(W0930-10-55) DNA-Based cGAS Agonists as a Novel Approach to cGAS-STING Activation for Cancer Immunotherapy

Purpose: Immune checkpoint blockade (ICB) is one of the current mainstream approaches to cancer immunotherapy. However, current ICB is associated with limitations such as the low response in the majority of cancer patients and elevated immune toxicity associated with the imbalance of immune homeostasis. Immunostimulants hold great potential to jump start the immune system and synergistically inhibit tumor growth with ICB. Recent studies have shown that cyclic GMP-AMP synthase-stimulated interferon genes (cGAS-STING) pathway plays a critical role in innate immune response and is essential for the anti-tumor effect of some of cancer therapies. In our study, we developed a cGAS agonist based on short DNA to activate cGAS-STING pathway for anti-tumor immune response.

Methods: We first investigated the immunostimulatory effect of DNA oligonucleotides, Svg3, in mouse and human immune cells. Specifically, we measured the expression levels of type I interferon in murine antigen presenting cells (APCs) after treatment with Svg3. We confirmed the type I interferon inducing effect was dependent on cGAS activation using a cGAS knock-out cell line. Co-stimulatory factors such as CD80 and CD86, CD40 and MHC II expressed on APCs were also measured, which are important signals of APC activation. We compared the immunostimulatory effect of Svg3 to that of cGAMP, a widely used STING agonist. To solve the stability issue with a DNA drug and improve its delivery efficiency, we formulated Svg3 into liposomes by ethanol evaporation method and characterized by dynamic light scattering. The improvement of cellular delivery and in vivo delivery of liposomal Svg3 was evaluated with fluorescent labeled Svg3 by flow cytometry and IVIS imaging, respectively. Finally, we tested Svg3’s immunotherapeutic efficacy combined with ICB and modulatory effect on immunosupressive tumor microenvironment in multiple murine tumor models, including 4T1 triple negative breast cancer mouse model, B16 melanoma mouse model and MOCII mouse oral cancer model.

Results: Immunostimulatory effects on antigen presenting cells induced by Svg3 - Our study showed that Svg3 can effectively promote the production of IFN-β in DC2.4 dendritic cells and RAW264.7 macrophages, but not cGAS-knockout macrophages, indicating that Svg3 is cGAS-specific. RNA sequencing Svg3 also elevated the expression of CD80, CD86, CD40, and MHC II on DC2.4. Furthermore, we found that Svg3 outperformed the existing STING agonist, cGAMP, inducing stronger immunostimulatory effects at a lower dose. Improved delivery efficiency of Svg3 using liposomes - Svg3 was efficiently loaded in liposomes with an average particle diameter of 200 nm and a slightly positive surface charge. Svg3 liposomes can be quickly taken up by immune cells and extend tumor retention time following a single dose of intratumoral injection. Therapeutic efficacy of Svg3 on mouse cancer model - In 4T1 triple negative breast cancer model, B16 melanoma model and MOC2 oral cancer model, Svg3 showed the ability to inhibit tumor progression combined with anti PD-1 treatment.

Conclusion: We developed a DNA-based immunotherapeutics as cGAS agonists, which showed a strong immunostimulatory effect in immune cells via cGAS-STING activation. Svg3 loaded into liposomes improved the immune delivery. Finally, Svg3 successfully inhibited tumor progression by modulating tumor microenvironment, which suggests its potential as a novel class of cancer immunotherapeutics.