O-13.04 SIlencing BRD4 to increase NK cell activity for adoptive cell therapy: a novel self-delivering RNAi approach

<h3>Background</h3> Natural killer (NK) cells are an essential part of the immune system and serve as the body’s first line of defense. They can recognize and kill tumor cells without prior exposure, making them an attractive option for adoptive cell therapy (NK-ACT). Unlike T cells, NK cells do not induce graft-versus-host disease, offering an advantage for ‘off-the-shelf’ cancer immunotherapies. Improving NK cell activity, however, is critical to enhance clinical efficacy. Targeting Bromodomain-containing protein 4 (BRD4) may enhance NK cell-mediated immune responses against tumors. BRD4 is an epigenetic regulator involved in controlling the transcription of genes related to cell proliferation and survival. BRD4 has a crucial role in processes such as inflammation, cancer, and immune cell regulation. Current BET inhibitors such as JQ1 and molibresib target BRD4 but lack specificity, leading to off-target effects that limit their clinical application. INTASYL™ offers a novel approach with a highly specific mechanism to target BRD4. These self-delivering RNAi compounds combine RNAi and antisense technologies, offering high potency, stability, and rapid cellular uptake without the need for additional delivery vehicles. This study investigates PH-894, an INTASYL compound that selectively inhibits BRD4 expression, with the potential to enhance NK cell function. <h3>Materials and Methods</h3> Primary human CD56+ NK cells were expanded using the ImmunoCult™ NK Cell Expansion Kit. After 14 days, cells were treated with PH-894. At the end of the culture period, cell viability, BRD4 mRNA, and protein expression levels were measured. Flow cytometry was performed to explore the impact on proliferation and activation makers. <h3>Results</h3> PH-894 treatment efficiently and selectively silenced BRD4 in NK cells without affecting other BET family members or compromising cell viability. Silencing BRD4 led to a significant decrease in CD94 expression and an increase in CD25, indicative of enhanced activation. Additionally, increased NK cell proliferation was detected using the CFSE dye dilution method. <h3>Conclusions</h3> Incorporating PH-894 treatment into ex vivo NK cell expansion protocols prior to ACT represents a promising strategy for improving NK cell efficacy. These findings suggest that BRD4 may play an important role in regulating NK cell activity and that its silencing may modulate NK cell function, potentially enhancing their antitumor activity. This study provides a foundation for further exploration of BRD4 as a target for enhancing NK cell-based immunotherapies. <b>M. Maxwell:</b> A. Employment (full or part-time); Significant; Phio Pharmaceuticals. <b>E. Noessner:</b> None.