Chemical and Biomolecular Strategies for STING Pathway Activation in Cancer Immunotherapy

Abstract

The stimulator of interferon genes (STING) cellular signaling pathway is a promising target for cancer immunotherapy. Activation of the intracellular STING protein triggers the production of a multifaceted array of immunostimulatory molecules, which, in the proper context, can drive dendritic cell maturation, antitumor macrophage polarization, T cell priming and activation, natural killer cell activation, vascular reprogramming, and/or cancer cell death, resulting in immune-mediated tumor elimination and generation of antitumor immune memory. Accordingly, there is a significant amount of ongoing preclinical and clinical research toward further understanding the role of the STING pathway in cancer immune surveillance as well as the development of modulators of the pathway as a strategy to stimulate antitumor immunity. Yet, the efficacy of STING pathway agonists is limited by many drug delivery and pharmacological challenges. Depending on the class of STING agonist and the desired administration route, these may include poor drug stability, immunocellular toxicity, immune-related adverse events, limited tumor or lymph node targeting and/or retention, low cellular uptake and intracellular delivery, and a complex dependence on the magnitude and kinetics of STING signaling. This review provides a concise summary of the STING pathway, highlighting recent biological developments, immunological consequences, and implications for drug delivery. This review also offers a critical analysis of an expanding arsenal of chemical strategies that are being employed to enhance the efficacy, safety, and/or clinical utility of STING pathway agonists and lastly draws attention to several opportunities for therapeutic advancements.

Figure 1:. The stimulator of interferon genes (STING) cellular signaling pathway.

The cGAS enzyme surveils the cytosol of cells for the accumulation of double-stranded DNA, which serves an indicator of cellular malfunction or infection. Notably, cytosolic double-stranded DNA may arise intrinsically (e.g. self-DNA leakage from nucleus or mitochondria) or extrinsically (e.g. pathogen-derived). Upon recognition (i.e. binding) of double-stranded DNA in the cytosol, cGAS oligomerizes into liquid-like droplets and catalyzes the production of 2′3′-cGAMP, which can bind and activate the STING protein on the endoplasmic reticulum to initiate downstream signaling, primarily through TBK1 and IKK. Notably, STING activation typically leads to the activation of the transcription factors, IRF3 and NF-κB1 as well as NF-κB2, which is known to partially inhibit the activity of NF-κB1. STING signaling results in the production of IFN-I and various other proinflammatory cytokines, the profile of which largely depends on context. Lastly, 2′3′-cGAMP can also vacate its cell of origin through various transport mechanisms and function as an immunotransmitter that can locally propagate STING signaling in neighboring cells. To pharmacologically activate the signaling pathway, STING pathway agonists (i.e. cGAS agonists and STING agonists) must cross the cell membrane, access the cytosol, and evade degradation by various deoxyribonucleases (DNases) and phosphatases. Due to its relatively large size and negative charge, exogenous DNA requires assistance (e.g. pathogen-mediated delivery) to penetrate cellular membranes and gain access the cytosol. Furthermore, DNA is highly susceptible to degradation by DNase I in the extracellular space, DNase II (i.e. Acid DNase) during natural endolysosomal trafficking, and DNase III (i.e. TREX1) in cytosols. Alternatively, CDNs can utilize various membrane channels and transporters to access the cytosol, though the use of such transfer modalities is relatively inefficient and typically requires high local concentrations of CDNs. Moreover, certain naturally occurring CDNs, including 2′3′-cGAMP, are highly susceptible to degradation by ENPP1 in the extracellular space. Figure created with biorender.com.

Figure 2:. Chemical structures of cyclic dinucleotide (CDN) STING agonists.

(A) Mammalian 2′3′-cGAMP. (B) Various naturally occurring or synthetic CDNs with the noncanonical 2′3′ linkage orientation that is produced by mammals. (C) Various naturally occurring CDNs with the canonical 3′3′ linkage orientation that is produced by bacteria. (D) Synthetic 2′2′-cGAMP with the noncanonical 2′2′ linkage orientation that has not yet been found in nature. (E) Naturally occurring 3′2′-cGAMP with the noncanonical 3′2′ linkage orientation that is produced by Drosophila melanogaster (i.e. fruit flies).

Figure 3:. Crystal Structures of symmetrical human STING dimers.

