Novel EBV LMP-2-affibody and affitoxin in molecular imaging and targeted therapy of nasopharyngeal carcinoma

Autoři: Shanli Zhu aff001;  Jun Chen aff001;  Yirong Xiong aff001;  Saidu Kamara aff001;  Meiping Gu aff001;  Wanlin Tang aff001;  Shao Chen aff001;  Haiyan Dong aff001;  Xiangyang Xue aff001;  Zhi-Ming Zheng aff002;  Lifang Zhang aff001
Působiště autorů: Institute of Molecular Virology and Immunology, Department of Microbiology and Immunology, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China aff001;  Tumor Virus RNA Biology Section, RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland, United States of America aff002
Vyšlo v časopise: Novel EBV LMP-2-affibody and affitoxin in molecular imaging and targeted therapy of nasopharyngeal carcinoma. PLoS Pathog 16(1): e32767. doi:10.1371/journal.ppat.1008223
Kategorie: Research Article


Epstein-Barr virus (EBV) infection is closely linked to several human malignancies including endemic Burkitt’s lymphoma, Hodgkin’s lymphoma and nasopharyngeal carcinomas (NPC). Latent membrane protein 2 (LMP-2) of EBV plays a pivotal role in pathogenesis of EBV-related tumors and thus, is a potential target for diagnosis and targeted therapy of EBV LMP-2+ malignant cancers. Affibody molecules are developing as imaging probes and tumor-targeted delivery of small molecules. In this study, four EBV LMP-2-binding affibodies (ZEBV LMP-212, ZEBV LMP-2132, ZEBV LMP-2137, and ZEBV LMP-2142) were identified by screening a phage-displayed LMP-2 peptide library for molecular imaging and targeted therapy in EBV xenograft mice model. ZEBV LMP-2 affibody has high binding affinity for EBV LMP-2 and accumulates in mouse tumor derived from EBV LMP-2+ xenografts for 24 h after intravenous (IV) injection. Subsequent fusion of Pseudomonas exotoxin PE38KDEL to the ZEBV LMP-2 142 affibody led to production of Z142X affitoxin. This fused Z142X affitoxin exhibits high cytotoxicity specific for EBV+ cells in vitro and significant antitumor effect in mice bearing EBV+ tumor xenografts by IV injection. The data provide the proof of principle that EBV LMP-2-speicifc affibody molecules are useful for molecular imaging diagnosis and have potentials for targeted therapy of LMP-2-expressing EBV malignancies.

Klíčová slova:

Binding analysis – Cancer detection and diagnosis – Cell fusion – Cell staining – Cytotoxicity – Epstein-Barr virus – Nasopharyngeal carcinoma – Phage display


1. Thorley-lawson DA, Hawkins JB, Tracy SI, Shapiro M. The Pathogenesis of Epstein-Barr Virus Persistent Infection. Curr OpinVirol. 2013;3(3):227–32. doi: 10.1016/j.coviro.2013.04.005 23683686

2. Young LS, Yap LF, Murray PG. Epstein-Barr virus: more than 50 years old and still providing surprises. Nat Rev Cancer. 2016;16(12):789. doi: 10.1038/nrc.2016.92 27687982

3. Tsao SW, Tsang CM, To KF, Lo KW. The role of Epstein-Barr virus in epithelial malignancies. J Pathol. 2015;235(2):323–333. doi: 10.1002/path.4448 25251730

4. Wolf H, Zur HH, Becker V. EB viral genomes in epithelial nasopharyngeal carcinoma cells. Nat New Biol.1973; 244(138):245–247. doi: 10.1038/newbio244245a0 4353684

5. Pathmanathan R, Prasad U, Sadler R, Flynn K, Raabtraub N. Clonal proliferations of cells infected with Epstein-Barr virus in preinvasive lesions related to nasopharyngeal carcinoma. N Engl J Med. 1995;333(11):693–698. doi: 10.1056/NEJM199509143331103 7637746

6. de Schryver A, Friberg S Jr, Klein G, Henle W, Henle G, De-Thé G, et al. Epstein-Barr virus-associated antibody patterns in carcinoma of the post-nasal space. Clin Exp Immunol. 1969;5(5):443–459. 4902604

7. Henle W, Henle G, Ho HC, Burtin P, Cachin Y, Clifford P, et al. Antibodies to Epstein-Barr virus in nasopharyngeal carcinoma, other head and neck neoplasms, and control groups. J Natl Cancer Inst.1970;44(1):225–31. 11515035

8. Price AM, LuftigMA. To be or not IIb: a multi-step process for Epstein-Barr virus latency establishment and consequences for B cell tumorigenesis. PLoS Pathog. 2015;11(3):e1004656. doi: 10.1371/journal.ppat.1004656 25790223

