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Fibroblasts Genetically Engineered to Secrete Interleukin 12 Can Suppress Tumor Growth and Induce Antitumor Immunity to a Murine Melanoma in Vivo¹

Department of Surgery [H. T., H. J. Z., W. J. S., I. P., M. T. L.], Molecular Genetics and Biochemistry [H. T., W. J. S., P. D. R., M. T. L.], and Cell Biology and Physiology [S. C. W.], University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Department of Inflammation/Autoimmune Diseases, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey [U. G.]; Genetics Institute, Inc., Cambridge, Massachusetts [S. F. W.]

² To whom requests for reprints should be addressed, at Section of Oncologic Surgery, Department of Surgery, University of Pittsburgh, W1543 Biomedical Science Tower, Pittsburgh, PA 15261.

Interleukin 12 (IL-12), a disulfide-linked heterodimeric cytokine produced primarily by macrophages, is composed of light (p35) and heavy (p40) chains. It binds to a receptor on T-cells and natural killer cells, promoting the induction of primarily a T_H1 response in vitro and in vivo. To determine whether paracrine IL-12 secretion can alter tumor cell growth or promote antitumor immunity, we have developed a delivery system using genetically engineered fibroblasts in murine tumor models. NIH3T3 cells were stably transfected to express 100–240 units/10⁶ cells/48 h of IL-12 using expression plasmids carrying both the murine p35 and p40 genes of murine IL-12. The effects of paracrine secretion of IL-12 on tumor establishment and vaccination models were examined using the poorly immunogenic murine melanoma cell line (BL-6) in C57BL/6 mice. To determine the effects of IL-12 on tumor formation, nonirradiated BL-6 cells were inoculated s.c. into C57BL/6 mice admixed with NIH3T3 cells transfected with both subunits of mIL-12 (3T3-IL-12) or with cells transfected with only the neomycin phosphotransferase gene (3T3-Neo). Compared to mice given injections of BL-6 alone, the day of emergence of detectable tumors was significantly delayed in mice given injections of BL-6 admixed with 3T3-IL-12, but not in mice with BL-6 admixed with 3T3-Neo. Effectiveness in this system was related to the amount of IL-12 expressed by the 3T3-IL-12. To determine the ability of locally secreted IL-12 at the tumor site to induce antitumor immunity, 10⁶ irradiated tumor cells mixed with 3T3-IL-12 or 3T3-Neo were injected as a vaccine, and the response to a tumor challenge was subsequently examined. With a tumor challenge of less than 1 x 10⁵ nonirradiated BL-6 cells, significant delay of establishment of tumor was noted with a relatively small amount of IL-12 secretion (1.2 units/5 x 10⁵ cells/48 h). Larger amounts of secreted IL-12 provided no additional therapeutic benefit. Histological examination of tumor inoculum with 3T3-IL-12 secreting a high level of IL-12 showed peritumoral accumulation of macrophages, a characteristic capsule around the tumor composed of palisades of fibroblasts, and decreased numbers of CD4+ cells in the tumor. These results suggest that local delivery of IL-12 inhibits tumor growth in a dose dependent manner but leads to the development of an antitumor immune response when IL-12 is expressed at the tumor site at the relatively small amount indicated above. These results suggest that IL-12, like IL-2, -4, -6, and -7 and granulocytemacrophage colony-stimulating factor, can induce an immune response against poorly immunogenic tumors.

¹ This work was supported in part by USPHS Grant PO1 CA59371 to M. T. L. H. J. Z. was supported by a grant from Genetic Therapy, Inc.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9/21/93. Accepted 10/28/93.

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