Abstract 2092: Characterizing NIR dye-IL13RA2 antibody conjugates for biodistribution studies in xenograft tumor models by fluorescence molecular tomography (FMT)
Authors: Gupta Parul , Ma Dangshe , Roach Rachel , Spilker Mary , Leal Mauricio , Bagi Cedo , Giddabasappa Anand 
Published: July 2016

Abstract
Background: The antibody based therapies are promising anti-cancer therapeutic modalities with minimal toxicity and maximum efficacy. The efficacy of these agents is regulated by their biodistribution and targeting. The contemporary methods of testing the biodistriution of large molecule drugs are expensive and tedious. The development of simple and rapid methodology, such as optical imaging, can enable effective screening of a larger number of compounds. Here we have used Fluorescence Molecular Tomography (FMT), an optical imaging technique to study biodistribution and tumor targeting of IL3RA2 antibody (Ab). Methods: Different near infrared (NIR) fluorophores (λmax:650-800nm) were conjugated to the Ab. The fluorophore conjugation protocol was optimized to achieve a degree of labeling (DOL) of 1-3 for all the conjugates. The properties of Ab-fluorophore conjugate (Ab-F) were compared to unlabeled Ab using SEC-HPLC and cell binding assays in three cell lines with varying expression of IL13RA2: A375(+++), U87MG(+) and H460(-). For in vivo evaluation, Ab-F conjugates were administered intravenously at a dose of 2 nmol fluorophore to nu/nu mice bearing A375. Similar studies were also conducted in U87MG and H460 xenografts with selected Ab-F. The mice were imaged longitudinally (6 time-points) for up to 96 hrs using FMT4000 and the data were analyzed using TrueQuant software. Results: The SEC-HPLC and flow cytometry studies demonstrated that conjugation of Ab with most of the fluorophores did not change its stability or functionality. The in vivo fluorescence data from all the NIR dyes showed a peak tumor accumulation of the Ab-F at 6h and was maintained until 96 hrs. Quantitation of various Ab-F conjugated revealed that 2-6% of the injected dose was accumulated in the A375 tumors. In contrast to tumor profile, there was a steep decline in heart signal (a surrogate for blood/ plasma concentration), suggesting fast clearance from blood. The in vivo and ex vivo data suggested that there was 15-80pmol of Ab-F conjugate accumulated in the tumors at 96h. In addition, Alexa Flour® (AF)680 and AF750 showed minimal non-specific accumulation in other organs, whereas VivoTag® (VT)680 and BODIPY®630 showed a significantly higher non-specific accumulation in liver. Conclusions: These results show that the biological properties of Ab were not changed by conjugation with various NIR fluorophores at DOL &lt;3. Optical imaging using fluorescent tags can effectively track and quantitate the tumor targeting/ biodistribution of large molecule drugs.
Citation Format: Parul Gupta, Dangshe Ma, Rachel Roach, Mary Spilker, Mauricio Leal, Cedo Bagi, Anand Giddabasappa. Characterizing NIR dye-IL13RA2 antibody conjugates for biodistribution studies in xenograft tumor models by fluorescence molecular tomography (FMT). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2092.