Cancer Immunotherapy Using a DNA Vaccine Encoding the Translocation Domain of a Bacterial Toxin Linked to a Tumor Antigen

Chien-Fu Hung, Wen-Fang Cheng, Keng-Fu Hsu, et al. Cancer Res 2001;61:3698-3703. Published online May 1, 2001.

Updated Version

Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/61/9/3698

Cited Articles Citing Articles

This article cites 49 articles, 22 of which you can access for free at: http://cancerres.aacrjournals.org/content/61/9/3698.full.html#ref-list-1 This article has been cited by 16 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/61/9/3698.full.html#related-urls

E-mail alerts Reprints and Subscriptions Permissions

Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at pubs@aacr.org. To request permission to re-use all or part of this article, contact the AACR Publications Department at permissions@aacr.org.

Downloaded from cancerres.aacrjournals.org on August 18, 2011 Copyright 2001 American Association for Cancer Research

[CANCER RESEARCH 61, 3698 3703, May 1, 2001]

Cancer Immunotherapy Using a DNA Vaccine Encoding the Translocation Domain of a Bacterial Toxin Linked to a Tumor Antigen1
Chien-Fu Hung,2 Wen-Fang Cheng,2 Keng-Fu Hsu, Chee-Yin Chai, Liangmei He, Morris Ling, and T-C. Wu3
Departments of Pathology [C-F. H., W-F. C., K-F. H., C-Y. C., L. H., M. L., T-C. W.], Oncology [T-C. W.], Obstetrics and Gynecology [T-C. W.], and Molecular Microbiology and Immunology [T-C. W.], The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205

ABSTRACT
Certain domains of bacterial toxins have been shown to facilitate translocation from extracellular and vesicular compartments into the cytoplasm. This feature represents an opportunity to enhance class I presentation of exogenous antigen to CD8 T cells. We investigated this notion by creating a novel fusion of the translocation domain (domain II) of Pseudomonas aeruginosa exotoxin A (ETA(dII)) with a model tumor antigen, human papillomavirus type 16 E7, in the context of a DNA vaccine. Our in vitro studies indicated that cells transfected with ETA(dII)/E7 DNA or dendritic cells pulsed with lysates containing ETA(dII)/E7 protein exhibited enhanced MHC class I presentation of E7 antigen. Vaccination of mice with ETA(dII)/E7 DNA generated a dramatic increase in the number of E7-specific CD8 T cell precursors ( 30-fold compared with wild-type E7 DNA) and converted a less effective DNA vaccine into one with significant potency against human papillomavirus type 16 E7-expressing murine tumors via a CD8-dependent pathway. These results indicate that fusion of the translocation domain of a bacterial toxin to an antigen may greatly enhance vaccine potency.

INTRODUCTION CTLs are critical effectors of antitumor responses (reviewed in Refs. 13). Activated CTLs function directly as effector cells, providing antitumor immunity through the lysis of tumor cells or through the release of cytokines capable of interfering with the propagation of tumors. Furthermore, depletion of CD8 CTL led to the loss of antitumor effects in several cancer vaccines (4, 5). Therefore, the enhancement of antigen presentation through the MHC class I pathway to CD8 T cells has been a primary focus of cancer immunotherapy. Recently, naked DNA vaccines have emerged as attractive approaches for vaccine development (reviewed in Refs. 6 11). DNA vaccines have been shown to generate long-term cell-mediated immunity (reviewed in Refs. 12). In addition, DNA vaccines are capable of generating CD8 T cell responses in vaccinated humans (13). However, one of the limitations of these vaccines is their potency, because they do not have the intrinsic ability to amplify and spread in vivo as some replicating viral vaccine vectors do. Furthermore, some tumor antigens may be poorly immunogenic, such as HPV4 E7 (5). Therefore, strategies that enhance DNA vaccine potency may be applicable for the development of more effective cancer immunotherapy.

Increased understanding of the antigen presentation pathway creates the potential for designing novel strategies to enhance vaccine potency. One potential strategy to enhance the presentation of antigen through the MHC class I pathway to CD8 T cells is the use of the translocation features of certain bacterial toxins such as ETA (reviewed in Refs. 14). ETA is one of several secreted bacterial toxins that are able to covalently modify particular proteins in mammalian cells through the translocation of bacterial toxin. Molecular characterization of ETA has revealed its three functional domains (15). Domain I is responsible for binding to a cell surface receptor (16), domain II is responsible for translocation to the cytosol (1719), and domain III is responsible for the toxic capability of binding to ADPribosyl transferase (20). In particular, domain II of ETA has been used to engineer a chimeric multidomain protein to deliver DNA into the cytosol (21, 22). The ability of domain II of ETA to facilitate translocation from the endosomal/lysosomal compartments to the cytoplasm suggests that it may lead to the enhancement of MHC class I presentation of exogenous antigen. We therefore engineered a DNA vaccine encoding ETA(dII) linked to a model antigen, which we hypothesized would enhance MHC class I presentation of antigen to CD8 T cells and thereby enhance vaccine potency. We chose HPV-16 E7 as a model antigen for vaccine development because E7 is important in the induction and maintenance of cellular transformation and coexpressed in most HPV-containing cervical cancers and their precursor lesions (23). Therefore, vaccines targeting E7 provide an opportunity to prevent and treat HPV-associated cervical malignancies. Our data indicated that vaccination with the chimeric ETA(dII)/E7 DNA vaccine enhanced MHC class I presentation of E7, leading to a dramatic increase in the number of E7-specific CD8 T cell precursors. Furthermore, the ETA(dII)/E7 DNA vaccine generated potent antitumor effects against s.c. E7-expressing tumors and preestablished E7-expressing metastatic lung tumors. These results indicate that fusion of the translocation domain of ETA to an antigen may greatly enhance MHC class I presentation of antigen and represents a novel strategy to improve vaccine potency. MATERIALS AND METHODS

