The four generations of CAR-T cells. First-generation CARs were composed of antibody single-chain variable fragments (scFvs) fused through a TM domain to the cytoplasmic tail of the TCR signaling component CD3ζ. Second-generation CARs had improved CAR-T cell proliferation and cytotoxicity, thanks to the addition of co-stimulatory signaling domains (such as CD27, CD28, CD134, and CD137). Third-generation CAR-T cells were generated by adding a second co-stimulatory signaling domain (such as OX-40, also known as CD134, or 4-1BB, also known as CD137) to second-generation constructs. Fourth-generation CAR-T cells, also known as “T cells redirected for universal cytokine-mediated killing” (TRUCKs) were then engineered to secrete transgenic cytokines (like interleukin-12) within the targeted cancer and thus attract more immune cells (such as natural killer (NK) cells and macrophages). (Adapted from Smith et al., 2016).