9. Gewurz BE, Mar JC, Padi M, Zhao B, Shinners NP, Takasaki K,et al. Canonical NF-κB Activation Is Essential for Epstein-Barr Virus Latent Membrane Protein 1 TES2/CTAR2 Gene Regulation. J Virol.2011;85(13):6764–73. doi: 10.1128/JVI.00422-11 21543491

10. Greenfeld H, Takasaki K, Walsh MJ, Ersing I, Bernhardt K, Ma Y, et al. TRAF1 Coordinates Polyubiquitin Signaling to Enhance Epstein-Barr Virus LMP1-Mediated Growth and Survival Pathway Activation. Plos Pathog.2015;11(5): e1004890. doi: 10.1371/journal.ppat.1004890 25996949

11. Young LS, Rickinson AB. Epstein-Barr virus: 40 years on. Nat Rev Cancer. 204; 4(10): 757–68. doi: 10.1038/nrc1452 15510157

12. Majerciak V, Yang W, Zheng J, Zhu J, Zheng ZM. A Genome-Wide Epstein-Barr Virus Polyadenylation Map and Its Antisense RNA to EBNA. J Virol. 2019;93(2). pii: e01593–18. doi: 10.1128/JVI.01593-18 30355690

13. Longnecker R. Epstein-Barr virus latency: LMP-2, a regulator or means for Epstein-Barr virus persistence? Adv Cancer Res.2000;79:175–200. doi: 10.1016/s0065-230x(00)79006-3 10818681

14. Sutkowski N, Chen G, Calderon G, Huber BT. Epstein-Barr virus latent membrane protein LMP-2A is sufficient for transactivation of the human endogenous retrovirus HERV-K18 superantigen. J Virol.2004; 78(14):7852–7860. doi: 10.1128/JVI.78.14.7852-7860.2004 15220463

15. Zhang L, Pagano JS. Interferon regulatory factor 7: a key cellular mediator of LMP-1 in EBV latency and transformation. Semin Cancer Biol.2011;11(6):445–53. doi: 10.1006/scbi.2001.0411 11669606

16. Pang MF, Lin KW, Peh SC. The signaling pathways of Epstein-Barr virus-encoded latent membrane protein 2A (LMP2A) in latency and cancer. Cell Mol Biol Lett. 2009; 14(2):222–247. doi: 10.2478/s11658-008-0045-2 19082921

17. Rechsteiner MP, Berger C, Zauner L, Sigrist JA, Weber M, Longnecker R, et al. Latent membrane protein 2B regulates susceptibility to induction of lytic Epstein-Barr virus infection. J Virol.2008;82(4):1739–1747. doi: 10.1128/JVI.01723-07 18057232

18. Rovedo M, Longnecker R. Epstein-Barr Virus Latent Membrane Protein 2B (LMP2B) Modulates LMP2A Activity. J Virol.2007;81(1):84–94. doi: 10.1128/JVI.01302-06 17035319

19. Löfblom J, Feldwisch J, Tolmachev V, Carlsson J, Ståhl S, Frejd FY. Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett. 2010; 584(12):2670–2680. doi: 10.1016/j.febslet.2010.04.014 20388508

20. Nord K, Nilsson J, Nilsson B, Uhlén M, Nygren PA. A combinatorial library of an alpha-helical bacterial receptor domain. Protein Eng. 1995;8(6):601–8. doi: 10.1093/protein/8.6.601 8532685

21. Frejd FY, Kim KT. Affibody molecules as engineered protein drugs. Exp Mol Med. 2017;49(3):e306. doi: 10.1038/emm.2017.35 28336959

22. Ståhl S, Gräslund T, Eriksson Karlström A, Frejd FY, Nygren PÅ, et al. Affibody Molecules in Biotechnological and Medical Applications. Trends Biotechnol.2017; 35(8):691–712. doi: 10.1016/j.tibtech.2017.04.007 28514998

23. Andersson KG, Oroujeni M, Garousi J, Mitran B, Ståhl S, Orlova A, et al. Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with99mTc using a peptide-based cysteine-containing chelator. Int J Oncol.2016; 49(6):2285–2293. doi: 10.3892/ijo.2016.3721 27748899

24. Oroujeni M, Garousi J, Andersson KG, Löfblom J, Mitran B, Orlova A, et al. Preclinical Evaluation of [68Ga]Ga-DFO-ZEGFR:2377: A Promising Affibody-Based Probe for Noninvasive PET Imaging of EGFR Expression in Tumors. Cells. 2018;18: 7(9). doi: 10.3390/cells7090141 30231504

