The extracellular antigen-binding domain is connected to the transmembrane domain through a hinge, also known as a spacer (Fig. 1). Most CAR designs use immunoglobulin (Ig)-like hinges, either derived from IgG or from native T-cell molecules. IgG-derived hinges can be targeted by myeloid cells or other lymphoid cells with Fc gamma receptors (FcγR) against the CH2 IgG domain. This interaction can lead to AICD of the CAR-T cell and decreased engraftment in animal models. However, mutating or removing the CH2 prevents interaction with FcγR and restores CAR-T cell function in vivo. On the other hand, T cell derived hinges are commonly composed of domains from CD28 or CD8.
Fig1. CAR domain components. Chimeric antigen receptor (CAR) proteins consist of an extra cellular antigen-binding domain, a hinge, a transmembrane domain, a co-stimulatory domain, and an activation domain. The extracellular portion of the CAR is most often composed of a single- chain variable fragment (scFv) molecule, though camelid nanobodies and humanized natural ligands or cytokines have been used. The intracellular regions, which function in activation upon antigen binding, typically harbor a T cell activation domain derived from the CD3ζ chain of the T cell receptor. Co-stimulatory domains often include CD28 or 4-1BB and can influence CAR-T cell memory, phenotype, and metabolism. Other co-stimulatory domains have been tested including OX40, CD27, and inducible T cell co-stimulator (ICOS). The activation domain, composed of CD3ζ, DAP12, or other sequences, contains immunoreceptor tyrosine-based activation motif (ITAM) regions that can be mutated to attenuate downstream activity
Hinge length and flexibility play important roles in CAR activation, since a specific distance between the interacting cells is required for an immune synapse to form. The length of the hinge influences flexibility. For example, longer hinges provide more flexibility to target epitopes of the target antigen that are closer to the cell membrane or with complex glycosylation. Alternatively, short hinges are best for tumor antigen epitopes near the amino terminal of the target protein, which are easily accessible.
The transmembrane portion anchors the CAR to the cell membrane through a hydrophobic α helix, usually derived from CD3ζ, CD28, CD4, or CD8α. Though it is the least studied of all the CAR components, the transmembrane domain has a significant role in the stability and function of the CAR-T cell. CD28 pro vides more stability, whereas a CD3ζ transmembrane domain can associate the CAR with other molecules of the native TCR/CD3 complex.
