Potent Bispecific Neutralizing Antibody Targeting Glycoprotein B and the gH/gL/pUL128/130/131 Complex of Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause developmental disorders following congenital infection and life-threatening complications among transplant patients. Potent neutralizing monoclonal antibodies (MAbs) are promising drug candidates against HCMV infection. HCMV can infect a broad range of cell types. Therefore, single neutralizing antibodies targeting one HCMV glycoprotein often lack either potency or broad cell-type coverage. We previously characterized two human-derived HCMV neutralizing MAbs. One was the broadly neutralizing MAb 3-25, which targets the antigenic domain 2 of glycoprotein B (gB). The other was the highly potent MAb 2-18, which specifically recognizes the gH/gL/pUL128/130/131 complex (pentamer). To combine the strengths of gB- and pentamer-targeting MAbs, we developed an IgG-single-chain variable fragment (scFv) bispecific antibody by fusing the 2-18 scFv to the heavy-chain C terminus of MAb 3-25. The resulting bispecific antibody showed high-affinity binding to both gB and pentamer. Functionally, the bispecific antibody demonstrated a combined neutralization breadth and potency of the parental MAbs in multiple cell lines and inhibited postinfection viral spreading. Furthermore, the bispecific antibody was easily produced in CHO cells at a yield above 1 g/liter and showed a single-dose pharmacokinetic profile comparable to that of parental MAb 3-25 in rhesus macaques. Importantly, the bispecific antibody retained broadly and potent neutralizing activity after 21 days in circulation. Taken together, our research provides a proof-of-concept study for developing bispecific neutralizing antibody therapies against HCMV infection.