Gene Therapy (2001) 8, 128–138 2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt RESEARCH ARTICLE Recombinant DNA vaccines protect against tumors that are resistant to recombinant vaccinia vaccines containing the same gene C-H Chen1,2, T-L Wang3,HJi3, C-F Hung3, DM Pardoll1, W-F Cheng3, M Ling3 and T-C Wu1,3,4,5 Departments of 1Oncology, 3Pathology, 4Obstetrics and Gynecology and 5Molecular Microbiology and Immunology, The Johns Hopkins Medical Institutions, Baltimore, MD, USA; and 2Department of Internal Medicine, National Taiwan University Hospital, National Taiwan University, Taipei, Taiwan Antigen-specific cancer immunotherapy involves the delivery ing against challenge with a more stringent subclone of TC-1 of tumor-associated antigen to the host for the generation of (TC-1 P2) established from TC-1 tumors that survived initial tumor-specific immune responses and antitumor effects. We Sig/E7/LAMP-1 vaccinia vaccination. Immunological assays hypothesized that different delivery systems may influence revealed that both vaccines induced comparable levels of the pattern of antigen-specific immune response and the CD8+ T cell precursors and anti-E7 antibody titers. Interest- outcome of antitumor effect. We therefore evaluated recom- ingly, Sig/E7/LAMP-1 vaccinia induced both E7-specific IFN- binant vaccinia virus and naked DNA for the generation of ␥- and IL4-secreting CD4+ T cell precursors while antigen-specific immune responses and antitumor effects. Sig/E7/LAMP-1 DNA induced only E7-specific IFN-␥- We previously found that recombinant vaccinia and naked secreting CD4+ T cell precursors. We also found that IL-4 DNA vaccines containing the chimeric Sig/E7/LAMP-1 gene knockout C57BL/6 mice vaccinated with Sig/E7/LAMP-1 were capable of controlling the growth of HPV-16 E7- vaccinia exhibited a more potent antitumor effect than vacci- expressing tumor cells (TC-1). In this study, we performed nated wild-type C57BL/6 mice in our tumor protection experi- a head-to-head comparison of optimized delivery of ments. These results suggest that IL-4 may play a detrimen- Sig/E7/LAMP-1 vaccinia and DNA vaccines using dose- tal role in the antitumor effect mediated by vaccinia vaccines. escalating tumor challenge. At a dose of 1 × 106 TC-1 cells Our findings suggested that DNA vaccines may provide bet- per mouse, Sig/E7/LAMP-1 DNA provided 100% protection ter tumor protection than vaccinia vaccines employing the against subcutaneous growth of tumors, while Vac- same gene, which may have implications in the future design Sig/E7/LAMP-1 protected only 40% of the mice. Further- of antigen-specific cancer immunotherapy. Gene Therapy more, Sig/E7/LAMP-1 DNA vaccines are capable of protect- (2001) 8, 128–138. Keywords: vaccinia vaccine; DNA vaccine; HPV-16; E7; cancer vaccine Introduction CD4+ and CD8+ T cell-mediated immune responses against tumors. The development of cancer vaccines has continued to In an attempt to enhance antigen presentation to CD4+ 1 show progress in recent years. Immunotherapy has been T cells, we have previously linked the sorting signals of shown to successfully control tumor growth in many the lysosome-associated membrane protein-1 (LAMP-1) murine tumor model systems. In addition, several phase to the human papillomavirus-16 (HPV-16) E7 antigen, I/II clinical trials using these vaccine strategies have creating the Sig/E7/LAMP-1 chimera. This specific link- yielded encouraging results in patients (for review, see age led to the targeting of HPV-16 E7 to the endosomal Ref. 2). Tumor-specific antigens, when efficiently + and lysosomal compartments and enhanced MHC class presented by antigen-presenting cells (APCs) to CD8 + 3 + II presentation of E7 to CD4 T cells. Furthermore, we cytotoxic T lymphocytes (CTLs) and CD4 helper T (Th) demonstrated that this strategy was not only capable of cells, are capable of inducing potent T cell-mediated activating CD4+ T helper cells, it also led to enhancement immunity – the most crucial component of antitumor of E7-specific CD8+ cytotoxic T cell activity. immunity. The field of cancer immunotherapy is there- The LAMP-1 targeting strategy was further investi- fore moving towards the development of antigen-specific gated in vivo using vaccinia in an E7-expressing tumor cancer vaccines, particularly those which enhance both model, TC-1. Our results indicated that intraperitoneal vaccination of Sig/E7/LAMP-1-containing recombinant vaccinia virus (Vac-Sig/E7/LAMP-1) can generate strong antitumor immunity against TC-1 inoculated subcutane- Correspondence: T-C Wu, Department of Pathology, the Johns Hopkins 4 University School of Medicine, Ross Research Building, Room 659, 720 ously. Subsequently, we have tested the Vac- 5 Rutland Avenue, Baltimore, Maryland 