ABSTRACT: Gamma delta T (γδ T) cells are among the most potent cytotoxic lymphocytes. Activating anti–butyrophilin 3A (BTN3A) antibodies prime diverse tumor cell types to be killed by Vγ9Vδ2 T cells, the predominant γδ T cell subset in peripheral circulation, by mechanisms independent of tumor antigen–major histocompatibility complex (MHC) complexes. In this report, we describe the development of a humanized monoclonal antibody, ICT01, with subnanomolar affinity for the three isoforms of BTN3A. We demonstrate that ICT01-activated Vγ9Vδ2 T cells kill multiple tumor cell lines and primary tumor cells, but not normal healthy cells, in an efficient process requiring approximately 20% target occupancy. We show that ICT01 activity is dependent on BTN3A and BTN2A but independent of the phosphoantigen (pAg)–binding B30.2 domain. ICT01 delays the growth of hematologic and solid tumor xenografts and prolongs survival of NOD/SCID/IL2rγnull (NSG) mice adoptively transferred with human Vγ9Vδ2 T cells. In single- and multiple-dose safety studies in cynomolgus macaques that received up to 100 mg/kg once weekly, ICT01 was well tolerated. With respect to pharmacodynamic endpoints, ICT01 selectively activated Vγ9Vδ2 T cells without affecting other BTN3A-expressing lymphocytes such as αβ T or B cells. A first-in-human, phase 1/2a, open-label, clinical study of ICT01 was thus initiated in patients with advanced-stage solid tumors (EVICTION: NCT04243499; EudraCT: 2019-003847-31). Preliminary results show that ICT01 was well tolerated and pharmacodynamically active in the first patients. Digital pathology analysis of tumor biopsies of a patient with melanoma suggests that ICT01 may promote immune cell infiltration within the tumor microenvironment.