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Steps toward mapping the human vasculature by phage display

Abstract

The molecular diversity of receptors in human blood vessels remains largely unexplored. We developed a selection method in which peptides that home to specific vascular beds are identified after administration of a peptide library. Here we report the first in vivo screening of a peptide library in a patient. We surveyed 47,160 motifs that localized to different organs. This large-scale screening indicates that the tissue distribution of circulating peptides is nonrandom. High-throughput analysis of the motifs revealed similarities to ligands for differentially expressed cell-surface proteins, and a candidate ligand–receptor pair was validated. These data represent a step toward the construction of a molecular map of human vasculature and may have broad implications for the development of targeted therapies.

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Figure 1: In vivo phage-display screening for peptides that home to human tissues through the systemic circulation.
Figure 2: Identification of extended homing motifs with the Clustal W program (European Molecular Biology Laboratory; EMBL).
Figure 3: Validation of the candidate receptor–ligand pairs resulting from the in vivo selection.
Figure 4: Characterization of CGRRAGGSC-displaying phage binding properties by using purified receptors in vitro.

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References

  1. Lander, E.S. et al. Initial sequencing and analysis of the human genome. Nature 409, 860–921 (2001).

    CAS  PubMed  Google Scholar 

  2. Venter, J.C. et al. The sequence of the human genome. Science 291, 1304–1351 (2001).

    CAS  PubMed  Google Scholar 

  3. Pasqualini, R., Arap, W., Rajotte, D. & Ruoslahti, E. In vivo phage display. In Phage display: a laboratory manual. (eds. Barbas, C. F., Burton, D.R., Scott, J.K. & Silverman, G.J.) 1–24 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2000).

    Google Scholar 

  4. Rajotte, D. et al. Molecular heterogeneity of the vascular endothelium revealed by in vivo phage display. J. Clin. Invest. 102, 430–437 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Pasqualini, R. & Ruoslahti, E. Organ targeting in vivo using phage display peptide libraries. Nature 380, 364–366 (1996).

    CAS  PubMed  Google Scholar 

  6. Ellerby, H.M. et al. Anti-cancer activity of targeted pro-apoptotic peptides. Nature Med. 5, 1032–1038 (1999).

    CAS  PubMed  Google Scholar 

  7. Koivunen, E. et al. Tumor targeting with a selective gelatinase inhibitor. Nature Biotechnol. 17, 768–774 (1999).

    CAS  Google Scholar 

  8. Burg, M.A., Pasqualini, R., Arap, W., Ruoslahti, E. & Stallcup, W.B. NG2 proteoglycan-binding peptides target tumor neovasculature. Cancer Res. 59, 2869–2874 (1999).

    CAS  PubMed  Google Scholar 

  9. Arap, W., Pasqualini, R. & Ruoslahti, E. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 279, 377–380 (1998).

    CAS  PubMed  Google Scholar 

  10. Pasqualini, R., Koivunen, E. & Ruoslahti, E. αv integrins as receptors for tumor targeting by circulating ligands. Nature Biotechnol. 15, 542–546 (1997).

    CAS  Google Scholar 

  11. Curnis, F. et al. Enhancement of tumor necrosis factor α antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N (CD13). Nature Biotechnol. 18, 1185–1190 (2000).

    CAS  Google Scholar 

  12. Hong, F. D. & Clayman, G. L. Isolation of a peptide for targeted drug delivery into human head and neck solid tumors. Cancer Res. 60, 6551–6556 (2000).

    CAS  PubMed  Google Scholar 

  13. Trepel, M., Grifman, M., Weitzman, M.D. & Pasqualini, R. Molecular adaptors for vascular-targeted adenoviral gene delivery. Hum. Gene Ther. 11, 1971–1981 (2000).

    CAS  PubMed  Google Scholar 

  14. Rajotte, D. & Ruoslahti, E. Membrane dipeptidase is the receptor for a lung-targeting peptide identified by in vivo phage display. J. Biol. Chem. 274, 11593–11598 (1999).

    CAS  PubMed  Google Scholar 

  15. Pasqualini, R. et al. Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis. Cancer Res. 60, 722–727 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Bhagwat, S.V. et al. CD13/APN is activated by angiogenic signals and is essential for capillary tube formation. Blood 97, 652–659 (2001).

    CAS  PubMed  Google Scholar 

  17. Kolonin, M.G., Pasqualini, R. & Arap, W. Molecular addresses in blood vessels as targets for therapy. Curr. Opinion. Chem. Biol. 5, 308–313 (2001).

    CAS  Google Scholar 

  18. Bacich, D.J., Pinto, J.T., Tong, W.P. & Heston, W.D. Cloning, expression, genomic localization, and enzymatic activities of the mouse homolog of prostate-specific membrane antigen/NAALADase/folate hydrolase. Mamm. Genome 12, 117–123 (2001).

