VHH212 nanobody targeting the hypoxia-inducible factor 1α suppresses angiogenesis and potentiates gemcitabine therapy in pancreatic cancer in vivo  

作　　者：Guangbo Kang Min Hu He Ren Jiewen Wang Xin Cheng Ruowei Li Bo Yuan Yasmine Balan Zixuan Bai He Huang 

机构地区：[1]Department of Biochemical Engineering,School of Chemical Engineering&Technology,Tianjin University,Tianjin 300350,China [2]Key Laboratory of Systems Bioengineering(Ministry of Education),Tianjin University,Tianjin 300072,China [3]Department of Gastroenterology,Center of Tumor Immunology and Cytotherapy,Medical Research Center of The Affiliated Hospital of Qingdao University,Qingdao 266003,China [4]Department of Chemical and Biological Engineering,University of Ottawa,Ottawa KIN 6N5,Canada

出　　处：《Cancer Biology & Medicine》2021年第3期772-787,共16页癌症生物学与医学（英文版）

基　　金：supported by grants from the National Key Research and Development Project(Grant No.2019YFA0905600);the Major State Basic Research Development Program of the Natural Science Foundation of Shandong Province in China(Grant No.ZR2020ZD11);the National Natural Science Foundation of China(Grant Nos.81772633 and 31470967);the Science and Technology Program of Tianjin,China(Grant No.19YFSLQY00110);the Taishan Scholars Program of Shandong Province。

摘　　要：Objective:We aimed to develop a novel anti-HIF-1αintrabody 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 affinity and specificity of anti-HIF-1αVHH212[a single-domain antibody(nanobody)].Molecular dynamics simulation was used to determine the protein-protein interactions between hypoxia-inducible factor-1α(HIF-1α)and VHH212.The real-time polymerase chain reaction(PCR)and Western blot analyses were performed to identify the expressions of HIF-1αand VEGF-A in pancreatic ductal adenocarcinoma cell lines.The efficiency of the VHH212 nanobody in inhibiting the HIF-1 signaling pathway was measured using a dual-luciferase reporter assay.Finally,a PANC-1 xenograft model was developed to evaluate the anti-tumor efficiency of combined treatment.Immunohistochemistry analysis was conducted to detect the expressions of HIF-1αand VEGF-A in tumor tissues.Results:VHH212 was stably expressed in tumor cells with low cytotoxicity,high affinity,specific subcellular localization,and neutralization of HIF-1αin the cytoplasm or nucleus.The binding affinity between VHH212 and the HIF-1αPAS-B domain was 42.7 n M.Intrabody competitive inhibition of the HIF-1αheterodimer with an aryl hydrocarbon receptor nuclear translocator was used to inhibit the HIF-1/VEGF pathway in vitro.Compared with single agent gemcitabine,co-treatment with gemcitabine and a VHH212-encoding adenovirus significantly suppressed tumor growth in the xenograft model with 80.44%tumor inhibition.Conclusions:We developed an anti-HIF-1αnanobody and showed the function of VHH212 in a preclinical murine model of PANC-1 pancreatic cancer.The combination of VHH212 and gemcitabine significantly inhibited tumor development.These results suggested that combined use of anti-HIF-1αnanobodies with first-line treatment may in the future be an effective treatment for pancreatic cancer.

关 键 词：Pancreatic cancer nanobody therapeutic intracellular antibody HIF-1αinhibitor GEMCITABINE CHEMOSENSITIZER 

分 类 号：R735.9[医药卫生—肿瘤]