Plasmid DNA Constructs and Preparation. The generation of pcDNA3-E7 has been described previously (5). For the generation of pcDNA3-ETA(dII), the pGW601 plasmid (Ref. 24; most kindly provided by Dr. Darrell R. Galloway, Ohio State University, Columbus, OH) was used as the template for Received 8/16/00; accepted 3/2/01. amplification of ETA(dII). The DNA fragment containing ETA(dII) was 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 generated using PCR with a set of primers: 5 -CCGGGAATTCATGCGCCT18 U.S.C. Section 1734 solely to indicate this fact. GCACTTTCCCGAGGGC-3 and 5 -CCGGAATTCGTTCTGCGTGCCGC1 Supported by U19 CA72108-02, RO1 CA72631-01, RO1 CA83706-01, Cancer GGGTGCTGAA-3 . The amplified DNA fragment was then cloned into the Research Institutes, and the Alexander and Margaret Stewart Trust Grant. This work was EcoRI site of pcDNA3 (Invitrogen, Carlsbad, CA). For the generation of supported by NIH 5 pol 34582-01, U19 CA 72108-02, and RO1 CA 72631-01, the Cancer Research Institute, and the Alexander and Margaret Stewart Trust Grant. pcDNA3-ETA(dII)/E7, the DNA fragment containing ETA(dII) DNA was 2 C-F. H. and W-F. C. contributed equally to this paper. cloned into the EcoRI site of pcDNA3-E7. For the generation of pcDNA33 To whom requests for reprints should be addressed, at Department of Pathology, The GFP, the DNA fragment encoding the GFP was first amplified with PCR using Johns Hopkins University School of Medicine, Richard Ross Research Building, Room pEGFPN1 DNA (Clontech, Palo Alto, CA) and a set of primers: 5 -ATCG659, 720 Rutland Avenue, Baltimore, MD 21205. Phone: (410) 614-3899; Fax: (410) 6143548; Email: wutc@jhmi.edu. GATCCATGGTGAGCAAGGGCGAGGAG-3 and 5 -GGGAAGCTTTAC4 The abbreviations used are: HPV, human papillomavirus; ETA, Pseudomonas TTGTACAGCTCGTCCATG-3 . The amplified product was then cloned into aeruginosa exotoxin A; GFP, green fluorescent protein; LDH, lactate dehydrogenase; DC, the BamHI/HindIII cloning sites of pcDNA3. For the generation of pcDNA3dendritic cell; aa, amino acid; MAb, monoclonal antibody; LAMP, lysosome associated E7/GFP, E7 was subcloned from pcDNA3-E7 into the EcoRI/BamHI sites of membrane protein. 3698

Downloaded from cancerres.aacrjournals.org on August 18, 2011 Copyright 2001 American Association for Cancer Research

CANCER IMMUNOTHERAPY USING A DNA VACCINE

pcDNA3-GFP. For the generation of pcDNA3-ETA(dII)/E7/GFP, the DNA fragment encoding ETA(dII) was amplified using pcDNA3-ETA(dII) DNA and a set of primers: 5 -GGGTCTAGAATGCGCCTGCACTTTCCCGAGGGC-3 and 5 -CCGGAATTCGTTCTGCGTGCCGCGGGTGCTGAA-3 . The amplified product was further cloned into the XbaI/EcoRI sites of pcDNA3-E7/GFP. The accuracy of all of the constructs was confirmed by DNA sequencing. DNA for vaccination was prepared using an endotoxin-free kit (Qiagen, Valencia, CA). Western Blot Analysis. Twenty g of DNA were transfected into 5 106 293 Db,Kb cells (25) using Lipofectamine 2000 (Life Technologies, Inc., Rockville, MD). Forty-eight h after transfection, medium from transfected culture was concentrated using a millipore filter (Millpore, Bedford, CT), and cells were lysed with protein extraction reagent (Pierce, Rockford, IL). Equal amounts of proteins (10 g) were loaded and separated by SDS-PAGE using a 10% polyacrylamide gel. The gels were electroblotted to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). Blots were blocked with PBS/ 0.05%, Tween 20 (TTBS) containing 5% nonfat milk for 2 h at room temperature. Membranes were probed with anti-GFP (Clontech, Palo Alto, CA) at 1:3000 dilution in TTBS for 2 h, washed four times with TTBS, and then incubated with goat antimouse IgG conjugated to alkaline phosphatase (Amersham, Piscataway, NJ) at 1:1000 dilution in TTBS containing 5% nonfat milk. Membranes were washed four times with TTBS and developed using enhanced Hyperfilm- enhanced chemiluminescence (Amersham, Piscataway, NJ). Mice. Female C57BL/6 mice 6 8 weeks of age from the National Cancer Institute (Frederick, MD) were purchased and kept in the oncology animal facility of the Johns Hopkins Hospital (Baltimore, MD). All animal procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals. CTL Assay Using Transfected 293 Db,Kb Cells as Target Cells. A human embryonic kidney 293 cell line expressing the Db and Kb (293 Db,Kb), two C57BL/6 mouse MHC class I molecules, was kindly provided by Dr. James C. Yang (National Cancer Institute, NIH, Bethesda, MD). Twenty g of pcDNA3 (no insert), ETA(dII), E7, or ETA(dII)/E7 DNA were transfected into 5 106 293 Db,Kb cells using Lipofectamine 2000 (Life Technologies, Inc., Rockville, MD). Cells were collected 40 44 h after transfection. Transfected 293 Db,Kb cells (25) were used as target cells, whereas a Db-restricted E7-specific CD8 T cell line (26) served as effector cells. Untransfected 293 Db,Kb cells were used as a negative control. Cytolysis was determined by quantitative measurements of LDH using CytoTox96 nonradioactive cytotoxicity assay kits (Promega, Madison, WI) according to the manufacturers protocol. CTL assays were performed with effector cells and targets cells (1 104 per well) mixed together at various ratios (1:1, 3:1, 9:1, and 27:1) in a final volume of 200 l. After a 5- incubation at 37C, 50 l of the cultured media were collected to assess the amount of LDH. The percentage of lysis was calculated from the following equation: 100 (A B)/(C D), where A is the reading of experimental-effector signal value, B is the effector spontaneous background signal value, C is maximum signal value from target cells, and D is the target spontaneous background signal value. CTL Assay Using DCs Pulsed with Lysates of Transfected 293 Db,Kb Cells as Target Cells. CTL assays were performed with freshly isolated bone marrow-derived DCs pulsed with cell lysates as target cells and E7-specific CD8 T cells as effector cells using a protocol similar to that described previously (27). The protein concentration was determined using the Bio-Rad protein assay (Bio-Rad) according to vendors protocol. 293 Db,Kb cells were transfected as described earlier. Cell lysates from E7 or ETA(dII)/E7 DNAtransfected 293 Db,Kb cells were standardized for E7 protein concentration using an ELISA. DCs were prepared by pulsing them with different concentrations of cell lysates of various DNA-transfected 293 Db,Kb cells (50 g/ml, 10 g/ml, 2 g/ml, and 0.4 g/ml) in a final volume of 2 ml for 16 20 h. CTL assays were performed at a fixed E:T (9:1) ratio with 9 104 E7-specific T cells mixed with 1 104 prepared DCs in a final volume of 200 l. Cytolysis was determined by quantitative measurements of LDH as described earlier. DNA Vaccination. Preparation of DNA-coated gold particles and gene gun particle-mediated DNA vaccination was performed using a helium-driven gene gun (Bio-Rad) according to a previously described protocol (5). DNA-coated gold particles (1 g DNA/bullet) were delivered to the shaved abdominal region of mice using a helium-driven gene gun (Bio-Rad) with a discharge pressure of 400 p.s.i.

