CARs are designed with one or multiple costimulatory domains intracellular to the transmembrane domain (Fig. 1). Costimulatory domains were added to second- generation CARs, since signaling from the CD3z domain alone was insufficient to activate resting T cells. As discussed previously, the primary costimulatory domains used in CARs are 4-1BB and CD28. Both are used in FDA-approved CAR-T cells and we now recognize that the activity and function of these products vary depending on which costimulatory domain is used. For example, CAR-T cells with a 4-1BB costimulatory domain tend to expand more slowly and persist longer compared to those with a CD28 costimulatory domain, which expand rapidly but are more prone to T cell exhaustion. In patients, CD28 CAR-T cells are usually undetectable within 3 months, while 4-1BB CAR-T cells are detectable for several years. This discrepancy is thought to be due to the constitutive association of the tyrosine kinase LCK with the CD28 costimulatory domain, resulting in a high magnitude of protein phosphorylation upon activation. In contrast, 4-1BB CAR-T cells show a lesser degree of phosphorylation, which may result in decreased sensitivity to triggering in the setting of low antigen density. The costimulatory domains also influence the CAR-T cell memory phenotype and metabolism. CAR-T cells bearing a 4-1BB domain have a more central memory phenotype and exhibit fatty acid metabolism while those with a CD28 domain have an effector like memory phenotype and primarily undergo glycolytic metabolism.
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
Most studies directly comparing costimulatory domains have been conducted in mice, though two small studies investigated products with different domains in the same patient. In the first of these clinical studies, patients with B cell malignancies were simultaneously injected with first and second generation CD19-targeted CAR-T cells. Serum FACS analyses revealed improved expansion and enhanced persistence among second-generation CAR-T cells, suggesting more favorable pharmacokinetics. The same group similarly compared second and third-generation CARs (with both CD28 and 4-1BB) in patients and showed further improved expansion and persistence with the addition of the 4-1BB domain.
Alternative costimulatory domains have been tested in CAR-T cells in preclinical models but have not yet been tested in clinical trials. These include OX40, CD27, ICOS, MYD88 and CD40, and killer cell immunoglobulin- like receptor SDS2 (KIR2DS2) combined with an immunotyrosine-based activation motif-containing adapter (DAP12). CD27 has been shown to enhance CAR-T cell survival compared to CD28. ICOS drives a Th1/ Th17 phenotype in CD4+ T cells and increases in vivo T cell persistence compared to CD28 or 4-1BB. When ICOS is used in combination with 4-1BB in a third generation CAR, T cells exhibit greater antitumor effects and increased persistence in vivo compared to second generation CAR-T cells.
