4- butanediol diacrylate, ethylene glycol di-methacrylate, maleic acid, and glutaraldehyde.

In some embodiments, the monomer and the cross-linker have a molar ratio of about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about 1.1:1, about 1.2:1, about 1.3:1, about 1.4:1, or about 1.5:1.

In some embodiments, the monomer and the cross-linker have a molar ratio of 1.1:1. In some embodiments, the polymer shell further comprises at least one capping moiety. In some embodiments, the capping moiety is selected from the group consisting of 1-(3- aminopropyl)-4-methylpiperazine) and polyethylene glycol. In some embodiments, the capping moiety is 1-(3-aminopropyl)-4-methylpiperazine). In some embodiments, the capping moiety is polyethylene glycol.

In some embodiments, the weight/weight ratio of the polymer shell to active agent is about 300:1, about 350:1, about 400:1, about 450:1, about 500:1, about 550:1, about 600:1, about 650:1, about 700:1, about 750:1, about 800:1, about 850:1, about 900:1, about 950:1, or about 1000:1. In some embodiments, the weight/weight ratio of the polymer shell to active agent is 500:1. In some embodiments, the active agent is encapsulated within the polymer shell. In some embodiments, the active agent is enmeshed within the polymer shell. In some embodiments the active agent is covalently coupled to the surface of the polymer shell.

In some embodiments, the nanoparticle has a zeta potential of about +5 mV, about +6 mV, about +7 mV, about +8 mV, about +9 mV, about +10 mV, about +11 mV, about +12 mV, about +13 mV, about +14 mV or about +15 mV. In some embodiments, the nanoparticle has a zeta potential of +10 mV.

In some embodiments, the diameter of the nanoparticle is about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 70, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm, about 155 nm, about 160 nm, about 165 nm, about 170 nm, about 175 nm, about 180 nm, about 185 nm, about 190 nm, about 195 nm, or about 200 nm.

In some embodiments, the diameter of the nanoparticle is about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 70, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, or about 100 nm.

In some embodiments, the diameter of the nanoparticle is about 1,000 nm, about 1,050 nm, about 1,100 nm, about 1,150 nm, about 1,200 nm, about 1,250 nm, about 1,300 nm, about 1,350 nm, about 1,400 nm, about 1,450 nm, or about 1,500 nm.

In some embodiments, the nanoparticle is formed by double emulsion or nanoprecipitation. Metallic Nanoparticles

In some embodiments, metallic nanoparticles can be used to deliver the active agents of the disclosure to the site of action. Analogous metallic nanoparticles can be synthesized by methods known to those skilled in the art, such as those described in WO/2000/002590, the contents of which is hereby incorporated by referenced in its entirety.

In some embodiments, the metal is iron, iron oxide, copper, copper oxide, gold, silver, silver oxide, nickel, aluminum, aluminum oxide, indium, cobalt, molybdenum, tin, titanium, titanium oxide, tungsten, zinc, or zinc oxide.

In some embodiments, the active agent is covalently coupled to the surface of the core of the nanoparticle. In some embodiments, the diameter of the nanoparticle is about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 70, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm, about 155 nm, about 160 nm, about 165 nm, about 170 nm, about 175 nm, about 180 nm, about 185 nm, about 190 nm, about 195 nm, or about 200 nm. In some embodiments, the diameter of the nanoparticle is about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 70, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, or about 100 nm.

Nanoparticles of Biomolecule-Agent Conjugates

In some embodiments, conjugates of biomolecules and active agents can be used to deliver the active agents of the disclosure to the site of action. Analogous conjugates can be synthesized by methods known to those skilled in the art, such as those described in US 5,672,683 and US 5,574,018, the contents of which are hereby incorporated by referenced in their entirety.

In some embodiments, the biomolecule is selected from the group consisting of an antibody, a protein fragment, a transferrin, and a protein.

In some embodiments, the protein is an interleukin. In some embodiments, the protein is selected from the group consisting of interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 3 (IL- 3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7) and albumin.

In some embodiments, the antibody is selected from is selected from intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments, single chain Fv (scFv) mutants, multispecific antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins including an antigen determination portion of an antibody, an anti-CD 28 antibody, an anti-CTLA-4 antibody and other modified immunoglobulin molecules including an antigen recognition site. In some embodiments, the antibody is selected from muromonab-CD3, abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab, etanercept, basiliximab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, alefacept, omalizumab, efalizumab, tositumomab-I ¹³¹ , cetuximab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, rilonacept, certolizumab pegol, romiplostim, belimumab, anti-CD20, tocilizumab, atlizumab, mepolizumab, pertuzumab, tremelimumab, ticilimumab, inotuzumab ozogamicin, aflibercept, catumaxomab, pregovomab, motavizumab, efumgumab, Aurograb®, raxibacumab, and veltuzumab.

In some embodiments, the protein fragment is a fragment of a diphtheria toxin.

In some embodiments the biomolecule is conjugated to the active agent via a thioether or an amide.

In some embodiments, the diameter of the nanoparticle is about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm, about 155 nm, about 160 nm, about 165 nm, about 170 nm, about 175 nm, about 180 nm, about 185 nm, about 190 nm, about 195 nm, or about 200 nm.

Liposome Based Nanoparticles

In some embodiments, liposomes comprising a membrane can be used to deliver the active agents of the disclosure to the site of action. Analogous liposomes can be synthesized by methods known to those skilled in the art, such as those described in US 8,663,599 and US/2013/0115273, the contents of which are hereby incorporated by referenced in their entirety.

In some embodiments, the membrane comprises hydrogenated soy phosphatidylcholine, cholesterol, distearoyl phosphatidylglycerol, distearoyl phosphatidylcholine, dioleoyl lecithin, dipalitoyl, triolein, tricaprylin, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn- glycero3-phosphoethanolamine sodium, sphingomyelin, muramyl tripeptide- phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine and 1,2-dioleoyl- sn-glycero-3-phospho-L-serine, or dimyristoyl-phosphatidylcholine.

In some embodiments, the membrane comprises hydrogenated soy phosphatidylcholine, cholesterol, and distearoyl phosphatidylglycerol in a molar ratio of 2:0.8:1. In some embodiments, the membrane comprises distearoyl phosphatidylcholine and cholesterol in a molar ratio of 2:1. In some embodiments, the membrane comprises dioleoyl lecithin, dipalmitoyl phosphatidylglycerol, cholesterol, and triolein. In some embodiments, the membrane comprises dioleoyl lecithin cholesterol, dipalmitoyl phosphatidylglycerol, tricaprylin, and triolein. In some embodiments, the membrane comprises N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero3- phosphoethanolamine sodium, fully hydrogenated soy phosphatidylcholine, and cholesterol. In some embodiments, the membrane comprises of sphingomyelin and cholesterol in a molar ratio of 6:4. In some embodiments, the membrane comprises of 1-palmitoyl-2-oleoyl-sn-glycerol-3- phosphocholine and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine.

In some embodiments, the diameter of the nanoparticle is about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm, about 155 nm, about 160 nm, about 165 nm, about 170 nm, about 175 nm, about 180 nm, about 185 nm, about 190 nm, about 195 nm, or about 200 nm.

Lipid Based Nanoparticles

In some embodiments, lipid bilayers comprising a plurality of bilayer units can be used to deliver the active agents of the disclosure to the site of action. Analogous lipid bilayers can be synthesized by methods known to those skilled in the art, such as those described in US 7,063,860 and US 5,576,016, the contents of which are hereby incorporated by referenced in their entirety.

In some embodiments, the bilayer units are independently selected from the group consisting of cholesteryl sulfate, 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine, and 1,2- dimyristoyl-sn-glycero-3-phosphorylglycerol sodium salt.

In some embodiments, the diameter of the nanoparticle is about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, or about 500 nm.

Surfactant Based Nanoparticles

In some embodiments, nanoparticles comprising a surfactant and an active agent can be used to deliver the active agents of the disclosure to the site of action. Analogous nanoparticles of surfactants and active agents can be synthesized by methods known to those skilled in the art, such as those described in US 7,030,155, the contents of which is hereby incorporated by referenced in its entirety.

In some embodiments, the surfactant is comprised of at least one of sodium deoxycholate, propofol, soybean oil, glycerol, egg lecithin, polysorbate 80, ethanol, or water.

In some embodiments, the diameter of the nanoparticle is about 50 nm , about 75 nm, about 100 nm, about 125 nm, about 150 nm, about 175 nm, about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, or about 500 nm. Nanocrystals

In some embodiments, nanoparticles comprising a nanocrystal of an active agent can be used to deliver the active agents of the disclosure to the site of action. Analogous nanocrystals can be synthesized by methods known to those skilled in the art, such as those described in US 7,229,497, US 9,056,057, and WO/2008/058755, the contents of which are hereby incorporated by referenced in their entirety.

In some embodiments, the diameter of the nanoparticle is about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 70, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, or about 100 nm.

Targeted Delivery

In some embodiments, the nanoparticles of the disclosure further comprise a targeting moiety that serves to target or direct the conjugate to a particular location (e.g., cell type, or diseased tissue) or interaction (e.g., a specific binding event).

In some embodiments, the targeting moiety is an antibody, a hormone, a hormone derivative, folic acid, a folic acid derivative, a biotin, a transferring, a small molecule, an oligopeptide, a sigma-2-ligand, or a sugar.

In some embodiments, the antibody is selected from intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments, single chain Fv (scFv) mutants, multispecific antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins including an antigen determination portion of an antibody, an anti-CD22 antibody or an anti-CD79b antibody. and other modified immunoglobulin molecules including an antigen recognition site.

In some embodiments, the antibody is selected from muromonab-CD3, abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab, etanercept, basiliximab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, alefacept, omalizumab, efalizumab, tositumomab-I ¹³¹ , cetuximab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, rilonacept, certolizumab pegol, romiplostim, belimumab, anti-CD20, tocilizumab, atlizumab, mepolizumab, pertuzumab, trastuzumab, tremelimumab, ticilimumab, inotuzumab ozogamicin, aflibercept, catumaxomab, pregovomab, motavizumab, efumgumab, Aurograb®, raxibacumab, and veltuzumab. In some embodiments, the steroid is an estrogen, an androgen, a cholesterol or any derivative thereof. In some embodiments, the hormone is estrogen, testosterone, dihydrotestosterone, ethisterone, or cholesterol.

In some embodiments, the targeting moiety is folic acid or any derivative thereof. In some embodiments, the targeting moiety is biotin. In some embodiments, the targeting moiety is a substituted benzodiazepine. In some embodiments, the targeting moiety is a glutamate-urea-lysine. In some embodiments, the targeting moiety is asparaginyl-glycinyl-aginine oligopeptide.

In some embodiments, the targeting moiety is an integrin ligand. In some embodiments, the integrin ligand is an RGD peptide. In some embodiments, the RGD peptide is an Arg-Gly-Asp oligopeptide.

In some embodiments, the targeting moiety is a sigma-2-ligand.

In some embodiments, the targeting moiety is a sugar. In some embodiments, the sugar is galactose. In some embodiments, the sugar is N-acetyl-galactosamine.

In some embodiments, the protein is an interleukin. In some embodiments, the interleukin is interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), or interleukin 7 (IL-7).

Exemplary Methods of Use

The present disclosure relates to methods for inducing the expression of a PRR (e.g., STING) in a subject through administration of an effective amount of nanoparticle disclosed herein or a composition thereof. In some embodiments, the subject may be suffering from a condition described below, e.g., a viral infection (e.g., viral latency), a bacterial infection, a cancer (e.g., a proliferative disease).

Treatment of Viral Infections

Pattern recognition receptors such as STING, RIG-I, and NOD2, have been shown to be an important factor in host recognition of a large number of RNA viruses from a variety of different viral families. In some embodiments, the methods of inducing expression of PRRs (e.g., STING) disclosed herein comprise administration of an effective amount of a nanoparticle disclosure herein or a composition thereof to a subject infected with a microbial infection. In some embodiments, the microbial infection is a virus. In some embodiments, the virus is a RNA virus (e.g., a double- stranded RNA (dsRNA) virus, a single-stranded RNA (ssRNA) virus (e.g., a positive-strand (sense) ssRNA virus or a negative-strand (antisense) ssRNA virus), or a ssRNA retrovirus) or a DNA virus (e.g., a dsDNA virus, ssDNA virus, or a dsDNA retrovirus). In some embodiments, the virus may be a Group I, Group II, Group III, Group IV, Group V, Group VI, or Group VII class of virus, e.g., according to the Baltimore classification system.

In some embodiments, the virus is dsRNA virus, e.g., a Group III virus. In some embodiments, expression of a PRR (e.g., STING) is induced through host-produced or viral- derived RNA. In some embodiments, the virus is a dsRNA virus, and is a member of the Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megabirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae, or Totiviridae families, or other family of dsRNA virus. Exemplary dsRNA viruses and virus genera include, but are not limited to, Picobirnavirus, Rotavirus, Seadornavirus, Coltivirus, Orbivirus, and Orthoreovirus, or a subtype, species, or variant thereof.

