A trophoblast glycoprotein specific 5T4-Vδ2 bispecific T cell engager recruits Vγ9Vδ2-T cells for tumor-selective cytotoxicity across solid malignancies

5 T4 is an oncofetal antigen that is aberrantly expressed across a wide range of malignancies, but only sparsely present in healthy adult tissues. Its tumor-preferential expression pattern has been linked to epithelial-to-mesenchymal transition, invasion, and poor clinical outcome in various tumors, making 5 T4 an attractive therapeutic target. Bispecific T-cell engagers (bsTCEs) have achieved major clinical success in hematological malignancies, yet efficacy in solid tumors has been limited by challenges including on-target off-tumor toxicity. Bispecific Vγ9Vδ2-T cell engagers may overcome this challenge by combining potent anti-tumor activity with reduced toxicity. Here, we developed high-affinity 5 T4-specific VHHs and linked them to a Vδ2 TCR-specific VHH to generate a bsTCE which engages Vγ9Vδ2-T cells to 5 T4-expressing tumors and triggers robust proinflammatory cytokine production and target cell lysis. We demonstrate that 5 T4 is expressed in the majority of evaluated solid malignancies and that the 5 T4-Vδ2 bsTCE elicits strong Vγ9Vδ2-T cell-mediated anti-tumor activity in both 2D and 3D patient-derived preclinical tumor models. Importantly, when tested against healthy 5 T4-expressing tissue, the bsTCE did not trigger Vγ9Vδ2-T cell cytotoxicity, underscoring its tumor-preferential activity. These findings support the 5 T4-Vδ2 bsTCE as a promising immunotherapeutic candidate for broad clinical application in (solid) tumors. • The oncofetal antigen 5 T4 is expressed across a broad range of solid malignancies. • High affinity 5 T4-specific VHHs were generated and showed no impact on CXCL12/CXCR4-mediated chemotaxis or tumor cell growth. • 5 T4-Vδ2 bsTCEs trigger potent Vγ9Vδ2-T cell-mediated cytotoxicity against 5 T4-expressing tumor cells in both 2D and 3D patient-derived organoid tumor models.