Small bispecific antibodies that induce T-cell–mediated cytotoxicity have the potential to damage late-stage tumor masses to a clinically relevant degree, but their cytotoxicity is critically dependent on their structural and functional properties. Here, we constructed an optimized procedure for identifying highly cytotoxic antibodies from a variety of the T-cell–recruiting antibodies engineered from a series of antibodies against cancer antigens of epidermal growth factor receptor family and T-cell receptors. By developing and applying a set of rapid operations for expression vector construction and protein preparation, we screened the cytotoxicity of 104 small antibodies with diabody format and identified some with 103-times higher cytotoxicity than that of previously reported active diabody. The results demonstrate that cytotoxicity is enhanced by synergistic effects between the target, epitope, binding affinity, and the order of heavy-chain and light-chain variable domains. We demonstrate the importance of screening to determine the critical rules for highly cytotoxic antibodies.

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.

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Production of various combinations of bispecific variable domain of heavy chain of heavy chain-only antibody (VHH) constructs to evaluate their therapeutic potential usually requires several gene-engineering steps. Here, we present an alternative method of creating bispecific VHH constructs in vivo through protein trans-splicing (PTS) reaction; this method may reduce the number of gene manipulation steps required. As a proof-of-concept, we constructed a bispecific antibody (bsAb) containing an anti-epidermal growth factor receptor VHH and anti-green fluorescent protein VHH, and we evaluated and confirmed its bispecificity. We also tested antibody labeling by fluorescent protein tagging using the PTS reaction. Compared with the conventional gene construction method, bsAb construction via PTS is a promising alternative approach for generating multiple bsAb combinations.