In some embodiments, the virus is ssRNA virus, e.g., a positive-strand (sense) ssRNA virus, e.g., a Group IV virus. In some embodiments, expression of a PRR (e.g., STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is a positive-strand (sense) ssRNA virus, and is a member of the Arteriviridae, Coronaviridae, Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnaviridae, Piconaviridae, Secoviridae, Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Alphatetraviridae, Alvernaviridae, Astroviridae, Barnaviridae, Bromoviridae, Caliciviridae, Carmotetraviridae, Closteroviridae, Flaviviridae, Leviviridae, Luteoviridae, Narnaviridae, Nodaviridae, Permutotetraviridae, Potyviridae, Togaviridae, or Virgaviridae families, or other family of positive-strand (sense) ssRNA virus. Exemplary positive-strand (sense) ssRNA viruses and virus genera include, but are not limited to, Yellow fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus, Rubella virus, Ross River virus, Sindbis virus, Chikungya virus, Norwalk virus, Japanese encephalitis virus, Tick-borne encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, Kyasanur Forest disease virus (e.g., Monkey disease virus), Western Equine encephalitis virus, Eastern Equine encephalitis virus, Venezuelan Equine encephalitis virus, Sapporo virus, Norovirus, Sapovirus, Calicivirus, Parechovirus, Hepatitis A virus, Rhinovirus (e.g., Rhinovirus A, Rhinovirus B, and Rhinovirus C), Enterovirus (e.g., Enterovirus A, Enterovirus B, Enterovirus C (e.g., poliovirus), Enterovirus D, Enterovirus E, Enterovirus F, Enterovirus G, or Enterovirus H), Apthovirus (e.g., Foot and mouth disease virus), Nidovirales (e.g., Cavally virus, Nam Dinh virus, Middle East respiratory syndrome coronavirus (MERS-CoV), Coronavirus HKU1, Coronavirus NL63, SARS-CoV, Coronavirus OC43, and Coronavirus 229E), Benyvirus, Blunevirus, Cilevirus, Hepevirus (e.g., Hepatitis E virus), Higrevirus, Idaeovirus, Negevirus, Ourmiavirus, Polemovirus, Sobemovirus, or Umbravirus, or a subtype, species, or variant thereof.

In some embodiments, the virus is a member of the genus Norovirus, or a subtype, species, or variant thereof. In some embodiments, the virus is the Norwalk virus, Hawaii virus, Snow Mountain virus, Mexico virus, Desert Shield virus, Southampton virus, Lordsdale virus, or Wilkinson virus, or a subtype or variant thereof. In some embodiments, the virus is a member of the genus Norovirus and can be classified as genogroup GI, genogroup GII, genogroup GIII, genogroup GIV, or genogroup GV.

In some embodiments, the virus is ssRNA virus, e.g., a negative-strand (antisense) ssRNA virus, e.g., a Group V virus. In some embodiments, expression of a PRR (e.g., STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is a negative-strand (antisense) ssRNA virus, and is a member of the Bornaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Nyamiviridae, Arenaviridae, Bunyaviridae, Ophioviridae, or Orthomyxoviridae families, or other family of negative-strand (antisense) ssRNA virus. Exemplary negative-strand (antisense) ssRNA viruses and virus genera include, but are not limited to, Brona disease virus, Ebola virus, Marburg virus, Measles virus, Mumps virus, Nipah virus, Hendra virus, Respiratory syncytial virus, Influenza and Parainfluenza viruses, Metapneumovirus, Newcastle disease virus, Deltavirus (e.g., Hepatitis D virus), Dichohavirus, Emaravirus, Nyavirus, Tenuivirus, Varicosavirus, or a subtype, species, or variant thereof.

In some embodiments, the virus is an ssRNA retrovirus (ssRNA RT virus), e.g., a Group VI virus. In some embodiments, expression of a PRR (e.g., STING) is induced through host- produced or viral-derived RNA. In some embodiments, the virus is an ssRNA RT virus and is a member of the Metaviridae, Pseudoviridae, or Retroviridae families, or other family of ssRNA RT virus. Exemplary ssRNA RT viruses and virus genera include, but are not limited to, Metavirus, Errantivirus, Alpharetrovirus (e.g., Avian leukosis virus, Rous sarcoma virus), Betaretrovirus (e.g., Mouse mammary tumor virus), Gammaretrovirus (e.g., Murine leukemia virus, Feline leukemia virus), Deltaretrovirus (e.g., human T-lymphotropic virus), Epsilonretrovirus (e.g., Walleye dermal sarcoma virus), Lentivirus (e.g., Human immunodeficiency virus 1 (HIV)), or a subtype, species, or variant thereof. In some embodiments, the virus is a DNA virus, e.g., a dsDNA virus or an ssDNA virus. In some embodiments, the virus is a dsDNA virus, e.g., a Group I virus, and expression of a PRR (e.g., STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is a dsDNA virus and is a member of the Myoviridae, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae, Malacoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfarviridae, Baculoviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae, Marseilleviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Polydnaviruses, Polymaviridae, Poxviridae, Sphaerolipoviridae, Tectiviridae, or Turriviridae families, or other family of dsDNA virus. Exemplary dsDNA viruses and virus genera include, but are not limited to, Dinodnavirus, Nudivirus, smallpox, human herpes virus, Varicella Zoster virus, polyomavirus 6, polyomavirus 7, polyomavirus 9, polyomavirus 10, JC virus, BK virus, KI virus, WU virus, Merkel cell polyomavirus, Trichodysplasia spinulosa- associated polyomavirus, MX polyomavirus, Simian virus 40, or a subtype, species, or variant thereof.

In some embodiments, the virus is an ssDNA virus, e.g., a Group II virus, and expression of a PRR (e.g., STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is an ssDNA virus and is a member of the Anelloviridae, Bacillariodnaviridiae, Bidnaviridae, Circoviridae, Geminiviridae, Inoviridae, Microviridae, Nanoviridae, Parvoviridae, or Spiraviridae families, or other family of ssDNA virus. Exemplary ssDNA viruses and virus genera include, but are not limited to, Torque teno virus, Torque teno midi virus, Torque teno mini virus, Gyrovirus, Circovirus, Parvovirus B19, Bocaparvovirus, Dependoparvovirus, Erythroparvovirus, Protoparvovirus, Tetraparvovirus, Bombyx mori densovirus type 2, lymphoidal parvo-like virus, Hepatopancreatic parvo-like virus, or a subtype, species, or variant thereof.

In some embodiments, the virus is a dsDNA reverse transcriptase (RT) virus, e.g., a Group VII virus, and expression of a PRR (e.g., STING) is induced through host-produced or viral- derived RNA. In some embodiments, the virus is a dsDNA RT virus and is a member of the Hepadnaviridae, or Caulimoviridae families, or other family of dsDNA RT virus. Exemplary dsDNA RT viruses and virus genera include, but are not limited to, Hepatitis B virus, or a subtype, species, or variant thereof. In some embodiments, the virus (e.g., a virus described herein) is latent, e.g., within a cell. In some embodiments, the virus is an RNA virus (e.g., a double-stranded RNA (dsRNA) virus, a single-stranded RNA (ssRNA) virus (e.g., a positive-strand (sense) ssRNA virus or a negative- strand (antisense) ssRNA virus), or a ssRNA retrovirus) or a DNA virus (e.g., a dsDNA virus, ssDNA virus, or a dsDNA retrovirus) and is latent, e.g., within a cell. In some embodiments, the virus is a Group I, Group II, Group III, Group IV, Group V, Group VI, or Group VII class of virus, e.g., according to the Baltimore classification system, and is latent, e.g., within a cell.

In some embodiments, the virus is an RNA virus (e.g., an RNA virus described herein) and is latent, e.g., within a cell. In some embodiments, the virus is an ssRNA retrovirus (ssRNA RT virus), e.g., a Group VI virus, and is latent, e.g., within a cell. In some embodiments, the virus is the human immunodeficiency virus 1 (HIV)), or a subtype, species, or variant thereof, and is latent, e.g., within a cell.

In some embodiments, the methods of inducing expression of a PRR (e.g., STING) in a subject suffering from a viral infection disclosed herein result in an increase in PRR expression (e.g., STING expression). In some embodiments, expression of a PRR (e.g., STING) is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more. In some embodiments, induction of expression of a PRR (e.g., STING) occurs within about 5 minutes of administration of a nanoparticle disclosed herein or a composition thereof. In some embodiments, induction of expression of a PRR (e.g., STING) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a nanoparticle disclosed herein or a composition thereof to a subject.

Treatment of Bacterial Infections

Recent studies have shown that PRRs (e.g., STING) play a critical role in host recognition of bacterial infections stemming from a variety of species (Dixit, E. and Kagan, J.C. Adv Immunol (2013) 117:99-125). In some cases, bacteria may secrete nucleic acids during the exponential growth phase (e.g., Listeria monocytogenes; Abdullah, Z. et al, EMBO J (2012) 31:4153-4164), which in turn are detected by PRRs such as RIG-I and thus promote the induction of further PRR expression. In other cases, such as for Legionella pneumophila, bacterial DNA enters into the cytosol over the course of infection and is transcribed into an RNA ligand for RIG-I (Chiu, Y. H. et al, Cell (2009) 138:576-591), thus triggering downstream PRR-mediated signaling events. PRR expression (e.g., STING expression) may further be induced upon recognition of RNA released during phagocytotic uptake of bacteria. Additionally, bacterial cell wall components such as peptidoglycans (e.g., muramyl dipeptide, i.e., MDP) may serve as ligands for activation and induction of PRRs, namely NOD2, and bacterial-derived nucleic acids such as cyclic dinucleotides (e.g., cyclic di-GMP) may bind to and activate PRRs, in particular STING. In some embodiments, the expression of one or more PRRs may be induced through other means not explicitly recited herein.

In some embodiments, the methods of inducing expression of a PRR (e.g., STING) disclosed herein comprise administration of an effective amount of a nanoparticle disclosed herein or a composition thereof to a subject infected with a microbial infection, e.g., a bacterial infection.

In some embodiments, the bacterium is a Gram-negative bacterium or a Gram-positive bacterium. Exemplary bacteria include, but are not limited to, Listeria (e.g., Listeria monocytogenes), Francisella (e.g., Francisella tularensis), Mycobacteria (e.g., Mycobacteria tuberculosis), Brucella (e.g., Brucella abortis), Streptococcus (e.g., group B Streptococcus), Legionella (e.g., Legionella pneumophila), Escherichia (e.g., Escherichia coli), Pseudomonas (e.g., Psuedomonas aeruginosa), Salmonella (e.g., Salmonella typhi), Shigella (e.g., Shigella flexneri), Campylobacter (e.g., Campylobacter jejuni), Clostridium (e.g., Clostrodium botulinum), Enterococcus (e.g., Enterococcus faecalis), Vibrio (e.g., Vibrio cholera), Yersinia (e.g., Yersinia pestis), Staphylococcus (e.g., Staphylococcus aureus), or other genera, species, subtypes, or variants thereof.

In some embodiments, the methods of inducing expression of a PRR (e.g., STING) in a subject suffering from a bacterial infection disclosed herein result in an increase in PRR expression (e.g., STING expression). In some embodiments, expression of a PRR (e.g., STING) is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more. In some embodiments, induction of expression of a PRR (e.g., STING) occurs within about 5 minutes of administration of nanoparticle disclosed herein or a composition thereof. In some embodiments, induction of expression of a PRR (e.g., STING) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a nanoparticle disclosed herein or a composition thereof.

Treatment of Cancer

Many patients with advanced solid tumors show a spontaneous T cell-inflamed tumor microenvironment, which is predictive of prognosis and clinical response to immunotherapies. Recent findings suggest the STING pathway of cytosolic DNA sensing is an important innate immune sensing mechanism driving type I IFN production in the tumor context. Knowledge of this pathway is guiding the further development of novel immunotherapeutic strategies.

In early-stage colorectal cancer, the presence of activated CD8+ T cells within the tumor microenvironment is prognostic of a positive outcome. Patients with other solid tumor histology also appear to have a spontaneous T cell infiltrate that may have similar positive prognostic value. These include breast cancer, renal cell carcinoma, melanoma, ovarian cancer, and gastrointestinal tumors. It is believed that T cell infiltrate includes tumor antigen-specific T cells that have been activated spontaneously in response to the growing tumor, perhaps through immune surveillance mechanisms. This attempted host immune response, even if it does not eliminate the tumor completely, is thought to delay tumor progression and thus yield improved clinical outcome. Furthermore, the innate immune mechanisms can lead to adaptive T cell response against tumor antigens even in the absence of exogenous infection. In this regard, human cancer gene expression profiling studies reveal an association between a type I IFN signature, T cell infiltration, and clinical outcome. Thus, innate immune sensing pathways that trigger type I IFN production might represent crucial intermediate mechanistic step. In gene expression profiling of melanoma, two major subsets of tumor microenvironment have been found that represent either the presence or absence of a transcriptional profile indicative of T cell infiltrate. In fact, CD8+ T cells, macrophages, as well as of some B cells and plasma cells in these lesions in melanoma metastases is similar to the phenotype described in early-stage colon cancer and other tumors in which activated T cells have been associated with favorable prognosis. CD8+ T cells were required for the up-regulation of all immune factors within the tumor micro-environment. Studies indicate that IFN production is necessary for optimal T cell priming against tumor antigens. There are many PRRs that trigger IFN- ^ ^production by host DCs in response to a growing tumor in^vivo including STING. STING is an adapter protein that is activated by cyclic dinucleotides generated by cyclic GMP-AMP synthase (cGAS), which in turn is directly activated by cytosolic DNA. In the presence of these cyclic dinucleotides and/or DNA, STING is translocated from the endoplasmic reticulum to various perinuclear components; for example, palmitoylation of STING at the Golgi has been shown to be essential for STING activation (Mukai, K. et al (2016) Nat Commun doi:10.1038/ncomms11932).

