The Role Of Recombinant Mutant VEGF-165b Protein In Tumor Immune Regulation

BackgroundTumor immune escape mechanism occurs in the process of development,such as induced by a variety of immunosuppressive cells,thereby inhibiting the immune response effectively.Previous studies have demonstrated that vascular endothelial growth factor-A(VEGF-A)exhibits immunosuppressive properties in addition to its pro-angiogenic activities.This inhibition of thymocyte maturation is caused through VEGFR2 pathway.Some anti-angiogenic molecules that target the VEGF-A/VEGFR axis could reverse tumor-induced immunosuppression.VEGF165b appears to be an endogenous antiangiog-enic agent and it was shown to significantly and dose dependently slow tumor growth.We investigated whether recombinant mVEGF165b had similar influences on MDSCs and Tregs as sunitinib in tumor-bearing mice.ObjectiveWe mutated by the cleavage site of VEGF165b,prolong the half-life of the drug,but retains its pharmaceutical activity.We show for the first time that VEGF165b inhibits the tumor-induced Tregs and MDSC proliferation and modulates tumor microenvironment for immune-based cancer therapies compared to a tyrosinase inhibitors(sunitinib).MethodsAfter mutated the cleavage site of VEGF165b,we transferred the correct plasmid into the carrier pPIC9k,and then integrated into the GS115 genome of yeast cells by means of electrical transfer.Then,high copy positive clones were screened by high density G418plate.The expression of exogenous proteins in the form of methanol induced by the highly-copied strains screened.The purity and properties of the protein are verified compared with the standard product by SDS-PAGE and western blot.Then in animal experiments,the tumor-bearing mice were treated with m VEGF165b protein and to determine whether VEGF165b can inhibit the proliferation of Tregs and MDSCs.In vitro experiments,Tregs in the spleen tumor-bearing mice were sorted by immunomagnetic beads and verify the impact on proliferation of Tregs.To further identify mVEGF165b inhibition of Tregs,we used the model mice.Results1.Successfully constructed and screened mutant VEGF165b(mVEGF165b)high expression of yeast strains.2.The expression of exogenous proteins in the form of methanol induced by the highly-copied strains screened.3.The cell level proved that mVEGF165b had better activity than the standard protein and could inhibit VEGF165 mediated huvec proliferation.4.mVEGF165b significantly and dose dependently slow tumor growth.5.mVEGF165b can reduce MDSC in spleen and tumor as compared to the control group.6.mVEGF165b can inhibit Tregs in spleen and tumor as compared to the control group.7.mVEGF165b can increase the proportion of CD4+and CD8+T cells in the spleen of tumor-bearing mice.8.mVEGF165b can reduce the proportion of RAW264.7 cell.9.mVEGF165b can inhibit Tregs proliferation in vitro.10.mVEGF165b can reduce the circulating Tregs levels in PBMC and tumor in eGFP-Foxp3 knock-in mice.Conclusions1.mVEGF165b protein suppress tumor growth,therefore mVEGF165b can be used as a kind of natural anticancer drug.2.mVEGF165b protein can reduce the proportion of Tregs and MDSCs,and increase the proportion of CD8~+and CD4~+T cells in tumor-bearing mice.Thus,mVEGF165b can be used as a drug to regulate immunosuppression.