Intracytoplasmic Cytokine Staining and Flow Cytometry Analysis. Cell surface marker staining of CD8 or CD4 and intracellular cytokine staining for IFN- and IL-4 as well as FACScan analysis were performed using conditions described previously (28). Before FACScan, splenocytes from nave or vaccinated groups of mice were incubated for 20 h with either 1 g/ml of E7 peptide (aa 49 57) containing MHC class I epitope for detecting E7-specific CD8 T cell precursors or 10 g/ml of E7 peptide (aa 30 67) containing MHC class II peptide for detecting E7-specific CD4 T cell precursors. ELISA. For detection of HPV-16 E7-specific antibodies in the sera of vaccinated mice, we performed a direct ELISA with 1:100, 1:500, and 1:1000 dilutions of sera in 1 PBS using a previously described protocol (29). Briefly, sera was added to microwell plates coated with bacteria-derived HPV-16 E7 proteins with subsequent incubation with peroxidase-conjugated rabbit antimouse IgG antibody (Zymed, San Francisco, CA). In Vivo Tumor Protection Experiments. For the tumor protection experiment, mice (five per group) were vaccinated via gene gun with 2 g of pcDNA3 without insert, ETA(dII) DNA, E7 DNA, ETA(dII) mixed with E7, or chimeric ETA(dII)/E7 DNA. One week later, the mice were boosted with the same regimen as that of the first vaccination. One week after the last vaccination, mice were s.c. challenged with 5 l04 cells/mouse TC-1 tumor cells (4) in the right leg and then monitored twice a week. In Vivo Tumor Treatment Experiments. Mice were i.v. challenged with 1 l04 cells/mouse TC-1 tumor cells via the tail vein on day 0. Three days after challenge with TC-1 tumor cells, mice were treated with 2 g of pcDNA3 without insert, ETA(dII) DNA, E7 DNA, or chimeric ETA(dII)/E7 DNA via gene gun. One week later, these mice were boosted with the same regimen as the first vaccination. Mice were killed on day 25. The number of pulmonary metastatic nodules of each mouse was evaluated and counted by experimenters blinded to sample identity. In Vivo Antibody Depletion Experiments. In vivo antibody depletions have been described previously (4). Briefly, mice were vaccinated with 2 g ETA(dII)/E7 DNA via gene gun, boosted 1 week later, and challenged with 5 l04 cells/mouse TC-1 tumor cells s.c. Depletions were started 1 week before tumor challenge. MAb GK1.5 was used for CD4 depletion, MAb 2.43 was used for CD8 depletion, and MAb PK136 was used for NK1.1 depletion. Depletion was terminated on day 63 after tumor challenge.

RESULTS Generation and Characterization of the ETA(dII)/E7 DNA Vaccine. A schematic diagram showing the domains of full-length ETA and the construct of chimeric ETA(dII)/E7 is presented in Fig. 1A. Chimeric ETA(dII)/E7 was created by linking ETA(dII) (aa 247 416) to the E7 protein. We performed a Western blot analysis to characterize protein expression in E7 and ETA(dII)/E7 DNA-transfected cells (Fig. 1B). Because we observed that Western blot analysis using available E7-specific mouse monoclonal antibodies generated significant background, we used green GFP linked to E7 as a tag for our assays. As shown in Fig. 1B, lysates from E7/GFP DNA-transfected 293 Db,Kb cells revealed a protein band with a size of approximately Mr 30,000 corresponding to E7/GFP protein in Lane 1. In comparison, lysates of ETA(dII)/E7/ GFP DNA-transfected 293 Db,Kb cells showed a protein band with a size of approximately Mr 56,000 corresponding to ETA(dII)/E7/ GFP protein in Lane 2. Our results indicated that E7 DNAtransfected cells exhibited comparable levels of E7 protein expression compared with ETA(dII)/E7 DNA-transfected cells. We also examined the secretion of E7/GFP protein in cells transfected with E7/GFP or ETA(dII)/E7/GFP DNA. As shown in Lanes 3 and 4 of Fig. 1B, E7/GFP protein was not detected in the culture medium of cells transfected with either E7/GFP or ETA(dII)/E7/GFP DNA. E7 secretion was detected from cells transfected with Sig/E7 DNA, which served as a positive control (data not shown).

