A bispecific antibody targeting a membrane-proximal ASGPR1 region promotes TNF-α recruitment and uptake

Abstract

Recruiting an endocytic receptor through an antibody offers a route to redirect extracellular cytokines into cells. Here, we developed BiAb-J31, an IgG-like bispecific antibody linking TNF-α recognition to a membrane-proximal region of asialoglycoprotein receptor 1 (ASGPR1). J31 was isolated by immunization with an engineered ASGPR1 extracellular domain followed by screening against wild-type ASGPR1 and HepG2 cells. Its variable domains were combined with unmodified adalimumab variable regions using knobs-into-holes and CrossMab engineering, with L234A/L235A/P329G Fc substitutions. BiAb-J31 retained HepG2 binding and engaged both antigens in bridging assays. Sequential binding experiments demonstrated recruitment of TNF-α to BiAb-J31-treated cells. Live-cell imaging showed uptake of fluorescent antibody-containing complexes and overlap with LysoTracker-positive compartments. In HepG2 cultures containing BiAb-J31, TNF-α and a PE-conjugated detection antibody, supernatant PE fluorescence fell by approximately 60% over 90 minutes. Truncation and synthetic-peptide assays localized J31 recognition to ASGPR1 residues 62–100, outside the carbohydrate-recognition domain, and AlphaFold 3 modeling proposed an Fv–peptide interface. These findings identify a membrane-proximal ASGPR1-binding antibody that is compatible with bispecific engineering and supports cellular recruitment and uptake of TNF-α-containing complexes. BiAb-J31 provides a molecular starting point for antibody-based cytokine-redirecting approaches.