[Construction, expression and activity test of a reshaping single-chain antibody against human CD3]

Monoclonal antibody (McAb) against human CD3 can adjust human body's immune statement in various ways, so that its clinical potential is highly regarded. In order to overcome the immunogenecity related to the murine McAb, this research effort was focused on constructing a reshaping single-chain antibody(scFv) against human CD3 employing antibody engineering. First, the CDRs of the murine McAb against human CD3 OKT3 was transplanted into the light-chain framework regions (FRs) of human McAb LS1 and the heavy-chain FRs of human McAb Nd respectively, spatial conformation was predicted by computer analysis. Then some particular residues were replaced in FRs basing on the result of conformational prediction to draw out the amino acid sequences of the reshaped VL and VH. The genes were chemically synthesize and inserted into an expression vector pROH80 to construct the reshaping scFv. Inducing the expression of reshaping scFv, the products are mainly as inclusion bodies. The reshaping scFv was expressed in another vector pALM. The inclusion bodies were denatured and then renatured by gel filtration. The renatured products were purified by immobilized metal affinity chromatograph (IMAC). Finally, the antigen-binding activily of the reshaping scFv against human CD3 was testified by the Compelitire in hibilory fluorescenceactivated cell sorting (FACS). The competitive inhibition rate is 18%.