Abstract 4290: In vitro & in vivo analysis of indocyanine green-labeled panitumumab for optical imaging: a cautionary tale
Authors: Zhou Yang , Kim Young-Seung , Milenic Diane E. , Baidoo Kwamena E. , Brechbiel Martin W. 
Published: May 2015

Abstract
Indocyanine green (ICG), the only clinically approved near-infrared fluorophore, is attractive to researchers for the development of targeted optical imaging agents when conjugated to monoclonal antibodies (mAbs) or their fragments. However, ICG is amphiphilic and readily facilitates aggregation of mAb that is not easily separable. Complications originating from this behavior are frequently overlooked by researchers. This study, for the first time, examines the chemical and biological characteristics of an ICG-labeled mAb (panitumumab). Reverse-phase HPLC and native polyacrylamide gel analysis indicated that the ICG-sulfo-OSu itself, the routinely used commercial product, forms aggregates in aqueous buffers. Size-exclusion HPLC analysis of the conjugation reactions performed at molar ratios of dye to mAb of 5, 10, or 20, indicated the percentages of ICG-panitumumab conjugate were ∼50, 30, 15, respectively with the remainder (∼30%, 50%, 60%, respectively) consisting of aggregates (&gt;150 kDa). The aggregates were successfully separated from the conjugated product and were found to be composed of ICG dye with 1 to 14 panitumumab units. The purified ICG-panitumumab products were analyzed by native- and SDS-PAGE followed by optical imaging. As evidenced by SDS-PAGE, free ICG remained associated with the purified ICG-panitumumab products, in increasing amounts with the increasing molar ratio of dye in the reaction. A competitive radioimmunoassay demonstrated that the targeting moiety of the ICG-mAb conjugates was conserved. Target-specific uptake of the purified bioconjugates was observed with excellent fluorescent intensity in in vitro and in vivo studies. In summary, the preparation of well-defined bioconjugate products labeled with commercial ICG-sulfo-OSu dye is not a simple process and controlling the conjugation reaction ratio and conditions is crucial. Furthermore, purification and characterization of the products is necessitated prior to in vivo optical imaging of tumors. Preliminary data indicate that the degree of aggregate formation during the conjugation reaction with ICG-sulfo-OSu is also dependent on the targeting vehicle, i.e., mAb, hence the need to empirically determine the conjugation conditions for each targeting vector. Using validated and characterized bioconjugate products should facilitate the development of clinically viable and reproducible ICG-conjugated mAb for optical imaging applications. The ability of SDS to dissociate the noncovalently bond ICG provides promise that this property can be exploited in the development of a purification scheme for ICG-conjugates.
Citation Format: Yang Zhou, Young-Seung Kim, Diane E. Milenic, Kwamena E. Baidoo, Martin W. Brechbiel. In vitro & in vivo analysis of indocyanine green-labeled panitumumab for optical imaging: a cautionary tale. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4290. doi:10.1158/1538-7445.AM2014-4290