Abstract
T cells and natural killer (NK) cells are major effector cells recruited by cancer therapeutic bispecific antibodies; however, differences in the populations of these cells in individual tumors limit the general use of these antibodies. In the present study, trispecific antibodies were created, namely T cell and NK cell engagers (TaKEs), that recruit both T cells and NK cells. Notably, three Fc‑fused TaKEs were designed, TaKE1‑Fc, TaKE2‑Fc and TaKE3‑Fc, using variable fragments targeting the epidermal growth factor receptor on tumor cells, CD3 on T cells, and CD16 on NK cells. Among them, TaKE1‑Fc was predicted to form a circular tetrabody‑like configuration and exhibited the highest production and greatest cancer growth inhibitory effects. TaKE1 was prepared from TaKE1‑Fc by digesting the Fc region for further functional evaluation. The resulting TaKE1 exhibited trispecificity via its ability to bind cancer cells, T cells and NK cells, as well as comparable or greater cancer growth inhibitory effects to those of two bispecific antibodies that recruit T cells and NK cells, respectively. A functional trispecific antibody with the potential to exert strong therapeutic effects independent of T cell and NK cell populations was developed.