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Preparation of human phage displayed single-chain antibody against hypoxia-inducible factor 1 alpha of laryngeal carcinoma and its effect on radiotherapy sensitization



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Preparation of human phage displayed single-chain antibody against hypoxia-inducible factor 1 alpha of laryngeal carcinoma and its effect on radiotherapy sensitization


ZHOU Hanjing LIU Jingshu LONG Shuzi TANG Cuiping ZHANG Tao

Department of Oncology, Chongqing Key Laboratory of Molecular Oncology and Epigenetics, Department of Radiation Medicine and Oncology, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China


phage-displayed single-chain variable fragment antibody laryngeal carcinoma hypoxia-inducible factor 1 alpha radiotherapy sensitization

R392-33; R392.11; R739.65

Objective     To construct a phage-displayed library and identify phage-displayed single-chain variable fragment (scFv) antibody against hypoxia-inducible factor 1 alpha (HIF1α) of laryngeal carcinoma. Methods      Total RNA of positive lymph node tissues adjacent to laryngeal carcinoma was extracted. ScFv gene fragments were amplified by RT-PCR and splicing-overlap-extension (SOE) PCR, then linked into the pCANTAB5E phagemid vector. The recombinant plasmids were transformed into E.coli TG1 cells in order to create the primary phage-displayed scFv library. The primary scFv library was performed by biopanning against HEP2 cells and HIF1α antigen. The properties of the product were identified by Western blotting, and its specificity was detected by ELISA and immunocytochemical assay. CCK8 assay was used to detect its effect on cell viability, and cell clone assay was used to detect its effect on survival fraction. Results     A phage-displayed scFv library targeting HIF1α antigen and HEP2 cells was successfully constructed. The prepared scFv antibody had an apparent molecular weight of 34×103 and down-regulated the expression of HIF1α protein in HEP2 cells. ELISA showed that the obtained scFv antibody had a positive recognition rate of 79% to HIF1α antigen. Immunocytochemical assay indicated that it could specifically bind to HEP2 cells. The obtained scFv antibody decreased the viability of HEP2 cells after X-ray radiation when compared with the cells treated by radiation alone (P<0.05). Colony formation test showed that the sensitization enhancement ratio (SER) of the antibody to radiation was 1.89. Conclusion    A human anti-HIF1α scFv antibody of laryngeal carcinoma is successfully prepared, and it can increase the radiation sensitivity of HEP2 cells.


