The Role of Natural Killer Cells in Adenovirus-mediated p53 Gene Therapy1

Adenovirus-mediated gene therapy is a promising new approach for treatment of ovarian cancer. In animal models, complete elimination of cancer cells is often achieved, although the therapeutic gene has not been delivered to all these cells. This is referred to as a bystander effect, because tumor cells near those that receive the therapeutic gene are also eliminated. Several mechanisms have been proposed for the bystander effect, including intercellular communication within the tumor via gap junctions, apoptosis, antiangiogenesis, cytokines or other soluble mediators, and immunological mechanisms. There are two well-documented antitumor effector cell populations in athymic nude mice: macrophages and natural killer (NK) cells. We hypothesize that peritoneal populations of NK cells in nude mice treated with adenoviruses are involved in the observed bystander effect in this in vivo model. We investigated the role of NK cells as immunological mediators for the bystander effect using the p53 tumor suppressor as the therapeutic anticancer gene. Most ovarian cancer cell lines tested were sensitive to lysis by NK cells, although different ovarian cancer cell lines exhibited different sensitivities to NK cell-mediated lysis. To determine the importance of NK cells in the overall efficacy and in the bystander effect of gene therapy, NK cells were depleted in mice by administration of anti-NK1.1 monoclonal antibodies. To study the efficacy of NK depletion, C57BL/6 ( nu/nu ) mice were given injections i.v. by a single tail vein injection or i.p. with increasing doses of anti-NK1.1 IgG. All doses of anti-NK1.1 antibody, from 100–500 μg, essentially eliminated cytotoxic NK activity. To assess the duration of depletion after a single dose of anti-NK1.1 IgG, a time-course experiment was performed. NK 1.1 antibody was effective in completely depleting cytotoxic NK cell activity in the mice for up to 7 days, whether given as 500 μg (i.p.) or 200 μg (i.v.). Flow cytometric analysis performed on peritoneal cell populations confirmed depletion of NK cells by ∼80%. Finally, a survival study was performed, in which animals were depleted of NK cells. In this experiment, NK cell-depleted mice were injected with anti-NK1.1 IgG, and control mice were mice were treated with normal saline. Two days later, all mice were inoculated with a lethal i.p. dose of NIH:OVCAR-3 ovarian cancer cells. After 3 days, the mice were divided into two treatment groups; one treatment group received three consecutive daily i.p. injections of Ad-CMV-p53 (SCH58500), and the second treatment group received three consecutive daily i.p. injections of control adenovirus construct, rAd-null. All of the NK cell-depleted animals, whether treated with rAd-null or with Ad-CMV-p53 (SCH58500) were dead of disease by 116 and 138 days, respectively, after initiation of adenovirus treatment, and no statistically significant difference in survival was observed ( P = 0.349). A significant survival advantage was seen in control (NK-competent) mice treated with rAd-null ( P = 0.04), although all were dead of disease by day 184. Importantly, control NK-competent mice treated with Ad-CMV-p53 (SCH58500) showed no tumor growth or ascites production, and all animals survived. These results indicate that immunological mechanisms involving natural killer cells play an important role in the bystander effect involving adenovirus- p53 gene therapy for ovarian cancer.