Phosphonate-antibody-drug conjugates, a novel immunostimulatory class of ADCs driving inside-out activation of Vgamma9Vdelta2 T cells leading to selective tumor cell killing

Gamma delta (gammadelta) T cells are cytotoxic effectors capable of recognizing and eliminating tumor cells independently of major histocompatibility complex (MHC) presentation. Their presence within tumors correlates with improved clinical outcomes across various cancer types. Vgamma9Vdelta2 T cells, the predominant gammadelta subset in human blood, recognize intracellular phosphoantigens (pAgs) through inside-out activation of the butyrophilin (BTN)3A/BTN2A complex. Clinical trials have previously been conducted using aminobisphosphonates or synthetic pAgs, either alone or in combination with low-dose interleukin-2 (IL-2); however, these compounds lack tumor specificity and are limited by a very short plasma half-life in vivo .​ To address the lack of tumor targeting and short half-life, we engineered antibody-drug conjugates (ADCs) that selectively deliver phosphonates to tumor cells, enabling inside-out activation of Vgamma9Vdelta2 T cells. These phosphonate-ADCs consist of a monoclonal antibody directed against a tumor-associated antigen (TAA), conjugated to a phosphonate payload via a linker that is cleavable by lysosomal proteases. We applied this strategy to various TAA-targeting antibodies, including CD123, CD20, TROP2 and HER2 directed IgG1-based monoclonal antibodies.​ TAA-positive tumor cell lines pretreated with phosphonate-ADCs effectively triggered Vgamma9Vdelta2 T cell activation through a BTN3A-dependent mechanism. This activation led to robust cytokine secretion, degranulation, and efficient tumor cell lysis. Vgamma9Vdelta2 T cell activation was successfully replicated with ex vivo patient tumoroids (EVPTs) and autologous peripheral blood mononuclear cells (PBMCs), supporting the translational relevance of this approach. In an acute myeloid leukemia (AML) adoptive transfer mouse model, Vgamma9Vdelta2 T cells significantly reduced the tumor burden in the presence of the tumor-targeting phosphonate-ADC. In cynomolgus monkeys, the lead phosphonate-ADC candidate displayed excellent tolerability at a single dose of up to 100 mg/kg, with no clinical signs of cytokine release syndrome (CRS). Immunostimulatory phosphonate-ADCs represent a novel strategy for targeted, physiologically relevant activation of Vgamma9Vdelta2 T cells by delivering phosphonates directly to tumor cells. This modular platform can be combined with virtually any tumor-targeting monoclonal antibody with the possibility to preserve Fcgamma receptor-mediated effector functions. The resulting dual mechanism, integrating the antibody's direct anti-tumor activity with the immune-stimulatory properties of the phosphonate payload, offers a versatile and powerful therapeutic modality. Overall, this platform holds significant promise not only for targeted cancer immunotherapy but also for potential applications in other disease areas.