Figure 3. Generations of CAR-T-cell construct designs. First generation CARs contained only the CD3ζ domain, the initiator of T-cell receptor intracellular signalling. However, these CARs demonstrated limited expansion and in vivo persistence due to lack of a costimulatory signal. Second generation CARs were engineered to contain CD3ζ and a co-stimulation signal such as CD28 or 4-1BB, thus conferring enhanced cytotoxicity, expansion, and persistence. Third generation CARs added another costimulatory domain with the first representing CD28 or 4-1BB and the second representing CD28, 4-1BB, or OXO40. These offer superior T-cell expansion and longer persistence through increased cytokine secretion, proliferation speed and survival rate of engrafted T cells. Fourth generation CARs, also called TRUCKs (T-cells redirected for universal cytokine-mediated killing), possess a cytokine induced domain which activates downstream transcription factor NFAT to induce cytokine production after antigen recognition, thus modulating immune effects. Fifth generation CARs, based on the second generation, require gene editing to inactivate the T-cell receptor alpha constant (TRAC) gene, leading to the removal of the TCR alpha and beta chains and the creation of a truncated cytoplasmic IL-2 receptor β-chain domain with a binding site for STAT3 transcription factor. Antigen activation triggers three synergistic signals through TCR CD3ζ, co-stimulatory CD28, and cytokine JAK–STAT3/5 signalling, which drive T-cell activation and proliferation (58). Adapted from (31).