3699

Downloaded from cancerres.aacrjournals.org on August 18, 2011 Copyright 2001 American Association for Cancer Research

CANCER IMMUNOTHERAPY USING A DNA VACCINE

Fig. 1. Chimeric ETA(dII)/E7 DNA construct and characterization of E7 protein expression. A, schematic diagram showing the constructs of full-length ETA and the chimeric ETA(dII)/E7 gene. The DNA fragment encoding ETA(dII) (aa 247 416) is depicted in the u. The fragment encoding HPV-16 E7 (aa 196) is depicted in the . B, Western blot analysis to characterize the expression of E7/GFP protein in cells transfected with E7/GFP or ETA(dII)/E7/GFP DNA. Lane 1, lysates from cells transfected with E7/GFP DNA; Lane 2, lysates from cells transfected with ETA(dII)/E7/GFP DNA; Lane 3, concentrated culture medium from cells transfected with E7/GFP DNA; Lane 4, concentrated culture medium from cells transfected with ETA(dII)/E7/GFP DNA; Lane 5, lysates from nontransfected 293 Db, Kb cells as a negative control. Note: lysates from E7/GFP DNA-transfected 293 Db, Kb cells revealed a protein band with a size of approximately Mr 30,000 corresponding to E7/GFP protein in Lane 1, as indicated by the short arrow. Meanwhile, lysates from ETA(dII)/E7/GFP DNA-transfected 293 Db, Kb cells generated a protein band with a size of approximately Mr 56,000 corresponding to ETA(dII)/E7/GFP protein in Lane 2, as indicated by the long arrow. E7/GFP DNAtransfected cells exhibited levels of protein expression comparable with that of ETA(dII)/ E7/GFP DNA-transfected cells.

Enhanced Presentation of E7 through the MHC Class I Pathway in Cells Transfected with ETA(dII)/E7 DNA. To demonstrate if the addition of the translocation domain of ETA to E7 can directly enhance MHC class I presentation of E7, we performed CTL assays to characterize the MHC class I presentation of E7 by 293 Db,Kb cells transfected with various DNA constructs. We chose 293 Db,Kb cells as target cells because they have been shown to have a stable high transfection efficiency (up to 80%) and expression of the C57BL/6 MHC class I Db molecule. A Db-restricted E7-specific CD8 T cell line (26) served as effector cells. As shown in Fig. 2A, 293 Db,Kb cells transfected with ETA(dII)/E7 DNA generated significantly higher

percentages of specific lysis at the 9:1 (33.26 3.34% versus 12.5 1.12%; P 0.001) and 27:1 (62.05 6.02% versus 22.6 3.0%; P 0.001) E:T ratios compared with cells transfected with wild-type E7 DNA. These results indicate that cells transfected with ETA(dII)/E7 DNA present E7 antigen through the MHC class I pathway more efficiently than cells transfected with wild-type E7 DNA. Enhanced Presentation of E7 through the MHC Class I Pathway in DCs Pulsed with Lysates of Cells Transfected with Chimeric ETA(dII)/E7 DNA. To demonstrate if the addition of the translocation domain of ETA to E7 can lead to enhanced MHC class I presentation of E7 via a cross-priming mechanism (30), we performed CTL assays to characterize the MHC class I presentation of E7 using bone marrow-derived DCs pulsed with cell lysates of 293 Db,Kb cells transfected with various DNA constructs. As shown in Fig. 2B, DCs pulsed with lysates of 293 Db,Kb cells transfected with ETA(dII)/E7 DNA generated significantly higher percentages of specific lysis as compared with DCs pulsed with lysates of 293 Db,Kb cells transfected with the other DNA constructs and nave DCs (P 0.001). These results revealed that the fusion of ETA(dII) to E7 may enhance MHC class I presentation of E7 via a cross-priming mechanism. Significant Enhancement of E7-Specific CD8 T Cell Precursors in Mice Vaccinated with ETA(dII)/E7 DNA. To determine whether mice vaccinated with various DNA vaccine constructs can generate E7-specific CD8 T cell precursors, we performed intracellular cytokine staining for E7-specific CD8 T cell precursors in spleens extracted from vaccinated mice (5). As shown in Fig. 3A, mice vaccinated with ETA(dII)/E7 DNA generated an 30-fold increase in CD8 T cell precursors (308/ the number of E7-specific IFN3 105 splenocytes) compared with mice vaccinated with E7 DNA (11/3 105 splenocytes; P 0.01). These results also indicated that fusion of ETA(dII) to E7 was required for enhancement of E7-specific CD8 T cell activity, because ETA(dII) mixed to E7 (ETA(dII) E7 DNA) did not generate enhancement of CD8 T cell activity. Furthermore, the linkage of irrelevant proteins (such as GFP and CTLA-4) to E7 did not generate enhancement of E7-specific CD8 T cell activity (data not shown). We found no significant difference in the number of E7-specific CD4 IFNT cells (Fig. 3B) or CD4 IL-4 T cells (data not shown) among each of the vaccination groups. Using an ELISA, we observed no significant enhancement of E7-specific antibody re-

Fig. 2. CTL assays. A, CTL assays to demonstrate enhanced presentation of E7 through the MHC class I pathway of cells transfected with ETA(dII)/E7 DNA. 293 Db, Kb cells transfected with various DNA constructs served as target cells. These CTL assays were performed with various E:T ratios using Db-restricted E7-specific CD8 T cells as target cells. B, CTL assays to demonstrate enhanced MHC class I presentation of E7 in bone marrow-derived DCs pulsed with cell lysates containing chimeric ETA(dII)/E7 protein. Bone marrow-derived DCs were pulsed with cell lysates from various DNA-transfected 293 Db, Kb cells at different concentrations as described in Materials and Methods. These assays were performed at a fixed E:T (9:1) ratio using Db-restricted E7-specific CD8 T cells as effector cells.

