Fusion Protein Vaccine by Domains of Bacterial Exotoxin Linked with a Tumor Antigen Generates Potent Immunologic Responses and Antitumor Effects

Research Article

Chao-Wei Liao,1 Chi-An Chen,2 Chien-Nan Lee,2 Yi-Ning Su,3 Ming-Cheng Chang,2 Ming-Houg Syu,2 Chang-Yao Hsieh,2 and Wen-Fang Cheng2

1Animal Technology Institute Taiwan, Miaoli, Taiwan and Departments of 2Obstetrics and Gynecology and 3Genetic Medicine, National Taiwan University Hospital, Taipei, Taiwan

Abstract cancer patients (3). The E7 protein interacts with the retinoblas- Antigen-specific immunotherapy represents an attractive toma tumor suppressor protein, impairing its function as a cell approach for cancer treatment because of the capacity to growth regulator (4). Because E7 expression is closely associated eradicate systemic tumors at multiple sites in the body while with neoplasia, and several lines of evidence suggest that cell- retaining the requisite specificity to discriminate between mediated immunity is important for control of both HPV infection and HPV-associated neoplasia, the E7 protein is considered a neoplastic and nonneoplastic cells. It has been shown that certain domains of bacterial exotoxins facilitate translocation potential target for cancer therapy. from extracellular and vesicular compartments into the Ideally, cancer treatment should eradicate systemic tumors at cytoplasm. This feature provides an opportunity to enhance multiple sites in the body while having the specificity to discriminate class I and/or II presentation of exogenous antigen to T between neoplastic and nonneoplastic cells. In this regard, lymphocytes. We investigated previously whether the translo- activation of antigen-specific T-cell–mediated immune responses cation domain (domain II) of Pseudomonas aeruginosa permits elimination of tumors associated with a specific antigen exotoxin A with a model tumor antigen, human papillomavirus (5, 6). Recently, many strategies, such as vaccines based on peptides type 16 E7, in the context of a DNA vaccine could enhance (7, 8), proteins (9, 10), DNA (11–13), naked RNA (14, 15), and vaccine potency. We then attempted to determine whether this recombinant viruses (16, 17), have emerged to be applied in the development of cancer treatment and immunotherapy. Protein- chimeric molecule could also generate strong antigen-specific + immunologic responses and enhance the potency of cancer based vaccines are reportedly capable of generating CD8 T-cell vaccine in the protein format. Our results show that vaccina- responses in vaccinated humans (18, 19). However, one of the tion with the PE(#III)-E7-KDEL3 fusion protein enhances MHC limitations of peptide and protein-based vaccines is their potency class I and II presentation of E7, leading to dramatic increases due to poor immunogenicity, especially against some tumor in the number of E7-specific CD8+ and CD4+ T-cell precursors antigens, such as E7 (20). Development of novel strategies that and markedly raised titers of E7-specific antibodies. Further- enhance protein vaccine potency is important for generation of more, the PE(#III)-E7-KDEL3 protein generates potent effective cancer vaccines and immunotherapies. One potential strategy for enhancement of antigen presentation through the MHC antitumor effects against s.c. E7-expressing tumors and pre- + established E7-expressingmetastaticlung tumors. Further,mice class I pathway to CD8 Tcells is the use of the translocation features immunizedwithPE(#III)-E7-KDEL3 protein vaccine also of certain bacterial toxins, such as exotoxin (21). Exotoxins are one of retained long-term immunologic responses and antitumor a class of several secreted bacterial toxins that are able to covalently effects. Our results indicate that retrograde-fusion protein via modify particular proteins in mammalian cells through the translocation of bacterial toxin. Exotoxin domain II is responsible the delivery domains of exotoxins with an antigen greatly enhances in vivo antigen-specific immunologic responses and for translocation to the cytosol (22), and its has been used to represents a novel strategy to improve cancer vaccine potency. engineer a chimeric multidomain protein to deliver DNA into the (Cancer Res 2005; 65(19): 9089-98) cytosol (23, 24). Our previous work has shown that a DNA vaccine encoding domain II of an exotoxin linked to the model antigen HPV 16 E7 enhances MHC class I presentation of E7 antigen to CD8+ T Introduction cells and thereby vaccine potency (11). The aim of this study was Cervical cancer is the second leading cause of cancer death in to test if the retrograde-delivery domains, including the I, II, and women worldwide, with an annual global incidence of f500,000 COOH-terminal KDEL domains of exotoxin A of Pseudomonas cases, of which about one-third are fatal (1). Human papillomavirus aeruginosa [PE(DIII)-KDEL3], could also enhance antigen-specific (HPV) has been recognized as the primary cause of cervical cancer, immunologic response and improve vaccine potency when linked and HPV DNA can be detected in f90% of all cases (2). HPV 16, with the same E7 antigen in the fusion protein format. We also which is the most prevalent strain, is detected in >50% of cervical attempted to distinguish any differences between the antitumor mechanisms of PE(DIII)-KDEL3 when linked with the E7 antigen in naked DNA and protein formats. D Note: C-W. Liao and C-A. Chen contributed equally to this work. Our data indicate that vaccination with the PE( III)-E7-KDEL3 Requests for reprints: Wen-Fang Cheng, Department of Obstetrics and fusion protein enhances MHC class I and II presentation of E7, Gynecology, National Taiwan University Hospital, Taipei, Taiwan. Phone: 886-2-2312- leading to a dramatic increase in the numbers of E7-specific CD8+ 3456, ext. 5166; Fax: 886-2-2393-4197; E-mail: [email protected]. + I2005 American Association for Cancer Research. and CD4 T-cell precursors and raised titers of E7-specific doi:10.1158/0008-5472.CAN-05-0958 antibodies. Furthermore, the PE(DIII)-E7-KDEL3 fusion protein www.aacrjournals.org 9089 Cancer Res 2005; 65: (19). October 1, 2005

