Generation and characterization of a novel tetravalent bispecific antibodythat binds to hepatitis B virus surface antigens

Hepatitis B virus (HBV) infection is a worldwide public health problem affe cting about 350 million people. HBV envelope contains three surface antigen s, called pre-S1, pre-S2 and S. For the prophylaxis of HBV infection, only an anti-S monoclonal antibody was tested for the protective efficacy agains t HBV infection, but it was shown to be incomplete. In addition, some immun e escape mutants carrying mutations on the S antigen were reported. Therefo re, a multivalent bispecific antibody rather than a single monoclonal antib ody would be more beneficial for the prophylaxis of HBV infection. We have generated a novel tetravalent bispecific antibody with two binding sites fo r each of the S and pre-S2 antigens. Each of the antigen-binding sites was composed of a single-chain Fv (ScFv). The tetravalent antibody was generate d by constructing a single gene encoding a single-chain protein. This prote in consisted of an anti-S ScFv whose carboxyl end was tethered, through a 4 5 amino acid linker, to the amino terminus of anti-preS2 ScFv that in turn was joined to the hinge region of human yl constant region. The single-chai n protein was expressed in Chinese hamster ovary cells and secreted in cult ure supernatant as a homodimeric molecule. The tetravalent bispecific antib ody showed both anti-S and anti-pre-S2 binding activities. In addition, the binding affinity of the bispecific antiboy for HBV particles was greater t han that of either parental antibody. The tetravalent bispecific antibody i s a potentially useful reagent for the prevention and treatment of HBV infe ction. (C) 2001 Elsevier Science Ltd. All rights reserved.