Investigation Of PD-1 Nb20 Nanobody Enhanced Tumor-specific CD8~+T Cells Responses In The Treatment Of Tumor

Background: CD8+ T cell adoptive therapy is a well-validated immunotherapy method for tumors.Traditional adoptive therapy with CD8+ T cells is not ideal,therefore,new strategies that enhance the antitumor activity of CD8+ T cells are required.Objective: To investigate the biological activity of PD-1 nanobody PD-1 Nb20.Further,to explore whether PD-1 Nb20 promotes the anti-tumor activity of CD8+ T cells,providing a new method for enhancing the anti-tumor effects of CD8+ T cells.Methods:1.Optimizing the prokaryotic expression conditions of PD-1 nanobody PD-1 Nb20,and induce PD-1 Nb20 to be expressed in large quantities.Identification by SDS-PAGE and Western blot.2.The binding ability of selected PD-1 Nb20 with PD-1 was detected by flow cytometry and ELISA.3.DC and tumor cells fusion cells DC/Tumor-FC(DC/HCC827-FC,DC/Hep G2-FC,DC/Tca8113-FC)were generated by fusing tumor cells HCC827,Hep G2,Tca8113)and human peripheral blood-drived DCs.Fusion efficiency of DC/Tumor-FC was detected by fluorescence microscope.The expression of CD80,CD86 and MHC-II molecules on the surface of the DC/Tumor-FC was detected by flow cytometry.4.The expression of CD25 and CD69 on the surface of the CD8+ T induced by DC/Tumor-FC+PD-1 Nb20 were detected by flow cytometry.5.Flow cytometry was used to detect the activation and proliferation of autologous CD8+ T lymphocytes stained by CFSE and induced by DC/Tumor-FC+PD-1 Nb20.6.Flow cytometry was used to detect the efficiency of tumor-specific CTLs induced by DC/Tumor-FC+PD-1-4 Nb20 against HCC827 cells,Hep G2 cells and Tca8113 cells.7.ELISPOT was used to count the IFN-γ+CD8+ T cells induced by DC/Tumor-FC+PD-1 Nb20.The cytokines secreted in the supernatant were detected by ELISA(IFN-γ,TNF-α,IL-2,IL-10).8.NOD SCID mice tumor xenograft tumor model(HCC827,Hep G2 and Tca8113 models)were constructed.Vein injection of CTL cells and normal T cells was processed every 7 days for a total of 4 times.Tumor size and the survival rate of mice was respectively measured and observed to evaluate the anti-tumor effects of DC/Tumor-FC+PD-1 Nb20 inducing tumor-specific CD8+ T cells in vivo.9.Xenograft tumors were fixed with formalin,embedded in paraffin,and sections were cut and processed for Immunohistochemical(IHC)staining.IHC staining was used to detect cell proliferation(ki67),vascular density(CD34).Apoptotic cells in tumor tissues were detected by Tunel method.Results:1.The optimal expression conditions of PD-1 Nb20 protein were as follows: temperature 28°C,IPTG concentration 1 mmol/L,induction time 6 h.The PD-1 Nb20 protein with a molecular weight of approximately 17 k Da and a purity of over 90%.2.Flow cytometry showed that PD-1 Nb20 could bind to PD-1.3.Fluorescence microscope showed that DC/Tca8113-FC,DC/HCC827-FC,DC/Hep G2-FC fusion cells were successfully prepared,and their fusion efficiency was approximately 50%.In vitro,experimental results showed that DC/Tumor-FC+PD-1 Nb20 induces tumor-specific CD8+ T cells to overexpress CD80,CD86 and MHC-II molecules.4.The levels of CD25 and CD69 induced by DC/Tumor-FC+PD-1 Nb20 inducing tumor-specific CD8+ T cells were significantly higher compared to other groups(p < 0.01).5.DC/Tumor-FC+PD-1 Nb20 could effectively stimulate CD8+ T cells proliferation(p < 0.01).6.With different effector-to-target ratios.The killing effect was enhanced with the increase of the effect-target ratios,and the killing efficiency of target cells with higher expression of PD-L1(HCC827)was higher than that with low expression of PD-L1(Hep G2,Tca8113).7.ELISPOT indicated that PD-1 Nb20 promoted the number of IFN-γ+CD8+ T cells(p < 0.01).The secretion of cytokines IFN-γ and TNF-α in DC/Tumor-FC+PD-1 Nb20 inducing tumor-specific CD8+ T cells increased,which was higher than that of the control groups(p < 0.01).There was no difference in the secretion of IL-10(p > 0.05).8.NOD SCID mice tumor xenograft tumor model(HCC827,Hep G2 and Tca8113 models)were constructed.Treatment with DC/Tumor-FC+PD-1 Nb20 inducing tumor-specific CD8+ T cells inhibited tumor growth and prolonged survival time(p < 0.05).9.Immunohistochemistry showed that DC/Tumor-FC+PD-1 Nb20 inducing tumor-specific CD8+ T cells could inhibit tumor cell proliferation,reduce vascular density in tumors,and promote expression of apoptotic protein in tumor-bearing mice.Conclusion: PD-1 Nb20 has a good activity in vitro and was shown to enhance the anti-tumor effects of tumor-specific CD8+ T induced by DC/Tumor-FC+PD-1 Nb20,providing a novel and promising approach for adoptive therapy of tumors.