Fig. 2

CAR structure for CAR T, CAR NK and CAR macrophage. CARs for CAR T, CAR NK and CAR macrophage have similar structures: the extracellular domain including the antigen binding domain and a spacer which is involved in engagement of target cells; transmembrane domain which docks CAR to immune cells and is also involved in other functions of CAR, such as stability and interaction with other membrane proteins; and the intracellular signaling domain which is involved in signaling transduction and activation of immune cells. For the target binding domain, in addition to scFv, native protein/peptide, cytokine and camelid nanobody have also been used. For the intracellular domain, in addition to the function to activate immune cells, other domains to regulate TME have also been used. Three generations of CAR structure are mainly determined by the difference of the intracellular domains. The first-generation CAR contains a single CD3ζ signaling domain. It has limited activities in CAR T cells as T cell activation requires a primary signal from T cell receptor complex with CD3 and a co-stimulatory signal from CD28. However, this generation of CAR has been used in CAR NK and CAR macrophage as a co-stimulatory signal is not required. The difference of the second- and third-generation CAR over the first-generation one is the addition of one and two co-stimulatory signaling domains. In the FDA-approved CAR T cells, these co-stimulatory domains are usually CD28 or 4-1BB. In CAR NK and CAR macrophage, their specific or other ITAM-containing domains are used for the intracellular signaling domain. (This figure was created at BioRender.com.)