21205, USA Sig/E7/LAMP-1 vaccine in a liver tumor and lung met- Received 24 February 2000; accepted 28 April 2000 astasis model.6 Our results indicated that this vaccine was capable of preventing and treating E7-expressing tumors. Treatment of vaccinia-resistant tumor cells by DNA vaccine C-H Chen et al 129 More recently, we have extended the LAMP-1 tar- trol mice grew tumors within 3 weeks, whereas all of the geting strategy to naked DNA vaccines. We found that mice vaccinated with either Sig/E7/LAMP-1 DNA or the Sig/E7/LAMP-1 DNA vaccine administered via gene Vac-Sig/E7/LAMP-1 remained tumor free 6 weeks after gun provided potent protection against the subcutaneous tumor challenge. In comparison, when mice were chal- growth of TC-1 cells and eradicated established TC-1 lenged with 2 × 105 TC-1 cells per mouse subcutaneously tumors.6 We also demonstrated a similarly potent anti- in the right leg, Sig/E7/LAMP-1 DNA generated 100% tumor immunity against TC-1 tumor metastases in the tumor protection for up to 55 days while Vac- liver and lungs.5 Sig/E7/LAMP-1 generated 80% tumor protection after 10 Since different vector delivery systems may influence days, falling to 60% after 21 days (Figure 1b). When the the pattern of antigen-specific immune response and the challenged tumor dose was increased to 1 × 106 TC-1 cells outcome of antitumor effect, it is important to discover per mouse, Sig/E7/LAMP-1 DNA still provided 100% which of these systems may lead to the best antitumor tumor protection against the subcutaneous growth of TC- effect. While vaccinia vaccines have the advantage of 1 tumor. In contrast, Sig/E7/LAMP-1 vaccinia vaccines efficient delivery of genes of interest to target cells, repeat could provide tumor protection in only 40% of the mice vaccination with this delivery system may be inhibited after 10 days (Figure 1c). These results indicated that due to antibodies generated against vaccinia or other tol- Sig/E7/LAMP-1 DNA generated a more potent anti- erance effects. On the other hand, DNA vaccines are safe, tumor effect against TC-1 compared with Vac- can be repeatedly administered, and can be expressed Sig/E7/LAMP-1 in our tumor protection experiments. over an extended period of time to enhance immunologic memory. The concern about DNA vaccines is their lim- Vaccination with either Sig/E7/LAMP-1 DNA or Vac- ited potency, an issue which has been addressed using Sig/E7/LAMP-1 and their therapeutic potential to the Sig/E7/LAMP-1 strategy. To determine if the choice eradicate established E7-expressing tumors in the lungs of vector delivery system for vaccination may influence To determine the therapeutic potential of Sig/E7/LAMP- vaccine potency, we performed a head-to-head compari- 1 DNA or Vac-Sig/E7/LAMP-1 in treating TC-1 tumor son of different vector delivery systems (naked DNA ver- metastases in the liver, mice were first challenged with 1 sus vaccinia) containing the same gene (Sig/E7/LAMP- × 104 TC-1 per mouse via intravenous tail vein injection. 1) for their ability to generate E7-specific immune Three days later, mice were vaccinated as described in responses and antitumor immunity against dose-escalat- Materials and methods. Mice were killed 28 days after ing challenge with TC-1 tumor cells and also against chal- tumor challenge. We determined that Sig/E7/LAMP-1 lenge using a more stringent tumor model, TC-1 P2. DNA and Vac-Sig/E7/LAMP-1 exhibited a significantly higher therapeutic effect compared with no treatment as assessed by counting the mean number of pulmonary Results nodules (Figure 2). Sig/E7/LAMP-1 DNA generated a slightly greater therapeutic effect compared with Vac- Vaccination with Sig/E7/LAMP-1 DNA generates a Sig/E7/LAMP-1, although this difference was not stat- stronger antitumor effect compared with vaccination with istically significant. These results indicated that both Sig/E7/LAMP-1 vaccinia Sig/E7/LAMP-1 DNA and Vac-Sig/E7/LAMP-1 can We have previously demonstrated that Sig/E7/LAMP-1 generate potent antitumor treatment in the lung vaccinia can generate a potent antitumor effect against a metastasis model using TC-1 tumor cells. tumor challenge of 5 × 104 TC-1 cells per mouse.4 In addition, we have demonstrated that Sig/E7/LAMP-1 Vaccination with Sig/E7/LAMP-1 DNA generates DNA is also capable of generating a potent antitumor stronger protection against a more stringent tumor effect against a tumor challenge of 5 × 104 TC-1 cells per model mouse.5,6 In order to conduct a head-to-head comparison Our previous immunotherapy studies demonstrated that of Sig/E7/LAMP-1 DNA and Vac-Sig/E7/LAMP-1, it Vac-Sig/E7/LAMP-1 can effectively prevent and treat was necessary to first optimize the dose and vaccination HPV-16 E7-expressing tumors (TC-1) in most cases.