Objective To design and prepare anti-GPC3/CD3 bispecific heavy chain antibody (BiHcAb) and analyze the difference of in vivo and in vitro anti-tumor activity between BiHcAb and bispecific T cell engager (BiTE).

Methods The sequences of anti-GPC3 and anti-CD3 single domain antibodies (sdAb) were obtained by gene synthesis, then cloned into an expression vector carrying IgG4 Fc segment with knob-into-hole (KIH) technology to produce stable bispecific heavy chain antibodies. Two plasmids were co-transfected into HEK-293F cells and cultured in suspension shake flasks to produce the target antibody. The molecular weight of productions was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining. The combination of antibodies and GPC3-positive tumor cells and peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometry. Cell cytotoxicity and cytokine release mediated by BiHcAb were measured under co-culture conditions. The tumor-inhibiting activity of BiHcAb in vivo was also tested in nude mouse xenograft model experiment.

Results The anti-GPC3/CD3 BiHcAb was successfully constructed and expressed, with a molecular weight of about 100 000. The molecular weight of BiTE was about 50 000. Compared with BiTE, anti-GPC3/CD3 BiHcAb has stronger killing activity on GPC3-positive tumor cells, and promotes the release of cytokines (IFN-γ, TNF-α, IL-2, and IL-6). It is shown that anti-GPC3/CD3 BiHcAb has a stronger tumor growth inhibition effect in vivo than BiTE in nude mice.

Conclusions We designed an anti-GPC3/CD3 bispecific heavy chain antibody with a stronger anti-liver cancer effect than BiTE in vivo and in vitro.