Cancer-Targeted BikDD Gene Therapy Elicits Protective ... · tumorigenic dose of parental tumor cells inoculated at distant sites in immunocompetent mice. In addition, this cancer-targeted

Therapeutic Discovery

Cancer-Targeted BikDD Gene Therapy Elicits ProtectiveAntitumor Immunity against Lung Cancer

Yuh-Pyng Sher1,2, Shih-Jen Liu3,4, Chun-Mien Chang1, Shu-Pei Lien4, Chien-Hua Chen1,Zhenbo Han5, Long-Yuan Li1,6, Jin-Shing Chen7,8, Cheng-Wen Wu9, and Mien-Chie Hung1,5,6,10

AbstractTargeted cancer-specific gene therapy is a promising strategy for treating metastatic lung cancer, which

is a leading cause of lung cancer–related deaths. Previously, we developed a cancer-targeted gene therapy

expression system with high tumor specificity and strong activity that selectively induced lung cancer cell

killing without affecting normal cells in immunocompromised mice. Here, we found this cancer-targeted

gene therapy, SV-BikDD, composed of the survivin promoter in the VP16-GAL4-WPRE integrated

systemic amplifier system to drive the apoptotic gene BikDD, not only caused cytotoxic effects in cancer

cells but also elicited a cancer-specific cytotoxic T lymphocyte response to synergistically increase the

therapeutic effect and further develop an effective systemic antitumoral immunity against rechallenges of

tumorigenic dose of parental tumor cells inoculated at distant sites in immunocompetent mice. In

addition, this cancer-targeted gene therapy does not elicit an immune response against normal tissues,

but CMV-BikDD treatment does. The therapeutic vector could also induce proinflammatory cytokines to

activate innate immunity and provide some benefits in antitumor gene therapy. Thus, this study provides

a promising strategy with benefit of antitumoral immune response worthy of further development in

clinical trials for treating lung cancer via cancer-targeted gene therapy. Mol Cancer Ther; 10(4); 637–47.

�2011 AACR.

Introduction

Lung cancer is the most common cause of cancerdeaths worldwide, and the metastatic form of the diseaseis a major cause leading to mortality (1). Targeted genetherapy is considered as a novel strategy for treatingmetastasized cancer or therapy refractory tumors owingto the benefit of tumor-specific expression of therapeutic

genes including an apoptosis-inducing gene to kill thetumor cells by a cancer-specific promoter (2). The successof this strategy also depends on suitable vectors that canefficiently deliver a therapeutic gene into target cells withminimal toxicity. However, most of the identified cancer-specific promoters that exhibit a high level of cancerspecificity are still much weaker than the nonspecificstrong cytomegalovirus (CMV) promoter (3). To over-come this issue, a promoter amplification system calledVP16-GAL4-WPRE integrated systemic amplifier (VISA)was developed to amplify the cancer-specific promoteractivity, and the activity in some cancer cell lines are evenable to reach a level comparable to that of CMV in cancercells though remaining inactive in normal tissues (4).

Previously, we showed that a lung cancer–specificpromoter, survivin, is highly active in lung cancer cellsbut not in other cell types, including immortalized nor-mal cell lines (5). Briefly, survivin is a member of theinhibitor of apoptosis gene family that mediates severalantiapoptotic functions (6, 7), and its expression is asso-ciated with disease progression and poor prognosis inmany cancer types (8). Moreover, survivin has also beenimplicated in the development of resistance to apoptosis-inducing agents (9) and promotion of metastasis (10). Weintegrated the survivin promoter into the VISA system toselectively enhance the expression of a therapeuticgene, BikDD, a mutant form of the proapoptotic Bcl2interacting killer (Bik), to generate survivin-VISA-BikDD

Authors' Affiliations: 1Center for Molecular Medicine, China MedicalUniversity Hospital; 2Graduate Institute of Clinical Medical Science and3Graduate Institute of Immunology, China Medical University, Taichung,Taiwan; 4Vaccine Research and Development Center, National HealthResearch Institutes, Miaoli, Taiwan; 5Department of Molecular and CellularOncology, The University of Texas MD Anderson Cancer Center, Houston,Texas; 6Graduate Institute for Cancer Biology, China Medical University,Taichung, Taiwan; 7Division of Thoracic Surgery, Department of Surgery,National Taiwan University Hospital and National Taiwan University Col-lege of Medicine, Taipei, Taiwan; 8Department of Surgery, National TaiwanUniversity Hospital Yun-Lin Branch, Yunlin, Taiwan; 9Institutes of Biome-dical Sciences, Academia Sinica, Taipei, Taiwan, and 10Department ofBiotechnology, Asia University, Taichung, Taiwan

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Yuh-Pyng Sher and Shih-Jen Liu contributed equally to this work.