Activated STING forms aggregates, activates TBK1, which in turn phosphorylates interferon regulatory factor 3 (IRF3) that directly contributes to type I IFN gene transcription. This pathway has been implicated in the sensing of DNA viruses, and also in selected autoimmune models. Moreover, activating mutations of STING have recently been identified in human patients with a vasculitis/pulmonary inflammation syndrome that is characterized by increased type I IFN production. Mechanistic studies using mouse transplantable tumor models revealed that STING‐ knockout mice, and IRF3-knockout mice showed defective spontaneous T cell priming against tumor antigens in^ vivo, and rejection of immunogenic tumors was ablated. Similarly, tumor- derived DNA was found within the cytosol of a major population of tumor-infiltrating DCs, and this was associated with STING pathway activation and IFN- ^^production. Therefore, the host STING pathway appears to be an important innate immune sensing pathway that detects the presence of a tumor and to drive DC activation and subsequent T cell priming against tumor- associated antigens in^ vivo. A functional role for the STING pathway in^ vivo^has also been reported in other mouse-tumor systems. An inducible glioma model was shown to result in induction of a type I IFN gene signature as part of the host response. This induction was substantially reduced in STING-knockout mice, and tumors grew more aggressively, leading to shorter mouse survival. Exogenous delivery of cyclic dinucleotides as STING agonists exerted a therapeutic effect in^vivo. A crucial role for host type I IFNs and the host STING pathway was also confirmed in the B16.OVA and EL4.OVA models in response to cryo-ablation. Interestingly, the mechanisms involved paralleled what was observed in the Bm12 mouse model of lupus because host STING was also required for maximal production of anti-DNA antibodies. Thus, the antitumor immune response triggered in part by tumor DNA has overlap with the mechanisms involved in autoimmunity driven by extracellular DNA. A role for STING also has been explored in an inducible colon cancer model. It seems likely that the ability of a cancer in an individual patient to support STING pathway activation is linked to the spontaneous generation of a T cell- inflamed tumor microenvironment. Because this phenotype is associated with improved prognosis of early-stage cancer patients, and also with clinical response to immunotherapies in the metastatic setting, failed STING activation may therefore represent an early functional block, and thus itself may have prognostic/predictive value as a biomarker. Second, strategies that activate or mimic the output of the host STING pathway should have immunotherapeutic potential in the clinic. In as much as non-T cell-inflamed tumors appear to lack evidence of a type I IFN transcriptional signature, strategies to promote robust innate signaling via APCs in the tumor microenvironment might facilitate improved cross-priming of tumor antigen-specific CD8+ T cells, and also augment chemokine production for subsequent oncolytic activity.

Recognition of nucleic acid ligands by a PRRs such as cGAS, RIG-I and/STING stimulates the production of type I interferons (e.g., IFN-α or IFN-β), thus triggering a series of downstream signaling events that may lead to apoptosis in susceptible cells. In recent years, a connection between the induction of PRR expression and a number of cancers has been discovered. For example, RIG-I expression has been shown to be significantly downregulated in hepatocellular carcinoma, and patients exhibiting low RIG-I expression in tumors had shorter survival and poorer responses to IFN-α therapy (Hou, J. et al, Cancer Cell (2014) 25:49-63). As such, it has been suggested that the level of RIG-I expression may be useful as a biomarker for prediction of prognosis and response to immunotherapy. In other cases, induction of RIG-I expression has been shown to induce immunogenic cell death of pancreatic cancer cells, prostate cancer cells, breast cancer cells, skin cancer cells, and lung cancer cells (Duewell, P. et al, Cell Death Differ (2014) 21:1825-1837; Besch, R. et al, J Clin Invest (2009) 119:2399-2411; Kaneda, Y. Oncoimmunology (2013) 2:e23566; Li, X.Y. et al, Mol Cell Oncol (2014) 1:e968016), highlighting a new approach in immune-mediated cancer treatment.

STING is recognized as the key adapter protein in the cGAS-STING-IFN cascade, although it is also reported to be a sensor for DNA. A role for STING in the stimulation of innate immunity in response to cancer has also been identified. Recent studies have revealed the presence of tumor-derived DNA in the cytosol of certain antigen-presenting cells, such as tumor-infiltrating dendritic cells, likely generated through tumor cell stress or cell death. This tumor-derived DNA is known to activate cGAS which causes the production of cyclic nucleotides that have been shown to activate STING, resulting in production of associated type 1 interferons (Woo, S.R. et al, Immunity (2014) 41:830-842). Stimulation of STING and resulting downstream signaling pathways also likely contributes to effector T cell recruitment into the inflamed tumor microenvironment (Woo, S. R. Trends in Immunol (2015) 36:250-256). STING activation in the tumor microenvironment can induce adaptive immune response leading to anti-tumor activity. Hence, in those tumors that are STING-deficient, the described herein can still have anti-tumor activity through activation of antigen-presenting cells and dendritic cells, (APCs and DCs) and induction of adaptive immune response.

In some embodiments, the methods of inducing expression of a PRR (e.g., a PRR described herein) comprise administration of an effective amount of a nanoparticle disclosed herein or a composition thereof to a subject suffering from cancer. In some embodiments, the methods of inducing expression of STING disclosed herein comprise administration of a nanoparticle of the disclosure to a subject suffering from cancer. In some embodiments, the methods of inducing expression of RIG-I disclosed herein comprise administration of nanoparticle disclosed herein or a composition thereof to a subject suffering from cancer. In some embodiments, the methods of inducing expression of NOD2 disclosed herein comprise administration of a nanoparticle disclosed herein or a composition thereof to a subject suffering from cancer. In some embodiments, the cancer is selected from a cancer of the breast, bone, brain, cervix, colon, gastrointestinal tract, eye, gall bladder, lymph nodes, blood, lung, liver, skin, mouth, prostate, ovary, penis, pancreas, uterus, testicles, stomach, thymus, thyroid, or other part of the body. In some embodiments, the cancer comprises a solid tumor (e.g., a carcinoma, a sarcoma, or a lymphoma). In some embodiments, the cancer is a hepatocellular carcinoma or other cancer of the liver. In some embodiments, the cancer is a leukemia or other cancer of the blood. In some embodiments, the cancer comprises breast cancer, renal cell carcinoma, colon cancer, melanoma, ovarian cancer, head and neck squamous cell carcinoma, pancreatic cancer, prostate cancer, lung cancer, brain cancer, thyroid cancer, renal cancer, testis cancer, stomach cancer, urothelial cancer, skin cancer, cervical cancer, endometrial cancer, liver cancer, lung cancer, lymphoma or gastrointestinal stromal cancer and solid tumors. In some embodiments, the cancer cells (e.g., tumor cells) comprise specific cancer-associated antigens that induce a T-cell-mediated anti-tumor response. In some embodiments, the methods of inducing expression of a PRR (e.g., STING, RIG-I, MDA5, LGP2) in a subject suffering from a cancer disclosed herein result in an increase in PRR expression (e.g., STING expression). In some embodiments, expression of a PRR (e.g., STING) is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more. In some embodiments, induction of expression of a PRRs e.g., STING) occurs within about 5 minutes of administration nanoparticle disclosed herein or a composition thereof. In some embodiments, induction of expression of a PRRs (e.g., STING) occurs within about 5 minutes of administration of a nanoparticle disclosed herein or a composition thereof. In some embodiments, induction of expression of a PRR (e.g., STING) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of nanoparticle disclosed herein or a composition thereof. It is recognized that activation of STING by nanoparticle disclosed herein or a composition thereof may lead to induction of expression of other PRRs such as RIG-I, MDA5, NOD2 etc. which may further amplify IFN production in the tumor microenvironment and prime T-cells for enhanced anti-tumor activity.

In some embodiments, the methods of inducing expression of a PRR (e.g., STING) in a subject suffering from a cancer disclosed herein result in an increase in PRR expression (e.g., STING expression). In some embodiments, expression of a PRR (e.g., STING) is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more. In some embodiments, induction of expression of a PRR (e.g., STING) occurs within about 5 minutes of administration of a nanoparticle of the disclosure. In some embodiments, induction of expression of a PRR (e.g., STING) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a nanoparticle of the disclosure.

Pharmaceutical Compositions

While a nanoparticle of the present disclosure may be administered alone, it is preferable to administer said nanoparticles as a pharmaceutical composition or formulation, where the nanoparticles are combined with one or more pharmaceutically acceptable diluents, excipients or carriers. The nanoparticles according to the disclosure may be formulated for administration in any convenient way for use in human or veterinary medicine. In certain embodiments, the nanoparticles included in the pharmaceutical preparation may be active itself, or may be a prodrug, e.g., capable of being converted to an active nanoparticles in a physiological setting. Regardless of the route of administration selected, the nanoparticles of the present disclosure, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present disclosure, are formulated into a pharmaceutically acceptable dosage form such as described below or by other conventional methods known to those of skill in the art.

The amount and concentration of nanoparticles of the present disclosure in the pharmaceutical compositions, as well as the quantity of the pharmaceutical composition administered to a subject, can be selected based on clinically relevant factors, such as medically relevant characteristics of the subject (e.g., age, weight, gender, other medical conditions, and the like), the solubility of nanoparticles in the pharmaceutical compositions, the potency and activity of the nanoparticles, and the manner of administration of the pharmaceutical compositions. For further information on Routes of Administration and Dosage Regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

Thus, another aspect of the present disclosure provides pharmaceutically acceptable compositions comprising a therapeutically effective amount or prophylactically effective amount of a nanoparticle described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present disclosure may be specially formulated for administration in solid or liquid form, including those adapted for oral, intratumoral, parenteral administration, for example, by subcutaneous, intramuscular, intraperitoneal, or intravenous injection as, for example, a sterile solution or suspension. However, in certain embodiments the subject nanoparticles may be simply dissolved or suspended in sterile water. In certain embodiments, the pharmaceutical preparation is non-pyrogenic, i.e., does not elevate the body temperature of a patient.

The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of the nanoparticles other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

The phrase "pharmaceutically acceptable" is employed herein to refer to those nanoparticles, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, stabilizing agent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject antagonists from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) ascorbic acid; (17) pyrogen-free water; (18) isotonic saline; (19) Ringer's solution; (20) ethyl alcohol; (21) phosphate buffer solutions; (22) cyclodextrins such as Captisol®; and (23) other non-toxic compatible substances such as antioxidants and antimicrobial agents employed in pharmaceutical formulations. As set out above, certain embodiments of the nanoparticles described herein may contain a basic functional group, such as an amine, and are thus capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of nanoparticles of the present disclosure. These salts can be prepared in situ during the final isolation and purification of the nanoparticles of the disclosure, or by separately reacting a purified nanoparticles of the disclosure in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like (see, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci.66:1-19).

In other cases, the nanoparticles of the present disclosure may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of the nanoparticles of the present disclosure (e.g., a nanoparticle disclosed herein). These salts can likewise be prepared in situ during the final isolation and purification of the nanoparticles, or by separately reacting the purified nanoparticles in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et al., supra).

Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha- tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

The pharmaceutically acceptable carriers, as well as wetting agents, emulsifiers, lubricants, coloring agents, release agents, coating agents, sweetening, flavoring agents, perfuming agents, preservatives, antioxidants, and other additional components may be present in an amount between about 0.001% and 99% of the composition described herein. For example, said pharmaceutically acceptable carriers, as well as wetting agents, emulsifiers, lubricants, coloring agents, release agents, coating agents, sweetening, flavoring agents, perfuming agents, preservatives, antioxidants, and other additional components may be present from about 0.005%, about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1%, about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 85%, about 90%, about 95%, or about 99% of the composition described herein.

Pharmaceutical compositions of the present disclosure may be in a form suitable for oral administration, e.g., a liquid or solid oral dosage form. In some embodiments, the liquid dosage form comprises a suspension, a solution, a linctus, an emulsion, a drink, an elixir, or a syrup. In some embodiments, the solid dosage form comprises a capsule, tablet, powder, dragée, or powder. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages. Pharmaceutical compositions may comprise, in addition to the nanoparticles described herein (e.g., a nanoparticle disclosed herein) or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and may optionally further comprise one or more pharmaceutically acceptable excipients, such as, for example, stabilizers (e.g., a binder, e.g., polymer, e.g., a precipitation inhibitor, diluents, binders, and lubricants.

In some embodiments, the composition described herein comprises a liquid dosage form for oral administration, e.g., a solution or suspension. In other embodiments, the composition described herein comprises a solid dosage form for oral administration capable of being directly compressed into a tablet. In addition, said tablet may include other medicinal or pharmaceutical agents, carriers, and or adjuvants. Exemplary pharmaceutical compositions include compressed tablets (e.g., directly compressed tablets), e.g., comprising a nanoparticle of the present disclosure or a pharmaceutically acceptable salt thereof.

Formulations of the present disclosure include those suitable for parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the nanoparticle which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about 99 percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent. Pharmaceutical compositions of this disclosure suitable for parenteral administration comprise nanoparticles of the disclosure in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a nanoparticles of the present disclosure, it may be desirable to slow the absorption of the drug from subcutaneous, intraperitoneal, or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered form of the nanoparticles of the present disclosure is accomplished by dissolving or suspending nanoparticles in an oil vehicle.

In some embodiments, it may be advantageous to administer the nanoparticle of the present disclosure in a sustained fashion. It will be appreciated that any formulation that provides a sustained absorption profile may be used. In certain embodiments, sustained absorption may be achieved by combining a nanoparticle of the present disclosure with other pharmaceutically acceptable ingredients, diluents, or carriers that slow its release properties into systemic circulation.