25. Orlova A, Magnusson M, Eriksson TL, Nilsson M, Larsson B, Höidén-Guthenberg I, et al. Tumor Imaging Using a Picomolar Affinity HER2 Binding Affibody Molecule. Cancer Res. 2006; 66(8):4339–4348. doi: 10.1158/0008-5472.CAN-05-3521 16618759

26. Sörensen J, Velikyan I, Sandberg D, Wennborg A, Feldwisch J, Tolmachev V, et al. Measuring HER2-Receptor Expression In Metastatic Breast Cancer Using [68Ga]ABY-025 Affibody PET/CT. Theranostics.2016; 6:262–71. doi: 10.7150/thno.13502 26877784

27. Malm M, Kronqvist N, Lindberg H, Gudmundsdotter L, Bass T, Frejd FY, et al. Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification. Plos One. 2013; 8: e62791. doi: 10.1371/journal.pone.0062791 23675426

28. Schardt JS, Oubaid JM, Williams SC, Howard JL, Aloimonos CM, Bookstaver ML, et al. Engineered Multivalency Enhances Affibody-Based HER3 Inhibition and Downregulation in Cancer Cells. Mol Pharm.2017; 14:1047–1056. doi: 10.1021/acs.molpharmaceut.6b00919 28248115

29. Fedorova A, Zobel K, Gill HS, Ogasawara A, Flores JE, Tinianow JN, et al. The Development of Peptide-Based Tools for the Analysis of Angiogenesis. Chem Biol. 2011;18(7):839–845. doi: 10.1016/j.chembiol.2011.05.011 21802005

30. Xue X, Wang B, Du W, Zhang C, Song Y, Cai Y,et al. Generation of affibody molecules specific for HPV16 E7 recognition. Oncotarget.2016;7(45):73995. doi: 10.18632/oncotarget.12174 27659535

31. Zhu S, Zhu J, Song Y, Chen J, Wang L, Zhou M, et al. Bispecific affibody molecule targeting HPV16 and HPV18E7 oncoproteins for enhanced molecular imaging of cervical cancer. Appl Microbiol Biotechnol.2018; doi: 10.1007/s00253-018-9167-2 29938318

32. Kreitman RJ, Pastan I. Importance of the glutamate residue of KDEL in increasing the cytotoxicity of Pseudomonas exotoxin derivatives and for increased binding to the KDEL receptor. Biochem J. 1995;307 (Pt 1):29–37.

33. Michalska M, Wolf P. Pseudomonas Exotoxin A: optimized by evolution for effective killing. Front Microbiol. 2015;6:963. doi: 10.3389/fmicb.2015.00963 eCollection 2015. 26441897

34. Mazor R, Onda M, Pastan I. Immunogenicity of therapeutic recombinant immunotoxins. Immunol Rev. 2016;270(1):152–64. doi: 10.1111/imr.12390 26864110

35. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin.2015;65(2):87–108. doi: 10.3322/caac.21262 25651787

36. Chia WK, Teo M, Wang WW, Lee B, Ang SF, Tai WM, et al. Adoptive T-cell Transfer and Chemotherapy in the First-line Treatment of Metastatic and/or Locally Recurrent Nasopharyngeal Carcinoma. Mol Ther.2014;22(1):132–139. doi: 10.1038/mt.2013.242 24297049

37. Chen MY, Jiang R, Guo L, Zou X, Liu Q, Sun R, et al. Locoregional radiotherapy in patients with distant metastases of nasopharyngeal carcinoma at diagnosis. Chin J Cancer. 2013; 32(11):604–613. doi: 10.5732/cjc.013.10148 24206918

38. Niedobitek G, Hansmann ML, Herbst H, Young LS, Dienemann D, Hartmann CA, et al. Epstein-Barr virus and carcinomas: undifferentiated carcinomas but not squamous cell carcinomas of the nasopharynx are regularly associated with the virus. J Pathol.1991;165(1):17–24. doi: 10.1002/path.1711650105 1659626

39. Longnecker R, Kieff E. A second Epstein-Barr virus membrane protein (LMP2) is expressed in latent infection and colocalizes with LMP1. J Virol.1990;64(5):2319–26. 2157888

40. Tomaszewski-flick MJ, Rowe DT. Minimal protein domain requirements for the intracellular localization and self-aggregation of Epstein-Barr virus latent membrane protein 2. Virus Genes.2007; 35(2):225–234. doi: 10.1007/s11262-007-0118-8 17564822

41. Feldwisch J, Tolmachev V. Engineering of affibody molecules for therapy and diagnostics. Methods Mol Biol. 2012;899:103–126. doi: 10.1007/978-1-61779-921-1_7 22735949

