Experimental Study On The Efficacy And Controllability Of EGFRvⅢ Targeted Splitting Receptor-modified T Cells (ssCAR-T)

BackgroundAdoptive cell immunotherapy（ACT）is a very potential treatment for tumor immunotherapy.ACT based on chimeric antigen receptor engineered T cell（CAR-T）is a new breakthrough in tumor immunotherapy in recent years.Observations from preclinical and clinical studies have shown that CAR-T has achieved significant efficacy in the treatment of hematological malignancies and some solid tumors.However,this treatment strategy has some inherent limitations.CAR is initiated by a constitutive promoter,and this type of promoter is continuously expressed without external influence,without tissue and spatiotemporal specificity.Once CAR-T is entered into the patient,as if the"switch"that was turned on is always"ON",it is not possible to regulate CAR-T cell activity by external factors.Several strategies have been proposed to improve CAR-T security.One strategy is to add an inducible suicide gene switch（iCaspase 9）to eliminate infused T cells.Once activated,this switch can quickly eliminate more than 90%of iCas9 transduced T cells.Other approaches have focused on regulating T cell activity through"non-fatal"switches,which may be beneficial for patients who require long-term treatment.Glioblastoma（GBM）is the most common malignant brain tumor in adults and one of the most deadly cancers.After radical surgery,radiotherapy,and chemotherapy,the median survival of patients is only 12-15 months,and it is urgent to explore new treatment strategies.The epidermal growth factor receptor type III mutant（EGFRvⅢ）is the most common mutant form of EGFR and is expressed only in tumor cells.In addition,EGFRvⅢ is closely associated with increased angiogenesis and invasiveness in glioblastoma.Therefore,EGFRvⅢ is an ideal target for tumor targeted therapy.In recent years,CAR-T therapy targeting EGFRvⅢ has attracted much attention in the treatment of GBM.Several immunotherapies under study are related to EGFRvⅢ,including peptide vaccine,dendritic cell vaccination therapy,monoclonal antibody and CAR-modified T cells..The constantly updated research observations,including our research group,demonstrate the efficacy of EGFRvⅢ CAR-T therapy,particularly in achieving long-term tumor suppression and reducing GBM-related mortality.At present,in the clinical trial of GBM,no obvious toxic side effects have been observed,which may be related to the lower degree of homing and proliferation of T cells.However,with the development of CARs,the safety of CAR-T cells is still worrying due to the lack of regulatory mechanisms.Therefore,the development of effective and regulatable CAR-T cells is a new idea for GBM treatment.ObjectiveTo prepare splitting-synthetic receptor-modified T cells（ssCAR-T）and explore the effectiveness and regulability of ssCAR-T.This experiment consists of the following three parts:PartⅠ:Construction and identification of human malignant glioma cell line U87MG stably expressing EGFRvⅢMethods（1）Lentiviral-mediated transfection of U87MG cells:Lentiviruses were packaged by calcium phosphate-mediated transfection of 293T cells with three plasmids.10⁵ U87MG cells were counted in 24-well plates and infected according to MOI=20.（2）Analysis of the lowest effective concentration of puromycin:Setting different concentrations of puromycin,observing the cell state at 72h,the lowest concentration of puromycin that can cause the cell to die is the selection concentration.（3）Selection of stably expressing EGFRvⅢ cell lines:First,an effective concentration of puromycin is used for primary screening,and then the wells of individual cells are selected by a 96-well plate dilution method,followed by expansion culture,and finally a cell line stably expressing EGFRvⅢ is obtained.（4）Identification of stably expressing EGFRvⅢ cell line:Flow cytometry,Western Blot,and cellular immunofluorescence were used to detect the expression of EGFRvⅢ.Results（1）The minimum effective concentration of puromycin against U87MG-EGFRv III is 2 ug/ml.（2）The results of flow cytometry showed that U87MG cells expressing EGFRvⅢ reached more than 90%after puromycin selection.（3）The results of Western Blot showed that the cells of EGFRvⅢ U87MG group and SMMC7721 group showed a band at a molecular weight of about 130KD,and the cells in the control MG63 group and U87MG group did not appear.Each group has a band at a molecular weight of about 37 KD,which is an internal referenceβ-actin.This result verified the expression of exogenous EGFRvⅢ in U87MG cells.