Corresponding Author: Mien-Chie Hung, Department of Molecular andCellular Oncology, University of Texas MD Anderson Cancer Center, 1515Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-3668; Fax:713-794-0209. E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-10-0827

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

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(SV-BikDD) and showed that systemic treatment withSV-BikDD DNA in liposome complexes controlled tumorgrowth and significantly prolonged survival in immuno-compromised mice with metastatic human lung cancerand produced virtually no toxicity compared with theCMV promoter vector (5).

As immune response is also considered critical fordetermining the success or failure of gene therapy clinicaltrials, we further examined the role of immune responseelicited under SV-BikDD gene therapy in immunocom-petent mice with lung cancer. Here, we show that SV-BikDD cancer-targeted gene therapy not only stimulatedinnate immunity to provide advantages in antitumortreatment but also elicited a cytotoxic T lymphocyte(CTL) response to increase the therapeutic effect andprotect against highly aggressive syngeneic TC-1 lungcancer when challenged again in animals, suggesting thatSV-BikDD gene therapy can elicit cancer-specific immu-nity and confer protection against lung cancer. Thus, thisstudy provides a promising cancer-targeted gene therapyworthy of further development in clinical trials for treat-ing lung cancer, especially the metastatic form of thedisease.

Materials and Methods

Cell lineThe mouse lung cancer cell line TC-1, which was

obtained by cotransformation of HPV-16 E6/E7 andactivated ras oncogene to primary mouse lung epithelialcells of C57BL/6 was kindly provided by T.C. Wu (11) in1997 and established aworking cell bank for tumormodelin 2008. Experiments were carried out within 6 months ofreceiving the cell lines from the established cell bank.The cancer cells were authenticated by their ability toform lung cancer tumors in C57BL/6 mice. Beforethis study, the cells were tested to determine if theywere mycoplasma free. TC-1 cells were grown inRPMI medium 1640 supplemented with 10% FBS and0.4 mg/mL G418.

Evaluation of promoter activity and cytotoxicityAll the plasmids used in this study were constructed as

previously described (5). To test promoter activity, weused dual-luciferase reporter assay (Promega) to normal-ize transfection efficiency according to the man-ufacturer’s instructions. Transfection of the promoterreporter plasmid was carried out with pRL-TK as aninternal control for transfection efficiency. The dual-luci-ferase ratio was presented as the luciferase activity of thetested plasmids divided by the luciferase activity of pRL-TK. For determining cytotoxicity, pRL-TK was also usedas a reporter gene in all experiments.

Lung cancer animal modelFemale C57BL/6 mice purchased from BioLASCO, at

6 to 8 weeks of age, were used for syngeneic lungcancer animal model. Mice were maintained in a spe-

cific pathogen-free environment in compliance withinstitutional policy and approved by the LaboratoryAnimal Care and Use Committee of the China MedicalUniversity (protocol 98-199-N). Each group has morethan 5 mice and each experiment was repeated. TC-1murine lung cancer cells (1 � 106) were inoculated inC57BL/6 mice through intravenous injection to estab-lish an experimental metastatic lung cancer animalmodel (12). Therapeutic plasmids were purified withan Endo-free Mega Prep Kit (Qiagen) according to thecommercial protocol. Plasmid DNA was dissolved inendotoxin-free TE buffer, and the amount of endotoxinwas determined to be less than 10 endotoxin units/mgof DNA by a chromogenic Limulus amoebocyte clot-ting assay (QCL-1000 kit; BioWhittaker). HLDC (HungLab–modified DOTAP:cholesterol) was produced inour laboratory according to Dr. Nancy Templeton’sprotocol (13). Plasmid DNA/HLDC complexes wereprepared as previously described (3). Briefly, HLDC(20 mmol/L) stock solution and stock DNA solutionwere diluted in 5% dextrose in water (D5W) and mixedin equal volumes to make a final solution of 4 mmol/LHLDC and 50 mg DNA in 100 mL of solution (liposome/DNA ratio 1:2.6). We used this concentration of DNA/HLDC complexes for detection of promoter specificityand acute toxicity in animals. The DNA/HLDC com-plexes were diluted in D5W to prepare 100 mL of DNA/HLDC complexes containing 25 mg of DNA in 2 mmol/LHLDC for each treatment in a mouse. We began treat-ing on day 7 after TC-1 cancer cells inoculation viaintravenous injection of 100 mL of DNA/HLDC com-plexes containing 25 mg of DNA which were adminis-tered to mice twice per week for a total of 6 treatmentsin the experimental metastatic lung cancer animalmodel. The lungs of mice were fixed in Bouin’s solutionand the lung tumor nodules were counted understereomicroscope (14). For the ex vivo experiment,TC-1 cells were transiently transfected with indicatedplasmids and 2 days later, equal number of transfectedcells were inoculated subcutaneously into 6-week-oldC57BL/6 mice. Tumor volume was monitored afterinoculation of tumor cells. Subcutaneous TC-1 cellsand normal lung tissue were dissected and total proteinlysate were extracted. Survivin protein was detectedwith anti-human survivin antibody (R&D System), andEF1a was used as total protein control with anti-EF1aantibody (Millipore) by Western blot analysis.

