Therapeutic targeting and biophysical characterization of CAR-mediated trogocytosis

About This Grant

PROJECT SUMMARY Chimeric Antigen Receptor (CAR) T cell therapy is an effective treatment for patients with blood cancers, that involves the genetic modification of patient T cells to express a CAR targeting an antigen expressed on the surface of cancer cells. CAR T cells are effective at treating various cancers but resistance to the therapy remains common and most patients who initially respond eventually relapse. A recently identified mechanism of treatment resistance is CAR-mediated trogocytosis (CMT). CMT is a process in which the targeted surface antigens are transferred from tumor cells to CAR T cells. This antigen transfer leads to rapid loss of the target antigen on the cancer cells, rendering them resistant to CAR T cell therapy. CMT also leads to killing of antigen-positive CAR T cells by other CAR T cells, resulting in reduced CAR T cell expansion/persistence and increased exhaustion. All of these consequences of CMT are associated with reduced clinical CAR T cell efficacy. Currently, the molecular basis of CMT remains unknown and there are no clinically approved, targeted approaches for the selective inhibition of CMT to enhance the therapeutic potential of CAR T cells. We have demonstrated that reducing the affinity of CARs for their target antigen significantly reduces CMT without altering anti-tumor activity. However, it remains unknown how, on the molecular and subcellular level, CAR affinity alters CMT. In addition, while reducing CAR affinity can be an effective strategy to reduce CMT, in some cases this approach may not be feasible, for example when antigens are expressed at low density on tumor cells. In search for alternative approaches, we found that inhibition of the cysteine protease cathepsin B (CTSB) expressed by T cells leads to substantially reduced CMT without altering short-term anti-tumor activity of CAR T cells. It remains unknown how CTSB facilitates CMT and whether CTSB inhibition could be an effective approach to increase the long-term anti-tumor activity of CAR T cells. In addition, it has previously been shown that altering cholesterol metabolism through overexpression of cholesterol 25-hydroxylase (CH25H) can reduce CMT and may be a promising approach for the therapeutic targeting of CMT. While all three approaches have shown to be able to alter CMT, the molecular mechanism of CMT remains unknown. In this project, we will carry out a quantitative characterization of CMT kinetics and its molecular drivers using state-of-the-art high-resolution live-cell imaging assays (Aim 1). In addition, this project will determine the effect of different approaches to target CMT in CAR T cells on their long-term in vitro and in vivo anti-tumor activity (Aim 2). If successful, this project will provide a mechanistic understanding of CMT and improve the efficacy of CAR T cell-based treatments.