Background Despite improved therapeutic outcomes for cancer patients with the advent of immunotherapy, not all patients respond to these agents. Some patients exhibit an ‘immune-cold’ or ‘excluded’ phenotype, with little to no infiltration of T cells into the tumor body. Lack of T-cell infiltration may result from an absence of T-cell recruiting chemokines in the tumor and its surrounding environment, such as CXCR3 ligands CXCL9 and CXCL10. Recent pan-cancer analyses have shown a strong positive correlation between levels of CXCL9 and CXCL10 in tumors and the response to checkpoint inhibitors (CPIs). Moreover, studies in animal models have demonstrated that increasing the levels of CXCL9 and/or CXCL10 can enhance anti-tumor activity, with or without concurrent CPI treatment. Here we explore the utility of epigenomic controllers to tunably upregulate CXCL9 and CXCL10 expression in hepatocellular carcinoma (HCC) and tissue resident cells (hepatocytes) as a potential strategy to create an immunologically-hot HCC tumor microenvironment (TME).

Methods We have developed epigenomic controllers (ECs), which are epigenomic mRNA therapeutics, that are designed to selectively target regulatory elements of target genes such as CXCL9 and CXCL10 and upregulate their expression. To assess the impact of CXCL9 and CXCL10 ECs in HCC cells and hepatocytes, we encapsulated the ECs in liver-targeted lipid nanoparticles (LNPs) and treated human primary hepatocytes and the HCC cell line, Hep3B. CXCL9/CXCL10 mRNA and protein expression levels were measured using qRT-PCR and ELISA, respectively, and changes in epigenetic markers were evaluated through ChIP-sequencing. Additionally, in vitro T-cell migration assays were conducted to test the effect of CXCL9 and/or CXCL10 upregulation on T-cell recruitment.

Results Our initial screening of CXCL9 and CXCL10 ECs encoding for various epigenetic activators revealed a broad spectrum of upregulation (ranging from 10- to 10⁶-fold), demonstrating, in this case, the broad upregulation capability of our platform. Treatment of human primary hepatocytes with CXCL9 and CXCL10 ECs resulted in the desired epigenetic modifications, leading to robust upregulation of CXCL9 and CXCL10 mRNAs and proteins. Importantly, the EC-mediated upregulation of CXCL9 or CXCL10 alone, or in combination, induced T-cell migration, providing in vitro proof-of-concept for this approach.

Conclusions Taken together, these findings suggest EC-driven upregulation of T-cell-recruiting chemokines CXCL9 and/or CXCL10 within the TME could enhance T-cell infiltration into tumors and support continued evaluation of this strategy as an approach to improve anti-tumor activity of immunological therapeutic agents in HCC and other cancers.