The extracellular segment of the CAR is classically composed of an scFv that binds to the target antigen (Fig. 1). The scFv contains variable light (VL) and variable heavy (VH) regions of an antibody specific to the target antigen connected by a flexible linker. The affinity of the scFv for its target antigen influences the strength of the activation signal relayed to the T cell, with higher affinity leading to greater activation. When the binding affinity is low, CAR-T cells can discern between cells with overexpression or normal expression of the target antigen. This is advantageous if the target is found on both normal and tumor cells. On the other hand, an scFv with high binding activity can lead to overactivation of the T cell, resulting in activation-induced cell death (AICD) or toxicity. The organization of the VL and VH within the CAR construct can also affect the binding activity of the scFv, with the VL-linker-VH orientation (versus VH-linker-VL) typically having greater binding activity.
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
The linker is traditionally composed of repeated glycine and serine residues, allowing for flexibility and solubility respectively. The length of the linker affects the binding activity of the scFv, since it determines the distance between the VL and VH. Depending on the composition, some scFvs have the tendency to dimerize or multimerize without interacting with their target antigen, leading to activation- independent or “tonic” signaling of the CAR. This can result in CAR-T cell exhaustion or activation-induced cell death (AICD). The length of the linker can also affect this clustering, as longer linkers tend to decrease dimerization. Linkers with different compositions, such as the Whitlow linker, have also been used to enhance the stability and affinity of the scFv.
The scFv is usually derived from monoclonal antibodies of mouse origin, and thus can be recognized as foreign by the immune system. In some cases, patients have developed immunity to the mouse portions of the CAR, thereby limiting the persistence of the CAR-T cells. To address this issue, several CARs were developed with a fully humanized scFv sequence that does not trigger rejection. However, some instances of anti-CAR immunity have been reported even with a human scFv. Camelid antibodies, composed of a single domain heavy chain (VHH, also known as nanobodies), have low immunogenic potential due to high sequence homology with human VH fragments and may circumvent immune rejection. CARs using VHH demonstrate high binding affinity to various tumor antigens and efficiently trigger T cell activation when used in a third generation CAR construct with CD28 and OX40 costimulatory domains or a second generation CAR with CD28.
Rather than using antibody fragments, the antigen binding domain can be engineered from natural ligands to receptors found on cancer cells. Natural ligands are attractive because they are also fully human and less likely to elicit rejection. Previously studied candidates include tumor receptor ligands, such as APRIL, which binds to BCMA and TACI on myeloma cells or T1E, which binds to ErbB receptors on various tumors. These CARs are referred to as “promiscuous” because they can bind multiple antigens with a single receptor. Immobilized cytokines (called zetakines) may also serve as the binding domain in the CAR con struct and have been shown to bind natural receptors expressed on tumor cells.
