Construction And Application Of Exosome-Mimetic Nanovesicles Targeting PD-1

BackgroundTumor antigen-specific T cell products are one of the options for the treatment of malignant tumors.PD-1⁺T cell subsets in tumor tissue and in peripheral blood of tumor patients were enriched with more tumor antigen-specific cells.We have previously used magnetic bead sorting to expand this population of cells,but this method is time-consuming,labor-intensive and expensive.Therefore,the establishing a selective amplification method without sorting can help reduce the disadvantages caused by sorting.Exosome can be targeted for delivery,but the yield is low and additional loading steps are required.Based on this,exploring the construction of PD-1-targeting exosome-mimetic nanovesicles is helpful to solve the shortcomings of low Exosome yield and additional loading steps,which is of great significance for the selective expansion of PD-1⁺T cells.ObjectiveTo establish the preparation method of NV targeting PD-1 with high yield and no additional loading steps,and to explore its targeted binding ability and effect on T cell proliferation.Methods1.Preparation and Characterization of NVNV was prepared by extrusion method.The particle size distribution was characterized by nanoparticle tracking analysis,and the morphology was portrayed by transmission electron microscope.The total protein concentration was measured by the BCA method.The related markers and loading capacity were detected by Western blotting.2.Functional analysis of OKT3-NVAnti-human CD3 monoclonal antibody（OKT3）is one of the commonly used stimulators for adoptive cell culture.The OKT3-loaded NV（OKT3-NV）was prepared by extrusion method.Peripheral blood mononuclear cells（PBMC）of healthy donors were obtained by density gradient centrifugation.The binding ability of OKT3-NV to T lymphocytes was detected by flow cytometry.And set up 4 cell culture groups:immobile-OKT3 group,free-OKT3 group,OKT3-NV group and Control-NV group.Cell expansion was observed by bright field microscope,cell count was performed by trypan blue exclusion method,and the characteristics of T cell products obtained by expansion were detected by flow cytometry.3.Functional analysis of a PD-1-OKT3-NVThe p MXs-HA-dis ND-p TM-IP plasmid vector containing the HA tag sequence,anti-PD-1（sc Fv）gene and puromycin selection gene was constructed by double digestion and ligation.The plasmid vector was transfected into HEK293T cells by Lipofectamine^TM2000,and screened with puromycin to obtain 293MP cells with high expression of anti-PD-1（sc Fv）.293MP cells were used to prepare a PD-1-OKT3-NV by extrusion method,and PBMCs from healthy donors were obtained by density gradient centrifugation.Cell counting was performed by trypan blue exclusion method to analyze the expansion ability of a PD-1-OKT3-NV on T lymphocytes,the ability of a PD-1-OKT3-NV to target PD-1⁺T lymphocytes was detected by flow cytometry.Results1.The extrusion method can prepare NV similar to Exosome characterization,but in high yield without additional loading steps.2.OKT3-NV has the ability to bind to T lymphocytes in a concentration and time dependent manner.3.OKT3-NV can rapidly expand human T lymphocytes in vitro.There is no difference in surface receptor expression（chemokine receptors,costimulatory and costinhibitory molecules）,cell activity（apoptotic cell ratio）and effector activity（IFN-γsecretion ability）between the T cell products obtained by OKT3-NV and by conventional culture methods.4.The 293MP cell with membrane expression of anti-PD-1（sc Fv）was successfully constructed.5.Using 293MP cells to prepare a PD-1-OKT3-NV,it was confirmed that the presence of anti-PD-1（sc Fv）did not affect the expansion ability of OKT3-NV.6.a PD-1-OKT3-NV has the ability to target PD-1 molecule.ConclusionsIn this study,the extrusion method was used to successfully prepare NV that is similar to the characterization of Exosome,but with a high yield and no additional loading steps.The OKT3 with high T lymphocytes expansion activity was cleverly loaded into the NV（OKT3-NV）to achieve rapid T lymphocytes amplification,and confirmed that the surface modification of loaded cells to prepare OKT3-loaded NV（a PD-1-OKT3-NV）has no effect on the ability of OKT3-NV to expand T lymphocytes,and can target and bind PD-1⁺T lymphocytes.It laid the foundation for the subsequent selective expansion of PD-1⁺T cells.