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

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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° C. However, the mean cell recovery was + 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 × 109 normal MNC, obtained by leukapheresis, yielded 5 × 108 A-LAK cells with a total of 5.7 × 105 lytic units of cytotoxicity against K562 and a total of 3.3 × 105 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.

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