Stimuli-responsive sting-activating polymer compositions and methods of use

Abstract

Compositions and methods for treating a cancer or an infectious disease by delivering a compound comprising a polymeric backbone and a STING agonist covalently linked by a stimulus responsive linker are provided.

Description

TITLESTIM ULI-RESPONSIVE STING-ACTIVATING POLYMER COMPOSITIONS ANDMETHODS OF USECROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 610,642, filed December 15, 2023, and titled “STIMULI-RESPONSIVE STINGACTIVATING POLYMER COMPOSITIONS AND METHODS OF USE,” which is incorporated by reference herein in its entirety.ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant Nos. CA244719 and CA216839 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND1. Field

[0003] The present disclosure is directed to compositions and methods for treating tumors or infectious diseases by activation of STING signaling.2. Discussion of Related Art

[0004] Stimulator of interferon genes (STING aka MITA, MYPS and encoded by TMEM 173 gene) is an endoplasmic reticulum (ER)-associated signaling protein that is essential for transcriptional regulation of numerous host defense genes against infection and cancer. STING is activated by 2’, 3’-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), an endogenous secondary messenger, which is produced by cGAMP synthase (cGAS) in response to microbial or self-DNA as a danger signal. Upon cGAMP binding, STING homodimer undergoes extensive conformational change that leads to oligomerization on the ER surface and translocation to the Golgi. STING oligomerization recruits and activates TANK- binding kinase 1 (TBK1) and inhibitor of KB kinases (IKKs), which in turn activate interferon regulatory factor 3 (IRF3) and nuclear factor KB (NFKB), respectively. IRF3 and NFKB cooperate to induce type I interferons and other inflammatory cytokines to mount a robust innate immune response. Besides immune activation, STING also triggers autophagy as a primordial function independent of TBK1 and IKK. Furthermore, high magnitude of STING signaling drives cell apoptosis in hepatocytes and lymphocytes through elevated ER stress. Understanding the cell context of STING activation and resulting molecular responses is paramount to mount a protective immune response against cancer.

[0005] This disclosure is based, in part, on the surprising discovery that small molecule STING agonists (e.g., MSA-2, diABZI) can be covalently conjugated to the ultra-pH sensitive PSC7A polymers through stimuli-responsive linkers, such as pH, hypoxia, or reduction. These dualSTI NG activating nanoparticles possess “AND” logic gate design that targets both tumor acidity and other cancer hallmarks. Only in the tumor microenvironment, tumor acidity will first trigger the dissociation of nanoparticle micelles to form individual unimers. Tumor specific enzymes or biomarkers will further cleave the stimuli responsive-linkers and release STING agonist to activate STING in situ. Compared to small molecular STING agonists, stimuli- responsive STING-activating nanoparticles reduced systemic toxicity by lowering the cytokine levels in the plasma after intravenous administration. In multiple immune cold tumor models (orthotopic LL / 2 lung, B16F10, 4T1 or HCC spontaneous liver), These STING nanoparticles dramatically decreased the number of tumor nodules and improved the overall survival.SUMMARY

[0006] The present disclosure is based, at least in part, on the discovery of a potent, targeted STING agonist that shows stronger efficacy over currently available STING agonists.

[0007] In some aspects, the disclosure provides a compound comprising (a) a biodegradable polymer that activates STING and (b) a small molecule STING agonist, wherein (a) is covalently conjugated to (b) via a stimulus-responsive linker.

[0008] In various aspects, (a) comprises a structure of Formula Iwherein R is branched amine or nitrogen containing heterocycle.

[0009] In still further aspects, the compounds provided herein may comprise a structure ofFormula II:

[0010] In various aspects, R is selected from:For example, in some aspects, R

[0011] In various aspects, the compound comprises a structure of Formula I l-A:wherein m is 3, 10, or 20, and n is 125-m.

[0012] In any of the foregoing or related aspects, the small molecule STING agonist may comprise MSA-2, diABZI, 2'3'-Cyclic GMP-AMP (cGAMP), or any combination thereof. For example, the small molecule STING agonist can comprise MSA-2. In another example, the small molecule STING agonist comprises diABZI. In another example, the small molecule STING agonist comprises 2'3'-Cyclic GMP-AMP (cGAMP).

[0013] In any of the foregoing or related aspects, the stimuli-responsive linker is responsive to pH, hypoxia, reduction, tumor-specific enzymatic cleavage or any combination thereof or wherein the stimuli-responsive linker is a cathepsin B cleavable linker, a cleavable ADC linker, or a dipeptide.

[0014] For instance, in an aspect, the stimuli-responsive linker may comprise a pH sensitive linker (

[0015] In another aspect, the stimuli-responsive linker is a redox sensitive linker (e.g.,

[0016] In still another aspect, the stimuli-responsive linker is a hypoxia-sensitive linker (e.g.,

[0017] In still another aspect, the stimuli-responsive linker is a cleavable ADC linker comprising Val-Cit.

[0018] In any of the foregoing or related aspects, the compound may be selected from:, wherein each m is3, 10, or 20 and each n is 125-m.

[0019] For example, in some aspects, the compound comprises, orwherein each m is 3, 10, or 20 and each n is 125-m.

[0020] In some aspects, each m is 10 and each n is 115. For example, in some aspects, the compound comprises

[0021] In other aspects, each m is 20 and each n is 105. For example, in some aspects the

[0022] Also provided herein are nanoparticles comprising a two or more compounds disclosed herein. In some aspects, these nanoparticles may further comprise a bioactive protein or compound, wherein the compounds encapsulate the bioactive protein or compound. The bioactive protein or compound can be immunogenic and / or antigenic. For example, the bioactive protein or compound may comprise an HPV E7, E2, E5 or E6 protein.

[0023] Also provided herein are compositions comprising one or more compounds disclosed herein and at least one carrier or excipient. Also provided are compositions comprising one or more nanoparticles disclosed herein and at least one carrier or excipient. In some aspects, the composition provided herein comprise a cancer vaccine. In further aspects, any of the compositions provided herein are formulated as a pharmaceutical composition.

[0024] In various aspects, the pharmaceutical compositions are formulated for systemic administration. For example, in some aspects, the pharmaceutical composition is formulated for administration orally, intraadiposally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularlly, intravitreally, liposomally, locally, mucosally, parenterally, rectally, subconjunctival, subcutaneously, sublingually, topically, transbuccally, transdermally, vaginally, in cremes, in lipid compositions, via a catheter, via a lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, or via localized perfusion.

[0025] In certain aspects, the pharmaceutical composition is formulated for administration via injection. In further aspects, the pharmaceutical composition is formulated for intraarterial administration, intramuscular administration, intraperitoneal administration, intratumoral administration, or intravenous administration.

[0026] In any of the compositions (e.g., pharmaceutical compositions) provided herein, the excipient can be a vehicle. For example, in some aspects, the compositions comprise a vehicle as the excipient. In some aspects, the vehicle is an aqueous solution suitable for injection.

[0027] Also provided are methods of treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition disclosed herein, wherein the therapeutically effective amount is sufficient to treat the cancer.

[0028] In various aspects, the methods further comprise administering at least one additional cancer therapy. The additional cancer therapy may comprise surgery, radiation therapy, chemotherapy, immunotherapy, hormone therapy, oncolytic viruses, targeted therapies, polysaccharides, neoantigens, vaccine, or any combination thereof.

[0029] In various aspects, the cancer comprises a solid tumor. In various aspects, the cancer is a metastatic cancer and wherein the therapeutically effective amount is sufficient to reduce metastasis. In still further aspects, the cancer comprises a non-small cell lung cancer, a head or neck cancer, melanoma, liver cancer, or cervical cancer. In various aspects, the cancer is caused by an infectious agent (e.g., human papilloma virus (HPV)).

[0030] In some specific aspects, the method of treating cancer comprises administering a composition comprising a compound having the structure of:

[0031] Also provided is a method of treating an infectious disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition provided herein, wherein the therapeutically effective amount is sufficient to treat the infectious disease. In various aspects, the infectious disease is a human papilloma virus (HPV).

[0032] In various aspects, the methods of treating an infection disease comprise administering to a subject in need thereof a composition comprising nanoparticle formed from a compound having a structure of:, wherein the nanoparticle encapsulates an bioactive protein or compound.

[0033] In various aspects, in any of the methods herein, the composition is administered intravenously, subcutaneously, or intratumorally. For example, in some aspects, the composition is administered intravenously.

[0034] In various aspects, in any of the methods herein, the subject in need thereof is a human.

[0035] Also provided is a kit comprising one or more compositions disclosed herein and at least one container.

[0036] Also provided is a use of any composition disclosed herein in the preparation of a medicament for treating cancer and / or an infectious disease.BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Embodiments of the present disclosure are illustrated by way of example in which like reference numerals indicate similar elements.

[0038] FIG. 1 is a schematic of general structures of various compounds of the present disclosure.

[0039] FIG. 2 is a schematic of a reaction scheme for synthesizing PSC7A-(pH)-mMSA-2.

[0040] FIG. 3 is a schematic of a reaction scheme for synthesizing PSC7A-(Re)-mMSA-2.

[0041] FIG. 4 is a schematic of a reaction scheme for synthesizing PSC7A-(Hy)-mMSA-2.

[0042] FIG. 5 is a schematic of a reaction scheme for synthesizing PSC7A-(Re)-mdiABZI.

[0043] FIG. 6 is a schematic of a reaction scheme for synthesizing PSC7A-(Hy)-mdiABZI.

[0044] FIGS. 7A-7C depict: HPLC spectra of PSC7A-(pH)-10MSA-2, PSC7A-(Re)-10MSA-2, PSC7A-(Hy)-10MSA-2 after incubated with acid, GSH(10 mM) & acid, NADPH (100 mM) and Azoreductase (0.5 mg / mL) for 24 h (FIG. 7A); and the release of MSA-2 in PSC7A-(Hy)- 10MSA-2 (FIG. 7B) or PSC7A-(Hy)-10diABZI ([MSA-2, diABZI]) (FIG. 7C), at 40 mM incubated with NADPH (100 mM) and Azoreductase (0.5 mg / mL) at 37 °C, pH 6.5 or pH 7.4, 24 h, monitored by HPLC.

[0045] FIGS. 8A-8I depict results from testing STING NPs in a metastasis model of Lewis lung carcinoma cells (LL / 2). FIGS. 8A-8B depict lung images (FIG. 8A) and nodule number (FIG. 8B) in a metastasis model of Lewis lung carcinoma cells (LL / 2) after treatment with various STING NPs (20 mg kg'1). FIG. 8C is a heatmap illustrating the count of lung metastasis nodules for 15 STING NPs(20 mg kg'1) screening. FIG. 8D is a heatmap representing cytokine levels and assessing liver and kidney function 6 hours after treatment with STING NPs (20 mg kg'1) for safety investigation. FIGS. 8E-8G depict lung images and nodule quantification in wildtype mice (FIG. 8E), STING'^ mice (FIG. 8F), and Batf3' / _mice (FIG. 8G) treated with PSC7A-Hy-20MSA-2 (20 mg kg'1). FIGS. 8H-8I depict overall survival after administration with aPD1(200 ug), PSC7A-Hy-20MSA-2(20 mg kg'1) (FIG. 8H) and LL / 2 subQ tumor growth curve after treated with different doses of PSC7A-Hy-20MSA2, PSC7A-Hy-10diABZI (FIG. 8I).

[0046] FIGS. 9A-9G depict results from testing STING NPs in a orthotopic triple negative 4T 1 breast tumor model. FIGS. 9A-9B depict primary tumor growth (FIG. 9A) and metastasis (FIG. 9B) to the lung in Balb / c mice bearing orthotopic triple negative 4T1 breast tumor after treatment with MSA-2 (1.3 mg / kg) and PSC7A-Hy-2 (10 mg / kg). FIGS. 9C-9D depict primary tumor growth (FIG. 9C) and overall survival (FIG. 9D) in Balb / c mice bearing orthotopic triple negative 4T 1 breast tumor after aPD1 (200 pg), PSC7A-Hy-20 MSA-2 (10 mg kg-1) treatment.FIGS. 9E-9G depict lung metastasis in C57BL / 6 mice bearing B16F10 tumor after MSA-2 (2.6 mg kg'1), PSC7A-Hy-20 MSA-2(20 mg kg_1) treatment (FIGS. 9E-9F) and overall survival after aPD1 (200 ug), PSC7A-Hy-20 MSA-2(20 mg kg'1) treatment (FIG. 9G).

[0047] FIGS. 10A-10D depict a schematic of experimental protocol (FIG. 10A) and measured pharmacokinetics after administration of PSC7A-Hy-20 MSA-2 : PSC7A-3 ICG (7:3) (total dose 20 mg kg-1) (FIG. 10B), as well as images thereof (FIG. 10C) and quantification (FIG. 10D) of biodistribution.

[0048] FIGS. 11A-11C depict STING NPs(20 mg kg-1) screening in an anti-HCC spontaneous tumor model (MYC / TP53). FIG. 11A is a schematic of an experimental protocol and FIGS. 11B-11C show quantification of liver / body mass ratios (FIG. 11B) and representative images of livers (FIG. 11C) from treated animals.

[0049] FIGS. 12A-12C depict STING NPs(20 mg kg-1) screening in an anti-HCC spontaneous tumor model (MYC / CTNNB1). FIG. 12A shows representative images of livers isolated from treated animals. FIGS. 12B-12C show liver / body mass ratios (FIG. 12B) and liver nodule numbers per mice (FIG. 12C) in treated animals.

[0050] FIGS. 13A-13C depict a scheme of a proposed nanovaccine using STING NPs (FIG. 13A), FPLC analysis of PSC7A-(Hy)-10diABZI encapsulating the E7 protein (FIG. 13B) and its DLS size (FIG. 13C).

[0051] FIGS. 14A-14C depict a pharmacokinetic (PK) and biodistribution (BD) data of E7- TMR(30 ug) and PSC7A-Cy5(15 mg kg'1). FIG. 14A is a plot of measured PK of the PSC7A- (Hy)-diABZI polymer alone or encapsulating E7. FIGS. 14B-14C are plots of biodistribution of BD E7-TMR (FIG. 14B) and PSC7A-(Hy)-diABZI-Cy5 (FIG. 14C).

[0052] FIGS. 15A-15F depict analysis of PolySTI NG nanovaccine efficacy in a TC1 tumor mouse model. FIGS. 15A-15B depict tumor growth (FIG. 15A) and overall survival (FIG. 15B) analysis of C57BL / 6 mice bearing TC1 tumors treated with STING NPs (15 mg kg'1) encapsulating the E7 protein (30 pg). FIGS. 15C-15D show analysis of tumor growth (FIG. 15C) and overall survival (FIG. 15D) in C57BL / 6 mice with TC1 tumors treated through different administration routes (i.t.(1.5 mg kg-1), i.v. (15 mg kg-1), subQ(1.5 mg kg-1) ) using PSC7A-Hy-10 diABZI encapsulating the E7 protein. FIGS. 15E-15F show primary (FIG. 15E) and distal (FIG. 15F) tumor growth in C57BL / 6 mice with TC1 tumors treated through different administration routes and doses (i.t.(L: 0.5, M: 5, H: 15 mg kg-1), i.v.(L: 0.5, M: 5, H: 15 mg kg-1), subQ.(L: 0.5, M: 5, H: 15 mg kg-1), i.p. (H: 15 mg kg-1)) using PSC7A-Hy-10 diABZI encapsulating the E7 protein.

[0053] FIGS. 16A-16E show screening of AND logic nanoparticles for their safety and antitumor efficacy in a LL / 2 lung metastatic model. FIG. 16A is a schematic of AND logic nanoparticle design and chemical structure of PEG-b-P(SC7A-L-MSA-2). FIG. 16B shows representative optical images of mouse lungs after single intravenous injection of different STING-activating nanoparticles in mice with LL / 2 metastatic nodules (see also FIG. 8A). FIG. 16C shows the number of LL / 2 nodules after i.v. injection of PBS, MSA-2 (2.3 mg / kg), PSC7A (20 mg / kg), and AND logic nanoparticles (20 mg / kg), n=4. FIG. 16D is a heatmap for the serum liver and kidney toxicity assessment and systemic cytokine levels from mice in treated in c, n=4. FIG. 16E shows evaluation of antitumor efficacy (x-axis) and toxicity (y-axis) using dimensionality reduction methods integrate for AND logic nanoparticles.

[0054] FIGS. 17A-17K show evaluation of AND logic design for the STING-activating nanoparticles. FIG. 17A is a schematic of PSC7A-Hy-MSA-2 (PHM NP) chemical structure. FIG. 17B shows an intended pH-hypoxia AND logic gate for PHM NP. FIG. 17C is an HPLC analysis of MSA-2 release from PHM NP (1 mg / mL) incubated in PBS at 37°C for 24 h, at pH 7.4 or 6.5, with or without NQO1 (100 pg / mL) / NADH (800 pM). FIG. 17D is a kinetic analysis of MSA-2 release over 24 h for PHM NP (1 mg / mL) under different environmental conditions. FIG. 17E shows release of MSA-2 for PHM NP (1 mg / mL) at pH 6.5 under various incubation conditions: GSH (10 mM), NADH (800 pM), NQO1 (100 pg / mL), NQO1 (100 pg / mL) / NADH (800 pM), and NQO1 (100 pg / mL) / NADH (800 pM) / Dicoumarol (100 pM). FIG. 17F shows dose dependent IFN-p secretion in THP1-ISG cells with PHM NP under normoxic (20% O2) or hypoxic (1 % O2) conditions after 24 h incubation. FIG. 17G shows IFN-p secretion in THP1- ISG cells co-incubated with PHM NP (50 pg / mL) with or without the inhibitors Dicoumarol (100 pM) or Bafilomycin (250 nM) under hypoxic conditions. FIG. 17H shows MSA-2 release for PLGA-Am-MSA-2 (4 mg / mL), PSC7A-Am- MSA-2 (1 mg / mL), PLGA-Hy-MSA-2 (4 mg / mL), and PSC7A-Hy-MSA-2 (1 mg / mL) incubated with pH 6.5 and NQO1 (100 pg / mL) / NADH (800 pM). FIG. 171 shows IFN-p secretion in THP1-ISG cells incubated with PLGA-Am-MSA-2 (0.4 mg / mL), PSC7A-Am-MSA-2 (0.1 mg / mL), PLGA-Hy-MSA-2 (0.1 mg / mL), and PSC7A-Hy- MSA-2 (0.4 mg / mL) under hypoxic conditions. FIG. 17J shows lung images from the LL / 2 lung metastasis model following treatment with PLGA-Am-MSA-2 (80 mg / kg), PSC7A-Am-MSA-2 (20 mg / kg), PLGA-Hy-MSA-2 (80 mg / kg), and PSC7A-Hy-MSA-2 (20 mg / kg). FIG. 17K shows lung metastasis nodule counts from the treatment in FIG. 17J (n = 3).

[0055] FIGS. 18A-18I show an analysis of pharmacokinetics, biodistribution and cell tropism of PHM NP. FIG. 18A shows a treatment plan of in vivo experiments. FIG. 18B shows pharmacokinetics of PSC7A-ICG / PHM mixed nanoparticles (mass ratio: 3 / 7) (20 mg / kg) injected and monitored over 24 h. FIG. 18C shows biodistribution in major organs at 24 h after injection of PSC7A-ICG / PHM mixed nanoparticles (20 mg / kg). FIG. 18D-18F shows celltropism in spleen (FIG. 18D), TDLN (FIG. 18E), and lung metastasis (FIG. 18F) at 24 h after i.v. injection of PSC7A-Cy5 / PHM NP (20 mg / kg). FIGS. 18G-18H show a western blot (FIG. 18G) and quantification data (FIG. 18H) showing NQO1 expression in DC2.4 cells incubated with or without PHM NP (0.1 mg / mL) under hypoxic or normoxic conditions. FIG. 181 shows IFN-p secretion from DC2.4 cells by ELISA assay.

[0056] FIGS. 19A-19K show data from a mechanistic investigation of immune cell dependence of STING-mediated rejection of lung metastasis. FIG. 19A is a diagram of the experimental setup. FIGS. 19B-19D depict lung images and lung metastasis nodule counts in WT (FIG. 19B), STING' / _(FIG. 19C) and Batf3-Z- (FIG. 19D) mice following treatment with 20 mg / kg PHM NP. FIG. 19E is a schematic of the immune cell depletion experiment. FIGS. 19F- 191 depict lung images and metastasis nodule counts in mice treated with 20 mg / kg PHM NP and no depletion (FIG. 19F), anti-CD8 antibody (200 mg / time) (FIG. 19G), anti-CD4 antibody (200 mg / time) (FIG. 19H), and anti-NK antibody (250 mg / time) (FIG. 191). FIG. 19J shows a schematic of the splenectomy experiment. FIG. 19K depicts lung images and lung metastasis nodule counts in mice treated with PBS or PHM NP (20 mg / kg).

[0057] FIGS. 20A-20D shows how multiplexed immunohistochemistry (mIHC) analysis reveals the elevation of cDC1-pSTING-CD8+T cell signature. FIG. 20A depicts a representative image of mIHC and H&E staining of LL / 2 lung metastasis from PBS, MSA-2 (2.5 mg / kg), PHM NP(20 mg / kg) treated mice. FIG. 20B depicts quantification of the percentage of cDC1 cells in the representative LL / 2 tumor region. FIG. 20C depicts quantification of the percentage of pSTING-activated cDC1 cells within the total cDC1 population in the representative region. FIG. 20D depicts quantification of the percentage of CD8+T cells in the representative region. n=10.

[0058] FIGS. 21A-21L shows PHM NP significantly enhances the therapeutic effect in metastatic tumors. FIG. 21 A is a schematic diagram of the experiment in orthotopic triplenegative 4T1 breast tumor and lung metastasis. FIG. 21 B shows tumor growth curve of the primary tumor after treatment with PBS, MSA-2 (1.25 mg / kg, same dose with that in PHM NP), and PHM NP (10 mg / kg). FIGS. 21C-21D show lung metastasis nodule counts (FIG. 21C) and lung images and H&E staining images (FIG. 21 D) on Day 24 post-treatment (Scale bar: 200 mm, n=5). FIG. 21 E is a schematic diagram of the combination of PHM NP with ICB treatments in mice bearing 4T1 tumors. FIGS. 21F-21G shows primary tumor growth curves (FIG. 21 F) and survival outcomes (FIG. 21 G) after treatment with PBS, aPD1 , PHM NP, and aPD1 / PHM NP. FIG. 21 H shows schematic diagram of the B16F10 metastatic tumor treatment experiment. FIGS. 211-21 J show lung metastasis nodule counts, lung images and representative H&E staining images on day 17 post-treatment of PBS, MSA-2, and PHM NP (Scale bar: 200 mm, n=5). FIG. 21 K is a schematic diagram of the combination of NP with ICBtreatments in mice bearing B16F10 metastatic tumor. FIG. 21L shows survival curves after treatment with PBS, aPD1 , PHM NP, and aPD1 / PHM NP.

[0059] FIGS. 22A-22C shows release mechanisms of MSA-2 from conjugated polymers with pH (FIG. 22A), redox (FIG. 22B), and hypoxia (FIG. 22C) linkers.

[0060] FIGS. 23A-23C shows UV-vis spectra of MSA-2 (left) and standard curve (right) for quantification of MSA-2 in polySTANDs: PSC7A-pH-mMSA-2 (FIG. 23A), PSC7A-Re-mMSA- 2 (FIG. 23B) and PSC7A-Hy-mMSA-2 (FIG. 23C).

[0061] FIG. 24 shows DLS of polySTAND NPs.

[0062] FIG. 25 shows lung metastasis nodules after treatments of PBS, free MSA-2, PSC7A, PSC7A-pH-10MSA-2, PSC7A-Re-10MSA-2, and PSC7A-Hy-10MSA-2 NPs. free MSA-2 and PSC7A NP were controlled the same dose with that in conjugated polymers.

[0063] FIG. 26 shows lung metastasis nodules after treatments of PBS, free MSA-2, PSC7A, PSC7A-pH-MSA-2, PSC7A- Re- MSA-2, and PSC7A-Hy-MSA-2 NPs with 3, 10 and 20 DPR. free MSA-2 and PSC7A were controlled the same dose with that in conjugated polymers. Bar legend: (A) PBS, (B) PSC7A, (C) MSA-2, (D) PSC7A-pH-3MSA-2, (E) PSC7A-pH-10MSA-2, (F) PSC7A-pH-20MSA-2, (G) PSC7A-Re-3MSA-2, (H) PSC7A-Re-10MSA-2, (I) PSC7A-Re- 20MSA-2, (J) PSC7A-Hy-3MSA-2, (K) PSC7A-Hy-10MSA-2, (L) PSC7A-Hy-20MSA-2.

[0064] FIG. 27 shows liver and kidney toxicity after treatments of PBS, free MSA-2, PSC7A, PSC7A-pH-MSA-2, PSC7A- Re- MSA-2, and PSC7A-Hy-MSA-2 NPs with 3, 10 and 20 DPR. free MSA-2 and PSC7A were controlled the same dose with that in conjugated polymers. Green background indicates the normal range. Bar legend: (A) PBS, (B) PSC7A, (C) MSA-2, (D) PSC7A-pH-3MSA-2, (E) PSC7A-pH-10MSA-2, (F) PSC7A-pH-20MSA-2, (G) PSC7A-Re- 3MSA-2, (H) PSC7A-Re-10MSA-2, (I) PSC7A-Re-20MSA-2, (J) PSC7A-Hy-3MSA-2, (K) PSC7A-Hy-10MSA-2, (L) PSC7A-Hy-20MSA-2.

[0065] FIG. 28 show cytokines after treatments of PBS, free MSA-2, PSC7A, PSC7A-pH- MSA-2, PSC7A-Re-MSA-2, and PSC7A-Hy-MSA-2 NPs with 3, 10 and 20 DPR. free MSA-2 and PSC7A were controlled the same dose with that in conjugated polymers. Bar legend: (A) PBS, (B) PSC7A, (C) MSA-2, (D) PSC7A-pH-3MSA-2, (E) PSC7A-pH-10MSA-2, (F) PSC7A- PH-20MSA-2, (G) PSC7A-Re-3MSA-2, (H) PSC7A-Re-10MSA-2, (I) PSC7A-Re-20MSA-2, (J) PSC7A-Hy-3MSA-2, (K) PSC7A-Hy-10MSA-2, (L) PSC7A-Hy-20MSA-2.

[0066] FIGS. 29A-29B shows efficacy (FIG. 29A) and toxicity score (FIG. 29B) after different treatments. Bar legend: (A) PBS, (B) PSC7A, (C) MSA-2, (D) PSC7A-pH-3MSA-2, (E) PSC7A- pH-10MSA-2, (F) PSC7A-pH-20MSA-2, (G) PSC7A-Re-3MSA-2, (H) PSC7A-Re-10MSA-2,(1) PSC7A-Re-20MSA-2, (J) PSC7A-Hy-3MSA-2, (K) PSC7A-Hy-10MSA-2, (L) PSC7A-Hy- 20MSA-2.

[0067] FIGS. 30A-30B shows experimental scheme in mice bearing LL / 2 metastatic tumors (FIG. 30A) and survival of mice after PBS, aPD-1, PSC7A-Hy-20MSA-2 NP, and aPD- 1 / PSC7A-Hy-20MSA-2 NP combination treatment (FIG. 30B). Survival curve legend: (1) PBS,(2) aPD1, (3) PSC7A-Hy-20MSA-2, (4) aPD1 / PSC7A-Hy-20MSA-2.

