Effective Melanoma Immunotherapy with Interleukin-2 Delivered by a Novel Polymeric Nanoparticle

2011;10:1082-1092. Published OnlineFirst April 25, 2011.Mol Cancer Ther Hong Yao, Samuel S. Ng, Long-Fei Huo, et al. Delivered by a Novel Polymeric NanoparticleEffective Melanoma Immunotherapy with Interleukin-2

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Preclinical Development

Effective Melanoma Immunotherapy with Interleukin-2Delivered by a Novel Polymeric Nanoparticle

Hong Yao1,2,3, Samuel S. Ng4, Long-Fei Huo3, Billy K. C. Chow4, Zan Shen3,5, Min Yang6, Johnny Sze2,3,Otis Ko6, Ming Li3, Alexander Yue3, Li-Wei Lu6, Xiu-Wu Bian1, Hsiang-Fu Kung1,7, and Marie C. Lin2,3

AbstractInterleukin-2 (IL-2) has been shown to possess antitumor activity in numerous preclinical and clinical

studies. However, the short half-life of recombinant IL-2 protein in serum requires repeated high-dose

injections, resulting in severe side effects. Although adenovirus-mediated IL-2 gene therapy has shown

antitumor efficacy, the host antibody response to adenoviral particles and potential biosafety concerns still

obstruct its clinical applications. Here we report a novel nanopolymer for IL-2 delivery, consisting of low

molecular weight polyethylenimine (600Da) linked by b-cyclodextrin and conjugated with folate (namedH1).

H1 was mixed with IL-2 plasmid to form H1/pIL-2 polyplexes of around 100 nm in diameter. Peritumoral

injection of these polyplexes suppressed the tumor growth and prolonged the survival of C57/BL6 mice

bearing B16–F1 melanoma grafts. Importantly, the antitumor effects of H1/pIL-2 (50 mg DNA) were similar to

those of recombinant adenoviruses expressing IL-2 (rAdv-IL-2; 2 � 108 pfu). Furthermore, we showed that

H1/pIL-2 stimulated the activation and proliferation of CD8þ, CD4þ T cell, and natural killer cells in

peripheral blood and increased the infiltration of CD8þ, CD4þ Tcells, and natural killer cells into the tumor

environment. In conclusion, these results show that H1/pIL-2 is an effective and safe melanoma therapeutic

with an efficacy comparable to that of rAdv-IL-2. This treatment represents an alternative gene therapy

strategy for melanoma. Mol Cancer Ther; 10(6); 1082–92. �2011 AACR.

Introduction

Melanoma is the most malignant skin cancer. Withthe frequency of cases rising rapidly, melanoma hasbecome one of the most fatal cancers in the past fewyears worldwide, especially in the white population(1, 2). Surgical resection, chemotherapy, and immu-notherapy are the conventional therapeutic strategiesfor patients with malignant melanoma. Although theefficacy of these therapies have been improved over thepast decade, the serious side effects induced by variouscytotoxic drugs and the low response rates observed in

melanoma patients have presented great challenges tophysicians (3, 4).

As an immune-modulating glycoprotein, interleukin-2 (IL-2) possesses strong antitumor activity by stimulat-ing the activity of T-lymphocytes and inducing thegrowth of natural killer (NK) cells and B cells (5, 6).Compared with other immunotherapeutic approaches,IL-2 has the best antitumor activity in patients withmalignant melanoma. Follow-up data through 2004confirmed the durability of responses produced byhigh-dose IL-2 regimen in patients with metastasismelanoma. However, the short half-life of IL-2 proteinin serum requires repeated high-dose injections, result-ing in severe side effects (7).

To overcome these limitations, intralesional delivery ofIL-2 gene by adenoviruses has been investigated in var-ious preclinical animal models (8–11). This approachenhanced tumor immunogenicity and led to tumorregression both at the injection site and distant sites,followed by the development of protective immunity.In particular, repeated intratumoral injections of recom-binant adenoviruses expressing IL-2 (rAdv-IL-2) devel-oped clinical responses in patients with metastaticmelanoma (12). However, the host antibody responseand potential biosafety issues associated with adenoviralvectors severely obstruct their clinical applications. Toaddress these problems, nonviral vectors, includingcationic lipids or polycationic polymers, are attractive

Authors' Affiliations: 1Institute of Pathology and Southwest CancerCenter, Southwest Hospital, Third Military Medical University, Chongqing,China; 2Department of Chemistry, The University of Hong Kong; 3BrainTumor Centre and Division of Neurosurgery, Department of Surgery, TheChinese University of Hong Kong, Shatin, Hong Kong; 4School of Biolo-gical Sciences, The University of Hong Kong; 5Department of Oncology,Affiliated 6th People's Hospital, Shanghai Jiao Tong University, Shanghai,China; 6Department of Pathology andCentre of Infection and Immunology,The University of Hong Kong; and 7Stanley Ho Center for EmergingInfectious Diseases, The State Key Laboratory in Oncology in South China,The Chinese University of Hong Kong, Hong Kong, China

