Clinical Efficacy of Humanized Anti-CD19 Chimeric Antigen Receptor T Cells in the Treatment of Acute B Lymphocytic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To study the safety and effectiveness of humanized CD19-targeted CAR-T cells (hCART19s) for treatment of patients with refractory/relapsed (R/R) B-ALL.@*METHODS@#The analyzed patients were 15 children and adults with relapsed/refractory B-ALL who not received treatment with murine CD19 CAR-T cells. The patients received a single dose (1×10/kg) of autologous hCART19 infusion after lymphodepletion chemotherapy based on cyclophosphamide and fludarabine.@*RESULTS@#Among the 15 patients, 13/14 (92.9%) evaluable patients achieved complete remission (CR) or CR with incomplete recovery of blood cells (CRi) on day 30 after hCART19s infusion. At day 180 after the infusion, the overall survival rate was 73.3%, and the leukemia-free survival rate was 69.2%. The cumulative incidence of relapse was 24.5% and non-relapse mortality rate was 7.7%. During treatment,12/15 patients (80%) developed cytokine release syndrome (CRS) of grade 1-2, and 3 patients (20.0%) developed CRS of grade 3-5. Only one patient (6.7%) suffered from the reversible neurotoxicity.@*CONCLUSION@#hCART19s can effectively treat refractory/relapsed (R/R) adult and children with B-ALL, and the incidence of treatment-related CRS and neurotoxicity is low.

Construction and in vitro verification of a new humanized anti-CD19 CAR-T cells with high affinity / 中华血液学杂志

Objective: To construct humanized anti-CD19 chimeric antigen receptor T cells and investigate its ability to kill leukemia cells in vitro and in vivo. Methods: Humanized anti-human CD19 antibody with a high affinity was obtained based on mouse anti-human CD19 antibody (FMC63). Humanized CD19 CAR-T cells (hCART19) were constructed through transfection of lentivirus carrying a CAR sequence of humanized anti-CD19 scFv into human peripheral CD3(+) T cells. The ability of hCART19 to kill leukemia cells and secrete cytokines was detected by LDH release assay and ELISA. The in vivo tumor-killing effect of hCART19 was evaluated in a leukemia mouse model. Results: Several different humanized CD19 single-chain antibodies which were constructed by IMGT database were expressed in the eukaryotic expression vector and purified followed by acquiring humanized CD19 antibody (Clone H3L2) with similar binding ability to FMC63. Humanized CD19 CAR lentivirus vector was constructed and transfected into T cells to obtain hCART19 cells. The LDH release experiment confirmed that the killing rate of target cells was increased gradually along with the increased E/T ratio. When the ratio of E/T was 10∶1, the killing rate of target cells by hCART19 reached a maximum. When Raji cells were used as target cells, the hCART19 cells group had a significantly higher kill rate [(87.56±1.99)%] than the untransduced T cells group [(19.31±1.16)%] and the control virus transduced T cells group [(21.35±1.19)%](P<0.001). ELISA analysis showed that the secretion of IL-2 [ (10.56±0.88) pg/ml] and IFN-γ [ (199.02±12.66) pg/ml] in the hCART19 cells group were significantly higher than those in the untransduced T cells group [IL-2: (3.55±0.26) pg/ml; IFN-γ: (37.63±0.85) pg/ml] and the control virus transduced T cells group [IL-2: (2.92±0.32) pg/ml; IFN-γ: (52.07±3.33) pg/ml](P<0.001). The above experiments also showed similar results when CHO-K1-CD19 cells were used as target cells. Moreover, in a human leukemia xenograft animal model, the results showed that mice in the untransduced T cells group and the control virus transduced T cells group all died within 20 to 30 days, and the hCART19 cell group survived >40 days, which was more than the survival time of the other two groups of mice. The difference was statistically significant (χ(2)=11.73, P=0.008). Conclusion: Humanized CD19 CAR-T cells with anti-leukemic activity have been successfully constructed, which will lay a foundation for clinical studies in the future.