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
Jf. Rippmann et al., 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, BIOCHEM J, 349, 2000, pp. 805-812
Solid tumours growing beyond a size of 1-2 mm in diameter induce supporting
connective tissue structures, the tumour stroma, comprising activated fibr
oblasts and newly formed blood vessels, embedded in an extracellular matrix
. The selective destruction of this tissue or the inhibition of its functio
n (e.g. tumour neoangiogenesis) may result in the destruction of tumour nod
ules, thus providing novel opportunities for tumour therapy. Our approach a
ims at an antibody-mediated induction of coagulation in tumour nodules to c
ut off their blood supply. As a target structure the fibroblast activation
protein (FAP) is used, which is specifically and abundantly expressed on th
e activated fibroblasts of the tumour stroma, We constructed a fusion prote
in comprising a single-chain module of a FAP-specific humanized antibody [s
ingle-chain fragment variable (scFv) OS4] and the extracellular domain of h
uman tissue factor. The fusion protein, designated TFOS4, was produced in t
he Proteus mirabilis protoplast expression system with a yield of 15 mu g/m
l. Biochemical characterization of TFOS4 revealed high-affinity binding to
cellular FAP. Further, TFOS4 bound to factor VIIa and also exerted alloster
ic activation of factor VIIa. A complex of TFOS4 and factor VIIa bound to F
AP-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 coa
gulation factors and their natural inhibitors. These findings suggest that
TFOS4 has the potential to increase the procoagulant state in a cell-type-s
pecific fashion. No systemic coagulation or side effects were observed when
TFOS4 was injected intravenously into normal mice, indicating the biosafet
y and specificity of the recombinant protein.