Novel tri-specific tribodies induce strong T cell activation and anti-tumor effects in vitro and in vivo

BACKGROUND: Immunotherapy based on Bi-specific T Cell Engagers (TCE) represents one of the most attractive strategy to treat cancers resistant to conventional therapies. TCE are antibody-like proteins that simultaneously bind with one arm to a Tumor Associated Antigen (TAA) on cancer cells and with the other one to CD3 complex on a T-cell to form a TCR-independent immune synapse and circumvent Human Leucocyte Antigen restriction. Among them, the tribodies, such as Tb535H, a bi-specific molecule, made up of a Fab and a scFv domain both targeting 5T4 and another scFv targeting CD3, have demonstrated anti-tumor efficacy in preclinical studies. METHODS: Here, we generated five novel tri-specific and multi-functional tribodies, called 53X tribodies, composed of a 5T4 binding Fab arm and a CD3 binding scFv, but differently from the parental Tb535H, they contain an additional scFv derived from an antibody specific for an immune checkpoint, such as PD-1, PD-L1 or LAG-3. RESULTS: Compared with the parental Tb535H bi-specific T cell engager targeting 5T4, the novel 53X tribodies retained similar binding properties of Tb535H tribody, but showed enhanced anti-tumor potency due to the incorporation of the checkpoint inhibitory moiety. In particular, one of them, called 53L10, a tri-specific T cell engager targeting 5T4, CD3 and PD-L1, showed the most promising anti-tumor efficacy in vitro and led to complete tumor regression in vivo. CONCLUSIONS: The novel tribodies have the potential to become strong and safe therapeutic drugs, allowing to reduce also the cost of production as one single molecule contains three different specificities including the anti-TAA, anti-CD3 and anti-IC binding arms. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13046-022-02474-3.