Optimization Of Lentivirus Transduction Conditions And Establishment Of CAR-T Killing Detection System

In 2017,the US FDA has approved the sale of two Chimeric Antigen Receptor T cells(CAR-T)products.After receiving CAR-T cell therapy which targets to CD19,the patients with extremely refractory-relapsed leukemia and lymphoma and with a median survival time of less than half a year are remitted completely by 50 %-80 %.CAR-T technology has thus opened a new era of tumor immunotherapy and is expected to become a significant method of mankind to overcome many diseases such as autoimmune diseases and viral infectious diseases.Additionally,unlike conventional small-molecule chemical medicines or antibody-based macromolecular biological medicines,the CAR-T cells are derived from human primary cells,and transfused back to patients after in vitro genetic modification and culture expansion.The CAR-T cell therapy is a brand-new cellular drug.In order to obtain high-quality CAR-T cells to improve their clinical efficacy,it is essential to perfect the genetic modification methods for CAR-T cells and to establish the laboratory methods which are able to assess the tumor-killing activity of CAR-T cells accurately.To this end,this study has carried out two aspects of work.First,in consideration of the technical difficulty in transducing lentivirus into primary T-lymphocytes,several key parameters were optimized in order to maximize the expression of CAR molecules.Second,in view of the "non-specific killing" phenomenon in reported methods,gene editing methods were used to separately knock out the HLA gene and CD19 gene.Eventually,four different types of cells were constructed to explore the mechanism of "non-specific killing",to optimize the calculation of killing activity and to improve the methods of detecting and evaluating CAR-T cell killing activity.Materials and MethodsPart One: Preparation and Optimization of CAR-T Cell1.Explore the optimal transduction titer of lentivirus LV_CAR19;2.Compare the effects of different activation modes of T-cells on the transduction efficiency of LV_CAR19;3.Explore the optimal transduction time of LV_CAR19 after T-cell activation;4.Compare the effects of different transduction reagents on the transduction efficiency of LV_CAR19;5.To determine whether the washing after transduction of LV_CAR19 can improve the transduction efficiency.Part Two: Construction and Identification of Target Cell1.Construct four different gene knocked-out Namalwa cells(HLA+\ CD19+,HLA-\CD19+,HLA+\ CD19-,HLA-\CD19-)with CRISPR/Cas9 and MACS(Magnetic Activated Cell Sorting)technology;2.Observe the changes of EGFP fluorescence signal levels in different Namalwa cell phenotypes;3.Observe the changes of luciferase signal in different Namalwa cell phenotypes;4.Observe the changes of proliferation activity in different Namalwa cell phenotypes.Part Three: Construction and Optimization of in Vitro Killing Activity Detect System for CAR-T Cell1.Assess the reported in vitro killing detect system for CAR-T cells;2.Optimize the in vitro killing detect system for CAR-T cells;3.Assess the optimized in vitro killing detect system for CAR-T cells;4.Detect the in vitro killing activity of CAR-T cell which optimized with new system.ResultsPart One: Preparation and Optimization of CAR-T CellThe optimized protocol of T-cell lentiviral LV_CAR19 transduction was: After 12 hours of OKT-activation,LV_CAR19 was added for transduction with MOI of 40,along with transduction-enhancing reagent Polybrene.Ultimately,CD19.CAR-T cells with CAR19 expression efficiency higher than 50% are obtained.Part Two: Construction and Identification of Target Cell1.Four different Namalwa cell phenotypes(HLA+\ CD19+,HLA-\ CD19+,HLA+\CD19-,HLA-\CD19-)were successfully prepared;2.Four different Namalwa cell phenotypes showed no significant differences in EGFP fluorescence level,luciferase signal,and cell proliferation activity.Part Three: Construction and Optimization of in Vitro Killing Activity Detect System for CAR-T Cell1.Reported CAR-T killing activity detect system tend to overestimate its killing activity,meanwhile with significant “non-specific killing” phenomenon of the T-cell;2.Effector cells inhibit the proliferation of target cell and HLA enhances CD19.CAR-T killing activity are the major causes of inaccurate detection by reported detect system;3.By introducing new target cells,optimizing the control groups and optimizing the killing calculation method,the problem of reported detect system were successfully solved,and the newly established detect system has improved the accuracy of CAR-T cell killing detection in vitro.ConclusionBy optimizing the parameters of transduction,the efficiency that LV_CAR19transducing into T-cell was successfully improved,and the expression efficiency of CAR19 in T cells was greater than 50%.A new in vitro CAR-T cell killing detect system was successfully established and optimized,with improved accuracy of CAR-T killing detection.