Corresponding Author: M. C. Lin, Brain Tumor Centre, Division ofNeurosurgery, Dept of Surgery, The Chinese University of Hong Kong,Shatin, Hong Kong. Phone: 852-2646-2713,E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-10-0717

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 10(6) June 20111082





candidates because of their low immunogenicity, relativesafety, and low cost. Direct intratumoral injection of IL-2DNA/cationic lipid complex has been shown to be safeand efficient in patients with metastatic melanoma ormetastatic renal cell carcinoma in clinical trials (13, 14).However, the efficiency of these nonviral vectors for IL-2gene delivery was lower than that of rAdv-IL-2.Recently, we have synthesized a novel polycationic

vector, H1, which consists of low molecular weightpolyethylenimine (600Da) linked by b-cyclodextrin andconjugated with folic acid. We have shown that H1 isa biodegradable, tumor specific, and efficient genedelivery vector in vitro and in vivo (15). Here, we reportthat subcutaneous administration of H1-human IL-2plasmid polyplexes (H1/pIL-2) could effectively reducemelanomagrowth andprolong the survival ofmelanoma-bearing mice. Importantly, the antitumor effects ofH1/pIL-2 (50 mg DNA) were similar to those of rAdv-IL-2 (2 � 108 pfu). Furthermore, we found that activation,proliferation, and infiltration of CD4þ, CD8þ T cells, andnatural killer (NK) cells were involved in the antitumoreffects ofH1/IL-2. Interestingly, thepercentage of Foxp3þin CD25þ/CD4þ cells inmice treatedwith H1/pIL-2wassignificantly lower than that of control mice, an observa-tion which is different from the IL-2 treatment.

Materials and Methods

Production of H1 and plasmidsH1 was synthesized using a method described by Yao

and colleagues (15). Enhanced green fluorescent protein(EGFP), firefly luciferase and human IL-2 cDNAs wereobtained by polymerase chain reaction (PCR) usingpEGFP-N1 (BD Biosciences), pGL3 Luciferase reportervector (Promega Corporation), and pDC-IL2-WRPE (con-structed by our laboratory) as templates, respectively.The cDNAs encoding EGFP, Luciferase, and human IL-2were subcloned into the pAM/CAG-WPRE-BGHpolyAplasmid and generate pAM/CAG-EGFP (pEGFP), pAM/CAG-Luciferase (pLuc), and pAM/CAG-IL-2 (pIL-2)plamids, respectively.

Production of rAdv-IL-2 or EGFPThe cDNAs encoding human IL-2 and EGFPwere sub-

cloned into pAd/CMV/DEST (Invitrogen) to generatepAd-IL-2 and pAd-EGFP, respectively. The pAd-IL-2 orpAd-EGFP plasmid was then transfected into 293A cellsusingLipofectamine 2000Reagent (Invitrogen) toproducerAdv-IL-2 or EGFP (rAdv-EGFP). The resulting viruseswere purified by cesium chloride gradient centrifugationand titteredusing tissue culture infectiousdose 50method(AdEasy Vector System Application manual, Qbiogene).The viruses were stored at �80�C before use.

Characterization of H1/pIL-2 polyplexesTo prepare H1/pIL-2 polyplexes, the H1 polymer solu-

tion was mixed with the pIL-2 plasmid solution (N/P

ratio of 20:1) with an equal volume of 5% glucose. TheH1/plasmid DNA polyplexes were characterized bytransmission electron microscopy. Briefly, the H1/pIL-2polyplexes (50 ml) were dropped to glow-discharged, 400-mesh carbon-coated copper grids. After drying at roomtemperature, images were recorded by a Philips 201electron microscope operated at 80 kV.

Animals and cellsFemale C57BL/6N mice (6–9 weeks old) were pur-

chased from Charles River Laboratories and werehoused under aseptic conditions and cared for accord-ing to the guidelines from the Laboratory Animal Unitof the University of Hong Kong. The mice were fed forthree days before the experiment. All experimentalprotocols were approved by the Department of Healthof the Government of Hong Kong Special Administra-tive Region and the Committee on the Use of LiveAnimals in Teaching and Research of the Universityof Hong Kong.

Mouse melanoma B16-F1 cells were obtained fromAmerican Type Culture Collection (CRL-6323), whichwas banked after receipt, and passaged for less than6 months before use in this study. The American TypeCulture Collection characterize cell lines using shorttandem repeat polymorphism analysis. The cells werecultured in DMEM supplemented with 10% heat inacti-vated fetal bovine serum, 100 U/ml penicillin, and 100mg/ml streptomycin (Invitrogen) and incubated at 37�Cin 5%CO2. Before the start of this study, tumorigenicity ofthe B16-F1 cells were tested by subcutaneous injection ofthe cells (2� 105 cells/mice) into the flank of 10 C57/BLNmice (6–9 weeks old). The cells were tumorigenic in 100%of the injected mice.

