Abstract
Chimeric antigen receptor (CAR) technologies have transformed treatment of haematological malignancies by directly targeting cancer-associated antigens. However, CAR T cell therapy is limited by T-cell exhaustion, antigen escape and toxicities. Recently, Natural Killer (NK) cells have been explored as a CAR candidate due to their easier ex vivo expansion and their inherent cytolytic abilities. To investigate whether NK cells can synergise with CAR T cells, we developed a dual targeting therapy combining CD19-specific CAR T cells with Natural Killer Group 2D (NKG2D)-based CAR NK cells to target B cell acute lymphoblastic Leukaemia (B-ALL) cell line NALM-6.
Third generation self-inactivating (SIN) lentiviral (LV) vectors encoding either a second generation CD19 CAR or NKG2D CAR were produced in Human Embryonic Kidney (HEK) 293T cells, concentrated, and titrated by flow cytometry. Primary human T and NK cells were activated and transduced at optimised multiplicities of infection (MOI) of 20 and 10, respectively. CAR expression was detectable on T cells by day 10 post transduction, with NK cells showing robust expression as early as day two, as confirmed by flow cytometry.
Functional activity was evaluated in vitro using luciferase-expressing NALM-6. CD19 CAR T cells outperformed untransduced T cells (p = <0.0001), while untransduced NK cells proved more effective than NKG2D CAR NK cells (p = 0.0004). In vivo efficacy was tested in NSG mice bearing NALM-6-Luc xenografts. Mice received a single intravenous dose of 1 × 106 NALM-6 cells, followed by CAR NK cells on day two and CAR T cells on day four, with tumour burden monitored by bioluminescent imaging. Combined therapy produced rapid tumour clearance of NALM-6-Luc xenografts, with majority of subjects surviving until the termination of the iiiexperiment at day 61. This outcome surpassed the average survival observed in both untreated control and CAR T monotherapy.
Dual-targeting approach has the potential to promote T cell persistence and NK cell cytotoxicity to address antigen loss and immune evasion, representing a versatile strategy to enhance the safety and efficacy of CAR-based immunotherapy in B-ALL.