Abstract 2674: Preparation and characterization of scFv antibody against tissue factor (TF) for the delivery of an MRI contrast agent.
Authors: Sato Ryuta , Tsumura Ryou , Obonai Toshifumi , Muneoka Satoshi , Tsumoto Kouhei , Koga Yoshikatsu , Yasunaga Masahiro , Matsumura Yasuhiro 
Published: August 2014

Background: There have been many clinical reports of the increased risk of thrombosis in cancer patients. Tumor cells and tumor vascular endothelial cells express tissue factor (TF) at high levels. This clinical evidence and various biochemical data suggest that TF is involved in the activation of cancer-related blood coagulation. We hypothesized that anti-TF antibody could be a useful tool for drug delivery to solid tumors. Antibodies of various sizes to suit the purpose could be used to deliver a payload to cancers. Here, we prepared an anti-TF single chain variable fragment (scFv) antibody as a potential delivery tool for diagnostic agents.

Method: Anti-TF IgG (clone name, 1157) was obtained from a hybridoma that produced IgG with high affinity for TF antigen. Products of the VL and VH genes of 1157 were obtained from the hybridoma cDNA and were constructed in both orientations, i.e. VH-linker-VL and VL-linker-VH. The scFv was produced in Escherichia coli and was purified by using a conventional method. The affinities of anti-TF IgG and scFv for TF protein were analyzed with flow cytometry (FCM). These antibodies were labeled with Alexa647 and given intravenously to mice bearing chemically induced cutaneous tumors with marked similarity to human solid cancers generally in terms of growth rate and stroma. Biodistribution of the antibodies was analyzed with an OV110 in vivo imaging system. The ratio of the concentration of each antibody in the tumor to that in the surrounding normal tissue area (T/N ratio) was calculated.

Results and Discussion: In vitro, the scFv gene VH-linker-VL was expressed as inclusion bodies in E. coli, whereas the scFv gene VL-linker-VH was expressed in the cytoplasm. scFv gene construction with VL-linker-VH was therefore more promising than that with VH-linker-VL, because scFv could be obtained from the former without the need to refold the scFv protein. FCM analysis revealed that the anti-TF scFv could bind to TF protein on the membrane of several cancer cell lines. In vivo, the T/N ratios of scFv in the mice 1, 3, and 6 h after injection were 1.02, 1.46, and 1.90, respectively. In contrast, those of the IgG were 1.02, 1.01, and 1.23, respectively. Because a higher T/N ratio can result in clearer contrast of the tumor, the scFv may be more useful than IgG as a molecular imaging tool in the 6 h after injection.

Conclusion and Future Plans: Anti-TF scFv accumulated selectively in tumor tissue for 6 h after iv injection. Our next step will be to quantify the ratio of the concentration of scFv in the tumor to that in the plasma (T/P ratio) by using radioisotopes. The scFv may be a useful tool for the delivery of MRI contrast agents.

Citation Format: Ryuta Sato, Ryou Tsumura, Toshifumi Obonai, Satoshi Muneoka, Kouhei Tsumoto, Yoshikatsu Koga, Masahiro Yasunaga, Yasuhiro Matsumura. Preparation and characterization of scFv antibody against tissue factor (TF) for the delivery of an MRI contrast agent. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2674. doi:10.1158/1538-7445.AM2013-2674