PD-1 Bispecific Killer Engager (PD-1 BiKE) effectively depletes effector T lymphocytes in experimental autoimmune encephalomyelitis

Lauren C NaatzShuyun DongYujia ZhaiBrian EvavoldMingnan Chen^*

• The University of Utah, Salt Lake City, United States

Bispecific killer engagers (BiKEs), which harness natural killer cells to deplete target cells, have garnered success in ablating tumor cells but have not been well explored in eliminating primary cells, such as effector cells. Previously, we reported a BiKE that targeted human lymphocytes expressing programmed death-1 (PD-1). The BiKE was shown to promote NK cellmediated depletion of PD-1 + cells in vitro. Here, we posited that a mouse-specific PD-1 BiKE could be used as a tool to deplete PD-1 + cells in vivo. PD-1 BiKE was first designed in an IgG-like format and demonstrated selective binding to PD-1 + T cells encompassing both a cell line (EL4) and primary cells. PD-1 BiKE simultaneously engaged its two targets, PD-1 + and NK cells, and mediated a 63% increase in cell-cell interactions between the two targets. In co-cultures of primary PD-1 + T cells and NK cells, the BiKE reduced the number of T cells by 28%. Importantly, PD-1 BiKE did not reduce PD-1 -T cells when co-cultured with NK cells. In vivo, PD-1 BiKE reduced the fraction of inoculated EL4 cells by ~53%. In EAE mice, PD-1 BiKE reduced the average number of primary PD-1 + T cells by 56% and 65% in the spinal cords and brains, respectively. Beyond the IgG-like BiKE, two non-IgG-like BiKEs were also designed and generated and demonstrated strong but distinct binding to PD-1 and CD16. Together, the work showcases the effectiveness of BiKE in depleting non-malignant cells.