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

Researchers have developed a novel bispecific antibody called BiAb J31, which binds to a specific region of the membrane-proximal ASGPR1. This antibody was created by genetically engineering an ASGPR1 extracellular domain and screening it against wild-type ASGPR1 and HepG2 cells. The variable domains of BiAb J31 were fused with the unmodified adalimumab variable regions using a technique known as knobs into holes and CrossMab engineering, with substitutions in the Fc region (L234A/L235A/P329G).

BiAb J31 maintains its ability to bind to HepG2 cells and simultaneously bridges both ASGPR1 and TNF. Sequential binding tests show that BiAb J31 triggers TNF recruitment to cells treated with the antibody. Live cell imaging reveals the uptake of antibody-containing complexes within cells and their overlap with compartments containing LysoTracker, a fluorescent dye that indicates the presence of cellular vesicles.

In cultures of HepG2 cells treated with BiAb J31, TNF and a detection antibody conjugated with PE (iodinated phosphate) show a 60% decrease in supernatant fluorescence over a 90-minute period.

Further research indicates that BiAb J31's recognition of ASGPR1 occurs at a specific region, from residues 62 to 100, located outside the carbohydrate-recognition domain. AlphaFold 3 modeling suggests that the bispecific antibody forms a peptide interface with ASGPR1. These findings demonstrate that BiAb J31 is compatible with the engineering of bispecific antibodies and supports the cellular recruitment and uptake of TNF-containing complexes.

This antibody serves as a promising starting point for developing new antibody-based approaches to redirect cytokines in the body.