IL-27 IN A FOURTH GENERATION CAR ENHANCES CAR-NK CELL EFFECTOR FUNCTION

Background & Aim

Interleukin 27 (IL-27), part of the IL-12 cytokine family, enhances NK cell cytotoxicity by inducing activating receptors (e.g., NKp46, NKG2D), and stimulating IFN-gamma production. While chimeric antigen receptor (CAR) has shown promise for NK cells in early-phase clinical trials, improving CAR-NK cell therapeutic effectiveness is critical. Although transgenic expression of cytokines (e.g., IL-15) in CAR-NK cells partially enhances pro-survival or proliferative properties, further research is needed to identify the optimal cytokine. Therefore, investigating the role of co-expressing IL-27 with a fourth-generation CD19-targeted CAR on NK cells is crucial.

Methods, Results & Conclusion

Lentiviral particles carrying CAR19 and CAR19-IL27 vectors were used to transduce NK-92 cell line. CAR+ cells were FACS-sorted and the potency of CAR-NK cells was evaluated against CD19+ cell lines (NALM-6 and Raji) both in vitro and in in vivo murine models. Tumor burden was quantified through bioluminescence. RNA from target-activated CAR19 and CAR19-IL27 was prepared following Illumina's TruSeq-stranded-mRNA protocol and conducted by LC Sciences. Our findings suggest that CAR19-IL27 enhances NK-92 inherent proliferative ability by about 4-fold compared to CAR19. Furthermore, CAR19-IL27 cells exhibited higher in vitro cytotoxicity against NALM-6 and Raji compared to CAR19, p<0.005 (NALM-6) and p<0.00005 (Raji); higher activation-induced degranulation (CD107a MFI), and IFN-gamma secretion; and decreased expression of inhibitory checkpoint CTLA-4 at both protein (-20-fold) and RNA levels (-1,45 log2FC). Potentially, IL-27 enhances NK cell cytotoxicity through MAPK-related pathways, as genes associated with this pathway were upregulated in CAR19-IL27 cells (e.g., CCR7, ICAM-1, IGF2, NTRK2, and NTRK3). Furthermore, in vivo experiments show that CAR19-IL27 NK cells, in contrast to CAR19 cells, exhibit an extended ability to control tumor growth, reducing tumor burden on day 21 (4 mice/group). Therefore, integrating IL-27 into CAR-NK cells is a promising strategy to boost therapeutic responses against cancer cells, supporting the evidence of fourth-generation CAR engineering in advancing NK cell-based immunotherapies. Financially supported by FAPESP 2020/07055-9, 2014/50947-7, 2013/08135-2; CNPq 440543/2022-3; CAPES 88887.817043/2023-00.