Recombinant Human Anti-TF Antibody Fab Fragment (D3H44)

Membrane TF was incubated with F.VIIa and different antibodies for 20 min. F.X or F.IX was added and reaction aliquots removed at different time points. Using specific chromogenic substrates, the concentrations of generated F.Xa and F.IXa were measured and the linear rates of substrate conversion calculated. Inhibition of the rates of (a) F.Xa formation and (b) F.IXa formation was expressed as fractional activities (vi /vo). The values are the average ± SD of three experiments. Circle, D3H44-F(ab')₂; square, D3H44- IgG2; filled diamond, D3H44-IgG4; , D3H44-IgG4b; triangle, humanized control antibody (anti-IgE E25)

Presta, L., Sims, P., Meng, Y. G., Moran, P., Bullens, S., Bunting, S., ... & Iverson, M. (2001). Generation of a humanized, high affinity anti-tissue factor antibody for use as a novel antithrombotic therapeutic. Thrombosis and haemostasis, 85(03), 379-389.

Presta, L., Sims, P., Meng, Y. G., Moran, P., Bullens, S., Bunting, S., ... & Iverson, M. (2001). Generation of a humanized, high affinity anti-tissue factor antibody for use as a novel antithrombotic therapeutic. Thrombosis and haemostasis, 85(03), 379-389.

The antibodies were incubated in human citrated plasma for 5 min and coagulation was initiated by addition of relipidated human tissue factor reagent (Innovin). The clotting times were monitored on an ACL 6000. Prolongation of clotting times are reported as the ratio of clotting times in the presence of antibody vs. baseline values. The results are the average ± SD of three experiments. Circle, D3H44-F(ab')₂; square, D3H44-IgG2; filled diamond, D3H44-IgG4; , D3H44-IgG4b; inverted triangle, D3H44-F(ab)

Presta, L., Sims, P., Meng, Y. G., Moran, P., Bullens, S., Bunting, S., ... & Iverson, M. (2001). Generation of a humanized, high affinity anti-tissue factor antibody for use as a novel antithrombotic therapeutic. Thrombosis and haemostasis, 85(03), 379-389.

Anti-TF blocking antibody (clone D3H44). A and B: SW480 colon cancer cells were grown to confluence in 96-well plates and pretreated with vehicle or a blocking anti-TF antibody (10 µg/ml) for 20 min. Subsequently, PPP pretreated with 20 µg/ml of anti-FXI antibody 1A6 or vehicle was added to SW480 cancer cells and fibrin generation was initiated with the addition of 8.3 mM CaCl₂. In A, fibrin formation was measured as change in turbidity at 405 nm. In B, time interval required for the solution turbidity to reach the half-maximal value was defined as THalf Max. For comparing treatments to vehicle, Student's t-test was used. *P < 0.05, statistically significant. C: immunofluorescent colocalization and ultrastructural verification of platelet adherence to cancer cells. Representative images of three independent experiments revealed cancer cells stained for epithelial cell adhesion molecule (EpCAM-APC-cy7; C,A), highlighting the plasma membrane (arrow) by confocal microscopy. C,B: tissue factor highlighted by FITC-conjugated antibody (arrowhead) showed diffuse staining of cancer cells. CC: composite of cancer cells stained for EpCAM, tissue factor, and platelets stained for CD41a-PE-cy7 (red) showed composite green: red (yellow) punctate pattern (asterisk). C,D and C,E: electron microscopy revealed clusters of platelets (asterisk) adherent to cancer cells (arrow). C,F: in contrast, no adherent platelet aggregates were observed when tissue factor is inhibited. Scale bar, 1 μm. PPP, platelet-poor plasma; TF, tissue factor.

Mitrugno, A., Tassi Yunga, S., Sylman, J. L., Zilberman-Rudenko, J., Shirai, T., Hebert, J. F., ... & Esener, S. (2018). The role of coagulation and platelets in colon cancer-associated thrombosis. American Journal of Physiology-Cell Physiology.