Enhanced Antimetastatic Activity of Lymphokine-activated Killer Cells Purified and Expanded by Their Adherence to Plastic1

We have recently reported a simple and reproducible technique for the purification and rapid expansion of homogeneous populations of large granular lymphocytes expressing a natural killer cell phenotype and high levels of broad antitumor cytotoxic activity [lymphokine-activated killer (LAK) activity]. This technique exploits the observation that, in the presence of recombinant interleukin 2 (rIL-2), large granular lympho cytes/natural killer cells become adherent to plastic surfaces, actively proliferate, and acquire high levels of LAK activity. Because of their adherent properties these cells have been termed adherent LAK or ALAK cells. The present studies investigate the antimetastatic effects of A-LAK cells in a syngeneic rat model of experimental pulmonary and hepatic métastases. For pulmonary métastases, F344 rats received i.v. injections with a natural killer-resistant mammary adenocarcinoma, MADB106, and, for hepatic métastases,animals received an intrasplenic injection of MADB106 tumor cells followed by surgical splenectomy. Three days later, the animals were treated with A-LAK cells alone, ALAK cells plus rIL-2, or rIL-2 alone. These treatments were compared to immunotherapy using standard cultures of LAK cells (unfractionated spleen cells) and rIL-2. The results indicate that the administration of unfractionated LAK cells plus interleukin 2 (II.-2) was effective in reducing established lung or liver métastasesin this rat model. However, the results also indicate that purified populations of A-LAK cells in combination with rIL-2 demonstrate dramatic and superior antimetastatic effects when compared to LAK cells cultured under standard conditions. The antimetastatic effects of standard LAK cells or A-LAK cells plus 1I.-2 translated into significant survival benefits compared to animals receiving no therapy or IL-2 therapy alone. Survival after therapy with A-LAK cells plus IL-2 was significantly prolonged compared to treatment with standard LAK cells. These data suggest that purified populations of LAK cells (derived from natural killer cells) may prove superior for adoptive immunotherapy in the clinical setting. INTRODUCTION AIT4 using LAK cells and rIL-2 has recently gained much attention due to the observed antimetastatic activity in experi mental animal models (1-4) and in some cancer patients (5-8). LAK cells are generated by the culture of lymphocytes derived from normal or cancer patients in crude IL-2 or rIL-2 (9, 10). Effective AIT using LAK cells plus rIL-2 requires the admin istration of large numbers of cultured lymphocytes and high (often toxic) levels of rIL-2. These limitations have compro mised the general use of this form of immunotherapy in the clinical setting. Moreover, because the actual frequency of the precursors of LAK effector cells within a population of periph eral blood lymphocytes is generally arond 10% (11-14), it has Received 6/6/88; revised 10/7/88; accepted 12/19/88. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported in part by Grants CA43765 and HL37638 from the NIH (J. C. H.) and by Grant Schw-368/1 from the Deutsche Forschungsge meinschaft (R. E. S.). 2On sabbatical leave from the Institute of Oncology and Radiology, Pasterova 14, 11000 Belgrade, Yugoslavia. 3To whom requests for reprints should be addressed, at Pittsburgh Cancer Institute, 3343 Forbes Avenue, Pittsburgh, PA 15213. 4The abbreviations used are: AIT, adoptive immunotherapy; LAK, lympho kine-activated killer; NK, natural killer; FCS, fetal calf serum; LGL, large granular lymphocyte; rIL-2, recombinant human interleukin 2; IL-2, interleukin 2; A LAK, plastic-adherent LGL/LAK; HBSS, Hanks1 balanced salt solution. not been possible to purify these cells to obtain sufficient numbers to be used for therapy. A major hypothesis has sug gested, however, that if unfractionated populations of IL-2activated lymphoid cells could mediate beneficial antitumor effects, then purified LAK effector cells should mediate even better effects. In addition, the use of purified populations of LAK cells would allow investigations into additional areas of study, such as /// vivo migration patterns, cytokine production, and cellular interactions responsible for the LAK effect. There fore, the development of simple and reproducible techniques in which the LAK effector cells could be purified and expanded to sufficient numbers to be used for therapy yet require a lower level of rIL-2 to support their antitumor activity would provide an alternative and presumably preferable technique to current culture methods. We have recently reported a simple technique for the isolation and rapid expansion of purified populations of LGL/NK cells with high levels of broad antitumor (LAK) cytotoxic activity (15). This technique involves the adherence of LGL to plastic surfaces induced by rIL-2. A-LAK cells expand rapidly in culture (up to 100-fold in 4 to 5 days) and generate exceedingly high levels of LAK cytolytic activity. The present study analyzes the antimetastatic activity of ALAK cells against 3-day established lung and hepatic métastases of an NK-resistant mammary adenocarcinoma, MADB106. The results demonstrate that A-LAK cells are superior to standard cultures of LAK cells in mediating the reduction of experimental métastasesas well as in conferring increased animal survival. MATERIALS AND METHODS