Radiation Therapy Sensitizes Head-and-Neck Cancer Cells to Killing by Chimeric Antigen Receptor (CAR)-NK Cells Targeting CD70.

PURPOSE/OBJECTIVE(S): CAR-T cell therapy is limited by toxicity, high cost, logistical manufacturing issues in the autologous setting and risk of GVHD in the allogeneic setting. Substitution of T cells with NK cells opens the possibility for an allogeneic off-the-shelf product with a better safety profile. However, the inadequate efficacy of CAR-NK cells against solid tumors can be extrapolated from experience with CAR-T cells. There is limited but promising preclinical evidence that radiation therapy (RT) enhances CAR-T cell tumoricidal activity against solid tumors. However, there is no data examining the potential synergy between RT and CAR-NK cell therapy.

MATERIALS/METHODS: We engineered CAR-NK cells with CD27 receptor as extracellular domain to target its natural ligand CD70, which is overexpressed in head-and-neck cancers (HNSCC). CAR-NK cell killing was assessed real-time through xCELLigence cytotoxicity assays. CD70+ OQ01 human HNSCCs were used for most experiments. FaDu is a CD70- HNSCC (negative ctrl). UMRC3 is a CD70+ kidney cancer cell line (positive ctrl). CD70 expression pre- and post-RT was assessed by flow cytometry and Western blot. Ionizing RT was compared at 5 doses: 0, 1.75, 3.5, 7, and 14 Gy. A single dose of 3.5 Gy was used for most experiments. Post-radiation effects were generally assessed at 3 days or 9 days post-RT. Intracellular staining was used to assess NK cell expression of IFN-γ, CD107a, and TNF-α by flow cytometry. CD27/CD70 interaction blockade was through α-CD27 pre-treatment of CAR-NK cells.

RESULTS: OQ01 HNSCCs heterogeneously express CD70 and are killed by CD70 CAR-NK cells in vitro. Pre-conditioning low-dose RT of 3.5 Gy applied to OQ01 HNSCCs 3 days prior to coculture with NK cells enhances CD70 CAR-NK cell killing, with ∼30% increased cytotoxicity against the tumor cells. Low-dose RT by itself did not induce acute cytolysis. As a possible mechanism for the increased sensitivity of irradiated OQ01 cells to CD70 CAR-NK cells, we found that RT enhances CD70 expression among HNSCCs in a dose-dependent manner. There was no increase in NK cell expression of IFN-γ, CD107a, and TNF-α with exposure to irradiated target cells. CD27/CD70 blockade does not solely abrogate RT-induced sensitization toward CAR-NK cell killing. Despite RT induction of transient increased expression of CD70, which normalizes by 9 days post-RT, there is persistent increase in RT-synergized target cell killing even at this later timepoint. Thus, altogether, RT sensitizes CD70-expressing HNSCC cells to CAR-NK cell killing in vitro.

CONCLUSION: This work represents the first preclinical study to identify the synergy of RT and CAR-NK cell therapy in solid tumors and is the first demonstration of CAR-NK cell activity against human HNSCCs. We show significantly enhanced potency of CAR-NK cells against irradiated tumor cells in vitro. Collectively, this research will be vital to guide efforts expanding into other target antigens and tumor types.