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Cancer Research vaccine generated potent antitumor effects against s.c. E7-expressing bone marrow cells in the presence of granulocyte macrophage colony- tumors and preestablished E7-expressing metastatic lung tumors. stimulating factor as described previously (15). The BMDCs pulsed with Further, mice immunized with PE(DIII)-E7-KDEL3 fusion protein various concentrations of E7 peptide (amino acids 49-57), E7 protein, or j vaccine also retained long-term immunologic responses and PE(DIII)-E7-KDEL3 fusion protein and incubated at 37 C for 16 hours were used as feeder cells. Then, the E7-specific CD8+ Tcells and peptide or protein- antitumor effects. These results indicate that fusion protein by pulsed BMDCs were cocultured for a further 24 hours. Golgistop was added 6 exotoxin domains with an antigen greatly enhances in vivo antigen- hours before harvesting the cells from the culture. Cell surface marker specific immunologic response and represents a novel strategy for staining for CD8+ and intracellular cytokine staining for IFN-g as well as improvement of vaccine potency. FACScan analysis were done using conditions described previously (27). Tumor cell line. The production and maintenance of TC-1 cells has been described previously (28). On the day of tumor challenge, tumor cells Materials and Methods were harvested by trypsinization, washed twice with 1Â HBSS, and finally Preparation of various DNA constructs. E7 is a gene encoding a 98– resuspended in 1Â HBSS to the designated concentration for injection. amino acid nucleocapsid protein in HPV 16 (HPV NC001526). The gene Mice. Six- to 8-week-old female C57BL/6J mice were purchased from the sequence was obtained from the National Center Biotechnology Information National Taiwan University (Taipei, Taiwan) and bred in the animal facility of Web site.4 The modified E7 polynucleotide sequence, with additional EcoRI- the National Taiwan University Hospital. All animal procedures were done SalI and XhoI restriction sites at two site arms, was produced by running according to approved protocols and in accordance with recommendations multiple-step PCR. The PCR products were purified using 5% polyacrylamide of the Committee for the Proper Use and Care of Laboratory Animals. gel; then, the bands corresponding to each product were cut out for elution. Cell surface marker staining and flow cytometric analysis. In the The E7 PCR fragment was inserted into pETand pPE(DIII) vectors as detailed first experiment, mice were immunized with 0.1 mg/mouse E7, PE(DIII), in our previous reports (25, 26). The E7 fragment was inserted into the EcoRI/ PE(DIII)-E7, or PE(DIII)-E7-KDEL3 mixed with 10% ISA206 adjuvant as XhoI cloning site of the pET15 or pET15-PE(DIII) plasmid to generate the described earlier. These animals were then boosted s.c. 1 and 2 weeks later plasmid encoding E7 or PE(DIII)-E7 (named pE7 and pETA-E7, respectively). using the same regimen. One week after the last immunization, the mice For increased antigenicity, the pE7-E7–encoded E7 dimer was generated by were sacrificed and the splenocytes were prepared as described previously insertion of an E7 DNA fragment cutting with SalI and PstI from the pE7 into (29). Before intracellular cytokine staining, 3.5 Â 105 pooled splenocytes another pE7 containing the E7 DNA fragment and cutting with XhoI and PstI. from each vaccinated group were incubated for 16 hours with either 1 Ag/ The DNA fragment containing the polynucleotide encoding EcoRI-SalI mL E7 peptide (amino acids 49-57) containing a MHC class I epitope (30) restriction enzyme site, KKDEL-RDEL-KDEL-stop codon-XhoI sequence for detecting E7-specific CD8+ T-cell precursors or 10 Ag/mL E7 peptide (named pKDEL3 encoding KDEL3 signal), was generated using PCR with the (amino acids 30-67) containing a MHC class II epitope (16) for detecting primers: 5V-AGAATTCGTCGACACCTCAAAAAAGACGAACTGAGAGAT- E7-specific CD4+ T-cell precursors. Cell surface marker staining for CD8+ GAACTG-3V (forward primer) and 5V-GTGGTGGTGCTCGAGTCATTA- or CD4+ and intracellular cytokine staining for IFN-g as well as FACScan CAGTTCGTCTTTCAGTTCATCTCTCAGTT-3V (reverse primer). These two analysis were done as described earlier. primers were able to anneal at the complement sequences of the forward and In the second experiment, mice were immunized with PE(DIII)-E7- reverse primers 5V-AACTGAGAGATGAACTG-3V and 5V-CAGTTCATCTCT- KDEL3 fusion protein vaccine once, twice, or thrice as described earlier. CAGTT-3V at the annealing temperature. Therefore, the KDEL3 DNA One week after the last immunization, the mice were sacrificed and the fragment could be generated by running PCRs without an additional splenocytes were prepared, stained, and analyzed as described earlier. template. The PCR product of the KDEL3 sequence was further inserted into In the third experiment, the mice were immunized with PE(DIII)-E7- pET23 using the recombinant technique. For generation of PE(DIII)-E7- KDEL3 thrice as described earlier. The mice were then sacrificed 7, 14, 21, KDEL3, the DNA fragment containing pKDEL3 was cut with PstI and XhoI 30, or 60 days after the last vaccination. The splenocytes were prepared, and then cloned into the PstI and XhoI sites of pETA-E7. The accuracy of all of stained, and analyzed as described earlier. the constructs was confirmed by DNA sequencing. ELISA for anti-E7 antibody. In the first experiment, mice were Generation and preparation of various protein vaccines. To induce vaccinated with various protein vaccines (five animals per group) a total the expression and production of the various recombinant proteins, the host- of three times as described earlier. Sera were prepared from the mice 14 vector system in Escherichia coli BL21(DE3)pLys was used as described days after the last immunization. E7-specific antibody titers were detected previously (25). The proteins were then purified under the His-Tag system in by direct ELISA as described previously (16). the denatured condition according to the manufacturer’s manual (Novagen). In the second experiment, mice were immunized s.c. with PE(DIII)-E7- The denatured samples in 8 mol/L urea were loaded into a column packed KDEL3 fusion protein once to thrice. Sera were prepared 14 days after the with a NTA-Ni2+–bound agarose resin. The bound proteins were then eluted last immunization, and the E7-specific antibody titers were also detected with different buffers (pH 8.0, 7.0, 6.5, 6.0, 5.0, 4.0, and 3.5) containing 4 mol/L using ELISA. urea, 0.3 mol/L NaCl, 20 mmol/L Tris-HCl, and 20 mmol/L phosphate buffer. In the third experiment, PE(DIII)-E7-KDEL3 fusion protein vaccine was After purification, the protein elution fractions were analyzed for purity and injected into the mice thrice as described earlier. The sera of the vaccinated quantification by SDS-PAGE analysis as described previously (26). mice were collected 7, 14, 21, 30, or 60 days after the last vaccination. The Preparation and vaccination of protein vaccines. The stock E7, sera anti-E7 antibody titers were also detected using ELISA. PE(DIII), PE(DIII)-E7, and PE(DIII)-E7-KDEL3 were diluted with PBS (1:10) In vivo tumor protection experiments. For the first experiment, mice 10 times and incubated for 2 hours in 37jC. The activated proteins were were vaccinated s.c. with 0.1 mg of various protein vaccines (five animals further mixed with 10% ISA206 (Seppic, Inc., Paris, France) throughout the per group) and then boosted 1 and 2 weeks later using the same regimen. in vivo experiments. The various ISA206 mixed-protein vaccines were then One week after the last vaccination, the mice were challenged with 5 Â 104 s.c. injected into the backs of the mice. TC-1 tumor cells by s.c. injection in the right leg. Naive mice received the Immunologic response of E7 and PE(#III)-E7-KDEL3 proteins by same number of TC-1 cells to assess natural tumor growth control. Tumor titration assay. Protein titration assays were done to test the ability of the growth was monitored by visual inspection and palpation twice weekly until E7 and PE(DIII)-E7-KDEL3 proteins to induce the E7-specific immunologic 60 days after the tumor challenge. response. The E7-specific CD8+ T cell line (kindly provided by Dr. T.C. Wu, For the second experiment, mice were vaccinated with PE(DIII)-E7- Johns Hopkins University, Baltimore, MD) was used for the effector cells. KDEL3 fusion protein once to thrice. One week after the last vaccination, Bone marrow–derived dendritic cells (BMDC) were generated from murine the mice were challenged with 5 Â 104 TC-1 tumor cells injected s.c. Tumor growth was monitored as described. For the third experiment, the mice were vaccinated with PE(DIII)-E7- 4 http://www.ncbi.nlm.nih.gov KDEL3 fusion protein a total of three times. The mice were then challenged

