Intra-Tumor Injection of CAR-Engineered NK Cells Induces Tumor Regression and Protection Against Tumor Re-Challenge

In this era of cancer immunotherapy, the long-term goal of treatment is to eliminate tumor cells anywhere in the body through induction of a specific immune memory response. In addition to spontaneous cytotoxicity, ADCC, and cytokine release, NK cells contribute to an adoptive immune response through crosstalk with dendritic cells and T cells. To further characterize the NK cell-induced adoptive response, aNK cells (formerly NK-92) were transduced with a retrovirus construct coding for a second-generation anti-murine CD19-CAR to create targeted activated NK-92 cells (mCD19.taNK). mCD19.taNK cells were injected intra-tumorally (two injections of 5x10⁶ cells, three days apart) into a murine model of syngeneic subcutaneous lymphoma, created by injecting 1x10⁶ A20 cells into BALB/c mice. Tumor size was monitored over time, and mice showing complete tumor regression were re-challenged with another subcutaneous contralateral injection of A20 cells.

mCD19.taNK cells effectively killed murine cancer cells in vitro (>60% killing at E:T ratio of 5:1). In vivo, intra-tumor injection of mCD19.taNK induced significant tumor regression versus saline (tumor volumes of 342 mm³ and 936 mm³, respectively at day 16, p<0.05) and significantly improved survival, with 75% of the mice showing complete tumor regression at day 32 (p<0.05). In contrast, injection of parental aNK cells did not significantly affect tumor size in mice (815 mm³at day 16). Upon re-challenge with A20 lymphoma cells, >80% mice remained free of tumor after 14 days.

In conclusion, the human aNK cell line expressing an anti-murine CD19-CAR (mCD19.taNK) can effectively kill CD19-positive murine cancer cells. Moreover, intra-tumor injections of mCD19.taNK into a fully immunocompetent mouse model can induce tumor clearance and protection from tumor re-challenge, suggesting induction of a memory ("vaccine") effect after the injection of mCD19.taNK.