Routes of Administration

The nanoparticles and compositions used in the methods described herein may be administered to a subject in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. Exemplary routes of administration of the compositions used in the methods described herein include topical, enteral, or parenteral applications. Topical applications include but are not limited to epicutaneous, inhalation, enema, eye drops, ear drops, and applications through mucous membranes in the body. Enteral applications include oral administration, rectal administration, vaginal administration, and gastric feeding tubes. Parenteral administration includes intravenous, intraarterial, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrastemal, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time. In certain embodiments of the disclosure, a composition described herein comprising a nanoparticle is administered orally. In other embodiments of the disclosure, a composition described herein comprising a nanoparticle is administered parenterally (e.g., intraperitoneally). It is recognized that for treatment of solid tumors, direct injection of the nanoparticles into the tumor may also be carried out (e.g., intratumoral administration). It is recognized that for treatment of solid tumors, direct injection of the nanoparticles into the tumor may also be carried out (e.g., intratumoral administration).

For intravenous, intraperitoneal, or intrathecal delivery or direct injection (e.g., intratumoral), the composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

The choice of the route of administration will depend on whether a local or systemic effect is to be achieved. For example, for local effects, the composition can be formulated for topical administration and applied directly where its action is desired. For systemic, long term effects, the composition can be formulated for enteral administration and given via the digestive tract. For systemic, immediate and/or short term effects, the composition can be formulated for parenteral administration and given by routes other than through the digestive tract.

Dosages

The compositions of the present disclosure are formulated into acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the compositions of the present disclosure (e.g., a nanoparticle of the present disclosure) may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of absorption of the particular agent being employed, the duration of the treatment, other drugs, substances, and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the composition required. For example, the physician or veterinarian can start doses of the substances of the disclosure employed in the composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the disclosure will be that amount of the substance which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Preferably, the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

Preferred therapeutic dosage levels are between about 0.1 mg/kg to about 1000 mg/kg (e.g., about 0.2 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg) of the composition per day administered (e.g., orally or intraperitoneally) to a subject afflicted with the disorders described herein (e.g., HBV infection). Preferred prophylactic dosage levels are between about 0.1 mg/kg to about 1000 mg/kg (e.g., about 0.2 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg) of the composition per day administered (e.g., orally or intraperitoneally) to a subject. The dose may also be titrated (e.g., the dose may be escalated gradually until signs of toxicity appear, such as headache, diarrhea, or nausea).

The frequency of treatment may also vary. The subject can be treated one or more times per day (e.g., once, twice, three, four or more times) or every so-many hours (e.g., about every 2, 4, 6, 8, 12, or 24 hours). The composition can be administered 1 or 2 times per 24 hours. The time course of treatment may be of varying duration, e.g., for two, three, four, five, six, seven, eight, nine, ten, or more days, two weeks, 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, or more than one year. For example, the treatment can be twice a day for three days, twice a day for seven days, twice a day for ten days. Treatment cycles can be repeated at intervals, for example weekly, bimonthly or monthly, which are separated by periods in which no treatment is given. The treatment can be a single treatment or can last as long as the life span of the subject (e.g., many years).

Patient Selection and Monitoring

The methods of the present disclosure described herein entail administration of a nanoparticle or a pharmaceutically acceptable salt thereof to a subject to activate the PRR for IFNs, ISGs and cytokines production or additionally induce the expression of PRRs (e.g., RIG-I, STING etc.). In some embodiments, the subject is suffering from or is diagnosed with a condition, e.g., a proliferative disease, e.g., cancer. Accordingly, a patient and/or subject can be selected for treatment using a nanoparticle or a pharmaceutically acceptable salt thereof by first evaluating the patient and/or subject to determine whether the subject is infected with a proliferative disease, e.g., cancer. A subject can be evaluated as infected with a proliferative disease (e.g., cancer) using methods known in the art. The subject can also be monitored, for example, subsequent to administration of a nanoparticle described herein or a pharmaceutically acceptable salt thereof.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is an adult. In some embodiments, the subject has a proliferative disease, e.g., cancer. In some embodiments, the subject has a cancer of the of the breast, bone, brain, cervix, colon, gastrointestinal tract, eye, gall bladder, lymph nodes, blood, lung, liver, skin, mouth, prostate, ovary, penis, pancreas, uterus, testicles, stomach, thymus, thyroid, or other part of the body. In some embodiments, the subject has a cancer comprising a solid tumor (e.g., a carcinoma, a sarcoma, or a lymphoma). In some embodiments, the subject has a hepatocellular carcinoma or other cancer of the liver. In some embodiments, the subject has a leukemia or other cancer of the blood. In some embodiments, the subject has a breast cancer, renal cell carcinoma, colon cancer, melanoma, ovarian cancer, head and neck squamous cell carcinoma, pancreatic cancer, prostate cancer, lung cancer, brain cancer, or gastrointestinal stromal cancer. In some embodiments, the subject has cancer cells (e.g., tumor cells) comprising specific cancer- associated antigens that induce a T-cell response.

In some embodiments, the subject is treatment naïve. In some embodiments, the subject has been previously treated for a proliferative disease (e.g., a cancer). In some embodiments, the subject has relapsed. Combination Therapies

A nanoparticle described herein may be used in combination with other known therapies. Administered“in combination”, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or“concurrent delivery”. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

A nanoparticle described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the nanoparticle described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.

In some embodiments, the combination of a nanoparticle disclosed herein or a pharmaceutically acceptable salt thereof and the additional agent has a synergistic or additive effect. In some embodiments, the term“additive” refers to an outcome wherein when two agents are used in combination, the combination of the agents acts in a manner equal to but not greater than the sum of the individual activity of each agent.

In some embodiments, the term“additive” refers to an outcome wherein when two agents are used in combination, the combination of the agents acts in a manner equal to but not greater than the sum of the individual activity of each agent. In some embodiments, the terms“synergy” or“synergistic” refer to an outcome wherein when two agents are used in combination, the combination of the agents acts so as to require a lower concentration of each individual agent than the concentration required to be efficacious in the absence of the other agent. In some embodiments, a synergistic effect results in a reduced in a reduced minimum inhibitory concentration of one or both agents, such that the effect is greater than the sum of the effects. A synergistic effect is greater than an additive effect. In some embodiments, the agents in the composition herein may exhibit a synergistic effect, wherein the activity at a particular concentration is greater than at least about 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 10, 12, 15, 20, 25, 50, or 100 times the activity of either agent alone.

For example, any of the methods described herein may further comprise the administration of a therapeutically effective amount of an additional agent. Exemplary additional pharmaceutical agents include, but are not limited to, anti-proliferative agents, anti-cancer agents, anti-diabetic agents, anti-inflammatory agents, immunosuppressant agents, and a pain-relieving agent. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In some embodiments, the additional agent is an anti-cancer agent, e.g., an alkylating agent (e.g., cyclophosphamide).

In an embodiment, the additional agent is an immunooncology agent, for example, an agent that activate the immune system, e.g., making it able to recognize cancer cells and destroy them. Exemplary immonooncology compounds are compounds that inhibit the immune checkpoint blockade pathway. In an embodiment, the compound is an antibody such as a PD-1 or PD-L1 antibody or a co-stimulatory antibody. In some embodiments, the compound is an anti-CTLA4 antibody. In another embodiment, the agent is a cell-based agent, such as CAR-t therapy. EXAMPLES

The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Preparation of Exemplary Compounds of Formula I

Step 1: Synthesis of 5’-OH-3’-Levulinyl-2’F-dA (2):

Levulinic acid (2.148 g, 18.5 mmol) was dissolved in dry-dioxane (50 mL) and the solution was cooled to 5-10 ^o C on an ice-water bath. DCC (1.939 g, 9.4 mmol) was added portion wise over 1 h. The ice-water bath was removed and the mixture was allowed to warm to room temperature and stirred for 2 hours. The dicyclohexyl urea precipitate was filtered off, and the precipitate washed with dry-dioxane (10 mL). The filtrate was then added to a solution of 5’DMT- 2’F-3’OH-dA ((1), 5.0 g, 7.4 mmol) in dry pyridine (50 mL) and catalytic amount of DMAP was added under argon. After stirring for 2 hours at room temperature the mixture was evaporated to dryness. The residue was dissolved in DCM (150 mL) and washed with 5% NaHCO3 (100 mL) and brine (100 mL). The organic phase was separated, dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the desired product (2) as a white solid. The product was carried onto the next step without further purification.

Step 2 (Tritylation): Synthesis of (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2- (hydroxymethyl) tetrahydrofuran-3-yl 4-oxopentanoate (3):

5’-OH-3’-Levulinyl-2’F-dA (1) was dissolved in DCM (100 mL) and water (1.33 mL, 74 mmol) was then added. 6% DCA in DCM (100 mL) was added, reaction mixture was stirred at room temperature for 10-15 min. The reaction mixture was quenched by addition of methanol (25 mL). The resulting mixture was washed with 5% NaHCO ₃ solution (150 mL) and brine (150 mL). The organic layers were separated, dried over Na ₂ SO ₄ and concentrated under reduced pressure to give a crude residue. The crude residue was purified on combi-flash silica gel column chromatography eluting with 0-5% MeOH in DCM to give 3.45 g (62% yield) of the desired product (3) as a white solid. Step 3: (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((2- cyanoethoxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidi n-1(2H)-yl)-4-fluoro-2- (hydroxymethyl)tetrahydrofuran-3-yl)oxy)phosphorothioyl)oxy) methyl)-4- fluorotetrahydrofuran-3-yl 4-oxopentanoate (5)

(1) (i)(Coupling):

5’OH-3’-Levulinylated-2’F-deoxy-Adinosine ((3), 700 mg, 1.48 mmol) and 5’DMT-2’F- 3’CED-Phosphoamidite-deoxy-Uridine ((4), 1.66 g, 2.22 mmol) mixture was dried under high vacuum for 1-2 hours. Argon was flushed over R.B. flask containing reaction mixture. Anhydrous acetonitrile (40 mL) was added to reaction mixture followed by ETT (279 mg, 2.146 mmol) in acetonitrile solution (5.0 mL) under an atmoshell of argon. The resulting mixture was stirred at room temperature under argon for 2 h. Once TLC analysis showed completion of the reaction, water was added (80 µL, 2 equivalents to amidite). The resulting mixture was carried onto the next step.

(ii) (Sulfurization): In a silanized flask, Beaucage reagent (3H-BD) (592 mg, 2.96 mmol) was dissolved in acetonitrile (5.0 mL). The above coupling reaction mixture containing (5) was added to the solution of sulfurizing reagent (3H-BD) in acetonitrile under an atmoshell of argon and the resulting mixture was stirred at room temperature for 45 minutes to allow the sulfurization reaction to go to completion. Methanol (10 mL) was then added and the resulting mixture was stirred for 30 min. The reaction mixture was concentrated under reduced pressure. The crude residue was dissolved in DCM (100 mL) and washed with water (75 mL). The organic layer was separated, dried over Na ₂ SO ₄ , and used in the next step (detritylation).

(2) Detritylation: The DCM solution containing the product from the previous step was cooled to ice-water bath in a R.B. flask.5% PTSA solution in DCM:MeOH (7:3, 100 mL) was added and the resulting mixture stirred for 15 minutes to allow the detritylation reaction to go to completion. Water (50 mL) was then added and the resulting mixture was stirred for another 15 minutes. The reaction mixture was transferred to separatory funnel water and the phases were separated. The organic layer was washed 5% NaHCO3 solution (100 mL) until the pH of the aqueous layer was adjusted to above 7.0. The organic layer was then dried over Na ₂ SO ₄ and concentrated under reduced pressure. The crude product was purified using Combiflash silica gel column chromatography eluting with 0-5% MeOH in DCM to give 960 mg of the desired product (5) as white solid.

Step 4 (Levulinyl group deprotection): O-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4- fluoro-3-hydroxytetrahydrofuran-2-yl)methyl) O-(2-cyanoethyl) O-((2R,3R,4R,5R)-5-(2,4- dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-2-(hydroxymeth yl)tetrahydrofuran-3-yl) phosphorothioate (6)

3’-Leculinyl protected dinucleotide thiophosphate was treated with 0.5M hydrazine monohydrate in a mixture of pyridine:acetic acid (3:2) and the resulting mixture was stirred at room temperature for 15 minutes. Once TLC analysis showed reaction completion, 2,4- pentanedione (2.0 mL) was then added to the reaction mixture in order to quench unreacted hydrazine hydrate. The volatiles were removed under reduced pressure and the resulting mixture was partitioned between 25% IPA in DCM (50 mL) and water (50 mL). The organic layers were separated and concentrated to dryness under reduced pressure to give a thick liquid, which was co- evaporated with toluene (2 x 15 mL) to give a crude residue. The crude product was purified using Combiflash silica gel column chromatography eluting with 0-10% MeOH in DCM to give 725 mg of the desired product (6) as white solid.

Step 5a: N-(9-((2R,3R,3aR,7aR,9R,10R,10aR,14aR)-5,12-bis(2-cyanoethox y)-9-(2,4-dioxo- 3,4-dihydropyrimidin-1(2H)-yl)-3,10-difluoro-5,12-disulfidoo ctahydro-2H,7H-difuro[3,2- d:3',2'-j][1,3,7,9]tetraoxa[2,8]diphosphacyclododecin-2-yl)- 9H-purin-6-yl)benzamide (7)

(i) Cyclization: Dinucleotide phosphorothioate trimester (6) (1 equivalent) and 2- cyanoethyl tetra isopropyl phosphorodiamidite (bisamidite) (1 equivalent) were dissolved in a mixture of dry acetonitrile and dry DCM (2:1, 30 mL). Disopropylaminotetrazolide (1 equivalent) was then added to the reaction mixture in 4 portions over a period of 1 hour under an inert atmoshell. The solution was stirred for an additional 2 h at r.t. ETT (2.0 equivalent) was then added and the reaction mixture stirred overnight. Deoxygenated water (29 μL) was then added to reaction mixture. The crude product was carried on to the next step without further purification.

