CHARACTERIZATION OF TUMOR-INFILTRATING LYMPHOCYTES DERIVED FROM HUMANTUMORS FOR USE AS ADOPTIVE IMMUNOTHERAPY OF CANCER

From 1991 to 1995, we initiated cultures of 94 fresh tumor samples of various histologies in an effort to grow tumor-infiltrating lymphocyte s (TIL) using flasks and subsequent expansion in semipermeable bags. T he five most prevalent tumor types from which TIL were successfully in itiated were melanoma (25 successful initiates in 34 tumor samples, 74 % success rate), colorectal cancer (12 of 18, 67%), renal cell carcino ma (9 of 12, 75%), breast (4 of 5, 80%), and sarcoma (5 of 7, 71%). Th e overall success rate for all tumors was 67 of 94 (71%). There were n o instances of contamination from the time of culture initiation throu gh harvesting of the final cell product for clinical use. The mean num ber of days to reach successful initiation (> 5 x 10(8) cells) was 35 +/- 24 days (mean +/- SD). TIL were then expanded from these successfu l initiates for either a repeated low-dose therapy (TIL reinfusion num bers of 5 x 10(8)-5 x 10(9)) or for a repeated high-dose therapy (> 5 x 10(9)-5 x 10(10)). The mean number of days to expand a TIL culture f rom the time of initiation to treatment for a first low-dose TIL was 5 9 days (range, 27-94 days) compared with 80 days (range, 33-209 days) for high-dose TIL. For patients who received a second or third high-do se TIL treatment, the average number of days needed to expand TIL was 39 days (n = 10) if there was no intervening cryopreservation of TIL, compared with 49 days (n = 10) if the culture had to be reestablished from cryopreserved TIL. For patients who received a second or third lo w-dose TIL, the mean number of days needed to expand TIL was 23 days ( n = 3) if there was no intervening cryopreservation compared with 42 d ays (n = 17) if cultures had to be reestablished after cryopreservatio n of TIL. Low-dose TIL displayed predominantly CD4(+) phenotype in 76% of 42 cultures, whereas high-dose TIL displayed predominantly CD8(+) phenotype in 84% of 44 cultures. Cells bearing the natural killer (NK) phenotype (CD3(-), CD56(+)) and the lymphokine activated killer (LAK) phenotype (CD3(+), CD56(+)) were present in both low-and high-dose TI L cultures, but these phenotypes were never predominant. Cytotoxicity testing consistently demonstrated the persistence of NK and LAK activi ty in addition to the killing of allogeneic and autologous melanoma tu mor targets. This work confirms that TIL cultures from most tumor type s can be successfully established and expanded for therapeutic use, an d repeated expansion from continuous TIL culture or cryopreserved TIL for repeated treatments is feasible, Such cultures are predominantly T lymphocytes that are phenotypically heterogeneous, and these phenotyp es do not remain constant during prolonged time in culture.