(I-332) Development of Macromolecular Polymeric STING Agonists for Cancer Immunotherapy Applications

Cancer immunotherapies have revolutionized the treatment of many cancer types, offering patients hope for complete tumor regression and life-long immunological memory to prevent disease recurrence. One promising cancer immunotherapy target is the Stimulator of Interferon Genes (STING) pathway, as it plays a crucial link between innate and adaptive antitumor immunity. Activation of the STING protein triggers a type-I interferon (IFN-I) inflammatory response which provides the proper immunological context to activate antigen presenting cells (APC), T cells, and natural killer cells, leading to potent antitumor responses. Unfortunately, systemic administration of endogenous STING agonists (e.g., 2′3′-cGAMP) are limited by poor stability and cytosolic delivery, which has prompted the development of non-nucleotide, synthetic STING agonists. Of the non-nucleotide STING agonists in use, recently described diamidobenzimidazole (diABZI) compounds have been shown to facilitate potent STING activation and antitumor activity but they pose significant formulation challenges due to their hydrophobicity and short half-life. Consequently, we have developed RAFT-compatible diABZI-based chain transfer agents (CTAs) and monomers and have leveraged these to create macromolecular polymeric STING agonists that retain potent STING activation. Our polymer engineering approach enables exquisite control over monomer composition, molecular weight, diABZI valency, linker chemistry, and downstream immunological signaling.

We co-polymerized methacrylate diABZI-based monomers with hydrophilic monomers (PEGMA or DMA) and polymerized diABZI-based CTAs with hydrophilic monomers (DMA or PEGMA) to create a library of macromolecular STING agonists. For all polymerizations, a 15 to 30% w/v solution of monomer(s), initiator, and chain transfer agent in DMF was prepared. A 5:1 CTA:initiator molar ratio and various molar equivalents of methacrylate diABZI-based monomers and hydrophilic monomers were used to generate the polymers. The reaction container was sealed, purged with argon for 15 minutes, and then allowed to react with stirring for 24 hours at 40 C or 70 C depending on the initiator. The reaction was quenched via exposure to oxygen and the polymer was characterized for percent conversion via H-NMR. All polymers were dialyzed, lyophilized, evaluated for purity via H-NMR, and stored at -20 C until use. STING activation of the diABZI-based polymers were evaluated in cell lines engineered to quantify interferon regulatory factor (IRF) pathway activation through monitoring the activity of secreted Lucia luciferase (e.g., THP-1 Dual reporters) and compared to relevant controls. A cell seeding density between 25k to 35k cells per well in a 96 well plate was used for all dose-response studies and luminescence readouts were performed 24 hours post-plating.

Overall, we synthesized and evaluated the in vitro STING activation of two main groups of diABZI-based compounds and polymers: 1) those based on an alkane-linked diABZI compound (diABZI-Alkane), which contain a butane linker between amidobenzimidazole groups and 2) those based on an alkene-linked diABZI compound (diABZI), which contain a trans-but-2-ene linker between amidobenzimidazole groups. The first category of macromolecular STING agonists evaluated were 25 kDa, 50 kDa, and 175 kDa p(DMAs) synthesized with a diABZI-Alkane-CTA (Figure 1) or a diABZI-CTA (Figure 2). As can be seen, trans-but-2-ene-linked diABZI compounds elicit more potent STING activation than diABZI-Alkane compounds. Furthermore, p(DMA) polymers made with both diABZI-Alkane-CTA and diABZI-CTA exhibit molecular weight-independent STING activation. Not shown is in vitro STING activation from diABZI-Alkane and diABZI methacrylate monomers, but these polymer constructs also demonstrate potent STING activation. In conclusion, we have developed a library of macromolecular STING agonists that improve formulation challenges associated with diABZI compounds and maintain potent STING activation in vitro. In the time leading up to the conference, I will further characterize these polymers with more in depth in vitro assays (e.g., STING-induced RNA production kinetics and surface expression of co-stimulatory/MHC molecules on APCs) and incorporate peptide antigens for cancer vaccine applications.