Systemic combinatorial peptide selection yields a non-canonical iron-mimicry mechanism for targeting tumors in a mouse model of human glioblastoma
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
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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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 3

In vivo targeting of Tf/TfR.

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In vivo targeting of Tf/TfR. (A) Schematic representation of the brain s...
(A) Schematic representation of the brain slicing technique and quantitative real-time PCR used to demonstrate that CRTIGPSVC-phage crosses the BBB. In step 1, phage was injected i.v. into tail veins of mice. In step 2, brain and control organs were collected. Coronal brain slices, 1-mm thick, were obtained with the aid of a brain-slicer mold. H&E-stained sections illustrate prominent tissue components: the lateral ventricle (LV; slice 2), hippocampus (Hip; slices 4 and 6), and cerebellum (primary fissure [prf]; slice 9). Insets show larger views; the boxes represent the area shown in high magnification in the larger images. In step 3, total DNA of each brain slice was purified and used as template for phage DNA amplification and quantification by quantitative real-time PCR. Original magnification, ×10; ×4 (insets). (B–D) Quantification of phage homing to tumor and normal brain after (B) 10 minutes, (C) 30 minutes, and (D) 1 hour of systemic circulation.