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 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 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 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 containing complexes. BiAb J31 provides a molecular starting point for antibody based cytokine redirecting approaches.