Transfection efficiency of H1/pIL-2 in B16-F1 cellsin vitro

B16-F1 cells were seeded at a density of 1 �105 cells/well in a 24-well plate for 24 hours. The previouslyprepared H1/pIL-2 polyplexes solution was added ineach well (1mg DNA, N/P ratio 20:1). At 48 hours aftertransfection, the total RNA of transfected cells wasextracted. The following primer sequences were usedto detect the cDNA of the human IL-2 gene: Sense:50-ATGTACAGGATGCAACTCCTGTCT-30 Anti-Sense:50- TCAAGTCAGTGTTGAGATGATGCTT-30.

Animal model and treatmentsB16-F1 cells were harvested at the exponential growth

phase and one hundred microliters of the cell suspension(2 � 105 cells) was subcutaneously injected in the rightflank of each C57BL/6 mouse. When the tumor grew toabout 0.2–0.4 cm, the mice were treated by peritumoralinjection with H1/pIL-2 (50 mg DNA), H1/pIL-2 (25 mgDNA), H1/pEGFP (50 mg DNA), 5% glucose and IL-2plasmid alone (50 mg), respectively. Each mouse wastreated once every two days till the desired endpoint.To compare the antitumor efficacy between rAdv-IL-2

Melanoma Immunotherapy with IL-2 Delivery by Nanopolymer

www.aacrjournals.org Mol Cancer Ther; 10(6) June 2011 1083





and H1/pIL-2, the tumor bearing mice were grouped asbelow: (i) rAdv-IL-2 (2 � 108 pfu) injection once every 5days; (ii) rAdv-IL-2 (2� 108 pfu) single injection; (iii) H1/pIL-2 (50 mg DNA) once every 2 days; (iv) rAdv-EGFP (2� 108 pfu); and (v) PBS, respectively. Tumor growth wasmonitored by measuring the two maximum perpendicu-lar tumor diameters with a caliper. Tumor volume (V)was calculated by the formulaV¼ 1/2� LS2, where L andS are the longest and the shortest diameters of the tumor,respectively.

In vivo imagingMicewere anesthetized and thenD-luciferin (Xenogen)

at a dose of 150 mg per gram mouse body weight wasinjected intraperitoneally. After that, wait for 5 minutesand take picture. The images were obtained at exposuretime from 1 to 10 minutes depending on the intensity ofthe emitted photons. The total photon flux (photons/second) in a region of interest (ROI) was quantitated bythe Living Image Software (Xenogen).

Histological distribution of H1/pEGFP polyplexesThe mice were injected with H1/pEGFP (50 mg DNA,

N/P 20:1) by peritumoral injection. At 24 hours postinjection, the tumor tissues were excised, fixed, andembedded in optimum cutting temperature medium.The embedded tissues were sectioned at 6 mm by afreezing microtome. The specimen was mounted withpropidium iodide solution and then directly examined byfluorescence microscope.

Immune cell and cytokine profiles in bloodThe peripheral blood was collected from mice and

then mixed with anticoagulant immediately. Serumand plasma samples were separated by centrifugationat 300 � g for 10 minutes at 4� C. The serum cytokineprofiles were analyzed by Th1/Th2 10plex kit andFlowCytomixPro 1.0 Software (Bender MedSysterms)according to the manufacturer’s instructions. Theimmune cell profiles of the plasma samples were ana-lyzed by flow cytometry. To detect the expression ofCD69 in T cells and NK-cells, plasma samples werelabeled with PE-CD4, PE/Cy5-CD8, PE-conjugated ratantimouse pan-NK and FITC-CD69 cocktail of antibo-dies. To detect Tregs in the plasma samples, a mouseregulatory T-cell staining kit (eBioscience) was used.Treg staining was carried out by following the protocol(eBioscience). To detect Ki67 antigen in CD4þ andCD8þ T cells, an intracellular staining protocol recom-mended by BD Pharmingen was used. Flow cytometryanalysis was carried out by Epics Altra (BechmanCoulter) and WinMDI (version 2.9) was used for dataanalysis.

Immunohistochemical staining of tumor tissueThe specimens of tumor tissues were incubated by the

antimousemonoclonal antibody of CD4-PE/Foxp3-FITC;CD8-FITC; and CD49-PE alone, CD8-FITC/CD25-PE and

CD49b-PE, respectively. CD16/CD32 antibody was usedto block nonspecific binding. 40, 6 diamidino 2 phenylin-dole (DAPI) solution was used for nuclear staining beforemounting the slides.

Stereologic quantification of tumor-infiltratingT cells

To quantify tumor-infiltrating T cells, the stereologicmethod depicted by Henrik and colleagues was usedwith minor modifications (16). Briefly, immunohisto-chemical fluorescence images taken from tumor sectionswere analyzed by the software of Image–Pro Plus 6.0(Media Cybernetics) at�20 magnification. The density oftumor-infiltrating lymphocytes was scored by countingall positive cells intratumorally and relating them to anarea of interest, representing the total tumor area mea-sured stereologically, excluding necrotic tumor tissueand nontumor tissue such as peritumoral connectivetissue. One section of each sample was examined inthe stereologic study.

