Engineering a bivalent anti-CsGRP78 nanobody via CDR grafting: in silico design and cytotoxic validation

GRP78 is a central endoplasmic reticulum (ER) chaperone and the primary sensor for the Unfolded Protein Response (UPR). Under cellular stress, GRP78 translocates to the cell surface (CsGRP78), where it functions as a signal-transducing receptor. CsGRP78 acts as a port of entry for viruses and plays a critical role in cancer pathogenesis by promoting tumor cell proliferation, survival, stemness, invasion, and metastasis. Its dysregulation in diseases like diabetes and cardiovascular disorders makes it a significant therapeutic target. Anti-GRP78 antibodies have therapeutic potential. However, conventional full-length antibodies face challenges, including insufficient tissue penetration and production difficulties. To overcome these, we engineered a novel, nanobody-based antagonist. Using CDR grafting and subsequent point mutations, we transferred the specificity of a parental antibody onto two nanobody scaffolds to create a bivalent construct, termed “CsGRP78-diaNb.” In silico docking and molecular dynamics simulations suggested promising target engagement. CsGRP78-diaNb was expressed, and its cytotoxicity was evaluated using an MTT assay. It demonstrated selective cytotoxicity against B16F10 cancer cells that overexpress CsGRP78, compared with the negative control CHO-S cells. These preliminary findings suggest that CsGRP78-diaNb is a promising candidate for further development as a targeted therapeutic, pending direct validation of cell-surface binding and in vivo efficacy.