Construction and characterization of a truncated tissue factor‑coagulation‑based composite system for selective thrombosis in tumor blood vessels

Int J Oncol. 2019 Oct;55(4):823-832. doi: 10.3892/ijo.2019.4855. Epub 2019 Aug 12.

Abstract

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.

MeSH terms

  • Animals
  • Antineoplastic Agents, Immunological / administration & dosage*
  • Antineoplastic Agents, Immunological / chemistry
  • Antineoplastic Agents, Immunological / pharmacology
  • Factor X / metabolism
  • Female
  • Hep G2 Cells
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Liver Neoplasms / blood supply
  • Liver Neoplasms / drug therapy*
  • Liver Neoplasms / metabolism
  • Mice
  • Neuropilin-1 / immunology*
  • Recombinant Fusion Proteins / administration & dosage
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / pharmacology
  • Streptavidin / chemistry
  • Thromboplastin / administration & dosage*
  • Thromboplastin / chemistry
  • Thromboplastin / pharmacology
  • Thrombosis / chemically induced*
  • Thrombosis / metabolism
  • Xenograft Model Antitumor Assays

Substances

  • Antineoplastic Agents, Immunological
  • NRP1 protein, human
  • Recombinant Fusion Proteins
  • Neuropilin-1
  • Factor X
  • Streptavidin
  • Thromboplastin