[0068] FIG. 31 shows pH titration of PEG-b-P(SC7A-Hy-20MSA-2) polymer and parent PEG- b-PSC7A polymer. Both polymers display ultra-pH sensitivity and strong buffer capacity at their pKa’s of 6.9.

[0069] FIG. 32 shows MSA-2 release under different concentration of NQO1 treatments.

[0070] FIGS. 33A-33B shows MSA-2 releases in PSC7A-pH-MSA-2 (FIG. 33A) under pH 7.4 (gate = 0) and pH 6.5 (gate = 1) and PSC7A-Re-MSA-2 (FIG. 33B) under pH 7.4 without 800 pM GSH (gate = 0, 0), pH 6.5 without 800 pM GSH (gate = 1 , 0), pH 7.4 with 800 pM GSH (gate = 0, 1), and pH 6.5 with 800 pM GSH (gate = 1, 1). The releases were determined by HPLC. Peaks in 7.44 and 8.13 min are the peaks of released MSA-2 from polymer NPs.

[0071] FIGS. 34A-34E shows an experimental schematic (FIG. 34A), biodistribution (FIG. 34B) and cell tropism in tumor (FIG. 34C), TDLN (FIG. 34D) or spleen (FIG. 34E) of PHM NP in 4T1 bearing mice.

[0072] FIG. 35 shows individual tumor growth curves of 4T 1 tumor bearing mice treated with different therapeutic combinations (n = 5). Mice were inoculated with 4T1 cells on the right flank on day 0. PHM NP, MSA-2 and PBS were i.v. administered on day 9, 12 and 15, respectively.

[0073] FIG. 36 shows individual tumor growth curves of 4T 1 tumor bearing mice treated with different therapeutic combinations (n = 5). Mice were inoculated with 4T1 cells on the right flank on day 0. aPD-1 was i.p. administered on day 8, 11 , and 14. PHM NP, MSA-2 and PBS were i.v. administered on day 9, 12, and 15, respectively.

[0074] FIG. 37 is an illustrative schematic of a STING nanovaccine is designed to deliver tumor antigens (Ag) and STING agonists (diABZI, PSC7A) to the tumor and secondary lymphoid organs to activate antitumor immunity. The nanovaccine stays in the assembled ‘OFF’ state to minimize systemic toxicity, and becomes activated in the antigen-presenting cells in tumors and secondary lymphoid organs to boost T cell immunity.

[0075] FIG. 38A-38G show production and characterization of STING nanovaccine. FIG. 38A depicts a chemical structure of PEG-b-P(SC7A-azo-diABZI) or STING polymer and formulation of STING nanovaccine. FIG. 38B shows dynamic light scattering analysis ofSTING NP (blue) and STING nanovaccine (red). FIG. 38C depicts fast protein liquid chromatograms (FPLC) of E7 antigen, STING NP, and STING nanovaccine. FIGS. 38D-38E show the kinetics of diABZI release (FIG. 38D) and high performance liquid chromatograms (HPLC, FIG. 38E) of STING NP under different incubation conditions (at pH 6.5 or 7.4 with or without NQO1 / NADH) at 37 °C after 24 h. FIG. 38F shows IFN-b signal in THP1-ISG cells in response to STING NP pretreated with or without NQO1 / NADH at pH 6.5 or 7.4. FIG. 38G shows that pretreatment of PSC7A NP encapsulated with diABZI (PSC7A / diABZI) at pH 6.5 or 7.4 showed comparable levels of IFN-b responses to free diABZI. All pretreatments were conducted in PBS (pH 6.5 or 7.4) for 24 h, and then solutions were added into THP1-ISG cell culture medium. Statistical significance was calculated by one-way ANOVA, ns: no significant difference, ****: p<0.0001.

[0076] FIGS. 39A-39I show PKBD and cell tropism analysis of STING nanovaccine after i.v. administration. FIG. 39A depicts an experimental scheme in mice bearing TC-1 tumors. FIG. 39B shows a PK profile of E7 antigen and STING polymer. FIG. 39C shows quantification of biodistribution of STING nanovaccine in selected organs and tumor tissues 24 h after i.v. injection. FIGS. 39D-39F shows cell tropism of STING nanovaccine in TC-1 tumors (FIG. 39D), tdLN (FIG. 39E), and spleen (FIG. 39F). FIG. 39G and FIG. 39H show NQO1 expression levels and function in RAW264.7 and DC2.4 determined by western blot (FIG. 39G) and absorption decrease of methyl red (FIG. 39H) after PBS, PSDBA NP, diABZI, PSC7A NP and STING NP treatment over 24 h. The concentration of all NPs is 50 pg / mL, equivalent diABZI concentration (7.85 pg / mL) were used in these studies. FIG. 39I shows IFN-p levels in RAW264.7 and DC2.4 after PBS, STING NP and STING NP with and without dicoumarol treatment over 24 hours. All the data are represented as mean ± sem. Statistical significance was calculated by two-way ANOVA, ns: not significant, ****: p<0.0001.

[0077] FIGS. 40A-40F shows evaluation of antitumor efficacy of STING nanovaccine against TC-1 tumors. FIG. 40A is a timeline for TC-1 tumor inoculation and treatments in C57BL / 6 mice. FIG. 40B shows tumor growth curves in mice after i.v. injection of STING nanovaccine at different doses (1.5, 5, and 15 mg / kg, n=5). PBS was used as a non-treated control. FIG. 40C and FIG. 40D show tumor growth of C57BL / 6 mice bearing TC-1 tumors after i.v. administration of vehicle, PSC7A, diABZI or STING NP (FIG. 40C) or vehicle + E7, PSC7A+ E7, diABZH- E7 or STING NP (FIG. 40D). FIG. 40E and FIG. 40F shows long-term survival of C57BL / 6 mice bearing TC-1 tumors after i.v. administration of vehicle, PSC7A, diABZI or STING NP (FIG. 40E) or vehicle + E7, PSC7A+ E7, diABZH- E7 or STING NP (FIG. 40F). All tumor growth data are represented as mean ± sem. Statistical significance was calculated by two-way ANOVA, ns: no significant difference, *: p<0.05, **: p<0.01 , ****: p<0.0001.

[0078] FIGS. 41A-41 D shows evaluation of administration routes of STING nanovaccine in the TC-1 distal tumor model. FIG. 41A shows a timeline for primary and distal TC-1 tumor inoculations and treatments in C57BL / 6 mice (n = 5). Mice were inoculated with TC-1 cells at the right flank on day 0 then at left flank on day 2. STING nanovaccine was injected through different methods (s.c., i.t. , and i.v.) on days 10 and 15. FIGS. 41 B-41 D show survival (FIG. 41 B), primary tumor growth (FIG. 41 C) and distal tumor growth (FIG. 41 D) in C57BL / 6 mice bearing TC-1 tumors after injection of STING nanovaccine. All tumor growth data are represented as mean ± sem. Statistical significance was calculated by two-way ANOVA, ns: no significant difference, **: p<0.01 , ***: p<0.001 , ****: p<0.0001.

[0079] FIGS. 42A-42J show STING nanovaccine activates DCs, macrophages, and lymphocytes in spleen, tdLN and TC-1 tumor. FIG. 42A shows a timeline of experimental design using TC-1 tumor bearing C57BL / 6 mice (n = 3 or 5). FIGS. 42B-42C show representative flow cytometry histogram (left) and quantification (right) of CD86 expressions in DCs (FIG. 42B) and macrophages (FIG. 42C) in the spleen. FIGS. 42D-42E show representative flow cytometry histogram (left) and quantification (right) of CD86 expressions in DCs (FIG. 42D) and macrophages (FIG. 42E) in the tdLN. FIG. 42F shows the ratio of M1 over M2-like macrophages within the spleen and tdLN. FIGS. 42G -42I show representative flow cytometry dot plots (top) and quantifications (bottom) of E7 specific CD8+T cells in TC-1 tumor (FIG. 42G), spleen (FIG. 42H) and tdLN (FIG. 42I), respectively. FIG. 42J shows total immune cell, CD8+T, CD4+T, NK, and NKT cells per milligram of tumor within the TC-1 tumor. All the data are represented as mean ± sem. Statistical significance was calculated by oneway ANOVA, ns: no significant difference, *: p<0.05, **: p<0.01 , ***: p<0.001 , ****: p<0.0001.

[0080] FIGS. 43A-43D shows treatment of MLM3 metastatic tumors by STING nanovaccine and anti-PD1 therapy. FIG. 43A shows a timeline for MLM3 metastasis formation and treatments in C57BL / 6 mice (n =4 5 or 7). FIG. 43B shows the number of lung metastatic nodules after PBS, aPD-1 , STING nanovaccine or a combination of STING nanovaccine and aPD-1 treatment (n = 5). FIG. 43C shows representative photographs (up) and hematoxylin and eosin (H&E, down) of mice after different treatments. Scale bar: 1 mm. FIG. 43D shows survival outcomes of mice with MLM3 tumor metastases after different treatments (n = 7). All the data are represented as mean ± sem. Statistical significance was calculated by one-way ANOVA, ns: no significant difference, *: p<0.05, ***: p<0.001 , ****: p<0.0001.

[0081] FIG. 44 is a schematic of a reaction scheme for synthesizing PEG-b-P(SC7A-azo- diABZI) polymer (see also FIG. 6).

[0082] FIG. 45 depicts results from a1H NMR of PEG-b-P(SC7A-azo-diABZI) polymer in CDCI3.

[0083] FIG. 46 depicts pH titration of PEG-b-P(SC7A-azo-diABZI) polymer and parent PEG- 6-PSC7A polymer. Both polymers display ultra-pH sensitivity and strong buffer effect at their pKa’s of 6.9.

[0084] FIG. 47 depicts the change in body weight of TC-1 tumor-bearing mice after two doses of STING nanovaccine treatments. Mice were subcutaneously inoculated with TC-1 cells on the right flank, and two treatments were i.v. administered on day 10 and 15. All the data are represented as mean ± sem, n = 5.

[0085] FIG. 48 depicts individual growth curves of TC-1 tumors treated with different therapeutic combinations as indicated (n = 5). Mice were inoculated with TC-1 cancer cells on the right flank on day 0. PBS, PSC7A NP, diABZI, and STING NP were i.v. administered on day 10 and 15.

[0086] FIG. 49 depicts individual growth curves of TC-1 tumors treated with different therapeutic combinations as indicated (n = 5). Mice were inoculated with TC-1 cells on the right flank on day 0. PBS+E7, PSC7A NP encapsulated with E7, diABZI+E7, and STING nanovaccine were i.v. administered on day 10 and 15.

[0087] FIG. 50 depicts individual growth curves of primary and distal TC-1 tumors after treatment with STING nanovaccine (n = 5). Mice were inoculated with TC-1 cells at the right flank on day 0 then at abscopal left flank on day 2. STING nanovaccine (15 mg / kg) were s.c., i.t., and i.v. administered on day 10 and 15.

[0088] FIG. 51 depicts flow cytometry gating strategy for DC and macrophage analyses.

[0089] FIG. 52 depicts flow cytometry gating strategy for lymphocyte (T, NK, NKT) analyses.

[0090] FIGS. 53A-53J depict CD86 expressions in DCs (FIG. 53A) and macrophages (FIG. 53B) in the spleen and tdLN in TC-1 bearing mice one day after treatments of E7, E7- capsulated PSC7A NP (PSC7A / E7), diABZI mixed with E7 (diABZI + E7), and STING nanovaccine (STING NVax); E7-specific CD8+T cell percentage in TC-1 tumor (FIG. 53C), spleen (FIG. 53D) and tdLN (FIG. 53E), and total immune cell (FIG. 53F), CD8+T (FIG. 53G), CD4+T (FIG. 53H), NK (FIG. 53I), and NKT (FIG. 53J) cells per unit mass of tumor within the TC-1 tumor three days after the second treatment with E7, PSC7A / E7, diABZI + E7, and STING nanovaccine. All the data are represented as mean ± sem. Statistical significance was calculated by one-way AN OVA, ns: no significant difference, **: p<0.01 , ****: p<0.0001.

[0091] FIGS. 54A-54B depict an analysis of PD-1+(FIG. 54A) and PD-1+Tim3+CD8+(FIG. 54B) T cell populations in TC-1 tumor, spleen and tdLN in tumor-bearing mice after PBS, E7, STING NP and STING nanovaccine treatments. All the data are represented as mean ± sem.Statistical significance was calculated by one-way ANOVA, ns: no significant difference, **: p<0.01 , ****: p<0.0001.

[0092] FIGS. 55A-55I depict cytokines, liver and kidney toxicity of STING NP after i.v. administration. FIG. 55A shows a timeline for toxicity evaluations following the STING NP, PSC7A NP, free diABZI, and PBS administration in tumor-free mice. FIGS. 55B- 55E depict results from an analysis of systemic cytokine levels (IL-6 (FIG. 55B), IFN-y (FIG. 55C), MCP- 1 (FIG. 55D) and TNF-a (FIG. 55E)) in plasma 6 h and 24 h post-administration. FIGS. 55F- 55I show AST (FIG. 55F), ALT (FIG. 55G), CREA (FIG. 55H), and BUN (FIG. 55I) levels in plasma 24 h post-administration. The green dotted background indicates normal range of these signals. All the data are represented as mean ± sem. Statistical significance was calculated by one-way ANOVA, ns: no significant difference, *: p<0.05, **: p<0.01 , ****: p<0.0001.

[0093] FIGS. 56A-56B depict IFN- (FIG. 56A) and CXCL10 (FIG. 56B) expressions in TC-1 tumors and other organs 6 hours after treatments with STING nanovaccine and diABZI / E7 control.

[0094] The drawing figures do not limit the present disclosure to the specific embodiments disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain embodiments of the present disclosure.DETAILED DESCRIPTION

[0095] The following detailed description references the accompanying drawings that illustrate various embodiments of the present disclosure. The drawings and description are intended to describe aspects and embodiments of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized, and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.

[0096] Conventional cancer chemotherapy targets cell proliferation in neoplasm, but it suffers from severe toxicity. Molecular targeted therapy (e.g., kinase inhibitors) shows improved therapeutic window but drug resistance rapidly develops. In recent years, checkpoint blockade therapy (e.g., anti-PD-1) has revolutionized cancer care with durable responses in a subset (20-30%) of patient population. However, immune related toxicities are still limiting factors while these treatments are not effective in immune desert tumors that lack T cell infiltration. Current therapies have several flaws in drug development. First, most clinically approved drugs are always ON, even in healthy tissues. In oncology, ‘on-target’ toxicity in normal tissues remains a major limiting factor which decreases therapeutic window. Second, current therapeutics lack a holistic design that addresses both pharmacokinetics (adsorption,distribution, metabolism, excretion) and pharmacodynamics (dose-response, mechanism of action). Single agent (either small molecule or protein macromolecule)-based approach is unlikely to meet the divergent PK / PD requirements for a safe and efficacious therapy. Systembased, cooperative nanotherapeutic approach that allows precision cancer targeting both pharmacokinetically and pharmacodynamically may prove essential for cancer drug development. The inventors have developed a series of stimuli-responsive STING activating nanoparticles for systemic administration of immuno-therapeutics. Small molecule STING agonists (e.g., MSA-2, diABZI) can be covalently conjugated to the ultra-pH sensitive PSC7A polymers through stimuli-responsive linkers, such as pH, hypoxia, or reduction. These dualSTI NG activating nanoparticles possess “AND” logic gate design that targets both tumor acidity and other cancer hallmarks. Only in the tumor microenvironment, tumor acidity will first trigger the dissociation of nanoparticle micelles to form individual unimers. Tumor specific enzymes or biomarkers will further cleave the stimuli responsive-linkers and release STING agonist to activate STING in situ. Compared to small molecular STING agonists, stimuli- responsive STING-activating nanoparticles reduced systemic toxicity by lowering the cytokine levels in the plasma after intravenous administration. In multiple immune cold tumor models (orthotopic LL / 2 lung, B16F10, 4T1 or HCC spontaneous liver) these STING nanoparticles dramatically decreased the number of tumor nodules and improved the overall survival.

[0097] Provided herein are compositions and methods for treating a subject with cancer by targeting STING signaling.I. Terminology

[0098] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[0099] As used in the specification, articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0100] “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result. The term “about” in association with a numerical value means that the numerical value can vary plus or minus by 5% or less of the numerical value.

[0101] Throughout this specification, unless the context requires otherwise, the word “comprise” and “include” and variations (e.g., “comprises,” “comprising,” “includes,” “including”) will be understood to imply the inclusion of a stated component, feature, element, or step or group of components, features, elements, or steps but not the exclusion of any other integer or step or group of integers or steps.

[0102] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0103] As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

[0104] Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0105] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise- Indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1 % to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0106] As used herein, “treatment,” “therapy” and / or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and / or the remission of the disease, disorder, or condition.

[0107] As used herein, “prevent” or “prevention” refers to eliminating or delaying the onset of a particular disease, disorder, or physiological condition, or to the reduction of the degree ofseverity of a particular disease, disorder or physiological condition, relative to the time and / or degree of onset or severity in the absence of intervention.

[0108] The term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results. The term “therapeutically effective amount,” as used herein, means an amount of a compound or combination of compounds that ameliorates, attenuates, or eliminates one or more symptoms of cancer or prevents or delays the onset of one or more symptoms of cancer as defined herein.

[0109] As used herein, “individual,” “subject,” “host,” and “patient” can be used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, prophylaxis, or therapy is desired, for example, humans, pets, livestock, horses or other animals. As used herein, the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals. The term “nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. In some embodiments, the subject can be a human. In other embodiments, the subject can be a human in need of treating a cardiomyopathy (e.g., DCM).II. Compositions

[0110] The present disclosure provides for compositions for treating a cancer or an infectious disease by activating STING (Stimulator of interferon genes) in receptive immune cell populations, thereby increasing targeted immune response against the tumor or infectious pathogen. In various aspects, the compositions herein can comprise a compound which comprises (a) a biodegradable polymer covalently linked or bonded to (b) a small molecule STING agonist via a stimulus-responsive linker. In various aspects, the biodegradable polymer also activates STING. In this way, the STING agonist may be dissociated from the polymer in a targeted environment (e.g., an environment having a suitable pH, oxygen level and / or presence of any other specific enzymes that would facilitate cleavage of the linker).

[0111] In various aspects, a biodegradable polymer of (a) that may be covalently bonded to a small molecule STING agonist may comprise a structure of Formula I:wherein R is a branched amine or nitrogen containing hetorocycle.

[0112] Suitable biodegradable polymers are described in detail in WO2020263733A1 and Wang X, Polycarbonate-based ultra-pH sensitive nanoparticles improve therapeutic window. Nat Commun. 2020 Nov 17;11(1):5828 which are both incorporated herein by reference in their entirety. For instance, in some aspects, R may be selected from, ,

[0113] Accordingly, in some aspects, a compound of the present disclosure (e.g., comprising (a) a biodegradable polymer covalently linked or bonded to (b) a small molecule STING agonist via a stimulus-responsive linker) may comprise a compound of Formula II:where R is as defined, m is 3, 10 or 20, and n is 125-m. In some aspects, n is 10 and m is 115. In some aspects, n is 20 and m is 105.

[0114] In some aspects, the biodegradable polymer comprises PEG-6- P(MAC-SC7A)(PSC7A) (e.g., when R is. In such aspects, the compound disclosed herein may comprise a structure of Formula ll-A:wherein m is 3, 10, or 20, and n is 125-m. In certain aspects, as described further below, m is 10 and n is 115. In other aspects, m is 20 and n is 105.

[0115] Any suitable small molecule STING agonist may be covalently linked to the biodegradable polymers described above. In some aspects, the STING agonist may be selected from MSA-2, diABZI, 2' 3'-cyclic GMP-AMP (cGAMP), or any combination thereof. The structures of these STING agonists are provided herein below for reference.

[0116] In various aspects, the small molecule STING agonist may comprise MSA-2.

[0117] In various aspects, the small molecule STING agonist may comprise diABZI.

[0118] In various aspects, the small molecule STING agonist may comprise 2' 3'-cyclic GMP- AMP (cGAMP).

[0119] As noted, the small molecule STING agonist may be covalently linked to the biodegradable polymer by a stimulus responsive linker. The term “stimulus responsive” as used herein means that the linker is cleaved or otherwise degrades when in the presence or when acted upon by a given stimulus. The stimulus here can be pH (e.g., proton concentration), oxygen levels (e.g., hypoxic conditions), reducing conditions, or can be a sitespecific enzyme or protein that acts on the linker. A site-specific enzyme can be, for instance, a tumor specific enzyme that is only expressed in a tumor environment. Accordingly, the linker herein may be responsive to pH, hypoxia, reduction, a tumor-specific enzymatic cleavage, or any combination thereof. In some aspects, the stimuli-responsive linker is a cathepsin B cleavable linker, an ADC cleavable linker (e.g., Val-Cit (CAS No: 159858-33-0)), or a dipeptide.

[0120] In accord with the foregoing, the linker may in some instances be selected from z°(pH sensitive), (Redox sensitive), , (Redox sensitive)(hypoxia sensitive), and(hypoxia sensitive). For example, in some aspects, the linkermay be a pH sensitive and, optionally, comprise . In some aspects, the linker maybe Redox sensitive and, optionally, be selected from and. In some aspects, the linker may be hypoxia sensitive and, optionally, be selected fromand . In certain aspects, the linker comprises. In still other aspects, the linker comprises

[0121] In some aspects, the linker comprises Val-Cit (CAS No: 159858-33-0) having the

[0122] Use of these linkers is particularly advantageous in the tumor microenvironment. Forinstance, regarding a pH sensitive linker ( ) at acidic pH, such as the environment found in tumor intracellular endosomes and lysosomes (pH<5.4), the hydrazone bond undergoes protonation, which increases the electrophilicity of carbonyl carbon, making the hydrazone bond susceptible to hydrolysis. As another example, for the redox sensitive linkers), disulfide bonds are stable under extracellular conditions but can be cleaved in the presence of reducing agents. The tumor intracellular environment is more reductive due to the presence of reducing agents like glutathione (GSH). In the cytoplasm (GSH ~10 mM), GSH react with disulfide bonds by thiol-disulfide exchange reaction, leading to the release of the small molecule STING agonist. As yet another example,for the hypoxia sensitive linkers (Qandmany types of tumors, including those of the breast, lung, colon, and liver, overexpress NQO1. This overexpression is often associated with the need for tumor cells to manage increased oxidative stress and maintain redox homeostasis. The reduced NQO1 , after obtaining two electrons from NADH, catalyzes a four-electron reduction reaction to cleave the azobenzene bond. Subsequently, the resulting unstable aniline undergoes a 1 ,6-elimination reaction, leading to the self-immolation of the remaining residue.

[0123] Accordingly, in these illustrative embodiments, the compositions herein may comprise PEG-6-P(MAC-SC7A) (PSC7A) covalently linked to MSA-2, diABZI, or cGAMP via a stimulus responsive linker. Accordingly, in these illustrative embodiments, the compositions herein may comprise PEG-6-P(MAC-SC7A) (PSC7A) covalently linked to either MSA-2 or diABZi via a stimulus responsive linker. In various aspects, the compositions herein comprise PEG-6-P(MAC-SC7A) (PSC7A) covalently linked to MSA-2 or diABZI via a hypoxia sensitive linker(e.g.,

[0124] In view of the foregoing, in various aspects, an exemplary compound of Formula II may be PSC7A-(pH)-mMSA-2 as depicted herein., wherein n is 3, 10, or 20 and m is 125-n. In some aspects, n is 10 and m is 115. In some aspects, n is 20 and m is 105.

[0125] In various aspects, another exemplary compound of Formula II may be PSC7A-(Re)- mMSA-2, shown below:PSC7A-(Re)-mMSA-2 , wherein n is 3, 10, or 20 and m is 125-n. In some aspects, n is 10 and m is 115. In some aspects, n is 20 and m is 105.

[0126] In still further aspects, another exemplary compound of Formula II may be PSC7A- (Hy)-m MSA-2, shown below:psc7A-(Hy)-mMSA-2 , wherein n is 3, 10, or 20 and m is 125-n. In some aspects, n is 10 and m is 115. In some aspects, n is 20 and m is 105.

[0127] In particular aspects, a compound of Formula II may comprise a PSC7A-(Hy)-mMSA-2 compound as provided below (e.g., when n is 20 and m is 105):

[0128] In still further aspects, another exemplary compound of Formula II may be PSC7A- (Re)-mdiABZI, shown below:, wherein n is 3, 10, or 20 and m is 125-n. In some aspects, n is 10 and m is 115. In some aspects, n is 20 and m is 105.

[0129] In still further aspects, another exemplary compound of Formula II may be PSC7A- (Hy)-mdiABZI, shown below:, wherein n is 3, 10, or 20 and m is 125-n. In some aspects, n is 10 and m is 115. In some aspects, n is 20 and m is 105.

[0130] In particular aspects, a compound of Formula II may comprise a PSC7A-(Hy)-mdiABZI compound as provided below (e.g., when n is 10 and m is 115):

[0131] Also provided herein are compositions (e.g., pharmaceutical compositions) comprising more than one of the compounds provided herein. In various aspects, the compositions may be formulated as nanoparticles - that is the polymeric backbone of these compounds self assembles into a micelle formation capable of encapsulating a desired molecule. Suitable methods for preparing nanoparticles of the compounds disclosed herein (e.g., PSC7A-(Hy)- mdiABZI or PSC7A-(Hy)-mMSA-2 as described above) are known in the art. For example, nanoparticles may be prepared using a pH inversion method as described in the Examples below. In some aspects, a composition comprising nanoparticles formed from PSC7A-(Hy)- mMSA-2 is provided. In some aspects, a composition comprising nanoparticles formed from PSC7A-(Hy)-mdiABZI is provided.

[0132] In some aspects, a composition comprising nanoparticles formed from selfencapsulating compounds described above are provided. In various aspects, the nanoparticles may encapsulate a bioactive protein or compound and / or may encapsulate additional STING agonists (e.g., a cyclic dinucleotide (CDN) such as cGAMP). These bioactive proteins or compounds may be a therapeutic or an immunogenic protein that could be used to treat or prophylactically trigger an immune response (i.e., such as a vaccine) to treat cancer or an infectious disease (e.g., HPV) in a patient. Non-limiting examples of bioactive proteins or compounds that could be incorporated into the compositions herein include an HPV E7, E2, E5 or E6 protein. Other bioactive proteins or compounds that are contemplated include tumor antigens that are known in the art.

[0133] In view of the foregoing, in certain embodiments, a composition is provided comprising a compound of Formula ll-A (e.g., PSC7A-(pH)-mMSA-2, PSC7A-(Re)-mMSA-2, PSC7A- (Hy)-mMSA-2, PSC7A-(Re)-mdiABZI, or PSC7A-(Hy)-mdiABZI) and an HPV protein (e.g., E7, E2, E5, or E7) where the compound of Formula II encapsulates the HPV protein. In certain embodiments, a composition is provided comprising a compound of Formula ll-A (e.g., PSC7A-(pH)-mMSA-2, PSC7A-(Re)-mMSA-2, PSC7A-(Hy)-mMSA-2, PSC7A-(Re)-mdiABZI, or PSC7A-(Hy)-mdiABZI) and an E7 protein where the compound of Formula ll-A encapsulates the H7 protein. In certain embodiments, a composition is provided comprising PSC7A-(Hy)-mdiABZI and an E7 protein where PSC7A-(Hy)-mdiABZI encapsulates the H7 protein. In certain embodiments, a composition is provided comprising PSC7A-(Hy)-mMSA-2 and an E7 protein where PSC7A-(Hy)-mMSA-2 encapsulates the H7 protein. Such a composition may be used, according to the following embodiments, as a prophylactic therapy for an HPV infection and related complications (i.e., cervical cancer). Alternatively, the compound of Formula II may encapsulate a cyclic dinucleotide (e.g., cGAMP) for targeted delivery and additional activation of the STING pathway in a desired location (i.e., in a tumor microenvironment).

