Antibodies which have high affinity for a specific antigen are promising biomolecules as molecular target drugs in the medical field, and the bispecific antibodies designed from the anti-epidermal growth factor receptor (EGFR) and anti-CD3 single chain fragments of variable region (scFvs) make linkages between cancer cells and T-lymphocytes and they show significant damage on cancer cell. Recently, we found spontaneous dimerization of the scFvs from anti-CD3 antibody OKT3, and the dimerization let us design multispecific and multivalent antibody on the dimeric form. In this study, we fused the anti-EGFR single variable domain of the heavy chain of a heavy-chain camel antibody (VHH) at the terminus of OKT3 scFv to design a bispecific and bivalent small antibody.Here, we fused the anti-CD3 scFv at the C-terminus of VHH fragment via an IgA peptide linker, and the recombinant proteins are expressed in E. coli and purified by means of metal chelate affinity and gel-filtrated chromatography. The VHH-fused scFv forms monomer and dimer, and the dimer had higher affinity for cancer cell and T-lymphocyte than monomer. Further, analysis of in vitro cytotoxicity against cancer cell showed that dimeric VHH-fused scFv had 1000-times cytotoxicity than monomeric form. We also compared the cytotoxicity with normal bispecific small antibody (diabody) designed from anti-EGFR scFV and anti-CD3 scFv; consequently, the dimeric VHH-fused scFv also showed 1000-times cytotoxicity than the diabody.

Design of smart antibody with bispecific and bivalent function by fusing single camel antigen-binding fragment onto dimerized single-chain Fv

248th National Meeting (2014)

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