Engineering stem cell-derived NK cells with chimeric antigen receptors for immunotherapy of acute myeloid leukemia
Acute Myeloid Leukemia (AML) continues to be a challenging malignancy, with 32% of adult patients surviving beyond five years post-diagnosis. In children and adolescents, the five-year survival rate reaches 70%, but still 20-30% of patients still succumb to relapse and treatment-related toxicities. Therefore, new therapies are needed to improve the outcome of AML patients of all ages whilst minimizing treatment-related side effects. Chimeric Antigen Receptor T cell (CAR-T) therapies have demonstrated promise in hematological malignancies, but AML remains particularly difficult to treat due to prolonged on-target, off-tumor toxicities. Allogeneic natural killer (NK) cell therapies offer a potential alternative, with evidence of favorable tolerability and complete remission rates of up to 47% in patients with relapsed/refractory AML.
To optimize NK cell adoptive immunotherapy for AML, we have developed a Good Manufacturing Practice (GMP)-compliant protocol for generating highly pure and potent NK cells from CD34+ hematopoietic stem and progenitor cells (HSPCs). To further augment the expansion capacity and functionality of our HPSC-NK cells, we employ the expression of transpresented IL-15 tethered to IL-15 receptor alpha (tIL-15). Additionally, we have engineered anti-CD33, CD123, and CLL1 CAR constructs to improve targeting to prevent antigen-negative escape. Moreover, we assessed various intracellular domain combinations for optimized transgene expression and functionality. These were used to generate CAR-HSPC-NK cells by transducing the HSPCs with viral constructs, followed by differentiation into NK cells.
Umbilical cord blood-derived HSPCs were successfully transduced with IL-15 and CAR constructs, achieving up to 80% transduction efficiency. CD34+ stem cells demonstrated improved capacity to differentiate into CD56+CD3- HSPC-NK cells without exogenous IL-15 supplementation. In addition, the tIL-15+ fraction of fully differentiated HSPC-NK cells was enriched in percentage and MFI when cultured for up to 6 days without IL-15, indicating a survival and proliferative advantage of the transduced cells. Furthermore, CAR-HSPC-NK cells exhibited superior potency compared to wildtype HSPC-NK cells, with a 3-fold increase in CD107a expression for CAR-HSPC-NK cells, indicating enhanced degranulation and cytotoxicity, compared to non-engineered cells. Furthermore, the intracellular domain modifications of the CAR enhanced its antigen-specific activity, which we further validated in the NK-92 cell line model.
Our data demonstrate that the HSPC-NK cell platform can be genetically modified to generate highly potent NK cells for the treatment of AML. By introducing tIL-15 and optimal CAR constructs, we significantly enhanced the functionality and fitness of these cells, positioning this platform as a promising candidate for next-generation immunotherapy in AML.