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Vaccine 28 (2010) 2769–2774

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

mmunogenicity and efficacy of a DNA vaccine encoding a human anti-idiotypeingle chain antibody against nasopharyngeal carcinoma

hen Luo1, Jia-Jia Wang1, Yue-Hui Li, Jin-Yue Hu, Guan-Cheng Li ∗

umor Immunobiology Laboratory of Cancer Research Institution, Central South University, Changsha 410078, Hunan, China

r t i c l e i n f o

rticle history:eceived 6 December 2009eceived in revised form 14 January 2010ccepted 16 January 2010vailable online 29 January 2010

a b s t r a c t

G22, an anti-idiotype single chain antibody screened from human nasopharyngeal carcinoma phageanti-idiotype antibody library, has been already identified by He et al.

G22 DNA vaccine was produced by cloning G22 gene and inserting the cloned gene into pcDNA3.1.To investigate the immunogenicity of pcDNA3.1-G22, C57BL/6 mice were immunized with the vaccine,pcDNA3.1 and PBS individually and the antibody response, T cell phenocyte at the 15th, 22th, 29th, 36th

eywords:NA immunitynti-idiotype antibodyasopharyngeal carcinomaaccine

day after the last immunity were detected. In the tumor protection experiment, the immunized mice werethen challenged with CMT-93-G22 cells or CMT-93-mock cells. The tumor size and the survival time ofthe animals were compared between these groups. The results showed that DNA vaccine pcDNA3.1-G22could raise G22-specific humoral and cellular immune responses. Furthermore, pcDNA3.1-G22 couldprolong the survival time and lessen the tumor size of the CMT-93-G22-bearing mice but had no protec-

ttackeclini

tion effect on the mice afor further studies on the

. Introduction

Nasopharyngeal carcinoma (NPC) is an Epstein–Barr virusEBV)-associated malignant disease with high prevalence inouthern Chinese [1]. Despite favourable response to radio-hemotherapy in most patients with early disease, it seems thathe conventional therapy has reached a therapeutic plateau and aignificant number of patients present with metastatic or refractoryisease or develop relapses [2]. Therefore new treatment strategiesre clearly needed for this disease.

According to Niels Jerne’s idiotypic network hypothesis, anti-enic epitope structures can be mirrored through an anti-idiotypicascade of antibodies. Of these, the so-called internal imagenti-idiotypic (Id) antibodies can mimic epitopes of self-antigensnd serve for tumor vaccination strategies [3,4]. Some experi-nce in human trials using anti-Id to stimulate immunity againstumors has shown that immunization with true anti-Id reagentsAb2�) can induce both cellular and humoral immunity, fre-uently when the original antigen does not, or when a state

f anergy to the self-expressed tumor-associated antigen exists5,6].

During the past two decades, a number of Ab2� monoclonalntibodies (mAbs) had been produced and applied into the therapy

∗ Corresponding author. Tel.: +86 731 84805445; fax: +86 731 82355042.E-mail address: libsun@163.com (G.-C. Li).

1 Both authors contributed equally to this work.

264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2010.01.033

d by CMT-93-mock cells. These results were expected to lay foundationcal application of pcDNA3.1-G22 DNA vaccine.

© 2010 Elsevier Ltd. All rights reserved.

of cancer patients [7–9]. However, therapy with these mAbs waslimited by development of a human anti-mouse antibody response(HAMA). With the development of phage display technology, theantibody fragments have attracted considerable attention, sincethese products can be easily engineered for specific purposes, suchas therapy of cancer without invoking HAMA response [10]. Inour previous work, we had constructed NPC phage anti-Id anti-body library successfully and screened five Ab2� anti-Id antibodieswhich binded specifically to Ab1(FC2) [11,12]. Among these Ab2�antibodies, single chain antibody (scFv) G22 had the strongest inhi-bition of binding of Ab1(FC2) to the surface antigen of HNE2 cellswhich could be used as a potential vaccine candidate. To assessthe immunogenicity of G22 anti-Id scFv, we had successfully con-structed an eukaryotic expression vector pcDNA3.1-G22 by geneticengineered method [13].

