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DOI: 10.1055/s-2005-872520
© Georg Thieme Verlag Stuttgart · New York
Ex vivo Expansion of Highly Purified NK Cells for Immunotherapy after Haploidentical Stem Cell Transplantation in Children
Ex-vivo-Expansion hochaufgereinigter NK-Zellen zur Immuntherapie nach haploidenter Stammzelltransplantation bei KindernPublication History
Publication Date:
24 November 2005 (online)
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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 postransplant 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 flowcytometric analyses. Results: Purification led to an absolute number of 234-1 237 × 106 CD56+CD3- NK cells from leukapheresis harvests with a median purity of 95 % and a 4 to 6œ 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 inbalanced chimerism post haplo-SCT as we could show for the first three patients [12].
Zusammenfassung
Hintergrund: Allogene Natürliche Killerzellen (NK) zeigen eine mittlere bis hohe zytotoxische Aktivität gegenüber HLA-nicht-identen Leukämie- und Tumorzellen. Um diesen Effekt für eine Immuntherapie mit NK-Zellen nach haploidenter Stammzelltransplantation (haplo-SZT) immuntherapeutisch zu nutzen, haben wir ein Verfahren zur Aufreinigung von NK-Zellen unter Good Manufacturing Practice (GMP) im klinischen Maßstab entwickelt. Methode: NK-Zellen allogener Spender wurden aus 15 unstimulierten Leukapheresen durch einen doppelten immunomagnetischen T-Zell-Depletionsschritt mit anschließender NK-Selektion gewonnen. Danach erfolgte die Stimulierung und Expansion der CD56+CD3- NK-Zellen in vitro unter GMP. Als Qualitätskontrolle wurde die Reinheit der NK-Zellen, der Gehalt residualer T-Zellen und die Zytotoxizität mittels multiparametrischer Durchflusszytometrie bestimmt. Ergebnisse: Durch die Aufreinigung konnte eine Absolutzellzahl von 234-1 237 × 106 CD56+CD3- NK-Zellen mit einer medianen Reinheit von 95 % und einer T-Zell-Depletion von 4 bis 6œ log-Stufen erreicht werden. Nach 14-tägiger Stimulierung mit IL-2 expandierten die NK-Zellen um das Fünffache mit einer T-Zell Kontamination unter 0,1 %. Die mediane Zellvitalität lag bei 95 % nach Selektion und bei 99 % nach Expansion. Die stimulierten NK-Zellen zeigten eine hohe Zytotoxizität gegenüber der MHC-I-defizienten K562-Zelllinie im Vergleich zu frisch isolierten NK-Zellen und eine mittlere Zytotoxizität gegenüber malignen Leukämiezellen von Patienten. Schlussfolgerung: Wir konnten zeigen, dass die Aufreinigung und Aktivierung von NK-Zellen allogener Spender im klinischen Maßstab möglich ist. Die Applikation einer großen Anzahl NK-Zellen bietet die Basis für ein neues Behandlungskonzept für pädiatrische Patienten mit Leukämien und soliden Tumoren bei minimaler Resterkrankung oder fallendem Spenderchimärismus nach haplo-SZT, was wir vorerst für 3 Patienten zeigen konnten [12].
Key words
NK cells - haploidentical stem cell transplantation - CD56 selection - CD3 depletion
Schlüsselwörter
NK-Zellen - haploidente Stammzelltransplantation - CD56-Selektion - CD3 Depletion
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Ulrike KoehlPh. D.
University Hospital Frankfurt · Pediatric Hematology and Oncology
Theodor Stern Kai 7
60596 Frankfurt
Germany
Phone: +49/69/63 01 49 18
Fax: +49/69/63 01 53 88
Email: koehl@em.uni-frankfurt.de