Bioluminescent imaging and quantificationTo detect promoter specificity, promoter constructs

were intravenously delivered to mice by liposome, andthe luciferase activity was imaged by IVIS Imaging sys-tem (Xenogen) 2 days after injection. Mice were anesthe-tized with a mixture of oxygen and isoflurane andintraperitoneally injected with 100 mL of D-luciferin(Xenogen; 30 mg/mL in PBS). Mice were imaged 10minutes after luciferin injection, and signals were ana-lyzed by the Living Image software.

Sher et al.

Mol Cancer Ther; 10(4) April 2011 Molecular Cancer Therapeutics638




Immunohistochemical stainingThe lung tumor and other tissues dissected from mice

were fixed in 10% neutral buffered formalin andembedded in paraffin wax. Five-micrometer sectionswere cut and stained with hematoxylin and eosin. Immu-nostaining for firefly luciferase was done as previouslydescribed (4) by using the goat anti-firefly luciferaseantibody (Abcam), horseradish peroxidase-conjugatedavidin–biotin complex from Vectastain Elite ABC Kit(Vector Laboratories), and AEC chromogen (VectorLaboratories). The sections were counterstained withhematoxylin and mounted. The intensity score wasjudged by a pathologist.

Cytometric bead array immunoassay analysisThe proinflammatory cytokines in sera were quantified

by using a mouse inflammation cytometric bead array kit(BD Biosciences) following the manufacturer’s instruc-tions. In brief, serum samples were diluted to 50 mL withassay diluents and added into a mixture of 6 differentantibody-coated beads. After addition of 50 mL of amixture of PE-conjugated antibodies against the cyto-kines, the mixture was incubated for 2 hours at the roomtemperature. The mixture was then washed with washbuffer, transferred to a 96-well microtiter plate, andanalyzed by BD FACSArray Bioanalyzer (BD Bio-sciences). The concentration of interleukin (IL)-6, IL-10,monocyte chemotactic protein-1 (MCP-1), IFN-g , TNF-a,and IL-12p70 was determined by FACSArray software.

ELISpot assayThe mouse IFN-g ELISpot assay was done as pre-

viously described (15). In brief, 2 � 105 splenocytes wereincubated with 1 � 105 of irradiated (30 Gy) TC-1 cells orsynthetic HPV16 E7 peptide (49–57, RAHYNIVTF; ref. 16)or synthetic survivin peptide (mouse survivin 57–64,CFFCFKEL; ref. 17) in anti–IFN-g coated PVDF platefor 48 hours. After incubation, the cells were removed,and biotinylated anti–IFN-g antibody (eBioscience) wasthen added to each well. The plates were again incubatedat 37�C for 2 hours. After 1-hour incubation at roomtemperature with an avidin–horseradish peroxidasecomplex reagent (eBioscience), the plates were washed,and then a 100 mL aliquot of 3-amine-9-ethyl carbazole(Sigma-Aldrich) staining solution was added to each wellto develop the spots. The reaction was stopped after 4 to6 minutes by placing the plate under tap water. Thespots were then counted by an ELISpot reader (CellularTechnology).

Cytotoxic T lymphocyte assayCytolytic activity was measured by standard 4-hour

chromium release assay as previously described (15). Inbrief, every 2 weeks after the last treatment, erythrocyte-depleted splenocytes (1 � 106 cells/mL) were cultured invitrowith irradiated TC-1 (30Gy; 1� 105) and 10U/mL ofrecombinant human IL-2 in 24-well plates for 5 days aseffector cells. The TC-1 cells (5 � 105) were then pulsed

with 100 mCi of 51Cr (Na251CrO4; PerkinElmer) at 37�C for

1 hour to serve as target cells. The irradiated TC-1 cell–stimulated splenocytes were mixed with labeled targetcells at various effector-to-target ratios as indicated. After4-hour incubation at 37�C, the radioactivity of the super-natant wasmeasured by a gamma counter (PerkinElmer).The percentage of specific lysis was calculated by usingthe following equation: 100 � [(experimental release �spontaneous release)/(maximal release � spontaneousrelease)].

Preparation of normal lung cells from mouseIsolation of normal lung cells from mice was done as

previously described (18, 19). Lung tissue was separatedand flushed with 10 mL of 1% of penicillin-streptomycinin PBS. The separated lung tissues were cut into 1-mm3

pieces and incubated with 0.2% of trypsin and 2 mg/mLof collagenase (Sigma) in pretreated medium (PM) for 30minutes with 100 rpm rotation. The tissues were thenplaced in PM with 1,000 units DNase I at room tempera-ture for 5 minutes. The isolated cells were then filteredthrough 70 and 40 mmmesh (BD Bioscience). The filtrateswere centrifuged and the pellet was resuspended in redblood cell lysis buffer for 2 minutes. The cell pellet wasadded into anti-CD45 (45 mg/dish) and anti-CD16/32(16.5 mg/dish) coated dish at 37�C for 2 hours. Thenonattached cells were collected and suspended withculture medium to 5 � 105 cells/well (6-well plate) for2 days. These cells were irradiated with 2.5 Gy as a singledose from an RS2000 X-ray irradiator (Rad Source Tech-nologies). The irradiated normal lung cells were used tostimulate splenocytes from mice that has been treatedwith gene therapy.

