BackgroundAllogeneic Natural Killer (NK) cells are emerging as a safe and effective modality for the treatment of cancer, overcoming several limitations associated with adoptive T cell therapies. Cytokine induced memory-NK cells offer several advantages over conventional NK cells, including enhanced functional persistence, efficacy, and metabolic fitness. Additionally, unlike iPSC and cord blood derived NK cells, they do not require engineering to enable functionality. Here we describe the use of WU-PRIME, a GMP-grade fusion protein complex to generate memory NK cells, and WU-EXPAND, a feeder cell free expansion system to expand memory-NK cells and create WU-NK-101. Further cryopreservation enables the large-scale, off-the-shelf manufacture of memory NK for cancer immunotherapy, with high anti-tumor activity.MethodsNK cells derived from healthy donor leukopheresate were either activated with WU-PRIME and then expanded with WU-EXPAND to form WU-NK-101 or immediately expanded with WU- EXPAND as controls and then cryopreserved. We compared NK cell expansion as well as post- thaw NK cell functionality as assessed by cytokine secretion and short-term and long-term anti- tumor functionality, long-term persistence in NSG mice, as well as anti-tumor activity in vivo.ResultsNK cells activated with WU-PRIME followed by WU-EXPAND (WU-NK-101), expand robustly in large-scale reactions, over 250-fold in 14 days. The cells maintain durable expression of CD25 after expansion, as well as several other hallmarks of the memory-NK phenotype as assessed by mass cytometry. As compared to cells expanded with WU-EXPAND only, WU-NK-101 cells have improved in vitro activity against K562 cells, as well as AML cell lines (TF-1, THP-1, and HL-60). Notably, this functionality is maintained long-term upon repeated challenge. In vivo, WU-NK-101 cells, compared to expanded NK cells have improved in vivo persistence (figure 1; 50,290 v. 9,623, p<0.0001). In vivo anti-tumor activity was also assessed in leukemia models, where Memory NK cells demonstrate superior anti-tumor activity compared to expanded NK cells.Abstract 188 Figure 1NK cell persistence in tumor-bearing mice. 10e6 cryopreserved NK cells were injected into K562 tumor-bearing mice, and supported with 50,000IU human IL-2 every other day. After 9 days, blood was harvested by cheek bleed and assessed for NK cells (hCD45+, CD56+, CD3) in the blood by flow cytometry.ConclusionsThe data demonstrate that WU-NK-101 generated using a feeder cell-free expansion system has a memory phenotype and improved in vitro and in vivo anti-tumor activity compared to conventional NK cells. This activation and expansion platform will enable the development and clinical translation of multiple allogeneic NK cell therapies.

Background Cytokine-induced memory NK cells, like WU-NK-101, offer several advantages over conventional NK cells. 1,2 These advantages include enhanced functional persistence, efficacy, and metabolic fitness that improve their survival and activity in liquid and solid tumor microenvironments. 3,4 Additionally, unlike conventional NK (cNK) cells derived from iPSC, cord blood, or adult peripheral blood, WU-NK-101 does not require engineering to enable anti-tumor activity.The Moneta TM Platform is a feeder cell-free system of fusion protein complexes to generate, expand, phenotypically maintain, and cryopreserve memory NK cells for an 'off-theshelf ' allogeneic drug product. Here we define the molecular characteristics of WU-NK-101, through RNA and protein phenotyping, and elucidate pathways involved in memory NK cell expansion and activity. Methods WU-NK-101 was generated from previously frozen NK cells derived from healthy donor whole blood using the Moneta TM platform. We evaluated pre-and post-expansion phenotype by flow cytometry, CyTOF, and RNA-Seq. We further assessed post-thaw functionality by cytokine secretion and cancer cell cytotoxicity in the absence or presence of monoclonal antibodies. Results CyTOF analysis revealed that WU-NK-101 have an expression profile distinct from cNK. Compared to cNK, WU-NK-101 have elevated expression of Memory NK markers (CD25, CD69, NKG2A), cytotoxic molecules (GzmB, TRAIL), activating receptors (NKp30, NKp44, NKG2D), and nutrient transporters (CD71, CD98), providing mechanistic rationale for their enhanced anti-tumor activity and metabolic flexibility. Flow cytometry and RNAseq analysis confirm this phenotype. When stimulated by cancer cells, WU-NK-101 have enhanced secretion of IFN-g, MIP-1a, and TNFa; and also demonstrate improved cytotoxicity compared to cNK. WU-NK-101 is able to utilize monoclonal antibodies to effectively drive antibodydependent cellular cytotoxicity against solid tumor cancer cell lines. Conclusions The Moneta TM platform expands memory NK cells while maintaining their cytokine-induced memory phenotype, as identified by RNA-Seq, flow cytometry, and CyTOF. As a result, WU-NK-101 demonstrates improved anti-tumor activity compared to cNK cells, even within the adverse tumor microenvironment. These data support the clinical development of WU-NK-101, an allogeneic Memory NK cell therapy in both liquid and in solid tumors, as a monotherapy and in combination with monoclonal antibodies, solid tumor engagers, or other anti-tumor modalities.