(ii) Sulfurization (Synthesis of protected cyclic phosphorothiodiphosphate): Beaucage reagent (3H-BD) (2.0 equivalent) was dissolved in acetonitrile in a silanized flask. One portion of above cyclization product (two thirds) was added to sulfurizing reagent under an atmoshell of argon. The resulting mixture was stirred at room temperature for 45 minutes. Methanol (10 mL) was then added and the reaction mixture was stirred for 30 minutes. The solvents were removed under reduced pressure and the crude residue was dissolved in DCM (50 mL) and washed with water (50 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified using Combiflash silica gel column chromatography eluting with 0-10 MeOH in DCM to give 150 mg of desired product (7).

Step 5b (Oxidation): N-(9-((2R,3R,3aR,7aR,9R,10R,10aR,14aR)-5,12-bis(2-cyanoethox y)-9- (2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,10-difluoro-5-ox ido-12-sulfidooctahydro- 2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9]tetraoxa[2,8]diphosphacy clododecin-2-yl)-9H-purin-6- yl)benzamide (8)

TBHP (4.0 equivalent) was added to a stirred solution of second portion of cyclization product from Step 5(a) (i) (one third) at 0 ^o C and the reaction mixture was warmed to r.t. over 15 minutes. Excess TBHP was then quenched by the addition of saturated sodium bisulfite solution. The resulting mixture was evaporated under reduced pressure. The resulting residue was dissolved in DCM (25 mL) and washed with water (20 mL). The organic layers were separated, dried over Na ₂ SO ₄ , and concentrated under reduced pressure to give crude product. The crude product was purified using Combiflash silica gel column chromatography eluting with 0-10% MeOH in DCM to give 60 mg of desired product (8).

Synthesis of Compound 2: Ammonium (2R,3R,3aR,7aR,9R,10R,10aR,14aR)-2-(6-amino- 9H-purin-9-yl)-9-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3 ,10-difluorooctahydro- 2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9]tetraoxa[2,8]diphosphacy clododecine-5,12-bis(thiolate) 5,12-dioxide

Protected cyclic phosphorothiodiphosphate (8) (60 mg) was dissolved in conc. NH4OH (2.0 mL) and stirred at r.t. overnight. Once LCMS showed reaction completion, reaction mixture was evaporated under reduced pressure to remove the ammonia. The water layer was washed with ethyl acetate (5 x 5 mL), separated and lyophilized to provide 100 mg of Compound 2 as white fluffy solid.

Synthesis of Compound 4: ((((2R,3R,3aR,7aR,9R,10R,10aR,14aR)-2-(6-amino-9H-purin-9- yl)-9-(2,6-dioxo-2H-1,3-oxazin-3(6H)-yl)-3,10-difluoro-5,12- dioxidooctahydro-2H,7H- difuro[3,2-d:3',2'-j][1,3,7,9]tetraoxa[2,8]diphosphacyclodod ecine-5,12- diyl)bis(sulfanediyl))bis(methylene))bis(4,1-phenylene) bis(4-(decyloxy)benzoate)

Compound 2 (25 mg) was dissolved in water (250 µL) and a solution of 4- (iodomethyl)phenyl 4-(decyloxy)benzoate (42 mg) in a mixture of THF:Acetone (1:1, 2.0 mL) was then added. The pH of the reaction mixture was approximately 3.5-4.0. The reaction mixture was then stirred at r.t. for 40 hours. The crude product was purified using Combiflash silica gel column chromatography eluting with 0-10% IPA in DCM to give 25 mg of Compound 4 as yellowish brown solid. Synthesis of Compound 3: Ammonium (2R,3R,3aR,7aR,9R,10R,10aR,14aR)-2-(6-amino- 9H-purin-9-yl)-9-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3 ,10-difluoro-12- sulfidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9]tetraox a[2,8]diphosphacyclododecin-5- olate 5,12-dioxide

Protected cyclic phosphoro monothiodiphosphate (8) (60 mg) was dissolved in conc. NH ₄ OH (5.0 mL) and stirred at r.t. overnight. Once LCMS showed reaction completion, the reaction mixture was concentrated under reduced pressure to remove the ammonia. The water layer was washed with ethyl acetate (5 x 5 mL), separated, and lyophilized to provide 50 mg of Compound 3 as a white fluffy solid.

Synthesis of Compound 9: Ammonium (2R,3R,3aR,7aR,9R,10R,10aR,14aR)-2-(6-amino- 9H-purin-9-yl)-12-((4-((4-(decyloxy)benzoyl)oxy)benzyl)thio) -9-(2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-3,10-difluorooctahydro-2H,7H-difu ro[3,2-d:3',2'- j][1,3,7,9]tetraoxa[2,8]diphosphacyclododecin-5-olate 5,12-dioxide

Compound 3 (20 mg) was dissolved in water (200 µL) and a solution of 4-(iodomethyl) phenyl 4-(decyloxy)benzoate (18 mg), in a mixture of THF:Acetone (1:1, 1.4 mL) was added. The pH of the reaction mixture was approximately 4.0. The reaction mixture then stirred at r.t. overnight. The solvent was removed under reduced pressure and the crude product was redissolved in water:acetonitrile (1:1, 2.0 mL). The resulting precipitate (unreacted alkylating reagent) was then removed by centrifugation. The mother liquor was lyophilized to provide 12 mg of crude product which was purified by using C18 Sep pack column (Waters, 4.0 g) using 0.2 M ammonium acetate buffer. The compound was eluted with acetonitrile:water (1:1). The pure fractions were collected and lyophilized to provide 5-6 mg of Compound 9 as a white fluffy solid.

Alkylation of cyclic phosphoromonothio diphosphate [Synthesis of Cmd 1`]: Cyclic phosphoromonothio diphosphate (20 mg) was dissolved in water (200 µL). A solution of 4- (iodomethyl)phenyl 4-(decyloxy)benzoate (18 mg) in a mixture of THF:Acetone (1:1, 1.4 mL) was then added. The reaction mixture pH was approximately 4.0. The reaction mixture stirred at r.t. overnight and solvents were removed under reduced pressure. The resulting crude residue was redissolved in water:acetonitrile (1:1, 2.0 mL). A precipitate (unreacted alkylating reagent) formed and was removed by centrifugation. The mother liquor was lyophilized and the crude product was purified by using C ₁₈ sep pack column (Waters, 4.0 g) with 0.2M ammonium acetate buffer. The compound was eluted with acetonitrile:water (1:1). The pure fractions were collected and lyophilized to provide 5-6 mg of pure desired product as a white fluffy solid. In vitro induction of IRF and NF-κβ in THP1 cells with compounds of Formula I Table 3: EC ₅₀ values for exemplary compounds of Formula I In Table 3,“A” represents an EC50 of less than 50 nM;“B” an EC50 of between 50 nM and 500 nM;“C” an EC ₅₀ of between 500 nM and 1 µM;“D” an EC ₅₀ of between 1 µM and 2 µM; and“E” an EC ₅₀ of greater than 2 µM. Data are shown as fold induction over cells that received DMSO (compound carrier) alone as the mean, +/- standard deviation of duplicate wells per stimulant. In vitro activation of ISG54 and NF-κβ in HEK293 cells with compounds of formula I

In this experiment, HEK293 cells (SZ14) stably expressing either the ISG54 ISRE-luc reporter or the NF-κβ-luc reporter gene were treated in duplicate with an exemplary compound of the disclosure (e.g., compound 1, compound 2, and compound 3) or 2’,3’-cGAMP as a control, each in digitonin buffer for 5 hours, in order to screen for potential STING agonists. ISG54 or NF-κβ activity was determined using the Steady-glo buffer system (Promega), and are summarized in FIGS. 7A-7B and FIG. 8. Data are shown as fold induction over cells that received DMSO (compound carrier) alone as the mean, +/- standard deviation of duplicate wells per stimulant.

In general, half maximal effective concentration (EC ₅₀ ) refers to the concentration of a drug that induces a response halfway between the baseline and maximum after a specified exposure time. This calculation is applicable for compounds with enzyme inhibition activity, as the baseline for an untreated sample may be set at 100% enzymatic activity, and therefore % inhibition is evaluated based on this 100% maximal basis. For these studies, the EC ₅₀ value relates to the concentration required to achieve a value 50% activity level above the untreated sample set at 0% In Table 4,“A” represents an EC ₅₀ of less than 50 nM;“B” an EC ₅₀ of between 50 nM and 500 nM;“C” an EC ₅₀ of between 500 nM and 1 µM;“D” an EC ₅₀ of between 1 µM and 2 µM; and“E” an EC ₅₀ of greater than 2 µM. Data are shown as fold induction over cells that received DMSO (compound carrier) alone as the mean, +/- standard deviation of duplicate wells per stimulant. Table 4: EC50 values for exem lar com ounds of the disclosure

Evaluation of IRF-type I IFN activity in THP cells and Raw-Lucia cells with compounds of Formula I

THP1-dual cells were treated in triplicate with exemplary compounds of the disclosure in lipofectamine (e.g., compound 2 or compound 3) or 2’,3’-cGAMP in lipofectamine as a control at varying concentrations for 22 hours. Levels of IRF-inducible luciferase reporter activity in the cell culture supernatants were assayed using the Quanti-luc reagent, and are summarized in FIG. 9. Data are shown as fold induction over cells that received DMSO (compound carrier) alone as the mean, +/- standard deviation of duplicate wells per stimulant. Alternatively, THP1-Dual cells (Human monocytes)) and Raw-Lucia cells (Mouse macrophages (RAW)) in 96-well plate were stimulated in triplicate with a compound disclosed herein alone for 24 hrs. Activity of secreted luciferase in cell culture supernatant was measured using Invivogen’s Quanti-luc. Data are shown as fold induction over DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant).

As shown in FIGs.31A-31B, Cmd 1, Cmd 5, Cmd 12, Cmd 13, Cmd 14, and Cmd 15 are more active in human monocytes (FIG.31A) and mouse macrophages (FIG.31B) than the natural STING ligand 3’,3’-cGAMP.

Efficacy of exemplary compounds of Formula I against norovirus, RSV, Junin Virus, Dengue Virus and HCV.

A Replicon of Norovirus strain GI NoV in HG23 (hepatoma) cell line was used and activity assessed by RNA hybridization and quantitative PCR. Cytotoxicity was measured through neutral red method. Infected cells were treated with a compound disclosed herein or 2’-C-methylcytidine (positive control). The results are shown in Table 3 below.

Table 4: Antiviral activity of the compounds against Norovirus

Compound 1 showed a high selectivity index, almost of 300, against norovirus strain GI NoV. CC ₅₀ was 100 µM while EC ₅₀ resulted of 0.342 µM for the HG23 cell line. Efficacy of Compound 1 against RSV. RSVA2-infected (0.5 MOI) A549 cells were used and viral titer was estimated by viral plaque assays. The RSV infected cells were treated with DMSO or 50 ^M, 100 ^M, or 200 ^M of a compound disclosed herein. RSV percentage infection was calculated based on the viral titer values. 100% infection represents RSV infection in vehicle treated cells. For vehicle vs. a compound of the disclosure treated cells p≤ 0.05 using a Student’s t test.

Treatment of RSVA-2 A549 cells with 50 ^M, 100 ^M, and 200 ^M of Compound 1 all decreased RSV titer and RSV percentage infection compared to vehicle (FIGs.12A and 12B) Efficacy of Compound 1 against Junin Virus and Dengue Virus. Activity against Junin (JUNV) and Dengue virus serotype 2 (DSV-2) was conducted using strain JV 4454 and DENV-2 (strain NGC) respectively in Vero cells and extracellular DENV/JUNV yields were determined by plaque assays. Cytotoxicity assays were done in parallel by neutral red, MTT or MTS methods. Infected cells were treated with vehicle or a compound disclosed herein.

As shown in FIG. 15A, virus yield diminished 1 log in A549 infected cells treated with Compound 1 compared to untreated A549 infected cells, both at 24 and 48 hours post infection (h p.i.). The virus yield of Dengue virus serotype 2 (DSV2) diminished 1 log in A549 cells infected with DSV2 compared to untreated A549 infected cells at 24 hours post infection (h p.i.). At 48 hours post infection, no significant difference was found. (FIG.15B)

Efficacy of Compound 1 against HCV Activity against HCV genotypes 1a and1b was tested using a capture fusion assay. THP-1 cells were briefly exposed to donor serum fused with Huh7 derivative cells and qPCR was used to assess HCV replication. Cells were treated with various concentrations of a compound disclosed herein.

As shown in FIGs. 13A-13G, HCV RNA replication was decreased upon treatment with increasing concentrations of Compound 1.

Compound 1 elicited potent antiviral activity against all tested RNA viruses with EC50 ranging from 0.34 to 5.5 μM, and with high selectivity index. Consistent with its mechanism of action, the STING agonist Compound 1 showed potent antiviral activity against several RNA viruses including hemorrhagic fever viruses.

Evaluation of Induction of IRF and NF-KB with compounds of Formula I THP1 dual cells grown in complete media were treated with various concentrations of a compound of the present disclosure or DMSO control. Dual cells carry both secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of an IFN-β minimal promoter fused to five copies of the NF-kB consensus transcriptional response element to measure NF-kB activity and Lucia reporter gene under the control of an ISG54 minimal promoter to measure IRF activity. After 20 h incubation, IRF activity was assessed using QUANTI-luc to measure levels of Lucia and NF-kB activity was determined by measure SEAP levels at 620-655 nm. % induction was calculated from fold change in luminescence/absorbance compared to DMSO treated sample. Any negative values were given base value 1 for plotting data in log scale for accurate demonstration of dose response. EC50 values were generated by curve fit in Xlfit.