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Fusion Protein Vaccine by Domains of Bacterial Exotoxin with 5 Â 104 TC-1 tumor cells injected s.c. 7, 30, or 60 days after the last exotoxin and the constructs for E7, chimeric PE(DIII)-E7, and vaccination. Tumor growth was monitored as described earlier. PE(DIII)-E7-KDEL3 are presented in Fig. 1A. The SDS-PAGE of In vivo antibody depletion experiments. In vivo antibody depletions PE(DIII), PE(DIII)-E7, PE(DIII)-E7-KDEL3, and E7 dimer are were done as described previously (31). Briefly, mice (five per group) were shown in Fig. 1B. vaccinated s.c. with 0.1 mg/mouse of PE(DIII)-E7-KDEL3 fusion protein, # 4 PE( III)-E7-KDEL3 fusion protein stimulates E7-specific boosted 1 and 2 weeks later, and challenged with 5 Â l0 TC-1 tumor cells + ; per mouse 1 week after the last immunization. Depletion was started 1 CD8 T cells secreting IFN- more efficiently than E7. Firstly, week before tumor challenge, with monoclonal antibodies GK1.5, 2.43, and we evaluated the relative efficacy of the cross-priming effects of + PK136 used for CD4+, CD8+, and NK1.1 depletion, respectively. Depletion PE(DIII)-E7-KDEL3 and E7. E7-specific CD8 T cells were cocultured was terminated on day 40 after the tumor challenge. with BMDCs pulsed with various concentrations of E7 or PE(DIII)- In vivo tumor treatment experiments. In vivo tumor treatment E7-KDEL3 protein, stained with the anti-IFN-g antibody, and experiments were done using a previously described lung hematogenous analyzed with flow cytometry as described in Materials and spread model (14). In the first experiment, the animals (five per group) were Methods. As shown in Fig. 2, there were more IFN-g-secreting 4 challenged with 5 Â l0 TC-1 tumor cells per mouse via the tail vein. Two days CD8+ Tcells where high to low concentrations of PE(DIII)-E7-KDEL3 after tumor challenge, the animals received 0.1 mg/mouse of E7, PE(DIII), fusion protein were used for pulsing in comparison with the E7 PE(DIII)-E7, or PE(DIII)-E7-KDEL3 protein vaccines s.c. every 7 days for protein. 2 more weeks (a total of three times; 0.3 mg protein). Unvaccinated mice + were used as negative controls. The animals were sacrificed and their lungs Our results show a more potent E7-specific CD8 T-cell response were explanted on day 30. Pulmonary tumor nodules in each mouse were with PE(DIII)-E7-KDEL3 fusion protein compared with E7 protein. # evaluated and enumerated by experimenters blinded to sample identity. PE( III)-E7-KDEL3 fusion protein increases the quantities + + In the second experiment, mice were challenged with TC-1 tumor cells of E7-specific CD4 and CD8 T-cell precursors and raises the and then received 0.1 mg PE(DIII)-E7-KDEL3 protein once to thrice as titers of the anti-E7 antibodies. The ability of the PE(DIII)-E7- described earlier. The animals were sacrificed on day 30 and pulmonary KDEL3 fusion protein to influence in vivo E7-specific immunologic tumor nodules were evaluated and enumerated as described earlier. responses was then evaluated. The numbers of E7-specific CD4+ F Statistical analysis. All data are expressed as mean SE. Statistical and CD8+ T-cell precursors for each group are presented in Fig. 3A. Package for Social Sciences software (SPSS 9.0, SPSS, Inc., Chicago, IL) was The number of E7-specific IFN-g-secreting CD4+ T-cell precursors used for ANOVA to compare the experimental groups. Differences were in the PE(DIII)-E7-KDEL3 group was the highest of all the considered significant if P < 0.05. vaccinated and naive groups (Fig. 3B). Further, the number of E7-specific IFN-g-secreting CD8+ T-cell precursors in the PE(DIII)- Results E7-KDEL3 group was also higher than all the other groups Generation and characterization of the PE(#III)-E7-KDEL3 (Fig. 3C). Mice vaccinated with PE(DIII)-E7-KDEL3 fusion protein fusion protein vaccine. A schematic of the domains of full-length generated f20- and 40-fold increases in the number of E7-specific

Figure 1. Chimeric PE(DIII)-E7-KDEL3 DNA construct and characterization of PE(DIII)-E7-KDEL3 fusion protein expression. A, schematic diagram showing the constructs for full-length exotoxin and PE(DIII), PE(DIII)-E7, and PE(DIII)-E7-KDEL-3 genes. B, SDS-PAGE for various proteins. Lane 1, bovine serum albumin; lane 2, PE (exotoxin); lane 3, PE(DIII); pJJ9; lane 4, PE(DIII)-E7; lane 5, PE(DIII)-E7-KDEL3; lane 6, E7-E7, E7 dimer.

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Figure 2. Protein titration assays of E7 and PE(DIII)-E7-KDEL3. E7-specific CD8+ T cell line as effector cells were cocultured with BMDCs pulsed with various concentrations of E7 or PE(DIII)-E7-KDEL3 protein, stained with the anti-CD8+ and anti-IFN-g antibodies, and analyzed using flow cytometry as described in Materials and Methods. The numbers of IFN-g-secreting CD8+ T cells, when pulsed from high to low concentrations of PE(DIII)-E7-KDEL3 fusion protein, were higher than those pulsed with E7 protein, respectively (10 Amol/L, 1,361.0 F 42.0 versus 319.5 F 10.5; 1 Amol/L, 596.0 F 21.0 versus 212.0 F 11.0; 100 nmol/L, 491.0 F 16.0 versus 152.5 F 10.5; 10 nmol/L, 397.5 F 12.5 versus 94.0 F 10.5; 1 nmol/L, 195.5 F 10.5 versus 77.0 F 9.0; 0.1 nmol/L, 122.0 F 14.5 versus 52.5 F 10.5. P < 0.001, one-way ANOVA.

