Abstract
A novel protein scaffold based on the cystine knot domain of the agouti-related protein (AgRP) has been used to engineer mutants that can bind to the αvβ3 integrin receptor with high affinity and specificity. In the current study, an 18F-labeled AgRP mutant (7C) was prepared and evaluated as a positron emission tomography (PET) probe for imaging tumor angiogenesis. AgRP-7C was synthesized by solid phase peptide synthesis and site-specifically conjugated with 4-nitrophenyl 2-18/19F-fluoropropionate (18/19F-NFP) to produce the fluorinated peptide, 18/19F-FP-AgRP-7C. Competition binding assays were used to measure the relative affinities of AgRP-7C and 19F-FP-AgRP-7C to human glioblastoma U87MG cells that overexpress αvβ3 integrin. In addition, biodistribution, metabolic stability, and small animal PET imaging studies were conducted with 18F-FP-AgRP-7C using U87MG tumor-bearing mice. Both AgRP-7C and 19F-FP-AgRP-7C specifically competed with 125I-echistatin for binding to U87MG cells with half maximal inhibitory concentration (IC50) values of 9.40 and 8.37 nM, respectively. Non-invasive small animal PET imaging revealed that 18F-FP-AgRP-7C exhibited rapid and good tumor uptake (3.24 percentage injected dose per gram [% ID/g] at 0.5 h post injection [p.i.]). The probe was rapidly cleared from the blood and from most organs, resulting in excellent tumor-to-normal tissue contrasts. Tumor uptake and rapid clearance were further confirmed with biodistribution studies. Furthermore, co-injection of 18F-FP-AgRP-7C with a large molar excess of blocking peptide c(RGDyK) significantly inhibited tumor uptake in U87MG xenograft models, demonstrating the integrin-targeting specificity of the probe. Metabolite assays showed that the probe had high stability, making it suitable for in vivo applications. 18F-FP-AgRP-7C exhibits promising in vivo properties such as rapid tumor targeting, good tumor uptake, and excellent tumor-to-normal tissue ratios, and warrants further investigation as a novel PET probe for imaging tumor angiogenesis.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Backberg M, Madjid N, Ogren SO, Meister B (2004) Down-regulated expression of agouti-related protein (AGRP) mRNA in the hypothalamic arcuate nucleus of hyperphagic and obese tub/tub mice. Brain Res Mol Brain Res 125(1–2):129–139. doi:10.1016/j.molbrainres.2004.03.012
Beer AJ, Niemeyer M, Carlsen J, Sarbia M, Nahrig J, Watzlowik P, Wester HJ, Harbeck N, Schwaiger M (2008) Patterns of alphavbeta3 expression in primary and metastatic human breast cancer as shown by 18F-Galacto-RGD PET. J Nucl Med 49(2):255–259. doi:10.2967/jnumed.107.045526
Cheng Z, Xiong Z, Subbarayan M, Chen X, Gambhir SS (2007a) 64Cu-labeled alpha-melanocyte-stimulating hormone analog for microPET imaging of melanocortin 1 receptor expression. Bioconjug Chem 18(3):765–772. doi:10.1021/bc060306g
Cheng Z, Zhang L, Graves E, Xiong ZM, Dandekar M, Chen XY, Gambhir SS (2007b) Small-animal PET of melanocortin 1 receptor expression using a F-18-labeled alpha-melanocyte-stimulating hormone analog. J Nucl Med 48(6):987–994. doi:10.2967/jnumed.107.039602
Cheng Z, De Jesus OP, Namavari M, De A, Levi J, Webster JM, Zhang R, Lee B, Syud FA, Gambhir SS (2008) Small-animal PET imaging of human epidermal growth factor receptor type 2 expression with site-specific 18F-labeled protein scaffold molecules. J Nucl Med 49(5):804–813. doi:10.2967/jnumed.107.047381
Cheng Z, De Jesus OP, Kramer DJ, De A, Webster JM, Gheysens O, Levi J, Namavari M, Wang S, Park JM, Zhang R, Liu H, Lee B, Syud FA, Gambhir SS (2010) 64Cu-labeled affibody molecules for imaging of HER2 expressing tumors. Mol Imaging Biol 12(3):316–324. doi:10.1007/s11307-009-0256-6
Daly NL, Craik DJ (2011) Bioactive cystine knot proteins. Curr Opin Chem Biol. doi:10.1016/j.cbpa.2011.02.008
Haubner R, Wester HJ, Weber WA, Mang C, Ziegler SI, Goodman SL, Senekowitsch-Schmidtke R, Kessler H, Schwaiger M (2001) Noninvasive imaging of alpha(v)beta3 integrin expression using 18F-labeled RGD-containing glycopeptide and positron emission tomography. Cancer Res 61(5):1781–1785
Haubner RH, Wester HJ, Weber WA, Schwaiger M (2003) Radiotracer-based strategies to image angiogenesis. Q J Nucl Med 47(3):189–199
Haubner R, Kuhnast B, Mang C, Weber WA, Kessler H, Wester HJ, Schwaiger M (2004) [18F]Galacto-RGD: synthesis, radiolabeling, metabolic stability, and radiation dose estimates. Bioconjug Chem 15(1):61–69. doi:10.1021/bc034170n