（4）The results of cellular immunofluorescence showed that the cells of the EGFRvⅢ-U87 group almost all emitted green fluorescence compared with the control group U87MG cells.This indicates that exogenous EGFRvⅢ has been successfully expressed in U87MG cells.PartⅡ:Preparation and functional study of chimeric antigen receptor-modified T cells（CAR-T）targeting EGFRvⅢMethods（1）Lentiviral packaging:A plasmid expressing the traditional second-generation CAR（cCAR）has been constructed previously.Lentiviruses were packaged by calcium phosphate-mediated transfection of 293T cells with three plasmids.The lentivirus is concentrated by ultracentrifugation.（2）Preparation of CAR-T cells:Human peripheral blood lymphocytes were separated by density gradient centrifugation,lymphocytes were activated with anti-human CD3/CD28 immunomagnetic beads,and then transfected with Lentivirus-mediated CAR.（3）Flow cytometry was used to analyze transfection efficiency.（4）Flow cytometry was used to detect the subtype of cells after amplification.（5）The non-radioactive cytotoxicity test kit was used to detect the killing ability of EGFRvⅢ⁺CAR-T cells.（6）The ability of EGFRvⅢ⁺CAR-T cytokine secretion was detected by ELISA.（7）The anti-tumor effect of EGFRvⅢ⁺CAR-T in vivo was detected by nude mouse xenograft model:tumor volume monitoring,immunohistochemical analysis.（8）Data were processed using SPSS 17.0 statistical software for statistical analysis.Results（1）The lentivirus titer detection kit showed that the prepared lentivirus droplets were（5-10）×10⁶ TU/ml.（2）Transfection efficiency was detected by flow cytometry.The results showed that the transfection efficiency of CAR was above 50%,and lymphocytes were successfully transfected with lentivirus.（3）Cell subtypes were detected by flow cytometry.The results showed that central memory T cells(T_CM)were the main cells after expansion,and these cells showed CD45RA^-CCR7⁺.In addition,less-differentiated lymphocytes accounted for（54.3±11.4%）,indicating that the CAR-T cells we prepared have good activity.（4）The cytotoxicity assay of EGFRvⅢ⁺CAR-T showed that EGFRvⅢ⁺CAR-T could specifically recognize EGFRvⅢ-positive U87 cells,and the killing ability of EGFRvⅢ⁺CAR-T cells increased with the increase of the effective target ratio.At a target ratio of 10:1,EGFRvⅢ⁺CAR-T cells were able to kill more than 50%of target cells.However,when co-cultured with EGFRvⅢ-negative U87,there was no significant difference between the T cells in each group at the same target-to-target ratio.（5）The results of EGFRvⅢ⁺CAR-T cytokine secretion assay showed that the level of IFN-γ（1972.4±115.4）secreted by EGFRvⅢ⁺CAR-T cells was higher than that of the control NT T group（274.5±7.3）when co-cultured with EGFRvⅢ-positive U87)and CD19⁺CAR-T group（217.2±39.0）cells,the difference was statistically significant（P<0.001）.Similarly,the levels of TNF-α（847.6±108.3）secreted by EGFRvⅢ⁺CAR-T cells were higher than those of control NT T（80.2±21.4）and CD19⁺CAR-T（100.8±18.6）cells,and the difference was statistically significant（P<0.001）.When co-cultured with EGFRvⅢ-negative U87,there was no significant difference in cytokine IFN-γand TNF-αsecretion between T cells in each group.（6）The tumor growth curve showed that the tumor volume decreased from 3weeks after injection of EGFRvⅢ⁺CAR-T cells,while the tumors in the control group（PBS group and NT T cell group）continued to grow rapidly,the difference was statistically significant（P<0.01）..（7）Immunohistochemical results of transplanted tumors showed that EGFRvⅢ-positive U87 cells were significantly reduced in xenografts of nude mice infused with EGFRvⅢ⁺CAR-T cells compared with the control group（PBS group,NT T cell group）.PartⅢ:Preliminary study on the efficacy and controllability of splitting receptor-modified T cells（ssCAR-T）Methods（1）Design of the splitting synthetic receptor（ssCAR）structure:The intracellular costimulatory domain and CD3ζdomain of the traditional second generation CAR（cCAR）were split into two separate domains,which were recombined by the introduction of chemically induced dimerization（CID）structures.Only in the presence of chemical molecules（AP21967）can ssCAR-T be activated to form a complete structure.In order to enable the recombined ssCAR to achieve comparable anti-tumor function to cCAR-T,two isolated synthetic receptor structures（structures I+IIa and I+IIb）containing different amounts of costimulatory molecules were designed.Flow cytometry was used to detect the expression of T cell activation molecule CD69 under different conditions to select a better ssCAR structure.（2）Preparation of Jurkat cells expressing ssCAR:ssCAR lentivirus was transfected into Jurkat cells,and ssCAR expression was verified by flow cytometry and Western blot.