Fig. 2.

Schematic representation of the four generations of CARs. The first-generation CAR contains one intracellular signaling domain, most commonly the signaling domain of the CD3 TCRζ chain. A second intracellular co-stimulatory domain, typically consisting of either a CD28 or 4-1BB domain, was added in the second-generation CARs, enhancing CAR-T cell activation and proliferation. Third- generation CARs utilize dual co-stimulatory intracellular signaling, classically consisting of either a CD28 or a 4-1BB domain followed by either a CD28, a 4-1BB or an OX40 domain. Fourth-generation CARs co-express, on top of a second-generation CAR construct, an antitumor cytokine gene (i.e., IL-12) or costimulatory ligands (i.e., 4-1BBL). CAR chimeric antigen receptor, ScFV single-chain variable fragment, TCR T cell receptor, IL-12 interleukin-12, 4-1BBL 4-1BB ligand