Off-the-Shelf Natural Killer Cells with Multi-Functional Engineering Using a Novel Anti-CD19 Chimeric Antigen Receptor Combined with Stabilized CD16 and IL15 Expression to Enhance Directed Anti-Tumor Activity

T lymphocytes that express an anti-CD19 chimeric antigen receptor (CAR) to redirect target specificity exhibit remarkable remissions in B cell malignancies. However, these cells are typically produced in a patient-specific manner that is relatively inefficient and expensive. Additionally, CAR-T cell treatment can lead to severe adverse events (SAEs) such as cytokine release syndrome (CRS) and neurotoxicity, as well as graft versus host disease (GvHD) when given in allogeneic setting. To circumvent these safety issues while maintaining multi-faceted anti-tumor activity, we developed an off-the-shelf natural killer (NK) cell consisting of a novel CAR combined with other effector mechanisms to enhance targeted cytotoxicity.

NK cells are potent anti-tumor effector cells that play an important role in innate and adaptive immunity. Multiple clinical studies have demonstrated that adoptive transfer of allogeneic NK cells can induce durable remissions in patients with cancers that have relapsed or are refractory to standard therapies without detection of SAEs such as CRS. However, NK cells are challenging to genetically engineer, and are dependent on cytokine support for persistence and exhibit donor-to-donor variability factors that make it difficult to create a consistent clinical product from NK cells.

Here we report the use of human induced pluripotent stem cells (iPSCs) to produce a renewable source of precisely engineered NK cells. This iPSC platform was utilized to evaluate a combination of CARs comprised of distinct NK-cell specific signaling and transmembrane domains with an autonomous protein to create a persistent and targeted NK cell immunotherapy. The selected NK cell optimized CAR (NK-CAR) backbone contains an NKG2D transmembrane domain, a 2B4 co-stimulatory domain, and a CD3ζ signaling domain to mediate a potent NK cell activating signal. To provide directed anti-tumor activity, anti-CD19 scFv was added to the NK-CAR backbone, engineered at the iPSC stage and subsequently differentiated on-demand to produce a uniform population of CAR-expressing NK cells. In addition, an IL-15RF fusion transgene was introduced to provide self-stimulating signals to support NK cell function and persistence. The IL-15RF construct was created by fusing IL-15Rα to IL-15 at the C-terminus through a flexible linker. As a third modality, a metalloprotease ADAM17-resistant version of the high-affinity CD16a (hnCD16) 158V variant was introduced at the iPSC stage to augment antibody-dependent cellular-cytotoxicity (ADCC) when used in combination with monoclonal antibodies. The selected iPSC clone exhibits stable expression of all three modalities and represents a renewable source of starting material for the reproducible generation of NK cells consisting of NK-CAR, IL-15RF and hnCD16 with product purity that is greater than 95% CD45+CD56+ with a product expansion greater than one million-fold over the course of the manufacturing process. In preclinical studies, these multi-functional engineered NK cells demonstrated enhanced directed cytotoxicity against CD19+ tumor targets when compared to non-engineered NK cells or iPSC-derived NK cells engineered with other CAR constructs. Additionally, the multi-functional engineered iPSC-CAR-NK cells significantly reduced tumor burden in a xenograft model of B acute lymphoblastic leukemia (p<0.05 at day 28). NK cells with IL-15RF inclusion demonstrated improved proliferation in the absence of cytokine support, as well as improved potency when immediately thawed and tested for efficacy. NK-CAR + IL-15RF eliminated 94% and 86% of target cells in the presence and absence of IL-2, respectively, while NK-CAR efficacy was reduced from 98% target elimination in the presence of IL-2 to 56% in the absence of IL-2. In conclusion, these studies demonstrate iPSCs serve as an optimal platform to provide a renewable multi-engineered NK cell product suitable for an "off-the-shelf" approach and serve as preclinical proof of concept for program FT519, a standardized CAR-targeted NK cell immunotherapy against B cell malignancies.