Systemic combinatorial peptide selection yields a non-canonical iron-mimicry mechanism for targeting tumors in a mouse model of human glioblastoma
Fernanda I. Staquicini, … , Renata Pasqualini, Wadih Arap
Fernanda I. Staquicini, … , Renata Pasqualini, Wadih Arap
Published December 22, 2010
Citation Information: J Clin Invest. 2011;121(1):161-173. https://doi.org/10.1172/JCI44798.
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Systemic combinatorial peptide selection yields a non-canonical iron-mimicry mechanism for targeting tumors in a mouse model of human glioblastoma

Abstract

The management of CNS tumors is limited by the blood-brain barrier (BBB), a vascular interface that restricts the passage of most molecules from the blood into the brain. Here we show that phage particles targeted with certain ligand motifs selected in vivo from a combinatorial peptide library can cross the BBB under normal and pathological conditions. Specifically, we demonstrated that phage clones displaying an iron-mimic peptide were able to target a protein complex of transferrin and transferrin receptor (TfR) through a non-canonical allosteric binding mechanism and that this functional protein complex mediated transport of the corresponding viral particles into the normal mouse brain. We also showed that, in an orthotopic mouse model of human glioblastoma, a combination of TfR overexpression plus extended vascular permeability and ligand retention resulted in remarkable brain tumor targeting of chimeric adeno-associated virus/phage particles displaying the iron-mimic peptide and carrying a gene of interest. As a proof of concept, we delivered the HSV thymidine kinase gene for molecular-genetic imaging and targeted therapy of intracranial xenografted tumors. Finally, we established that these experimental findings might be clinically relevant by determining through human tissue microarrays that many primary astrocytic tumors strongly express TfR. Together, our combinatorial selection system and results may provide a translational avenue for the targeted detection and treatment of brain tumors.

Authors

Fernanda I. Staquicini, Michael G. Ozawa, Catherine A. Moya, Wouter H.P. Driessen, E. Magda Barbu, Hiroyuki Nishimori, Suren Soghomonyan, Leo G. Flores 2nd, Xiaowen Liang, Vincenzo Paolillo, Mian M. Alauddin, James P. Basilion, Frank B. Furnari, Oliver Bogler, Frederick F. Lang, Kenneth D. Aldape, Gregory N. Fuller, Magnus Höök, Juri G. Gelovani, Richard L. Sidman, Webster K. Cavenee, Renata Pasqualini, Wadih Arap

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Figure 1

Targeting the specificity of phage displaying the peptide CRTIGPSVC in normal mouse brain.

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Targeting the specificity of phage displaying the peptide CRTIGPSVC in n...
(A) Overall structure of the N-lobe half-molecule of Tf. Domains N1 and N2 are indicated. The numbers represent the 6 consensus disulfide bonds, Cys10–Cys49, Cys20–Cys40, Cys119–Cys195, Cys159–Cys175, Cys162–Cys180, and Cys172–Cys178, respectively. (B) Ribbon diagram showing the structural organization of iron-loaded Tf and location of the peptide clusters. (C) Side view of the Tf/TfR molecular complex. N- and C-lobes are shown. (D) Homing specificity of CRTIGPSVC-phage and insertless phage in normal brain compared with that in non-brain control organ (muscle is shown). CRTIGPSVC-phage and insertless phage were administered i.v. into mice. Phage was allowed to circulate for 10 minutes, 30 minutes, and 24 hours, and tissues were recovered and processed as described in Methods. (E) Fractionation of brain microvessels and parenchyma confirmed the ability of the CRTIGPSVC-phage to cross the intact BBB. (F) Binding of phage to rat gliosarcoma 9L cells and 9L cells expressing human TfR (9L3.9) in the presence of apo-Tf and holo-Tf. *P < 0.05. (G) Binding assays reveal that CRTIGPSVC-phage target human apo-Tf in vitro. Insertless phage and BSA served as negative controls. (H and I) The effect of iron on the binding of phage to apo-Tf was reversed by EDTA, but no effect was observed with holo-Tf. In vitro binding assays were performed in 96-well plates coated with either human apo-Tf (H) or with human holo-Tf (I), in the presence of iron and increasing amounts of EDTA.