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.
- cancer therapy