Bispecific light T-cell engagers for gene-based immunotherapy of epidermal growth factor receptor (EGFR)-positive malignancies

The recruitment of T-cells by bispecific antibodies secreted from adoptively transferred, gene-modified autologous cells
has shown satisfactory results in preclinical cancer models. Even so, the approach’s translation into the clinic will require
incremental improvements to its efficacy and reduction of its toxicity. Here, we characterized a tandem T-cell recruiting
bispecific antibody intended to benefit gene-based immunotherapy approaches, which we call the light T-cell engager (LiTE),
consisting of an EGFR-specific single-domain VHH
antibody fused to a CD3-specific scFv. We generated two LiTEs with the
anti-EGFR VHH
and the anti-CD3 scFv arranged in both possible orders. Both constructs were well expressed in mammalian
cells as highly homogenous monomers in solution with molecular weights of 43 and 41 kDa, respectively. In situ secreted
LiTEs bound the cognate antigens of both parental antibodies and triggered the specific cytolysis of EGFR-expressing cancer
cells without inducing T-cell activation and cytotoxicity spontaneously or against EGFR-negative cells. Light T-cell engagers
are, therefore, suitable for future applications in gene-based immunotherapy approaches.