|dc.description.abstract||Coagulative abnormalities are a well-recognised feature of malignancy. The risk of developing thrombotic disorders is increased four-fold in cancer patients and more than six-fold higher in those receiving chemotherapy. Consequently, coagulative disorders are the second leading cause of death in cancer patients. One of the principal mechanisms through which cancer cells promote coagulation is through the upregulation of tissue factor (TF), a transmembrane receptor expressed on cells surrounding the blood vessels and a potent initiator of coagulation. Upon binding to circulating coagulation factor VIIa (FVIIa), TF and FVIIa form a catalytic complex, initiating the extrinsic coagulation pathway and resulting in the rapid formation of blood clots.
Chimeric Antigen Receptor (CAR) T cell therapy is an emerging form of immunotherapy which combines the specificity of antibodies with the cytolytic activity of T cells, enabling potent killing of tumours in an antigen-antibody defined interaction. TF has been explored as a therapeutic target for a range of cancer treatments, however its potential as a target antigen for CAR T cell therapy is yet to be realised. The anti-TF monoclonal antibody, TF8-5G9, has been shown to effectively bind to and inhibit the procoagulant function of human TF making it an ideal candidate for a novel CAR structure. The efficacy of the construct would first need to be demonstrated in a pre-clinical setting. However, human TF is highly efficient at complexing with murine FVIIa. Thus, expression of the antigen in a live model could result in major coagulopathies.
This research tackles this issue by generating a truncated form of human TF, using inverse PCR, thereby deleting the FVIIa binding sites while disabling any TF-driven coagulation. Flow cytometric and western blot analyses showed that the expression of the truncated TF was mainly intracellular. However, no coagulation was triggered by the low-level surface expression of the truncated TF, as shown by our in vitro coagulation assay. Failure to efficiently export TF to the surface may be attributed to protein misfolding and thus demands different strategies to truncate the TF gene. Together, these results provide the model framework for TF as a novel target in immunotherapy.||