Evaluation of an anti-CD3 VHH and construction of an anti-CD3/anti-EGFR bispecific tandem VHH as a cancer cell targeting drug construct

Recently, the development of T-cell engager cancer therapeutics, consisting of anticancer and anti-T-cell antibody parts to engage the T-cell to the cancer site, has gained interest. Anti-CD3 antibodies are predominantly used to achieve specific binding to T-cells for the T-cell engager construction. Various kinds of anti-CD3 IgG clones have been developed and engineered, but the available anti-CD3 VHH, a single variable domain of a dromedary heavy-chain antibody, clones are limited in number. Thus, the assessment of the available anti-CD3 VHHs is important for therapeutic applications. Here, we demonstrated the expression and characterization of an anti-CD3 VHH clone, 117G03, and evaluated this T-cell engager with an anti-EFGR VHH in a bispecific format to assess the developability of this anti-CD3 VHH clone. 117G03 was expressed as a monomer in the soluble fraction and demonstrated decent thermal stability with binding activity against a CD3-positive cell line. The T-cell engager construct was prepared using the refolding method and exhibited enhanced cytotoxic activity against the epidermal growth factor receptor (EGFR)-positive cell line mediated by activated T-cells. These results indicate that 117G03 can be utilized for T-cell engager applications.