[0134] Any of these compounds may be formulated into pharmaceutical compositions and / or formulations as described further below.

[0135] In certain embodiments, any one or more active agents disclosed herein (e.g., a compound of Formula II) may be provided per se or as part of a pharmaceutical composition, where the active agent(s) can be mixed with suitable carriers or excipients. As used herein a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0136] Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0137] In certain embodiments, compositions disclosed herein may further compromise one or more pharmaceutically acceptable diluent(s), excipient(s), or carrier(s). As used herein, a pharmaceutically acceptable diluent, excipient, or carrier, refers to a material suitable for administration to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Pharmaceutically acceptable diluents, carriers, and excipients can include, but are not limited to, physiological saline, Ringer’s solution, phosphate solution or buffer, buffered saline, and other carriers known in the art. Pharmaceutical compositions may also include stabilizers, antioxidants, colorants, other medicinal or pharmaceutical agents, carriers, adjuvants, preserving agents, stabilizing agents, wetting agents, emulsifying agents, solution promoters, salts, solubilizers, antifoaming agents, antioxidants, dispersing agents, surfactants, and combinations thereof. Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in REMINGTON’S PHARMACEUTICAL SCIENCES, Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0138] In certain embodiments, pharmaceutical compositions described herein may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries to facilitate processing of genetically modified endothelial progenitor cells into preparations which can be used pharmaceutically. In other embodiments,any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art.

[0139] In certain embodiments, pharmaceutical compositions described herein may be an aqueous suspension comprising one or more polymers as suspending agents. In some aspects, polymers that may comprise pharmaceutical compositions described herein include: water-soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose; water-insoluble polymers such as cross-linked carboxyl-containing polymers; mucoadhesive polymers, selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran; or a combination thereof. In other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of polymers as suspending agent(s) by total weight of the composition.

[0140] In certain embodiments, pharmaceutical compositions disclosed herein may comprise a viscous formulation. In some aspects, viscosity of the composition may be increased by the addition of one or more gelling or thickening agents. In other aspects, compositions disclosed herein may comprise one or more gelling or thickening agents in an amount to provide a sufficiently viscous formulation to remain on treated tissue. In still other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of gelling or thickening agent(s) by total weight of the composition. In yet other aspects, suitable thickening agents can be hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate. In other aspects, viscosity enhancing agents can be acacia (gum arabic), agar, aluminum magnesium silicate, sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, Carbopol, xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia, chitin, carboxymethylated chitosan, chondrus, dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthan gum, gum tragacanth, ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether / maleic anhydride copolymer (PVM / MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethylcellulose (HPMC), sodium carboxymethyl-cellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® (dextrose, maltodextrin and sucralose), orcombinations thereof. In some embodiments, suitable thickening agent may be carboxymethyl cellulose.

[0141] In certain embodiments, pharmaceutical compositions disclosed herein may comprise additional agents or additives selected from a group including surface-active agents, detergents, solvents, acidifying agents, alkalizing agents, buffering agents, tonicity modifying agents, ionic additives effective to increase the ionic strength of the solution, antimicrobial agents, antibiotic agents, antifungal agents, antioxidants, preservatives, electrolytes, antifoaming agents, oils, stabilizers, enhancing agents, and the like. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more agents by total weight of the composition. In other aspects, one or more of these agents may be added to improve the performance, efficacy, safety, shelf-life and / or other property of the muscarinic antagonist composition of the present disclosure. In s aspects, additives will be biocompatible, and will not be harsh, abrasive, or allergenic.

[0142] In certain embodiments, pharmaceutical compositions disclosed herein may comprise one or more acidifying agents. As used herein, “acidifying agents” refers to compounds used to provide an acidic medium. Such compounds include, by way of example and without limitation, acetic acid, amino acid, citric acid, fumaric acid and other alpha hydroxy acids, such as hydrochloric acid, ascorbic acid, and nitric acid and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic acid may be used. In other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more acidifying agents by total weight of the composition.

[0143] In certain embodiments, pharmaceutical compositions disclosed herein may comprise one or more alkalizing agents. As used herein, “alkalizing agents” are compounds used to provide alkaline medium. Such compounds include, by way of example and without limitation, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, and trolamine and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic base can be used. In other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more alkalizing agents by total weight of the composition.

[0144] In certain embodiments, pharmaceutical compositions disclosed herein may comprise one or more antioxidants. As used herein, “antioxidants” are agents that inhibit oxidation andthus can be used to prevent the deterioration of preparations by the oxidative process. Such compounds include, by way of example and without limitation, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophophorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate and sodium metabisulfite and other materials known to one of ordinary skill in the art. In some aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more antioxidants by total weight of the composition.

[0145] In certain embodiments, pharmaceutical compositions disclosed herein may comprise a buffer system. As used herein, a “buffer system” is a composition comprised of one or more buffering agents wherein “buffering agents” are compounds used to resist change in pH upon dilution or addition of acid or alkali. Buffering agents include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate and other materials known to one of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic buffer can be used. In another aspect, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more buffering agents by total weight of the composition. In other aspects, the amount of one or more buffering agents may depend on the desired pH level of a composition. In some embodiments, pharmaceutical compositions disclosed herein may have a pH of about 6 to about 9. In other embodiments, pharmaceutical compositions disclosed herein may have a pH greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6. In a preferred embodiment, compositions disclosed herein may have a pH greater than about 6.8.

[0146] In certain embodiments, pharmaceutical compositions disclosed herein may comprise one or more preservatives. As used herein, “preservatives” refers to agents or combination of agents that inhibits, reduces, or eliminates bacterial growth in a pharmaceutical dosage form. Non-limiting examples of preservatives include Nipagin, Nipasol, isopropyl alcohol and a combination thereof. In some aspects, any pharmaceutically acceptable preservative can be used. In other aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more preservatives by total weight of the composition.

[0147] In certain embodiments, pharmaceutical compositions disclosed herein may comprise one or more surface-acting reagents or detergents. In some aspects, surface-acting reagents or detergents may be synthetic, natural, or semi-synthetic. In other aspects, compositionsdisclosed herein may comprise anionic detergents, cationic detergents, zwitterionic detergents, ampholytic detergents, amphoteric detergents, nonionic detergents having a steroid skeleton, or a combination thereof. In still other aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more surface-acting reagents or detergents by total weight of the composition.

[0148] In certain embodiments, pharmaceutical compositions disclosed herein may comprise one or more stabilizers. As used herein, a “stabilizer” refers to a compound used to stabilize an active agent against physical, chemical, or biochemical process that would otherwise reduce the therapeutic activity of the agent. Suitable stabilizers include, by way of example and without limitation, succinic anhydride, albumin, sialic acid, creatinine, glycine, and other amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycols, sodium caprylate and sodium saccharin and others known to those of ordinary skill in the art. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more stabilizers by total weight of the composition.

[0149] In certain embodiments, pharmaceutical compositions disclosed herein may comprise one or more tonicity agents. As used herein, a “tonicity agents” refers to a compound that can be used to adjust the tonicity of the liquid formulation. Suitable tonicity agents include, but are not limited to, glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose, and others known to those or ordinary skill in the art. Osmolarity in a composition may be expressed in milliosmoles per liter (mOsm / L). Osmolarity may be measured using methods commonly known in the art. In preferred embodiments, a vapor pressure depression method is used to calculate the osmolarity of the compositions disclosed herein. In some aspects, the amount of one or more tonicity agents comprising a pharmaceutical composition disclosed herein may result in a composition osmolarity of about 150 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 280 mOsm / L to about 370 mOsm / L or about 250 mOsm / L to about 320 mOsm / L.

[0150] In certain embodiments, the present disclosure provides compositions formulated for one or more routes of administration. Suitable routes of administration may, for example, include oral, rectal, transmucosal, transnasal, intestinal, and / or parenteral delivery. In some embodiments, compositions herein formulated can be formulated for parenteral delivery. In some embodiments, compositions herein formulated can be formulated intramuscular, subcutaneous, intramedullary, intravenous, intraperitoneal, and / or intranasal injections.

[0151] In certain embodiments, one may administer a composition herein in a local or systemic manner, for example, via local injection of the pharmaceutical composition directly into a tissue region of a patient. In some embodiments, a pharmaceutical composition disclosed herein can be administered parenterally, e.g., by intravenous injection, intracerebroventricular injection, intra-cisterna magna injection, intra-parenchymal injection, or a combination thereof. In some embodiments, a pharmaceutical composition disclosed herein can administered to subject as disclosed herein. In some embodiments, a pharmaceutical composition disclosed herein can administered to human patient. In some embodiments, a pharmaceutical composition disclosed herein can administered to a human patient via at least two administration routes. In some embodiments, the combination of administration routes by be intracerebroventricular injection and intravenous injection; intrathecal injection and intravenous injection; intra-cisterna magna injection and intravenous injection; and / or intra-parenchymal injection and intravenous injection.

[0152] In certain embodiments, pharmaceutical compositions of the present disclosure may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0153] In certain embodiments, pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the active ingredients of a pharmaceutical composition herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, physiological salt buffer, or any combination thereof.

[0154] In certain embodiments, pharmaceutical compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection herein may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. In some embodiments, compositions herein may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and / or may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0155] In certain embodiments, pharmaceutical compositions herein formulated for parenteral administration may include aqueous solutions of the active preparation (e.g., a bisphosphonate, such as risedronate) in water-soluble form. In some embodiments,compositions herein comprising suspensions of the active preparation may be prepared as oily or water-based injection suspensions. Suitable lipophilic solvents and / or vehicles for use herein may include, but are not limited to, fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. In some embodiments, compositions herein comprising aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, and / or dextran. In some embodiments, compositions herein comprising a suspension may also contain one or more suitable stabilizers and / or agents which increase the solubility of the active ingredients (e.g., a STING agonist) to allow for the preparation of highly concentrated solutions.

[0156] In some embodiments, compositions herein may comprise the active ingredient in a powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water-based solution, before use.

[0157] Pharmaceutical compositions suitable for use in context of the present disclosure may include compositions wherein the active ingredients can be contained in an amount effective to achieve the intended purpose. In some embodiments, a therapeutically effective amount means an amount of active ingredients (e.g., a STING agonist) effective to prevent, slow, alleviate or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.

[0158] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0159] For any preparation used in the methods of the present disclosure, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays and or screening platforms disclosed herein. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0160] In some embodiments, toxicity and therapeutic efficacy of the active ingredients disclosed herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. In some embodiments, data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in a human subject. In some embodiments, a dosage for use herein may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient’s condition. (See e.g., Fingl, et al., 1975, in “THE PHARMACOLOGICAL BASIS OF THERAPEUTICS”).

[0161] In certain embodiments, dosage amounts and / or dosing intervals may be adjusted individually to brain or blood levels of the active ingredient that are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). In some embodiments, the MEC for an active ingredient (e.g., a STING agonist) may vary for each preparation but can be estimated from in vitro data. In some embodiments, dosages necessary to achieve the MEC herein may depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0162] In certain embodiments, depending on the severity and responsiveness of the condition to be treated, dosing with compositions herein can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is affected or diminution of the disease state is achieved.

[0163] In certain embodiments, amounts of a composition herein to be administered will be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and the like. In some embodiments, effective doses may be extrapolated from dose-responsive curves derived from in vitro or in vivo test systems.

[0164] In certain embodiments, the amount of the bioactive composition contained in the dosage forms of the present disclosure will depend on the specific STING agonist composition selected and the continuous dosing schedule upon which the STING agonist is dosed to the subject. Continuous dosing schedules of daily, weekly, twice monthly, three times per month, and once monthly are non-limiting examples of dosing regimens suitable for use with the dosage forms of the present disclosure. The terms “three times per month” or “thrice monthly” mean that an oral dosage form is administered thrice, i.e., three times, during a monthly calendar period. In a thrice monthly schedule, the dosage forms disclosed herein may be administered on three consecutive days, or once about every nine to eleven days. The terms “twice per month” or “twice monthly” mean that an oral dosage form is administered twice, i.e., two times, during a monthly calendar period. In a twice monthly regimen, the dosage forms disclosed herein may be administered on consecutive days or once about every fourteen to sixteen days. The terms “monthly” or “once monthly” mean that a dosage form disclosed herein is administered once, i.e., one time during a monthly calendar period, that is, about every 28 to 31 days.III. Methods of Use

[0165] The present disclosure provides for methods of treating, attenuating, and preventing various diseases or conditions in a subject in need thereof. Together these methods all provide for more targeted, effective therapies for a variety of the diseases or conditions.

[0166] In various aspects, the compounds and compositions herein are useful for targeted delivery of a STING agonist to a target in vivo. These may allow for targeted delivery of the STING agonist to treat a variety of diseases or conditions. Accordingly, a method for treating and / or preventing a disease or condition in a subject is provided, the method comprising administering to a subject a composition comprising a compound (e.g., a compound Formula II), comprising a STING activating biodegradable polymer covalently linked to a small molecule STING agonist via a stimulus-responsive linker, as provided herein.

[0167] In various aspects, the disease or disorder is an infectious disease. In this way, the compositions may in some aspects be delivered as a vaccine (i.e. , as a prophylactic treatment for the infectious disease or as an oncovaccine). In some cases, the infectious disease can be a bacterial infection, a viral infection, a fungal infection, a parasitic infection. In some aspects, the infectious disease may comprise human papilloma virus (HPV), influenza (e.g., influenza A or B), a coronavirus infection (e.g., COVID-19), a streptococcal infection (e.g., streptococcal pharyngitis), meningitis (e.g., viral or bacterial meningitis), tuberculosis, or any other infectious disease. In some instances, the infectious disease is HPV. In certain aspects, a method of treating or preventing HPV is provided comprising administering an HPV antigenic peptide (i.e., E7, E2, E5 or E6 peptide) to a subject via the compositions (nanoparticles) disclosed herein. In some aspects the HPV antigenic peptide is encapsulated in a nanoparticle formed from a compound having the structure of:

[0168] In various aspects, the disease or condition can include a cancer. In various aspects, the cancer can include a solid tumor. In various aspects the cancer comprises a non-small cell lung cancer, a head or neck cancer, melanoma, liver cancer, or cervical cancer. In various aspects, the tumor can be a non-small cell lung carcinoma. In various aspects, the tumor can be a squamous cell carcinoma or an adenocarcinoma. In various aspects the tumor can be a cervical carcinoma (e.g., a squamous cell carcinoma or an adenocarcinoma of the cervix). In some aspects, the cancer is caused by an infectious agent (e.g., human papilloma virus (HPV)).

[0169] In further aspects, methods above further comprise administering an additional cancer therapy. The cancer therapy can comprise a composition disclosed herein, or can include any one or more of surgery, radiation therapy, chemotherapy, immunotherapy, hormone therapy, oncolytic viruses, targeted therapies, polysaccharides, neoantigens, vaccine, or combinations thereof. Suitable examples of each of these cancer therapies are described further below.(a) Immunotherapy

[0170] In some embodiments, one or more cancer therapies may be immunotherapy. Immunotherapy may comprise, for example, use of cancer vaccines, an immune checkpoint inhibitor, a cytokine therapy, sensitized antigen presenting cells, or other modified immune cells (e.g., CAR-T cells). The immunotherapy can involve passive immunity for short-term protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen (e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen). Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines.

[0171] In some aspects, the immunotherapy comprises a checkpoint inhibitor. In some aspects, the checkpoint inhibitor is a PD-1 or PDL-1 inhibitor. PD-1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1 , B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7-DC, Btdc, and CD273. In some embodiments, PD-1 , PDL1 , and PDL2 are human PD-1 , PDL1 and PDL2.

[0172] In some embodiments, the PD-1 inhibitor is a molecule that inhibits the binding of PD- 1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and / or PDL2. In another embodiment, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and / or B7-1 . In another embodiment, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S.Patent Application Nos. US2014 / 0294898, US2014 / 022021 , and US2011 / 0008369, all incorporated herein by reference.

[0173] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD- 1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, the PD-1 inhibitor is nivolumab. In some embodiments, the PD-1 inhibitor is pidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some embodiments, the PDL1 inhibitor comprises AMP- 224. Nivolumab, also known as MDX- 1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009 / 114335. Pidilizumab, also known as CT-011 , hBAT, or hBAT-1 , is an anti-PD-1 antibody described in W02009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-FC fusion soluble receptor described in W02010 / 027827 and WO2011 / 066342. Additional anticancer PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0174] In some embodiments, the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHlgM12B7.

[0175] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1 , CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1 , CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another embodiment, the antibody competes for binding with and / or binds to the same epitope on PD-1 , PDL1 , or PDL2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.

[0176] Another immune checkpoint that can be targeted in the methods provided herein as an additional anti-cancer therapy is the cytotoxic T-lymphocyte-associated protein 4 (CTLA- 4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbankaccession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off” switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell costimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA-4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1 , and / or B7-2 activity. In some embodiments, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some embodiments, the inhibitor blocks the CTLA- 4 and B7-2 interaction.

[0177] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0178] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA- 4 antibodies disclosed in: US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. W02001 / 014424, W02000 / 037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.

[0179] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1 , MDX- 010, MDX- 101 , and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WOO 1 / 14424).

[0180] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1 , CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1 , CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another embodiment, the antibody competes for binding with and / or binds to the same epitope on PD- 1 , B7-1 , or B7-2 as the above- mentioned antibodies. In another embodiment, the antibodyhas at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.

[0181] In certain aspects, the immunotherapy comprises an adoptive T-cell therapy. Adoptive T cell therapy is a form of passive immunization by the transfusion of T-cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically, they activate when the T-cell’s surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune- mediated tumor death.

[0182] Multiple ways of producing and obtaining tumor targeted T-cells have been developed. T-cells specific to a tumor antigen can be removed from a tumor sample (TILs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the results reinfused. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens.

[0183] In some aspects, the immunotherapy comprises a CAR-T cell therapy. Chimeric antigen receptors (CARs, also known as chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy.

[0184] The basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions. The general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on cancer cells. Scientists can remove T-cells from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells. Once the T cell has been engineered to become a CAR-T cell, it acts as a “living drug”. CAR-T cells create a link between an extracellular ligand recognition domain to an intracellular signaling molecule which in turn activates T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of molecule that is targeted.

[0185] In some aspects, the immunotherapy comprises a cytokine therapy. Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immuneresponses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.

[0186] Interferons are produced by the immune system. They are usually involved in anti-viral response, but also have use for cancer. They fall in three groups: type I (IFNa and I FN p) , type II (IFNy) and type III (IFNA).

[0187] Interleukins have an array of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.(b) Chemotherapy

[0188] In some embodiments, one or more cancer therapies may be chemotherapy. Chemotherapeutic agents may be selected from any one or more of cytotoxic antibiotics, antimetabolities, anti-mitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof. Suitable classes of chemotherapeutic agents include (a) Alkylating Agents, such as treosulfan, trofosfamide and nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin), (c) Natural Products, such as vinca alkaloids (e.g., vinblastine, vincristine), other plant alkyloids (paclitaxel, docetaxol), epipodophylotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitoxanthrone), enzymes (e.g., L- asparaginase), and biological response modifiers (e.g., Interferon-a), (d) DNA topoisomerase inhibitors (doxorubicin, epirubicin, etoposide, camptothecin, topotecan, irinotecan, teniposide, crisnatol, and mitomycin), (e) DNA antimetabolites (2'-deoxy-5-fluorouridine, aphidicolin glycinate, and pyrazoloimidazole) and antimitotic agents (halichondrin, colchicine, and rhizoxin) and (e) Miscellaneous Agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydiazine derivatives (e.g., procarbazine), and adreocortical suppressants (e.g., taxol and mitotane). In some embodiments, cisplatin is a particularly suitable chemotherapeutic agent. Compositions comprising one or more chemotherapeutic agents (e.g., FLAG, CHOP) may also be used. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone.

[0189] Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, with efficacious doses used in clinical applications including about 15 mg / m2 to about 20 mg / m2 for 5 days every three weeks for a total of three courses being contemplated in certain embodiments. In some embodiments, the amount of cisplatin delivered to the cell and / or subject in conjunction with the construct comprising an Egr-1 promoter operably linked to a polynucleotide encoding the therapeutic polypeptide is less than the amount that would be delivered when using cisplatin alone.

[0190] Other suitable chemotherapeutic agents include antimicrotubule agents, e.g., Paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”). The combination of an Egr- 1 promoter / TNFa construct delivered via an adenoviral vector and doxorubicin was determined to be effective in overcoming resistance to chemotherapy and / or TNF-a, which suggests that combination treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNF-a.

[0191] Doxorubicin is absorbed poorly and is preferably administered intravenously. In certain embodiments, appropriate intravenous doses for an adult include about 60 mg / m2 to about 75 mg / m2 at about 21 -day intervals or about 25 mg / m2 to about 30 mg / m2 on each of 2 or 3 successive days repeated at about 3 week to about 4 week intervals or about 20 mg / m2 once a week. The lowest dose should be used in elderly patients, when there is prior bone-marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs.

[0192] Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the disclosure. A nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®) (available from Mead Johnson) and NEOSTAR® (available from Adria), is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, intravenous doses include, for example, initially about 40 mg / kg to about 50 mg / kg in divided doses over a period of about 2 days to about 5 days or about 10 mg / kg to about 15 mg / kg about every 7 days to about 10 days or about 3 mg / kg to about 5 mg / kg twice a week or about 1.5 mg / kg / day to about 3 mg / kg / day. Because of adverse gastrointestinal effects, the intravenous route is preferred. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities.

[0193] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluouracil; 5-Fll) and floxuridine (fluorodeoxyuridine; FudR). 5-Fll may be administered to a subject in a dosage of anywhere between about 7.5 to about 1000 mg / m2. Further, 5-Fll dosing schedules may be for a variety of time periods, for example up to six weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.

[0194] Gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., “gemcitabine”), another suitable chemotherapeutic agent, is recommended for treatment of advanced and metastatic pancreatic cancer, and will therefore be useful in the present disclosure for these cancers as well.

[0195] The amount of the chemotherapeutic agent delivered to the patient may be variable. In one suitable embodiment, the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapy is administered with the construct. In other embodiments, the chemotherapeutic agent may be administered in an amount that is anywhere between 2 to 10,000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. For example, the chemotherapeutic agent may be administered in an amount that is about 20 fold less, about 500 fold less or even about 5000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. The chemotherapeutics of the disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct, as well as for determination of effective dosages. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc. In vitro testing may also be used to determine suitable combinations and dosages, as described in the examples.(c) Radiation Therapy

[0196] In some embodiments, one or more cancer therapies may be radiation therapy. The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays, X-rays, or proton beams. Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and / or total abdominal and pelvic radiation therapy. In some aspects, the radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. In other aspects, the radiation treatment can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass. Alsoencompassed is the use of photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A; and 2BA-2-DMHA.

[0197] In some embodiments, the amount of ionizing radiation is greater than 20 Gy and is administered in one dose. In some embodiments, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some embodiments, the amount of ionizing radiation is at least, at most, or exactly 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 18, 19, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 40 Gy (or any derivable range therein). In some embodiments, the ionizing radiation is administered in at least, at most, or exactly 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 does (or any derivable range therein). When more than one dose is administered, the does may be about 1 , 4, 8, 12, or 24 hours or 1 , 2, 3, 4, 5, 6, 7, or 8 days or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any derivable range therein.

[0198] In some embodiments, the amount of IR may be presented as a total dose of IR, which is then administered in fractionated doses. For example, in some embodiments, the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each. In some embodiments, the total dose is 50-90 Gy, administered in 20-60 fractionated doses of 2-3 Gy each. In some embodiments, the total dose of IR is at least, at most, or about 20, 21 , 22, 23, 24, 25, 26, 27,28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52,53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77,78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 ,102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, or 150 (or any derivable range therein). In some embodiments, the total dose is administered in fractionated doses of at least, at most, or exactly 1 , 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein). In some embodiments, at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 fractionated doses are administered (or any derivable range therein). In some embodiments, at least, at most, or exactly 1 , 2, 3, 4, 5, 6, 7,8, 9, 10, 11 , or 12 (or any derivable range therein) fractionated doses are administered per day. In some embodiments, at least, at most, or exactly 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range therein) fractionated doses are administered per week.(d) Hormone Therapy

[0199] In some embodiments, one or more cancer therapies may be hormonal therapy, Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LLIPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).(e) Targeted Therapies

[0200] In some embodiments, the one or more cancer therapies comprise a targeted therapy. Targeted therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules (“molecular targets”) that are involved in the growth, progression, and / or spread of cancer. Targeted cancer therapies are sometimes called “molecularly targeted drugs,” “molecularly targeted therapies,” “precision medicines,” or similar names. Non-limiting examples of targeted therapies include hormone therapies, signal transduction inhibitors, gene expression modulators, apoptosis inducers, angiogenesis inhibitors, immunotherapies, toxin delivery molecules, and the like. In particular embodiments, the targeted therapy may be a poly ADP ribose polymerase (PARP) inhibitor (e.g., niraparib). PARP (e.g., PARP-1 and / or PARP-2) inhibitors are well known in the art (e.g., Olaparib, ABT- 888, BSI-201 , BGP-15, INO-1001 , PJ34, 3-aminobenzamide, 4-amino-1 ,8- naphthalimide, 6(5H)-phenanthridinone, benzamide, NLI1025).(f) Oncolytic virus

[0201] In some embodiments, one or more cancer therapies comprise an oncolytic virus. An oncolytic virus is a virus that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumor. Oncolytic viruses are thought not only to cause direct destruction of the tumor cells, but also to stimulate host anti-tumor immune responses for long-term immunotherapy.(g) Polysaccharides

[0202] In some embodiments, one or more cancer therapies comprise polysaccharides. Certain compounds found in mushrooms, primarily polysaccharides, can up-regulate the immune system and may have anti-cancer properties. For example, beta-glucans such as lentinan have been shown in laboratory studies to stimulate macrophage, NK cells, T cells and immune system cytokines and have been investigated in clinical trials as immunologic adjuvants.(h) Neoantigens

[0203] In some embodiments, one or more cancer therapies comprise neoantigen administration. Many tumors express mutations. These mutations potentially create new targetable antigens (neoantigens) for use in T cell immunotherapy. The presence of CD8+ T cells in cancer lesions, as identified using RNA sequencing data, is higher in tumors with a high mutational burden. The level of transcripts associated with cytolytic activity of natural killer cells and T cells positively correlates with mutational load in many human tumors.(i) Surgery

[0204] In some aspects, the one or more cancer therapy may comprise surgery. Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).

[0205] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1 , 2, 3, 4, 5, 6, or 7 days, or every 1 , 2, 3, 4, and 5 weeks or every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months. These treatments may be of varying dosages as well.

