CD28 and cancer: Unlike TGN1412, however, VHH38 exhibits a lateral paratope orientation that likely spatially hinder the cross-linking of CD28 molecules on the same T cell, probiding a structural basis for its co-stimulatory activity rather than superagonistic activity.<h4>Discussion</h4>In summary, through an integrated process of affinity screening, functional agonism ranking, and therapeutic efficacy evaluation, we have identified a panel of co-stimulatory anti-CD28 VHHs with strong potential as functional modules in Tri-TCEs for cancer immunotherapy.