Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Peptide targeting of adenoviral vectors to augment tumor gene transfer

Cancer Gene Therapy volume 19, pages 476–488 (2012)Cite this article

Subjects

Abstract

Adenovirus serotype 5 remains one of the most promising vectors for delivering genetic material to cancer cells for imaging or therapy, but optimization of these agents to selectively promote tumor cell infection is needed to further their clinical development. Peptide sequences that bind to specific cell surface receptors have been inserted into adenoviral capsid proteins to improve tumor targeting, often in the background of mutations designed to ablate normal ligand:receptor interactions and thereby reduce off target effects and toxicities in non-target tissues. Different tumor types also express highly variable complements of cell surface receptors, so a customized targeting strategy using a particular peptide in the context of specific adenoviral mutations may be needed to achieve optimal efficacy. To further investigate peptide targeting strategies in adenoviral vectors, we used a set of peptide motifs originally isolated using phage display technology that evince tumor specificity in vivo. To demonstrate their abilities as targeting motifs, we genetically incorporated these peptides into a surface loop of the fiber capsid protein to construct targeted adenovirus vectors. We then systematically evaluated the ability of these peptide targeted vectors to infect several tumor cell types, both in vitro and in vivo, in a variety of mutational backgrounds designed to reduce CAR and/or HSG-mediated binding. Results from this study support previous observations that peptide insertions in the HI loop of the fiber knob domain are generally ineffective when used in combination with HSG detargeting mutations. The evidence also suggests that this strategy can attenuate other fiber knob interactions, such as CAR-mediated binding, and reduce overall viral infectivity. The insertion of peptides into fiber proved more effective for targeting tumor cell types expressing low levels of CAR receptor, as this strategy can partially compensate for the very low infectivity of wild-type adenovirus in those cells. Nevertheless, the incorporation of relatively low affinity peptide ligands into the fiber knob, while effective in vitro, has only minimal targeting efficacy in vivo and highlights the importance of high affinity ligand:receptor interactions to achieve tumor targeting.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Huard J, Lochmüller H, Acsadi G, Jani A, Massie B, Karpati G . The route of administration is a major determinant of the transduction efficiency of rat tissues by adenoviral recombinants. Gene Ther 1995; 2: 107–115.

    CAS  PubMed  Google Scholar 

  2. Li Q, Kay MA, Finegold M, Stratford-Perricaudet LD, Woo SL . Assessment of recombinant adenoviral vectors for hepatic gene therapy. Hum Gene Ther 1993; 4: 403–409.

    Article  CAS  PubMed  Google Scholar 

  3. van der Eb MM, Cramer SJ, Vergouwe Y, Schagen FH, van Krieken JH, van der Eb AJ et al. Severe hepatic dysfunction after adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene and ganciclovir administration. Gene Ther 1998; 5: 451–458.

    Article  CAS  PubMed  Google Scholar 

  4. Herz J, Gerard RD . Adenovirus-mediated transfer of low density lipoprotein receptor gene acutely accelerates cholesterol clearance in normal mice. Proc Natl Acad Sci U S A 1993; 90: 2812–2816.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Tomko RP, Xu R, Philipson L . HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses. Proc Natl Acad Sci U S A 1997; 94: 3352–3356.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Nemerow GR, Pache L, Reddy V, Stewart PL . Insights into adenovirus host cell interactions from structural studies. Virology 2009; 384: 380–388.

    Article  CAS  PubMed  Google Scholar 

  7. Martin K, Brie A, Saulnier P, Perricaudet M, Yeh P, Vigne E . Simultaneous CAR- and alpha V integrin-binding ablation fails to reduce Ad5 liver tropism. Mol Ther 2003; 8: 485–494.

