In vitro affinity maturation to improve the efficacy of a hypoxia-inducible factor 1α single-domain intrabody.

Affinity is an important property of therapeutic antibodies, so improving affinity is critical to the biological activity and clinical efficacy. An anti–HIF–1α nanobody, VHH212, was screened via a native ribosome display library with a 26.6 nM of K D value was used as the parent. In this paper, a Venn-intersection of multi-algorithms screening (VIMAS) strategy for computer-aided binding affinity prediction was designed. Homology modeling and protein docking methods were used to substitute the need for a crystal structure. Finally, a mutant with a 17.5-fold enhancement in binding affinity (1.52 nM) was obtained by using the VIMAS strategy. Furthermore, the biological activity of mutants was verified at the cellular level. Targeting HIF-1α can sensitize PDAC (pancreatic ductal adenocarcinoma) tumors to gemcitabine, which is a potential co-treatment method for pancreatic cancer patients. Our results showed that the cytotoxicity of gemcitabine on pancreatic cancer cell lines increased with the enhanced-affinity of an intrabody under combined treatment. • Provide a strategy for in silico affinity maturation of nanobodies. • Affinity of mutants increased via computer-aided design. • Nanobody-based intrabodies provide a specific strategy for combined therapy. • Targeting HIF-1α with nanobody increased the cytotoxicity of gemcitabine to MIA PaCa-2 and BxPC-3 cell lines. [ABSTRACT FROM AUTHOR]