Engineering Strategies for CAR-T Cells
As briefly discussed above, five generations of CAR-T cells have been classified based on modifications to the intracellular signaling domain(s). Due to its modular design, however, the other elements within the CAR can be optimally designed to engineer safer and more effective CAR-T cells.
Hinge Region and Transmembrane Domain
Engineering of the hinge region and transmembrane domain influences the stability and function of the CAR-T cells. The hinge provides flexibility to the antigen-binding domain to overcome steric hindrances and facilitate access to the target antigen; therefore, an optimal hinge length is required for effective antigen binding and signaling. Previously, amino acid sequences from CD8, CD28, IgG1 or IgG4 have all been used to derive different hinge regions.
Different transmembrane domains, which are typically derived from type 1 proteins such as CD3ζ, CD28, CD4 or CD8α, have been exploited in the engineering of CARs. As the cell membrane anchor, the type of transmembrane domain used influences the stability of the CAR, and its expression on the T cell. For example, CARs with CD28 transmembrane domains are more stable than those with CD3ζ transmembrane domains.
Traditionally, antigen-binding domains of CARs consist of scFvs that target extracellular antigens of cell-surface proteins expressed by cancer cells. This enables the activation of T cells independent of the major histocompatibility complex (MHC). Various efforts have demonstrated the versatility of engineering scFvs or the antigen-binding domain, as discussed below.
CARs mimicking MHC-dependent T cell receptors (TCRs) that recognize intracellular tumour-associated antigens have been engineered by incorporating scFvs derived from TCR-like fragment antigen-binding (Fab) regions of antibodies. CARs have also been engineered with scFvs that bind to soluble ligands found within the tumour microenvironment. Finally, nanobodies containing the VH domains of camelid antibodies, which inherently lack light chains and thus VL domains, have also been used to engineer CARs.
Modifications to the scFv can influence CAR function beyond antigen recognition and binding. The relative positioning of the complementarity determining region in the scFv can affect the specificity and affinity of the CAR. This ultimately determines the effectiveness of the CAR in T cell signaling and activation, and if not controlled can lead to cell death and other toxicities.
Beyond scFvs, alternative molecules have been exploited as antigen-binding domains for CARs. These include cytokines, such as IL-13 zetakine, ligands, such as a proliferation-inducing ligand (APRIL), and peptides, such as designed ankyrin repeat proteins (DARPins). CARs containing these scFv-alternatives have been tested, or are being tested, in preclinical and clinical studies across a range of malignancies.