Distant site tumor challenge in treated animalsOneweek after the final treatment course, animals in all

treatment groups were challengedwith tumorigenic doseof the TC-1 parental tumor cells by injecting 5 � 105 cellssubcutaneously at a single site on the right flank of theanimals. The presence of subcutaneous tumors in animalswas observed and calculated by using the formula, V(mm3) ¼ a � b2/2, where a is the largest dimension and bis the perpendicular diameter. The results were analyzedstatistically by the Fisher exact test.

Results

Cancer-specific targeting of survivin promoter toTC-1 cells in vitro and in vivo

To assess whether the host immune system plays someroles in our cancer-targeted gene therapy, a mouse lungcancer cell line, TC-1, was used to establish syngeneiclung cancer animal model in immunocompetent mice(11). Using a series of expression vectors that were pre-viously constructed (5) to drive expression of luciferase(Fig. 1A, top), we found that survivin combined withVISA system consistently enhanced the survivin promo-ter activity in TC-1 lung cancer cells. Specifically, SV

Gene Therapy Induces Cancer-Specific Immunity

www.aacrjournals.org Mol Cancer Ther; 10(4) April 2011 639




exhibited a 17-fold increase in promoter activity com-pared with the survivin promoter alone, but the increasein activity was only approximately half of the CMVpromoter (Fig. 1A, bottom). To further examine whetherthe activity and specificity of SV is still retained in vivo,luciferase expression constructs driven by CMV and SV(Fig. 1A), complexed with liposomes, were administeredto tumor-free (without cancer cell inoculation) andtumor-bearing (with cancer cell inoculation) C57BL/6mice via intravenous injection (Fig. 1B). Bioluminescentimaging revealed a strong signal in the thoracic area ofmice treated with CMV–luciferase (CMV-Luc) constructin both tumor-free and tumor-bearing mice, but a lowsignal was observed in mice treated with SV-Luc con-struct (Fig. 1B, top). There was no signal observed fromother areas of the body of the mice. To precisely monitorthe source of signal, these mice were sacrificed immedi-ately after imaging, and their organs were dissected for exvivo imaging (Fig. 1B, bottom). Quantified photon signalshowed high values in CMV-Luc–treated but not SV-Luc–treated groups in the lungs of tumor-free and

tumor-bearing mice. Consistent with ex vivo imaging,immunohistochemical analysis also showed strong luci-ferase expression in both normal lung and tumor tissuesin the CMV-Luc–injected group with intensity scores of1þ and 3þ, respectively, whereas in the SV-Luc–injectedgroup the signal was only detectable in tumor tissueswith intensity score 2þ, but with intensity score 0 innormal lung tissue by a pathologist’s judgment(Fig. 1C). The IHC results show a positive signal in bothnormal and tumor tissue in the CMV-Luc–injected group(Fig. 1C), thus it caused both strong bioluminescenceimaging in tumor-free and tumor-bearing mice(Fig. 1B). However, there was only a strong signal intumor tissue and no signal in normal tissue in SV-Luc–injected group (Fig. 1C). As the luciferase signal was onlylocated in the tumor nodules that were in a small part,and not all, of the lungs, the overall bioluminescenceimaging in lungs reflect slightly higher in SV-Luc–injected tumor-bearingmouse compared with tumor-freemouse, but is incomparable to the CMV-Luc–injectedgroup. Together, consistent with our previous study

Figure 1. SV vector is active in vitro and in vivo in mouse lung cancer. A, schematic diagram of engineered promoter driving luciferase constructs.The VISA system enhances transgene expression driven by the survivin promoter in a mouse lung cancer cell lines, TC-1. The transcriptional activity of thesurvivin, SV, and CMV promoters was measured in a mouse lung cancer cell line cotransfected with indicated plasmid DNA and pRL-TK in dual luciferaseassay. The relative luciferase activity shown here represents the dual luciferase activity ratio (firefly versus Renilla luciferase) by setting CMV activity as 100%.B, SV vector selectively expressed luciferase in the lung tumor tissue of syngeneic lung cancer animal model whereas CMV promoter strongly expressedluciferase in both of lung and lung tumor tissue of tumor-bearingmice. HLDC liposome/DNA complexes containing 50 mg plasmid were administered into miceby tail vein injection and luciferase activity was detected by using a noninvasive imaging system (IVIS imaging system; Xenogen) after 48 hours. The promoterspecificity of CMV and SV was detected by driving luciferase expression in tumor-free and tumor-bearing mice (top). The organs from mice were thendissected for ex vivo imaging (bottom). The quantified signal from lungs and livers is shown at the bottom. C, lungs from tumor-bearing mice were fixed andprocessed for immunohistochemical analysis for firefly luciferase expression using anti-Luc antibody. The luciferase protein was stained in red and nucleus inblue. The intensity score was judged by a pathologist and is shown in the bottom of each picture.

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(5), the in vivo data here also showed that SV vector–driven transgene expression is more lung cancer–specificthan CMV vector and seems to be suitable for genetherapy in syngeneic lung cancer animal model in immu-nocompetent mice.

SV-BikDD treatment inhibited mouse lung cancercells growth in vitro and ex vivoAs mentioned earlier in the text, we have previously

designed a therapeutic vector that expresses a potentinducer of apoptosis, BikDD, for lung cancer. Bikmutants, BikDD in which threonine 33 and/or serine35 were changed to aspartic acid to mimic the phosphor-ylation at these two residues, enhanced their bindingaffinity with the antiapoptotic proteins Bcl-XL and Bcl-2 and were more potent than wild-type Bik in inducingapoptosis and inhibiting cell proliferation in varioushuman cancer cells (20). BikDD expression increasedthe protein level of cleaved caspase-3, which is one ofthe key executioners of apoptosis by Western blot (Sup-plementary Fig. 1). To determine the cytotoxicity effect ofthe therapeutic plasmids in mouse lung cancer cells, 3treatments—SV (vector control without the BikDD gene),CMV-BikDD, and SV-BikDD—were used to determinetherapeutic effect in vitro and ex vivo. We first cotrans-fected TC-1 cells transiently with these plasmids pluspRL-TK (internal control) and measured the relativeluciferase activity with the control vector set at 100%.In vitro cytotoxicity assay showed that SV-BikDD nearlykilledmore than 80% of TC-1 cells as indicated by the lossof luciferase activity compared with the control (Fig. 2A).We then subcutaneously inoculated TC-1 cells that weretransiently transfected with therapeutic plasmids in vitroand measured the tumor volume in C57BL/6 mice(Fig. 2B). Tumors removed from mice in each group onday 33 clearly showed that pretreatment of TC-1 cellswith SV-BikDD significantly reduced tumor growth inmice compared with untreated cells (Fig. 2B, bottom).Interestingly, although in vitro cytotoxicity assay (Fig. 2A)showed that CMV-BikDD exhibited better cell killingeffect than SV-BikDD, the reverse was observed in theex vivo experiment in which pretreatment of SV-BikDDshowed stronger tumor growth suppression than CMV-BikDD. In addition, mice that received cells pretreatedwith SV also showed significant initial tumor suppressionwith smaller tumor size than untreated group beforeday 25 after tumor inoculation (P < 0.01), suggesting thatour VISA cassette might play a role in stimulating hostimmune response, which in turn inhibits tumor growthandmay partially explain the differential killing effect weobserved between SV-BikDD and CMV-BikDD in vitroand ex vivo.

SV-BikDD inhibited tumor growth and prolongedmice survival time in an experimental metastaticlung cancer animal modelTo evaluate the antitumor effect of SV-BikDD treat-

ment in mouse suffering from metastatic lung cancer,

TC-1 cells were intravenously injected to mimic meta-static circulating cancer cells in immunocompetent micewhich were then given treatment by systemic delivery ofliposome/DNA complexes 1 week later for a total of 6treatments. On day 35 after tumor inoculation, fewer lungtumor nodules were found in mice from SV, CMV-BikDD, and SV-BikDD treatment groups compared with

Figure 2. The cytotoxic effects of BikDD expression in mouse lung cancercells. A, in vitro cell killing effect of BikDD. Indicated therapeutic plasmidand pRL-TK were cotransfected into cells by Lipofetamine 2000. TheRenilla luciferase activity was detected 48 hours posttransfection, andrelative cell viability was measured by setting control as 100%. B,expression of BikDD driven by SV vector inhibits tumor growth morestrongly than by CMV promoter in mouse TC-1 lung cancer ex vivosyngeneic models. Mouse lung cancer cells, TC-1 transfected withindicated plasmids treatment, were harvested and inoculatedsubcutaneously into 6-week-old C57BL/6 mice. Tumor volume wasmonitored after inoculation of tumor cells. The tumors frommice were thendissected on day 33 after tumor inoculation (bottom). *, P < 0.05;**, P < 0.01.






uncountable nodules in the lungs of PBS-treated mice(P < 0.01; Fig. 3A). Although SV, CMV-BikDD, and SV-BikDD treatment all prolonged survival time comparedwith PBS treatment (P < 0.01), mice from CMV-BikDD–treated and SV-BikDD–treated groups still had signifi-cantly longer survival time than SV group (P < 0.05;

Fig. 3B). Overall, mice that were administered SV-BikDDmaintained a 20% survival rate over 17 months but not inthe other treatment groups.

