Construction and characterization of a truncated tissue factor‑coagulation‑based composite system for selective...

The selective induction of tumor vascular thrombosis using truncated tissue factor (tTF) delivered via a target ligand is a promising novel antitumor strategy. In the present study, an anti‑neuropilin‑1 (NRP‑1) monoclonal antibody (mAb)‑streptavidin (SA):tTF‑biotin (B) composite system was established. In this system, anti‑NRP‑1‑mAb located tTF to the tumor vascular endothelial cell surface and induced vascular embolization. Due to their high binding affinity, SA and B were used to enhance thrombogenic activity. mAb was conjugated with SA using a coupling method with water‑soluble 1‑ethyl‑3‑(3‑dimethylaminopropyl) carbodiimide and N‑hydroxysulfosuccinimide. Biotinylated tTF (tTF‑B) was prepared using a B‑labeling kit subsequent to the generation and purification of fusion protein tTF. Confocal microscopy and flow cytometry indicated that the anti‑NRP‑1‑mAb‑SA conjugate retained mAb targeting activity. The preservation of B‑conjugate binding capacity was confirmed using a competitive ELISA, and factor X‑activation analysis revealed that tTF‑B retained the procoagulant activity exhibited by tTF. Live imaging was performed to assess mAb‑SA distribution and tumor‑targeting capability, and this yielded promising results. The results of in vivo studies in mice with subcutaneous xenografts demonstrated that this composite system significantly induced tumor vascular thrombosis and inhibited tumor growth, whereas these histological changes were not observed in normal organs.