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

Abstract

Hepatitis B virus (HBV) infection is a worldwide public health problem affecting about 350 million people. HBV envelope contains three surface antigens, 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 against HBV infection, but it was shown to be incomplete. In addition, some immune escape mutants carrying mutations on the S antigen were reported. Therefore, a multivalent bispecific antibody rather than a single monoclonal antibody would be more beneficial for the prophylaxis of HBV infection. We have generated a novel tetravalent bispecific antibody with two binding sites for 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 generated by constructing a single gene encoding a single-chain protein. This protein consisted of an anti-S ScFv whose carboxyl end was tethered, through a 45 amino acid linker, to the amino terminus of anti-preS2 ScFv that in turn was joined to the hinge region of human γ1 constant region. The single-chain protein was expressed in Chinese hamster ovary cells and secreted in culture supernatant as a homodimeric molecule. The tetravalent bispecific antibody showed both anti-S and anti-pre-S2 binding activities. In addition, the binding affinity of the bispecific antiboy for HBV particles was greater than that of either parental antibody. The tetravalent bispecific antibody is a potentially useful reagent for the prevention and treatment of HBV infection.

Introduction

Hepatitis B virus (HBV) infection is a worldwide public health problem affecting about 350 million people. The HBV envelope contains three surface glycoproteins called the small (S), middle (M), and large (L) proteins, and these proteins contain three different surface antigens, preS1, preS2, and S (Heerman et al., 1984). All three antigens were shown to elicit virus-neutralizing and protective antibodies (Stevens et al., 1984, Itoh et al., 1986; Neurath et al., 1989). Several human anti-S monoclonal antibodies (mAbs) have been produced and tested for the prophylaxis of HBV infection (Harada et al., 1989, Ehrlich et al., 1992). However, a single anti-S mAb did not efficiently protect against HBV infection (Ogata et al., 1993). In addition, some immune escape mutants carrying mutations on the HBV S protein were reported (Carman et al., 1990, Yamamoto et al., 1994). These results suggest that antibodies against more than one surface antigen may be needed for better protection against HBV infection. One approach to increase the protective efficacy may involve developing a multivalent bispecific antibody against the S and preS antigens.

Bispecific antibodies, which have two binding specificities within a single molecule, have been constructed and used for cancer therapy, immunoassays, and immunomodulation (reviewed by Van de Winkel et al., 1997). A variety of techniques, including chemical cross-linking (Glennie et al., 1987, Stickney et al., 1989, Repp et al., 1995), hybridoma technology (Suresh et al., 1986, Staerz and Bevan, 1986), and genetic engineering (Holliger et al., 1993, Kurucz et al., 1995, Mack et al., 1995, Hayden et al., 1996, Coloma and Morrison, 1997) have been used to generate bispecific antibodies. The first two approaches generate a heterogeneous mixture of molecules consisting of bispecific antibody, parental antibodies and mismatched pairs of the heavy and light chains. Thus, it requires labor-intensive methods to purify the desired bispecific antibody. To circumvent the problems, bispecific antibodies have been genetically engineered by using various strategies. Bispecific immunoglobulin (Ig) molecules lacking constant regions, such as F(ab′)₂ or a pair of single-chain Fv (ScFv) molecules, (ScFv)₂, have been produced by associating two molecules through dimerization domains, polypeptide chain, or cross-linking hinge cysteins (reviewed by Holliger and Winter, 1993). A novel bispecific antibody with effector functions was produced by fusing a ScFv of one specificity at the carboxy terminus of an antibody with a different specificity. Generation of this novel Ig molecule required two genes encoding the heavy and light chains (Coloma and Morrison, 1997).

In this study, we have constructed a single gene encoding a single-chain bispecific antibody that is composed of two consecutive ScFvs, specific to the S and preS2 antigens of HBV, fused to the Fc region of human IgG1. The single-chain bispecific antibody was expressed in Chinese hamster ovary (CHO) cells and secreted in the culture supernatant, as a homodimeric molecule formed via the disulfide bonds between the hinge regions of the two chains. The tetravalent bispecific antibody showed both anti-S and anti-preS2 binding activities and exhibited greater binding activity for HBV particles than that of each of the parental antibodies.