42. Mitran B, Altai M, Hofström C, Honarvar H, Sandström M, Orlova A, et al. Evaluation of 99mTc-Z IGF1R:4551-GGGC affibody molecule, a new probe for imaging of insulin-like growth factor type 1 receptor expression. Amino acids.2015;47(2):303–15. doi: 10.1007/s00726-014-1859-z 25425114

43. Wikman M, Rowcliffe E, Friedman M, Henning P, Lindholm L, Olofsson S, et al. Selection and characterization of an HIV-1 gp120-binding affibody ligand. Biotechnol Appl Biochem.2006; 45(Pt 2):93–105. doi: 10.1042/BA20060016 16712522

44. Sörensen J, Sandberg D, Sandström M, Wennborg A, Feldwisch J, Tolmachev V, et al. First-in-human molecular imaging of HER2 expression in breast cancermetastases using the 111In-ABY-025 affibody molecule. J Nucl Med. 2014;55(5):730–5. doi: 10.2967/jnumed.113.131243 24665085

45. Cheng Q, Wållberg H, Grafström J, Lu L, Thorell JO, HäggOlofsson M, et al. Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake. EJNMMI Res. 2016;6(1):58. doi: 10.1186/s13550-016-0213-8 27388754

46. Oroujeni M, Garousi J, Andersson KG, Löfblom J, Mitran B, Orlova A, et al. Preclinical Evaluation of [68Ga]Ga-DFO-ZEGFR:2377: A Promising Affibody-Based Probe for Noninvasive PET Imaging of EGFR Expression in Tumors. Cells. 2018;7(9). pii: E141. doi: 10.3390/cells7090141 30231504

47. Rosestedt M, Andersson KG, Mitran B, Tolmachev V, Löfblom J, Orlova A, et al. Affibody-mediated PET imaging of HER3 expression in malignant tumours.Sci Rep. 2015;5:15226. doi: 10.1038/srep15226 26477646

48. Baum RP, Prasad V, Müller D, Schuchardt C, Orlova A, Wennborg A, et al. Molecular imaging of HER2-expressing malignant tumors in breast cancer patients using synthetic 111In- or 68Ga-labeled affibody molecules. J Nucl Med.2010;51(6):892–897. doi: 10.2967/jnumed.109.073239 20484419

49. Zielinski R, Lyakhov I, Jacobs A, Chertov O, Kramer-Marek G, Francella N, et al. Affitoxin- A Novel Recombinant, HER2-Specific, Anti-Cancer Agent for Targeted Therapy of HER2-Positive Tumors. J Immunother.2009;32(8):817–25. doi: 10.1097/CJI.0b013e3181ad4d5d 19752752

50. Zielinski R, Lyakhov I, Hassan M, Kuban M, Shafer-Weaver K, Gandjbakhche A, et al. HER2-Affitoxin: A Potent Therapeutic Agent for the Treatment of HER2-Overexpressing Tumors. Clin Cancer Res. 2011;17(15):5071–5081. doi: 10.1158/1078-0432.CCR-10-2887 21791637

51. Iglewski BH, Liu PV, Kabat D. Mechanism of action of Pseudomonas aeruginosa exotoxin Aiadenosine diphosphate ribosylationof mammalian elongation factor 2 in vitro and in vivo. Infect Immun. 1977;15:138–144. 188760

52. Ekerljung L, Lindborg M, Gedda L, Frejd FY, Carlsson J, Lennartsson J. Dimeric HER2-specific affibody molecules inhibit proliferation of the SKBR-3 breast cancer cell line. Biochem Biophys Res Commun. 2008; 377(2):489–494. doi: 10.1016/j.bbrc.2008.10.027 18930032

53. Göstring L, Malm M, Höidén-Guthenberg I, Frejd FY, Ståhl S, Löfblom J, et al. Cellular effects of HER3-specific affibody molecules. PLoS One. 2012;7(6):e40023. doi: 10.1371/journal.pone.0040023 22768204

54. Xue X, Zhu S, Li W, Chen J, Ou Q, Zheng M,et al. Identification and characterization of novel B-cell epitopes within EBV latent membrane protein 2 (LMP2). Viral Immunol. 2011; 24(3):227–236. doi: 10.1089/vim.2010.0092 21668364

55. Aavula SM, Nimmagadda SV, Biradhar N, Sula S, Chandran D, Lingala R, et al. Generation and Characterization ofan scFv Directed against Site II of Rabies Glycoprotein. Biotechnol Res Int. 2011;2011:652147. doi: 10.4061/2011/652147 22007309

56. Song S, Xue J, Fan K, Kou G, Zhou Q, Wang H, et al. Preparation and characterization of fusion protein truncated Pseudomonas Exotoxin A (PE38KDEL) in Escherichia coli. Protein Expr Purif. 2005;44(1):52–7. doi: 10.1016/j.pep.2005.04.004 15922623

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