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Diabody 31, a bispecific therapeutic antibody, is a heterodimer comprised of two types of chimeric single-chain variable fragments (scFv), that has been identified as a clone with high cytotoxicity against cancer cells. Diabody 31 is inactive as a homodimer and monomer, yet active as a heterodimer in solution. Herein, we examined the association between two kinds of diabodies, LH- and HL type, based on the behavior of four kinds of their constituent chimera scFvs, by size analysis. More than half of the LH diabody fraction, including two types of chimeric scFV that were equally expressed, and nearly all HL diabody fraction components, including one chimeric HL-L scFv, were in inactive form and changed components dynamically as time passed. The association of these diabodies corresponded to that of the scFv chimera, indicating that analysis of these chimeras could predict diabody quality. Furthermore, addition of the deficient HL-R chimera scFv, comprised of an unbalanced chimeric scFv ratio, to the HL diabody fraction increased cancer cell cytotoxicity, with maximal effects obtained upon repeating the process five times. These findings provide insights into the efficient construction of a functional diabody by noting the characteristics of associated diabodies and the molar ratio of expressed chimera scFvs.

The cytotoxicity of T cell-recruiting antibodies with their potential to damage late-stage tumor masses is critically dependent on their structural and functional properties. Recently, we reported a semi-high-throughput process for screening highly cytotoxic small bispecific antibodies (i.e., diabodies). In the present study, we improved the high-throughput performance of this screening process by removing the protein purification stage and adding a stage for determining the concentrations of the diabodies in culture supernatant. The diabodies were constructed by using an Escherichia coli expression system, and each diabody contained tandemly arranged peptide tags at the C-terminus, which allowed the concentration of diabodies in the culture supernatant to be quantified by using a tag-sandwich enzyme-linked immunosorbent assay. When estimated diabody concentrations were used to determine the cytotoxicity of unpurified antibodies, results comparable to those of purified antibodies were obtained. In a surface plasmon resonance spectroscopy-based target-binding assay, contaminants in the culture supernatant prevented us from conducting a quantitative binding analysis; however, this approach did allow relative binding affinity to be determined, and the relative binding affinities of the unpurified diabodies were comparable to those of the purified antibodies. Thus, we present here an improved high-throughput process for the simultaneous screening and determination of the binding parameters of highly cytotoxic bispecific antibodies.

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.

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.

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.