IFN-g+CD4+ and IFN-g+CD8+ T-cell precursors compared with mice assessed by performing an in vivo tumor treatment experiment vaccinated with the E7 protein. using a previously described lung hematogenous spread model We further evaluated whether the PE(DIII)-E7-KDEL3 fusion (29). The representative figures of pulmonary tumor nodules for protein could also enhance the anti-E7 antibody titer. The animals various protein-vaccinated groups are shown in Fig. 4C. The mean vaccinated with the PE(DIII)-E7-KDEL fusion protein generated the numbers of pulmonary tumor nodules for mice treated with highest sera anti-E7 antibody titers compared with the other PE(DIII)-E7-KDEL3 fusion protein were also significantly lower vaccinated groups (Fig. 3D). than those for the animals treated with the E7 protein (Fig. 4D). The data for our intracellular cytokine staining and anti-E7 These results indicate that PE(DIII)-KDEL3-to-E7 fusion is antibodies showed that PE(DIII)-E7-KDEL3 fusion protein can required for antitumor immunity and prevention of E7-expressing enhance various E7-specific immunologic responses. TC-1 tumor cells and that treatment with PE(DIII)-E7-KDEL3 Vaccination with PE(#III)-E7-KDEL3 fusion protein gener- fusion protein could control established E7-expressing tumors in ates tumor protection in mice challenged with E7-expressing the murine lung. tumor cells. In vivo tumor protection experiments were done to Multiple immunization of PE(#III)-E7-KDEL3 fusion pro- determine if the observed enhancement in E7-specific CD4+ and tein enhances E7-specific immunologic responses. We further CD8+ T-cell–mediated immunity translated to a significant E7- evaluated whether the number of immunizations with PE(DIII)-E7- specific antitumor effect. All the mice receiving PE(DIII)-E7-KDEL3 KDEL3 protein would influence generation of E7-specific immu- fusion protein remained tumor-free 60 days after the TC-1 nologic responses. The numbers of IFN-g-secreting CD4+ and CD8+ challenge (Fig. 4A). In contrast, the unvaccinated E7, PE(DIII), T-cell precursors and the titers of anti-E7 antibodies increased and PE(DIII)-E7 groups developed tumors within 15 days of the significantly in mice vaccinated with PE(DIII)-E7-KDEL3 fusion tumor challenge. Further, all of the mice receiving E7 mixed with protein once to thrice (Fig. 5A-C). PE(DIII) protein also suffered tumorigenesis (data not shown). Our data showed that increasing numbers of vaccinations with CD4+ T, CD8+ T, and natural killer cells are essential for the PE(DIII)-E7-KDEL3 fusion protein can also enhance E7-specific antitumor effect generated by the PE(#III)-E7-KDEL3 fusion immunologic responses. protein vaccine. To determine the subset of lymphocytes that are Further immunization with PE(#III)-E7-KDEL3 protein important for the rejection of E7-positive tumor cells, in vivo vaccine improves antitumor effects in mice. In vivo prevention antibody depletion experiments were done. As shown in Fig. 4B, all and treatment experiments using mice immunized once to thrice naive mice and all those depleted of CD8+ T cells grew tumors with PE(DIII)-KDEL3 fusion protein were done (as described within 14 days of tumor challenge. Further, all mice depleted of earlier) to evaluate if the antitumor effects were reflected in the CD4+ T cells grew tumors within 40 days of challenge. Forty percent E7-specific immunologic profiles of the different subgroups. All of of the animals depleted of NK1.1 cells grew tumors within 60 days the naive mice and the animals immunized once with the PE(DIII)- of tumor challenge. By contrast, all of the nondepleted mice KDEL3 fusion protein grew tumors within 14 days of the tumor remained tumor-free for 60 days after the tumor challenge. challenge. By contrast, however, 60% or 100% of the mice These results suggest that CD4+ T, CD8+ T, and natural killer immunized twice or thrice, respectively, were tumor-free 60 days (NK) cells are all essential for the antitumor immunity generated by after the tumor challenge (Fig. 5D). the PE(DIII)-E7-KDEL3 fusion protein vaccine. Similar results were also observed in the tumor treatment Treatment with PE(#III)-E7-KDEL3 fusion protein leads to experiments. The number of pulmonary tumor nodules decreased significant reduction in pulmonary tumor nodules in C57BL/6 significantly after one to three injections of PE(DIII)-KDEL3 fusion mice. The therapeutic potential of each protein vaccine was protein (Fig. 5E).

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Our results indicate that multiple PE(DIII)-KDEL3 fusion protein although the precise mechanism of such translocation remains vaccinations are needed to generate the effectively preventative and unclear. The domain II of exotoxin without the KDEL signaling therapeutic antitumor effects against E7-expressing tumor cells. transducer is enough to link to a DNA-binding protein and facilitate Mice immunized with PE(#III)-E7-KDEL3 fusion protein DNA entry into the cytosol (23, 24). In our study, however, PE(DIII)- vaccine retain long-term immunologic responses and anti- E7 protein without the KDEL signaling transducer did not tumor effects. Finally, the mice were immunized with PE(DIII)- significantly enhance antigen-specific immunologic response E7-KDEL3 fusion protein vaccine and long-term immunologic (Fig. 3). Naked DNA vaccine functions without the KDEL signaling response was evaluated by comparing the various immunologic transducer (11). Thus, it seems reasonable to suggest that protein- assays (7-60 days after immunization) as described in Materials format vaccine needs the KDEL signaling transducer to facilitate the and Methods. The number of E7-specific CD4+ and CD8+ T-cell precursors peaked at 7 days after immunization (Fig. 6A and B). However, the number of E7-specific CD4+ T-cell precursors decreased gradually, whereas the numbers of E7-specific CD8+ T-cell precursors dropped dramatically 14 days after immunization. The titers of E7-specific antibodies increased from 14 days after immunization, with this elevation persisting as far out as 60 days after immunization (Fig. 6C). We further tested the relationship between long-term immunologic response and in vivo antitumor effect. Mice were immunized with PE(DIII)-E7-KDEL3 fusion protein thrice and then challenged with TC-1 tumor cells 7, 30, or 60 days after the last immunization. All of the mice receiving the PE(DIII)-E7-KDEL3 fusion protein 7 or 30 days after, when challenged with TC-1, remained tumor-free 60 days later (Fig. 6D). Only 60% of mice receiving the PE(DIII)-E7- KDEL3 fusion protein 60 days after their last immunization remained tumor-free 60 days after TC-1 challenge. Our results show that PE(DIII)-E7-KDEL3 fusion protein induces various E7-specific immunologic responses that persist for different periods after immunization. Additionally, PE(DIII)-E7-KDEL3 fusion protein seems to protect mice from tumor growth, at least partially, for an extended interval after vaccination. Discussion Our protein vaccine represents one successful example of using the translocation domains of a bacterial toxin for development of cancer vaccine and immunotherapy. Previous study has employed the translocation domain of an exotoxin linked to a targeted protein to facilitate the entry of protein and DNA into the cytosol, whereas the truncated forms of this chimeric protein that lacks the translocation domain failed to facilitate efficient protein or DNA transfer (23). This indicates that the bacterial domain may serve as a useful tool for introduction of exogenous protein into the cytosol,

