Expression Of The Human PD-1 Extracellular Domain And Its Nanobody Screening

Objective:Based on the breakthrough effect of anti-PD-1?immune checkpoint?antibodies in the field of cancer immunotherapies,China's NHFPC has been the anti-PD-1inhibitors as a new direction of National Significant New Drugs Development since2015.To date,regulatory approved anti-PD-1 antibodies have been monoclonal antibody.Nanobody?Nb?is more suitable for development as PD-1 inhibitor than monoclonal antibody with low molecular weight,high stability,strong tissue permeability,low immunogenicity and expressed by prokaryotic system.In addition,there has not been functional anti-PD-1 nanobody reported yet,so this study hopes to make up for this blankness,intending to exploit anti-PD-1nanobody candidates with blocking PD-1/PD-L1 pathway to provide material.Methods and Results:?1?We obtained the recombinant sequence of human PD-1 extracellular domain-His,which was inserted into the eukaryotic expression vector of pCMV.The recombinant plasmids?pCMV-PD-1 ECD?was transfected into HEK293F cells to express the PD-1 ECD fusion protein,and purified by Ni-NTA affinity chromatography.Since the PD-1 ECD fusion protein,purity of 91.1%,can binds to PD-L1 or commercial mouse anti-PD-1 mAb,it can be used for subsequent nanobody screening as an antigen.?2?By three rounds of panning and screening with periplasmic extracts-ELISA,we isolated six specific anti-PD-1 nanobodies?NbPD-1 ECD?,which were mainly different on the amino sequence and named Nb 1-Nb 6,from the largre na?ve phage display Nb library(6.68×10¹¹ CFU/Total).Based on the bioinformatics analysis,it was found that the molecular weights of NbPD-1 ECD were between 17.7 kDa and 18.4kDa;Their theoretical isoelectric points were 5.02⁸.85;As the GRAVY of NbPD-1 ECD were negative,all of them were hydrophilic protein.?3?pMECS-Nb_{PD-1 ECD} were introduced into E.coli WK6.After Nb_{PD-1 ECD}D-1 ECD were expressed in WK6 cells with IPTG and Low temperature induction,the NbPD-1ECD were extracted from the periplasm by osmotic shock and they were purified on a NI-NTA affinity chromatography.Finally,the purity of obtained NbPD-1 ECD were92⁹7%.And the yield of six NbPD-1 ECD were reached 2.75 mg/L to 6.54 mg/L.?4?Two NbPD-1 ECD?Nb 2 and Nb 3?with high binding specificity to human PD-1 ECD were identified as anti-PD-1 candidate nanobodies for following research.It was proved that Nb 2 and Nb 3 had high affinity,?2.28±1.38?×10⁷ L/mol and?4.57±1.64?×10⁷ L/mol.Besides,Nb 2 and Nb 3 showed higher affinity and antigen-binding thermostability at 60°C and 90°C compared to mouse anti-PD-1 mAb.?5?Furthmore,Nb 2 and Nb 3 were effective in blocking the interaction between PD-1 and PD-L1,while the binding epitope of Nb 2 and Nb 3 with PD-1 were overlap or similar to PD-L1's,or PD-1 mAb with PD-1's.?6?At the level of cells,HEK 293T/PD-1 stable cell strain was screened by puromycin,after we constructed the recombinant plasmid pCDH-CMV-MCS-EF1-Puro-PD-1 and transfected it into HEK 293T cells by lentivirus.The target specificity to natural transmembrane PD-1 protein of Nb 2 and Nb 3 were investigated by HEK293T/PD-1.Flow cytometry results show that both Nb 2 and Nb 3 specifically recognize PD-1 which was expressed on the cell surface and they have the same binding capacity as commercial PD-1 mAb.?7?In PBMC system,Nb 2 and Nb 3 could effectively block the PD-1/PD-L1 signaling pathway,and cause T cells to activate that releases IFN gamma.Conclusion:In this study,we constructed a eukaryotic expression plasmid encoding human PD-1 ECD fusion protein.After expression and purification we obtained PD-1 ECD fusion protein,purity 91.1%,with functional activity.The fusion protein as antigen,Two Nbs against human PD-1 ECD?Nb 2 and Nb 3?with small molecular weight,high binding specificity,high affinity and high thermal stability were isolated from the large na?ve phage display Nb library.In vitro assay,we have found that Nb 2 and Nb 3 could inhibit the interaction between PD-1 and PD-L1 molecules,specifically bind to the natural transmembrane PD-1 on cell surface and block the PD-1/PD-L1 pathway to maintain T cell activity.In summary,both Nb 2 and Nb 3 have the potential to develop inhibitors for immunological check points,providing material for cancer treatment candidates.