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Engineered knottin peptides: a new class of agents for imaging integrin expression in living subjects.

ABSTRACT: There is a critical need for molecular imaging agents to detect cell surface integrin receptors that are present in human cancers. Previously, we used directed evolution to engineer knottin peptides that bind with high affinity ( approximately 10 to 30 nmol/L) to integrin receptors that are overexpressed on the surface of tumor cells and the tumor neovasculature. To evaluate these peptides as molecular imaging agents, we site-specifically conjugated Cy5.5 or (64)Cu-1,4,7,10-tetra-azacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) to their N termini, and used optical and positron emission tomography (PET) imaging to measure their uptake and biodistribution in U87MG glioblastoma murine xenograft models. NIR fluorescence and microPET imaging both showed that integrin binding affinity plays a strong role in the tumor uptake of knottin peptides. Tumor uptake at 1 hour postinjection for two high-affinity (IC(50), approximately 20 nmol/L) (64)Cu-DOTA-conjugated knottin peptides was 4.47% +/- 1.21% and 4.56% +/- 0.64% injected dose/gram (%ID/g), compared with a low-affinity knottin peptide (IC(50), approximately 0.4 mumol/L; 1.48 +/- 0.53%ID/g) and c(RGDyK) (IC(50), approximately 1 mumol/L; 2.32 +/- 0.55%ID/g), a low-affinity cyclic pentapeptide under clinical development. Furthermore, (64)Cu-DOTA-conjugated knottin peptides generated lower levels of nonspecific liver uptake ( approximately 2%ID/g) compared with c(RGDyK) ( approximately 4%ID/g) 1 hour postinjection. MicroPET imaging results were confirmed by in vivo biodistribution studies. (64)Cu-DOTA-conjugated knottin peptides were stable in mouse serum, and in vivo metabolite analysis showed minimal degradation in the blood or tumor upon injection. Thus, engineered integrin-binding knottin peptides show great potential as clinical diagnostics for a variety of cancers.

SUBMITTER: Kimura RH

PROVIDER: S-EPMC2833353 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Engineered knottin peptides: a new class of agents for imaging integrin expression in living subjects.

Kimura Richard H RH Cheng Zhen Z Gambhir Sanjiv Sam SS Cochran Jennifer R JR

Cancer research 20090310 6

There is a critical need for molecular imaging agents to detect cell surface integrin receptors that are present in human cancers. Previously, we used directed evolution to engineer knottin peptides that bind with high affinity ( approximately 10 to 30 nmol/L) to integrin receptors that are overexpressed on the surface of tumor cells and the tumor neovasculature. To evaluate these peptides as molecular imaging agents, we site-specifically conjugated Cy5.5 or (64)Cu-1,4,7,10-tetra-azacyclododecan ...[more]

PMID: 19276378

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Project description:Photoacoustic imaging is a nonionizing biomedical imaging modality with higher resolution and imaging depth than fluorescence imaging, which has greater sensitivity. The combination of the 2 imaging modalities could improve the detection of cancer. Integrin αvβ6 is a cell surface marker overexpressed in many different cancers. Here, we report the development and evaluation of a dye-labeled cystine knot peptide, which selectively recognizes integrin αvβ6 with high affinity, for photoacoustic and fluorescence imaging. The new dual-modality probe may find clinical application in cancer diagnosis and intraoperative imaging of integrin αvβ6-positive tumors.MethodsAn engineered cystine knot peptide, R01, that recognizes integrin αvβ6 was labeled with Atto 740 (A740) and evaluated for its specific cell uptake and its sensitivity threshold. A740-R01 was injected via the tail vein into nude mice xenografted with A431 (integrin αvβ6-positive) or 293T (integrin αvβ6-negative) tumors. Photoacoustic and fluorescence scans of tumors were acquired before and at 0.5, 1, 2, and 4 h after injection of A740-R01. Dynamic photoacoustic scans of various normal organs were also acquired. Ex vivo fluorescence imaging of tissues was performed 1 h after injection.ResultsThe A740-R01 demonstrated integrin αvβ6-dependent binding to A431 cells in culture. Sensitivity studies indicated that the probe may potentially detect lesions as small as 1 or 6 mm3 by fluorescence or photoacoustic imaging, respectively. The photoacoustic and fluorescence signals of A431 xenografts at 1 h after injection were 1.87 ± 0.25 arbitrary units (AU) and 8.27 ± 0.87 AU, respectively. Target specificity was confirmed by low tumor uptake in 293T tumors at 1 h after injection (1.07 ± 0.15 AU and 1.10 ± 0.14 AU for photoacoustic and fluorescence signals, respectively). A740-R01 exhibited hepatobiliary clearance marked by high uptake in the liver, spleen, and intestine but low uptake in the kidneys.ConclusionA740-R01 specifically targeted integrin αvβ6 with low nanomolar affinity. A740-R01 was able to detect integrin αvβ6 both in vitro and in vivo by photoacoustic and fluorescence imaging. A740-R01 is able to detect αvβ6-positive tumors in living subjects and may have clinical application in cancer diagnosis and real-time image-guided surgery.

Dataset Information

Engineered knottin peptides: a new class of agents for imaging integrin expression in living subjects.

Publications

Engineered knottin peptides: a new class of agents for imaging integrin expression in living subjects.

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