3700

Downloaded from cancerres.aacrjournals.org on August 18, 2011 Copyright 2001 American Association for Cancer Research

CANCER IMMUNOTHERAPY USING A DNA VACCINE

Fig. 3. Intracellular cytokine staining with flow cytometry analysis. A, the number of IFN- -producing E7-specific CD8 T cells was determined using flow cytometry in the presence of MHC class I-restricted E7 peptide (aa 49 57; Ref. 48). B, the number of IFN- -producing E7-specific CD4 T cells was determined using flow cytometry in the presence MHC class II-restricted E7 peptide (aa 30 67; Ref. 49). The data presented in this figure are from one representative experiment of two performed.

using a previously characterized E7-expressing tumor model, TC-1 (4). As shown in Fig. 4A, 100% of those receiving ETA(dII)/E7 DNA vaccination remained tumor-free 56 days after TC-1 challenge, whereas all of the other groups of mice developed tumor growth within 15 days after tumor challenge. These results also indicated that fusion of ETA(dII) to E7 was required for antitumor immunity, because ETA(dII) mixed with E7 (ETA(dII) E7 DNA) did not generate enhancement of antitumor immunity. Furthermore, the linkage of irrelevant proteins (such as GFP and CTLA-4) to E7 did not enhance tumor protection (data not shown). Treatment with ETA(dII)/E7 Fusion DNA Eradicates Established E7-Expressing Tumors in the Lungs. To determine the therapeutic potential of chimeric ETA(dII)/E7 DNA in treating TC-1 tumor metastases in the lungs, an in vivo tumor treatment experiment was performed using a lung metastasis model (31). As shown in Fig. 4B, mice vaccinated with ETA(dII)/E7 DNA revealed the lowest mean number of pulmonary nodules (1.6 1.1) compared with mice vaccinated with wild-type E7 DNA (77.6 22.1), or ETA(dII) DNA (73.4 14.6; one-way ANOVA; P 0.001). These data indicated that mice treated with ETA(dII)/E7 could control established E7expressing tumors in the lungs. CD8 T Cells but not CD4 T cells or NK cells Are Essential for the Antitumor Effect Generated by Chimeric ETA(dII)/E7 DNA. To determine the subset of lymphocytes that are important for the rejection of E7-positive tumor cells, we performed in vivo antibody depletion experiments. As shown in Fig. 4C, all nave mice and all of the mice depleted of CD8 T cells grew tumors within 14 days after tumor challenge. In contrast, all of the nondepleted mice and all of the mice depleted of CD4 T cells or NK1.1 cells remained tumor-free for 60 days after tumor challenge. These results suggest that CD8 T cells are essential for the antitumor immunity generated by the ETA(dII)/E7 DNA vaccine.

sponses in mice vaccinated with ETA(dII)/E7 DNA compared with the other vaccination groups (data not shown). Vaccination with ETA(dII)/E7 Fusion DNA Enhances Protection of Mice against the Growth of E7-Expressing Tumors. To determine whether the observed enhancement in E7-specific CD8 T cell-mediated immunity translated to a significant E7-specific antitumor effect, we performed an in vivo tumor protection experiment

DISCUSSION In this study, we demonstrated that direct linkage of ETA(dII) to E7 can dramatically enhance the potency of HPV-16 E7-containing DNA vaccines. A DNA vaccine encoding ETA(dII) fused to HPV-16 E7 elicited strong E7-specific CD8 T cell-mediated immunity and generated significant CD8 T cell-dependent preventive effects against HPV-16 E7-expressing murine tumors. Furthermore, the chimeric

Fig. 4. In vivo tumor protection and treatment experiments against the growth of TC-1 tumors and the effect of lymphocyte subsets on tumor treatment. A, in vivo tumor protection experiment. Note: 100% of mice receiving ETA(dII)/E7 DNA vaccination remained tumor-free 60 days after TC-1 challenge. B, in vivo tumor treatment experiment. Note: the ETA(dII)/E7 group had the fewest pulmonary nodules compared with the other vaccinated groups (one-way ANOVA; P 0.001). Data are expressed as the mean number of lung nodules; bars, SE. C, in vivo antibody depletion experiments to determine the effect of lymphocyte subsets on the tumor protection of the ETA(dII)/E7 DNA vaccine. CD4, CD8, and NK1.1 depletions were initiated 1 week before tumor challenge and lasted 63 days after tumor challenge. The data presented in this figure are from one representative experiment of two performed.

3701

Downloaded from cancerres.aacrjournals.org on August 18, 2011 Copyright 2001 American Association for Cancer Research