The optimal DNA vaccine encoding anti-idiotype antibody ide-ally will generate both humoral and cellular immune responses bydelivering foreign antigen to APCs that stimulate CD4+ and CD8+ Tcells [14,15]. CD8+CTLs are an important effector arm in antitumorimmunity and CD4+ T cell help is critical to the maintenance of CD8+

T cell population [16,17]. To explore the potential of pcDNA3.1-G22 as antitumor vaccine, we had examine its immunogenicityby the detection of antibody response and phenotype of prolif-

erating splenocytes of G22-immunized mice. Several studies havedocumented that the expansion of natural Ab repertoire by Id vac-cination could participate in the lysis of tumor cells [18], and thus,the ability of pcDNA3.1-G22 DNA vaccine to protect mice againsttumor cells challenge had also been investigated.

2 ne 28 (2010) 2769–2774

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. Materials and methods

.1. FC2 mAb

One of the highly reactive hybridoma cell lines (FC2) [19] wasnjected intraperitoneally into female Balb/c mice (6–8 weeks old,hanghai Slac, China) and the ascites were collected, centrifuged toemove the debris and stored at 4 ◦C until use. Purified FC2 mAb wasabeled with horseradish peroxidase (HRP) by improved methodf NaIO4 [20]. HRP-FC2 was employed to analyse the inhibitoryctivity of Ab2� anti-Id antibodies.

.2. DNA vaccine construction and manufacturing

The pcDNA3.1-G22 plasmid encoding a human anti-idiotypeingle chain antibody was constructed as described by Luo et al.13]. CMT-93 cells (CCL-223, ATCC, USA) were transiently trans-ected with pcDNA3.1-G22 using Lipofectamine 2000 ReagentInvitrogen, San Diego, CA, USA) according to the manufacturer’specifications, and cells were harvested 18 h post transfection. Toetect the expression and expression site of G22 protein, West-rn blot and immunocytochemistry method were employed withC2 as primary antibody and HRP-coupled goat anti-mouse IgGNovagen, Germany) as secondary antibody.

.3. Mice and tumor cells

Female C57BL/6 mice (6–8 weeks old) were purchased fromhanghai Slac animal center. All experiments were undertaken inccordance with ethical guidelines for care and use of laboratorynimals in central south university. The CMT-93 is a tumorigenicurine colonic epithelial cell line. It produces rapidly growing

ocal tumors after subcutaneous (s.c.) inoculation. The G22 genepcDNA3.1-G22) was stably transfected into the CMT-93 cell line.fter selection with G418 (Dingguo, China), clones that expressed22 were obtained by the limiting dilution method. Control clonesMT-93-mock were obtained by CMT-93 cells transfected withhe pcDNA3.1 vector alone. These cell lines were maintained inMEM containing 10% heat-inactivated bovine serum (FBS) (all

rom Hyclone, USA).

.4. Immunization schedule and tumor challenge studies

The mice were divided randomly into 3 groups (n = 17). A total of00 �g pcDNA3.1-G22 in 100 �l of phosphate-buffered saline (PBS)as injected into the anterior tibialis muscle of the mice (every 2eeks, 3 times). The mice treated with either the empty plasmidcDNA3.1 or PBS were used as the negative control. 15, 22, 29, 36ays after the last immunization, the mice were sacrificed (n = 3)nd sera, spleens were harvested for the assessment of humoralnd cellular immunity responses.

To further investigate the efficacy of the DNA vaccine, a tumorhallenge assay was performed. At 9 week after the first vaccina-ion, mice were inoculated with CMT-93-G22 cells or CMT-93-mockells subcutaneously in each group (5 from each group). The tumorevelopment in individual mice was monitored every 2–3 days andhe tumor size (in mm3) was calculated by the following formula:.5 × length (mm) × width (mm)2. The survival time (until deathr when the tumor volume was over 1000 mm3) after the tumorhallenge was recorded.

