MIDGE/hNIS vaccination generates antigen-associated CD8 1 IFN-c 1 T cells and enhances protective antitumor immunity Yun Choi 1–4 , Yong-Hyun Jeon 2–5 , Joo-Hyun Kang 2–5 , June-Key Chung 2–5 , Manuel Schmidt 6 and Chul-Woo Kim 1–4 * 1 Department of Pathology, Seoul National University College of Medicine, Seoul, Korea 2 Department of Tumor Biology, Seoul National University College of Medicine, Seoul, Korea 3 Tumor Immunity Medical Research Center, Seoul National University College of Medicine, Seoul, Korea 4 Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea 5 Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, Korea 6 Mologen AG, Berlin, Germany Human sodium iodide symporter (hNIS) is a transmembrane pro- tein that actively transports iodide ions into thyroid cells. hNIS is over-expressed in some cases of the thyroid cancers compared with the surrounding normal tissues and has been considered to be an attractive target for immunotherapy. The aim of this study is to determine the feasibility of utilizing the hNIS antigenic pro- tein in enhanced-antigen-associated immunotherapy using image analysis with a gamma counter. To accomplish this, minimalistic immunogenically defined gene expression (MIDGE), either plain or coupled to a nuclear localization signal (NLS) peptide, was used as a vector system. Vaccination with MIDGE/hNIS, MIDGE/ hNIS-NLS and pcDNA3.1/hNIS produced a significant increase in the number of hNIS-associated IFN-c-secreting CD8 1 T cells, with MIDGE/hNIS having the strongest effect. In addition, immu- nization with the hNIS encoding vectors induced antigen-mediated antitumor activity against NIS-expressing CT26 tumors in vivo, with the highest tumor free rate (100%) and lowest tumor growth being observed up to 40 days after the CT26/NIS tumor challenge with MIDGE/hNIS than those resulting from other immunization groups. Tumor progression could be followed noninvasively and repetitively by monitoring levels of hNIS gene expression in the tumors using scintigraphic image analysis. Overall, hNIS has a potential use as an antigen for immunization approaches, and vaccination with MIDGE/hNIS vectors is an effective means of generating hNIS-associated immune responses in mice. ' 2007 Wiley-Liss, Inc. Key words: hNIS; antitumor vaccination; MIDGE vectors; moni- toring technique; CD8 1 T cells; CT26 tumor model DNA vaccines can induce both cellular and humoral immune responses and have become an attractive immunization strategy for protecting the host in various experimental models against diseases, such as infections, cancer and autoimmunity. 1–3 It is believed that DNA vaccines are potentially safer than traditional vaccines, having the advantages of stability and cost effectiveness in terms of manufacturing and storage. In addition, multiple anti- gens could be combined into a single plasmid to target multiple pathogens or multiple components of a single pathogen. 4 How- ever, thus far, DNA vaccines have shown low immunogenicity when tested on large animals and humans. 5 There is a need for strategies that can enhance the immunoge- nicity elicited by DNA vaccines expressing the antigens of inter- est. The potency of a DNA vaccine in terms of eliciting an effec- tive immune response is associated with the levels the encoded proteins in eukaryotic cells. 6 Several vector systems have been used in clinical gene transfer trials to increase the efficiency of DNA delivery with retroviral and adenoviral vectors being most common. 7,8 Although the viral vectors can effectively and stably transduce the target cells, they are also associated with risks, such as recombination with wild-type viruses and the activation of protooncogenes. 9 A plasmid vector, which is comprised of double-stranded circular DNA, can avoid these risks. However, the efficiency of DNA delivery is usually lower than those of viral vectors. 7 The expression plasmids used for DNA based vaccina- tion usually contain a transcription unit as well as bacterial sequences, and the presence of constitutive genes in a plasmid can have a strong influence on the outcome of the immunization. 10 Furthermore, the application of plasmid DNA as a vaccine can dis- seminate antibiotic resistance genes. The minimalistic immuno- genically defined gene expression (MIDGE) vector system can address both issues by reducing the sequence content of the trans- fected DNA to the expression cassette encoding the antigen and eliminating the antibiotic resistance gene as well as other nones- sential sequences. 10 The MIDGE vector is linear plasmid vector containing only the expression cassette (CMV enhancer/promoter region, antigen encoding DNA and a chimeric intron and poly- adenylation sequences) and lacks selection marker genes. Nuclear localization signal (NLS) peptides can be conjugated to DNA- based vectors in order to increase their transfection efficiency. 11–14 Previously Moreno et al. 15 and Schirmbeck et al. 16 reported the expression level of encoded antigens along with the immune responses obtained using the MIDGE vector administered through various injection sites. It was shown that MIDGE-NLS vectors are as efficient as plasmids in transferring genes into the various cell lines or through an in vivo injection. Moreover, immunization with MIDGE-NLS vectors has been reported to generate more antigen specific CD8 1 T cells than immunization with the plasmid or MIDGE. 15,16 The human sodium-iodide symporter (hNIS) belongs to the sodium/glucose cotransporter family that can transport iodide into thyroid cells. 17,18 Most methods used for imaging gene expression from transplanted ÔreporterÕ genes in rodents, primates and humans require the tissue to be obtained postmortem. However, new meth- ods using hNIS as a reporter gene labeled with 124 I, 125 I, 131 I or Tc-99m have allowed the visualization of various biochemical processes in the tissues of living subjects. As with other reporter genes, the hNIS gene has also been examined in the context of the long-term imaging of gene therapy experiments. 19–22 If a proper immune response can be induced through in vivo vaccination using the hNIS gene, this vaccination strategy would be helpful for 2 reasons, i.e., targeting immunotherapy against NIS expressing cancer and as an innovative and objective tool for evaluating the efficacy of a vaccine using scintigraphic imaging. The first two authors contributed equally to this paper. Grant sponsor: Korean Science & Engineering Foundation (KOSEF). *Correspondence to: Department of Pathology, Tumor Immunity Med- ical Research Center and Cancer Research Institute, Seoul National Uni- versity College of Medicine, 28 Yongon-Dong, Jongno-Gu, Seoul 110- 799, Korea. Fax: 182-2-3673-0662. E-mail: cwkim@plaza.snu.ac.kr Received 28 August 2006; Accepted after revision 4 December 2006 DOI 10.1002/ijc.22567 Published online 31 January 2007 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 120, 1942–1950 (2007) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer