Compact Seahorse-Shaped T Cell–Activating Antibody for Cancer Therapy

Abstract

The vast information available on hierarchically structured proteins enables the creation of novel proteins with customized functions through the assembly of independent functional component modules. Here, a compact T cell–activating antibody is constructed from the antigen-binding modules of variable domains. Genetic fusion of a single variable domain of the heavy chain of a heavy chain llama antibody (VHH) to the human single-chain variable region of an antigen-binding fragment (scFv), which is designed to be dimerized, yields a compact bispecific and bivalent antibody (BiBian) with a seahorse-shaped structure. BiBian recognizes epidermal growth factor receptor (EGFR) on cancer cells and CD3 receptors on T cells; the two VHHs and dimerized scFv are structurally independent and positioned such that they are easily accessible to each target. BiBian adhered strongly to both cancer cells and T cells, promoted T cell activation (due to the bivalent CD3 modules), and induced dramatic cytotoxicity against tumor spheroids in vitro and in vivo. This compact structure is proposed as a fundamental format for homogeneous, highly cytotoxic, bacterially expressed antibodies.