ENHANCED ANTI-TUMOR ACTIVITY OF CD19.CAR-NK CELLS BY INCORPORATING IL-27

Abstract

Autologous T-cells expressing Chimeric Antigen Receptor (CAR) have shown great efficacy against hematologic malignancies, however, severe side effects limit their clinical use. Natural Killer (NK) cells are an attractive alternative to T-cells as carriers for CARs, since they could be used as an allogeneic treatment, and have no, or minimal, risk for Cytokine Release Syndrome (CRS). CAR-NK cells have recently shown promising effect against leukemia without serious side effects after adoptive transfer. Possible allogeneic application of CAR-NK cells could dramatically reduce the costs of such adoptive cell therapy, nevertheless sufficient ex vivo expansion of NK cells remains challenging. Transgenic expression of cytokines in CAR-NK cells was shown to partially improve pro-survival or proliferative properties (e.g., IL-15), however what is the optimal cytokine for CAR NK cells is not clear to date. Here, we compared 2nd generation CD19-targeted CAR (CAR.19) with a fourth generation CAR.19 coexpressing Il-27 (CAR.19-IL27) in promoting NK-92 cell line proliferation, activation, and cytotoxic activity against B-cell cancers. CAR constructs were inserted into lentiviral vectors and subsequently transduced into the NK-92 cell line. Effects of CAR-NK cells were assessed against CD19-expressing B-cell lines NALM-6 or Raji in vitro and in vivo in a murine model of B-cell neoplasia with Raji cells. Tumor burden was measured by bioluminescence and by the curve of survival. We have demonstrated that CAR.19-IL27 exhibits a superior expansion capacity in comparison to both NK-92wt and CAR.19 cells. Moreover, CAR.19-IL27 cells showed higher cytotoxicity towards NALM-6 and Raji compared to CAR.19 in vitro (**p < 0.001). Degranulation assay, assed by CD107a, showed that CAR.19-IL27 have higher expression of CD107a compared to CAR.19 cells (**p < 0.001), which indicates increased secretion of perforin and granzymes. Our systematic transcriptome analysis of activated NK-92 CAR variants shows that IL-27 may be involved in the PI3K/AKT, and MAPK signaling pathways, as genes related to proliferation from these pathways were increased, e.g., PDK1, RPS6, MYC, and PGK1. In vivo studies with a xenograft mouse model of B-cell neoplasia showed that CAR.19-IL27 reduced tumor burden compared to CAR.19-treated. Altogether, CD19-CAR NK cells co-expressing Il-27 showed potent and selective antitumor activity with the potential to improve clinical use of CAR NK cells.