[0206] In various aspects, the cancer therapy can comprise chemotherapy, immunotherapy, or a combination thereof. In various aspects, the chemotherapy can comprise a neoadjuvant chemotherapy. In various aspects the immunotherapy can comprise a PD-1 inhibitor such as Pembrolizumab.

[0207] In certain embodiments, the duration and / or dose of treatment with anticancer therapies may vary according to the particular anti-cancer agent or combination thereof. An appropriate treatment time for a particular cancer therapeutic agent will be appreciated by the skilled artisan. In some embodiments, the continued assessment of optimal treatment schedules for each cancer therapeutic agent is contemplated, where the genetic signature of the cancer of the subject as determined by the methods of the invention is a factor in determining optimal treatment doses and schedules.

[0208] In various aspects, the cancer therapy can comprise administering to a subject in need thereof a compound provided herein (e.g., biodegradable STING activating polymer covalently linked to a STING agonist via a stimulus-responsive linker such as Formula II). In various aspects, the cancer therapy comprises administering one or more nanoparticles formed from a compound provided herein (e.g., biodegradable STING activating polymer covalently linked to a STING agonist via a stimulus-responsive linker such as Formula II). In one non-limiting example, the cancer therapy may comprise a nanoparticle formed from

[0209] In any of these aspects the compound comprising a biodegradable polymer covalently linked or bonded to a STING agonist via a stimulus-responsive linker are administered orally, intraadiposally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularlly, intravitreally, liposomally, locally, mucosally, parenterally, rectally, subconjunctival, subcutaneously, sublingually, topically, transbuccally, transdermally, vaginally, in cremes, in lipid compositions, via a catheter, via a lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, or via localized perfusion. For instance, in certain aspects the compound comprising a biodegradable polymer covalently linked or bonded to a STING agonist via a stimulus-responsive linker is administered via intraarterial administration, intramuscular administration, intraperitoneal administration, intratumoral administration, or intravenous administration.

[0210] Suitable tumors that may be targeted according to the methods herein can include any solid tumor. In some embodiments, a solid tumor can be an abnormal mass of tissue that is devoid of cysts or liquid regions within the tumor. In some embodiments, solid tumors can be benign (not progressed to a cancer), a malignant or metastatic tumor. In some embodiments, a solid tumor herein can be a malignant cancer that has metastasized. In other embodiments, solid tumors contemplated herein can include, but are not limited to, sarcomas, carcinomas, lymphomas, gliomas or a combinational thereof. In accordance with some embodiments herein, tumors resistant to platinum-based chemotherapy can include, but are not limited to, a testicular tumor, ovarian tumor, cervical tumor, a kidney tumor, bladder tumor, head-and- neck tumor, liver tumor, stomach tumor, lung tumor, endometrial tumor, esophageal tumor,breast tumor, cervical tumor, central nervous system tumor, germ cell tumor, prostate tumor, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, neuroblastoma, sarcoma, multiple myeloma, melanoma, mesothelioma, osteogenic sarcoma or a combination thereof. In some embodiments, a targeted tumor contemplated herein can include a solid tumor such as ovarian tumors, breast tumors, or any combination thereof. For instance, in some aspects, the tumor is a lung tumor, a pancreatic tumor, a skin tumor, or any other solid tumor. In some aspects, the tumor is a non-small cell lung carcinoma, head or neck cancer, melanoma, liver cancer, or cervical cancer.

[0211] In certain embodiments, compositions disclosed herein can treat and / or prevent cancer in a subject in need. In some embodiments, compositions disclosed herein can impair tumor growth compared to tumor growth in an untreated subject with identical disease condition and predicted outcome. In some embodiments, tumor growth can be stopped following treatment with compositions disclosed herein. In other embodiments, tumor growth can be impaired at least about 5% or greater to at least about 100%, at least about 10% or greater to at least about 95% or greater, at least about 20% or greater to at least about 80% or greater, at least about 40% or greater to at least about 60% or greater compared to an untreated subject with identical disease condition and predicted outcome. In other words, tumors in subject treated using a composition of the disclosure have tumors that grow at least 5% less (or more as described above) when compared to an untreated subject with identical disease condition and predicted outcome. In some embodiments, tumor growth can be impaired at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, at least about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, at least about 100% compared to an untreated subject with identical disease condition and predicted outcome. In some embodiments, tumor growth can be impaired at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% compared to an untreated subject with identical disease condition and predicted outcome.

[0212] In some embodiments, treatment of tumors with compositions disclosed herein can result in a shrinking of a tumor in comparison to the starting size of the tumor. In some embodiments, tumor shrinking is at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% (meaning that the tumor is completely gone after treatment) compared to the starting size of the tumor.

[0213] In certain embodiments, compositions disclosed herein can improve patient life expectancy compared to the cancer life expectancy of an untreated subject with identical disease condition and predicted outcome. As used herein, “patient life expectancy” is defined as the time at which 50 percent of subjects are alive and 50 percent have passed away. In some embodiments, patient life expectancy can be indefinite following treatment with a composition disclosed herein. In other aspects, patient life expectancy can be increased at least about 5% or greater to at least about 100%, at least about 10% or greater to at least about 95% or greater, at least about 20% or greater to at least about 80% or greater, at least about 40% or greater to at least about 60% or greater compared to an untreated subject with identical disease condition and predicted outcome. In some embodiments, patient life expectancy can be increased at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, atleast about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, at least about 100% compared to an untreated subject with identical disease condition and predicted outcome. In some embodiments, patient life expectancy can be increased at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% compared to an untreated patient with identical disease condition and predicted outcome.

[0214] In some embodiments, the methods of the present disclosure increase anti-tumor activity (e.g., reduce cell proliferation, tumor growth, tumor volume, and / or tumor burden or load or reduce the number of metastatic lesions over time) by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels prior to treatment or in a control subject. In some embodiments, reduction is measured by comparing cell proliferation, tumor growth, and / or tumor volume in a subject before and after administration of the pharmaceutical composition. In some embodiments, the method of treating or ameliorating a cancer in a subject allows one or more symptoms of the cancer to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, methods disclosed herein may include administration of the compositions herein to reduce tumor volume, size, load or burden in a subject to an undetectable size, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the subject’s tumor volume, size, load or burden prior to treatment. In other embodiments, methods disclosed herein may include administration of the compositions herein to reduce the cell proliferation rate or tumor growth rate in a subject to an undetectable rate, or to less than about 1 %, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the rate prior to treatment.

[0215] In some embodiments, a subject to be treated by any of the methods and / or compositions herein can present with one or more cancerous solid tumors, metastatic nodes, of a combination thereof. In some embodiments, a subject herein may have a cancerous tumor cell source that can be less than about 0.2 cm3to at least about 20 cm3or greater, at least about 2 cm3to at least about 18 cm3or greater, at least about 3 cm3to at least about 15 cm3or greater, at least about 4 cm3to at least about 12 cm3or greater, at least about 5 cm3to at least about 10 cm3or greater, or at least about 6 cm3to at least about 8 cm3or greater.

[0216] In certain embodiments, the compositions disclosed herein can be effective for treating at least one tumor cell in a solid tumor from a subject in need. In some embodiments, the amount of viable tumor cells may be reduced by at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about25% or greater, at least about 30% or greater, at least about 35% or greater, at least about40% or greater, at least about 45% or greater, at least about 50% or greater, at least about55% or greater, at least about 60% or greater, at least about 65% or greater, at least about70% or greater, at least about 75% or greater, at least about 80% or greater, at least about85% or greater, at least about 90% or greater, at least about 95% or greater, at least about100% compared to an untreated subject with identical disease condition and predicted outcome.

[0217] Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the compositions disclosed herein to a subject, depending upon the type of disease to be treated or the site of the disease. In some embodiments, compositions herein can be administered to a subject by intravenous infusion, by subcutaneous administration, by inhalation, by intranasal administration or other mode of administration. In some embodiments, compositions herein can be administered to a subject via intra-tumoral injection.

[0218] In some embodiments, any of the methods disclosed herein can further include monitoring occurrence of one or more beneficial or adverse effects in the subject. Exemplary adverse effects include, but are not limited to, hepatic impairment, hematologic toxicity, neurologic toxicity, cutaneous toxicity, gastrointestinal toxicity, or a combination thereof. When one or more adverse effects are observed, the method disclosed herein can further include reducing or increasing the dose of the compound comprising a biodegradable polymer covalently linked or bonded to a STING agonist via a stimulus-responsive linker disclosed herein (or other cancer therapy) depending on the adverse effect or effects in the subject.

[0219] A suitable subject includes a human, a livestock animal, a companion animal, a lab animal, or a zoological animal. In one embodiment, the subject may be a rodent, e.g., a mouse,a rat, a guinea pig, etc. In another embodiment, the subject may be a livestock animal. Nonlimiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas, and alpacas. In yet another embodiment, the subject may be a companion animal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, the subject may be a zoological animal. As used herein, a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In a specific embodiment, the animal is a laboratory animal. Non-limiting examples of a laboratory animal may include rodents, canines, felines, and non-human primates. In certain embodiments, the animal is a rodent. Non-limiting examples of rodents may include mice, rats, guinea pigs, etc. In preferred embodiments, the subject is a human.IV. Kits

[0220] The present disclosure provides kits for use in treating a tumor. Such kits can include one or more containers including one or more disclosed active agents (e.g., a compound comprising a biodegradable polymer covalently linked or bonded to a STING agonist via a stimulus-responsive linker). In some embodiments, kits can include one or more containers including one or more one or more disclosed active agents.

[0221] In some embodiments, the kits herein can include instructions for use in accordance with any of the methods described herein. The included instructions can have a description of administration of the one or more disclosed active agents to treat, delay the onset, or alleviate a target disease as those described herein, or a combination thereof. In some embodiments, the kit can further include a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease, e.g., applying a diagnostic method as described herein. In still other embodiments, the instructions can have a description of administering any one of the compositions described herein to an individual at risk of the target disease.

[0222] In some embodiments, kit instructions relating to the use of one or more disclosed active agents can generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers can be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0223] The label or package insert indicates that the composition is used for treating, delaying the onset and / or alleviating the disease and / or symptom thereof (e.g., cancer). In some embodiments, instructions are provided for practicing any of the methods described herein.

[0224] The kits of this disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. In some embodiments, a kit has a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some embodiments, the container also has a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).

[0225] In some embodiments, kits herein can optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides articles of manufacture comprising contents of the kits described above.

[0226] Having described several embodiments, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the present disclosure. Additionally, a number of well-known processes and elements have not been described in order to avoid unnecessarily obscuring the present disclosure. Accordingly, this description should not be taken as limiting the scope of the present disclosure.

[0227] Those skilled in the art will appreciate that the presently disclosed embodiments teach by way of example and not by limitation. Therefore, the matter contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the method and assemblies, which, as a matter of language, might be said to fall there between.EXAMPLES

[0228] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments whichare disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Example 1 : Preparation of Stimulus Responsive STING agonist compounds

[0229] In a first example, a series of compounds were prepared each containing a polymeric backbone, a stimulus responsive linker and a STING agonist. FIG. 1 illustrates a general structure (Formula ll-A) of these compounds. As shown in the figure, the polymeric backbone is connected to the STING agonist via a linker, which can be stimulus responsive. Illustrative stimulus - responsive linkers (i.e. , those responsive to pH, redox, or hypoxia) and suitable STING agonists (e.g., MSA-2 and diABZI) are also shown in FIG. 1.

[0230] Using these linkers and STING agonists a series of compounds were synthesized. The general synthesis schemes for each of the following compounds are described below and depicted in FIG. 2, FIG. 3, FIG. 4, FIG. 5, and FIG. 6.

[0231] Synthesis of PSC7A(pH)-mMSA-2

[0232] PSC7A-(pH)-mMSA-2 is a compound of Formula I where the linker is pH sensitive, and the STING agonist is MSA-2. Referring to FIG. 2, PSC7A-(pH)-mMSA-2 is synthesized via the two intermediates, 1-1 and 1-2, described herein.

[0233] Synthesis of 1-1 (tert-butyl 2-(3-mercaptopropanoyl)hydrazine-1-carboxylate)

[0234] In a solution containing 3-mercaptopropionic acid (1.0 g, 9.4 mmol) in 10 mL of dichloromethane (DCM), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (3.6 g, 18.8 mmol), and NHS (N-hydroxysuccinimide) (1.3 g, 11.3 mmol) were introduced. The reaction mixture was stirred at room temperature for 1 hour, followed by the addition of tert-butyl carbazate (1.5 g, 11.3 mmol), and stirring was continued at room temperature overnight. The resulting crude product was subjected to washing with DCM / water and drying over sodium sulfate. The final product was isolated through column chromatography on silica gel, yielding 1-1 (1 g, 50%).1H NMR (400 MHz, CDCI3) 5 2.77 (t, 2H, -CH2-), 2.55 (t, J = 6.8 Hz, 2H, -CH2-), 1.43 (s, 9H, -3(CH3)3). LC-MS: [M+H]+: 221.1.

[0235] Synthesis of 1-2 (PEG-b-pMAC-m(tert-butyl 2-(3-mercaptopropanoyl)hydrazine-1- carboxylate))

[0236] PEG-b-pMAC (1 g) and 1-1 (m=3: 21.4 mg, m=10: 71.0 mg, m=20: 142.0 mg) were dissolved in 50 mL of dimethylformamide (DMF). Subsequently, 2,2-dimethoxy-2- phenylacetophenone (DMPA) (19.4 mg, 75.7 pmol) was added, and the reaction proceeded under UV light for 2 hours at room temperature. Following this, C7AS HCI (12.3 g, 62.8 mmol) was introduced into the reaction solution, and the remaining DMPA (19.4 mg, 75.7 pmol) was added under UV light. The reaction further continued for 16 hours at room temperature. Theresulting crude product was subjected to dialysis in water, followed by lyophilization, yielding the final product 1-2.1H NMR (400 MHz, CDCI3) 54.24 (d, J = 20.0 Hz, 6H, -CH2CCH2-, -CH2- ), 3.62 (s, 2H, -OCH2CH2O-), 3.35 (s, 3H, -CH3), 3.24 (s, 6H, -CH2-, -CH2NCH2-), 3.01 (s, 2H, -SCH2-), 2.62 (d, J = 6.8 Hz, 2H, -CH2-), 2.14 (s, 2H, -CH2-), 1.94 (t, J = 7.0 Hz, 4H, -CH2-, - CH2-), 1.68 (d, J = 15.4 Hz, 4H, -CH2-, -CH2-), 1.41 (s, 9H, -3(CH3)3), 1.25 (d, J = 4.1 Hz, 3H, -CH3).

[0237] Synthesis of PSC7A-(pH)-mMSA-2

[0238] 1-2 (100 mg) and MSA-2 (m=3: 10 mg, m=10: 30 mg, m=20: 60 mg) were dissolved in 5 mL of DCM with 0.5 mL trifluoroacetic acid (TFA), and the reaction proceeded overnight. The resulting reaction mixture was then dialyzed with methanol to obtain the final product.1H NMR (400 MHz, CDCI3) 5 7.90 (s, 1 H, Ar-H), 7.24 (s, 2H, Ar-H), 4.26 (d, J = 20.0 Hz, 6H, - CH2CCH2-, -CH2-), 3.98 (d, J = 9.4 Hz, 6H, -CH3), 3.64 (s, 2H, -OCH2CH2O-), 3.37 (s, 3H, - CH3), 3.23 (s, 6H, -CH2-, -CH2NCH2-), 3.02 (s, 2H, -SCH2-), 2.63 (d, J = 6.8 Hz, 2H, -CH2-), 2.16 (s, 2H, -CH2-), 1.95 (t, J = 7.0 Hz, 4H, -CH2-, -CH2-), 1.68 (d, J = 15.4 Hz, 4H, -CH2-, - CH2-), 1.44 (s, 9H, -3(CH3)3), 1.26 (d, J = 4.1 Hz, 3H, -CH3).

[0239] Synthesis of PSC7A-(Re)-mMSA-2

[0240] PSC7A-(Re)-mMSA-2 is a compound of Formula I where the linker is redox sensitive, and the STING agonist is MSA-2. Referring to FIG. 3, PSC7A-(Hy)-mMSA-2 is synthesized via four intermediates (2-1 , 2-2, 2-3, and 2-4), described herein below.

[0241] Synthesis of 2-1 (2-(pyridin-2-yldisulfaneyl)ethan-1-ol)

[0242] 1 ,2-Di(pyridin-2-yl)disulfane (5.6 g, 25.6 mmol) was dissolved in 50 mL of ethanol (EtOH) with stirring. Subsequently, 2-mercaptoethanol (1.0 g, 12.8 mmol) was slowly added dropwise to the solution, and the reaction proceeded for 6 hours at rt. The solvent was then evaporated, and the resulting mixture was washed with ethyl acetate and water. Afterward, the crude product was dried over sodium sulfate. Purification of the crude product was acomplished through silica gel column chromatography, resulting 2-1 (1.5 g, 62.5%).1H NMR (400 MHz, CDCI3) 5 7.66 - 7.52 (m, 2H, Py-H), 7.39 (t, J = 7.9 Hz, 1 H, Py-H), 6.77 (t, J = 6.7 Hz, 1 H, Py-H), 3.91 (t, J = 5.8 Hz, 2H, -CH2-), 2.88 (t, J = 5.8 Hz, 2H, -CH2-). LC-MS: [M+H]+: 188.0.

[0243] Synthesis of 2-2 (2-(pyridin-2-yldisulfaneyl)ethyl 4-(5,6-dimethoxybenzo[b]thiophen-2- yl)-4-oxobutanoate)

[0244] MSA-2 (157.1 mg, 534.0 mmol) and 2-1 (100.0 mg, 534.0 mmol) were dissolved in 50 mL of DCM. Subsequently, DMAP (4-dimethylaminopyridine) (13.1 mg, 106.8 mmol) and EDCI (204.0 mg, 1068.0 mmol) were added to the solution, and the reaction proceeded for 6hours. The solvent was then evaporated, and the residue was washed with DCM / water. The crude product was further purified through a silica gel column, yielding 2-2 (195.2 mg, 78.9%).1H NMR (400 MHz, CDCI3) 5 8.46 (ddd, J = 4.8, 1.8, 1.0 Hz, 1 H, Py-H), 7.89 (d, J = 0.6 Hz, 1 H, Ar-H), 7.68 (s, 1 H, Py-H), 7.66 (dd, J = 7.1 , 1.8 Hz, 1 H, Py-H), 7.25 (d, J = 2.0 Hz, 2H, Ar- H), 7.09 (ddd, J = 7.1 , 4.8, 1.4 Hz, 1 H, Py-H), 4.37 (t, J = 6.4 Hz, 2H, -CH2-), 3.96 (d, J = 10.0 Hz, 6H, -CH3), 3.31 (t, J = 6.7 Hz, 2H, -CH2-), 3.05 (t, J = 6.4 Hz, 2H, -CH2-), 2.78 (t, J = 6.7 Hz, 2H, -CH2-). LC-MS: [M+H]+: 464.1.

[0245] Synthesis of 2-3 (3-((2-((4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4- oxobutanoyl)oxy)ethyl)disulfaneyl)propanoic acid )

[0246] 2-2 (100 mg, 215.7 pmol) was dissolved in a mixture of 50 mL DCM and ethanol (EtOH). Subsequently, 3-mercaptopropionic acid (27.5 mg, 258.9 pmol) was added dropwise to the solution, and the reaction was allowed to proceed for 6 hours. The solvent was then evaporated, and the resulting residue was washed with DCM / water. The crude product underwent further purification through a silica gel column, affording 2-3 (79.12 mg, 80.0% yield).1H NMR (400 MHz, CDCI3) 5 7.89 (s, 1 H, Ar-H), 7.25 (s, 2H, Ar-H), 4.37 (t, J = 6.6 Hz, 2H, -CH2-), 3.96 (d, J = 9.6 Hz, 6H, -CH3), 3.32 (d, J = 6.7 Hz, 2H, -CH2-), 2.99 - 2.88 (m, 4H, -CH2-), 2.86 - 2.75 (m, 4H, -CH2-). LC-MS: [M+H]+: 458.1.

[0247] Synthesis of 2-4 (2-((3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)disulfaneyl)ethyl 4- (5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate)

[0248] 2-3 (50.0 mg, 109.0 pmol) and EDCI (41.7 mg, 218.1 pmol) were dissolved in 50 mL of DCM. Subsequently, NHS (15.1 mg, 130.8 pmol) was added to the solution, and the reaction proceeded for 2 hours. The solvent was then evaporated, and the resulting residue was washed with DCM / water, yielding 2-4 (50.1 mg, 82.5%).1H NMR (400 MHz, CDCI3) 5 7.89 (s, 1 H, Ar-H), 7.25 (s, 2H, Ar-H), 4.37 (t, J = 6.4 Hz, 2H, -CH2-), 3.96 (d, J = 9.4 Hz, 6H, -CH3), 3.32 (t, J = 6.7 Hz, 2H, -CH2-), 3.09 - 3.03 (m, 2H, -CH2-), 3.03 - 2.97 (m, 2H, -CH2-), 2.94 (t, J = 6.5 Hz, 2H, -CH2-), 2.88 - 2.71 (m, 6H, -CH3), 1 .33 - 1.20 (m, 2H, -CH2-). LC-MS: [M+H]+: 556.1.

[0249] Synthesis of PSC7A-(Re)-mMSA-2

[0250] PSC7A-mNH2 (100 mg) and 2-4 (m=3: 10 mg, m=10: 30 mg, m=20: 60 mg) were dissolved in 5 mL of dimethylformamide (DMF), then K2CO3 (5 mg) was added to the solution, and the reaction proceeded overnight. The reaction mixture was then dialyzed with methanol to obtain the final product.1H NMR (400 MHz, CDCI3) 5 7.89 (s, 1 H, Ar-H), 7.25 (s, 2H, Ar-H), 4.21 (d, J = 20.0 Hz, 6H, -CH2CCH2-, -CH2-), 3.96 (d, J = 9.4 Hz, 6H, -CH3), 3.59 (s, 2H, - OCH2CH2O-), 3.33 (s, 3H, -CH3), 3.27 - 3.04 (s, 2H, -SCH2-), 2.62 (d, J = 6.8 Hz, 2H, -CH2-),2.14 (s, 2H, -CH2-), 1.94 (t, J = 7.0 Hz, 4H, -CH2-, -CH2-), 1.68 (d, J = 15.4 Hz, 4H, -CH2CH2- ), 1.25 (d, J = 4.1 Hz, 3H, -CH3).

[0251] Synthesis of PSC7A-(Hy)-mMSA-2

[0252] PSC7A-(Hy)-mMSA-2 is a compound of Formula I where the linker is hypoxia sensitive, and the STING agonist is MSA-2. Referring to FIG. 4, it is synthesized via four intermediates (3-1 , 3-2, 3-3, and 3-4) as detailed below.

[0253] Synthesis of 3-1 (tert-butyl 2-(4-nitrosophenyl)acetate)

[0254] Tert-butyl 2-(4-aminophenyl)acetate (1 g, 4.8 mmol) was dissolved in 50 mL of DCM and added to a solution of Oxone® (3.0 g, 7.7 mmol) in water. Subsequently, the reaction mixture was stirred for 4.5 hours at room temperature. Afterward, the deep green organic layer was separated, and the aqueous solution was neutralized by the addition of a saturated solution of NaHCO3 (50 mL). The mixture was then washed with DCM / water, dried over Na2SO4, resulting in the product of 3-1.1H NMR (400 MHz, CDCI3) 5 8.18 (d, 2H, J = 8.7, Ar- H), 7.44 (d, 2H, J = 8.7, Ar-H), 3.63 (s, 2H, Ar-CH2), 1.44 (s, 9H, -(CH3)3). LC-MS: [M+H]+: 222.1.

[0255] Synthesis of 3-2 (tert-butyl 2-(4-((4-(hydroxymethyl)phenyl)diazenyl)phenyl)acetate)

[0256] 3-1 (500 mg, 2.71 mmol) was dissolved in 10 mL of ethanol (EtOH) containing acetic acid (AcOH, 0.5 mL) with stirring at 0 °C. Subsequently, 4-aminobenzyl alcohol (334.0 mg, 2.71 mmol) was added slowly to the solution, and the reaction proceeded for 2 hours at room temperature. The solvent was then evaporated, and the resulting residue was washed with DCM / water. The crude product underwent further purification through a silica gel column, yielding 3-2 (650.0 mg, 88.1%).1H NMR (400 MHz, CDCI3) 5 8.25 (d, J = 8.6 Hz, 2H, Ar-H), 8.16 (d, J = 8.6 Hz, 2H, Ar-H), 7.42 - 7.35 (m, 4H, Ar-H), 3.61 (s, 2H, ArCH2-), 3.58 (s, 2H, ArCH2-), 1.44 (s, 9H, -(CH3)3). LC-MS: [M+H]+: 327.2.

[0257] Synthesis of 3-3 (4-((4-(2-(tert-butoxy)-2-oxoethyl)phenyl)diazenyl)benzyl 4-(5,6- dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate)

[0258] MSA-2 (50.0 mg, 170.0 pmol) and 3-2 (66.5 mg, 203.9 pmol) were dissolved in 15 mL of DCM. Subsequently, 4-dimethylaminopyridine (DMAP) (4.2 mg, 34.0 pmol) and 1-ethyl-3- (3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (65.0 mg, 340.0 pmol) were added to the solution, and the reaction proceeded for 6 hours at room temperature. The solvent was then evaporated, and the resulting residue was washed with DCM / Water. The crude product was further purified through a silica gel column, yielding 3-3 (90.0 mg, 87.9%).1H NMR (400 MHz, CDCI3) 5 7.94 - 7.78 (m, 5H, Ar-H), 7.48 (d, J = 8.4 Hz, 2H, Ar-H), 7.43 (d, J = 8.4 Hz, 2H, Ar-H), 7.24 (d, J = 6.0 Hz, 2H, Ar-H), 5.22 (s, 2H, ArCH2-), 3.96 (d, J = 9.7Hz, 6H, -CH3), 3.61 (s, 2H ArCH2-), 3.35 (t, J = 6.7 Hz, 2H, -CH2-), 2.88 (t, J = 6.7 Hz, 2H, - CH2-), 1.45 (s, 9H, -(CH3)3). LC-MS: [M+H]+: 603.2.

[0259] Synthesis of 3-4 (4-((4-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2- oxoethyl)phenyl)diazenyl)benzyl 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate)

[0260] 3-3 (50.0 mg, 83.0 pmol) was dissolved in 10 mL of DCM, then 10 mL of trifluoroacetic acid (TFA) was added with further stirring for 15 minutes at 0 °C. Subsequently, the solvent was evaporated, and the resulting residue was dissolved in 10 mL of DCM without requiring further purification, proceeding directly to the next step. EDCI (31.7 mg, 166.0 pmol) and NHS (11.5 mg, 99.6 pmol) were added to the solution, and the reaction proceeded for 2 hours at rt. The solvent was then evaporated, and the resulting residue was washed with DCM / water, yielding 3-4 (50.0 mg, 93.6%).1H NMR (400 MHz, CDCI3) 5 8.02 - 7.81 (m, 5H, Ar-H), 7.49 (dq, J = 11.0, 5.9, 4.1 Hz, 4H, Ar-H), 7.23 (d, J = 5.6 Hz, 2H, Ar-H), 5.21 (s, 2H, ArCH2-), 4.11 (q, J = 7.2 Hz, 4H, -CH2-), 4.01 (s, 2H, ArCH2-), 3.96 (d, J = 9.7 Hz, 6H, -CH3), 3.95 (dd, J = 10.2, 2.9 Hz, 4H, -CH2-). LC-MS: [M+H]+: 644.2.

