Ex vivo expansion of highly purified NK cells for immunotherapy after haploidentical stem cell transplantation in children.
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 .
Koehl
U
U
University Hospital Frankfurt, Pediatric Hematology and Oncology, Frankfurt, Germany. koehl@em.uni-frankfurt.de
Esser
R
R
Zimmermann
S
S
Tonn
T
T
Kotchetkov
R
R
Bartling
T
T
Sörensen
J
J
Grüttner
H-P
HP
Bader
P
P
Seifried
E
E
Martin
H
H
Lang
P
P
Passweg
J R
JR
Klingebiel
T
T
Schwabe
D
D
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
D018941:Antigens, CD19; D017252:CD3 Complex; D019002:CD56 Antigen