IL15 (N-803) and IL21 (oHSV-21) Significantly Enhance ROR1 CAR NK Cells Against Pediatric Neuroblastoma

Background Metastatic and relapsed/refractory neuroblastoma (NB) has a very poor diagnosis. Novel therapies are desperately needed. ROR1 is overexpressed in a variety of human cancers including NB. Our group has successfully expanded peripheral blood NK cells (exPBNKs) and modified exPBNK cells to express chimeric antigen receptor (CAR) (Chu/Cairo, et al, Cancer Immuno Res. 2015). N-803 is a superagonist of an IL15 variant bound to an IL-15RαSu-Fc fusion with enhanced biological activity (Liu/Wong, et al, Cytokine, 2018). The N-803 combined with dinutuximab and exPBNK cells significantly extended the survival of NB xenografts (Chu/Cairo, et al, JITC. 2021). C021 is a selective replication competent oncolytic herpes simplex virus that can secrete human IL21 (oHSV-21) after infection of NB cells. Objective To determine if novel cytokine-based therapy (N-803 or C021) can overcome NB tumor resistance and relapse of anti-ROR1 CAR exPBNK (CAR NK) cells in vitro and improve the survival of human NB xenografted NSG mice. Methods ExPBNK cells were expanded with lethally irradiated K562-mbIL21cells (Chu/Cairo, et al, JITC, 2021). ExPBNK cells were electroporated with anti-ROR1-CAR mRNA using Maxcyte electroporator. N-803 was kindly provided by ImmunityBio, Inc. C021 was generated by modifying C134 to express human IL21 gene. In vitro cytotoxicity of CAR NK against NB cell lines were examined at different E:T ratios. IFN-γ, granzyme and perforin levels were evaluated by ELISA assays. In vivo survival and anti-tumor effect of 6 doses of CAR NK cells with or without the N-803 or C021 was examined utilizing human NB xenografted NSG mice (Chu/Cairo, et al, JITC. 2021). Results CAR NK cells had significantly enhanced in vitro cytotoxicity compared to Mock CAR NK against ROR1+ SKNBE(2)N, CHLA-255, and SKNFI NB cells at different E:T ratios (p<0.001) regardless of MYCN amplification. N-803 significantly enhanced ROR1 CAR NK in vitro cytotoxicity with significantly enhanced perforin and granzyme release compared to control groups (p<0.05). In vivo studies showed that the combination of N-803 and CAR NK significantly enhanced the survival of NB xenografts compared to the ROR1 CAR NK alone group (p=0.05) (Fig.1).C021 significantly enhanced ROR1 CAR NK in vitro cytotoxicity against NB with significantly increased IFN-g, granzyme B and perforin secretion compared to control groups (p<0.05). Our in vivo animal study showed that C021 infected NB cells in NB xenografted NSG mice secreted human IL21 at day 1 and day 3 post infection. The combination of C021 and ROR1 CAR NK significantly enhanced the survival of NB xenografts compared to the ROR1 CAR NK alone group (p<0.01) (Fig.2). Conclusion Our data demonstrated IL15 or IL21 based novel cytokine therapy (N-803 or C021) significantly enhanced the anti-tumor efficacy of ROR1 CAR NK targeting NB in vitro and in vivo (Funded by U54 CA232561).