Development and Scale-up of a Novel GMP Method for Enrichment and Expansion of Terminally Differentiated Adaptive Natural Killer Cells (FATE-NK100) with Enhanced Anti-Tumor Function

Natural killer (NK) cells are innate lymphoid cells that mediate immune responses against pathogens and cancer. Human NK cells are distinguished by the surface phenotype CD3-CD56+ and differential expression of the CD56 surface antigen defines subsets. CD56bright NK cells are presumed to be precursors of CD56dim NK cells, and terminal maturation of CD56dim NK cells is associated with acquisition of CD57. Rather than being an immunosenescence marker CD57 acquisition represents a shift toward greater effector function, including increased CD16 signaling (Fc receptor responsible for triggering antibody-dependent cellular cytotoxicity), enhanced cytotoxicity and decreased responsiveness to interleukin (IL)-12 and IL-18 stimulation. Cytomegalovirus (CMV) infection is uniquely associated with expansion of CD57+ NK cells expressing the activating receptor NKG2C.We have reported that in vivo expanded of CD57+NKG2C+ NK cells (referred to as adaptive NK cells) persist for over one year and are directly associated with reduced leukemia relapse after reduced intensity hematopoietic cell transplantation. Ex vivo expansion to enrich the subset of cells with the adaptive NK cell phenotype represents a new strategy to obtain high numbers of NK cells with enhanced effector function for use in adoptive transfer to treat cancer patients. The main challenge in enriching for CD57+ NK cells using current ex vivo expansion protocols is that IL-15, the cytokine that drives NK cell proliferation and is critical for NK cell survival preferentially expands less mature NK subsets that fail to terminally differentiate in culture. Our group has developed a novel NK cell expansion method that overcomes this barrier. Peripheral blood mononuclear cells from CMV seropositive donors are depleted of CD3+ T cells and CD19+ B cells and cultured for 7-9 days with IL-15 and a small molecule inhibitor of glycogen synthase kinase 3-beta (GSK3β), a multifunctional kinase downstream of the PI(3)K pathway. Compared to vehicle control, addition of the GSK3β inhibitor led to a substantial increase (2.2-fold ± 0.19, n=23, p Disclosures Cichocki: Fate Therapeutics, Inc: Research Funding. Valamehr:Fate Therapeutics, Inc: Employment. Cooley:Fate Therapeutics: Research Funding. Bjordahl:Fate Therapeutics, Inc: Employment. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Rogers:Fate Therapeutics, Inc: Employment. Green:Fate Therapeutics, Inc: Employment. McKenna:Fate Therapeutics, Inc: Research Funding. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Wolchko:Fate Therapeutics: Employment. Miller:Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.