Circumventing Resistance within the Ewing Sarcoma Microenvironment by Anti-MCAM Chimeric Antigen Receptor Modified Ex-vivo Expanded Natural Killer Cells Combinatorial Therapy

Pediatric patients with recurrent metastatic Ewing sarcoma (ES) have a dismal 5-year survival of < 30% largely secondary to treatment resistance in the tumor microenvironment (TME). Novel therapeutic approaches are desperately needed and represent an unmet need. Here, we developed a chimeric antigen receptor (CAR) expressing natural killer (NK) cell targeting melanoma cell adhesion molecule (MCAM) by electroporation of CAR mRNA into ex-vivo expanded NK cells. Expression of anti-MCAM CAR significantly and specifically enhanced NK cytotoxic activity compared to mock NK cells against MCAMhigh ES cells in vitro, and significantly reduced lung metastasis and extended animal survival in an orthotopic xenograft mouse model of ES. However, anti-MCAM CAR NK cells had minimal effects on decreasing primary tumor growth in vivo. We performed transcriptome profiling in patient-derived xenografts to identify the underlying mechanisms of NK cell resistance in ES TME. Luciferase expressing patient-derived ES A673 cells were orthotopically injected into NSG mice. When the tumors reach the size of 0.5 cm, PBS or ex vivo expanded NK cells were injected via tail vein. Tumors were harvested 24 hr after NK injection and dissected into peripheral and central tumor sections. Total RNA was extracted separately from the NK treated (T) or untreated (U) peripheral (P) or central (C) tumor sections and subjected to bulk RNA-Seq on NovaSeq 6000.