Abstract 2062: The biodistribution pattern depending on the size of anti-tissue factor antibodies
Authors: Tsumura Ryo , Sato Ryuta , Koga Yoshikatsu , Yamamoto Yoshiyuki , Fujiwara Yuki , Yasunaga Masahiro , Matsumura Yasuhiro 
Published: May 2015

Abstract
[Introduction] It has been known for a long time that cancer induces blood hypercoagulability. Recent clinical data have indicated that most cancer patients are at a high risk for thrombosis. Tissue factor (TF), a transmembrane protein, initiates extrinsic coagulation by forming a complex with factor VIIa. High expression levels of TF are seen in many types of tumor cells, including pancreatic cancer and glioma cells. TF is also reported to be strongly expressed in the tumor stromal cells and endothelium of tumor vessels. Furthermore, many studies indicate the roles of TF in cell signaling, inflammation, angiogenesis, and tumor growth and metastasis. Based on this evidence, we propose that TF could be a promising target to deliver therapeutic or diagnostic agents into tumor tissues. In the present study, we investigated the in vivo characteristics of the whole IgG of 1849, a high-affinity rat anti-human TF monoclonal antibody (mAb) developed by us, and also those of its F(ab')2 and Fab fragments. Based on the results of this analysis, we determined which fragment might be the most suitable for use in imaging and/or therapy.
[Methods] F(ab')2 and Fab fragments of 1849 and its isotype control mAb were produced through enzyme digestion, respectively. The TF-binding activity of each fragment was evaluated by flow-cytometric analysis using BxPC3, a human pancreatic cancer cell line that strongly expresses TF. In vivo fluorescence imaging and ex vivo studies were carried out in BxPC3 xenograft models. Immunofluorescence staining was also conducted to evaluate the distribution of each of the antibody fragments in the tumor tissues.
[Results] The results of the flow-cytometric analysis revealed that all of the fragments of 1849 examined showed a high affinity for BxPC3 cells, whereas the isotype control fragments did not recognize the BxPC3 cells. In vivo fluorescence imaging revealed prolonged retention and intratumor accumulation of the whole IgG of 1849, while in contrast, the Fab fragment was rapidly cleared from the blood via the kidneys. The results of the ex vivo study were also consistent with those of the in vivo fluorescence imaging. The results of immunofluorescence staining revealed that the fragments of 1849 antibody showed enhanced penetration into the tumor tissues from an early time-point as compared to whole IgG.
[Conclusions] Imaging applications commonly require tumor deposition combined with rapid blood clearance and minimal accumulation in non-tumor tissues. The results of in vivo fluorescence imaging and ex vivo studies suggest that the Fab fragment of the 1849 antibody may be the most suitable for application to imaging diagnosis. On the other hand, the prerequisites for application to therapeutics include longer circulation times with minimal toxicity of the anticancer drugs to normal tissues by reduced delivery to non-tumor tissues. Thus, our results suggest that the whole IgG of 1849 antibody may be the most suitable for application to therapy.
Citation Format: Ryo Tsumura, Ryuta Sato, Yoshikatsu Koga, Yoshiyuki Yamamoto, Yuki Fujiwara, Masahiro Yasunaga, Yasuhiro Matsumura. The biodistribution pattern depending on the size of anti-tissue factor antibodies. [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 2062. doi:10.1158/1538-7445.AM2014-2062