| Background The main cause of low life quality of nasopharyngeal carcinoma(NPC)as the most common malignant tumor in otolaryngology head and neck surgery is local recurrence and distant metastasis.At present,radiotherapy is the first choice for the therapy of nasopharyngeal cancer.Because the early symptoms of nasopharyngeal carcinoma are not typical,patients often miss the best stage of treatment,and it is difficult to completely remove the nasopharyngeal carcinoma cells.With the development of chimeric antigen receptor(CAR)T cell therapy based on T cell adoptive therapy,it provides a new strategy to cure NPC for the targeting and specificity.At present,anti-CD19 chimeric antigen receptor(CAR)modified T cells which are demonstrated to have obvious therapeutic effects on hematological malignancy have approved for the clinical application by FDA.In most of solid tumors,lack of specific targets and the immune escape in vivo are barriers of CAR modified T cell therapy,despite anti-CD19 CAR modified T cells have successfully cured a patient with recurrent glioma in a case report.Studies have showed that genesis and development of NPC closely correlate to the infection of Epstein-Barr virus(EBV).EBV mediated the loss of NPC cell polarization,induced the transformation of epithelial cells of nasopharynx through the regulation of epithelialmesenchymal transition(EMT)and promoted the proliferation,migration,invasion,and inhibiting apoptosis of NPC cells.Latent membrane protein 1(LMP1)which is generally expressed in NPC cells is an ideal target of diagnosis and treatment.Based on the humanized anti-LMP1 antibody,this study is aim to prepare anti-LMP1 CAR modified T cells and verify the proliferation inhibition of anti-LMP1 CAR modified T cells to NPC cells in vitro and vivo.Objective 1.To prepare anti-LMP1 CAR modified T cells.2.To optimize the proliferation,differentiation and expression efficiency of anti-LMP1 CAR modified T cells.3.To explore the proliferation inhibition of anti-LMP1 CAR modified T cells to NPC cells in vitro and vivo.Methods 1.Based on the humanized anti-LMP1 antibody with a high affinity which prepared in the previous study,primers were designed to obtain the VH and VK genes of LMP1 sc Fv and overlap PCR was used to construct LMP1 sc Fv.Genetic engineering technology was used to construct LMP1 CAR lentiviral expression vector by connecting the LMP1 sc Fv and lentiviral vector with CD8?,CD28,CD137 and CD3?.2.LMP1 CAR lentiviral expression vector was transfected to 293 T cells by PEI.Western blot assay was to test the expression of LMP1 CAR in 293 T cells with CD3? antibody as primary antibody.3.PBMC isolated from peripheral blood of healthy volunteers by Ficoll were activated by CD3/CD28 antibody and amplified by IL-2 to achieve enough T cells.T cells were infected by virus which was packaged by LMP1 CAR lentiviral expression vector to prepare anti-LMP1 CAR modified T cells.4.Nasopharyngeal carcinoma cells(CNE1 and CNE2,HONE1,C666-1 and SUNE1)were cultured to detect the expression of LMP1 of nasopharyngeal carcinoma cell by flow cytometry,to screen LMP1 high,low and no expression of nasopharyngeal carcinoma cell lines,respectively.5.NPC cells were collected and co-cultured with anti-LMP1 CAR modified T cells for 6 hours.Cell counting kit-8(CCK-8)assay was to detect the lethal effect of anti-LMP1 CAR modified T cells to NPC cells(CNE1,CNE2,HONE1)at 20:1,10:1,5:1 and 2:1 ratio of effective cells to target cells compared to anti-CD19 CAR modified T cells and T cells.6.CCK8 assay was to monitor the lethal effect of anti-LMP1 CAR modified T cells to NPC cells(CNE1,CNE2,HONE1)at 10:1 ratio of effective cells to target cells.7.NPC cells were collected and co-cultured 24 hours with anti-LMP1 CAR modified T cells for the supernatants.Enzyme-linked immunosorbent assay(ELISA)assay was to detect the secretion of cytokines(IL-2 and IFN-?)of anti-LMP1 CAR modified T cells with the stimulation of NPC cells.8.CNE1 cells were marked by luciferase.CNE1 cells were subcutaneously injected together with engineered T cells into BALB/c nude mice in one side of oxter.Before the injection of engineered T cells,Fluorescent imaging system was performed to verify the proliferation of NPC cells and the inhibition of anti-LMP1 CAR modified T cells in vivo.9.CNE1 cells were subcutaneously injected together with engineered T cells into BALB/c nude mice in both sides of oxter.Fluorescent imaging system was performed to verify the tumor-specific cytotoxicity to LMP1-positive NPC cells but not LMP1-negative NPC cells.Results 1.PCR assay showed the fragment had no differences with the theoretical value.And the result of sequencing showed the correctness of sequence.2.Western blot assay was used to test the expression of CD3? and showed the band size was consistent with the theoretical value.The result revealed LMP1 CAR lentiviral expression vector can be expressed in X-293 T cells.3.LMP1 high-expression nasopharyngeal carcinoma cell line CNE1,LMP1 low expression nasopharyngeal carcinoma cell line CNE2 and LMP1 no-expression nasopharyngeal carcinoma cell line HONE1 were screened Successfully.4.CCK8 assay showed there was statistic difference of the killing effect of anti-LMP1 CAR modified T cells to LMP1-positive NPC cells at 20:1 and 10:1 ratio of effective cells to target cells(P<0.05).At 10:1 ratio of effective cells to target cells,the killing rate of LMP1 CAR-T cells to CNE1 and CNE2 was(66.51 ± 4.06)% and(49.91± 6.81)% respectively,which was statistically different from CD19 CAR-T cells and T cells(P<0.05).The killing rate of LMP1 CAR-T cells to HONE1 was(33.33 ± 0.91)%,which was no statistical difference compared with CD19 CAR-T cells and T cells(P<0.05).5.ELISA assay showed the IL-2 and IFN-? secretion in the co-culture of LMP1 CAR-T cells and CNE1 were(1,962.58±54.65)pg/ml and(2,229.73 ± 45.69)pg/ml,respectively.IL-2 and IFN-? secretion in the co-culture of LMP1 CAR-T cells and CNE2 were(550.86 ± 98.64)pg/ml and(438.21 + 44.52)pg/ml,respectively,with a statistically significant difference(P<0.05)compared with CD19 CAR-T cells and T cells.IL-2 and IFN-? secretion in the co-culture of LMP1 CAR-T cells and HONE1 were(95.31 ± 30.60)pg/ml and(201.50 ±20.47)pg/ml,with no statistical difference compared with CD19 CAR-T cells and T cells(P>0.05).LMP1-positive cells promoted the secretion of IL-2 and IFN-? of anti-LMP1 CAR modified T cells.6.Fluorescent imaging assay of tumors in one side showed anti-LMP1 CAR modified T cells inhibited the proliferation of NPC cells in vivo.7.Fluorescent imaging assay of tumors in both sides showed anti-LMP1 CAR modified T cells inhibited the proliferation of LMP1-positive NPC cells in vivo but not LMP1-negative NPC cells.Conclusion In this study,we constructed LMP1 CAR lentiviral expression vector,prepared anti-LMP1 CAR modified T cells successfully and verified the proliferation inhibition of anti-LMP1 CAR modified T cells to LMP1-positive NPC cells in vitro and vivo. |
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