    CAS  PubMed  Google Scholar 

  19. Chang, S.S. et al. Five different anti-prostate-specific membrane antigen (PSMA) antibodies confirm PSMA expression in tumor-associated neovasculature. Cancer Res. 59, 3192–3198 (1999).

    CAS  PubMed  Google Scholar 

  20. St Croix, B. et al. Genes expressed in human tumor endothelium. Science 289, 1197–1202 (2000).

    CAS  PubMed  Google Scholar 

  21. Carson-Walter, E.B. et al. Cell surface tumor endothelial markers are conserved in mice and humans. Cancer Res. 61, 6649–6655 (2001).

    CAS  PubMed  Google Scholar 

  22. Vendruscolo, M., Paci, E., Dobson, C.M. & Karplus, M. Three key residues form a critical contact network in a protein folding transition state. Nature 409, 641–645 (2001).

    CAS  PubMed  Google Scholar 

  23. Ruoslahti, E. RGD and other recognition sequences for integrins. Annu. Rev. Cell Dev. Biol. 12, 697–715 (1996).

    CAS  PubMed  Google Scholar 

  24. Koivunen, E. et al. Inhibition of β2 integrin-mediated leukocyte cell adhesion by leucine-leucine-glycine motif-containing peptides. J. Cell Biol. 153, 905–916 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Thompson, J.D., Higgins, D.G. & Gibson, T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Daluiski, A. et al. Bone morphogenetic protein-3 is a negative regulator of bone density. Nature Genet. 27, 84–88 (2001).

    CAS  PubMed  Google Scholar 

  27. Mahboubi, K., Biedermann, B.C., Carroll, J.M. & Pober, J.S. IL-11 activates human endothelial cells to resist immune-mediated injury. J. Immunol. 164, 3837–3846 (2000).

    CAS  PubMed  Google Scholar 

  28. Campbell, C.L., Jiang, Z., Savarese, D.M. & Savarese, T.M. Increased expression of the interleukin-11 receptor and evidence of STAT3 activation in prostate carcinoma. Am. J. Pathol. 158, 25–32 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Lin, B.Z., Pilch, P.F. & Kandror, K.V. Sortilin is a major protein component of Glut4-containing vesicles. J. Biol. Chem. 272, 24145–24147 (1997).

    CAS  PubMed  Google Scholar 

  30. Nugent, M.A., Nugent, H.M., Iozzo, R.V., Sanchack, K. & Edelman, E.R. Perlecan is required to inhibit thrombosis after deep vascular injury and contributes to endothelial cell-mediated inhibition of intimal hyperplasia. Proc. Natl Acad. Sci. USA 97, 6722–6727 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Wu, K.K. et al. Thrombomodulin Ala455Val polymorphism and risk of coronary heart disease. Circulation 103, 1386–1389 (2001).

    CAS  PubMed  Google Scholar 

  32. Barrow, P.A. & Soothill, J. S. Bacteriophage therapy and prophylaxis: rediscovery and renewed assessment of potential. Trends Microbiol. 5, 268–271 (1997).

    CAS  PubMed  Google Scholar 

  33. Latham, P.W. Therapeutic peptides revisited. Nature Biotechnol. 17, 755–757 (1999).

    CAS  Google Scholar 

  34. Wijdicks, E.F. The diagnosis of brain death. N. Engl. J. Med. 344, 1215–1221 (2001).

    CAS  PubMed  Google Scholar 

  35. Implementing Human Research Regulations; in President's Commission for the Study of Ethical Problems in Medicine and Biomedical and Behavioral Research. (The United States of America Government's Printing Office, Washington, DC; 1983).

Download references

Acknowledgements

We thank R.C. Bast, Jr., R.R. Brentani, W.K. Cavenee, A.C. von Eschenbach, I.J. Fidler, W.K. Hong, D.M. McDonald, J. Mendelsohn and L.A. Zwelling for comments on the manuscript; W.D. Heston for sharing unpublished data; C.L. Cavazos, P.Y. Dieringer, R.G. Nikolova, C.A. Perez, B.H. Restel, C.P. Soto and X. Wang for technical assistance. This work was funded in part by grants from NIH (CA90270 and CA8297601 to R.P., CA90270 and CA9081001 to W.A.) and awards from the Gilson–Longenbaugh Foundation and CaP CURE (to R.P. and W.A.). M.G.K., J.L. and P.J.M. received support from the Susan G. Komen Breast Cancer Foundation, R.J.G. from FAPESP (Brazil), M.C.V. from the Department of Defense, L.C. from the NCCRA and E.K. from the Academy of Finland.

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Correspondence to Wadih Arap or Renata Pasqualini.

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The University of Texas and W. Arap and R. Pasqualini have equity in NTTX Biotechnology, which is subject to certain restrictions under university policy. The terms of these arrangements are being managed by the university in accordance with its conflict-of-interest policies.

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Arap, W., Kolonin, M., Trepel, M. et al. Steps toward mapping the human vasculature by phage display. Nat Med 8, 121–127 (2002). https://doi.org/10.1038/nm0202-121

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