A novel single-vector featuring bidirectional promoters for production of bispecific antibody against rabies virus

The World Health Organization (WHO) recommends combination therapy with two non-overlapping monoclonal antibodies (mAbs) to enhance protection in Rabies post-exposure prophylaxis (PEP). While bispecific antibodies (bsAbs) provide superior protection over monoclonal antibodies (mAbs) by simultaneously targeting two distinct epitopes, their development is hindered by complex and inefficient vector design. In this study, we aimed to develop a novel single-vector expression system to efficiently produce potent anti-rabies virus (RABV) neutralizing bsAbs. We engineered a minimalistic peaSKYA single-vector featuring bidirectional dual-CMV promoters to enable synchronous co-expression of antibody heavy (HC) and light chains (LC) within a single construct. By eliminating redundant backbone elements, this optimized vector increased the expression-cassette ratio by 17% and improves transfection efficiency by 32% compared to dual-vector systems. The platform demonstrated 2-fold greater yields of bispecific antibodies in HEK293F transient expression, with the tetravalent DVD-Ig format antibody HHDVD showing significantly enhanced neutralization potency versus parental monoclonal antibodies in both pseudovirus-based neutralization assays (PBNA, 2193.19 IU/mg) and rapid fluorescent focus inhibition tests (RFFIT, 1679.52 IU/mg). We developed peaSKYA single-vector expression system for robust production of bispecific antibodies, with candidate HHDVD exhibiting potential in Rabies PEP. This streamlined vector design approach exhibited remarkable potential in bispecific neutralizing antibody development, offering a transferable platform for other engineered bispecific antibodies.