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Background Gamma delta (γδ) T cells are a distinct subset of T cells with features of both innate and adaptive immunity. Based on their TCR δ chain usage, γδT cells can be divided into Vδ1 cells present in tissues, Vδ2 cells predominately in blood, and rarer Vδ3 cells found in the liver and gut. These cells recognize a wide range of antigens in an MHC-independent manner, including phosphoantigens via interaction of Vγ9Vδ2 and BTN3A1 or stress-induced ligands (MICA/B, ULBPs, etc.) via expression of innate receptors (NKG2D, DNAM-1, etc.). Activated γδT cells display exceptional cytolytic activity against tumor cells through innate sensing and secretion of perforin, granzymes, and pro-inflammatory cytokines. Intratumoral γδT cells are highly correlated with favorable prognosis in multiple cancers, underscoring their critical role in tumor control. 1 Thus, engaging γδT cells presents a promising strategy for treatment in hematologic and solid malignancies. We developed an anti-pan γδ x CD123 bispecific antibody that leverages the cytotoxic potential and innate discriminatory capacity of γδT cells by engaging all γδT cell subsets to kill CD123+ tumor cells for treatment of AML.Methods An anti-pan γδ x CD123 bispecific antibody was generated by combining γδ TCR and CD123 VHHs using knobs-in-holes technology. Anti-tumor activity was assessed in cytotoxicity assays using either Vδ1, Vδ2, or PBMCs as effector cells and compared to bispecifics that engage solely Vδ1 or Vδ2. Proliferation, activation, and enhancement of innate sensing by γδT cells were also evaluated. To assess their inherent ability to discriminate tumor versus normal cells, a cytotoxicity assay was conducted against healthy CD123+ monocytes compared to anti-CD3xCD123 bispecifics.Results Anti-pan γδ x CD123 bound to γδ TCR and CD123 and induced potent γδT cell-mediated killing of AML cells expressing a range of CD123. Compared to bispecifics that engage solely Vδ1 or Vδ2, anti-pan γδ x CD123 demonstrated equivalent tumor killing in the presence of Vδ1 or Vδ2 T cells, respectively, but exhibited significantly improved anti-tumor activity when PBMCs were used as effector cells. Treatment of γδT cells with anti-pan γδ x CD123 also resulted in proliferation, activation, and enhancement of innate sensing. Importantly, anti-pan γδ x CD123 did not induce killing of healthy CD123+ monocytes in contrast to anti-CD3xCD123 bispecifics.Conclusions Our data demonstrate that an anti-pan γδ x CD123 bispecific antibody engages all γδT cell subsets to effectively kill tumor cells without affecting CD123+ healthy cells and may provide a safer and more effective treatment option for AML.Reference Gentles AJ, Newman AM, Liu CL, Bratman SV, Feng W, Kim D, Nair VS, Xu Y, Khuong A, Hoang CD, Diehn M, West RB, Plevritis SK, Alizadeh AA. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nat Med. 2015; 21:938–945.