(A) The resting ‘Open Lid’ configuration of an apo (i.e. unbound) human STING dimer. Adapted with permissions from reference. Copyright © 2020 American Association for the Advancement of Science; permission conveyed through Copyright Clearance Center, Inc. (PDB ID: 4F9E). Copyright © 2012 Elsevier Science & Technology Journal; permission conveyed through Copyright Clearance Center, Inc. (B) The ‘Closed Lid’ configuration of a holo (i.e. ligand bound) human STING dimer bound to 2′3′-cGAMP. Adapted with permissions from reference. Copyright © 2020 American Association for the Advancement of Science; permission conveyed through Copyright Clearance Center, Inc. (PDB ID: 4KSY). Copyright © 2013 Elsevier Science & Technology Journal; permission conveyed through Copyright Clearance Center, Inc. (C) The ‘Open Lid’ configuration of a holo (i.e. ligand bound) human STING dimer bound to 3′3′-diGMP. Adapted with permissions from reference. Copyright © 2020 American Association for the Advancement of Science; permission conveyed through Copyright Clearance Center, Inc. (PDB ID: 4F9G). Copyright © 2012 Elsevier Science & Technology Journal; permission conveyed through Copyright Clearance Center, Inc.

Figure 4:. Intracellular delivery challenges for STING pathway agonists.

Exogenous DNA and CDNs are negatively charged and hydrophilic and consequently cannot readily access the cytosol to activate the STING pathway. While both natural and synthetic CDNs are small enough to infiltrate the cytosol through the use of membrane channels and transporters, these transport modalities are inefficient. Furthermore, extracellular nuclease and phosphatases quickly degrade exogenous DNA and natural CDNs, respectively. Accordingly, relatively high concentrations of CDNs are required to elicit measurable STING activation. Non-nucleotide, small molecule agonists of the STING pathway have potential to passively diffuse across the cell membrane and therefore are an attractive alternative to the natural agonists. Lastly, certain nanocarriers can improve the efficacy and safety of STING pathway agonists by promoting intracellular delivery. Figure created with biorender.com.

Figure 5:. The importance of STING signaling kinetics.

The distinct outcomes of STING activation are balanced by signal persistence. Chronic STING signaling, which is quite often the result of genetic mutations, can lead to numerous IFN-driven inflammatory diseases, autoimmunity, and even cancer metastasis. Conversely, transient STING signaling, which can be induced by the acute STING activation from STING pathway agonists, can galvanize robust antiviral and/or anticancer immunity. Figure created with biorender.com.

Figure 6:. STING and the Cancer Immunity Cycle.

STING can promote antitumor immunity via the Cancer Immunity Cycle by promoting each of the following steps: 1) Antigen processing and presentation, 2) Lymphatic trafficking, 3) T cell priming and activation, 4) Systemic trafficking of T cells, 5) Infiltration of T cells into tumors, 6) Immune recognition of cancer cells, and 7) Killing of cancer cells / antigen release. Figure created with biorender.com.

Figure 7:. Cancer therapies that can iatrogenically activate the STING pathway.

STING activation is a known biological consequence of many classical cancer treatments, including DNA-damaging chemotherapies, therapies that compromise the DNA damage response, and radiotherapy. While the effects of classical cancer treatments are multifaceted, therapies that also induce STING signaling have potential to enhance overall therapeutic efficacy by providing a supportive inflammatory context for generating antitumor immunity. Figure created with biorender.com.

Figure 8:

Chemical structures of non-nucleotide, small molecule STING agonists.

Figure 9:. Development of the dimeric amidobenzimidazole (diABZI) STING agonist.

(A) Chemical structure of the monomeric ABZI STING agonist, Compound 1. (B) Chemical structure of the dimeric ABZI (diABZI) STING agonist, Compound 2. (C) Chemical structure of the fully optimized diABZI STING agonist, Compound 3. (D) The ‘Open Lid’ configuration of a holo (i.e. ligand bound) hSTING dimer bound to diABZI Compound 2. Adapted with permissions from reference. Copyright © 2020 American Association for the Advancement of Science; permission conveyed through Copyright Clearance Center, Inc. (PDB ID: 6DXL). Copyright © 2018 Springer Nature BV; permission conveyed through Copyright Clearance Center, Inc.

Figure 10:. Development of the MSA-2 (i.e. benzothiophene oxobutanoic acid) STING agonist.

(A) Chemical structure of MSA-2. (B) Self-dimerization of MSA-2 (PDB ID: 6UKM). (C) The ‘Closed Lid’ configuration of a holo (i.e. ligand bound) hSTING dimer bound to MSA-2 (PDB ID: 6UKM). Adapted with permission from reference. Copyright © 2020 American Association for the Advancement of Science; permission conveyed through Copyright Clearance Center, Inc.

Figure 11:. Development of the SR-717 STING agonist.

(A) Chemical structures of SR-001 (i.e. the prodrug screening hit), SR-012 (i.e. the elucidated STING agonist), SR-717 (i.e. the optimized STING agonist), and SR-301 (i.e. orally bioavailable analog of SR-717). (B) The ‘Closed Lid’ configuration of a holo (i.e. ligand bound) hSTING dimer bound to SR-717. Adapted with permission from reference (PDB ID: 6XNP). Copyright © 2020 American Association for the Advancement of Science; permission conveyed through Copyright Clearance Center, Inc.

