Ultrasound-Guided STING Activation by Nucleotide Nanocomplex-Decorated Microbubbles for Cancer Immunotherapy

The recent success of immunotherapy for treatment-refractory metastatic melanoma, lung cancer and renal cell carcinoma has provided a new hope that immunotherapy can be generalized to a broader range of cancers. However, many cancers do not respond to immune checkpoint inhibitors, which has limited their use to a subset of patients. There is now greater recognition that to generate optimal antitumor immunity requires activation of both innate and adaptive immune systems in patients. Innate immune cells, including macrophages and dendritic cells (DCs), possess phagocytic and antigen presenting capabilities that enable the initial immune recognition of tumor cells and serve as a link between the innate and adaptive immune systems through antigen cross-presentation and subsequent priming of T cells. In order for these professional antigen-presenting cells (APCs) to perform these immune functions, several cellular processes must be activated, including the innate immune sensor cyclic GMP–AMP synthase–stimulator of interferon genes (cGAS-STING). Agents that target the innate immune regulators, such as STING agonists are now being investigated as potential therapeutics for a wide range of human cancers. However, because STING is a cytosolic innate immune sensor, the intracellular delivery of agonists such as the negatively charged 2'3'-cyclic-GMP-AMP (cGAMP) is very challenging. Equally important, non-specific global STING activation can cause systemic inflammatory responses and toxicity. Systemic administration of STING agonists leads to dose-dependent T cell death that facilitates tumor immune evasion, further hindering the translation of STING agonists into the clinic.<br/><br/>To address this technical challenge and meet the clinical need, we developed a technology termed MUSIC (<u>M</u>icrobubble-assisted <u>U</u>ltra<u>s</u>ound-guided <u>I</u>mmunotherapy of <u>C</u>ancer) that utilizes gas-filled microbubbles (MBs) conjugated with APC-targeting antibodies, and cationic polymers to efficiently load cGAMP via formation of nanocomplexes on the MB surface.<br/><br/>Our preliminary results show that, upon exposure to US, MUSIC produces robust STING activation and type I interferon responses in APCs and more efficiently primes antigen-specific CD4⁺ and CD8⁺ T cells <i>in vitro</i>. These immune stimulatory effects of MUSIC directly translated into antitumor responses <i>in vivo</i>, where we showed that the MUSIC was able to generate antitumor effects against syngeneic orthotopic primary (EO771) and metastatic (4T1) murine breast cancer models. Both models showed dramatic antitumor responses following local treatment of the primary tumor. Specifically, 6 out of 10 EO771 tumor-bearing animals were tumor-free after 50 days while 4T1 tumor-bearing mice exhibited a significant decrease of the systemic disease burden including lung metastases. We also confirmed the establishment of systemic immune memory following MUSIC treatments as mice rejected tumor cells upon re-challenge. MUSIC was also evaluated in two syngeneic murine melanoma models (B16-F10 and D4M) with 100% of D4M-tumor bearing animals and 50% of B16-tumor-bearing animals achieving complete remission when treated with a combination of MUSIC and immune checkpoints inhibitors.<br/><br/>MUSIC showed efficient antitumor immune responses in localized and metastatic murine breast cancer models as well as in two melanoma models leading to the establishment of immune memory (<i>i.e.</i>, <i>in situ</i> vaccination). This nanocomplex-conjugated microbubble platform enables more efficient loading and release of cGAMP and provides a new drug delivery vehicle for further functional modifications including for loading drug or gene delivery for cancer treatment and/or vaccines.