W-NK1 Choreographs Innate and Adaptive Immune Responses to Provide a Robust and Durable Anti-AML Response
Authors: Leedom Tom , Muz Basia , Magee Kristann , Vadakekolathu Jayakumar , Arthur Laura , Tran Melissa , Luukkonen Lena , Mahajan Nitin , Hamil Alexander , Muth John , Berrien-Elliott Melissa M , Fehniger Todd A , Rutella Sergio , Davidson-Moncada Jan K 
Published: December 2024

Memory-like natural killer (ML-NK) cells represent a promising advancement in cancer immunotherapy, offering superior anti-tumor activity compared to conventional NK (cNK) based cell therapies. Overall, adoptive cell therapy for cancer immunotherapy has been hampered by immunosuppressive signals within the tumor micro-environment (TME), effectively abrogating proliferation, infiltration and antigen presentation of innate and adaptive immune cells. WU-NK-101 (W-NK1) is a cytokine-reprogrammed, cryopreserved, off-the-shelf adoptive ML-NK cell therapy, derived from cNK cells, with a unique phenotype consisting of enhanced levels of activation markers and immune cell engagement receptors. We have previously shown that these molecular characteristics circumvent hurdles encountered within the TME (Rutella, et al. ASH 2023). Herein, detailed mechanisms through which W-NK1 engages the endogenous immune system, enhancing recruitment and activation of immune cells in the TME resulting in deep and durable anti-AML responses are reported.

Using flow cytometry, we confirmed that W-NK1 drug product has higher expression of NKG2A, 2B4, and DNAM-1 as well as the immune engaging receptors CD100, CD86 and CD337 compared to donor-matched cNK cells. Upon activation by target cell killing, W-NK1 cells release a variety of cytokines and chemokines which lead to maturation and activation of dendritic cells (DC) (TNFa, IFNg, MIP-1b/CCL4, RANTES/CCL5, MIP-3a/CCL20), migration and activation of T cells (CXCL9, CXCL10, CXCL11), and maturation of macrophages (CCL2, CCL5). Indeed, we show that co-culture of immature DC, with W-NK1 in the presence of AML cells caused increased expression of the activation markers CD40, CD80, CD83 and CD86 leading to enhanced phagocytosis of target cells (~3-fold greater than when incubated with cNK). Additionally, W-NK1 secreted chemokines have the ability to recruit T-cells in a transwell assay, suggesting that W-NK1 are able to recruit T-cells into the TME. In mixed lymphocyte reactions combining NK with PBMCs and donor matched monocyte-derived DC, T cell activation (determined by CD25 expression) and proliferation was significantly enhanced when W-NK1 was compared to cNK (P &lt;0.01). In the absence of matured DC T-cell proliferation was much lower (P &lt;0.01). Overall, this demonstrated coordination between NK cells and DC toward T-cell activation that was significantly enhanced by W-NK1. Finally, we assessed the impact of W-NK1 on monocyte-derived M1 vs. M2 macrophages in a co-culture assay. W-NK1 demonstrated selective cytotoxicity against pro-tumorigenic M2 macrophages while sparing M1 macrophages (P &lt; 0.001) indicating an ability to tilt the TME toward a less immunosuppressive environment.

These findings were confirmed in multiple humanized AML xenograft NCG models (THP-1 and HL-60). In vivo, W-NK1 synergized with the human immune system to coordinate robust anti-AML responses. In concert with human immune cells, W-NK1 treatment led to deeper (&gt;10-fold) and more durable anti-tumor responses compared to humanization or W-NK1 treatment alone (P &lt;0.05). Immune engagement was confirmed by immunofluorescence staining of the tumor samples on a GeoMx Digital Spatial Profiling platform (NanoString Technologies, San Diego, CA). Furthermore, splenocyte-derived T-cells extracted from HL-60 humanized NCG mice treated with W-NK1-demonstrated a significantly enhanced ability to kill the parental HL-60 tumor cells ex vivo (~2-fold vs. vehicle at E:T 3:1; P &lt;0.001). Importantly, the splenocyte-derived T-cells appear to retain specificity as T-cells ‘educated’ against the HL-60 tumor cell line were less effective against an alternate THP-1 AML tumor type (1.0-fold vs. vehicle at E:T 3:1) indicating potentiation of allo-response.

In summary, our studies demonstrate that the unique properties of W-NK1 leverages potent engagement with the endogenous immune system through multiple mechanisms. This exquisite synergy between innate and adaptive mechanisms is enabled through W-NK1 reconditioning of the adaptive immune response, effectively enhancing recruitment, proliferation, activation state and antigen presentation leading to a deep and durable adaptive response. In this manner the pharmacodynamic effect of W-NK1 extends far beyond the pharmacokinetic effect. These data augur positively for patients to be treated with W-NK1 in future studies.