Gene Therapy of Anaplastic Thyroid Carcinoma with a Single-Chain Interleukin-12 Fusion Protein

Abstract

Anaplastic thyroid carcinoma is the most aggressive type of thyroid malignancy with a mean survival time of less than 8 months. No effective therapeutic approach is currently available, making the development of novel treatments necessary. Interleukin (IL)-12 is a proinflammatory heterodimeric cytokine with strong antitumor activity. In the present study, we investigated the potential of IL-12 gene therapy for anaplastic thyroid carcinoma in BALB/c (nu/nu) nude mice. A single-chain IL-12 fusion protein construct was created to assure equal expression of its p35 and p40 subunits. Human anaplastic thyroid carcinoma cell line ARO was stably transfected with an IL-12 expression plasmid under the control of cytomegalovirus (CMV) promoter (scIL-12/CMVpDNA). High levels of functional IL-12 (26.78 ± 4.11 ng/ml per 10⁶ cells per 48 hr) were produced by scIL-12-transfected ARO cells (ARO/IL-12). Tumorigenicity in nude mice was completely lost in scIL-12-transfected ARO cells, as demonstrated by the lack of tumor formation after subcutaneous injection of 2 × 10⁶ ARO/IL-12 cells, even though there was no difference in cell proliferation between ARO and ARO/IL-12 cells. Tumor growth was observed after challenge with ARO tumor cells, indicating that protective immunity had not developed against the parental cells. Furthermore, the growth rate of established subcutaneous ARO tumors was significantly reduced by either subcutaneous injection of 2 × 10⁶ ARO/IL-12 cells weekly or intramuscular injection of 50 μg scIL-12/CMVpDNA twice weekly. The antineoplastic activity of ARO/IL-12 cells was, however, abrogated by intraperitoneal injection of anti-natural killer (NK) cell antibody. Moreover, significantly higher number of ARO/IL-12 cells and ARO cells were killed by splenocytes from nude mice previously treated with ARO/IL-12 compared to those treated with ARO cells (32% vs. 9% when ARO were used as target cells, 43% vs. 17% when ARO/IL12 were used as target cells; p < 0.01) in an in vitro cytotoxicity assay. Again, tumor cell killing was neutralized by the addition of anti-NK cell antibody in the assay. In conclusion, we have demonstrated successful gene therapy with a scIL-12 fusion protein against anaplastic thyroid carcinoma in an in vivo model. The immune response against ARO/IL-12 cells is mediated by NK cells. These results may set the stage for clinical application of IL-12 gene therapy for poorly differentiated thyroid carcinoma.