C.C. Leong

Cytokine gene therapy or infusion as treatment for solid human cancer

In the induction of tissue-directed immune responses, cytokines tend to be released within the affected tissues. We used two strategies to expose tumor tissues to continuous high levels of cytokines: First, a vaccinia interleukin (IL)2 recombinant was injected directly intratumorally 3-weekly at 10(7) pfus/dose in six patients with the solid tumor malignant mesothelioma (MM). No virus excretion was detectable. At each cycle vaccinia-IL-2 mRNA (SQ [semi-quantitative] reverse transcription polymerase chain reaction) was maximal 24-72 h following injection reduced at 8 days and faded by 21 days. No tumor regression occurred. Second, based on the success of granulocyte macrophage colony-stimulating factor (GM-CSF) in gene transfer experiments, we conducted a study using continuous intratumoral GM-CSF infusion in eight patients with MM using a portable pump at doses of 10 micro/cg/24 h over 8 weeks. Systemic neutrophil agglutination and local catheter-related difficulties occurred. Two patients demonstrated tumor necrosis, one of whom had a marked progressive mononuclear cell infiltration of the tumor associated with a partial response (>50% reduction in tumor area). Murine studies using our MM model in CBA and BALB/C mice have demonstrated that B7-1 and allo-class I transfections induce strong tumor-specific cytotoxic T lymphocyte responses: GM-CSF, IL-12, and IL-2 induced mixed nonspecific plus specific responses, whereas B7-2 and class II transfections were not effective. We conclude that increased intratumoral cytokine concentrations can be generated using both gene transfer and cytokine infusion approaches; however, both have their limitations and, at this stage, have not produced dramatic antitumor effects in humans.

The immune anti-tumor effects of GM-CSF and B7-1 gene transfection are enhanced by surgical debulking of tumor

Malignant mesothelioma (MM) is a solid tumor largely unresponsive to conventional therapies. Immunological gene therapy shows promise in murine models and human clinical trials; however, the role of surgery in combination with gene therapy has not been widely studied. The aim of this study was to determine if debulking surgery improved the effectiveness of gene therapy in a murine MM model. Mice were subcutaneously inoculated with the MM cell line, AC29, at two different sites, 4 days apart, to allow a surgical and distal site tumor to develop. Once tumors were established, the surgical site tumor was debulked and vaccination of syngeneic tumor transfectants encoding genes for IL-4, IL-2, GM-CSF, B7-1 or allogeneic MHC molecules commenced at a site away from both tumors, and tumor growth was measured. Neither debulking surgery nor gene therapy alone delayed tumor growth. However, there was a clear delay of tumor growth when debulking surgery was combined with vaccination of tumor transfectants expressing B7-1 or high levels of GM-CSF. Combinations of these two transfectants did not lead to a synergistic effect. This study demonstrates that debulking surgery can augment the immunostimulatory effects of immunological gene therapy and can delay tumor growth. This has implications for the future design of human gene therapy trials for solid tumors such as MM. Cancer Gene Therapy (2001) 8, 580–588

Transfection of the gene for B7-1 but not B7-2 can induce immunity to murine malignant mesothelioma

Transfection of the genes encoding the co‐stimulatory molecules B7‐1 and B7‐2 has enhanced the development of immunity to a variety of experimental tumors, although most of these were inherently immunogenic. We have determined the effect of expression of these genes on the induction of immunity to 2 non‐immunogenic murine malignant mesothelioma (MM) cell lines (AC29 and AB1). We had previously shown that B7‐1 transfection into AC29 delayed but did not prevent tumor development by certain of the transfectant clones. Here we demonstrate that over‐expression of B7‐1 can inhibit tumor development by certain AB1‐B7‐1 clones, that inhibition of transfectant growth is dependent on CD4+ and CD8+ T cells and that mice that reject some of these transfectant clones are capable of rejecting subsequent inocula of the parental cell line, AB1. The transfectant clones can generate tumor‐specific cytotoxic T cells. By contrast, expression of B7‐2 in several clones derived from either AB1 or AC29 had no significant effect on the development of tumors in vivo. Our data are consistent with data from other systems that show differences in the effect of modification by B7‐1 or B7‐2 on the modulation of anti‐tumor immune responses. They demonstrate that such modifications can induce protective immunity against an MM cell line but confirm the intra‐ and inter‐tumoral heterogeneity in the effect of genetic modification on the induction of immunity. Our observations are relevant to human MM because these cell lines have been derived from asbestos‐induced tumors and share many properties with human cell lines of the same histological type. Int. J. Cancer 71:476‐482, 1997.

The induction of immune responses to murine malignant mesothelioma by IL-2 gene transfer

Stable IL‐2 transfectant clones have been derived from two non‐immunogenic murine malignant mesothelioma (MM) cell lines to investigate the induction of protective antitumour immunity to MM. AC29‐IL‐2 transfectant clones grew at a slower rate in vivo than the parental cell line or a transfectant control clone but all inoculated mice developed tumours despite the continued ability of the tumour cells to express IL‐2. Tumour development after inoculation of AB1‐IL 2 transfectants varied, the degree of in vivo inhibition (40–100%) being directly related to the rate of IL‐2 secretion of the transfectants. When mice which had rejected the ABl‐lL‐2 transfectants were challenged with parental AB1 cells, a proportion (16–70%) of mice from each group remained tumour free at least 45 days after challenge (naive mice developed tumours within 26 days). The inhibition of growth of the initial inoculum of ABl‐IL‐2 transfectants was independent of CD4+ and CD8+ cells, consistent with the demonstration of non‐specific cytotoxic activity by splenocytes from mice inoculated with the IL‐2 transfectants. These data suggest that IL‐2 expression by MM cells is capable of generating in vivo immunity to the tumour. This immunity may be relatively weak or may be subject to downregulation so that consistent rejection of unmodified tumour cells is not achieved. Genetic modification with combinations of genes, including lL‐2 and B7‐1.will be necessary for reliable generation of protective immunity to MM.