Enhanced Anti-Tumor Activity of Off-the-Shelf NK Cells with Multi-Functional Engineering and Novel Anti-CD19 CAR

The research focuses on developing a novel, ready-to-use natural killer (NK) cell therapy to address limitations associated with anti-CD19 chimeric antigen receptor (CAR) T cells. Traditional CAR-T cell production is patient-specific, inefficient, and costly, and can lead to serious side effects such as cytokine release syndrome and neurotoxicity. To overcome these challenges, a new NK cell therapy was engineered with a unique CAR and additional mechanisms to boost its targeted cytotoxicity.

NK cells are critical in the body's innate and adaptive immune responses, and clinical studies have shown that transferring allogeneic NK cells can lead to lasting remissions in cancer patients without severe adverse events. However, the genetic engineering of NK cells is complex, and their effectiveness can vary due to the need for cytokine support and donor variability.

The study introduces the use of induced pluripotent stem cells (iPSCs) to create a consistent and renewable source of engineered NK cells. The iPSC platform was used to test various CAR combinations with NK-cell specific signaling and transmembrane domains, along with an autonomous protein to ensure the NK cells' persistence and targeted action. The resulting NK cell CAR (NK-CAR) features an NKG2D transmembrane domain, a 2B4 co-stimulatory domain, and a CD3ζ signaling domain, all designed to activate NK cells effectively.

To enhance the anti-tumor capabilities, an anti-CD19 scFv was integrated into the NK-CAR backbone, allowing for the production of a uniform CAR-expressing NK cell population. Additionally, an IL-15RF fusion transgene was introduced to support NK cell function and persistence without the need for external cytokines. The IL-15RF fusion was created by linking IL-15Rα to IL-15. Furthermore, a metalloprotease ADAM17-resistant high-affinity CD16a (hnCD16) 158V variant was added to enhance antibody-dependent cellular cytotoxicity when used with monoclonal antibodies.

The selected iPSC clone stably expresses all three components, providing a consistent starting material for generating NK cells with NK-CAR, IL-15RF, and hnCD16, with high purity and significant expansion during the manufacturing process. Preclinical studies showed that these engineered NK cells were more effective against CD19+ tumor targets and significantly reduced tumor burden in a model of B acute lymphoblastic leukemia. The inclusion of IL-15RF improved NK cell proliferation and potency, even without cytokine support. The NK-CAR + IL-15RF combination was particularly effective, eliminating a high percentage of target cells both with and without IL-2.

The research concludes that iPSCs are an excellent platform for creating a standardized, multi-engineered NK cell product suitable for an "off-the-shelf" approach, providing a proof of concept for a standardized CAR-targeted NK cell immunotherapy against B cell malignancies.