A Novel Method to Expand Large Numbers of CD56+Natural Killer Cells from a Minute Fraction of Selectively Accessed Cryopreserved Cord Blood for Immunotherapy Post-transplantation

Umbilical cord blood transplantation (UCBT) is increasingly used to treat acute leukemias. UCB-units are thawed and infused in their entirety at transplant, precluding later use as immunotherapy to prevent or treat leukemia relapse. We developed a device which selectively thaws only 1-ml of the UCB-unit leaving the remaining UCB-unit cryopreserved for subsequent transplantation. We also show that large numbers of CD56 + NK cells can be expanded from these 1-ml fractions of selectively-accessed UCB. Immunomagnetic depletion of CD3 + cells of the 1-ml fraction was performed, and the cells were subsequently stimulated with irradiated Epstein-Barr virus-transformed lymphoblastoid cell line (EBV-LCLs) and set to culture in media containing IL-2. When a 1: 20 ratio of TNCs to EBV-LCL feeder cells was used, day 21 and day 35 NK cell cultures initiated from 1 ml of UCB contained a median of 430 ×10 6 (range: 44–4321 ×10 6 ) and 6092 ×10 6 (range: 165–20947× 10 6 ) CD3 − CD56 + NK cells. These cells expressed high levels of CD161, LFA-1, CD69, NKG2D, NKp30, NKp44, NKp80 and NKp46. UCB-derived NK cells were highly cytotoxic against K562 leukemia cells, although cytotoxicity was slightly lower than expanded PBMC-derived NK cells. In conclusion, we have developed and optimized a strategy to selectively access a small fraction from cryopreserved UCB and show that large numbers of CD56 + cells can be expanded from this selectively-accessed fraction. This strategy presents a method to explore whether early adoptive transfer of NK cells expanded from the same UCB-unit used for transplantation can prevent leukemic relapse and decrease graft versus-host disease following UCBT. Large numbers of cytotoxic CD56 + NK cells can be expanded from 1ml of cord blood