Optimal Xenogeneic Adoptive Transfer of Human NK Cells: Fresh NK Cells and IL-15 Administration Are Superior to Frozen NK Cells and IL-2
Authors: Tolar Jakub , Curtsinger Julie , McElmurry Ron , McCullar Valarie , Verneris Michael R. , van Rhee Frits , Lapteva Natalia , Rooney Cliona M , Wagner John E. , Blazar Bruce R. , Miller Jeffrey S. 
Published: October 2019

Abstract
Abstract 346
Expansion of NK cells after adoptive transfer is a major determinant of their anti-tumor efficacy but the mechanisms of their expansion are not completely understood. To study in vivo NK cell expansion and the relative merits of fresh versus frozen NK cell products, we used xenotransplantation of human GMP NK cell products (provided by two PACT centers funded by the NHLBI) into immune deficient mice. NOD/IL-2Rγc/Rag (NOG) that lack T-, B-, and NK cells and that have a macrophage defect that renders recipients highly amenable to human cell engraftment, were given 250 cGy total body irradiation, and infused with fresh or frozen NK cells. Two products were tested: 1) an enriched fresh NK cell product obtained by CD3 and CD19 depletion followed by overnight activation with IL-2 or IL-15 (U. of Minnesota) or 2) ex vivo expanded NK cells using membrane bound IL-15 and 41BB-L transduced K562 cells with 10 U/ml IL-2 in G-Rex devices for 10 days (Baylor). NK cells (1–2 million NK cells for each individual experiment) were given IV and mice were treated with no cytokines, 5 or 10 mg of IL-2 (Novartis), or 5 mg IL-15 (produced by the NCI for clinical use) as 3 injections per week for 2 weeks. Peripheral blood (Table), spleen and marrow were evaluated on days 7, 14, 21 and 28 after adoptive transfer. Counts were converted to absolute cell numbers per 100 μL of blood or the number of cells recovered per spleen or after flushing of a single femur. On day 7, the number of huNK cells in blood in the absence of cytokine administration was low with both cell products and numbers continued to diminish by day 14. In marked contrast, every other day IL-2 or IL-15 significantly increased huNK cells in murine blood. On day 7 and 14, ex vivo expanded NK cell products resulted in significantly higher numbers of huNK in mice receiving 5μg IL-2 compared to mice who received fresh NK cells activated overnight with IL-2. With 5μg IL-2, huNK number decreased between day 7 and 14 irrespective of cell product. In contrast, only IL-15 lead to increased numbers of huNK cells between days 7 and 14. Previously cryopreserved NK cells showed significantly worse survival for both cell products, with a larger fold decrease seen with ex vivo expanded cells. Blood collected from cytokine treated mice at day 14 after infusion of fresh products contained huNK cells that were fully functional as assessed by potent CD107a degranulation, TNF and IFN production after exposure to K562 target cells as well as augmented IFN production induced by IL-12 and IL-18. On day 8, Ki67+ (proliferating) huNK cells were significantly higher with IL-15 compared to IL-2 (both at 5μg) in marrow for fresh (76% vs 35%, p=<0.001) and ex vivo expanded (88% vs 53%, p=0.003) NK cells. Ki67+ proliferating NK cells had decreased by day 14 under all conditions. With fresh NK cells and IL-15 on day 8, there were 1371 huNK relative counts recovered from spleen compared to 450 recovered from marrow (p=0.01). In marked contrast, ex vivo expanded NK cells (that express a lower percentage of CD62L compared to fresh NK cells) had a reverse homing pattern with 874 huNK cells in spleen versus 2146 in marrow (p=0.07). There was a significant difference between the two cells products in terms of the absolute numbers of huNK cells found in marrow at day 8 (p=0.0007). We conclude that 1) ex vivo expanded NK cells home better to BM which may be useful in treating AML localized to BM, whereas fresh NK cells may be preferable for homing to lymphoid tissue in the periphery; 2) at early time points fresh huNK cells were less abundant than expanded cells but catch up at late time points; and 2) IL-15 is more effective than IL-2 on a mcg basis. Overall, fresh and ex vivo expanded NK cells have different properties and kinetics after infusion. This xenogeneic model results in definitive readouts to optimize the best characteristics of each cell product to tailor a product to have maximal efficacy for clinical testing.
Results of experiments with fresh or ex vivo expanded NK cells
Exp. Activated NK source In vivo cytokine # mice Avg hu NK Day 7/100 μL muBlood Avg hu NK Day 14/100 μL muBlood 1 Fresh NK none 6 117 24 IL2-5 ug 6 238 53 Ex vivo expanded none 6 28 1 IL2-5 ug 6 760 235 2 Fresh NK none 4 212 16 IL2-5 ug 4 465 93 IL15-5 ug 4 540 1151 Ex vivo expanded none 4 63 9 IL2-5 ug 4 888 181 IL15-5 ug 4 3221 2930 3 Fresh NK none 12-16 141 55 IL2-5 ug 12-16 331 169 IL2-10 ug 12-16 197 182 IL15-5 ug 12-16 266 1177 Ex vivo expanded none 12-16 22 3 IL2-5 ug 12-16 323 106 IL2-10 ug 12-16 372 673 IL15-5 ug 12-16 735 1955

Disclosures:
Miller: Celgene: Membership on an entity's Board of Directors or advisory committees; Coronado Bioscience: Membership on an entity's Board of Directors or advisory committees.