Abstract
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.
Highlights
Antibody molecules with high molecular recognition ability and cellular cytotoxicity have been extensively used as molecular-targeting agents
For briefly constructing the expression vectors of diabodies, 4 VH genes containing were prepared from the vectors of anti-CD3 or -CD28 single-chain Fv (scFv), and they were simultaneously ligated into the 13 linearized vectors where VH genes were removed from anti-cancer scFvs with variable domains in the order VH-VL (Fig. S1A)
The vectors containing hetero scFv genes with an anti-epidermal growth factor receptor (EGFR)-family VH domain followed by an anti-CD3 or -CD28 VL domain were produced in the same way, and all of the gene fragments encoding hetero scFv were ligated into the linear vector carrying the complementary hetero scFv to produce the expression vectors of HL-type diabodies
Summary
Antibody molecules with high molecular recognition ability and cellular cytotoxicity have been extensively used as molecular-targeting agents Their functions are independently localized in different fragments of the antibody: the fragment of variable region (Fv) has high specificity for the binding to target antigen or epitope, and the fragment of crystallizable region induces the activation of immune cells. These small antibodies have the potential to be produced by bacterial expression systems[16], which would enable low-cost production of therapeutic antibodies These potential advantages of small T-cell–recruiting antibodies have driven researchers to generate a large number of these antibodies with different cancer targets and bispecific structure formats; the studies have shown www.nature.com/scientificreports/. The relationships between these factors are complicated, and we have no optimized approach for choosing the appropriate Fvs and domain arrangements to construct bispecific antibodies with sufficiently high cytotoxicity to be clinically effective
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