Recombinant Endoglin-scFv/IP10 Fusion Protein Potently Boosts The Anti-tumor Efficacy Of Adoptively Transferred Specific CD8~+CD28~+ Cytotoxic T Lymphocytes In Mice

Background Endoglin is a specific marker for proliferating endothelial cells in cancer.Interferon gamma-induced protein 10(IP 10)is a potent chemokine acting on activated CD8+T cells.Tumor antigen-specific cytotoxic T lymphocytes(cytotoxic T lymphocytes,CTLs)has become a hot tumor biological treatment.Objective In this study,we aim to investigate the therapeutic efficacy of a newly designed fusion protein containing Endoglin single-chain fragment and IP 10(Endoglin-scFv/IP 10),together with our recently generated TRP2-specific CD8+CD28+CTLs in controlling melanoma growth in mice.Methods Firstly,the reconbinant Endoglin-scFv/IP10 was expressed in E.coil.purfied by affinity chromatography,and characterized in vitro for its chemotactic activity and immunoreactivity with endoglin-expressing cells.In vivo,secondly,transplanted mouse melanoma were established in C57BL/6 mice using B16 cells.Mice were then treated with intravenous injections of vehicle(PBS),Endoglin-scFv/IP10 alone,CD8+CD28+CTLs alone,or Endoglin-scFv/IP10+CD8+CD28+CTLs.The theraputic efficacy was assessed by monitoring tunor growth,mouse survival and multiple changes on cellular levels.Results Endoglin-scFv/IP10 fusion protein potently stimulated the chemotaxis of CD8+T lymphocytes and specifically bound to endoglin-expressing cells in vitro.In vivo,Endoglin-scFv/IP10 fusion protein inhibited tumor growth ande prolonged the survival times of mice inhibited angiogensis and tumor proliferation,enhanced apoptosis of tumor cells,increased the amount of CXCR3+CD8+cells in tumor and IFN-y-positive cells in spleen,decreased the number of MDSCs and Tregs in the tumor-bearing mice.Fuether study demonstrated that Endoglin-scFv/IP10 fusion protein together with CD8+CD28+CTLs significantly inhibited tumor growth and improved mouse survival.On the cellular level,the combination treatment dramatically reduced the number of systemic and tumor-associated myeloid-derived suppressor cells(MDSCs),increased that of tumor-responsive interferon-y-producing lymphocytes in spleen,and inhibited the proliferative but stimulated apoptotic activity within melanoma tissue.Conclusions This study demonstrates the therapeutic potential of Endoglin-scFv/IP10 fusion protein in combination with CD8+CD28+CTLs in melanoma treatment.