| Objective: Although the therapeutic effects of acute B lymphocytic leukemia and B cell lymphoma have been greatly improved,some patients are still relapsed and refractory.Clinical trials of second-generation CD19-targeted chimeric antigen receptor-engineered(CAR19)T-cell therapy have elicited unprecedented responses.However,CD19-negative relapse represents a most frequent cause of treatment failure and confers dismal outcomes in patients post CD19-directed therapies,so we need to find novel therapeutic strategies.We constructed a third-generation anti-CD19 and anti-CD22 CAR,respectively,then demonstrated their efficacy and safety in vitro and in vivo.We conducted an open-label,single-center and single-arm pilot study to explore the efficacy and safety of sequential infusion of anti-CD19 and anti-CD22 CAR-T cells in patients with r/r B-ALL/NHL.Methods: Anti-CD19 and anti-CD22 were cloned to a lentivirus expression vector.Vectors were co-transfected into 293 T cells to package lentiviruses.CD3+ T cells of sorted from donors were transducted with lentivirus to express anti-CD19 and anti-CD22 CAR.The transduction and apoptosis ratio were tested.Tumoricidal activity,cytokine secretion and CD107 a expression were performed to verify the in vitro function of CD19 and CD22 CAR-T cells and the anti-tumor effect in vivo was verified in a NSG xenograft mouse model.Eighty-nine patients with refractory B-ALL/B-NHL were enrolled in the clinical trial of sequential infusion of anti-CD19 and anti-CD22 CAR-T cells.The patient’s diagnosis and treatment was statistically analyzed to systematically evaluate the safety and efficacy of the clinical trial.The patient’s genetic factors and other clinical parameters were analyzed to explore its prognostic value for CAR-T cell therapy.Results: The third generation anti-CD19 CAR and CD22 CAR-T were successfully constructed,and stably and efficiently expressed on T cells,and the apoptosis level was not significantly increased.CAR-T cells had normal function of tumoricidal activity,cytokine secretion,and degranulation in vitro.Anti-CD19 CAR-T and anti-CD22 CAR-T cells also exhibited a good anti-tumor effects in vivo and did not cause treatment-related toxicity.There was no significant difference between CD19 CAR-T and CD22 CAR-T cells.Among the 51 patients with acute lymphoblastic leukemia,the minimal residual disease-negative response rate was 96.0%(95% confidence interval(CI),86.3 to 99.5).With a median follow-up of 7.6 months(range,1.3 to 22.2),the median progression-free survival(PFS)was 12.0 months(95% CI,5.4 to 18.6),and the median overall survival(OS)was not reached(NR)(95% CI,10.1 to NR).Among the 38 patients with non-Hodgkin lymphoma,the overall response rate was 72.2%(95% CI,54.8 to 85.8),with a complete response rate of 50.0%(95% CI,32.9 to 67.1).With a median follow-up of 5.3 months(range,0.4 to 16.2),the median PFS was 5.8 months(95% CI,3.3 to NR),and the median OS was NR(95% CI,6.1 to NR).Loss of CD19 or CD22 was not detected after CAR19/22 T-cell therapy.High-grade(≥ grade 3)cytokine release syndrome and neurotoxicity occurred in 22.4% and 13.5% patients,respectively.All except one were reversible.Our results indicated that sequential infusion of CAR19/22 T-cell was efficient and safe to circumvent antigen escape relapse in B-cell malignancies.Conclusion: The third generation anti-CD19 CAR-T and CD22 CAR-T cells are effective and specific to kill tumor cells in vitro and in vivo.Our results indicated that sequential infusion of CAR19/22 T-cell was efficient and safe to circumvent antigen escape relapse in B-cell malignancies. |
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