[0261] Synthesis of PSC7A-(Hy)-mMSA-2

[0262] PSC7A-mNH2 (100 mg) and 3-4 (m=3: 10 mg, m=10: 30 mg, m=20: 60 mg) were dissolved in 5 mL of dimethylformamide (DMF), then K2CO3 (5 mg) was added to the solution, and the reaction proceeded overnight at rt. The reaction mixture was then dialyzed with methanol to obtain the final product.1H NMR (400 MHz, CDCI3) 5 8.02 - 7.81 (m, 5H, Ar-H), 7.49 (dq, J = 11.0, 5.9, 4.1 Hz, 4H, Ar-H), 7.23 (d, J = 5.6 Hz, 2H, Ar-H), 5.21 (s, 2H, ArCH2- ), 4.24 (d, J = 20.0 Hz, 6H, -CH2CCH2-, -CH2-), 3.96 (d, J = 9.4 Hz, 6H, -CH3), 3.62 (s, 2H, - OCH2CH2O-), 3.35 (s, 3H, -CH3), 3.24 (s, 6H, -CH2-, -CH2NCH2-), 3.01 (s, 2H, -SCH2-), 2.62 (d, J = 6.8 Hz, 2H, -CH2-), 2.14 (s, 2H, -CH2-), 1.94 (t, J = 7.0 Hz, 4H, -CH2-, -CH2-), 1.68 (d, J = 15.4 Hz, 4H, -CH2-, -CH2-), 1.25 (d, J = 4.1 Hz, 3H, -CH3).

[0263] Synthesis of PSC7A-(Re)-mdiABZI

[0264] PSC7A-(Re)-mdiABZI is a compound of Formula I where the linker is redox sensitive and the STING agonist is diABZI. Referring to FIG. 5, PSC7A-(Re)-mdiABZI is synthesized via four intermediates (4-1 , 4-2, 4-3, and 4-4), as described herein below.

[0265] Synthesis of 4-1

[0266] 1 ,2-Di(pyridin-2-yl)disulfane (3.7 g, 16.6 mmol) was dissolved in 20 mL of ethanol (EtOH) with stirring. Subsequently, 4-mercaptobutyric acid (1 .0 g, 8.3 mmol) was slowly added dropwise to the solution, and the reaction proceeded for 6 hours. The solvent was then evaporated, and the resulting mixture was washed with ethyl acetate and water. Afterward, the crude product was dried over sodium sulfate. Purification of the crude product wasachieved through silica gel column chromatography, yielding 4-1 (1.1 g, 60.1 %). 1 H NMR (400 Hz, CDCI3) 5 10.01 (s, 1 H, COOH), 8.45-8.46 (m, 1 H, Py-H), 7.69-7.72 (m, 1 H, Py -H), 7.61-7.66 (m, 1 H, Py -H), 7.06-7.09 (m, 1 H, Py -H), 2.78 (t, J = 6.63 Hz, 2H, -CH2-), 2.34 (t, J = 6.64 Hz, 2H, -CH2-), 1.71-1.73 (m, 2H, -CH2-). LC-MS: [M+H]+: 230.0.

[0267] Synthesis of 4-2

[0268] diABZI (100.0 mg, 128.1 umol) and 4-1 (35.2 mg, 153.7 umol) were dissolved in 10 mL of DCM. Subsequently, DMAP (4-dimethylaminopyridine) (13.1 mg, 106.8 mmol) and EDCI (204.0 mg, 1068.0 mmol) were added to the solution, and the reaction proceeded for 6 hours at rt. The solvent was then evaporated, and the residue was washed with DCM / water. The crude product was further purified through a silica gel column, yielding 4-2 (110.1 mg, 86.7%).1 H NMR (400 MHz, DMSO) 58.45 (m, 1 H, Py-H) 8.00 (d, J = 11.9 Hz, 4H, -NH2), 7.73 (m, 1 H, Py -H), 7.69 - 7.57 (m, 3H, Ar-H, Py-H), 7.42 (s, 2H, Pyr-H), 7.33 (s, 4H, Ar-H), 7.66 (m, 1 H, Py -H), 6.50 (d, J = 2.4 Hz, 2H, -CH=CH-), 5.82 (s, 2H, Ar-CH2), 4.90 (d, J = 6.3 Hz, 4H, -CH2-), 4.58 - 4.48 (m, 4H, -CH2-), 4.06 (t, J = 6.4 Hz, 2H, -CH2-), 3.74 (s, 3H, -CH3), 3.31 (s, 2H, Ar-CH2), 2.80 (t, J = 6.63 Hz, 2H, -CH2-), 2.36 (t, J = 6.64 Hz, 2H, -CH2-), 2.09 (d, J =2.6 Hz, 6H, -CH3), 1.70-1.73 (m, 4H, -CH2-), 1.20 (t, J = 7.3 Hz, 6H, -CH3). LC-MS: [M+H]+: 1084.4

[0269] Synthesis of 4-3

[0270] 4-2 (100 mg, 100.8 pmol) was dissolved in a mixture of 10 mL DCM and ethanol (EtOH). Subsequently, 3-mercaptopropionic acid (12.8 mg, 130.0 pmol) was added dropwise to the solution, and the reaction was allowed to proceed for 6 hours at rt. The solvent was then evaporated, and the resulting residue was washed with DCM / water. The crude product underwent further purification through a silica gel column, affording 2-3 (85.1 mg, 85.5% yield).1H NMR (400 MHz, DMSO) 5 8.31 (d, J = 7.6 Hz, 2H, Ar-H), 8.19 (s, 4H, -NH2), 8.02 (s, 1 H, Ar-H), 7.64 (d, J = 4.2 Hz, 1 H, Ar-H), 7.34 (s, 2H, Pyr-H), 7.02 (d, J = 7.6 Hz, 3H, Ar-H), 5.34 - 5.22 (m, 2H, -CH2-), 4.56 (d, J = 5.8 Hz, 2H, -CH2-), 4.55 - 4.49 (m, 2H, -CH2-), 4.40 (t, J =6.7 Hz, 3H, -CH3), 3.91 (q, J = 7.2 Hz, 2H, -CH2-), 3.59 (s, 3H, -CH3), 3.57 (s, 3H, -CH3), 2.68 (s, 3H, -OCH3), 2.59 (d, J = 4.1 Hz, 2H, -CH2-), 2.31 (dd, J = 16.6, 4.5 Hz, 2H, -CH2-), 2.23 (t, J = 7.1 Hz, 2H, -CH2-), 2.09 (d, J = 2.9 Hz, 2H, -CH2-), 2.02 - 1.93 (m, 2H, -CH2-), 1.89 - 1.80 (m, 2H, -CH2-), 1.70 (t, J = 6.1 Hz, 2H, -CH2-), 1.07 - 1.04 (m, 3H, -CH3), 0.84 (t, J = 6.4 Hz, 2H, -CH2-). LC-MS: [M+H]+: 987.3.

[0271] Synthesis of 4-4

[0272] 4-3 (50.0 mg, 50.7 pmol) and EDCI (19.4 mg, 101.4 pmol) were dissolved in 50 mL of DCM. Subsequently, NHS (7.0 mg, 60.8 pmol) was added to the solution, and the reaction proceeded for 2 hours at rt. The solvent was then evaporated, and the resulting residue waswashed with DCM / water, yielding 4-4 (50.2 mg, 91.4%).1H NMR (400 MHz, DMSO) 5 8.33 (d, J = 7.6 Hz, 2H, Ar-H), 8.21 (s, 4H, -NH2), 8.04 (s, 1 H, Ar-H), 7.66 (d, J = 4.2 Hz, 1 H, Ar-H), 7.36 (s, 2H, Pyr-H), 7.05 (d, J = 7.6 Hz, 3H, Ar-H), 5.32 - 5.21 (m, 2H, -CH2-), 4.58 (d, J = 5.8 Hz, 2H, -CH2-), 4.55 - 4.49 (m, 2H, -CH2-), 4.40 (t, J = 6.7 Hz, 3H, -CH3), 3.91 (q, J = 7.2 Hz, 2H, -CH2-), 3.59 (s, 3H, -CH3), 3.58 (s, 3H, -CH3), 2.69 (s, 3H, -OCH3), 2.59 (d, J = 4.1 Hz, 2H, -CH2-), 2.31 (dd, J = 16.6, 4.5 Hz, 2H, -CH2-), 2.23 (t, J = 7.1 Hz, 4H, -CH2-), 2.09 (d, J = 2.9 Hz, 2H, -CH2-), 2.00 - 1.92 (m, 4H, -CH2-), 1.86 - 1.81 (m, 2H, -CH2-), 1.74 (t, J = 6.1 Hz, 2H, -CH2-), 1.09 - 1.05 (m, 3H, -CH3), 0.89 (t, J = 6.4 Hz, 2H, -CH2-). LC-MS: [M+H]+: 1084.4.

[0273] Synthesis of PSC7A-(Re)-mdiABZI

[0274] PSC7A-mNH2 (100 mg) and 4-4 (m=3: 10 mg, m=10: 30 mg, m=20: 60 mg) were dissolved in 5 mL of dimethylformamide (DMF), then K2CO3 (5 mg) was added to the solution, and the reaction proceeded overnight. The reaction mixture was then dialyzed with methanol to obtain the final product.1H NMR (400 MHz, CDCI3) 5 8.34 (d, J = 7.6 Hz, 2H, Ar-H), 8.22 (s, 4H, -NH2), 8.06 (s, 1 H, Ar-H), 7.68 (d, J = 4.2 Hz, 1 H, Ar-H), 7.38 (s, 2H, Pyr-H), 7.08 (d, J = 7.6 Hz, 3H, Ar-H), 5.34 - 5.22 (m, 2H, -CH2-), 4.62 (d, J = 5.8 Hz, 2H, -CH2-), 4.51 - 4.46 (m, 2H, -CH2-), 4.21 (d, J = 20.0 Hz, 6H, -CH2CCH2-, -CH2-), 3.59 (s, 2H, -OCH2CH2O-), 3.33 (s, 3H, -CH3), 3.27 - 3.04 (s, 2H, -SCH2-), 2.62 (d, J = 6.8 Hz, 2H, -CH2-), 2.14 (s, 2H, -CH2-), 1.94 (t, J = 7.0 Hz, 4H, -CH2-, -CH2-), 1.68 (d, J = 15.4 Hz, 4H, -CH2CH2-), 1.25 (d, J = 4.1 Hz, 3H, -CH3), 0.86 (t, J = 6.4 Hz, 2H, -CH2-).

[0275] Synthesis of PSC7A-(Hy)-mdiABZI

[0276] PSC7A-(Hy)-mdiABZI is a compound of Formula I where the linker is hypoxia sensitive and the STING agonist is diABZI. Referring to FIG. 6, PSC7A-(Hy)-mdiABZI is synthesized via three intermediates (5-1 , 5-2, and 5-3), as described herein below.

[0277] Synthesis of 5-1

[0278] 3-2 (500 mg, 1.53 mmol) and DMAP (37.5 mg, 306.0 pmol) were dissolved in 10 mL of dichloromethane (DCM) with stirring at 0 °C. 4-Nitrophenyl chloroformate (370.5 mg, 1.84 mmol) was slowly added to the solution, and the reaction proceeded for 1 hour at room temperature. The solvent was then evaporated, and the resulting residue was washed with DCM / water. The crude product underwent further purification through a silica gel column to obtain the final product 5-1 (650.0 mg, 86.3%).1H NMR (400 MHz, CDCI3) 5 8.15 (dd, J = 9.9, 2.9 Hz, 2H, Ar-H), 7.85 - 7.75 (m, 4H, Ar-H), 7.50 - 7.43 (m, 2H, Ar-H), 7.35 - 7.24 (m, 4H, Ar-H), 5.25 (s, 2H, ArCH2-), 3.50 (s, 2H, ArCH2-), 1.34 (s, 9H, -(CH3)3). LC-MS: [M+H]+: 492.2.

[0279] Synthesis of 5-2

[0280] diABZI (50.0 mg, 64.0 pmol) and 5-1 (47.2 mg, 95.1 pmol) were dissolved in 5 mL of DCM. Subsequently, 4-dimethylaminopyridine (DMAP) (1.6 mg, 12.8 pmol) was added to the solution, and the reaction proceeded for overnight at room temperature. The solvent was then evaporated, and the resulting residue was washed with DCM / water. The crude product was further purified through a silica gel column, yielding 5-2 (41.6 mg, 56.8%).1H NMR (400 MHz, DMSO-cfe) 6 8.44 (d, J = 5.8 Hz, 4H, -NH2), 8.06 (d, J = 8.1 Hz, 2H, Ar-H), 7.77 (d, J = 11.9 Hz, 2H, -CH=CH-), 7.66 (dd, J = 18.7, 8.1 Hz, 4H, Ar-H), 7.21 (s, 2H, Ar-H), 6.92 (s, 2H, Pyr- H), 6.78 (d, J = 6.0 Hz, 4H, Ar-H), 5.94 (s, 2H, ArCH2-), 5.38 (s, 2H, -CH2-), 5.12 (s, 3H, -CH3), 4.87 (s, 3H, -CH3), 4.33 (s, 2H, ArCH2-), 3.84 (s, 2H, -CH2-), 3.72 (s, 2H, -CH2-), 3.47 (s, 3H, -OCH3), 2.31 (s, 2H, -CH2-), 1.95 (s, 2H, -CH2-), 1.69 (s, 6H), 1.64 (d, 6H, -CH3), 1.51 (s, 2H, -CH2-). LC-MS: [M+H]+: 1133.5.

[0281] Synthesis of 5-3

[0282] 5-2 (50.0 mg, 44.1 pmol) was dissolved in 10 mL of DCM, then 10 mL of trifluoroacetic acid (TFA) was added with further stirring for 15 minutes at 0 °C. Subsequently, the solvent was evaporated, and the resulting residue was dissolved in 10 mL of DCM without requiring further purification, proceeding directly to the next step. EDCI (8.4 mg, 88.2 pmol) and NHS (6.1 mg, 52.9 pmol) were added to the solution, and the reaction proceeded for 2 hours at rt. The solvent was then evaporated, and the resulting residue was washed with DCM, yielding 5-3 (35.5 mg, 68.5%).1H NMR (400 MHz, DMSO-cfe) 6 8.46 (d, J = 5.8 Hz, 4H, -NH2), 8.08 (d, J = 8.1 Hz, 2H, Ar-H), 7.78 (d, J = 11.9 Hz, 2H, -CH=CH-), 7.66 (dd, J = 18.7, 8.1 Hz, 4H, Ar- H), 7.24 (s, 2H, Ar-H), 6.94 (s, 2H, Pyr-H), 6.79 (d, J = 6.0 Hz, 4H, Ar-H), 5.95 (s, 2H, ArCH2- ), 5.40 (s, 2H, -CH2-), 5.17 (s, 3H, -CH3), 4.89 (s, 3H, -CH3), 4.33 (s, 2H, ArCH2-), 3.84 (s, 2H, -CH2-), 3.72 (s, 2H, -CH2-), 3.47 (s, 3H, -OCH3), 2.35-2.37 (m, 4H, -CH2-), 1.94-1.98 (m, 4H, - CH2-), 1.68 (s, 6H), 1.66 (d, 6H, -CH3), 1.55 (s, 2H, -CH2-). LC-MS: [M+H]+: 1174.4.

[0283] Synthesis of PSC7A-(Hy)-mdiABZI

[0284] PSC7A-mNH2(100 mg) and 5-4 (m=3: 10 mg, m=10: 30 mg, m=20: 60 mg) were dissolved in 5 mL of dimethylformamide (DMF), then K2CO3(5 mg) was added to the solution, and the reaction proceeded overnight. The reaction mixture was then dialyzed with methanol to obtain the final product.1H NMR (400 MHz, CDCI3) 5 8.48 (d, J = 5.8 Hz, 4H, -NH2), 8.09 (d, J = 8.1 Hz, 2H, Ar-H), 7.80 (d, J = 11.9 Hz, 2H, -CH=CH-), 7.65 (dd, J = 18.7, 8.1 Hz, 4H, Ar-H), 7.25 (s, 2H, Ar-H), 6.96 (s, 2H, Pyr-H), 6.77 (d, J = 6.0 Hz, 4H, Ar-H), 5.96 (s, 2H, ArCH2-), 5.41 (s, 2H, -CH2-), 5.18 (s, 3H, -CH3), 4.90 (s, 3H, -CH3), 4.35 (s, 2H, ArCH2-), 4.51 - 4.46 (m, 2H, -CH2-), 4.22 (d, J = 20.0 Hz, 6H, -CH2CCH2-, -CH2-), 3.59 (s, 2H, -OCH2CH2O- ), 3.33 (s, 3H, -CH3), 3.27 - 3.04 (s, 2H, -SCH2-), 2.62 (d, J = 6.8 Hz, 2H, -CH2-), 2.14 (s, 2H,-CH2-), 1.94 (t, J = 7.0 Hz, 4H, -CH2-, -CH2-), 1.68 (d, J = 15.4 Hz, 4H, -CH2CH2-), 1.25 (d, J = 4.1 Hz, 3H, -CH3).Example 2: In vitro Release Studies

[0285] Each of the following compounds: PSC7A-(pH)-10MSA-2, PSC7A-(Re)-10MSA-2, PSC7A-(Hy)-10MSA-2, were incubated with acid, GSH (10 mM plus acid), NADPH (100 mM) or Azoreductase (0.5 mg / mL) for 24 hours and then measured using HPLC. The HPLC spectra for each of the compounds is shown in FIG. 7A and the release of each of the STING agonists (e.g., MSA-2 or diAPZI) was quantified in each condition in FIG. 7B and FIG. 7C, respectively. It was shown that the STING agonist was preferentially released at pH 6.5 and in the presence of azoreductase in both compounds.

[0286] As shown in FIG. 7A, PSC7A-(pH)-10MSA-2 was incubated at pH values of 4.5, 5.5, 6.5, and 7.4. After 24 hours, the investigation revealed that MSA-2 release occurred specifically under pH=4.5 conditions from PSC7A-(pH)-10MSA-2. Also shown in FIG. 7A, PSC7A-(Re)-10MSA-2 was incubated with glutathione (GSH) at a concentration of 10 mM under various pH conditions. The HPLC results indicate that the drug release is independent of changes in pH and is solely influenced by the presence or absence of glutathione (GSH). Interestingly, the final released drug is not a complete MSA-2 molecule but rather MSA-2 conjugated with mercaptoethanol. Finally, FIG. 7A also shows that PSC7A-(Hy)-10MSA-2 was incubated with Azoreductase / NADH at pH=6.5 or pH=7.4. The efficient release of MSA-2 was observed only when the pH=6.5 and in the presence of Azoreductase / NADH. This observation supports the characterization of PSC7A-(Hy)-10MSA-2 as a STING NP with an AND logic response gate.

[0287] As shown in FIG. 7B-7C, the time course of drug release from PSC7A-(Re)-10MSA-2 and PSC7A-(Hy)-10diABZI revealed that no payloads release was observed in the absence of Azoreductase / NADH. However, upon the addition of Azoreductase / NADH, the release of MSA-2 at pH=6.5 was 61%, and the release of diABZI was 69%. In contrast, the release of MSA-2 at pH=7.4 was only 8%, and diABZI was 11 %. These results further validate the AND logic drug delivery system.Example 3: Reducing metastasis and tumor growth in a mouse model of metastasis (Lewis lung carcinoma cells (LL / 2))

[0288] At day 0, LL / 2 (Lewis lung carcinoma) 106cells were tail vein injected into 6-8 week- old C57B16 mice. Different drugs were intravenously injected at day 5, maintaining a consistent drug-to-polymer ratio (10:1). The mice were euthanized at day 14, and lung samples were collected (FIG. 8A). After fixation, the nodules were counted (FIG. 8B). PSC7A- (pH)-10MSA-2, PSC7A-(Hy)-10MSA-2, PSC7A-(Re)-10diABZI, and PSC7A-(Hy)-10diABZIexhibited the best tumor suppress effect (FIG. 8B). Subsequently, the screening was expanded to include all STING NPs, revealing that PSC7A-(Hy)-10MSA-2, PSC7A-(Re)- 10diABZI, and PSC7A-(Hy)-10diABZI were the top three groups among all drugs (FIG. 8C). Further testing of cytokine toxicity and biochemical indicators demonstrated that the toxicity of free drugs is significantly higher than that of polymer drugs (FIG. 8D). Comparing drug safety and efficacy data, PSC7A-(Hy)-20MSA-2 and PSC7A-(Hy)-10diABZI emerged as the main candidate drugs due to their superior efficacy and safety profiles, making them suitable for subsequent animal experiments.

[0289] In another set of experiments to test the mechanism behind the tumor suppression above, wild-type, STING-KO, and Batf3-K0 tumor-bearing mice were used. Following treatment with PSC7A-(Hy)-20MSA-2 (20 mg / kg), a loss of therapeutic efficacy (as measured by lung nodule formation) was observed in the STING-KO, Batf3-K0 group compared to the Wild Type group of mice. This is shown in FIG. 8E-8G which show representative lung images and nodule quantification for wildtype, STING- / - and Batf3- / - mice, respectively. This underscores the crucial roles played by STING activation and CDC1 activation in in vivo tumor suppression.

[0290] In a third set of experiments (anti-PD1 / STING combination experiments), PSC7A-(Hy)- 20MSA-2 / aPD1 demonstrated a 100% survival rate compared to PSC7A-(Hy)-20MSA-2 80% survival rate at day 36 (FIG. 8H). In the dose-dependent tumor suppression experiment, 106LL / 2 tumor cells were subcutaneously inoculated, then treatments of PSC7A-(Hy)-20MSA-2 and PSC7A-(Hy)-10diABZI (2, 5, 10, 20 mg / kg) two times were initiated when the tumor size reached 60 mm3, with continuous monitoring of changes in tumor size. It was observed that the changes in tumor size were STING NPs dose-dependent, showing the best tumor inhibition ability at a dose of 20 mg / kg (FIG. 8I). Additionally, PSC7A-(Hy)-10diABZI exhibited a slightly better tumor inhibition ability than PSC7A-(Hy)-20MSA-2.

[0291] Together these experiments demonstrate that illustrative STING NPs of the present disclosure successfully reduce metastasis and tumor growth and increase survival in a mouse model of metastatic lung cancer.Example 4: Treatment of 4T1 andB16F10 breast tumor model mice.

[0292] 5X1054T1 tumor cells was inoculated into mammary fat pad of BABL / C female mice at day 0, PSC7A-(Hy)-20MSA-2 treatment was given when the tumor size was 60mm3. The changes in tumor volume were continuously monitored (FIG. 9A), and the mice were euthanized on day 24 to collect the lungs and count the metastasis nodules (FIG. 9B). It can be seen from FIG. 9A-9B that PSC7A-(Hy)-20MSA-2 exhibits the best tumor suppression ability and rarely produces lung metastases. A separate set of Balb / c mice bearing orthotopictriple negative 4T1 breast tumor were treated with aPD1 (200 ug), PSC7A-Hy-20 MSA-2(10 mg kg-1) and primary tumor growth and overall survival recorded. In these later experiments, when PSC7A-(Hy)-20MSA-2 was combined with aPD1 , because 4T1 is insensitive to aPD1 , PSC7A-(Hy)-20MSA-2 alone showed a major tumor suppressor factor (FIG. 9C) and greatly extended the survival time (FIG. 9D) of mice.

[0293] 105B16F10 tumor cells were tail vein injected into 6-8-week-old C57B16 mice at day 0. PSC7A-(Hy)-20MSA-2 treatment was administered on day 7, and lungs were collected on day 17 to count the number of metastases (FIG. 9E). PSC7A-(Hy)-20MSA-2 demonstrated a notable ability to inhibit B16F10 lung metastases, as measured by lung nodules (FIG. 9F). Additionally, in the PSC7A-(Hy)-20MSA-2 combined with aPD1 experiment, PSC7A-(Hy)- 20MSA-2 / aPD1 significantly improved the survival rate of early-stage mice (FIG. 9G).Example 5: Pharmacokinetics and Biodistribution of STING NPs

[0294] 5X1054T1 tumor cells was inoculated into mammary fat pad of BABL / C female mice at day 0 (FIG. 10A). The formulation of PSC7A-(Hy)-20MSA-2: PSC7A-Cy5(7:3) was given when the tumor size was 100 mm3. Blood samples were collected and quantified using ICG fluorescence, and a fitting analysis resulted in a calculated half-life (TI / 2) fast=0.1859 h, (TI / 2)siow=166 h (FIG. 10B). Simultaneously, image data and fluorescence quantitative data obtained after homogenization of isolated tissues were captured (FIG. 10C). The NPs was primarily taken up by the liver and tumors also (FIG. 10D).Example 6: Anti-HCC Spontaneous Tumor Model (MYC / TP53) and HCC Spontaneous Tumor Model (MYC / CTNNB1)

[0295] MYC / TP53 (FIGS. 11A-11C) or MYC / CTNNB1 (FIGS. 12A-12C) plasmid was administered via tail vein injection on day 0, STING NPs were injected at the third week, then the mice were euthanized in the fifth (MYC / CTNNB1 , FIG. 12A-12C) and sixth weeks (MYC / TP53, FIGS. 11A-11C). Liver and mouse weights were measured since tumor growth would be evident in the liver weight (FIG. 11 B and FIG. 12B). Additionally, livers were collected to count the number of nodules (FIG. 11C, FIG. 12A and FIG. 12C). From the liver images, it is evident that the PSC7A-(Hy)-10diABZI group exhibits clean and small livers, with the majority of liver-to-weight ratios being lower than 0.05 (FIG. 11 B, FIG. 11C, FIG. 12A, FIG. 12C).Example 7: Development of a PolySTING nanovaccine

[0296] In another experiment, a polySTING nanovaccine was derived as shown in FIG 13A. This nanovaccine contained PSC7A-(Hy)-10diABZI encapsulating an E7 protein and was prepared by two methods: (a) “physical mixing” where a mixture of the E7 protein and PSC7A-(Hy)-1 OdiABZI was adjusted to pH 7.4 before they were vigorously mixed and (b) “pH titration” where the protein and STING NPs were adjusted to pH 6.5, before mixing and then titrating the pH to 7.4.

