Canine MGA271 CAR T cells are efficient at killing canine osteosarcoma spheroids. A, Four CAR constructs were used in this study. Each construct consists of a scFv, a hinge, a transmembrane domain (tm), a canine costimulatory signaling domain, a canine CD3 signaling domain, T2A, and a truncated human EGFR sequence. B, Flow plots of day 14 transduction efficiencies of different constructs by EGFR staining. C, Longitudinal images of GFP-transduced canine osteosarcoma Moresco spheroids (green) cocultured with sorted canine CAR T cells (light gray) at E:T ratio of 2:1. Untransduced and TCR-transduced canine cells were used as negative controls. All conditions were measured in triplicates. D, Summary of relative GFP intensity of Moresco spheroids cocultured with different T cells. Graph shows mean ± SD. Samples were measured in triplicate. Statistical analysis was performed using the multiple group comparison Holm-Šídák test; ***, P < 0.001; **, P < 0.01; ns, not significant.