（3）Optimization of experimental conditions based on ssCAR⁺Jurkat cells:The amount of cytokine IL-2 secreted by ssCAR⁺Jurkat was detected by ELISA in the presence of different doses of AP21967.When 500 nM AP21967 was present at 1,3,5,12 and 24 h,changes in CD69 expression by ssCAR⁺Jurkat T cells were detected by flow cytometry.（4）Preparation of primary lymphocytes（ssCAR-T）expressing ssCAR:Lentivirus was transfected into T lymphocytes,and the expression efficiency of CAR was detected by flow cytometry.（5）ELISA was used to detect the effects of different doses of small molecules on the secretion of ssCAR-T cytokines IFN-γand TNF-α.（6）Non-radioactive cytotoxicity test kit analyzes the effect of different doses of small molecules on the killing function of ssCAR-T cells（7）Flow cytometry was used to analyze the effect of different administration time of small molecules on the function of ssCAR-T cells.（8）Data were processed using SPSS 17.0 statistical software for statistical analysis.Results（1）The results of flow cytometry showed that compared with the structure of I+IIa（31.6±5.1）%,the expression of CD69 in cells transfected with structure I+IIb was（72.2±4.8）%,the difference was statistically significant.（P<0.01）.The expression of CD69 in cells transfected with structure I+IIb was not significantly different from that of cCAR-T（78.2+5.6）%（P>0.05）.Furthermore,in the absence of small molecules,only cells expressing the traditional second-generation CAR（cCAR）structure are activated.（2）The results of flow cytometry showed that the efficiency of Jurkat cells transducing ssCAR was 85.2%.（3）The results of Western blot showed that bands in the corresponding lanes with a molecular weight of approximately 38 kD（Pant II of ssCAR）and 54 kD（Pant I of ssCAR）were detected,but not observed in untransfected T cells.This indicates that ssCAR has successfully transduced Jurkat cells.（4）The amount of cytokine IL-2 secreted by ssCAR⁺Jurkat was detected by ELISA.The results showed that the secretion of cytokine IL-2 increased with the increase of small molecule dose,when the concentration of AP21967 was At 500 nM,Jurkat T cells produced strong IL-2 secretion.（5）Flow cytometry was used to analyze the response of ssCAR⁺Jurkat to small molecule doses.The results showed that with the prolonged action of small molecules,the number of activated cells increased.When treated with AP21967 for 5 h,the expression of CD69 was significantly increased（63.4%）.The percentage of activated T cells was 66.3%and 69.5%at 12 h and 24 h,respectively.Furthermore,ssCAR-T cells cannot be activated in the presence of only target antigen（EGFRvⅢ）or only small molecule stimulation.（4）Flow cytometry results showed that the efficiency of ssCAR transduction of T lymphocytes exceeded 50%.（5）The results of ELISA showed that the release of IFN-γand TNF-αincreased synchronously with the increase of AP21967 concentration.The level of cytokine release by ssCAR-T cells was similar to that of cCAR-T cells under the action of 500nM AP21967.（6）The results of ssCAR-T cytotoxicity test showed that the ssCAR-T killing ability was positively correlated with the concentration of AP21967.SsCAR-T cells treated with higher doses（100 nM）had significantly increased cytotoxicity compared to lower dose（10 nM）AP21967 treated cells.The ssCAR-T cytotoxicity was comparable to cCAR-T cells in the presence of 500 nM AP21967.As a control,neither untransduced T cells nor cCAR-T cells showed an increase in tumor cell killing when co-cultured with EGFRvⅢ-negative U87 cells.（7）The effect of small molecule administration duration on the cytotoxicity of ssCAR-T was detected by flow cytometry.The results showed that small molecule treatment induced more than 35%of target cell apoptosis in 3 h,but 66%at 5 h.In the absence of AP21967 treatment,only 12.5%of EGFRvⅢ⁺U87 cells were apoptotic.Conclusion（1）U87MG cell line（EGFRvⅢ⁺U87MG）stably expressing EGFRvⅢ was successfully constructed by lentiviral-mediated transgenic technique followed by puromycin selection and 96-well plate limiting dilution.（2）The second generation of CAR-modified T cells targeting EGFRvⅢ can specifically and effectively kill EGFRvⅢ⁺U87MG cells and effectively inhibit the growth of xenografts in nude mice.（3）Splitting synthetic receptor-modified T cells（ssCAR-T）can specifically and efficiently recognize EGFRvⅢ-positive tumor cells in the presence of small molecules,which are characterized by tumor antigen specificity and small molecule-dependent properties.And T cell activation and anti-tumor activity can be regulated by ssCAR in a small molecule doses and time-dependent manners.In the presence of sufficient small molecules,ssCAR-T is able to achieve comparable anti-tumor function to cCAR-T.