Significant Ex-Vivo Expansion of Cord Blood (CB) Natural Killer (NK) Cells and Concomitant Decrease in CB T-Cells by Genetically Reengineered K562 Cells (K562-mbIL15-41BBL)

The limitations of the use of NK cells in adoptive tumor immunotherapy include lack of tumor recognition by NK cells and limited numbers of functionally active NK cells (Shereck/Cairo et al, Pediatr Blood Cancer, 2007). To circumvent these limitations, methods to expand and activate peripheral blood NK cells have been developed (Imai et al, Blood, 2005). We previously reported the successful expansion and functional activation of cord blood NK cells by ex-vivo cellular engineering with a cocktail of antibody and cytokines (Ayello/Cairo et al, BBMT, 2006). In this study we sought to develop an improved CB NK cell expansion system for use in adoptive cellular immunotherapy strategies. Freshly isolated CB mononuclear cells (CBMC) were cultured with modified K562 cells expressing membrane bound IL15 and 4-1BB ligand (K562-mbIL15-41BBL; Imai et al, Blood, 2005). After irradiation with 100Gy, K562-mbIL15-41BBL cells were incubated in a 1:1 ratio with CBMC + 10 IU/mL rhIL-2 for 7–14 days. CD3 and CD56 expression was determined by flow cytometry at Days 0, 7 and 14. On Day 0, CBMC included a population of CD56+/CD3- NK cells of 3.9% ± 1.3%. After 7 days of culture with K562-mbIL15-41BBL cells the percentage of NK cells increased to 71.7% ± 3.9%, as compared to 9.7% ± 2.4% in cultures with media alone and 42.6% ± 5.9% in cultures with wild-type K562 cells (p<0.01). Concomitantly, there was a significant decrease in CB T-Cells vs culture with wild-type K562 or media alone (15.1 ± 1.7% vs 35.7 ± 2.4% vs 51 ± 7.1%, respectively) (p<0.001). Overall, this represented a 35-fold or 3374% ± 385% increase of the input NK cell number. This was significantly increased compared to culture with wild-type K562 (1771% ± 300%, p<0.05). On Day 14, there remained a significant increase in NK cell populations between CBMC incubated with modified K562 cells compared to wild-type K562 cells and media alone (62.0% ± 2.1% vs 27.9% ± 2.4% vs 5.5% ± 0.4%, p<0.001). A standard cryopreserved UCB unit (25 mL) contains approximately 750 × 106 MNCs. By using the smaller 5mL aliquot (20%) of a two-aliquot bag (150 × 106 MNCs x 3.9% = 5.8 × 106 NK cells), this expansion method would hypothetically yield approximately 200 × 106 CB NK cells after 7 days of culture. In summary, we have demonstrated CBMC can be ex-vivo expanded by K562 cells expressing membrane bound IL15 and 4-1BB ligand (K562-mbIL15-41BBL) resulting in specific expansion of CB NK cells and decrease in CB T-Cells.