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Optimal methods for generating expanded lymphokine activated killer cells capable of reducing established murine tumors in vivo.

The systemic administration of lymphokine activated killer (LAK) cells and recombinant interleukin-2 (RIL-2) is effective in reducing the number of established pulmonary and hepatic metastases from multiple murine tumors and has recently been shown to be effective in mediating the regression of metastatic cancer in humans as well. The generation of sufficient numbers of LAK cells for the effective therapy of human tumors remains a major obstacle to the widespread application of this immunotherapeutic approach. We have thus studied methods for the in vitro expansion of LAK cells effective in immunotherapy. Our previous studies used LAK cells generated in culture with RIL-2 for 3 days. LAK cells cultured in RIL-2 for 5 or 7 days were not significantly different from cells cultured for 3 days either in the number of cells obtained, their in vivo cytotoxicity or their in vivo therapeutic effectiveness. When day 3 LAK cells were transferred to fresh culture medium containing 1000 U/ml of RIL-2, a highly reproducible expansion of these cells was obtained. By day 5, cell numbers expanded 9.6 +/- 0.8-fold (mean +/- SEM; n = 36) and by day 8, cells expanded 15.1 +/- 1.0-fold (n = 19). In 4 h 51Cr release assays against fresh tumor target cells, day 3 LAK cells had a mean of 13 lytic units/10(6) cells in 24 experiments. Day 5 expanded LAK cells had a mean of 30 lytic units/10(6) cells in 13 experiments (P less than 0.05 compared to day 3 LAK cells) and day 8 expanded LAK cells had a mean of 11 lytic units/10(6) cells in 6 experiments (P = NS compared to day 3 LAK cells) When day 5 and day 8 expanded LAK cells were infused in vivo with RIL-2, they were found to significantly reduce the number of experimentally induced pulmonary metastases as effectively as non-expanded conventional day 3 LAK cells. Similar findings were documented in experiments against hepatic metastases. These experiments demonstrate that LAK cells could expand a mean of 15-fold in vitro in RIL-2 and maintain their anti-tumor therapeutic effectiveness when adoptively transferred. These experiments suggest methods for generating increased numbers of cells for use in the adoptive immunotherapy of human cancers and may substantially reduce the need for repeated leukophereses of cancer patients undergoing this therapy.

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