Combining ROR1- Specific Chimeric Antigen Receptor (CAR) NK Cells with IL-15 Superagonist (N-803/ALT-803) to Target Chemotherapy Resistant Neuroblastoma (2019)

BackgroundNeuroblastoma is the most common malignant extracranial solid tumor in children (Park/Eggert Pediatr Clin North Am., 2011). Despite significant survival improvements in multiple pediatric malignancies, children with relapsed neuroblastoma (NB) (London/Matthay, Journal of Clinical Oncology, 2011) continue to have dismal outcomes. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is highly expressed in the majority of neuroblastoma (Rebagay/Yan Front Oncol, 2012). ALT-803 is a novel IL-15 superagonist complex where mutant IL-15N72D is bound to an IL-15RαSu-Fc fusion protein (Xu/Wong, Cancer Res, 2013). ALT-803 (N-803) has been shown to have superior biological activity, higher potency, and a longer half-life compared to IL-15 (Han/Wong, Cytokine, 2011), and is currently being tested in several clinical trials.ObjectivesWe hypothesized that ROR1-CAR NK cells by mRNA electroporation will be highly effective against neuroblastoma alone, and in combination with ALT-803.MethodsPBMCs were isolated and expanded ex vivo with lethally irradiated K562-mbIL21cells (Denman/Lee Plos One, 2012). ExPBNK cells were isolated using Miltenyi NK cell isolation kits as previously described (Chu/Cairo, Can Imm Res, 2015). ExPBNK cells (aNK) were electroporated with anti-ROR1-CAR mRNA with a 4-1BB signaling domain which is synthesized in vitro (ROR1-CAR was generously provided by Riddell S, MD, Fred Hutch, WA). N-803 was generously provided by Altor BioScience. CHLA-136, CHLA-255 cells were generously provided by Seeger R.C. MD, UCLA). Functional cytotoxic activity of anti-ROR1-CAR NK cells against neuroblastoma cell lines were examined via LDH-based and luciferase-based assays at different E:T ratios. IFN-g levels were evaluated by ELISA assays.ResultsFlow cytometry demonstrated that ROR1 is highly expressed in different neuroblastoma cell lines (CHLA-136, CHLA-255, SKN-BE-(2)). Anti-ROR1-CAR was expressed on up to 90% of anti-ROR1 mRNA-transfected NK cells at 24 hrs. Anti-ROR1-CAR expressing NK cells had significantly higher in vitro cytotoxicity compared to mock NK cells at 6 hours (p< 0.05) against ROR1 positive neuroblastoma cells at different E:T ratios. Combination of N-803 with anti-ROR1-CAR NK cells significantly improved in-vitro cytotoxicity compared to anti-ROR1-CAR NK alone (p<0.001). Release of IFN-g was significantly higher in anti-ROR1-CAR NK group compared to mock NK against ROR1 positive CHLA-136 neuroblastoma cells (p<0.005). Combination of N-803 with anti-ROR1-CAR NK further increased the IFN-g release from CHLA-136 neuroblastoma cells (p<0.005).ConclusionThese data demonstrated that N-803 significantly increases in vitro cytotoxicity of anti-ROR1-CAR NK cells against ROR1 neuroblastoma cells. In vivo studies using human NB xenograft mouse model are currently in progress. Neuroblastoma is the most common malignant extracranial solid tumor in children (Park/Eggert Pediatr Clin North Am., 2011). Despite significant survival improvements in multiple pediatric malignancies, children with relapsed neuroblastoma (NB) (London/Matthay, Journal of Clinical Oncology, 2011) continue to have dismal outcomes. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is highly expressed in the majority of neuroblastoma (Rebagay/Yan Front Oncol, 2012). ALT-803 is a novel IL-15 superagonist complex where mutant IL-15N72D is bound to an IL-15RαSu-Fc fusion protein (Xu/Wong, Cancer Res, 2013). ALT-803 (N-803) has been shown to have superior biological activity, higher potency, and a longer half-life compared to IL-15 (Han/Wong, Cytokine, 2011), and is currently being tested in several clinical trials. We hypothesized that ROR1-CAR NK cells by mRNA electroporation will be highly effective against neuroblastoma alone, and in combination with ALT-803. PBMCs were isolated and expanded ex vivo with lethally irradiated K562-mbIL21cells (Denman/Lee Plos One, 2012). ExPBNK cells were isolated using Miltenyi NK cell isolation kits as previously described (Chu/Cairo, Can Imm Res, 2015). ExPBNK cells (aNK) were electroporated with anti-ROR1-CAR mRNA with a 4-1BB signaling domain which is synthesized in vitro (ROR1-CAR was generously provided by Riddell S, MD, Fred Hutch, WA). N-803 was generously provided by Altor BioScience. CHLA-136, CHLA-255 cells were generously provided by Seeger R.C. MD, UCLA). Functional cytotoxic activity of anti-ROR1-CAR NK cells against neuroblastoma cell lines were examined via LDH-based and luciferase-based assays at different E:T ratios. IFN-g levels were evaluated by ELISA assays. Flow cytometry demonstrated that ROR1 is highly expressed in different neuroblastoma cell lines (CHLA-136, CHLA-255, SKN-BE-(2)). Anti-ROR1-CAR was expressed on up to 90% of anti-ROR1 mRNA-transfected NK cells at 24 hrs. Anti-ROR1-CAR expressing NK cells had significantly higher in vitro cytotoxicity compared to mock NK cells at 6 hours (p< 0.05) against ROR1 positive neuroblastoma cells at different E:T ratios. Combination of N-803 with anti-ROR1-CAR NK cells significantly improved in-vitro cytotoxicity compared to anti-ROR1-CAR NK alone (p<0.001). Release of IFN-g was significantly higher in anti-ROR1-CAR NK group compared to mock NK against ROR1 positive CHLA-136 neuroblastoma cells (p<0.005). Combination of N-803 with anti-ROR1-CAR NK further increased the IFN-g release from CHLA-136 neuroblastoma cells (p<0.005). These data demonstrated that N-803 significantly increases in vitro cytotoxicity of anti-ROR1-CAR NK cells against ROR1 neuroblastoma cells. In vivo studies using human NB xenograft mouse model are currently in progress.