Mentor Name: Yael Mossé

Heritable genetic mutations in the receptor tyrosine kinase domain of ALK are the major cause of familial neuroblastoma and are the most frequently somatically acquired mutations in sporadic forms of the disease. Small molecule inhibition of mutant ALK has demonstrated clinical activity, but resistance inevitably arises and this modality is only suitable for 25% of patients. The Mossé lab has shown that native ALK is expressed on the cell surface of the majority of neuroblastoma tumors, and that expression of this lineage-restricted antigen is not on normal post-fetal tissues. CAR T cells have shown efficacy in hematologic malignancies and early evidence suggests early signs of activity in solid tumors but has been limited by identifying a tumor-restricted target antigen. As ALK has been shown to be a tumor restricted antigen without expression on healthy tissue, we hypothesize that CAR T cell therapy directed against the ALK oncofetal protein will provide a novel treatment modality for patients with neuroblastoma. To test this hypothesis, we will develop and validate a third-generation CAR T cell construct that includes CD28 and 41BB costimulatory domains that may enhance CAR T cell persistence and activity at lower target antigen density, while reducing T cell exhaustion and increasing persistence. This investigation will be completed by plasmid design, lentivirus production, transduction of human donor T cells, followed by in vitro cytotoxicity assays with T cell activation and cytokine production analysis. Plasmid design encoding the CAR construct will be completed using SnapGene software, followed by lentivirus production via transfection of LentiX 293T cells. Healthy human donor T cells will be transduced with the lentiviral particles using lipofectamine to express the ALK-specific CAR. The CAR T-cell functionalities will be assessed through in vitro co-culture assays using GFP-labeled neuroblastoma cell lines with variable surface ALK expression to measure cytotoxicity and T-cell activation through flow cytometry and cytokine production through immunosorbent assays. Additionally, flow cytometry and RNA sequencing methods will be used to characterize T-cell phenotypes, and Seahorse assays which will further define their metabolic fitness, in comparison to other generations of T Cell therapy. The project aims to address therapeutic barriers of CAR T cells in solid tumors, such as low antigen density and the immunosuppressive tumor microenvironment. Ultimately, this project will advance the development of an ALK-directed cellular therapy to enhance treatment efficacy and reduce toxicity for patients with neuroblastoma.