Generation of a CD70-Specific Fusion Nanobody with IgG Recruiting Capacity for Tumor Killing

Due to its competitive advantages such as small size, high stability, easy production, and good tissue penetration compared with monoclonal antibodies (mAb), nanobodies (Nbs) were considered the next generation of therapeutics. However, the absence of Fc fragments and Fc-triggered immune effectors limits their clinical applications. In order to overcome these limitations, we develop a novel approach by attaching an IgG binding domain (IgBD) to Nbs for recruiting endogenous IgG and recovering the immune effectors for tumor killing.

We linked a Streptococcal Protein G-derived IgBD, termed C3Fab, at the C-terminus of a CD70-specific Nb 3B6 to construct an endogenous IgG recruitment antibody (termed EIR). The recombinant Nb3B6-C3Fab was expressed in E. coli BL21 (DE3) and purified by nickel affinity chromatography. We further evaluated the binding, recruitment of IgG, and the serum half-life of Nb3B6-C3Fab. The tumor-killing effects on CD70 positive cells mediated by antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity were also detected.

We successfully constructed a IgBD fused Nb3B6-C3Fab with high affinity for CD70 and mouse IgG (mIgG). Nb3B6-C3Fab can specifically bind to CD70 positive tumor cells and recruit mIgG on the cell surface. Ligating of Nb3B6 with C3Fab increased its serum half-life in mice almost 39-fold from 0.96 h to 37.67 h. Moreover, we demonstrated remarkable cytotoxicity of Nb3B6-C3Fab to CD70 positive tumor cells via C3Fab by immune effector cells.

Our study demonstrates that IgBD fusion endows Nbs with the ability for endogenous IgG recruitment and half-life promotion. Linking IgBD to Nbs is an effective strategy to recovering immune effectors for tumor killing.