VEGF165b and its mutant demonstrate immunomodulatory, not merely anti-angiogenic functions, in tumor-bearing mice.

A great deal of evidence has shown that anti-angiogenic molecules and antibodies targeting the VEGF-A/VEGFRs signal pathway can also reverse tumor-induced immunosuppression to an extent. VEGF165b, an anti-angiogenic VEGF-A isoform, has demonstrated capacity as an efficacious anti-tumor therapy in mice as an anti-angiogenic agent. However, whether VEGF165b also plays an immunomodulatory role in anti-tumor field remains unclear. mVEGF165b effect on regulatory T cells (Tregs) in vitro were evaluated using flow cytometry and Cell Counting Kit-8 (CCK-8) methods. Its effects on Tregs (or Foxp3 expressing cells) and myeloid-derived suppressor cells (MDSCs) were analyzed in vivo using flow cytometry and immunostaining techniques. In this study, we found VEGF165b and its mutant (its half-life in plasma was extended 10 times while retaining its bioactivity; the VEGF165b mutant is called mVEGF165b for short) inhibited the proliferation of Tregs in vitro. In addition, mVEGF165b dramatically inhibited the accumulation of MDSCs and Tregs (or Foxp3 expressing cells) in the spleen and tumor in tumor-bearing mice. In conclusion, our findings demonstrated for the first time that VEGF165b and its mutant has immunoregulatory functions. It may be used as a potential immunomodulatory agent, beyond its anti-angiogenic capacities, in cancer therapies.

Zhang H ，Xia W ，Liang C ，Wang X ，Zhi L ，Guo C ，Niu Z ，Zhu W ... -  《-》

VEGF165b Mutant Promotes the Apoptosis of Murine Breast Cancer Cells Induced by Paclitaxel by Inducing Tumor Vessel Maturation.

The anti-angiogenic agent vascular endothelial growth factor 165b (VEGF165b) mutant (mVEGF165b), which was developed by our laboratory, has superior antitumor activity to that of native VEGF165b; however, its mechanism of action and druggability need further exploration. Using the commercial anti-angiogenic drug bevacizumab as a positive control, the mechanism and developability of mVEGF165b were evaluated and explored. The Cell Counting Kit-8 assay was performed to evaluate the effects of mVEGF165b and bevacizumab alone on the proliferation of human umbilical vein endothelial cells (HUVECs). Meanwhile, the inhibitory effects of mVEGF165b and bevacizumab combined with paclitaxel in a mouse model of breast cancer were assessed. Immunohistochemistry was used to detect their effects on tumor vascular maturation, and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to detect the apoptosis of tumor cells. In vitro cell experiments confirmed that mVEGF165b inhibited the proliferation of HUVECs with an efficacy equivalent to that of bevacizumab. mVEGF165b and bevacizumab combined with paclitaxel significantly delayed the growth of breast cancer in mice. Immunohistochemistry and the TUNEL assay showed that mVEGF165b and bevacizumab combined with paclitaxel-induced higher vascular maturity and more apoptosis than paclitaxel alone. mVEGF165b showed similar efficacy and mechanism of action as bevacizumab, indicating its potential to be developed into a safe and effective anti-angiogenic drug.

Liang C ，Li Y ，Guo E ，Bai S ，Wang Y ，Zhang H ... -  《-》

VEGF165b augments NK92 cytolytic activity against human K562 leukemia cells by upregulating the levels of perforin and granzyme B via the VEGR1-PLC pathway.

Pro-angiogenic Vascular endothelial growth factors (VEGFs) exert immunosuppressive functions on some immune cells by interacting with VEGF receptors. Blocking the VEGF/VEGFR pathway could reverse the tumor immunosuppressive microenvironment to some degree. We recently demonstrated that the anti-angiogenic VEGF isoform VEGF165b, similar to other anti-angiogenic agents, inhibit the accumulation immunosuppressive cells such as Tregs and MDSCs. However, whether VEGF165b affects the functions of immune effector cells remain unclear. Here, NK92 cell line was utilized as an immune effector cell model. Our results verified that NK92 cells endogenously express VEGF165 and VEGFR1. Further investigation showed that NK92 treatment with VEGF165b augments its killing ability against human K562 leukemia cells by upregulating perforin and granzyme B through the VEGFR1-PLC pathway, whereas VEGF165b had no impact on the proliferation of NK92 cells in vitro. The results of this study improve our understanding of the immunomodulatory function of VEGF165b, which may help in enhancing the efficacy of NK92-based cancer immunotherapy.

Wang X ，Liang C ，Xia W ，Guo C ，Niu Z ，Zhu W ，Zhang H ... -  《-》

VEGF165b mutant can be used as a protein carrier to form a chimeric tumor vaccine with Mucin1 peptide to elicit an anti-tumor response.

Liang C ，Geng L ，Dong Y ，Zhang H ... -  《-》

VEGF165b mutant with a prolonged half-life and enhanced anti-tumor potency in a mouse model.

VEGF165b has been shown to be an effective anti-cancer agent; however, its short half-life limits further application in the clinical field. The development of a mutant VEGF165b with a prolonged half-life is urgently needed for its future application. A mutant VEGF165b was generated by inactivation of its plasmin cleavage site. The mutant and native VEGF165b proteins without purification tags were expressed via the Pichia pastoris expression system followed by purification with a HiTrap heparin affinity chromatography column through optimization of the purification conditions. Furthermore, its binding affinity with VEGF Receptors and its functions in vitro and in vivo were examined. Results showed that the half-life of mutant VEGF165b increased to approximately 10 times (Intravenous), 9.1 times (Intraperitoneal) and 5.4 times (Subcutaneous) greater than that of VEGF165b, and the mutation did not cause significant alteration of VEGFR1 and VEGFR2 binding affinity. Mutant VEGF165b inhibited the proliferation and migration of HUVECs in vitro, similar to the native VEGF165b. In a mouse melanoma model, mutant VEGF165b exhibited stronger anti-tumor activity in comparison with its native counterpart. These results indicate that the mutant VEGF165b had a prolonged half-life and retained the anti-angiogenic activity of the native VEGF165b, suggesting that this novel mutant VEGF165b may be a stronger anti-cancer agent.

Zhang H ，Jia E ，Xia W ，Lu C ，Zhu W ... -  《-》