Results

Characterization of H1/pIL-2 polyplexes andexpression of IL-2 in B16-F1 cells transfected withH1/pIL-2

The schematic diagrams of pEGFP, pIL-2, and pLucplasmids are shown in Fig. 1A. As shown in Fig. 1B, H1could condense and package the pIL-2 plasmids to acompact globular structure with a particle size of around100 nm. The expression of IL-2 in H1/pIL-2-transfectedB16-F1 cells was determined by reverse transcription–PCR at 48 hours posttransfection. A high level of IL-2mRNA expression was detected in B16-F1 cells trans-fected with H1/pIL-2, whereas IL-2mRNAwas absent inthe control B16-F1 cells transfected with H1/pEGFP(Fig. 1C).

Expression and histological distribution oftransgene induced by H1/pLuc or H1/pEGFPpolyplexes

To evaluate the dynamic change of transgene expres-sion over time in mice injected with H1/pLuc poly-plexes, C57/BL6 mice bearing B16-F1 tumors wereinjected with naked pLuc (50 mg), H1/pLuc (25 mgDNA, N/P ratio 20:1) or H1/pLuc (50 mg DNA, N/Pratio 20:1) by peritumoral injection. The expression ofluciferase was then monitored by a cooled charge-coupled device camera from day 1 to day 5. As shownin Fig. 1D, the peak value of luciferase activity inducedby H1/pLuc (50 mg, DNA) occurred at 24 hours post-transfection. It was then reduced by 40% and 60% at48 hours and 72 hours, respectively. The peak lucifer-ase activity of H1/pLuc (25 mg DNA) also appeared at24 hours posttransfection, but the value was only onethird of that of H1/pLuc (50 mg DNA) and the biolu-minescent signals were observed only for 3 days. Theseindicated that efficiency and duration time of gene

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Mol Cancer Ther; 10(6) June 2011 Molecular Cancer Therapeutics1084





expression were dependent on the dosage of H1/pLucinjected. For the mice injected with naked pLuc (50 mgDNA), the signals could be observed only when theexposure time was extended to 10 minutes, indicating

that the luciferase activity produced by naked pLucwas very weak.

On day 3 after the first injection, the second injectionwas carried out in the samemice. Twenty-four hours after

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Figure 1. Expression and histological distribution of IL-2 delivered by H1. A, schematic diagrams of pAM/CAG-EGFP (pEGFP), pAM/CAG-luciferase(pLuc), and pAM/CAG-interleukin-2 (pIL-2) plasmids. B, transmission electron microscopy image showing the globular morphology of H1/pIL-2 polyplexesat an N/P ratio of 20. Scale bar, 200 nm. C, RT–PCR showing the mRNA expression of IL-2 and GAPDH in B16-F1 cells at 48 hours after transfectionwith H1/pIL-2 or H1/pEGFP. D, representative images of C57BL/6 mice with B16-F1 grafts displaying in vivo bioluminescence at 24, 48, and 72 hoursafter peritumoral injectionwith H1/pLuc (50 mgDNA, N/P ratio 20:1). The total photon flux (photons/second) in a region of interest (ROI) is shown in boxes. E, thedynamic change of gene expression transduced by peritumoral injection with H1/pLuc (50 mg DNA), H1/pLuc (25 mg DNA), and pLuc (50 mg DNA) alone inmelanoma grafts in mice. The total flux of ROI over time (ROI/minute) was used as a parameter to compare their transfection efficiency. The arrow indicatesthe time point when the second injection was carried out (n ¼ 4). F, representative histological sections of the tumor tissues collected from the miceperitumorally injected with H1/pEGFP (50 mg DNA) and the 5% glucose, respectively. The nuclei were stained by propidium iodide (PI). TB, tumor border;SC, subcutaneous connective tissue.







the second injection, the luciferase activity reboundedandwas almost similar to the first injection in all 3 groups(Fig. 1E). We also examined the tissue distribution oftransgene expression in the mice injected with H1/pEGFP polyplexes. Histological analysis showed thatEGFP expression was localized at the tumor border,subcutaneous fibro connective tissue, and muscle fibrotissue near the tumor mass (Fig. 1F).

H1/pIL-2 and rAdv-IL-2 effectively reduced B16-F1melanoma growth

Next, we tested whether H1/pIL-2 is efficaciousin treating melanoma in a mouse model. Comparedwith the control mice injected with H1/pEGFP, pIL-2plasmid alone, or 5% glucose, the growth of subcuta-neously inoculated B16-F1 melanoma was effectivelyinhibited in the mice receiving H1/pIL-2 (25 mg DNA)or H1/pIL-2 (50 mg DNA). The tumor growth was muchslower in the group treated with H1/pIL-2 (50 mg DNA)than the group treated with H1/pIL-2 (25 mg DNA)(Fig. 2A). In addition, the survival of the mice treatedwith H1/pIL-2 (25 mg DNA) or H1/pIL-2 (50 mg DNA)

was markedly prolonged as compared with that of thecontrol mice (Fig. 2B).

