Angelats, L.; Marzal, B.; Rodriguez-Garcia, A.; Espanol-Rego, M.; Lobo-Jarne, T.; Hernandez-Sanchez, M.; Cascallo, G.; Colell, S.; Gimenez-Alejandre, M.; Colell, G.; Castellsague, J.; Andreu-Saumell, I.; Calderon, H.; Galvan, P.; Urbano-Ispizua, A.; Delgado, J.; Gonzalez-Navarro, E. A.; Prat, A.; Juan, M.; Guedan, S.

The development of clinically effective CAR-T cell therapies for solid tumors requires careful optimization of receptor design, functional fitness, and manufacturability. While advancing low-affinity HER2-targeting CAR-T cells toward clinical application, we found that the candidate with the strongest in vivo antitumor activity--comprising a CD8 hinge and transmembrane region and a 4-1BB co-stimulatory domain--exhibited measurable tonic signaling. This basal antigen-independent signaling, likely driven by high CAR surface expression, was associated with increased apoptosis and reduced ex vivo expansion under research-grade manufacturing conditions. Modification of the transmembrane domain reduced CAR surface expression but did not alleviate tonic signaling and instead impaired antitumor activity. By contrast, transient pharmacologic inhibition of CAR signaling with dasatinib rescued expansion and reduced apoptosis in small-scale research cultures. Notably, these tonic-signaling-associated defects were largely absent during large-scale, GMP-compliant manufacturing, which enabled robust CAR-T cell expansion without additional benefit from dasatinib supplementation. Together, these findings show that tonic signaling is not inherently detrimental to CAR-T cell performance and that its functional consequences are highly dependent on manufacturing context. Our study underscores the importance of evaluating CAR candidates within clinically relevant production platforms and supports the advancement of this 4-1BB-based HER2-specific CAR-T cell product toward clinical testing.