T-cells resist CD5 CAR mediated fratricide by continuously degrading CD5 protein

Abstract

Abstract Developing chimeric antigen receptor (CAR) T cells for patients with T cell malignancies remains challenging as most CARs targeting T-lineage antigens induce T cell fratricide. A notable exception is a CD5-specific CAR, which does not produce extensive fratricide, coinciding with a loss of detectable surface CD5 by an unknown mechanism. In this study, we used live imaging fluorescent microscopy, Western Blot, and flow cytometry to show CD5 protein is rapidly internalized and degraded in transduced T cells following CD5 CAR expression. Experiments with C-and N-tagged CD5 confirmed complete degradation of the entire protein in CD5 CAR T cells. Physical ligation of CD5 antigen by the CAR in cis is sufficient to drive loss of CD5 protein, independently of either CAR or CD5 signaling, resulting in full protection from CD5-directed fratricide. In an ongoing Phase I clinical trial (NCT03081910), CD5 CAR T cells expand and produce robust anti-tumor activity in patients with T cell malignancies. However, patients exhibit incomplete aplasia of non-transduced normal T-cells and a population of circulating T cells remain resistant to CD5-directed elimination and persistalongside CAR T cells. These resistant T cells are surface CD5-negative suggesting that in the presence of CD5 CAR T cells there can be removal of the target antigen as a means of evading killing. Indeed, in vitro experiments showed normal T cells degrade CD5 protein upon coculture with CD5 CAR T cells indicating antigen down modulation occurs on target cells not only in cis but also in trans. These data identify a possible mechanism of resistance of normal T cells to CD5-directed CAR T-cell elimination. Supported by CAGT T32 Training Grant NIH NCI SPORE P2