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

Hanjing ZHOU; Jingshu LIU; Shuzi LONG; Cuiping TANG; Tao ZHANG

doi:10.16016/j.1000-5404.201612056

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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

更新时间：2026-01-09

- Preparation of human phage displayed single-chain antibody against hypoxia-inducible factor 1 alpha of laryngeal carcinoma and its effect on radiotherapy sensitization
- Journal of Third Military Medical University Vol. 39, Issue 16, Pages: 1625-1630(2017)
- 作者机构：
- 作者简介：
- 基金信息：
  
  the Projects of Traditional Chinese Medicine Science and Technology of Chongqing Municipal Health and Family Planning Commission(ZY20150245);the National Clinical Key-discipline Construction Program of China(国卫办医函(2013)544号)
- DOI：10.16016/j.1000-5404.201612056
  CLC：
- Received：08 December 2016，
  
  Published：30 August 2017
- 稿件说明：
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Hanjing ZHOU, Jingshu LIU, Shuzi LONG, et al. Preparation of human phage displayed single-chain antibody against hypoxia-inducible factor 1 alpha of laryngeal carcinoma and its effect on radiotherapy sensitization[J]. Journal of Third Military Medical University, 2017, 39(16): 1625-1630.
DOI：

Hanjing ZHOU, Jingshu LIU, Shuzi LONG, et al. Preparation of human phage displayed single-chain antibody against hypoxia-inducible factor 1 alpha of laryngeal carcinoma and its effect on radiotherapy sensitization[J]. Journal of Third Military Medical University, 2017, 39(16): 1625-1630. DOI： 10.16016/j.1000-5404.201612056.

摘要

Abstract

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 34×10

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 (

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.

关键词

Keywords

references

SUZUKI G YAMAZAKI H OGO E Predisposing Factors for Larynx Preservation Strategies with Non-surgical Multimodality Treatment for Locally Advanced (T3-4) Larynx, Hypopharynx and Cervical Esophageal Disease Anticancer Res 2014 34 9 5205 5210 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.

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 Clin Otolaryngol 2010 35 5 373 382 10.1111/j.1749-4486.2010.02191.x 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.

LAI S Z LI W F CHEN L How does intensity-modulated radiotherapy versus conventional two-dimensional radiotherapy influence the treatment results in nasopharyngeal carcinoma patients? Int J Radiat Oncol Biol Phys 2011 80 3 661 668 10.1016/j.ijrobp.2010.03.024 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.

KIM D H GONG E J JUNG H Y Clinical significance of intensive endoscopic screening for synchronous esophageal neoplasm in patients with head and neck squamous cell carcinoma Scand J Gastroenterol 2014 49 12 1486 1492 10.3109/00365521.2013.832369 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.

SEMENZA G L Hypoxia-inducible factors: mediators of cancer progression and targets for cancer therapy Trends Pharmacol Sci 2012 33 4 207 214 10.1016/j.tips.2012.01.005 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.

罗弋庞华李淑杰人源抗PeroxiredoxinⅠ肺腺癌噬菌体抗体的制备及鉴定南方医科大学学报 2010 36 5 841 846 10.13481/j.1671-587x.2010.05.050 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 Preparation and characterization of human phage display antibody against peroxiredoxin Ⅰ of lung adenocarcinoma J Southern Med Univ 2010 36 5 841 846 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.

ZHAO W S LUO Y LI B Y Anti-ABCG2 scFv antibody of lung adenocarcinoma increases chemosensitivity and induces apoptosis through the activation of mitochondrial pathway Am J Cancer Res 2016 6 5 1026 1039 http://www.ncbi.nlm.nih.gov/pubmed/27293996 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.

OVERGAARD J Hypoxic modification of radiotherapy in squamous cell carcinoma of the head and neck--a systematic review and meta-analysis Radiother Oncol 2011 100 1 22 32 10.1016/j.radonc.2011.03.004 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.

GODA N RYAN H E KHADIVI B Hypoxia-inducible factor 1α is essential for cell cycle arrest during hypoxia Mol Cell Biol 2003 23 1 359 369 10.1128/MCB.23.1.359-369.2003 GODA N, RYAN H E, KHADIVI B, et al . Hypoxia-inducible factor 1α is e ssential for cell cycle arrest during hypoxia[J ] . Mol Cell Biol, 2003, 23(1): 359-369. DOI: 10.1128/ MCB.23.1.359-369.2003.

HAN S X ZHU Q MA J L Apoptin sensitizes radiation-induced cell death via classic mitochondrial, caspase and p53-dependent signaling in HepG2 cells Mol Med Rep 2011 4 1 59 63 10.3892/mmr.2010.391 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.

OU G ITASAKA S ZENG L Usefulness of HIF-1 imaging for determining optimal timing of combining bevacizumab and radiotherapy Int J Radiat Oncol Biol Phys 2009 75 2 463 467 10.1016/j.ijrobp.2009.02.083 OU G, ITASAKA S, ZENG L, et al . Usefulness of HIF-1 imaging for determining optimal timing of co mbining bevacizumab and radiotherapy[J ] . Int J Radiat Oncol Biol Phys, 2009, 75(2): 463-467. DOI: 10.1016/j.ijrobp.2009.02.083.

CHEN G LI X YANG J Prognostic significance of cyclooxygenase-2 expression in patients with hepatocellular carcinoma: a meta-analysis Arch Med Sci 2016 12 5 1110 1117 10.5114/aoms.2016.61916 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.

DE CECCO L BOSSI P LOCATI L Comprehensive gene expression meta-analysis of head and neck squamous cell carcinoma microarray data defines a robust survival predictor Ann Oncol 2014 25 8 1628 1635 10.1093/annonc/mdu173 DE CECCO L, BOSSI P, LOCATI L, et al . Comprehensive gene expression meta-analysis of head and neck squamous cell carci noma microarray data defines a robust survival predictor[J ] . Ann Oncol, 2014, 25(8):1628-1635. DOI: 10.1093/annonc/mdu173.

NELSON A L DHIMOLEA E REICHERT J M Development trends for human monoclonal antibody therapeutics Nat Rev Drug Discov 2010 9 10 767 774 10.1038/nrd3229 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.

FARAJNIA S AHMADZADEH V TANOMAND A Development trends for generation of single-chain antibody fragments Immunopharmacol Immunotoxicol 2014 36 5 297 308 10.3109/08923973.2014.945126 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.

QIU Q WANG Q DENG C Small molecular peptide-ScFv αvβ3 conjugates specifically inhibit lung cancer cell growth in vitro and in vivo Am J Cancer Res 2016 6 12 2846 http://www.ncbi.nlm.nih.gov/pubmed/28042504 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.

PAN X Y LIU X J LI J The ANTITUMOR efficacy of anti-p21Ras scFv mediated by the dual-promoter-regulated recombinant adenovirus KGHV300 Gene Ther 2017 24 1 40 48 10.1038/gt.2016.74 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.

MOHAMMADI M NEJATOLLAHI F GHASEMI Y Anti-Metastatic and Anti-Invasion Effects of a Specific Anti-MUC18 scFv Antibody on Breast Cancer Cells Appl Biochem Biotechnol 2017 181 1 379 390 10.1007/s12010-016-2218-1 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.

HAN D WU J HAN Y A novel anti-PSMA human scFv has the potential to be used as a diagnostic tool in prostate cancer Oncotarget 2016 7 37 59471 59481 10.18632/oncotarget.10697 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.

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