Background NK cells act as the body’s first line of defense against cancer cells. They quickly recognize and kill tumor cells without prior exposure. Adoptive cell therapy (ACT) using NK cells shows promise against hematological cancers. Cytotoxic activity of these cells is restricted by inhibitory receptors that reduce NK cell-mediated cytotoxicity. Overcoming this inhibition would allow for a more potent antitumor response following ACT and potential application against solid tumors. We have developed a new class of stable, self-delivering RNAi compounds (INTASYL) that incorporate features of RNAi and antisense technology. INTASYL compounds demonstrate potent activity, stability, and are rapidly and efficiently taken up by cells. INTASYL PH-804 targeting the inhibitory receptor TIGIT enhances the cytotoxic activity of expanded human NK cells in vitro.

Methods Primary human CD56⁺ NK cells were expanded using the ImmunoCult™ NK Cell Expansion Kit from StemCell Technologies. Following the 14-day expansion protocol, cells were collected, and the cell density was adjusted to 0.5 x 10⁶ cells/mL in culture media containing IL-2. Cells were seeded directly into 24-well plates containing PH-804 ranging in final concentration from 1 μM to 5 μM. Taqman gene expression assays were used to determine expression levels of TIGIT following the RNA-to-Ct 1-step protocol. In addition, cells were stained using fluorescently labeled antibodies for flow cytometry. Cytotoxic capabilities of the PH-804 transfected NK cells against the K562 (Chronic Myelogenous Leukemia) cancer cell line were tested in a DELFIA cell cytotoxicity assay.

Results Transfection with PH-804 results in consistent mRNA and protein silencing without negative impact on NK cell viability. For example, treatment with 5 μM PH-804 results in a 60% reduction in TIGIT mRNA. The reduction is seen at >7 days post-transfection and results in a 45% reduction in surface expression of TIGIT by flow cytometry. Silencing of TIGIT with PH-804 resulted in increased expression of markers of NK cell activation and increased cytotoxic capabilities of NK cells against K562 cancer cells in the DELFIA cell cytotoxicity assay.

Conclusions Here, we demonstrate the potential of PH-804 to improve NK cell potency in ACT. By treating NK cells with INTASYL targeting the inhibitory receptor TIGIT ex vivo, during NK cell expansion, the anti-tumor response of these cells was enhanced potentially resulting in a more effective cell therapy for solid tumors and hematological malignancies.