A novel CD3ε fusion receptor allows use of T-cell engagers on TCR-less allogeneic CAR T cells to improve activity and prevent antigen escape

Abstract

Chimeric antigen receptor (CAR) T-cell therapies have shown clinical success in treating hematologic malignancies. However, heterogenous target antigen expression can impair the durability of response. Combining CAR and T-cell engagers (TCEs) targeting additional tumor antigens can address tumor heterogeneity and antigen escape. In allogeneic settings, eliminating the T-cell receptor (TCR) of the adoptive T-cell therapy prevents graft versus host disease. However, the absence of TCR leads to loss of surface CD3 expression, preventing cooperative activity with CD3-directed TCEs. We utilized induced pluripotent stem cells (iPSCs) to support the required multiplexed editing, establish a renewable starting material for off-the-shelf manufacture, and create the desired TCR-less CAR⁺ CD3⁺ T cells. Here we illustrate surface expression of a CD3ε fusion receptor (CD3FR) in iPSC-derived CAR T (CAR iT) cells, enabling TCE-mediated targeting of diverse antigens. In vitro and in vivo, CD3FR⁺ CAR iT cells demonstrated potent cytotoxic response and cooperative activity against mixed tumor lines and multiple antigens. CD3FR⁺ iT cells were further engineered to secrete TCEs, eliminating the need for extra supplementation with TCEs. Collectively, the data highlights the ability to integrate TCEs with allogeneic CAR iT cells for multi-antigen targeting, overcoming tumor relapse, and supporting off-the-shelf therapy for patient access.