Hersey P, Sosman J, O’Day S, Richards J, Bedikian A, Gonzalez R, Sharfman W, Weber R, Logan T, Buzoianu M, Hammershaimb L, Kirkwood JM (2010) A randomized phase 2 study of etaracizumab, a monoclonal antibody against integrin alpha(v)beta(3), + or − dacarbazine in patients with stage IV metastatic melanoma. Cancer 116(6):1526–1534. doi:10.1002/cncr.24821
Huang L, Gainkam LO, Caveliers V, Vanhove C, Keyaerts M, De Baetselier P, Bossuyt A, Revets H, Lahoutte T (2008) SPECT imaging with 99mTc-labeled EGFR-specific nanobody for in vivo monitoring of EGFR expression. Mol Imaging Biol 10(3):167–175. doi:10.1007/s11307-008-0133-8
Jiang L, Kimura RH, Miao Z, Silverman AP, Ren G, Liu H, Li P, Gambhir SS, Cochran JR, Cheng Z (2010) Evaluation of a (64)Cu-labeled cystine-knot peptide based on agouti-related protein for PET of tumors expressing alphavbeta3 integrin. J Nucl Med 51(2):251–258. doi:10.2967/jnumed.109.069831
Kimura RH, Cheng Z, Gambhir SS, Cochran JR (2009a) Engineered knottin peptides: a new class of agents for imaging integrin expression in living subjects. Cancer Res 69(6):2435–2442. doi:10.1158/0008-5472.CAN-08-2495
Kimura RH, Levin AM, Cochran FV, Cochran JR (2009b) Engineered cystine knot peptides that bind alphavbeta3, alphavbeta5, and alpha5beta1 integrins with low-nanomolar affinity. Proteins 77(2):359–369. doi:10.1002/Prot.22441
Kimura RH, Miao Z, Cheng Z, Gambhir SS, Cochran JR (2010) A dual-labeled knottin peptide for PET and near-infrared fluorescence imaging of integrin expression in living subjects. Bioconjugate Chem 21(3):436–444. doi:10.1021/Bc9003102
Kimura RH, Jones DS, Jiang L, Miao Z, Cheng Z, Cochran JR (2011) Functional mutation of multiple solvent-exposed loops in the Ecballium elaterium trypsin inhibitor-II cystine knot miniprotein. PLoS One 6(2):e16112. doi:10.1371/journal.pone.0016112
Kolmar H (2010) Engineered cystine-knot miniproteins for diagnostic applications. Expert Rev Mol Diagn 10(3):361–368. doi:10.1586/erm.10.15
Li RH, Hoess RH, Bennett JS, DeGrado WF (2003) Use of phage display to probe the evolution of binding specificity and affinity in integrins. Protein Eng 16(1):65–72. doi:10.1093/proeng/gzg002
Liu S, Liu Z, Chen K, Yan Y, Watzlowik P, Wester HJ, Chin FT, Chen X (2010) 18F-labeled galacto and PEGylated RGD dimers for PET imaging of alphavbeta3 integrin expression. Mol Imaging Biol 12(5):530–538. doi:10.1007/s11307-009-0284-2
Miao Z, Ren G, Liu H, Kimura RH, Jiang L, Cochran JR, Gambhir SS, Cheng Z (2009) An engineered knottin peptide labeled with 18F for PET imaging of integrin expression. Bioconjug Chem 20(12):2342–2347. doi:10.1021/bc900361g
Miao Z, Levi J, Cheng Z (2010) Protein scaffold-based molecular probes for cancer molecular imaging. Amino Acids. doi:10.1007/s00726-010-0503-9
Ollmann MM, Wilson BD, Yang YK, Kerns JA, Chen Y, Gantz I, Barsh GS (1997) Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein. Science 278(5335):135–138
Ren G, Zhang R, Liu Z, Webster JM, Miao Z, Gambhir SS, Syud FA, Cheng Z (2009) A 2-helix small protein labeled with 68 Ga for PET imaging of HER2 expression. J Nucl Med 50(9):1492–1499. doi:10.2967/jnumed.109.064287
Shutter JR, Graham M, Kinsey AC, Scully S, Luthy R, Stark KL (1997) Hypothalamic expression of ART, a novel gene related to agouti, is up-regulated in obese and diabetic mutant mice. Gene Dev 11(5):593–602
Silverman AP, Levin AM, Lahti JL, Cochran JR (2009) Engineered cystine-knot peptides that bind alpha(v)beta(3) integrin with antibody-like affinities. J Mol Biol 385(4):1064–1075. doi:10.1016/j.jmb.2008.11.004
Acknowledgments
This work was supported, in part, by National Cancer Institute (NCI) 5R01 CA119053, NCI In Vivo Cellular Molecular Imaging Center (ICMIC) grant P50 CA114747, NCI 5K01 CA104706, and a Stanford Molecular Imaging Scholars postdoctoral fellowship R25 CA118681. SJM has been supported by an NSF Graduate Research Fellowship, the Medtronic Stanford Graduate Fellowship, and a Siebel Scholars Fellowship. This work is also partly sponsored by Grants from the Zhejiang Provincial Natural Science Foundation of China (Z2110230), Health Bureau of Zhejiang Province (2010ZA075, 2011ZDA013), National Science Foundation of China (NSFC) (no. 81101023, 81170306, 81173468), and Ministry of Science and Technology of China (2012BAI13B06). The authors acknowledge Tamara Locke for help on editing the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Jiang, H., Moore, S.J., Liu, S. et al. A novel radiofluorinated agouti-related protein for tumor angiogenesis imaging. Amino Acids 44, 673–681 (2013). https://doi.org/10.1007/s00726-012-1391-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00726-012-1391-y