CANCER IMMUNOTHERAPY USING A DNA VACCINE

ETA(dII)/E7 DNA vaccine was capable of controlling lethal E7expressing metastatic lung tumors. Our vaccine represents one successful example of using the translocation domain of a bacterial toxin for the development of gene therapy. Previous studies have used the translocation domain of ETA linked to a DNA-binding protein to facilitate the entry of DNA into the cytosol (21, 22). Truncated forms of this chimeric protein lacking the translocation domain failed to facilitate efficient DNA transfer (21). Therefore, these studies indicated that domain II may serve as a useful tool to introduce exogenous protein into the cytosol, although the precise mechanism of such translocation remains unclear. One of the potential explanations that may account for the observed enhancement of E7-specific CD8 T-cell activity in mice vaccinated with ETA(dII)/E7 DNA may be the enhanced MHC class I presentation of E7 in cells expressing this chimeric protein. Indeed, in our in vitro assays, we found that cells transfected with ETA(dII)/E7 DNA presented E7 through the MHC class I pathway more efficiently than cells transfected with wild-type E7 DNA (Fig. 2A). Because ballistic DNA delivery can introduce DNA directly into dermal professional APCs, ETA(dII)/E7 DNA-transfected APCs may directly enhance the presentation of E7 through MHC class I pathway to CD8 T cells and contribute to the generation of E7-specific CD8 T cell precursors in vivo. Another important mechanism that may contribute to the enhanced E7-specific CD8 T cell immune responses in vivo is the so-called cross-priming effect of ETA(dII)/E7 protein, whereby release of ETA(dII)E7 antigen can lead to uptake and processing by other APCs via the MHC class I-restricted pathway (30). Although Western blot analysis on the supernatant of transfected cells failed to detect secretion of ETA(dII)/E7 from ETA(dII)/E7-DNA transfected cells, we could not rule out the possibility that ETA(dII)/E7 protein was eventually released from ETA(dII)/E7 DNA-transfected cells after lysis. Our results suggested that the linkage of ETA(dII) to E7 may lead to enhanced stimulation of E7-specific CD8 T cells via a cross-priming mechanism. One previous study found that exogenous ETA (domains I and II) chimerically linked to influenza A protein or nucleoprotein resulted in MHC class I processing and presentation of the antigen to CTLs (32). However, our data suggested that linkage to domain II of ETA alone is sufficient for delivering exogenous antigen into the MHC class I presentation pathway. Another important factor for the enhancement of antigen-specific CD8 T cell activity and the antitumor effect generated by chimeric ETA(dII)/E7 DNA may be the biology of professional APCs at the vaccination sites. It is now clear that bacterial DNA can contain immunostimulatory elements such as CpG islands (33, 34), which have been shown to cause simultaneous maturation and activation of murine DCs (35). Furthermore, CpG islands may elicit the secretion of IL-12 and IFN- (34, 36), which are related to the T-helper-1-like inflammatory response and important for the antitumor effect. However, it is not clear whether vaccination with chimeric ETA(dII)/E7 DNA leads to greater maturation of DCs or secretion of Th1-related cytokines compared with vaccination with the other DNA constructs. Thus, it would be interesting to investigate further whether chimeric ETA(dII)/E7 DNA influences the biology of DCs or the cytokine profile. The success of the ETA(dII)/E7 DNA vaccine and the importance demonstrated by the ETA domain II component warrants consideration of strategies that use the translocation domains of other bacterial toxins to enhance vaccine potency. The translocation domains for several bacterial toxins have been reported in previous studies, including diphtheria toxin (37, 38), clostridial neurotoxins such as tetanus neurotoxins and botulinum neurotoxins (39, 40), anthrax toxin lethal factor (41, 42), shiga toxin (43), Escherichia coli heat-labile

toxin (44), yersinia cytotoxins (YopE and YopH; Ref. 45), listeria toxin (listeriolysin O; Ref. 46), and pertussis adenylate cyclase toxin (47). The understanding of these translocation domains may allow such molecules to be incorporated into vaccine designs similar to the one we describe in this study. ETA(dII)/E7 generates potent E7-specific CD8 T cell responses through enhanced MHC class I presentation, and the antitumor effect was completely CD4-independent. Interestingly, these features resemble were observed in a previously described chimeric DNA vaccine using Mycobacterium tuberculosis heat shock protein 70 linked to the E7 antigen (5). In the future, it would be important to perform a head-to head comparison to determine which vaccine is more suitable for clinical application. Furthermore, continued investigation of the mechanisms that account for the efficient antigen presentation of these vaccines would help in the development of better vaccines. Although the ETA(dII)/E7 targets antigen to the MHC class I presentation pathway for the enhancement of CD8 T cell activity, other constructs that target antigen to MHC class II presentation pathways may provide enhanced CD4 T cell responses. This realization raises the notion of coadministration of vaccines that directly enhance MHC class I- and class II-restricted pathways. Previously we had developed a chimeric Sig/E7/LAMP-1 DNA vaccine that uses the LAMP-1 endosomal/lysosomal targeting signal for enhancing the MHC class II presentation pathway of E7 (28). The ETA(dII)/E7 vaccine described here in conjunction with a MHC class II-targeting vaccine such as Sig/E7/LAMP-1 may activate multiple arms of the immune system in a synergistic fashion, leading to significantly enhanced CD4 and CD8 T-cell responses and potent antitumor effects. In summary, our findings illustrate the promise of enhancing vaccine potency by the linkage of ETA(dII) to antigen, leading to enhanced antigen-specific CD8 T-cell activity and potent antitumor effects in vivo. Because a majority of cervical cancers express HPV E7, our vaccine can potentially be used for the prevention and treatment of HPV-associated tumors. Furthermore, the strategy of using the translocation property of bacterial toxins may be promising in future vaccine development for the control of cancers and infectious diseases. ACKNOWLEDGMENTS
We thank Drs. Robert J. Kurman and Drew Pardoll for insightful discussions. We also thank Dr. Richard Roden at the Johns Hopkins Medical Institutions, Baltimore, Maryland for critical review of the manuscript.