    Article  CAS  PubMed  Google Scholar 

  8. Einfeld DA, Schroeder R, Roelvink PW, Lizonova A, King CR, Kovesdi I et al. Reducing the native tropism of adenovirus vectors requires removal of both CAR and integrin interactions. J Virol 2001; 75: 11284–11291.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Koizumi N, Mizuguchi H, Sakurai F, Yamaguchi T, Watanabe Y, Hayakawa T . Reduction of natural adenovirus tropism to mouse liver by fiber-shaft exchange in combination with both CAR- and alphav integrin-binding ablation. J Virol 2003; 77: 13062–13072.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bayo-Puxan N, Cascallo M, Gros A, Huch M, Fillat C, Alemany R . Role of the putative heparan sulfate glycosaminoglycan-binding site of the adenovirus type 5 fiber shaft on liver detargeting and knob-mediated retargeting. J Gen Virol 2006; 87 (Pt 9): 2487–2495.

    Article  CAS  PubMed  Google Scholar 

  11. Nicklin SA, Wu E, Nemerow GR, Baker AH . The influence of adenovirus fiber structure and function on vector development for gene therapy. Mol Ther 2005; 12: 384–393.

    Article  CAS  PubMed  Google Scholar 

  12. Waddington SN, McVey JH, Bhella D, Parker AL, Barker K, Atoda H et al. Adenovirus serotype 5 hexon mediates liver gene transfer. Cell 2008; 132: 397–409.

    Article  CAS  PubMed  Google Scholar 

  13. Kalyuzhniy O, Di Paolo NC, Silvestry M, Hofherr SE, Barry MA, Stewart PL et al. Adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo. Proc Natl Acad Sci U S A 2008; 105: 5483–5488.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Vigant F, Descamps D, Jullienne B, Esselin S, Connault E, Opolon P et al. Substitution of hexon hypervariable region 5 of adenovirus serotype 5 abrogates blood factor binding and limits gene transfer to liver. Mol Ther 2008; 16: 1474–1480.

    Article  CAS  PubMed  Google Scholar 

  15. Dechecchi MC, Melotti P, Bonizzato A, Santacatterina M, Chilosi M, Cabrini G . Heparan sulfate glycosaminoglycans are receptors sufficient to mediate the initial binding of adenovirus types 2 and 5. J Virol 2001; 75: 8772–8780.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Dechecchi MC, Tamanini A, Bonizzato A, Cabrini G . Heparan sulfate glycosaminoglycans are involved in adenovirus type 5 and 2-host cell interactions. Virology 2000; 268: 382–390.

    Article  CAS  PubMed  Google Scholar 

  17. Smith TA, Idamakanti N, Marshall-Neff J, Rollence ML, Wright P, Kaloss M et al. Receptor interactions involved in adenoviral-mediated gene delivery after systemic administration in non-human primates. Hum Gene Ther 2003; 14: 1595–1604.

    Article  CAS  PubMed  Google Scholar 

  18. Smith TA, Idamakanti N, Rollence ML, Marshall-Neff J, Kim J, Mulgrew K et al. Adenovirus serotype 5 fiber shaft influences in vivo gene transfer in mice. Hum Gene Ther 2003; 14: 777–787.

    Article  CAS  PubMed  Google Scholar 

  19. Hogg RT, Thorpe P, Gerard RD . Retargeting adenoviral vectors to improve gene transfer into tumors. Cancer Gene Ther 2011; 18: 275–287.

    Article  CAS  PubMed  Google Scholar 

  20. Ruoslahti E . Targeting tumor vasculature with homing peptides from phage display. Semin Cancer Biol 2000; 10: 435–442.

    Article  CAS  PubMed  Google Scholar 

  21. Koivunen E, Wang B, Ruoslahti E . Phage libraries displaying cyclic peptides with different ring sizes: ligand specificities of the RGD-directed integrins. Biotechnology (N Y) 1995; 13: 265–270.

    CAS  Google Scholar 

  22. Pasqualini R, Koivunen E, Ruoslahti E . Alpha v integrins as receptors for tumor targeting by circulating ligands. Nat Biotechnol 1997; 15: 542–546.

    Article  CAS  PubMed  Google Scholar 

  23. Curnis F, Arrigoni G, Sacchi A, Fischetti L, Arap W, Pasqualini R et al. Differential binding of drugs containing the NGR motif to CD13 isoforms in tumor vessels, epithelia, and myeloid cells. Cancer Res 2002; 62: 867–874.

