Various types of the recombinant antibody format have been designed from the domains. One of them, Bispecific diabody is designed only from two kinds of the fragments of variable region (Fvs). The diabody composed of the Fvs from anti-cancer and anti-lymphocyte antibodies can recruit cancer cells and T-lymphocyte, and induce T-cell–mediated cytotoxicity (Fig. 1). However, 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 the epidermal growth factor receptor family for cancer and antibodies against CD3 and CD28 for T-cell retargeting. By developing and applying a set of rapid operations for expression vector construction and protein preparation (scheme. 1), 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.
In conclusion, a domain library approach generated various bispecific diabodies with a wide-range of cytotoxicity. The results of this screening process demonstrate that cytotoxicity changes drastically according to the Fv used and the domain order, and provide critical rules for the designing diabody with high cytotoxicity, effective Target on the T-lymphocyte is CD3, the domain order is LH-type than HL-type, effective target on the TFK-1 cancer cells is EGFR, using anti-EGFR antibody recognizing EGF-bound area on folded EGFR with high affinity. In general, parental antibodies binding the desired target are selected from large-scale libraries, but combinatorial optimization of the choice of Fv fragments and domain order to construct highly cytotoxic bispecific antibodies has not been attempted previously. Our results reveal that the construction of a diabody library from parental antibodies, used in combination with our novel screening method enables the rapid selection of Fvs suitable for constructing highly cytotoxic bispecific antibodies.
