Modular\nSemisynthetic Approach to Generate T Cell-Dependent\nBispecific Constructs from Recombinant IgG1 Antibodies

Redirecting T cells\nto tumor cells by bispecific antibodies is\nan effective approach to treat cancer, and T cell-dependent bispecific\nantibodies (TDBAs) are an emerging class of potent immunotherapeutic\nagents. By simultaneously targeting antigens on tumor cells and T\ncells, T cells are activated to kill tumor cells. Herein, we report\na platform to generate a novel class of 2:1 structure of T cell-dependent\nbispecific antibody with bivalency for HER2 receptors on tumor cells\nand monovalency for CD3 receptors on T cells. For this, we use a biogenic\ninverse electron-demand Diels–Alder (IEDDA) click reaction\non genetically encoded tyrosine residues to install one TCO handle\non therapeutically approved antibody trastuzumab. Subsequent TCO-tetrazine\nclick with a tetrazine-functionalized CD3-binding Fab yields a 2:1\nHER2 × CD3 TDBA that exhibits a tumor-killing capability at picomolar\nconcentrations. Monovalency toward the CD3 receptor on T cells can\nlower the chances of cytokine release syndrome, which is a common\nside effect of such agents. Our semisynthetic approach can generate\nhighly potent TDBA constructs in a few chemoenzymatic and synthetic\nsteps.