[0297] The FPLC data (FIG. 13B) reveals that the retention volume of PSC7A-(Hy)-1 OdiABZI is 9.11 , and that of E7 is 15.62. However, the retention volume of the nanovaccine, produced using two different methods, increased to 8.88 (Physical mix) and 8.52 (pH titration) (FIG. 13C). Simultaneously, the nanoparticle size also exhibits an increase. This indicates that the E7 protein can be effectively encapsulated by PSC7A-(Hy)-1 OdiABZI, forming a nanovaccine.Example 8: Pharmacokinetics and Biodistribution of nanovaccine

[0298] 2x105TC-1 tumor cells were subcutaneously inoculated when tumor volume around 100 mm3. Mix PSC7A-Cy5 and PSC7A-DIABZ in a 3:7 ratio and then add E7-TMR to form a nano-vaccine with dual fluorescence, which is injected into mice through the tail vein. Simultaneously, blood samples were collected for pharmacokinetic (PK) research, revealing a significant increase in the PK lifespan compared to nanoparticles alone (FIG. 14A). Furthermore, major organs were extracted at different time points for quantitative analysis, revealing a substantial targeting of nanovaccines to the tumor-draining lymph nodes (tDLN) (FIGS. 14B-14C).Example 9: Treatment of mice with PolySTING nanovaccine

[0299] 2x105TC-1 tumor cells were subcutaneously inoculated into C57 B16 female mice. When the tumor size reached 120 mm3, different nanovaccines were injected by the tail vein. The E7@PSC7A-(Hy)-1 OdiABZI group showed the best tumor suppression ability (FIG. 15A), with a 100% survival rate of mice on day 60 (FIG. 15B). To explore the impact of different administration methods on tumor suppression ability, E7-PSC7A-(Hy)-10diABZI was administered through different administration routes (i.t.(1.5 mg kg-1), i.v. (15 mg kg-1), subQ(1.5 mg kg-1)). The IV group exhibited the best therapeutic effect, followed by the IT group and SLIBQ group, validating the importance of systemic drug administration (FIG. 15C- 15D). To investigate the effects of different administration methods on distal tumors, TC-1 tumors (2x105cells) were inoculated subcutaneously on day 0 and re-inoculated TC-1 (1x105cells) tumors on the opposite side on day 2. When the primary tumor size reached 120 mm3, E7-PSC7A-(Hy)-10diABZI NPs were administered through IT, IV, SubQ, and IP routes (i.t.(L: 0.5, M: 5, H: 15 mg kg-1), i.v.(L: 0.5, M: 5, H: 15 mg kg-1), subQ.(L: 0.5, M: 5, H: 15 mg kg-1), i.p. (H: 15 mg kg-1)). The results indicated that the effects of IV and high doses of IT were similar, but the high dose of IT had a significantly lower efficacy on distal tumors compared to the high dose of IV (FIG. 15E-15F). Additionally, in the IP high-dose group, significant tumor suppression ability was observed in both primary and distal tumors.Example 10: Introduction to Examples 11-17

[0300] Stimulator of interferon genes (STING) agonists are promising immune drugs for activating innate immunity and driving robust antitumor responses. However, their clinical potential in systemic immunotherapy for metastatic cancers is limited by poor bioavailability and off-target toxicity, which reduce their therapeutic window. To overcome these challenges, dual-stimuli-responsive AND logic nanoparticles were developed that enable precise spatiotemporal control of STING activation within the tumor microenvironment. By conjugating the STI NG agonist 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (MSA-2) to the pH-responsive polymer PSC7A via different stimuli-responsive linkers, it was identified that PSC7A-Hy- MSA-2 (PHM NP) with hypoxia responsive linker and 20:1 drug-to-polymer molar ratio showed excellent antitumor efficacy and minimal systemic toxicity. PHM NP follows the AND logic gate response to activate STING pathway under hypoxic and acidic conditions. Compared to single stimuli-responsive NP controls, PHM NP demonstrated robust STING activation in THP1-ISG cells and dramatically decreased metastatic foci in LL / 2 lung metastatic tumor model. Furthermore, mechanistic studies revealed that the therapeutic efficacy of PHM NPs was abrogated in STING / _, Batf3 / _, or CD8+T cell depleted mice, underscoring the critical role of STING-conventional type 1 dendritic cells (cDC1s)- CD8+T axis in tumor rejection. In multiple immune-cold metastatic tumor models (LL / 2, 4T1 , and B16F10), PHM NP treatment led to marked inhibition of metastatic foci and increased longterm survival, establishing the AND logic platform as a transformative approach to systemic immunotherapy. By exploiting the metabolic vulnerabilities of tumors — acidosis and hypoxia — the AND logic platform introduces a safe and efficacious approach for systemic therapy of metastatic cancers.

[0301] Metastatic disease is the major cause of mortality in cancer patients, responsible for as much as 90% of cancer-related deaths. Across all cancer types, the five-year survival rate for patients with metastatic disease is less than 20%. Conventional treatments, such as chemotherapy and radiation, offer limited efficacy and are often associated with high toxicity and adverse effects. Targeted therapies and immunotherapies have shown promise but still struggle to effectively control or eliminate metastatic cancer cells, underscoring an urgent need for innovative therapeutic strategies that can activate a robust, systemic antitumor response.

[0302] In recent years, immunotherapy has dramatically changed the landscape of cancer care by harnessing the host immunity against cancer. The stimulator of interferon genes (STING) pathway has emerged as a critical component in the activation of innate immune responses and plays a pivotal role in antitumor immunity. By detecting cytosolic DNA — often released from tumor cells — the cGAS-STING pathway initiates a cascade that activates immune cells and promotes a multifaceted type-l interferon (IFN-I) response that promotesthe maturation and migration of dendritic cells (DCs), and primes cytotoxic T lymphocytes and natural killer (NK) cells for spontaneous immune responses. However, despite its potential, STING-based therapies face significant challenges, including poor bioavailability, inefficient delivery, and the risk of excessive immune activation that can cause toxicity in healthy tissues.

[0303] To address these challenges, stimuli-responsive designs have been developed to enhance the delivery and tumor specificity of STING agonists. These systems can respond to tumor microenvironment factors (pH, enzymes etc.) to release therapeutic agents selectively within tumors. While single-stimulus-responsive designs have shown promise, they are often insufficient in the context of metastatic cancers due to the heterogeneity and complex nature of tumor microenvironments, such as endoprotease in reticuloendothelial system cells, glutathione (GSH) in the bloodstream, and acidic conditions in inflamed tissues complicate the selective activation of therapeutic agents. XMT-2056, a systemically administered antibody- diABZI (a potent STING agonist) conjugates with an endoprotease cleavable linker that target a novel HER2 epitope for local activation of STING in tumors encountered fatal toxicity in a Phase I trial, leading to its suspension. Such setbacks highlight the need for precise control over the location, intensity, and duration of STING activation to maximize antitumor immunity while minimizing off-target effects. AND logic nanoparticles represent a significant advancement in this field. By requiring the simultaneous presence of two distinct stimuli for drug release, these systems improve spatiotemporally precision, reduce off-target effects, and enhance therapeutic outcomes. This dual-stimulus approach is particularly advantageous for systemic therapies treating metastatic tumors, where minimizing immune-related toxicity in healthy tissues is as crucial as achieving tumor-specific activation.

[0304] In this study, the design and evaluation of a novel AND logic nanoparticle for systemic STING therapy of metastatic cancers is presented. This platform, initially described in Examples 1-9 above, utilizes a dual stimuli-responsive mechanism using the biodegradable, ultra-sensitive pH-responsive polymer PEG-b-P(SC7A) (pKa = 6.9), which utilizes a “shock- and-lock” dual STING signaling mode with STING activation in cDC1s for tumor rejection. .A STING agonist MSA-2 was conjugated on the pH-sensitive polymer through a linker that responds to either acidic pH, hypoxia or redox. The AND logic design ensures drug release only when dual stimuli are present: nanoparticle disassembly at acidic pH and the presence of a secondary tumor microenvironment stimulus. Initial screening of those AND logic STING nanoparticles with different MSA-2 ratio and 3 linkers combination showed anti-tumor immunity in LL / 2 metastatic tumors after systemic administration but with varying profiles of efficacy and toxicity. Among them, PSC7A-Hy-20MSA-2 (PHM NP) with hypoxia responsive linker and 20:1 drug-to-polymer ratio exhibited optimal therapeutic performance, showing myeloid cell tropism and selective STING activation in hypoxic conditions with high NQO1expression. Furthermore, cDC1 cells were identified as key mediators of STING activation, promoting crosstalk with CD8+T cells for effective tumor rejection. Collectively, this work highlights the potential of AND logic-based nanoplatforms as a paradigm shift approach for systemic STING therapy in metastatic cancers, offering a promising strategy for precision immunotherapy.Example 11 : Design of AND Logic Nanoparticles and screening for tumor efficacy

[0305] Earlier work by the inventors developed “shock-and-lock” PolySTI NG nanoparticles using the PSC7A polymer for intratumoral STING agonist delivery, demonstrating their potential for localized immunotherapy (see WO2020263733A1 which is incorporated herein by reference in its entirety). Building on this foundation, the inventors then sought to develop AND logic nanoparticles for systemic therapy by conjugating the STING agonist MSA-2 to the polymer backbone through stimuli-responsive linkers (FIG. 16A). Among the widely used stimuli for tumor-targeted drug release, pH, redox, and hypoxia were targeted because they are hallmark features of the tumor microenvironment.

[0306] T o achieve precise control over MSA-2 release, three polymer variants were designed, each functionalized with linkers sensitive to these stimuli: carbonyl hydrazone (acidic pH), disulfide (redox), and azobenzene (hypoxia). The synthetic schemes for these compounds (PSC7A-pH-mMSA-2, PSC7A-Re-mMSA-2, and PSC7A-Hy-mMSA-2) are described Example 1 and shown in FIGS. 2-4. These linkers enable controlled MSA-2 release through distinct chemical mechanisms upon nanoparticle disassembly, operating as an AND logic gate. Specifically, FIGS. 22A-22C show a schematic of chemical reactions releasing MSA-2 from compounds synthesized in FIGS. 2-4 in certain physiological conditions. FIG. 22A refers to PSC7A-pH-mMSA-2, synthesized as shown in FIG. 2. At acidic pH, such as the environment found in tumor intracellular endosomes and lysosomes (pH<5.4), hydrazone bond undergoes protonation, which increases the electrophilicity of carbonyl carbon, making the hydrazone bond susceptible to hydrolysis. FIG. 22B refers PSC7A-Re-mMSA-2 synthesized as shown in FIG. 3. Disulfide bonds are stable under extracellular conditions but can be cleaved in the presence of reducing agents. The tumor intracellular environment is more reductive due to the presence of reducing agents like glutathione (GSH). In the cytoplasm (GSH ~10 mM), GSH react with disulfide bonds by thiol-disulfide exchange reaction, leading to the release of MSA-2. FIG. 22C refers to PSC7A-Hy-mMSA-2 synthesized as shown in FIG. 4. Many types of tumors, including those of the breast, lung, colon, and liver, overexpress NQO1. This overexpression is often associated with the need for tumor cells to manage increased oxidative stress and maintain redox homeostasis. The reduced NQO1 , after obtaining two electrons from NADH, catalyzes a four-electron reduction reaction to cleavethe azobenzene bond. Subsequently, the resulting unstable aniline undergoes a 1 ,6- elimination reaction, leading to the self-immolation of the remaining residue.

[0307] To modulate the density of conjugated MSA-2, the number of side-chain amino groups was adjusted by varying the ratio of cysteamine on the PSC7A backbone. This strategy allowed for tuning drug-to-polymer ratios (3, 10, and 20), which were quantified via UV-vis spectroscopy (Table 1 and FIGS. 20A-20D). These optimizations enabled precise control over the therapeutic index of the AND logic nanoparticles, paving the way for safe and effective systemic delivery of STING agonists.Table 1 : The actual drug conjugating number for all nanoparticles. DPR= drug to polymer ratio.

[0308] AND logic nanoparticles incorporating various stimuli-responsive linkers and drug-to- polymer ratios were formulated by a pH inversion method, achieving a consistent hydrodynamic diameter of ~30 ± 3 nm (Table 2 and FIGS. 21 A-21 L). Specifically, 5 mg of the polymer was dissolved in 1 mL of PBS buffer (pH 5.0). Sodium hydroxide (0.1 M) was then added dropwise under continuous stirring until the pH reached 7.4, facilitating micelle formation. The resulting nanoparticles were characterized using dynamic light scattering (DLS; Zetasizer, Malvern Instruments) equipped with a He-Ne laser (A = 633 nm). Measurements included hydrodynamic size, size distribution, and zeta potential to confirm particle properties.Table 2: The hydrodynamic diameter and polydispersity index (PDI) of all nanoparticles.

[0309] To assess tumor-specific STING activation for immunotherapy, a highly invasive, immune-cold Lewis lung carcinoma (LL / 2) lung metastasis model was used. For the efficacy study, mice received intravenous injections of nanoparticles with a 10:1 drug-to-polymer ratio five days after tumor inoculation (FIG. 16B). On day 14, mouse lungs were harvested and number of metastatic nodules were quantified to evaluate therapeutic outcomes. All AND logic nanoparticles demonstrated suppression of metastatic lung nodules compared to the PBS and free MSA-2 treatment. Among the formulations, the hypoxia-responsive nanoparticles showed the most pronounced therapeutic effect, reducing metastatic foci to just 2-3 nodules per lung, whereas the PBS group exhibited over 30 nodules (FIG. 25). This highlights the superior efficacy of hypoxia-responsive linkers in leveraging the tumor microenvironment for targeted immunotherapy.

[0310] Next, the efficacy and toxicity across three drug-to-polymer ratios (3, 10, and 20) were evaluated for each linker type to determine their therapeutic window for systemic therapy. Following the protocol used in the linker screening, mice were treated intravenously with nanoparticles five days post-tumor inoculation, and lung metastases were quantified on Day 14. Within each linker series, increasing the drug-to-polymer ratio improved antitumor efficacy, with PSC7A-Hy-20MSA-2 demonstrating the most significant reduction in lung metastases (FIG. 16C, FIG. 26). Toxicity was assessed by analyzing liver (ALT, AST) and kidney (CREA, BUN) biomarkers 24 hours post-injection and cytokine expression levels (I L-12P70, IL-6, IL- 10, MCP-1 , IFN-y, TNF) six hours post-injection. While nanoparticles with pH-responsive linkers exhibited increased toxicity at higher drug-to-polymer ratios, as evidenced by elevated liver enzyme and systemic cytokine levels, those with redox- and hypoxia-responsive linkers demonstrated significantly lower toxicity than free MSA-2, comparable to PBS (FIG. 16D, FIG. 26, FIG. 27).

[0311] To identify the optimal formulation, both efficacy (nodule count) and toxicity (biomarker levels) were standardized, assigning equal weight to each parameter. PSC7A-Hy-20MSA-2 achieved the best balance of high efficacy and low toxicity (FIG. 16E, FIGS. 29A-29B), emerging as the lead candidate. Combining PSC7A-Hy-20MSA-2 with aPD1 further enhancedlong-term survival of the animals, highlighting the synergistic potential of this combination therapy (FIGS. 30A-30B).Example 12: MSA-2 release from PHM NP and downstream STING activation

[0312] Building on the tumor regression effects observed with PSC7A-Hy-20MSA-2 (hereafter referred to as PHM NP, FIG. 17A) in the LL / 2 lung metastasis model, further in vitro and in vivo studies on AND logic design were conducted using this formulation. To investigate MSA- 2 release triggered by the AND logic gate mechanism (FIG. 17B), its response was measured under conditions mimicking the tumor microenvironment: hypoxia (NADH quinone oxidoreductase 1 or NQO1 , 100 pg / mL, NADH 800 pM) and acidic pH (pH 6.5). PHM NP retained the pKa of the parent polymer PSC7A at 6.9 (FIG. 31). MSA-2 was efficiently released, reaching 82% after 6 hours and 92% after 24 hours, but only when both hypoxia and acidic pH were present (FIG. 17C, FIG. 17D). The release increased with higher concentrations of NQO1 , plateauing at 100 pg / mL (FIG. 32). Notably, the addition of dicoumarol (100 pM), an NQO1 inhibitor, significantly inhibited release (FIG. 17E).

[0313] In contrast, PSC7A-pH-20MSA-2 with the acyl hydrazone linker released MSA-2 solely under acidic conditions (pH 4.5, FIG. 33A), while PSC7A-Re-20MSA-2 depended on the presence of glutathione (GSH) and was independent of pH (FIG. 33B). Additionally, MSA-2 released from PSC7A-Re-20MSA-2 contained residual mercaptoethanol, potentially impairing its therapeutic efficacy. The AND logic gate in PHM NP thus ensures a highly regulated release of MSA-2, requiring both hypoxia and acidic conditions.

[0314] To assess STING activation, PHM NP was co-incubated with THP1-ISG cells under normoxic (20% O2) or hypoxic (1% O2) conditions. PHM NP exhibited a 10-fold increase in IFN-p production under hypoxia environment compared to normoxia conditions (FIG. 17F). To validate the AND logic mechanism, inhibitors were used: dicoumarol (10 pM) to suppress hypoxia responsiveness and bafilomycin (100 nM), a vacuolar H+ATPase (V-ATPase) inhibitor, to suppress pH in the endosomes and lysosomes. Both inhibitors significantly reduced STING activation (FIG. 17G), confirming the necessity of both hypoxia and acidic conditions for optimal activation.

[0315] To further evaluate the role of dual stimuli-response, four polymers were synthesized either from pH-insensitive PEO-b-poly(glycolic-co-lactic acid) (PLGA) copolymer or using hypoxia-insensitive amide (Am) linkers: PLGA-Am-MSA-2 (pH OFF, hypoxia OFF), PSC7A- Am-MSA-2 (pH ON, hypoxia OFF), PLGA-Hy-MSA-2 (pH OFF, hypoxia ON), and PSC7A-Hy- MSA-2 (pH ON, hypoxia ON). Only PSC7A-Hy-MSA-2 released MSA-2 under hypoxic and acidic conditions (FIG. 17H), attributed to its precise combination of pH-responsive polymers and hypoxia linkers. In contrast, PLGA-Am-MSA-2 and PSC7A-Am-MSA-2 showed no releasedue to the stability of the amide bond, while PLGA-Hy- MSA-2 failed to release MSA-2 due to lack of micelle disassembly, which hindered NQ01 accessibility to the hypoxia linker. In hypoxic conditions, only PSC7A-Hy-MSA-2 induced STING activation in THP1-ISG cells (FIG. 171). This was further corroborated by in vivo mouse studies, where the PSC7A-Hy-MSA-2 group exhibited a significant reduction in lung metastases (~2 nodules) in the LL / 2 model, compared to over 20 nodules in all other treatment groups (FIGS. 17J-17K). These findings validate the strategic design of AND logic STING nanoparticles, combining pH-responsive polymers with hypoxia-responsive linkers for precise and effective drug delivery.Example 13: Pharmacokinetic, biodistribution and cell tropism studies

[0316] For pharmacokinetics and biodistribution studies, the polymer PSC7A was labeled with the fluorescent dye indocyanine green (ICG). In a LL / 2 lung metastasis model (FIG. 18A), PSC7A-ICG and PHM NP were formulated into nanoparticles with 3:7 ratio and then were administered intravenously into mice, followed by blood sample collection at various time points to assess the concentration of nanoparticles in the plasma. The data showed that the half-time in blood were 0.09 h for a-elimination phase and 7.44 h for p-phase (FIG. 18B). Organ distribution analysis 24 h post-injection (FIG. 18C) show the nanoparticles were primarily taken up by the spleen (0.15 ± 0.014 pg / mg), liver (0.08 ± 0.019 pg / mg), lungs (0.03 ± 0.009 pg / mg), and lymph nodes (0.04 ± 0.013 pg / mg). Similar biodistribution results were observed in the Balb / c 4T1 orthotopic solid tumor model (FIG. 34B). High accumulation in spleen facilitates immune surveillance against metastasis as metastatic tumor cells often spread antigens through the blood.

[0317] To further analyze the cellular distribution of nanoparticles, the PSC7A polymer was labeled with the fluorescent dye Cy5. This fluorescent polymer was then tracked in spleen, tumor-draining lymph nodes (TDLN), and lungs in the LL / 2 lung metastasis model (FIGS. 18D- 18F), and spleen, TDLN, tumors in the 4T1 orthotopic tumor model (FIGS. 34C-34E). The results indicated that nanoparticles were predominantly taken up by immune cells (CD45+) instead of cancer cells (CD45j in 4T 1 tumor model (FIG. 34C). Within the immune cells, more than 60% dendritic cells (DCs) endocytosed nanoparticles in the spleen, tumors, and lung metastasis, followed by macrophages, and natural killer (NK) cells. A negligible number of nanoparticles was endocytosed by the T cells (FIG. 18D-18F and FIGS. 34C-34E). This highlights the selective uptake of nanoparticles by antigen-presenting cells, whether in the tumor or secondary lymphoid organs.Example 14: PSC7A polymer increases NQO1 expression in DC2.4

[0318] NQO1 plays a crucial role in the activation of PHM NP within antigen-presenting cells (APCs). Studies have shown that NQO1 is predominantly present in various solid tumors andexhibits high activity under hypoxic conditions. As shown in FIGS. 18G-18H, DC2.4 cells exhibit high expression of NQO1 under hypoxic conditions. Interestingly, the addition of PHM NP also induces a modest expression of NQO1 under normoxic condition. Further, the secretion of IFN-p from DC2.4 cells with or without PHM NP treatment was investigated in either normoxic or hypoxic condition (FIG. 181). It was found that PHM NP treatment under hypoxic condition leads to a significant production of IFN-p (1.3 ng / mL), and a moderate IFN- P (0.5 ng / mL) is produced after PHM NP incubation under normoxic condition. Both are significantly higher than that without PHM NP incubation.Example 15: cDC1 drives STING-mediated tumor suppression

[0319] For immune mechanism, NP-mediated antitumor effects were investigated in WT, STING'', and Baft3-- (cDC1 KO) mice bearing LL / 2 lung metastasis (FIGS. 19A-19D). Compared to wildtype mice, PHM NP treatment lost their antitumor efficacy in mice lacking STING or cDC1 (FIGS. 19C-19D). It should be noted reduced number of lung nodules was observed from both the treatment and control group of STING'- mice because STING knockout diminished activity of indoleamine 2,3-dioxygenase (IDO), which was responsible for impaired formation of LL / 2 lung metastases.

[0320] Subsequently, the impact of depleting NK, CD4+, and CD8+T cells on nanoparticle- mediated tumor suppression (FIGS. 19E-19I) was investigated. Depletion of CD8+T (FIG. 19G, red curve, 29 ± 2.2 nodules) and NK (FIG. 191, 11.8 ± 1.7 nodules) cells resulted in complete and partial loss of antitumor efficacy, respectively, suggesting a major role of CD8+T and a minor role of NK cells in tumor rejection. CD4+T cells play a minimal role in antitumor efficacy as indicated by robust metastasis suppression after their depletion (FIG. 19H, red curve 1.3 ± 1.3 nodules). Overall, these data demonstrate that the nanoparticles prime CD8+T cells against tumor cells through STING activation and cDC1 priming.

[0321] Finally, the contribution of the spleen to the generation of antitumour efficacy was investigated. LL / 2 metastatic tumors were introduced into splenectomized mice and treated with PHM NP, 5 days after inoculation (FIG. 19J). The nodules in the lungs showed that splenectomy resulted in more metastatic tumor formation compared to WT mice (FIG. 19K and FIG. 19F, gray curve, 44 ± 9 nodules vs. 30 ± 3 nodules, respectively). PHM NP inhibited the formation and growth of metastases in splenectomized mice (FIG. 19K), but not as significant as in WT mice (red curve, 26 ± 4 nodules vs. 1.5 ± 1.0 nodules).Example 16: Elevated cDC1-pSTING-CD8+T signature in lung metastasis

[0322] Based on the mechanistic data, multiplex immunohistochemistry (mIHC) on formalin- fixed and paraffin-embedded (FFPE) LL / 2 lung metastasis tissue was used to examine STING activation and immune cell profiles. Using XCR1+, p-STING+and CD8+biomarkers, STING-activated cDC1 cells were identified as well as their physical interactions with CD8+T cells within the tumor microenvironment (FIGS. 20A-20D). In the PHM NP-treated group, cDC1 cells on lung metastasis sites exhibited robust STING activation, as evidenced by strong p- STING signals. Notably, direct physical contact between cDC1 and CD8+ T cells was observed in multiple tumor regions, highlighted by white-box annotations. In contrast, such interactions were absent from both the PBS and MSA-2 control groups. Quantitative analysis revealed that -0.5% of immune cells in the PHM NP group were cDC1 cells, with 95% of those exhibiting STING activation. This activation correlated with the recruitment of CD8+ T cells, which accounted for 5.9% of immune cells in the tumor regions.

[0323] These findings emphasize the pivotal role of STING activation in cDC1 cells. By enhancing cDC1 recruitment and promoting their interactions with CD8+T cells, STING activation facilitates a robust antitumor immune response, highlighting its potential as a mechanism for effective immunotherapy.Example 17: Therapeutic efficacy of PHM NP in treating metastatic tumors

[0324] To assess the therapeutic efficacy of PHM NP, its performance was assessed in two additional metastatic tumor models. The triple-negative breast cancer 4T1 orthotopic (metastatic foci in the lung) model, known for spontaneously producing lung metastases by the second week post-inoculation, was used to examine the effect of PHM NP. Intravenous injections were administered three times when tumors reached -60-70 mm3in the mammary pad. Primary tumor growth was tracked, and lung metastatic nodules were quantified on Day 24 (FIG. 21A). PHM NP displayed significant regression of primary tumor growth, while free MSA-2 showed minimal effect (FIG. 21 B, FIG. 35). Furthermore, systemic immunity induced by PHM NP reduced lung metastatic nodules to 4.2 ± 1.6, a striking improvement compared to free MSA-2 (48 ± 2.4) and PBS (72.6 ± 5.9) (FIG. 21C, FIG. 21 D). PHM NP treatment also significantly increased survival rates compared to PBS and anti-PD1 treatment alone (FIG. 21 E, FIG. 21 F, FIG. 36). However, combining PHM NP with anti-PD1 offered no additional benefit in this model (FIG. 21 G).

[0325] To further evaluate its efficacy, PHM NP was tested in the B16F10 melanoma lung metastasis model. Mice received intravenous injections of 2 x 10® B16F10 cells, and lungs were harvested on day 17 to assess metastatic nodule formation (FIG. 21H). PHM NP significantly reduced lung nodules to 10 ± 1.1 , compared to 22.3 ± 1.8 for free MSA-2 and 35.3 ± 4.3 for PBS (FIG. 211, FIG. 21 J). When combined with anti-PD1 , PHM NP further extended survival times, demonstrating a synergistic effect (FIG. 21 K, FIG. 21 L). These results highlight the efficacy of PHM NP in suppressing tumor growth and metastasis across multiple models. Its ability to trigger systemic antitumor immune responses and enhance survival underscoresits potential as a promising therapeutic strategy for treating metastatic and immune-cold tumors.Example 18: Discussion of Examples 11-17

[0326] Metastasis, unlike primary tumors, which are often manageable through localized treatments like surgery or radiation, requires systemic approaches for prevention and treatment. STING pathway has emerged as a critical mediator in cancer therapy, playing a pivotal role in both traditional treatments and immunotherapies. It has also been implicated in the reactivation of dormant metastases in lung adenocarcinoma. However, systemic administration of STING agonists is limited by severe side effects.

[0327] The development of polymer nanoparticles (PHM NP) that activate STING via an AND logic gate marks a significant breakthrough in the systemic therapy of metastatic cancers. By conjugating the STING agonist MSA-2 to the pH-sensitive polymer PSC7A through hypoxia- responsive linkers, these nanoparticles enable precise, controlled release of MSA-2 only under tumor-specific conditions where pH and hypoxia coexist. Systematic screening identified PHM NP as the optimal formulation, balancing high antitumor efficacy with minimal toxicity.