We also compared the efficacy of H1/pIL-2 andrAdv-IL-2 in suppressing the growth of melanoma inmice bearing the B16-F1 tumor grafts. In mice receivingsingle or multiple intratumoral injections of rAdv-IL-2(2 � 108 pfu) or peritumoral injection of H1/pIL-2(50 mg DNA, N/P ratio 20:1), tumor growth wasgreatly inhibited as compared with those treated withrAdv–EGFP (2 � 108 pfu) or PBS. In particular, thetumor growth of mice treated with H1/pIL-2 wasslower than that of mice treated with a single injectionof rAdv–IL-2. Furthermore, H1/pIL-2-injected micehad tumor sizes almost similar to those receivingmultiple injections of rAdv–IL-2, which elicitedthe most potent antitumor effects among the fiveexperimental groups (Fig. 3A and 3B).

To evaluate the antitumor function of peritumoral H1/pIL-2 treatment on the metastatic melanoma, we estab-lished an additional melanoma mouse model by inocu-lating melanoma cells subcutaneously into both left andright flanks of the mice. The left-flank tumor was treatedwith peritumoral injection of H1/pIL-2 (50 mg DNA)or H1/pEGFP (50 mg DNA), whereas the right-flanktumor (distal tumor) was kept untreated. We found thatH1/pIL-2, but not the control H1/pEGFP, significantlysuppressed the growth of the untreated distal tumors.However, in theH1/pIL-2-treatedmice, the growth of theuntreated distal tumors was still faster than that of thetreated tumors (Fig. 3C).

Stimulation of activation and proliferation of CD4þT cells, CD8þ T cells, and NK-cells in peripheralblood after administration of H1/pIL-2

We used flow cytometry to analyze the immuneresponses elicited in the H1/pIL-2-treated mice. CD69is recognized as a cell surface antigen expressed byactivated NK cells, T cells, B cells, and other subsets ofimmune cells. It is a pleiotropic molecule for the media-tion of T-cells and NK-cells, which lead to the secretion ofTh1/Th2 cytokines and activation of immune cell pro-liferation and cytotoxicity (17). On day 3 after the firsttreatment, the percentage of CD49þ/CD69þ cells in theperipheral blood lymphocytes (PBL) of the H1/pIL-2-treated mice was 5-fold and 30-fold of that in the controlH1/pEGFP-treated and 5% glucose-treated mice, respec-tively. Similarly, the percentage of CD4þ/CD69þ andCD8þ/CD49þ cells in the H1/pIL-2-treated mice wassignificantly higher than that in the control mice. (Fig. 4Aand 4B; **, P < 0.05; n ¼ 4; Student’s t-test). Theseresults suggested that CD4þ, CD8þ T cells, and NKcells were activated in peripheral blood of mice treatedby H1/pIL-2. Meanwhile, we also detected Ki67 expres-sion in theCD4þ andCD8þT-cells of PBL by intracellularstaining. The percentage of Ki67þ/CD4þ and Ki67þ/CD8þ T cells of mice treated by H1/pIL-2 was 2.6-foldand 3.7-fold of that in the control H1/pEGFP-treatedmice, respectively (Fig. 4C and 4D), suggesting that the

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Figure 2. Effects of H1/pIL-2 polyplexes on tumor growth and survival ofmicebearingB16-F1grafts.A, thevolumeofmelanomagrafts inmice treatedwith H1/pIL-2 (50 mg DNA), H1/pIL-2 (25 mg DNA), 5% glucose, H1/pEGFP(50 mg DNA), and pIL-2 (50 mg DNA), respectively. B, survival of melanomagrafts in mice treated with the same reagents mentioned previously.

Yao et al.






CD4þ and CD8þ effectors Tcells were proliferative afterH1/pIL-2 treatment.In addition to thementionedCD8þ, CD4þ, andCD49þ

effector cells, we also investigated the effect of H1/pIL-2

treatment on Tregs. The percentage of CD25þ/CD4þcells in the PBL of the mice treated with H1/pIL-2 was3.1-fold and 3.7-fold higher than that of mice treated withthe H1/pEGFP and 5% glucose-treatedmice, respectively(Fig. 4E).Whenwe gated onCD4þ cells, the percentage ofCD25þ/Foxp3þ cells of the mice treated by H1/pIL-2were 0.9-fold and 1.3-fold lower than that of H1/pEGFP-treated and 5% glucose-treated mice, respectively(Fig. 4F). By contrast, CD25þ/Foxp3-population wassignificantly increased and CD25-/Foxp3-populationwas not changed by the H1/pIL-2 treatment.