REFERENCES
1. Chen, C. H., and Wu, T. C. Experimental vaccine strategies for cancer immunotherapy. J. Biomed. Sci., 5: 231252, 1998. 2. Pardoll, D. M. Cancer vaccines. Nat. Med., 4: 525531, 1998. 3. Wang, R. F., and Rosenberg, S. A. Human tumor antigens for cancer vaccine development. Immunol. Rev., 170: 85100, 1999. 4. Lin, K-Y., Guarnieri, F. G., Staveley-OCarroll, K. F., Levitsky, H. I., August, T., Pardoll, D. M., and Wu, T-C. Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res., 56: 2126, 1996. 5. Chen, C-H., Wang, T-L., Hung, C-F., Yang, Y., Young, R. A., Pardoll, D. M., and Wu, T-C. Enhancement of DNA vaccine potency by linkage of antigen gene to an HSP70 gene. Cancer Res., 60: 10351042, 2000. 6. Hoffman, S. L., Doolan, D. L., Sedegah, M., Gramzinski, R., Wang, H., Gowda, K., Hobart, P., Margalith, M., Norman, J., and Hedstrom, R. C. Nucleic acid malaria vaccines. Current status and potential. Ann. NY Acad. Sci., 772: 88 94, 1995. 7. Robinson, H. L. Nucleic acid vaccines: an overview. Vaccine, 15: 785787, 1997. 8. Donnelly, J. J., Ulmer, J. B., Shiver, J. W., and Liu, M. A. DNA vaccines. Annu. Rev. Immunol. 15: 617 648, 1997. 9. Klinman, D. M., Verthelyi, D., Takeshita, F., and Ishii, K. J. Immune recognition of foreign DNA: a cure for bioterrorism? Immunity, 11: 123129, 1999. 10. Restifo, N. P., Ying, H., Hwang, L., and Leitner, W. W. The promise of nucleic acid vaccines. Gene Ther., 7: 89 92, 2000.

3702

Downloaded from cancerres.aacrjournals.org on August 18, 2011 Copyright 2001 American Association for Cancer Research

CANCER IMMUNOTHERAPY USING A DNA VACCINE

11. Gurunathan, S., Klinman, D. M., and Seder, R. A. DNA vaccines: immunology, application, and optimization. Annu. Rev. Immunol., 18: 927974, 2000. 12. Gurunathan, S., Wu, C. Y., Freidag, B. L., and Seder, R. A. DNA vaccines: a key for inducing long-term cellular immunity. Curr. Opin. Immunol., 12: 442 447, 2000. 13. Wang, R., Doolan, D. L., Le, T. P., Hedstrom, R. C., Coonan, K. M., Charoenvit, Y., Jones, T. R., Hobart, P., Margalith, M., Ng, J., Weiss, W. R., Sedegah, M., de Taisne, C., Norman, J. A., and Hoffman, S. L. Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine. Science (Wash. DC), 282: 476 480, 1998. 14. Goletz, T. J., Klimpel, K. R., Leppla, S. H., Keith, J. M., and Berzofsky, J. A. Delivery of antigens to the MHC class I pathway using bacterial toxins. Hum. Immunol., 54: 129 136, 1997. 15. Hwang, J., Fitzgerald, D. J., Adhya, S., and Pastan, I. Functional domains of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli. Cell, 48: 129 136, 1987. 16. Guidi-Rontani, C., and Collier, R. J. Exotoxin. A of Pseudomonas aeruginosa: evidence that domain I functions in receptor binding. Mol. Microbiol., 1: 6772, 1987. 17. Jinno, Y., Ogata, M., Chaudhary, V. K., Willingham, M. C., Adhya, S., FitzGerald, D., and Pastan, I. Domain II mutants of Pseudomonas exotoxin deficient in translocation. J. Biol. Chem., 264: 1595315959, 1989. 18. Siegall, C. B., Ogata, M., Pastan, I., and FitzGerald, D. J. Analysis of sequences in domain II of Pseudomonas exotoxin A, which mediate translocation. Biochemistry, 30: 7154 7159, 1991. 19. Prior, T. I., FitzGerald, D. J., and Pastan, I. Translocation mediated by domain II of Pseudomonas exotoxin A: transport of barnase into the cytosol. Biochemistry, 31: 35553559, 1992. 20. Chaudhary, V. K., Jinno, Y., FitzGerald, D., and Pastan, I. Pseudomonas exotoxin contains a specific sequence at the carboxyl terminus that is required for cytotoxicity. Proc. Natl. Acad. Sci. USA, 87: 308 312, 1990. 21. Fominaya, J., and Wels, W. Target cell-specific DNA transfer mediated by a chimeric multidomain protein. Novel nonviral gene delivery system. J. Biol. Chem., 271: 10560 10568, 1996. 22. Fominaya, J., Uherek, C., and Wels, W. A chimeric fusion protein containing transforming growth factor- mediates gene transfer via binding to the EGF receptor. Gene Ther., 5: 521530, 1998. 23. Wu, T. C. Immunology of the human papilloma virus in relation to cancer. Curr. Opin. Immunol., 6: 746 754, 1994. 24. Wozniak, D. J., Hsu, L. Y., and Galloway, D. R. His-426 of the Pseudomonas aeruginosa exotoxin A is required for ADP- ribosylation of elongation factor II. Proc. Natl. Acad. Sci. USA, 85: 8880 8884, 1988. 25. Bloom, M. B., Perry-Lalley, D., Robbins, P. F., Li, Y., el-Gamil, M., Rosenberg, S. A., and Yang, J. C. Identification of tyrosinase-related protein 2 as a tumor rejection antigen for the B16 melanoma. J. Exp. Med., 185: 453 459, 1997. 26. Wang, T-L., Ling, M., Shih, I-M., Pham, T., Pai, S. I., Lu, L., Kurman, R. J., Pardoll, D. M., and Wu, T-C. Intramuscular administration of E7-transfected dendritic cells generates the most potent E7-specific antitumor immunity. Gene Ther., 7: 726 733, 2000. 27. Lu, Z., Yuan, L., Zhou, X., Sotomayor, E., Levitsky, H. I., and Pardoll, D. M. CD40-independent pathways of T cell help for priming of CD8( ) cytotoxic T lymphocytes. J. Exp. Med., 191: 541550, 2000. 28. Ji, H., Wang, T-L., Chen, C-H., Hung, C-F., Pai, S., Lin, K-Y., Kurman, R. J., Pardoll, D. M., and Wu, T-C. Targeting HPV-16 E7 to the endosomal/lysosomal compartment enhances the antitumor immunity of DNA vaccines against murine HPV-16 E7expressing tumors. Hum. Gene Ther., 10: 27272740, 1999. 29. Wu, T-C., Guarnieri, F. G., Staveley-OCarroll, K. F., Viscidi, R. P., Levitsky, H. I., Hedrick, L., Cho, K. R., August, T., and Pardoll, D. M. Engineering an intracellular pathway for MHC class II presentation of HPV-16 E7. Proc. Natl. Acad. Sci., 92: 1167111675, 1995. 30. Huang, A. Y., Golumbek, P., Ahmadzadeh, M., Jaffee, E., Pardoll, D., and Levitsky, H. Role of bone marrow-derived cells in presenting MHC class I-restricted tumor antigens. Science (Wash. DC), 264: 961965, 1994.

