Identification and characterization of blocking nanobodies against human CD70

Abstract

CD70 is overexpressed in a variety of solid and hematological tumors and plays a role in tumor proliferation and evasion of immune surveillance. Targeting and blocking its binding to the receptor CD27 have the potential to treat CD70-dependent tumors. To generate novel CD70 blocking agents, we screen a human CD70-immunized camel VHH phage display library and isolate two blocking nanobodies against human CD70 targeting different epitopes. Upon enrichment by three rounds of biopanning, two strategies are employed to identify CD70 blockers. One named affinity selection is used for detecting clones with CD70 binding by conventional PE-ELISA. However, no clone with a blocking effect is obtained from 188 enriched clones by this method. The alternative strategy named competitive selection is based on the inhibiting capacity of CD70-CD27 binding by enriched VHHs. By this method, two clones, Nb-2B3 and Nb-3B6, with strong blocking capacity are obtained from 20 enriched VHHs, suggesting the efficiency of this strategy. Furthermore, Nb-2B3 and Nb-3B6 specifically bind to CD70-positive SKOV3 and Raji cells at low concentrations. Meanwhile, Nb-2B3 has no competitive effect on the binding of Nb-3B6 to CD70, and vice versa, indicating that they target two different epitopes on CD70. Our data show that nanobodies Nb-2B3 and Nb-3B6 are potential attractive theranostic agents for CD70-expressing cancers.

Keywords

• CD70
• CD27
• nanobody
• phage display
• blocking antibody