Cells grown in complete media were treated with various concentrations of a compound of the disclosure or DMSO control. Dual cells carry both secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of an IFN-β minimal promoter fused to five copies of the NF-kB consensus transcriptional response element to measure NF-kB activity and Lucia reporter gene under the control of an ISG54 minimal promoter to measure IRF activity. After 20 h incubation, IRF activity was assessed using QUANTI-luc to measure levels of Lucia and NF-kB activity was determined by measure SEAP levels at 620-655 nm. % induction was calculated from fold change in luminescence/absorbance compared to DMSO treated sample. EC ₅₀ values are generated by curve fit in Xlfit.

Cmd 1, Cmd 1A, Cmd 1B, Cmd 12, Cmd 13, Cmd 14, Cmd 15 all show induction of IRF and NF-kB. (See FIGS.17A,17B, 18A-18D, 19A-19B, 20A-20D, 21A-21D, 22A-22D, 23A-23D, and 24A-24B) The results indicate that Cmd 1, Cmd 1A, Cmd 1B, Cmd 12, Cmd 13, Cmd 14, Cmd 15 are taken up by cells without the use of transfection agents. Cmd 3 showed no NF-kB activity (FIGS.19A-19B).

FIGS. 27A-27B and 35A-35B compare the induction of IRF (FIGS. 27A and 35A) and NF-κB (FIGS. 27B and 35B) by Cmd 15, Cmd 15-A and Cmd 15-B (isomers of Cmd 15), and Cmd 16.

Determination of Stability of exemplary compounds of Formula I

Serum Stability Study: 0.5 mM of a compound disclosed herein is incubated with Rabbit Serum for various time points at 37 ^o C. The reactions are quenched with addition of 1 mL Acetonitrile. The supernatant with compound was collected after snap freezing and centrifuging @ 4 ^o C for 5 min. The supernatant with compound was later analyzed in HPLC.

Microsome Stability Study: 0.5 mM of a compound disclosed herein is incubated with Human microsomes for various time points at 37 ^o C. The reactions are initiated with 20 mM NADPH, incubated, then quenched with addition of 1 mL Acetonitrile. The supernatant with compound was collected after snap freezing and centrifuging at 4 ^o C for 5 min. The supernatant with compound was later analyzed in HPLC.

As can be seen in FIGS.25A-25B, the isomers of Cmd 1, Cmp1-A and Cmd 1-B, are stable in Rabbit serum and Human microsomes. Peak 1 and peak 2 represent Cmds 1-A and 1-B. Cmd 15 is also stable in Rabbit serum and Human microsomes. FIGS. 26A-26B show the stability of the isomers of Cmd 1, Cmd 151-A and Cmd 15B in Rabbit serum and Human microsomes.

Determination of Cytotoxicity of exemplary compounds of Formula I

The cytotoxicity of exemplary compounds in THP1 cells was assessed using Cell titer Glo Assay (Promega). THP1 dual cells grown in complete media were treated with various concentrations of compounds or DMSO control. The CellTiter-Glo® Luminescent Cell Viability/cytotoxicity was a determined by assessing number of viable cells in culture based on quantitation of the ATP present through a“glow-type" luminescent signal, produced by the luciferase reaction. % apoptosis was calculated from fold change in luminescence compared to DMSO treated sample.

FIG.28 shows the induction of apoptosis through % cytoxicity of THP1 cells when treated with various concentrations (5 ^ ^M, 14 ^ ^M, 41 ^ ^M, 123 ^ ^M, 370 ^ ^M, 1111 ^ ^M, 3333 ^ ^M, and 1000 ^M) of Cmd 15 and its isomers, Cmd 15-A and Cmd 15-B.

Quantification of STING binding with compounds of Formula I

SZ14 HEK293 cells stably expressing the ISG54 ISRE-luc reporter gene were treated with a compounds disclosed herein, 2`3`-cGAMP (natural STING ligand), or DMSO in the presence of digitonin for 5-6 hrs. ISRE-luciferase activity was determined and normalized to DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant).

As shown in FIGS. 29A-29B, the binding of Cmd 1 to STING activates type 1 IFN signaling, similar to the activation of type 1 IFN signaling observed with 2’,3’-cGAMP.

Alternatively, raw-ISG-Dual cells in 96-well plates were stimulated in triplicate with compound/lipo, cGAMP/lipo complex or compound alone for 22-24 hours at 37⁰C, 5% CO ₂ . Activity of secreted luciferase in cell culture supernatant was measured using Invivogen Quanti- luc. Data are shown as fold induction over DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant). As shown in FIG 30, Cmd 1 is highly active in mouse macrophages in activating type 1 IFN signaling, similar to the activation of type 1 IFN signaling observed with 2’,3’-cGAMP.

Induction of Type III IFN (IL-29) production in THP cells by exemplary compounds of Formula I

THP1-Dual (WT) cells were treated in triplicate with an exemplary compound alone or cGAMP/lipo for 21 hrs. Level of IL-29 in culture supernatant was determined using ELISA. Results shown are the average ± standard deviation of duplicate wells.

Treatment of cells with Cmd 1 and Cmd 15 induced type III interferon (IL-29) production in THP1 cells (FIG.33A). This indicates that both Cmd 1 and Cmd 15 are taken up by cells without use of a transfection reagent (FIG.33B).

Induction of Type I IFN production in THP cells by exemplary compounds of Formula I

SZ14 cells (HEK293 stably expressing ISG54 ISRE-luc reporter gene) were treated in triplicate with compound/digitonin buffer for 5 hrs. ISG54 ISRE-luc activity was determined using Promega Steady-Glo luciferase assay buffer and normalized to DMSO treated cells (mean ± standard deviation of triplicate wells).

Alternatively, THP1-Dual (WT) cells were treated in triplicate with compound alone for 3-22h. IRF-type I IFN activity was determined using Quanti-luc buffer and normalized to DMSO treated cells (mean ± standard deviation of triplicate wells). FIG.32A-B and FIG.34

FIGS.32A-32B and 34A-34B show the induction of type I IFN signaling in HEK293 (FIG. 32A) and THP1 (FIG. 32B) cells treated with Cmd 1 and its isomers Cmd 1A (Cmd 1-PK1) and Cmd 1B (Cmd 1-PK2). Cmd 1, Cmd 1-A, Cmd 1-B, Cmd 13 and Cmd 15 all induce type I IFN signaling compared to control.

Fig.44 shows that Cmd 1 causes cell death by apoptosis.

The Apoptosis in THP1 cells was assessed using Caspase-Glo® 3/7 Assay (Promega). THP1 dual cells grown in complete media were treated with various concentrations of Cmd 1 or 2’3-cGAMP or DMSO control with Lipofectamine LTX. The caspase-3 and -7 activity was measured by using a pro-luminescent caspase-3/7 substrate that contains the tetrapeptide sequence DEVD which is cleaved to release amino-luciferin, a substrate of luciferase used in the production of light. After incubation for 20 h, Apoptotic activity was assessed by measuring levels of amino- luciferin. % Apoptosis was calculated from fold-change in luminescence compared to DMSO- treated sample. CC50 values are generated by curve fit in Xlfit.

Fig.45 shows the selective induction of apoptosis by Cmd 1 in acute monocytic leukemia cell line (THP1) vs. PBMCs.

The Apoptosis in THP1 cells and PBMCs was assessed using Caspase-Glo® 3/7 Assay (Promega). THP1 cells and PBMCs grown in complete media were treated with various concentrations of Cmd 1 or 2’3-cGAMP or DMSO control with Lipofectamine LTX. The caspase- 3 and -7 activity was measured by using a proluminescent caspase-3/7 substrate that contains the tetrapeptide sequence DEVD which will be cleaved to release aminoluciferin, a substrate of luciferase used in the production of light. After incubation for 20 h, Apoptotic activity was assessed by measure levels of aminoluciferin. % Apoptosis was calculated from fold change in luminescence compared to DMSO treated sample.

Fig.46 shows that the Cmd 1 causes selective and enhanced induction of ISG and PRR-associated genes in acute monocytic leukemia cell line (THP1) compared to primary cells PBMCs. Gene expression analysis in THP1 and PBMCs

THP1 cells and PBMCs grown in complete media were treated with 5 uM of either Cmd 1 or 2’3-cGAMP or DMSO control with Lipofectamine LTX. After incubation for 20 h, RNA was extracted and gene expression of different Interferon Stimulated Genes (ISGs) and various Pattern Recognition Receptors (PRRs) was evaluated by real time PCR. Fold Induction was calculated by ΔΔct method.

Fig.47 shows that Cmd 1 inhibits tumor cell growth.

Tumor cells in 96-well plate were treated once daily with Cmd 1 (no lipofectamine) or recombinant IFN (U-IFN) for 3 days. Cells were fixed with 1% paraformaldehyde and stained with DAPI. Cells were automatically imaged on ImageXpress and total number of survival cells were analyzed using MetaXpress software. Results are shown as total number of cells per group or % reduction calculated by normalizing to DMSO treated cells.

Fig.55 shows that Cmd 4 has enhanced activity in acute monocytic leukemia cell line (THP1) compared to primary cells PBMCs.

Gene expression analysis in THP1 and PBMCs: THP1 cells and PBMCs grown in complete media were treated with 5 uM of either Cmd 4 or 2’,3’-cGAMP or DMSO control with Lipofectamine LTX. After incubation for 20 h, RNA was extracted and gene expression of different Interferon Stimulated Genes (ISGs) and various Pattern Recognition Receptors (PRRs) was evaluated by real time PCR. Fold Induction was calculated by ΔΔct method. Efficacy of exemplary compounds via intraperitoneal administration in a breast carcinoma model.

The efficacy of intraperitoneal administration of Cmd 1 was investigated in the 4T1.luc2 orthotopic murine breast carcinoma model. Thirty female BALB/c mice between 7-10 weeks old were randomized into four treatment groups based on Day 1 body weight, and the treatment was carried out according to the regimen outlined in Table 4 below. Cmd 1 was dissolved in saline and administered at 10 mL/kg (0.200 mL/20 g mouse), with a cell injection volume of 0.05 mL/mouse.

Table 5. IP administration in breast carcinoma model: study regimen

Each animal was monitored individually. The endpoint of the experiment was a tumor volume of 2000 mm2 or 45 days. Animals in Groups 1 and 2 were subjected to whole body bioluminescent imaging starting on Day 5 and once a week thereafter (Days 12, 19, 26, 33, and 41). At the endpoint, blood and tissue (lung, lymph nodes, spleen, and tumor) was analyzed for presence of metastases and biomarker (CD45, CD3, CD4, CD8, CD11b, CD25, Ly-6G, Ly-6C, FoxP3) levels. As seen in FIG.59, mice treated with Cmd 1 showed a significant decrease in tumor growth compared with control

Determination of maximum tolerated dose of orally administered exemplary compounds.

In order to investigate the maximum tolerated dosage of orally administered compounds, 15 female BALB/c mice between 7-10 weeks old were split into three treatment groups. Each group was administered either Cmd 1 or vehicle orally, according to the schedule outlined in Table 5 below. Cmd 1 was provided at 10 mL/kg (0.200 mL/20 g mouse). Upon oral administration of Cmd 1, once daily or twice daily up to 60 mgkg/day, there were no adverse clinical signs and the compound was well tolerated as shown in Table 5. Table 6. Oral MTD study regimen and results

Preparation of Exemplary Compounds of Formula II

Synthesis of Allyl ((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2- (hydroxymethyl)tetrahydrofuran-3-yl) (2-cyanoethyl) phosphate (C)

Step 1: 5’O-DMT-2’F-3’Phosphramidite-dA (15.0 g, 17.12 mmol) was co-evaporated with anhydrous acetonitrile (2 x 100 mL), and dried under high vacuum for 1 h. Argon was flushed over the residue in the flask. Acetonitrile (150 mL, anhydrous) was added to residue under argon. Allyl alcohol (Aldrich, 99%) (2.32 mL, 34.24 mmol) was added to the solution followed by ETT (2.22 g, 17.12 mmol) in acetonitrile (20 mL). The reaction mixture was stirred at room temperature under argon for 2.5 hours. TLC analysis (98:2 DCM: MeOH, multiple runs) showed completion of the reaction. It was then cooled in an ice water bath to 0-5C. Tert-butyl hydroperoxide (TBHP, 5-6 M solution in nonane, 2.0 equivalents) was added to the reaction mixture dropwise at 0-5 ^o C (ice water bath). The mixture was allowed to warm to room temperature and stirred for an additional 30 minutes at room temperature. Excess TBHP was quenched by cooling the solution followed by the addition of saturated thiosulfate solution (10 mL). Reaction mixture was warmed up to room temperature and solvents were evaporated under reduced pressure to remove acetonitrile. The reaction mixture was partitioned between DCM (150 mL) and water (100 mL). The organic layer was separated and water layer was extracted with DCM (50 mL). The combined organic layers were dried over Na ₂ SO ₄ and filtered to remove the Na ₂ SO ₄ salt.

