Synthesis of a Phosphoantigen Prodrug that Potently Activates Vγ9Vδ2 T-Lymphocytes

• A charge-neutral phosphoantigen prodrug exhibits enhanced cellular efficacy
• A phosphoantigen prodrug is stronger than HMBPP and more selective than risedronate
• A phosphonate binds the BTN3A1 B30.2 domain more tightly than the diphosphate IPP
• Phosphoantigen binding alters the structure of the BTN3A1 membrane proximal region

SummaryPhosphoantigen-sensitive Vγ9Vδ2 T cells are important responders to infections and malignancy. However, the mechanisms by which phosphoantigens stimulate Vγ9Vδ2 T cells are unclear. Here, we synthesized phosphoantigen prodrugs and used them to demonstrate that intracellular delivery of phosphoantigens is required for their activity. The pivaloyloxymethyl prodrug is the most potent phosphoantigen described to date, with stronger stimulation of Vγ9Vδ2 T cells from human peripheral blood and greater ability to induce lysis of Daudi lymphoma cells relative to the previously most potent compound, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP). We demonstrate high binding affinity between phosphoantigens and the intracellular region of butyrophilin 3A1 (BTN3A1), localized to the PRY/SPRY (B30.2) domain, but also affecting the membrane proximal region. Our findings promote a phosphoantigen prodrug approach for cancer immunotherapy and unravel fundamental aspects of the mechanisms of Vγ9Vδ2 T cell activation.

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