[CANCER RESEARCH 58. 1225-1230. March 15. 1998| Interleukin 12 Gene Therapy of MHC-negative Murine Melanoma Métastases1 Patrizia Nanni,2 liarÃ-aRossi, Carla De Giovanni, Lorena Landuzzi, Giordano Nicoletti, Antonella Stoppacciaro, Marii-Ila Parenza, Mario P. Colombo, and Pier-Luigi Lollini Institute for Cancer Research, University of Bologna, 1-40126 Bologna [P. N., I. /?., C. D. G., P- L. L.]: National Cancer Institute of Genoa. Biolechnoh>x\ Satellite Unit of Bologna, 1-40126 Bologna ¡L.L. G. N.]: Department of Experimental Medicine, University of Rome, 1-()Õ)I61Rome ¡A.S.l; and Experimental Oncologv [). Natitmal Cancer Institute, 1-20133 Milan ¡M.P., M. P. C.]: Italy ABSTRACT Immunological gene therapy of cancer relies heavily on the activation of T cells, but tumors with defects in MHC gene expression are not recog nized by MHC-restricted T cells. To investigate the potential of cytokine genes for the therapy of MHC-negative tumors, we transduced B78H1, a class I-negative murine melanoma clone, with a polycistronic vector car rying murine interleukin (II.I-I2 genes. The clones studied produced 400—25,000 pg/ml 11-12 their in vitro growth properties were similar to those of parental cells. A complete inhibition of growth was observed in vivo both after s.c. and i.v. administration of all II.-12 clones. II ,-12- transduced cells were also used as a therapeutic vaccine in mice bearing micrometastases by nontransduced parental cells. A significant (80-90%) reduction in the number of lung nodules was obtained. Immunohisto- chemical analysis and studies in immunocompromised hosts showed that T cells and natural killer cells had a significant role in the elimination of IL-12-releasing cells. In situ hybridization with cytokine probes detected a strong increase in the proportion of leukocytes positive for IFN-y, tumor necrosis factor a, H.-l/i. and IFN-inducible protein 10 at the site of rejection of IL-12-engineered tumor cells. However, it was clear that the loss of in vivo growth was also due to T-cell- and natural killer cell- independent factors, possibly related to the antiangiogenic properties of 11-12. In conclusion, tumor therapy based on 11-12 gene transduction was effective on a MHC-negative metastatic tumor, suggesting a possible application to MHC-defective human neoplasms. INTRODUCTION The use of recombinant cytokines and of cells transduced with cytokine genes, in association with the cloning of specific tumor rejection antigens, has considerably increased the possibilities to induce or to boost an antitumor immune response based mainly on T lymphocytes. On the other hand, detailed studies of MHC gene products revealed that defects in class I MHC expression are wide spread among human tumors. In fact, according to some estimates, most human tumors fail to express one or more class I MHC glyco- proteins on their membrane (1, 2). A causal link between specific MHC class I loss and immunoselec- tion by T lymphocytes has not yet been established; however, it is certain that the absence of a given MHC gene product prevents the recognition by T lymphocytes of the antigenic peptides for which the MHC product represents the restriction element (1). The high prevalence of tumors with class I MHC defects indicates that specific strategies should be devised to cope with tumors that cannot be efficiently killed by MHC-restricted CTLs. A good candi date for this type of application is IL-12,3 which is able to activate both specific TH1 responses and nonrestricted NK effectors (3). Received 10/15/97; accepted 1/16/98. 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. 1 Supported by grants from the Italian Association for Cancer Research, the Italian Ministry for University and Research, and the National Research Council. I. R. is the recipient of an Italian Association for Cancer Research fellowship. 