Anti-tumor Immune Effect Of NK-92 Cells Modified By Chimeric Costimulatory Converting Receptor

BackgroundIn recent years,cell-based immunotherapy for cancer has drawn much attention in cancer therapy due to its remarkable curative effect.Particularly,chimeric antigen receptor(CAR)�modified adoptive T-cell therapy has been successfully applied to the treatment of hematologic malignancies.Because of its good targeting,lethality and persistence,CAR-T has become the most convincing breakthrough in the field of cancer immunotherapy.While there are some obstacles which need to be tackled for CAR-T based tumor treatments,one of the major obstacles is that CAR-T,despite its strong ability to kill cancer cells,has potential to respond to its own excess immune response threatening patient's life.And no matter CAR-T or TCR-T(T cell receptor chimeric T cell),the treatment of solid tumors rarely works.NK cells are important immune cells in vivo and play an important role in the early immune response and immune surveillance of the development of solid tumors.Compared to T cells,NK cells have many immune characteristics and advantages,which brings many new ideas for the immunotherapy of tumors.ObjectiveBased on the above ideas,we can invent a new type of anti-tumor NK cells.This study is on the basis of CAR design theory,constructing a chimeric costimulatory converting receptor(CCCR)to modify NK92 cell,so that it can enhance the anti-tumor immune effect.MethodsThe chimeric costimulatory converting receptor(CCCR)gene PD1-NKG2D-41 BB was synthesized by PCR,then the PD1-NKG2D-41 BB fragment was cloned into a new generation of lentiviral vector pCDH-CMV-MCS-EF1-CopGFP.After recombinant lentivirus was prepared,NK-92 cells was transduced by lentivirus to obtain anti-tumor NK-92 cells,which stably expressing the chimeric costimulatory converting receptor.The expression of PD-1,CD56 and CD16 on CCCR-NK92 cells was detected by flow cytometry.The rate of CCCR-NK92 and NK-92 cells proliferation was evaluated by CCK8 assay.The ability of CCCR-NK92 cells specifically kill H1299,EMT6 tumor targets was assessed by LDH assay.To establish the model of NOG mice borne subcutaneously transplanted human lung cancer to observe the anti-tumor effect of CCCR-NK92 cells in vivo.Results1.Successfully constructed anti-tumor NK-92 cells(ie,CCCR-NK92 cells)that stably express the chimeric costimulatory converting receptor PD1-NKG2D-41 BB.2.The results of flow cytometry showed that the expression of PD-1 on CCCR-NK92 cells was 57.4%,while PD-1 in parental NK92 cells was almost not expressed.3.The results of flow cytometry showed that CD56 was expressed on both parental NK-92 cells and CCCR-NK92 cells,while CD16 was not expressed,which proved that CCCR-NK92 cells did not change its phenotype in this project.4.The proliferation of CCCR-NK92 cells showed no significant difference compared with the parental NK-92 cells,which proves that the modification of NK-92 did not affect the proliferation rate of NK-92 cells.5.In the detection of LDH cytotoxicity,the cytotoxicity of CCCR-NK92 cells were higher than parental NK-92 cells under different effective target ratios,whereas the cytotoxicity of CCCR-NK92 was even more potent at a ratio of 5: 1.6.In the apoptosis experiment,the number of apoptotic cells in CCCR-NK92 cells after killing the tumor cells was significantly higher than that of the parental NK-92 cells.7.In the human lung cancer NOG mouse subcutaneous xenograft model,the tumor volume of mice receiving CCCR-NK92 cell treatment was smaller than that of the mice receiving the parental NK-92 treatment.ConclusionsNK-92 cells containing the chimeric costimulatory converting receptor PD1-NKG2D-41BB(ie,CCCR-NK92 cells)were successfully constructed and proved by in vitro and in vivo experiments that the antitumor effect of CCCR-NK92 cells were significantly higher than parental NK-92 cells.