Large-scale preparation of adherent lymphokine-activated killer (A-LAK) cells for adoptive immunotherapy in man

Abstract

Stepwise counterflow centrifugal elutriation of leukapheresed human mononuclear cells (MNC) in a Beckman JE-6B rotor and J6-M/E centrifuge yielded a population highly enriched in natural killer (NK) cells (70-75% large granular lymphocytes with 10-13 times greater NK activity) at a flow rate of 38-44 ml/min using a fixed rotor speed of 3000 rpm at 27 degrees C. However, the mean cell recovery was less than 1%. To obtain sufficient numbers of purified NK cells for adoptive immunotherapy, a strategy combining counterflow centrifugal elutriation with adherence of recombinant interleukin-2(rIL-2)-activated NK cells to plastic was developed. First, MNC were elutriated to give a twofold enrichment in NK cells, containing 22% Leu19+ cells, 18% large granular lymphocytes and 51 lytic units of activity against K562 targets as opposed to the unfractionated MNC containing 10% Leu19+ cells, 7% large granular lymphocytes and 26 lytic units of activity. The mean recovery was 80 +/- 15% (n = 10). Further enrichment was obtained by isolation of the elutriated cells that adhered to plastic after culture for 24 h in the presence of 1000 U/ml rIL-2. The initial adherent lymphokine-activated killer (A-LAK) cells represented 1-4% of total MNC, but their subsequent expansion was at least 10-22-fold during 8-14 days in culture with 1000 U/ml rIL-2. Using this strategy, 2 x 10(9) normal MNC, obtained by leukapheresis, yielded 5 x 10(8) A-LAK cells with a total of 5.7 x 10(5) lytic units of cytotoxicity against K562 and a total of 3.3 x 10(5) lytic units against Daudi targets. This enrichment method has yielded sufficient numbers of A-LAK cells to form the basis for a phase I clinical trial of adoptive immunotherapy in patients with advanced cancer.