[0328] The release of MSA-2 and subsequent STING activation by PHM NPs follow the AND logic mechanism (pH ON and hypoxia ON), ensuring robust antitumor responses. Replacing either the pH-responsive polymer PSC7A or the hypoxia linker disrupted STING activation and significantly impaired tumor suppression, underscoring the critical role of this dualresponsiveness.

[0329] The study also highlights the importance of systemic immunity in metastatic tumor rejection. PHM NP demonstrated preferential accumulation in secondary lymphoid organs, such as the spleen and tumor-draining lymph nodes (TDLNs), which are essential for T cell priming and activation. Interestingly, the nanoparticles showed specific tropism for myeloid cells and upregulated NQO1 protein expression in dendritic cells (DCs), facilitating controlled MSA-2 release and mild STING activation. This mechanism likely underpins the superior performance of the pH-Hypoxia AND logic gate compared to other 2 strategies, by employing the most stringent release conditions to mitigate systemic toxicity, and enhanced tumor suppression through more precise and effective STING agonist delivery to DCs. This mechanism differs from STING activation by cancer-cell-associated STING agonists and underscores the selective immune modulation achieved by PHM NPs.

[0330] Mechanistically, therapeutic efficacy was abolished in STING / _and Batf3 / _(cDC1- deficient) mice, confirming the central role of STING activation in cross-presenting cDC1 cells. Depletion studies identified CD8+ T cells as primary mediators of the antitumor response, withNK cells playing a supportive role. Splenectomy experiments further demonstrated the spleen’s critical role in achieving the therapeutic potential of PHM NP, emphasizing its importance in immune surveillance and activation. Direct interactions between STING- activated cDC1 cells and CD8+T cells were observed, highlighting the licensing of cDC1 cells as pivotal in mounting a robust antitumor immune response.

[0331] Anti-metastatic tumor models, including the LL / 2 lung metastasis model, 4T1 orthotopic breast cancer model, and B16F10 lung metastasis model, showcased the therapeutic potential of PHM NP. These findings demonstrate that the AND logic system enhances the delivery and activation of STING within the tumor microenvironment and immune organs, resulting in significant reductions in lung metastases.

[0332] In conclusion, the AND logic platform offers a promising approach for metastatic cancer therapy by combining dual tumor-stimuli response with precise immune activation. By selectively targeting STING activation to immune-favorable sites, this strategy enhances antitumor efficacy while minimizing systemic side effects. This platform not only advances systemic immunotherapy but also holds potential for combination with other immunotherapeutic strategies, paving the way for innovative treatments of metastatic cancers, particularly those resistant to current therapies.Example 19: Introduction to Examples 11-18

[0333] Vaccines play a pivotal role in public health, particularly in their prophylactic use against infectious diseases. Recently, therapeutic vaccines against cancer have been actively pursued to stimulate T cell immunity against various tumors. Despite the progress, the effectiveness of these vaccines remains modest across many oncologic indications. Although depot-forming water-in-oil adjuvant and antigen systems can improve immunogenicity, booster immunizations can cause T-cell sequestration at the vaccine site, leading to T-cell exhaustion and deletion. Inefficient co-delivery of adjuvants and antigens to secondary lymphoid organs and tumor microenvironment, and subsequent low accumulation in antigen presenting cells, may lead to weak immune stimulation or even immune tolerance. To optimize T cell priming and vaccine efficacy, simultaneous innate immune activation and antigen processing and presentation are essential for creating the immunological synapse.

[0334] To bolster the vaccine efficacy, many agonists of pathogen recognition receptors have been investigated as adjuvants. Among these, stimulator of interferon genes (STING) has emerged as a potent immune target for vaccine development and cancer immunotherapy. Activation of the STING pathway initiates a cascade of host defense responses, including the production of type I interferons and pro-inflammatory cytokines, leading to enhanced antigen presentation, T cell priming, and ultimately, antitumor immunity. For treatment of advanceddiseases, systemic administration is desirable but it has been limited due to off-target toxicity. Balancing immune activation with circumvention of immune-related toxicity is important to achieve a safe and efficacious immunotherapy.

[0335] In earlier studies (Li, S. et al. Prolonged activation of innate immune pathways by a polyvalent STING agonist. Nat Biomed Eng 5, 455-466 (2021), incorporated herein by reference in its entirety), it was found that ultra-pH sensitive polymers, including PC7A and PSC7A, enhance the immune response through a STING-dependent pathway. These polymers bind to different sites on the STING protein compared to small molecule agonists such as cGAMP, leading to a prolonged activation. Combination of polymer and small molecule agonists is able to potentiate the immune response and enhance the efficacy of the treatment. In addition, polymeric nanoparticles are found to deliver antigen with high efficiency. Current research is dedicated to developing a more potent systemic tumor vaccine that not only targets tumors but also activates secondary lymphoid organs, thus maximizing therapeutic outcomes by ensuring a virtuous cancer-immunity cycle (FIG. 37).

[0336] Here, the development of a stimuli-responsive nanovaccine composed of a STING- activating polymer and HPV16 E7 protein for the treatment of late-stage HPV-positive tumors is described. To boost STING activation, diABZI is conjugated to the biodegradable, STING- activating polymer (PSC7A) using an azobenzene linker. Under normal physiological conditions (e.g., blood), the E7 protein is encapsulated in the polymeric micelles with masked immune activity. Upon reaching tumors and secondary lymphoid organs, the nanovaccine is selectively taken up by antigen presenting cells (dendritic cells, macrophages), where acidic pH and azoreductase enzymes (e.g., NQO1 , which is elevated under tumor hypoxic conditions and breaks azo bonds) selectively dissociate micelles and cleave diABZI from the polymers for cooperative antigen release and STING activation. Systemic administration of the nanovaccine incurred mild toxicity but dramatically boosted tumor-specific T cell immunity against late-stage HPV-positive tumors with distal and metastatic foci, where local administration (e.g., subcutaneous or intratumoral injection) is less effective. The integration of stimuli-responsive nanotechnology with antigen delivery and innate activation may prove important in harnessing the STING pathway for cancer immunotherapy.Example 20: Materials and Methods for Examples 12-18

[0337] Cell culture: THP1-ISG cells were provided by Dr. Z. J. Chen (UT Southwestern). TC- 1 cells were provided by Dr. T. C. Wu (John Hopkins University). MLM3 cells were purchased in Applied Biological Materials Inc. (Cat. No. T8311). All cells were cultured at 37° C in an atmosphere of 5% (v / v) CO2. THP1-ISG cells were cultured in RPMI 1640 supplemented with 10% (v / v) heat inactivated fetal bovine serum (FBS) and 1 % (v / v) penicillin-streptomycinantibiotics. TC-1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v / v) FBS and 1 % (v / v) penicillin-streptomycin antibiotics. MLM3 cells were cultured in a mixture of DMEM and Ham’s Nutrient Mixture F12 Media (Hams F12) with 3:1 (v:v) ratio with 10% FBS, 0.5 pg / ml hydrocortisone, 5 pg / ml transferrin, 5 pg / ml insulin, 1.36 ng / ml Tri-lodo-Thyronine, 5 ng / ml mouse EGF and 1 % penicillin-streptomycin antibiotics.

[0338] Formulation and characterization of STING nanovaccine: PEG-b-P(SC7A-azo-diABZI) NP (STING NP) was used as an example. In a typical procedure, 1 mg PEG-b-P(SC7A-azo- diABZI) polymer was dissolved in PBS at pH 5.0. NaOH solution was added to adjust the final pH to ~7.4 under stirring. The solution was further stirred for 30 mins followed by addition of 100 pg E7 protein (GenScript USA, Inc.) and kept for additional 30 mins to form PEG-b- P(SC7A-azo-diABZI) / E7 nanovaccine (STING nanovaccine). The obtained STING NP or STING nanovaccine were analyzed using dynamic light scattering (Zeta Sizer, Malvern, He- Ne laser, A = 633 nm) to measure the hydrodynamic diameter and size distribution. The E7 loading efficiency was analyzed by fast protein liquid chromatography (AKTA pure™ protein purification system). The release efficiency and kinetics of diABZI from the STING NP was quantified by high-performance liquid chromatography (1260 Infinity II, Agilent).

[0339] Western blot analysis: All solutions were purchased from Bio-Rad and antibodies anti- NQO1 C-terminal (N5288, rabbit polyclonal, 1 :1000-1 :2000) were obtained from Sigma / Aldrich. RAW264.7 and DC2.4 cells were treated with PBS, PSDBA NP, PSC7A NP, diABZI and STING NP for 24 h. The concentration of all NPs was at 50 pg / mL, and equivalent diABZI concentration (7.85 pg / mL) were used in these studies. Growth medium was replaced with complete medium containing 10 pM p-lapachone for 2 h after which the medium was removed, and the plates washed twice with PBS. Cells were collected and lysed in SDS sample buffer (with protease and phosphatase inhibitor cocktail) and heated for denaturation. Supernatant was loaded onto a 4-15% Mini-PROTEAN gel (Bio-Rad) and run at 50 V for 20 min followed by 100 V for 60 min. Electrotransfer was performed using 100 V for 60 min on ice. After transfer, the membrane was blocked either in 5% non-fat milk or BSA (phosphorylated protein) for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. Goat anti-rabbit IgG HRP-linked secondary antibody (1 :3,000, Bio legend) was used for 1 h at room temperature before detection on gel imaging system (GelDoc Go Gel Imaging System, Bio-Rad). The membrane was stripped in stripping buffer for 30 min and reused for p-actin (A2228, Sigma / Aldrich, mouse mAb, 1 :5000) detection.

[0340] Methyl Red assay: RAW264.7 and DC2.4 cells were cultured in a 6-well plate and treated with PBS, PSDBA NP, PSC7A NP, diABZI and STING NP for 24 h. The concentration of all NPs was 50 pg / mL and equivalent diABZI concentration (7.85 pg / mL) were used in these studies. Cells were collected and lysed with 60 pL RIPA buffer on ice for 30 min and thesupernatants were collected for assay. In a Methyl Red assay, Methyl Red (50 pM) and NADH (800 pM) were dissolved into a mixed solvent of DMSO and H2O with 1 :10 ratio. 5 pL supernatants were added into Methyl Red assay and incubated for 2 h at 37°C. The decrease in absorbance (Amax at 430 nm) were used to quantify NQO1 using a calibration curve. Total proteins were determined by BCA assay (Thermo Scientific, 23227).

[0341] IFN-B assay: RAW264.7 and DC2.4 cells were treated with PBS, STING NP (50 pg / mL), and STING NP with dicoumarol (10 pM) for 24 h. The supernatants were collected. IFN-p levels were measured by Mouse IFN-beta DuoSet ELISA (DY8234-05, R&D).

[0342] Pharmacokinetic analysis: C57BL / 6 mice bearing TC-1 tumor were intravenously administered with dye-labeled nanovaccine, in which a hybrid nanoparticle consisting of PEG- b-P(SC7A-azo-diABZI) and PEG-b-PSC7A-Cy5 (each PSC7A polymer is conjugated with -3 Cy5 dye) with a molar ratio of 7:3 and TMR-labelled E7 (10%) was formulated. The injected dose was 15 mg / kg for the nanoparticles. At predefined time points following injection, mice were bled to collect 50 pl of blood (n=5 for each group). The plasma fraction was collected after centrifugation, diluted 5-fold with PBS buffer containing 5 mM EDTA at pH 6.0, and the fluorescence was quantified using a plate reader. Fluorescent nanoparticles were quantified using a 640 nm (for Cy5) and 540 nm (for TMR) excitation and 670 (for Cy5) and 570 nm (for TMR) emission wavelength by a plate reader. Data are presented as percent injected dose (% ID) and plasma collected at 5 min was taken to represent the maximum injected dose (100% ID). Data were fitted using nonlinear regression and a two-phase decay model using GraphPad Prism software.

[0343] Organ and cell distribution: C57BL / 6 mice bearing TC-1 tumors (-120 mm3) were intravenously injected with dye-labelled STING NP as described in the previous section. Mice were euthanized 24 h after i.v. injections and different tissues (tumor, tdLN, liver, lung, heart, kidney and spleen) were collected and weighted. Tissues were mechanically dissociated and homogenized with in lysis buffer (2 wt% Triton X-100 + 100 mM HEPES + 5 mM EDTA, pH 7.1) using tissue grinder tubes (Precellys Lysing Kits). Next, tissues were centrifuged at 500g for 3 mins. The supernatants were transferred to a black 96 well plate for quantification using a 640 nm excitation and 670 emission wavelengths by a plate reader. The concentration of nanoparticle was determined using a tissue-specific standard curve prepared with tissue digests from untreated mice. Organ uptake was reported as the percentage of injected dose per gram of tissue.

[0344] For cell distribution studies, mice were euthanized 24 h after injections. Tissues including tumor, tdLN, and spleen were collected and dissociated into single-cell suspensions, and then stained, washed, and resuspended in a fluorescence-activated cell sorting (FACS)buffer. The following antibodies were used: CD45 PerCP (clone 30-F11 , BioLegend), MHC-II AF700 (clone M5 / 114.15.2, Invitrogen), CD11c BV605 (clone N418, BioLegend), CD11 b BV605 (clone M1 / 70, BioLegend), CD4 FITC (clone RM4-5, BioLegend), CD8a PE (clone 53- 6.7, BioLegend), CD3e BV786 (clone 145-2C11 , BD BioSciences), F4 / 80 PE (clone BM8, Miltenyi Biotec), NK1.1 PE-Cy7 (clone PK136, BD Biosciences), B220 APC-Cy7 (clone RA3- 6B2, BioLegend). The LIVE / DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen, L34966) was used. Data were collected on BD LSRFortessa or Beckman CytoFLEX flow cytometer and analyzed by FlowJo (Tree Star Inc., Ashland, OR) software.

[0345] Tumor treatment by STING nanovaccine: In TC-1 tumor model, C57BL / 6 mice were subcutaneously inoculated with TC-1 (2 x 105cells) that were suspended in 100 pl sterile PBS on the right shaved flank on day 0. For distal tumor experiments, mice were inoculated with TC-1 (1 x 105cells) on the left flank opposite to the initial tumor inoculation. Mice were intravenously treated on day 10 and 15 after tumor inoculation. For the administration method study, subcutaneous treatment was near the tail base, intratumoral treatment was injected in the right primary tumor, and intravenous treatment was through the tail vein. For all studies, mice were randomized into groups before treatment initiation. T umor size was measured using calipers, and the tumor volume was calculated with the formula V = (lengthxwidthxwidth) / 2, where length is the longest dimension and width is the perpendicular dimension. Mice were sacrificed when the tumor volume exceeded 1500 mm3or tumor ulceration became severe.

[0346] In MLM3 tumor model, C57BL / 6 mice were intravenously inoculated with MLM3 (1 x 106cells) that were suspended in 100 pl sterile PBS on day 0. Mice were intravenously treated with STING nanovaccine or other control groups on day 10 and 16 after tumor inoculation. Anti-PD-1 (200 pg) was intraperitoneally injected on day 10, 13 and 16. Mice were euthanized, and their lungs were collected and fixed in Bouin’s solution on day 35 and further analyzed by histology analysis. Additional mice were also included for long-term survival studies.

[0347] Safety evaluations: Tumor free C57BL / 6 mice were intravenously injected with the STING NP, PSC7A NP, free diABZI, and PBS. Mice (n=4 for each group) were bled to collect 500 pl of blood 6 or 24 h after injections. The plasma fraction was collected after centrifugation. The systemic concentrations of interleukin-6 (IL-6), interleu kin- 10 (IL-10), monocyte chemoattractant protein-1 (MCP-1), interferon-y (IFN-y), tumor necrosis factor-a (TNF-a), and interleukin-12p70 (IL-12p70) were measured using a mouse inflammation kit (BD™ Cytometric Bead Array, 552364). The activities of aspartate aminotransferase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatinine (SCEA) were measured in the Metabolic Phenotyping Core at UTSW. Statistical analysis was performed using GraphPad Prism 7.

[0348] Flow cytometry: C57BL / 6 mice bearing TC-1 tumors were intravenously injected with STING nanovaccine or other control groups on day 10 and 15. Spleen and tdLN were harvested 1 day after the first injection for DC and macrophage analysis, and tumor, spleen and tdLN were harvested 3 days after the second injection for T, NK and NKT cell analysis. Tissues were digested using collagenase IV (160 pg / ml; Sigma-Aldrich) and DNase I (20 pg / ml; Sigma-Aldrich) in RPMI 1640 medium at 37°C for 30 min and then washed and strained through a 70 pm filter (BD Falcon). The single cells were resuspended in a FACS buffer. The following antibodies were used: MHC-II AF700 (clone M5 / 114.15.2, Invitrogen), CD45 PerCP (clone 30-F11 , BioLegend), CD11 b PB (clone M1 / 70, BioLegend), CD11c BV605 (clone N418, BioLegend), F4 / 80 APC-cy7 (clone BM8, BioLegend), CD86 APC (clone GL-1 , BioLegend), CD80 PE-CY7 (clone 16-10A1 , BioLegend), CD206 PE (clone C068C2, BioLegend), CD8a AF700 (clone 53-6.7, BD BioSciences), E7 Tetramer APC (clone 53-6.7, MBL International Corporation), CD4 Pacific blue (clone GK1.5, BioLegend), CD3e bv786 (clone 145-2C11 , BD BioSciences), NK1.1 FITC (clone PK136, BioLegend). All gating strategies were shown in FIG. 51 and FIG. 52.

[0349] H&E analysis: Fresh tissues were first fixed in 10% formalin solution, and then were embedded in paraffin, sectioned and H&E stained by the Molecular Pathology Core at UTSW. The slides were scanned using Hamamatsu Nanozoomer 2.0HT and the images were analyzed using NDP.view 2.7.25 software.

[0350] Statistical analysis: Statistical analysis was performed using GraphPad Prism Version 9.0. Statistical tests used are indicated in the figure legends. Data were considered statistically significant if p < 0.05. *: p<0.05, **: p<0.01 , ***: p<0.001 , ****: p<0.0001.Example 21 : Engineering nanovaccine for antigen and STING agonist delivery

[0351] As noted in Example 1 , above, a small molecule STING agonist (dABZI) (IC50 ~20 nM) was chemically conjugated to the biodegradable STING-activating polymer poly(ethylene oxide)-b-PSC7A (PSC7A, Kd ~30 nM) using an azobenzene linker. This conjugate, known as “PSC7A-(Hy)-10diABZ” above, is now referred to as “PEG-b-P(SC7A-azo-diABZI) polymer” or “STING polymer” in Examples 22 -28 herein below. FIG. 44 provides a summary of the synthesis of this polymer according to the methods described in Example 1. For ease of reference, the synthetic protocol and characterization of the key intermediates is provided again herein below.

[0352] Tert-butyl 2-(4-nitrosophenyl)acetate (1). Tert-butyl 2-(4-aminophenyl)acetate (1 g, 4.8 mmol) was dissolved in 50 mL of dichloromethane (DCM) and added to a solution of Oxone® (3.0 g, 7.7 mmol) in water. Subsequently, the reaction mixture was stirred for 4.5 h at room temperature. Afterward, the deep green organic layer was separated, and theaqueous solution was neutralized by the addition of a saturated solution of NaHCO3(50 mL). The mixture was then washed with DCM / water, dried over Na2SO4, resulting in the product of 1.1H NMR (400 MHz, CDCI3) 5 8.18 (d, 2H, J = 8.7, Ar-H), 7.44 (d, 2H, J = 8.7, Ar-H), 3.63 (s, 2H, Ar-CH2), 1.44 (s, 9H, -(CH3)3). LC-MS: [M+H]+: 222.1.

[0353] Tert-butyl 2-(4-((4-(hydroxymethyl)phenyl)diazenyl)phenyl)acetate (2).Compound 1 (500 mg, 2.71 mmol) was dissolved in 10 mL of ethanol (EtOH) containing acetic acid (AcOH, 0.5 mL) with stirring at 0 °C. Subsequently, 4-aminobenzyl alcohol (334 mg, 2.71 mmol) was added slowly to the solution, and the reaction proceeded for 2 h at room temperature. The solvent was then evaporated, and the resulting residue was washed and extracted with DCM / water. The crude product underwent further purification through a silica gel column, yielding 2 (650 mg, 88%).1H NMR (400 MHz, CDCI3) 5 8.25 (d, J = 8.6 Hz, 2H, Ar-H), 8.16 (d, J = 8.6 Hz, 2H, Ar-H), 7.42 - 7.35 (m, 4H, Ar-H), 3.61 (s, 2H, ArCH2-), 3.58 (s, 2H, ArCH2-), 1.44 (s, 9H, -(CH3)3). LC-MS: [M+H]+: 327.2.

[0354] Tert-butyl 2-(4-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)diazenyl) phenyl )acetate (3). Compound 2 (500 mg, 1.53 mmol) and 4-dimethylaminopyridine (DMAP) (37.5 mg, 306 pmol) were dissolved in 10 mL of DCM with stirring at 0 °C. 4-Nitrophenyl chloroformate (370.5 mg, 1.84 mmol) was slowly added to the solution, and the reaction proceeded for 1 hour at room temperature. The solvent was then evaporated, and the resulting residue was washed with DCM / water. The crude product underwent further purification through a silica gel column to obtain the final product 3 (650 mg, 86%).1H NMR (400 MHz, CDCI3) 5 8.15 (dd, J = 9.9, 2.9 Hz, 2H, Ar-H), 7.85 - 7.75 (m, 4H, Ar-H), 7.50 - 7.43 (m, 2H, Ar-H), 7.35 - 7.24 (m, 4H, Ar-H), 5.25 (s, 2H, ArCH2-), 3.50 (s, 2H, ArCH2-), 1.34 (s, 9H, - (CH3)3). LC-MS: [M+H]+: 492.2.

[0355] Tert-butyl 2-(4-((4-((((3-((5-carbamoyl-1 -((E)-4-(5-carbamoyl-2-(1 -ethyl-3-methyl- 1 H-pyrazole-5-carboxamido)-7-methoxy-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -y l)-2-( 1 - ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-7- yl)oxy)propoxy)carbonyl)oxy)methyl)phenyl)diazenyl)phenyl)acetate (4). DiABZI (50.0 mg, 64.0 pmol) and Compound 3 (47.2 mg, 95.1 pmol) were dissolved in 5 mL of DCM. Subsequently, DMAP (1.6 mg, 12.8 pmol) was added to the solution, and the reaction proceeded for overnight at room temperature. The solvent was then evaporated, and the resulting residue was washed with DCM / water. The crude product was further purified through a silica gel column, yielding 4 (41.6 mg, 56.8%).1H NMR (400 MHz, DMSO-cfe) 6 8.44 (d, J = 5.8 Hz, 4H, -NH2), 8.06 (d, J = 8.1 Hz, 2H, Ar-H), 7.77 (d, J = 11.9 Hz, 2H, -CH=CH-), 7.66 (dd, J = 18.7, 8.1 Hz, 4H, Ar-H), 7.21 (s, 2H, Ar-H), 6.92 (s, 2H, Pyr-H), 6.78 (d, J = 6.0 Hz, 4H, Ar-H), 5.94 (s, 2H, ArCH2-), 5.38 (s, 2H, -CH2-), 5.12 (s, 3H, -CH3), 4.87 (s, 3H, -CH3), 4.33 (s, 2H, ArCH2-), 3.84 (s, 2H, -CH2-), 3.72 (s, 2H, -CH2-), 3.47 (s, 3H, -OCH3), 2.31 (s, 2H,-CH2-), 1.95 (s, 2H, -CH2-), 1.69 (s, 6H), 1.64 (d, 6H, -CH3), 1.51 (s, 2H, -CH2-). LC-MS: [M+H]+: 1133.5.

[0356] 2,5-Dioxopyrrolidin-1-yl 2-(4-((4-((((3-((5-carbamoyl-1-((E)-4-(5-carbamoyl-2-(1- ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-1 H-benzo[d]imidazol-1-yl)but- 2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-7- yl)oxy)propoxy)carbonyl)oxy)methyl)phenyl)diazenyl)phenyl)acetate (5). Compound 4 (50.0 mg, 44.1 pmol) was dissolved in 10 mL of DCM, then 10 mL of trifluoroacetic acid (TFA) was added with further stirring for 15 minutes at 0 °C. Subsequently, the solvent was evaporated, and the resulting residue was dissolved in 10 mL of DCM without requiring further purification, proceeding directly to the next step. EDCI (8.4 mg, 88.2 pmol) and NHS (6.1 mg, 52.9 pmol) were added to the solution, and the reaction proceeded for 2 hours at rt. The solvent was then evaporated, and the resulting residue was washed with DCM, yielding 5 (35.5 mg, 68.5%).1H NMR (400 MHz, DMSO-cfe) 6 8.46 (d, J = 5.8 Hz, 4H, -NH2), 8.08 (d, J = 8.1 Hz, 2H, Ar-H), 7.78 (d, J = 11.9 Hz, 2H, -CH=CH-), 7.66 (dd, J = 18.7, 8.1 Hz, 4H, Ar-H), 7.24 (s, 2H, Ar-H), 6.94 (s, 2H, Pyr-H), 6.79 (d, J = 6.0 Hz, 4H, Ar-H), 5.95 (s, 2H, ArCH2-), 5.40 (s, 2H, -CH2-), 5.17 (s, 3H, -CH3), 4.89 (s, 3H, -CH3), 4.33 (s, 2H, ArCH2-), 3.84 (s, 2H, - CH2-), 3.72 (s, 2H, -CH2-), 3.47 (s, 3H, -OCH3), 2.35-2.37 (m, 4H, -CH2-), 1.94-1.98 (m, 4H, - CH2-), 1.68 (s, 6H), 1.66 (d, 6H, -CH3), 1.55 (s, 2H, -CH2-). LC-MS: [M+H]+: 1174.4.

[0357] Synthesis of PEG-b-P(SC7A-azo-diABZI) polymer. PSC7A-mNH2 (100 mg) and Compound 5 (m=3: 10 mg, m=10: 30 mg, m=20: 60 mg) were dissolved in 5 mL of dimethylformamide (DMF), then K2CO3(5 mg) was added to the solution, and the reaction proceeded overnight. The reaction mixture was then dialyzed with methanol to obtain the final product.1H NMR (400 MHz, CDCI3) 5 8.48 (d, J = 5.8 Hz, 4H, -NH2), 8.09 (d, J = 8.1 Hz, 2H, Ar-H), 7.80 (d, J = 11.9 Hz, 2H, -CH=CH-), 7.65 (dd, J = 18.7, 8.1 Hz, 4H, Ar-H), 7.25 (s, 2H, Ar-H), 6.96 (s, 2H, Pyr-H), 6.77 (d, J = 6.0 Hz, 4H, Ar-H), 5.96 (s, 2H, ArCH2-), 5.41 (s, 2H, - CH2-), 5.18 (s, 3H, -CH3), 4.90 (s, 3H, -CH3), 4.35 (s, 2H, ArCH2-), 4.51 - 4.46 (m, 2H, -CH2- ), 4.22 (d, J = 20.0 Hz, 6H, -CH2CCH2-, -CH2-), 3.59 (s, 2H, -OCH2CH2O-), 3.33 (s, 3H, -CH3), 3.27 - 3.04 (s, 2H, -SCH2-), 2.62 (d, J = 6.8 Hz, 2H, -CH2-), 2.14 (s, 2H, -CH2-), 1.94 (t, J = 7.0 Hz, 4H, -CH2-, -CH2-), 1.68 (d, J = 15.4 Hz, 4H, -CH2CH2-), 1.25 (d, J = 4.1 Hz, 3H, -CH3).