Figure 3. Immunologic profiles for vaccinated mice using intracellular cytokine staining and ELISA. Mice were immunized with various protein vaccines and the splenocytes, and the sera were prepared as described in Materials and Methods. A, number of IFN-g-secreting CD4+ T cells in the presence (white columns)or absence (black columns) of the corresponding E7 peptide (amino acids 30-67). The numbers of E7-specific IFN-g-secreting CD4+ T-cell precursors in the PE(DIII)-E7-KDEL3 group were highest of all vaccinated or naive groups [9.5 F 2.1, 12.5 F 3.5, 16.5 F 3.5, 19.5 F 4.5, and 232.5 F 17.7 for the naive, E7, PE(DIII), PE(DIII)-E7, and PE(DIII)-E7-KDEL3 groups, respectively]. P < 0.01, one-way ANOVA. B, number of IFN-g-secreting CD8+ T-cell precursors in the presence (white columns) or absence (black columns) of the corresponding E7 peptide (amino acids 49-57). The numbers of E7-specific IFN-g-secreting CD8+ T-cell precursors in PE(DIII)-E7-KDEL3 group were highest of all the other groups [10.0 F 1.4, 14.0 F 2.1, 12.0 F 2.1, 36.0 F 2.8, and 564.0 F 28.0 for the naive, E7, PE(DIII), PE(DIII)-E7, and PE(DIII)-E7-KDEL3 groups, respectively]. P < 0.01, one-way ANOVA. Columns, mean number of IFN-secreting CD4+ and CD8+ T cells per 3.5 Â 105 splenocytes; bars, SE. C, ELISA shows E7-specific antibodies in mice vaccinated with various protein vaccines. Columns, mean absorbance (OD450 ; nm) from the 1:100, 1:500, and 1:1,000 dilution; bars, SE. For 1:100 dilution: 0.629 F 0.093, 0.882 F 0.086, 0.690 F 0.06, 0.930 F 0.06, and 3.593 F 0.54 for the naive, E7, PE(DIII), PE(DIII)-E7, and PE(DIII)-E7-KDEL3 groups, respectively. P < 0.01, one-way ANOVA. www.aacrjournals.org 9093 Cancer Res 2005; 65: (19). October 1, 2005

Figure 4. In vivo tumor protection and treatment experiments and in vivo antibody depletion experiments using mice vaccinated with various protein vaccines and PE(DIII)-E7-KDEL3 protein, respectively. A, in vivo tumor protection experiments. Mice were immunized with various protein vaccines and challenged with TC-1 tumor cells as described in Materials and Methods. All mice receiving the PE(DIII)-E7-KDEL3 protein remained tumor-free 60 days after TC-1 challenge. In contrast, all of the unvaccinated mice and those receiving E7, PE(DIII), and PE(DIII)-E7 protein developed tumors within 15 days of the tumor challenge. B, in vivo antibody depletion experiments. Mice were immunized with PE(DIII)-KDEL3 fusion protein, injected with respective antibodies, and challenged with TC-1 tumor cells to determine the effect of lymphocyte subsets on the potency of the PE(DIII)-E7-KDEL3 fusion protein vaccine as described in Materials and Methods. All mice depleted of CD8+ T cells grew tumors within 14 days of tumor challenge. Further, all mice depleted of CD4+ T cells grew tumors within 40 days of tumor challenge. Forty percent of the animals depleted of NK1.1 cells grew tumors within 60 days of tumor challenge. C, representative figures for pulmonary tumor nodules in the treatment experiments for each protein vaccinated group. Mice were challenged with TC-1 tumor cells and subsequently treated with various protein vaccines as described in Materials and Methods. 1, naive; 2, E7; 3, PE(DIII); 4, PE(DIII)-E7; 5, PE(DIII)-E7-KDEL3. D, in vivo tumor treatment experiments using mice treated with various protein vaccines. The mean number of pulmonary tumor nodules in the mice treated with PE(DIII)-E7-KDEL3 fusion protein (0.6 F 0.4) was significantly lower than those for analogues treated with PE(DIII)-E7 fusion protein (80.7 F 3.5) or E7 protein (109.7 F 5.0). processing and presentation of E7 antigen protein in vivo. The KDEL I–restricted pathway (35). Indeed, in our in vitro assays, we found signal enhances protein retention in the endoplasmic reticulum (32) that BMDCs pulsed with PE(DIII)-E7-KDEL fusion protein and/or controls glycosylation of the therapeutic proteins for presented E7 through the MHC class I pathway more efficiently antitumor effect (33). These may account for the fact that KDEL than BMDCs pulsed with the E7 protein (Fig. 2). These PE(DIII)-E7- signaling is required for the protein vaccine to enhance the processes KDEL protein-phagocytosed APCs may directly enhance E7 of antigen processing and presentation. presentation through the MHC class I pathway to CD8+ T cells The PE(DIII)-E7-KDEL protein enhances MHC class I presenta- and contribute to the generation of E7-specific CD8+ T-cell tion of E7 in cells expressing this fusion protein to enhance E7- precursors in vivo. specific CD8+ T-cell activity in vivo. One of the potential Another important factor for the enhancement of antigen- explanations is that linkage of PE(DIII) to E7 may lead to enhanced specific CD8+ T-cell activity and the antitumor effect generated by stimulation of E7-specific CD8+ T cells in vivo via the so-called PE(DIII)-E7-KDEL3 protein may be CD4+ T-cell–mediated immu- cross-priming mechanism. The PE(DIII)-E7-KDEL protein mixed nity. It is clear that CD4+ T cells play a major facilitative role in with ISA206 adjuvant may facilitate antigen uptake, transport, and B-cell antibody production. More recently, it has become evident presentation by antigen-presenting cells (APCs; ref. 34) and lead to that CD4+ T-helper cells also control induction and maintenance of antigen uptake and processing by these APCs via the MHC class CD8+ T cells (36) and help other CD4+ T cells by a process of linked

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T-cell help (37). The pivotal position of CD4+ T-helper cells has toxin (44), E. coli heat-labile toxin (45), Yersinia cytotoxins (46), stimulated our focus on their importance in protein vaccination Listeria toxin (47), and pertussis adenylate cyclase toxin (48). It will and motivated design of fusion proteins for induction of activating be interesting to discover whether these bacterial toxins can also Th responses (38). In addition to the helper effect of CD4+ T cells generate comparatively potent vaccine effects when linked with the for CD8+ CTLs, the former can also induce direct killing of tumor HPV 16 E7 antigen. cells (39, 40). Thus, the role of E7-specific CD4+ T lymphocytes The mechanisms underlying the antitumor effects of the available generated by the PE(DIII)-E7-KDEL3 fusion protein in our vaccine formats differ. We tested previously the bacterial toxin investigation might attribute to provision of the requisite signals PE(DIII) strategy in a mammalian expression DNA vector (pcDNA3; for the priming of MHC class I–restricted CD8+ CTLs and/or ref. 11). We found that naked DNA or protein vaccine enhanced E7- directly attack E7-expressing tumor cells. specific CD8+ T-cell–mediated immune responses and antitumor The success of the PE(DIII)-E7-KDEL3 fusion protein vaccine, as effects. Our studies have shown that CD8+ T cells are important for shown by the exotoxin retrograde-delivery domains component, both naked DNA and protein vaccines. By contrast, CD4+ T cells are suggests that consideration of strategies that use the domains of essential only for the antitumor effect generated by the protein other bacterial toxins to enhance vaccine potency is warranted. vaccine. Depletion of CD4+ T or NK cells did not lead to any Several studies have investigated the retrograde-delivery domains significant loss in antitumor effect in mice vaccinated with naked for several bacterial toxins, including diphtheria toxin (41), DNA vaccine (11). However, depletion of CD4+ T cells led to a clostridial neurotoxins (42), anthrax toxin lethal factor (43), Shiga complete loss of antitumor effect in mice vaccinated with the

