Directed Differentiation of Mobilized Hematopoietic Stem and Progenitor Cells into Functional NK Cells with Enhanced Antitumor Activity

Abstract

Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.

Current Perspectives on “Off-The-Shelf” Allogeneic NK and CAR-NK Cell Therapies

“…Peripheral blood contains NK cells and is a reliable source of CD34 progenitor cells from individuals undergoing GCS-F mobilization ( 34 ). Isolating large numbers of PB NK cells and hematopoietic stem cells is difficult as the percentage derived from leukapheresis can be low and highly variable ( 22 , 35 , 36 ).…”

Enhancing a Natural Killer: Modification of NK Cells for Cancer Immunotherapy

“…Membrane IL-21 alone [ 39 ] and IL-21 in combination with IL-15 [ 40 , 41 , 42 ] promotes the expansion of NK cells ex vivo with preclinical studies establishing efficacy against solid tumors. Several clinical trials also show the value of using IL-21 alone against metastatic melanoma, but drug combinations have yielded mixed outcomes [ 43 , 44 ].…”

“…A recent study by Oberoi et al [ 42 ] showed that healthy donor PB can serve as a valuable starting material for creating hematopoietic stem and progenitor cells (HSCs), despite earlier studies indicating poor and donor-dependent differentiation [ 172 , 173 , 174 , 175 , 176 ]. This was due to a refined approach to differentiation and expansion protocols that utilized optimized cytokine cocktails and expansion enhancement with K562 feeder cells, co-expressing 4-1BB ligand and mbIL-15 and IL-21 [ 42 ] (see Section 3.1 ). As healthy PB is readily available and easier to access compared to umbilical cord blood (UCB) and human embryonic stem cells (hESCs), this protocol may serve as an economically viable option for NK cell generation.…”

HLA Class I Knockout Converts Allogeneic Primary NK Cells Into Suitable Effectors for “Off-the-Shelf” Immunotherapy

“…However, two major aspects of human NK cells, the marked resistance to standard genetic modifications with lentiviral vectors and the limited ex vivo expansion capacities, have hampered their use for both allogeneic and also autologous CAR-redirected immunotherapies. Hence, researchers have resorted to stable NK cell lines, such as NK-92 (36)(37)(38)(39)(40)(41)(42), or to using NK cells differentiated from CD34+ hematopoietic stem and progenitor cells (HSPCs) or pluripotent stem cells (PSCs) (43)(44)(45)(46) with subsequent expansion using feeder cells (47)(48)(49)(50).…”