    CAS  PubMed  Google Scholar 

  24. Pasqualini R, Koivunen E, Kain R, Lahdenranta J, Sakamoto M, Stryhn A et al. Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis. Cancer Res 2000; 60: 722–727.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Arap W, Pasqualini R, Ruoslahti E . Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 1998; 279: 377–380.

    Article  CAS  PubMed  Google Scholar 

  26. Garde SV, Forté AJ, Ge M, Lepekhin EA, Panchal CJ, Rabbani SA et al. Binding and internalization of NGR-peptide-targeted liposomal doxorubicin (TVT-DOX) in CD13-expressing cells and its antitumor effects. Anticancer Drugs 2007; 18: 1189–1200.

    Article  CAS  PubMed  Google Scholar 

  27. Pastorino F, Brignole C, Marimpietri D, Cilli M, Gambini C, Ribatti D et al. Vascular damage and anti-angiogenic effects of tumor vessel-targeted liposomal chemotherapy. Cancer Res 2003; 63: 7400–7409.

    CAS  PubMed  Google Scholar 

  28. Pastorino F, Brignole C, Di Paolo D, Nico B, Pezzolo A, Marimpietri D et al. Targeting liposomal chemotherapy via both tumor cell-specific and tumor vasculature-specific ligands potentiates therapeutic efficacy. Cancer Res 2006; 66: 10073–10082.

    Article  CAS  PubMed  Google Scholar 

  29. Mizuguchi H, Koizumi N, Hosono T, Utoguchi N, Watanabe Y, Kay MA et al. A simplified system for constructing recombinant adenoviral vectors containing heterologous peptides in the HI loop of their fiber knob. Gene Ther 2001; 8: 730–735.

    Article  CAS  PubMed  Google Scholar 

  30. Majhen D, Gabrilovac J, Eloit M, Richardson J, Ambriović-Ristov A . Disulfide bond formation in NGR fiber-modified adenovirus is essential for retargeting to aminopeptidase N. Biochem Biophys Res Commun 2006; 348: 278–287.

    Article  CAS  PubMed  Google Scholar 

  31. Jullienne B, Vigant F, Muth E, Chaligné R, Bouquet C, Giraudier S et al. Efficient delivery of angiostatin K1-5 into tumors following insertion of an NGR peptide into adenovirus capsid. Gene Ther 2009; 16: 1405–1415.

    Article  CAS  PubMed  Google Scholar 

  32. Bayo-Puxan N, Gimenez-Alejandre M, Lavilla-Alonso S, Gros A, Cascallo M, Hemminki A et al. Replacement of adenovirus type 5 fiber shaft heparan sulfate proteoglycan-binding domain with RGD for improved tumor infectivity and targeting. Hum Gene Ther 2009; 20: 1214–1221.

    Article  CAS  PubMed  Google Scholar 

  33. Rein DT, Breidenbach M, Wu H, Han T, Haviv YS, Wang M et al. Gene transfer to cervical cancer with fiber-modified adenoviruses. Int J Cancer 2004; 111: 698–704.

    Article  CAS  PubMed  Google Scholar 

  34. Li P, Liu Y, Maynard J, Tang Y, Deisseroth A . Use of adenoviral vectors to target chemotherapy to tumor vascular endothelial cells suppresses growth of breast cancer and melanoma. Mol Ther 2010; 18: 921–928.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Singh R, Kostarelos K . Designer adenoviruses for nanomedicine and nanodiagnostics. Trends Biotechnol 2009; 27: 220–229.

    Article  CAS  PubMed  Google Scholar 

  36. Kritz AB, Nicol CG, Dishart KL, Nelson R, Holbeck S, Von Seggern DJ et al. Adenovirus 5 fibers mutated at the putative HSPG-binding site show restricted retargeting with targeting peptides in the HI loop. Mol Ther 2007; 15: 741–749.

    Article  CAS  PubMed  Google Scholar 

  37. Roelvink PW, Mi Lee G, Einfeld DA, Kovesdi I, Wickham TJ . Identification of a conserved receptor-binding site on the fiber proteins of CAR-recognizing adenoviridae. Science 1999; 286: 1568–1571.

