Abstract 4935: Hypoxia targeting fluorescent nanobodies for optical molecular imaging of preinvasive breast cancer
Authors: Brussel Aram SA van , Adams Arthur , Oliveira Sabrina , Khatabbi Mohamed E. , Vermeulen Jeroen F. , Wall Elsken Van der , Mali Willem PThM , Derksen Patrick W. , Diest Paul J. Van , Henegouwen Paul MP Van Bergen en 
Published: May 2015

Abstract
Introduction:
Optical molecular imaging is a novel strategy in diagnosis and therapy of breast cancer. Essential for succesful implementation of this technology is a high tumor-to-normal tissue ratio (TNR), which requires tumor-specific probes/tracers. However, most tumor markers are expressed in a minority of tumors only. Therefore, we have developed a novel probe binding to the membrane-bound protein carbonic anhydrase IX (CAIX), which is commonly expressed in solid tumors as a result of hypoxic conditions. The probe is based on nanobodies (15 kDa, variable domains obtained from heavy-chain antibodies present in animals from the Camelidae family), that has a faster tumor uptake and body clearance than conventional (monoclonal) antibodies, and can be applied for molecular imaging of pre-invasive breast cancer.
Methods:
High affinity CAIX-specific nanobodies were selected using a modified phage display technology and site-directed conjugated to IRDye800CW. Mice bearing orthotopically transplanted MCF10DCIS (DCIS) and CAIX-overexpressing MCF10DCIS (DCIS+CAIX) xenografts were injected with fluorescent CAIX-specific nanobody (B9-IR), or non-specific control nanobody (R2-IR) and imaged up to 48 hours non-invasively. Tumors were resected under image-guidance. Nanobody uptake was assessed in tissue sections and quantified using a biodistribution assay.
Results:
A highly specific nanobody for CAIX was obtained. In DCIS+CAIX xenografts, mean TNR was 4.3±0.6 (8 mice) already two hours after injection. In DCIS tumors, mean TNR was 1.8±0.1 (8 mice), which was higher than TNR obtained after injection with R2-IR (1.4±0.2, 4 mice). After three hours, probe accumulation was sufficient to allow imag-guided tumor resection. Biodistribution studies showed that B9-IR uptake was 4.6±0.8% of injected dose per gram in DCIS tumor tissue (%I.D./g), while 2.0±0.2 %I.D./g was found with R2-IR. Upon fluorescence imaging of tissue sections, B9-IR was present in perinecrotic areas, while R2-IR showed no accumulation in these regions.
Conclusion:
We have developed a novel IRDye800CW-labeled anti-CAIX-nanobody that can be used for rapid optical molecular imaging of (pre-)invasive breast cancer before and during surgery. Furthermore, the stability of the conjugate allowed for ‘molecular fluorescence pathology’, which might results in better contrast than conventional chromagen based immunohistochemistry at the pathology department.
Acknowledgement:
This research was supported by the Center for Translational Molecular Medicine (MAMMOTH).
Citation Format: Aram SA van Brussel, Arthur Adams, Sabrina Oliveira, Mohamed El Khatabbi, Jeroen F. Vermeulen, Elsken Van der Wall, Willem PThM Mali, Patrick WB Derksen, Paul J. Van Diest, Paul MP Van Bergen en Henegouwen. Hypoxia targeting fluorescent nanobodies for optical molecular imaging of preinvasive breast cancer. [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 4935. doi:10.1158/1538-7445.AM2014-4935