Systematic single amino acid affinity tuning of CD229 CAR T cells retains efficacy against multiple myeloma and eliminates on-target off-tumor toxicity.

T cells expressing chimeric antigen receptors (CARs) have shown remarkable therapeutic activity against different types of cancer. However, the wider use of CAR T cells has been hindered by the potential for life-threatening toxicities due to on-target off-tumor killing of cells expressing low amounts of the target antigen. CD229, a signaling lymphocyte-activation molecule (SLAM) family member, has previously been identified as a target for CAR T cell-mediated treatment of multiple myeloma (MM) due to its high expression on the surfaces of MM cells. CD229 CAR T cells have shown effective clearance of MM cells in vitro and in vivo. However, healthy lymphocytes also express CD229, albeit at lower amounts than MM cells, causing their unintended targeting by CD229 CAR T cells. To increase the selectivity of CD229 CAR T cells for MM cells, we used a single amino acid substitution approach of the CAR binding domain to reduce CAR affinity. To identify CARs with increased selectivity, we screened variant binding domains using solid-phase binding assays and biolayer interferometry and determined the cytotoxic activity of variant CAR T cells against MM cells and healthy lymphocytes. We identified a CD229 CAR binding domain with micromolar affinity that, when combined with overexpression of c-Jun, confers antitumor activity comparable to parental CD229 CAR T cells but lacks the parental cells' cytotoxic activity toward healthy lymphocytes in vitro and in vivo. The results represent a promising strategy to improve the efficacy and safety of CAR T cell therapy that requires clinical validation.