[0358] The average repeating unit of PSC7A polymer is 125 and each polymer contains approximately 7 molecules of diABZI based on the UV-Vis and1H NMR analysis (FIG. 45). pH titration experiments show the PEG-b-P(SC7A-azo-diABZI) polymer (or STING polymer) maintained the ultra-sensitive pH response inherent to the parent polymer PSC7A (FIG. 46). At pH above the apparent pKa(6.9), the polymers self-assemble into micelle nanoparticles (STING NP) with a hydrodynamic diameter of 24 ± 3 nm by dynamic light scattering analysis (FIG. 38B). HPV16 E7 protein (98 amino acid, isoelectric point 5.4) was physically mixed withSTING NP at room temperature for 1 h with a 10% w / w loading ratio. This process resulted in the quantitative encapsulation of E7 proteins within the STING NP as indicated by the disappearance of E7 peak by FPLC analysis (FIG. 38C). Encapsulation of E7 protein slightly increased the hydrodynamic diameter of the nanoparticles (28 ± 3 nm) with a polydispersity index of 0.33 (FIG. 38B).

[0359] Further to the experiments described in Example 2, above, the stimuli-responsive release of diABZI was investigated from the STING NPs (FIG. 38D). Acidic pH (6.5) and NQO1 / NADH (200 pg / mL and 1 mM, respectively) were used as stimulants. DiABZI was efficiently released (85% in the first 6 h, 98% after 24 h incubation) when both acidic pH and NQO1 / NADH signals were present (FIG. 38D, FIG. 38E). In contrast, less than 5% of diABZI was cleaved even after 24 h when only one of the stimulus conditions was met (i.e. , pH 6.5 solution without NQO1 / NADH, or pH 7.4 solution with NQO1 / NADH). When neither condition was present, negligible release of diABZI was observed. STING activation was also evaluated in THP-1-ISG cells after STING NPs were pretreated under different conditions. Specifically, STING NPs were incubated at pH 6.5 or 7.4 in the presence or absence of NQO1 / NADH, after which the medium was added to THP-1-ISG cell culture to measure interferon-b (IFN-b) signals. PSC7A NP physically encapsulated with diABZI (PSC7A / diABZI, without covalent conjugation) was used as a control. Data show STING NP resulted in ~70-fold I FN-p elevation when both pH 6.5 and NQO1 / NADH dual stimuli exist, which is markedly higher than the other conditions (FIG. 38F). In contrast, PSC7A / diABZI NP showed strong STING activation when incubated at pH 6.5 or 7.4 with comparable I FN-p response to free diABZI, suggesting the premature release of diABZI from PSC7A NP even at neutral pH (FIG. 38G). This dual-stimuli responsive design of STING NP is intended to mask the immune-related toxicity under normal physiological conditions, while under acidic and hypoxic conditions in tumors (where NQO1 level is high), STING signaling is selectively activated for immune stimulation.Example 22: Pharmacokinetic, biodistribution and cell tropism studies

[0360] For pharmacokinetic and biodistribution (PKBD) studies, E7 protein and PSC7A polymer were fluorescently labeled with tetramethyl rhodamine (TMR) and Cy5 dyes, respectively. HPV16 E6 / E7-induced TC-1 tumors (-120 mm3) in C57BL / 6 mice were used in these studies (FIG. 39A). E7-TMR / PSC7A-Cy5 STING nanovaccine was intravenously administered in mice and blood was collected at different time points to evaluate the plasma concentrations. Data show almost identical blood clearance kinetics for E7-TMR and Cy5- labelled polymer (FIG. 39B), suggesting the E7 antigen was stably encapsulated in the polymer nanoparticle during blood circulation. The blood half-lives for a- and b-elimination phases are 0.06 and 3.1 h for E7-TMR, and 0.06 and 3.3 h for STING polymer, respectively. The organ distribution of STING nanovaccine at 24 h after intravenous (i.v.) injection showsthe highest uptake in the spleen (40 ± 3 %l D / g), followed by liver (21 ± 3% I D / g), tumor (14 ± 5 %l D / g) and tumor draining lymph nodes (13 ± 2 %l D / g) (FIG. 39C).

[0361] The cellular distribution of the STING nanovaccine was further evaluated in the TC-1 tumors, tumor-draining lymph nodes (tdLNs) and spleen (FIGS. 39D-39F). Results show that the STING nanovaccine was preferentially taken up by immune cells (CD45+) over tumor cells (CD45‘) in the TC-1 tumors. Within the immune cell populations, NK cells, macrophages and dendritic cells (DCs) are the predominant cell types that took up the nanoparticles with negligible uptake by the T cells. In the spleen and tumor-draining lymph nodes, STING nanovaccine is mostly internalized by DCs and macrophages (FIGS. 39D-39F). These results highlight the STING nanovaccine’s selective accumulation in antigen presenting cells in the tumor and secondary lymphoid organs for T cell activation.Example 23: PSC7A polymer increases NQO1 expression in APCs for elevated STING activation

[0362] NQO1 is a homodimeric, two-electron oxidoreductase with the ability to reduce a broad range of quinone and organic substrates. In the hypoxic environment often found in solid tumors, it contributes to cellular adaptation by regulating redox homeostasis and protecting cells from oxidative damage. While the expression and functional implications of NQO1 are well-documented in tumors, its expression and regulatory mechanisms in secondary lymphoid tissues are less understood. Based on the cell tropism of STING NP uptake in myeloid cells in the spleen and tdLNs, the expression levels of NQO1 were next investigated in a representative macrophage (RAW264.7) and dendritic cell lines (DC2.4). These cells were treated with STING NP and several controls. PSDBA NP (pKa = 5.2) serves as a nanoparticle control with buffering ability for endosomes but no endosomolytic or STING activating properties. Free diABZI and PSC7A NP (without diABZI conjugation) are also included.

[0363] After 24 h incubation, the cells were collected, lysed, and analyzed by Western blot for protein expression and Methyl Red assay for NQO1 reducing functions. Data show treatment with PSDBA NP and diABZI did not lead to elevated NQO1 expressions in RAW264.7 and DC2.4 cells compared to the PBS control (FIG. 39G). In contrast, PSC7A NP and STING NP treatments increased the NQO1 expressions in these cell lines with a more pronounced effect from PSC7A NP. Protein expression by Western blot analysis is further corroborated by the Methyl Red assay where PSC7A NP and STING NP induced high NQO1 expressions compared to PBS, PSDBA, and diABZI (FIG. 39H). These data indicate PSC7A NP and STING NP stimulate NQO1 expressions and their reducing capacities in these cell lines. Further experiments investigated the downstream IFN-p levels after treating these cells with STING NP. Data show IFN-p levels increased 40 folds for DC2.4 and 8 folds RAW264.7 afterSTING NP treatment over PBS control, and the effect can be abrogated by the co-treatment with dicoumarol, an inhibitor of NQO1 (FIG. 39I).Example 24: STING nanovaccine elicits potent antitumor efficacy in TC-1 tumors

[0364] To assess the effect of the STING nanovaccine, HPV16 E6 / E7 transfected TC-1 tumor model was tested in C57BL / 6J mice. When tumors reached volumes of 100-150 mm3(10-12 days after inoculation), the mice were intravenously injected with different doses of STING nanovaccine (1.5, 5, and 15 mg / kg, 10% E7 loading density) in the phosphate buffered saline (PBS) on day 10 and 15 post-tumor cell inoculation (FIG. 40A). The highest dose of 15 mg / kg displays the best tumor growth inhibition and complete tumor regressions (FIG. 40B). Body weight measurements show weight loss for the PBS and 1.5 mg / kg group, likely due to the tumor burden, whereas no significant weight changes were observed for the 5 and 15 mg / kg dose groups (FIG. 47).

[0365] Several control groups were included to evaluate the antitumor efficacy of STING nanovaccine over its components (FIGS. 40C-40F, FIG. 48 and FIG. 49). Without E7 antigen, diABZI or PSC7A NP alone resulted in a small degree of tumor growth inhibition compared to PBS control, whereas STING NP displayed marked improvement over either agonist alone (FIG. 40C). E7 antigen alone (same antigen dose as in STING nanovaccine) had negligible effect on tumor growth over PBS control (FIG. 40D). Mixing of E7 with diABZI and PSC7A NP showed improved antitumor response over diABZI and PSC7A NP alone, with stronger effect from the E7-PSC7A NP group, likely due to the improved E7 antigen delivery by the nanoparticle. E7-encapsulated STING NP, or STING nanovaccine, illustrates the best tumor regression and long-term survival over STING NP and the other E7-adjuvant controls (FIGS. 40D-40F). These results highlight the cooperative design of the STING nanovaccine, in which each of its component, PSC7A NP, diABZI, and E7 antigen, plays an irreplaceable role in eliciting robust antitumor effects against the aggressive TC-1 tumor.

[0366] Different administration routes (subcutaneous = s.c., intratumoral = i.t., and intravenous = i.v.) were also tested for their antitumor effect in the primary and distal TC-1 tumor models. Primary and distal tumors were inoculated on the right and left flanks of mice on day 0 and day 2, respectively (FIG. 41 A). Nanovaccines were administered subcutaneously (s.c.) near the tail base, in the right primary tumor (i.t.), or through the tail vein (i.v.) at the same vaccine dose (15 mg / kg) on day 10 and 15. All the administration methods inhibited tumor growth in both primary and distal sites compared to the PBS control. The s.c. and i.t. treatments are effective in treating primary tumors but less effective in distal tumors (FIGS. 41C-41D), whereas i.v. administration achieved the greatest efficacy in reducing the growthof both primary and distal tumors with significantly improved survival outcomes (FIGS. 41 B- 41 D and FIG. 50).Example 25: STING nanovaccine activates antigen presenting cells for T cell priming

[0367] Next, immune cell activations in tumor and secondary lymphoid organs were investigated after treatment with STING nanovaccine or its molecular components. The potentiation of myeloid cell and lymphocyte compartments were systematically evaluated in these tissues by flow cytometry (gating strategies are shown in FIG. 51 and FIG. 52, respectively). In the first cohort of experiments (FIG. 42A-42J), the immune cell profiles from STING nanovaccine were evaluated over E7 antigen and STING NP (without E7); in the second cohort, the STING nanovaccine was compared to E7-encapsulated PSC7A NP or E7 mixture with diABZI to evaluate the combined adjuvant effect of P(SC7A-azo-diABZI) polymer over either component alone (FIGS. 53A-53J).

[0368] In the first cohort of study, dramatic elevation of CD86 expressions were observed in the DC and macrophage populations after treatment with either STING nanovaccine or STING NP (FIGS. 42A-42E). Over 50% of DC and macrophage populations are CD86 positive in the spleen after either treatment, compared to less than 10% for E7 or PBS control. A slightly less percentage (40-50%) of DCs and macrophages are CD86 positive in the tdLN than spleen after STING nanovaccine and STING NP treatment. Furthermore, the repolarization of M2- like macrophages (CD206hiCD86low) to a more M1-like state (CD206lowCD86hi) was observed in mice treated with STING nanovaccine in the spleen and the tdLN (FIG. 42F). Despite similar myeloid cell activation by the STING NP and STING nanovaccine groups, STING nanovaccine drove a markedly higher level of antigen-specific CD8+T cells over STING NP and other groups (FIGS. 42G-42I). In TC-1 tumors, over 80% of CD8+T cells are E7-specific, compared to less than 20% for STING NP, E7 and PBS control (FIG. 42G). This difference persisted in the spleen and tdLN (FIGS. 42H-42I). In the tumor microenvironment, the CD8+T, CD4+T, NK and NKT cells were all significantly increased in the STING nanovaccine group over control groups (FIG. 42J), which may cooperatively contribute to the antitumor immunity. These data suggest E7-specific CD8+T cells are the driving factor for elevated antitumor immunity, which is corroborated by tumor regression and long-term survival outcomes (FIGS. 40A-40F and FIGS. 41A-41 D).

[0369] In the second cohort of study, the effect of immune cell activation from combined diABZI-PSC7A activation was compared to the individual component alone. At 24 h after the first vaccination, a higher level of CD86 co-stimulation was observed on both DC and macrophage populations by diABZI+E7 over STING nanovaccine group (FIG. 53A, FIG. 53B). PSC7A NP group displayed a mild level of CD86 co-stimulation mostly in the spleen. Despitethe highest level of DC and macrophage potentiation, diABZI+E7 treatment did not significantly increase the formation of E7-specific CD8+T cells in TC-1 tumors, spleen or tdLN compared to the PBS group (FIG. 53C-53E). In contrast, STING nanovaccine treatment yielded the highest level of E7-specific CD8+T cells. Despite the mild effect in myeloid cell activation, PSC7A-E7 NP group produced higher level of E7-specific CD8+T cells in TC-1 tumors and tdLN than diABZI+E7 group. Interestingly, PSC7A-E7 NP group also led to significantly increased CD4+T cells and NKT cells over PBS control (whereas diABZI+E7 group did not) (FIG. 53F-53J), which correlates with slightly improved tumor growth inhibition by PSC7A-E7 NP over diABZI+E7 group (FIG. 40D). Overall, these data highlight the importance of cooperative antigen delivery with robust STING activation to achieve efficient antigen-specific T cells to drive antitumor immunity.

[0370] Lastly, PD-1 and Tim-3 expressions on the CD8+T cells after STING nanovaccine treatment were evaluated and compared them with E7 or STING NP group. Data show significantly higher percentage of PD-1+CD8+T cells in the tumor, spleen and tumor-draining lymph nodes over the other treatment groups (FIG. 54A). Interestingly, STING nanovaccine group also showed the highest percentage (14%) of terminally exhausted PD-1+Tim-3+CD8+T cells in the TC-1 tumors over control groups, but not in the spleen or tumor-draining lymph nodes (FIG. 54B). These data suggest a potential synergy in combining STING nanovaccine with checkpoint blockade therapy to avoid T cell exhaustion in the tumor microenvironment.Example 26: STING nanovaccine clears MLM3 metastases in the lung

[0371] The STING nanovaccine was also evaluated in a metastatic MLM3 tumor model in the lung. In this model, male C57BL / 6J mice were inoculated i.v. with the mEERL lung metastasis cell line clone 3 (MLM3), which was transfected by HPV16 E6 / E7. According to the treatment regimen outlined in FIG. 43A, mice treated with PBS showed notably large metastatic nodules on day 35. Anti-PD1 treatment (i.p. injection, 200 mg for three times) did not significantly reduced the number of lung nodules, however, it showed improved survival over the PBS control. STING nanovaccine showed the dramatic reduction of metastatic burden over both the PBS and anti-PD1 groups (FIG. 43B), which is further supported by histological analysis (FIG. 43C). The survival outcome is further improved when STING nanovaccine is combined with anti-PD-1 (aPD-1) over STING nanovaccine alone, where all and 71 % of the mice are still alive at 60 days post-treatment, respectively (FIG. 43D).Example 27: Safety evaluations of STING nanovaccine

[0372] To assess the systemic toxicity of STING NPs, plasma levels of several cytokines were measured at 6 and 24 h and liver and kidney functions at 24 h after i.v. administration (FIGS. 55A-55I). The cytokine panel included interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), interferon-v (I FN-y) , and tumor necrosis factor-a (TNF-a). As expected, free diABZI (1 .5 mg / kg) triggered significant elevations of IFN-y, IL-6, MCP-1 , and TNF-a at 6 h but went down at 24 h after injection. In contrast, STING NP (15 mg / kg) and PSC7A NP (15 mg / kg) induced minimal or undetectable levels of inflammatory cytokines comparable to PBS control (FIGS. 55B-55E). Liver and kidney functions were also measured, including aspartate aminotransferase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatinine (CREA). Data indicate comparable levels of AST and ALT in the group that received STING NP and PBS. Significant increase of AST and ALT levels was observed with free diABZI group over the PBS control (FIGS. 55F-55G). The levels of CREA and BUN for all groups were within the expected span of variations (FIGS. 55H-55I). IFN-p and CXCL10 expressions in tumors and different organs were analyzed to evaluate STING activations in mice bearing TC-1 tumors treated with STING nanovaccine. Free diABZI mixed with E7 antigen (diABZI and E7 doses match those in STING nanovaccine) was used as a control. Free diABZI / E7 group showed about 2 times higher IFN-p and CXCL10 levels in the kidney and heart, with comparable inductions in tumors, spleen, and liver compared to STING nanovaccine (FIGS. 56A-56B). These results illustrate a reduced systemic toxicity for stimuli- responsive design of STING NP, which is further supported by the lack of weight loss in the mice treated with STING nanovaccine (FIG. 47).Example 28: Discussion of Examples 12-18

[0373] Nanoparticles are of great interest as carriers of subunit vaccines owing to their ability to co-deliver antigen and immune adjuvant, promote cross-presentation of antigens in antigen presenting cells, and augment cellular and humoral immunity. In conventional vaccines, inadequate co-delivery of antigens and adjuvants to the secondary lymphoid organs and tumors may lead to suboptimal antitumor response while producing strong immune-related toxicity. The Examples above report a stimuli-responsive nanoparticle-based vaccine design (FIG. 37 and FIG. 38A). This construct leverages the STING signaling pathway, known for its role in innate immune responses through the production of type I interferons and pro- inflammatory cytokines. Earlier work on STING nanoparticle design consisted of physical encapsulation of cGAMP inside the PSC7A polymeric micelles. Although this formulation is sufficiently stable for intratumoral administration, it is not optimal for intravenous usage because of premature release of cGAMP during blood circulation. In these Examples, diABZI was chosen for its high potency (EC50 =20 nM) and ease of chemical modification for covalent conjugation to the PSC7A polymer. The introduction of stimuli-responsive linkers further improved the stability and safety of the nanovaccine design. STING activation through PSC7A- azo-diABZI polymer in coordination with antigen delivery addresses the need for a balanced immune activation. Through the stimuli-responsive design, the inherent limitations of alwaysON STING agonists were overcome (e.g., triggering cytokine release syndrome but with limited or insufficient T cell priming (e.g., as indicated by the diABZI+E7 control)). This systemic approach not only treats primary tumor sites but is also effective at eradicating distant metastases (FIGS. 41A-41D and FIGS. 43A-43D), showcasing a broadened therapeutic window.

[0374] This approach introduces an activation mechanism involving NQO1 — an enzyme that is elevated in hypoxic tumor microenvironment and plays important roles in redox biology and detoxification. It was serendipitously and surprisingly discovered that PSC7A polymer is able to elevate NQO1 expressions in the antigen-presenting cells, which resulted in diABZI release and STING activation which can be blocked by dicoumarol, an NQO1 inhibitor (FIGS. 39E- 39G). Earlier work by the inventors has shown that besides STING activation, PC7A / PSC7A polymers are disruptive to endosomal membranes allowing efficient cytosolic delivery of tumor antigens upon pH activation in the early endosomes. To investigate whether STING activation or endosomolytic properties of PSC7A is driving the NQO1 expression, the Examples above also investigated free diABZI that activates STING but does not disrupt endosomes, and PSDBA polymer that buffers endosomal pH but does not damage endosomal membranes. Data show neither free diABZI nor PSDBA induced high level of NQO1 expressions in the DC2.4 or RAW264.7 cells, suggesting endosomolytic property of PSC7A is responsible for the transcriptional activation of NQO1. These unexpected findings illustrates the synergy of azobenzene linker with PSC7A polymer in NQO1-mediated release of diABZI, which may be extendable to other polymer-drug conjugates to improve the therapeutic window.

[0375] A key element of the disclosed nanovaccine design herein is the engagement of secondary lymphoid organs (SLOs), such as the spleen or tumor-draining lymph nodes, where nanovaccines are readily taken up by the APCs (FIGS. 39C-39D). Since the biodistribution studies were performed at 24 h, which is longer than the circulation half-lives of STING nanovaccine (i.e., -3 h for the b-phase), it is conceivable that STING activation at earlier times may affect metabolism and clearance mechanism to boost their uptake in the immune organs. Through combined antigen delivery and STING activation, significant control and elimination of distal or metastatic tumors are achieved from systemic vaccination but not by local administration (FIGS. 41A-41D and FIGS. 43A-43D). These data suggest the benefit of targeting the STING pathway not only in tumors, but also in secondary lymphoid organs for the generation of antigen-specific cytotoxic T cells. Data revealed spleen contained higher percentage (-30%) of E7-specific CD8+T cells over that (-5%) in the tumor-draining lymph nodes (FIGS. 42G-42I). By applying a stimuli-responsive strategy to mitigate systemic toxicity, the current design offers a viable strategy against late-stage metastatic tumors. This approach, when combined with immune checkpoint blockade therapy, can further improve the antitumorresponse, as demonstrated in a preclinical MLM3 tumor model (FIGS. 43A-43D). The current E7 nanovaccine is designed to treat HPV-induced cancers, the incidence of which especially head and neck oropharyngeal cancer is growing in the US and world-wide. The related nanoparticle design is expected to be applicable to other cancer indications by the choice of different tumor-specific antigens.

[0376] In conclusion, systemically administered, stimuli-responsive STING nanovaccine is effectively boosting the tumor-specific T cell immunity against late-stage cancers. By targeting and activating the immune system’s natural processes in a controlled manner, the nanovaccine paves the way for developing safer, more effective therapeutic options for a range of cancers, potentially leading to better patient outcomes and broader applicability of immunotherapeutic strategies.

Claims

1. A compound comprising (a) a biodegradable polymer that activates STING and (b) a small molecule STING agonist, wherein (a) is covalently conjugated to (b) via a stimulus-5 responsive linker.

2. The compound of claim 1, wherein (a) comprises a structure of Formula Icontaining heterocycle.wherein R is branched amine or nitrogen3. The compound of claim 2, having a structure of Formula II:4-oh 'm, , STING Stimukss-rssponsivei___! A . . , . , r       1 > AgomstLmker      '   \10wherein R is as defined, m is 3, 10, or 20, and n is 125-m.

4. The compound of claim 2 or 3, wherein R is selected from:15   6. The compound of any one of claims 1 to 5, having a structure of Formula Il-A:wherein m is 3, 10, or 20, and n is 125-m.

7. The compound of any one of claims 1 to 6, wherein the small molecule STING agonist comprises MSA-2, diABZI, 2'3'-Cyclic GMP-AMP (cGAMP), or any combination thereof.

8. The compound of claim 7, wherein the small molecule STI NG agonist comprises MSA-2.

9. The compound of claim 7, wherein the small molecule STING agonist comprises diABZI.

10. The compound of claim 7, wherein the small molecule STING agonist comprises 2'3'-Cyclic GMP-AMP (cGAMP).

11. The compound of any one of claims 1 to 10 wherein the stimuli-responsive linker is responsive to pH, hypoxia, reduction, tumor-specific enzymatic cleavage or any combination thereof or wherein the stimuli-responsive linker is a cathepsin B cleavable linker, a cleavable ADC linker, or a dipeptide.

12. The compound of claim 11, wherein the stimuli-responsive linker is a pH sensitive linker.HN.N13. The compound of claim 12, wherein the pH sensitive linker comprises H .

14. The compound of claim 11, wherein the stimuli-responsive linker is a redox sensitive linker.

15. The compound of claim 14, wherein the redox sensitive linker comprises:

16. The compound of claim 11, wherein the stimuli-responsive linker is a hypoxia-sensitivelinker.

17. The compound of claim 16, wherein the hypoxia-sensitive linker comprises:,5   18. The compound of claim 11, wherein the stimuli-responsive linker is a cleavable ADClinker comprising Val-Cit.

19. The compound of any one of claims 1 to 18, wherein the compound is selected from:wherein each m is 3, 10, or 20 and each n is 125-m.

21. The compound of claim 19 or 20, wherein each m is 10 and each n is 115.

22. The compound of claim 21, wherein the compound comprises o         o23. The compound of claim 19 or 20, wherein each m is 20 and each n is 105.10   25. A nanoparticle comprising a two or more compounds of any one of claims 1 to 24.

26. The nanoparticle of claim 25 further comprising a bioactive protein or compound, wherein the compounds encapsulate the bioactive protein or compound.

27. The nanoparticle of claim 26, wherein the bioactive protein or compound is immunogenic and / or antigenic.

28. The nanoparticle of claim 26 or 27, wherein the bioactive protein or compound comprises an HPV E7, E2, E5 or E6 protein.

29. A composition comprising one or more compounds of any one of claims 1 to 24 and at least one carrier or excipient.

30. A composition comprising one or more nanoparticles of any one of claims 25 to 28 and at least one carrier or excipient.

31. The composition of claim 30, wherein the composition comprises a cancer vaccine.

32. The composition of any one of claims 29 to 31, wherein the composition is formulatedas a pharmaceutical composition.

33. The composition of claim 32, wherein the pharmaceutical composition is formulated for systemic administration.

34. The composition of claim 33, wherein the pharmaceutical composition is formulated for administration orally, intraadiposally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularlly, intravitreally, liposomally, locally, mucosally, parenterally, rectally, subconjunctival, subcutaneously, sublingually, topically, transbuccally, transdermally, vaginally, in cremes, in lipid compositions, via a catheter, via a lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, or via localized perfusion.

35. The composition of claim 34, wherein the pharmaceutical composition is formulated for administration via injection.

36. The composition of claim 35, wherein the pharmaceutical composition is formulated for intraarterial administration, intramuscular administration, intraperitoneal administration, intratumoral administration, or intravenous administration.

37. The composition of any one of claims 29 to 36, wherein the excipient is a vehicle.

38. The composition of claim 37, wherein the vehicle is an aqueous solution suitable forinjection.

39. A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of any one of claims 30 to 38, wherein the therapeutically effective amount is sufficient to treat the cancer.

40. The method of claim 39, further comprising administering at least one additional cancer therapy.

41. The method of claim 40, wherein the cancer therapy comprises surgery, radiation therapy, chemotherapy, immunotherapy, hormone therapy, oncolytic viruses, targeted therapies, polysaccharides, neoantigens, vaccine, or any combination thereof.

42. The method of any one of claims 39 to 41, wherein the cancer comprises a solid tumor.

43. The method of any one of claims 39 to 42, wherein the cancer is a metastatic cancer and wherein the therapeutically effective amount is sufficient to reduce metastasis.

44. The method of any one of claims 39 to 43, wherein the cancer comprises a non-small cell lung cancer, a head or neck cancer, melanoma, liver cancer, or cervical cancer.

45. The method of any one of claims 39 to 44, wherein the cancer is caused by an infectious agent.

46. The method of claim 45, wherein the infectious agent is human papilloma virus (HPV).

47. The method of any one of claims 39 to 46, wherein the composition comprises acompound           having           the           structureof:

48. A method of treating an infectious disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of any one of claims 30 to 38, wherein the therapeutically effective amount is sufficient to treat the infectious disease.

49. The method of claim 48, wherein the infectious disease is a human papilloma virus (HPV).

50. The method of claim 48 or 49, wherein the composition comprises a nanoparticle formed from a compound having a structure of:5   51. The method of any one of claims 39 to 50, wherein the composition is administeredintravenously, subcutaneously, or intratumorally.

52. The method of claim 51, wherein the composition is administered intravenously.

53. The method of any one of claims 39 to 52, wherein the subject in need thereof is ahuman.10   54. A kit comprising one or more compositions of claims 29 to 38 and at least onecontainer.

55. Use of a composition of any one of claims 29 to 38 in the preparation of a medicament for treating cancer and / or an infectious disease.

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