    Article  CAS  PubMed  Google Scholar 

  38. Chartier C, Degryse E, Gantzer M, Dieterle A, Pavirani A, M Mehtali Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli. J Virol 1996; 70: 4805–4810.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Gerard RD, Meidell RS . Adenovirus vectors. In: Hames BD, Glover D, (eds). DNA Cloning: A Practical Approach. Oxford University Press: Oxford, 1995 pp 285–307.

    Google Scholar 

  40. Von Seggern DJ, Huang S, Fleck SK, Stevenson SC, Nemerow GR . Adenovirus vector pseudotyping in fiber-expressing cell lines: improved transduction of Epstein-Barr virus-transformed B cells. J Virol 2000; 74: 354–362.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Hogg RT, Garcia JA, Gerard RD . Adenoviral targeting of gene expression to tumors. Cancer Gene Ther 2010; 17: 375–386.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Henry LJ, Xia D, Wilke ME, Deisenhofer J, Gerard RD . Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli. J Virol 1994; 68: 5239–5246.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Sharma A, Li X, Bangari DS, Mittal SK . Adenovirus receptors and their implications in gene delivery. Virus Res 2009; 143: 184–194.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Xia D, Henry LJ, Gerard RD, Deisenhofer J . Crystal structure of the receptor-binding domain of adenovirus type 5 fiber protein at 1.7 A resolution. Structure 1994; 2: 1259–1270.

    Article  CAS  PubMed  Google Scholar 

  45. Pandha HS, Stockwin LH, Eaton J, Clarke IA, Dalgleish AG, Todryk SM et al. Coxsackie B and adenovirus receptor, integrin and major histocompatibility complex class I expression in human prostate cancer cell lines: implications for gene therapy strategies. Prostate Cancer Prostatic Dis 2003; 6: 6–11.

    Article  CAS  PubMed  Google Scholar 

  46. Wu Z, Li ZB, Chen K, Cai W, He L, Chin FT et al. microPET of tumor integrin alphavbeta3 expression using 18F-labeled PEGylated tetrameric RGD peptide (18F-FPRGD4). J Nucl Med 2007; 48: 1536–1544.

    Article  CAS  PubMed  Google Scholar 

  47. Wu H, Seki T, Dmitriev I, Uil T, Kashentseva E, Han T et al. Double modification of adenovirus fiber with RGD and polylysine motifs improves coxsackievirus-adenovirus receptor-independent gene transfer efficiency. Hum Gene Ther 2002; 13: 1647–1653.

    Article  CAS  PubMed  Google Scholar 

  48. Bilbao R, Reay DP, Hughes T, Biermann V, Volpers C, Goldberg L et al. Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors. Gene Ther 2003; 10: 1821–1829.

    Article  CAS  PubMed  Google Scholar 

  49. Koizumi N, Mizuguchi H, Utoguchi N, Watanabe Y, Hayakawa T . Generation of fiber-modified adenovirus vectors containing heterologous peptides in both the HI loop and C terminus of the fiber knob. J Gene Med 2003; 5: 267–276.

    Article  CAS  PubMed  Google Scholar 

  50. Hidaka C, Milano E, Leopold PL, Bergelson JM, Hackett NR, Finberg RW et al. CAR-dependent and CAR-independent pathways of adenovirus vector-mediated gene transfer and expression in human fibroblasts. J Clin Invest 1999; 103: 579–587.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Reynolds P, Dmitriev I, Curiel D . Insertion of an RGD motif into the HI loop of adenovirus fiber protein alters the distribution of transgene expression of the systemically administered vector. Gene Ther 1999; 6: 1336–1339.

    Article  CAS  PubMed  Google Scholar 

  52. Assa-Munt N, Jia X, Laakkonen P, Ruoslahti E . Solution structures and integrin binding activities of an RGD peptide with two isomers. Biochemistry 2001; 40: 2373–2378.