Step 2 Detritylation: The solution of crude DMT-N-bz-3'-O-Allyl-2'-FdA obtained above (200 mL) in DCM was cooled in an ice-water bath. Para-Toluene Sulfonic Acid (PTSA) (10.0 g) was dissolved in MeOH (60 mL) and diluted with DCM (140 mL) to make 5% PTSA solution in DCM : MeOH (7:3, 200 mL) and added to DMT-N-bz-3'-O-Allyl-2'-FdA. This was stirred at 0- 5C for about 30 minutes and checked for reaction completion by TLC (95:5 DCM: MeOH, Rf = 0.2). When DMT deprotection was completed, water (100 mL) was added and stirred for 15 min whilst the reaction was allowed to warm to room temperature. The mixture was transferred to a separatory funnel and the layers were separated. The aqueous layer was extracted with DCM (25 mL) and the combined organic layers were washed with aq. NaHCO3 (5%, 2 x 100 mL). The organic layer was then washed with saturated brine (100 mL) and dried over Na2SO4. After filtering the salts, the solution was concentrated in vacuo to yield the crude product which was dried under high vacuum to yield a foamy solid. The crude product was dissolved in DCM (30 mL) and added to t-butyl methyl ether (180 mL) to yield a white precipitate, which was collected by filtration. After the first isolation, the product was triturated with t-butyl methyl ether (150 mL) and filtered to obtain a white powder, which was dried under high vacuum for overnight to get 9.3 g (99% yield) of pure product C as white solid.

Synthesis of allyl ((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((2- cyanoethoxy)(((2R,3S,4R,5R)-2-(2,4-dioxo-3,4-dihydropyrimidi n-1(2H)-yl)-4-fluoro-5- (hydroxymethyl)tetrahydrofuran-3-yl)oxy)phosphorothioyl)oxy) methyl)-4- fluorotetrahydrofuran-3-yl) (2-cyanoethyl) phosphate (F)

Step 1 Coupling Reaction for Synthesis of Phosphorothioate Dimer: A mixture of C (1.09 g, 2.0 mmol) and E (1.5 g, 2.0 mmol) was co-evaporated with anhydrous acetonitrile (2 x 40 mL) and dried under high vacuum for 1 h. Argon was flushed over the round bottom flask and anhydrous acetonitrile (40 mL) was added to reaction mixture. ETT (260 mg, 2.0 mmol) in acetonitrile (2.0 mL) was added to the mixture of C and E, under argon. The mixture was stirred at room temperature under argon for 2h. TLC analysis (95:5 DCM: MeOH, Rf = 0.5) indicated reaction completion. Deoxygenated water was added to the reaction mixture (72 µL, 2 equivalents to E).

Step 2 Sulfurization: In a silanized flask, Iyer-Beaucage reagent (3H-BD) (800 mg, 4.0 mmol) was dissolved in acetonitrile (10.0 mL). The reaction mixture of C and E from above was added to a solution of sulfurizing reagent (3H-BD) under argon and stirred at room temperature for 45 minutes to complete the sulfurization reaction. Methanol (10 mL) was added to reaction mixture and it was stirred for 30 min followed by concentration under reduced pressure until dryness. The dried residue was dissolved in DCM (50 mL) and washed with water (50 mL). The DCM layer was collected and dried over Na ₂ SO ₄ and filtered.

Step 3 Detritylation: The dried DCM solution (50 mL) was cooled to approximately 0°C in a round bottom flask. PTSA (2.5 g) was dissolved in methanol (15 mL) and diluted with DCM (35 mL) to make 5% PTSA solution in DCM:MeOH (7:3, 50 mL) which was added to DCM reaction mixture solution and stirred for 15-20 min in an ice water bath. Reaction progress was monitored by TLC (95:5 DCM : MeOH, Rf = 0.15). Water (50 mL) was added and mixed for another 15 minutes. The mixtures were transferred to separator funnel, the water layer was separated and the organic layer was collected. The water layer was extracted with DCM (25.0 mL). The combined organic layers were washed with 5% NaHCO ₃ solution (2 x 50 mL) to ensure the pH of the aqueous layer was > 7.0. The organic layers were then washed with saturated brine and dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give crude product, which was dried under high vacuum. Crude product was purified by combiflash silica gel column chromatography using 0-5% MeOH in DCM to give 550 mg of the desired product F as off white solid.

Synthesis of (2S,3S,4S,5S)-5-(6-benzamido-9H-purin-9-yl)-2-((((2- cyanoethoxy)(((2S,3R,4S,5S)-2-(2,4-dioxo-3,4-dihydropyrimidi n-1(2H)-yl)-4-fluoro-5- (hydroxymethyl)tetrahydrofuran-3-yl)oxy)phosphorothioyl)oxy) methyl)-4- fluorotetrahydrofuran-3-yl (2-cyanoethyl) hydrogen phosphate (G)

To a solution of 3’-Allyl protected dimer (500 mg, 0.565 mmol) in acetone (10 mL) was added sodium iodide (810 mg, 5.41 mmol) and the resulting solution was stirred at 60 ^o C for 1 h. TLC analysis (80:20 DCM : MeOH, Rf = 0.15) showed completion of the reaction. The reaction mixture was cooled to room temperature. DCM (10 mL) was added to the suspension to precipitate of the product. Product was collected by centrifugation, which was triturate with DCM (25 mL) then centrifuged a second time to obtain the product. The product was dried under high vacuum to yield an off white solid. This solid was triturated with 20% MeOH in DCM:tButyl methyl ether (1:1, 25 mL) and collected by centrifugation, which was dried under high vacuum to get 500 mg of product as off white solid. Synthesis of (H)

Dinucleotide G (500 mg, 0.565 mmol) was co-evaporated with anhydrous pyridine (2 x 20 mL), dried under high vacuum, flushed with argon (3 times) and dissolved in anhydrous pyridine (20 mL).1-Mesitylene-2-sulfonyl-3-nitro-1,2,4-triazole (MSNT) (0.838 g, 2.82 mmol) was added to the solution of G at room temperature. The resulting mixture was stirred at room temperature for 1.5 h. Reaction progress was monitored by TLC analysis (90:10 DCM: MeOH) showed completion of the cyclization after 1.5 hours. Toluene (20 mL) was added to the reaction mixture. Solvents were evaporated under reduced pressure to give crude product. The resulting mixture was dissolved in 25% IPA in DCM (50 mL) and washed with water (50 mL). The aqueous layer was extracted with 25% IPA in DCM (50 mL), and the combined organic layers were washed with saturated aq. NaHCO3 (10 mL) and brine (10 mL). Organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give crude product. Crude product was dissolved in 10% MeOH in DCM (5 mL) and precipitated by adding to t-butyl methyl ether (10 mL) (to remove colored impurities). The precipitate was collected by centrifugation. Product was triturated with DCM: t-butyl methyl ether (1:1, 15 mL) and product was collected by centrifugation to give light yellow product. Crude product was purified by combiflash silica gel column chromatography (gradient 0-10% MeOH in DCM) to yield 80 mg of product H as off white solid

Synthesis of 4-(Iodomethyl)phenyl 4-(decyloxy)benzoate (I)

Step 1: To a suspension of benzoic acid derivative (10 g, 0.054 mol) in a 250 mL single neck flask in toluene, thionyl chloride (7.8 mL) was added slowly and stirred at r.t. for 15 minutes followed by heating in an oil bath at 80-85°C to obtain a clear solution that was maintained for ~ 3h. The reaction mixture was cooled to RT and excess thionyl chloride was removed in vacuo. The toluene was concentrated using a rotovap at 40-45°C. It was then co-evaporated twice with ethyl acetate (25 mL). The residue was taken up in ethyl acetate (15ml).4-Hydroxybenzyl alcohol (4.5 g, 0.054 mol) was suspended in ethyl acetate (25 mL) and cooled in an ice bath. TEA (5.5 mL) was added with stirring followed by the addition of the ethyl acetate solution of acid chloride. A suspension forms and this was stirred overnight. The insoluble solids were removed by filtration and the filtrate was transferred to a separatory funnel. The filtrate was diluted with ethyl acetate (200 mL), washed with water (50 mL), and the organic layer washed with brine (50 mL). Concentration after drying gave the crude product, which was taken up in 200 mL of 4:1 Hexane (or Heptanes):EtOAc, and stirred for 2 h to precipitate the product. The precipitated product was filtered and the solid dried under high vacuum to yield 9.0 g (67% yield) of the desired product.

Step 2: To a suspension of 4- hydroxyl benzyl alcohol coupled derivative (9.0 g, 0.026 mol) in a mixture of anhydrous acetonitrile (80 mL) and anhydrous dichloromethane (30 mL) in a 250 mL single neck flask, was added CsI (18.2 g, 0.078 mol) in one portion. To this, BF3.Et ₂ O (8.7 mL) was added slowly and stirred in the dark (covered with aluminum foil) under argon overnight at room temperature. The reaction was found to be complete by TLC Hex: EtOAc (7:3). The product was concentrated and the reaction mixture was worked up by adding water (50 mL) followed by extraction with DCM (200 mL) in a separatory funnel. The organic layer was washed with saturated sodium bicarbonate (25 mL), followed by washing with NaHSO3 (5%, 30 mL). The organic layer was dried over anhydrous Na2SO4, filtered, concentrated to a film and later dried in high vacuum for two days to give 9.6 g (85% yield) the desired product I.

Synthesis of Example 4*

Step 1 Deprotection of cyclic phosphoromonothio diphosphate: Fully protected cyclic phosphoro monothio diphosphate (70 mg) was dissolved in a mixture of conc. NH4OH (2.0 mL) and DCM (5.0 mL) stirred at room temperature overnight. LC-MS analysis showed completion of the reaction. Reaction mixture was transferred to separatory funnel and the DCM layer was removed. The aqueous layer was evaporated under reduced pressure to remove ammonia and was then washed with ethyl acetate (3 x 5 mL) to remove benzamide byproduct completely. The product was isolated from the aqueous layer by lyophilization to yield 60 mg of as white solid.

Step 2: Cyclic phosphoromonothio diphosphate (50 mg, 0.072 mmol) was dissolved in water (500 uL). A solution of I (53 mg, 0.108 mmol) in a mixture of THF: Acetone (1:1, 3.5 mL) was added to the reaction mixture. The solution was stirred at room temperature for two days. Solvents were removed under reduced pressure. The crude product was re-dissolved in THF: acetone (1:1, 5.0 mL) and precipitated by adding to diethyl ether (10 mL) to remove unreacted iodo-compound. The precipitate was collected by centrifugation to yield product as an off white solid. This was re-dissolved in IPA:DCM (1:1, 20 mL) and mixed with water (20 mL), which was formed as a single-phase solution. Saturated sodium chloride (5 mL) was added to achieve separation of the two phases. The organic layer was collected (lower layer) and aqueous layer was re-extracted with IPA:DCM (1:1, 2x10 mL). The combined organic layers were dried over Na ₂ SO _4, filtered, and concentrated under reduced pressure to yield the product as off white solid. The product was re-dissolved in 5% acetonitrile in water (2.0 mL) and lyophilized to obtain 76 mg of product 4 as off white solid.

In vitro induction of IRF and NF-κβ in THP1 cells with exemplary compounds of Formula II

EC ₅₀ values for exemplary compounds of the disclosure.“A” represents an EC50 of less than 50 nM;“B” an EC50 of between 50 nM and 500 nM;“C” an EC50 of between 500 nM and 1 μM;“D” an EC50 of between 1 μM and 2 μM;“E” an EC50 of greater than 2 μM.

Table 5

Evaluation of Induction of IRF and NF-KB with exemplary compounds of Formula II THP1 dual cells grown in complete media were treated with various concentrations of a compound of the present disclosure or DMSO control. Dual cells carry both secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of an IFN-β minimal promoter fused to five copies of the NF-kB consensus transcriptional response element to measure NF-kB activity and Lucia reporter gene under the control of an ISG54 minimal promoter to measure IRF activity. After 20 h incubation, IRF activity was assessed using QUANTI-luc to measure levels of Lucia and NF-kB activity was determined by measure SEAP levels at 620-655 nm. % induction was calculated from fold change in luminescence/absorbance compared to DMSO treated sample. Any negative values were given base value 1 for plotting data in log scale for accurate demonstration of dose response. EC50 values were generated by curve fit in Xlfit. Cells grown in complete media were treated with various concentrations of a compound of the disclosure or DMSO control. Dual cells carry both secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of an IFN-β minimal promoter fused to five copies of the NF-kB consensus transcriptional response element to measure NF-kB activity and Lucia reporter gene under the control of an ISG54 minimal promoter to measure IRF activity. After 20 h incubation, IRF activity was assessed using QUANTI-luc to measure levels of Lucia and NF-kB activity was determined by measure SEAP levels at 620-655 nm. % induction was calculated from fold change in luminescence/absorbance compared to DMSO treated sample. EC ₅₀ values are generated by curve fit in Xlfit.

Prophetic Example of Preparation of Nanoparticles

Copolymers of polyethylene glycol (PEG), and an alkenyl amine (e.g., 2- (hexamethyleneimino)ethyl methacrylate) for use in nanoparticles can be synthesized by methods analogous to those described in this prophetic example. See Nature Nanotechnology, 2017, 12, pp 648–654.

The alkenyl amino monomers can be prepared by the following method. 2- (pentamethyleneimino)ethanol (12.9 g, 0.1 mol), triethylamine (10.1 g, 0.1 mol), and inhibitor hydroquinone (0.11 g, 0.001 mol) can be dissolved in 100 mL THF. Following this methacryloyl chloride (10.4 g, 0.1 mol) can be added dropwise to a three necked flask. The solution can then be refluxed in THF for 2 hours. After reaction, the solution can be filtered to remove the precipitated triethylamine-HCl salts, and THF can be removed by concentration under reduced pressure. The resulting residue can be distilled in vacuo as to yield a colorless liquid. After the synthesis, the monomers can be characterized by 1H NMR.

