RESUMO
@# Objective: To design and prepare a novel bi-specific chimeric antigen receptor (CAR)-T cell targeting both CD20 and CD19 antigen on B lymphocyte surface, and to detect its killing effect on B lymphocyte tumors as well as its treatment efficacy on immunodeficiency B-NSG mouse with leukemia. Methods: Bi-specific CAR molecule of CD20 (human originated)/CD19 (murine originated) scFv was constructed and packaged into lentiviral vector in 293 cells, and then transfected into T lymphocytes from healthy donors to prepare BiCAR-T cells. K562-CD19-GFP cells (with positive CD19 expression), K562-CD20-GFP cells (with positive CD20 expression) and Nalm6-Luc-GFP cells expressing luciferase were constructed as target cells. After being co-incubated with above mentioned targets cells, the cytotoxic effects of BiCAR-T cells on target cells were evaluated via LDH release assay, and the secretion of IFN-γ by BiCAR-T cells was evaluated by ELISA. Nalm6-Luc-GFP cells were used to construct the mouse model of leukemia and BiCAR-T cells were transfused via tail veins; the treatment efficacy of BiCAR-T cells on tumor bearing mice was evaluated with small animal imaging method. Results: After 7 days’incubation, the BiCAR-T cells originated from healthy donors amplified about 20-50 times with a positive rate of 10%~92%, indicating successful preparation of BiCAR-T cells. Under an effector∶target ratio of 10∶1, the killing rates of BiCAR-T cells against Nalm-6, K562-CD19-GFP and K562-CD20-GFP cells were significantly higher than that of control cells [(76.7±7.4)% vs (8.7±2.4)%, (93.3±5.2)% vs (46.7±6.2)%, (51.0±0.8) vs (30.7±0.5)%, all P<0.01]. Compared with control group, BiCAR-T cells co-incubated with Nalm-6 cells also secreted significantly more IFN- γ [(872.7±7.7) vs (101.0±5.3) pg/ml, P<0.01). Animal experiment showed that BiCAR-T cells had significant efficacy on B-NSG mice with leukemia; NSG leukemia mice treated with BiCAR-T cells all lived up to 70 days (till they were mercy killed) and leukemia cells disappeared at about 50 days, while the mice in PBS and T lymphocytes group all died at (19±3) d and (20±1) d, respectively. Conclusion: Bi-specific CAR molecules expressing CD19 and CD20 were successfully designed and BiCAR-T cells were successfully prepared. The BiCAR-T cells can effectively kill CD19 and/or CD20 tumor cells and secret large amounts of IFN-γ after co-incubation with target cells, exerting significant treatment efficacy on B-NSG immunodeficiency mouse with leukemia.