Membrane bound IL-21 based NK cell feeder cells drive robust expansion and metabolic activation of NK cells

Abstract

NK cell adoptive therapy is a promising cancer therapeutic approach, but there are significant challenges that limiting its feasibility and clinical efficacy. One difficulty is the paucity of clinical grade manufacturing platforms to support the large scale expansion of highly active NK cells. We created an NK cell feeder cell line termed ‘NKF’ through overexpressing membrane bound IL-21 that is capable of inducing robust and sustained proliferation (>10,000-fold expansion at 5 weeks) of highly cytotoxic NK cells. The expanded NK cells exhibit increased cytotoxic function against a panel of blood cancer and solid tumor cells as compared to IL-2-activated non-expanded NK cells. The NKF-expanded NK cells also demonstrate efficacy in mouse models of human sarcoma and T cell leukemia. Mechanistic studies revealed that membrane-bound IL-21 leads to an activation of a STAT3/c-Myc pathway and increased NK cell metabolism with a shift towards aerobic glycolysis. The NKF feeder cell line is a promising new platform that enables the large scale proliferation of highly active NK cells in support of large scale third party NK cell clinical studies that have been recently intiatied. These results also provide mechanistic insights into how membrane-bound IL-21 regulates NK cell expansion.

Automated Manufacture of Autologous CD19 CAR-T Cells for Treatment of Non-hodgkin Lymphoma

“…Cytotoxicity assays were performed by co-culturing CAR-T cells with target cells at various effector to target ratios as previously reported (21). Briefly, target cells were pre-labeled with 1 µg/mL calcein-AM (Life Technologies, Carlsbad, CA, United States) for 30 min at 37 • C. 10,000 target cells were plated per well of a 96well plate and various ratios of CAR-T cells were added.…”

“…While a full assessment of patient responses is currently in progress, here we present the persistence of the manufactured CAR-T cells. Peripheral blood samples were obtained at 6,14,21,30,60,90,180, and 365 days after CAR-T cell infusion. Analyses were conducted by qPCR and flow cytometry to quantify CAR-T cell persistence.…”

Use of Cell and Genome Modification Technologies to Generate Improved “Off-the-Shelf” CAR T and CAR NK Cells

“…For CAR NK cells, the cell product should contain mostly CD56 + cells (≥90%), and be free of CD3 + cells (e.g., ≤0.2%) and CD14 + cells (e.g., ≤5%). In the case that CAR NK cells are expanded via co-culture with irradiated feeder cells, for example, membrane bound IL-15 and 4-1BB ligand expressing K562 cells or membrane bound IL-21 expressing OCI-AML3 cells, the final CAR NK cell product should be demonstrated to be free from contamination of co-cultured cells (e.g., ≤1%) (48,49). Contamination of primary NK cell therapeutics with feeder cells may be mitigated by alternative expansion methods, such as use of coated beads or cytokine combinations to expand NK cells.…”

Novel cytokine–antibody fusion protein, N-820, to enhance the functions of ex vivo expanded natural killer cells against Burkitt lymphoma

“… 13 19 It is both cumbersome and expensive to isolate enough primary NK cells from patients for adoptive cell therapy since NK cells make up only 10%–15% of the lymphocytes in peripheral blood. 20 Our group and others have successfully expanded active NK cells in vitro by short-term culture with cytokines alone and co-culture with engineered feeder cells. 21 22 A genetically engineered feeder cells expressing membrane bound IL-21 and 41BBL (K562-mbIL21-41BBL) has been developed by our group to successfully expand peripheral blood mononuclear cells (PBMCs) into NK cells.…”