Abstract 4931: ImmunoPET and fluorescence imaging with Zirconium-89 and IRDye 800CW labeled glycoengineered epidermal growth factor receptor antibody GA201

Abstract

Abstract Background: GA201 is a novel epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), glycoengineered for enhanced antibody dependent cell-mediated cytotoxicity (ADCC). ADCC response is influenced by the amount of antibody bound to the membrane. Therefore we investigated specific accumulation and membrane binding of GA201 on tumor cells in vitro and in human cancer xenografts in vivo using Zirconium-89 (89Zr) and IRDye 800CW (800CW ) labeled GA201. Furthermore, 89Zr-GA201 and 800CW-GA201 were tested in human tumor bearing mice in order to visualize EGFR tumors expression in vivo. Methods: GA201 was conjugated with N-Suc-TFP-desferal and labeled with 89Zr or 800CW-NHS. EGFR membrane binding and internalization of GA201 was determined in vitro using 800CW-GA201 immunofluorescence microscopy and radio-immuno assay (RIA) with 89Zr-GA201 during 4 h at 37 ° C on the human cancer cell line A431 (epidermoid; EGFR overexpressing). 89Zr-GA201 and 800CW-GA201 were validated in human tumor bearing mice for quantification and visualization of EGFR-driven tumor uptake and biodistribution using micro positron emission tomography (microPET), near infrared (NIR) imaging and microscopic evaluation of tumor distribution. Biodistribution of 89Zr-GA201 was performed 6 days post injection (pi) using 10, 25 and 100 μg doses (1 MBq) in A431 subcutaneous(s.c.) xenografts. Serial micro positron emission tomography (microPET) scans were made on day 1, 3 and 6 pi in A431 s.c. xenografts, followed by biodistribution. All mice were co-injected with an equal dose of 111In-DTPA-ITC-IgG (1 MBq) as aspecific control. NIR imaging in A431 s.c. xenografts was performed daily up to 6 days pi using 100 μg 800CW-GA201, co-injected with 100 μg IRDye 680RD-IgG as aspecific control. At 1, 3 and 6 days pi mice were sacrificed for microscopic assessment. Results: Immunofluorescence shows membrane binding and internalization of 800CW-GA201. RIA showed 41.8 ± 3.3 % of 89Zr-GA201 internalized after 4 h incubation. Biodistribution showed tumor uptake of 89Zr-GA201 (specific) vs 111In-IgG (aspecific) of 9.5 ± 4.5 vs 6.4 ± 2.5 (ns, 10 μg dose), 13.5 ± 5.5 vs 5.7 ± 1.2 (p < 0.05, 25 μg dose) and 10.2 ± 3.5 vs 4.7 ± 1.0 (p < 0.05, 100 μg dose) %ID/g. The optimal dose was selected as 25 μg. Preferential tumor uptake of 89Zr-GA201 was seen on microPET scans. 800CW-GA201 (specific) vs 680RD-IgG (aspecific) NIR Imaging 6 days pi resulted in a tumor-to-background ratio (TBR) of 2.9 ± 0.3 vs 2.1 ± 0.4 (P < 0.05). 800CW-GA201 was mainly detected on the cell membrane in tumor tissue slices. Conclusion: GA201 binds to the outer cell membrane and internalizes in vitro. 89Zr-GA201 effectively accumulates in and visualizes EGFR expressing tumors, whereas 800CW-GA201 can be used to visualize EGFR expressing tumors locally. Acknowledgments: Supported by ERC Advanced Grant, OnQview and Hoffmann-La Roche AG. Citation Format: Martin Pool, Arjan Kol, Marjolijn N. Lub-De Hooge, Christian A. Gerdes, Steven de Jong, Elisabeth G.E. de Vries, Anton G.T. Terwisscha van Scheltinga. ImmunoPET and fluorescence imaging with Zirconium-89 and IRDye 800CW labeled glycoengineered epidermal growth factor receptor antibody GA201. [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 4931. doi:10.1158/1538-7445.AM2014-4931