.5. Measurement of antibody response by ELISA

The presence of Ab3 in sera was determined in a solid-hase ELISA (enzyme-linked immunosorbent assay) [21]. In brief,b2–Ab3 interaction was determined by coating the microtiter

Fig. 1. Western blot analysis of monoclonal cell line. Lane 1: CMT-93-mock, lane2: CMT-93-G22. CMT-93-G22 was transfected with pcDNA3.1-G22; while CMT-93-mock was transfected with pcDNA3.1. The cell line CMT-93-G22 expressed the G22protein.

plates (Sigma, St. Louis, MO) with HNE2 cells (QK10903, cell library,China), blocking with 2% bovine serum albumin (BSA; v/v; Ding-guo) in PBS, pH 7.2 for 2 h at 37 ◦C. After three washes with PBSwith 0.05% (v/v) Tween20 (PBST), 100 �l of serial two-fold dilu-tions of mouse sera in PBS were added and incubated at 37 ◦C for2 h. The plates were then washed three times with PBST, and 100 �lof HRP-labeled goat anti-mouse IgG (Novagen) diluted 1:1000 wasadded. After an hour of incubation at 37 ◦C, 100 �l of fresh ABTS(2′.2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) substratesolution (0.3 g/l ABTS and 0.03% H2O2 in a glycine/citric acid buffer,pH 4.0; w/v, v/v) was added for color development. The opticaldensity (OD) of the plate was measured at 405 nm.

Inhibition of Ab1 (HRP-FC2) binding to the surface antigen ofHNE2 cells by Ab3 sera was measured by competitive ELISA, where50 �l of different sera at sequential dilutions were incubated for2 h at 37 ◦C in HNE2 cells coated microtiter plates (Sigma), togetherwith 50 �l of pre-determined dilution of HRP-FC2 which had givenan absorbance value of 0.8–1.2 in a preliminary titration. Thebinding of HRP-FC2 was detected by adding ABTS complex anddeveloped as described above. The adsorbance was measured at405 nm in an ELISA reader (Bio-Tek, USA). The inhibitory activitywas expressed as percentage of inhibition and determined as fol-lows: percentage of inhibition = 100 × (A405 nm of HRP-FC2 − A405 nmof HRP-FC2 bound to the surface antigen in the presence of Ab3sera)/A405 nm of HRP-FC2 [22].

2.6. Flow cytometric analysis (FCM)

The collected splenocytes (106 cells) were resuspended in 100 �lPBS and incubated with FITC-conjugated anti-mouse CD4 and PE-conjugated anti-mouse CD8 mAbs at a concentration of 0.5 mg per106 cells in an ice bath. After 30 m incubation, the cells were washedand analysed using a BD-LSR cytofluorimeter using CellQuest soft-ware (BD Biosciences).

2.7. Statistical analysis

All data were presented as mean ± SD. ANOVA was used toidentify significant differences in multiple comparisons. Statis-tical differences between two groups were evaluated by theunpaired Student’s t-test. The survival time was calculated by theKaplan–Meier method. The survival rates were compared by thelog-rank test. A level of p <0.05 was considered statistically signif-icant.

3. Results

3.1. Monoclonal cell line verifications

The monoclonal cell line CMT-93-G22 could express G22 proteinwhile the control cell line CMT-93-mock showed no expression ofthe G22 protein (Fig. 1). The cell line CMT-93-G22 was used in sub-

C. Luo et al. / Vaccine 28 (2010) 2769–2774 2771

Fig. 2. Immunocytochemistry staining of monoclonal cell line (×1200). CMT-93-G22 couhad no expression of G22.

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ig. 3. Induction of specific antibody responses. Specific antibody production inhe sera of three different groups as detected by ELISA at different time pointsfter immunization. The G22-specific antibodies in sera of different groups wereetermined at a 1:100 dilution by ELISA. The data were expressed as the meanbsorbances (OD) at 405 nm. Error bars represent the standard deviation.

equent experiments, while the cell line transfected with pcDNA3.1as named CMT-93-mock. G22 was expressed in the monoclonal

ell line CMT-93-G22, especially on the cytoplasm and cytoblast,owever, the cell line CMT-93-mock showed no expression of G22Fig. 2).