Figure 5. Immunologic profiles and antitumor effects for mice vaccinated with PE(DIII)-E7-KDEL3 fusion protein. Mice were immunized with one, two, or three injections of PE(DIII)-E7-KDEL3 fusion protein and splenocytes were prepared as described in Materials and Methods. A, number of IFN-g-secreting CD4+ T cells in the presence (white columns) or absence (black columns) of the corresponding E7 peptide (amino acids 30-67). The numbers of IFN-g-secreting CD4+ T-cell precursors increased significantly in mice with increasing numbers of vaccinations with PE(DIII)-E7-KDEL3 protein (22.5 F 4.2, 102.0 F 8.5, and 232.5 F 17.7 for one, two, and three injections, respectively; P < 0.001, ANOVA). B, number of IFN-g-secreting CD8+ T-cell precursors in the presence (white columns) or absence (black columns) of the corresponding E7 peptide (amino acids 49-57). The number of IFN-g-secreting CD8+ T-cell precursors also increased in mice vaccinated with PE(DIII)-E7-KDEL3 protein once to thrice (36.5 F 7.8, 96.0 F 8.5, and 547.5 F 21.5 for one, two, or three injections, respectively; P < 0.001, one-way ANOVA). C, ELISA shows E7-specific antibodies in mice vaccinated with one, two, or three injections of PE(DIII)-E7-KDEL3 fusion protein. The titers of anti-E7 antibodies in the sera of the vaccinated mice also increased as the number of immunizations increased (1:100 dilution: 0.917 F 0.045, 1.912 F 0.061, and 3.593 F 0.45 for one, two, and three injections, respectively; P < 0.01, one-way ANOVA). D, in vivo tumor protection experiments using mice vaccinated with one, two, or three injections of PE(DIII)-E7-KDEL3 fusion protein. Mice were immunized with PE(DIII)-KDEL3 fusion protein once, twice, or thrice and challenged with TC-1 tumor cells as described in Materials and Methods. All of the naive mice and those immunized only once with PE(DIII)-KDEL3 fusion protein vaccine grew tumors within 14 days of the tumor challenge. However, 60% and 100% of the animals immunized twice and thrice, respectively, with the PE(DIII)-KDEL3 fusion protein vaccine were tumor-free 60 days after tumor challenge. E, in vivo tumor treatment experiments using mice vaccinated with one, two, or three injections of PE(DIII)-E7-KDEL3 protein. Mice were challenged with TC-1 tumor cells and subsequently treated with PE(DIII)-KDEL3 fusion protein once, twice, or thrice as described in Materials and Methods. The number of pulmonary tumor nodules decreased significantly comparing subgroups treated with PE(DIII)-KDEL3 fusion protein vaccine (103.0 F 3.8, 28.8 F 6.1, and 0.6 F 0.4 for one, two, or three injections, respectively; P < 0.001, one-way ANOVA). www.aacrjournals.org 9095 Cancer Res 2005; 65: (19). October 1, 2005

Figure 6. Kinetic immunologic responses and in vivo tumor protection experiments using mice immunized with the PE(DIII)-E7-KDEL3 fusion protein vaccine. Mice were immunized with PE(DIII)-E7-KDEL3 fusion protein thrice, and the splenocytes and sera of the vaccinated mice were then collected 7, 14, 21, 30, or 60 days after the last vaccination to detect the immunologic profiles as described in Materials and Methods. A, number of E7-specific CD4+ T-cell precursors. The numbers of E7-specific CD4+ T-cell precursors decreased gradually from day 7 to day 60 after immunization (264.5 F 7.5, 233.5 F 19.5, 198.5 F 12.5, 169.5 F 6.5, and 19.0 F 2.0 on days 7, 14, 21, 30, and 60, respectively). B, number of E7-specific CD8+ T-cell precursors. The numbers of E7-specific CD8+ T-cell precursors dropped dramatically 14 days after immunization (524.0 F 26.5, 94.0 F 12.0, 73.0 F 10.5, 54.0 F 6.0, and 12.5 F 1.5 on days 7, 14, 21, 30, and 60, respectively). C, titers of E7-specific antibodies from ELISA. The E7-specific antibodies of the PE(DIII)-E7-KDEL3 fusion protein vaccinated mice were significantly enhanced from 14 days after immunization, with the effect persisting as far out as 60 days (1:100 dilution: 1.040 F 0.045, 3.451 F 0.027, 3.621 F 0.014, 3.505 F 0.020, and 3.070 F 0.058 on days 7, 14, 21, 30, and 60, respectively). D, in vivo tumor protection experiments. Mice were immunized with PE(DIII)-E7-KDEL3 fusion protein thrice and challenged with TC-1 tumor cells 7, 30, or 60 days after the last immunization as described in Materials and Methods. All of the mice receiving the PE(DIII)-E7-KDEL3 fusion protein 7 or 30 days later, when challenged with TC-1, remained tumor-free 60 days after the TC-1 challenge. In the group receiving the PE(DIII)-E7-KDEL3 fusion protein 60 days later, when challenged with TC-1, 60% remained tumor-free 60 days after the TC-1 challenge.

PE(DIII)-E7-KDEL3 fusion protein. Thus, the CD4+ T cells were the pathway of CD4+ T-helper cells. Although it has not been shown important for the antitumor effect generated by the PE(DIII)-E7- that antibody-mediated responses play an important role in KDEL3 fusion protein because the vaccine actively induced E7- controlling HPV-associated malignancies, antigen-specific antibod- specific CD4+ T cells (Fig. 4), suggesting that CD4+ T cells contribute ies are significant in other tumor models, such as breast cancer, with to the antitumor effect via an antigen-specific mechanism. Further, the HER-2/neu antigen. It has also been shown that HER-2/neu– we discovered that NK cells also play a role in the antitumor effect specific antibodies induce growth arrest where high levels of HER-2/ generated by the PE(DIII)-E7-KDEL3 fusion protein but not the neu are expressed on the surface of breast and ovarian cancer cells naked DNA vaccine. It seems reasonable to suggest, therefore, that (49, 50). The fusion protein vaccine strategy by linked with the different types of vaccines encoding the same construct may activate domains of P. aeruginosa exotoxin A may also be used to generate different subsets of effector cells in the vaccinated host and have HER-2/neu antibodies for treatment of other cancers. specific immunologic or antitumor mechanisms. Protein vaccines can eliminate the concern associated with We observed that E7-specific antibody titers and the number of DNA vaccines of DNA integration. Our previous studies have E7-specific CD4+ T-helper cells were significantly enhanced by shown that many chimeric E7 DNA vaccines linked to E7 hold PE(DIII)-E7-KDEL protein vaccination. We propose that the promise for mass immunization in the murine model (11–13, 29, mechanism for enhancement of antibody responses operates via 51). Certain safety issues still need to be resolved, however.

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Notably, the concern that DNA may integrate into the host Memory T cells are increased in frequency and have distinct genome, although it is estimated that the frequency of such activation requirements and cell-surface proteins that distinguish integration is much lower than that of spontaneous mutation and them from effector T cells (53, 54). Secondary and subsequent should not, therefore, pose a significant risk (52). The concern responses to the antigens are mediated solely by memory with respect to DNA integration is avoided with protein vaccines. lymphocytes and not by naive lymphocytes (55). In terms of future The PE(DIII)-E7-KDEL3 fusion protein vaccine may be used for clinical utilization of the PE(DIII)-E7-KDEL3 fusion protein vaccine, future human clinical trials into prevention of HPV 16 infection knowledge of the capacity of vaccines to induce these memory and therapy for HPV 16–related cervical cancer. responses and the frequency and phenotype of the generated The PE(DIII)-E7-KDEL3 fusion protein could induce long-term effector and central memory lymphocytes is important. protective antitumor effects as well as antigen-specific T-cell and antibody memory responses. Immunologic memory, the most Acknowledgments important consequence of adaptive immunity, is the capacity of the immune system to respond more rapidly and effectively to antigens Received 3/22/2005; revised 6/20/2005; accepted 7/14/2005. The costs of publication of this article were defrayed in part by the payment of page that have been encountered previously. It reflects the preexistence charges. This article must therefore be hereby marked advertisement in accordance of a clonally expanded population of antigen-specific lymphocytes. with 18 U.S.C. Section 1734 solely to indicate this fact.

References 16. Cheng WF, Hung CF, Hsu KF, et al. Enhancement of vaccine encoding calreticulin linked to a tumor antigen. Sindbis virus self-replicating RNA vaccine potency by J Clin Invest 2001;108:669–78. 1. IARC. Monograph on cancer. Human papillomavirus. targeting antigen to endosomal/lysosomal compart- 30. Feltkamp MC, Smits HL, Vierboom MP, et al. Geneva (Switzerland): WHO; 1995. ments. Hum Gene Ther 2001;12:235–52. Vaccination with cytotoxic T lymphocyte epitope- 2. zur Hausen H. Human papillomaviruses in the 17. Marshall JL, Hoyer RJ, Toomey MA, et al. Phase I containing peptide protects against a tumor induced pathogenesis of anogenital cancer. Virology 1991;184: study in advanced cancer patients of a diversified prime- by human papillomavirus type 16-transformed cells. Eur 9–13. and-boost vaccination protocol using recombinant J Immunol 1993;23:2242–9. 3. Lorincz AT, Reid R, Jenson AB, Greenberg MD, vaccinia virus and recombinant nonreplicating avipox 31. Cheng WF, Hung CH, Chai CY, et al. Enhancement of Lancaster W, Kurman RJ. Human papillomavirus virus to elicit anti-carcinoembryonic antigen immune Sindbis virus self-replicating RNA vaccine potency by infection of the cervix: relative risk associations of responses. J Clin Oncol 2000;18:3964–73. linkage of herpes simplex virus type 1 VP22 protein to 15 common anogenital types. Obstet Gynecol 1992;79: 18. Vantomme V, Dantinne C, Amrani N, et al. Immuno- antigen. J Virol 2001;75:2368–76. 328–37. logic analysis of a phase I/II study of vaccination with 32. Bjorndahl M, Cao R, Eriksson A, Cao Y. Blockage of 4. Munger K, Werness BA, Dyson N, Phelps WC, MAGE-3 protein combined with the AS02B adjuvant in VEGF-induced angiogenesis by preventing VEGF secre- Harlow E, Howley PM. Complex formation of human patients with MAGE-3-positive tumors. J Immunother tion. Circ Res 2004;94:1443–50. papillomavirus E7 proteins with the retinoblastoma 2004;27:124–35. 33. Tekoah Y, Ko K, Koprowski H, et al. Controlled tumor suppressor gene product. EMBO J 1989;8: 19. Zhang Y, Chaux P, Stroobant V, et al. A MAGE-3 glycosylation of therapeutic antibodies in plants. Arch 4099–105. peptide presented by HLA-DR1 to CD4+ T cells that were Biochem Biophys 2004;426:266–78. 5. Boon T, Cerottini JC, Van den Eynde B, van der isolated from a melanoma patient vaccinated with a 34. Schijns VE. Immunological concepts of vaccine Bruggen P, Van Pel A. Tumor antigens recognized by MAGE-3 protein. J Immunol 2003;171:219–25. adjuvant activity. Curr Opin Immunol 2000;12:456–63. T lymphocytes. Annu Rev Immunol 1994;12:337–65. 20. Cheng WF, Hung CF, Pai SI, et al. Repeated DNA 35. Huang AY, Golumbek P, Ahmadzadeh M, Jaffee E, 6. Chen CH, Wu TC. Experimental vaccine strategies for vaccinations elicited qualitatively different cytotoxic Pardoll D, Levitsky H. Role of bone marrow-derived cells cancer immunotherapy. J Biomed Sci 1998;5:231–52. T lymphocytes and improved protective antitumor in presenting MHC class I-restricted tumor antigens. 7. Brossart P, Heinrich KS, Stuhler G, et al. Identification effects. J Biomed Sci 2002;9:675–87. Science 1994;264:961–5. of HLA-A2-restricted T-cell epitopes derived from the 21. Goletz TJ, Klimpel KR, Leppla SH, Keith JM, 36. Janssen EM, Lemmens EE, Wolfe T, Christen U, von MUC1 tumor antigen for broadly applicable vaccine Berzofsky JA. Delivery of antigens to the MHC class I Herrath MG, Schoenberger SP. CD4+ T cells are required therapies. Blood 1999;93:4309–17. pathway using bacterial toxins. Hum Immunol 1997;54: for secondary expansion and memory in CD8+ T 8. Vonderheide RH, Hahn WC, Schultze JL, Nadler LM. 129–36. lymphocytes. Nature 2003;421:852–6. The telomerase catalytic subunit is a widely expressed 22. Jinno Y, Ogata M, Chaudhary VK, et al. Domain II 37. Gerloni M, Xiong S, Mukerjee S, Schoenberger SP, tumor-associated antigen recognized by cytotoxic mutants of Pseudomonas exotoxin deficient in translo- Croft M, Zanetti M. Functional cooperation between T lymphocytes. Immunity 1999;10:673–9. cation. J Biol Chem 1989;264:15953–9. T helper cell determinants. Proc Natl Acad Sci U S A 9. Marchand M, Punt CJ, Aamdal S, et al. Immunisation 23. Fominaya J, Uherek C, Wels W. A chimeric fusion 2000;97:13269–74. of metastatic cancer patients with MAGE-3 protein protein containing transforming growth factor-a medi- 38. Zhu D, Stevenson FK. DNA gene fusion vaccines combined with adjuvant SBAS-2: a clinical report. Eur ates gene transfer via binding to the EGF receptor. Gene against cancer. Curr Opin Mol Ther 2002;4:41–8. J Cancer 2003;39:70–7. Ther 1998;5:521–30. 39. Huang H, Li F, Gordon JR, Xiang J. Synergistic 10. Gerard CM, Baudson N, Kraemer K, et al. Therapeu- 24. Fominaya J, Wels W. Target cell-specific DNA transfer enhancement of antitumor immunity with adoptively tic potential of protein and adjuvant vaccinations on mediated by a chimeric multidomain protein. Novel transferred tumor-specific CD4+ and CD8+ Tcells tumour growth. Vaccine 2001;19:2583–9. non-viral gene delivery system. J Biol Chem 1996;271: and intratumoral lymphotactin transgene expression. 11. HungCF,ChengWF,HsuKF,etal.Cancer 10560–8. Cancer Res 2002;62:2043–51. immunotherapy using a DNA vaccine encoding the 25. Chen JR, Liao CW, Mao SJ, Weng CN. A recombinant 40. Frey AB. Rat mammary adenocarcinoma 13762 translocation domain of a bacterial toxin linked to a chimera composed of repeat region RR1 of Mycoplasma expressing IFN-g elicits antitumor CD4+ MHC class tumor antigen. Cancer Res 2001;61:3698–703. hyopneumoniae adhesin with Pseudomonas exotoxin: II-restricted T cells that are cytolytic in vitro and 12. Hung CF, Cheng WF, He L, et al. Enhancing major in vivo evaluation of specific IgG response in mice and tumoricidal in vivo. J Immunol 1995;154:4613–22. histocompatibility complex class I antigen presentation pigs. Vet Microbiol 2001;80:347–57. 41. Oh KJ, Senzel L, Collier RJ, Finkelstein A. Transloca- by targeting antigen to centrosomes. Cancer Res 2003; 26. Liao CW, Hseu TH, Hwang J. A target-specific tion of the catalytic domain of diphtheria toxin across 63:2393–8. chimeric toxin composed of epidermal growth factor planar phospholipid bilayers by its own T domain. Proc 13. Hung CF, Hsu KF, Cheng WF, et al. Enhancement of and Pseudomonas exotoxin A with a deletion in its Natl Acad Sci U S A 1999;96:8467–70. DNA vaccine potency by linkage of antigen gene to a toxin-binding domain. Appl Microbiol Biotechnol 1995; 42. Pellizzari R, Rossetto O, Schiavo G, Montecucco C. gene encoding the extracellular domain of Fms-like 43:498–507. Tetanus and botulinum neurotoxins: mechanism of tyrosine kinase 3-ligand. Cancer Res 2001;61:1080–8. 27. Cheng WF, Hung CF, Hsu KF, et al. Cancer action and therapeutic uses. Philos Trans R Soc Lond B 14. Cheng WF, Hung CF, Lee CN, et al. Naked RNA immunotherapy using Sindbis virus replicon particles Biol Sci 1999;354:259–68. vaccine controls tumors with down-regulated MHC encoding a VP22-antigen fusion. Hum Gene Ther 2002; 43. Collier RJ. Mechanism of membrane transloca- class I expression through NK cells and perforin- 13:553–68. tion by anthrax toxin: insertion and pore forma- dependent pathways. Eur J Immunol 2004;34:1892–900. 28. Lin KY, Guarnieri FG, Staveley-O’Carroll KF, et al. tion by protective antigen. J Appl Microbiol 1999; 15. Cheng WF, Hung CF, Chai CY, et al. Enhancement of Treatment of established tumors with a novel vaccine 87:283. Sindbis virus self-replicating RNA vaccine potency by that enhances major histocompatibility class II presen- 44. Sandvig K, Garred O, Prydz K, Kozlov JV, Hansen SH, linkage of Mycobacterium tuberculosis heat shock tation of tumor antigen. Cancer Res 1996;56:21–6. van Deurs B. Retrograde transport of endocytosed Shiga protein 70 gene to an antigen gene. J Immunol 2001; 29. Cheng WF, Hung CF, Chai CY, et al. Tumor-specific toxin to the endoplasmic reticulum. Nature 1992;358: 166:6218–26. immunity and antiangiogenesis generated by a DNA 510–2. www.aacrjournals.org 9097 Cancer Res 2005; 65: (19). October 1, 2005

