Fusion of the tissue factor extracellular domain to a tumour stroma specific single-chain fragment variable antibody results in an antigen-specific coagulation-promoting molecule.

摘要

Solid tumours growing beyond a size of 1-2 mm in diameter induce supporting connective tissue structures, the tumour stroma, comprising activated fibroblasts and newly formed blood vessels, embedded in an extracellular matrix. The selective destruction of this tissue or the inhibition of its function (e.g. tumour neoangiogenesis) may result in the destruction of tumour nodules, thus providing novel opportunities for tumour therapy. Our approach aims at an antibody-mediated induction of coagulation in tumour nodules to cut off their blood supply. As a target structure the fibroblast activation protein (FAP) is used, which is specifically and abundantly expressed on the activated fibroblasts of the tumour stroma. We constructed a fusion protein comprising a single-chain module of a FAP-specific humanized antibody [single-chain fragment variable (scFv) OS4] and the extracellular domain of human tissue factor. The fusion protein, designated TFOS4, was produced in the Proteus mirabilis protoplast expression system with a yield of 15 microg/ml. Biochemical characterization of TFOS4 revealed high-affinity binding to cellular FAP. Further, TFOS4 bound to factor VIIa and also exerted allosteric activation of factor VIIa. A complex of TFOS4 and factor VIIa bound to FAP-expressing cells efficiently generated activated factor X. Finally, cell-bound TFOS4 selectively induced plasma coagulation, implying its activity under physiological conditions, notably with relevant concentrations of coagulation factors and their natural inhibitors. These findings suggest that TFOS4 has the potential to increase the procoagulant state in a cell-type-specific fashion. No systemic coagulation or side effects were observed when TFOS4 was injected intravenously into normal mice, indicating the biosafety and specificity of the recombinant protein.