Technical aspects of lymphokine‐activated killer cell production

Abstract

Adoptive immunotherapy is a novel approach to treating patients with cancer, utilizing as therapy a patient's own peripheral blood lymphocytes that have been activated by incubation with interleukin‐2 (IL‐2). These cells develop the ability to mediate tumor regression in vivo and are referred to as lymphokine‐activated killer (LAK) cells. The production of LAK cells is a complex and labor‐intensive process. Lymphocytes are collected by continuous‐flow centrifugation, purified on Ficoll‐Hypaque (FH) density gradients, incubated in vitro with IL‐2, and then harvested for infusion into the patient. An automated approach to LAK cell generation has been developed using the Fenwal CS‐3000 cell separator and polyolefin PL‐732 blood storage bags. Lymphocyte concentrates (LC) containing 6.5 × 10⁹ mononuclear cells per pack were obtained using standard leukapheresis techniques. Disposable apheresis kits were then modified to allow the LC to be pumped into the separation chamber along with a counter‐centrifugal flow of saline, removing the platelets and plasma by elutriation. The remaining cells were underlaid with FH, displacing the lymphocytes into a collection bag, where they were washed and concentrated. Mean leukocyte recovery was 59.2% {99.9% lymphocytes, (n=14). The final product contained 6.7% of the initial platelets and had a hematocrit of 0.05) and platelet contamination was significantly reduced (2.7% ± 0.5% vs. 26.6% ± 5.7% residual platelets, P < 0.001). Cells were first diluted in the collection bag with RPMI 1640 culture medium supplemented with 2% human AB serum and then dispensed via a media pump into polyolefin storage bags. The final cell concentration was 1.5–3.0 × 10⁶/ml and was supplemented with IL‐2, 1,000–1,500 U/ ml. After 3–4 days of culture in 5% CO₂ at 37°C, LAK cells were harvested and washed in an automated manner using the CS‐3000 device. The cells were infused at 88 ml/min into each of two independent collection chambers (total flow rate 176 ml/min), pelleted against the bag wall, and concentrated. Residual culture media was removed by a final wash with 2 liters of saline. Cells were resuspended in normal saline supplemented with human serum albumin plus IL‐2 and diverted into a transfer pack. In paired studies, cell yields using the automated harvest technique compared favorably with those obtained using the standard manual harvests (79.2% ± 5.4% vs. 64.9% ± 5.0%, P < 0.01). Lytic capacity of the cells against fresh human tumor was assessed in a 4‐h⁵¹Cr release assay. The cytotoxicity of cells prepared using the automated system was equivalent to that obtained using standard manual methods. Production of LAK cells in the automated system reduces the complexity of the process, decreases the potential for bacterial contamination, and decreases manpower needs.