.2. Humoral immune responses

Because specific antibodies also play a role in antitumor immu-ity, we measured the anti-G22 titres in the serum of immunizedice by ELISA. Sera were obtained from the animals 15, 22, 29, 36

ays after the last immunization, respectively. As shown in Fig. 3,era from animals of pcDNA3.1 and PBS group did not show anypecific reactivity. By contrast, animals of pcDNA3.1-G22 groupemonstrated specific antibodies only 15 days after the last immu-ization. Along with the schedule, the antibody level decreased to

ome extent but was still at a high level. The difference between thecDNA3.1-G22 and negative control group were of statistically sig-ificance (p < 0.05). These results showed that the humoral immuneesponse had been primed and possessed the ability to recognizend react to stimulation of pcDNA3.1-G22 DNA vaccine.

able 1hange of T lymphocyte phenotype of pcDNA3.1-G22 group after immunization for 3 tim

15th day 22th day

CD8+ (46.20 ± 11.52) % (29.91 ± 9.85CD4+ (66.28 ± 10.09)% (48.28 ± 12.2CD4+/CD8+ 1.46 ± 0.15 1.64 ± 0.14

ld express G22 protein especially on the cytoplasm and cytoblast; CMT-93-mock

To confirm that a specific response against FC2 idiotype wasgenerated by the immunization, the same aliquot of HRP-FC2 andmouse sera competed for the surface antigen of HNE2 cells, andthe remnant reactivity against the surface antigen was measuredby ELISA. As shown in Fig. 4, sera from animals of pcDNA3.1-G22group exhibited high inhibitory activity during the schedule andthe inhibitory activity decreased gradually along with the schedule.The percentage of inhibition of pcDNA3.1-G22 group decreased to32.67% at the 36th day after the last immunization which was stillhigher than that of pcDNA3.1 and PBS group (p < 0.05).

3.3. The effect of G22 DNA vaccine on the T cell phenotype

The mice were sacrificed at 15th, 22th, 29th, 36th day afterthe third immunization and spleen cells were harvested for theassessment of T cell phenotype. At the 15th day after the thirdimmunization, the percentage of CD4+ and CD8+T cells of G22-immunized mice was higher than that of PBS, pcDNA3.1 group andthe differences were of statistically significance (Tables 1 and 2,p < 0.05). Along with the schedule, the percentage of CD4+ andCD8+T cells of G22-immunized mice decreased gradually, how-ever, the ratio of CD4+/CD8+ increased gradually. Compared withpcDNA3.1, PBS group, the ratio of CD4+/CD8+ of G22-immunizedgroup at 29th, 36th day after the third immunization were higher(Tables 1 and 2, p < 0.05). These results indicated that pcDNA3.1-G22 was capable of inducing both CD4+ T helper activity as well asCD8+ cytolytic activity and CD4+ T cells was the predominant cellsubset.

3.4. Vaccination with DNA vaccine expressing G22 generatessignificant protection and anti-tumor effect

In order to assess the anti-tumor immunity generated bypcDNA3.1-G22 vaccine, we performed an in vivo tumor protec-tion assay. C57BL/6 mice (5 per group) were immunized with

pcDNA3.1-G22 3 times at a two-week interval. Seven days after thelast immunization, the mice were challenged subcutaneously with5 × 106 CMT-93-G22 or CMT-93-mock tumor cells. Tumor growthwas monitored by visual inspection and palpation twice a week.As shown in Fig. 5A, immunization with pcDNA3.1-G22 resulted in

es (�̄ ± SD).

