CARs usually consist of a monoclonal antibody-derived single-chain variable fragment (scFv) linked by hinge and transmembrane domains to a variable number of intracellular signaling domains. As our understanding of the CAR-T system has evolved so has the overall structure of the CARs themselves. First-generation CARs contain the CD3-zeta activation domain, whereas second-generation CARs also contain a CD28 or CD137/4-1BB costimulatory domain.
Third-generation CARs contain multiple costimulatory domains, secrete high amounts of IL-2, exhibit increased cytolytic activity, upregulate anti-apoptotic genes and demonstrate improved persistance in vivo. Future CARs may include other signaling domains or may finetune the domains of existing CARs, i.e. CD28 domains lacking lck binding regions that appear to reduce the numbers of regulatory T cells within the tumor.