Study Of CTL’s Enhanced Antitumor Effect Induced By PD-1Antibody Expressed From Reconbinant Adenovirus Ad35-anti-PD-1

Cytotoxic T lymphocyte is a class of major histocompatibility complex restricted andantigen-specific T cell.It plays an important role in tumor immunotherapy clinical applicationsbecause of its characteristic of effective targeted killing of tumor cells. Immunologists hope tobreak through basic reseach to improve the clinical efficacy of adoptive cell therapy of CTL.Fortunately, as the reaserch on the mechanism of tumor immune suppression deepening,especially immune checkpoint PD-1(programmed cell death protein1) and CTLA4(cytotoxic TLymphocyte Antigen4) discovery, which provides a new idea for improving the efficacy ofACT of CTL.Both PD-1and CTLA4are negative regulator proteins on T cell activation whichcan inhibit the activity of T cells and reduce the antitumor function of T cells.Our project focuses on changes of antitumor function effect with the blockade of CTL’sPD-1signaling. First, we constructed type5/35recombinant adenovirus Ad35-anti-PD-1containing PD-1antibody genes.Second,we identified four correct virus clones by PCR, then used them to infect293cells toexpress PD-1antibody. The supernatant was collected at different time points for ELISA assay.Results showed that PD-1antibody expression of the3rdAd35-anti-PD-1virus clone was thehighest at72hour, which is approximately600ng/mL. Next, Westernblotting indicated that PD-1antibody comprised a right heavy chain (about50kD) and a right light chain (about25kD).Third, we choosed the3rdAd35-anti-PD-1virus clone for large scale PD-1antibodyexpression, after72hs, the supernatant was cellected for Protein G affinity chromatographypurification. Coomassie blue staining confirmed that the purified PD-1antibody has high purity. ELISA assay showed total of45μg purified PD-1antibodies from100mL cell supernatant andthe concentration was about40μg/mL after enrichment.Finally, we isolated and cultured DCs and T cells from human peripheral bloodmononuclear cells. Then, DCs were infected with recombinant adenovirus Ad35-TP53. Next, theDCs were used to initially sensitize T cells to obtain the antigen-specific CTLs.We used theantigen-specific CTLs to test cytotoxic effect on tumor cells in vitro: for the experiment group,we added purified PD-1antibody to block the PD-1pathway while for the controll group, weadded equal volume PBS. The CCK8kit was used to caculate the tumor cell lysis percentage.Results showed that the lysis percentage in the experiment group was higher than that in thecontroll group.At present, the project has been completed from the constuction of Ad35-anti-PD-1to killthe tumor cells by CTLs with purified PD-1antibody in vitro.Next, T cells are infected by Ad35-anti-PD-1to secrete PD-1antibody that can block the PD-1signaling of T cells and DCs or Tcells and tumor cells,which can improve the efficacy of ACT of CTL comprehensively.