29th day 36th day

)% (21.25 ± 6.16)% (13.63 ± 5.75)%4)% (36.71 ± 9.19)% (25.43 ± 8.76)%

1.74 ± 0.08 1.92 ± 0.18

2772 C. Luo et al. / Vaccine 28 (2010) 2769–2774

Fig. 4. Specific inhibition of binding of Ab1(FC2) to the surface antigen of HNE2 cells by mice sera. Binding of FC2 to HNE2 cells coated plates was tested in the presenceof serial dilutions of pooled mice sera at 15th (A), 22th (B), 29th (C), 36th (D) day after the last immunization. The percentage of inhibition was relative to the binding ofHRP-FC2 mAb to the surface antigen of HNE2 cells in the absence of serum.

Table 2Change of T lymphocyte phenotype of control group after immunization for 3 times (�̄ ± SD).

PBS pcDNA3.1

15th day 22th day 29th day 36th day

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CD8+ (34.21 ± 10.58)% (33.29 ± 10.28)%CD4+ (49.79 ± 13.75)% (50.45 ± 12.37)%CD4+/CD8+ 1.47 ± 0.05 1.53 ± 0.11

significant protection compared to pcDNA3.1 and PBS (p < 0.05).urthermore, the protection against the tumor was G22-specific,ince the mice that were immunized with pcDNA3.1-G22, followedy challenging with CMT-93-mock tumor cells were not protectedFig. 5B, p < 0.05). These results demonstrated that the pcDNA3.1-22 vaccine could induce G22-specific tumor protection.

Long-term survival of pcDNA3.1-G22-vaccinated mice wasssessed. Immunized mice were tumor-challenged and monitoredor tumor growth until death. As shown in Fig. 6A, the micemmunized with pcDNA3.1-G22 were protected better against theMT-93-G22 tumor cells than the mice in the pcDNA3.1 and PBSroups, because the mice in this group survived longer in com-arison with the mice in the other two groups. Moreover, thisffective protection was G22-specific, because the survival timef the mice immunized with pcDNA3.1-G22 and challenged withMT-93-mock tumor cells did not increase (Fig. 6B).

. Discussion

Among the vaccine strategies developed to overcome immuneolerance to self-proteins, vaccination with anti-Id antibodies haseen described as a promising approach for treatment of severalalignant diseases [23]. Up to now, anti-Id vaccine has successfully

een applied into the therapy of lymphoma [24], colorectal car-

(33.98 ± 11.27)% (30.27 ± 9.85)% (35.71 ± 8.94)%(50.19 ± 11.28)% (46.78 ± 10.19)% (52.45 ± 10.27)%1.52 ± 0.18 1.58 ± 0.19 1.49 ± 0.08

cinoma [25], melanoma [26], ovarian cancer [27], non-small lungcancer [28], but few report on the therapy of nasopharyngeal carci-noma. A clinical trial of active immunotherapy with anti-idiotypeantibodies 2H4 and 5D3 in nasopharyngeal carcinoma patientshad been carried out in our laboratory, however, HAMA responseaffected their curative efficacy severely [29]. Antibody-variableregion fragments, obtained by phage display methodology haveattracted considerable attention, since these products can be easilyengineered for specific purposes such as therapy of cancer withoutinvoking HAMA response.

Numerous vaccination strategies against tumors have yieldedencouraging results in mice. Among them, DNA immunization hasemerged as an promising approach to developing effective vac-cines. In the clinical situation, vaccination protocols that can elicitcellular and humoral responses are expected to be more efficient fortumor protection and therapy. In our previous study, we had devel-oped a new anti-idoytpe vaccine, designated as pcDNA3.1-G22. Toevaluate the immune responses of the DNA vaccine pcDNA3.1-G22, C57BL/6 mice received three intramuscular injections of

pcDNA3.1-G22, pcDNA3.1 and PBS individually. Our observationsindicated that DNA vaccine pcDNA3.1-G22 could elicit strong anti-body response and the anti-anti-idiotypic antibodies elicited bypcDNA3.1-G22 were true Ab3 antibodies, as evidenced by thestrong inhibition of Ab1(FC2) binding toHNE2 cells by mice immune