Figure 12:. Chemical structures of modified CDN STING agonists.

Chemical structures of various CDN STING agonists that have been chemically modified for improved stability, activity, and cell permeability. The CDNs modified to include fluorine substitutions for 2′-hydrogens or 2′-hydroxyl groups on the pentose rings (i.e. 2′-F-c-di-GMP, Dithio-2′-F-cAIMP (Compound 53), and the 3′3′-c-Di(2′F,2′dAMP) Prodrug) exhibit improved membrane permeability as well as stability against enzymatic degradation. The carbocyclic STING agonist, 15a, which comprises carbocyclic nucleotides, cyclopentane instead of ribose, and the imidazole portion of adenine replaced with a pyrimidine ring, exhibits significantly improved STING binding, cellular activity, and membrane permeability.

Figure 13:. Utility and challenges facing the administration of STING pathway agonists.

The route of therapeutic administration can significantly impact the efficacy of STING pathway agonists. Administration can be local (e.g. intratumoral) or systemic (e.g. intravenous, oral), and each delivery route is associated with unique utility and challenges. Figure created with biorender.com.

Figure 14:. Opportunities for nanotechnology in the delivery of STING pathway agonists.

Nanotechnology can be employed to overcome many drug delivery challenges and therefore has potential to greatly improve the therapeutic efficacy of STING pathway agonists. Notably, nanotechnology can exploit dysfunctional tumor vasculature, protect drug cargo, promote lymphatic drainage, enable cellular targeting, facilitate cytosolic delivery, and allow for cellular co-delivery of various drugs. Figure created with biorender.com.

Figure 15:. Lipid-based CDN delivery systems.

(A) Schematic of a cationic liposomal 2′3′-cGAMP formulation, which comprises DOTAP, cholesterol, and DSPE-PEG(2000), and the proposed mechanism of intracellular 2′3′-cGAMP delivery. Reproduced with permission from reference. Copyright © 2017 John Wiley & Sons - Books; permission conveyed through Copyright Clearance Center, Inc. (B) Schematic demonstrating that nanoparticles can enhance drug delivery to draining lymph nodes via lymphatic transport. Figure created with biorender.com. (C) By exploiting the lymphatic transport of nanocarriers, CDN (i.e. cdGMP) concentration increases in the draining lymph nodes of the injection site and decreases in the blood stream when delivered with a lipid nanoparticle. Reproduced with permission from reference. Copyright © 2015 American Society for Clinical Investigation; permission conveyed through Copyright Clearance Center, Inc.

Figure 16:. Polymeric CDN delivery systems.

(A) Formulation of cGAMP and ovalbumin (OVA) into linear polyethyleneimine / hyaluronic acid (LPEI/HA) hydrogels for enhanced STING activation and antigen presentation. Adapted with permission from reference. Copyright © 2015 Elsevier Science & Technology Journals; permission conveyed through Copyright Clearance Center, Inc. (B) Nanoparticle assembly and cytosolic delivery of CDNs using cationic poly(beta-amino ester) (PBAE) nanoparticles to induce STING activation. Reproduced with permission from reference. Copyright © 2017 Elsevier Science & Technology Journals; permission conveyed through Copyright Clearance Center, Inc. (C) Chemical structure, formulation strategy, and intracellular delivery mechanism for STING-NPs (i.e. endosomolytic polymersomes for cytosolic delivery of 2′3′-cGAMP). Reproduced with permission from reference. Copyright © 2019 Springer Nature BV; permission conveyed through Copyright Clearance Center, Inc. (D) Antitumor effect and prolonged survival of mice with B16-F10 melanoma treated with intravenous administration of STING-NPs alone and in combination with ICB (i.e. anti-PD-1 and anti-CTLA-4). Reproduced with permission from reference. Copyright © 2019 Springer Nature BV; permission conveyed through Copyright Clearance Center, Inc.

Figure 17:. Biologically-derived CDN carriers.

(A) Schematic of the SYNB1891 bacteria strain, which has been engineered to localize in the hypoxic tumor environment, activate STING in tumor APCs through enzymatic production of c-di-AMP, and trigger complementary proinflammatory pathways through additional PRR activation. Reproduced with permission from reference. Copyright © 2020 Springer Nature. Distributed under a CC BY 4.0 license http://creativecommons.org/licenses/by/4.0/. (B) Schematic of iExo^STINGa exosomes, which have been engineered to deliver 2′3′-cGAMP. Reproduced with permission from reference. Copyright © 2021 The Authors. Published by Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology. Distributed under a CC BY 4.0 license http://creativecommons.org/licenses/by/4.0/. (C) Representative TEM image of extracellular vesicles used for CDN delivery. Reproduced with permission from reference. Copyright © 2021 Codiak BioSciences, Inc. Distributed under a CC BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/. (D) Schematic illustrating the delivery of 2′3′-cGAMP using recombinant, transmembrane-deficient STING to induce type I interferon responses. Reproduced with permission from reference. Copyright © 2020 The Authors, some rights reserved; exclusive licensee AAAS. Distributed under a CC BY-NC 4.0 license http://creativecommons.org/licenses/by-nc/4.0/.