Targeted gene therapy elicits cancer-specificimmune response

Next, we determined whether any cancer-specificimmunity was elicited by our therapeutic plasmidstreatments in vivo. After a total of 6 treatments, IFN-gELISpot assays were done to detect T-cell responseagainst TC-1 cells in mice bearing circulating tumorcells. Two weeks after the final treatment, spleen cellswere harvested and restimulated with irradiated TC-1cells in vitro to detect the IFN-g secretion. As shown inFig. 4A, mice that were treated with SV, CMV-BikDD,and SV-BikDD induced an increased number of IFN-g–secreting cells (111 � 34, 205 � 37, and 242 � 13spots per 1 � 106 cells, respectively) compared withthe naive group (59 � 10 spots per 1 � 106 cells),indicating that all the treated plasmids can stimulateT-cell activation against TC-1 cells. As the oncogenicE7 protein is overexpressed in TC-1 cancer cells, IFN-gELISPOT assays were done to detect T-cell responseagainst E7 peptides (Fig. 4B). The results in T-cellresponse against E7 peptides showed similar patternas against irradiated TC-1 cells. Next, we determinedwhether CTL response against TC-1 cells in the sple-nocytes obtained from mice with different treatmentswas elicited in 2 or 4 weeks after last treatment bychromium-51 (51Cr) release assay (Fig. 4C). In the firstdetection point at 2 weeks after last treatment, weobserved a significant increase in specific CTL activitydirected against TC-1 cells from mice in the SV andSV-BikDD groups, but only a slight increase in theCMV-BikDD group compared with those from naivemice. However, in the second detection point at4 weeks after last treatment, all the treatment groupsshowed significantly increased CTL activity, suggest-ing that CTL activity against TC-1 cells in CMV-BikDD group was elicited more slowly compared withSV-BikDD group. These results indicate that both SVand SV-BikDD vectors induced tumor-specific cyto-toxic activities faster than CMV-BikDD.

As survivin protein is overexpressed in TC-1 tumor butnot in normal lung tissue, it can serve as cancer-specificantigen (Fig. 4D, Western blot). A study recently identi-fied a survivin-derived H-2Kb–restricted CTL epitopesthat could inhibit tumor growth in mouse model (17). Weused the survivin-derived peptides as stimulator to acti-vate survivin-specific T-cell population in the splenocytesin ELISpot assay. The result showed that survivin pep-tides induced a larger number of IFN-g–secreting T cellsin SV-BikDD–treated mice compared with naive mice(Fig. 4D). Together, the results from both IFN-g ELISpotassay from TC-1 cells and survivin peptide stimulationshowed that SV-BikDD treatment could induce strongercancer-specific T-cell immunity compared with CMV-BikDD treatment.

Figure 3. The therapeutic gene, BikDD driven by SV promoter, inhibitedtumor growth and prolonged survival in syngeneic metastatic lung canceranimal model. A, C57BL/6 mice that received intravenous injection of1 � 106 TC-1 lung cancer cells were treated with 25 mg of liposome/DNAcomplexes by intravenous injection for a total 6 times within 3 weeks. Lungtumor nodules were counted under stereomicroscope on day 35 aftertumor inoculation. The tumor nodules in PBS treatment group wereuncountable, and thus were set at 300 (maximum) for countingconvenience. Each group contained 5 mice. B, Kaplan–Meier survivalanalysis. The mean survival time is 1.04 � 0.04, 2.85 � 0.25, 5.27 � 1.07,and 6.24 � 1.72 (limited to 17) months in PBS, SV, CMV-BikDD, andSV-BikDD groups, respectively. Each group has 10 mice. *, P < 0.05;**, P < 0.01.

Sher et al.





SV-BikDD does not elicit T-cell response againstnormal cells, but elicits antitumor immunity toprotect secondary tumor challengeA previous study indicated that CMV-BikDD treat-

ment caused cytotoxicity in normal tissue such as lungand liver in BALB/c mice as measured by terminaldeoxynucleotidyl transferase-mediated dUTP nick endlabeling assay, but not in SV-BikDD treatment (5). Here,we further conducted an acute liver toxicity study indetection of serum level of aspartate aminotransferaseand alanine aminotransferase and showed significantlyincreased cytotoxicity in C57BL6mice treated with CMV-BikDD (P ¼ 0.03 and 0.04, respectively), but not with SV-BikDD (Supplementary Fig. S2). We next investigatedwhether normal tissue damage from CMV-BikDD treat-ment stimulates T-cell response against normal tissues.To address this, two types of normal cells, splenocytesand normal lung cells, were used as stimulator to eval-

uate the normal cell–specific T-cell responses in micetreated with the therapeutic plasmids. As shown inFig. 5A, CMV-BikDD induced the highest number ofIFN-g–secreting T cells against normal splenocytes com-pared with the naive, SV, or SV-BikDD group. In aparallel experiment using normal lung cells, we showedthat CMV-BikDD also induced a substantial increase inIFN-g–secreting T cells against these normal cells(Fig. 5B). The results indicate that CMV-BikDD treatmentinduced stronger immunity against normal cells thanother treatments.