    Article  CAS  PubMed  Google Scholar 

  53. Kirby I, Lord R, Davison E, Wickham TJ, Roelvink PW, Kovesdi I et al. Adenovirus type 9 fiber knob binds to the coxsackie B virus-adenovirus receptor (CAR) with lower affinity than fiber knobs of other CAR-binding adenovirus serotypes. J Virol 2001; 75: 7210–7214.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Niu G, Xiong Z, Cheng Z, Cai W, Gambhir SS, Xing L et al. In vivo bioluminescence tumor imaging of RGD peptide-modified adenoviral vector encoding firefly luciferase reporter gene. Mol Imaging Biol 2007; 9: 126–134.

    Article  PubMed  PubMed Central  Google Scholar 

  55. Xiong Z, Cheng Z, Zhang X, Patel M, Wu JC, Gambhir SS et al. Imaging chemically modified adenovirus for targeting tumors expressing integrin alphavbeta3 in living mice with mutant herpes simplex virus type 1 thymidine kinase PET reporter gene. J Nucl Med 2006; 47: 130–139.

    CAS  PubMed  Google Scholar 

  56. Minea RO, Helchowski CM, Zidovetzki SJ, Costa FK, Swenson SD, Markland FS##Jr . Vicrostatin - an anti-invasive multi-integrin targeting chimeric disintegrin with tumor anti-angiogenic and pro-apoptotic activities. PLoS One 2010; 5: e10929.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Demaegdt H, Lenaerts PJ, Swales J, De Backer JP, Laeremans H, Le MT et al. Angiotensin AT4 receptor ligand interaction with cystinyl aminopeptidase and aminopeptidase N: [125I]Angiotensin IV only binds to the cystinyl aminopeptidase apo-enzyme. Eur J Pharmacol 2006; 546: 19–27.

    Article  CAS  PubMed  Google Scholar 

  58. Garrigues HJ, Rubinchikova YE, Dipersio CM, Rose TM . Integrin alphaVbeta3 Binds to the RGD motif of glycoprotein B of Kaposi's sarcoma-associated herpesvirus and functions as an RGD-dependent entry receptor. J Virol 2008; 82: 1570–1580.

    Article  CAS  PubMed  Google Scholar 

  59. Shim JS, Kim JH, Cho HY, Yum YN, Kim SH, Park HJ et al. Irreversible inhibition of CD13/aminopeptidase N by the antiangiogenic agent curcumin. Chem Biol 2003; 10: 695–704.

    Article  CAS  PubMed  Google Scholar 

  60. Terauchi M, Kajiyama H, Shibata K, Ino K, Nawa A, Mizutani S et al. Inhibition of APN/CD13 leads to suppressed progressive potential in ovarian carcinoma cells. BMC Cancer 2007; 7: 140.

    Article  PubMed  PubMed Central  Google Scholar 

  61. Freedland SJ, Seligson DB, Liu AY, Pantuck AJ, Paik SH, Horvath S et al. Loss of CD10 (neutral endopeptidase) is a frequent and early event in human prostate cancer. Prostate 2003; 55: 71–80.

    Article  PubMed  Google Scholar 

  62. Liu AY . Differential expression of cell surface molecules in prostate cancer cells. Cancer Res 2000; 60: 3429–3434.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors wish to thank Julie Poirot and Jessica Mullens for technical help with recombinant virus construction and preparation. Angie Bookout provided advice on analysis and presentation of the integrin expression data. Paul Card helped prepare the manuscript for publication, and Steve Kliewer provided a critical reading. Glen Nemerow was kind enough to share the 633 cell line with us. Optical imaging was facilitated by the UT Southwestern Small Animal Imaging Research Program funded by NCI U24 CA126608. This work was supported by a Texas Higher Education Coordinating Board Advanced Technology Program grant and by NIH R01 CA115935 to RDG.

Author information

Authors and Affiliations

  1. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA

    E N Ballard, V T Trinh, R T Hogg & R D Gerard

Authors
  1. E N Ballard
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. V T Trinh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. R T Hogg
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R D Gerard
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to R D Gerard.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on Cancer Gene Therapy website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ballard, E., Trinh, V., Hogg, R. et al. Peptide targeting of adenoviral vectors to augment tumor gene transfer. Cancer Gene Ther 19, 476–488 (2012). https://doi.org/10.1038/cgt.2012.23

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/cgt.2012.23

Keywords

This article is cited by

Search

Advanced search

Quick links