ADENOVIRUS-MEDIATED INTERLEUKIN-2 PRODUCTION BY TUMORS INDUCES GROWTHOF CYTOTOXIC TUMOR-INFILTRATING LYMPHOCYTES AGAINST HUMAN RENAL-CELL CARCINOMA

Combination therapy with interleukin-2 (IL-2) and tumor-infiltrating l ymphocytes (TILs) demonstrates significant clinical activity in patien ts with metastatic renal cell carcinoma (RCC). To investigate whether local delivery of IL-2 via gene transfer is capable of improving the p otency and efficacy of in vitro propagated TILs as compared with stand ard growth conditions [400 BRMP U (BU)/ml], a replication-deficient ad enovirus expressing the human IL-2 gene under control of the cytomegal ovirus (CMV) promoter (Ad-IL-2) has been constructed in our laboratory . RCC-TIL cultures were initiated by directly infecting RCC tumor susp ension with Ad-IL-2 at a multiplicity of infection of 10:1. Subsequent ly the TIL cultures were restimulated with nonirradiated autologous RC C infected with Ad-IL-2 (RCC-Ad-IL-2) every 10 days (TIL/tumor = 50:1) . Cell growth, phenotype, cytotoxicity, and cytokine messenger RNA (mR NA) expression were analyzed and compared with TIL growth stimulated w ith exogenous IL-2 (400 BU/ml), All five TILs tested responded to RCC- Ad-IL-2 activation, and a completed clearance of tumor cells was obser ved in cultures within 7-10 days. Lysis of nonirradiated RCC-Ad-IL-2 c ells by TILs also was observed in cultures 3-5 days after restimulatio n. The IL-2 concentration in cell culture supernatants was maintained between 10 BU and 35 BU/ml (2 and 7 ng/ml), respectively. When compare d with exogenous IL-2, RCC-Ad-IL-2 induced less growth expansion of TI Ls whereas a reduced CD56(+) (23 +/- 14% vs. 44 +/- 13%: p < 0.05) but increased CD3(+)CD4(+) cell population (32 +/- 11% vs. 15 +/- 6%; p < 0.05) with enhanced T cell-receptor use (59 +/- 10% vs. 42 +/- 7%; p < 0.005) was determined. An augmented human leukocyte antigen (HLA)-re stricted and tumor-specific cytotoxicity was detected in RCC-Ad-IL-2-e xpanded TILs (day 35, 15.3 +/- 4.2 LU vs. 4.6 +/- 1.8 LU; p < 0.005). These properties were mediated by the CD8(+) and CD4(+) T-cell populat ions, as demonstrated by antibody-blocking assays. A unique cytokine p rofile also was detected in RCC-Ad-IL-2-induccd TILs, which demonstrat ed an upregulation of both GM-CSF and IL-6 mRNA as compared with TILs expanded in the presence of exogenous IL-2. These data suggest that RC C-Ad-IL-2 is a potent immune stimulant that can be used in vitro as an immunogen to propagate cytotoxic RCC-TILs for adoptive immunotherapy or potentially in vivo by direct injection as a live tumor vaccine.