On day 7 after the first treatment, peripheral bloodsamples were collected respectively from mice receivingsubcutaneous administration of H1/pIL-2 (25 mg DNA),H1/pIL-2 (50 mg DNA), or H1/pEGFP (50 mg DNA). Wefound that the populations of CD4þ and CD8þ T-cellsfrom mice treated with H1/pIL-2 (25 mg DNA) increasedslightly, while the mice treated with H1/pIL-2 (50 mgDNA) increased significantly compared with those trea-ted with H1/pEGFP (Fig. 5A and 5B). The population ofCD49þ NK-cells in mice treated with H1/pIL-2 (50 mgDNA) and H1/pIL-2 (25 mg DNA) was 2-fold and 1-foldhigher than those treated with H1/pEGFP, respectively(*, P < 0.05; **, P < 0.001; n¼ 4; Student’s t-test). On day 14posttreatment, higher levels of CD4þ and CD49þ cellswere also detected in the mice treated with H1/pIL-2,whereas the population of CD8þ cells declined and therewas no significant difference between the percentage ofCD8þ cells in the PBL of H1/pIL-2-, H1/pEGFP-, and 5%glucose-treated mice (Fig. 5C).

Increase of Th1, Th2, and Th17 cytokine secretioninduced by H1/pIL-2 administration

To investigate the changes in the blood cytokine levelsofmice treatedwithH1/pIL-2 polyplexes, serum samplesfrommice treated with H1/pIL-2 (25 mg DNA), H1/pIL-2(50 mg DNA), or H1/pEGFP (50 mg DNA) were collectedand analyzed on day 7 after the first treatment. The serumTh1 cytokines (e.g., IL-2, IFN-g , and TNF-beta), Th2cytokines (IL-4, IL-6) and IL-17 in the mice treated withH1/pIL-2 (25 mg DNA) and H1/pIL-2 (50 mg DNA)increased significantly compared with those mice treatedwithH1/pEGFP. IL-2, IFN-g , TNF-beta, IL-4, IL-6, and IL-17 in the mice treated with H1/pIL-2 (50 mg DNA) were2.0-, 2.6-, 2.7-, 4.5-, 3.5-, and 2.1-fold higher than the micetreated with H1/pEGFP (50 mg DNA), respectively(Fig. 5D; *, P < 0.05; **, P < 0.001; n ¼ 4; Student’s t-test).The increase of Th1 and Th2 cytokines in peripheralblood of mice suggested that H1/pIL-2 treatment acti-vated the cell-mediated and humoral immune responsesof tumor bearing mice.

H1/pIL-2 increased the density of tumor-infiltratingT cells and NK cells

We also assessed the effects of H1/pIL-2 administra-tion on the degree of CD4þ/Foxp3þ, CD8þ T cells, andNK cells infiltration into the tumor. Immunofluorescencestaining was used to stain B16 tumor specimens taken out

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Figure 3. The comparison of antitumor effects of H1/pIL-2 and rAdv-IL-2treatments. A, survival of melanoma grafts in mice treated with PBS,rAdv–EGFP (2� 108 pfu), rAdv-IL-2 (2� 108 pfu) single injection, H1/pIL-2(50 mg DNA), and rAdv-IL-2 (2� 108 pfu) multiple injection, respectively. B,the volume of melanoma grafts in mice treated with the same reagentsmentioned previously. C, the volume of themelanoma grafts in mice whereboth flanks were inoculated with melanoma cells (**, P < 0.05; n ¼ 4;Student's t-tests).







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Figure 4. Activation of CD8þ T cells, CD4þ T cells, NK cells, and CD4þ/CD25þ T cells (but not Foxp3þ/CD4þ/CD25þ T cells) in the peripheral blood of tumorbearing mice treated with H1/pIL-2. A, the percentage of CD49þ/CD69þ, CD4þ/CD69þ, and CD8þ/CD69þ lymphocytes in H1/pIL-2-, H1/pEGFP-,and 5% glucose-treated mice, respectively. B, representative density plots showing the percentage of CD49þ/CD69þ, CD4þ/CD69þ, and CD8þ/CD69þcells in the peripheral blood of mice treated with H1/pIL-2, H1/pEGFP, and 5% glucose, respectively. C and D, Ki67 expression in CD4þ and CD8þ T cellsof H1/pIL-2-, H1/pEGFP-, and 5% glucose-treated mice, respectively. E, the percentage of CD4þ/CD25þ cells in H1/pIL-2-, H1/pEGFP-, and 5%glucose-treated mice, respectively. Bottom, the relative representative density plots are shown. The square frames in the density plots representthe CD4 gated region for subsequent analysis of Foxp3þ/CD25þ cells. F, the percentage of Foxp3þ/CD25þ cells when we gated on CD4þ lymphocytes.Bottom, the representative density plots are shown.

Yao et al.






on day 3 after the first H1/pIL-2 treatment. One sectionfrom each tumor sample was stained and the density oftumor-infiltrating lymphocytes (TIL) was scored bycounting positively stained cells in a stereologically mea-sured area of interest, which excluded peritumoral con-nective tissue and necrotic tissue. The density of CD8þTIL in the mice treated with H1/pIL-2 was 4-fold higherthan that of 5% glucose-treated group (Fig. 6A and 6B). Inall sections stained by Foxp3-FITC/CD4- PE cocktailantibodies, Foxp3 positive signals were absent in thetumor cells and only a low level of Foxp3 could bedetected in the peritumoral connective tissue, whichdid not merge with CD4 positive signals. We thereforedefined those CD4 positive cells as CD4þ/Foxp3-cells.As shown in Fig. 6C and 6D, the density of CD4þ/Foxp3-TIL in themice treatedwithH1/pIL-2was 1.4-fold higherthan that of 5% glucose-treated group. We also usedCD49-PE monoclonal antibody to detect NK cells inthe tumor tissue. We found that the density of tumor-infiltrating NK-cells in the mice treated with H1/pIL-2was 4.1-fold higher than 5% glucose treatedmice. (Fig. 6Eand 6F; n ¼ 4; ***, P < 0.001; Student’s t-test).