31. Ji, H., Chang, E. Y., Lin, K. Y., Kurman, R. J., Pardoll, D. M., and Wu, T. C. Antigen-specific immunotherapy for murine lung metastatic tumors expressing human papillomavirus type 16 E7 oncoprotein. Int. J. Cancer, 78: 41 45, 1998. 32. Donnelly, J. J., Ulmer, J. B., Hawe, L. A., Friedman, A., Shi, X. P., Leander, K. R., Shiver, J. W., Oliff, A. I., Martinez, D., Montgomery, D., et al. Targeted delivery of peptide epitopes to class I major histocompatibility molecules by a modified Pseudomonas exotoxin. Proc. Natl. Acad. Sci. USA, 90: 3530 3534, 1993. 33. Sato, Y., Roman, M., Tighe, H., Lee, D., Corr, M., Nguyen, M. D., Silverman, G. J., Lotz, M., Carson, D. A., and Raz, E. Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science (Wash. DC), 273: 352354, 1996. 34. Klinman, D. M., Yamshchikov, G., and Ishigatsubo, Y. Contribution of CpG motifs to the immunogenicity of DNA vaccines. J. Immunol., 158: 36353639, 1997. 35. Sparwasser, T., Koch, E. S., Vabulas, R. M., Heeg, K., Lipford, G. B., Ellwart, J. W., and Wagner, H. Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur. J. Immunol., 28: 20452054, 1998. 36. Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K., and Akira, S. A Toll-like receptor recognizes bacterial DNA. Nature (Lond.), 408: 740 745, 2000. 37. Umata, T., and Mekada, E. Diphtheria toxin translocation across endosome membranes. A novel cell permeabilization assay reveals new diphtheria toxin fragments in endocytic vesicles. J. Biol. Chem., 273: 8351 8359, 1998. 38. Oh, K. J., Senzel, L., Collier, R. J., and Finkelstein, A. Translocation of the catalytic domain of diphtheria toxin across planar phospholipid bilayers by its own T domain. Proc. Natl. Acad. Sci. USA, 96: 8467 8470, 1999. 39. Finkelstein, A. Channels formed in phospholipid bilayer membranes by diphtheria, tetanus, botulinum, and anthrax toxin. J. Physiol., 84: 188 190, 1990. 40. Pellizzari, R., Rossetto, O., Schiavo, G., and Montecucco, C. Tetanus and botulinum neurotoxins: mechanism of action and therapeutic uses. Philos. Trans. R. Soc. Lond. B Biol. Sci., 354: 259 268, 1999. 41. Arora, N., and Leppla, S. H. Fusions of anthrax toxin lethal factor with shiga toxin and diphtheria toxin enzymatic domains are toxic to mammalian cells. Infect. Immun., 62: 4955 4961, 1994. 42. Collier, R. J. Mechanism of membrane translocation by anthrax toxin: insertion and pore formation by protective antigen. J. Appl. Microbiol., 87: 283, 1999. 43. Sandvig, K., Garred, O., Prydz, K., Kozlov, J. V., Hansen, S. H., and van Deurs, B. Retrograde transport of endocytosed Shiga toxin to the endoplasmic reticulum. Nature (Lond.), 358: 510 512, 1992. 44. Sixma, T. K., Pronk, S. E., Kalk, K. H., van Zanten, B. A., Berghuis, A. M., and Hol, W. G. Lactose binding to heat-labile enterotoxin revealed by X-ray crystallography. Nature (Lond.), 355: 561564, 1992. 45. Sory, M. P., Boland, A., Lambermont, I., and Cornelis, G. R. Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach. Proc. Natl. Acad. Sci. USA, 92: 11998 2002, 1995. 46. Parrisius, J., Bhakdi, S., Roth, M., Tranum-Jensen, J., Goebel, W., and Seeliger, H. P. Production of listeriolysin by -hemolytic strains of Listeria monocytogenes. Infect. Immun., 51: 314 319, 1986. 47. Karimova, G., Fayolle, C., Gmira, S., Ullmann, A., Leclerc, C., and Ladant, D. Charge-dependent translocation of Bordetella pertussis adenylate cyclase toxin into eukaryotic cells: implication for the in vivo delivery of CD8( ) T cell epitopes into antigen-presenting cells. Proc. Natl. Acad. Sci. USA, 95: 1253212537, 1998. 48. Feltkamp, M. C., Smits, H. L., Vierboom, M. P., Minnaar, R. P., de, J. B., Drijfhout, J. W., Ter, S. J., Melief, C. J., and Kast, W. M. Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells. Eur. J. Immunol., 23: 22422249, 1993. 49. Tindle, R. W., Fernando, G. J., Sterling, J. C., and Frazer, I. H. A. Public T-helper epitope of the E7 transforming protein of human papillomavirus 16 provides cognate help for several E7 B-cell epitopes from cervical cancer-associated human papillomavirus genotypes. Proc. Natl. Acad. Sci. USA, 88: 58875891, 1991.

3703

Downloaded from cancerres.aacrjournals.org on August 18, 2011 Copyright 2001 American Association for Cancer Research