Specific tumor targeting and activation of Vγ9Vδ2 T cells by bi-specific nanobodies

Gamma delta T cells expressing the Vg9Vδ2 T cell receptor (TCR) are the most predominant gδ-T cell subset in peripheral blood accounting for approximately 1-5 % of all T cells. Vg9Vδ2 T cells recognize phosphoantigens (pAg) such as isopentenyl pyrophosphate (IPP), a naturally occurring pAg that can accumulate in tumor cells, resulting in activation, cytokine release and anti-tumor activity of Vg9Vδ2 T. The use of Vg9Vδ2 T cells in clinical trials, either via adoptive transfer of ex vivo expanded Vg9Vδ2 T cells or through in vivo activation by aminobisphosphonates or synthetic pAg, has led to promising results. Antitumor responses were observed in some patients, but overall results lack consistency. This might be related to systemic activation of Vg9Vδ2 T cells in these trials, not providing a specific trigger for these cells to accumulate at the tumor site. In order to improve the efficacy of Vg9Vδ2 T cell based immunotherapy, we focused on the design of a tumortargeting construct that binds both the TCR of Vg9Vδ2 T cells and the Epidermal Growth Factor Receptor (EGFR), which is over-expressed by many tumor types, including Vg9Vδ2 T cell susceptible tumors like colon carcinoma and head and neck cancer. For this bi-specific construct an antagonistic anti-EGFR single domain antibody fragment (VHH or Nanobody) and an agonistic anti-Vg9Vδ2 TCR VHH were identified, characterized and constructed into a bi-specific targeting molecule. Only when bound to both EGFR expressing tumor cells and Vg9Vδ2 T cells, this bi-specific targeting molecule induced Vg9Vδ2 T cell activation, release of IFN-g and TNF-a as well as upregulated expression of cytolytic molecules such as perforin-and granzyme B. Importantly, tumor targeted Vg9Vδ2 T cells were able to efficiently lyse EGFR expressing tumor cells in vitro. This study shows that bi-specific anti-Vg9Vδ-T-antiEGFR-nanobodies can specifically and efficiently lyse EGFR-expressing tumor cells and are promising candidates for cancer immunotherapy.