PSCA-CAR-NK cells exert cytotoxic activity against PSCA-expressing tumor cells and are characterized by a specific chemokine profile.

Abstract

Genetic modification of NK cells with chimeric antigen receptors (CARs) is a rapidly evolving approach to treating tumor diseases. CAR-NK technology is being developed for various antigens associated with both hematologic and solid tumors. In this study, peripheral blood NK cells overexpressing CAR against prostate stem cell antigen (PSCA) were obtained using retroviral transduction. To assess the specific functional activity of the PSCA-CAR-NK cells, we used cell lines modified to express surface PSCA via lentiviral transduction. The increased specific cytotoxic responses of PSCA-CAR-NK cells against these PSCA-positive cell lines were demonstrated using two alternative in vitro methods: a degranulation assay measuring CD107a expression level on the NK cell surface and a cytotoxicity test assessing fluorescent dye release from dying target cells. The specific cytotoxicity of PSCA-CAR-NK cells was confirmed using the BxPС-3 tumor cell line spontaneously expressing PSCA. In addition, profiles of chemokine receptors involved in the antitumor response, including CCR7, CXCR1, CXCR3 and CXCR4, were analyzed in NK cell subsets ex vivo, in IL-2/feeder cell-activated NK cells, and in transduced NK cells. We observed that the expression levels of CCR7 and CXCR4 on CD56⁺ NK cells and in the CD56⁺CD57⁺ fraction were highest in the activated state, decreasing after transduction. Proportion of NK cells expressing the CXCR1 receptor markedly decreased after activation while that of CXCR3 increased, the transduction procedure had no significant influence on the CXCR1 and CXCR3 levels. Following transduction, PSCA-CAR-NK cells showed increased levels of CCR7 expression compared to unmodified GFP-NK cells, most marked in more mature cells expressing CD57 or KIR2DL2/3. Moreover, an increased CXCR3 expression was noted in CD57⁺ subsets of the PSCA-CAR-NK cells. Since the ability of NK cells to migrate to lymph nodes and reach tumor sites depends on the presence of CCR7 and CXCR3 receptors, respectively, the patterns we have identified in the distribution of these chemokine receptors on cytotoxic PSCA-CAR-NK cells are particularly interesting and may be useful in the context of fighting solid tumors.