Abstract

Recent advances in bispecific agents targeting both CD19 and CD20 highlight their potential as an effective strategy for improving clinical outcomes in B-cell malignancies. Herein, we developed a CD19/CD20 bispecific nanobody, 19Nb1-20Nb2, by fusing the anti-CD19 nanobody 19Nb1 to the N-terminus of the anti-CD20 nanobody 20Nb2. To reconstitute missing Fc effector functions, 19Nb1-20Nb2 was site-specifically conjugated with a tetravalent rhamnose (Rha) derivative via sortase A-mediated ligation. The resulting conjugate, 19Nb1-20Nb2-(Rha)₄, exhibited good affinity for CD19/CD20 dual-positive cancer cells, and was capable of triggering potent antibody-dependent cell-mediated phagocytosis and complement-dependent cytotoxicity for B cell killing through recruiting high level of anti-Rha antibodies. Furthermore, the bispecific conjugate 19Nb1-20Nb2-(Rha)₄ demonstrated higher anti-tumor activity in vitro compared to monospecific therapeutics. This study provides a novel perspective for the development of CD19/CD20 dual-targeting therapeutic strategies.