The ability of the immune system to respond morestrongly to the exposure of the same antigen is a hallmarkof T-cell–mediated immunity. To test whether the anti-tumoral immunity in the treated animals provides anyprotection against secondary tumor challenge, TC-1 cellswere inoculated again subcutaneously after the treatmentcourse in mice bearing circulating tumor cells on day 35

Figure 4. Targeted gene therapy elicited antitumor immunity in mice. A, IFN-g ELISpot assay was done to detect T-cell activation against TC-1cancer cell in mice with different treatments. Two weeks after the last treatment, spleen cells were harvested and analyzed for IFN-g by an ELISpot assay.Irradiated TC-1 cells were used as stimulators. Each group has 5 mice. B, synthetic HPV16 E7 peptides (49–57, RAHYNIVTF) were used as stimulator toactivate E7-recognized T-cell population. C, every 2 weeks after the last treatment, effector cells (splenocytes) from treated mice were cocultured withirradiated TC-1 cells for 5 days and subjected to a standard 51Cr-release assay with TC-1 cells as targets. CTL assays were done with 3 mice per group andobserved with effector/target (E/T) ratios of 11 and 33. The data shown represent 1 of 2 separate experiments with similar results. D,Western blot (WB) analysisshowed that survivin protein is highly expressed in TC-1 tumor but not in normal lung tissue. EF1a protein was detected as total protein control. Syntheticsurvivin peptides (mouse survivin 57–64, CFFCFKEL) were used as stimulator to activate survivin-recognized T-cell population. Each grouphas 6 mice. The bars represent mean values for each group. Error bars indicate SEM. *, P < 0.05; **, P < 0.01.






(Fig. 5C). All animals in the naive group, which neverreceived any tumor injection prior to implantation ofsubcutaneous tumors at the challenge sites, showed atumorigenic dose of 5 � 105 TC-1 cells. In CMV-BikDDand SV-BikDD treatment groups, the percentage oftumor-free mice was more than 50%, which was signifi-cantly more than that of the naive group on day 50 afterfirst cancer cells inoculation. Moreover, the tumorvolume measured at the challenge site in mice wassmaller in CMV-BikDD and SV-BikDD treatment groupscompared with the naive and SV groups (Fig. 5D). Theseresults indicate that mice gained protection against sec-ondary distant site tumor challenge after administeringcancer-targeted gene therapy.

Induction of innate immunity by gene therapyTo investigate whether the therapeutic plasmids con-

taining CpG motifs stimulate innate immunity in hostand mediate tumor remission, we analyzed the levels of

proinflammatory cytokines from the serum after intra-venous administration of plasmids in immunocompetentmice. The frequency of CpG motifs was similar in SV,CMV-BikDD, and SV-BikDD with 19, 17, and 19 in eachplasmid, respectively (Table 1). The proinflammatorycytokines, IL-6, MCP-1, IFN-g , TNFa, and IL-12, wereelicited quickly 8 hours after DNA inoculation for all 3plasmids, but these cytokines decreased to basal levelafter 24 hours (Fig. 6). In contrast, IL-10 level did notchange significantly between 8 and 24 hours after injec-tion. These results suggest that all 3 plasmids could elicitproinflammatory cytokines to activate innate immunityand provide some benefits in antitumor gene therapy.

Discussion

Here, we report that the cancer-targeted SV-BikDDsystem, which selectively enhanced BikDD expressionto induce apoptosis in lung cancer cells, also elicited host

Figure 5. SV-BikDD did not elicit T-cell response against normal cells, but elicited protective antitumor immunity. A, IFN-g ELISpot assay was done usingirradiated splenocytes as stimulators to detect the T-cell response against normal cells. Each group has 5 mice. B, using irradiated normal lung cells asstimulators to detect the T-cell response against normal cells. Each group has 5 mice. C, systemic antitumoral immunity against parental tumor cellschallenged at distal sites. Cancer cells (5� 105) were inoculated subcutaneously (rechallenged) 1 week after the last treatment in C57BL/6mice withmetastaticlung cancer cells. Naive group is a control group to show the tumorigenic dose of cancer cells for inoculation of 5� 105 cancer cells. Subcutaneous tumor sizewas measured on day 50 after first tumor inoculation. The number shown above each column indicates the subcutaneous tumor-free events over total micenumber. D, the subcutaneous tumor volume was measured on day 50 after first tumor inoculation. Data spots for individual animal in each treatment groupwere plotted. The bars represent mean values for each group. Error bars indicate SEM. *, P < 0.05; **, P < 0.01.