Discussion

Our in vivo imaging data indicated that transgeneexpression induced by H1/pLuc peaked at 24 hourspostinjection and subsequently decreased. When the sec-

ondary injection was carried out, a high-level transgeneexpression recurred. Based on these results, to maintain ahigh level of IL-2 expression, we injected the tumor-bearing mice once every two days with H1/pIL-2 byperitumoral injection. Antitumor activities wereobserved in mice treated with H1/pIL-2 (50 mg DNA)or H1/pIL-2 (25 mg DNA), but not in those treated with5% glucose, H1/pEGFP (50 mg DNA) or IL-2 (50 mg DNA)plasmid alone.We also found that the antitumor effects ofH1/pIL-2 (50 mg DNA) were better than H1/pIL-2 (25 mgDNA). These results indicate that a high level of IL-2induced by H1/pIL-2 injection plays a major role ininhibiting the growth of melanoma.

It has been documented that rAdv-IL-2 can induceregression and immunity in melanoma (8, 9, 11, 12).Hence, we used rAdv-IL-2 as a benchmark to comparethe efficacy of H1/pIL-2 to inhibit melanoma growth. Wefound that the antitumor efficacy of H1/pIL-2 (50mgDNA) was better than that of rAdv-IL-2 (2 � 108 pfu)with single injection and comparable with that of rAdv-IL-2 (2 � 108 pfu) with multiple injections. Our previousstudy showed that the level of luciferase gene expressiontransduced by a single injection of rAdv–luc peaked at 48hours, declined subsequently, and then maintained at aconstant level for 7–10 days (15). Thus, the amount of IL-2produced by a single injection of rAdv-IL-2 could inducetumor suppression only at an early stage. The decline ofthe luciferase gene expression after 48 hours is likely due

Figure 5. Lymphocyte andcytokine profiles in the peripheralblood of tumor bearing micetreated with H1/pIL-2 and H1/pEGFP, respectively. A, thepercentage of CD4þ T cells,CD8þ T cells, and CD49þ NKcells of H1/pIL-2 (50 mg DNA)-, H1/pIL-2 (25 mg DNA)-, and H1/pEGFP (50 mg DNA)-treated mice,respectively, on day 7posttreatment. B, representativehistograms of flow cytometricanalyses showing the percentageof CD4þ, CD8þ, and CD49þ cellsin PBL of the treated mice. C, thepercentage of CD4þ, CD8þ, andCD49þ cells of H1/pIL-2-, 5%glucose- and H1/pEGFP-treatedmice, respectively, on day 14posttreatment. D, cytokinesprofiles of the peripheral blood inmice treated with H1/pIL-2 (25 mgDNA), H1/pIL-2 (50 mg DNA), andH1/pEGFP (50 mg DNA),respectively.

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to the presence of antiadenoviral neutralizing antibodiesin the mice. On the other hand, repeated peritumoralinjection of H1/pIL-2 could maintain IL-2 at a constantlyhigh level for tumor suppression, and the polyplexeswould not be attenuated by specific host antibodies.Therefore, the antitumor efficacy of H1/pIL-2 was com-parable with that of rAdv-IL-2 with multiple injections.Together, these results show that a continued expressionof IL-2 is necessary for its antitumor activity in ourmelanoma model.

We also analyzed the local and systemic immuneresponses elicited by H1/pIL-2 treatment. We found thatH1/pIL-2 significantly increased the density of CD8þ,CD4þ/Foxp3-, and CD49þ tumor infiltrating lympho-cytes, suggesting that the antitumor effects induced byH1/pIL-2 may be attributed to the activated and tumorinfiltrating CD8þ, CD4þ/Foxp3- T cells, and NK cells.Furthermore, peritumoral injection of H1/pIL-2 couldprovoke a systemic antitumor response by increasingthe activation and proliferation of the circulatingCD8þ, CD4þ/Foxp3- T cells, and NK cells, which may

be responsible for inhibiting the growth of the untreateddistal tumor in our mouse melanoma model.