[1]SUZUKI G, YAMAZAKI H, OGO E, et al. Predisposing Factors for Larynx Preservation Strategies with Nonsurgical Multimodality Treatment for Locally Advanced (T34) Larynx, Hypopharynx and Cervical Esophageal Disease[J]. Anticancer Res, 2014, 34(9): 5205-5210.
[2]SPIELMANN P M, MAJUMDAR S, MORTON R P. Quality of life and functional outcomes in the management of early glottic carcinoma: a systematic review of studies comparing radiotherapy and transoral laser microsurgery[J]. Clin Otolaryngol, 2010, 35(5): 373-382. DOI: 10.1111/ j.1749-4486.2010.02191.x.
[3]LAI S Z, LI W F, CHEN L, et al. How does intensitymodulated radiotherapy versus conventional two-dimensional radiotherapy influence the treatment results in nasopharyngeal carcinoma patients?[J]. Int J Radiat Oncol Biol Phys, 2011, 80(3): 661-668. DOI: 10.1016/ j.ijrobp.2010.03.024.
[4]KIM D H, GONG E J, JUNG H Y, et al. Clinical significance of intensive endoscopic screening for synchronous esophageal neoplasm in patients with head and neck squamous cell carcinoma[J]. Scand J Gastroenterol, 2014, 49(12): 1486-1492. DOI: 10.3109/00365521.2013.832369.
[5]SEMENZA G L. Hypoxiainducible factors: mediators of cancer progression and targets for cancer therapy[J]. Trends Pharmacol Sci, 2012, 33(4): 207-214. DOI: 10.1016/ j.tips.2012.01.005.
[6]罗弋, 庞华, 李淑杰, 等. 人源抗PeroxiredoxinⅠ肺腺癌噬菌体抗体的制备及鉴定[J]. 南方医科大学学报, 2010, 36(5): 841-846. DOI:10.13481/j.1671-587x.2010.05.050.
LUO Y, PANG H, LI S J, et al.Preparation and characterization of human phage display antibody against peroxiredoxin I of lung adenocarcinoma[J]. J Southern Med Univ, 2010, 36(5): 841-846. DOI:10.13481/j.1671-587x.2010.05.050. 
[7]ZHAO W S, LUO Y, LI B Y, et al. AntiABCG2 scFv antibody of lung adenocarcinoma increases chemosensitivity and induces apoptosis through the activation of mitochondrial pathway[J]. Am J Cancer Res, 2016, 6(5): 1026-1039.
[8]OVERGAARD J. Hypoxic modification of radiotherapy in squamous cell carcinoma of the head and neck-a systematic review and meta-analysis[J]. Radiother Oncol, 2011, 100(1): 22-32. DOI: 10.1016/j.radonc.2011.03.004.
[9]GODA N, RYAN H E, KHADIVI B, et al. Hypoxiainducible factor 1α is essential for cell cycle arrest during hypoxia[J]. Mol Cell Biol, 2003, 23(1): 359-369. DOI: 10.1128/ MCB.23.1.359369.2003.
[10]HAN S X, ZHU Q, MA J L, et al. Apoptin sensitizes radiationinduced cell death via classic mitochondrial, caspase and p53dependent signaling in HepG2 cells[J]. Mol Med Rep, 2011, 4(1): 59-63. DOI: 10.3892/mmr.2010.391.
[11]OU G, ITASAKA S, ZENG L, et al. Usefulness of HIF-1 imaging for determining optimal timing of combining bevacizumab and radiotherapy[J]. Int J Radiat Oncol Biol Phys, 2009, 75(2): 463-467. DOI: 10.1016/j.ijrobp.2009.02.083.
[12]CHEN G, LI X, YANG J, et al. Prognostic significance of cyclooxygenase-2 expression in patients with hepatocellular carcinoma: a meta-analysis[J]. Arch Med Sci, 2016, 12(5): 1110-1117. DOI: 10.5114/aoms.2016.61916.
[13]DE CECCO L, BOSSI P, LOCATI L, et al. Comprehensive gene expression metaanalysis of head and neck squamous cell carcinoma microarray data defines a robust survival predictor[J]. Ann Oncol, 2014, 25(8):1628-1635. DOI: 10.1093/annonc/mdu173.
[14]NELSON A L, DHIMOLEA E, REICHERT J M. Development trends for human monoclonal antibody therapeutics[J]. Nat Rev Drug Discov, 2010, 9(10): 767-774. DOI: 10.1038/nrd3229.
[15]FARAJNIA S, AHMADZADEH V, TANOMAND A, et al. Development trends for generation of single-chain antibody fragments[J]. Immunopharmacol Immunotoxicol, 2014, 36(5): 297-308. DOI: 10.3109/08923973.2014.945126.
[16]QIU Q, WANG Q, DENG C, et al. Small molecular peptide-ScFv αvβ3 conjugates specifically inhibit lung cancer cell growth in vitro and in vivo[J]. Am J Cancer Res, 2016, 6(12): 2846.
[17]PAN X Y, LIU X J, LI J, et al. The ANTITUMOR efficacy of antip21Ras scFv mediated by the dualpromoterregulated recombinant adenovirus KGHV300[J]. Gene Ther, 2017, 24(1): 40-48. DOI:10.1038/gt.2016.74.
[18]MOHAMMADI M, NEJATOLLAHI F, GHASEMI Y, et al. Anti-Metastatic and Anti-Invasion Effects of a Specific AntiMUC18 scFv Antibody on Breast Cancer Cells[J]. Appl Biochem Biotechnol, 2017, 181(1): 379-390. DOI:10.1007/s12010-016-2218-1.
[19]HAN D, WU J, HAN Y, et al. A novel antiPSMA human scFv has the potential to be used as a diagnostic tool in prostate cancer[J]. Oncotarget, 2016, 7(37): 59471-59481. DOI:10.18632/oncotarget.10697.


Last Update: 2017-08-23