Harnessing an optimized cellular therapy platform for ALK-expressing pediatric and adult solid tumors

Background: Patients with high-risk neuroblastoma and fusion-positive rhabdomyosarcoma face poor survival outcomes and significant treatment-related co-morbidities, necessitating precision targeted therapies. The Anaplastic Lymphoma Kinase (ALK) is a tumor-restricted oncofetal protein highly expressed in these pediatric tumors and select adult malignancies. Methods: We developed 56 ALK-directed chimeric antigen receptor (CAR) T cell architectures constructed from a library of 11 highly active and specific scFvs produced by Retained Display TM screen against ALK antigen encompassing the extracellular domain. CAR architectures included varying scFv orientations, CD8 or CD28 transmembrane domains, and CD28 or 41BB co-stimulatory domains. We systematically screened these CAR constructs with multiple T cell donors against neuroblastoma cell lines and spheroid models with a range of ALK surface expression: NB-1 (high), IMR-32 (moderate), NGP (moderate), and CHP134 (low). Lead candidates were tested with two different T cell donors in vivo against NB-1 (ALK high) and Felix (ALK moderate) xenograft models with 10x10 6 CAR+ cells. Peripheral blood was analyzed weekly for CAR+ cells. Results: Three ALK-directed scFvs significantly increased T cell activation (CD69) and degranulation (CD107a, p<0.01) with elevated IL-2, IFNgamma and granzyme B production (p<0.05) in an ALK-dependent manner after 6 and 24 hours of co-culture. The lead constructs exhibited potent cytotoxicity with tumor clearance in both 2D and spheroid co-cultures confirmed by live imaging of GFP-labeled cell lines and Caspase-3/7 red dye assay across effector-to-target cell ratios of 4:1 to 1:4. Antigen specificity was validated by the absence of activation, cytokine production or cytotoxicity against ALK knockout NGP cells. In xenograft models, these CAR T cell constructs achieved complete and durable tumor regression in NB-1 and transient-to-durable responses in Felix tumors. Peak circulating CAR+ T cells occurred at day 14 in NB-1 and day 7 in Felix models with enrichment of central-memory phenotypes (CD62L+ and IL7Ralpha+, low CD45RA and LAG3). Constructs incorporating CD28 transmembrane and costimulatory domains conferred enhanced activation, cytokine release and efficacy at moderate and low ALK density. Conclusions: These findings validate ALK as a selective cellular immunotherapy target and highlight novel CAR architectures integrating CD28 transmembrane and costimulatory domains to achieve high antigen sensitivity and sustained functional persistence. These optimized CAR designs enable variable ALK recognition and may improve clearance of minimal residual disease. Next-generation constructs incorporating FOXO1 overexpression and NFAT-inducible IL-15 expression via bicistronic vectors will further address resistance mechanisms within the immunosuppressive solid tumor microenvironment.