Activate Antitumor Immunity by Polymer-Induced STING Condensation

Suxin Li and Jinming Gao

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The STimulator of INterferon Genes (STING) is an endoplasmic reticulum-bound protein which has received extensive interest as a therapeutic target for infectious diseases and cancer. STING protein, discovered in 20081, acts as first responders when a danger signal such as DNA from cancer cells or virus is detected in the cell cytosol2. Activation of STING mediates a multifaceted type I interferon responses that promote the maturation and migration of dendritic cells and prime cytotoxic T lymphocytes. Early phase clinical trials involving first generation small molecule STING agonists, however, have shown limited antitumor efficacy and dose-limiting toxicity.

Originally, our team aimed to build a nanoparticle vehicle to deliver the natural STING ligand, cyclic GMP-AMP (cGAMP) to cell cytosol in tumor tissues and protect it from enzymatic degradation. We employed a library of ultra-pH-sensitive polymeric nanoparticles (UPS NPs) that display a cooperative all-or-nothing response across a narrow pH range (<0.3 pH unit)3. Micelles are formed in the physiological pH environment (i.e., blood, lymphatic fluid) to carry the payload, while they will disassemble once endocytosed into acidic endosomes, and release payload to reach STING molecules4. But one polymer with 7-membered cyclic amine side chains we synthesized, PC7A, had an unexpected novel effect: it generated interferon responses even without cGAMP. When packed with an antigen, the cytotoxic T lymphocytes response was not dependent on Toll like receptor or RNA-induced innate pathways.  Surprisingly, STING knockout mice abolished the antitumor effect. We reported the initial results in 20175, not knowing at the time exactly how PC7A worked; the polymer does not resemble any known cyclic dinucleotide drugs that activate STING.

 

Figure 1. (a) Confocal image of a mouse embryonic fibroblast with green fluorescent protein labelled-STING. PC7A polymer induces STING condensation through polyvalent interactions. Scale bar, 5 μm. PC7A polymer (b) displays prolonged STING activation in THP1 macrophages compared to cGAMP (c).

 

In this work, we elucidated the biochemical mechanism of STING activation by PC7A and furthermore, designed a nanoparticle-based STING agonist for cancer immunotherapy. We first determined the binding affinity between PC7A and STING by isothermal calorimetry, and a strong binding was observed (apparent Kd=72 nM). Attempts at forming a co-crystal of PC7A and STING complexes to determine the specific binding site failed because of polyvalent nature of these interactions (see below). Based on computational modeling, we identified a few potential binding sites on the STING that would engage optimal electrostatic and hydrophobic interactions with the PC7A polymer. Accordingly, we constructed STING mutants with several negatively charged amino acids (glutamate or aspartate) in the α5-β5-α6 region replaced by neutral alanine residues and investigated their PC7A binding affinity and STING activation in live cells. Strikingly, mutation of two acidic residues (E296A/D297A) on the α5 helix was sufficient to abolish polymer binding and STING activation, whereas two other mutants (D319A/D320A and E336A/E337A/E339A/E340A) exhibited marginal effects. Because the distinct binding site compared with the natural ligand, we hypothesized PC7A might work in patients who are resistant to cyclic dinucleotide drugs. According to report, up to 20 percent of people have inherited a slightly different gene (R232H) for STING. This mutation makes cGAMP and its derivative drugs ineffective, but PC7A still works.

As a new concept in cell biology, polyvalent phase condensation has been shown to regulate diverse biological processes, including ribosome assembly, gene expression, and signal transduction. By forming biomolecular condensates, proteins involved in signaling cascades can be easily enriched in membrane-less assemblies and amplify responses to small changes in the environment. In the case of interaction between PC7A and STING, because each polymer chain consists of multiple binding units, and each STING protein dimer has two binding sites, we hypothesized such polyvalent interactions could multimerize STING molecules and trigger biomolecular phase condensation (Fig. 1a). In fact, we did observe higher degree of phase condensation and stronger STING activation when longer polymers (thus more binding units) were used.

Interestingly, the temporal profile of PC7A-induced STING activation is different from those induced by cGAMP or other existing drugs (Fig. 1b,c). While cGAMP activates the protein over the course of about 6 hours, PC7A sustains STING activation over 48 hours, which leads to a more effective immune T cell response against solid tumors. In addition, combining cGAMP with PC7A synergizes STING activation in resected human tumors and lymph nodes and improves survival in animal tumor models.

This study generated new mechanistic insights and novel therapeutic strategies that exploit noncanonical STING activation for cancer immunotherapy. The research team is highly interdisciplinary, covering polymer chemistry, nanotechnology, molecular biology, cell biology, and tumor immunology, which allowed the synthesis of information from a diverse background.  Our ultimate goal is to utilize nano-immune-engineering approaches that incorporate nature’s design with engineering ingenuity to improve the safety and efficacy of cancer immunotherapy. Now we’re looking forward to testing this novel nanoparticular therapeutics in clinical translation.

 

References:

  1. Ishikawa, H., Barber, G.N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature455, 674-678 (2008).
  2. Wu, J., et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826-830 (2013).
  3. Ma, X., et al. Ultra-pH-sensitive nanoprobe library with broad pH tunability and fluorescence emissions. Am. Chem. Soc. 136, 11085-11092 (2014).
  4. Wang, Z., et al. A redox‐activatable fluorescent sensor for the high‐throughput quantification of cytosolic delivery of macromolecules. Chem. Int. Ed. 129, 1339-1343 (2017).
  5. Luo, M., et al. A STING-activating nanovaccine for cancer immunotherapy. Nanotechnol. 12, 648-654 (2017).

Jinming Gao

Professor, UT Southwestern Medical Center