2 To whom requests for reprints should be addressed, at Istituto di Cancerologia. Viale Filopanti 22.1-40126 Bologna, Italy. Fax: 39-51-242-169; E-mail: nanni@cancer.unibo.it. 3 The abbreviations used are: IL. interleukin; rIL. recombinant IL: IP10. IFN-inducible protein 10; TNF. tumor necrosis factor; NK. natural killer: niAb. monoclonal antibody. IL-12 is a heterodimeric cytokine produced by antigen-presenting cells, phagocytes, and granulocytes (3). A strong antitumor efficacy of rIL-12 or of cells engineered to produce IL-12 has been reported in a great variety of tumors (4-11). Antitumor activity of IL-12 is thought to be mediated mainly by in vivo induction of IFN--y, given that the therapeutic efficacy of IL-12 is reduced in mice treated with anti- IFN-y antibodies (5). The IFN-inducible chemokine IP10 has been indicated in turn as a further mediator of the antitumor activity of IL-12 (7). In this paper, we show that IL-12 gene transduction of a MHC- negative murine melanoma clone results in a complete loss of malig nancy, and that IL-12-transduced cells can be used as a therapeutic vaccine against métastasesproduced by nontransduced MHC-nega tive parental cells. MATERIALS AND METHODS Cells. B78H1 is an amelanotic clone of B16 melanoma (12). B78H1 cells were cultured in DMEM (Lite Technologies. Inc.. Milan. Italy) supplemented with 10% fetal bovine serum (Life Technologies. Inc.). Gene Transduction. IL-12-producing B78H1 cells were obtained by trans duction with the polycistronic Lp4()Ip'5SN retroviral vector coding for both p35 and p4() murine IL-12 subunits as described previously (10). Single G418-resistant colonies were isolated, expanded into cell lines, and subjected to further analysis. The IL-12 concentration was determined by a two-site sandwich ELISA using 9A5 mAb to p70 and peroxidase conjugate mAb 5C3-POD to p40, kindly provided by Dr. Luciano Adorini (Roche Milano Ricerche. Milan. Italy). Mice. Eight-week-old C57BL/6NCrlBR (referred to as C57BL/6) male mice and 5-week-old nu/nu male mice on Swiss GDI background were purchased from Charles River Laboratories (Calco. Italy) and treated according to European Community guidelines. To obtain NK-depressed animals, we injected some groups of mice i.v. with 0.4 ml of a 1:25 dilution of anti-asialo CM, antiserum (WAKO. Dusseldorf. Germany) 24 h before cell injection. Some mice were treated every 3 days with anti-asialo GM, at the concentration described above and with 100 /j.g of anti-IFN-y mAb (AN 18 hybridotna; kindly provided by Dr. G. Carotta. Roche. Basel. Switzerland). Tumor Growth and Metastasis. Mice were challenged s.c. with 0.2 ml of a single-cell suspension containing 5 X IO5 (C57BL/6) or IO6 (nude mice) tumor cells: cell doses were chosen to obtain tumors with a similar incidence and growth curve in C57BL/6 and in nude mice. The incidence and the growth of tumors were evaluated twice weekly. Neoplastic masses were measured with calipers: tumor volume was calculated as 77/6 X [(V (a x b)]}. in which o and h are two perpendicular major diameters. Experimental métastaseswere evaluated 28 days after the injection in a lateral tail vein of 5 x IO5 (C57BL/6). IO6 (nude mice), or 2.5 x IO5 (anti-asialo GM,-treated C57BL/6) tumor cells suspended in PBS. Lung nodules were contrasted with black India ink; all metastasis counts were performed on dissected lung lobes under a stereoscopic microscope. Therapeutic Vaccination. Therapeutic vaccination started I day after B78H1 parental cell challenge and consisted of seven injections of 10" viable or mitomycin C (80 fig/ml at 37°Cfor 45 min: Sigma)-treated cells 3-4 days apart. Administration of Exogenous Mouse IL-12. Murine rIL-12. kindly pro vided by Dr. Maurice Gately (Hoffmann LaRoche. Nutley, NJ). was injected (0.1 /j.g/day diluted in PBS containing 100 fig/ml murine serum albumin) i.p. or at the site of s.c. vaccination. Starting 1 day after i.v. injection of tumor cells, mice received two courses of five daily i.p. injections with a 2-day 1225 Research. on February 5, 2016. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from