Sher et al.





antitumor immunity to synergistically block tumorgrowth in a syngeneic lung cancer animal model. Theresults show that SV vector without apoptotic geneexpression has therapeutic effect through eliciting anti-tumor immunity (Figs. 2B, 3, and 4).We proposed that theexpression of exogenous protein from the VISA vectorsuch as Gal4-VP16 fusion proteinmight serve as an tumorantigen in cancer cells (21). Similar observations werenoted, in which cancer cell lines modified to expressforeign protein such as EGFP and b-galactosidase elicitedcellular antitumor immunity when transplanted intoimmunocompetent mice (22–24). In addition, the pre-

sence of CpGs within DNA sequences has also beenlinked to stimulation of the mammalian immune system.One study has shown that this form of antitumor immu-nity can be generated by using null vector approachesthrough activation of the innate immune system (25).Moreover, a rapid elevation in the serum proinflamma-tory cytokines in mice after systemic delivery of lipo-some/DNA complexes within 8 hours, followed by areduction (Table 1; Fig. 6) also indicated the innateimmune response elicited posttreatment (26). Theseobservations partially explained why treatment evenwith just vector alone led to a certain reduction of tumorgrowth compared with the untreated group. Further-more, our cancer-targeted gene therapy may provide asimilar function as cancer vaccine by introducing a sur-rogate antigen, Gal4-VP16 protein, driven by a cancer-specific promoter in cancer cells to raise antitumor immu-nity. This strategy would also prevent injury to normaltissue in response to the exogenous antigen expressiondue to control by the cancer-specific promoter.

Although previous reports have found that apoptoticcells alone were not sufficient to provide the inflamma-tory signals required to induce dendritic cell maturation

Table 1. Induction of proinflammation cyto-kines in gene therapy: the CpG content oftherapeutic plasmids

Size (bp) CpG Frequency

SV 7,405 393 19CMV-BikDD 4,785 288 17SV-BikDD 7,936 416 19

Figure 6. Induction ofproinflammation cytokines in genetherapy. Profile of theproinflammatory cytokinesinduced by SV, CMV-BikDD, andSV-BikDD. The proinflammatorycytokines in sera were quantifiedby using a mouse inflammationcytometric bead array kit.






for activation of T cells (27), recent studies have shownthat apoptosis induced by pathogens does elicit potentimmune responses associated with induction of IFN- gand the generation of specific CTLs (28, 29). Our resultsshow that therapeutic effects of plasmids are different in3 types of assay systems (in vitro, ex vivo, and in vivo). Itshows that SV-BikDD treatment has more antitumorability in ex vivo and in vivo than CMV-BikDD treatment,whereas CMV-BikDD has better in vitro cytotoxicitythan SV-BikDD. Although the promoter activity of SVis only half of the CMV promoter activity, the antitumorability of SV-BikDD was stronger than CMV-BikDD inanimal model, indicating stronger synergistic antitumoreffects with immune system in SV-BikDD treatment. Inour study, we showed that SV-BikDD elicits T-cellresponse against TC-1 cancer cells, especially in survi-vin-overexpressed TC-1 cancer cells (Fig. 4D), but notagainst normal tissues such as splenocytes and lungcells, suggesting SV-BikDD treatment induced morecancer-specific immunity to kill tumor cells thanCMV-BikDD. However, although CMV-BikDD treat-ment also elicits strong T-cell response against TC-1cancer cells in a comparable level to SV-BikDD, it elicitssome T-cell response against normal cells as well.Because normal cells and TC-1 cancer cells might pre-sent a similar cellular antigen, some active T cellsagainst TC-1 cancer cells might be from the T cellsagainst normal cells and cause the value to be over-estimated in CMV-BikDD treatment. Therefore, basedon our result, the therapeutic effect in SV-BikDD treat-ment was better than CMV-BikDD treatment in vivo and

this specific antitumoral immunity in mice also pro-vided protection against secondary tumor challenge.Whether nonspecific tissue damage such as CMV-BikDD treatment may decrease the specific antitumorimmunity still need further investigation. Collectively,our results showed that SV-BikDD vector not onlyinduced apoptosis but also elicited an innate immuneresponse and specific antitumor immunity, supportingit as an excellent candidate for alternative cancer treat-ment option in future clinical trials.

Disclosure of Potential Conflicts of Interest

M-C. Hung is an inventor of patents covering BikDD as a therapeuticagent filed with the University of Texas MD Anderson Cancer Center.

Acknowledgments

Inmemoriam,Mr. Nan-TuHuang for his courageous fight against lungcancer.

Grant Support

This work was supported by grants DOH99-TD-G-111-027 and DOH-TD-C-111-005 (M-C. Hung) and NSC98-2320-B-039-044 (to Y-P. Sher) from NationalResearch Program for Genomic Medicine, MD Anderson—China Medical Uni-versity and Hospital Sister Institution Fund (to M-C. Hung), and Taiwan CancerResearch Center (to M-C. Hung).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 1, 2010; revised November 15, 2010; acceptedDecember 7, 2010; published OnlineFirst January 31, 2011.

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2011;10:637-647. Published OnlineFirst January 31, 2011.Mol Cancer Ther Yuh-Pyng Sher, Shih-Jen Liu, Chun-Mien Chang, et al. Antitumor Immunity against Lung CancerCancer-Targeted BikDD Gene Therapy Elicits Protective

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Cancer-Targeted BikDD Gene Therapy Elicits Protective ... · tumorigenic dose of parental tumor cells inoculated at distant sites in immunocompetent mice. In addition, this cancer-targeted