C. Luo et al. / Vaccine 28 (2010) 2769–2774 2773

Fig. 5. Tumor protection in mice immunized with pcDNA3.1-G22. (A) Mice wereimmunized with either pcDNA3.1-G22, or the empty vector pcDNA3.1, or PBS, asindicated in the legends, followed by challenge with CMT-93-G22 tumor cells (n = 5).Each plasmid was administrated at 100 �g doses. The mean tumor volume of themice immunized with pcDNA3.1-G22 was significantly smaller than that in the micein the other two groups (PBS and pcDNA3.1) (p < 0.05). (B) Mice were immunizedwith pcDNA3.1-G22, and then challenge with CMT-93 tumor cells (n = 5). The meantw

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Fig. 6. Survival analysis of vaccinated mice challenged with tumor cells. (A) Micewere immunized with either pcDNA3.1-G22, or the empty vector pcDNA3.1, or PBS,as indicated in the legends, followed by challenge with CMT-93-G22 tumor cells(n = 5). Mice were monitored for tumor development until death. As shown in thefigure, the mice immunized with pcDNA3.1-G22 survived longer compared to the

[3] Baral R, Sherrat A, Das R, Foon KA, Bhattacharya-Chatterjee M. Murine mono-clonal anti-idiotypic antibody as a surrogate antigen for human Her-2/neu. Int

umor volume in these mice was significantly different from that in mice immunizedith pcDNA3.1-G22 and challenged with CMT-93-G22 (p < 0.05).

era which indicated the presence of antibodies sharing idiotypesith FC2 mAb.

Many studies have shown that anti-idiotypes are powerful stim-lators of CD4 responses, while the evidence of for stimulation ofD8 responses has been less convincing. In our study, we had inves-igated the T lymphocyte phenotypes at the 15th, 22th, 29th, 36thay after the third immunization, the results showed that bothD4+ and CD8+T cells had been activated and the predominantell subset was CD4+T cells. Traditionally, CD8+CTLs are an impor-ant effector arm in antitumor immunity, however, several recenttudies suggest that both CD4+ and CD8+ T cells are required tochieve tumor protection [30]. We could conclude from our resultshat both CD4+ T helper activity as well as CD8+ cytolytic activ-ty might have been elicited by three injections of pcDNA3.1-G22

hich might protect mice against tumor cells challenge.Theoretically, any kind of vaccine should be able to eradicate

he tumor. Few studies, however, demonstrated the effect of DNAaccine on the therapy of tumor, and most studies have focusedn the preventive effect of the DNA vaccine. Because HNE2 cellsere of human origin, the inoculation of HNE2 cells into C57BL/6ice would be rejected. Therefore, to investigate the protective

ffect of the DNA vaccine pcDNA3.1-G22, we first constructed aurine colonic cancer cell which could express the human G22

rotein and mainly on the cytoplasm and cytoblast (Figs. 1 and 2).hree intramuscular injections of pcDNA3.1-G22 suppressed, in a22-specific manner, the development of cancer expressing G22

n C57BL/6 mice (Fig. 5). The mice immunized with pcDNA3.1-G22ollowed with the challenge of CMT-93-G22 had a much longer lifepan than the mice in the control groups (Fig. 6).

mice in the other two groups (p < 0.05). (B) Mice immunized with pcDNA3.1-G22,followed by challenge with CMT-93 cells had a much shorter survival time comparedwith CMT-93-G22 (p < 0.05).

Many studies have shown that the antibody responses play littlerole in tumor protection, if any [31,32]. However, antibody responseprimed in vivo by G22 vaccination may be important to medi-ate tumor protection. Traditionally, CD4+T cells function as helpercells for antibody production, however, these cells have been alsoshown to have cytolytic functions inducing apoptotic and necroticcell death [33]. Therefore, G22 vaccination may prime Thl-typehelper cells which may enhance the cytolytic activity of CD8+T cellsby secretion of cytokines, such as IFN-�. Taken together, both thehumoral immunity and cellular immunity have been primed whichmay play an important role in mediating tumor protection.

Acknowledgements

The authors would like to thank Shuqian Dong for excellenttechnical assistance and Mingqing Chang for figure preparation.We also thank Weijia Zhang for critical and constructive reading ofthe manuscript.

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