The co-polymers can be synthesized by an atom transfer radical polymerization (ATRP) method described herein. Alkenyl amino monomers, and polyethylene glycol monomers (e.g., MeO-PEG114-Br) can be charged in a polymerization tube. Then a mixture of solvents such as 2- propanol and DMF can be added to dissolve the monomers. After three cycles of freeze-pump- thaw to remove oxygen, CuBr (14 mg, 0.1 mmol) can be added into the reaction tube under a nitrogen atmosphere, and the tube can be sealed in vacuo. The polymerization can then be carried out at 40°C for 8 hours. After polymerization, the reaction mixture can be diluted with a solvent, such as THF, and passed through an Al2O3 column to remove the catalyst. The solvent can then be removed by rotovap. The residue can then be dialyzed in distilled water and lyophilized to obtain the purified copolymer. The resulting copolymers can be characterized by NMR or gel permeation chromatography. The active agents of the disclosure can be coupled to the copolymers.

Polymeric nanoparticles can be prepared from these copolymers utilizing a solvent evaporation method. For example, the copolymer can be dissolved in a solvent (e.g., THF) and then added to distilled water dropwise under sonication. The solvent can then be removed through ultrafiltration with a 100 kD membrane. Distilled water can then be added to adjust the polymer concentration for use as a stock solution. After micelle formation, the nanoparticles can be characterized by transmission electron microscopy.

Prophetic Example of Preparation of Nanoparticles

Poly(beta-amino ester)s (PBAEs) for use in nanoparticles can be synthesized in a two-step Michael addition reaction. See Nanomedicine: Nanotechnology, Biology, and Medicine 14 (2018) 237–246; Mol. Pharm.2012, 9, pp 3375-83. For example, PBAEs can be synthesized with a molar ratio of 1.1:1 1,4-butanediol diacrylate:4-amino-1-butanol and then end capped with a 0.2 M solution of 1-(3-aminopropyl)-4-methylpiperazine in anhydrous tetrahydrofuran. The PBAEs can then be precipitated twice in a 10× volume of anhydrous diethyl ether, and isolated via centrifugation. They can then be stored under vacuum 2-aminoethyl methacrylatein in order to remove excess diethyl ether. PBAEs can then be dissolved in anhydrous DMSO at a concentration of 100 μg/μL and stored at -20 °C in individual use aliquots. PBAE molecular weight can be determined via gel permeation chromatography.

Diluted solutions of compound and polymer in sodium acetate buffer (25 mM, pH 5.0), can then be mixed in a 1:1 volume ratio and incubated for 10 min to formulate nanoparticles. A instrument such as a Nanosight NS500 (Malvern nstruments, UK) can be used to determine number-averaged nanoparticle hydrodynamic diameter. An instrument such as a Zetasizer Nano ZS (Malvern Instruments, UK) can be used to determine the Z-average hydrodynamic diameter and zeta potential of particles.

Study to Determine the Efficacy of Compound 4* in a CT26 Murine Colon Carcinoma Model Using Female BALB/c Mice

Mice

Female BALB/c mice (BALB/c AnNcr1, Charles River) were eight weeks old on Day 1 of the study and had a body weight range of 15.1 to 19.7 g. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl) and a NIH 31 Modified and Irradiated Lab Diet consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber.

Tumor Cell Culture

CT26 murine colon carcinoma cells were grown in RPMI-1640 medium containing 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin G sodium, 100 μg/mL streptomycin _{sulfate, and 25 μg/mL gentamicin. The cells were cultured in tissue culture} ^{flasks in a humidified} incubator at 37 ^C, in an atmosphere of 5% CO ₂ and 95% air.

In Vivo Implantation and Tumor Growth On the day of implantation, cultured CT26 cells were harvested during log phase growth and resuspended in phosphate buffered saline, pH 7.4 (PBS) at a concentration of 3 x 10 ⁶ cells/mL. Each mouse was injected subcutaneously in the right flank with 3 x 10 ⁵ tumor cells (0.1 mL cell suspension) and tumors were monitored as their volumes approached the target range of 80 to 120 mm ³ . Eleven days after tumor cell implantation, on Day 1 of the study, animals were sorted into three groups (n=8/group) with individual tumor volumes of 63 to 126 mm ³ , and a group mean tumor volume of 105 mm ³ . Tumors were measured with a caliper twice weekly for the duration of the study. Tumor size was calculated using the formula:

wherein w is width and l is length, in mm, of a tumor. Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm3 of tumor volume. Test Articles Compound 4* was dissolved by adding the appropriate volume of sterile saline (vehicle) into each tube, vortexing, incubating at 37 °C for 2-5 minutes, followed by sonication, if needed. Preparations of Compound 4* resulted in the appropriate 0.2 and 0.6 mg/mL dosing solutions which provided 1 and 3 mg/kg doses in a dosing volume of 5 mL/kg, adjusted to the body weight of the animal. A fresh vial was prepared on each day of dosing. Treatment On Day 1 of the study, three groups of BALB/c mice (n = 8) began dosing according to the protocol in Figure 137. Compound 4* and vehicle were administered intravenously (i.v.). Group 1 received vehicle on Days 1, 5, 9, and 14. Groups 2 and 3 received CMD 4 at 1 and 3 mg/kg, respectively, on Days 1, 5, 9, and 14.

Tumor Growth Delay Endpoint

The study endpoint was a tumor volume of 2000 mm ³ or Day 30, whichever came first. The study ended on Day 29. The study protocol specified a tumor growth delay assay based on the median time-to-endpoint (TTE) in a treated group versus the control group. Tumors were measured using calipers twice per week, and each animal was euthanized for tumor progression (TP) when its tumor reached the 2000 mm ³ volume endpoint. The TTE for each mouse was calculated with the following equation:

where b is the intercept and m is the slope of the line obtained by linear regression of a log- transformed tumor growth data set. The data set is comprised of the first observation that exceeded the study endpoint volume and the three consecutive observations that immediately preceded the attainment of the endpoint volume. Any animal that did not reach endpoint was euthanized at the end of the study and assigned a TTE value equal to the last day of the study (Day 29). In instances in which the log-transformed calculated TTE preceded the day prior to reaching endpoint or exceeded the day of reaching tumor volume endpoint, a linear interpolation was performed to approximate TTE.

On Day 29, MTV (n) was defined as the median tumor volume of the number of animals, n, that survived to the last day and whose tumors had not reached the volume endpoint. Any animal determined to have died from treatment-related (TR) causes was to be assigned a TTE value equal to the day of death. Any animal that died from non- treatment-related (NTR) causes was to be excluded from the analysis. Treatment outcome was evaluated from tumor growth delay (TGD), which was defined as the increase in the median TTE for a treatment group compared to the control group:

TGD = T– C expressed in days, or as a percentage of the median TTE of the control group:

wherein T is the median TTE for a treatment group and C is the median TTE for the control group. Tumor Growth Inhibition (TGI) Analysis

The study endpoint was defined as a mean tumor volume of 2000 mm ³ in the control group (sum of both flank tumors) or 30 days, whichever came first. The study reached TGI endpoint on Day 18. Treatment efficacy was determined using data from the final day that all control animals remained on study (Day 18). The MTV (n), the median tumor volume for the number of animals, n, on the final day, was determined for each group. Percent tumor growth inhibition (%TGI) was defined as the difference between the MTV of the designated control group (Group 1) and the MTV of the drug-treated group, expressed as a percentage of the MTV of the control group:

The data set for TGI analysis includes all animals in a group, except those euthanized for sample collection (ES) and those that die due to treatment-related (TR) or non-treatment- related (NTR) causes.

Criteria for Regression Responses

Treatment efficacy was also determined from the number of regression responses. Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal. In a PR response, the tumor volume is 50% or less of its Day 1 volume for three c _{onsecutive measurements during the course of the study, and equal to or greater than 13.5 mm} ³ for one or more of these three measurements. In a CR response, the tumor volume is less than 13.5 m _m ³ _{for three consecutive measurements during the course of the study. Animals were scored} only once during the study for a PR or CR event and only as CR if both PR and CR criteria were satisfied. Any animal with a CR response on the last day of the study is additionally classified as a tumor-free-survivor (TFS).

Toxicity Animals were weighed daily for the first five days of the study and twice weekly thereafter. The mice were observed frequently for health and overt signs of any adverse treatment related (TR) side effects, and noteworthy clinical observations were recorded. Individual body weight loss was monitored per protocol, and any animal with weight loss exceeding 30% for one measurement, or exceeding 25% for three measurements, was to be euthanized for health as a TR death. If group mean body weight recovered, dosing may resume in that group, but at a lower dose or less frequent dosing schedule. Acceptable toxicity was defined as a group mean BW loss of less than 20% during the study and not more than one TR death among ten treated animals, or 10%. Any dosing regimen resulting in greater toxicity is considered above the maximum tolerated dose (MTD). A death was to be classified as TR if it was attributable to treatment side effects as evidenced by clinical signs and/or necropsy, or may also be classified as TR if due to unknown causes during the dosing period or within 14 days of the last dose. A death was classified as NTR if there was evidence that the death was related to the tumor model, rather than treatment-related. NTR deaths are further categorized as NTRa (due to accident or human error), NTRm (due to necropsy-confirmed tumor dissemination by invasion or metastasis), and NTRu (due to unknown causes).

Study Design

Table 6: Protocol Design for the CT26 Study

Table 6 displays the study design as of Day 1 of the study. Vehicle is saline. Exemplary Nanoparticle Formulations of STING Agonists

Different biodegradable polymers were evaluated to assess the entrapment of STING agonists (e.g., Compound 1) in the polymer. Table 1 briefly summarizes the polymers evaluated during this development study and a note on the entrapment efficiency of each polymer. It was evident from the studies that PLGA (50:50) grades and PCL would be suitable for entrapment of Compound 1. Table 7: Exemplary List of polymers evaluated for understanding entrapment of CMD1

Entrapment via Double emulsion (w/o/w)

The drug was first dissolved in WFI (water for injection); polymer was separately dissolved in solvent (DCM/EtOAc). Drug solution was added into the polymer solution under high homogenization. The mixture obtained was added into aqueous solution containing a surfactant (e.g., Polyvinyl alcohol (PVA)/Tocopheryl Polyethylene Glycol Succinate (TPGS)) and homogenized. This was followed by the evaporation of solvent at room temperature, centrifugation and washing to remove the obtained nanoparticles. The nanoparticles obtained were lyophilized for long-term stability.

Entrapment via Nanoprecipitation

The drug was dissolved in a water-miscible solvent (e.g., acetone) or solvent mixtures (e.g., acetone/benzyl alcohol or DMSO) along with polymer. The solution obtained was added slowly into aqueous solution containing surfactant (like PVA) and homogenized for 15-20 min. The solvent was then evaporated, the sample centrifuged and washed to obtain nanoparticles. The nanoparticles thus obtained were lyophilized for long-term stability. In certain cases, where centrifugation did not yield any particles, the sample was lyophilized as such.

In-vitro evaluation and SEM study

Different nanoparticle batches were evaluated for in-vitro analysis and SEM morphological studies. Samples manufactured via double emulsion were discrete spherical round particles. Samples manufactured via nanoprecipitation showed smaller round particles; these may form aggregates.

In-vitro evaluation of these double emulsion batches vs. the separated nanoprecipitation batch did not show leakage from the nanoparticles and the particles appeared to be similar.

The amount of Compound 1 in each NP sample was calculated by leaching out Compound 1 from NPs by dissolving in acetonitrile followed by sonication.

Nanoparticles were re-suspended directly in cell culture media before adding to cells. Nanoparticle amount added to cells is normalized to Compound 1 concentration within the nanoparticles. The starting concentration of Compound 1 in each nanoparticle after adding to cells was 2.5 µM. THP-1 cells were suspension cells and RAW macrophages are adherent cells; the uptake of nanoparticles may be different between suspension and adherent cells. The EC50 was calculated by measuring IRF induction which is a measure of STING inducing activity. CC50 was calculated by measuring the number of dead cells, which is a measure of cytotoxicity.

Table 8: Exemplary Activity of STING Agonist Containing Nanoparticles

Table 8: Exemplary Size of STING Agonist Containing Nanoparticles

PLGA (50:50) was the preferred polymer for the nanoparticles. Resomer RG 503 was evaluated for current in-vitro and SEM studies.

Double emulsion produced well separated discrete spherical particles, however, they larger in size (~1 µm) as compared to nanoprecipitation. Nanoprecipitation produced smaller sized spherical particles (~300 nm), however, particles may aggregate.

Both processes (i.e., double emulsion and separated nanoprecipitation) showed retention of entrapped Compound 1 (i.e., no leakage of entrapped drug) in in-vitro studies.

Exemplary Biological Activity of Nanoparticle Formulations of STING Agonists THP-1 dual cells and RAW-ISG cells (Invivogen) were cultured under 5% CO2 at 37 °C in RPMI and DMEM containing 10% fetal bovine serum (FBS), 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin respectively. THP-1 cells were seeded into the 96 well assay plate on the day of assay whereas RAW cells were seeded into the 96 well assay plate 18 hours before the assay.50,000 cells seeded in a 96-well flat bottom plate were treated with different concentrations of nanoparticles (NP1, NP2, NP3) or Cmd 1 alone. The cells were then incubated for 20 hours @ 37°C in CO2 incubator before measuring IRF activation by using Quanti-Luc (Invivogen). The % induction was calculated as {[(luminescence of COI treated well/ luminescence of non-COI treated well)/100] X 100}-100. EC50 was calculated by plotting in Xlfit.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. EQUIVALENTS

While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.