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

• Published in: Biochemical and biophysical research communications Vol. 529; no. 4; pp. 936 - 942
• Main Authors: Hu, Min, Kang, Guangbo, Cheng, Xin, Wang, Jiewen, Li, Ruowei, Bai, Zixuan, Yang, Dong, Huang, He
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.09.2020
• Subjects: Affinity maturation; Algorithms; Antibody Affinity; Antibody Specificity; Antimetabolites, Antineoplastic - pharmacology; Antineoplastic Agents, Immunological - chemistry; Antineoplastic Agents, Immunological - metabolism; Antineoplastic Agents, Immunological - pharmacology; Binding Sites; Cell Line, Tumor; Cell Survival - drug effects; Cell Survival - genetics; Combined treatment; Deoxycytidine - analogs & derivatives; Deoxycytidine - pharmacology; Homology modeling; Humans; Hypoxia-inducible factor; Hypoxia-Inducible Factor 1, alpha Subunit - antagonists & inhibitors; Hypoxia-Inducible Factor 1, alpha Subunit - genetics; Hypoxia-Inducible Factor 1, alpha Subunit - immunology; Molecular Docking Simulation; Molecular Dynamics Simulation; Mutation; Nanobody; Pancreatic Ducts - immunology; Pancreatic Ducts - pathology; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Single-Domain Antibodies - chemistry; Single-Domain Antibodies - genetics; Single-Domain Antibodies - pharmacology; Structural Homology, Protein; User-Computer Interface; Hypoxia-inducible factor; Nanobody; Combined treatment; Affinity maturation; Homology modeling
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2020.06.097

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 KD 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.