The diagnosis of early and aggressive types of cancer is important for providing effective cancer therapy. Cancer-induced fibrin clots exist only within lesions. Previously, we developed a monoclonal antibody (clone 102-10) that recognizes insoluble fibrin but not fibrinogen or soluble fibrin and confirmed that fibrin clots form continuously in various cancers. Here, we describe the development of a Fab fragment probe of clone 102-10 for tumour imaging. The distribution of 102-10 Fab was investigated in genetically engineered mice bearing pancreatic ductal adenocarcinoma (PDAC), and its effect on blood coagulation was examined. Immunohistochemical and ex vivo imaging revealed that 102-10 Fab was distributed selectively in fibrin clots in PDAC tumours 3 h after injection and that it disappeared from the body after 24 h. 102-10 Fab had no influence on blood coagulation or fibrinolysis. Tumour imaging using anti-fibrin Fab may provide a safe and effective method for the diagnosis of invasive cancers by detecting fibrin clots in tumour stroma.

Tissue factor (TF), which serves as the initiator of the extrinsic blood coagulation cascade, has been found to be overexpressed in various solid tumors, especially brain tumors, pancreatic cancer, and gastric cancer. Overexpression of TF is considered to contribute to the high incidence of thrombotic complications and poor prognosis in patients with such cancers. Therefore, detection or targeting of TF may be a promising approach for the diagnosis and treatment of solid tumors that are known to overexpress the protein. Here, we used the recombinant DNA technology to develop an anti-TF single-chain Fv (scFv) of small size and high affinity for its target. The biochemical characteristics of the anti-TF scFv were evaluated using surface plasmon resonance (SPR) sensing and flow cytometry. The data obtained showed that the affinity of the anti-TF scFv was 2.04 × 10−8 (KD), and that the protein showed significant binding to the cancer cells. Then, Alexa 647-labeled anti-TF scFv and anti-TF IgG were administered to mice bearing chemically induced spontaneous tumors. The maximum tumor to background ratios of anti-TF scFv and anti-TF IgG were obtained 3 and 24 h after the injections, respectively. This study indicates anti-TF scFv may be suitable as an imaging probe for the diagnosis of solid tumors.

Background: CD73 is expressed on certain types of leukocytes and tumor cells and is known as a poor prognostic factor in patients. CD73 catalyzes conversion of extracellular adenosine monophosphate (AMP) to adenosine (Ado) which accumulates in the tumor microenvironment (TME). Ado suppresses immune activation through Ado receptors, allowing tumors to escape from the host immune system. Suppressing CD73 activity provokes T-cell activation and reverses immune suppression exerted by Ado. Thus, the inhibition of CD73 by anti-CD73 antibodies may provide potent cancer immunotherapy options.Methods: We performed a large screen of anti-human CD73 mouse antibodies, and two clones were humanized using human IgG1 framework with Fc silent. Binding activity was confirmed by flow cytometry (FCM) and surface plasmon resonance. Tcell proliferation was observed using human PBMC with CD3/CD28-agonistic beads and the antibodies in the presence of ATP. Internalization of CD73 was assessed by immunocytochemistry and FCM. Anti-tumor effect was investigated with immunehumanized mouse model (xenogenic grafted MDA-MB-231 in human CD34+ cellengrafted NSG mice) and human CD73 knock-in mouse (subcutaneous MC38-hCD73 in hCD73 konck-in Mice).Results: Humanized anti-CD73 antibody (BP1200) binds to both human and cynomolgus CD73 expressing on cell surface with KD value of 0.2 nM. BP1200 blocked AMP hydrolysis by CD73 on cell surface with an inhibition rate like benchmark antibody. BP1200 was internalized into T lymphocytes besides tumor cell lines by binding to CD73. BP1200 enhanced T-cell proliferation several folds compared to competitive antibody and increased cytokines production. Finally, we examined the anti-tumor effect with humanized immune system mice and human CD73 knock-in mice. In both model mice, BP1200 augmented the effect of anti-PD-1 immunotherapy on tumor growth.Conclusions: BP1200 is a humanized anti-CD73 antibody that attenuates the AMP hydrolysis activity of CD73. By blocking AMP hydrolysis, BP1200 ameliorates adenosine mediated immunosuppressive TME and activates T-cell activity. The combination of BP1200 and immunocheckpoint antibody for cancer treatment will be a promising regimen in clinical practice.Legal entity responsible for the study: BrightPath Biotherapeutics Co., Ltd.

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