(228ew) Domain Library Approach for Designing Therapeutic Small Antibody with High Cytotoxicity for Tumor Cells

Antibody has a hierarchical structure constructed from structurally-independent domains, and various recombinant antibodies with artificial structural formats are designed from the domains. Bispecific Diabody (BsDbs) is a small antibody designed only from two kinds of the fragments of variable region (Fvs), and the diabody composed of the Fvs from anti-cancer and anti-lymphocyte antibodies can form a bridge between cancer and lymphocyte cells to make an effective damage on cancer cells. The cytotoxicity of diabody is dependent on the characters of Fv used, that is, targets on cancer cell / T-lymphocyte and the epitopes of the targets. Here, we propose a methodology for rapid screening of the most effective bispecific antibody of more than 100 diabodies designed from several anti-cancer and anti-lymphocyte IgG type antibodies.

We applied each medium fraction to immobilized metal affinity chromatography (IMAC) to purify expressed diabodies. We step-wisely measured the cytotoxicity of IMAC-refined diabodies at the various diabody concentrations by MTS assay. In this assay, all samples were prepared to less than 50 mM imidazole concentration, because imidazole showed the cytotoxicity against cancer cells at more than 75 mM. In the result of step-wise screening, we got 6 kinds high cytotoxic diabodies which damaged at cancer cells at 100 fM concentration

By means of the screening, we found some rules of high cytotoxicity, specific domain order, target molecules, and specific antibodies for highly active diabody. These results imply that high cytotoxic diabodies have universal rules of structure and using antibodies for diabody.