Abstract B25: Development of 5B1, an anti-CA19.9 monoclonal antibody, as a near-infrared fluorescent probe for intraoperative imaging of pancreatic cancer
Authors: Houghton Jacob L. , Abdel-Atti Dalya A. , Sawada Ritsuko , Scholz Wolfgang W. , Lewis Jason S. 
Published: October 2015

Abstract
Background: For patients diagnosed with pancreatic ductal adenocarcinoma (PDAC), resection offers the best chance of survival. However, for those 20% of patients who qualify for resection, the 5-year survival rate is only 25% due to the high incidence of undiscovered metastases. Along with identifying metastases, properly defining the margin between tumor and healthy tissue is also a crucial determinant of the success of resection. The intraoperative use of near-infrared fluorescence (NIRF) imaging agents to facilitate the identification of previously undetected metastases and to define proper resection margins has the potential to improve the outcomes in patients. Monoclonal antibodies (mABs) are one promising platform for the development of NIRF imaging agents due to their high-specificity and low background in healthy tissues. CA19.9 is a well-established biomarker for PDAC, and 5B1, a fully human, anti-CA19.9 antibody has already shown promise both as a potential therapeutic and as a platform for development of a positron emission tomography agent. Based on previous studies we investigated 5B1’s potential as a NIRF imaging agent for use in an intraoperative setting.
Methods: IRDye800CW-NHS and IRDye650-NHS were conjugated to 5B1 and a mouse IgG via lysine residues to generate 5B1-FL800, 5B1-FL650, and IgG-FL800. Each construct and the free acid of the IR800Dye were characterized using in vitro binding assays with two CA19.9-positive cell pancreatic cancer derived lines (BxPC3 and L3.6pl) and a negative cell line (PanC-1). The in vivo dose needed achieve an optimal signal to background ratio was determined in female, athymic nude mice with varying doses of 5B1-FL800. The potential of the constructs for use as NIRF agents for image-guided tumor identification and surgery was examined in a subcutaneous xenograft model using the previously mentioned cell lines as well as an L3.6pl orthotopic pancreas xenograft model. Images were obtained at 24, 48, 96, and 144 hours (750nm excitation/800nm emission). Post mortem, NIRF image-guided surgery was performed to identify and remove the tumor tissue and the relative uptake was compared to healthy tissue. Immunohistochemistry (IHC) as well as hematoxylin and eosin staining (H&E) were performed on excised tumors to confirm specific uptake in tumor tissue.
Results: The NIRF dyes were conjugated to 5B1 and a non-specific mouse IgG with a dye:mAB ratio of 1.1−3.2 (&gt;98% purity). In vitro binding assays showed negligible binding to any cell line by the NIRF-labeled IgG and unconjugated dye. The two NIRF 5B1 constructs bound to both CA19.9 positive cell lines with low nanomolar affinity while no specific binding to PanC1 was observed. Specific binding could be inhibited by addition of excess 5B1. The binding was visualized in BxPC3 and L3.6pl cells by fluorescence microscopy. Pilot imaging studies in a small cohort of mice showed that 5B1-FL800 was superior to 5B1-FL650 in achieving a high signal-to-background ratio in vivo and that 0.5 nmol per mouse dose was an optimal dose for in vivo imaging. Imaging studies showed excellent tumor uptake in CA19.9-positive tumor bearing mice with an optimal tumor:background ratio being achieved at 96 hours post injection. The ratio of NIRF signal in the excised tumors was 8-30x higher than any healthy organ. The 5B1-800FL signal was used to successfully identify and guide the removal of tumor tissue from mice, including small portions of the tumor that were invisible to the naked eye. IHC and HE staining confirmed specific binding of the NIRF-conjugated 5B1 constructs to CA19.9 positive tumor tissue.
Conclusions: IRDye800CW and IRDye650 were conjugated to the anti-CA19.9, fully-human mAB, 5B1, which was successfully used for in vivo NIRF imaging in both subcutaneous and orthotopic xenograft models of PDAC. 5B1 demonstrated exceptional potential for use in delineation of pancreatic tumor margins, NIRF guided-resection, and identification of metastases overlooked by other imaging techniques.
Citation Format: Jacob L. Houghton, Dalya A. Abdel-Atti, Ritsuko Sawada, Wolfgang W. Scholz, Jason S. Lewis. Development of 5B1, an anti-CA19.9 monoclonal antibody, as a near-infrared fluorescent probe for intraoperative imaging of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B25.