Besides inducing these antitumor immune effectors,IL-2 also regulates the Treg lymphocyte balance, whichplays an important role in peripheral tolerance to self-antigen and suppress antitumor immune response (18,19). Treg cells are generally defined as CD4þ/CD25þ Tcells with an expression of transcription factor Foxp3.Foxp3 acts as a key regulator for the development andimmune repression function of Tregs (20, 21). In thisstudy, we found that H1/pIL-2 significantly upregulatedthe expression of CD25 in CD4þ T cells but the frequencyof Foxp3 in CD4þ/CD25þ cells was reduced. Theincrease of CD25 expression was consistent with theactivation of IL-2 on T cells (22). However, reductionof Foxp3þ Tregs population by H1/pIL-2 treatment wasdifferent from the systemic increase in Tregs observed inIL-2 therapy. We speculate that more complex mechan-isms are involved in regulating Foxp3 expression in Tregspresent in theH1/pIL-2-treatedmice. In this study, a highlevel of IL-6, IL-4, and IL-17 cytokines were detected in

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Figure 6. Immunohistochemicalstaining of tumor tissues. A, C,and E, representativeimmunofluorescence photosshowing CD8þ, CD4þ/Foxp3- Tcells and NK cells in the tumortissues excised from the mice onday 3 after first H1/pIL-2 treatment(magnification of A and C, �200;magnification of E, �320). CD8-FITC, CD4-PE, and CD49-PEsignals are CD8þ, CD4þ T, andNK cells, respectively. The cellnuclei are stained by DAPI.B, D, and F, stereologicalanalysis showed that the densityof TIL such as CD8þ, CD4þ/Foxp3- T cells and NK cells inthe mice treated by H1/pIL-2and 5% glucose, respectively(n ¼ 4; ***, P < 0.001;Student's t-test).

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the peripheral blood ofmice injectionwithH1/pIL-2. IL-6and IL-4 are proinflammatory cytokines, which havebeen documented to decrease Foxp3 expression anddownregulate the functions of Tregs in vivo and in vitro(23–26). IL-17 is also a proinflammatory cytokine, whichis secreted by Th17 cells. The high level of IL-17 and lowlevel of Tregs existing in peripheral blood of H1/pIL-2treated mice is in line with the previous findings that IL-6can promote the differentiation of naive T cells into Th17cells rather than Foxp3þ Tregs (27).Our immunofluorescence staining showed that little

CD4þ/Foxp3þ Tregs were rarely found within the H1/pIL-2-treated tumors, indicating that mechanisms otherthan Tregs are responsible for the regulating the anti-tumor effects induced by H1/pIL-2. Based on our datashowing the increased infiltration of CD8þ T cells andNK cells into the tumor, it is also likely that innateimmune cells like plasmacytoid dentritic cells andNKT cells played important roles in this process. Liuand colleagues reported that CpG-activated plasmacy-toid dendritic cells could activate NK cells, conventionaldentritic cells and CD8þ T cells to initiate effective andsystemic antitumor immunity (28). Zaini and colleaguesshowed that NKT cells were responsible for the antitu-mor immunity induced by activated DC cells (29). In theirB16 melanoma transplant model, these innate immunecells, when activated by Toll-like receptor (TLR) ligands,could produce chemokines and cytokines which in turnactivate CD8þ T cells. The plasmid used in our experi-ments is a bacterially derived plasmid, which containsunmethylated CpG dinucleotides (30, 31). We speculatethat the CpG dinucleotides present in the plasmid couldbe the potential TLR ligands, which stimulate dentriticcells and macrophages by the Toll pathway and even-tually activate plasmacytoid dentritic cells andNKT cells.Meanwhile, these activated antigen-presenting cells alsoproduced proinflammatory cytokines (e.g., IL-6 and IL-4)(32–33), which could decrease Foxp3 expression and thepopulation of Foxp3þ Tregs in circulating CD4þ T-cells.

Because the H1/pEGFP polyplexes did not induce anypotent antitumor response, the plasmid CpG alone couldnot account for the observed antitumor activity in theH1/pIL-2-treated mice. We hypothesize that the innateimmune cells may act as an initiating factor to break theimmune tolerance induced by tumor cells, and tip theimmune balance regulated by IL-2 to antitumor immu-nity rather than immune tolerance.

In conclusion, we have established that peritumoralinjection of H1/pIL-2 suppressed tumor growth andprolonged the survival in C57/BL6 mice bearing B16-F1 melanoma grafts. The antitumor effect of H1/pIL-2can be attributed mainly to the activation, proliferation,and infiltration of effector T cells and NK cells intotumor. The therapeutic efficacy of H1/pIL-2 wasdose-dependent, of low toxicity, and similar to that ofrAdv-IL-2 (2 � 108 pfu). Overall, our data show thatH1/pIL-2 represents a promising gene therapeuticstrategy for melanoma.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Wewould like to thankMr. Hong–Ping Xia, Mr. Yan–Xin Ren, andMr.Su-Guang Zuo for their support in the animal study.

Grant Support

The work was supported by the funding from the Innovation and TechnologyFund (ITS/243/09 to M.C. Lin and S.S. Ng) of the Government of the Hong KongSpecial Administrative Region, the National Natural Science Foundation ofChina (PC: 81001023), and the Natural Science Foundation of ChongQing (CSPC:2010BC5007).

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 August 2, 2010; revised March 11, 2011; accepted March 29,2011; published OnlineFirst April 25, 2011.

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Effective Melanoma Immunotherapy with Interleukin-2 Delivered by a Novel Polymeric Nanoparticle

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