Tanushree Ganguly1, Sarah Y Tang1, Nadine Bauer1, Julie L Sutcliffe2,3,4. 1. Department of Internal Medicine, Division of Hematology & Oncology, University of California, Davis, CA, USA. 2. Department of Internal Medicine, Division of Hematology & Oncology, University of California, Davis, CA, USA. jlsutcliffe@ucdavis.edu. 3. Department of Biomedical Engineering, University of California, Davis, CA, USA. jlsutcliffe@ucdavis.edu. 4. Center for Molecular and Genomic Imaging, University of California, Davis, CA, USA. jlsutcliffe@ucdavis.edu.
Abstract
PURPOSE: The purpose of this study was to develop and evaluate two αvβ6-targeted fluorescent imaging agents. The integrin subtype αvβ6 is significantly upregulated in a wide range of epithelial derived cancers, plays a key role in invasion and metastasis, and expression is often located at the invasive edge of tumors. αvβ6-targeted fluorescent imaging agents have the potential to guide surgical resection leading to improved patient outcomes. Both imaging agents were based on the bi-PEGylated peptide NH2-PEG28-A20FMDV2-K16R-PEG28 (1), a peptide that has high affinity and selectivity for the integrin αvβ6: (a) 5-FAM-X-PEG28-A20FMDV2-K16R-PEG28 (2), and (b) IRDye800-PEG28-A20FMDV2-K16R-PEG28 (3). PROCEDURES: Peptides were synthesized using solid-phase peptide synthesis and standard Fmoc chemistry. Affinity for αvβ6 was evaluated by ELISA. In vitro binding, internalization, and localization of 2 was monitored using confocal microscopy in DX3puroβ6 (αvβ6+) and DX3puro (αvβ6-) cells. The in vivo imaging and ex vivo biodistribution of 3 was evaluated in three preclinical mouse models, DX3puroβ6/DX3puro and BxPC-3 (αvβ6+) tumor xenografts and a BxPC-3 orthotopic pancreatic tumor model. RESULTS: Peptides were obtained in > 99% purity. IC50 values were 28 nM (2) and 39 nM (3). Rapid αvβ6-selective binding and internalization of 2 was observed. Fluorescent intensity (FLI) measurements extracted from the in vivo images and ex vivo biodistribution confirmed uptake and retention of 3 in the αvβ6 positive subcutaneous and orthotopic tumors, with negligible uptake in the αvβ6-negative tumor. Blocking studies with a known αvβ6-targeting peptide demonstrated αvβ6-specific binding of 3. CONCLUSION: Two fluorescence imaging agents were developed. The αvβ6-specific uptake, internalization, and endosomal localization of the fluorescence agent 2 demonstrates potential for targeted therapy. The selective uptake and retention of 3 in the αvβ6-positive tumors enabled clear delineation of the tumors and surgical resection indicating 3 has the potential to be utilized during image-guided surgery.
PURPOSE: The purpose of this study was to develop and evaluate two αvβ6-targeted fluorescent imaging agents. The integrin subtype αvβ6 is significantly upregulated in a wide range of epithelial derived cancers, plays a key role in invasion and metastasis, and expression is often located at the invasive edge of tumors. αvβ6-targeted fluorescent imaging agents have the potential to guide surgical resection leading to improved patient outcomes. Both imaging agents were based on the bi-PEGylated peptide NH2-PEG28-A20FMDV2-K16R-PEG28 (1), a peptide that has high affinity and selectivity for the integrin αvβ6: (a) 5-FAM-X-PEG28-A20FMDV2-K16R-PEG28 (2), and (b) IRDye800-PEG28-A20FMDV2-K16R-PEG28 (3). PROCEDURES: Peptides were synthesized using solid-phase peptide synthesis and standard Fmoc chemistry. Affinity for αvβ6 was evaluated by ELISA. In vitro binding, internalization, and localization of 2 was monitored using confocal microscopy in DX3puroβ6 (αvβ6+) and DX3puro (αvβ6-) cells. The in vivo imaging and ex vivo biodistribution of 3 was evaluated in three preclinical mouse models, DX3puroβ6/DX3puro and BxPC-3 (αvβ6+) tumor xenografts and a BxPC-3 orthotopic pancreatic tumor model. RESULTS: Peptides were obtained in > 99% purity. IC50 values were 28 nM (2) and 39 nM (3). Rapid αvβ6-selective binding and internalization of 2 was observed. Fluorescent intensity (FLI) measurements extracted from the in vivo images and ex vivo biodistribution confirmed uptake and retention of 3 in the αvβ6 positive subcutaneous and orthotopic tumors, with negligible uptake in the αvβ6-negative tumor. Blocking studies with a known αvβ6-targeting peptide demonstrated αvβ6-specific binding of 3. CONCLUSION: Two fluorescence imaging agents were developed. The αvβ6-specific uptake, internalization, and endosomal localization of the fluorescence agent 2 demonstrates potential for targeted therapy. The selective uptake and retention of 3 in the αvβ6-positive tumors enabled clear delineation of the tumors and surgical resection indicating 3 has the potential to be utilized during image-guided surgery.
Authors: Eben L Rosenthal; Jason M Warram; Esther de Boer; Thomas K Chung; Melissa L Korb; Margie Brandwein-Gensler; Theresa V Strong; Cecelia E Schmalbach; Anthony B Morlandt; Garima Agarwal; Yolanda E Hartman; William R Carroll; Joshua S Richman; Lisa K Clemons; Lisle M Nabell; Kurt R Zinn Journal: Clin Cancer Res Date: 2015-04-22 Impact factor: 12.531
Authors: N Ahmed; F Pansino; Riley Clyde; P Murthi; M A Quinn; G E Rice; M V Agrez; S Mok; M S Baker Journal: Carcinogenesis Date: 2002-02 Impact factor: 4.944
Authors: Anissa N Elayadi; Kausar N Samli; Ludmila Prudkin; Ying-Horng Liu; Aihua Bian; Xian-Jin Xie; Ignacio I Wistuba; Jack A Roth; Michael J McGuire; Kathlynn C Brown Journal: Cancer Res Date: 2007-06-15 Impact factor: 12.701
Authors: B Sipos; D Hahn; A Carceller; J Piulats; J Hedderich; H Kalthoff; S L Goodman; M Kosmahl; G Klöppel Journal: Histopathology Date: 2004-09 Impact factor: 5.087
Authors: Jonathan L Hecht; Brian M Dolinski; Humphrey A Gardner; Shelia M Violette; Paul H Weinreb Journal: Appl Immunohistochem Mol Morphol Date: 2008-12
Authors: Ruimin Huang; Jelena Vider; Joy L Kovar; D Michael Olive; Ingo K Mellinghoff; Philipp Mayer-Kuckuk; Moritz F Kircher; Ronald G Blasberg Journal: Clin Cancer Res Date: 2012-08-22 Impact factor: 12.531