[CANCER RESEARCH 63, 6378 – 6386, October 1, 2003] Intratumoral Delivery of Dendritic Cells Engineered to Secrete Both Interleukin (IL)-12 and IL-18 Effectively Treats Local and Distant Disease in Association with Broadly Reactive Tc1-type Immunity 1 Tomohide Tatsumi, Jian Huang, William E. Gooding, Andrea Gambotto, Paul D. Robbins, Nikola L. Vujanovic, Sean M. Alber, Simon C. Watkins, Hideho Okada, and Walter J. Storkus 2 Departments of Surgery [T. T., A. G., W. J. S.], Immunology [J. H., W. J. S.], Biostatistics [W. E. G.], Molecular Genetics and Biochemistry [A. G., P. D. R.], Pathology [N. L. V., S. M. A., S. C. W.], and Neurosurgery [H. O.], University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute [A. G., P. D. R., N. L. V., W. J. S.], and University of Pittsburgh Molecular Medicine Institute [A. G., P. D. R., W. J. S.], Pittsburgh, Pennsylvania 15213 ABSTRACT Dendritic cells (DCs) were adenovirally engineered to constitutively and durably secrete the potent Th1-biasing cytokines interleukin (IL)-12 (AdIL12DC) and/or IL-18 (AdIL18DC) and evaluated for their ability to promote therapeutic antitumor immunity in murine sarcoma models. Injection of either AdIL12DC or AdIL18DC into day 7 CMS4 or MethA tumors resulted in tumor rejection or slowed tumor growth when com- pared with control cohorts. Importantly, intratumoral injection with DCs engineered to secrete both IL-12 and IL-18 (AdIL12/IL18DC) resulted in complete and the most acute rejection of any treatment group analyzed. This strategy was also effective in promoting the regression of contralat- eral, untreated tumors. Both CD4 and CD8 T cells were required for tumor rejection. CD8 splenic T cells from mice treated with AdIL12/ IL18DC produced the highest levels of IFN- in response to tumor re- challenge in vitro and displayed the broadest repertoire of Tc1-type reac- tivity to acid-eluted, tumor-derived peptides among all treatment cohorts. This apparent enhancement in cross-presentation of tumor-associated epitopes in vivo may result from the increased capacity of engineered DCs to kill tumor cells, survive tumor-induced apoptosis, and present immu- nogenic MHC/tumor peptide complexes to T cells after intratumoral injection. In support of this hypothesis, cytokine gene-engineered DCs expressed higher levels of MHC and costimulatory molecules, as well as Fas ligand and membrane-bound tumor necrosis factor , with the latter markers associated with elevated tumoricidal activity in vitro. Cytokine gene-engineered DCs appeared to have a survival advantage in situ when injected into tumor lesions, to be found in approximation with regions of tumor apoptosis, and to have the capacity to ingest apoptotic tumor bodies. These results support the ability of combined cytokine gene trans- fer to enhance multiple effector functions mediated by intralesionally injected DCs that may concertedly promote cross-priming and the accel- erated immune-mediated rejection of tumors. INTRODUCTION DCs 3 effectively elicit primary and boost secondary immune re- sponses to self and foreign antigens (1, 2). Because these specialized antigen-presenting cells can induce the generation of both antigen- specific CTLs and T helper cells, DC-based vaccines are attractive strategies for the treatment of cancer. In this regard, DCs pulsed with tumor-associated antigens in various forms, including whole cell lysates (3, 4), peptides (5, 6), proteins (7), RNA (8), or DNA (9, 10), have proven effective in eliciting protective and therapeutic antitumor immunity in murine models. The results of several DC-based tumor vaccine trials have also recently been reported in the setting of B-cell lymphoma, melanoma, prostate cancer, and renal cell carcinoma, among others (11–14). Although tumor-specific T cells were pro- moted by vaccination in most patients, objective clinical responses have thus far only been observed in a minority of treated individuals. These modest current clinical successes for DC-based cancer vaccines would be expected to improve if study designs were modified for optimal DC promotion of Th1-type immunity in cancer-bearing hosts. IL-12 exhibits a number of immunologically important activities, including the ability to enhance natural killer and CTL activities (15–17) and polarize CD4 + T-cell responses by supporting Th1/Tc1- type and suppressing or repolarizing Th2-type immunity (18, 19). We and others have reported potent antitumor effects associated with IL-12 gene therapy using IL-12 gene-modified tumor cells (20 –22) and DCs (23) or systemic administration of IL-12 protein (24, 25) in murine tumor models. Based on these results, Phase I/II clinical trials of IL-12 gene therapy have been performed, with significant but transient objective clinical responses reported to date (26). IL-18 is a member of the IL-1 family of proinflammatory cytokines, produced by activated macrophages and DCs, that also appears to play an important role in driving Th1/Tc1-dominated immune responses (27–29). Recently, IL-18 has also demonstrated potential as a biolog- ical “adjuvant” in murine tumor models, with systemic administration of recombinant IL-18 or direct intratumoral injection of IL-18 adeno- viral vector inducing significant antitumor effects in multiple murine tumor models (30 –32). Indeed, we have recently reported that intra- tumoral delivery of IL-18 gene-transduced DCs can elicit antitumor Th1-type immunity in association with enhanced therapeutic efficacy in the CMS4 tumor model (33). IL-12 acts synergistically with IL-18 by enhancing IFN- produc- tion from Th1/Tc1-type T cells (34, 35), thereby providing a strong rationale for the use of these factors in combined CGT approaches. Whereas the coordinate administration of these two cytokines (as recombinant proteins) in murine tumor models has resulted in more potent antitumor responses than that observed for the single agents, coadministration has also been associated with lethal organ damage and septic shock-like toxicities that appear attributable to the ex- tremely high systemic levels of IFN- evoked by this strategy (31). To overcome such systemic toxicities, we examined the effectiveness of therapies based on the injection of genetically transduced DCs to provide paracrine secretion of IL-12 and/or IL-18 in the tumor- associated microenvironment. We demonstrate that intratumoral de- livery of DCs genetically modified to secrete both IL-12 and IL-18 safely induces accelerated tumor rejection, in association with stron- Received 11/6/02; revised 6/3/03; accepted 7/22/03. 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 NIH Grants CA 63350 (to W. J. S.) and DE 14775 (to N. L. V.). T. T. is supported in part by the Yamanouchi Foundation for Research on Metabolic Disorders (Ibaraki, Japan). 2 To whom requests for reprints should be addressed, at Department of Surgery, University of Pittsburgh School of Medicine, L1.32e The Hillman Cancer Center, 5117 Center Avenue, Pittsburgh, PA 15213-1863. Phone: (412) 623-3240; Fax: (412) 623- 7709; E-mail: storkuswj@msx.upmc.edu. 3 The abbreviations used are: DC, dendritic cell; CGT, cytokine gene therapy; HPLC, high-performance liquid chromatography; IL, interleukin; pfu, plaque-forming unit(s); FasL, Fas ligand; TNF, tumor necrosis factor; Ad, adenovirus; CM, complete media; MOI, multiplicity of infection; PE, phycoerythrin; Ab, antibody; TRAIL, TNF-related apopto- sis-inducing ligand; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; MFI, mean fluorescence intensity; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- nyltetrazolium bromide; BM, bone marrow; Tg, transgenic; EGFP, enhanced green fluorescent protein; ATCC, American Type Culture Collection; mAb, monoclonal Ab; mIL, murine IL; LT, lymphotoxin; rIL, recombinant IL. 6378 Research. on May 23, 2015. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from