A Modular Semi-Synthetic Approach to Generate T cell-Dependent Bispecific Constructs from Recombinant IgG1 Antibodies

Redirecting T cells to tumor cells by bispecific antibodies is an effective approach to treat cancer and T cell-dependent bispecific antibodies (TDBAs) are an emerging class of potent immunotherapeutic agents. By simultaneously targeting antigens on tumor cells and that on T cells, T cells are activated to kill tumor cells. Herein, we report a platform to gener-ate a novel class of 2:1 structure of T cell-dependent bispecific antibody with bivalency for HER2 receptors on tumor cells and monovalency for CD3 receptors on T cells. For this, we use a biogenic inverse electron-demand Diels-Alder (IEDDA) click reaction on genetically encoded tyrosine residues to install one TCO-handle on the therapeutically ap-proved antibody trastuzumab. Subsequent TCO-tetrazine click with a tetrazine-functionalized CD3-binding Fab yields a 2:1 HER2×CD3 TDBA that exhibits tumor killing capability at picomolar concentrations. Monovalency towards CD3 recep-tor on T cells can lower chances of cytokine release syndrome, which is a common side effect of such agents. Our semi-synthetic approach can generate highly potent TDBA constructs in a few chemoenzymatic and synthetic steps.