VHH212 nanobody targeting the hypoxia-inducible factor 1α suppresses angiogenesis and potentiates gemcitabine therapy in pancreatic cancer <i>in vivo</i>
Guangbo Kang, Min Hu, He Ren, Jiewen Wang, Xin Cheng, Ruowei Li, Bo Yuan, Yasmine Balan, Zixuan Bai, He Huang
Abstract
Objective: We aimed to develop a novel anti-HIF-1α intrabody to decrease gemcitabine resistance in pancreatic cancer patients. Methods: Surface plasmon resonance and glutathione S-transferase pull-down assays were conducted to identify the binding affinityand specificity of anti-HIF-1α VHH212 [a single-domain antibody (nanobody)]. Molecular dynamics simulation was used todetermine the protein-protein interactions between hypoxia-inducible factor-1α (HIF-1α) and VHH212. The real-time polymerasechain reaction (PCR) and Western blot analyses were performed to identify the expressions of HIF-1α and VEGF-A in pancreatic ductaladenocarcinoma cell lines. The efficiency of the VHH212 nanobody in inhibiting the HIF-1 signaling pathway was measured using adual-luciferase reporter assay. Finally, a PANC-1 xenograft model was developed to evaluate the anti-tumor efficiency of combinedtreatment. Immunohistochemistry analysis was conducted to detect the expressions of HIF-1α and VEGF-A in tumor tissues. Results: VHH212 was stably expressed in tumor cells with low cytotoxicity, high affinity, specific subcellular localization, andneutralization of HIF-1α in the cytoplasm or nucleus. The binding affinity between VHH212 and the HIF-1α PAS-B domain was42.7 nM. Intrabody competitive inhibition of the HIF-1α heterodimer with an aryl hydrocarbon receptor nuclear translocator wasused to inhibit the HIF-1/VEGF pathway in vitro. Compared with single agent gemcitabine, co-treatment with gemcitabine and aVHH212-encoding adenovirus significantly suppressed tumor growth in the xenograft model with 80.44% tumor inhibition. Conclusions: We developed an anti-HIF-1α nanobody and showed the function of VHH212 in a preclinical murine model ofPANC-1 pancreatic cancer. The combination of VHH212 and gemcitabine significantly inhibited tumor development. These resultssuggested that combined use of anti-HIF-1α nanobodies with first-line treatment may in the future be an effective treatment forpancreatic cancer.