Figure 18:. Controlled-release delivery systems for local delivery of STING pathway agonists.

(A) Schematic and fabrication of PLGA microparticles for temporally programable pulsatile cargo release. Reproduced with permission from reference. Copyright © 2020 American Association for the Advancement of Science; permission conveyed through Copyright Clearance Center, Inc. (B) Cumulative in vivo release of AF647 from microparticles in the B16-F10 tumor model. Reproduced with permission from reference. Copyright © 2020 American Association for the Advancement of Science; permission conveyed through Copyright Clearance Center, Inc. (C) A nanotube hydrogel for TME regulation and chemoimmunotherapy tumor sensitization. A peptidedrug conjugate was created by linking the hydrophilic tumor-penetrating peptide, iRGD to the hydrophobic anti-cancer drug, camptothecin (CPT). The diCPT-iRGD conjugates self-assembled into cationic supramolecular nanotubes, which electrostatically bound anionic c-di-AMP (i.e. CDA) and enabled localized and sustained drug release within the tumor microenvironment for a combination of cancer immunotherapy and chemotherapy. Reproduced with permission from reference. Copyright © 2020 Springer Nature BV; permission conveyed through Copyright Clearance Center, Inc.

Figure 19:. Biomaterials that can intrinsically activate the STING pathway.

There is a growing list of biomaterials known to activate the STING pathway, either directly or indirectly. Direct activation of the STING pathway involves molecules that can bind to and functionally activate either cGAS or STING proteins. Indirect activation of the STING pathway most commonly involves endogenous cGAS activation and is typically achieved by inducing the cytosolic relocation of DNA from mitochondria and/or nuclei. Figure created with biorender.com.

Figure 20:. Biomaterials that can directly bind and activate STING.

(A) Relative IFNB1 and CXCL10 mRNA levels over time in THP1 cells treated with 2′3′-cGAMP or the synthetic diblock copolymer, PC7A, along with the chemical structure of PC7A. Reproduced with permission from reference. Copyright © 2021 Springer Nature BV; permission conveyed through Copyright Clearance Center, Inc. (B) PC7A led to sustained TBK1/IRF3 phosphorylation and slower STING degradation compared to 2′3′-cGAMP in THP1 cells. Reproduced with permission from reference. Copyright © 2021 Springer Nature BV; permission conveyed through Copyright Clearance Center, Inc. (C) Schematic of STING oligomerization and the uncharacteristic immunostimulatory condensation (i.e. unlike that of the natural STING phase-separator, which negatively regulates STING-driven gene expression) induced by PC7A. Reproduced with permission from reference. Copyright © 2021 Springer Nature BV; permission conveyed through Copyright Clearance Center, Inc. (D) Schematic of mRNA-encapsulating LNPs incorporating STING-activating ionizable lipidoids. A18 was selected as the lead cyclic lipid candidate. Reproduced with permission from reference. Copyright © 2019 Springer Nature BV; permission conveyed through Copyright Clearance Center, Inc.

Figure 21:. Strategies for potentiating STING signaling.

The magnitude of STING-driven gene expression and/or profile of the resultant immune response can be modulated by many different biochemical agents (i.e. potentiators). Depicted in this figure are some notable potentiators of the cGAS/STING pathway. These potentiators include: certain metal ions (e.g. Mn²⁺ and Mg²⁺), which can amplify STING signaling through a variety of mechanisms; cGAS-binding proteins, which can augment 2′3′-cGAMP production by sensitizing cGAS to dsDNA; inhibitors of DNA methyltransferases, which can restore the activity of epigenetically silenced cGAS and STING proteins; various inhibitors of NF-κB signaling, which can influence downstream gene expression; inhibitors of the STING phase-separator, which have potential to prevent the inhibition of STING signaling induced by the liquid-liquid phase condensation of STING; VRAC agonists, which may enable enhanced transmission of CDN STING agonists; and inhibitors of ENPP1, which can be used to increase local 2′3′-cGAMP concentrations by deterring the degradation of 2′3′-cGAMP. Figure created with biorender.com.

Figure 22:. Rate of Publications.

Google Scholar search results for: “stimulator of interferon genes” “cancer immunotherapy”.