CD2 costimulation bridges potent CAR-induced cytolysis and durable persistence

Background Current second-generation CAR T cell products rely on CD28 or 4-1BB costimulatory domains, additions that respectively favor rapid cytolysis or long-term persistence, but rarely both. Preclinical modeling and retrospective analysis have linked CD2–CD58 engagement to superior preclinical and clinical responses, yet the direct contribution of CD2 intracellular signaling remains undefined. Methods We replaced the costimulatory domain of anti-mesothelin (SS1) and anti-TnMUC1 (5E5) CARs with the human CD2 cytoplasmic tail (CD2z) and benchmarked them against 28z and BBz formats. Transient mRNA expression was used to profile proximal Ca 2+ flux and degranulation free of tonic viral signals; durable functional assays employed lentiviral CARs. Cytokine release, genome-wide transcriptional programs, and anti-tumor activity were assessed in vitro and in NSG xenograft models. Results CD2z CAR T cells degranulated as efficiently as other z-containing CARs and generated a Ca 2+ flux signal intermediate to 28z and BBz CARs. Lentiviral CD2z CARs released a Th1-skewed cytokine panel and matched 28z cytolysis despite a lower acute cytokine release. Transcriptomic analysis characterized CD2z cells in an early effector-memory state: glycolytic, mTORC1, and TNFa–NF-κB hallmarks were upregulated, whereas exhaustion-up signatures were selectively depleted vs 28z. In vivo, a single CD2z infusion induced deep and durable tumor regressions over the 60-day observation period in subcutaneous mesothelin-positive mesothelioma and orthotopic TnMUC1-positive pancreatic tumor models, achieving tumor control comparable to 28z and more rapid early tumor clearance than BBz, while supporting peripheral T cell persistence similar to BBz. Conclusions The CD2 cytoplasmic tail, in combination with CD3z, delivers balanced costimulation that couples brisk tumor debulking to T cell persistence. CD2z therefore may provide a simple, versatile alternative to canonical CD28 and 4-1BB modules for next-generation CAR T therapies.