45. Sixma TK, Pronk SE, Kalk KH, van Zanten BA, cells: implication for the in vivo delivery of CD8(+) T cell 52. Nichols WW, Ledwith BJ, Manam SV, Troilo PJ. Berghuis AM, Hol WG. Lactose binding to heat-labile epitopes into antigen-presenting cells. Proc Natl Acad Potential DNA vaccine integration into host cell enterotoxin revealed by X-ray crystallography. Nature Sci U S A 1998;95:12532–7. genome. Ann NY Acad Sci 1995;772:30–9. 1992;355:561–4. 49. Nahta R, Hung MC, Esteva FJ. The HER-2-targeting 53. Bachmann MF, Kundig TM, Hengartner H, Zinker- 46. Sory MP, Boland A, Lambermont I, Cornelis GR. antibodies trastuzumab and pertuzumab synergistically nagel RM. Protection against immunopathological Identification of the YopE and YopH domains inhibit the survival of breast cancer cells. Cancer Res consequences of a viral infection by activated but not required for secretion and internalization into the 2004;64:2343–6. resting cytotoxic T cells: T cell memory without cytosol of macrophages, using the cyaA gene 50. Cuello M, Ettenberg SA, Clark AS, et al. Down- ‘‘memory T cells’’? Proc Natl Acad Sci U S A 1997;94: fusion approach. Proc Natl Acad Sci U S A 1995; regulation of the erbB-2 receptor by trastuzumab 640–5. 92:11998–2002. (herceptin) enhances tumor necrosis factor-related 54. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia 47. Parrisius J, Bhakdi S, Roth M, Tranum-Jensen J, apoptosis-inducing ligand-mediated apoptosis in breast A. Two subsets of memory T lymphocytes with distinct Goebel W, Seeliger HP. Production of listeriolysin by and ovarian cancer cell lines that overexpress erbB-2. homing potentials and effector functions. Nature 1999; h-hemolytic strains of Listeria monocytogenes. Infect Cancer Res 2001;61:4892–900. 401:708–12. Immun 1986;51:314–9. 51. Hung CF, Cheng WF, Chai CY, et al. Improving 55. Stevenson PG, Belz GT, Castrucci MR, Altman JD, 48. Karimova G, Fayolle C, Gmira S, Ullmann A, Leclerc vaccine potency through intercellular spreading and Doherty PC. A g-herpesvirus sneaks through a CD8(+) T C, Ladant D. Charge-dependent translocation of Borde- enhanced MHC class I presentation of antigen. J cell response primed to a lytic-phase epitope. Proc Natl tella pertussis adenylate cyclase toxin into eukaryotic Immunol 2001;166:5733–40. Acad Sci U S A 1999;96:9281–6.

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