Abstract 2864: Tumor targeting and imaging using 64Cu labeled cyclic RGD conjugated human serum albumin via click chemistry
Authors: Park Cho Rong , Song Myung Geun , Park Ji-Yong , Youn Hyewon , Chung June-Key , Jeong Jae Min , Lee Yun-Sang , Kang Keon Wook 
Published: July 2017

Abstract
PURPOSE: The RGD specifically recognizes the integrin αvβ3 which is overexpressed on various malignant tumors. One major drawback of small peptide such as RGD, however, is short half-life in the blood, which greatly compromises their targeting efficacy. To improve blood circulation time and targeting efficacy, we developed cyclic RGDyK (cRGDyK) conjugated human serum albumin (HSA) using click chemistry reaction.
METHODS: HSA was conjugated with DBCO-NHS (dibenzyl cyclooctyne) under physiologically favorable reaction condition for the preparation of strain promoted azide-alkyne reaction. Using this reaction group, DBCO-HSA was conjugated with N3-c RGDyK (azido cRGDyK) for integrin αvβ3 targeting, N3-FNR648 for fluorescence labeling and 64Cu labeled N3-NOTA (3-azidopropyl-NOTA) for radiolabeling. Cell binding of cRGDyK-HSA were analyzed with FNR648 labeled probes. Cellular uptake of 64Cu-cRGDyK-HSA were tested for integrin αvβ3 specific binding at cell level. PET images were acquired after tail vein injection of 64Cu-HSA, 64Cu-cRGDyK-HSA in integrin αvβ3 positive tumor (SK-OV3, ovarian cancer cell line) bearing BALB/c nude mice. PET signals were quantitatively analyzed with PET image analysis program, AMIDE.
RESULTS: The number of cRGDyK on DBCO-HSA conjugates was confirmed using
MALDI-TOF MS. cRGDyK-HSA was successfully conjugated with 64Cu labeled N3-NOTA. Integrin αvβ3 mRNA and protein was highly expressed in SK-OV3 cell line. At fluorescence labeled probes were treated in SK-OV3, cRGDyK-HSA were highly bound to cell membrane and this pattern were decreased with pre-treatment of excess cRGDyK. When compared to cellular uptake level, 64Cu-cRGDyK-HSA were more accumulated at integrin αvβ3 positive cells (SK-OV3) than integrin αvβ3 negative cells (P &lt; 0.05) and there were no difference in 64Cu-HSA. Serial PET images were acquired 0, 4, 24, 48 hours after tail vein injection of 64Cu-cRGDyK-HSA. Radioactivity of 64Cu-cRGDyK-HSA in SK-OV3 tumor was higher than that of 64Cu-HSA. 64Cu-labeled-cRGDyK-HSA can be observed at 48 hours after injection, which shows longer circulation time in mice.
CONCLUSION: We successfully conjugated cyclic RGDyK to HSA using click chemistry approach. We demonstrated that cRGDyK-HSA specifically bind to integrin αvβ3 in in vitro and in vivo model. And in animal model, 64Cu-labeled-cRGDyK-HSA can be observed at 48 hours after injection, which shows longer circulation time. Our results indicated that cRGDyK-HSA have a potential to diagnosis and therapy response monitoring of tumor expressing integrin αvβ3.
Citation Format: Cho Rong Park, Myung Geun Song, Ji-Yong Park, Hyewon Youn, June-Key Chung, Jae Min Jeong, Yun-Sang Lee, Keon Wook Kang. Tumor targeting and imaging using 64Cu labeled cyclic RGD conjugated human serum albumin via click chemistry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2864. doi:10.1158/1538-7445.AM2017-2864