(184aa) Design and Delivery of a Sting Mimicking Peptide-Polyanion Conjugate for Cancer Immunotherapy

Therapeutic activation of stimulator of interferon genes (STING) signaling shows promise as a method of inflaming the tumor microenvironment to generate an anti-cancer immune response. However, STING signaling is frequently inactivated in human cancer, commonly by epigenetic silencing of the STING gene. This is a hypothesized mechanism of resistance to STING agonist immunotherapy, as agonists have no effect in patient cancer cells. The ability to activate STING signaling in cancer lacking STING protein expression would overcome this challenge, motivating the development of strategies to directly activate downstream signaling proteins TBK1 and IRF3. Our group previously reported that delivering cargos that mimic the multivalent active state of STING protein to the cytosol can activate TBK1 and IRF3, initiating the desired innate immune response even in STING-silenced cancer.

In this work, we develop a multivalent-peptide polyanion conjugate material that can easily be delivered to the cytosol, interacts with TBK1 and IRF3, and potently activates a type I interferon response. While existing strategies deliver the entire STING protein or remove only the transmembrane domain, truncation experiments demonstrate that delivering a short fragment from the disordered C-terminal tail of STING is sufficient to activate downstream signaling. This fragment is only 40 amino acids, just one tenth of the STING protein, yet it contains the protein interaction motifs required to activate TBK1 and IRF3. Using copper catalyzed click chemistry, we synthesize conjugates with precisely controlled, high-valency peptide display on a polymer backbone. High-valency display better mimics the multivalent active state of STING protein, allowing for greatly improved potency and therapeutic window compared to a previously developed STING mimetic therapeutic STINGΔTM. The conjugate material was designed with electrostatic complexation in mind, the negative charge of the polyanion backbone enables encapsulation by existing nanocarriers designed to deliver negatively charged nucleic acids. A functional delivery assay demonstrates that the peptide-polyanion conjugate can be delivered to the cytosol using a variety of nucleic acid carriers with little modification, including polyethyleneimine, poly(beta-amino esters), and lipid nanoparticles. Finally, a lipid nanoparticle formulation was optimized to maximize conjugate encapsulation, transfection ability, and incorporate an ovarian cancer targeting polymer surface coating. Overall, this work serves as an in vitro demonstration that multivalent display of a short peptide containing key protein interaction motifs can mimic the active state of the STING protein, enabling type I interferon response activation even in STING-silenced cancer cells. Strong preliminary results motivate ongoing evaluation in vivo, highlighting potential for the treatment of STING-silenced cancers.