title: Ex vivo expansion of highly purified NK cells for immunotherapy after haploidentical stem cell transplantation in children.
abstract: BACKGROUND
  Allogeneic natural killer (NK) cells are known to show medium to high cytotoxic activity against HLA-nonidentical leukemia or tumor cells. For a possible benefit of post transplant treatment with NK cells after haploidentical stem cell transplantation (haplo-SCT) we developed a clinical scale procedure for NK cell processing observing Good Manufacturing Practice (GMP).
  
  
  METHODS
  Allogeneic donor NK cells were selected from 15 unstimulated leukaphereses using two rounds of immunomagnetic T cell depletion, followed by an NK cell enrichment step. CD56 (+)CD3 (-) NK cells were stimulated and expanded in vitro according to GMP. Quality control of NK cell purity, residual T cells and cytotoxic activity was done by multi-coloured flow cytometric analyses.
  
  
  RESULTS
  Purification led to an absolute number of 234-1 237 x 10 (6) CD56 (+)CD3 (-) NK cells from leukapheresis harvests with a median purity of 95 % and a 4 to 6(1/2) log depletion of T cells. After two weeks stimulation with IL-2 a five-fold expansion of NK cells with a T cell contamination below 0.1 % was reached. Median cell viability was 95 % after purification and 99 % after expansion. The IL-2 stimulated NK cells showed a highly increased lytic activity against the MHC-I deficient K562 cells compared to freshly isolated NK cells and a medium cytotoxicity against patients' leukemic cells.
  
  
  CONCLUSIONS
  Clinical scale enrichment and activation of allogeneic donor NK cells is feasible. High dose NK cell application may be a new treatment option for pediatric patients with leukemia or solid tumors in case of minimal residual disease or unbalanced chimerism post haplo-SCT as we could show for the first three patients .
authors.affiliation: University Hospital Frankfurt, Pediatric Hematology and Oncology, Frankfurt, Germany. koehl@em.uni-frankfurt.de
authors.forename: U
authors.initials: U
authors.lastname: Koehl
authors.forename: R
authors.initials: R
authors.lastname: Esser
authors.forename: S
authors.initials: S
authors.lastname: Zimmermann
authors.forename: T
authors.initials: T
authors.lastname: Tonn
authors.forename: R
authors.initials: R
authors.lastname: Kotchetkov
authors.forename: T
authors.initials: T
authors.lastname: Bartling
authors.forename: J
authors.initials: J
authors.lastname: Sörensen
authors.forename: H-P
authors.initials: HP
authors.lastname: Grüttner
authors.forename: P
authors.initials: P
authors.lastname: Bader
authors.forename: E
authors.initials: E
authors.lastname: Seifried
authors.forename: H
authors.initials: H
authors.lastname: Martin
authors.forename: P
authors.initials: P
authors.lastname: Lang
authors.forename: J R
authors.initials: JR
authors.lastname: Passweg
authors.forename: T
authors.initials: T
authors.lastname: Klingebiel
authors.forename: D
authors.initials: D
authors.lastname: Schwabe
mesh_terms: D018941:Antigens, CD19 / Q000276:immunology*; D017252:CD3 Complex / Q000032:analysis; D019002:CD56 Antigen / Q000032:analysis; D002460:Cell Line; D002470:Cell Survival / Q000276:immunology; D002648:Child; D015925:Cryopreservation; D003601:Cytotoxicity Tests, Immunologic; D005434:Flow Cytometry; D006238:Haploidy; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006801:Humans; D016219:Immunotherapy, Adoptive; D066298:In Vitro Techniques; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000276:immunology*; D007937:Leukapheresis; D007938:Leukemia / Q000276:immunology / Q000628:therapy*; D007945:Leukemia, Lymphoid / Q000276:immunology / Q000628:therapy*; D018655:Lymphocyte Count; D008212:Lymphocyte Depletion
chemical_list: D018941:Antigens, CD19; D017252:CD3 Complex; D019002:CD56 Antigen