Novel α-BTN Antibodies Modulate Vγ9Vδ2 Activation and Reveal Insights into the Extracellular Architecture of the Phospho-Antigen Signaling Complex

Infected and malignant target-cells trigger Vγ9Vδ2 T cell activation by signaling pathogenic intracellular metabolite accumulation through Butyrophilin (BTN)-3A1 and BTN2A1 proteins to the Vγ9Vδ2 T cell receptor (TCR). An incomplete understanding of the molecular dynamics in this signaling complex precludes Vγ9Vδ2 T cell immunotherapeutic efficacy. Here, we develop a panel of engineered α-BTN3A1 and α-BTN2A1 antibody (mAb) reagents to probe the roles of BTN3A1 and BTN2A1 in pAg signaling. By modifying agonist α-BTN3A1 mAb 20.1 with increased inter-Fab distances, we establish that tight clustering of BTN3A1 is not necessary to stimulate Vγ9Vδ2 T cell activation. By similarly modifying antagonist α-BTN3A1 mAb 103.2, we demonstrate that 103.2 does not antagonize Vγ9Vδ2 T cell activation through conformational constraint but likely through steric occlusion of a critical binding interaction between BTN3A1 and a yet unknown co-receptor. Finally, we developed a panel of α-BTN2A1 mAb Vγ9Vδ2 antagonists that utilize different biophysical mechanisms to compete with the Vγ9Vδ2 TCR for BTN2A1 binding. Our 2.8Å structure of BTN2A1 ectodomain in complex with the Fab of potent antagonist, 2A1.9, provides molecular insights into BTN2A1 residues critical for pAg-signaling. 2A1.9 may be developed into an immunotherapy adjuvant to sensitize tumors in which high BTN2A1 expression is a poor prognostic marker to αβ T cell immunotherapies.