Shaping Functional Avidity of CAR T Cells: Affinity, Avidity, and Antigen Density That Regulate Response

Abstract

Chimeric antigen receptors (CARs) are immunoreceptors which redirect T cells to selectively kill tumor cells. Given their clinical successes in hematological malignancies there is a strong aspiration to advance this immunotherapy for solid cancers, hence molecular CAR design and careful target choice are crucial for their function. To evaluate the functional significance of the biophysical properties of CAR binding, i.e. affinity, avidity and antigen density, we generated an experimental system in which these properties are controllable. We constructed and characterized a series of CARs, which target the melanoma tumor-associated antigen Tyr/HLA-A2, and in which the affinity of the single-chain Fv binding domains ranged in KD from 4 to 400 nM. These CARs were transduced and each CAR-T cell population was sorted by the level of receptor expression. Finally, the various CAR-T cells were encountered with target cells that present different levels of the target antigen. We detected nonmonotonic behaviors of affinity and antigen density, and an interrelation between avidity and antigen density. Anti-tumor activity measurements in vitro and in vivo corroborated these observations. Our study contributes to understanding of CAR-T cell function and regulation, having the potential to improve therapies by the rational design of CAR-T cells.